4.

967

Review

Nature-Based Biomaterials and

Their Application in Biomedicine

Eoin Troy, Maura A. Tilbury, Anne Marie Power and J. Gerard Wall

Special Issue
Polymeric Biomaterials for Biomedical Applications
Edited by
Dr. Catalina Vallejo-Giraldo

https://doi.org/10.3390/polym13193321
 polymers
Review
Nature-Based Biomaterials and Their Application
in Biomedicine
Eoin Troy 1 , Maura A. Tilbury 1,2 , Anne Marie Power 3 and J. Gerard Wall 1,2, *

1 Microbiology, College of Science and Engineering, National University of Ireland, NUI Galway,
H91 TK33 Galway, Ireland; [email protected] (E.T.); [email protected] (M.A.T.)
2 SFI Centre for Medical Devices (CÚRAM), NUI Galway, H91 TK33 Galway, Ireland
3 Zoology, School of Natural Sciences, NUI Galway, H91 TK33 Galway, Ireland;
[email protected]
* Correspondence: [email protected]

Abstract: Natural polymers, based on proteins or polysaccharides, have attracted increasing interest
in recent years due to their broad potential uses in biomedicine. The chemical stability, structural
versatility, biocompatibility and high availability of these materials lend them to diverse applications
in areas such as tissue engineering, drug delivery and wound healing. Biomaterials purified from
animal or plant sources have also been engineered to improve their structural properties or promote
interactions with surrounding cells and tissues for improved in vivo performance, leading to novel
applications as implantable devices, in controlled drug release and as surface coatings. This review
describes biomaterials derived from and inspired by natural proteins and polysaccharides and
highlights their promise across diverse biomedical fields. We outline current therapeutic applications
of these nature-based materials and consider expected future developments in identifying and





 utilising innovative biomaterials in new biomedical applications.
Citation: Troy, E.; Tilbury, M.A.;
Keywords: biomaterial; scaffold; tissue engineering; drug delivery; collagen; gelatine; silk; cellulose;
Power, A.M.; Wall, J.G. Nature-Based
Biomaterials and Their Application in
chitosan; alginate
Biomedicine. Polymers 2021, 13, 3321.
https://doi.org/10.3390/
polym13193321
1. Introduction
Academic Editor: Traditional biomaterials used in biomedicine, such as gelatine, silk and collagen, were
Catalina Vallejo-Giraldo derived from natural sources [1], with their first clinical applications dating to the 1950s [2].
While they have had enormous impact on patient quality of life to date, they are being
Received: 6 August 2021
continuously modified, exploiting advances in the fields of molecular and cellular biology
Accepted: 17 September 2021
and polymer chemistry, to improve their material properties, bioactivities and suitability
Published: 28 September 2021
for therapeutic applications [3]. Furthermore, as biomaterials expand into new applications
such as drug delivery, tissue engineering, scaffolds and bioprinting [4], new and modified
Publisher’s Note: MDPI stays neutral
materials are being developed that can remain in intimate and productive contact with
with regard to jurisdictional claims in
tissues in the body for long periods [5].
published maps and institutional affil-
Biologically inert materials were originally favoured for biomedical applications on
iations.
the basis of safety and stability. Years of clinical use have identified that even inert mate-
rials may elicit damaging cellular and immunological responses, however [6,7]. As a re-sult,
biomaterials must now, at a minimum, interact with their surrounding tissues, while in
more advanced applications, they may be designed to interact with surrounding cells
Copyright: © 2021 by the authors.
and tissues to promote tissue healing and regeneration. Biomaterials that are biologically
Licensee MDPI, Basel, Switzerland.
inert and passive are augmented with drugs, growth factors or gene delivery vectors to
This article is an open access article
manipulate cellular responses in vivo for greater therapeutic effect [8,9].
distributed under the terms and
This review describes the broad range of current biomaterials that are derived from, or
conditions of the Creative Commons
Attribution (CC BY) license (https://
inspired by, natural proteins and polysaccharides. We summarise the diverse biomedical
creativecommons.org/licenses/by/
and biotechnological roles successes of these materials to date, in fields such as regen-
4.0/).
erative medicine and therapeutics. Finally, we consider potential future directions for

Polymers 2021, 13, 3321. https://doi.org/10.3390/polym13193321 https://www.mdpi.com/journal/polymers

Polymers 2021, 13, 3321 2 of 37

the development and modification of novel biomaterials with broader and more effective
biomedical applications.

1.1. Which Biomaterial?

Choosing a suitable biomaterial for, e.g., scaffold construction is critical for its success.
In vivo target sites at which biomaterials are used differ greatly and include soft (ligament,
skin, cartilage, muscle, nerve, tendon, vascular sites) [10] and hard (bone and teeth) [11]
tissues with very different biological and physicochemical properties [12]. Traditional
alloys, metals and ceramics with limited functionality are increasingly being replaced by
more versatile materials, while polymeric biomaterials are replacing permanent prosthetics
due to concerns about the lower biocompatibility of the latter and the need for revision
surgeries [13]. Increasing numbers of polymeric biomaterials are emerging from synthetic
and natural sources, with diverse applications in tissue engineering and regeneration, as
well as in specialist fields such as drug delivery, nanotechnology and gene therapy [14].
Biodegradable polymeric biomaterials can be divided into natural and synthetic
materials, based on their origin and whether they contain naturally occurring extracellular
matrix (ECM) [15]. Commonly used synthetic polymers, such as polyglycolide (PGA),
polylactic acid (PLA) and poly(lactic-co-glycolic acid) (PLGA), are often cheaper to produce
at scale and have more homogeneous structures, mechanical strengths and degradation
rates [16]. They lack natural binding sites for cells, however, resulting in lower bioactivities
and increased in vivo rejection rates than natural biomaterials [17]. Natural biomaterials
are sub-classified based on their composition and include protein-based materials such as
collagen, gelatine, silk and fibrin, and polysaccharide-based materials such as cellulose,
chitosan and alginate [5,15], as described in detail below.

1.2. Biomaterials in Biomedicine

Scaffolds that act as templates for tissue regeneration and guide the development of
new tissues can be produced from a diverse range of materials (Table 1) [18]. Natural ECM
can act as a porous 3D microenvironment “scaffold”, with its multitude of growth factors,
effector molecules, enzymes and cellular adhesion motifs influencing cell proliferation,
gene expression and intracellular signalling [19–21]. Due to the complexity and variability
of ECM, however, scaffolds used in tissue engineering are typically more tailored to
particular functions, such as promoting cell adhesion or differentiation [22,23]. Regardless
of the clinical objective or environment, all biomaterial scaffolds share the following
essential properties.

1.2.1. Biocompatibility
Scaffolds should allow or promote cells to adhere, proliferate and spread before
creating a new matrix, and must not elicit an inflammatory response that could lead to
infection, longer healing times or patient discomfort [18].

1.2.2. Biodegradability
Scaffolds should ideally be temporary templates which will be replaced by newly
regenerated tissue [24]. Therefore, they should be biodegradable, resulting in non-toxic
breakdown products which are safely excreted without interfering with normal bodily
functions [18].

1.2.3. Structure
Scaffolds must be highly porous to promote cell migration, waste dispersal, scaffold-
tissue interaction and nutrient and fluid permeability [25]. Cell-binding ligands may be
naturally present in ECM-derived scaffolds or incorporated into synthetic materials [18].
Pores must be large enough to allow infiltration of cells but small enough to establish a
suitable cell density attached to the scaffold [18,26].
Polymers 2021, 13, 3321 3 of 37

1.2.4. Mechanical Properties

Scaffolds must have sufficient mechanical strength to maintain their structural integrity
—including during transport, surgical handling and implantation [27–29]. Engineered scaf-
folds should typically mimic the mechanical properties of their target tissue [27] and
therefore vary greatly between, e.g., soft tissue applications and bone or cartilage scaf-
folds [30,31].

1.2.5. Manufacturing Technology

Scaffolds must be cost-effective to produce and easily scalable from laboratory pro-
duction. Production must also be compatible with good manufacturing practice (GMP)
standards [32] for translation from the laboratory to the clinic [33].

Table 1. Summary of the different protein and polysaccharide-based biomaterials, their sources, main properties, structural
forms used in biomedicine and biomedical applications.

Material Source Properties Structures Biomedical Applications

Scaffolds
Weakly immunogenic
Sponges Tissue repair [34–38]
Natural ECM or Cross-linked to increase
Collagen Hydrogels Wound care [39–41]
recombinant strength, stability
Films/membranes Drug delivery [42–45]
Cell binding
Bioinks
Biocompatible
Biodegradable Drug delivery
Bovine or porcine
Gelatine Cross-linked to increase Microparticles [46–52]
collagen
strength, stability Tissue regeneration [53]
Cell binding
High
Butterflies/moths, Wound dressings [22,54,55]
strength-to-density Films
Silk spiders Suturing [56,57]
Insoluble in water Woven meshes
or recombinant Device coatings [58,59]
Highly stable
Tissue engineering [60,61]
Biocompatible
Nanofibres Artificial blood vessels
Combine stiffness and
Cellulose Plants, bacteria Gels [62–64]
flexibility
Nanocrystals Drug delivery [65–67]
Tuneable properties
Wound repair [68–77]
Rigid structure Films
Exoskeleton of Insoluble in water Fibres Wound healing [78]
Chitosan crustaceans; plant cell Biodegradable Scaffolds Anti-microbial coatings [79,80]
envelopes Antimicrobial Hydrogels Drug delivery [81–84]
Versatile Nanoparticles
Widely available Hydrogels Wound healing [85–89]
Inexpensive Sponges Drug delivery [90–95]
Alginate Brown algae
Biodegradable Films Tissue engineering
Excellent gelating Microparticles [94,96–100]

2. Protein-Based Biomaterials
In nature, an array of proteins play vital structural roles in living organisms, which has
led to their incorporation in recent years into protein/polypeptide-based biomaterials based
on their structural chemistry, cellular interactions or cell communication properties [101].
Non-structural proteins are also gaining attention due to their ability to modulate the
functional properties of biomaterials. In this section, we review protein-based materials
derived from natural sources and consider their increasing impact in biomedicine.

2.1. Collagen
Collagen is the most abundant structural protein in humans and animals. It makes
up approximately 30% of all mammalian proteins and is an essential component of the
Polymers 2021, 13, 3321 4 of 37

ECM [102]. By virtue of its characteristic fibrillar structure, it provides structural support to
hard and soft tissues, including cartilage, tendon, bone, ligament and blood vessels [103].
The collagen family consists of 29 distinct collagen types which are divided into four
classes based on their composition and structural properties [102]. All types exhibit a
characteristic triple helix structure, consisting of three α-chains comprised of more than
1000 amino acids and a repeating Gly-X-Y sequence. The glycine residues allow tight
inter-molecular packaging of the α-chains while the X and Y positions are typically filled
by proline and 4-hydroxyproline, respectively [104]. Of the 29 types, only types I, II, III, V
and XI are known to form collagen fibres [103] and these are favoured in collagen-based
biomaterials [105].

2.1.1. Biological Characteristics

Collagen is weakly immunogenic, with fibrillose collagen exhibiting lower immuno-
genicity than smaller molecules due to the burial of potentially antigenic sites during
its auto-polymerisation [103]. Removal of non-helical regions and cross-linking of colla-
gen chains can be carried out to further reduce antibody recognition [106]. Cross-linking
also provides stability and increases resistance to collagenase activity—properties which
contribute to its biocompatibility and degradability in biomedical applications [103,107].

2.1.2. Cell Binding

Specific peptide sequences in collagen bind four different types of cell-surface recep-
tor. GPO (Gly-X-Y) motifs, where X and Y can be any amino acid residue but are most
commonly proline [108], bind type 1 receptors, which includes glycoprotein VI [109]. GFO
(Gly-Phe-Hyp) inter-act with type 2 receptors, which consist of collagen-binding integrin
proteins and discoidin domain 1 and 2 [110,111]. Small molecule-binding cryptic domains
in collagen [112] bind various integrins in type 3 receptors, while type 4 cell receptors
bind non-collagenous domains within the protein [113]. Indirect cell-collagen interactions
also promote cellular adhesion to the ECM, often via fibronectin (an ECM glycoprotein).
Fibronectin was the first molecule on which the integrin-binding sequence RGD (Arg-Gly-
Asp) was identified, which has since been found in many protein types, explaining their
ability to bind collagen [103,114]. Receptor-binding motifs make an important contribution
to the success of collagen as a biomaterial scaffold by aiding in cell seeding, adhesion and
promoting cell differentiation and migration.

2.1.3. Obtaining Collagen

Collagen can be obtained for biomedical applications from mammalian sources such
as cows, pigs, rats and sheep [105,115], as well as human peripheral nerve tissue [116] or
human placenta [117]. It is purified by decellularisation, in which cellular antigens from
the natural collagen matrix are removed while maintaining the ECM shape and structure,
or by an extraction, purification and polymerisation approach which yields more refined
scaffolds [103]. Decellularisation can be achieved by physical, chemical and enzymatic
processes [118]. Physical decellularisation disrupts cell membranes and promotes cell
lysis by the use of rapid freezing or high pressure approaches, and may be combined
with chemical steps to increase tissue penetration [118,119]. Chemical decellularisation
involves the addition of alkalines, acids, detergents and chelating agents to remove the
cellular components of ECM while enzymatic decellularisation utilises the proteolytic
enzyme trypsin, nuclease enzymes and ethylenediaminetetraacetic acid (EDTA) to remove
proteins and DNA and RNA [103,118]. As none of the methods yields an ECM entirely
free of cellular material, several techniques are typically combined to produce a pure,
decellularised ECM [103] suitable for use as ligament prostheses or cardiac valves.
Extraction and purification of collagen from natural sources can also be carried out
by solubilisation and purification. Due to its covalent cross-linking, collagen exhibits low
solubility in organic solvents and is typically solubilised in acidic (0.5 M acetic acid), neutral
salt (0.15–0.20 M NaCl) or proteolytic solutions.
Polymers 2021, 13, 3321 5 of 37

2.1.4. Cross-Linking Collagen

Unlike decellularised collagen which has been naturally polymerised in vivo, ex-
tracted collagen must be cross-linked to increase its mechanical strength and resistance
to enzymatic degradation. There are several well-established methods of cross-linking,
including physical processes such as ultra-violet (UV) or thermal treatment [120], via
chemicals such as formaldehyde and glutaraldehyde [121], and by the use of cross-linking
enzymes such as transglutaminase [122]. The addition of biomolecules such as elastin [123],
chitosan [124] and glycosaminoglycans (GAG) [125,126] during cross-linking can be used
to improve the cell differentiation, migration, or proliferation characteristics, or mechanical
properties of the resultant scaffolds [103,107].

2.1.5. Sterilisation of Collagen

Whether purified via decellularisation or solubilisation, extraction and cross-linking,
collagen must be sterilised for in vivo use. Due to its relatively fragile, temperature-
sensitive structure, sterilisation methods can alter its molecular properties. Even low-dose
gamma irradiation reduces its enzymatic resistance and mechanical strength, though the
addition of glucose can mitigate these effects by mediating cross-linking [127]. β- and
electron-beam irradiation, while less damaging than gamma irradiation, have still been
reported to cause structural degradation, and reduced mechanical strength and resistance
to enzymatic degradation [103]. Immersion of collagen in low concentrations of sterilant
is becoming a popular sterilisation approach, with low concentration pancreatic acid
effective for decellularised collagen, and an ethanol/antibiotic mix for extracted, cross-
linked collagen. As no approach can completely avoid altering the collagen structure,
however, it is essential to thoroughly investigate the effect of each method on the properties
of the resultant material relative to its intended application [103].

2.1.6. Recombinant Production

As collagen is primarily obtained from animal sources, the transmission of infectious
disease is a concern in therapeutic applications. The recent COVID-19 pandemic exemplifies
the dangers of zoonotic infections, while cases of bovine spongiform encephalopathy have
resulted from the use of prion-contaminated bovine scaffolds [128]. Meanwhile, religious
constraints surround the use of porcine and bovine materials, and up to 2–4% of the world’s
population may be allergic to porcine and bovine-derived collagen [129]. These factors, as
well as the heterogeneity of natural collagen preparations, have led to the development of
approaches to produce recombinant human collagen (rhCOL) [130,131].
Escherichia coli is the best-established expression system for recombinant proteins due
to its ease of genetic manipulation, rapid growth rate, track record in protein production
and suitability to scale-up [132]. It can produce a protein similar to human collagen which
contains the characteristic Gly-X-Y sequences but differs from the native collagen through
its lack of proline and lysine hydroxylation [133]. The failure of E. coli to carry out these post-
translational modifications (PTMs) results in a collagen with reduced thermostability and
limited fibre assembly, thereby restricting its usefulness in tissue engineering [131]. Cloning
of prolyl and lysyl hydroxylase genes from the aquatic giant Mimiviridae virus family
into E. coli has enabled the successful production of molecules with hydroxyproline and
hydroxylysine patterns that are characteristic of human collagen and capable of supporting
the growth of human endothelial cells [134], but despite this success, eukaryotic systems
continue to dominate recombinant production of collagen.
Since the expression of human interferon in Saccharomyces cerevisiae in the 1980s [135],
yeasts have been very successfully used in recombinant protein production. S. cerevisiae
and Pichia pastoris are the two most commonly used yeast hosts [136] and their utility is
due to their eukaryotic protein folding mechanisms and ability to carry out PTMs required
for many proteins’ functioning. Like prokaryotic systems, they lack native lysyl and propyl
hydroxylases [132] but co-expression of human hydroxylases enables them to produce
rhCOL closer in structure and properties to native human collagen that that expressed
Polymers 2021, 13, 3321 6 of 37

in E. coli [132]. rhCOL from both S. cerevisiae and P. pastoris has been used to produce
hydrogels for wound healing applications [131].
A variety of mammalian systems have also been investigated for the accurate produc-
tion of human collagen. Chinese hamster ovary (CHO) cell-derived rhCOL was shown
to reverse the disease phenotype of dystrophic epidermolysis bullosa (characterised by
collagen deficiency within the skin) when administered intravenously, without eliciting
an immune response in mice models of the disease [137]. Human HeLa cells [138] and
embryonic kidney cells [139] have also been used to produce rhCOL types I, V and VII
identical to native human collagen produced in vivo. Yields are much lower than from
other expression systems, however, so non-human animal platforms are typically preferred.
rhCOL has also been produced in transgenic animals, with mouse embryos transfected
with a COL1A1 gene found to secrete correctly folded rhCOL type I through their mammary
glands [140]. Transgenic animal production systems are considerably more specialised and
expensive than cell-based systems, however [132].

2.1.7. Collagen-Based Scaffolds

Upon obtaining collagen, scaffolds of pure collagen, a collagen/natural polymer blend
or a collagen/synthetic polymer blend can be produced [141].
Collagen types I, II and III have been electrospun into fibres at submicron to nanometre
scale that replicate the biological properties of ECM. Collagen fibres with a 67-nm binding
pattern, similar to native collagen, have been formed from electrospinning collagen type
I [142], leading to the production of biomimetic scaffolds with tuneable porosity, mechanical
strength and fibre alignment to topographically guide tissue formation. Electrospun
collagen scaffolds have also been demonstrated to support cellular growth [142–144]. Pure
collagen scaffolds have weak structural stability and mechanical strength, however. While
this can be improved by cross-linking fibres using UV irradiation or dehydrothermal
treatment (high temperature exposure under a vacuum), care must be taken to ensure no
residues of toxic cross-linking chemicals such as glutaraldehyde remain in the scaffold [141].
Scaffolds can also be constructed by blending natural or synthetic polymers with
collagen for improved mechanical properties. Natural polymers already established in
tissue engineering include fibroin, chitosan and silk. Chitosan is a non-immunogenic,
biodegradable, positively charged polymer which has been combined with collagen to
form scaffolds with excellent mechanical and biological properties, as well as excellent
compatibility with a range of seeded cell types [145–148]. Synthetic polymers utilised
in blended collagen scaffolds include PLA and polyethylene glycol (PEG) [141]. In this
scenario, the synthetic polymer typically improves the mechanical properties and struc-
ture of the scaffold while collagen provides cell signalling and binding sites crucial for
tissue repair.

Collagen Sponges
Collagen sponges are produced from insoluble collagens extracted from cows or
pigs. The scaffold is created by freeze-thawing alkali and aqueous acid collagen solutions
containing up to 5% dry matter, with the rate and temperature of freezing determining
the pore size and structure: rapid freezing at extremely low temperatures cause cracking
to occur in the collagen, resulting in small channels and a highly fibrous structure, while
slower freezing and higher temperatures causes the collagen to have large, non-uniform
pores and more continuous channels [103].
Sponges are ideal for use in wound care as they adhere smoothly to the wound bed, are
capable of absorbing large volumes of exudate, maintain a moist environment and shield
against physical trauma and bacterial infection [104]. Cross-linking with glutaraldehyde or
other polymers can be used to increase their mechanical strength. As collagen promotes
invasion of immune/inflammatory cells such as neutrophils, sponges have potential uses in
treating burns, diabetic ulcers and at donor sites [39]. Loading of sponges with exogenous
growth factors can also be used to improve wound healing, such as platelet-derived growth
Polymers 2021, 13, 3321 7 of 37

factor (PDGF) and fibroblast growth factor (FGF) to promote capillary formation and
epidermal wound healing, respectively [40,41]. Collagen sponges have also been used to
provide sustained delivery of antibiotics such as vancomycin [42] and gentamycin [43]
to treat sepsis, and intra-vaginal delivery of retinoic acid to avoid systemic effects in the
treatment of cervical dysplasia [44].

Collagen Hydrogels
A hydrogel is a 3D network of polymers which can hold significant volumes of
fluids. The main therapeutically-relevant properties of polymeric hydrogels are their water
retention, due to the hydrophilic functional groups on their polymeric backbone, their
resistance to dissolution due to their cross-linking and their similar flexibility to natural
ECM [149]. Due to their structural similarity to tissue, collagen hydrogels are frequently
investigated as biomimetic 3D scaffolds to support cell growth [150].
The amphoteric (adsorbs to both anions and cations) nature of collagen type I fibres
enables them to form hydrophobic, dipole-dipole, electrostatic and hydrogen interactions
which lead to gel formation in aqueous systems [150]. The gels can typically be dissociated
by collagenases or changes in temperature or pH [151]. While the natural cross-linking
of collagen confers proteolytic resistance and mechanical strength, additional physical or
chemical cross-linking can be necessary to prevent enzymatic degradation [152]. Of these
approaches, glutaraldehyde cross-linking via lysine and hydroxy-lysine residues [153],
sometimes used in combination with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
(EDC) cross-linking of carboxyl and amine groups [150], yields the most stable hydrogels
and can be designed to vary the mechanical properties and function of the hydrogel [150],
though some cytotoxicity has been reported [154].
Figure 1 illustrates non-cross-linked collagen compared with glutaraldehyde cross-
linked and EDC cross-linked collagens.

Figure 1. Collagen hydrogels: (a) non-cross-linked, (b) glutaraldehyde cross-linked and

(c) EDC/NHS cross-linked [150].

Collagen hydrogels are attractive scaffolds in tissue engineering due to their retention
of cells and bioactive molecules. They have found widespread application in cartilage
and bone tissue engineering, such as acting as carriers for bovine chondrocytes [34] to
provide structural support and pain-free articulation of cartilage [35]. As chondrocytes
produce cartilage ECM, their transplantation in collagen hydrogels can be used to treat
a variety of articular cartilage defects [36] or to engineer bone tissue [37]. Osteoblasts
derived from calf metacarpus periosteum, have also been demonstrated to proliferate and
migrate within a 3D collagen hydrogel without any loss of viability over three weeks, as
well forming a bone-like ECM containing osteonectin, osteocalcin and new collagen type
I [37]. Collagen type I hydrogel-mediated treatment of bone defects in rat dorsal nasal
bones was demonstrated to lead to the growth of a thin layer of bone after six weeks [155].
Blending of synthetic polymers such as polyvinyl alcohol (PVA) and polyacrylic acid (PAC)
with collagen in hydrogels can be used to improve the mechanical strength of the natural
polymers and the biocompatibility of the synthetic molecules [1]. In one such example,
PVA was blended with collagen and used to form sponges, films and hydrogels, which
Polymers 2021, 13, 3321 8 of 37

were loaded with growth hormone. Release of the hormone was monitored in vitro by
enzyme-linked immunosorbent assay and could be tuned by altering the collagen content
of the gels [156].
Collagen type I is commonly used as in bioprinting but its slow gelation rate at physio-
logical temperatures means that it must typically be used in combination with biomolecules
that improve its structural integrity [4] (Figure 2). Collagen-alginate bioinks have been
demonstrated to have increased mechanical strength and to accelerate the proliferation
of human chondrocytes for articular cartilage repair, [38] while printed collagen-alginate
hydrogels have also demonstrated sustained release of antibacterial drugs [45].

Figure 2. Bioprinting of natural polymers, frequently in combination with cells and/or biomolecules
to fine-tune or increase in vivo activity, has potential to provide carefully designed, highly structured
materials for tissue and organ engineering applications.

Collagen Films/Membranes
Collagen films of 0.01–0.5 mm thickness can be produced by drying bovine collagen
that has had its telopeptides (nonhelical regions that flank collagen’s triple helix) removed,
followed by a series of enzymatic and chemical cross-linking steps [39,157]. Their main
applications are as barriers to protect wounds or ulcers, while simultaneously releasing
therapeutic drugs. Drugs can be loaded onto films via covalent or hydrogen bonding or
through simple entrapment and the films can be easily sterilised without affecting their
mechanical properties [39]. Collagen films are well established in wound dressing applica-
tions, as well as reinforcing compromised tissues and guiding tissue regeneration. They
have also been used to deliver antibiotics [158,159] and, as collagen-coated polyurethane
(PU) films, to promote attachment and proliferation of fibroblasts [160], which stimulates
further collagen synthesis and the formation of new connective tissue and ECM. Individual
films can also be easily combined into multiple-layered membranes which can release
molecules such as PDGF at constant rates for up to 100 h in vivo to aid wound healing and
tissue regeneration [107], or human growth factors to support healing of diabetic ulcers
in murine models [161]. Collagen-based scaffolds have also been utilised as a resorbable
template, alone or with additional components such as hyaluronic acid [162] or synthetic
polymers [163,164], to regenerate the meniscal template of the knee, with promising in vivo
outcomes [165].
Polymers 2021, 13, 3321 9 of 37

Collagen-based materials are currently to the fore amongst biomaterials used in

regenerative medicine and tissue engineering, based largely on their low immunogenicity,
high biocompatibility and structural versatility. Advances in extraction and scaffold
formulation have led to increasingly diverse applications of collagen in fields such as
wound healing, drug delivery and tissue regeneration. Future research is likely to focus
on improving the mechanical strength, drug delivery capabilities and biodegradability of
collagen-based scaffolds in order to enhance their in vivo efficacies.

2.2. Gelatine
Gelatine is a well-characterised, biocompatible and biodegradable polymer which
is formed by disintegration and denaturation of natural collagen, typically of bovine or
porcine origin [166]. It is commonly utilised in food and cosmetic production as a cheaper
alternative to collagen and has extensive pharmaceutical applications. It is a derivative
of type I fibrillar collagen and contains up to 92% pure protein, as well as mineral salts
and water [46]. It exhibits several advantages over its parent collagen in therapeutic
applications, including reduced immunogenicity [167], increased solubility in aqueous
systems and ease of transition from solution to gel at temperatures of 30 ◦ C [168].

2.2.1. Gelatine Structure

The structure of gelatine depends on the source and method of denaturation of the
parent collagen [168]. This leads to heterogeneity of gelatine and batch-to-batch variabil-
ity in its molecular weight from a thousand to greater than a million Dalton [166,169].
Denaturing collagen forms a gelatine solution with very low viscosity, which forms a
gel at temperatures below 37 ◦ C. This reversible process of thermo-responsive gelation
results from peptide coils transitioning into helices due to the high numbers of Gly-Pro
bonds within the structure [170]. As the gels return to a liquid state at temperatures above
37 ◦ C, this makes natural gelatine unstable for in vivo use, though numerous cross-linking
approaches have been developed to stabilise the macromolecular structure and avoid its
rapid degradation in host tissues [169], as outlined below.
Mammalian gelatine is preferred for in vivo applications due to its high concentration
of cell-binding domains, which create an excellent substrate for recruitment and attachment
of adherent cells. Meanwhile, gelatine is less antigenic than collagen due to its lower
composition of phenylalanine and absence of tyrosine and tryptophan which form aromatic
rings and radicals that can promote an antigenic response [171].

2.2.2. Obtaining Gelatine

Gelatine is produced by thermal denaturation and hydrolysis of collagen. Heating to
only 40 ◦ C disrupts the interior structure of newly formed or highly soluble collagen [169],
whereas non-soluble collagen requires an additional hydrolysis treatment using acid or
alkali solutions [169]. Soaking in dilute acidic solutions yields gelatine type A while
submersion in alkali solutions produces gelatine type B, with the former more similar to
collagen in isoelectric point and amino acid composition [172].

2.2.3. Cross-Linking Gelatine

As with collagen, gelatine can be cross-linked using chemical, physical or enzymatic
approaches. Physical cross-linking involves physical gelation via heat and pH changes
as described above. To avoid the reversibility of physical gelation upon temperature or
pH changes, chemical methods have been developed to produce a more stable polymer.
Glutaraldehyde cross-linking is typically the preferred strategy as glutaraldehyde is easily
accessible, cheap and rapidly boosts the mechanical strength of gelatine [151]. Chemical
cross-linking can leave behind traces of potentially toxic chemical agents, however, leading
to investigation of natural cross-linking agents such as caffeic acid, tannic acid, genipin and
grape seed proanthocyanidin. Genipin, derived from the fruits of Gardenia jasminoides [173],
is far less toxic than glutaraldehyde and achieves comparable mechanical properties in
Polymers 2021, 13, 3321 10 of 37

the cross-linked gelatine, but is limited by its high cost and formation of a dark blue
pigment which can constrain its biomedical use [46,174]. Enzymatic approaches have been
used to synthesise highly stable gelatine structures, with transglutaminases in particular
favoured due to their abundance in nature and ability to produce a mechanically strong
product [175].

2.2.4. Gelatine Biomaterials

Gelatine is favoured in biomedical applications due to its commercial availability,
low price, high solubility, biocompatibility, biodegradability, the presence of cell-binding
domains and its lack of antigenicity or toxicity to cells [46]. Its disadvantages, however,
surround its poor mechanical properties, lack of thermal stability, greater susceptibility
to some proteases than collagen and faster degradation [176]. To overcome these disad-
vantages, advanced manufacturing techniques and cross-linking approaches are used to
improve the thermal and mechanical stability, biocompatibility and overall bioactivity of
native gelatine.

Gelatine Microparticles
The ability of gelatine to form a gel, its biocompatibility and its biodegradability
make it ideal for the production of microparticles. Gelatine microparticles are extensively
used as drug carriers due to their ease of production, stability and lack of toxicity, as
well as their ability to interact with multiple bioactive compounds [177]. While smaller
particles are used to protect and control the release of bioactive molecules in vivo [47],
such as growth factors to stimulate cell proliferation and differentiation [46,48], larger
microparticles, with modified surfaces for improved cell attachment and differentiation,
can be used as “microcarriers” of cells, e.g., delivery of embryonic stem cells to aid bone
regeneration [53].
Gelatine microparticles are traditionally produced by techniques such as solvent
evaporation, spray drying and precipitation, though these may cause denaturation [47] or
leave solvent traces in the final product [178] (Figure 3). Therefore, improved production
methods such as water-in-water emulsification have been developed [177].

Figure 3. Loading of gelatine microparticles with growth factors and/or cells to direct cellular
differentiation (based on [46]).

Drug Delivery
The ability of gelatine microparticles to deliver anti-inflammatory [49], antibacte-
rial [50] and antineoplastic [51,52] agents is well established. While encapsulation in the
microparticle typically improves a drug’s pharmacokinetic profile and efficacy [179], a
Polymers 2021, 13, 3321 11 of 37

major challenge associated with nano- and micro-scale drug carriers is that they are com-
monly phagocytosed in vivo [51]. PEGylation has shown promise in protecting gelatine
microparticles against opsonisation (particle attack from phagocytic immune response)
and reducing their immunogenicity [51], as demonstrated in their improved delivery of
doxorubicin and reduced cytotoxicity in a mouse model of pulmonary metastasis [180], and
in sustaining release of ibuprofen in vivo, reducing the need for repeated injections [181].
Gelatine microparticles have also been used in optimised targeting of drug delivery,
which is particularly important to avoid side effects and toxicity of oncology treatments.
Magadala and Amiji [182] introduced an epidermal growth factor receptor (EGFR) recogni-
tion sequence into the gelatine amino acid backbone, leading to gene delivery to and uptake
by EGFR-expressing Panc-1 human pancreatic adenocarcinoma cells, in an approach with
clear potential to improve safety and efficacy of pancreatic cancer treatment [182].
Overall, gelatine is a versatile, biocompatible and widely available biomaterial which
offers versatility across a range of tissue engineering and drug delivery applications. Given
recent advances in targeted drug delivery, it is easy to envisage the continued development
of gelatine-based systems in oncological applications.

2.3. Silk
Silk is a fibrous protein which has been used in the textile industry for centuries. It is
naturally produced by arthropods, including silk “worms” which in fact are butterflies and
moths (order Lepidoptera), and members of the class Arachnida (approx. 34,000 species
of spiders). These organisms produce silk in specialised endothelial cells, followed by
secretion into the lumen of their glands and spinning into fibres to build cocoons, nets,
traps and webs [183]. The best-characterised silks are from the orb spiders Nephila clavipes
and Araneus diadematus and the silkworm Bombyx mori [184]. The mechanical strength
of spider silks is much greater than that of silkworm silks, but difficulties in cultivating
predatory spiders has led to silkworm silk being more commonly used in the commercial
silk industry, with spider silks (particularly their spidroin components) generally produced
recombinantly [54]. Like collagen, silk is a fibrous protein and characterised by a repetitive
primary sequence, which in turn leads to homogeneity in its secondary structure.

2.3.1. Fibroins from B. mori Silk

Naturally produced B. mori silks are produced by larvae which grow for up to six
weeks before they spin silk fibres to form cocoons which protect the larvae against pre-
dation, moisture and microorganisms (Figure 4) [185]. Harvesting and unravelling of
the cocoons is then carried out to obtain the silk fibres, of which silk fibroin, the main
component protein, is of particular interest in drug delivery, wound healing and tissue
engineering applications [54].
Silks contain two or more fibroin proteins containing hydrophilic light (25 kDa) and
hydrophobic heavy (325 kDa) chains linked by disulphide bonds [54]. The proteins are
enclosed in a glue-like coat known as sericin which fuses the fibres to form the cocoon
casing and must be removed by boiling in sodium carbonate [54]. The fibroins occur
in a series of interlocked nanofibrils which combine to form microfibrils of 20–200 nm
in diameter, with friction between the nanofibrils leading to the impressive mechanical
strength of the silk fibres. Glycoprotein P25 is also non covalently attached and plays an
essential structural role in the silk fibroin [186]. The hydrophilic light chains, dominated by
alanine (14%), serine (10%), glycine (9%) and N-terminal acetylated serine residues [54],
and the hydrophobic heavy chains, featuring glycine (46%), alanine (30%), serine (5.5%)
and valine (2%), form a crystalline, “anti-parallel” β-sheet, fishnet-like structure, which is
fundamental to the high mechanical strength of the silk nanofibre [187].
Once sericin has been removed, silk-based materials are usually produced for biomed-
ical applications by the dissolution of the fibrils/threads in calcium nitrate, lithium thio-
cyanate and 4-methylmorpholine N-oxide, followed by dialysis against pure water to
Polymers 2021, 13, 3321 12 of 37

remove electrolytes. The final fibroin solution can be stored at low temperature (months)
or room temperature (weeks) prior to the production of scaffolds or other biomaterials [54].
The nanofibrillar silk structure provides excellent strength, toughness, weight, flexibil-
ity and extensibility [188]. Its extremely high strength-to-density ratio is ideally suited to
biomedical applications. Remarkably, silk fibres have been reported to be stronger than
Kevlar and of equal strength to steel and nylon [189,190]. Silk fibroins are insoluble in
water and most organic solvents, and very stable at high temperatures, with side-chain
amino acid groups and peptide bonding reportedly stable up to 200 ◦ C [191]. They carry
no infection risk and, while some materials have failed to gain clinical approval due to
immunogenicity, this may have been due to the outer sericin layer as the core silk materials
invoke very mild inflammatory or immune responses while sericin can trigger allergic and
immune reactions, and the release of tumour necrosis factor-alpha (TNF-α) [192]. Removal
of sericin reduces the immunogenic response to silk fibroin to less than that of collagen or
synthetic PLGA [54]. Silk fibroins exhibit a slow loss of mechanical strength in vivo due
to enzymatic degradation, with non-toxic degradation products [54]. Overall, silk-based
biomaterials can be completely biodegradable several weeks post-implantation and 100%
bioresorbable following 11–12 months [193].

Figure 4. Silk spinning process (upper level) with liquid crystal and micelle theories of fibre formation
(lower panels). A solution of silk protein is secreted into the spinning gland. During thread formation,
the solution is passed through a channel in which ion exchange and phase separation occurs. Drawing
of the thread causes fibre formation, which may be assembled by crystalline alignment of proteins
(left) or micelle formation and assembly (right) (based on [189]).

2.3.2. Silk Fibroin Biomaterials

The properties of silks mean that they are outgrowing their traditional applications in
textiles and expanding into proof of concept experiments and pre-clinical investigations in
areas such as wound healing and gene therapy.
Polymers 2021, 13, 3321 13 of 37

Silk films can be easily produced from fibroin stock solutions by spin-coating or
layer-by-layer methods and have been utilised as scaffolds in numerous tissue engineering
applications. Silk films functionalised through chemical binding of RGD domains promoted
bone formation when scaffolds were seeded with osteoblasts [194], while silk and collagen
films exhibited equivalent abilities to support cell binding, differentiation and physiological
morphology in human cell culture [195,196]. A silk/chitosan composite film containing
40–50% chitosan was demonstrated to be a promising matrix for use in wound dressing
and artificial skin material [55], and seeding with human adipose-derived stem cells may
promote repair of soft tissue wounds in murine models [22].
Electrospinning of silk fibres can generate fibres with diameters from sub-micron
to nano-scale, with large surface areas capable of incorporating nano-sized molecules.
Spinning is usually carried out using the initial silk fibroin solution and fibres are treated
with methanol and washed with deionised water to form the characteristic β-sheet struc-
tures [54,197]. Electrospun silk fibroin fibres can be woven or compiled (non-woven) into
biomedical scaffolds which are gaining interest in wound healing due to their oxygen per-
meability, fluid drainage and water retention [54]. By incorporating silver nanano-particles,
a silk fibroin non-woven mat exhibited antimicrobial properties against skin commensals
Staphylococcus aureus and Pseudomonas aeruginosa that were equivalent to commercially
available wound dressings containing a 20× higher silver concentration [198]. Growth
factors can also be loaded onto mats to stimulate epithelialisation. Loaded mats were
shown to provide sustained release of epidermal growth factor (EGF) in a 3D model of
human skin, leading to 98% wound closure after 48 h compared with 8% in non-treated
control and 26% in silk only groups [199].
The analogous natural biosynthetic process (Figure 4) and shear thinning properties
of silk fibroin also make it suited to extrusion bioprinting [4]. A silk–gelatin combination
hydrogel, in which gelatin was provided bulk [200], was used to print a human ear which
exhibited in vitro and in vivo biocompatibility and maintained its shape and volume for
three months while promoting cellular infiltration and tissue integration [201].

2.3.3. Spider Silks (Spidroins)

Spider silk is one of the toughest known biomaterials. It owes its mechanical properties
to the organisation of the large spider silk proteins, one of which is spidroin [202]. While
natural fibres typically display lower strength and stiffness than synthetic materials, spider
silks are capable of absorbing up to three times more weight than Kevlar [203] —and have
been utilised in biomedicine for centuries, with ancient Greeks and Romans using spider
silk poultices to cover wounds and act as styptics (anti-antihaemorrhagics) [56]. They have
been more challenging to commercialise than insect (B. mori) silks, however, as they are
produced in much smaller quantities and spiders’ predatory nature makes them more
difficult to grow in large numbers. There are numerous types of spider silks produced from
separate glands on the body, with some spiders producing several forms for distinct prey
capture, shelter and dragline functions, but the best characterised is the major ampullate
gland (MA) silk produced by spiders in the Nephila genus, notably Nephila clavipes. Spider
silk is also much more elastic than insect silk and can resist more mechanical stress caused
by bending which, in addition to its shape memory, allows spider silk to outperform many
natural and synthetic fibres in specific biomedical applications [189], as outlined below.
The lack of sericin proteins in spider silk may also present some advantages.

Structure of Spider Silk (N. clavipes)

MA silk is one of six silk fibres produced by the genus Nephila and has a very defined
nanostructure composed of the two main structural proteins, major ampullate spidroin-1
(MaSp1) and -2 (MaSp2) [203]. The primary structure consists mainly of proteins with large
numbers of hydrophobic and non-polar amino acids [204] and highly repetitive amino acid
sequences of up to 50 residues which make up 90% of the overall silk protein [189,205]. An
alternating hydrophilic and hydrophobic pattern in the core domain dominates the primary
Polymers 2021, 13, 3321 14 of 37

structure and is thought to be necessary for phase separation during the silk spinning
process [189]. There are four oligopeptide motifs which are often repeated within MA silks:
(i) (GA)n/(A)n; (ii) GPGGX/GPGQQ; (iii) GGX; and (iv) a “spacer” sequence of charged
amino acids [206]. Structural and functional analysis of these sequences is ongoing but it is
clear that (GA)n/(A)n sequences form α-helices in solution and β-sheet structures when
assembled into fibres [189], while the GPGGX/GPGQQ and GGX sequences may encode
amorphous rubber-like structures within the protein [207]. Intermolecular disulphide
bonds stabilise protein dimers and trimers and may contribute to silk protein assembly [58].
When first secreted from glands, spider silks exhibit no stable secondary or tertiary
structure [208]. Long repetitive sequences allow intra- and inter-molecular interactions
with other proteins and enable the secondary, tertiary and quaternary structures to form
through the silk spinning process. As the final silk thread structure contains high electron
density areas within areas of noticeably lower densities [209], it is postulated that the
former areas, corresponding to a high concentration of β-sheets, promote mechanical
strength, while the latter, found in regions with amorphous structures, provide elasticity to
the silk fibres [189,210].

Spider Silk Assembly

Within the glands, spider silks have a random coil structure which quickly assembles
and becomes water-insoluble when secreted into the spinning duct [211]. Assembly in-
volves folding of the protein and is based on aligning and packing of single silk proteins,
with the alignment of repetitive hydrophobic core regions and multimerisation through
shear forces and the proteins’ terminal domains.
A number of alternative mechanisms of silk fibre assembly have been proposed
(Figure 4). The first indicates that silk proteins form micelles of up to 200 nm in diameter
due to their amphiphilic (both hydrophilic and hydrophobic) domains [212]. Dimerisation
of these proteins, with each dimer forming a hydrophobic tail, can prime self-organsiation
into micelles, followed by the combination of micelles into larger-diameter globules which
pass through the spinning duct to elongate and form fibres [189]. The second hypothesis
suggests that fibre assembly is based on the crystalline alignment of proteins within the
laminar flow of the spinning ducts, with high concentrations of monomeric and multimeric
proteins passing through the spinning duct and alignment of these proteins causing liquid-
crystalline behaviour in the spinning dope (polymer solution) [189], with fibres drawn
from the spinning duct through solvent loss.

Obtaining MA Silk
Due to the predatory nature of spiders and their low silk yields in captivity, recom-
binant expression approaches have been developed to obtain larger quantities of spider
silk. Initial expression attempts in E. coli [18] were typically limited by low yields, poor
solubility of the translated products and inclusion body formation, translation inefficiencies
due to differences in codon usage between the spider genes and the bacterial host, and
depletion of tRNA pools [213].
As mammalian cells can typically express and process much larger proteins than
prokaryotes, recombinant MaSp1 and MaSp2 silk genes were expressed in murine mam-
mary glands [214]. While use of a goat signal sequence enabled production of MA silk in
the milk of the transgenic mice, yields were too low for large-scale exploitation [214,215].
Silkworms have also been investigated as expression hosts for spider silk [216]. Using
the well-established Baculovirus gene expression system, spider silk protein was success-
fully produced in B. mori larvae [217]. Yields were again limited by the poor solubility of
the protein [217] though this could be overcome using a chimeric N. clavipes MaSp1 pro-
tein, resulting in expression and spinning of composite B. mori silk fibres with mechanical
strength equal to native MA silk [218].
Polymers 2021, 13, 3321 15 of 37

2.3.4. Spider Silk Biomaterials

Spider silks have found application in suturing, in coating of implants and as foams
and hydrogels due to their versatility, mechanical and environmental stability, and bio-
compatibility [56]. MA silk fibres have been used to suture severe tendon ruptures, which
are notoriously slow to regenerate. Traditional suture materials are not compatible with
interior suturing, can cause immune reactions and may lose mechanical stability over
time [56]. Silk fibres from N. clavipes were braided and used to secure tendon injuries [57],
with tensile strength equal to traditional sutures and no reduction of strength after more
than 1000 rounds of fatigue testing. No immune response was detected, emphasising the
potential of spider silk threads to replace many traditional suturing methods.

Organ Reconstruction
MA silk from Nephila species has been investigated in pre-clinical trials for use in
bladder reconstruction [219]. Single silk threads were cross-woven to form a mesh which
supported primary human urothelial cell (HUC) adhesion and growth in vitro without
additional biological stimuli. No changes were detected in epithelial-to-mesenchymal
transition or expression of fibrosis-associated genes while the HUCs could elongate on the
material and form a bladder mucosal layer without any toxicity [219], demonstrating the
potential of this silk type in bladder reconstruction.

Spider Silk Coatings

Coatings made from recombinant spider silks have also been deployed as “bioshields”
to reduce the foreign body reaction associated with the use of silicone in medical and
cosmetic procedures [59]. This formation of a capsule around the implanted material causes
contraction of the implant, leading to distortion of surrounding tissues [220] and infection,
pain and unsightly cosmetic results in up to 10–26% of silicone implant recipients [221],
particularly as biocompatibility of the implanted material decreases over time [59]. Coating
of silicone implants with a micro-thin layer of recombinant Araneus diadematus spider silk
protein eADF4(C16) [58] significantly increased their biocompatibility in the critical initial
months post-implantation [59]. This lack of immunogenicity of the eADF4(C16) spider silk
makes it an ideal candidate for improving the biocompatibility of implanted silicones and
other implanted devices and prosthetics currently hindered by immunological reactions.
Silk-based biomaterials have proved themselves one of the most versatile and promis-
ing protein-based biomaterial categories for biomedical applications. They have exceptional
biocompatibility, mechanical properties, biodegradability and cell-binding capabilities.
Silks from both silkworm and spider sources have excelled across multiple applications in
bone regeneration, tissue engineering/regeneration, suturing and improving the biocom-
patibility of implanted devices. Future research may focus on translating that success into
medical devices and prostheses, which may lower incidences of rejection and infection,
resulting in improved outcomes and postoperative patient comfort.

3. Polysaccharide-Based Biomaterials
Polysaccharide biomaterials can be obtained from a wide variety of sources and
have found application in cosmetics, food industries, biomedicine and pharmaceuti-
cals [222]. The biomaterials include alginates, the structural cell wall constituent of many
seaweeds [223], chitin and its derivative chitosan from crustacean and fungal sources [224],
and cellulose, the most abundant polysaccharide on earth [225]. Many polysaccharides
exhibit biocompatibility, stability and biodegradability, as well as widespread natural
abundance, which lends them potential in biomaterials applications [226,227].

3.1. Cellulose
Cellulose can be sourced from cotton, wood and other plant-based sources, as well
as from some bacterial sources [228]. It is the most abundant biopolymer in nature, with
almost 30 billion tonnes of natural cellulose biomass produced annually [229]. Plant cellu-
Polymers 2021, 13, 3321 16 of 37

lose (PC) exists as a primary material of the plant cell wall while bacterial cellulose (BC) is
chemically identical to PC but secreted by bacterial from genera such as Acetobacter, Pseu-
domonas and Sarcina [225]. Both forms of cellulose have tuneable biochemical, mechanical
and physical properties, as well as biocompatibility, good mechanics and bioactivity [230].

3.1.1. Molecular Structure of Cellulose

Cellulose is an unbranched carbohydrate polymer in which β-D-glucopyranose
residues are linked by β-1,4-glycosidic bonds [225]. Different crystalline forms can be
distinguished by their overall structure: cellulose I is the native form of cellulose and is
defined by inter- and intra-sheet hydrogen bonds and van der Waals interactions, parallel
β-(1,4′ )-D-glucopyranose chains and two main crystalline structures—triclinic cellulose
Iα and monoclinic cellulose Iβ [230]. Cellulose II, meanwhile, is produced by alkali treat-
ment of cellulose I and exhibits altered hydrogen bonding and antiparallel cellulose I
subunits [231,232]. Cellulose III is produced by ammonia or amide treatment of cellu-
lose I or II and defined by the hydrogen bonding between sheets [233]. The molecular
structures of the cellulose classes greatly affect their physical and mechanical properties.
Cellulose I is the strongest, with a Young’s modulus of 138 GPa, and cellulose III the
weakest (58 GPa) [234], while stability also decreases from cellulose I through to cellulose
III due to the different microfibrillar arrangements within the molecules [230].

3.1.2. Plant and Bacterial Celluloses

While PC and BC are both cellulose I, their differences in structure and microfibre
arrangement confer different properties on the molecules, including their purity, ability
to retain water, mechanical strength, porosity and crystallinity [230]. BC is considered the
“pure” cellulose as PC typically contains impurities in the form of residues from protective
hemicellulose [235], while it also contains a higher proportion of cellulose Iβ, making it
less crystalline [236]. BC microfibrils are noticeably smaller than those of PC, resulting
in BC having a higher capacity for water retention and being much more porous than
PC, though the properties of each material are influenced by their source and production
methods [230].

3.1.3. Nanocellulose
For biomedical applications, celluloses formed as nanofibre networks are preferred [235].
This increases the surface area of the celluloses and strengthens interactions with poly-
mers and biomaterials [237]. Nanocelluloses are cellulose extracts composed of structural
nanoscale materials which take advantage of the properties of both cellulose and nano-
materials [238]. While the health and environmental risks of nanoparticles are well estab-
lished [239,240], nanocellulose fibres are irreversibly fixed within the molecular structure of
cellulose. Nanocelluloses have been reported to exhibit very low toxicity to date but toxicol-
ogy studies are continuing, and inhalation of cellulose nanocrystals (CNC) has been reported
to cause pulmonary inflammation in animals due to self-aggregation of nanocellulose and its
slow degradation [238].
There are three different types of nanocellulose described to date: CNC and cellulose
nanofibrils (CNF), both of which are produced by the breakdown of PC using refinery
techniques and shear forces, and bacterial nanocellulose (BNC) which is produced by
bacteria [235,238].

Obtaining Nanocellulose
CNC and CNF are produced from plant sources such as wood, hemp, cotton, algae and
tunicin. Chemically induced destructuring such as by acid hydrolysis removes the amor-
phous cellulose regions while preserving their highly crystalline structure of CNCs [238].
For CNF, production involves mechanical destructuring of the cellulose structure by grind-
ing or homogenisation, followed by chemical and enzymatic treatments [238], which can
completely remove cellulose nanofibrils from microfibres within the cellulose structure.
Polymers 2021, 13, 3321 17 of 37

Unlike CNCs, CNFs retain both amorphous and crystalline cellulose regions and so appear
as longer—up to 10–100 nm, depending on the source, mechanical defibrillation process
and chemical treatment—and more flexible chains than CNCs [241].
While CNCs and CNFs are both produced via destructive processes, BNC is synthe-
sised by bacterial species in a form which requires very little processing [242]. Chains of
glucose are produced within the bacteria and secreted through pores in the cell envelope.
The glucose chains combine to form microfibrils which aggregate further to create BNC
with typical diameters of 20–100 nm [242].

Properties of Nanocellulose
Nanocellulose is characterised by its ordered crystalline and disordered amorphous
regions, with cellulose chains within the crystalline regions providing stiffness, and chains
within the disordered amorphous regions adding flexibility to the molecule. CNC is
stiffer and more rigid than CNF or BNC, with a Young’s modulus of 100–200 GPa, which
compares favourably to that of steel (200–220 GPa) [243]. CNF and BNC, meanwhile, exhibit
lower Young’s moduli of approximately 100 GPa due to their amorphous regions [238].
Nevertheless, these values indicate potential for application of all three nanocellulose types
as load-bearing biomaterials.
During formation of nanocrystals, cellulose chains orient in a unidirectional, parallel
manner within the fibrils, resulting in a robust hydroxyl functionality at one end and a
reducing moiety at the other. Meanwhile, glucose units display multiple active hydroxyl
groups, which are responsible for hydrogen bonding between glucose chains and can be
up to 10 times more reactive than regular OH groups [244]—and can be functionalised to
add surface properties to nanocellulose [245].
There have been very few biocompatibility studies on cellulose nanofibrils. Some stud-
ies report only evaluations of material biocompatibility in terms of cultivation, growth and
activity of cells, such as with CNC hydrogels [238], while others have focused on haemo-
compatibility: 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)-oxidised CNCs could regu-
late variables of blood metabolites while achieving acceptable haemocompatibility [246].
Subcutaneous injection of BNC in rats, including with infiltrated fibroblasts, identified
no detectable immune foreign body response after 12 weeks [247]. Cellulose degrades
slowly within the body, however, due to the absence of cellulolytic enzymes [238]. Oxidised
cellulose is more vulnerable to hydrolysis, leading to attempts to enhance BNC degradation
by oxidation, which proved promising in in vitro analyses [248].

3.1.4. Nanocellulose as a Biomaterial

In recent years, significant progress has been made in the use of nanocellulose in lesion
repair, as scaffolds to support cell culture and in tissue repair/regeneration (Figure 5), with
a predicted annual market value of approximately $97 billion [238].

Nanocellulose in Lesion Repair

Bodin et al. [60] compared the mechanical properties of a BNC gel with collagen
implants commonly used to treat degenerative meniscal lesions. The BC gel, grown to a
thickness of 5–15 mm, exhibited much greater load-bearing ability than the collagen, as
well as an ability to be moulded into a meniscus shape and promote cell migration [60]. An
ear-shaped BNC prototype was also produced using a reverse ear mould, demonstrating
that BC could successfully mimic the mechanical properties of native ear cartilage and
could be moulded to produce ear replacements in a patient-specific manner [61].
Polymers 2021, 13, 3321 18 of 37

Figure 5. Examples of the uses of cellulose as a biomaterial (based on [249]).

Nanocellulose as Artificial Blood Vessels

The production of artificial blood vessels is an area of interest (with BNC in particular)
due to the mechanical strength and blood biocompatibility of nanocellulose-based materi-
als [238]. Zang et al. [62] produced a BNC artificial blood vessel of 100 mm length and 1 mm
thickness using Gluconacetobacter xylinum. Extensive physical and structural analysis of the
tubular scaffolds indicated that they displayed satisfactory thermal stability, an extensive
nanofibre architecture and an adaptability to in vivo environments suited to use as blood
vessel replacements [62]. CNC or CNF cannot be fabricated directly into tubular scaffolds
and usually require the use of a matrix. CNC/fibrin composites have been reported to
form small diameter, tubular scaffolds, with the fibrin matrix adding strength in vitro [63].
Tubular scaffolds of 0.7–1.0 mm wall thickness have also been fabricated from PU/CNF
biocomposites, with CNF used to reduce the thrombogenicity and increase elasticity of
PU [64]. The scaffolds were successfully utilised as prosthetic blood vessels in the carotid
artery of an endocrine neoplasia patient [64]. Given their success in this instance, it is
surprising that PU/CNF tubular scaffolds have not been investigated further. Neverthe-
less, nanocellulose has clear potential application in creating artificial blood vessels but
large-scale in vivo testing is required to address critical, still unanswered questions about
their long term stability, potential postoperative complications and thrombogenicity [238].

Nanocellulose as a Drug Excipient and Drug Delivery

Celluloses have been used as excipients for condensing drug-loaded matrices into
pharmaceutical tablets for oral administration for many years. Despite this, nanocelluloses
remain a relatively new biomaterial in the field. Pure CNC prepared by acid hydrolysis
has been shown to bind large quantities of water-soluble tetracycline and doxorubicin,
followed by sustained release over 24 h [65]. By modifying the crystalline regions of
CNC via binding of cetyltrimethylammonium bromide (CTAB) to give the nanocellulose a
neutral charge, CNC could also be used to bind and release, over 48 h, the hydrophobic
anticancer drugs paclitaxel, docetaxel and etoposide. CTAB-modified CNC could also
bind and be taken up by KU-7 (urothelial carcinoma) cells, indicating their potential use
in targeted drug delivery [65]. CNF and BNC were al-so found to be better suited to
spray drying of tablets than commercially available micro-crystalline cellulose, with much
lower porosity [66] and improved flexibility, foldability and mechanical properties [67],
respectively, than traditional tablet coatings.
Polymers 2021, 13, 3321 19 of 37

Nanocellulose Wound Dressings

BNC, in particular, has attracted a lot of interest in wound care as it appears to
stimulate the proliferation of a number of human cell types, including human adipose-
derived stem cells, while exhibiting minimal cytotoxicity. Tissue regeneration and capillary
formation were accelerated in wound beds treated with BNC-based biomaterials compared
to commercial wound dressings (Figure 5) [68,69]. BNC wound dressing materials have
been also demonstrated to promote accumulation of ECM in rat ulceration models, leading
to contraction of the wound and improved healing [70], and to adhere extremely well
to burn sites, limit “dead space” and create an ideal healing environment [71]. Given
their promise in promoting wound healing, nanocelluloses have also been combined with
materials such as cotton gauze [72], gelatine [73] and collagen [74] to create biocomposites
with enhanced healing capabilities.
Unlike most applications of nanocelluloses, BNC-based biomaterials have moved past
clinical trials and into common clinical practice. Their primary use is in the treatment
of non-healing lower extremity ulcerations, where the use of BNC biomaterials has been
reported to reduce healing times from a mean of 240–390 days to 80 days [238]. Many
BNC-based wound healing products are currently available on the market with some, such
as BioFil (burn and ulcer therapy) and Gengiflex (periodontal disease treatment), having
dual functions in hydration and absorption to maintain an ideal healing environment [238].
Nanocellulose wound dressings with antimicrobial properties have also been developed by
binding antimicrobial agents to nanocellulose by physical or chemical approaches. Silver
and lysozyme are the most commonly used antimicrobials, with the former capable of
being incorporated into nanocellulose by simple chemical reduction and impregnation
techniques to yield materials which display strong antimicrobial activity while maintaining
the biocompatibility necessary for practical applications [75–77].
CMC and cellulose nanocrystals have also been incorporated into numerous bioinks
to improve their viscosity for printing and to provide mechanical strength to printed
hydrogels [250]. This has enabled materials suited to wound dressing applications to be
printed [4], as well as more anatomically complex structures such as a human ear and sheep
meniscus, in a study in which alginate was incorporated for cross-linking, and human
chondrocytes bioprinted in the ink exhibited 86% viability after 7 days of 3D culture [251].
Cellulose and nanocellulose clearly have widespread applications across wound
healing, drug delivery and as substitute implants, due largely to their biocompatibility,
versatility and tuneable chemical and physical characteristics. Their inherent lack of degrad-
ability when used as scaffolds may be the main limitation of nanocellulose biomaterials,
so that their greatest promise may reside in drug delivery and wound healing, especially
when combined with antimicrobial agents for improved effectiveness.

3.2. Chitosan
Chitosan is a unique biopolymer, derived from chitin, the second most abundant
natural polymer on earth, which has gained significant attention in recent years for its
potential in tissue engineering applications [252]. Both chitin and chitosan are found in the
exoskeleton of crustaceans such as crabs and in the cell envelope of plants such as fungal
hyphae (mushrooms) [250]. Chitin is a semi-crystalline linear polysaccharide copolymer
composed of repetitive β-(1-4)-2-acetamido-2-deoxy-D-glucopyranose units in which the
amines are completely acyetlated. Chitosan is a derivative of chitin with repeating β-(1-4)-2-
amino-2-deoxy-D-glucopyranose units in which the N-acetyl glucosamine residues of chitin
are completely deacetylated, giving rise to N-glucosamine [250]. Chitosan usually occurs
as a copolymer of (1-4)-2-acetamido-2-deoxy-β-D-glucan (N-acetyl D-glucosamine) and
(1-4)-2-amino-2-deoxy-β-D-glucan (D-glucosamine) units (Figure 6) [253]. To be classified
as chitosan, chitin must have a deacetylation degree (DDA) of at least 60%, i.e., should
contain at least 60% D-glucosamine residues [254]. Although discovered in the 1800s, chitin
has been much less developed in biomedicine than structurally similar cellulose, which
is largely attributable to its rigid structure and insolubility [250]. Chitosan is soluble in
Polymers 2021, 13, 3321 20 of 37

mild acidic solutions, however, and this, together with its amenability to modification,
biodegradability and biocompatibility, has led to its extensive application in ophthalmology,
wound healing/dressing and tissue engineering [250].

3.2.1. Obtaining Chitosan

Chitosan is produced by deacetylation of its parent molecule chitin. While typically
carried out using concentrated sodium hydroxide and sodium borohydride to prevent
reactive species generation and chitin depolymerisation [250], numerous modifications
have been described which alter the DDA of the resultant molecule [255], leading to
changes in its physical and chemical properties [256,257]. Its chemical properties can then
be further modified by adding functionalities to the abundant hydroxyl and amine groups
on the chitosan backbone, such as addition of lactobionic acid to cause chitosan to become
fully water-soluble [258], or oligopeptide addition [259] or carboxylation [260] to develop
functionalised chitosans for tissue engineering applications.

Figure 6. Composition of the chitosan polymer and examples of properties that influence its biomed-
ical uses (based on [261]).

3.2.2. Biological Properties

Though a polysaccharide, chitosan exhibits multiple amino groups which render it
degradable by human proteases such as lysozyme in vivo [262]. In addition, eight human
chitinases have been described to date, of which three can degrade chitosan in vivo [263].
The byproducts of degradation are non-toxic oligosaccharides of varied sizes which are
excreted via normal metabolic processes [264].
Chitosan is well known for its antimicrobial and antifungal properties, which make it
attractive for use in biomedical scaffolds. The basis for its antimicrobial activity is uncertain
but its positive charge is believed to interact with the negatively charged cell surfaces of
microorganisms and inhibit both uptake and excretion of materials [265]. Low molecular
weight chitosan is also reported to penetrate bacterial cell walls and bind to DNA via its
protonated amino groups, leading to disruption of essential microbial activities such as
RNA synthesis [266].
Recent in vitro and in vivo studies have highlighted the antitumor activity of chitosan,
which is thought to result from an increase in interleukin (IL)-1 and IL-2 secretion due to
Polymers 2021, 13, 3321 21 of 37

the infiltration of mature cytotoxic T lymphocytes [267,268]. Chitosan has also been shown
to be involved in inducing apoptosis and thus influencing the direct killing of tumour
cells: it prevented adhesion and proliferation of primary melanoma A375 cells and caused
apoptosis of metastatic melanoma RPMI7951 cells through inhibiting caspase activity and
upregulating apoptosis regulatory genes such as Bax [269]. Expression of CD95 receptors
was also upregulated on RPMI7951 cells following chitosan administration, which made
the cells highly vulnerable to FasL-induced apoptosis [269].
Chitosan can aid blood clotting and reduce pain via blocking of nerve endings [270].
These haemostatic properties are attributed to negatively charged red blood cells being
attracted to the protonated amine groups, leading to blood cell aggregation and clot for-
mation, which quickly halts bleeding [270,271] Aside from stimulating red blood cell
coagulation, studies have shown that chitosan can enhance platelet activation and aggrega-
tion [271] via a mechanism which has not been elucidated but is dependent on chitosan’s
positive charge [272]. Chitosan is also well known for its mucoadhesive properties, and its
ability to bind many surfaces within the body [273].

3.2.3. Chitosan Biomaterials

The biodegradability and antimicrobial, anti-tumour, haemostatic and mucoadhesive
properties of chitosan have led to it finding numerous applications in biomedicine and
pharmaceuticals, as outlined below.

Chitosan in Wound Healing

Chitosan is used in wound healing due to its versatility and the fact that it can be
readily manufactured both into 2D films and fibres and 3D scaffolds such as hydrogels and
sponges [274]. As well as its haemostatic capabilities, chitosan induces neutrophil migra-
tion [275] and stimulates proliferation of dermal fibroblasts to accelerate re-epithelialisation
in wound healing applications [276]. Antimicrobial chitosan hydrogels developed as a
coating for vascular prosthetic grafts have been demonstrated to provide resistance to
E. coli in vitro and in vivo [79,80]. Meanwhile electrospun chitosan/poly (vinyl alcohol)
composite nanofibre wound dressings were found to stimulate fibroblast attachment and
proliferation with a complete absence of cellular toxicity [78].

Chitosan-Based Molecule Delivery

The exceptional mucoadhesive properties of chitosan also lend the material to drug
delivery applications. Nasal administration of chitosan microspheres containing peptides
has been shown to promote transport of the encapsulated peptides through the nasal barrier,
leading to enhanced adhesion and absorption on the nasal epithelium [81]. Delivery of
diphtheria toxoid in chitosan microparticles in this manner significantly improved systemic
and local immune responses to the toxoid in mice models through enhanced absorption
arising from chitosan’s mucoadhesive properties [81]. Chitosan is also increasingly used
to deliver micronutrient polyphenols which exhibit pharmacological potential as anti-
inflammatory agents in Alzheimer’s disease, cardiovascular disease and some forms of
cancer [82]. Encapsulation of tea-derived polyphenols in chitosan nanoparticles for oral
administration protected the polyphenols against degradation and oxidation within the
gastrointestinal tract while enhancing their uptake by endothelial cells [83]. Chitosan
nanoparticles have similarly been shown to protect peptide drugs such as insulin and
enhance their stability and controlled release [84].
Chitosan’s excellent biodegradability and antimicrobial, anti-tumour, haemostatic
and mucoadhesive properties enable it to create suitable environments to promote wound
healing. It has also been shown to have potential in the delivery of drugs and vaccines.
While chitosan nanoparticles look particularly promising, additional studies are required
on their toxicity to humans in particular.
Polymers 2021, 13, 3321 22 of 37

3.3. Alginate
Alginates are anionic and hydrophilic polymers which are amongst the most widely
available biosynthesised materials on earth [85]. They occur as cell wall constituents of
brown algae such as Laminaria hyperborea and Macrocystis pyrifera, and Pseudomonas and
Azotobacter bacterial species [277]. They are linear biopolymers consisting of homoge-
nous (poly-G, poly-M), heterogeneous (MG) or block-like arrangements of 1,4-linked β-D
mannuronic acid (M) and 1,4 α-L-guluronic acid (G) residues [277]. Their widespread
availability, low cost, minimal toxicity, biodegradability and profound gelating ability
have led to them being approved by the U.S. FDA for use as a biopolymer in regenerative
medicine and tissue engineering [278].

3.3.1. Structure and Properties

Alginates linear copolymers consist of blocks of residues of both (1,4)-linked β-D-
mannuronate (M) and α-L-guluronate (G) which can occur as continuous G or M residues,
i.e., (GGGGGG) or (MMMMMM), or as alternating residual patterns, i.e., (GMGMGM)
(Figure 7) [85]. There are currently over 200 unique alginates produced for different
purposes with varying G and M contents and overall block lengths [223].
G/M ratios, length of G blocks and the sequence pattern of GM block repeats all
affect the physical properties of alginate hydrogels [279]. Alginates from different sources
produce highly variable gels [85], with many bacterial alginates, such as those extracted
from Azotobacter species, extremely rigid due to very high concentrations of G-blocks [280],
which in turn impacts their potential application in drug release and cell encapsulation.
Despite extensive research on the biocompatibility of alginates, the effect of the molec-
ular structure on biocompatibility remains uncertain. While alginates high in M blocks have
been reported to be highly immunogenic and to stimulate cytokine production at 10-fold
higher levels than alginates with a high G block content [281], other in vivo studies have
detected no immunogenic or inflammatory reaction to implanted alginates [282–284]. It is
likely that inflammatory reactions may be due to impurities within the alginate preparation
rather than the material itself, with potential impurities including endotoxins, polyphenolic
compounds, non-algal proteins, arsenic and heavy metals [285].

Figure 7. Alginate is extracted and purified from a wide variety of brown algae. It is composed
of α-L-guluronic acid (G) and β-D-mannuronic acid (M) blocks, the precise combination of which
impact the properties of the alginate material and its potential biomedical utility. Image is based
on [286].
Polymers 2021, 13, 3321 23 of 37

Alginate is not degraded by mammals as they lack the alginase enzyme responsible
for cleaving the polymer chains [85]. Additionally, mammals lack receptor sites for alginate
polymers, rendering alginate gels inert [287], though hydrogels with increased biological
activity can be produced, as outlined below.

3.3.2. Obtaining Alginate

Most commercially available alginates are derived from the above kelp species, typ-
ically harvested from natural populations as cultivation is considered too expensive for
industrial production. While alginate biopolymers can make up almost 40% of the dry
weight of the brown seaweeds [288], their quantity and quality vary with species, season
and age of the seaweed. Alginate is typically extracted in aqueous alkali [85], filtered,
precipitated and recovered as a water-soluble sodium alginate powder [224]. While modifi-
cation of the natural structure to improve its physical and chemical properties is gaining
attention [289,290], this review focuses on natural alginates from brown algae given their
current dominance in the market.

3.3.3. Alginate Hydrogels

While other biomaterials are utilised in many different formats, such as sponges, films
and microparticles, alginate is primarily used in the form of hydrogels. These hydrogels
can be formed by a variety of gelling mechanisms to modify their functionality.

Ionic Cross-Linking Gelling Strategies

Alginate hydrogels are most commonly prepared by the addition of ionic cross-linking
agents such as divalent Ca2+ cations, typically in the form of calcium chloride [85]. The
cations form highly coordinated bonds with the G blocks of the alginate chains, leading
to alignment of the G blocks of adjacent chains into a cross-linked “egg-box” formation
which provides the gel structure [291]. Buffers rich in phosphate groups, or lowering the
reaction temperature, are often utilised to slow the reaction, which enhances the ordering
of cross-links and increases the mechanical stability of the hydrogel [291,292]. As noted
above, the origin and structure of the alginate influences cross-linking, with alginates with
higher G-block contents undergoing more extensive cross-linking and yielding more rigid
gels [293].
Stability issues may arise when gels formed through ionic cross-linking are incubated
under physiological conditions for extended periods of time, however, due to exchange
reactions with surrounding monovalent cations [85]. Ca2+ ions released via this process
can promote haemostasis, while the resultant poorly cross-linked gel may also lead to
aggregation of red blood cells and platelets [294]. Therefore, some focus has been turned to
cross-linking gels through covalent means for increased structural stability.

Covalent Cross-Linking
Cross-linking of alginates using PEG amines of different molecular weights has been
investigated to produce gels with a broader range of mechanical properties. Increasing
the cross-linking density/amount (weight) of PEG led to improved mechanical strength
and increased Young’s moduli in gels [85,295]. Controlling the cross-linking density and
using multiple cross-linking agents can regulate the overall mechanical properties and
swelling of gels [295]. As an example, poly(aldehyde guluronate) (PAG) gels cross-linked
using multifunctional agents, such as poly(acrylamide-co-hydrazide) (PAH), displayed
significantly enhanced mechanical properties and lower degradation than similar gels
cross-linked using the bi-functional cross-linker adipic acid dihydrazide (AAD), apparently
due to greater binding of PAH to the PAG gel [296].

Enhancing Biodegradation Properties

As mammals lack the enzymatic capacity to break down alginate chains, this jeopar-
dises the potential biomedical usefulness of alginates, as biodegradation and safe excretion
Polymers 2021, 13, 3321 24 of 37

from the body are primary concerns for biomaterials in vivo [287]. While ionically-linked
alginate gels dissolve over time due to the release of divalent ions into the surrounding
medium, many commercial alginate gels have molecular weights larger than the threshold
for renal clearance, which disrupts their excretion from the body [85,297].
Multiple approaches have been developed to produce biodegradable alginates for
in vivo applications. Partial oxidation of alginate leads to increased degradability in
aqueous environments [287] due to changing the conformation of the alginate backbone to
an open-chain adduct without disrupting its gelling characteristics. In another approach,
alginates can also be constructed entirely from G-blocks isolated from whole alginates,
resulting in a degradable gel [298] better suited to biomaterial production.

Enhancing Cellular Interaction

As mammals lack cell receptors for alginate polymers, coupling of molecules such as
fibronectin [299] and collagen [300] may be necessary to promote adhesion [287]. While
coupling of whole molecules has proved difficult, attaching short amino acid chains
which promote ECM adhesion has been more successful. RGD-modified gels promote
increased proliferation of myoblasts [301], with RGD concentration and M/G ratio of
the gels affecting cell behaviour and cell phenotype development [302]. Altering ligand
densities can modify biological effects, with endothelial cell differentiation enhanced in
gels containing intermediate concentrations of ligands [303] and hepatocyte differentiation
stimulated only at much lower ligand densities [304]. Gel stiffness and rigidity also
influence ligand clustering and cell adhesion [287], proliferation and differentiation [305]
and morphology [306].

3.3.4. Alginate-Based Biomaterials

Alginate gels have been widely applied across various fields of biomedicine, including
wound healing, tissue/bone regeneration and as model systems to study mammalian cell
culture [85].

Alginates as Wound Dressings

Ionically cross-linked alginate gels can be processed by freeze-drying to produce
foam-like porous sheets and fibrous dressings which provide a moist environment more
suited to wound healing than traditional dressings [86]. The dressings begin dry but
quickly absorb excaudate fluid from the wound bed to begin a “re-gelling” transition
and form a gel which provides excellent healing conditions and supports formation of
granulation tissue and re-epithelialisation. Commercial alginate wound dressings include
Algisite™ (Smith and Nephew) and Comfeel Plus™ (Coloplast) [85], the success of which
has led to the development of alginate-based dressings designed to release bioactive
compounds. Incorporation of a regulator of human keratinocyte proliferation, dibutyryl
cAMP (DBcAMP), to oxidised alginate gels allowed for its sustained release into the
surrounding medium, leading to full wound healing and complete re-epithelialisation
over 10 days in rat models, compared to 15 days with control alginate gels [87]. Similarly,
incorporation of stromal cell-derived factor-1 (SDF-1) to alginate dressings accelerated
healing in porcine wound models—and with reduced scar formation [88]. Incorporation of
silver into alginate-based dressings improved antimicrobial activity and inhibited infection,
as well as enhancing binding of matrix metallo-proteases-2 (MMP-2) and pro-inflammatory
cytokines TNF-α and IL-8 [89].

Alginates in Drug Delivery

Alginate gels have been used for sustained delivery of both low molecular weight
chemical and protein drugs. The former diffuse rapidly from nanoporous (pore size
~5 nm) [90] alginate gels, e.g., anti-inflammatory drug flurbiprofen is readily released from
partially oxidised and ionically cross-linked alginate gels but its release is significantly
delayed from ionic and covalently cross-linked alginate [91]. While the chemical structure
Polymers 2021, 13, 3321 25 of 37

of the drug predominantly dictates its release kinetics, some drugs, such as a number of
anthracenediones used in chemotherapy, can interact with alginate or its cross-linkers and
only be released upon breakdown and complete dissociation of the gel; others, however,
such as methotrexate, form no interactions with alginate and are released almost immedi-
ately via diffusion [52,85]. In an innovative approach that holds promise for transporting
and safely releasing a broad array of hydrophobic drugs, composite alginate hydrogels
were produced by encapsulating oil-in-water microemulsions in hydrophilic gels [93],
enabling the hydrophobic oil core to solubilise the hydrophobic drug ketoprofen and the
Ca2+ cross-linked outer gel matrix to act as a delivery vehicle.
Alginate has also proven an excellent delivery vehicle for protein-based drugs as
proteins are easily incorporated into alginate gels without denaturation and the gels protect
the proteins from degradation and allow their controlled release. Protein release rates are
typically fast due to the gels’ hydrophilic properties and porous structures [85], though
some growth factors—in particular molecules such as vascular endothelial growth factor
(VEGF) and FGF which bind heparin—interact with the gels, leading to a more sustained
release [94,95]. Much current research is dedicated to controlling the release of proteins
from gel vehicles, particularly molecules which do not have heparin-binding properties.
The fast release of most protein drugs from alginate gels can be slowed by gel cross-
linking techniques which can increase the incidence and strength of protein-gel interac-
tions [85]. Insulin, for example, is completely released from alginate gel micro-spheres
outside the gastrointestinal environment due to dissociation of the carrier vehicle at pH
6.8 [307]. By developing alginate/polyanionic polymer (cellulose acetate phthalate) blends
followed by chitosan coating, however, insulin is protected in gastric pH 1.2 conditions and
can be successfully delivered to the intestine. Similarly, layering B. mori silk fibroin over
alginate microspheres has been utilised to achieve a mechanically rigid structure which
protects enclosed proteins and prevents their diffusion [308].

Alginates as Protein and Cell Carriers for Tissue Engineering

Alginate gels have been widely utilised to transport proteins and cells for regeneration
or engineering of tissues and organs in the body. As molecules smaller than 5 nm are
quickly released from the porous alginate gel whereas those larger than the pore size are
only released following gel dissociation, plasmid DNA molecules of approximately 100 nm
in diameter are released from alginate gels only upon degradation [309].
Alginate gels have been used to stimulate angiogenesis and partly relieve ischemia in
murine tissue through sustained and localised release of growth factors such as VEGF [94].
Gels have also been used to effect sequential delivery of growth factors for angiogenesis,
with VEGF released prior to PDGF to stimulate early angiogenesis and blood vessel forma-
tion, followed by their enhanced formation and functionality in ischemic mice models [96].
Alginate gels are also attractive in bone regeneration as they can fill irregular shapes
that other scaffolds may not and can be introduced into the body non-invasively and easily
modified to carry cell-adhesion ligands or growth factors. Their inability to bear weight is
of some concern [287] but they have nonetheless successfully guided bone regeneration
in rat models in combination with RGD delivery [97] and in vitro with DNA encoding
bone morphogenetic proteins [98]. They have also been used to transport whole cell
populations (rat calvarial osteoblasts in a murine model [99], and osteoblasts with primary
chondrocytes [100]) to take a direct part in new bone formation.
Alginate is also one of the most commonly used biomolecules in 3D bioprinting
due to its compatibility with extrusion and inkjet printing processes [310], and its ability
to maintain cell growth due to its similar properties to natural ECM [85,309]. As noted
above, the ability of alginate hydrogels to bear weight and maintain their shape is limited,
however, and so they are typically reinforced with natural and/or synthetic molecules
such as fibrin or fibrinogen, nanocellulose, gelatin and nano-hydroxyapatite (n-HAP) to
improve their structure [311].
Polymers 2021, 13, 3321 26 of 37

Alginate has shown considerable potential as a biomaterial, particularly in wound

healing, guided tissue engineering and drug delivery. While in hydrogel form, it exhibits
very promising gelling capability, ease of modification and biocompatibility, the poor
mechanical strength of other alginate forms limits their potential applications. Future
research is likely to see the development of dressings with much higher bioactivities, as
well as modified carrier vehicles for drug and bioactive molecules. Functionalisation with a
wider range of molecules and engineering the molecules’ physical and chemical properties
will be key to developing application-specific, tissue-interactive alginate biomaterials.

4. Conclusions and Future Perspectives

Natural polymers, both protein- and polysaccharide-based, display enormous poten-
tial in tissue engineering, drug delivery and diverse biomedical applications. Traditional
materials previously designed to be inert in order to be “biologically safe” no longer meet
the needs of modern medicine, where biomaterials are designed to make extensive biologi-
cal and physical interactions with tissues and cells for improved in vivo performance. The
ECM-like characteristics of natural polymers, their similarities to many human proteins
and their natural versatility have led to the modification of well-established biomaterials,
as well as the development of new materials, that have found novel applications in tissue
engineering, as implantable structures and as vehicles for controlled drug release.
Though enormous progress has been made in the field of nature-based biomaterials
in recent decades, room for improvement remains. Novel biomaterials which outperform
current state-of-the-art offerings in terms of reduced side effects, lower purification or
manufacturing costs and greater therapeutic efficacies will continue to be developed.
Researchers continue to search for new natural sources of biomaterials which, together
with innovative scaffold fabrication techniques, will lead to more effective biomaterials
and new applications. The recent successes with natural materials and the broad array
of materials currently available will inspire researchers to continue to look to nature for
novel biomaterials, however, with fundamental biological and chemical understanding of
such materials remaining central to the emergence of new, innovative biomaterials for new
biomedical applications.

Author Contributions: Conceptualization, A.M.P. and J.G.W.; writing—original draft preparation,

E.T.; writing—review and editing, E.T., M.A.T., A.M.P. and J.G.W.; writing—figure preparation,
M.A.T. and E.T.; supervision, A.M.P. and J.G.W. All authors have read and agreed to the published
version of the manuscript.
Funding: MT and GW were supported by Science Foundation Ireland (SFI) co-funded under the
European Regional Development Fund (grant no. 13/RC/2073) and Health Research Board Ireland
COVID-19 Pandemic Rapid Response Funding Opportunity 2020 (COV19-2020-081). AMP was
supported by the European Network of Bioadhesion Expertise (COST Action CA15216). ET was
supported by the MSc Biotechnology programme at NUI Galway.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: The authors thank Rob Gaughan for assistance in generating Figures. Figures 2,
3, 5 and 7 were created with BioRender (www.biorender.com).
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or
in the decision to publish the results.
Polymers 2021, 13, 3321 27 of 37

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