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

496 1369-7021/06 ß 2013 Elsevier Ltd. Open access under CC BY-NC-ND license. http://dx.doi.org/10.1016/j.mattod.2013.11.017
Materials Today  Volume 16, Number 12  December 2013
without any remnant [8]. A minimum pore size between 100 and
150 mm is needed for bone formation [4,9]; however, enhanced
bone formation and vascularization are reported for scaffolds with
pore size larger than 300 mm [9–11]. Pore size also plays an impor-
tant role in ECM production and organization. Poly(D,L-lactic acid)
(PDLLA) scaffolds with pore size 325 and 420 mm led to well-
organized collagen I network; whereas, smaller pore size of
275 mm prevented the human osteosarcoma-derived osteoblasts
to proliferate, differentiate and produce functional ECM [12]. Pore
volume also controls the permeability of nutrients to the scaffold
and their mechanical properties. Permeability in poly-e-caprolac-
tone (PCL) increased with higher pore volume and resulted in
better bone regeneration, blood vessel infiltration, and compres-
sive strength in vivo, when other pore parameters were kept the
same [13]. Apart from biological performance, the initial mechan-
ical properties and strength degradation rate should match that of
the host tissue for optimum bone healing [14]. The strength
degradation kinetics of porous scaffolds are highly affected by
pore size, geometry, and strut orientation with respect to the
loading direction [15,16]. Finally, surface properties such as chem-
istry, surface charge and topography also influence hydrophilicity
and in turn cell–material interactions for bone tissue ingrowth
[17–19].
Porous bone scaffolds can be made by a variety of methods.
Chemical/gas foaming [20], solvent casting, particle/salt leaching
[12,21], freeze drying [22], thermally induced phase separation
[23], and foam-gel [24] are some of those that have been used
extensively. However, pore size, shape, and its interconnectivity
cannot be fully controlled in these approaches. Moreover scaffolds
with tailored porosity for specific defects are difficult to manufac-
ture with most of these approaches [21–24]. Such scaffolds can be
designed and fabricated using additive manufacturing (AM)
approaches. Different AM approaches, for example, 3D printing
(3DP), solid freeform fabrication (SFF), rapid prototyping (RP), are
approaches that allow complex shapes for scaffolds’ fabrication
directly from a computer aided design (CAD) file [25–27]. The
concept of AM was first introduced by Chuck Hull in 1986 via a
process known as ‘stereolithography (SLA)’ [28,29].
Some of the commercially available AM techniques are 3DP
(ExOne, PA), fused deposition modeling (FDM, Stratasys, MN),
selective laser sintering (SLS, 3D Systems, CA), stereolithography
(3D Systems, CA), 3D plotting (Fraunhofer Institute for Materials
Research and Beam Technology, Germany), as well as various forms
of direct writing [27]. In all these AM approaches, 3D scaffolds are
created layer-by-layer without any part specific tooling or dies
[30,31]. These AM techniques can be classified as – (a) extrusion
(deformation + solidification), (b) polymerization, (c) laser-assisted
sintering, and (d) direct writing-based processes. Table 1 [32–59]
summarizes some of the AM techniques toward bone tissue engi-
neering applications including their advantages and disadvantages.
3D printing (3DP) – history and methodology
3DP, a technology developed in the early 1990s at MIT (Cam-
bridge, MA) by Sachs et al. [60], is a powder-based freeform
fabrication method in which using a regular ink-jet print-head,
binders are printed on to loose powders in a powder bed. Early
research in this area was focused on rapid tooling using metals and
ceramics [61].
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Fig. 1 shows a schematic representation of the 3DP process [62].
For bone tissue engineering, 3DP is useful for the direct fabrication
of scaffolds with tailored porosity from a CAD file. Before printing,
essential parameters such as powder packing density, powder
flowability, layer thickness, binder drop volume, binder saturation
and powder wettability need to be optimized to improve the
quality of the resultant part. Packing density is the relative density
of the powder bed after uniform spreading. To start a build,
enough powder should be packed homogeneously in a feed bed.
A set of rollers spread a layer of powder to a predetermined
RESEARCH: Review
thickness to create a powder bed. Powder flowability is critical
in this process as it determines the spreading ability. Flowability is
primarily determined by particle size, size distribution, surface
roughness and shape. The desired layer thickness is in part deter-
mined by geometry and powder characteristics. Thinner layers
cause binder penetration and excess spreading to other sites
resulting in poor resolution and tolerance. However, thick layers
need high saturation for the powders to bind [62].
The printhead sprays the binder across the build layer in several
passes, based on the instructions in the tool path file created
according to the CAD file. The binder, which can be organic or
water-based, locally binds the particles and hardens the wetted
area, or results in a reaction similar to the hydraulic setting
reaction in cements [63–65]. The binder drop volume and satura-
tion play crucial roles. The binder drop volume is the amount of
binder released from each nozzle per drop during printing, which
depends on the binder density and viscosity. By coordinating the
powder packing density and the drop volume, the binder satura-
tion data required for printing is obtained. For a constant packing
density, a higher drop volume demands a lower binder saturation
[62]. Low saturation can cause layer displacement during proces-
sing. The binder saturation also depends on the powder wettabil-
ity. The powder wettability, which is related to the powder particle
chemistry and surface energy, determines the printing accuracy
and the achievable tolerance [66]. While high wettability results in
extensive binder spreading, low wettability causes week powder-
binder integration [67].
After printing, the printed layer is moved under a strip heater to
allow the binder to dry out and prevent spreading between layers
[62]. This process is repeated until the printing of the designed part
is complete. Heat treatment is needed to complete the binder
reaction and increase the part green strength. Next step is depow-
dering, that is, the removal of loose powder from the printed body.
This is one of the major challenges for porous scaffolds in 3DP due
to the low green density of the part. Loose powder removal from
fine pores can easily crack a green part [68].
In general, a large variety of ceramic, metallic, polymeric, and
composite materials can be processed using 3DP; however, binder
selection and process parameter optimization are the keys to
successful part fabrication. In bone tissue engineering the advan-
tages of this method arise through the control of fine features
including interconnected porosity, no contamination issues
related to any second material for support structures and the direct
printing ability with both metallic and ceramic biomaterials
[65,69]. Fig. 2a shows some examples of 3D printed scaffolds with
different pore sizes. It is important to note that extensive optimi-
zation is needed to process good quality parts with 3DP for any
new material, a fundamental drawback for this approach.
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TABLE 1
RP techniques for bone scaffold fabrication.
Technique Process details
3D Plotting/direct ! Strands of paste/viscous material
ink writing (in solution form) extrusion based
on the predesigned structure
! Layer by layer deposition of
strands at constant rate,
under specific pressure
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! Disruption of strands according to
the tear of speed
Laser-assisted ! Coating the desired material on
bioprinting transparent quartz disk (ribbon)
(LAB) ! Deposition control by laser
pulse energy
! Resolution control by distance
between ribbon/substrate, spot
size and stage movement
SLS ! Preparing the powder bed
! Layer by layer addition of powder
! Sintering each layer according to
the CAD file, using laser source
SLA ! Immersion of platform in a
photopolymer liquid
! Exposure to focused light according
to desired design
! Polymer solidifying at focal point,
non-exposed polymer remains liquid,
! Layer by layer fabrication by platform
moving downward
FDM ! Strands of heated polymer/ceramics
extrusion through nozzle
Robotic assisted ! Direct writing of liquid using a
deposition/ nozzle
robocasting ! Consolidation through liquid-to-
gel transition
3D printed bone scaffolds
Table 2 summarizes a few selected material-binder system combi-
nations for bone scaffolds using 3DP. Starch-based binders are one
of the candidates for bone replacement applications. These binders
are biocompatible and produce structures that have a mechanical
strength close to trabecular bone [71,72]. Structural designs and
post processing conditions both can influence the mechanical
properties of 3D-printed starch-based scaffolds [73]. 3D-printed
polyethylene (PE) scaffolds with 22.3–49.7% porosity have shown
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Materials Today  Volume 16, Number 12  December 2013
Processed materials for bone Advantages (+) and Reference
tissue engineering disadvantages ()
! PCL +: [32–38]
! Hydroxyapatite (HA) ! Mild condition of process allows
! Bioactive glasses drug and biomolecules (proteins
! Mesoporous bioactive and living cells) plotting
glass/alginate composite :
! Polylactic acid (PLA)/polyethylene ! Heating/post-processing
glycol (PEG) needed for some materials restricts
! PLA/(PEG)/G5 glass the biomolecule incorporation
! Poly(hydroxymethylglycolide-
co-e-caprolactone) (PHMGCL)
! Bioactive 6P53B glass
! HA +: [39–42]
! Zirconia ! Ambient condition
! HA/MG63 osteoblast-like cell ! Applicable for organic,
! Nano HA inorganic materials and cells
! Human osteoprogenitor cell ! Quantitatively controlled
! Human umbilical vein ! 3D stage movement
endothelial cell :
! Homogeneous ribbons needed
! PCL +: [43–48]
! Nano HA ! No need for support
! Calcium phosphate (CaP)/poly ! No post processing is needed
(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
! Poly(propylene fumarate) +: [49–52]
(PPF)/diethyl fumarate (DEF) ! Complex internal features can
! PPF/DEF-HA be obtained
! PDLLA/HA ! Growth factors, proteins and cell
! b-TCP patterning is possible
:
! Only applicable for photopolymers
! Tricalcium phosphate (TCP) +: [26,30,31,
! TCP/polypropylene (PP) No need for platform/support 53–58]
! Alumina (Al2O3) :
! PCL ! Material restriction due to need
! TCP/PCL for molten phase
! HA/PLA +: [59]
! HA/PCL ! Independent 3D nozzle movement
! 6P53B glass/PCL ! Precise control on thickness
! No need for platform/support
:
! Material restriction
a tensile strength up to 4 MPa, and no toxicity to human osteo-
blasts [74].
CaP ceramics are widely used in bone tissue engineering due to
their excellent bioactivity, osteoconductivity, and similarities in
composition to bone. Capillaries and vessel formation, and homo-
geneous osteoconduction from central channels, have previously
been observed in 3D-printed HA blocks [75]. The effect of pore size
on human fetal osteoblasts (hFOB) was studied with 3D-printed
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.
3D printed and microwave sintered b-TCP scaffolds fabricated
using 3DP are shown in Fig. 2a, showing interconnected macro
porosity across the sample. Fig. 2c (i–iv) presents the morphologies
of hFOB cells on scaffold surfaces and pore walls after 3 days of
culture showing good cell adherence and cell ingrowth into the
pores, suggesting that the scaffolds were non-toxic. A secondary
electron microscopy (SEM) image of the surface morphology and
the designed macro pore distribution in a pure TCP scaffold is
shown in Fig. 2d. New bone formation was observed at the
implant/host bone interface as well as inside the interconnected
macro and intrinsic micro pores after 4 and 8 weeks in both pure
and doped TCP as shown in Fig. 2e. However, more osteoid like
new bone formation was observed in SrO–MgO doped TCP scaffold
as shown in Fig. 2f. Histological evaluation and histomorpho-
metric analysis reveal that the treatment group (doped TCP scaf-
folds) facilitated higher osteoid like bone at an early stage, and
completely mineralized bone later, which could be essential for
fast bone healing and mineralization in vivo [62,84].
Further studies have shown that the addition of SiO2–ZnO
dopants to TCP scaffolds increases cell viability in different pore
size ranges [76]. The biocompatibility of 3D printed CaP ceramics
has also been studied using osteoclasts. Tartrate resistant acid
phosphatase (TRAP) staining, lacunae formation and microscopic
images confirmed the monocyte differentiation to multinuclear
osteoclast-like cells on a wide range of compositions [69]. It has
been shown that the use of phosphoric acid instead of polymeric
binders can improve both resolution and compressive strength
[64]. HA scaffolds with high surface areas showed no cytotoxicity
and adequate cell adhesion with MC3T3-E1 fibroblast cells [65]. In
addition to in vitro experiments, in vivo biocompatibility and
osteoconductivity of 3D-printed scaffolds showed that the 3D-
printed brushite and monetite cements with controlled open
porosity increased osteoconduction in vivo in a goat model [8].
3D-printed TCP samples with micro and macro-porosity also
facilitated osteogenesis in a rat femur model [53]. Cytotoxicity
results of MC3T3-E1 cells on two different bone cement based
compositions of TTCP/b-TCP and TTCP/calcium sulfate dihydrate
have been reported for bone tissue engineering. A wide range of
binders were used. It has been reported that the shortest hardening
time can be obtained between 20–40% of citric acid, and 30–40%
of lactic acid; however, a lower range of those binders and a
different concentration of sodium hydrogenphosphate with sul-
furic and phosphoric acids can be used to increase the hardening
time for the cements [68]. Fig. 3a and b show patient specific 3D
printed CaP implants. These results point to the application of 3DP
in a large variety of materials and structures for bone tissue
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
Low mechanical strength is a major challenge in porous scaffolds,
and is primarily controlled by pore volume. This is also true for 3D
printed ceramic scaffolds and limits their use only in non-load
bearing and low-load bearing applications. Optimized post proces-
sing approaches and compositional modifications can improve
mechanical properties of ceramic scaffolds. The compressive
strength of 3D printed TCP sintered scaffolds is shown in Fig. 2b.
In agreement with observed shrinkage and increased density, micro-
wave sintering results in a higher compressive strength. The
strength of the scaffold increases with decreasing pore size or
volume, and a maximum strength of 10.95  1.28 MPa has been
observed for scaffolds with 500 mm pores, with 42% total open
porosity, when sintered at 1250 8C for 1 h in a microwave furnace
[62]. In another study, when a mixture of TTCP/b-TCP was sintered
at 1400 8C, it increased the strength of the 3D printed scaffold.
However, sintering a TTCP/calcium sulfate dihydrate composite
caused a decrease in the strength due to water release [68]. Tarafder
et al. reported an effective densification approach, using microwave
sintering compared to conventional heating, and improved the
mechanical properties of 3D-printed TCP scaffolds [62]. Bioactive
liquid phase sintering aids have also been reported to increase
strength. 3D printed HA/A-W glass, where the glassy phase is added
as a liquid phase sintering aid, showed an increase in strength from
1.27 MPa to 76.82 MPa when sintered at 1300 8C for 3 h [83]. The
enhancement of tensile properties was also found in PE scaffolds as a
result of thermally induced densification and binder degradation
[77]. To increase the strength of ceramic scaffolds without impairing
biological properties of scaffolds, another approach is monomer or
polymer infiltration. A mixture of bismethacrylated oligolactide
macromer (DLM-1), containing 10 wt% of 2-hydroxyethyl metha-
crylate has been used to increase the strength of scaffolds before and
after sintering [68]. The immersion of HA scaffolds in triethylene
glycol dimethacrylate (TEGDMA), 2,2-bis[4 (2-hydroxy-3thacryloy-
loxypropyloxy)-henyl] propane (bis-GMA) resulted in an increase of
the flexural strength by at least 20 times [85]. Table 3 summarizes the
mechanical properties of 3D printed scaffolds tailored for bone
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
Apart from inorganic scaffold manufacturing, AM approaches are
also used to explore possibilities in fabricating scaffolds with live
cells and tissues. Organogenesis of liver tissue using 3D printed
PLLA/poly(lactic-co-glycolic acid) (PLGA) scaffolds has been inves-
tigated in vitro. It was shown that culturing a mixture of hepato-
cytes and endothelial cells on a channeled biodegradable scaffold
results in the desired tissue structure intrinsically [86]. In 3D fiber
deposition, a cell-laden viscous polymer paste was prepared and
printed using a syringe dispenser. Alginate hydrogel-embedded
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multipotent stromal cells (MSCs)/chondrocytes were printed with
a high cell viability using this method. The incorporation of MSCs
and chondrocytes resulted in distinctive ECM formation both in
vitro and in vivo. In addition, an increase in strand distance was
shown to increase porosity with a lower elastic modulus. However,
changing the strand orientation from 908 to 458 increased the
elastic modulus [87]. Unique distribution and organization of
human umbilical vein endothelial cells (HUVECs) and mouse
embryonic fibroblast cells was obtained using gelatin methacrylate
scaffolds prepared by SLA [88,89]. 3D patterned human osteopro-
genitors (HOPs) and HA/HOPs fabricated by laser-assisted bio-
printing maintained osteoblastic phenotype and functionality
as evidenced by alkaline phasphatase (ALP) expression [39].
Growth factor and drug delivery using 3D printed
scaffolds
There are many growth factors such as vascular endothelial growth
factor (VEGF), fibroblast growth factors (FGFs) and bone morpho-
genic proteins (BMPs) that are important in bone tissue engineer-
ing. VEGF is an angiogenic protein which regulates endothelial
cell proliferation. FGFs are group of proteins, essential for the FGF
signaling pathway that induces angiogenesis through endothelial
and osteoblast cell proliferation, respectively [90]. BMPs, however,
induce osteogenesis through osteoprogenitors and mesenchymal
stem cell (MSC) differentiation to osteoblasts or binding to col-
lagen [62,91]. Bioprinting has become a versatile method in recent
years to create protein-based arrays. Bioprinting methods allow the
study of the effects of microenvironment changes due to the
aligned configuration to determine cell differentiation and align-
ment [92]. Muscle derived stem cells (MSDCs) cultured on printed
ECM containing BMP-2 indicated differentiation to osteogenic
lineage under myogenic conditions [91]. BMP-2 printed fibrin-
coated spun fibers regulated ALP as an osteoblast marker during
mouse myoblast cell line (C2C12) culture [91]. However, a delayed
BMP-2 administration from a 3D-printed HA block did not
enhance osteoinduction due to soft tissue ingrowth [75].
3D printed scaffolds have also been used for growth factor and
drug delivery to enhance bone growth in scaffolds. The localized
delivery of growth factors and drugs has attracted significant
attention due to the potential for dose reduction, controlled
release pattern, and the negligible side effects compared to sys-
temic delivery [93]. For scaffolds, pore size, connectivity and
geometry are effective parameters to control drug loading as well
as release rates in vivo [94]. Three different calcium phosphates
(CaPs) – brushite, monetite, and HA – were fabricated using 3DP as
shown in Table 2. Vancomycin hydrochloride, ofloxacin and
tetracycline hydrochloride were loaded onto these compositions
via immersion/vacuum impregnation. Drug absorption was
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Materials Today  Volume 16, Number 12  December 2013
dependent on the specific surface area and the release followed
an exponential pattern. In addition, drug immersion in a poly-
lactide–polyglycolide (PLA/PGA) 50:50 polymer resulted in a
delayed release profile [82]. It was also shown that polymer incor-
poration in 3D-printed scaffolds could retard drug release kinetics
from first to zero order. In addition, vancomycin, heparin and
rhBMP-2 incorporation during printing revealed a reduction in
biological activity due to the degradation of drugs during spraying
through the nozzles [93]. Use of copper in DCPD scaffolds proved
that incorporation and release of copper can induce angiogenesis,
vasculogenesis and osteogenesis, as shown in Fig. 3c [80]. Fig. 4a
and b show a hexagonal gelatin methacrylate (GelMa) scaffold
seeded with HUVEC-green fluorescent protein (GFP), showing cell
spreading and organization [89]. Fig. 4c and d show vancomycin
release from 3D printed brushite and brushite/chitosan scaffolds
when a drug is loaded homogeneously, while Fig. 4e and f show
the release behavior from similar scaffolds when drug is loaded at
the center of scaffold [93]. Fig. 4g and h show various antibiotics
released from 3D printed calcium phosphate and brushite
matrices after immersion in a PBS buffer. The influence of PLA/
PGA polymer impregnation on vancomycin release from brushite
matrices is shown in Fig. 4i [82]. Overall, these studies show that
3D printed scaffolds can be used in drug delivery. There is a lot of
potential for direct printing of bone tissue engineering scaffolds,
but only if the reduction in biological activity of drugs/growth
factor can be minimized, and reproducibility and stability can be
assured. Today, bioprinting, and drug and growth factor delivery
using 3D printed tissue engineering scaffolds are still in their
infancies.
Future direction and challenges
AM offers unique advantages toward part fabrication that are
needed for the production of small volumes or one of a kind
product manufacturing. Among the different AM techniques,
3DP is a versatile tool that has become popular for making scaf-
folds for bone tissue engineering. 3DP can fabricate scaffolds with
defined shapes, with controlled and interconnected porous struc-
tures. Although the process characteristics provide the opportu-
nity for the fabrication of almost all types of materials, the
selection of a suitable binder for 3DP is still a challenge, and
extensive optimization may be needed before high quality parts
can be made. Among different binders, organic binders work well,
however, they can affect the plastic parts of 3DP machines during
long term operation. The residue from binders may be difficult to
remove during sintering, an issue that may need special attention
for biomaterials. Moreover, to achieve the desired accuracy and
resolution in 3DP, a minimum distance between pores is necessary
which is dependent on powder characteristics and the build
parameters. The minimum distance requirement for a powder
based process makes it difficult to print highly porous scaffolds
with a sintered pore size below 300 mm [68].
Post processing is always required for 3DP processed parts.
Sintering or densification at high temperature is just one of them.
During sintering, parts shrink and the shrinkage is not necessarily
uniform throughout the part. Non-uniform shrinkage can cause
extensive cracking in parts and make them unusable. This is a
particular challenge for porous scaffolds. Since the outside part of
bone is a dense structure with 10% or less porosity while inside it
Materials Today  Volume 16, Number 12  December 2013 RESEARCH
[(Figure_4)TD$IG]

RESEARCH: Review
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
very difficult using 3DP due to challenges related to non-uniform
shrinkage during sintering. Another post-processing challenge is
the removal of loose powders from interconnected pores inside the
part. This problem is magnified for parts with small pores, in
particular below 600 mm. Trapped powders inside the pores may
well sinter with the porous part making it less interconnected than
the designed part. Such problems with loose powders can reduce
the dimension of the pores after sintering.
Demand for processes such as 3DP will increase in the coming
years due to their ability to make custom medical devices that can
be tailored for patient specific and defect specific clinical needs.
Extensive process-property optimization is still needed to accom-
plish this goal. For ceramics, the most critical issue that needs
attention is the mechanical properties of porous scaffolds. Increas-
ing the porosity will decrease the strength of the scaffolds. Low
strength along with brittleness makes these scaffolds difficult to
even handle during processing. Resorbable polymer infiltration to
enhance strength and toughness in these scaffolds is one way to
minimize this problem; the use of resorbable glassy materials can
also help. Finally, printing live cells or adding growth factors/drugs
is another fascinating area of growth. However, most of the
challenges here are limited to survivability of the cells, viability
of the growth factors and drugs after printing. Although current
techniques let us build structures with similar composition to that
of tissue, we are still a long way from completely printing func-
tioning tissue [95]. More process-property optimization, in vitro
and in vivo research are needed in that direction to make any of
those approaches useful toward bone tissue engineering.
Acknowledgements
Financial support from the US National Institute of Health under
the Grant Number (R 01 EB-007351) is acknowledged. Authors like
to also acknowledge Dr. Solaiman Tarafder for his help.
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