Progress in Polymer Science 53 (2016) 86–168

Contents lists available at ScienceDirect

Progress in Polymer Science

journal homepage: www.elsevier.com/locate/ppolysci

Advancing biomaterials of human origin for tissue

engineering
Fa-Ming Chen a,b,∗ , Xiaohua Liu c
a
State Key Laboratory of Military Stomatology, Department of Periodontology, School of Stomatology, Fourth Military Medical
University, Xi’an 710032, PR China
b
State Key Laboratory of Military Stomatology, Translational Research Team & Biomaterials Unit, School of Stomatology, Fourth
Military Medical University, Xi’an 710032, PR China
c
Department of Biomedical Sciences, Texas A&M University Baylor College of Dentistry, Dallas, TX 75246, USA

a r t i c l e i n f o a b s t r a c t

Article history: Biomaterials have played an increasingly prominent role in the success of biomedical
Received 1 June 2014 devices and in the development of tissue engineering, which seeks to unlock the regen-
Received in revised form 14 January 2015 erative potential innate to human tissues/organs in a state of deterioration and to restore
Accepted 27 February 2015
or reestablish normal bodily function. Advances in our understanding of regenerative bio-
Available online 28 March 2015
materials and their roles in new tissue formation can potentially open a new frontier in the
fast-growing field of regenerative medicine. Taking inspiration from the role and multi-
Keywords:
component construction of native extracellular matrices (ECMs) for cell accommodation,
Raw materials
the synthetic biomaterials produced today routinely incorporate biologically active compo-
Biopolymers
Extracellular matrix nents to define an artificial in vivo milieu with complex and dynamic interactions that foster
Tissue decellularization and regulate stem cells, similar to the events occurring in a natural cellular microenviron-
Biomimetic design ment. The range and degree of biomaterial sophistication have also dramatically increased
Blood-derived biomaterials as more knowledge has accumulated through materials science, matrix biology and tis-
Transplantation sue engineering. However, achieving clinical translation and commercial success requires
regenerative biomaterials to be not only efficacious and safe but also cost-effective and
convenient for use and production. Utilizing biomaterials of human origin as building
blocks for therapeutic purposes has provided a facilitated approach that closely mimics the
critical aspects of natural tissue with regard to its physical and chemical properties for the

Abbreviations: 3D, three-dimensional; ACI, autologous chondrocyte implantation; ACS, absorbable collagen sponge; AM, amniotic membrane; ASI,
alternating solution immersion; ASCs, adipose-derived stem cells; BBIM, bioactive bone-inducing material; BM, basement membrane; BMC, bone marrow
concentrate; BMPs, bone morphogenetic proteins; CaP, calcium phosphate; CBD-BMP-2, collagen-binding domain bone morphogenetic protein-2; CCC,
cortical cancellous chips; CMC, carboxymethylcellulose; CNTF, ciliary neurotrophic factor; CS, chondroitin sulfate; CTA, complex tissue allotransplanta-
tion; DBM, demineralized bone matrix; DDM, demineralized dentin matrix; Dex-GMA, glycidyl methacrylate-derivatized dextran; DFDBAs, demineralized
freeze-dried bone allografts; DMD, Duchenne muscular dystrophy; ECGF, epithelial cell growth factor; ECM, extracellular matrix; EDTA, ethylenediaminete-
traacetic acid; EGF, epidermal growth factor; EGTA, ethylene glycol tetraacetic acid; EPCs, endothelial progenitor cells; ESCs, embryonic stem cells; FAM,
fiber-assisted molding; FBS, fetal bovine serum; FDA, Food and Drug Administration; FDBAs, freeze-dried bone allografts; FGG, free gingival graft; GAGs,
glycosaminoglycans; GMP, good manufacturing practice; HA, hyaluronic acid; HGF, hepatocyte growth factor; HIV, immunodeficiency virus; HLA, human
leukocyte antigen; HS, heparin sulfate; HSCs, hematopoietic stem cells; ICBG, iliac crest bone graft; IGF, insulin-like growth factor; IVD, intervertebral
disc; MMP2, matrix metalloproteinase 2; MSCs, mesenchymal stem cells; NCPs, non-collagen proteins; NF-␬B, nuclear factor-␬B; NF-gelatin, nanofibrous
gelatin; NP, nucleus pulposus; PCL, poly(␧-caprolactone); PDAF, platelet-derived angiogenesis factor; PDEGF, platelet-derived endothelial growth factor;
PDGFs, platelet-derived growth factors; PEG, polyethylene glycol; PF-4, platelet factor-4; PGA, polyglycolic acid; PL, platelet lysate; PLA, polylactic acid;
PLGA, poly(lactic-co-glycolic acid); PRF, platelet-rich fibrin; PRGF, plasma rich in growth factor; PRP, platelet-rich plasma; RGD, arginine–glycine–aspartic
acid; rhBMP-2, recombinant human bone morphogenetic protein-2; rhELR, recombinant human elastin-like polymer; SDF-1, stromal cell-derived factor-1;
SDS, sodium dodecyl sulfate; SEM, scanning electron microscopy; SF, silk fibroin; SIS, small intestinal submucosa; SM, stromal matrix; SVF, stromal vascular
fraction; TCP, tricalcium phosphate; TGF-␤, transforming growth factor-␤; VEGFs, vascular endothelial growth factors.
∗ Corresponding author at: State Key Laboratory of Military Stomatology, Department of Periodontology, School of Stomatology, Fourth Military Medical
University, 145th West Changle Road, Xi’an 710032, PR China. Tel.: +86 29 84776093/29 84776096; fax: +86 29 84776096.
E-mail address: [email protected] (F.-M. Chen).

http://dx.doi.org/10.1016/j.progpolymsci.2015.02.004
0079-6700/© 2015 Elsevier Ltd. All rights reserved.
 F.-M. Chen, X. Liu / Progress in Polymer Science 53 (2016) 86–168 87

orchestration of wound healing and tissue regeneration. In addition to directly using tissue
transfers and transplants for repair, new applications of human-derived biomaterials are
now focusing on the use of naturally occurring biomacromolecules, decellularized ECM
scaffolds and autologous preparations rich in growth factors/non-expanded stem cells to
either target acceleration/magnification of the body’s own repair capacity or use nature’s
paradigms to create new tissues for restoration. In particular, there is increasing interest
in separating ECMs into simplified functional domains and/or biopolymeric assemblies so
that these components/constituents can be discretely exploited and manipulated for the
production of bioscaffolds and new biomimetic biomaterials. Here, following an overview
of tissue auto-/allo-transplantation, we discuss the recent trends and advances as well
as the challenges and future directions in the evolution and application of human-derived
biomaterials for reconstructive surgery and tissue engineering. In particular, we focus on an
exploration of the structural, mechanical, biochemical and biological information present
in native human tissue for bioengineering applications and to provide inspiration for the
design of future biomaterials.
© 2015 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
2. Biomaterials for tissue engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
2.1. Roles of biomaterials in tissue engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
2.2. Naturally derived biomaterials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
2.3. Synthetic polymer biomaterials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
2.4. Challenges in biomaterial design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
3. Tissue grafts of human origin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
3.1. Autologous tissue grafts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
3.1.1. Soft-tissue grafts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
3.1.2. Bone grafts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
3.2. Allogenic tissue grafts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
3.2.1. Corneal and skin grafts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
3.2.2. Composite tissue allotransplantation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
3.2.3. Allogenic bone grafts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
3.2.4. Human dentin matrix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
3.3. Tissue engineering: the state of the art in transplantation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
4. Human tissue ECM-based biomaterials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
4.1. Biomaterials for tissue engineering inspired by the ECM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
4.2. ECM constituents for scaffolding biomaterials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
4.2.1. Collagen I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
4.2.2. Collagen II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
4.2.3. Collagen IV, laminin and entactin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
4.2.4. Glycosaminoglycans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
4.2.5. Fibronectin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
4.2.6. Elastin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
4.2.7. ECM assemblies as scaffold building blocks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
4.3. Decellularized ECMs for biomaterials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
4.3.1. Rationale and methods for decellularization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
4.3.2. Applications of decellularized ECMs in tissue engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
4.3.3. Whole-organ decellularization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
5. Preparations containing non-expanded autologous stromal cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
5.1. Bone marrow concentrate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
5.2. Stromal vascular fraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
6. Formulations enriched with endogenous growth factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
6.1. Platelet-rich plasma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
6.2. Platelet-rich fibrin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
6.3. Platelet lysate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
7. Future directions and outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
7.1. Design of ECM-mimicking biomaterials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
7.2. Revisiting ECM influences for information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
7.3. Cell-formed decellularized matrices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
7.4. ECM–stem cell interactions: signposts in advanced biomaterials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
8. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
88 F.-M. Chen, X. Liu / Progress in Polymer Science 53 (2016) 86–168

1. Introduction Although biotechnology that can produce complex

organs de novo is not yet available [11], mounting evidence
The human body has a limited ability to correctly auto- suggests that, at least to a certain degree, the body’s innate
regenerate most, if not all, of its major tissues and organs powers of regeneration can be augmented by replacing
in the event that the original tissue integrity has been seri- sections of tissue and enhancing the regenerative cascade
ously damaged as a result of medical disorders involving [4,12]. The current strategy for tissue engineering typically
tissue dysfunction or devastating deficits [1,2]. Faced with entails the ex vivo expansion of multipotential cell popu-
an ever-increasing burden of trauma, congenital abnor- lations, such as mesenchymal stem cells (MSCs), followed
malities and degenerative diseases, tissue engineering and by their transplantation into damaged areas [10]. Due
regenerative medicine promise to develop new biological to their unique regenerative potential and immunomod-
therapeutics to treat a diverse range of diseases that are ulatory properties, MSCs hold great promise in tissue
currently intractable. Additionally, in most cases, this type engineering and reconstructive therapies, not only directly
of research seeks to assist and accelerate the regenerative participating in wound healing and regeneration but also
process by stimulating the patient’s own inherent healing modulating the host foreign-body immunogenic reaction
potential or, alternatively, to create replacement biological to transplants [13]. These cells are normally transplanted
tissues (or, more challengingly, whole organs) to replace within a biomaterial-cell construct based on a biodegrad-
damaged, deteriorated or lost body parts [3–5]. These ther- able 3D matrix that provides the requisite extracellular
apeutic strategies regulate physiological conditions in a milieu, which contains physical and chemical cues for
spatial and temporal manner and mimic the mechanisms of cell-driven tissue development and regeneration [10,14].
normal tissue repair and regeneration in different parts of Although a wide variety of therapeutic strategies based on
the human body, and endeavors in this field have sparked different types of biomaterials and stem cells have been
a revolution in current and emerging trends in medical and are still being explored, in practice, modern tissue
science [4]. engineering is not an easily accessible approach to achieve
Although bold steps have been made toward creat- regeneration in a clinical setting [15]. In particular, several
ing tissue constructs that could serve as integral parts biological (e.g., a poor understanding of underlying mech-
of the clinical toolbox, many of these engineered tis- anisms), technical (e.g., the large-scale expansion of stem
sues fail to fully match the functional properties of their cells) and regulatory (e.g., cost and safety) hurdles relating
native counterparts. This failure is partially due to our to the use of exogenously manipulated stem cells and engi-
poor quantitative understanding of the mechanisms of the neered constructs for human therapeutics have yet to be
adaptive responses (i.e., the growth and remodeling pro- overcome [16,17]. In addition, a thorough understanding
cesses) that modify the architecture of engineered tissues of the normal physiological processes in tissue develop-
following in vivo transplantation [6]. Considering that most ment and of the mechanisms underlying the interactions
living tissues are composed of numerous repeating units between stem cells and biomaterials during the cascade of
that are hierarchically assembled across multiple length new tissue formation will be required to advance this field,
scales and possess well-defined three-dimensional (3D) as many crucial details remain unclear [18].
microarchitectural features and tissue-specific functional Biomaterials play a pivotal role in the success of tissue
properties, the production of micron-sized tissue modules engineering, though this is not to say that traditional syn-
has attracted increasing interest in the fast-growing field thetic biomaterials must always be used [19]. However,
of tissue engineering [5,7]. These modules can be used to either create living neo-tissues in vitro that are similar
alone as living materials (fillers) to repair wounded tis- or identical to their native body counterparts or facilitate
sues at the sites of injury or can serve as building blocks in situ tissue regeneration by controlled presentation
for the generation of large tissue grafts or whole-organ and on-demand release of specific chemokines at sites
implants through a so-called “bottom-up” approach [8]. of injury, temporary biodegradable support matrices
In light of these applications, in vitro, it is indispensable with natural, tissue-resembling structural and functional
to recapitulate not only the structural organization but attributes are generally necessary, if not indispensable, for
also the cellular and molecular composition of a native cell attachment and housing [20–23]. Similar to a blood
tissue to enhance the biological performance and the over- clot serving as a natural polymeric scaffold in the cascade
all therapeutic outcome of such engineered tissues upon of wound healing events, these matrices should have a
in vivo transplantation [9]. Such modular tissues could desirable shape that provides functionality and supports
be extraordinarily useful when used as injectable living tissue regrowth until sufficient new tissue is formed [21].
microtissues for repair at sites of injury. Alternatively, Therefore, from a fundamental perspective, the goal of
if assembled into large 3D tissues, these modules could biomaterial design in tissue engineering is to identify or
also be used as a patch for a large number of types of fabricate a substance that is innately able (or has been
hitherto intractable extended damage to restore tissue engineered) to assume a desirable form that can be applied
function [7]. In the future, an increased availability of to both synthesize a 3D cellular microenvironment for cell
engineered “living” tissue or organ substitutes could sig- accommodation and guide new tissue formation [24,25].
nificantly reduce the demand for organ replacement and The material should be able to maintain its structure and
dramatically expedite the development of new therapeu- integrity for predictable periods of time to ensure new
tics that can cure patients with revivable organ failure, tissue formation and maturation, even under load-bearing
eliminating the need for organ allotransplantation alto- circumstances [26,27]. In recent years, the development
gether [10]. of regenerative biomaterials has rapidly evolved to allow
 F.-M. Chen, X. Liu / Progress in Polymer Science 53 (2016) 86–168 89

the sequestration and controlled release of growth factors to facilitate their translation into clinical settings to help
that work in concert with materials to achieve tailored the greatest number of patients, leading to an ineluctable
biological properties and improved functionalities, which, dichotomy between the need for an appropriate level of
in a precise and near-physiological fashion, can control sophistication (integrating complex information into scaf-
stem cell fate under niche-mimicking, recognizable con- folds) and the ease of scaffold production (bypassing device
ditions both in vitro and in vivo [28–30]. The key concept over-engineering and keeping material complexity to a
of these designs is the recreation of myriad cellular and minimum) [15,20,38].
molecular events involved in the regeneration of a new Unfortunately, if we wait for every aforementioned
tissue/organ [31]. Therefore, the design of material devices question to be answered, the timely introduction of novel
that approximate many of the critical features of normal treatments based on tissue engineering into human health-
cellular matrices in human tissue and thus foster and care will be impossible [16]. In pursuing a perfect synthetic,
direct the formation of target tissues lies at the forefront of tissue-engineered template, it is important to consider
biomaterials science and tissue engineering and is indeed whether we have looked too far ahead and missed the
the epitome of the fields’ present motivation [26]. readily available building blocks, such as naturally derived
No longer simply a non-viable material used in a med- biomacromolecules required to create biomaterials for
ical device that is generally used as a “filler”, a biomaterial this purpose [39]. For decades, scientists have learned
is now defined as “a substance that is able, or has been that the human body is a tremendous potential source
engineered, to take a form which, alone or as part of a com- of bioscaffolds and biopolymers for therapeutics; these
plex system, is used to direct, by control of interactions with biomaterials have attracted considerable attention in the
components of living systems, the course of any therapeutic tissue engineering and regenerative medicine communi-
or diagnostic procedure, in human or veterinary medicine” ties [40,41]. For instance, certain therapeutic biomaterials
[21,23]. The clinical benefit of bioengineering technolo- may be produced from human blood. Several types of
gies, based to some extent on the use of biomaterials, blood-derived bioscaffolds are utilized in clinical situations
in an increasing number of patients places exponentially demanding a high fibrinogen content, whereas platelet-
growing demands on scaffolding materials. The search for rich formulations are used because they contain multiple
an “excellent” tissue engineering template has remained platelet-derived growth factors [42]. Based on the latest
a research hotspot as the rapidly growing multidisci- definition of biomaterials, from 2009 [21,23], in the present
plinary area of tissue engineering continues to advance, review, we define human-derived biomaterials much more
along with its intertwined field of “regenerative medicine” broadly than we are accustomed to doing. We specifi-
[20,22,32–34]. The term “regenerative medicine” was ini- cally define these biomaterials as those existing (e.g., donor
tially considered to encompass many more disciplines and organs and tissue grafts) or originally found (e.g., decellu-
fields of medicine than the traditional concept of “tis- larized ECM materials and ECM components/constituents)
sue engineering” did, but today, the two terms are often within our bodies, along with a large variety of cell popu-
used interchangeably [10]. Therefore, the philosophy of lations obtained from human materials and active proteins
the emerging discipline of current tissue engineering and, (e.g., mitogenic, chemotactic, adhesive, angiogenic and
indeed, of regenerative medicine is that rather than aim- antiangiogenic proteins) of human origin (Fig. 1). In par-
ing to develop a complex living-tissue replacement ex ticular, the use of human-derived biomaterial scaffolds,
vivo, concerted efforts must focus on creating extracellular naturally occurring proteins, ECM components and prepa-
matrix (ECM)-mimicking biomaterials or modulating stem rations rich in growth factors or non-expanded stem cells
cell niches that recapitulate pivotal interactions with host for tissue engineering provides new approaches for the
cells to unlock the patient’s own regenerative ability for redesign of clinically translatable regenerative therapies.
organization and self-repair [22,24,25]. Biomaterials scientists aim to recreate the intrinsic prop-
Although the structural, mechanical and biochemical erties of human-derived biomaterials in next generation
information coded within the native ECM directs the design of regenerative biomaterials tailored to specific applica-
of new types of tissue-engineering templates, unfortu- tions [39]. Those properties include, but are not limited to,
nately, the biological properties and network architecture the provision of a structural support for resident cells and
of currently available porous synthetic scaffolds fall short the establishment of physical integrity in the tissue. Addi-
of the criteria for the creation of a complex human tissue tionally, these properties have a profound influence on cell
[16,18,35,36]. It is now widely recognized that this gap fate through the regulation of cell proliferation, migration
has largely arisen because the study of porous scaffolds and gene expression and the maintenance of functional
in vivo is often limited by the experimenter’s inabil- homeostasis [26,43]. Research in this area is growing very
ity to control all of the technical parameters, several of rapidly thanks to the combined efforts of the multidis-
which rely on the systemic responses of the living organ- ciplinary biomaterials and bioengineering communities.
ism [18,27,37]. Furthermore, the regulation of structural As will be detailed in this article, it is likely that the
parameters in the development of fully synthetic bio- use of these biomaterials as biocompatible, biodegradable
materials and their bioactivation, achieved through the and versatile matrices in tissue engineering will circum-
integration of key biomacromolecules and signals capa- vent many biological and technical problems in the design
ble of directing cell and tissue fate in vivo, represent a and development of synthetic biomaterials, hence opening
great challenge in practice [35,36]. Most importantly, in medically exploitable avenues for the translational suc-
the hope of commercial success, regenerative biomate- cess of tissue engineering solutions [44]. In the future,
rials must be not only efficacious but also cost-effective these exciting human “raw materials” will be essential to
90 F.-M. Chen, X. Liu / Progress in Polymer Science 53 (2016) 86–168

Fig. 1. Schematic representation of the “biomaterials” of human origin frequently used as therapeutics in medicine or as potential building blocks in
the specific context of advancing the “next generation” of tissue engineering templates for clinical use and commercial production. From tissue/organ
transplantation to the use of either naturally occurring biopolymers or biomimetic materials inspired by nature in tissue engineering and regenerative
medicine, the terms may change, but the essential goals remain the same. Examples are given where appropriate (please see the text for additional details).
Although ex vivo-expanded cells derived from human tissues/organs for therapeutic use may be broadly classified as human-derived “biomaterials”, they are
not included in the general definition of biomaterials and hence will not be detailed in this review. Abbreviations appearing in the figure: ECM, extracellular
matrix; GAGs, glycosaminoglycans; HA, hyaluronic acid; HS, heparin sulfate; CS, chondroitin sulfate; PRP, platelet-rich plasma; PRF, platelet-rich fibrin; PL,
platelet lysate; BMC, bone marrow concentrate; SVF, stromal vascular fraction.

help reconcile the pressures facing tissue engineering with largest limitations of current regenerative biomaterials. A
respect to commercial production and clinical translation major priority is to involve clinicians who practice regener-
[39]. ative medicine and who regularly encounter the problems
Following an overview of tissue and organ auto-/allo- that they aim to solve in the design and creation of
transplantation, which represents the original practice in advanced biomaterials and tissue-engineered constructs
this field, this review will discuss recent new insights into for clinical use. If more international research efforts are
and expanding applications of human-derived biomacro- made in this direction, the unleashed potential of bioma-
molecules and biomaterials in tissue engineering for terials of human origin will benefit more and more clinical
the management of human tissue-destructive conditions, patients each year.
recalcitrant chronic wounds and persistent/deteriorating
organ failure. This review will also highlight recent 2. Biomaterials for tissue engineering
approaches and expanding opportunities to exploit the
molecular mechanisms of these “raw materials” to cre- The staggering potential of living tissues for auto-
ate bioscaffolds with a wide range of material properties regeneration may be restricted/impaired by an age-related
and applications, even though these biomaterials are in decline in the number and quality of host stem
their early stages of development. The potential chal- cell/progenitor populations, by the innately low regener-
lenges facing the field and the obstacles that must be ative capacity of certain tissues or by the negative effect of
addressed to explore and develop truly clinically viable inflammation on wound repair [20]. In an effort to compen-
biomaterials and regenerative therapies are also discussed sate for such poor healing capacity, tissue engineering has
in detail. Although concerted efforts have been and still been established as a potential therapeutic option to recre-
are being made in the field of synthetic biomaterials in ate several of the biological processes that occur during
an attempt to develop advanced devices that mimic the tissue development and in the native wound healing cas-
critical aspects of natural ECMs and to shed light on their cade in microcosms [3–5]. For this purpose, a harmonious
potential translation to clinical settings, we believe that combination of a scaffold/supporting materials, adequate
the development and use of biomaterials of human ori- target cells and growth-stimulating bioactive factors is
gin is an equally inevitable trend. We therefore present a used to promote the regeneration of damaged tissues or to
call to action for biological and materials scientists, fund- replace failing or malfunctioning/deteriorating organs [17].
ing agencies and professionals in reconstructive medicine Therefore, tissue-engineered constructs have to mimic a
to pool their resources to hasten this development through certain degree of the native complexity of a tissue to assist
a highly multidisciplinary approach that addresses the in the restoration of the full structure and functionality
 F.-M. Chen, X. Liu / Progress in Polymer Science 53 (2016) 86–168 91

of the tissue. This approach is the first medical therapy Ideally, a scaffold should serve as a transient structure
wherein engineered tissues can potentially become fully that, over an extended period, will be degraded or reab-
integrated into the patient, thus conferring a permanent sorbed in a controlled manner that is in accordance with
cure for a diverse range of diseases that are not curable the regrowth rate of new tissue [51,52]. Consequently, the
today [45]. Notably, biomaterials that support and foster biomaterial template is correctly replaced with naturally
regenerative cell growth have played a considerable role deposited ECM and the newly formed tissue of interest. In
in the tissue engineering design paradigm and in the suc- the host environment, a biomaterial’s ability to orchestrate
cess of numerous medical devices for clinical regenerative human host responses to exogenously transplanted cells
therapies [36,39]. may positively influence cell behavior and function and
Broadly speaking, biomaterials can be defined as mate- ultimately dramatically affect the desired tissue formation
rial devices or implants used to repair/replace native body [53]. Fortunately, advances in biomaterials science, com-
tissues or as scaffolding materials adopted to construct bined with recently increasing knowledge of ECM biology
manmade tissues and organs [19]. Commonly, therapeutic and the role of environmental cues in tissue development,
biomaterials can be classified into two main categories: (I) have led to the redesign of material templates that are
living or once-living material of animal or human origin; modified to provide appropriate structural support and,
and (II) other materials, including materials from vegetal in certain cases, biological and mechanical cues to pro-
sources and synthetic materials and their composites that mote the safe and effective reconstruction of a functional
are biocompatible and can be applied for tissue regenera- tissue in vivo [36,37,54]. Moreover, scaffolding biomate-
tion. For over two decades, progress in polymer science and rials can be tailored to mobilize and present biologically
tissue engineering has paved the way for the generation active molecules, including cell adhesion peptides, cell
of sophisticated and ingenious biomaterials to optimize homing factors and numerous growth/differentiation and
existing clinical treatments and to develop more safe and mechanical signals; to expand or recreate the stem cell
effective cures for a higher quality of human life. compartment to facilitate the recruitment of stem cells
and their subsequent differentiation into a large number of
2.1. Roles of biomaterials in tissue engineering daughter cells; and finally, to direct new tissue formation
and integration [12,22,54–57]. For damaged sites featuring
We preface the following discussion with a brief enough repair cells in the local microenvironment, scaf-
description of the multifaceted roles of biomaterials in folds mainly serve to promote the homing of the host’s
tissue engineering, particularly their tasks as tissue- own cells for in situ tissue regeneration, whereas other
templates that home, foster and coax stem cells to form approaches leverage material templates for the delivery
new tissue [23]. The basic role of biomaterials in tissue of exogenously expanded cell populations to supplement
engineering is to provide temporary mechanical support the body’s cell niche [22,57,58]. In either case, tissue engi-
and mass transport to encourage cell adhesion, prolif- neering scaffolds seek to mimic the natural ECM, at least
eration, and differentiation and to control the size and partially, and to create a favorable microenvironment to
shape of the regenerated tissue [45]. Moreover, biomate- support and induce tissue formation [35]. Therefore, the
rials, usually described as scaffolds, may present physical identification of adequate biomaterials for cell accommo-
and chemical signals with spatiotemporal accuracy, which dation and mass transport is a pivotal step in any tissue
are of great importance to the modulation of cell perfor- engineering design. A wide range of options exist for
mance and function and in the guidance of correct tissue designing a specific biomaterial to be used as a matrix
regeneration, as an ECM contains the intrinsic signals piv- template, including natural biomaterials, synthetic bioma-
otal to communicating with and controlling niche cells terials, and composites composed of two or more material
[46]. Instead of an inert structure temporarily employed to types/classes. The advantages and disadvantages of apply-
construct inanimate objects, this new concept of a tissue ing these biomaterials and their suitability for application
engineering “template” incorporates the sense of a struc- must be determined [48,59] (Fig. 2).
ture that is actively involved in delivering cues to cells
and that takes part in the formation and characteristics 2.2. Naturally derived biomaterials
of the engineered/regenerated tissue [47]. These design
requirements stem from the recognition that mimicking Natural biomaterials present a crucial subset of bio-
the in vivo cell-supporting niche (i.e., the ECM) with regard materials for use as tissue engineering templates due to
to its structural, mechanical and biochemical properties their bioactivity, biocompatibility, tunable degradation and
will coax niche cells into behaving similarly to their nat- mechanical kinetics and their intrinsic structural resem-
ural in vivo counterparts [48]. Recent insights into ECM blance of native tissue ECM. Natural biopolymers are often
mimics have already enriched our understanding of how to processed using environmentally–friendly aqueous-based
explore/harness the regenerative potential of various cell methods. Upon application within biological systems, they
types via a well-designed cellular matrix-scaffold to cre- do not release cytotoxic products during degradation, and
ate an artificial tissue/organ and to dramatically enhance their degradation rates may be adjusted by altering the
the engraftment of ex vivo-expanded progenitor/stem cells starting formulation and/or processing conditions [60]. An
[35]. To this end, scaffolding templates provide a 3D matrix advantage of natural biomaterials is their innate ability to
that replicates, as far as possible, the niche of the target promote biological recognition, which may positively sup-
cells, defining an artificial niche with complex and dynamic port cell adhesion and function [39]. In addition, in nature,
regulation, in which a target tissue can form [49,50]. helical macromolecules such as collagen, cellulose and
92 F.-M. Chen, X. Liu / Progress in Polymer Science 53 (2016) 86–168

Fig. 2. Schematic representation of pivotal factors (structural, mechanical, biochemical and biological) involved in the design of biomaterials (templates)
for tissue engineering that coax cells to behave in the same or a similar manner as their natural in vivo counterparts.

chitin are critical for the morphogenesis and functionality Due to the key advantage of these materials in sup-
of a large variety hierarchically structured materials [61]. porting the attachment, proliferation and differentiation of
Naturally derived biomaterials may typically be divided cells, natural polymers have been extensively explored in
into two groups: protein-based biomaterials (e.g., collagen, the development of tissue engineering templates, often in
silk fibroin, gelatin, fibronectin, keratin, fibrin and eggshell combination with molecular and mechanical signals, for
membrane) and polysaccharide-based biomaterials (e.g., applications ranging from tissue repair to functional organ
hyaluronan, cellulose, glucose, alginate, chondroitin, and replacement [63]. For therapeutic applications, these poly-
chitin and its derivative, chitosan). Protein-based bioma- mers are generally processed for implantation as porous
terials are typically obtained from animal and human scaffolds, hydrogels, particulates or thin membranes and
sources and include bioactive molecules that mimic the are typically enzymatically degradable into nontoxic end-
extracellular environment, whereas polysaccharide-based products in vivo. Although the kinetics of the degradation
biomaterials are mostly obtained from algae, as in the of these biomaterials may not be easily controlled or
case of agar and alginate, or from microbial sources, predicted, they are still effective if local, short-term respon-
as in the case of dextran and its derivatives [40,41,52]. sive action is sufficient. Additionally, special forms of
Another class of natural biomaterials is termed decel- natural polymers (e.g., injectable hydrogel) may be admin-
lularized tissue-derived biomaterials, which are created istered noninvasively to a target site of tissue damage
by the elimination of all cellular and nuclear materials [24,52,64,65].
from native tissues/organs, as in decellularized dermis, The disadvantages of naturally derived biomaterials
heart valves, blood vessels, small intestinal submucosa include generally weak mechanical strength and inconsis-
(SIS) and liver, among others. These decellularized tissue- tency in compositions and properties, which are associated
derived biomaterials contain a variety of different organic with batch production due to their origin in living beings
and/or inorganic components. If the tissue/organ is from [66]. To overcome these limitations, recent advances in tis-
a human, the resulting decellularized materials can be sue engineering template redesign and fabrication have led
considered as human-derived biomaterials, which will be to a paradigm shift toward the development of biomimetic
detailed in Section 4.2. Certain natural polymers also con- scaffolds that incorporate ligands imitating the native ECM.
tain surface ligands or motifs required for cell adhesion These scaffolds are often utilized in vitro as analogs of the
and proliferation. In particular, cell adhesion and subse- natural ECM to facilitate investigations of cell–ECM inter-
quent cell activity are mediated by specific integrin–ligand play and other intricate processes [67,68]. Another concern
interactions between cells and their surrounding ECMs with naturally derived polymeric materials is the variabil-
[62]. ity inherent in the production of the materials and the
 F.-M. Chen, X. Liu / Progress in Polymer Science 53 (2016) 86–168 93

potential, albeit small, of the materials to evoke an immune adhesion or direct phenotypic expression, as a natural poly-
response [35]. mer would. However, various synthesis techniques have
been developed and optimized to incorporate biologically
2.3. Synthetic polymer biomaterials active domains into synthetic polymer templates, thereby
enabling the production of biomimetic scaffolds with a
The use of synthetic polymers as matrices and tem- defined and tunable composition [82]. For example, syn-
plates in bioengineering presents several key advantages thetic polymeric scaffolds with a collagen or serum coating
relative to naturally derived polymers, offering attractive are usually sufficient to permit initial cell attachment and
options for the control of shape, architecture and chem- ECM deposition, whereas coating synthetic polymeric scaf-
istry to generate reasonable alternatives to or mimics of folds with ceramic (calcium phosphate, or CaP) is crucial
ECM systems of human origin that emulate or control for bone tissue engineering applications [34,83]. In other
biomaterial functions [69,70]. The most widely used syn- cases, synthetic polymeric scaffolds have been fabricated
thetic polymers for tissue regeneration are poly(␣-hydroxy and modified through the covalent immobilization of ECM-
acids), which include polylactic acid (PLA), polyglycolic derived moieties to enable the presentation of biologics
acid (PGA) and their copolymer, poly(lactic-co-glycolic with spatiotemporal accuracy, to promote cell attachment
acid) (PLGA) [71,72]. These polymers’ nontoxic degrada- and to enhance the directed differentiation of progeni-
tion products (lactic acid and glycolic acid) are generated tor cell populations [84]. Presenting bioactive agents on
via simple chemical hydrolysis of the polymers and are synthetic polymer template surfaces is the most efficient
cleared away by normal metabolic pathways [73]. Given way to elicit desired cell–material interactions [85]. The
the lack of dependence on local enzyme concentrations, ability to devise these polymer systems to influence cell
chemical hydrolysis may be more readily predicted and behaviors and interplay is another crucial feature that
controlled than enzymatic degradation in vivo [63,71]. The provides both fundamental insights into the chemistry of
properties of synthetic polymers, such as tensile strength, structure–function relationships and enormous potential
the mechanical modulus and the degradation rate, can be to directly utilize these biomaterials as cellular scaffolds
easily tailored for target applications by altering the lac- [86].
tide/glycolide proportions and polymerization parameters.
Indeed, these materials were successfully applied in the 2.4. Challenges in biomaterial design
clinic for the creation of urethral tissue as well as for blad-
der replacement in patients with idiopathic detrusor or Each tissue commonly presents a unique cascade of
neurogenic bladder [74–77]. In addition, in situ-forming wound healing processes following injury due to disease or
hydrogels based on synthetic polymers can be engineered trauma; however, common cellular and molecular events
to locally deliver a wide range of bioactive agents in a during tissue repair exist. Most tissue-healing phases
controlled and sustained manner to regulate stem cell involve multiple signaling components that coax cells
fates encapsulated within the 3D polymer network, such under tight spatial and temporal control, leading to opti-
as polyethylene glycol (PEG) [78]. Due to its exceptional mal tissue regeneration [2]. Ideally, a cellular scaffold, in
qualities, such as its biocompatibility, low immunogenic- addition to being biocompatible, should be a biomaterial
ity, hydrolysis under physiological conditions, and FDA device with physical and mechanical properties that match
approval for clinical use, poly(␧-caprolactone) (PCL) is those of the target tissue and that contain a multitude of
another synthetic polyester based on hydroxyalkanoic cytokines, growth factors and cell adhesion molecules that
acids that has attracted intense attention in tissue engi- can promote a regenerative microenvironment for appro-
neering. This polymer is used either alone, as hydrophobic priate cell populations and induce their behavior [87]. Far
PCL, or as a PCL-containing amphiphilic block copoly- more often than expected, a single-component template
mer when in combination with other agents, resulting in does not meet the requirements for a regenerative bio-
improved performance in certain applications [70,79,80]. material matrix due to a lack of a controlled degradation
Many synthetic polymers (e.g., PLGA, PEG, PCL, poly- rate; a lack of desired mechanical properties and bioactiv-
acrylic acid, polyvinyl alcohol and polyvinylpyrrolidone) ity; and, more importantly, a lack of the desired cell–matrix
owe their broad biomedical application to their biomimetic interactions to control gene expression, cytoskeletal struc-
ECM-like micro/nanoscale fibers, attractive processability ture and dynamics [33,34]. A combination of two or more
and biocompatibility. Although synthetic polymer bio- types of biomaterials into a medical device may overcome
materials can be manufactured into scaffolds with fully several of these limitations. Whereas composite biomate-
interconnected pores, certain classes, such as poly(␣- rials from the same class will generate a certain degree of
hydroxy esters), may produce acidic degradation products regulation, mixing biomaterials from multiple classes may
that can alter the pH of their surrounding tissues [51]. In confer a greater level of control over the overall material
turn, this pH change can affect cell behavior and survival properties for cell guidance. For example, hybrid hydro-
and cause adverse tissue and inflammatory reactions [81]. gel scaffolds synthesized from selected biopolymers may
Nevertheless, synthetic polymers themselves typically do provide opportunities to closely mimic the key character-
not carry a risk of inducing an immune response because of istics of the native ECM, including by displaying adhesion
a lack of biologically functional domains. This feature is also sites and presenting growth factors, which not only induces
a limitation because the lack of peptide side-chain reac- reparative cells but also triggers and governs specific
tivity for binding regulatory peptides, growth factors and events at the cellular and tissue levels [48,88]. In particu-
other biological signals does not allow the facilitation of cell lar, the addition of natural components, with their natural
94 F.-M. Chen, X. Liu / Progress in Polymer Science 53 (2016) 86–168

ratios, into synthetic polymers, followed by the incorpo- that more effectively generate multi-material and cell-
ration of biochemical and biophysical cues, mirroring the laden scaffolds with less effort. In this respect, polymer
chemistry as well as the nanofibrous network of the native brushes with various structures and chemistries, as well
matrix, has emerged as a leading strategy in scaffold- as diverse brush-based strategies, which are both passive
ing design [52,89]. Such materials chemistry has made a and bioactive, may be utilized for biomaterial modifica-
fundamental and an increasingly crucial impact on mate- tion. In particular, these features may make the material
rials science, showing significant promise in replicating surfaces biocompatible and non-fouling, which passively
the morphologies, nanostructures and functional building prevents subsequent undesirable host responses [93]. In
blocks of a large variety of human tissues or in fully recre- addition, fiber-assisted molding (FAM) has been shown to
ating these building blocks using integrated reparative cell be a simple and robust method to create biomimetic 3D
populations [90]. surfaces with controllable curvature and a helical twist.
Alongside these positive developments based on Such modified surfaces are able to guide cell alignment
biomimetic materials chemistry, there is growing recogni- and the assembly of helically patterned ECM, demonstrat-
tion that the physical properties of the cell’s environment ing the potential of FAM for materials science and tissue
are also crucial to a broad spectrum of cell biological func- engineering applications [100].
tions that must be carefully taken into account in the In addition to presenting interconnected pores with a
design of biomaterials [34]. Unfortunately, however, there tunable pore size and biomimetic surfaces with control-
has been a surprising paucity of biomaterial templates lable curvature and a helical twist, scaffolds are commonly
that are designed to accurately mimic the architectures engineered and used for the presentation or controlled
and functions of the structural fabric of native tissues, delivery of bioactive agents to accelerate and orchestrate
ensuring precise tissue regeneration [90]. Indeed, phys- tissue regeneration [22,101–103] (Fig. 3). For this purpose,
ical attributes, such as scaffold shape, size, architecture, significant efforts have been made to create biodegrad-
structure, mechanics, porosity, surface texture and com- able polymeric scaffolds with functional groups on the
partmentalization, can profoundly affect the biological material surfaces that are coupled with biological cues
functions of biomaterials once they are placed into an and delivered to biological compartments, hence eliciting
in vivo cellular microenvironment [33,34]. For example, desired cell–material interactions [85]. The modification
cells use compartmentalization to control various bio- of biomaterials for the recapitulation of the native tissue
chemical reactions in space and time [91], and the way in healing cascade and other variables using a wide range
which cells migrate is directed by the physical aspects of of bioactive agents allows host cells to interface with the
their surroundings, and particularly the properties of the engineered environment and hence leads to better cell
ECM [92]. Notably, the surprising properties of biomaterials propagation and tissue regeneration [12,28,55]. To be effec-
largely result from their surfaces as well as their sophis- tive as therapeutics, bioactive agents have to reach their
ticated hierarchical bulk structures [93,94]. For example, sites of action without damage or degradation. Addition-
scaffolding biomaterials must possess biocompatible (and ally, they have to maintain their effective concentrations
ideally antibacterial) surfaces to reduce or eliminate unde- in the target area sufficiently long enough to exert desired
sirable host responses, mimic the structure of the target biological functions [56]. When labile drugs are delivered
living organism in one to three dimensions, exhibit inter- in their native form, without control over their localiza-
connected porosity to support cell/tissue penetration and tion or rate of release, high doses are generally needed
be capable of resorption over time to create space for and are indeed frequently adopted to ensure the required
new tissues [18,45,46]. Although fabrication techniques therapeutic effect. Beyond generating additional waste and
(e.g., scaffold sheet design strategies, particulate leach- extra expense, these supraphysiological doses may result
ing techniques and electrospinning methods) have been in increased toxic responses or undesirable side effects,
proposed to enable the fabrication of porous 3D bioma- such as inflammation, dangerous tissue over-growth and
terial templates with an appropriate porosity and pore even tumor formation [104,105]. To this end, localized
size, controlling the pore geometry and architecture of drug delivery systems have been developed to act as a
these templates to match the native tissue has been a depot of biologics targeted to damaged areas for controlled
daunting, largely unpredictable task [95–97]. Moreover, release, which can considerably improve therapeutic effi-
the requisite mechanical and compositional standards of cacy and safety and also offer protection to labile factors
tissue engineering templates for clinical translation are [105,106]. In contrast, a number of sophisticated drug
complicated by the anisotropic nature of human tissues, delivery devices that circumvent challenges associated
such as the concentrically layered sheets of the inter- with traditional delivery systems have been engineered to
vertebral disc (IVD) and the parallel-arranged collagen exert control over the precise spatial and temporal pre-
fibers within tendons [33]. Recently, 3D printing methods sentation of a complex array of bioactive agents, including
have emerged to enable the fabrication of scaffolds with growth factors and therapeutic cells, in a tailored man-
defined scaffold geometries while precisely controlling the ner [28,29,101,105]. As our ever-expanding fundamental
arrangement of cells and bioactive nanomaterials through- knowledge of the cell and molecular biology of human
out the structure; however, in certain cases, printing physiology and disease reveals new therapeutic targets
complex organ-targeted templates with clinically relevant that require more advanced strategies to control cell behav-
dimensions, such as whole hearts or livers, may be too time ior and to address specific pathological situations, the
consuming for widespread application [98,99]. A grow- importance of sophisticated material devices in medicine is
ing goal in this field has been to explore new strategies expected to increase [30,107]. Through the development of
 F.-M. Chen, X. Liu / Progress in Polymer Science 53 (2016) 86–168 95

Fig. 3. The presentation of growth factors or other therapeutic agents via biomaterials engineering. Cargo presentation via (A) adsorption or embedding.
(B) Non-covalent immobilization (e.g., forming ionic complexes with the polymer backbone). (C) Covalent immobilization (e.g., tethering of cues to the
polymer chains by linking via cleavable bonds). (D) Pre-encapsulation into a well-defined particulate system.
Source: [22], Copyright 2011. Reproduced with permission from Elsevier Ltd.

smart biomaterials into a specific assembled system, a cer- overall outcomes of therapy [12]. For the implementation
tain amount of cargo can be delivered to meet an individual of these distinct requirements for tissue engineering use,
patient’s therapeutic requirements in spatially, tempo- biomaterial platforms must offer an increasing number
rally and dosage-controlled fashions [38,55,108,109]. To of sophisticated strategies for controlled release, ensur-
achieve such stimuli-responsive drug delivery systems ing that under adverse conditions, an optimized ratio of
requires the selection of biocompatible biomaterials that multiple biologics, each acting in a specific spatiotempo-
can respond to a specific stimulus or that are particularly ral pattern, is delivered solely to the location where the
susceptible to specific physical incitement, undergoing factors are required and only at the levels, dosages and
protonation, a (supra) molecular conformational change times at which they are needed [12,55,101,104]. Clearly, it
or hydrolytic cleavage [29,106,110]. In addition to tem- is highly challenging to integrate all of these functionalities
plates for tissue engineering, many recent advances have into a single medical device. Unfortunately, with respect
shed light on more sophisticated devices with one or to the dichotomy between the pursuit of sophistication
more characteristics, such as efficient drug protection, and the feasibility of commercialization and regarding the
accurately controlled release, localized drug targeting, critical aspects of the healing cascade, it remains unclear
permeation enhancement, expanded self-modulated ther- how much extrinsic physiochemical information is indis-
apeutic action, enzyme inhibition, reporting or imaging pensable to coax endogenously residing or exogenously
[12,87,111]. transplanted cells into generating a complex tissue for a
The crucial challenges related to drug delivery in the specific purpose and, in particular, what minimum levels
design of biomaterials arguably include the selection of of biomaterial complexity are necessary for a given task
not only the appropriate factor or combination of fac- [16,20,29,37,56,102].
tors necessary to induce a desired response but also the Recently, however, a wealth of research has revealed
dose and spatiotemporal delivery needed for proper tis- that the development of tissue engineering templates
sue regeneration [55,105]. Furthermore, modulation of the might be experiencing the emergence of a diverse and
exuberant host response to transplants and microbial con- powerful set of new concepts for biomaterials design
tamination, which directs the in vivo milieu against tissue [34]. Advanced biomaterial technologies for creating artifi-
regeneration, has not attracted enough attention. Scien- cial refined cell-instructive platforms based on knowledge
tists therefore must explore the combined administration obtained from materials science, biology and engineer-
of anti-infective agents or host modifiers to optimize the ing have heralded a new era in the redesign of cellular
96 F.-M. Chen, X. Liu / Progress in Polymer Science 53 (2016) 86–168
 F.-M. Chen, X. Liu / Progress in Polymer Science 53 (2016) 86–168 97

scaffolds [37,96]. In this era, the architecture of the stem biomaterials in the past. Consequently, as “living tissue
cell milieu or niche can be replicated in terms of its bio- replacements”, tissue grafts (autogenous or allogenic tis-
chemical, mechanical, structural and component details sue grafts) and donor organs of human origin can be
to manipulate cell fate, including cell migration, gene considered as the gold standard “biomaterials” for recon-
expression and the maintenance of functional homeo- structive therapies [115,116] (Fig. 4). The last century
stasis [48]. To design cell-based therapeutics for tissue experienced remarkable advances in the science of recon-
defects with complex shapes, an injectable cell scaffold structive surgery, and over the span of the past two
integrating an ECM-resembling structural feature for cell decades, surgical technology as well as graft safety and fea-
residence is desirable to achieve a precise anatomic fit sibility have largely improved, and soft- and hard-tissue
and to minimize the complexity of the surgical proce- grafts have grown in popularity for tissue reconstruction
dure [65,112]. Unfortunately, although a superabundance in routine clinics. This growth has been driven, in part,
of robust material devices exists to analyze the effects of by a desire to restore the patients’ impaired, damaged or
the physical and chemical properties of stem cell microen- lost body parts and hence to improve the patients’ quality
vironments, these devices have only just started to be used of life. Endeavors in the field of transplantation, alongside
to instruct stem cell behavior, and they often fail in regard the shortage of tissue grafts and donated organs avail-
to “biointegration”. Using the technologies established to able for use in patients, have prompted the use of cells
date, it is still impossible to achieve an optimized protein- and biomaterials for the creation of lab-grown tissue/organ
releasing mode that mimics naturally occurring events replacements that mimic, at least to a large extent, the com-
[34,113]. New developments stemming from biological plexity and functionality of a native tissue [117]. Human
disciplines are actively directing the redesign of ingenious MSCs can be obtained from patient-derived tissue mate-
biomaterials that work using nature’s own mechanisms rials of mesenchymal origin or tissues derived therefrom,
for regeneration; however, much remains unclear about such as bone marrow, blood, adipose tissue, and, recently,
the underlying events during which tissues heal and form clinically discarded dental-related tissues that have long
[20]. Clearly, much work needs to be done before we can been considered to be of no use [118–122]. In the last case,
rationally design synthetic materials with attached func- a broad spectrum of research has suggested that dental
tional groups, similar to native ECMs that are capable of pulp tissue, periodontal ligaments and gingiva can be envis-
providing autonomous direction to pluripotent stem cell aged as suitable and the most accessible sources of stem
populations in routine clinical therapies [37,114]. Crucial cells, whether in a healthy or inflamed state [123–131].
to current endeavors in this field is further collaboration At the same time, other cell populations, such as chon-
between cell biologists and biomaterials scientists, which drocytes, can be separated from autologous cartilage and
promises to foster intense effort in tissue engineering multiplied using a strictly controlled cell culture system
and to offer new insights into cell-instructive niches that for the development of new cartilage repair techniques.
will advance cell-based strategies for clinical tissue recon- In this context, autologous chondrocyte implantation (ACI)
struction [15,16]. Until ECM-mimicking material devices and matrix-supported ACI have been demonstrated to be
are successfully commercialized and readily available for practical clinical techniques for the repair of full-thickness
widespread application, the use of biomaterials derived chondral defects in the knee [132–134]. Although tissue
from human tissues/organs provides an option to clini- engineering emerged as a field at the intersection of numer-
cians, who have a moral obligation not only to address ous disciplines over 20 years ago, tissue engineers have
problems that may benefit patients in the future but also largely stood on the shoulders of giants, relying on those
to develop therapeutics that can be immediately translated who have worked in related fields, such as tissue/organ
into routine clinical practice to assist patients today. bioreactors, preservation and transplantation [135]. This
previous work was conducted over several decades, and
3. Tissue grafts of human origin several of the principles established by those pioneering
explorers are still followed by the scientists working in
As described in Section 1, our original concept of bio- current bioengineering disciplines; be it tissue (or organ)
materials for use in the biomedical arena has changed; auto-/allo-transplantation or tissue engineering (or regen-
biomaterials can now include many substances, such as erative medicine), the essential goals remain the same.
engineered constructs, therapeutic cells and indeed, a Through the involvement of living substances in bioma-
number of living tissues or organs used for transplan- terials science, and particularly native tissues and organs
tation [21,23], that may generally not be considered as that exceed the traditional aspects of materials science, and

Fig. 4. Schematic representation of tissue grafts and organs of human origin for clinical therapeutics (examples are given where appropriate; illustrations are
not to scale). Autologous tissue grafts include soft tissues, such as free gingival grafts; fat, fascial, skin (partial-thickness or full-thickness) and myocutaneous
flaps; and bone grafts, including block bone and cancellous bone. Allogenic tissues for transplantation include corneal grafts; skin grafts; and certain
composite (organ-level) tissues, such as a full hand or a near-total face transplant. In addition, organ allotransplantation is often performed for the kidney,
liver, lung and heart, among others. Bone grafting materials and dentin matrix can be produced from the bone and teeth of human cadavers. The images
used here are selected samples for schematic representation only; they do not represent any particular preference by clinicians.
Source: Figure components (6) and (11): [115], Copyright 2011, and [116], Copyright 2009, respectively. Reproduced with permission from Elsevier Ltd;
Component (10) courtesy of Jewish Hospital, Kleinert, Kutz and Associates Hand Care Center and University of Louisville; the remaining components are
by the authors, or from unpublished resources from the corresponding author’s institution (provided by Dr. Guicai Liu in the Department of Oral and
Maxillofacial Surgery).
98 F.-M. Chen, X. Liu / Progress in Polymer Science 53 (2016) 86–168

the accompanying inspiration in and evolution of bioma- Nevertheless, given the relative abundance and accessibil-
terials, we are now able to illustrate the best characteristic ity of adipose tissue due to its proximity to the surface
for an engineered medical device to guarantee a maximally of the skin, this graft appears to be an option for the
predictable outcome following clinical transplantation. The management of both acquired and congenital soft-tissue
pivotal steps, or a roadmap, for device implementation also defects. Additionally, autologous fat grafting remains an
need to be carefully developed, hence providing an intel- appropriate material choice for myringoplasty, limited
lectual challenge that did not exist when we simply focused soft-tissue augmentation and the obliteration of frontal
on material replacement for physical support and/or geo- sinuses in head and neck surgery, albeit being associ-
metrical reconstruction. An overview of the advantages and ated with limitations such as unpredictability in certain
disadvantages of each tissue resource (external or inter- situations [141]. In most medical uses, the expected resorp-
nal) and types of grafts will provide surgeons validated tion of adipose transplants can be estimated, and this
principles regarding graft choice during clinical practice. phenomenon theoretically may be compensated for by ini-
Furthermore, understanding the native tissue composition tial overcorrection. Moreover, the use of adjuvants such as
and structure and revisiting the observed clinical benefits autologous platelet-rich formulations and cell-containing
related to tissue grafts can offer practicing tissue engineers products, which will be addressed in Sections 5 and 6 in this
important information on state-of-the-art biomaterial evo- review, may decrease the rate of adipose graft resorption
lution and design inspiration [115,136]. and hence ameliorate the overall clinical outcome.
Human amniotic membrane (AM) has been used as
3.1. Autologous tissue grafts a grafting material for over 100 years, either directly or
following decellularization. This material exceeds several
The use of biomaterials of human origin for therapeu- qualities of common materials, indicating great poten-
tics was first rooted in tissue grafts transferred from one tial to treat a variety of medical conditions, including
site to another site within the same individual. Even today, corneal defects, diabetic foot ulcers and severe skin burns
many clinicians still consider harvested autologous tissue [142,143]. For example, it has long been suggested that
to be the best material for the reconstruction of most, nonpreserved human AM transplantation in patients with
if not all, tissue defects (Fig. 4). Autologous tissue grafts, acute chemical eye burns may reduce surface inflamma-
also called autografts, are the gold standard with which tion, increase patient comfort and decrease the extent and
all other implantable biomaterials are compared because severity of vascularization [144]. Additionally, an auto-
these grafts maintain large masses of living cells and pos- graft of amniotic tissue can be used as an autologous
sess all of the properties required for new tissue regrowth grafting material in a variety of pediatric neurosurgical
and structural reconstruction. Most importantly, an autol- procedures, such as for repair of myelomeningocele, with
ogous graft, whether of hard or soft tissue, is taken from a no risks of rejection, foreign-body reactions or transmis-
patient’s own body; hence, antigenicity is absent following sion of slow virus infection [145]. For covering venous
transplantation [137,138]. Indeed, the ultimate goal for tis- ulcers that do not respond to conventional treatment,
sue engineering strategies is to develop a tissue construct human AM demonstrates excellent therapeutic potential
that has biological performance identical or similar to that for re-epithelialization but is less expensive than other
of an autologous tissue graft upon implantation. skin substitutes [146]. Recently, it is becoming increas-
ingly evident that human AM may also be used as a
3.1.1. Soft-tissue grafts cost-effective wound dressing for split-thickness skin-graft
Regarding soft-tissue grafts, there has been consider- donor sites [147]. When serving as an adjunctive ther-
able interest in the use of autologous adipose grafts for apy after primary pterygium excision, AM grafts have
the management of cutaneous injuries, the treatment of been demonstrated to be as effective as standard con-
soft-tissue volume deficiencies and the reconstruction of junctival autografts in preventing pterygium recurrence
missing parts of the human body since the late 19th cen- [148]. Moreover, as an effective procedure with a low
tury. Indeed, autologous fat grafting has many clinical uses, rate of recurrence, sutureless human AM transplantation
ranging from routine facial rejuvenation, breast surgery, combined with a narrow-strip conjunctival autograft is
buttock augmentation and treatment for Romberg syn- considered as a preferred grafting strategy for primary
drome to a tool for treating liposuction sequelae. However, pterygium, although further randomized controlled trials
concerns about graft survival following in vivo transplanta- involving larger populations remain to be performed [149].
tion have significantly limited the method’s use. Although Additionally, to use this human material as an advanced
refinements in procuring and grafting technologies have biomedical product containing viable stem cells and bio-
considerably improved the overall clinical outcomes of logics for reconstructive surgery, much more work remains
autologous fat transplantation, the MSCs contained within to be conducted to shed light on the influences of tissue
adipose tissue (e.g., adipose stem cells) may offer unex- culture and/or cryopreservation conditions on cell viabil-
pected opportunities for tissue repair and regeneration ity, to identify easy and practical processes to store human
[139]. The placement of mature adipocytes and adipocyte- AM containing robust cells and to verify the quality of the
derived stem cells into the hormonally active environment tissue transferred before its clinical application [143].
of the breast for breast augmentation raises the possibil- In dentistry, the management of gingival recessions is a
ity of inducing a breast tumor. However, no clinical trial universal request from patients due to its significant influ-
has demonstrated this potential, and a consensus on the ence on both dentin hypersensitivity and esthetics. In this
fundamental knowledge remains in development [140]. respect, a free gingival graft (FGG) can be used either alone
 F.-M. Chen, X. Liu / Progress in Polymer Science 53 (2016) 86–168 99

or, most often, adjuvanted with a coronally positioned flap grafts can satisfy all of the clinical requirements for an
as an effective treatment for gingival recession. As first optimal transplant. Identifying the indications and each
described by Sullivan and Atkins (1968), an FGG can be tissue’s advantages and disadvantages will extend the clin-
directly utilized to cover the denuded root and restore ician’s armamentarium when soft-tissue correction using
the gingival margin to its correct position [150]. Today, grafting materials is required [141]. The use of fascia grafts
modified techniques based on FGGs have been demon- has proven very reliable for soft-tissue augmentation, par-
strated to be successful for the management of isolated and ticularly when tensile strength is a requirement for the
multiple gingival recessions along both the upper and the transplant. Generally, a fascia graft permits more accurate
lower incisors and premolars to ameliorate root coverage and predictable reconstruction than does a fat graft, as evi-
potential and to improve mucogingival junction alignment denced by the observation that the majority of fascia grafts
[151,152]. This successful application is particularly true in can survive as living tissue and retain their native charac-
the specialties of periodontics and implant surgery. When teristics. However, a relative lack of a blood supply or 3D
placing dental implants in partially edentulous areas, FGG, bulk limits the biological potential of the fascia for healing
whether alone or in combination with another tissue aug- and reconstruction [159]. In contrast, the transplantation
mentation technique, is the best-documented and most of free muscle grafts leads to muscle cell death and sub-
successful surgical procedure for increasing the width of sequent partial fibrous tissue replacement in most, if not
keratinized mucosa and augmenting the soft-tissue volume all, cases due to the enormous metabolic needs of this type
around implants and in the esthetic zone [153]. of graft. Nonvascularized muscle grafts are therefore gener-
The capacity of the skin to heal itself by intrinsic ally, if not only, used under conditions in which the desired
mechanisms after injury is vital to human survival, but result is the obliteration of fibrous tissue in a small defect
the process of cutaneous wound repair is disrupted in a (such as in the Eustachian tube or nasofrontal duct). If the
spectrum of disorders. Indeed, a large skin defect would bulk of the transplant or maintenance of the volume is of
not heal properly without a medical intervention such as the utmost importance, the transfer of a vascularized tissue
skin transplantation [154]. Of note, either “full-thickness” should instead be the first consideration. To this end, a wide
or “partial-thickness”, accessible skin grafts can be har- variety of simple and composite flaps of vascularized fat,
vested to treat small- to medium-sized superficial defects. fascia, muscle and other tissues have been designed to meet
Both types of skin grafts involve the entire epidermis the requirements of different specific applications [160].
and offer minimal postoperative pain and scar formation In contrast to free tissue grafts, these flaps are harvested
at the donor site; however, full-thickness grafts include in such a way that their blood supply is maintained, and
all dermal components and appendages (e.g., hair folli- hence, they can maintain their structure following trans-
cles or, if present, sweat glands), whereas partial-thickness fer to a recipient site. Importantly, tissue grafts can be
grafts leave the deeper reticular dermis (including dermal advanced or rotated into position and can retain a good
appendages) in place because these grafts are harvested blood and nerve supply via their pedicle if the donor tis-
at the level of the more superficial papillary dermis [155]. sues for local flaps are located close to their recipient area.
Full-thickness grafts are also esthetically superior and have Many local flaps (e.g., the temporalis muscle flap) have been
less postoperative shrinkage. Recently, Thangavelu et al. applied for the correction of facial and oral defects. Typical
(2011) reported the merit of using an autologous, full- examples are flaps involving the lip (i.e., Abbe flaps) and
thickness subcutaneous adipose composite graft isolated those within the oral cavity (i.e., tongue flaps and palatal
from the patient’s abdomen as an interpositional biomate- flaps) [155]. In contrast, for single-stage restoration of a
rial in the treatment of temporomandibular joint ankylosis complex soft-tissue defect, the anterolateral thigh flap with
in seven patients (eight joints) [156]. However, a careful vascularized fascia lata may offer a relatively reliable fascial
examination of the literature soon reveals that following component [161]. However, vascularized tissue transfer
temporomandibular joint discectomy, there is still no per- is certainly not a solution to all reconstructive needs. For
fect interpositional biomaterial that favors all of the criteria example, an Achilles tendon rupture is often complicated
for the repair of a damaged/missing articular disc [157]. by skin substance loss around the tendon, which is a poorly
Nevertheless, experience with soft-tissue correction using vascularized site. Soft-tissue repair at this site is a crucial
dermal fat grafts in the temporal fossa to augment temporal reconstructive problem and becomes very complex if skin
hollowing has been expanding, suggesting a treatment that reconstruction has to be associated with complex tendon
appears to have good long-term esthetic outcomes [158]. repair [162]. Although each graft has its own limitations,
Future research endeavors, such as hormonal amendment the use of adipose, fascia and occasionally muscle tissue
of adipose grafts and advances in preadipocyte transplants, grafts remains a prevailing choice for soft-tissue recon-
will perhaps significantly ameliorate the overall outcomes struction when properly selected and applied in head and
of soft-tissue transplantation [141]. neck surgery [141]. In addition to recipient-site character-
In addition to fat or dermofat, very different autolo- istics, the function and esthetics of both the donor and the
gous soft tissues, such as dermal fascia and muscle, used in recipient sites must be taken into account in the selection of
the form of flaps according to the requirements of the tis- musculocutaneous or perforator flaps for application. Clin-
sue defects caused by trauma, autoimmune disease, cancer ically, muscle flaps are applied for the obliteration of deep
or infection, have also been used for facial augmentation. spaces because they offer a well-vascularized, pliable tissue
The survival of a graft relies on a well-vascularized recipi- replacement, whereas fasciocutaneous flaps are typically
ent site, and the graft can remain immobile in its nutrient utilized for the treatment of flatter and more superficial
bed [155]. Clearly, none of the aforementioned soft-tissue wounds [163].
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3.1.2. Bone grafts rapidly revascularized and easily incorporated into the
Most, though not all, bone-devastating deficits can lead bone bed at the recipient site and ultimately achieve
to significant alterations in function and appearance and strength equivalent to that of cortical grafts by 6–12
can prove difficult to remedy, which may have signifi- months post-transplantation [138]. Theoretically, both the
cant implications for patients and pose serious clinical remaining viable cells within the graft itself and the recipi-
dilemmas for clinicians [164]. Bone tissue and materi- ent site cells participate in the incorporation of an autograft
als derived from bone have a long and successful history following transplantation. When an autologous fresh graft
of use as bone grafting materials to treat selected con- is transplanted into a recipient site, several osteoprogen-
ditions, such as small- or medium-sized bone defects itors that have acquired the ability to create sufficient
[165]. To replace bone tissue with a material that will daughter bone-forming cells (e.g., osteoblasts), and, hence,
eventually become bone, surgeons’ first choice is to use a significant amount of new bone, accompany the auto-
pieces of the patient’s own bone. Autologous bone grafts graft and are transferred to the recipient site [170]. Because
are the predominantly considered osteoconductive mate- solely the osteoblasts and endosteal lining cells on the
rials for bone replacement, with success rates of well surface of the autograft may survive the transplant, a
over 90% [166]. Similar to autologous soft tissues, bone cancellous bone graft, acting primarily as an osteoconduc-
autografts from a patient’s own body deliver no risk of tive substrate upon application, may support the effective
immunological rejection; possess complete histocompat- penetration of new osteoblasts and osteoblast precursors
ibility; and offer superior osteogenic, osteoconductive and and facilitate ingrowth of new blood vessels [172]. More-
osteoinductive performance compared with other clin- over, osteoinductive molecules and other growth signals
ically available grafting materials [167]. By their very released from the autograft during the resorptive process,
nature, as mineralized scaffolds, living bone grafts can as well as cytokines produced during the inflammatory
deliver an optimized combination of cellular components, phase, can contribute to healing of the autograft [173].
including, but not limited to, differentiated osteoblasts, Based on the amount and shape of a bone autograft
an appropriate matrix of cancellous bone, and a mix- needed for a specific application, bone particulates or
ture of bone growth factors at a physiological level. This blocks are commonly harvested from non-essential bones,
combination supports bone regrowth and integration into such as the iliac crest, tibia, fibula, chin, ribs, mandible
the surrounding bone, normally through creeping sub- and even parts of the skull [174]. However, the iliac crest
stitution, and ultimately rebuilds mechanically efficient is the most common area from which a cancellous auto-
bone structures [168]. However, clinical benefits are not graft is harvested, although an iliac crest bone graft (ICBG)
guaranteed, and these autografts still suffer from draw- is occasionally also obtained from the distal part of the
backs such as resorption, limited availability, short-term radius/tibia. Cancellous bone is widely utilized for the
viability and unpredictable graft resorption. Most impor- reconstruction of depressed fractures of the lateral tibial
tantly, the extraction of an autograft is essentially a second plateau and, more often, the delayed union of long-bone
surgery. The harvesting procedure can include pain follow- fractures [175,176]. The principal merits of cancellous
ing surgery and numbness at the extraction site, in addition grafts are their safety, including a low risk of transplant
to the potential attendant risks and postoperative compli- rejection and disease transmission, and their excellent clin-
cations [169]. Fortunately, recent minimally invasive and ical success rate. However, the supply of donor bone grafts
innovative harvesting tools and techniques have largely is restricted, and donor site morbidity increases if a larger
decreased historical issues such as donor site morbidity, autograft is harvested, in addition to other disadvantages,
making the acquisition of the required amount of bone such as increased blood loss, potential wound infection
simpler and easier [170]. Hence, clinical surgeons have and the prolonged anesthetic time [137,177]. In particu-
renewed interest in choosing autologous bone grafts as a lar, although the incidence is low, complications associated
preferred source of bone reconstructive materials. with the harvesting of iliac crest bone, such as persistent
Native bone is a mineralized matrix consisting of postoperative pain and nerve/arterial injury, have been
biopolymers (mostly collagen I and certain minor but reported [170].
important noncollagenous proteins) and biominerals. The Autologous cortical grafts are osteoconductive but
transplantation of a fresh bone autograft is an attempt to lack osteoinductive properties; however, the surviving
achieve rapid bone restoration because living bone can osteoblasts within the transferred bone do provide cer-
survive well and add to bone volume at a recipient site tain osteogenic properties [178,179]. Several parameters
and eventually maintain bone strength. Compared with contribute to the success rate of such autografts, includ-
cortical bone grafts, cancellous bone autografts are con- ing the stability of the bone and the prevailing situation
sidered to be more osteogenic because the existence of within the host recipient area [155]. A cortical graft may
native spaces within their structure permits the diffu- not be revascularized as rapidly or properly as cancellous
sion of nutrients necessary for new bone formation and bone. The structure of the cortical bone does not permit
allows limited revascularization through the microanas- a large contact area between the autograft and the host
tomosis of circulating vessels [138,170,171]. The vascular for vascular penetration, so its revascularization generally
response in autologous cancellous grafts is much greater requires approximately 2 months. However, nonvascular-
than that in cortical autografts. As a result, within l–2 ized bone autografts can provide a relatively reliable choice
weeks, the entire cancellous bed can be completely revas- for osseous defect filling, and following bone remodeling,
cularized. Although cancellous grafts, as good space fillers, new bone growth and complete integration of the graft into
do not provide immediate structural support, they are the host site occur [155]. Compared with cancellous grafts,
 F.-M. Chen, X. Liu / Progress in Polymer Science 53 (2016) 86–168 101

nonvascularized cortical autografts may offer immediate Not surprisingly, autologous ICBGs have been and will
structural support, but they appear mechanically weak continue to be the gold standard for grafting in many
during the first 6 weeks post-transplantation due to resorp- surgical procedures, including procedures for repairing
tion and revascularization [178–180]. Revascularization bone defects, treating bone fractures, promoting non-
is completed through the old Haversian and Volkmann union healing and alleviating severe back pain through
canals. After the revascularization of the periphery of the spinal fusion, but their use necessitates harvesting autol-
transplants, interior revascularization rapidly follows suit. ogous bone from a separate area, which may involve an
The coverage of the nonvascularized bone and the pres- additional surgery and donor site morbidity after harvest-
ence of an optimized soft-tissue recipient bed are generally ing [137,138]. In this respect, recombinant human bone
required to decrease healing complications with respect to morphogenetic protein-2 (rhBMP-2) on an absorbable col-
infection and wound dehiscence and to ensure the survival lagen sponge (ACS) or other bone grafting materials have
of the osteogenic cells within the transplanted bone [181]. been suggested as an alternative to an autologous bone
The shape and size of vascularized cortical autografts are graft for bone reconstruction. In particular, several tri-
largely dictated by the morphology of the donor site, but als have reported statistically superior or similar clinical
compared with nonvascularized grafts, vascularized cor- outcomes for these bone substitutes compared with auto-
tical autografts are less dependent on sufficient soft-tissue grafts transferred from the iliac crest following lumbar
bed vascularity at the recipient site. At the host–graft inter- fusion procedures or maxillary reconstruction in patients
face, vascularized cortical autografts heal quickly, and their with a cleft lip and palate in terms of inducing new bone
remodeling is typically similar to the biological process of formation, achieving fusion success and avoiding reopera-
normal bone turnover [182]. Because vascularized auto- tion [189–191]. Therefore, bone substitutes incorporating
grafts do not undergo revascularization and resorption, osteoinductive protein(s), whether alone or associated
they may offer superior initial strength during the initial 6 with autologous bone, have been considered as part of
weeks post-implantation. These grafts, however, still must the orthopedic surgeon’s treatment options [192,193].
be supported by external or internal fixation to protect Interestingly, in the treatment of long-bone nonunion,
them from fracture [179]. The excess soft tissue associated rhBMP-2/ACS mixed with a cancellous allograft showed
with the transferred vascularized cortical bone often must possible advantages, including a shorter operative time
be removed by a second operation because such a graft is and reduced intraoperative blood loss compared with an
initially transplanted with a periosseous cuff of soft tissue autologous iliac bone graft [192]. Similarly, rhBMP-2-aided
containing its blood supply [183]. In oral and maxillofa- bone tissue engineering has been demonstrated to be as
cial applications, additional adjuvant procedures may also effective as traditional autologous bone grafting for the
be needed to increase the grafted bone volume to allow treatment of tibial fractures associated with extensive trau-
for immediate or subsequent dental implant rehabilitation matic diaphyseal bone loss [194] and for reconstruction of
[184]. In fact, vascularized bone transplants, along with the alveolar cleft in patients with a cleft lip, alveolus and
soft-tissue grafts, are now routinely applied by clinical sur- palate [193]. In implant dentistry, it appears that a vari-
geons for the restoration of large composite tissue defects, ety of bone materials in association with growth factors
through the more difficult and technically demanding may be an alternative/complement to autografts, leading
method of microvascular composite tissue transfer [155]. to significant bone formation in the floor of the maxillary
In this regard, cortical bone autografts are good choices for sinus or to lateral bone augmentation of the alveolar ridge
the treatment of bone defects requiring immediate struc- [195]. Despite considerable interest, however, the clinical
tural support, such as segmental bone defects of more than experience so far has been unsatisfactory, if not relatively
5–6 cm. disappointing. At the least, the field has not lived up to
In oral and maxillofacial therapies, historically, the best expectations, and to date, no well-documented prospective
bone grafting procedures have been particulate cancellous clinical studies have been performed. A clearly increased
bone/bone marrow autografts, which can offer a rich source risk of cancer and other adverse events among patients
of bone and marrow cells that have osteogenic potential receiving recombinant human protein treatments and a
[185]. Histologic findings from case reports substantiate lack of reproducibility have been major problems. Most
the potential for autologous bone/bone marrow grafts to importantly, a carefully review of data published thus far,
support periodontal regeneration in humans [186,187]. and particularly those data from the original industry-
Multiple clinical concerns, however, have largely limited sponsored BMP trials, reveals a possible study design bias
the transfer of extraoral autografts, particularly from the in the execution of these studies, thus weakening our con-
iliac crest, for intraoral therapies, including the possibil- fidence about the reported benefits of BMP administration
ity of surgical complications and pain associated with the in the treatment of orthopedic disorders [196]. Indeed,
donor site. Therefore, for the treatment of oral diseases, rhBMP-2 has no proven advantage over autografts based on
autologous bone is frequently harvested from intraoral critical reviews of currently available evidence, and more
sites, often in the same quadrant as the regenerative work remains to be done to standardize and optimize the
surgery [188]. Intraoral donor sites, however, typically technique. Additionally, more focused post-surgery evalu-
yield comparatively limited graft volume. Harvesting suf- ations of the complications and adverse events related to
ficient donor bone, therefore, as an osseous coagulum of clinical BMP application are needed to prevent clinician and
cortical or cortical-cancellous bone, can necessitate the cre- patient biases from affecting the functional outcome, thus
ation of additional intraoral surgical sites, thereby increas- providing surgeons and the public with more reliable and
ing the potential for surgical morbidity and discomfort. useful information [197].
102 F.-M. Chen, X. Liu / Progress in Polymer Science 53 (2016) 86–168

3.2. Allogenic tissue grafts histocompatibility barriers undergo rejection in approx-

imately 100% of hosts [205]. Nevertheless, the temporary
Although an autograft is the best choice for the man- coverage of a third-degree facial burn with allograft skin
agement of bone defects, the disadvantages of autologous following debridement has been evidenced to facilitate
grafts are the limited amount of available graft material initial vascularization of the wound bed [206]. Six days
and the morbidity associated with their harvest. The limita- later, the allograft skin is replaced with a split-thickness
tions related to the procurement of autologous tissue grafts skin autograft because the allograft usually undergoes
may be addressed using a myriad of other grafting materi- rejection within 2 weeks, although the possible repopula-
als, which can be classified into the following categories: tion of a skin allograft with host cells may make it persist
(i) allografts, also called allogenic or homologous grafts in nonimmunosuppressed burn patients for as long as 7
or homografts, which are composed of materials acquired weeks [207]. Further, recent evidence suggests that human
from another individual of the same species (Fig. 4); (ii) dermal/epidermal cell fractions can be used directly from
xenografts, also known as heterografts or xenogenic grafts, isolation and safely and conveniently transplanted in one
which are materials acquired from another species; and (iii) single surgical intervention for wound closure toward
alloplastic grafts or synthetic grafts, which are artificial or improved healing [208]. Although allogenic skin grafts
manufactured materials that can be subdivided based on can be used directly for soft-tissue augmentation and
their origin and chemical composition [198]. With respect have been described as having satisfactory efficacy when
to biomaterials of human origin, this review will focus its appropriately applied in optimized settings, a unique
detailed discussion on allografts in this section. The suc- understanding of the components of human skin has led
cessful transplantation of allogenic materials (e.g., tissues to the development of acellular human dermal matri-
and organs) into patients depends on not only the benefits ces through decellularization [206,207,209,210]. These
but also the mitigation of risks [199]. Rejection continues to human-derived materials offer a viable and more feasible
be the main risk factor in allograft transplantation, and the solution for soft-tissue reconstruction and the manage-
immune response is one of the pivotal determinants of the ment of difficult wounds, especially in situations in which
development of rejection. However, the allograft outcome allografts are not readily available or extensive surgery for
can be improved with an optimized immunosuppressive acquiring sufficient autograft material might be hazardous
medication and enhanced human leukocyte antigen (HLA) to the patient [211].
matching between donors and recipients [200].
3.2.2. Composite tissue allotransplantation
3.2.1. Corneal and skin grafts Recent breakthroughs in the field of complex tis-
Since the rise of modern corneal graft surgery, allogenic sue allotransplantation (CTA) have enabled surgeons to
corneal transplantations have been successfully performed address several complex problems that exceed the pos-
in human subjects for nearly a century and continue to sibilities of traditional tissue transplantation [212]. As a
be the most commonly performed allotransplants because pioneer of complex tissue allotransplantation (CTA), the
they enjoy an immune privilege that is unrivaled in the field hand allograft was successfully established near the end
of allotransplantation [201]. The capacity of corneal allo- of the last century and, despite arguments related to
grafts to evade immune rejection is attributable to multiple its practicality and methods, has been unabatedly used
anatomical, physiological and immunoregulatory condi- for over 20 years [213]. Shortly after the report of the
tions that collaborate to escape the induction of alloim- first case of hand allotransplantation, CTA, such as upper-
munity [202]. To determine whether the donor or tissue extremity and face transplantation, has evolved into an
characteristics of corneas for transplantation are predictive exciting and promising subset of reconstructive transplant
of adverse events reported to occur in the early postopera- surgery, along with advances in immunotherapy. This sub-
tive period, Ple-Plakon et al. (2013) recently compared the set involves a variable combination of upper-extremity (i.e.,
preoperative donor and tissue characteristics of corneal tis- various levels) and facial (myocutaneous versus osteomy-
sues with or without reported adverse events from 2007 ocutaneous) composite subtypes [214]. In particular, tissue
to 2011. It was observed that adverse events more com- damage/loss occurring in the craniofacial area may cause
monly occurred after endothelial keratoplasty and that an serious physiological and psychological consequences in
increased rate of primary graft failure was associated with affected patients. In November 2005, the first successful
male donors and donors with a cancer history [203]. partial facial transplantation was performed in Amiens,
In patients under close observation, however, over- France [215], and a similar case was also successfully
all, corneal grafts have exhibited an excellent survival performed in China 5 months later [216]. Furthermore,
rate. Of course, proper donor cornea handling and in December 2008, the first near-total face transplanta-
patient review/selection and thorough preoperative and tion was performed in the United States, with 80% of the
postoperative assessment are pivotal for the successful patient’s traumatic facial deficit replaced by a composite
allotransplantation of corneal tissue and, indeed, of any allograft from a brain-dead donor [116,217]. Recent evi-
other tissue [204]. Generally, orthotopic corneal allografts dence suggests that the transplantation of an entire face,
experience long-term survival in 50% to more than 90% of along with the auricles and scalp, is technically practi-
hosts, depending on the histocompatibility barriers that cal and feasible in humans [218]. However, reconstruction
confront the host. of the face to a functional and esthetic level presents a
In contrast, skin allografts transplanted across var- formidable challenge, even though it is desired by most,
ious major histocompatibility complexes or minor if not all, affected patients. Apart from the technical
 F.-M. Chen, X. Liu / Progress in Polymer Science 53 (2016) 86–168 103

hurdles and difficult ethical/psychological concerns related perceived risks of disease transmission still exist; in partic-
to such a complex surgical procedure, the recipient patient ular, the risk of transmission of human immunodeficiency
must take immunosuppressive agents for his or her entire virus (HIV) is presumed to be 1 in 1.6 million [136,224]. Fur-
life, similar to those patients who have undergone solid- thermore, there have been certain case reports of hepatitis
organ allotransplantation [219]. Although progress in CTA B and C transmission through the transplantation of mus-
could provide new treatment for patients with severe tis- culoskeletal allografts [225]. Despite these risks and given
sue disfigurements, much remains to be done to make the advantages of bone grafting using allograft material,
this technique more safe and practical. For example, it is bone grafting procedures expanded from 5000 to 10,000
arguable that logistical and immunologic challenges cur- cases in 1985 to approximately 150,000 in 1996 and more
rently restrict the widespread clinical application of CTA. In than 1 million in 2004, and the number of cases continues
addition to careful oversight and individualized screening to increase [141,226–228].
procedures, further investigations are required for tech- Both cortical and corticocancellous bone allografts are
nical renovation to minimize the levels of surgery risk commercially available in various forms, such as partic-
and to identify optimal immunomodulating protocols for ulates, chips and blocks, and these allografts have been
specific patients as they seek improved quality of life elucidated by basic science and validated for clinical use;
[220,221]. Specifically, the use of bioreactors for tissue cul- the industries built around these items are more success-
ture may extend ex vivo allograft survival times and enable ful and in demand than ever before [229]. Under United
allograft modulations that enhance graft function while States Food and Drug Administration (FDA) regulations,
mitigating immunogenicity following allotransplantation. facilities engaged in procuring and processing human tis-
Approaches utilizing bioreactor systems have expanded sues for transplantation must ensure that infectious disease
the reconstructive potential and applications of CTA and testing (i.e., for HIV-1, HIV-2 and hepatitis B and C in the
could one day enable organ-level engineering of cus- United States) and specified minimum medical screen-
tomized complex tissue grafts and organs [212]. However, ing have been performed and that records exist and are
donor tissue/organ shortages and the adverse effects maintained to document the testing and screening of each
of chronic immunosuppression imply that alternatives human tissue. In addition, tissue processing (i.e., washing
to allogeneic organ transplantation (e.g., bioengineered to ensure the removal of blood components, freeze dry-
organs based on patient-derived stem cells and decellu- ing or gamma irradiation) is applied by bone banks and
larized organ templates) may hold greater promise for other commercial vendors to ensure the safe clinical use
patients suffering from end-stage organ failure in the future of the resultant allografts [224]. Because of these mea-
[222]. Nevertheless, information gathered during the prac- sures, immunological rejection, depending on the method
tice of CTA as well as solid-organ allotransplantation will applied for graft preservation and the potential risk of viral,
definitely offer signposts in the future design and develop- bacterial or prion transmission associated with allograft
ment of biological devices or tissue-engineered constructs transplantation, has been well controlled at a clinically
or organs for transplantation. acceptable level. During the 30-year history of the use
of freeze-dried allograft bone in reconstructive surgery,
3.2.3. Allogenic bone grafts there have been very few reports of disease transmission
In contrast to complex tissues/organs, allogenic bone [141,227,230]. Tissue banks process bone allografts using
grafts that are cadaveric in origin and are available from various methods, with several based on proprietary tech-
commercial vendors (e.g., bone banks) have been widely niques; however, most are based on similar underlying
used in reconstructive surgery. In this case, the expense and concepts that address cleansing, decontamination, antimi-
trauma associated with autograft harvesting, the quantity crobial treatment, dehydration, graft sizing and terminal
and size limitations of grafts and donor site morbidity are sterilization [230]. Allografts typically arrive in a form in
no longer concerns. Although lacking osteogenic properties one of two main categories, i.e., demineralized freeze-dried
due to the absence of living cells, allografts are in posses- bone allografts (DFDBAs) or mineralized freeze-dried bone
sion of both osteoconductive and weakly osteoinductive allografts (FDBAs). Both allograft bone types can be in the
abilities upon implantation because they release bone mor- form of matrices, cancellous or morselized chips, cortical
phogenetic proteins (BMPs) that coax bone-forming cells or corticocancellous grafts or whole-bone or osteochondral
[223]. In addition to its advantages, such as its ready avail- segments. It is suggested that FDBAs may yield a satisfac-
ability in the required sizes and shapes, its lack of donor tory outcome in socket and sinus augmentation, although
site morbidity and its avoidance of the need to sacrifice host a long time is needed to achieve a suitable amount of new
bone structures for harvesting autografts, this type of graft- bone formation [231,232]. However, large cortical allograft
ing material is attractive because it closely matches the bone undergoes minimal remodeling and revasculariza-
recipient in constitutional elements and architecture and is tion following implantation. The persistence of a nonvital
theoretically available in unlimited quantities. The funda- graft at the site of a bone defect is incapable of physiolog-
mental problems of this grafting material are antigenicity ical adaptation to functional loads and hence leads to the
and the potential for infectious agent transmission, which accumulation of microfractures over time [233].
is a major consideration that is in fact minimized by recent DFDBAs exhibit the capacity to induce bone formation
strategies associated with tissue processing, sterilization at nonorthotopic sites, such as muscle, and are considered
and freezing. However, these procedures in turn decrease to be osteoinductive. Levels of approximately 2% residual
graft properties with regard to osteoinduction, osteocon- calcium in DFDBAs have been shown to provide max-
duction and mechanical strength, and indeed, real and imum osteoinductive potential by assay systems [234],
104 F.-M. Chen, X. Liu / Progress in Polymer Science 53 (2016) 86–168

presumably due to the exposure of BMPs [235]. It was number of clinical circumstances to fill defects caused by
proposed that removal of the mineral component allows bone cysts and cavities for craniomaxillofacial reconstruc-
greater exposure of osteoinductive proteins [226,236,237]; tion and for the bridging of large bone defects, despite
however, allografts are predominately space-occupying the potential to transmit disease [239,248–250]. When
osteoconductive lattices or frameworks. The rigorous pro- applied, DBM can be used in association with a cancellous
cesses involved in the removal of potential antigenicity and bone autograft when the defect is very large and the
pathogenicity, e.g., ethylene oxide sterilization or higher supply of autologous bone is insufficient. Additionally,
levels of gamma irradiation, lead to a low concentration DBM has been combined with several different types of
of bone growth factors, biological proteins and bioactive materials, such as glycerol (Grafton from Osteotech, USA),
materials that are necessary for osteoinduction in the hyaluronate (i.e., DBX from Synthes, USA), poloxamer
resultant grafts, and hence, the grafts present minimal (DynaGraft from GenSci Regeneration Sciences, Canada),
osteoinductivity and no osteogenicity [238]. Additionally, gelatin (Regenafil from Regeneration Technologies, USA)
certain processing techniques have been associated with and calcium sulfate (Allomatrix from Wright Medical
detrimental impacts on the biological and mechanical Technology, USA), to facilitate clinical handling and to
properties of cortical bone allografts. For example, freeze improve surgical outcomes, leading to a variety of new
drying may cause microcracks in the bone, and gamma products offered by various commercial vendors [251]. The
irradiation increases bone brittleness [227]. use of patient-derived allografts, however, also introduces
The biological performance of bone allografts may be potential challenges regarding the potential transmission
ameliorated by the incorporation of recombinant human of infectious agents, the graft formats in which assets
growth factors, autologous bone (or exogenously cultured can be properly maintained and the ability to retrieve
autologous bone-forming cells), enamel matrix derivative the grafts effectively, in addition to potential regulatory
or various platelet-rich preparations [176,239–241]. Alter- hurdles and damage to material components after months
natively, the treatment of mineralized bone allografts with to years of storage.
a 1:1 formic acid–citric acid mixture or with hydrochloric
acid (0.5–0.6 M) can remove inorganic elements, yielding 3.2.4. Human dentin matrix
a natural polymer product that is generally referred to as As a mineralized connective tissue, dentin is well
demineralized bone matrix (DBM) [50,242]. As an acellular adapted to its role as a major structural and functional
organic matrix, DBM mimics the microstructure of native component of the tooth. Although similar in composition
bone but is less immunogenic and possesses good osteoin- to bone, the dentin matrix is not remodeled physiolog-
ductive and osteoconductive performance [227,242]. DBM ically, and this matrix has been traditionally considered
contains the major protein components of bone, such as to be a relatively inert tissue [252]. In general, removed
adhesion ligands and osteoinductive growth factors that human teeth are considered as infective medical waste.
may contribute to new bone formation [243]. However, However, biomaterials based on teeth, as an important
DBM cannot provide structural support, so its primary native resource, contain native growth factors and several
application is in structurally stable places such as the important functional sequences that support cell adhe-
sites of bone defects. Although current clinical DBM deliv- sion as an anchorage matrix. The use of human teeth
ery generally requires the incorporation of DBM particles removed for orthodontic, impaction-related or irreversible
within a carrier liquid, recent evidence suggests that it periodontic reasons as grafting materials addresses sev-
is possible to produce a soluble form of ECM materials eral of the problems with the bone grafting technique,
or DBM product that could be induced to form a hydro- such as the limited availability of bone mass, the risk of
gel scaffold [244]. These bone matrix-based materials with donor site infection and significant resorption of the grafted
distinct structural, mechanical and biological properties bone, although there are still concerns about the residual
can rapidly revascularize and act as suitable carriers for infective risks [253]. To assess the extent to which deminer-
autologous bone marrow for medical use. As the antigenic alized dentin matrix (DDM), prepared by a process similar
surface structure of the graft is demolished during dem- to how DBM is obtained, induces osteochondral regener-
ineralization, the graft generally evokes no appreciable ation, DDM from bovine teeth was implanted into rabbit
local foreign-body immunogenic reaction and facilitates knees with surgery-created full-thickness articular carti-
cell attachment and growth (Fig. 5) [245]. Recently, it has lage defects. It was found that the DDM led to active new
become evident that DBM can be remineralized based bone formation early in the postoperative period, indicat-
on the alternating solution immersion (ASI) technique, ing that the DDM acted as a suitable scaffolding material for
resulting in mechanically stiff, strong and biocompati- osteochondral regeneration [254]. Recently, it was demon-
ble allografts that facilitate tissue engineering and clinical strated that without causing an inflammatory reaction or
applications [246]. The biological activity of these products infection, allogenic DDM significantly increased bone mass
may be attributed to the various proteins and growth fac- and improved bone quality when it was used as a bone
tors present within the mineral component of the product grafting material to treat surgically created bone defects
and to the demineralization process, which makes these on the skull of rabbits [255]. Further evidence suggests
factors available in the host environment. that DDM increases the expression of vascular endothelial
The osteoinductive properties of DBM are not consis- growth factors (VEGFs) and accelerates the healing pro-
tent; can be influenced by processing, sterilization and cess by stimulating bone deposition and vessel formation
storage methods; and can change from donor to donor [256]. Based on these findings, DDM has been successfully
[247]. Of note, DBM has been successfully applied in a applied as an osteoinductive/osteoconductive material for
 F.-M. Chen, X. Liu / Progress in Polymer Science 53 (2016) 86–168 105

Fig. 5. Representative scanning electron microscopy (SEM) images of the attachment and growth of bone marrow mesenchymal stem cells on demineralized
bone matrix (DBM) for a 48-h period. (A) 12 h: cells attached onto the material; (B) 24 h: cells spread across the gap; (C) 48 h: constituting cell-cell
interactions; (D) 72 h: protein and matrix production; (E) 96 h: cell penetration into the material and cell sheet formation; (F) 120 h: cell–matrix layer
infiltration and enwrapping of the material.

bone reconstruction in the clinic for many years (Fig. 6). dentin scaffold and dentin-forming cells are important not
Considering that the particulate morphology of DDM limits only for biocompatibility but also for the potential for the
its applications at sites requiring structural support, col- material to skew the cell response toward correct differ-
lagen has been used in combination with DDM to form a entiation [260]. In this regard, DDM has been evidenced to
composite DDM–collagen material that has a significant be an appropriate scaffold that provides an inductive envi-
clinical advantage over DDM alone and the potential to be ronment for both dentin regeneration [261,262] and tooth
used in bone and orthopedic surgeries [257]. root construction [263,264]. It is speculated that materials
Accumulating laboratory studies have supported the science will develop the next generation of regenerative
use of human DDM in both bone regeneration and procedures by using the human dentin matrix to treat
tooth tissue engineering. It is widely recognized that the patients in the near future.
dentin–pulp complex demonstrates strong regenerative
potential due to the many bioactive molecules bound 3.3. Tissue engineering: the state of the art in
within the dentin matrix. Following dental injury, the transplantation
release of dentin matrix components and many other sig-
nals can contribute to the angiogenic and cell-recruitment Although treatments by autogenic and allogenic tissue
events necessary for regeneration of the dentin–pulp com- transplantation have been successfully applied in clini-
plex [258,259]. The outcomes of the interplay between a cal procedures for numerous medical conditions, these
106 F.-M. Chen, X. Liu / Progress in Polymer Science 53 (2016) 86–168

Fig. 6. Representative scanning electron microscopy (SEM) images of demineralized dentin matrix (DDM) (images from a human tooth-derived sample).
From (A) to (D), gradually magnified views of the DDM bioscaffold showing the arrangement of tubules in human dentin.

therapies are largely impeded by their disadvantages, such or emergency situations [115,136,266]. From the inception
as the limited tissue available, considerable donor site mor- of tissue engineering as a field, research on the regrowth
bidity and risks of disease transmission [140,162,198,228]. of most, if not all, types of human tissues has been and
A brief overview of conventional strategies for treat- still is being conducted, and as a result of interdisciplinary
ing disfiguration and large composite tissue defects by endeavors by biologists, materials scientists, engineers and
applying human-derived tissue materials directly illus- physicians, tissue-engineered products for bone, cartilage
trates that in addition to autologous tissues, all of the and skin repair have been approved for clinical applica-
grafts and techniques currently available for clinical tion by the United States FDA. Several of these products
application fall short of achieving the complete func- have been produced using biomaterials of human ori-
tional and esthetic replacement of lost/damaged tissues gin as scaffolds [155]. Techniques for producing bioactive
[137,157,178,182,187,218,219,265]. However, close coor- synthetic bone scaffolds have the potential to signifi-
dination between biologists and surgeons offers a critical cantly improve the performance of bone graft replacement
step for clinical success, and the implantation of specimens materials, and knowledge generated from tissue/organ
as rapidly as possible may improve the overall engraftment allotransplantation, ranging from technique innovation to
rate. Similarly, recent advances in quality management transplant design to immunomodulating protocols, will
systems, coupled with new insights into the preserva- instruct the future development, optimization and applica-
tion and storage of human materials, have been shown tion of tissue-engineered products [135,267,268]. Instead
to improve the safety and quality of human materials of the long-standing goal of merely replicating natural tis-
applied in transplantation [199]. As we continue to face sue regeneration, in the future, manmade materials and
severe shortages of organs (or certain specific tissues) for structures could be utilized to exceed the body’s natural
transplantation throughout the world, we need to consider healing response and, for the first time, to offer a bone
innovative solutions to decrease the numbers of patients graft replacement material with a clear advantage over
on waiting lists, which are ever growing because of the traditional autografts; however, current biotechnology for
expanding aging population and the severe shortage of converting grafting materials to functional bone tissues
suitable donor tissues and/or organs available [266]. The remains in its infancy [136,165]. The recreation of more
approach that is most likely to make a real difference complex human tissue replacements remains challenging,
in transplantation in the long term is tissue engineering, although the production of complex tissues has recently
a field that has emerged from the selective conjunction appeared in the literature in many forms [5,35], such as in
of stem cells, biomaterial scaffolds, gene therapy and the form of a tissue-engineered bladder [74,269], rendering
chemical/mechanical molecules for new implantable tis- tissue engineering a potential candidate to revolutionize
sue/organ production that may be used in either planned our current clinical strategies for reconstructive surgery
 F.-M. Chen, X. Liu / Progress in Polymer Science 53 (2016) 86–168 107

and greatly enhance the lives of patients. The selection of molecules (i.e., growth factors and chemokines), increas-
a perfect biomaterial can significantly contribute to this ing the local concentrations of agonists to which target
innovation, not only by serving as a carrier device for sig- cell populations in the niche are exposed [277]. Accord-
naling molecules and responsive cells but also by offering ing to recent findings, however, as a highly dynamic entity,
a platform that interacts with and guides tissue forma- the ECM has been increasingly shown to exert an intense
tion. Mass transport and the regulation of cell-material impact on cell behavior and function via its matrix stiffness,
interplay are two of the crucial parameters that must be its ligand types and its degree of coupling of fibrous protein
taken into consideration during design [31,50]. The similar- to the surface of the underlying substrate (i.e., protein teth-
ity of the microstructure, composition and biomechanical ering and matrix porosity); such biological influence can be
and biological properties of decellularized materials and explored for use in the design of new ingenious biomateri-
substrates to those of native human tissues and organs als [278–283]. It is now well known that the living-tissue
is spurring interest in the direct use of these materials ECM not only acts as a reservoir for morphogens while
for tissue repair and in recreating human-derived tis- providing mechanical support to resident cells but also par-
sues/organs in simplified forms for tissue engineering ticipates in defining the stability and shape of tissues and
applications, such as the reduction of ECM into short in facilitating most cell communication activities associ-
functional domains to influence stem cell differentiation ated with tissue development, turnover and regeneration
[222]. In particular, key ECM components, including chem- [276,284] (Fig. 7).
ical macromolecules, physical parameters (i.e., shear stress
and tissue stiffness) and microenvironmental signals (i.e., 4.1. Biomaterials for tissue engineering inspired by the
hypoxia), can be advantageous for therapeutic applica- ECM
tions because human cells already have a predisposition
to recognize them and because these components’ usage ECMs are the focus of intensive research endeavors
has a low potential to induce negative immune responses worldwide that are directed not only at illustrating ECMs’
[270–273]. For many years, it has been recognized that nature and unique properties in biology and materials sci-
both simple tissues and complicated organs may be decel- ence but also at mining ECMs or ECM-like biomaterials for
lularized for tissue engineering use, and decellularization tissue engineering and regenerative medicine applications
methods have been optimized to completely remove cel- [276,285]. As a multi-component structural meshwork
lular components while keeping the ECM intact [274,275]. that is assembled into unique tissue-specific architec-
The following section provides examples of human tissue tures, the ECM provides dynamic signaling cues that
ECM components that are currently being tested as scaf- modulate various aspects of cell fate commitment in part
folds for tissue engineering and regenerative therapies. In through its physical, chemical and mechanical properties
particular, the section highlights different classes of ECM [26,286,287]. Over the past few decades, a considerable
components and different strategies that are commonly amount of attention has been focused on exploring the
applied or are promising in their possibility of future appli- biological information and components of native ECM for
cation and development in making the transition from a biomaterials design. Based on its spatial patterning, chem-
human tissue-derived ECM to a tissue engineering scaffold. ical composition and functionality, ECM can typically be
divided into 2 categories of components, namely, the base-
4. Human tissue ECM-based biomaterials ment membrane (BM) and the stromal matrix (SM) (Fig. 8).
In epithelia, BMs are specialized ECM assemblies (contain-
The ECM, or the extracellular macromolecule network ing type IV collagens, laminins, perlecan, agrin, nidogen
between and around niche cells, is a multi-component and other macromolecules) that play a key organizing role,
structural element that is synthesized and assembled by providing a film-like substrate for a tissue’s peripheral
the resident cells and that combines ubiquitous structural cells, which includes wrapping around the vasculature as a
biomacromolecules, including an array of multidomain supporting substratum for epithelial sheets and maintain-
biomacromolecules (e.g., collagens, glycoproteins, proteo- ing cell polarity [26]. However, in tissues such as tooth,
glycans and elastic fibers); its protein network remains in bone, cartilage, muscle and tendons, in which BMs play
equilibrium with the surrounding cells and tightly reg- an obvious mechanical role, the SM is comprised of larger,
ulates the fiber diameter, composition and organization fibrous structures and constitutes the bulk of the ECM,
[271]. As a complex, fibrillar 3D network of proteins and serving as the main structural support of the ECM. Quan-
polysaccharides, the ECM has a highly regulated, tissue- titatively, BM is a major component that dictates overall
specific composition and set of physical properties in the mechanical characteristics. The organization and com-
majority of tissues and organs in the human body, and the position of the ECM in these tissues reflect evolutionary
nature of its contact with stem cells also varies consider- adaptation to mechanical load, and each ECM component
ably [276]. The ECM of living organisms may be dispersed is unique in its interplay with the tissue-forming cells
as an amorphous “ground substance” and/or organized that have been studied for use in various regenerative
into interacting fibrous structures arranged in a cell/tissue- procedures [288]. Indeed, in the development of new bio-
specific manner. The general functionality of the native materials by combining different molecular components
ECM is to impart physical cohesiveness on a tissue with at tailored concentrations and geometries, a wide range of
regard to the provision of a structural function and an tissue-unique structural requirements can be met [276].
anchoring support for cells. Under certain conditions, the Along with recent advances in ECM science and develop-
ECM also anchors and acts as a reservoir for various soluble mental biology, concentrated efforts on the exploration
108 F.-M. Chen, X. Liu / Progress in Polymer Science 53 (2016) 86–168

Fig. 7. Schematic representation of the extracellular matrix (ECM) functions and crosstalk at the cell–cell and cell–matrix interfaces (illustrations are
not to scale). Crosstalk between stem cells and niche cells is mediated by soluble ECM growth factors (secreted by producer cells via autocrine and/or
paracrine routes) and the properties of the surrounding ECM. Through cell membrane growth factor receptors and a complex signal transduction network,
the outside instructions are conveyed to the stem cells, resulting in specific biological cellular responses and functionalities, such as cell differentiation
and gene expression [12]. Aside from acting as a mediator of mechanical constraints applied to cells, the ECM also affects cells via its architecture and
overall biological and mechanical properties [278]. Soluble and matrix-binding factors combine with cell–matrix adhesion, cell–cell contact and signaling
gradients to determine and control the most fundamental behaviors and characteristics of stem cells, including polarity, adhesion, anchorage, proliferation,
migration, differentiation and apoptosis. In turn, cells contribute to the complex cell–matrix feedback loop, with their overall functionalities resulting in
proteolytic turnover and ECM structural integrity [276]. The design of new ingenious biomaterials must consider these functions of the native ECM, at least
to a certain degree, to mimic the natural environment to regulate stem cell fate decisions.

of human-derived biomaterials for therapeutics have now and cell–matrix interactions and communication [48,287].
moved from the direct use of autogenic tissue grafts, A prefabricated material device must assume this instruc-
allogenic tissues/organs from donors and a wide variety of tive role to a certain degree to ensure cell viability and
allografts from cadavers toward the recreation of human- dictate cell behavior following cell seeding. Efforts to engi-
derived extracellular influences in simplified forms, the neer such “cell-instructive” materials, regardless of their
decellularization of tissues and organs for scaffolding use composition, by incorporating well-defined physical and
and the incorporation of short functional domains derived chemical properties designed to affect surrounding cells,
from human tissue into ECM-mimicking biomaterials to biological signals and tissues in a specific manner are
manipulate cell fate commitment [52,289,290]. therefore largely inspired by the native ECM of different
The selection and design of biomaterials are key steps tissues and organs [291]. In a recent study, non-collagen
in advancing medical devices for regenerative medicine. proteins (NCPs) from bone ECM combined with 3D nanofi-
In general, excellent biomaterials, as mentioned in Section brous gelatin (NF-gelatin) scaffolds were used to form a
2, should be of favorable biocompatibility and nontoxic, material device mimicking both the chemical composition
should coax appropriate cell-material interactions toward and the nanostructured architecture of natural bone ECM.
new tissue regeneration and should possess adequate The incorporation of NCPs into the surfaces of the device
physical and mechanical properties until the new tissue was found to result in significant osteogenesis and min-
structure is constructed [52]. As noted previously in this eralization, leading to new bone regeneration, suggesting
section, the ECM was once considered to offer only struc- that biomimics are a new signpost for future cell scaffolds
tural support to tissues by serving as a substrate/template and tissue engineering templates [292]. Of note, clues for
for cell binding; it is now, however, widely accepted that how to construct ECM-mimicking biomaterials arise from
the ECM can additionally provide vital mechanical and ECM assemblies/components, including collagen types I
chemical information to the cells that mediate cell–cell and III, fibronectin, elastin, proteoglycans, laminin and
 F.-M. Chen, X. Liu / Progress in Polymer Science 53 (2016) 86–168 109

Fig. 8. Schematic representation of the location of selected endogenous extracellular matrix (ECM) components within the basement membrane (BM) or
stromal matrix (SM) that have been and are still being investigated for use in regenerative therapies and tissue engineering biomaterials (schematic is not
to scale).
Source: modified from Ref. [288].

many others. Naturally, the production and accumulation and biological performance of the resultant ECM, and (ii)
of these ECM assemblies at structural, chemical and phys- the maintenance of the ECM structural components allows
ical levels yields an optimized milieu that mimics the the remaining matrix to offer biomechanical strength and
actual in vivo microenvironment to instruct tissue forma- structural integrity to newly formed tissues and to enable
tion and to maintain homeostasis. Taking inspiration from substantial cell rebinding [26]. The hypothesis here is that
such physiological events, a practical paradigm has been such a decellularized ECM, acting as a native scaffolding
to procure the main ECM constituents and to use them as matrix, would preserve both biological information, which
building blocks for biomaterials after purification. Given would play an instructive role in cell interactions, and
recent advances in the fields of matrix biology, surface other physicochemical features, which would maintain the
chemistry and biopolymer science, purified ECM macro- correct spaces and microstructures for new tissue develop-
molecules of human or animal origin have served us well ment following cell reseeding. These benefits would help
in the production of regenerative biomaterials for recon- to overcome one of the crucial disadvantages of synthetic
structive surgery and tissue engineering [293]. polymers, namely, a lack of cell recognition signals, and
During the same period, considerable efforts have been could help to provide a tissue-specific template with nat-
made to synthesize biomaterials based on the function- ural microstructures that facilitate new tissue formation
ality of natural ECM molecules to guide morphogenesis [45].
in tissue engineering and regenerative medicine [267].
Decellularized ECMs can be used in tissue engineering 4.2. ECM constituents for scaffolding biomaterials
directly, with or without further modifications, because
(i) current methodologies are now able to remove nearly Recent decades have witnessed growing interest in
all cellular and nuclear material, while minimizing any the composition of the ECM of a given human tissue or
adverse impacts on the composition, mechanical integrity organ as well as in the developmental and physiological
110 F.-M. Chen, X. Liu / Progress in Polymer Science 53 (2016) 86–168

Fig. 9. Schematic representation of four representative tissue-specific stem cell niches and their cellular and extracellular matrix (ECM) components.
Examples are given where appropriate. (A) Hematopoietic stem cell (HSC) niche; (B) hair follicle stem cell niche; (C) satellite cell niche; and (D) neural
stem cell niche (SVZ). Readers are directed to the original article for more information.
Source: [97], Copyright 2014, Reproduced with permission from Elsevier Ltd.

roles of each matrix constituent (Fig. 9). In accordance structural and regulatory roles in tissue development
with their unique molecular structures, these matrix and function and exceptionally strong relevance to and
components can generate biomaterials with various translational implications for human disease diagnosis
well-defined 3D configurations, such as fibrillar meshes, and treatment [271,276,296,297]. In particular, the use
and the resulting physical and biological properties of these macromolecules, whether natively derived or
are linked to each macromolecule’s structure–function produced by decellularization, can mimic many features
relationships [294,295]. Many ECM-embedded and cell of the native ECM, offering a simple way to design and
surface-associated assemblies/constituents have a highly synthesize biomimetic materials for tissue engineering
organized spatiotemporal pattern, suggesting crucial and regenerative medicine. Advanced materials either
 F.-M. Chen, X. Liu / Progress in Polymer Science 53 (2016) 86–168 111

composed of naturally occurring macromolecular assem- sources and reduced bioactivity due to production by
blies or including adjuvant ECM functional domains in a recombinant techniques [305]. Thus far, most collagen has
controlled 3D configuration have been demonstrated to been extracted from tissues of animal or human cadaveric
regulate the healing cascade by modulating host immune origin, such as the skin or tendons, or from discarded
responses, facilitating host cell homing, resisting bacterial human tissues, such as the placenta or extracted adipose
infections and infiltrating and reestablishing homeo- tissue; human collagen is more attractive for therapeutic
stasis in the damaged areas targeted for regeneration applications [306]. Decades of research have uncovered
[298]. Many ECM constituents and naturally derived more than 20 distinct forms of collagen, most of which
proteins/substrates that could enable the application of have been purified for biomedical use. Various amount of
research on scaffolding biomaterials have been character- types I, II, III, IV, V and VI are present in mammalian tissues,
ized at the structural, chemical and physical levels, and the among which type I is the most abundant (approximately
overall outcomes have been very successful in regenera- 90%) [267,306]. Other types, however, are only present
tive medicine. Broadly speaking, two classes of structural in relatively minor amounts; their roles in the in vivo cell
and functional macromolecules are yielded by the ECM: milieu remain poorly understood. The attractive proper-
fibrous proteins (mainly collagen, laminin and elastin) and ties of collagen for use in tissue engineering biomaterials
glycosaminoglycans (GAGs) [26,266]. Here, we provide a include, but are not limited to, its good biocompatibility;
detailed discussion of several representative protein-based low antigenicity; and tailored mechanical, degradation
biomacromolecules (constituents) isolated from ECMs of and water uptake properties due to its ability to be
human origin (Fig. 8), and their enormous potential for crosslinked through chemical glycation procedures or heat
future scaffold design and development is detailed. treatments [267]. Compared with protein constituents
extracted from animal tissues, human-derived collagen for
4.2.1. Collagen I scaffold production in tissue engineering lowers the risk
Collagen is the most abundant protein (approximately of hypersensitivity and immunogenicity, but the poten-
30% of the total protein content) in the ECM and is arguably tial to cause pathogenic contamination and/or disease
the most dominant in many types of human soft and hard transmission still exists [307]. In this respect, recombinant
connective tissues [299]. Collagen specifically comprises human collagen based on plant materials provides an
a right-handed bundle of three parallel, left-handed alternate natural collagen source without the risk of
polyproline II-type helices (Fig. 10). This protein not disease transmission or concerns regarding variability
only constitutes a key fibrous structural component in the [308]. Currently, growing evidence suggests that synthetic
human body but also helps in manufacturing the structural polymer nanofibers may play the similar role as natural
proteins necessary for the macromolecular composition ECM collagen in the tissue regeneration process, thus
and structural architecture of the skin, the skeletal systems allowing the design of biomimetic materials in a way that
(e.g., bone, cartilage, joints, ligaments, tendons and blood regulates cell incorporation and behavior toward desired
vessels) and various internal organs [300]. Collagen plays overall differentiation and function [309–311]. The use
the role of a “mattress” that glues our body cells together, of plant-derived and recombinant collagens for materials
links all of the tissues/organs and supports the entire engineering has thus presented a new way of not only
body framework, in which tough bundles of collagen expanding our insights into raw materials science but also
are normally termed collagen fibers. Collagen, along exploring the short functional domains of human proteins
with elastin and keratin, specifically constitutes fibrous for the molecular design of biomimetic materials [312].
extracellular networks that enable tissues or organs to Over the last few decades, accumulated knowledge
withstand repetitive stresses and high tensile stress with- about the complex hierarchical structure of native collagen
out plastic deformation or rupture [301]. At the cellular molecules and the role of collagen’s physicochemical prop-
level, the intracellular bonds formed by collagen fibers erties in tissue development and regeneration has inspired
provide the cementing mechanisms required for linkages, biomaterials scientists to design innovative biomimetic
reinforcement and protection, and even the supply of materials that mirror the native nanofibrous collagen net-
oxygen and nutrients. Insufficiency of collagen will hence work to instruct cell behavior and guide tissue formation
naturally result in an unhealthy cellular microenviron- [61,89,313]. In particular, human tissue-derived collagen I
ment, which eventually takes its toll on our overall health is a ubiquitous ECM protein and one of the most common
(http://vitaking.kurazmotorsports.com/what-is-collagen). structural elements in a number of tissues. This protein
Based on its functional and bioactive properties, collagen adheres to various cell types and may provide a satisfac-
has been demonstrated to be a versatile biomaterial that tory scaffolding material upon which cells may thrive via
can be formed into highly organized 3D matrices (e.g., capillary formation and cell chemotaxis [288]. Purified col-
sponges, films, skin grafts and dressings) endowed with lagen I isolated from human tissues (e.g., connective tissue
high tensile strength and intrinsically biocompatible, and the BM) has been an appropriate choice in a variety
biodegradable, and nontoxic properties upon exogenous of restorative applications, and in recent years, there has
application. These attributes make collagen the biopoly- been broad and intense interest in this protein’s utility
mer of choice in many biomedical fields, such as drug in a variety of biological and tissue engineering applica-
delivery and regenerative medicine [302–304]. However, tions, partially due to its unique properties and relative
controversy persists about the perfect source of collagen abundance in living tissue [295]. Interestingly, this type
for these applications, given possible disease transmis- of material is now also employed to generate 3D systems
sion and immunogenicity related to animal or allogenic for the culture and directed differentiation of a number of
112 F.-M. Chen, X. Liu / Progress in Polymer Science 53 (2016) 86–168

Fig. 10. Overview of the collagen triple helix. (A) First high-resolution crystal structure of a collagen triple helix, formed from
(ProHypGly)4 –(ProHypAla)–(ProHypGly)5 . (B) View down the axis of a (ProProGly)10 triple helix, with the three strands depicted in space-filling,
ball-and-stick and ribbon representations. (C) Ball-and-stick image of a segment of a collagen triple helix, highlighting the ladder of interstrand hydrogen
bonds. (D) Staggering of the three strands shown in panel (C).
Source: [300], Copyright 2009. Reproduced with permission from Annual Reviews.

cell populations and, indeed, to investigate the signaling on capitalizing on the protein’s distinctive ability to form
interactions between these cells and the matrix, which is biomimetic hydrogels with well-defined fibrous architec-
a fundamental issue in cell biology and materials research tures, biological properties, stable topographies that can
[314]. As a cell culture system, it has been suggested that withstand mechanical loading and the ability to modu-
collagen I hydrogel supports long-term in vitro cell expan- late cell and tissue responses against inflammation and
sion and the maintenance of fully elaborated human small oxidative stress [316,317]. The reconstitution of collagen
intestinal epithelium for at least one month. This system into hydrogel scaffolds can be specifically controlled to
gives rise to a new pattern of sheet-like growth at the achieve desired hierarchical structures from the nanoscale
gel–liquid interface as well as familiar enteroid structures, to the microscale to the macroscale, leading to a highly
with polarized columnar cells with basolaterally located relevant biomaterial for regulating cell function and mim-
nuclei and apical brush borders [315]. Years of efforts to icking tissue properties [61]. In this regard, collagen I offers
harness isolated collagen matrix as a substrate for cultivat- a structural framework that determines the morphologi-
ing cells have expanded into a large body of work focused cal characteristics of human connective tissues and plays a
 F.-M. Chen, X. Liu / Progress in Polymer Science 53 (2016) 86–168 113

dominant role in the temporal cascade of myriad cellular lamellar structure characterized by tunable co-aligned
and molecular events leading to the regeneration of new micro- and macropores and have great potential to be
bone from osteoblastic progenitors [295]. applied in bone tissue engineering applications due to their
If we reconsider specific molecular structures, the biomimetic architectures, favorable mechanical strength
potential of the P-15 cell-binding domain of collagen I to and desirable biocompatibility [323]. More specifically,
create desirable biomimetic environments for osteoblasts a three-component, biomimetic, injectable hydrogel
offers the possibility of exploring ECM cues for osteogen- composite composed of triblock PEG–PCL–PEG copolymer,
esis and, subsequently, bone repair and regeneration. To collagen and nano-hydroxyapatite was found to have
this end, the introduction of this peptide into cell scaffolds good thermo-sensitivity, biodegradability and biocompat-
has been demonstrated to largely enhance the attachment, ibility, leading to favorable performance in guided bone
proliferation and differentiation of human MSCs [318]. In regeneration applications [317,324]. Furthermore, growth
an in vitro microenvironment without any other verte- factors and other bioactive agents can be incorporated
brate ECM components of calcifying tissues, the collagen into collagen-based systems, such as gels and scaffolds, to
I matrix is able to initiate and orientate the generation of modulate their release rates and improve their therapeutic
the mineral carbonated apatite based on a collagen/apatite effects as tissue engineering strategies [52,325]. In this
self-assembly process. Additionally, collagen I can influ- regard, a type I collagen matrix was found to be an effective
ence and control not only the structural characteristics vehicle for growth factor delivery in vascular tissue engi-
of apatite on the atomic scale but also its size and the neering [314,326], and a collagen-based biomatrix-scaffold
3D distribution on a larger scale, likely through orches- composed of DBM and collagen-binding domain BMP-2
trated signal cascades and cellular events modulated by (CBD-BMP-2) has been used as a bioactive bone-inducing
the collagen I matrix [319]. Although collagen I does not material (BBIM) for bone regeneration [327]. Additionally,
signal MSC migration on its own, when cells are severely collagen can be used to minimize unwanted progenitors
stressed, collagen I can induce a potent migratory response. localizing to non-target sites and causing undesirable
Such findings suggest that cells may secrete a substance, tissue formation, addressing one major issue associated
which appears to be a protease, upon injury or when in with stem cell therapy. The incorporation of MSCs into
a disease state. The substance interacts with the collagen a collagen scaffold was found to reduce the dispersal of
matrix to release cell homing agents, and hence, a chemo- transplanted MSCs into the surrounding non-infarcted
tactic signal emerges to recruit stem cells to damaged or myocardium and the relocation of those cells to remote
injured areas to exert therapeutic function; the interplay organs after intramuscular injection. More specifically, no
appears to be due to the digestion of the protease into frag- relocated cells were found in the liver of animals receiving
ments that are chemotactic [273,320]. For several decades, this combination treatment, which is, however, normally
it has been recognized that many other ECM components a sink for the relocation of injected cells [328]. Similarly,
in addition to collagen I that also specifically modulate cell another study demonstrated improved early engraftment
activities, including the migration, adhesion, proliferation and directed localization of transplanted endothelial
and osteogenic differentiation of MSCs of human origin, can progenitor cells (EPCs) post-transplantation via enhancing
be used as a cell vehicle for bone tissue engineering appli- progenitor cell retention and limiting distribution to
cations (Fig. 11). For example, fibronectin facilitates cell nonspecific tissues when the cells were impregnated in an
adhesion, migration and proliferation, but not osteogenic injectable collagen matrix [329]. ECM-mimetic hydrogels
differentiation, whereas fibrinogen may enhance cell pro- containing the collagen I-derived peptide GPQGIAGQ were
liferation and adhesion, but not migration [273]. These also demonstrated to induce endothelial cell adhesion
properties of ECM components offer the possibility of and capillary-like network formation [330]. Furthermore,
exploring naturally derived biomaterials that can instruct the conjugation of a collagen-mimetic protein with a PEG
stem cell fate decisions. hydrogel is able to generate a bioactive hydrogel that can
Furthermore, collagen I-based biomaterials have been bind to endothelial cells and resist platelet adhesion, form-
applied to cell therapeutics in vivo, and recently, for tissue ing a vascular graft with a potential multilayer design that
engineering of whole menisci, a high-density form of can achieve rapid endothelialization of the conduit while
type I collagen hydrogel has been used as an injectable minimizing the risk of thrombosis, intimal hyperplasia and
scaffold [321]. In other applications, combinations of mechanical failure [331]. Taken together, these findings
collagen with other biomaterials have also been tested as suggest multiple important roles for collagen-containing
bone substitutes and skin replacements and, indeed, for materials in enhancing cell adhesion, locally retaining cells
the engineering of manmade valves and blood vessels, and regenerating capillary-like networks, which may have
wherein collagen I and laminins are crucial for vessel significant implications for both cell-based therapy and
structural integrity and deliver the contrasting signals vessel tissue engineering paradigms.
necessary for angiogenesis [322]. To increase mechanical
performance, collagen microsponges may be used for 4.2.2. Collagen II
the modification of previously prepared synthetic poly- Collagen II is the main protein present in the ECM of
meric materials; these sponges are easily impregnated hyaline cartilage and the nucleus pulposus (NP), within
into the structures of the scaffolds [52]. Meanwhile, which little to no collagen I is found. As a potential autoanti-
biomimetic collagen-apatite scaffolds can be engineered gen in inflammatory synovial disease, collagen II may
via a self-assembly process in a simulated body fluid be useful for limited applications. For example, increas-
system. These scaffolds possess a unique multi-level ing evidence suggests that small doses of collagen II can
114 F.-M. Chen, X. Liu / Progress in Polymer Science 53 (2016) 86–168

Fig. 11. Representative scanning electron microscopy (SEM) images of the attachment and growth of human bone marrow-derived stem cells on collagen
fibers derived from human skin [58]. (A) Before cell seeding; (B) 8 h after cell seeding: cell attachment on collagen fibers; (C) 24 h after cell seeding:
extracellular matrix formation and cell ingrowth into collagen fiber structures; (D) 48 h after cell seeding: cell–matrix–collagen integration.

modulate joint health in both rheumatoid arthritis and differential moduli of the cartilaginous and osseous com-
osteoarthritis [332,333]. The ingestion of non-denatured partments, these layered scaffolds are capable of exhibiting
type II collagen (microgram quantities) in an animal model compressive deformation performance, similar to that
of collagen-induced arthritis specifically caused a dramatic observed in natural articular joints [337]. Similarly, with
reduction in the circulating levels of pro-inflammatory sufficient crosslinking, a composite hydrogel composed
mediators (e.g., IL-2 and IL-17), along with an increase of collagen II and hyaluronic acid (HA) at a ratio equiv-
in anti-inflammatory molecule (e.g., IL-4 and transform- alent to that in the native tissue ECM of the NP can
ing growth factor-␤ (TGF-␤)) production, hence decreasing serve as a potential candidate for IVD regeneration [338].
both the severity and the incidence of arthritis [334]. This Recently, photocrosslinked carboxymethylcellulose (CMC)
finding indicates that collagen II plays an essential role hydrogels have been demonstrated to direct human MSC
in orchestrating the immune balance of Th17/Treg and differentiation with respect to chondrogenic or NP-like
Th1/Th2 in mice [334]. ECM elaboration; however, the mechanical properties of
Due to its presence in significantly fewer ECMs of tis- these IVD constructs need to be improved. Interestingly,
sues in the body, collagen II has not been applied as the macromer concentration of photocrosslinked CMC
frequently as collagen I in raw biomaterial approaches for hydrogels was found to direct functional NP-like matrix
generating tissue engineering constructs, even for carti- accumulation and organization, which is likely attributable
lage tissue regeneration [335]. Nevertheless, collagen II to the diffusive properties of the various hydrogel for-
has also been used for the production of matrix-scaffolds, mulations and the quantifiable differences in polymer
including hydrogels, sponges and microspheres, but has crosslinking density. In particular, a lower polymer con-
mainly been utilized in cartilage and NP tissue engineering centration allowed for a greater NP-like ECM assembly and
applications [39]. To mimic the native compositions asso- hence improved the mechanical functionality of these IVD
ciated with the transition of tissue types at the interface constructs, approaching the values of native NP over time
of cartilage, an osteochondral template based on colla- [339].
gen I/CaP, with an interfacial layer that connected to a The association between a microfracture of the sub-
collagen II/chondroitin sulfate (CS) layer, was designed chondral plate and a coverage scaffold has emerged as
and developed. These designs are combined to enable a promising treatment for the management of cartilage
the resultant biomaterials to be embedded into an osteo- lesions via a one-step procedure. Although a type I collagen
chondral defect site in the subchondral bone, with no scaffold is most often used for this purpose, biomatrix-
demand for glue, sutures or screws [336]. To this end, scaffolds made of mixed type I and II collagen, which defied
a highly interconnected porous network can be inserted the immunological reaction of the synovial tissue, exhib-
throughout the entire osteochondral defect. Due to the ited good biocompatibility in vivo and favored cartilage
 F.-M. Chen, X. Liu / Progress in Polymer Science 53 (2016) 86–168 115

restoration by the collagen membrane when associated in the ischemic heart, without noted myocyte regeneration.
with a microfracture [340]. The limited use of collagen II The functional findings were considered to be caused by
in scaffolding materials may be potentially due to its lim- increased vascularity, indicating a pro-angiogenic impact
ited availability and high cost, the lack of substantial data to of specific collagen IV domains that could be explored for
support its application, limited consciousness of its utility the management of a large variety of ischemic vascular dis-
or a combination of these factors [39]. However, the strat- eases. Similar to collagen I, as mentioned in Section 4.2.1,
egy for mimicking the native ECM composition in hyaline collagen IV by itself is not a chemotactic agent for MSCs;
cartilage and the NP has increased the utility of collagen however, under stress conditions, collagen IV may also
II, whether alone or in combination with other ECM com- induce a potent migratory response [320].
ponents, in tissue engineering, although, more in-depth Laminins are a large glycoprotein family that con-
investigations are required to demonstrate its potential. tains at least 16 isoforms; these isoforms associate with
different heterotrimers composed of globular, laminin-
4.2.3. Collagen IV, laminin and entactin type epidermal growth factor (EGF)-like repeats and
BMs are cell surface-associated ECMs that are fun- ␣-helical domains. Laminins specifically consist of ␣, ␤
damental to tissue physiology and organization in all and ␥ polypeptide chains, and the triple-helical coiled-
metazoans, with roles ranging from structuring, polariz- coil domain in the center of each chain may combine to
ing, protecting and compartmentalizing cells to supplying form distinct assembled structures that are involved in
them with growth factors [341]. Although BMs are com- diverse cellular events, such as adhesion, survival, migra-
monly composed of collagen IV and laminin networks that tion and differentiation [272,299]. The use of genetics
are stabilized by entactin/perlecan bridges, their precise to probe the functions of BM laminins with regard to
composition is as unique as the tissues to which they are their structure and binding and self-assembly activities
localized, with entactin, proteoglycans, perlecan and colla- has indicated that various laminin subunits profoundly
gen VII and other macromolecules also present in the native influence tissue morphogenesis by inducing and maintain-
membrane-like structures of BM [272,342]. Collagen IV and ing cell polarity, establishing tissue compartment barriers,
laminin are capable of connecting a cell to and procur- organizing cells into tissues and protecting adherent cells
ing a cell from its microenvironment, suggesting that the from detachment-induced cell death and anoikis, start-
production and maintenance of both macromolecules play ing around the embryonic stage and extending through
a central role in endothelial cell function and the regen- organogenesis and into the postnatal period [342]. It is
eration of different tissues [343]. Indeed, the BM is well evident that laminins are also crucial for the initial genera-
known to support an atheroprotective endothelium, and tion of the polymeric scaffolding structure of cell-attached
collagen VII has been validated as a critical player in the matrices. As additional matrix components are gradu-
physiological wound healing cascade in humans [344]. In ally integrated into the scaffold, it becomes more and
addition, the presence of collagen IV and laminin has been more mature in terms of ligand diversity, matrix sta-
found in mature and developing articular cartilage and has bility and functional complexity. These findings suggest
also been observed in tissue-engineered cartilaginous con- that diverse BM components differentially promote cell
structs ex vivo and in cartilage repair implants transplanted polarization, compartmentalize and organize developing
into in vivo defect sites [345]. Meanwhile, increasing evi- tissues, and indeed maintain adult tissue function [342].
dence indicates that chondrocytes in articular cartilage are Interestingly, recent research found that a matrix metallo-
surrounded by a narrow pericellular matrix that serves as a proteinase 2 (MMP2)-cleaved laminin-111 fragment was
transducer of microenvironmental signals to the chondro- highly up-regulated during the early stage of stem cell dif-
cyte and that is biochemically defined by distinct molecular ferentiation, suggesting a previously unidentified role of
components, such as type VI collagen and perlecan [346]. laminin that goes far beyond BM assembly and a mech-
These findings may have implications for the tacit under- anism by which a biologically active laminin fragment
standing of the function of human ECMs’ BM molecules modulates the dynamic epithelial-to-mesenchymal tran-
in chondrogenesis during cartilage repair and regenera- sition of embryonic stem cells (ESCs) [350].
tion, in the control of the mechanical environment and For the regeneration of peripheral nerves after injury,
mechanobiology of cells in articular cartilage and in the the switching of Schwann cells to a proliferative state,
physiology and pathology of articular cartilage [345]. the secretion of trophic factors and the presence of ECM
As a non-fibrillar collagen, collagen IV within small molecules (such as laminin, collagen and fibronectin) in the
sheets represents a predominant component of the BM distal stump are necessary elements in creating a permis-
and plays a structural role in its assembly, suggesting an sive environment for axons to grow [270]. In this respect,
approach mimicking this protein’s organization for vascu- bioscaffolds yielded by cryogelation of gelatin or dextran
lar regeneration [347]. Collagen type IV is the main ECM linked to laminin [351] or PCL-chitosan scaffolds with sur-
constituent in the BM of blood vessels and plays crucial faces modified via the crosslinking of laminin [352] could
roles in regulating the cellular balance to minimize the risk serve as versatile substrates with excellent mechanical
involved in the use of vascular implants [348]. Two specific and surface properties for in vivo cell delivery, resulting
interacting domains of collagen IV, Hep I and Hep III, have in highly neuroregenerative properties for nerve tissue
been purified and investigated for their biological potential engineering applications. Furthermore, the application
[349]. Although these peptides were not observed to induce of laminin-modified linear ordered collagen biomaterials
migratory responses in vitro, treatment with the peptides loaded with laminin-binding ciliary neurotrophic factor
resulted in improved functional recovery of cardiac muscle (CNTF) was beneficial for sciatic nerve regeneration and
116 F.-M. Chen, X. Liu / Progress in Polymer Science 53 (2016) 86–168

functional recovery when tested in a rat sciatic-nerve tran- inherent bioactive properties, retaining delivered cells,
section model [353]. Data obtained from this study suggest promoting cell survival and maintaining or promoting a
that the modification of linear ordered collagen scaffolds specific cell phenotype in vivo. For example, a methylcel-
with laminin guided axon growth, as the laminin-binding lulose material functionalized with laminin-1 was found
domain fused to the N-terminus of CNTF retained more to provide a biomimetic microenvironment that may
CNTF on the bioscaffolds and additionally enhanced nerve modulate neural stem cell survival, apoptosis, migration,
regeneration and functional recovery. Recently, a grow- differentiation and matrix production [360]. Similarly,
ing body of evidence has suggested that in addition to when a PEG-based hydrogel was functionalized with the
collagen IV, collagens V, VI and XV are all key compo- laminin-derived adhesion peptide YIGSR, the resulting bio-
nents of peripheral nerves, in which they jointly not only material significantly enhanced intracellular triglyceride
provide structural support for neurite outgrowth but also accumulation in encapsulated adipocytes and ultimately
affect Schwann cell function and myelination by trigger- promoted the formation of coherent adipose tissue-like
ing intracellular signals [354]. Therefore, the effects and structures featuring many mature unilocular fat cells [361].
molecular mechanisms of different collagens in peripheral Although MSCs have been suggested to be a potential
nerve myelination and function must be carefully evalu- source for disc tissue regeneration, these cells neither dif-
ated and taken into account when designing a scaffold for ferentiate into NP-like cells nor regenerate matrix with
nerve tissue engineering application. unique characteristics matching that of immature NP tis-
Because laminin-1 can induce endothelial differentia- sues of the IVD unless co-cultured with human NP cells
tion in vitro and increase the formation of new blood vessels or placed in a laminin-rich culture environment [362,363].
in vivo, it is considered to be a stimulator of angiogenesis Indeed, substantial evidence suggests that NP cell-laminin
[322,355] and may support vasculogenesis via the guid- interplay is unique to the immature disc because immature
ance of smooth muscle cell proliferation [356]. It is now is NP cells were found to express specific laminin isoforms
commonly accepted that a laminin-rich microenvironment and laminin-binding receptors [364,365]. This evidence
plays a critical role in muscle regeneration. In this regard, includes higher expression of levels of the laminin ␣5
the treatment of dystrophic muscles with the laminin-111 and ␥1 chains, laminin receptors (the integrin ␣3, ␣6
isoform in the mdx mouse model of Duchenne muscular and ␤4 subunits and CD239) and their related binding
dystrophy (DMD) significantly reduced the amount of mus- proteins in NP cells compared with cells from the neighbor-
cle damage (protecting the muscle from exercised-induced ing annulus fibrosus [365,366]; soft-laminin-containing
damage) [357]. In addition to being a powerful stimulator ECM substrates promoting immature NP cell morphology,
of myoblast migration and proliferation in vitro, an intra- cell–cell interactions and proteoglycan synthesis in the
muscular injection of laminin-111 resulted in increased cells of the NP [367]; and immature porcine NP cells adher-
strength and resistance in treated muscles. When laminin- ing to laminins in higher numbers compared with cells
111 was used as a coadjuvant for myoblast transplantation, from the adjacent annulus fibrosus [368]. In this respect,
this protein was found to considerably improve the over- a soft, laminin-functionalized hydrogel was developed as a
all therapeutic outcomes in the mdx mouse model of DMD biomaterial carrier for cell delivery to the pathological IVD
[358]. These findings indicate that laminin-111 may serve to enhance IVD regeneration. The findings from this study
as an unexpected and highly potent therapeutic agent demonstrate the ability of laminin-111-functionalized PEG
for patients with congenital myopathies, representing a (PEG-LM111) hydrogels to crosslink under physiological
paradigm for the systemic administration of ECM proteins conditions, without the need for an initiator. Addition-
as therapeutics for genetic diseases [357,358]. Based on its ally, delivery within a PEG-LM111 hydrogel significantly
capability to direct myoblast migration, laminin has been improved primary NP cell retention in the disc space com-
proven to be a powerful signaling molecule in endogenous pared with the retention of cells delivered without a carrier
regenerative therapies for guiding skeletal myoblasts to in an organ culture model [369].
damaged areas to foster the in situ regeneration of skele- Entactin, also termed nidogen, is another widespread
tal muscles [288]. In this context, it has also been found BM constituent of 150 kDa that primarily binds to laminin
that collagen VI is a key component of the satellite cell and collagen IV; multiple component interactions, con-
niche (including myofibers and ECM), where it plays essen- sisting of inter-protein binding, self-polymerization and
tial roles in the regulation of satellite cell self-renewal and cell surface adhesion, facilitate BM assembly and integrity
regeneration of skeletal muscles, as evidenced by reduced [342]. In humans as well as in all other mammals, there
satellite cell self-renewal capability and impaired muscle are two ubiquitous entactins (entactin-1 and entactin-2)
regeneration after injury in Col6a1−/− mice as a result of the encoded by distinct genes, whose specific interplay with
lack of collagen VI. Further investigation has revealed that collagen IV, laminin and perlecan is considered to be impor-
collagen VI plays an unforeseen role in regulating satellite tant for organizing basal laminae, including those in the
cell activity, through which the biomechanical proper- muscle, the skin and the nervous system [370]. Struc-
ties of skeletal muscles are modulated and satellite cell turally, entactins are sulfated glycoproteins that involve
homeostasis is regulated, suggesting a potential therapeu- three globular domains (G1–G3) separated by rod-like
tic strategy for the rescue of collagen VI-related muscular domains. Whereas G3 is at the C-terminal domain, con-
dystrophies [359]. nected by a rod-like region that contains EGF repeats,
Recently, the cell-laminin interplay has been demon- G1 and G2 are at the N-terminus, connected by a small
strated to be useful for biomaterial design, allowing a linkage region [322]. Entactin is reported to be respon-
material to serve as an optimal platform by possessing sible for the long-term maintenance and maturation of
 F.-M. Chen, X. Liu / Progress in Polymer Science 53 (2016) 86–168 117

contractile skeletal myotubes, indicating a biological func- studies will provide important information for the devel-
tion for entactin in myogenesis [371]. Recent evidence also opment of self-assembled biopolymer networks for tissue
shows that entactin is an ECM protein that regulates the engineering with good biological compatibility as well as
proliferation and migration of Schwann cells and induces close chemical, structural and mechanical similarities to
elongation in the regenerative axon growth of adult sen- native ECMs [385–387].
sory neurons, indicating a critical role of entactin in proper
peripheral nerve regeneration [372]. 4.2.4. Glycosaminoglycans
The interactions between entactin and laminin are GAGs, including HA, heparin, heparin sulfate (HS) and
arguably of crucial importance to the assembly of BMs. CS, are linear, anionic and highly heterogeneous carbohy-
Investigations using recombinant entactin to probe the drate polymers composed of repeating disaccharide units,
calcium binding potential of various entactin domains which are commonly a uronic acid component and a hex-
and to examine the binding of entactin to various BM osamine (glucosamine or galactosamine). These polymers
proteins indicated a large number of BM targets, demon- are ubiquitously present at the cell surface and in the
strating the capacity of entactin to perform a connective ECM and may regulate matrix assembly and remodeling
function and to mediate the formation of ternary com- and cell–matrix and cell–cell interactions via interact-
plexes between laminin and collagen IV and between ing with various structural proteins (e.g., fibronectin and
laminin and HS proteoglycan while integrating other BM collagen) and signaling molecules (e.g., growth factors
members into the ECM [373]. For example, recombinant and chemokines) [388]. The supramolecular presentation
domain IV of perlecan, consisting of 14 immunoglobu- of GAG chains, along with other cell surface or ECM
lin superfamily modules, was found to bind to entactins molecules, is likely to be functionally important for cell
(entactin-1 and entactin-2), the laminin-1/entactin-1 com- adhesion, which is broadly applicable to the creation of
plex, fibronectin, fibulin-2 and heparin [374]. Entactin-1 multifunctional biomimetic surfaces in biomaterials [389].
is one of the central BM elements that play an essen- The chemical characteristics of GAGs specifically enable
tial role in hippocampal synaptic plasticity and excitability them to be highly hydrated, endowing them with gelati-
[375]. This entactin binds several BM macromolecules via nous properties or making them what has been termed
domain-specific, well-defined interplay, and the highest- a “ground substance” [322]. Except for HA, such chains
affinity cell-binding site of entactin-1 is positioned on one are bound to a central protein, such as glypican, perlecan
of the EGF repeats in the laminin ␥1 chain, which is crucial or syndecan, to form proteoglycans. In particular, heparin
for nerve guidance and for kidney development [376,377]. plays an important role in many biological processes via its
Entactin-2 is evidently more adhesive than entactin-1 in interplay with various proteins, and hydrogels composed
certain cell lines and is mainly mediated by ␣3 ␤1 and of heparin exhibit attractive properties, such as anticoag-
␣6 ␤1 integrins, as shown by antibody inhibition [378]. The ulant activity, growth factor binding and antiangiogenic
nature of the nidogen-binding epitope on the laminin ␥1 and apoptotic effects, making them great candidates for
chain suggests much more complementary functions of emerging applications [390]. Whether the molecule is a
entactin-1 and nidogen-2 in modulation of the endothelial heparin or CS proteoglycan is determined by the types of
phenotype as well as vascularization and implies extensive GAG side-chain residues, which are sulfated to different
co-regulation of the expression of the two nidogens than extents, depending on the location, source of production
was previously recognized [377,379]. Further elucidation and physiological conditions; much of the proteoglycan
of the potential roles of entactin and specific laminin chains heterogeneity may be attributed to the broad range of
not only will clarify important aspects of BM regeneration GAG sulfation patterns [288]. Because of their ionic prop-
but also may provide a better understanding of BM dis- erties, GAGs can absorb a mass of water, and this osmotic
orders (e.g., hepatic cirrhosis, skin-blistering diseases and swelling confers compressive strength on an ECM prod-
inherited kidney syndromes) and even suggest therapeutic uct, within which the amount of GAGs present significantly
approaches [380–383]. depends on the decellularization methods applied for
Evidence that BM assembly and turnover depend on the tissue processing. Additionally, sulfated GAGs are promis-
composition and mechanical characteristics of the adjacent ing constituents for functional scaffolds because sulfate
ECM and the dynamics of molecular self-polymerization groups determine growth factor binding and thereby affect
is steadily accumulating [282]. In addition to collagen IV wound repair [391]. By immobilizing chemokines either
and laminin, minor local components such as perlecan and in the ECM or on cell surfaces or by generating the stable
entactins are known to play crucial roles in the orchestra- haptotactic gradients required for directional cell migra-
tion of matrix assembly and remodeling; herein, perlecan tion under shear flow, GAGs also modulate the in vivo
functions as a bridging stabilizer, whereas entactins largely bioactivity of chemokines [392]. In this regard, positively
serve as molecular adaptors or catalysts [384]. The BM charged chemokines not only bind to receptors whose
composition and the functions of its macromolecules in different oligomeric forms can induce different but interre-
embryonic development as well as in the homeostasis of lated signaling responses but also interact with GAGs that
adult tissues are increasingly being analyzed by structural are negatively charged. Mimicking this phenomenon has
investigations at atomic resolution and by recombinant the potential to yield GAG-based biomatrices that induce
techniques, opening the possibility of inducing distinct desired cell activities. The degree of GAG sulfation within
effects through changes in BM composition [272,341]. these matrices can be systematically manipulated via
Investigations of mutated genes identified in inherited bioinspired alterations in the GAG content (increased sulfa-
disorders and new insights obtained from gene-targeting tion from HA to CS to heparin) to regulate the sequestration
118 F.-M. Chen, X. Liu / Progress in Polymer Science 53 (2016) 86–168

of growth factor signals and, indeed, their subsequent func- (SF)-based biomaterials, allowing the combination of the
tion to influence cell fate within the biomatrix [393]. The biological characteristics of HA with the superior mechan-
strong interactions between chemokines and GAGs can ical properties of SF. Following MSC seeding and in vitro
protect chemokines from proteolysis and stabilize the for- culture, histological investigations of the constructs after
mation of a large variety of chemokine oligomers and other a 3-week incubation revealed improved cellular penetra-
structures that would not otherwise form in solution [394]. tion and ingrowth into SF/HA composites compared with
Thus, both the interaction of chemokines with GAGs (GAG plain SF materials. Furthermore, in vitro stem cell cultures
binding) and the ability to form higher-order oligomers on SF/HA composites in the presence of tissue-inductive
contribute significantly to the overall chemotactic function stimuli showed more efficient tissue-forming potential in
of specific chemokines [395]. Furthermore, interactions terms of GAG and collagen I/III gene expression compared
between GAGs and stromal cell-derived factor-1␣ (SDF- with plain SF scaffolds, which were used as control materi-
1␣, or CXCL12) isoforms not only play a distinct role in the als [404]. Collectively, these findings indicate that HA may
retention of hematopoietic stem cells (HSCs) in the bone be an appropriate ECM biomaterial for use in revasculariza-
marrow under homeostatic conditions but also contribute tion techniques and may provide anti-apoptotic, protective
to stem cell recruitment and appropriate tissue revascular- functions while enhancing cell growth and differentiation
ization after acute ischemia [392,396]. [288].
HA, also known as hyaluronan, is one of the primary Of note, during long-term in vitro culture, MSCs undergo
components of the ECM and is present as high- cellular senescence, accompanied by a loss of cell migratory
molecular-weight chains. This linear GAG is synthesized by and homing abilities. It has been suggested that the migra-
membrane-bound hyaluronan synthases and is composed tory capability of ex vivo-expanded cells can be reversed
of disaccharide units containing N-acetyl-d-glucosamine if HA is used as an adjuvant supplement for cell cultures
and glucuronic acid, which distinguish HA from other [405,406]. Because HA is a native element of cartilage,
GAGs that are produced in the Golgi apparatus [397]. HA MSCs can interact with HA materials through cell sur-
can be harvested from many tissues by enzymatic diges- face receptors, leading to stem cell differentiation and,
tion or extraction and is increasingly being utilized in hence, cartilage formation [405]. Consequently, the incor-
biomedical applications due to both its ability to serve poration of MSCs into HA-based biomaterials has been
as a blank slate and its biological activity. In particular, demonstrated to improve chondrogenic differentiation and
HA is commonly used to create hydrogel scaffolds for cartilage-like ECM deposition. The use of chondrocytes in
tissue engineering, which may in turn be applied for local- an HA material for the treatment of focal lesions of the artic-
ized drug delivery purposes [398]. At the cellular level, ular cartilage of the knee has already been tested in clinical
high-molecular-weight HA and low-molecular-weight HA trials [407]. Additionally, recently, as a natural lubricant in
exhibit opposite effects on orchestrating cell function. In the body, HA was used to enhance lubrication on tissue and
addition to inhibiting cell proliferation, high-molecular- biomaterial surfaces through a polymer-peptide surface-
weight HA is anti-angiogenic. For example, HA acts as a coating platform, offering a potential strategy to coat
high-molecular-weight barrier that blocks endothelial cell medical devices and to treat tissue-lubricating dysfunction
migration and subsequent angiogenesis in the fetal devel- [408]. In addition, HA hydrogels have been widely engi-
opment of rat follicles. However, cleaving the polymer into neered and researched due to their biocompatibility and
shorter fragments with hyaluronidase enables endothe- their ability to incorporate a wide variety of cues to modify
lial cells to migrate and activate angiogenesis [399]. HA cell-material interplay and to ultimately affect and adapt
is expressed on the BM of human sinusoidal endothelium to greater control over the behavior of cells [398]. When
and endosteum. Interestingly, SDF-1 is also constitutively the effect of HA on the physicochemical characteristics
expressed at high levels in these regions and plays a distinct of collagen–gelatin composites and its cytocompatibility
role in maintaining the marrow HSC pool in a quiescent were investigated, the addition of HA at less than 15% to
state. It has been suggested that as a major GAG com- composites composed of collagen and gelatin (ratio of 1:9)
ponent of bone marrow ECM, hyaluronan is an in vivo resulted only in incremental improvement in the physical
priming factor for the SDF-1-dependent transendothelial structure and cytocompatibility of the resultant biomate-
migration of human CD34+ stem/progenitor cells to sites rials with a human intestinal epithelial cell line. However,
with low CXCL12 concentrations and also contributes to increasing the proportion of HA in the scaffolds to 25%
the cells’ final anchorage within specific niches in the resulted in a dramatic improvement in the scaffolds’ prop-
BM [400]. It is now widely recognized that native HA erties, including their support of cell adhesion, growth and
exhibits a pro-survival effect on contacting cells by acti- viability, as well as their structural characteristics [409].
vation of cell anti-apoptotic Akt pathways [401] or by Recent studies have also demonstrated that incorporat-
protection of the cells against toxic insults [402]. How- ing various chemical, mechanical and spatial cues into HA
ever, the HA-induced pro-survival effect on contacting cells hydrogels can lead to tuned unicellular and multicellu-
is reversed when HA–receptor interactions are inhibited lar responses. The ability to control other HA gel-specific
[403]. cellular functions, such as cell-mediated hydrogel degra-
The design of HA-based hydrogel scaffolds to elicit dation, is useful in designing systems via multipronged
highly controlled and tunable cell responses and behavior approaches that offer a range of applications. As research
is a major area of interest in developing tissue engineering on HA hydrogels continues to progress, more intelligent
and regenerative medicine applications [398]. In partic- systems with clearer, more cell-directed purposes will be
ular, HA has been tested as a copolymer for silk fibroin developed [398].
 F.-M. Chen, X. Liu / Progress in Polymer Science 53 (2016) 86–168 119

HS, a carbohydrate–protein complex, is a highly sulfated HS chains exhibit both gender- and tissue-specific diversity
proteoglycan that contains heparin chains or glucosamine in biochemical composition that straightforwardly reflect
and glucuronic/iduronic acid repeating disaccharide units. their biological activities, as demonstrated by the potential
HS proteoglycans are major components of the ECM that benefit of gender-specific liver HS in manipulating human
are required for the insolubility, self-assembly and bar- MSC properties, including cell expansion, multipotentiality
rier characteristics of BMs. As a component of endothelial and subsequent matrix production [418].
cell membranes and ECMs, HS proteoglycans are involved Considering their role in endothelial cell and skeletal
in many critical functions of the endothelium and of muscle proliferation and progenitor cell mobilization, HS
antigen-presenting cells [410]. A potent cell-mobilizing proteoglycans can play important roles in recruiting angio-
and cell-recruiting molecule used in biomaterial design, genic stem cells to a site of ischemia for tissue repair [419].
SDF-1, is well recognized to be bound to cell surfaces by Notably, a consistent body of in vivo evidence suggests the
HS proteoglycans and is believed to significantly affect necessity of a specific HS proteoglycan for the successful
the chemokine’s biological properties, yet its role remains regeneration of skeletal muscles, a highly regulated and
largely unexplored [411,412]. Interactions with HS pro- complex process that involves muscle precursor prolifer-
teoglycans are supposed to provide chemokines with the ation and division and most likely demands the synergism
capacity to bind to the ECM and cell surface to trig- of a vast wealth of heparin-binding growth factors, such
ger cell signaling, through which HS plays an important as hepatocyte growth factor (HGF), FGFs and TGF-␤ [420].
role in the functions of CXCL12 isoforms both during Although a broad range of heparin-based biomaterials has
homeostasis and in physiopathological settings [396]. The been developed and used in the clinic, materials must be
polysaccharide side chains of HS proteoglycans differ in designed to maintain the desired level of stability in vivo
the structure and composition of their sulfated domains and to be effectively eliminated from the body, without the
among various tissue types, resulting in selective protein formation of undesired metabolites. As these challenges
binding. By selective accumulation on the cell’s macro- are addressed, heparin-based materials are likely to have
molecular GAG coating, namely, the cell glycocalyx, HS an increasingly significant impact on the management of
may also enhance the specificity of chemokine function various diseases and damaged tissues [390].
[392]. For example, when cellular HS from bone marrow CS is a ubiquitous component of proteoglycans that
endothelial cells and human umbilical vein endothelial is present both in the ECM and at the cell surface in
cells is characterized, differences in the glycocalyx GAG various tissues. Arranged in an alternating unbranched
pattern and in SDF-1 binding between the two cell types sequence, CS is composed of hexuronic acid (d-glucuronic
are observed. The highly sulfated domains present in HS acid) and hexosamine (d-galactosamine) units. Function-
chains from bone marrow endothelial cells are required ally, CS is largely covalently bound to core proteins (i.e.,
to deliver chemokines that coax stem cells to roll during proteoglycans), such that it displays specific interplay
transendothelial migration. These proteoglycans are also with proteoglycans in cell proliferation, differentiation and
critical for the binding of chemokines (e.g., SDF-1) and migration decisions. As a main component of the brain
the adhesion of hematopoietic progenitor cells following ECM, CS proteoglycans are involved in neural develop-
cell transplantation [413]. Furthermore, soluble HS was ment; axon pathfinding and guidance; and post-injury
observed to enhance SDF-1-driven migration in a dose- nerve regeneration, plasticity and rehabilitation in the
dependent manner, indicating that SDF-1 presentation nervous system [421]. CS is well known as an integral
may be optimized by using SDF-1–HS complexes instead of cartilage component in addition to HA, whose physical
SDF-1 alone. These findings imply that in the subendothe- and mechanical strength is due to its CS component. CS
lial matrix, proteoglycans can create an SDF-1 gradient also has profound effects on proliferative and adhesive
that directs hematopoietic progenitor cell homing to the phenotypes in MSCs and in the chondrogenesis of vari-
bone marrow [414]. It is also believed that this strategy ous stem cells and chondrocytes [273]. To this end, CS is
for SDF-1 immobilization can be used to recruit progen- being extensively examined for use in cartilage tissue engi-
itor cells toward a targeted place (e.g., ischemic muscle), neering approaches. Hydrogels containing CS may establish
where angiogenic cells are required for the restoration of a niche-mimicking microenvironment that plays a mor-
perfusion [415]. In addition to SDF-1, mounting evidence phogenetic role in directing cells toward a chondrogenic
is also revealing the molecular mechanisms by which HS phenotype in terms of the temporal pattern of cartilage-
proteoglycans bind other cytokines, including, but not lim- specific gene expression and in subsequently promoting
ited to, VEGFs, platelet-derived growth factors (PDGFs) and new matrix deposition during MSC chondrogenesis. In
fibroblast growth factors (FGFs), to modulate progenitor addition, this microenvironment may promote the inhi-
cell migration, recruitment and angiogenesis. This evidence bition of further MSC differentiation into hypertrophic
may result in a novel means to develop strategies that chondrocytes [422].
implement HS-induced control of cell fate commitments In non-weight-bearing defects in a rat model, HA
[416]. Following demonstration of the necessity of HS for hydrogels were also demonstrated to support cartilage
the growth factor-stimulated differentiation of osteopro- regeneration by human ESCs and to promote the inte-
genitor cells, the significance of HS for endogenous FGF-2 gration of neocartilage into surrounding native cartilage
signaling (as a coreceptor) was also confirmed, suggesting [423]. Recently, the encapsulation of human chondrocytes
that purified GAGs may be promising alternatives to certain in gelatin-methacrylamide-based hydrogels demonstrated
growth factors for enhancing the ex vivo proliferation and that with the addition of a relatively small proportion
differentiation of MSCs [417]. Moreover, it is suggested that of photocrosslinkable HA-methacrylate and, to a lesser
120 F.-M. Chen, X. Liu / Progress in Polymer Science 53 (2016) 86–168

extent, CS-methacrylate, chondrogenesis and mechanical opted to procure and apply specific bioactive domains
properties may be potentiated for potential cartilage tis- that have been identified in the fibronectin molecule. Of
sue regeneration. The encapsulated chondrocytes remain note is the arginine–glycine–aspartic acid (RGD) tripep-
viable for as long as 8 weeks in culture, and the incor- tide, or arginine–glycine–aspartic acid, in the tenth Fn3
poration of HA-methacrylate into gelatin-methacrylamide module, which participates in multiple cell fate decisions
cell constructs enhances chondrogenesis and facilitates [431]. The modification of 3D scaffolding materials with
matrix distribution, whereas the incorporation of CS- RGD sequences can increase the degree to which and rate
methacrylate enhances chondrocyte differentiation in at which MSCs migrate into and populate the constructs
certain ways. These phenomena highlight the potential [432]. A 3D cell culture system was created and used to
for multiple-component photocrosslinkable hydrogels to assess the effects of both substrate stiffness and integrin
enhance chondrocyte behavior and facilitate new matrix binding density on the morphology of human MSCs and on
formation by incorporating GAGs into hydrogels [424]. reconstruction of the microvascular network of endothe-
Recent evidence suggests that biomaterials comprising lial cells. When endothelial cells were encapsulated into
a mixture of collagen, HA and CS can sequester and/or 3D hydrogel scaffolds without binding sites, no network
activate growth factors and thereby establish an even formation and little cell elongation occurred, regardless of
better biomimetic environment that mimics natural car- the matrix stiffness. However, hydrogels containing RGD
tilage ECM, enhancing chondrogenesis and promoting binding sites induced robust microvascular network for-
cartilage-specific matrix deposition among loaded cells mation, the extent of which was inversely proportional to
[425]. Therefore, the combination of collagen with HA the matrix stiffness. In addition, the presence of the matri-
and CS has the potential to result in biomaterials with ces attracted an increased number of MSCs and resulted
appropriate scaffolding properties for cartilage bioengi- in longer cellular projections at higher stiffness. In con-
neering and hence warrants further scientific exploration trast, the absence of RGD induced round morphology at
[426]. This combination has also been successful in cell all rigidities. Overall, these findings reveal the potential to
delivery and implantation within target ischemic tissue control both the binding site density and the substrate stiff-
using a collagen I/CS tissue-engineered matrix [427]. Other ness within 3D cell-populated hydrogels and demonstrate
such biomaterials with well-defined chemical, topograph- the crucial influence of both adhesion and stiffness on cell
ical, and mechanical cues and even gradients of these type-specific cellular behaviors [433].
physicochemical cues may also enhance endogenous pro- Fibronectin-coated surfaces are important in bioengi-
genitor cell homing and engrafting to sites of ischemia neering and other approaches involving cell contact;
[428] and may serve as novel substrates for human cir- however, the random distribution of molecular orien-
culating angiogenic cells to augment angiogenesis for the tations resulting from many immobilization strategies
revascularization of ischemic and infarcted tissue [290]. represents a problem. To address this issue, one potential
However, native GAGs derived from human tissue are het- strategy is the deposition of layers of oriented fibronectin,
erogeneous and structurally complex, and specific GAG which could enhance the availability of the cell-binding
moieties have been demonstrated to trigger specific cellu- sites in the layer. It was found that human umbilical vein
lar responses during cell division, motility and migration. endothelial cells spread much faster and in a more spheri-
Although the specific structure–function relationships are cally symmetrical manner on an oriented fibronectin layer
difficult to clarify, the identification and use of selected sul- (i.e., in the presence of bound monoclonal antibodies) com-
fation patterns and chain lengths of GAG subfractions to pared with a control fibronectin layer (i.e., in the absence
enhance cell fate determination is an exciting new avenue of immobilized antibodies) [434].
for devising new biomaterials for target applications [429]. Of note, the BM of the corneal epithelium presents bio-
physical cues in the form of compliance and topography,
4.2.5. Fibronectin which can modulate cells’ phenotypes and behaviors and
Fibronectin, an elongated 45 nm protein, consists of their nuclei based on the presence of surface-associated
2 nearly identical outer globular domains (subunits of ECM proteins [435]. Recently, the effects of a combination
approximately 250 kDa), and an ␣-helical coiled-coil seg- of exogenous fibronectin-collagen coatings with substra-
ment is covalently connected to each end of the central tum topography on cytoskeletal architectures and on the
domain (C-terminus) by a pair of disulfide bonds. As a migration and alignment of immortalized corneal epithe-
ubiquitous and important ECM protein, fibronectin is a lial cells were investigated. It was observed that in the
multifunctional component residing in the SM that is absence of a fibronectin-collagen coating, a considerably
known to regulate cell behavior via its cell-binding site and greater number of cells aligned parallel with the long
related synergy sites. In addition to being very crucial for axis of the underlying anisotropically ordered topographic
vertebrate development, fibronectin regulates diverse cel- features, but their migratory capability was impaired.
lular functions (e.g., cell adhesion, growth, migration and In addition, the surface area, orientation and elongation
differentiation) among multiple cell types, as confirmed of cytoskeletal elements were variously affected by the
by the early embryonic lethality of targeted inactivation absence or presence of fibronectin-collagen, suggesting
of the fibronectin gene in mice [430]. Based on the bio- that the impacts of topographic cues on cell behaviors are
logical activity of its several modules, this glycoprotein regulated by the presence of surface-associated ECM pro-
may serve as a substrate for cell attachment and adhe- teins [436].
sion. Because of the numerous responses that fibronectin Fibronectin has a remarkably broad variety of func-
can elicit from diverse cell types, many investigations have tional bioactivities, in addition to binding to cell surface
 F.-M. Chen, X. Liu / Progress in Polymer Science 53 (2016) 86–168 121

integrins. Fibronectin binds to numerous biologically and proline) and crosslinked hydrophilic domains (mainly
significant biomolecules, including heparin, fibrin and col- lysine and alanine), is synthesized by cells into the extracel-
lagen/gelatin. These interactions are modulated by several lular space, where the polymerization of tropoelastin into
distinct functional and structural domains, which have a fibrillar biomatrix occurs in a process called elastogenesis
been determined by recombinant DNA analyses or prote- [443]. The correct crosslinking and deposition of secreted
olytic fragmentation [437]. As a provisional matrix during tropoelastin as well as its temporal and spatial arrange-
wound healing and tissue repair, fibrin is highly promis- ment are thought to be vital steps in elastic fiber formation.
cuous in its growth factor-binding capacity, which may Beginning with tethering to the elastogenic cell surface,
be one of fibrin’s main physiological functions, and the water-soluble tropoelastin interacts with multiple proteins
coordinated interplay between the matrix and growth found in or colocalized with microfibrils. Subsequently, the
factors plays a crucial and ubiquitous role in regulating macromolecule undergoes the complex stepwise process
tissue regeneration. The reproduction of growth factor- of elastogenesis (crosslinking, self-association and matu-
ECM interactions within a fibrin-mimetic matrix could be ration) and finally aggregates into organized spheres for
clinically useful and has the significant benefit of a more self-assembly and incorporation into growing elastic fibers
direct regulatory pathway in relation to chemical synthe- in a rubber- or sheet-like network [444–446]. Based on
sis, in contrast to human-sourced material [438]. Notably, ultrastructural and biochemical analyses, these fibers have
fibronectin facilitates the migration, adhesion and prolifer- been revealed to be primarily composed of two distinct
ation of MSCs, but not osteogenic differentiation, whereas types of small segments that alternate along a polypep-
fibrinogen enhances cell proliferation and adhesion, but tide chain. These segments are an abundant amorphous
not migration. Consequently, the integrin expression pat- segment that is ␣-helix rich and composed of alanine and
tern of MSCs on specific matrix components has been lysine chains, which are where the crosslinks form between
associated with cell fate decisions [273]. Similarly, RGD- the molecules, and a 10–12 nm microfibrillar segment that
modified fibronectin hydrogels have been demonstrated is mainly located around the periphery of the amorphous
to offer anti-apoptotic properties to cells that migrate into component and composed of highly insoluble amorphous
the scaffolds. Apparent apoptosis was shown in unmodified segments that are responsible for the elastic proper-
scaffolds, indicating that cell adhesion via the fibronectin ties [447]. The formation of tetravalent bonding between
RGD sequence is one of the important methods for cell elastin and 3 lysine-derived products, namely, lysinonor-
preservation and survival. In this respect, the immobiliza- leucine, desmosine and isodesmosine, via a crosslinking
tion of the RGD peptide on non-ECM component-based reaction leads to the polymerization of tropoelastin into
biomaterials, such as macroporous alginate scaffolds, has insoluble elastin [444]. Although it is likely that the random
also proven to be a necessary parameter in cardiac tis- coil structure of the molecules crosslinked into a network
sue engineering, contributing to the better preservation of offers the ability to stretch similarly to a rubber band, the
regenerated tissue in culture and the formation of func- contribution of the elastin fiber structural conformation to
tional cardiac muscle tissue [439]. These results suggest the functional elasticity of the fibers remains unclear [447].
that RGD modification is an omnipotent strategy for the As another main component of elastic fibers, the microfib-
design of regenerative biomaterials. Recently, the incor- ril segments found either in association with elastin or
poration of fibronectin into multilayer elastin-like protein independently contain a variety of distinct and different
biomaterials was observed to enhance overall cytocom- glycoproteins [448]. Fibrillins, a family of such microfibril
patibility for tissue engineering; the high cell viability in resident proteins, were first harvested from the media of
the resultant 3D constructs indicated the applicability of human fibroblast cell cultures; its members precede elastin
fibronectin to the creation of resilient, strong manmade in developing tissues and yield a matrix scaffold to which
vessels and other soft-tissue replacements [440]. It would elastin fibers subsequently attach [447,449]. Fibrillin is a
not be surprising if, in the future, fibronectin attracts 350 kDa protein that is periodically arrayed along individ-
increasingly concentrated attention in the design of bio- ual microfibrils that may be aligned within bundles. Type VI
material strategies. collagen interweaving among large, banded collagen fibers
is not associated with the microfibril system identified by
4.2.6. Elastin the presence of fibrillin, although it has been proposed as
Elastin is a hydrophobic macromolecule of the ECM a possible microfibrillar component [449].
that is found throughout the vertebrate kingdom, includ- In major arteries, elastin is secreted and synthesized by
ing in humans. This macromolecule possesses a unique vascular smooth muscle cells. Elastin is the most promi-
chemical composition that is rich in proline, glycine and nent ECM protein deposited in the arterial wall, comprising
hydrophobic amino acids, consonant with its character- up to 50% of the nonhydrated mass of the vessel [450].
istic physical properties [441]. The presence of highly The elastic matrix imparts vessel integrity, extensibility
crosslinked elastic fibers in the extracellular space, whose and arterial elasticity with regard to the native structural
main components are elastin and microfibrils, allows a configurations of vascular tissues and their ability to recoil
range of tissues, such as the large arteries, the liga- after stretch under pulsatile flow. Via biomechanical trans-
ments, the dermis, the tendons, elastic cartilage and the duction, elastin plays an equally critical role in regulating
lung parenchyma, to have the required elastic ability to cell signaling pathways (e.g., those of smooth muscle cells
transiently stretch [322,442]. Tropoelastin, a 60–72 kDa and luminal endothelial cells in the arterial wall) involved
biosynthetic precursor form of the elastin protein that in morphogenesis, inflammation and injury response
consists of hydrophobic domains (mainly valine, glycine [451,452]. Moreover, it has been demonstrated that elastin
122 F.-M. Chen, X. Liu / Progress in Polymer Science 53 (2016) 86–168

is an important autocrine factor that ensures vascular By alteration of the composition and length of these
homeostasis via a combination of biologic signaling and repeats, the mechanical properties of the biopolymers
biomechanical support. The elastic lamina specifically are tunable, meaning that these properties may be con-
alternates with smooth muscle rings due to elastin-smooth trolled for specific tissue engineering applications [462].
muscle cell interaction to promote cellular synthesis and Notably, their mechanical stiffness, chemical composi-
the assembly of elastic matrix superstructures, forming a tion and even fate within cells can also be controlled
flexible, strong part of the arterial wall [450,453]. at the gene level [461]. The use of an rhELP containing
For a tissue-engineered replacement to maintain vas- the RGD tripeptide sequence can generate a 3D tissue
cular homeostasis, it is therefore important to ensure that equivalent derived from human oral epithelial cells and
the elastic matrix superstructures unique to its target tis- lamina propria fibroblasts. This tissue equivalent may
sue can be generated. However, the use of cells, scaffolds contain as many highly proliferative and self-renewing
and growth factors based on strategies such as dynamic cells as the native tissue itself and displays mechani-
stretch and contact guidance to produce the vascular elas- cal strength, stiffness and resilience resembling those of
tic matrix is particularly difficult because adult vascular native tissue [463]. Moreover, recent evidence suggests
cells inherently possess a very poor ability to synthe- that porous scaffolds with surface elastin and poly-l-lysine
size elastin precursors. Replication of the process during can maintain active chondrocytic proliferation and ECM
development such that elastin precursors organize into secretion by cryopreserved chondrocytes [464]. Similarly,
the mature elastic matrix in terms of its structures and coating PLGA biomaterials with an elastin-like polypep-
biocomplexity represents an even more challenging task tide has been demonstrated to improve neural progenitor
[451]. However, the incorporation of elastin into engi- cell adhesion and proliferation in an elastin concentration-
neered scaffolds is not a typical choice due to a lack of dependent manner, and in combination with retinoic
access to pure, homogeneous human elastin, although a acid, these scaffolds may stimulate the differentiation of
limited yield of natural elastin can be extracted from tis- these progenitor cells into neuronal and astroglial lin-
sues by harsh alkaline treatments [454]. Recently, it has eages [465]. Additionally, although injectable hydrogels for
been suggested that the crosslinking of tropoelastin in tissue engineering biomaterials generally lack mechani-
the developing elastin matrix may be simulated using cal strength, synthetic human elastin scaffolds reinforced
short elastin-mimetic peptide sequences engineered to with collagen microfibers or injectable hydrogels modi-
mimic the active motifs of human elastin. The potential fied by elastin incorporation have demonstrated tunable
for increased cell adhesion with appropriate cell-binding gelation and biodegradation properties, tailorable poros-
motifs and the engineering of elastin chains into synthetic ity and pore size, and favorable mechanical characteristics
polymer biomaterials is a new concept for elastin tissue and/or structural stability, favoring various biomedical
engineering [455]. With remarkable advances in the field applications [466–469]. Interestingly, nanocarriers and
of genetic engineering, the design and biosynthesis of an injectable hydrogels based on silk-elastin-like proteins,
ECM analog using human polypeptide sequences as its a family of genetically engineered recombinant protein
building blocks is possible [456]. To this end, recombinant polymers, may possess properties allowing controlled
human tropoelastin, the monomeric precursor of elastin, therapeutic release due to their rational design, tunable
can be chemically crosslinked to form a polymer with structure–function relationships, stimuli-responsive fea-
unique properties, termed a recombinant human elastin- tures and target specificity [470]. Despite its growing utility
like polymer (rhELP); both the full-length monomer and as a scaffolding material in tissue engineering and its
elastin-like polypeptides can be applied to yield biomate- well-recognized function in the vasculature, the hemo-
rials with physical properties resembling those of native compatibility of elastin is often overlooked. However, as
polymeric elastin [442,457–459]. In particular, artificial elastin scaffolds and coatings display increased hemocom-
elastomeric proteins that mimic the molecular architec- patibility [465,469], the potential of decellularized elastin
ture of titin, a giant muscle protein in intact myofibrils that and arterial elastin as nonthrombogenic biomaterials has
governs the passive elasticity of muscle, have been used begun to be recognized [452]. Thorough comprehension
to develop new muscle-mimetic biomaterials. Following of developmental elastogenesis and the subsequent ability
photochemical crosslinking into solid form, the resulting to mimic the spatiotemporal alterations that occur dur-
biomaterial may behave as a shock-absorber-like mate- ing that phase in the cellular environment will enable us
rial at high strain by effectively dissipating energy and to design elastogenic therapies to restore homeostasis in
as a rubber-like material showing high resilience at low de-elasticized vessels and to develop clinically translatable
strain. By adjusting the composition of the elastomeric pro- elastic vascular tissue grafts [451].
teins, the mechanical properties of such a biomaterial can
be fine-tuned to develop biomaterials that mimic various 4.2.7. ECM assemblies as scaffold building blocks
muscle types [441]. Modeled after the naturally occurring The ECM biomacromolecules, such as collagens, GAGs,
tropoelastin, rhELPs have emerged as inspired synthetic laminins, entactin, fibronectin and elastin, involved in the
biopolymers for the engineering of compliant, resilient matrix and the structure, organization and manner in
soft tissues due to these polymers’ non-immunogenic, which they are assembled determine the biochemical and
biocompatible and biodegradable properties [460,461]. biophysical properties of the resultant ECM [299]. The
For example, based on recombinant DNA technology, use of ECM assemblies directly or indirectly derived from
recombinant silk-elastin polymers are engineered to be human tissues/organs for tissue engineering applications
composed of tandem repeats of silk and elastin units. opens a new route for scaffolding biomaterial design. On
 F.-M. Chen, X. Liu / Progress in Polymer Science 53 (2016) 86–168 123

the one hand, efforts should in fact be made to establish of these artificial scaffold-based constructs, at least in part,
or optimize new or combined processing approaches to fail to match the sophisticated properties of their native
yield robust biopolymers based on human ECM compo- counterparts in terms of structure, dynamics, biocompati-
nents that are likely to help to reconcile commercial and bility and function. Clearly, we have thus far been unable
clinical pressures on regenerative medicine [471]. On the to develop a cell-friendly bioactive material template with
other hand, new insights into the physical and molecu- an architecture similar to that of a complex native tis-
lar information coded within the human ECM milieu are sue/organ, which involves an extensive vasculature system
already informing the redesign of the “next generation” of and contains intricate information within its physical and
advanced cell-instructive scaffolds for the clinical manage- chemical structures [5,6]. Recent advances in tissue/organ
ment of recalcitrant chronic wounds [20]. Investigations in decellularization offer acellular human-derived bioma-
highly regenerative organisms have revealed a specialized terials that preserve the natural architecture from the
formulation of powerful ECM molecules that dynamically whole-organ level to the microstructural scale and even
respond to cellular and soluble components to dictate the down to the nanoscale [287,475]. The use of various forms
distribution and function of tissue-specific cells and to of ECM scaffolds, with recent implementations including
support repair and regeneration while avoiding acellular whole organs, derived from decellularized allogenic tis-
fibrotic scar tissue formation; several of these molecules sues/organs that retain structurally organized entities such
have already been adopted in scaffolding science, whereas as collagen, GAGs and fibronectin, is increasingly routine,
many others remain unidentified. By comparing matrix enabling natural templates that accommodate tissue engi-
contexts that feature scarring resulting from fibrotic repair neering and regenerative approaches [266,274] (Fig. 12).
with matrices conducive to scar-less healing and full Even with a lack of living cellular components, the decellu-
regeneration, biomaterials modified using specific ECM larized ECM may be regarded as a physiological depot for
components were found to significantly enhance func- various signaling molecules, which retain their functional-
tional outcomes upon application [472]. This phenomenon ity, at least in part. Upon application, these bioactive agents
is spurring interest in advancing our knowledge of tis- are released and play their natural roles in cell regulation,
sue and ECM science, thus instructing the intentional thus offering the specific ECM the information neces-
design of porous biopolymer-based scaffolds incorporating sary to conduct repair and regeneration [102]. The gentle
ECM assemblies that constitute controlled morphologies removal of cells from human tissue or organs can leave
at various scales (e.g., a combination of pores or micro- behind a “footprint” within the ECM scaffold that directs
and nano-elements). These scaffolds comprise a variety cell ingrowth and repopulation; the biological components
of structural and matrix proteins that are spatially orga- remaining in the intact matrix may specifically enhance cell
nized and have the ability to bind relevant informative activities such as adhesion, proliferation, migration and dif-
signals, such as growth factors and cell homing agents, ferentiation in a way that reflects the biological identity and
in a tailorable manner. However, exact control over the functional requisites of the original tissues [274]. Specifi-
sequence composition of these biomacromolecules and cally, human-derived decellularized matrices can also be
their self-assembled superstructures to generate multi- considered biologically active scaffolds capable of recruit-
tudes of well-defined protein-based biohybrid materials ing endogenous host stem/progenitor cells and stimulating
poses a major challenge in bionanotechnology and mate- in vivo cell proliferation and differentiation toward the for-
rials science [52,473]. Based on a careful review of the mation of a biointegrated tissue [476]. Decellularized ECM
literature, a step forward could involve moving from has thus attracted increased attention in tissue engineering
the reduction of ECM into short functional domains and as an “off-the-shelf” and immune-compatible biomaterial
biomacromolecules for biomaterial functionalization to the that may be used to create tissue-engineered alternatives
exploration of basic material chemistries that dictate cell to living tissue grafts for tissue replacement and repair
fate commitments and more closely reproduce the dynam- [477,478].
ically evolving in vivo milieu occurring in the natural ECM
[92,282,299,474]. This field is wide open for new creative 4.3.1. Rationale and methods for decellularization
scientists to make certain approaches practical for actual Decellularization is the process of stripping a donor tis-
clinical use. sue or organ of its resident cells while maintaining the
native ultrastructure, ECM components and chemical cues
4.3. Decellularized ECMs for biomaterials that are essential for cell preservation and homeostasis in
the remaining matrix template [475]. The removal of cells
Through the use of synthetic biomaterials in association and a large proportion of the major histocompatibility com-
with stem cells and/or growth factors, tissue engineering plex from the ECM templates eliminates the inflammatory
has adopted an interdisciplinary approach to the design response, the foreign-body reaction and the potential for
of new biological therapeutics, and recent advances in immune rejection and thus favors repopulation by new
this field have enabled the creation of certain functional cells [274,479]. The effectiveness of a technique for the
tissue replacements in the laboratory [4]. Early attempts decellularization of a specific tissue or organ is dictated
at engineering certain tissues (e.g., skin and cartilage) by many factors, such as tissue organization, cell den-
have achieved considerable success thanks to their simple sity, the biological and geometric properties required in
architectures, which subsequently fueled enthusiasm for the post-processed product and the targeted tissue engi-
applying these same or similar approaches to the fabrica- neering and clinical uses (Fig. 13) [298]. Because human
tion other complex tissues and organs [3]. However, many ECM molecules are highly conserved among tissues and
124 F.-M. Chen, X. Liu / Progress in Polymer Science 53 (2016) 86–168

Fig. 12. Decellularized matrices from tissues (e.g., small intestinal submucosa (SIS)) or organs (e.g., kidney, heart and liver) that have native-like extracellular
matrix (ECM) microstructures, compositions and biomechanical properties (schematic is not to scale). These decellularized ECMs may maintain the shapes
of the original tissues and organs when used as scaffolding materials in tissue engineering approaches for new tissue/organ regeneration. Alternatively,
decellularized matrices derived from tissues and organs can be made into different types, such as a patch or particle, for tissue engineering scaffolding
biomaterials or can be designed as an injectable gel for cell culture substrates [477].

organs, antigenic epitopes must be completely removed structures but also via the physiological involvement of
from intra- and extracellular components when preparing a vast wealth of regulatory factors in a physically and
decellularized scaffolds. If this removal can be achieved, mechanically appropriate microenvironment [480,481].
then adverse immunological responses can theoretically be Another prerequisite is the preservation of the complex
circumvented [274,288]. Decellularized ECM is expected to composition and 3D ultrastructure of decellularized ECM
direct cellular activities and regenerative events of breath- and the activity of its functional components. Therefore, as
taking complexity not only via specific “organomorphic” mentioned previously in this review, the general purpose of

Fig. 13. The production of extracellular matrix (ECM) products for targeted biomedical use is influenced by many factors, including the properties of
the donor tissue or organ (e.g., tissue type, architecture, cellularity and dimension), the method, agent and protocol for decellularization (e.g., chemical,
enzymatic and physical treatments) and the desired biological and geometric properties of the post-processed product. It is worth noting that every cell
decellularization treatment will alter the ECM composition, damage the biochemical features and disrupt the native ultrastructure and architecture to
some degree; the selection of an appropriate strategy for the decellularization of a particular tissue/organ for a target application is important to minimize
rather than to completely avoid these undesirable effects [274].
 F.-M. Chen, X. Liu / Progress in Polymer Science 53 (2016) 86–168 125

all proposed and established decellularization methods is decellularization and the intended use of the decellular-
to minimize the cellular and nuclear materials in the matrix ized ECM in tissue engineering and reconstructive surgery
as efficiently as possible, while avoiding any potentially [274,489] (Fig. 14). Each of these treatments can disrupt
adverse impacts on the biological activity, composition the tissue ultrastructure, biochemical composition and
and mechanical integrity of the ECM product [482]. Unfor- mechanical behavior of the end-product to a certain degree,
tunately, no decellularization methodology is absolutely which in turn affects host responses to the ECM scaffold
effective in removing all cellular components and DNA [486]. These frequently used decellularization techniques
materials from matrix constituents (1 mg dry weight of and their impact on the structure and composition of the
matrix material contains less than 50 ng of dsDNA), and resulting ECMs have been reviewed elsewhere [274]. To
in terms of the biochemical and structural properties of simplify the cell removal process, undesirable excess tis-
the resultant product, all decellularization strategies are sue is generally removed prior to using decellularization
highly variable in their results [275,483]. Moreover, long agents. However, much attention should be given to reten-
periods of decellularizing, as well as certain detergents, tion of the main structure and ECM components, such as
can adversely influence GAG and collagen components and the BM. For thin tissue laminates, such as intestine, uri-
then ECM antigenicity and/or integrity, resulting in both nary bladder, amnion or pericardium, the most frequently
disruption of the construction and potential loss of the sur- applied decellularization technique is freezing and thaw-
face composition and structure [484,485]. ing [269]. Undesirable layers, such as submucosa or muscle,
The efficiency of cell elimination from a tissue is deter- are first mechanically removed, and the resulting tissue is
mined by not only the characteristics of the tissue and its briefly exposed to easily removed acids or detergents for a
origin but also the specific physical, chemical and enzy- relatively short time, followed by rinsing. More extensive
matic strategies that are applied for decellularization [486]. biochemical exposure and longer rinse times are required
Decellularization strategies can be reasonably chosen if for thicker tissue laminates, such as dermis or cardiac mus-
the principles of the disruptive process are thoroughly cle tissue (Fig. 14). In contrast, the adjuvant use of lipid
understood and contemplated [275,485,487]. The struc- solvents such as alcohols is required for amorphous tis-
tures and arrangement of the various ECM proteins in sues and organs, such as adipose tissue, pancreas and brain
the resulting acellular matrices are largely conserved by (reviewed in Ref. [274]).
recent new technologies for decellularization, resulting Although a wide range of decellularization tech-
in general retention of the mechanical properties of the niques for both tissue parts and whole organs have been
original tissue (reviewed in Ref. [274]) (Fig. 14). The post- established and successfully used in many biomedical
processed matrix may enable efficient cell reseeding and applications, routine protocols are inadequate for yield-
offer biomechanical strength and structural integrity for ing an intact, elegant scaffolding ECM for the targeted
new tissue formation. For example, retrograde or antegrade tissue/organ that can then be revitalized by repopula-
perfusion has been applied as a method for whole-organ tion with ex vivo-expanded cells. By making educated
decellularization, in which the 3D architecture of the organ decisions about the techniques and agents used during pro-
is largely preserved [93,486]. The decellularized matrices cessing, the preservation of ECM bioactivity and integrity
maintain the shape of the original organ and can be either during tissue decellularization can be optimized to pro-
directly used as scaffolding materials in tissue engineering duce an application-specific standard product [298,487].
approaches for organ regeneration or made into different However, there is no established “gold standard” protocol
types (e.g., patch, particle or gel) of tissue engineering scaf- for decellularization thus far, and scientists must there-
folding biomaterials [488]. fore select an effective technique or different technique
Biochemical methodologies for tissue/organ decel- combinations based on the requirements for a targeted tis-
lularization include solvent extraction; osmotic shock; sue/organ as well as continually develop new techniques
acid/alkaline treatments; ionic and nonionic detergent to refine these protocols [475]. Not surprisingly, the util-
treatments; and enzymatic digestion with lipase, pro- ity of apoptosis as a decellularization method goes beyond
teases, DNase and RNase. Recently, the pH of certain the production of ECM materials with improved perfor-
decellularization solutions was found to influence ECM mance. Thanks to the potential to specifically target the
retention (e.g., elastin, fibronectin and laminin) and cell- cellular component of a tissue, the deliberate activation of
removal efficacy of the resulting product [489,490]. Direct programmed cell death is expected to better maintain the
force can also be used to aid in tissue decellularization. structural, biochemical and/or biomechanical features of
Commonly, biochemical agents are employed in combi- the decellularized ECM [491]. In principle, by correlating
nation with physical techniques to lyse cells, and the cell regenerative potential with a particular ECM composi-
remnants are then removed by rinsing [298]. Of course, tion, this concept could also provide the unprecedented
any biochemical procedure applied to remove cellular and potential to observe the properties of decellularized but
nuclear materials may also slightly damage or alter the theoretically intact ECM and to identify a set of signals
native 3D architecture of the resulting ECM template, necessary to elicit specific functions. The identification
and thus, during the decellularization process, achieving of such a relationship would then subsequently allow
a balance between chemical and physical treatments is a transition from the paradigm of decellularized tissue-
indispensable [274]. The optimal protocol for the use of engineered constructs to entirely synthetic scaffolding
agents for decellularization is determined by the charac- biomaterials devised to contain the sufficient and critical
teristics and functions of the detergents commonly being set of cues needed for specific tissue/organ regeneration
applied, the thickness and density of the tissue targeted for [491].
126 F.-M. Chen, X. Liu / Progress in Polymer Science 53 (2016) 86–168

Fig. 14. Representative decellularization protocols for (A) thin laminates (e.g., intestine, pericardium, amnion and urinary bladder); (B) thicker laminates
(e.g., dermis); (C) amorphous, fatty tissues (e.g., adipose tissue, pancreas and brain); (D) composite tissues (e.g., trachea and urogenital tract) or whole simple
organs (e.g., bladder and intestine); and (E) whole vital organs (e.g., heart, lung and liver). (F) Representative images of the gross appearance of an intact rat
liver before (I), during (II), after (III) decellularization and of a decellularized liver following blue dye perfusion (IV). (G) Representative photomicrographs
(scale bars are 50 ␮m) of hematoxylin-eosin staining of a native rat liver (I) and a decellularized liver-extracellular matrix (ECM) (II) or fluorescent staining
with DAPI (4 ,6-diamidino-2-phenylindole) in a native rat liver (III) and liver-ECM (IV).
Source: [274], Copyright 2011, Reproduced with permission from Elsevier Ltd.

Regardless of which decellularization method is used, 4.3.2. Applications of decellularized ECMs in tissue
cell residues remaining in ECM materials should be accu- engineering
rately assessed using a quantitative definition because of Decellularized ECMs, such as those of SIS, arteries, the
the expanding list of clinical applications and the rapid urinary bladder and heart valves, are common sources
diversification of both decellularization techniques and of instructive scaffold materials enriched in collagen and
source tissues [298]. It is reasonable to establish standards endogenous proteins [492–494]. Under the assumption
for tissue/organ decellularization based on readily deter- that these matrices are capable of dictating the differen-
mined quantitative criteria for the remaining cell remnants tiation decision of seeded cells, ECM materials may be
within ECM biomaterials, additional data related to the host revitalized by living cells before implantation [495–497].
tissue response upon the in vivo transplantation of these Alternatively, decellularized ECM can be directly applied to
biological scaffolds and the observed regenerative capacity recruit resident host cells for endogenous tissue regenera-
of a decellularized but theoretically intact ECM with a spe- tion based on the leveraging principles of morphogenesis
cific composition [274]. The concept would also provide the [22,55,477,498]. In this regard, scaffolds based on decel-
unprecedented potential to evaluate the impacts of certain lularized adipose tissue may be endowed with inherent
cell remnants and nuclear materials on the host response adipo-inductive properties to facilitate adipose tissue
and to identify a set of cues critical to elicit certain functions growth in vivo, as demonstrated by a recent study that
[491]. revealed that bFGF-binding, heparinized decellularized
 F.-M. Chen, X. Liu / Progress in Polymer Science 53 (2016) 86–168 127

adipose tissue is an efficient, biocompatible and injectable is a reality. However, clinical experience has been far
adipogenic system for de novo adipogenesis and in vivo adi- from successful, especially when longer-term benefits
pose tissue engineering [499]. Indeed, adipose ECM-based are taken into account. It behooves us to return to the
biomaterials may facilitate endogenous tissue regenera- bench in order to elucidate the macromolecular chemistry
tion, even without the delivery of exogenous cells, which is and biological roles of human tissue-derived ECM during
an attractive solution for the management of a number of decellularization–recellularization so that the ultimate
soft-tissue defects [500]. The use of decellularized adipose goal of solving the problem of on-demand tissue/organ
tissues of allogenic origin, for example, may offer clin- assembly can be successfully realized [17,26,286].
icians “off-the-shelf” products for corrective procedures Of all potential tissues, SIS is one of the raw bioma-
to restore contour, offering an ideal alternative to autol- terials that is most often investigated and is used as a
ogous tissue transfer for patients in need of soft-tissue prototypical ECM scaffold in a wide variety of applica-
reconstruction [501]. Of note, acellular tissue matrices tions because this material exhibits the key features of
have recently evolved from space fillers and mechani- a highly supportive scaffold and presents growth factors
cal supports to biological tissue replacements that have and adhesion peptide sequences, which facilitate integra-
been demonstrated to support the penetration of cells and tion with surrounding tissue [26,477,492]. SIS tissue is
tissues in several applications, without inducing a gross degradable and is known to display excellent biocompati-
immune response, while guiding endogenous tissue regen- bility in clinical use; even xenogenic SIS possesses low or
eration [500,502]. Indeed, given the natural origin of the no immunogenicity, and when used as a wound-dressing
matrices, ECM scaffolds degrade slowly after implantation material, SIS may provide defense against infections. In
and are correctly replaced by or remodeled with a new certain applications, SIS is crosslinked to reduce its hemo-
matrix produced by cells [35]. compatibility or to modulate the mechanical properties or
ECM-based biomaterials that retain the native materials degradation rate of a scaffold [523]. Both SIS and another
and proteins of a living tissue/organ, along with the innate small-intestine-derived preparation termed the biological
spatial arrangements in certain cases, provide appealing vascularized matrix (BioVaM) have been found to sup-
tissue engineering templates on which either exoge- port endogenous cell homing after implantation, and the
nously transplanted or endogenously recruited cells may matrix may be subsequently remodeled to produce func-
adhere, proliferate, differentiate and ultimately integrate tional tissue [492]. By dry weight, an SIS-ECM biomaterial is
to form functional tissues [266,286,287]. The clinical use composed of more than 90% collagen, and the collagen fiber
of these materials to replace missing and compromised orientation can be retained during the decellularization
tissues/organs has dramatically enhanced the practice process [26]. Depending on the type of decellularization
and philosophy of reconstructive surgery. Currently, both method used, the material can contain various GAGs that
SIS and human DBM have been approved by the FDA for existed in the living tissue, including heparin, HS, CS and
clinical uses; DBM has been investigated for more than 30 HA [298]. However, in the decellularization process, ionic
years for application in bone grafting procedures (noted in detergents are commonly applied, which can remove GAGs
Section 3.2.3). A number of ECM products based on tissues from the post-processed ECM product [486,489]. More-
or organs of human origin are already commercially avail- over, a pool of adhesion molecules (e.g., fibronectin and
able (Table 1). Since the first use of decellularized bone as laminin), the proteoglycan decorin and the glycoproteins
a prototype ECM grafting material [503], this fast-growing biglycan and entactin are present in a well-prepared SIS-
field has gained convincing proof-of-principle evidence of ECM scaffold [26]. Most importantly, a pool of growth
this approach’s efficacy for bone [504], vaginal [505,506], factors and other bioactive molecules also exists in a well-
epithelial [507], skin [206,207,209–211], musculoskeletal prepared SIS-ECM, such as TGF-␤, b-FGF and VEGF. Even
[508,509], corneal [201,204,510] and vascular [511] tissue after terminal sterilization and long-term storage, these
repair as well as for tissue engineering of pulmonary factors have been shown to maintain their bioactivity [492].
[512–514], myocardial [488], airway [515,516], liver [517], All of these inherent advantages have sparked strategies
renal [518] and pancreatic [519] implants. Most studies that apply SIS-ECM as a scaffolding material in applications
reported complete and functional organ regeneration for soft-tissue reconstruction and cardiovascular, neural
in small-animal models, and early clinical successes and urogenital tissue engineering [39]. SIS is currently
with complex tissues in preclinical studies and certain approved by the FDA for several urogenital uses, including
individuals have served as proof of concept [266]. In bladder augmentation, hernia repair and urethral stricture
particular, urinary bladder and lung matrices, arteries repair [26,76,77]. Although SIS-ECM is evolving and slowly
and heart valves from allogenic human sources have been becoming a new standard for bladder augmentation [269],
applied in preclinical and clinical procedures over the last collagen-composite biomaterials that consist of nanofiber
decade, although the outcomes of large-animal models networks highly resembling natural ECMs populated with
and human clinical trials have varied (Table 2) (reviewed in self-assembled fibroblast sheets or the patient’s own mus-
Refs. [26,76,266,298,477,520,521]). However, we must be cle and urothelial cells are another attractive strategy that
mindful of the fact that our understanding of the cellular is showing optimistic clinical outcomes [74,524,525]. Other
processes involved in the recellularization and revital- defects of the urogenital system, such as incontinence and
ization of these bioscaffolds to form practical tissues is vesicoureteral reflux, can also be managed by injection of
still incomplete [522]. The last two decades have included collagen-based SIS-ECM biomaterials. To modify naturally
quick translation of decellularized matrices from the bench derived SIS biomaterials in regenerative medicine, HA-
to the clinic, and today, their clinical use in reconstruction PLGA nanoparticles were synthesized and were observed
128 F.-M. Chen, X. Liu / Progress in Polymer Science 53 (2016) 86–168

Table 1
Partial list of commercially available extracellular matrix (ECM)-based products of human origin for tissue reinforcement or replacement.a

Product Company ECM source Application focus Product form (brief description)

AlloDerm® LifeCell Corp. Skin Soft-tissue augmentation, Dry sheet (an intact acellular matrix of
reinforcement or replacement (e.g., natural biological components)
abdominal wall, breast)
GraftJacket® Wright Medical Skin Soft-tissue augmentation, chronic Dry sheet (a thin fenestrated acellular
Technology Inc. wound treatment matrix)
AxisTM dermis Mentor Worldwide LLC Dermis Pelvic floor repair, horizontal and Dry sheet (allograft dermis consisting
vertical soft-tissue augmentation in of solvent-dehydrated,
thickness and length gamma-irradiated, preserved human
collagen)
NeoFormTM Mentor Worldwide LLC Dermis Breast augmentation Dry sheet (acellular human dermal
collagen retaining its constituent
elastin fibers)
DermaMatrixTM Synthes Inc. Dermis Replacement, repair, or reinforcement Dry sheet (an allograft derived from
of soft tissue in grafting procedures donated human skin)
such as root coverage and soft-tissue
ridge augmentation
SuspendTM Mentor Worldwide LLC Fascia lata Pelvic floor reconstruction Dry sheet (a dense matrix of collagen
bundles and transverse fasciculi)
®
AlloPatch Musculoskeletal Fascia lata Rotator cuff augmentation Dry sheet (human fascia lata from
Transplant Foundation iliotibial band)
AlloPatch HDTM Musculoskeletal Dermis Tendon reconstruction Dry sheet (an intact or fenestrated
Transplant Foundation dermal graft that preserves and
maintains the natural biomechanical
and biochemical matrix properties)
FlexHD® Musculoskeletal Dermis Breast augmentation, hernia repair Fully hydrated matrix (a strong,
Transplant Foundation versatile, ready-to-use acellular dermal
matrix)
IOPatchTM IOP Inc. Pericardium Ophthalmologic repair Dry sheet (an acellular pericardial
matrix)
Puros® DBM Zimmer Bone Bone repair (e.g., reconstruction of Allograft demineralized bone matrix
cavitary bone deficiency) (DBM) putty (a natural polymer
product produced by a mineralized
bone allograft following the removal of
its inorganic elements using a
demineralizing agent; 100% derived
from allograft tissue)
AlloMatrix® Wright Medical Bone Bone repair (e.g., reconstruction of Injectable or formable putty (DBM
Technology Inc. cavitary bone deficiency, augmentation combined with surgical-grade calcium
in situations of segmental bone loss sulfate that can be formed into an onlay
and interbody spinal fusion) or injected directly into a defect site)
AlloFuse® AlloSource Bone Bone repair (e.g., reconstruction of Injectable gel or putty (DBM combined
cavitary bone deficiency) with heat-sensitive copolymer)
InterGro® Biomet Osteobiologics Bone Bone repair (e.g., reconstruction of Paste, putty or mix containing
cavitary bone deficiency and hydroxyapatite/calcium carbonate
segmental bone loss) composite granules (DBM in a lecithin
carrier)
Grafton® Osteotech Inc. Bone Bone repair (e.g., reconstruction of Gel (DBM combined with glycerol)
cavitary bone deficiency)
Osteofil® /Regenafil® Regeneration Bone Bone repair (e.g., reconstruction of Injectable paste or putty, strips and
Technologies cavitary bone deficiency, augmentation blocks with cortical cancellous chips
in situations of segmental bone loss (CCC) (DBM combined with a non-toxic
and interbody spinal fusion) natural gelatin carrier)
Optefil® Exactech Inc. Bone Bone repair (e.g., reconstruction of Injectable bone paste/dry powder
cavitary bone deficiency and ready to be hydrated (DBM suspended
segmental bone loss) in a gelatin carrier)
Opteform® Exactech Inc. Bone Bone repair (e.g., reconstruction of Formable putty or dry powder ready to
cavitary bone deficiency and be hydrated (DBM and CCC suspended
segmental bone loss) in a gelatin carrier)
Optecure® Exactech Inc. Bone Bone repair (e.g., reconstruction and Dry mix delivered with buffer solution
augmentation of deficient maxillary (an optimal concentration of DBM
and mandibular alveolar ridges and suspended in a resorbable hydrogel
dental intraosseous defects) carrier)
Optecure® + CCC Exactech Inc. Bone Bone repair (e.g., reconstruction of the 3D matrix delivered with buffer
spine, pelvis and extremities) solution (DBM and CCC suspended in a
hydrogel carrier)
a
The product list does not represent any particular preference by the authors.
 F.-M. Chen, X. Liu / Progress in Polymer Science 53 (2016) 86–168 129

Table 2
Partial list of human tissue/organ-derived decellularized matrices that may be used for tissue engineering applications.

Tissue source Decellularized matrix Application options Partial list of Refs.

Skin Dermal tissue Abdominal wall, breast, ear, nose and throat/head and neck [206,207,209]
reconstruction, grafting
Bone Bone matrix Bone repair and regeneration [245,247,248,503]
Cornea Corneal stroma Corneal transplantation [201,204,510]
Adipose tissue Adipose tissue Adipose tissue regeneration [499,501]
Vagina Amniotic membrane Vaginal/cervical reconstruction [505,506]
Small intestine Small intestinal submucosa Reconstruction of integument, body wall, urinary bladder, [26,286,477]
rotator cuff, intestine, urethra, ureter and diaphragm
Trachea Trachea matrix Replacement of the main left bronchus [515,516]
Heart Heart matrix Engineering of a bioartificial heart for heart tissue engineering [488]
Lung Lung matrix Lung regeneration, orthotopic transplantation [514]

to stabilize the porous structure of SIS and hence to cartilage substitution, the biomatrix-scaffold was observed
improve the surface biocompatibility and performance of to exhibit desirable compatibility in both in vitro and in vivo
the resultant composite scaffolds in tissue regeneration tests, offering a product for application in osteochondral
[526]. The presence of endogenous growth factors and defect repair [530,532]. In investigating the possibility of
aligned collagen fibers in an acellular SIS-ECM has also an acellular cartilage material for application as a scaffold
sparked considerable research interest in the bone tissue in cartilage tissue engineering, this type of biomatrix dis-
engineering community [39]. As an adjuvant material to played good biocompatibility with cultured rabbit BMSCs,
DBM, SIS/DBM has a tendency to promote more cellu- with no indications of cytotoxicity in either contact or
lar proliferation and osteoblast differentiation than does extraction assays. Compared with control groups at 6 and
DBM alone. In one study, although SIS without cell seeding 12 weeks, the repair of cartilage defects in rabbit knees
resulted in no new bone formation in vivo, whereas DBM utilizing the cell–matrix constructs showed a significant
alone demonstrated new bone formation along the edge of improvement in histological scores [533]. Although cellular
old DBM particles, an SIS/DBM composite exhibited higher cartilage can offer a native ECM for cartilage regenera-
osteoinductivity. Moreover, the residual SIS/DBM was sur- tion, it is difficult, if not impossible, for cells to penetrate
rounded by an osteoid-like matrix and newly formed bone the biomatrix due to its nonporous structure. To address
[527]. The capacity for localized collagen formation and this limitation in engineering cartilage, a sandwich model
osteocalcin deposition by SIS-ECM was demonstrated in containing chondrocytes and acellular cartilage slices was
a full-thickness bilateral bone-defect model in rat crania; established; porcine chondrocytes were seeded in between
the defects were managed with SIS sponges, and the pres- each layer of cartilage, and the cartilage had a designed
ence of bone marrow-derived stem cells (BMSCs) resulted shape, achieved by pre-shaping the slices before in vivo
in significantly greater bone formation [528]. Similarly, implantation. This strategy for using nonporous matrices
a tissue-engineered periosteum fabricated from osteoin- appears promising and warrants future deeper investi-
duced rabbit BMSCs and porcine SIS was demonstrated gations [534]. Although many of the examples discussed
to be superior to a structural allograft in the repair of an herein describe animal sources, the principles may apply
allogenic segmental bone defect [529]. Overall, the use to human ECM-based biomaterials with variations depend-
of SIS-ECM may provide a new dimension to raw bioma- ing on both the processing methods and the tissue source
terials in tissue engineering; the combination of various of the ECM.
molecules and native aligned collagen fibers fosters the cel- Decellularized matrices may also be processed to form
lular processes necessary for the optimal function of the particulates that can be used either alone or in combi-
tissue and organs from which they are isolated. Although nation with other biomaterials to promote tissue repair,
the essence of an ECM obtained from an individual tis- and scaffolds may be prepared in many forms, including
sue can function as a bioscaffold for the same tissue type sheets, powders and hydrogels [535]. Loai et al. (2010) com-
because the compositions and structures of different tis- bined particles of porcine bladder acellular biomatrix with
sues may vary from one another, the modification of an SIS polymeric materials (e.g., HA) loaded with VEGF in the fab-
biomatrix-scaffold with other materials may expand its use rication of scaffolds with biological activity and tunable
in diverse tissue engineering applications [39]. properties for the generation of a vascularized bladder in
One scarcely explored decellularized ECM material is murine and porcine preclinical models [536]. Alternative
decellularized hyaline cartilage, which would be expected approaches have seeded cell populations onto scaffolds and
to offer a material rich in collagen type II, aggrecan and leveraged culture conditions to drive the differentiation of
native growth factors [530]. As a significant source of cells and the concomitant production of ECM. Along with
morbidity in lower back pain, treatments for facet joint fluid shear stresses, the inherent osteoinductive potential
osteoarthritis have generally focused on reduction of the of bone-like ECM has recently been used to synergistically
pain associated with this disease. However, recent efforts improve the osteodifferentiation of MSCs, which may have
have shifted to the creation of decellularized articular profound implications for bone tissue engineering uses
cartilage that is utilized as a replacement for diseased [537]. Moreover, by culturing MSCs within an electrospun,
facet cartilage [531]. When a decellularized porcine car- biodegradable PCL fiber mesh material in a flow perfusion
tilage bone construct was used as an ECM material for bioreactor, an osteogenic ECM construct with a temporal
130 F.-M. Chen, X. Liu / Progress in Polymer Science 53 (2016) 86–168

composition that may be useful for bone repair because of as a result of advances in various immunosuppressive drugs
its ability to mineralize and capabilities for future remod- to control rejection, this paradigm has fallen victim to
eling may be generated [538]. More recently, thanks to its own success given the growing disparity between the
advances in directing cell differentiation toward specific numbers of patients on the organ transplantation waiting
lineages, tissue-engineered constructs are also being used list and the donor organ pool available for such proce-
as a substrate for decellularization. This strategy opens a dures; this disparity leads to a substantial number of
new route for the production of large quantities of cus- patient deaths each year [26,284]. In light of the shortage
tomized and standardized ECM-based materials [491]. of donor organs, decellularized solid organs can be used
During regeneration, cells rely on environmental input to create potentially functional organ constructs in a short
for direction. ECM from an individual tissue therefore has a period of time, which may perform organ-specific func-
crucial role in aiding in skewing cell differentiation toward tions following recellularization, and surgical implantation
the specific cell phenotypes that normally reside in the and preliminary animal studies have delivered encourag-
native ECM of that tissue [539]. For example, myoblasts ing outcomes for this proof-of-concept [11,43,222,545].
seeded in skeletal muscle ECM extract displayed acceler- The use of bioreactor-based strategies for cell seeding
ated proliferation and differentiation, potentially because of organ-level bioscaffolds provides a blueprint for the
they were in an environment in which they would be de novo fabrication of complex tissues and could allow
observed endogenously, offering an optimal and natural the generation of customized organ grafts, irrespective of
space for their maturation [509]. Based on this concept, a their natural geometry [212,546–548]. Although culturing
decellularized arterial scaffold that includes various ECM cells within a decellularized organ prior to implantation
agents has been used to overcome limitations such as rejec- offers scientists a high degree of control over the fates of
tion, thrombosis, calcification, intimal hyperplasia, chronic the residing cells, in most cases, multiple cell types are
inflammation, infection and a lack of growth potential asso- required to recellularize a complex organ system (e.g., a
ciated with vessel scaffolds composed of synthetic poly- mixture of cardiomyocytes, endothelial cells and smooth
mers. In vitro, the endothelial cell matrix contains factors muscle cells is likely to be necessary for heart recellular-
that are observed to instruct MSC differentiation into both ization) [549]. The diverse range of different in vitro cell
endothelial cells and smooth muscle cells, and in turn, these culture parameters is very difficult to balance, and cultur-
factors are modified by MSC-secreted agents after the seed- ing more than one cell type in the same decellularized ECM
ing of autologous bone marrow onto the scaffold. By study- template presents its own unique pitfalls and challenges
ing the framework by which an endothelial cell matrix [475]. Recent studies suggest, however, that functional
coaxes MSC differentiation, a feedback system has been tissues or organs may also be obtained via the in vivo
uncovered, by which MSCs are able to alter the very matrix cell repopulation of an implanted decellularized matrix
cues acting upon them [540]. Similarly, a tissue-engineered based on cell recruitment from the neighboring tissue
heart valve was developed utilizing umbilical cord blood- and circulation, thus indicating significant clinical poten-
derived EPCs of human origin and decellularized valve scaf- tial, although work in this field thus far has only resulted
folds. The EPCs can differentiate into endothelial cells and in short-term functionality [276,277,486,504,543]. For a
form a functional endothelium atop the scaffolds, similar to deeper understanding of the interactions between the
that formed by normal endothelial cells on healthy human host cells and an implanted matrix to maximize recruiting
heart valves [541]. Currently, decellularized xenogenic efficacy and the propensity to spontaneous in body self-
heart valves have been used as a starter ECM material regeneration, more systematic studies of different types
for the tissue engineering of valve grafts, with promising of decellularized matrix systems both in vitro and in vivo
preclinical and clinical outcomes [520,542]. Interestingly, are required [550]. The complexity of the decellularization
when seeded with cardiac progenitor cells, a human technique and the length of the decellularization proto-
pericardium-derived biomatrix with well-defined archi- col are commonly proportional to the degree of biological
tecture and interconnected pores, which mimics the nat- and geometric conservation required in the resultant tis-
ural myocardial extracellular surroundings, holds tremen- sue with respect to its composition (BM components
dous promise in the treatment of ischemic heart diseases and bioactive molecules) and architecture (macrostructure
[539]. Based on these investigations, it is concluded that the and ultrastructure), especially for composite tissues and
application of native decellularized tissues as biomaterials whole organs [26,284,543,545]. Today, decellularization
has the potential to open a new avenue for guiding seeded has become a reliable methodology for the production of
cells toward their normal activity and function. complex 3D biomatrix scaffolds that maintains the intrin-
sic vascular network by removing residing cells from whole
4.3.3. Whole-organ decellularization organs while retaining the original architecture [11,222].
Techniques of tissue decellularization and their impacts This method is optimal for already existing biomaterials
on the properties of post-processed ECM materials were because the scaffolds do not only maintain the structure
reviewed in 2006 by Gilbert et al. (2006) [486] and recently of the original organ but also contain native biomolecules
by He and Callanan (2013) [543]. In recent years, the within their ECM that direct correct cellular function [486].
development of novel decellularization methodologies for For example, as a native and functional decellularized ECM
effective 3D composite tissue and whole-organ decel- biomaterial, ovine forestomach matrix has suitable bio-
lularization has attracted increasing attention [475,544]. physical properties for clinical uses in which the grafted
Although the treatment of end-stage organ failure using scaffold is under load [486,543]. Clinical materials such as
organ transplantation is now a well-established procedure surgical mesh products based on ECM are obtained from
 F.-M. Chen, X. Liu / Progress in Polymer Science 53 (2016) 86–168 131

diverse allogenic or xenogenic tissue sources, including such as Triton X-100, for the removal of cell nuclei from
the urinary bladder, dermis, small intestine, pericardium, dense tissues and organs while maintaining their native
mesothelium and heart valves, and from several differ- mechanics, but SDS is also more likely to induce ultra-
ent species [11,76,222]. In 2008, the main left bronchus structure disruption in and growth factor elimination from
in a young man was replaced with a decellularized cadav- the resulting ECM [531]. Whether for a simple tissue or
eric trachea seeded with the patient’s own cells [515]. a complex whole organ, a standard protocol for tissue
Since then, similar strategies have been clinically utilized to decellularization offers many benefits that aid in advanc-
replace organs in patients with a tracheal/bronchial tumor ing biomaterials of human origin for tissue engineering.
or congenital tracheal stenosis [516,551], and many groups First, the standardization of post-processing ECM prod-
have investigated the production of decellularized organs ucts enables the possibility of a congruous comparison
for the replacement of the heart [488,520], liver [517] and of various ECM scaffolds and allows both researchers and
lung [512,513] in small and large animals. Thus far, ECM manufacturers to appraise the efficacy and effectiveness of
and cells procured from the above-mentioned tissues and an established protocol when developing new decellular-
organs have been used to study the potential advantage of ization methodologies or characterizing a particular ECM
tissue specificity in maintaining selected cell functions and product derived from a particular decellularized tissue or
phenotypes, and these ECMs have been demonstrated to organ. Second, standardized decellularization minimizes
affect cell chemotaxis and mitogenesis, to guide cell differ- the existence of and variation in residual cell and DNA
entiation and to induce constructive host tissue remodeling material in a post-processing product, thereby potentially
responses. The 3D ultrastructure, composition and sur- eliminating adverse cell and host responses to ECM scaf-
face topology of the organ ECM all likely contribute to folds and facilitating the comparison and interpretation of
these effects [284,476,552]. In contrast, the remaining in vitro and in vivo findings [11,222,476]. Thus, the stan-
residual cellular material within the biological scaffold dardization of the current decellularization protocol and
materials attenuates or fully negates the constructive tis- its post-processing product are prerequisites to expanding
sue remodeling advantages in vivo. Thus, tissue-processing the clinical application of this technique and to promoting
strategies and decellularization techniques appear to be the rapid and effective development of more biomaterials
critical determinants of the clinical success of organ ECM of human origin for tissue engineering and regenerative
products [274]. Although the general focus on structural medicine [274].
relevance in the ECM research field is of fundamental Overall, whole-organ decellularization provides a
importance, there is now a strong emphasis on several scaffolding platform on which to establish a strong
other new areas. In particular, the cell–matrix interface translational pipeline for future organ tissue engineering,
offers a pivotal signaling nexus that controls all aspects of although organ-level decellularized matrices have not yet
cell functions [276]. reached the stage of clinical adoption [545]. In principle,
The complete decellularization of most, if not all, organs the integrity of the post-processed material following
commonly necessitates a synergism of physical, chem- decellularization supports the reproducible, predictable
ical and enzymatic treatments [274]. It is necessary to and effective clinical application of the ECM product [544].
select the mildest approach that can result in an acellu- From a scientific standpoint, the constituents remaining
lar scaffold without damage to the functional constituents in the ECM are instrumental to possibly identifying the
and structural geometry of the ECM [43,543]. A typi- mechanisms by which specific biomolecules and their
cal progressive protocol would begin with treatment in organization elicit tissue regeneration processes [26].
a hypertonic or hypotonic solution, followed by a mild However, in the production of readily available, patient-
zwitterionic or nonionic detergent. If needed, enzymatic specific ECM equivalents, the typical procedures applied
treatment with trypsin, either alone or in combination with for the efficient removal of cellular and nuclear materials
a chelating agent such as ethylenediaminetetraacetic acid result in a simultaneous and at least partial impairment of
(EDTA) and ethylene glycol tetraacetic acid (EGTA), can ECM integrity and its constituents (e.g., a decline in soluble
be used as an adjuvant prior to the detergent treatment type II collagen and GAG content), especially in whole-
to aid in disrupting the bonds between cell membranes organ template generation [274]. Although those bioactive
and the ECM. It is likely that EDTA or EGTA contributes materials have provided substantial evidence supporting
to cell dissociation from ECM proteins and to subtle dis- recellularization and cell propagation, the cues from the
ruptions in protein–protein interactions by sequestering “matrix footprint” that are critical in cell adhesion and
metal ion mechanisms. Finally, an ionic detergent such viability remain unidentified. Additionally, the materials’
as sodium dodecyl sulfate (SDS), Triton X-100 or deoxy- allogenic nature has raised clinically relevant concerns,
cholate may be applied in the decellularization protocol particularly with regard to both their safety and ethical
if the prior treatments are still inadequate to remove all issues, suggesting that host responses to residual cell debris
of the cellular and nuclear residues from the tissue or and matrix constituents should be carefully characterized
organ of interest [43,475,486]. An objective evaluation of before therapies based on these matrices can be used in
the effects of the agents that are used to yield a particu- the clinic [475]. Upon application, critical challenges and
lar ECM structure and composition can assist in the design hurdles remain associated with the following: sterilization
of an optimal decellularization protocol [489]. For tissue without compromising the protein framework, selection
delipidation, ionic detergents such as deoxycholate typi- of the optimal cell source required to restore tissue
cally appear to be less effective than nonionic detergents. functionality, acquisition and growth of patient-specific
SDS appears to be more effective than other detergents, cells on decellularized materials without contamination,
132 F.-M. Chen, X. Liu / Progress in Polymer Science 53 (2016) 86–168

reintroduction of these cells into their proper location (e.g., crest, these cells can differentiate into multiple types of
denuded vascular structures in a whole-organ scaffold) tissue-forming cells, such as chondrocytes and osteoblasts.
through seeding methods or induced migration, cell distri- However, the in vitro expansion of MSCs encounters vari-
bution or propagation following reseeding and subsequent ous problems, such as problems regarding the sterility of
organ revitalization, achievement of the functional prop- the cell culture; the risk associated with the use of fetal
erties of organs, characterization of regenerated organs bovine serum (FBS) (this risk may be bypassed via the use
and prevention of thrombosis [266,521,552]. Even when of PL); and the length of time required for cell cultivation,
an organ ECM material is correctly recellularized with as cell transplantation normally requires a time-delayed
sufficient cell numbers, it is difficult to predict whether second operation. The need for advanced laboratory and
the engineered organ, as a defective transplant, is going technical support, as well as regulatory issues and high
to work with unpredictable fate or as a less-than-perfect costs, are also major hurdles that need to be addressed.
transplant but with “self-repairing” and remodeling A possible alternative could be the use of a perioperative
potential that will eventually restore the impaired bodily stem cell concentrate in a single-step procedure using den-
function [11,222]. Finally, in addition to the scientific and sity gradient centrifugation of autologous bone marrow
technical bases of decellularized matrices, the demand [555]. Indeed, bone marrow has been directly applied to
for clinical-grade bioreactors, the identification of the induce bone formation in skeletal defects and non-unions.
appropriate population of patients, regulatory issues and The cells contained in the bone marrow may participate
the clinical logistics of the transplantation approach should in the wound healing cascade, serving as building blocks
be considered to facilitate the clinical translation of these for or directing regeneration via the secretion of growth or
matrices for genuine real-world applications in the future cellular signals instead of, or in addition to, directly partici-
[476]. From a therapeutic prospective, regulation of the pating in regrowth of the tissue [553]. The main advantage
production and management of donor tissues and organs, of using bone marrow is that this technique can be accom-
their effectiveness and safety evaluations, quality control, plished percutaneously in routine clinical practice, free of
application protocols and guidelines, standardization and nearly any patient morbidity. Technically, the centrifuga-
ethics are all critical issues that must be carefully taken tion of aspirated bone marrow at 400 times gravity for
into account in the development and commercialization 10 min may completely separate the marrow cells from
of decellularized ECM templates of human origin as “off- the plasma to decrease the volume of material injected,
the-shelf” substitutes or, following recellularization and resulting in the marrow product termed BMC. It has been
revitalization, as fully functional organs similar to donor demonstrated that the osteogenic potential of the cells can
organs that can be transplanted into patients suffering be well preserved in a well-prepared BMC product [558].
from end-stage organ failure (Fig. 15) [274]. BMC contains a mixture of cell populations, such as
MSCs and HSCs, as well as other substances, such as
5. Preparations containing non-expanded platelets and cytokines; all can facilitate the regenera-
autologous stromal cells tion of numerous tissues as part of reconstructive surgery
and tissue engineering strategies [176]. BMC represents a
Recently, autologous preparations, such as bone mar- new frontier in cell-based therapeutics for an unexpectedly
row concentrate (BMC) and the stromal vascular fraction wide variety of human diseases, including those involv-
(SVF), have been used in regenerative procedures because ing autoimmunity, inflammation and tissue damage, due
they contain uncultured stromal cells that may partic- to the multi-differentiation and the immunomodulatory
ipate in the wound healing cascade during therapeutic and anti-inflammatory properties of BMC preparations. In
regeneration (Fig. 16). As a new concept in using bioma- particular, an intraoperative, one-step procedure for the
terials of human origin, the use of non-expanded BMCs clinical application of progenitor cells from bone marrow
and SVFs offers interesting alternatives to therapeutic cell has shown promising results in musculoskeletal tissues
populations, carefully circumventing translational barriers [559] (Fig. 16). Following bone marrow aspiration, BMC is
regarding cell expansion and delivery. Although concerted easily prepared using density gradient centrifugation and
efforts have been and are still being made in the identifica- is available for a same-day procedure with minimal manip-
tion and delivery of ex vivo-expanded cell populations for ulation of the cells, thus complying with FDA restrictions.
tissue engineering, these cell populations’ economic and Future advances in this field will be the development of an
clinical feasibility continues to present formidable chal- easy procedure for harvesting (e.g., by vacuum aspiration)
lenges. By providing an overview of BMC and the SVF, each from the iliac crest to facilitate the availability of autolo-
an important but currently overlooked aspect of patient- gous bone marrow and the establishment of a standardized
derived biomaterials, we hope to present a call for action dose of stromal cells and mononuclear cells in a well-
to develop these therapies for routine clinical use. For prepared BMC product [560]. For many years, it has been
more detailed information, the reader is directed to many recognized that BMC may possess high potency in cartilage
focused reviews published elsewhere, several of which are and osseous defect healing when used in combination with
cited in this section [553–557]. grafting materials and occasionally PRP [561–565]. How-
ever, there are no published randomized controlled trials
5.1. Bone marrow concentrate on the efficacy of autologous BMC intra-articular injections
performed as a same-day, in-office procedure for treat-
MSCs represent a promising cell source for osteochon- ing patients with cartilage or bone disease [566]. In any
dral regeneration because once obtained, e.g., from the iliac case, the exponential rate of progress in biotechnology has
 F.-M. Chen, X. Liu / Progress in Polymer Science 53 (2016) 86–168 133

Fig. 15. Schematic representation of the distinct and crucial challenges related to the production and application of extracellular matrix (ECM)-based
biomaterials in regenerative medicine and tissue engineering.

Fig. 16. Schematic of the preparation of human-derived bone marrow concentrate (BMC) and a stromal vascular fraction (SVF) from non-expanded cell
populations for cell therapy and tissue engineering applications (schematic is not to scale). BMC is a rich source of the regenerative cells needed for bone
formation and angiogenesis, including mesenchymal stem cells (MSCs, which convert to osteoblasts in support of new bone formation), hematopoietic stem
cells (HSCs, which orchestrate bone formation) and endothelial progenitor cells (EPCs, which stimulate angiogenesis). In addition, BMC includes platelets,
which mediate cell-to-cell adhesion via the release of multiple growth factors; lymphocytes, which support the migration and proliferation of EPCs; and
granulocytes, which release vascular endothelial growth factors (VEGFs) in support of angiogenesis. The use of BMC therefore offers the potential to bridge
the gap between stem cells and signaling factors in a traditional tissue engineering triad. The SVF is the product of a lipoaspirate, which is obtained from
liposuction of excess adipose tissue. The SVF contains a large population of mature cells, progenitors and stem cells. Adipose-derived stem cells (ASCs)
share many similarities with bone marrow-derived stem cells, including self-renewal and multilineage differentiation capacity.
134 F.-M. Chen, X. Liu / Progress in Polymer Science 53 (2016) 86–168

allowed for the immediate application of myriad novel translational obstacles related to ex vivo cell cultivation
therapies before clear evidence of benefit from random- and transplantation, allowing the development of one-step
ized clinical trials. In addition to its fundamental science, surgical procedures due to the high frequency and abun-
the ease of on-site preparation of bone marrow-derived dant availability of SVF cells [574]. Of note, these cells can
cells within the operating theater in the routine clinical set- be added to transplants of human origin, such as puri-
ting, minimizing the specific risk of contamination and cell fied adipose tissue or bone grafts, or to a material carrier
changes during ex vivo cell manipulation, suggests the great such as an ECM scaffold or an alloplastic material, stimulat-
therapeutic potential of BMC for autologous cell-based ing long-term cell retention and subsequent colonization
therapy for bone or other tissue repair and regeneration and providing a cure for various tissue injuries/damages.
[555,559,567]. Based on this concept, non-expanded SVF cells (freshly
procured without cell culture) were used to form new
5.2. Stromal vascular fraction adipose tissue in vitro using a porous collagen/gelatin
sponge as a scaffold [575]. Recent evidence also suggests
Human adipose tissue is becoming an increasing focus that SVF transplantation combined with a chitosan con-
of tissue engineering due to the abundance of its tissue duit may be considered as a readily available stromal cell
source, its relatively easy retrieval and the intrinsic bio- source for ameliorating the functional recovery of the sci-
logical properties of MSCs residing in its stroma [568,569]. atic nerve [576]. Furthermore, an intratendinous injection
Beyond its use as a fat grafting material, knowledge of of uncultured adipose-derived SVF cells results in improved
the cell biology, isolation/manipulation and differentia- structural and mechanical properties in tendon repairs and
tion, and regenerative and immunomodulatory properties could be an effective modality for the treatment of tendon
of adipose-derived cells has increasingly advanced in the injury [577]. In addition, the administration of SVF cells has
past 10 years. In particular, concerted research efforts have been shown to ameliorate chronic experimental autoim-
yielded a wealth of basic science-based and preclinical mune encephalomyelitis in animal models, demonstrating
evidence regarding the properties of both heterogeneous the potential immunomodulatory, anti-inflammatory and
SVF cells and more homogeneous adipose-derived stem regenerative effects of non-expanded SVF cells. Interest-
cells (ASCs) derived from the patient’s own tissue [568]. ingly, it has also been revealed that SVF cells effectively
There is also mounting evidence demonstrating that the inhibit disease severity and are significantly more effec-
human SVF compartment contains multipotent MSCs that tive than culture-expanded ASCs [578]. Similarly, when
may differentiate into smooth and cardiac muscle cells, osteochondral defects in medial condyles and trochlear
osteocytes, neural cells, adipocytes and chondrocyte pre- grooves in the knees of goats were treated with a freshly
cursors and that are capable of generating tube-like cellular procured SVF or cultured ASCs, the SVF cells tended to
structures in 3D culture in vitro [569–571]. In addition perform better on all parameters, including the forma-
to being a multipotent cell population amenable to soft- tion of type II collagen and hyaline-like cartilage and the
and hard-tissue repair, the human adipose SVF cell popu- elastic modulus [579]. These findings suggest that freshly
lation represents a complex mixture of HSCs, endothelial procured, heterogeneous SVF cells, including a mixture
cells, pericytes, T regulatory cells and anti-inflammatory of multiple cell types with both immunomodulatory and
M2 macrophages, indicating that this population is a useful regenerative properties, can dictate a more effective cure
source of cells for treating ischemic insults and autoim- upon application compared with culture-expanded and
mune/inflammatory disease [571]. However, the reason relatively homogeneous ASCs. More often than expected,
why the adipose-tissue SVF represents a hot topic in stem bone tissue engineering based on the SVF has emerged as
cell research is that this non-expanded tissue compart- a promising approach to manage the structure and func-
ment offers a rich source of multipotent ASCs (Fig. 16). tion of bone compromised by disease or injury [580,581].
Prior to cell transplantation, ASCs are readily accessible in Indeed, as a practical, promising candidate for cell-based
human autologous fat tissue and have significant potential therapy, the SVF has also attracted increasing attention for
for tissue repair in scenarios of heart failure, myocardial application in clinical reconstructive surgery [573].
infarction, hind limb ischemia and inflammation [572]. Over the past several years, considerable advances have
Indeed, ASCs display comparatively stable growth and pro- been made in the science and technology related to the
liferation kinetics and can differentiate into chondrogenic, use of SVF cells in tissue engineering and regenerative
osteogenic, adipogenic, myogenic or neurogenic lineages medicine. Of note, the clinical utility of cell-based thera-
when cultivated under lineage-specific conditions. More- peutics for tissue repair and regeneration has encountered
over, ASCs have been proven to induce substantial tissue numerous translational barriers [582]. In light of these
formation for several biomedical uses. In this regard, cer- barriers, an uncultured SVF in possession of a pool of
tain clinical trials on the utility of human ASCs in bone regenerative cells may help to avoid an additional culture
reconstruction have been concluded and have indicated period, to reduce the risk of extensive cell contamina-
efficacy [573]. tion and to increase cost-effectiveness [583]. Recent data
Human SVF cells can be easily procured by centrifuga- have revealed promising outcomes when a freshly pro-
tion of collagenase-digested adipose tissue of human origin cured SVF was used as a non-expanded stem cell source
and are then ready for biomedical application, without for advanced cartilage therapy [584] and adipose forma-
the need for cell culture [556,557]. As such, non-expanded tion [568]. Furthermore, methods for the cryopreservation
SVFs provide an alternative to cell products in regenera- of the SVF in a serum-free freezing medium have been
tive medicine. This alternative may bypass many of the tested, and the findings indicated that the cell viability
 F.-M. Chen, X. Liu / Progress in Polymer Science 53 (2016) 86–168 135

and differentiability of the SVF can be preserved [585]. harvested from an individual patient, and a personalized
Overall, the application of autologous SVFs in cell-based formulation rich in growth factors can be obtained by
therapy not only is easy and effective but also facilitates simply controlling the degree of coagulation of the samples
their translation into human healthcare [574]. However, and the elaboration protocol designed for production [591]
more investigations are required to identify whether the (Fig. 17). Here, an overview of these products’ roles and
techniques described in recent studies will still work on the implications in future tissue engineering is intended to
larger scale of tissue defect models and whether autologous shed light on the various prospects of these formulations
SVF transplants can maintain their dimensions and shape and on clinical insights into regenerative medicine.
over time at defect sites in humans. Additionally, the degree
or longevity of engraftment of ASCs following SVF use has
not been measured by external investigations independent 6.1. Platelet-rich plasma
of commercial organizations. It is also not clear whether
the reported positive outcome of SVF cell administra- PRP is a platelet concentrate in a small volume of
tion was the result of local immunosuppression, paracrine plasma that is typically developed from autologous blood
expression of anti-apoptotic/angiogenic factors, adipose [589,590]. Ranging from two- to several-fold above phys-
cell differentiation, or a combination of these or other iological levels, the platelet count in a PRP product varies
unidentified effects [586]. Therefore, the stage has been according to the preparation protocol. Upon activation, the
set for the clinical translation of SVF cells from the bench platelets contained within a PRP product release the con-
to the bedside, but this process will involve “developmen- tents of their granules, which consist of a complex array of
tal” steps that fall outside of the traditional paradigm of growth factors, cytokines and chemokines, such as PDGFs
the mechanism-based and hypothesis-driven experimen- (i.e., PDGF-␣␣, PDGF-␤␤ and PDGF-␣␤), TGF-␤ (e.g., TGF-
tal design common in the stem cell literature [587]. It ␤1 and TGF-␤2), insulin-like growth factor (IGF), VEGF,
is likely that several, if not all, of the above-mentioned platelet factor-4 (PF-4), platelet-derived angiogenesis fac-
questions must be addressed before clinicians can use tor (PDAF), IL-1, epidermal growth factor (EGF), epithelial
SVF products, whether harvested from the patient or pro- cell growth factor (ECGF), platelet-derived endothelial
vided by biotechnology companies. It should be noted that growth factor (PDEGF), osteonectin, osteocalcin, fibrino-
although a variety of commercially available systems may gen, fibronectin, vitronectin and thrombospondin-1 [592],
yield measurable amounts of SVF cells in a clinical out- many of which have been demonstrated to participate
patient surgical environment, significant variability exists in the wound healing and tissue regeneration processes
in the number, the identity and the safety profiles of the [593,594]. The interactions between these growth factors
recovered viable cells. The lack of preclinical and clini- and their surface receptors on responsive cells activate
cal data reported in peer-reviewed manuscripts that can the intracellular signaling pathways that enhance tissue
be used to objectively assess the overall performance of regenerative processes, such as cell proliferation and dif-
SVF products suggests that side-by-side clinical trials will ferentiation, matrix deposition, collagen synthesis and
be required to establish the relevance of these variations osteoid production [595]. Thus, the potential to deliver
[588]. these growth factors and matrix elements to a site of
injury is the basis for using PRP in regenerative medicine
6. Formulations enriched with endogenous growth (Fig. 17). In addition to its major roles in hemostasis and
factors cell fate commitment, PRP is involved in the inflamma-
tory and immunological aspects of wound healing. Platelets
In addition to the application of a human graft and its play a direct role in the inflammatory response through
ECM, the concept that biomaterials of human origin may the production and release of a vast wealth of inflamma-
be useful for regenerative therapy has been supported by tory mediators, including various cytokines, such as IL-1␤,
numerous pioneering studies that show that tissue regen- TGF-␤ and CD40L, as well as numerous chemokines, such
eration can be accelerated using formulations enriched as CXCL1, CXCL4, CXCL5, CXCL7, CXCL8, CXCL12, CCL2,
with growth factors from a patient’s blood, such as platelet- CCL3, CCL5 and CXCL4L1. Moreover, platelets express a
rich plasma (PRP) and platelet-rich fibrin (PRF) [589,590]. number of chemokine receptors, and particularly CCR1,
Although a vast wealth of recombinant proteins and CCR3, CCR4 and CXCR4 [596]. The collection of such
growth factors are now commercially available for diverse bioactive molecules in well-prepared PRP plays a syner-
applications, several of which have also been approved gistic role in the fundamental processes of tissue repair,
for testing and use in humans, unfortunately, their clinical including inflammation, angiogenesis, cell migration and
implementation hitherto has been disappointing. One piv- metabolism. In pathological conditions such as osteoarthri-
otal aspect of the development of preparations enriched tis, PRP exhibits anti-inflammatory properties through its
with endogenous growth factors has been transforming impacts on the canonical nuclear factor-␬B (NF-␬B) signal-
human platelets, which are reservoirs of a spectrum of ing pathway in multiple cell types, including synoviocytes,
autologous growth factors, into therapeutic preparations macrophages and chondrocytes. Analyzing the effect that
that can be easily handled, evaluated and adopted by each biological factor can have on tissue-specific cells
researchers and surgeons who practice regenerative and understanding PRP in molecular terms could help us
medicine [44]. In particular, PRP and PRF have undergone to exploit its therapeutic potential and could aid in the
clinical translation from bench to bedside in an easy, development of novel treatments and tissue engineering
simple and predictable way; blood samples are generally approaches [42,597].
136 F.-M. Chen, X. Liu / Progress in Polymer Science 53 (2016) 86–168

Fig. 17. Schematic of the preparation of platelet-rich plasma (PRP), platelet lysate (PL) and platelet-rich fibrin (PRF) for tissue engineering applications
(schematic is not to scale). PRP and PL may be used as autologous alternatives to recombinant human growth factors in a traditional tissue engineering
triad, whereas PRF offers the potential to bridge the gap between biomaterials and signals.

An attractive advantage of PRP lies in its easy and potentially toxic materials, such as bovine thrombin, is
rapid acquisition from a patient’s own blood, thus theo- avoided [602]. In addition, the calcium results in reduced
retically circumventing the risk of disease transmission or burst protein release compared with thrombin, leading to
an immunogenic reaction because the receptor and donor a more sustained delivery of growth factors [603]. Further-
are the same [44]. Due to its autologous origin and low more, leukocytes can be removed from PRGF to eliminate
cost, PRP has significant advantages over other therapies the pro-inflammatory impacts of the proteases contained
including recombinant growth factors. Although appeal- in white blood cells, such as acid hydrolases and metal-
ing, the autologous nature of PRP introduces variability loproteases, which may provoke tissue-destroying effects
into plasma preparations, creating challenges for both the [604]. Most importantly, it is possible to regulate the
researcher and the clinician [590]. Differences in patients platelet concentration and, therefore, the dose of endoge-
at the time of the blood draw result in plasma preparations nous growth factors within a target product by adjusting
that vary both within and between patients. This variability the processing parameters, among other variables. Finally,
is compounded by the multitude of protocols and devices predictable platelet dosing also allows final control of the
available for procuring PRP [598]. Criteria for PRP dosing molecule/protein ratio because PRP contains a mixture
and for tailoring preparations to each pathological situation of growth factors and bioactive agents involved in both
have yet to be established. The common need for bovine plasma and platelets [605].
plasma-derived thrombin, which triggers platelet activa- It has long been well demonstrated that platelet prepa-
tion through the thrombin receptor, present on the platelet rations promote tissue regeneration by inducing cell
surface, represents another limitation of current protocols migration to and proliferation and differentiation in the
for obtaining a PRP product [599]. Of note, the application of area of an injury, which are essential processes for regen-
thrombin is associated with potential immunological reac- eration [590]. The ability of PRP preparations that mimic
tions, and approximately 30% of patients exposed to bovine a native ECM milieu to recruit host progenitor/stem cells
thrombin may develop cross-reacting antibodies to certain to enhance endogenous regenerative processes has also
human plasma proteins. These antibodies have the poten- been proposed for further investigation [55]. In one study,
tial to result in life-threatening coagulopathies [600,601]. when used as an adjuvant agent in arthroscopic microfrac-
An appealing strategy to circumvent several of the chal- ture surgery for the management of osteochondral lesions
lenges is the use of an autologous plasma product obtained of the talus, PRP resulted in an improved functional sta-
from an individual patient by plasmapheresis. This prod- tus score in the medium term [606]. Additionally, when
uct, which is enriched in platelets, is termed “plasma rich used in lumbar spine surgery for patients with postero-
in growth factor (PRGF)”, and calcium chloride is used to lateral arthrodesis, a cancellous bone substitute soaked in
activate the platelets [591]. Because calcium chloride is PRP resulted in an enhanced fusion rate during the first
adopted to activate the coagulation cascade, the use of 6 months after surgery and increased bone density, thus
 F.-M. Chen, X. Liu / Progress in Polymer Science 53 (2016) 86–168 137

joining osteoinductive and osteoconductive effects [607]. biopolymer material for scaffolding purposes in tissue
Recently, it has also been shown that PRP derived from engineering. To this end, fibrin is a prime material of human
bone marrow aspirate promotes new cementum formation origin for producing auto-assembled 3D scaffolds for rapid
in rat periodontal fenestration defect models [608] and that clinical translation [611–614].
a single autologous PRP injection combined with a reha- Along with the ECM assemblies (e.g., collagen and GAGs)
bilitation program is an effective treatment for hamstring noted in Section 4.2, fibrous-type proteins present in blood
muscle injuries, one of the most common types of injury (e.g., fibrinogen and fibronectin) are currently considered
affecting athletes [609]. Although a growing body of evi- the ideal components to prepare bioscaffolds for tissue
dence supports the use of PRP as a clinical treatment for engineering. As a unique group of blood-derived products
bone, muscle, tendon, cartilage and periodontal injury in including platelet- and cell-derived active components,
reconstructive surgery, with short-term clinical benefits, fibrin polymers may be formed through self-assembly or
most of the studies published to date are of poor qual- following enzymatic activation (Fig. 18). The normal func-
ity and at high risk of bias, and indeed, improvements in tion of protein bioscaffolds in wound healing is to prevent
healing and clinical outcomes have not been universally the loss of body fluid and to provide stability to bio-
reported [44,574,590,591]. One reason for this poor qual- logical structures. In regenerative medicine, polymerized
ity may be that each PRP product varies from the others fibers elicit multiple physiological responses that ensure
and contains different cocktails of growth factors and reg- pivotal functions to provide mechanical and flexible sup-
ulatory molecules. Typically, the volume of whole blood port, as well as tensile strength, and hence reconstruct
collected, the efficacy of platelet recovery, the final concen- injured or pathologically abnormal tissues [42]. As a typical
tration of platelets in the plasma, the absence or presence bioscaffold composed of platelet concentrates, PRF con-
of white blood cells and the addition of xenogenic thrombin sists of a fibrin matrix polymerized in a tetra molecular
to activate the platelets or calcium chloride to induce fib- structure that contains all the beneficial constituents of
rin formation can all influence the character and potential a blood sample that are favorable to tissue regeneration
efficacy of the resulting PRP formulation [610]. It should be [42,614]. Because it features all the crucial parameters per-
noted that the lack of standardized and optimized proto- mitting optimal wound healing, the clinical experience
cols might partly account for the outcome variability across confirms that PRF can be considered a healing biomaterial.
patient populations receiving PRP-based therapies. There- PRF already has a list of clinical uses, and considering its
fore, it is still impossible to compare data from different advantages over traditionally prepared PRP, numerous bio-
investigations to draw a general conclusion. Further high- engineering applications can also be imagined, increasing
quality comparative studies with longer follow-up periods its popularity [615–617].
are needed to ascertain whether PRP is beneficial, either Prepared at the bedside from a small volume of the
alone or as an adjunct to other interventions during surgical patient’s own blood, PRF, consisting of a dense crosslinked
procedures. fibrin lattice itself, contains an abundant variety of signals
and growth factors that facilitate tissue repair [614]. Sim-
6.2. Platelet-rich fibrin ply by modulating the platelet activation process, PRGF
and PRF technologies have been established to yield a 3D
Although PRP use offers some efficacy in certain types fibrin material for the controlled delivery of growth fac-
of acute and chronic wounds, plasma-rich scaffold-like bio- tors [605]. Because autologous fibrinogen can be acquired
materials, such as fibrin and PRF, might be the best choice if from plasma, PRF avoids the risk of a foreign-body response
the aim is to retain growth factors from an excessive initial upon application [612]. To this end, scaffold-like PRF rep-
burst release upon implantation and to maintain the con- resents a new generation of formulations that have been
centration of these therapeutic agents at a site of injury for demonstrated to possess several benefits over traditional
a desired period (Fig. 17) [611]. As a major blood compo- PRP, such as their ease of production/application and
nent responsible for hemostasis, fibrin has a long history their lack of a need for biochemical handling of blood
of use for hemorrhage control. Thanks to fiber branching, [611]. Indeed, fibrin has been clinically employed as a safe
fibrin fibers are able to auto-assemble into a mesh scaffold and versatile biomaterial for stimulating and accelerat-
without the help of other proteins. However, this help is ing wound healing and tissue regeneration in numerous
required if the purpose is to generate an intricate network medical conditions [593,611,617]. Interestingly, recent evi-
based on collagen fibers [612]. At a damaged site in need of dence suggests that a 3D fibrin scaffold may mimic the
tissue regeneration, fibrin is a provisional matrix generated main components of the hematopoietic niche, thus meet-
by fibrinogen polymerization in the presence of thrombin, ing the optimal requirements of clinical protocols for cord
which is not a regular component of the ECM [613]. Because blood–hematopoietic stem cell expansion and transplan-
different bioactive molecules are enclosed within the fib- tation [618].
rin mesh, it offers a perfect tool to seal surgical defects PRF scaffolds containing a 3D macroscopic network are
while promoting the full re-epithelialization of soft tis- now easily generated from human blood by biotechno-
sues [44,591,604]. During the wound healing process, the logical methods without the use of exogenous thrombin
mesh is progressively replaced by structurally stabilized and may be applied in diverse situations to aid in tissue
ECM, and finally, new tissue is formed [613]. Thus, fibrin regeneration and to facilitate wound healing [619]. It is
plays a pivotal role in the physiological wound healing cas- recognized that the inherent properties of a prefabricated
cade. Recently, fibrin has also been extensively applied, PRF can determine the pattern of growth factor release,
either alone or in combination with other materials, as a including the establishment of a provisionally stable fibrin
138 F.-M. Chen, X. Liu / Progress in Polymer Science 53 (2016) 86–168

Fig. 18. Representative scanning electron microscopy (SEM) images of a well-prepared platelet-rich fibrin (PRF) generated by fibrinogen polymerization.
(A) A scaffold-like PRF derived from human blood; (B) a magnified view of a portion of (A) showing a dense crosslinked fibrin lattice; (C) a magnified view
of a portion of (B) showing plenty of platelets (white arrows) enclosed within the fibrin mesh structures; (D) a magnified view of a portion of (C) showing
the interconnected three-dimensional (3D) network of the PRF scaffold, which may facilitate cell penetration and accommodation.

matrix; the platelet concentration in the PRF; the concen- release of various bioactive agents important for tissue
tration and type of the platelet activator used; the rate and repair while retaining the agents’ bioactivity against prote-
amount of individual growth factors released from the acti- olytic degradation [42,617]. Recently, evidence has shown
vated platelets; the fibrin network and degradation rate; that well-prepared PRF can gradually release a pool of
and the specifications of the anatomical site targeted for endogenous growth factors for a period lasting more than
implantation, such as the fluid turnover rate [620]. During 20 days, implying a potentially durable effect on wound
the formation of a temporary matrix, several parameters repair [616]. Thus, autogenetic PRF may serve as a space-
can be modified to alter PRF’s mechanical properties, struc- filling matrix for implantable fillings or as a regenerative
ture and degradation. For instance, the ionic strength of material. The best characteristics of a PRF matrix for the
or thrombin concentration in the solution may affect the clinical demands of tissue engineering biomaterials include
crosslinking time of PRF, which in turn determines its its autologous nature, low cost, good manageability, func-
fiber diameter and pore size [599]. Additionally, the tensile tional flexibility and absence of any allergic reaction or
strength and shear modulus can be optimized by varying other adverse effects in the patient [612]. In this respect,
the concentration of two components, calcium and fibrino- PRF has been validated as an entirely autologous, injectable
gen, respectively. The inclusion of antifibrinolytic agents in cell delivery system that overcomes the histocompatibil-
formulations of fibrin glue may delay or slow fibrinolysis, ity issues related to synthetic scaffolds while ensuring a
a process leading to the destruction of PRF gel by impair- stable 3D matrix for residing cells both in vivo and ex
ing blood clot formation and stability [617]. It cannot be vivo [65,615]. Due to the non-cytotoxic, biocompatible
ignored that, in addition to variation in PRF products, the and non-immunogenic nature of these fibrin scaffolds, the
quantity of PRF produced from an autologous blood sam- combination of PRF with exogenously manipulated cells
ple is quite low, and only a small volume can be utilized. and recombinant human growth factors has opened new
An optimized and reproducible protocol for the prepara- avenues for various biomedical applications, especially for
tion of PRF with regard to its growth factor content and its bone tissue engineering and the regeneration of cartilage
structure for cell accommodation remains undefined. and periodontal tissues [56,104,621].
In recent years, fibrin-based scaffolds have paved the When used alone, PRF, which is composed of cellular
way to the regeneration of a large variety of human tis- and fibrillar components, not only acts as a vehicle for
sues, such as bone, cartilage, adipose tissue, cardiac tissue, proteins and growth factors but also permits cellular
ocular tissue, nervous tissue, liver, skin, ligaments and penetration and subsequent integration of newly formed
tendons [614]. In tissue engineering and clinical applica- tissue into the native tissue [605]. The versatility of PRF can
tions, PRF has been demonstrated to offer the sustained be broadened when the patient’s autologous tissues are
 F.-M. Chen, X. Liu / Progress in Polymer Science 53 (2016) 86–168 139

transferred along with PRF for grafting or when PRF is used the same manufacturer, coupled with concerns relating to
in association with other naturally derived materials, such its biosafety and clinical availability. Aside from ethical
as gelatin, alginate, collagen or CS, for scaffold modification considerations, the most important concern is the risk of
[241,622–626]. PRF combined with additional natural or zoonoses due to the transmission of bacteria, viruses and/or
synthetic materials can yield scaffolds that offer tighter prions and immunological reactions due to its xenogenic
control over their cargo biodistribution and pharmacoki- origin. Therefore, there is an increasing need to search for
netics [44,605]. For instance, the growth factors released xeno-free agents to replace FBS [619,620]. Unfortunately,
from PRF following its activation can be immobilized by alternatives to FBS that yield competitive results for both
the addition of an acidic gelatin material with an isoelectric cell isolation and cell expansion have not been identified
point of 5.0; the physicochemical interplay substantially thus far [628]. In addition, a chemically defined xeno-free
alters the growth factors’ kinetic profile because release nutritional supplement that is as good as FBS would per-
depends on hydrogel degradation [622]. Similar strategies haps be more expensive, which would ultimately hamper
have been reported using CS [627], alginate [623], collagen production on a large scale [629]. Human blood prepara-
[624] or PCL-tricalcium phosphate (TCP) composites [625] tions, such as autologous and allogenic human sera; PRP;
as the underlying biomaterials. Interestingly, via a heparin- cord blood serum; and human platelet derivatives, includ-
binding delivery system, the incorporation of exogenous ing platelet lysate (PL) and platelet-released factors, have
growth factors or other bioactive molecules into its mesh been efficiently implemented into MSC clinical manufac-
structures may additionally improve the functionality of turing and increasingly introduced into stem cell therapy
PRF as a scaffolding material [438]. To this end, recent tech- as a compelling substitute for FBS [630–632].
nologies, such as magnetically influenced self-assembly For the safe translation of stem cell therapy to the bed-
and inkjet printing, are able to predict the most appropri- side, the standardized, large-scale propagation of MSCs in
ate hierarchical structures of a fibrin structure for a given animal serum-free medium (without using animal-derived
target application. Based on the inkjet printing technique, components) is quite important and profoundly impacts
for example, PRF can be utilized as a printable gel to the overall safety of stem cell therapies [633]. It is sug-
generate specific cell patterns in a 3D construct, offering gested that cell culture with PL supplementation does
a milieu that mimics the highly hydrated state of a native not change the expression of surface markers on cultured
tissue, which renders this type of construct a promising cells compared to that in cultures supplemented with
candidate for cell delivery and tissue engineering [614]. FBS [634]. Most importantly, these cells retain their bio-
In addition to the sustained release of growth fac- logical features, such as their growth and differentiation
tors with spatiotemporal accuracy, many materials can potential favoring target tissue regeneration [634,635]. In
provide increased strength and stability to the fibrin scaf- recent years, cumulative evidence has strongly indicated
fold, resulting in increased mechanical stability [612,614]. that human PL-supplemented media not only preserve cell
PRF in turn may facilitate the manipulation and handling phenotypes and shorten culture time by increasing the
of a wide variety of polymers. In oral implantology, for cells’ growth rate but also maintain the cells’ multilineage
example, the handling and use of certain bone grafting differentiation capacity and can give rise to osteoblasts,
biomaterials and even bone autografts are relatively chal- chondrocytes, adipocytes, neurons and astrocytes, among
lenging. Because PRF acts as a biological glue to hold others [630,634–638]. Interestingly, accompanied by a
matrix particles together, it is possible to improve the more compact colony formation and a more spindle-
handling and adaptation of bone grafts by combining shaped morphology, PL-cultured BMSCs [639] and ADSCs
them with scaffold-like PRF prior to their transplantation [640] remained significantly smaller in size than did FBS-
into a defect site [44,591,605]. Moreover, the incorpora- cultured cells. These small-sized cells occupied less space
tion of PRF into alginate-based or hydrocolloid dressings in the culture, and hence their intravenous administra-
for the management of chronic ulcers, the combination tion would be safer and effective because smaller cells
of PRF with augmentation materials applied in orthope- facilitate cell penetration through capillary vessel systems,
dic surgery and the production and use of a hemostatic while larger cells can create obstructions in the cardiac
and elastic fibrin for a specific application have also been and pulmonary microcirculation [639,640]. Further evi-
tested in recent years and have yielded promising results dence suggests that PRP cultures significantly inhibit serum
[599]. Although PRF offers a versatile tool with great starvation- or TNF-␣/cycloheximide-induced apoptosis in
potential for use in biomaterials science and regenerative multipotential preadipocytes. These results imply that cell
medicine, more intensive investigations are required to culture with PL for clinical transplantation may increase the
illustrate the molecular roles that drive the varying bio- survival of stem cells [641]. In terms of chromosomal stabil-
logical performance of PRF and to identify potential new ity [637] and immunomodulatory capacity [630,633,635],
therapeutic indications for and applications of PRF tech- no differences were observed between stem cells cultured
nologies [620]. under standard conditions and those cultured with human
PL. All these findings recommend substituting FBS with
6.3. Platelet lysate human PL for cell expansion if the expanded cells are
for clinical use. The functional capacity of PL-expanded
In most clinical trials, FBS is used as the main nutritional cells, however, has only been partially explored. For exam-
supplement in cell culture media. Although FBS is a widely ple, impaired immunomodulatory properties have also
accepted standard, its use is discouraged by regulatory been observed for MSCs cultured with human PL, imply-
authorities due to its high lot-to-lot variability even from ing some potential limitations in the utility of PL-expanded
140 F.-M. Chen, X. Liu / Progress in Polymer Science 53 (2016) 86–168

MSCs as immunomodulators in clinical applications [642]. been previously thought to be solely dependent on the
Replacing FBS with human PL prevents bovine prion, viral presence of FBS [629,650].
and zoonotic contamination of the stem cell product, and MSC therapies are limited by the loss of self-renewal
there is evidence showing that good manufacturing prac- and cell plasticity associated with ex vivo expansion in cul-
tice (GMP)-compliant culture medium with human PL in a ture and, on transplantation, by increased immunogenicity
closed hollow-fiber-based bioreactor can be employed for due to xenogen exposure during culture. Recently, it has
large-scale MSC expansion for safe application in humans become increasingly clear that PL stimulation induces a
[643]. Recently, human PL was subjected to pathogen inac- transient increase in the inflammatory response in qui-
tivation by psoralen to further eliminate the possibility of escent human osteoblasts during bone regeneration. This
PL as a human-derived blood material to alleviate immuno- increase is mediated by NF-␬B activation, cyclooxygenase-
logic risks. The resulting product is an even safer alternative 2 induction, prostaglandin E2 production and the secretion
than PL to FBS, and is even more advantageous with regard of pro-inflammatory cytokines. Furthermore, long-term PL
to cellular growth and stemness [644]. The exploration of stimulation enhances the proliferation of actively repli-
human platelet derivatives for clinical-scale MSC expan- cating osteoblasts without affecting their differentiation
sion may represent a major step toward promising new potential, along with changes in cell morphology, result-
stem cell therapies [637]. ing in increased cell density at confluence [651]. Notably,
As a culture substitute for clinical-scale human MSC human PL cultures of aged and senescent MSCs demon-
expansion, PL may fully replace FBS, and its expansion- strate cellular rejuvenation, reflected by a decreased
enhancing effect is likely due to the high concentration doubling time and smaller cell size. These findings sug-
of native growth factors and bioactive molecules that PL gest that PL culture not only enhances MSC proliferation
may contain [636,637]. Human PL can be generated by but also positively affects the physiological properties of
subjecting common platelet units to several freeze and MSCs [652]. Aside from its use in cell processing, human
thaw cycles, which damages the membranes of platelets PL has been used to functionalize biomaterials for tis-
and releases their natural growth factors into the plasma. sue engineering [653] (Fig. 18). For example, a PL coating
The platelet fragments are removed by centrifugation to directly increases the chemoattraction and adhesion of
deplete potential antigens and to avoid extensive aggre- human MSCs and endothelial cells to a scaffold. More-
gate formation. The major advantage of using human donor over, such a coating was demonstrated to induce human
recipient-matched or autologous PL as an alternative to MSCs to produce and secrete pro-angiogenic proteins (e.g.,
FBS is the elimination of any risk of secondary impacts placental growth factor and VEGF), which may in turn posi-
that may be induced by FBS constituents in cell culture tively affect cell behavior, leading to synergistic effects that
[645]. However, the potential contamination associated enhance in vivo neovascularization and new bone forma-
with adventitious agents in blood-derived substituents tion [654].
remains a risk. Fortunately, such a risk may be decreased In sum, human PL has multiple beneficial effects on
by strict adherence to blood bank quality standards for therapeutic cells. In addition to PL’s clinical safety, the
blood collection, handling and processing. Another impor- significant increases in proliferation in culture with PL
tant point that should be noted is that the growth factor compared with standard FBS culture allow more rapid cul-
levels within human platelets vary significantly from donor ture expansion of MSCs to clinically relevant numbers ex
to donor; pooling many prescreened individual samples vivo, without compromising their genomic stability or dif-
may reduce the high variability among blood samples of ferentiation capacity [655]. Furthermore, PL cultures of
human origin [646]. Although the removal of platelet frag- high-passage and senescent MSCs may rejuvenate the cells,
ments creates PL for allogenic application, it is safer if allowing them to be expanded to clinically relevant num-
human PL from the same donor is used, which may fur- bers ex vivo, even after being cultured for a short duration
ther minimize the risk of immunological side effects and with conventional FBS supplementation [652]. The util-
viral infections [647,648]. ity of native growth factors presented in well-prepared PL
The transfusion of blood and blood components has in therapeutics is revolutionizing many biomedical areas,
been the standard of care for treating anemia for more cell biology and regenerative medicine. To succeed, scien-
than half a century. Platelets separated from the white tists are actively working in this field to characterize the
and red blood cell fractions are of specific interest because platelet secretome, to improve strategies for endogenous
they do not compete with the need for erythrocyte and growth factor administration and to design new regenera-
plasma preparations for the limited number of avail- tive biomaterials that increase the versatility of human PL
able blood donations. The buffy coat-derived platelets application.
used for PL preparation can be concentrated to at least
1 × 109 platelets/mL by centrifugation [646]. A cocktail of 7. Future directions and outlook
growth factors, cytokines and mitogens stored in the ␣
granules of platelets can be released following platelet Taking inspiration from native ECMs, scientists have
activation by thrombin or the disruption of platelets by on one hand sought help from chemistry in designing
freeze thawing, with the latter approach being more regenerative biomaterials and, on the other hand, are
attractive because the use of commercially available seeking physics-derived solutions for controlling biolog-
thrombin derived from bovine plasma is bypassed [649]. ical responses [34]. Recently, biomaterial strategies have
Hence, human PL may be an adequate non-xenogenic considerably advanced to closely mimic the constituents
alternative to FBS in many cell culture systems that have and framework of a native tissue and to incorporate
 F.-M. Chen, X. Liu / Progress in Polymer Science 53 (2016) 86–168 141

structural, biochemical and biomechanical signals that properties of the native ECM of human tissues remains an
are able to communicate with cells and with the elusive goal and a great challenge [32,61,97,299]. The tools
in vivo microenvironment in a biologically specific man- provided by synthetic biology and protein engineering, as
ner [48,656]. Future developments in biomaterials science, well as electrospinning, have offered an unprecedented
as well as in related fields (e.g., medicine, biology, chem- level of biomimicry, which is invaluable for the biomimetic
istry and engineering), will need a thoughtful approach to design of the next generation of advanced cell-instructive
ensure that tissue engineering fulfills its true clinical poten- biomaterials; these materials can provide attractive ECM
tial. To enter widespread clinical application, biomedical conditions conductive to specific cell behaviors, including,
engineering and regenerative paradigms are required to but not limited to, the anchorage, migration and differenti-
be not only scientifically appropriate but also inexpen- ation of different types of stem cells required for successful
sive, safe and clinically expeditious [16,582]. Expedited tissue regeneration [54,660–663].
strategies for tissue regeneration based on human-derived In fact, biomimetic biomaterials inspired by the dynam-
biomaterials are compatible with existing clinical modal- ics of the stem cell–niche interactions of the natural ECM
ities and have dramatically accelerated the pace of the have shown significant potential as instructive matri-
clinical translation of tissue engineering therapies. In par- ces or cell vehicles for tissue engineering that are also
allel, the natural ECM, with its multitude of evolved targeted for regeneration [67,112,292,294,661,664–666]
cell-instructive and cell-responsive properties, provides (Fig. 19). Regarding the physical properties of such mate-
inspiration and guidelines for the design of advanced rials, they are biochemically and mechanically defined
biomaterials that recapitulate a 3D, ECM-mimicking envi- by the tissue of origin, and their physical architecture
ronment to activate specific cell–material interactions for and chemical composition are organized in a nanoscale
the organization of individual cells into functional tissues manner, in addition to having the required nanotopo-
[279,293,398,471]. graphical surface features [34,667]. In general, synthetic
scaffolds exhibit uniformly distributed porosity, whereas
7.1. Design of ECM-mimicking biomaterials biomimetic materials may not need to be uniformly porous
because in natural tissue, porosity is not uniformly dis-
Together with new insights into ECM assembly and its tributed [668]. Based on recent innovations in scaffold
role during tissue development, expansion and regener- design and advanced scaffold-manufacturing technologies,
ation, advanced biomaterials for tissue engineering have a diverse distribution or gradient of porosity throughout
of late largely exceeded the requirements for passive the scaffold dimension can be achieved to mimic nature
biocompatibility that were previously considered accept- with respect to nanoscale topographical features, micro-
able for medical devices, and a new era of biomimicry and macroscale gradient structures, interconnectivity, pore
has been unveiled to yield synthetic ECM-mimicking bio- size and size distribution [97] (Fig. 20). Recently, biocom-
materials that have both multi-component frameworks posite nanofibrous scaffolds consisting of two or more
and high degrees of compositional and functional defini- polymeric blends were fabricated into uniform copolymers
tion [37,471,657,658]. Cells residing in living systems are with interconnected pores to form flexible, cell-supporting
extremely sensitive to their surrounding physicochemi- substrates with desired biofunctional and biomechanical
cal milieu, continually reading microenvironmental cues properties dependent on their applications [669]. The intri-
and responding to them to control the cellular pheno- cate and ingenious geometry is responsible for the overall
type and functions and to promote homeostasis [32,474]. performance of such tissue engineering templates [33,34].
Biomimicry, “a new science that studies nature’s models As the primary role of biomaterials, a certain level of physi-
and then imitates or takes inspiration from these designs cal support from the moment of implantation must also be
and processes to solve human problems”, has recently been ensured to guarantee mechanical integrity and assist tissue
introduced as a term to describe design innovations in function while new matrix is being deposited [34]. How-
biomaterials science [24,25,659]. Cells that grow in a natu- ever, establishing a clear and direct mechanistic correlation
ral niche incorporating blood vessels continuously receive between the nanomechanical properties of the individual
nutrients and oxygen, and their waste products, including constituent macromolecules and the emergent mechani-
CO2 , are continuously removed [26,286,287]. In contrast, cal performance of the resulting bulk polymeric materials
when cells are grown in a stagnant biomaterial-based as a guide for the biomimetic design of advanced materi-
scaffold without an ECM-mimicking nanoenvironment, als with well-defined bulk mechanical properties remains
nutrients and oxygen are not transported into the cells, and a challenge [33,670].
waste products are not removed from the cells. Thus, the Considering that the survival of cells following in vivo
nutrient concentration decreases and the concentration of transplantation is often poor when the cells are placed in
waste products increases until the artificial scaffold is even- a suboptimal environment with an absence of necessary
tually noxious for cell viability and function [45,114,293]. ECM macromolecules, such as a prefabricated scaffold
Such an artificial biomaterial is far from the ideal environ- without any biomimetic modifications, materials design
ment that these cells experience in their natural state and has recently moved from simple cell delivery and physical
hence is not suitable for new tissue growth and reconstruc- support to the creation of an artificial niche inspired by
tion. Although a particular cell behavior may be imitated tissue-specific niches [387,671]. Defining an artificial in
in vitro by mimicking the corresponding in vivo conditions, vivo milieu with complex and dynamic regulation where
the design of materials that closely mimic the hierar- cells interact and behave according to the surrounding
chical architecture and the biochemical and biophysical environmental cues, ECM-mimicking substrates and
142 F.-M. Chen, X. Liu / Progress in Polymer Science 53 (2016) 86–168

Fig. 19. Design of extracellular matrix (ECM)-mimicking scaffolds. (A) Scanning electron microscopy (SEM) micrograph of a three-dimensional (3D) nanofi-
brous gelatin (NF-gelatin) scaffold that mimics the physical architecture and chemical composition of type I collagen in the ECM. (B) Higher-magnification
image of (A), showing the nanofibrous pore walls of the gelatin scaffold. (C) Incorporation of bone-like apatite into the surface of the 3D NF-gelatin to
further mimic the inorganic components of bone ECM and improve the mechanical strength of the scaffolding. (D) Incorporation of non-collagen pro-
teins (NCPs) into the surface of the 3D NF-gelatin to form an artificial matrix (NF-gelatin-NCPs) mimicking both the nano-structured architecture and the
chemical composition of natural bone ECM. The NCPs were labeled with fluorescein isothiocyanate (FITC). (E) Design and synthesis of nanofibrous hollow
microspheres integrating the ECM-mimicking architecture that have a highly porous, injectable form, efficiently accommodating cells and enhancing tissue
regeneration. (F) Higher-magnification image of (A), showing the nanofibers of the injectable hollow microspheres.
Source: (A–C) [67], Copyright 2009, (D) [292], Copyright 2013, (E and F) [112], Copyright 2011. Reproduced, respectively, with permission from Elsevier
Ltd, Mary Ann Liebert Inc and Nature Publishing Group.

scaffolds are excellent candidates for regenerative change and remodel during tissue development and regen-
medicine because in our bodies, cells rest upon or are eration [26,276,474]. Ideally, the synthetic biomaterials
surrounded by ECM, which functions not only as a sup- used in tissue engineering and cell delivery should create
port material but also as a regulator of cellular events the same or similar microenvironment for the seeded cells
[18,37,297]. In this respect, today, the development of as that in an ECM existing in vivo. However, native ECMs
biomaterials for tissue engineering is increasingly con- have very intricate structures and are composed of numer-
sidering the growing knowledge of and new insights ous types of proteins, many of which remain unidentified
into ECM components and structures that affect tissue thus far [37,280]. Therefore, using conventional physic-
development and regeneration [165,522,672]. To provide ochemical methods, it is difficult to obtain a scaffold or
a strategy complementary to the ingenious provision of substratum that has the same composition and the com-
ECM-mimicking information to cells inside manmade plex microstructure and architecture of an in vivo ECM
scaffolds, structural, physical and biomechanical factors [477]. Biomimetic hydrogels formed by self-assembled
continue to be intensively exploited while attempting to biopolymer networks such as silk, keratin elastin, resilin
effectively present all of the necessary influences found and periodate oxidized alginate and gelatin display close
in the native milieu of a given tissue [20,113]. For clinical chemical, structural and mechanical similarities with the
translation and success, however, the complexity of the native ECM and have therefore been widely used as arti-
resulting biomaterials should be reduced to a commercially ficial cell niches with tunable properties that favor cell
acceptable level, regardless of the modes of action. To meet functions similar to the events occurring in natural extra-
this prerequisite, to date, most efforts have concentrated cellular microenvironments [64,317,387,469,673,674]. In
on distillation of the essential chemical character of ECM addition, these hydrogels, which are either derived from
influences into simple chemical functionalities for future naturally occurring molecules or are synthetic polymers
scaffolding production [20]. that recapitulate key motifs of biomolecules, typically have
In recent years, awareness that the ECM has a pivotal a highly interconnected porous network, good biological
regulatory function, directly contributing to fundamental compatibility and may be degraded by proteolytic enzymes
aspects of cell behavior and tissue formation, has increased in the body, holding great promise for various biomedical
[279]. The compositions, structures and biomechanical applications [88,675]. However, the reproducibility of cell
properties of ECM networks and molecules vary accord- constructs often remains complicated because of batch-
ing to each specific tissue and organ and also dynamically to-batch variation and the sensitivity of cells (especially
 F.-M. Chen, X. Liu / Progress in Polymer Science 53 (2016) 86–168 143

Fig. 20. Representative scanning electron microscopy (SEM) images of glycidyl methacrylate-derivatized dextran (Dex-GMA)/gelatin scaffolds with intri-
cate and ingenious hierarchical structures (a diverse distribution of porosity throughout the scaffold dimension), designed in our laboratory using a
computer-aided technique, that resemble naturally derived extracellular matrix (ECM) with regard to geometry, interconnectivity, pore size and size dis-
tribution (images from unpublished results; a sample with similar architecture has been shown in Ref. [56]). (A) Material niches with nano- and microscale
features; (B) a magnified view of the central region shown in (A); (C) a magnified view of a portion of (B); and (D) a magnified view of the marginal region
shown in (A).

progenitor or stem cells) to these differences. Addition- behind the healing processes that are required for success-
ally, due to their variable printability, the implementation ful tissue repair and regeneration [48].
of these biomaterials via biofabrication may be challeng- At the cellular level, recruiting cells out of their inherent
ing [676]. Furthermore, the changing biochemical cues in niches demands comprehensive insight into the biophys-
the hydrogels developed to date often induce simultane- ical and biochemical cues that control stem cell actions.
ous changes in the hydrogels’ mechanical properties, which Instead of an individual entity that probes its surrounding
does not support mechanistic investigations of the stem milieu and responds to the players to which it is exposed,
cell-niche interplay under highly controlled conditions. To the cell is a key part of the living system, residing within
address this concern, a PEG-based interpenetrating net- a complex and dynamic architecture generated by itself
work was recently developed as an artificial cell niche that [107,672]. The coordinated interplay between surround-
resembles the micro- and nanoenvironment found in a nat- ing cells, soluble morphogens and growth factors, as well
ural tissue. The resulting hydrogel was composed of two as insoluble biomacromolecules of the ECM, is orches-
polymer networks that could independently and simul- trated by spatiotemporal signaling patterns. In this context,
taneously crosslink to form hydrogels in a cell-friendly cells obtain and process information from their surround-
manner. Hence, the hydrogel allowed independently tun- ing microenvironment (i.e., the ECM) while they remodel
able biochemical and mechanical properties and stable its components and geometry [679]. A vital goal of tissue
and more homogeneous presentation of biochemical lig- engineering and scaffolding science is therefore to gener-
ands in 3D than allowed by currently available methods ate a manmade ECM that at least partially resembles the
[677]. Upon presentation of appropriate biological cues, a most critical aspects of such a complex native scenario,
transglutaminase factor XIII (FXIII)-crosslinked, PEG-based such that the processes controlling cell function and cell
biomimetic 3D matrix was also found to mobilize mes- commitment can be regulated and understood [294]. The
enchymal progenitor cells from the amnion to proliferate degradation of scaffolding materials into macromolecules
and secrete native ECM proteins for fetal membrane heal- and the subsequent release of cell-instructive signals into
ing [678]. More work must be performed to clarify the a target place have opened a biomedically exploitable
characteristics of a specific native tissue, to then design avenue toward modifying the in vivo cellular milieu into a
a scaffold with the proper mechanical properties and to more refined condition [680]. Nonetheless, our poor under-
ultimately define the cellular and molecular mechanisms standing of the signaling pathways that dictate cell biology,
144 F.-M. Chen, X. Liu / Progress in Polymer Science 53 (2016) 86–168

together with the complexity of the natural ECM sys- as biological mediators to control cell fate have paved the
tem and its dynamic interactions with niche cells, makes way for the development of a new generation of scaffolds
the design of appropriate ECM-mimicking biomaterials and medical devices for tissue engineering [20,293,694]. In
exceedingly difficult [37,672,681,682]. this context, for example, extracellular vesicles (EVs) that
Researchers have thus attempted to use ECM in tissues comprise a heterogeneous population of cell-derived lipid
and organs or ECM proteins produced by in vitro-cultured vesicles (e.g., exosomes, microvesicles and others) have
cells after decellularization treatment [477]. Meanwhile, recently emerged as patient-derived vehicles for targeted
the technology of scaffolding science has provided an drug delivery in regenerative applications. Moreover,
exciting opportunity to deconstruct this landscape to iden- these vesicles are recognized as playing crucial roles in
tify and evaluate the particular effects of a specific ECM cell-based therapies because they are primary biological
component on the hosted cells [113]. Moreover, microfab- mediators of intercellular information transfer in multiple
rication and, more recently, nanofabrication are allowing physiological systems. There is a growing need for biolo-
the creation of appropriate investigating models in which gists and material scientists to understand and exploit the
the necessary biologics may be assessed on the supra- bioactivity of EVs secreted by therapeutic cells, harness
millimeter to the nanometer scale [683–686]. Interestingly, such information for the design of artificial ECM materials
following recent mining of the diversity of functional and specifically control their biological performance to
peptide modules, the modular design of ECM-mimetic enhance the efficacy of regenerative therapies [695].
protein-based biomaterials has become possible, which If simplified into its essential mechanical elements,
involves the combination of multiple protein domains with the ECM is composed of several secreted proteins (col-
diverse functionalities into an individual, modular polymer lagens, fibronectin, elastin and fibrillins) that constitute
sequence or motif (generally termed protein engineering), macromolecular structures in their functional embodi-
leading to a multifunctional matrix endowed with single ments, such as fibrils, microfibrils or fibers [285]. This
functional domains that are independently tunable [661]. category of ECM components also includes enzymes
To this end, both decellularized scaffolds and synthetic that post-translationally modify these biomacromolecules,
matrices are being explored to satisfy the needs of the such as proteinases, which cleave peptide bonds (e.g., the
clinic, and the fundamental mechanisms by which stem matrix metalloproteinases), and lysyl oxidase, which forms
cells contribute to regeneration and homeostasis are begin- intermolecular crosslinks [20]. Another category of ECM
ning to be clarified. To advance the field, multifaceted molecules (termed matricellular proteins, such as throm-
technologies will be increasingly necessary to examine and bospondins and tenascins) is in charge of modulating
coax cells ex vivo, to engineer predictive cell and tissue con- cell functions and cell–matrix interplay, without a direct
structs and to ultimately improve stem cell integration and contribution to the generation or function of structural
tissue regeneration in vivo for therapeutic benefit [18]. complexes [276]. The design of new ECM-like biomate-
rials should begin with exploring the simplest functional
7.2. Revisiting ECM influences for information performance of several ECM components that are indis-
pensable to addressing a defined clinical question and
For all cells outside the circulation, the ECM consti- should then involve other elements and soluble factors
tutes the cellular milieu that is known to have a major that may act cooperatively or synergistically with these key
instructive or regulatory effect on cell fate commitments macromolecules to ensure that the extracellular microen-
[687]. Based on this principle, by introducing specific vironment can be properly recapitulated in 3D [28,279].
structural and molecular elements in varying geometries Of note, early ambitious approaches attempting to engi-
and at different concentrations, a range of biomaterials neer whole organs have mostly given way to smaller,
with tissue-specific structural properties and bioinspired more accessible and practical goals. This change explains
polymeric surface close to those of the native ECM can why clinical research on cardiac regeneration, for exam-
be created [687,688]. Upon application, penetrating cells ple, is now focusing on engineering coronary arteries,
may be surrounded by a dynamic pericellular matrix, myocardium and valves individually, rather than trying to
similar to a native matrix, that possesses considerable replace an entire heart [20,26,520].
regulatory potential [689–692]. This approach is spurring The GAG hyaluronan is the main nonproteinaceous
interest in the use of natural ECM proteins for scaffolding ECM component, and several core proteins of the ECM
production. To this end, most research has been focused may be amended by the linkage of different types of
on isolated ECM proteins and their combinations, although GAG chains to form proteoglycans. These carbohydrate-
the ECM has a complex composition in each specific rich components may be hydrated to exert a swelling
tissue. However, each tissue is often regionally specialized, pressure against the surrounding fibrous networks, which
regardless of its type and size. In this regard, a recent results in tissue turgidity and compressibility for molecu-
investigation of anatomically distinct cartilages presented lar transport. Based on a systems-level bioinformatics view
substantial evidence for intriguing tropism in diverse of ECM function and composition, Cromar et al. (2012)
patterns of joint pathology, highlighting distinct varia- defined a set of 357 proteins that represent core compo-
tions in protein patterns associated with different tissue nents of the ECM, together with an additional 524 genes
mechanical properties [693]. Nevertheless, biomaterial that modulate their associated functional roles, and gen-
strategies for the recreation of ECM influences in simplified erated a map illustrating their physical interactions [696].
forms, the reduction of biopolymers into short functional In biomaterial design, there is immense current interest
domains and the application of basic chemistries as well in the roles of the physical and mechanical properties
 F.-M. Chen, X. Liu / Progress in Polymer Science 53 (2016) 86–168 145

of ECM, such as stiffness and elasticity, in affecting cell of MSCs during in vitro expansion and to rejuvenate the
fate commitment, directly signaling to cells and modulat- cell functions of aging MSCs, indicating that cell-formed
ing soluble signals [697]. One role is signaling via matrix ECM is an appropriate culture substrate to improve the
adhesion proteins and receptors, such as discoidin domain bioactivity of scaffolding biomaterials to facilitate cell pen-
and integrin receptors [698]. Another role is the activa- etration [706]. These biomaterials can be stored until use in
tion and sequestration of signaling molecules, such as the engineering of autologous living tissues, such as blood
those in the TGF-␤ superfamily [699], and the modula- vessels, using autologous vascular cells, accelerating the
tion of morphogen gradients. With increasing elucidation production of vascular substitutes [707]. After the forma-
of the potential roles of cytokines and growth factors and tion of an ECM by cultured cells, cell-formed matrices can
their interplay with ECM components, advanced materi- be extracted without the use of detergents, sterilized, and
als are being devised that more closely mimic the healing then used to coat tissue culture plates or can be directly
microenvironments of native tissues, leading to increased used as a new cell culture substrate after a decellular-
efficacy in applications in wound healing and tissue regen- ization treatment; the resultant animal-free product can
eration [87]. The cell–matrix interface in particular offers a support the culture and differentiation of human stem cells
pivotal signaling nexus that controls all aspects of stem cell [708].
activity. Today, many types of predominantly cell-derived sub-
Alongside the traditional focus on the structural rel- strates and matrices are prepared under various culture
evance of the ECM, which has not diminished, there is conditions to satisfy specific application needs, includ-
now a huge emphasis on several new fields. For instance, ing, but not limited to, cell expansion; tissue engineering;
mimicking the organizational, biological and functional and, recently, growth factor delivery (Fig. 21). Along these
complexity of native tissue ECM at the molecular level is lines, a fibroblast-derived, ECM-mediated platform was
now regarded as the next challenge in biomaterial inno- successfully engineered for VEGF delivery; its capacity to
vation [676]. This emerging interdisciplinary research area convey VEGF in a tailored way resulted in an advanced
provides a platform on which to establish a tangible trans- angiogenic response that promoted blood vessel ingrowth
lational pipeline, such as by modifying chemical signals to and maturation [709]. In another study, to create a cell-
responsive cells and by creating manmade tissues or organs formed nanofibrous scaffold, a fibroblast cell sheet with
using mechanical and adhesive molecules as raw building highly aligned cells and ECM nanofibers was first gener-
blocks [39]. It is plausible that in the near future, exciting ated by directing the growth of human dermal fibroblasts
developments that rise to the demands and realities of the on synthetic nanogratings. A highly aligned nanofibrous
marketplace will inform us how these goals can be real- ECM scaffold was then obtained after removing the cellu-
ized at the clinical level and how even relatively practical lar components from the sheet. Due to the preservation
and simple regenerative solutions may render considerable of the highly aligned elastin fibers, the elastic modulus
functional benefits [16,38,700]. was well maintained. Reseeding of cells indicated the
excellent capacity of the scaffold to provide cell adhe-
7.3. Cell-formed decellularized matrices sion sites and to support and direct cell proliferation and
alignment along the underlying fibers, suggesting that a
Although decellularized matrices derived from tissues specific cell-formed matrix may be harnessed to direct
and organs have many advantages in terms of their compo- particular cell behaviors [710]. Indeed, when stem cells
sition, microstructure and biomechanical properties, their differentiate into somatic cells, their differentiation pro-
utility is limited by the availability, geometry and constitu- ceeds in step-by-step manner, and their cell-formed ECM
tive properties of the tissue or organ from which the ECM is is a crucial factor in instructing cell activities in tissue
harvested [701]. In particular, tissue and organ decellular- engineering design [711]. Far from being a static struc-
ization is not suitable for the preparation of decellularized ture, during this stepwise differentiation of stem cells,
matrices mimicking an ECM that is specific to a region the ECM surrounding the differentiating cells continues
of tissue, such as stem cell niches [477,671]. Knowing to undergo remodeling (i.e., assembly and degradation)
that the composition of the ECM depends on the types according to the stage of development, differentiation
of residing cells, the tissue type and the organ type and and tissue regeneration [296]. The chemical and phys-
that only native tissue-like matrices can have the greatest ical interactions of cells with the surrounding soluble
effect on cellular functions, matrices with unique physical and/or non-soluble messengers/components of the ECM
and chemical attributes formed by a variety of cell lin- play fundamental roles in these cellular processes. More-
eages (e.g., skin fibroblasts, brain-derived astrocytes and over, cells can sense the stiffness and nanoscale features
MSCs) have been developed for a variety of research and (e.g., ligand presentation and substrate topography) of the
medical applications [701–705]. This self-assembly strat- ECM and can deform it via generating cytoskeletal tension
egy stems from the capability of MSCs to secrete, deposit [712]. Given that mimicking the ECM at each maturational
and organize their own ECM. When these cells are cul- stage is one of the important approaches for directing
tured in vitro, ECM proteins are secreted from the cells stem cell differentiation, decellularized matrices mimick-
and then deposited beneath the cells. The decellulariza- ing the ECM during the osteogenesis or adipogenesis of
tion of these cell-formed matrices enables the production MSCs were developed as stepwise tissue development-
of autologous constructs from stem cells or from tissue- mimicking matrices [522,713]. These stepwise cell-formed,
specific cells [243]. Recently, ECM derived from MSCs has tissue development-mimicking matrices can be applied
been demonstrated to maintain the multipotent potential not only in regenerative medicine but also in basic
146 F.-M. Chen, X. Liu / Progress in Polymer Science 53 (2016) 86–168

Fig. 21. Schematic representation of several cell culture methods frequently used for the preparation of cell-formed decellularized extracellular matrices
(ECMs) that satisfy specific application needs [477].

biological studies. The fact that cells reside in their spe- bioreactor-based production of ECM-decorated materials
cific ECM within a complex in vivo milieu elicits the as “off-the-shelf” hybrid scaffolds (i.e., carefully engineered
necessity to further illustrate the impacts of ECMs pro- synthetic polymers modified by naturally derived ECM)
duced by various cell sources on overall cell fates [714]. combining selected structural and mechanical properties
More exciting than expected, a cohesive cell-formed sheet with the physiological presentation of native cell-secreted
can be layered into 3D tissues or organs with physiologi- ECM macromolecules and instructive biological signals
cal mechanical strength [715,716]; this concept has been [497,721]. One of the current strategies to enhance the
demonstrated to be feasible in clinical sittings [717]. In osseointegrative performance of titanium and its related
addition, there is a wealth of evidence that cell-formed implants relies on the regulation of their surface cell
ECM products can be applied to enhance the large-scale receptor–ligand interactions, in which surface-bound ECM
expansion of highly functional adult human MSCs [718] or proteins act as ligands, whereas integrins act as cell recep-
to decorate the surfaces of synthetic polymers and man- tors [665,722–724]. The surface modification technique has
ufactured metals on which subsequent cell adhesion is therefore been established as a reliable strategy for implant
regulated by adsorbed ECM proteins [96,184,719,720]. It is optimization. However, when linked to implant surfaces,
self-evident that the physical properties of synthetic bio- specific ECM proteins and peptide sequences appear to be
materials can be extensively tailored, but it is also clear limited in their ability to trigger native cell responses. To
that these materials often suffer from limited biological cope with this limitation, native or synthetic ECM is applied
functionalities. Fortunately, their biological performance to search for an optimal composition and structure that
in in vivo application can be enhanced by ECM deposition may help to overcome the difficulty [725].
by cultured cells in vitro and subsequent decellularization, As a scaffold-free cell delivery approach, as well as a
as demonstrated by the resulting product’s maintenance modern technique for the generation of functional tissue-
of its molecular functionality, retention of its structural or organ-like structures, cell sheet technology is also based
integrity and enhancement of tissue regeneration fol- on ECM formation by cultured cells [726]. In this regard,
lowing its in vivo transplantation [497]. Considering that a “cell sheet” composed of living cells and their secreted
polymers can be tailored for surface-driven ECM assem- ECM may be created and grafted onto the surface of a cell
bly, a microtextured polydimethylsiloxane scaffold was culture dish with the help of a temperature-responsive,
developed to induce cell-formed ECM deposition. When intelligent polymer. By altering the temperature to below
cultured on this scaffold, which had defined microto- 32 ◦ C, a contiguous cell sheet capable of maintaining
pography and chemistry, human fibroblasts underwent intact ECM components and cell–cell interactions can
significant changes in their morphology, adhesion and be obtained [726]. Such strategies are relatively com-
actin cytoskeleton, which finally led to the generation of plicated and time-consuming. To simplify the process,
compacted units of fibronectin on the surface of the scaf- modified protocols based on dexamethasone, vitamin C
fold [721]. These findings consolidate the vista of in vitro or osthole have been developed to create cell sheets that
 F.-M. Chen, X. Liu / Progress in Polymer Science 53 (2016) 86–168 147

Fig. 22. Representative scanning electron microscopy (SEM) images of cell sheet formation. Bone marrow mesenchymal stem cells were plated into 6-well
plates at a density of 1 × 106 cells/well and cultured until the cells reached 100% confluence, after which they were induced to form cell sheets using
cell-sheet-inducing medium (i.e., basal medium supplemented with 50 ␮g/mL vitamin C) for a 10-day period. (A) Three days following cell sheet induction:
extracellular matrix protein production; (B) 5 days following cell sheet induction: cell–cell and cell–matrix interactions established; (C) 5 days following
cell sheet induction: sheet-like structure formation; (D) 10 days following cell sheet induction: complete cell sheet formation.

do not require special materials (Fig. 22). The resulting 7.4. ECM–stem cell interactions: signposts in advanced
sheets can be used for cell transplantation, without biomaterials
the need for synthetic biomaterials, which has already
paved the way to clinical therapeutics and clinically The ECM–stem cell interface is a complex, dynamic
relevant animal experiments, such as studies on visual milieu wherein the cells bind to and contract against
acuity, cardiomyopathy, esophageal ulcerations, periodon- the ECM, commonly via transmembrane receptors of the
tics and type 1 diabetes, confirming the safety and efficacy integrin family and by means of a feedback-signaling pro-
of the treatment [726–728]. Recently, decellularized cell cess. This binding cooperatively dictates their respective
sheets retaining ECM integrity have emerged as an inter- fates via the interactions of nanotopographical features
esting cell-derived ECM product for diverse biomedical with integrin receptors in the cells’ focal adhesions
uses because they offer a naturally occurring matrix with a [325,667,712]. Tissue engineering approaches integrat-
complex set of physiologically functional biomolecules for ing material nanotopography, integrin–matrix interactions
cell repopulation and function [275,704,729,730]. More and soluble growth factors (e.g., presented by the materials)
specifically, such cell-formed constructs may possess could lead to the development of ingenious extracellu-
the ability to support allogenic recellularization; this lar niches that can alter how cells adhere to material
direction is in need of investigation of both techniques surfaces and regulate cellular differentiation commitment
and applications [730]. Compared with decellularized and functions through changes in both cell morphology
tissue- and organ-derived ECM templates, cell-formed and cell biochemistry [277,667,731,732]. Recent endeav-
decellularized matrices have the advantages of unlimited ors have tried to decipher the complex interaction between
availability and adaptability to different developmental ECM niches and stem cells, and it is increasingly evident
stages. However, in the self-assembly approach to ECM that inherent biomaterial properties (e.g., physicochemical
biomaterials, it is difficult to mimic the complicated native properties or nanoscale surface features) can be exploited
ECM-like architectures. Because the ECM secretion pattern to control stem cell fate commitment [667]. Thus, the abil-
of in vitro-cultured cells is different from that of cells ity to harness nanoscale and nanotopographical design
in vivo, the potential difference in ECM composition and and material–stem cell interactions is poised to have
structure between cell-formed decellularized matrices substantial implications for the redesign of the next gener-
and native ECMs should be considered when cell-formed ation of stem cell delivery systems and tissue engineering
decellularized matrices are used in tissue engineering templates [325,671,712,733]. Traditional cellular scaffolds
applications [477]. served as inert matrices that merely acted as a support
148 F.-M. Chen, X. Liu / Progress in Polymer Science 53 (2016) 86–168

for the attached cells; however, recently, more emphasis the characterization and quality control of a living cell-
has been placed on adding appropriate physical and chem- based therapeutic product. The approach also significantly
ical properties to these platforms for cell transplantation reduces the amount of time and effort required before
and tissue engineering [3]. In fact, several of these recent implantation, thereby offering the possibility of fostering
innovations have led to new insights into the design of attractive new therapeutic paradigms [18,277]. Over the
artificial niches encompassing a wide range of biophysical, past decade, mounting data have revealed the ability of
biochemical and biomechanical cues that are able to play BMSCs to mobilize from the bone marrow to the peripheral
a guiding role to elicit targeted cell functions [671]. More blood and to eventually enter an injured site to replen-
often than expected, the coordinated interplay between ish dying cells and regenerate damaged tissues. However,
resident cells and their surrounding natural or synthetic when the intrinsic biological response is inadequate, the
scaffolds, soluble factors and other niche cells can define stem cells naturally mobilized in response to injury (e.g.,
a local mechanical and biochemical microenvironment trauma and ischemic diseases) are generally quantitatively
that has been and still is being studied and exploited to insufficient and hence do not offer a universal regenera-
instruct an orchestrated set of cell events and reactions tive solution [277,392,743,744]. Worse, if the competing
[297,664,734,735]. balance of processes associated with tissue remodeling
The last several decades have witnessed remarkable and inflammation is perturbed, particularly irreversibly,
advances in the field of synthetic biology, with a sig- the resulting positive feedback loops can continue to exac-
nificant increase in the number of patients benefiting erbate tissue damage and deterioration and compromise
annually from the therapeutics that have arisen from this cell recruitment and regeneration [745]. Therefore, in most
field [107]. Today, synthetic biology is gaining increasing cases, interventions such as the administration of bio-
attention in materials science research, including research materials, small biomolecules, genes or other biological
into the signals that cells directly acquire via interactions agents may be required to ensure a satisfactory therapeu-
with scaffolds or bioactive molecules in their environment tic benefit. A question that arises here is thus how required
[20,70,736]. Notably, the inspiration that directs the devel- clinical efficacy can be achieved while the complexity of
opment of new materials science strategies is commonly the therapeutic interventions used is kept to a minimum;
drawn from the elucidation of the biomaterials organized indeed, such cell-free interventions are less complex than
naturally by living cells in tissue on different scales [737]. the transplantation of cells manipulated ex vivo [277].
Therefore, penetrating insight into thus far undiscovered The quest for clinical strategies that might improve the
areas motivates the development of new tools that allow body’s natural regeneration mechanisms must be based
for the more thorough exploration of cell–ECM interplay on a thorough understanding of the cellular and molecu-
and its effects in a feedback manner [738–740]. lar events underpinning tissue repair and its failure after
As mentioned previously, molecular-scale interactions surgery; trauma; and vascular disease, diabetes and other
between stem cells and the surrounding ECM can be disease conditions that are frequently associated with
exploited to modulate the stem cell niche for facilitated healing pathologies [746]. To date, however, complete
tissue regeneration because an in vivo milieu represents knowledge of in vivo cell repopulation has yet to be estab-
a significant entry point for the therapeutic regulation of lished with regard to what cell types need to be recruited;
stem cell function [477,502,741] (Fig. 23). Given that poten- what chemoattractant/signaling molecules are required to
tially available progenitor/stem cells already exist in the modulate the niche therapeutically and ensure success-
body, a particularly exciting area involves the manipulation ful homing; whether a precise chronological sequence of
of the cellular components of the stem cell microenvi- events occurs during such homing processes; and, if so,
ronment to target specific aspects of the niche, normally how each biological event and the human immune system
by the administration of chemokines or other chemoat- can be orchestrated to enable the successful regenera-
tractants that act cooperatively or synergistically with tion and integration of the targeted tissue via materials
chemokines, to induce the mobilization and homing of design [268,476]. Nevertheless, there is emerging recogni-
host reparative cells [277,741]. These cell populations tion that this approach is practical and effective in certain
can interpret and interact with multiple input signals ideal clinical scenarios. A clearer picture is emerging that
and hence can be recruited to areas of tissue damage a target-specific biomaterial scaffolding system that can
to acquire active biological functions, such as secreting effectively control the host microenvironment and mobi-
a cocktail of immune-regulating signals or growth fac- lize host stem/progenitor cells to the damaged areas is
tors instead of, or in addition to, directly participating in generally required to practice this concept [500]. Although
regeneration of the tissue (Fig. 24) [22,277,498]. As an alter- damage to cells at a site of injury can result in neighbor-
native or adjunct to cell transplantation therapy, which ing cells dedifferentiating to replace the damaged cells,
is inherently expensive and labor intensive, this strat- cell populations residing within tissues neighboring an
egy has the advantage of repairing damaged tissue with insult are generally too low in number to have a clin-
the patient’s own stem/progenitor cells, without the need ically meaningful therapeutic effect. In most cases, it is
for specialized facilities for ex vivo cell isolation, expan- therefore worthwhile to target stem cells from the circu-
sion and transplantation [742]. Compared with strategies lation (such as BMSCs) to be mobilized into the peripheral
that rely on transplanting stem cells or their differentiated blood system and, finally, directed to the target sites [747]
derivatives, this approach circumvents the current transla- (Fig. 24). A wide spectrum of cytokines and chemoattrac-
tional barriers associated with extensive cell manipulation tants contribute to the whole-cell mobilization, homing
steps and costly manufacturing challenges associated with and engraftment process, and the ability to control the
 F.-M. Chen, X. Liu / Progress in Polymer Science 53 (2016) 86–168 149

Fig. 23. Concept of the use of decellularized tissue (organ) as a tissue engineering biomaterial (schematic is not to scale). Decellularized tissue (organ) can
be obtained by the decellularization of living tissue (organ). The resulting cell-free ECM can be modified (e.g., by heparin crosslinking and growth factor
binding) before implantation. To this end, the ECM scaffold can be reseeded with ex vivo-cultured cells that “prime” the biomaterial (e.g., to enhance its
ability in vascularization or remodeling) and/or “get primed” toward a specific cell fate decision (e.g., to proliferate or differentiate). Such a cell–matrix
construct could induce tissue regeneration by the combined action of seeded and recruited cells in a functionalized native matrix. Alternatively, modified
ECM can be directly transplanted into a patient without cell seeding. In this case, tissue regeneration entirely relies on the capacity of the ECM material to
instruct resident cells toward target recruitment, specific differentiation and subsequent tissue formation (endogenous tissue regeneration).

spatiotemporal presentation of these bioactive agents may and stromal cells for the reconstruction of complex tissues
provide important clues as to how to devise biomaterials remains infeasible [392,476]. Second, endogenous regener-
that ensure new tissues can reliably be generated in this ation may still not be sufficient in many situations or could
way (reviewed in Refs. [22,500]). The use of biomateri- be further enhanced by the delivery of exogenous cells;
als alone to coax host stem cell homing for in situ tissue hence, the second strategy is the application of biomaterials
regeneration is a new concept in tissue engineering. In this as delivery vehicles for therapeutic cells [58]. In this con-
context, an appropriate combination of multiple growth text, exogenous cells are used for injection/transplantation
factors in acellular materials and their spatiotemporal or delivery via various types of material vehicles, either
coordination will provide a favorable microenvironment directly or in the form of cell sheets/pellets. The cells may be
that leads to more successful outcomes in tissue regen- autologously sourced, with or without outside expansion,
eration [29,30,57,105,108,109,502,748]. The design of cell or may be allogeneic; regardless, they can be genetically
recruitment to biomaterials, how these biomaterials may manipulated to over-express and secrete selected factors
establish the necessary interplay with endogenous cells or delivered in combination with bioactive agents. Upon
in a way that unlocks the body’s intrinsic capabilities of transplantation, these cells may be able to reverse the typ-
self-repair and organization and the implications in tissue ical course of cellular apoptosis and local inflammation
engineering have been reviewed elsewhere [57,498]. within an injury site and markedly accelerate regeneration
New insights into the complex interplay and pathways in certain types of tissue damage [105]. To engineer various
of the biomolecules that are involved in targeted tissue tissues, such as liver or heart tissue, cell-dense structures
repair are necessary to achieve effective therapeutic out- that are similar to the native geometry and architecture
comes for translation into the clinic [684]. Nevertheless, are necessary. Developments in this field have consider-
the enhancement of endogenous tissue repair by acellu- able potential to synthesize “intelligent” materials that can
lar materials or suitable chemokine delivery devices is not communicate with the matrix, cells and multiple signal-
a replacement for stem cell therapy or transplantation. ing factors [110]. Further investigations will be necessary
First, although an artificial increase in the concentrations to fully identify and capture the intricacy and role of this
of specific chemokines (such as SDF-1) at diseased sites cell–scaffold–cell feedback loop [276,278]. It is notable that
offers an up-to-date paradigm to enhance and potenti- as an established clinical technique, cell sheet engineering
ate the homing of host reparative cells and to amplify has been based on the fact that it possible to obtain a sheet-
in situ tissue regeneration processes, in vivo chemokine- like construct full of cells, along with their cell-formed ECM
guided recruitment of the whole spectrum of parenchymal and natural cell-to-cell junctions [725].
150 F.-M. Chen, X. Liu / Progress in Polymer Science 53 (2016) 86–168

Fig. 24. Schematic representation of cell migration mechanisms within the body that may be modulated to induce the recruitment or homing of endogenous
stem cells for wound healing and regeneration (illustrations not to scale). (A) Stem cells located in cell niches neighboring an injury can recognize and obey
the gradients of signals (e.g., directional cues) within the extracellular matrix (ECM) and reach the site of injury, independent of blood flow, normally via
active amoeboid movement or chemotaxis-guided interstitial migration. (B) Stem cells from central cell niches (i.e., the bone marrow) can be mobilized
to enter the blood (i.e., by signals produced in response to injury or systemically administered homing factors) and disseminated throughout the blood
circulation system until they reach the local capillary vessel network surrounding the diseased site. Here, the cells recognize and interact with microvascular
endothelial cells, after which they exit the blood to replenish and maintain the cell niche neighboring the injury and hence enhance the regenerative potential
of the injured tissue. Alternatively, the cells can escape from the circulation and directly migrate to the injury site to participate in wound healing and
tissue regeneration.
Source: modified from Refs. [57,498].

Fig. 25. Schematic representation of the main players within the extracellular matrix (ECM), including inputs derived from neural and supportive cells,
blood vessels and secreted and paracrine factors and ECM components that need to be captured in advanced biomaterial design to reestablish ECM-stem
cell interactions [297]. The ECM offers a highly specialized and dynamic microenvironment encompassing different length scales; in this context, the
matrix elasticity/topography and a number of inputs combine and control cell fate commitment. The ECM may act by soluble factor presentation (a) and
be remodeled by the action of enzymes that produce functional fragments (b). More importantly, the ECM may directly bind cell surface receptors or
co-receptors (c, d and e), thereby potentially regulating cell anchoring and mediating diverse pathways involved in mechanotransduction and intracellular
signaling.
 F.-M. Chen, X. Liu / Progress in Polymer Science 53 (2016) 86–168 151

Regardless of the application paradigm (with cells or tuned, dynamically controlled and evolutionarily directed
with cell removal via decellularization), the utility of an spatial periodicity and remodeling [755]. An area that war-
individual ECM assembly or a known combination of ECM rants further attention is native matrix biology during
components can be more advantageous than utilizing tissue development, homeostasis and regeneration, includ-
whole ECM products due to the potential to selectively ing, but not limited to, dynamic properties, biological tubes
regulate biological activities [298]. Scaffold development and the active site architecture. Insights into this area
in tissue engineering is rapidly advancing to display prop- will facilitate the generation of in vitro systems that accu-
erties that, in a physiological and precise fashion, could rately recapitulate the in vivo microenvironment and the
manipulate stem cell fate by recreating native ECM func- subsequent the biomimetic design of biomaterials that
tion to elaborate the full complexity of biological signaling can adapt to, or dynamically interact with, surrounding
both in vitro and in vivo [297,311,749] (Fig. 25). In an in vitro tissue, thereby promoting desirable cellular processes to
environment mimicking the in vivo extracellular fluid ensure that natural function can be immediately replicated
inherent to living bone tissue in terms of three major fea- after the biomaterials’ in vivo implantation [293,756,757].
tures (i.e., monodispersed fibrils, high fibrillar density and Meanwhile, a fundamental understanding of ECM storage
long-range hierarchical organization), collagen mineraliza- (cues and growth factors) could offer important cues about
tion can occur based on a collagen/apatite self-assembly the nature of molecule–matrix interactions and improve
process, even in the absence of Ca-rich NCPs, which were the potency of current biopolymers in the presentation and
previously thought to play an important role in bone forma- delivery of multiple therapeutic agents for tissue engineer-
tion [319]. Therefore, in bone tissue engineering, the design ing [101,102]. In the future, the design of next-generation
and processing of synthetic hybrid materials in which bio-inspired materials should attempt to recapitulate the
multiple polymer constituents or phases are organized natural events involved in the secretion, clearance and
across multiple length scales, together with environmen- interaction of endogenously produced molecules within
tal cues, have allowed replication of the desirable biological tissues under pathological/healing conditions, as well as
activity of bone via easy-to-fabricate polymeric materials during the regeneration of new tissues or organs [31].
with nanoscale features such as substrate topography and
surface ligand presentation [164,165,750,751]. The biolog- 8. Conclusions
ical response to these new tissue engineering templates
often exceeds that observed with scaffolds synthesized Increasing evidence suggests that biomaterials of
using biological materials, even if the scaffold’s content human origin are yielding an ever-growing list of products
and architecture, the host immune response and the scaf- and successful clinical approaches to maintain, enhance
fold’s functional response to mechanical stimulation have or restore tissues and organs. These “raw materials” con-
all been carefully taken into consideration [165,666,752]. tinue to demonstrate great potential and will have an
Based on the development of advanced biomaterials with increasingly remarkable impact on synthetic biology, tis-
defined micro- and nanostructures allowing for the presen- sue engineering and clinical regenerative therapies in the
tation of endogenous and/or exogenous bioactive factors future. Stem cells respond to these biomaterials containing
in a physiological manner, future advances in this dis- native ECM information via self-recognition and inter-
cipline should aim to create cell–material and cell–cell play, which are very unlikely to elicit severe negative
interactions that encourage overall improvement follow- immune responses upon medical application. Decellular-
ing regeneration [12,55]. However, the pursuit of effective ized materials are promising because they take advantage
cell and protein (gene) delivery strategies remains ham- of nature’s platform to create an intrinsically complex
pered by the lack of noninvasive imaging approaches to ECM template that is thought to better mimic the native
monitor transplant survival, particularly over a long period extracellular surroundings. In addition, synthesized bio-
[31]. Although recent material technologies enable the use materials are directing our nascent understanding of the
of sophisticated scaffolds to direct or guide highly specific cellular milieu and how the basic building blocks (e.g.,
and coordinated cellular events, these reductionist strate- biomacromolecules) of human systems are correctly inte-
gies generally cannot satisfy the complexity of cell–ECM grated into the dynamic landscape that represents tissue
interactions and stimuli necessary for tissue regeneration physiology. Unfortunately, the science of human ECM biol-
under natural conditions [753]. Moreover, the bidirectional ogy remains in its infancy, with all current and emerging
signaling between implanted biomatrices and their host macromolecular assemblies or ECM mimics having serious
environment, i.e., the dynamic reciprocity that drives the limitations. As we advance the science of stem cell–ECM
process of constructive remodeling to ensure successful nanointeractions, we will become more precise in our
graft integration, and how this signaling may be modulated ability to directly dictate regenerative processes based on
and exploited to enhance tissue regeneration are far from tissue ECM materials or information coded within the ECM,
fully characterized [754]. An increasing appreciation for the which provides signposts for advanced biomaterial design.
microstructure, biomechanical properties and functional- Elucidating the molecular pathways through which cells
ity of manmade biological systems has led biomaterials discriminate signals from their ECM will reveal important
scientists to reconsider nature, the master architect of signposts in designing new biomimetic polymers tuned
biomaterials, for design inspiration [293,657]. By support- for a more defined cellular response, allowing for more
ing microenvironmentally sensitive and cell-dependent controlled and efficient tissue regeneration if progress
binding affinity and integrin specificity, the natural ECM continues. Additionally, materials science strategies are
displays complex interactions with cells, leading to finely bridging the gaps in various but interrelated scientific
152 F.-M. Chen, X. Liu / Progress in Polymer Science 53 (2016) 86–168

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