Materials Today  Volume 16, Number 12  December 2013 RESEARCH

RESEARCH: Review

Bone tissue engineering using 3D

printing
Susmita Bose*, Sahar Vahabzadeh and Amit Bandyopadhyay
W. M. Keck Biomedical Materials Research Lab, School of Mechanical and Materials Engineering, Washington State University, Pullman, WA 99164, USA

With the advent of additive manufacturing technologies in the mid 1980s, many applications benefited
from the faster processing of products without the need for specific tooling or dies. However, the
application of such techniques in the area of biomedical devices has been slow due to the stringent
performance criteria and concerns related to reproducibility and part quality, when new technologies
are in their infancy. However, the use of additive manufacturing technologies in bone tissue engineering
has been growing in recent years. Among the different technology options, three dimensional printing
(3DP) is becoming popular due to the ability to directly print porous scaffolds with designed shape,
controlled chemistry and interconnected porosity. Some of these inorganic scaffolds are biodegradable
and have proven ideal for bone tissue engineering, sometimes even with site specific growth factor/drug
delivery abilities. This review article focuses on recent advances in 3D printed bone tissue engineering
scaffolds along with current challenges and future directions.

Introduction challenges related to other treatment options involving different

Osseous tissue, known as bone, is made of two different structures; materials such as autografts or allografts. Apart from material issues,
cancellous and cortical bone. Cancellous, or the inner part of bone, a clear understanding of biology involving cells, extracellular matrix
is spongy in nature having 50–90 vol% porosity. However, cortical (ECM) and growth factors are pivotal in bone tissue engineering [7].
bone is the dense outer layer of bone with less than 10 vol% Scaffolds are an integral part of bone tissue engineering. Scaf-
porosity. Both types of bone undergo dynamic remodeling, matura- folds are three dimensional (3D) biocompatible structures which
tion, differentiation, and resorption that are controlled via interac- can mimic the ECM properties (such as mechanical support,
tions among osteocyte, osteoblast, and osteoclast cells [1]. cellular activity and protein production through biochemical
Osteoblasts are primarily responsible for new bone formation while and mechanical interactions), and provide a template for cell
osteoclasts are responsible for the resorption of old bone. Such a attachment and stimulate bone tissue formation in vivo [3,5–7].
dynamic process involving osteoclasts and osteoblasts is known as Besides chemistry, pore size, pore volume and mechanical strength
bone remodeling, and is responsible for maintaining a healthy are critical parameters which define a scaffold’s performance. At an
bone. Bone is well known for its self-healing abilities [2]; however, early stage, bone ingrowth happens at the periphery of scaffolds
large-scale bone defects cannot be healed completely by the body with a negative gradient in mineralization toward the inner parts
[3,4], and in most cases, external intervention is needed to restore [4]. For continuous ingrowth of bone tissue, interconnected por-
normal operations. Among different treatment options such as osity is important. Open and interconnected pores allow nutrients
autografts (bone taken from the same person’s body) and allografts and molecules to transport to inner parts of a scaffold to facilitate
(bone tissue from a deceased donor), bone tissue engineering that is cell ingrowth, vascularization, as well as waste material removal
focused on methods to synthesize and/or regenerate bone to restore, [4,6,8]. Since higher porosity increases surface area per unit
maintain or improve its functions in vivo [5,6] is becoming popular. volume, the biodegradation kinetics of scaffolds can be influenced
Successful application of bone tissue engineering can avoid by varying pore parameters. Biodegradation through a cell-
mediated process or chemical dissolution are both important to
*Corresponding author:. Bose, S. ([email protected]) ascertain stabilized repair and scaffold replacement with new bone

496 1369-7021/06/$ - see front matter ß 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.mattod.2013.11.017
Materials Today  Volume 16, Number 12  December 2013 RESEARCH

without any remnant [8]. A minimum pore size between 100 and Fig. 1 shows a schematic representation of the 3DP process [62].
150 mm is needed for bone formation [4,9]; however, enhanced For bone tissue engineering, 3DP is useful for the direct fabrication
bone formation and vascularization are reported for scaffolds with of scaffolds with tailored porosity from a CAD file. Before printing,
pore size larger than 300 mm [9–11]. Pore size also plays an impor- essential parameters such as powder packing density, powder
tant role in ECM production and organization. Poly(D,L-lactic acid) flowability, layer thickness, binder drop volume, binder saturation
(PDLLA) scaffolds with pore size 325 and 420 mm led to well- and powder wettability need to be optimized to improve the
organized collagen I network; whereas, smaller pore size of quality of the resultant part. Packing density is the relative density
275 mm prevented the human osteosarcoma-derived osteoblasts of the powder bed after uniform spreading. To start a build,
to proliferate, differentiate and produce functional ECM [12]. Pore enough powder should be packed homogeneously in a feed bed.
volume also controls the permeability of nutrients to the scaffold A set of rollers spread a layer of powder to a predetermined

RESEARCH: Review
and their mechanical properties. Permeability in poly-e-caprolac- thickness to create a powder bed. Powder flowability is critical
tone (PCL) increased with higher pore volume and resulted in in this process as it determines the spreading ability. Flowability is
better bone regeneration, blood vessel infiltration, and compres- primarily determined by particle size, size distribution, surface
sive strength in vivo, when other pore parameters were kept the roughness and shape. The desired layer thickness is in part deter-
same [13]. Apart from biological performance, the initial mechan- mined by geometry and powder characteristics. Thinner layers
ical properties and strength degradation rate should match that of cause binder penetration and excess spreading to other sites
the host tissue for optimum bone healing [14]. The strength resulting in poor resolution and tolerance. However, thick layers
degradation kinetics of porous scaffolds are highly affected by need high saturation for the powders to bind [62].
pore size, geometry, and strut orientation with respect to the The printhead sprays the binder across the build layer in several
loading direction [15,16]. Finally, surface properties such as chem- passes, based on the instructions in the tool path file created
istry, surface charge and topography also influence hydrophilicity according to the CAD file. The binder, which can be organic or
and in turn cell–material interactions for bone tissue ingrowth water-based, locally binds the particles and hardens the wetted
[17–19]. area, or results in a reaction similar to the hydraulic setting
Porous bone scaffolds can be made by a variety of methods. reaction in cements [63–65]. The binder drop volume and satura-
Chemical/gas foaming [20], solvent casting, particle/salt leaching tion play crucial roles. The binder drop volume is the amount of
[12,21], freeze drying [22], thermally induced phase separation binder released from each nozzle per drop during printing, which
[23], and foam-gel [24] are some of those that have been used depends on the binder density and viscosity. By coordinating the
extensively. However, pore size, shape, and its interconnectivity powder packing density and the drop volume, the binder satura-
cannot be fully controlled in these approaches. Moreover scaffolds tion data required for printing is obtained. For a constant packing
with tailored porosity for specific defects are difficult to manufac- density, a higher drop volume demands a lower binder saturation
ture with most of these approaches [21–24]. Such scaffolds can be [62]. Low saturation can cause layer displacement during proces-
designed and fabricated using additive manufacturing (AM) sing. The binder saturation also depends on the powder wettabil-
approaches. Different AM approaches, for example, 3D printing ity. The powder wettability, which is related to the powder particle
(3DP), solid freeform fabrication (SFF), rapid prototyping (RP), are chemistry and surface energy, determines the printing accuracy
approaches that allow complex shapes for scaffolds’ fabrication and the achievable tolerance [66]. While high wettability results in
directly from a computer aided design (CAD) file [25–27]. The extensive binder spreading, low wettability causes week powder-
concept of AM was first introduced by Chuck Hull in 1986 via a binder integration [67].
process known as ‘stereolithography (SLA)’ [28,29]. After printing, the printed layer is moved under a strip heater to
Some of the commercially available AM techniques are 3DP allow the binder to dry out and prevent spreading between layers
(ExOne, PA), fused deposition modeling (FDM, Stratasys, MN), [62]. This process is repeated until the printing of the designed part
selective laser sintering (SLS, 3D Systems, CA), stereolithography is complete. Heat treatment is needed to complete the binder
(3D Systems, CA), 3D plotting (Fraunhofer Institute for Materials reaction and increase the part green strength. Next step is depow-
Research and Beam Technology, Germany), as well as various forms dering, that is, the removal of loose powder from the printed body.
of direct writing [27]. In all these AM approaches, 3D scaffolds are This is one of the major challenges for porous scaffolds in 3DP due
created layer-by-layer without any part specific tooling or dies to the low green density of the part. Loose powder removal from
[30,31]. These AM techniques can be classified as – (a) extrusion fine pores can easily crack a green part [68].
(deformation + solidification), (b) polymerization, (c) laser-assisted In general, a large variety of ceramic, metallic, polymeric, and
sintering, and (d) direct writing-based processes. Table 1 [32–59] composite materials can be processed using 3DP; however, binder
summarizes some of the AM techniques toward bone tissue engi- selection and process parameter optimization are the keys to
neering applications including their advantages and disadvantages. successful part fabrication. In bone tissue engineering the advan-
tages of this method arise through the control of fine features
3D printing (3DP) – history and methodology including interconnected porosity, no contamination issues
3DP, a technology developed in the early 1990s at MIT (Cam- related to any second material for support structures and the direct
bridge, MA) by Sachs et al. [60], is a powder-based freeform printing ability with both metallic and ceramic biomaterials
fabrication method in which using a regular ink-jet print-head, [65,69]. Fig. 2a shows some examples of 3D printed scaffolds with
binders are printed on to loose powders in a powder bed. Early different pore sizes. It is important to note that extensive optimi-
research in this area was focused on rapid tooling using metals and zation is needed to process good quality parts with 3DP for any
ceramics [61]. new material, a fundamental drawback for this approach.

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TABLE 1
RP techniques for bone scaffold fabrication.
Technique Process details Processed materials for bone Advantages (+) and Reference
tissue engineering disadvantages ()
3D Plotting/direct ! Strands of paste/viscous material ! PCL +: [32–38]
ink writing (in solution form) extrusion based ! Hydroxyapatite (HA) ! Mild condition of process allows
on the predesigned structure ! Bioactive glasses drug and biomolecules (proteins
! Layer by layer deposition of ! Mesoporous bioactive and living cells) plotting
strands at constant rate, glass/alginate composite :
under specific pressure ! Polylactic acid (PLA)/polyethylene ! Heating/post-processing
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! Disruption of strands according to glycol (PEG) needed for some materials restricts
the tear of speed ! PLA/(PEG)/G5 glass the biomolecule incorporation
! Poly(hydroxymethylglycolide-
co-e-caprolactone) (PHMGCL)
! Bioactive 6P53B glass
Laser-assisted ! Coating the desired material on ! HA +: [39–42]
bioprinting transparent quartz disk (ribbon) ! Zirconia ! Ambient condition
(LAB) ! Deposition control by laser ! HA/MG63 osteoblast-like cell ! Applicable for organic,
pulse energy ! Nano HA inorganic materials and cells
! Resolution control by distance ! Human osteoprogenitor cell ! Quantitatively controlled
between ribbon/substrate, spot ! Human umbilical vein ! 3D stage movement
size and stage movement endothelial cell :
! Homogeneous ribbons needed
SLS ! Preparing the powder bed ! PCL +: [43–48]
! Layer by layer addition of powder ! Nano HA ! No need for support
! Sintering each layer according to ! Calcium phosphate (CaP)/poly ! No post processing is needed
the CAD file, using laser source (hydroxybutyrate–co- :
hydroxyvalerate) (PHBV) ! Feature resolution depends
! Carbonated hydroxyapatite on laser beam diameter
(CHAp)/poly(L-lactic acid) (PLLA)
! PLLA
! b-Tricalcium phosphate (b-TCP)
! PHBV
SLA ! Immersion of platform in a ! Poly(propylene fumarate) +: [49–52]
photopolymer liquid (PPF)/diethyl fumarate (DEF) ! Complex internal features can
! Exposure to focused light according ! PPF/DEF-HA be obtained
to desired design ! PDLLA/HA ! Growth factors, proteins and cell
! Polymer solidifying at focal point, ! b-TCP patterning is possible
non-exposed polymer remains liquid, :
! Layer by layer fabrication by platform ! Only applicable for photopolymers
moving downward
FDM ! Strands of heated polymer/ceramics ! Tricalcium phosphate (TCP) +: [26,30,31,
extrusion through nozzle ! TCP/polypropylene (PP) No need for platform/support 53–58]
! Alumina (Al2O3) :
! PCL ! Material restriction due to need
! TCP/PCL for molten phase
Robotic assisted ! Direct writing of liquid using a ! HA/PLA +: [59]
deposition/ nozzle ! HA/PCL ! Independent 3D nozzle movement
robocasting ! Consolidation through liquid-to- ! 6P53B glass/PCL ! Precise control on thickness
gel transition ! No need for platform/support
:
! Material restriction

3D printed bone scaffolds a tensile strength up to 4 MPa, and no toxicity to human osteo-
Table 2 summarizes a few selected material-binder system combi- blasts [74].
nations for bone scaffolds using 3DP. Starch-based binders are one CaP ceramics are widely used in bone tissue engineering due to
of the candidates for bone replacement applications. These binders their excellent bioactivity, osteoconductivity, and similarities in
are biocompatible and produce structures that have a mechanical composition to bone. Capillaries and vessel formation, and homo-
strength close to trabecular bone [71,72]. Structural designs and geneous osteoconduction from central channels, have previously
post processing conditions both can influence the mechanical been observed in 3D-printed HA blocks [75]. The effect of pore size
properties of 3D-printed starch-based scaffolds [73]. 3D-printed on human fetal osteoblasts (hFOB) was studied with 3D-printed
polyethylene (PE) scaffolds with 22.3–49.7% porosity have shown TCP scaffolds [62]. The decrease in designed pore size from 1000 to

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FIGURE 1
(a) 3D printing schematic using an inkjet printing system. (b) 3D printed CaP sintered structures fabricated at WSU.

TABLE 2
3D printed materials for bone tissue engineering.
Material Layer thickness Binder Reference
TCP 20 mm Aqueous based [62]
a/b-TCP modified with 5 wt% hydroxypropymethylcellulose 100 mm Water [64]
CaP mixture with Ca/P ratio of 1.7 100 mm 10% phosphoric acid [64]
Tetracalcium phosphate (TTCP), dicalcium phosphate and TCP 100 mm 25% citric acid [64]
HA 300 mm Schelofix (water soluble polymeric compound) [65,75]
TTCP/b-TCP 100 mm 25 wt% of citric acid [68]
TTCP/calcium sulfate dihydrate 100 mm 25 wt% of citric acid [68]
HA 100 mm No information [69]
TCP 100 mm No information [69]
Biphasic calcium phosphate (BCP) 100 mm No information [69]
a/b-TCP (final product: dicalcium phosphate dihydrate (DCPD)) No information 20% phosphoric acid [70]
Starch/PLLA + PCL No information Distilled water + blue dye [73]
High density PE (HDPE) 0.175 mm Maltodextrin + polyvinyl alcohol (PVA) [74]
SiO2–ZnO-doped TCP 20 mm Aqueous based [76]
PE or HDPE 0.175 mm Water based binder [77,78]
PLA No information Chloroform [79]
TCP (final product:DCPD) 0.1 mm 20% phosphoric acid [80]
HA/maltodextrin 0.175 Water based binder [81]
TTCP (final product: HA) 100 mm 0.5 mol/l Ca(H2PO4)2 + 10% H3PO4 [82]
TCP (final product: brushite) 100 mm 0.5 mol/l Ca(H2PO4)2 + 10% H3PO4 [8,82]
HA/A-W glass 0.1 mm Water based [83]

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FIGURE 2
(a) Photograph of the sintered 3D printed TCP scaffolds for mechanical strength and in vivo testing (small samples) [62]. (b) Compressive strength
comparison of the scaffolds sintered at 1250 8C in conventional and microwave furnaces (**p < 0.05, *p > 0.05, n = 10) [62]; (c) SEM micrographs of hFOB
cells showing the cell adhesion and proliferation on the scaffold surface and inside the 3D interconnected macro pores after 3 days of culture (white arrows
indicate cells): 500 mm (i) & (ii), and 750 mm (iii) & (iv) [62]; (d) SEM image of the pure TCP scaffold showing the surface morphology and designed macro
pore distribution [84]; (e) photomicrograph of 3DP pure (TCP) implants (i and iii), and Sr–Mg doped TCP implants (ii and iv) showing the new bone
formation inside the interconnected macro and intrinsic micro pores of the 3DP scaffolds after 4 and 8 weeks in rat distal femur model. Modified Masson
Goldner’s trichrome staining of transverse section. OB: old bone, NB: new bone and BM: bone marrow. Color description: Dark gray/black = scaffold; orange/
red = osteoid; green/bluish = new mineralized bone (NMB)/old bone [84]; and (f ) histomorphometric analysis of osteoid area fraction (osteoid area/total area,
%) from 800 mm width and 800 mm height tissue sections (**p < 0.05, *p > 0.05, n = 8). Completely mineralized bone formation was observed in presence
of SrO and MgO in TCP after 12 weeks, hence no osteoid area was observed. All osteoid like bone was transformed into mineralized bone after 12 weeks in
doped TCP due to the presence of strontium and magnesium. Hence, there was no osteoid like bone left after 12 weeks in Sr–Mg-doped TCP [84].

750 and 500 mm resulted in an increase in proliferated cell density. images confirmed the monocyte differentiation to multinuclear
3D printed and microwave sintered b-TCP scaffolds fabricated osteoclast-like cells on a wide range of compositions [69]. It has
using 3DP are shown in Fig. 2a, showing interconnected macro been shown that the use of phosphoric acid instead of polymeric
porosity across the sample. Fig. 2c (i–iv) presents the morphologies binders can improve both resolution and compressive strength
of hFOB cells on scaffold surfaces and pore walls after 3 days of [64]. HA scaffolds with high surface areas showed no cytotoxicity
culture showing good cell adherence and cell ingrowth into the and adequate cell adhesion with MC3T3-E1 fibroblast cells [65]. In
pores, suggesting that the scaffolds were non-toxic. A secondary addition to in vitro experiments, in vivo biocompatibility and
electron microscopy (SEM) image of the surface morphology and osteoconductivity of 3D-printed scaffolds showed that the 3D-
the designed macro pore distribution in a pure TCP scaffold is printed brushite and monetite cements with controlled open
shown in Fig. 2d. New bone formation was observed at the porosity increased osteoconduction in vivo in a goat model [8].
implant/host bone interface as well as inside the interconnected 3D-printed TCP samples with micro and macro-porosity also
macro and intrinsic micro pores after 4 and 8 weeks in both pure facilitated osteogenesis in a rat femur model [53]. Cytotoxicity
and doped TCP as shown in Fig. 2e. However, more osteoid like results of MC3T3-E1 cells on two different bone cement based
new bone formation was observed in SrO–MgO doped TCP scaffold compositions of TTCP/b-TCP and TTCP/calcium sulfate dihydrate
as shown in Fig. 2f. Histological evaluation and histomorpho- have been reported for bone tissue engineering. A wide range of
metric analysis reveal that the treatment group (doped TCP scaf- binders were used. It has been reported that the shortest hardening
folds) facilitated higher osteoid like bone at an early stage, and time can be obtained between 20–40% of citric acid, and 30–40%
completely mineralized bone later, which could be essential for of lactic acid; however, a lower range of those binders and a
fast bone healing and mineralization in vivo [62,84]. different concentration of sodium hydrogenphosphate with sul-
Further studies have shown that the addition of SiO2–ZnO furic and phosphoric acids can be used to increase the hardening
dopants to TCP scaffolds increases cell viability in different pore time for the cements [68]. Fig. 3a and b show patient specific 3D
size ranges [76]. The biocompatibility of 3D printed CaP ceramics printed CaP implants. These results point to the application of 3DP
has also been studied using osteoclasts. Tartrate resistant acid in a large variety of materials and structures for bone tissue
phosphatase (TRAP) staining, lacunae formation and microscopic engineering scaffolds.

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FIGURE 3
(a) 3D printed cranial segment [68], (b) general view of the implant bearing skull. Implants are fixed with miniplate osteosynthesis respectively bicortical
osteosynthesis (mandibular defect). The drill holes for screw insertion were made after the positioning of the implants using a common bone drill [70], (c)
representative macroscopic views of one half of bioceramic implant at retrieval, loaded with 56 ng copper [80].

Mechanical properties of 3D printed scaffolds sintering compared to conventional heating, and improved the
Low mechanical strength is a major challenge in porous scaffolds, mechanical properties of 3D-printed TCP scaffolds [62]. Bioactive
and is primarily controlled by pore volume. This is also true for 3D liquid phase sintering aids have also been reported to increase
printed ceramic scaffolds and limits their use only in non-load strength. 3D printed HA/A-W glass, where the glassy phase is added
bearing and low-load bearing applications. Optimized post proces- as a liquid phase sintering aid, showed an increase in strength from
sing approaches and compositional modifications can improve 1.27 MPa to 76.82 MPa when sintered at 1300 8C for 3 h [83]. The
mechanical properties of ceramic scaffolds. The compressive enhancement of tensile properties was also found in PE scaffolds as a
strength of 3D printed TCP sintered scaffolds is shown in Fig. 2b. result of thermally induced densification and binder degradation
In agreement with observed shrinkage and increased density, micro- [77]. To increase the strength of ceramic scaffolds without impairing
wave sintering results in a higher compressive strength. The biological properties of scaffolds, another approach is monomer or
strength of the scaffold increases with decreasing pore size or polymer infiltration. A mixture of bismethacrylated oligolactide
volume, and a maximum strength of 10.95  1.28 MPa has been macromer (DLM-1), containing 10 wt% of 2-hydroxyethyl metha-
observed for scaffolds with 500 mm pores, with 42% total open crylate has been used to increase the strength of scaffolds before and
porosity, when sintered at 1250 8C for 1 h in a microwave furnace after sintering [68]. The immersion of HA scaffolds in triethylene
[62]. In another study, when a mixture of TTCP/b-TCP was sintered glycol dimethacrylate (TEGDMA), 2,2-bis[4 (2-hydroxy-3thacryloy-
at 1400 8C, it increased the strength of the 3D printed scaffold. loxypropyloxy)-henyl] propane (bis-GMA) resulted in an increase of
However, sintering a TTCP/calcium sulfate dihydrate composite the flexural strength by at least 20 times [85]. Table 3 summarizes the
caused a decrease in the strength due to water release [68]. Tarafder mechanical properties of 3D printed scaffolds tailored for bone
et al. reported an effective densification approach, using microwave tissue engineering.

TABLE 3
Mechanical properties of 3D printed scaffolds.
Material Compressive Compressive Compressive Bending Bending Reference
strength stiffness yield strength modulus strength
(MPa) (MPa) (MPa) (GPa) (MPa)
TCP-sintered conventionally at 1250 8C 6.4 [62]
TCP-sintered using microwave at 1250 8C 10.9 [62]
TTCP/b-TCP 0.7 [68]
DLM infiltrated TTCP/b-TCP 76.1 [68]
Brushite 5.2 [70]
Monetite 3.9 [70]
Starch 11.15 1.12 [73]
PLLA/PCL infiltrated starch 55.19 1.77 [73]
TCP-sintered conventionally at 1250 8C 5.5 [76]
SiO2–ZnO doped TCP-sintered conventionally at 1250 8C 10.2 [76]
HA/A-W glass 0.35 1.27 [83]
HA/A-W glass-sintered at 1300 8C 34.1 76.82 [83]
HA 0.4 0.69 [85]
HA/bis-GMA 6.2 50 [85]

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Bioprinting of tissue engineering scaffolds dependent on the specific surface area and the release followed
Apart from inorganic scaffold manufacturing, AM approaches are an exponential pattern. In addition, drug immersion in a poly-
also used to explore possibilities in fabricating scaffolds with live lactide–polyglycolide (PLA/PGA) 50:50 polymer resulted in a
cells and tissues. Organogenesis of liver tissue using 3D printed delayed release profile [82]. It was also shown that polymer incor-
PLLA/poly(lactic-co-glycolic acid) (PLGA) scaffolds has been inves- poration in 3D-printed scaffolds could retard drug release kinetics
tigated in vitro. It was shown that culturing a mixture of hepato- from first to zero order. In addition, vancomycin, heparin and
cytes and endothelial cells on a channeled biodegradable scaffold rhBMP-2 incorporation during printing revealed a reduction in
results in the desired tissue structure intrinsically [86]. In 3D fiber biological activity due to the degradation of drugs during spraying
deposition, a cell-laden viscous polymer paste was prepared and through the nozzles [93]. Use of copper in DCPD scaffolds proved
printed using a syringe dispenser. Alginate hydrogel-embedded that incorporation and release of copper can induce angiogenesis,
RESEARCH: Review

multipotent stromal cells (MSCs)/chondrocytes were printed with vasculogenesis and osteogenesis, as shown in Fig. 3c [80]. Fig. 4a
a high cell viability using this method. The incorporation of MSCs and b show a hexagonal gelatin methacrylate (GelMa) scaffold
and chondrocytes resulted in distinctive ECM formation both in seeded with HUVEC-green fluorescent protein (GFP), showing cell
vitro and in vivo. In addition, an increase in strand distance was spreading and organization [89]. Fig. 4c and d show vancomycin
shown to increase porosity with a lower elastic modulus. However, release from 3D printed brushite and brushite/chitosan scaffolds
changing the strand orientation from 908 to 458 increased the when a drug is loaded homogeneously, while Fig. 4e and f show
elastic modulus [87]. Unique distribution and organization of the release behavior from similar scaffolds when drug is loaded at
human umbilical vein endothelial cells (HUVECs) and mouse the center of scaffold [93]. Fig. 4g and h show various antibiotics
embryonic fibroblast cells was obtained using gelatin methacrylate released from 3D printed calcium phosphate and brushite
scaffolds prepared by SLA [88,89]. 3D patterned human osteopro- matrices after immersion in a PBS buffer. The influence of PLA/
genitors (HOPs) and HA/HOPs fabricated by laser-assisted bio- PGA polymer impregnation on vancomycin release from brushite
printing maintained osteoblastic phenotype and functionality matrices is shown in Fig. 4i [82]. Overall, these studies show that
as evidenced by alkaline phasphatase (ALP) expression [39]. 3D printed scaffolds can be used in drug delivery. There is a lot of
potential for direct printing of bone tissue engineering scaffolds,
Growth factor and drug delivery using 3D printed but only if the reduction in biological activity of drugs/growth
scaffolds factor can be minimized, and reproducibility and stability can be
There are many growth factors such as vascular endothelial growth assured. Today, bioprinting, and drug and growth factor delivery
factor (VEGF), fibroblast growth factors (FGFs) and bone morpho- using 3D printed tissue engineering scaffolds are still in their
genic proteins (BMPs) that are important in bone tissue engineer- infancies.
ing. VEGF is an angiogenic protein which regulates endothelial
cell proliferation. FGFs are group of proteins, essential for the FGF Future direction and challenges
signaling pathway that induces angiogenesis through endothelial AM offers unique advantages toward part fabrication that are
and osteoblast cell proliferation, respectively [90]. BMPs, however, needed for the production of small volumes or one of a kind
induce osteogenesis through osteoprogenitors and mesenchymal product manufacturing. Among the different AM techniques,
stem cell (MSC) differentiation to osteoblasts or binding to col- 3DP is a versatile tool that has become popular for making scaf-
lagen [62,91]. Bioprinting has become a versatile method in recent folds for bone tissue engineering. 3DP can fabricate scaffolds with
years to create protein-based arrays. Bioprinting methods allow the defined shapes, with controlled and interconnected porous struc-
study of the effects of microenvironment changes due to the tures. Although the process characteristics provide the opportu-
aligned configuration to determine cell differentiation and align- nity for the fabrication of almost all types of materials, the
ment [92]. Muscle derived stem cells (MSDCs) cultured on printed selection of a suitable binder for 3DP is still a challenge, and
ECM containing BMP-2 indicated differentiation to osteogenic extensive optimization may be needed before high quality parts
lineage under myogenic conditions [91]. BMP-2 printed fibrin- can be made. Among different binders, organic binders work well,
coated spun fibers regulated ALP as an osteoblast marker during however, they can affect the plastic parts of 3DP machines during
mouse myoblast cell line (C2C12) culture [91]. However, a delayed long term operation. The residue from binders may be difficult to
BMP-2 administration from a 3D-printed HA block did not remove during sintering, an issue that may need special attention
enhance osteoinduction due to soft tissue ingrowth [75]. for biomaterials. Moreover, to achieve the desired accuracy and
3D printed scaffolds have also been used for growth factor and resolution in 3DP, a minimum distance between pores is necessary
drug delivery to enhance bone growth in scaffolds. The localized which is dependent on powder characteristics and the build
delivery of growth factors and drugs has attracted significant parameters. The minimum distance requirement for a powder
attention due to the potential for dose reduction, controlled based process makes it difficult to print highly porous scaffolds
release pattern, and the negligible side effects compared to sys- with a sintered pore size below 300 mm [68].
temic delivery [93]. For scaffolds, pore size, connectivity and Post processing is always required for 3DP processed parts.
geometry are effective parameters to control drug loading as well Sintering or densification at high temperature is just one of them.
as release rates in vivo [94]. Three different calcium phosphates During sintering, parts shrink and the shrinkage is not necessarily
(CaPs) – brushite, monetite, and HA – were fabricated using 3DP as uniform throughout the part. Non-uniform shrinkage can cause
shown in Table 2. Vancomycin hydrochloride, ofloxacin and extensive cracking in parts and make them unusable. This is a
tetracycline hydrochloride were loaded onto these compositions particular challenge for porous scaffolds. Since the outside part of
via immersion/vacuum impregnation. Drug absorption was bone is a dense structure with 10% or less porosity while inside it

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FIGURE 4
(a) Hexagonal gelatin methacrylate (GelMa) scaffold seeded with HUVEC-green fluorescent protein (GFP), (b) cells spreading and organization on scaffold
shown in (a) [89], (c) and (d) vancomycin release from 3D printed brushite and brushite/chitosan samples, when the drug is loaded homogeneously, (e) and
(f ) vancomycin release from 3D printed brushite and brushite/chitosan samples, when the drug is loaded in the center of scaffold [93], (g) release of
vancomycin from different 3D printed calcium phosphate matrices after immersion in PBS buffer, (h) release of various antibiotics from brushite matrices
after immersion in PBS buffer, (i) influence of PLA/PGA polymer impregnation (10–50% polymer solution) on vancomycin release from brushite matrices [82].

is highly porous with >50% porosity, mimicking such structures is and in vivo research are needed in that direction to make any of
very difficult using 3DP due to challenges related to non-uniform those approaches useful toward bone tissue engineering.
shrinkage during sintering. Another post-processing challenge is
the removal of loose powders from interconnected pores inside the Acknowledgements
part. This problem is magnified for parts with small pores, in Financial support from the US National Institute of Health under
particular below 600 mm. Trapped powders inside the pores may the Grant Number (R 01 EB-007351) is acknowledged. Authors like
well sinter with the porous part making it less interconnected than to also acknowledge Dr. Solaiman Tarafder for his help.
the designed part. Such problems with loose powders can reduce
the dimension of the pores after sintering. References
Demand for processes such as 3DP will increase in the coming [1] A. Bandyopadhyay, S. Bose, Characterization of Biomaterials, Elsevier Inc., 2013p. 1.
years due to their ability to make custom medical devices that can [2] V. Mouriño, A.R. Boccaccini, J. R. Soc. Interface 7 (2010) 209–227.
be tailored for patient specific and defect specific clinical needs. [3] H. Seitz, et al. J. Biomed. Mater. Res. B 74B (2005) 782–788.
[4] A.C. Jones, et al. Biomaterials 28 (2007) 2491–2504.
Extensive process-property optimization is still needed to accom-
[5] K. Rezwan, et al. Biomaterials 27 (2006) 3413–3431.
plish this goal. For ceramics, the most critical issue that needs [6] B. Müller, et al. Proc. SPIE 7401 (2009).
attention is the mechanical properties of porous scaffolds. Increas- [7] A.J. Salgado, O.P. Coutinho, R.L. Reis, Macromol. Biosci. 4 (2004) 743–765.
ing the porosity will decrease the strength of the scaffolds. Low [8] P. Habibovic, et al. Biomaterials 29 (2008) 944–953.
[9] V. Karageorgiou, D. Kaplan, Biomaterials 26 (2005) 5474–5491.
strength along with brittleness makes these scaffolds difficult to
[10] W. Xue, et al. Acta Biomater. 3 (2007) 1007–1018.
even handle during processing. Resorbable polymer infiltration to [11] B. Otsuki, et al. Biomaterials 27 (2006) 5892–5900.
enhance strength and toughness in these scaffolds is one way to [12] M. Stoppato, et al. J. Bioact. Compat. Pol. 28 (2013) 16–32.
minimize this problem; the use of resorbable glassy materials can [13] A.G. Mitsak, et al. Tissue. Eng. Part A 17 (2011) 1831–1839.
[14] A. Bandyopadhyay, et al. J. Am. Ceram. Soc. 89 (2006) 2675–2688.
also help. Finally, printing live cells or adding growth factors/drugs
[15] S.S. Banerjee, et al. Acta Biomater. 6 (2010) 4167–4174.
is another fascinating area of growth. However, most of the [16] S. Bose, et al. Bone 48 (2011) 1282–1290.
challenges here are limited to survivability of the cells, viability [17] K. Das, S. Bose, A. Bandyopadhyay, Acta Biomater. 3 (2007) 573–585.
of the growth factors and drugs after printing. Although current [18] S. Bodhak, S. Bose, A. Bandyopadhyay, Acta Biomater. 5 (2009) 2178–2188.
[19] S. Tarafder, et al. Langmuir 26 (2010) 16625–16629.
techniques let us build structures with similar composition to that [20] M. Kucharska, et al. Mater. Lett. 85 (2012) 124–127.
of tissue, we are still a long way from completely printing func- [21] H. Cao, N. Kuboyama, Bone 46 (2010) 386–395.
tioning tissue [95]. More process-property optimization, in vitro [22] N. Sultana, M. Wang, J. Mater. Sci.: Mater. Med. 19 (2008) 2555–2561.

503
 RESEARCH Materials Today  Volume 16, Number 12  December 2013

[23] D.W. Hutmacher, Biomaterials 21 (2000) 2529–2543. [56] C.X.F. Lam, et al. Polym. Int. 56 (2007) 718–728.
[24] H. Yoshikawa, et al. J. R. Soc. Interface 6 (2009) S341–S348. [57] J.-T. Schantz, Tissue Eng. 9 (2003) S127–S139.
[25] S. Bose, S. Suguira, A. Bandyopadhyay, Scr. Mater. 41 (1999) 1009–1014. [58] C.X.F. Lam, et al. J. Biomed. Mater. Res. A 90A (2009) 906–919.
[26] S. Bose, et al. Mater. Sci. Eng. C 23 (2003) 479–486. [59] J. Russias, et al. J. Biomed. Mater. Res. A 83A (2007) 434–445.
[27] I. Gibson, et al., Additive Manufacturing Technologies: Rapid Prototyping to [60] E.M. Sachs, et al., Three-dimensional printing techniques, US Patent #
Direct Digital Manufacturing, Springer, 2009. 5,204,055.
[28] C.W. Hull, Apparatus for production of three-dimensional objects by stereolitho- [61] E. Sachs, M.J. Cima, J. Cornie, CIRP Ann. 39/1 (1990) 204–210.
graphy, US Patent # 4,575,330. [62] S. Tarafder, et al. J. Tissue Eng. Regen. Med. 7 (2012) 631–641.
[29] F.B. Prinz, et al. JTEC/WTEC Panel Report on Rapid Prototyping in Europe and [63] P.H. Warnke, et al. J. Biomed. Mater. Res. 93B (2010) 212–217.
Japa, vol. I, Rapid Prototyping Association of the Society of Manufacturing [64] E. Vorndran, et al. Adv. Eng. Mater. 10 (2008) B67–B71.
Engineers, Loyola College in Maryland, 1997. [65] B. Leukers, et al. Mater. Wiss. Werkstofftech. 36 (2005) 781–787.
[30] J. Darsell, et al. J. Am. Ceram. Soc. 87 (2003) 1076–1080. [66] S.A. Uhland, et al. J. Am. Ceram. Soc. 84 (2001) 2809–2818.
RESEARCH: Review

[31] S. Bose, et al. J. Mater. Sci.: Mater. Med. 13 (2002) 23–28. [67] S. Amirkhani, R. Bagheri, A. Zehtab Yazdi, Acta Mater. 60 (2012) 2778–2789.
[32] Y. Luo, et al. Biofabrication 5 (2013), http://dx.doi.org/10.1088/1758-5082/5/1/ [68] A. Khalyfa, et al. J. Mater. Sci.: Mater. Med. 18 (2007) 909–916.
015005. [69] R. Detsch, et al. J. Biomater. Appl. 26 (2011) 359–380.
[33] J.M. Sobral, et al. Acta Biomater. 7 (2011) 1009–1018. [70] U. Klammert, et al. J. Cranio Maxill. Surg. 38 (2010) 565–570.
[34] R. Detsch, J. Mater. Sci.: Mater. Med. 19 (2008) 1491–1496. [71] A.J. Salgado, O.P. Coutinho, R.L. Reis, Tissue Eng. 10 (2004) 465–474.
[35] C. Wu, et al. Acta Biomater. 7 (2011) 2644–2650. [72] A.L. Oliveira, R.L. Reis, J. Mater. Sci.: Mater. Med. 15 (2004) 533–540.
[36] T. Serra, J.A. Planell, M. Navarro, Acta Biomater. 9 (2013) 5521–5530. [73] C.X.F. Lam, et al. Mater. Sci. Eng. C 20 (2002) 49–56.
[37] H. Seyednejad, et al. Biomaterials 33 (2012) 4309–4318. [74] J. Suwanprateeb, et al. J. Porous Mater. 19 (2012) 623–632.
[38] Q. Fu, E. Saiz, A.P. Tomsia, Acta Biomater. 7 (2011) 3547–3554. [75] S.T. Becker, et al. Int. J. Oral Maxillofac. Surg. 41 (2012) 1153–1160.
[39] S. Catros, et al. Biofabrication 3 (2011), http://dx.doi.org/10.1088/1758-5082/3/2/ [76] G.A. Fielding, A. Bandyopadhyay, S. Bose, Dent. Mater. 28 (2012) 113–122.
025001. [77] J. Suwanprateeb, et al. Polym. Int. 60 (2011) 758–764.
[40] A. Doraiswamy, et al. J. Biomed. Mater. Res. A 80 (2007) 635–643. [78] J. Suwanprateeb, et al. J. Bioact. Compat. Pol. 26 (2011) 317–331.
[41] M.L. Harris, et al. Mater. Sci. Eng. C 28 (2008) 359–365. [79] R.A. Giordano, et al. J. Biomater. Sci.-Polym. E 8 (1996) 63–75.
[42] B. Guillotin, et al. Biomaterials 31 (2010) 7250–7256. [80] U. Gbureck, et al. Adv. Mater. 19 (2007) 795–800.
[43] J.M. Williams, et al. Biomaterials 26 (2005) 4817–4827. [81] J. Suwanprateeb, R. Chumnanklang, J. Biomed. Mater. Res. B 78B (2006) 138–145.
[44] C. Shuai, et al. Nanotechnology 22 (2011), http://dx.doi.org/10.1088/0957-4484/ [82] U. Gbureck, et al. J. Control. Release 122 (2007) 173–180.
22/28/285703. [83] J. Suwanprateeb, et al. J. Mater. Sci.: Mater. Med. 20 (2009) 1281–1289.
[45] B. Duan, et al. Acta Biomater. 6 (2010) 4495–4505. [84] S. Tarafder, et al. Biomater. Sci. (2013), http://dx.doi.org/10.1039/c3bm60132c.
[46] S.H. Lee, et al. J. Biomimetics Biomater. Tissue Eng. 1 (2008) 81–89. [85] J. Suwanprateeb, R. Sanngam, W. Suwanpreuk, J. Mater. Sci.: Mater. Med. 19
[47] H. Qingxi, et al. International Technology and Innovation Conference, 2006. (2008) 2637–2645.
[48] T.F. Pereira, et al. Virtual Phys. Prototyp. 7 (2012) 275–285. [86] L.G. Griffith, et al. Bioartif. Organs: Sci. Med. Technol. 831 (1997) 382–397.
[49] P.X. Lan, et al. J. Mater. Sci.: Mater. Med. 20 (2009) 271–279. [87] N.E. Fedorovich, et al. Tissue Eng. Part C 18 (2012) 33–44.
[50] J.W. Lee, et al. Microelectron. Eng. 86 (2009) 1465–1467. [88] A.P. Zhang, et al. Adv. Mater. 24 (2012) 4266–4270.
[51] A. Ronca, L. Ambrosio, D.W. Grijpma, Acta Biomater. 9 (2013) 5989–5996. [89] R. Gauvin, et al. Biomaterials 33 (2012) 3824–3834.
[52] W.G. Bian, et al. Biofabrication 3 (2011), http://dx.doi.org/10.1088/1758-5082/3/ [90] J.P. Bilezikian, et al., Principles of Bone Biology, Elsevier Inc., 2008p. 3.
3/034103. [91] J.A. Phillippi, et al. Stem Cells 26 (2008) 127–134.
[53] S.J. Kalita, et al. Mater. Sci. Eng. C 23 (2003) 611–620. [92] E.D.F. Ker, et al. Biomaterials 32 (2011) 8097–8107.
[54] V.L. Tsang, S.N. Bhatia, Adv. Drug Deliv. Rev. 56 (2004) 1635–1647. [93] E. Vorndran, et al. Adv. Funct. Mater. 20 (2010) 1585–2159.
[55] C.X.F. Lam, et al. Biomed. Mater. 3 (2008), http://dx.doi.org/10.1088/1748-6041/ [94] J. Schnieders, et al. J. Biomed. Mater. Res. B 99B (2011) 391–398.
3/3/034108. [95] B. Derby, Science 338 (2012) 921–926.

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