3D Printing of Scaffolds For Tissue Regeneration

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3D Printing of Scaffolds for Tissue Regeneration

Applications
Anh-Vu Do, Behnoush Khorsand, Sean M. Geary, and Aliasger K. Salem*
nonsteroidal anti-inflammatory drugs

The current need for organ and tissue replacement, repair, and regeneration (e.g., aspirin and ibuprofen),[7] are given
for patients is continually growing such that supply is not meeting demand to patients suffering from osteoarthritis
primarily due to a paucity of donors as well as biocompatibility issues leading (degenerative joint disease) primarily for
pain relief, however, they play a negligible
to immune rejection of the transplant. In order to overcome these drawbacks,
role in tissue regeneration/repair. As a
scientists have investigated the use of scaffolds as an alternative to trans- result, patients are obliged to live with
plantation. These scaffolds are designed to mimic the extracellular matrix chronically damaged tissues which leads
(ECM) by providing structural support as well as promoting attachment, to a lower quality of life and contributes
proliferation, and differentiation with the ultimate goal of yielding functional to an increased healthcare cost.[8] The aim
of regenerative medicine is to restore or
tissues or organs. Initial attempts at developing scaffolds were problematic
replace damaged or diseased tissues with
and subsequently inspired an interest in 3D printing as a mode for generating healthy, functioning tissue.
scaffolds. Utilizing three-dimensional printing (3DP) technologies, ECM-like Tissue engineering requires an under-
scaffolds can be produced with a high degree of complexity, where fine details standing of the biological processes
can be included at a micrometer level. In this Review, the criteria for printing required for cellular proliferation and dif-
viable and functional scaffolds, scaffolding materials, and 3DP technologies ferentiation.[9–12] The process of tissue
engineering often begins with a scaffold,
used to print scaffolds for tissue engineering are discussed. Creating biofunc-
which is a three-dimensional support
tional scaffolds could potentially help to meet the demand by patients for tis- medium essential for the appropriate
sues and organs without having to wait or rely on donors for transplantation. proliferation and differentiation of cells
embedded in, or infiltrating, the scaffold.
Conventional techniques used for scaf-
fold fabrication include solvent-casting particulate-leaching, gas
1. Introduction foaming, fiber meshes/fiber bonding, phase separation, melt
molding, emulsion freeze drying, solution casting, as well as
Each year, the number of people in the United States suffering freeze drying, and these are discussed further elsewhere.[13,14]
from organ dysfunction or organ failure due to damaged or These conventional methods have many limitations since
diseased tissue is increasing because of the aging popula- they are often inadequate at fabricating precise pore size, pore
tion.[1] Illnesses or traumas, such as heart attacks,[2] strokes,[3] geometry, high levels of interconnectivity, and high mechanical
and joint degeneration[4] can drastically reduce the quality of strength.[13,14] Other limitations of these conventional tech-
life for the victims as well as causing levels of tissue damage niques also include suboptimal distribution of cells due to the
that current medicine is incapable of adequately repairing. This inaccuracies inherent in the process of seeding cells manually.
lack of therapeutic efficacy is primarily due to the fact that cur- This becomes problematic since cells may need to be precisely
rent treatments are aimed at merely preventing or reducing arranged according to the need and function of the tissue such
further tissue damage rather than contributing to the repair or as endothelial cells aligning to form vessels, or osteoblasts
regeneration of the tissue. Medications such as anticoagulants forming mineralized clusters.[14] Three dimensional printing
(warfarin) and antiplatelet agents (aspirin) for heart attacks has been developed as an advanced technology to overcome the
and strokes primarily function by preventing blood clots and limitations of these conventional methods and may ultimately
do not contribute to any form of tissue regeneration.[5] Simi- lead to the production of matrix scaffolds capable of more effec-
larly, analgesics, such as acetaminophen (paracetamol)[6] and tively promoting the regeneration of functional tissue.
Three-dimensional printing technology has emerged as a
promising tool to fabricate scaffolds with high precision and
A.-V. Do, B. Khorsand, Dr. S. M. Geary, Prof. A. K. Salem
accuracy, creating intricately detailed biomimetic 3D struc-
Department of Pharmaceutical Sciences and
Experimental Therapeutics tures.[15] The techniques currently being used to achieve 3D
College of Pharmacy, University of Iowa printing of scaffolds involve a layer-by-layer process, which
Iowa City, IA 52242, USA includes, but is not limited to, direct 3D printing, fused dep-
E-mail: aliasger-salem@uiowa.edu osition modeling, stereolithography, and selective laser sin-
DOI: 10.1002/adhm.201500168 tering. These techniques have been used to produce scaffolds
1742 wileyonlinelibrary.com © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Adv. Healthcare Mater. 2015, 4, 1742–1762
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REVIEW
ranging from millimeter to nanometer sized scaffolds. It is also
Anh-Vu Do received his
important to note that solid free-form fabrication, additive man-
B.S. Degrees in Psychology
ufacturing and 3D printing have become synonymous over the
and Biology as well as a
past decade and are now used interchangeably. Advantages of
B.S.Bch.E.in Biochemical
using 3D printing include the ability to fabricate versatile scaf-
Engineering from the
folds with complex shapes capable of homogenous cell distribu-
University of Georgia in 2012.
tion, and the ability to imitate the extracellular matrix (ECM).
He is currently pursuing a PhD
However, the availability of biomaterials with the stability and
in Chemical and Biochemical
desired properties for 3D printing of scaffolds is restricted
Engineering under the supervi-
depending on the printing technology used. Another disadvan-
sion of Prof. Aliasger Salem
tage is the production time that it takes to fabricate scaffolds,
at the University of Iowa. His
which dramatically increases as the scaffold design becomes
research interests include 3D
more precise and intricate.[16] This is especially the case for
printing and controlled drug
conventional methods which involve a lot of manual labor
delivery, specifically in conjunction with designing functional
compared to an automated process.[17] With increased research
and novel 3D printed devices for tissue engineering and
and understanding of 3D printing, the use of hybrid materials
cancer vaccinations.
and multiple printing technologies may lead to the fabrication
of ECM-like scaffolds capable of overcoming current limita-
tions. Evolving from conventional techniques, 3D printing pro- Behnoush Khorsand received
vides tissue engineers with a way to design scaffolds capable her M.Sc. in Polymer
of mimicking the complex structures of the ECM and thereby Chemistry at Concordia
providing a microenvironment for cell attachment, prolifera- University (Canada) in 2013
tion, distribution and differentiation with the potential to form for development of stimuli-
functional tissue. In this Review, we will assess the current responsive degradable block
3D printing materials and technologies being implemented to copolymer micelles as smart
design scaffolds. drug delivery nanocarriers. In
2014, she began her PhD in
Pharmaceutical Sciences and
2. Materials Used for 3D Printing of Scaffolds Experimental Therapeutics
under the supervision of Pr.
Important criteria to consider when fabricating suitable scaf- Aliasger Salem at the University of Iowa College of Pharmacy.
folds are biocompatibility, biodegradability, pore intercon- Her research is focused on tissue engineering using scaffolds
nectivity, pore size, porosity, and mechanical properties. and novel drug carrier formulations.
Biocompatibility and biodegradability are important properties
for scaffold materials to possess, ensuring they are degraded
into nontoxic products while leaving behind only the desir- Aliasger K. Salem is the Bighley
able living tissue. In addition, the material should induce Professor of Pharmaceutical
minimal inflammatory responses, thereby reducing the likeli- Sciences and Head of the
hood of rejection by the host’s immune system. It would also Division of Pharmaceutics and
be beneficial if scaffold materials could behave as substrates Translational Therapeutics at
that promote cellular attachment, proliferation and differen- the University Of Iowa College
tiation. Furthermore, as cells proliferate and differentiate, the Of Pharmacy. Salem is also
scaffold must be able to withstand the forces being applied by the leader of the Experimental
the cells otherwise its collapse would result in poor diffusion Therapeutics program at the
of oxygen, nutrients and waste, leading to inefficient tissue for- Holden Comprehensive Cancer
mation. Finally, the mechanical stability of the scaffold must be Center, University of Iowa
structurally sound so as to withstand daily activity and normal Hospitals and Clinics. Salem’s
body movements.[18] Naturally derived materials such as algi- primary appointment at the University of Iowa is based in
nate, chitosan, collagen, fibronectin, and hyaluronic acid have the College of Pharmacy, with additional secondary appoint-
an advantage over synthetic materials as they provide more ments in the College of Dentistry, Department of Chemical
innately biological functions. Using naturally derived materials, and Biochemical Engineering, Department of Biomedical
that normally constitute or inhabit the ECM, results in a better Engineering, College of Engineering and the Holden
mimicking of genuine ECM and this therefore enhances cell Comprehensive Cancer Center.
attachment and regulates cellular proliferation more efficiently
than synthetic polymers.[19] Although natural materials are
beneficial for cellular processes, the use of synthetic polymers
such as poly(e-caprolactone) (PCL) and poly(D,L-lactic-co-glycolic rates.[19,20] However, these synthetic polymer scaffolds have
acid) (PLGA) for scaffolding has yielded higher mechanical relatively low biological activity, in terms of promoting tissue
strengths, higher processability, and controllable degradation regeneration, compared to naturally derived ECM polymers.
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REVIEW
In addition to being less biologically active, the intrinsic hydro- trace amounts of metals to increase the mechanical strength
phobicity of synthetic polymers, such as polyesters, generally of current scaffolds has shown some promise.[36–38] The limita-
results in poor cell adhesion,[21] which results in suboptimal tion of using metals is that they are not typically biodegradable,
proliferation and differentiation, ultimately leading to sub- which provides a large disadvantage when trying to design an
standard tissue formation.[19] ECM-like scaffold for tissue regeneration. Nevertheless, biode-
For 3D printing systems utilizing powder beds, grain size gradable metals have emerged as prominent candidates for 3D
and grain size distributions must be taken into account to pro- printing of scaffolds.
duce porous scaffolds,[22] as these factors have a direct influ- Recently, the term “biodegradable metals” (BMs) has been
ence on microporosity which has been seen to influence cell used to describe degradable metallic biomaterials used for
distribution, attachment, proliferation, and differentiation.[23,24] medical applications. Zheng et al. defines BMs as “metals
To achieve biomimicry of the ECM, scaffolds need to be bio- expected to corrode gradually in vivo, with an appropriate host
logically active, have high mechanical strengths, be easy to response elicited by released corrosion products, then dissolve
process, and have controllable degradation rates. To create completely upon fulfilling the mission to assist with tissue
these complex scaffolds, hybrid systems comprising both syn- healing with no implant residues”.[39] Degradable metal mate-
thetic and natural polymers have been used and are likely to rials need to fulfill two criteria in order to be legitimately classi-
be used in the future.[25–27] It is important to keep in mind that fied as BMs. The first of these is the requirement for the metal
different powdered combinations, materials, and structure size material to possess suitable degradation rates in vivo, whilst the
have direct effects on the scaffold printability, as is the case for second criterion is that they must be comprised of an essential
most materials in 3D printing. To be a viable option for tissue metallic element that can be readily metabolized by pathways in
regeneration, the materials used for 3D printing of scaffolds the human body. Examples of BMs include those that are mag-
for tissue engineering should be printable with a high degree nesium-based, iron-based, zinc-based, or calcium-based and
of reproducibility. Such materials should also be cost-effective which consist of the pure metals themselves, alloys, or metal
and malleable to form the desired morphology of the designed matrix composites. Specifically, Mg[40] and Fe[32,41] based BMs
scaffold. are already being used for scaffold fabrication. A more expan-
sive list of BMs and their feasibility for use in medical applica-
tions has been published elsewhere.[39]
2.1. Metals Fe-based BMs were amongst the first used in 3D printing of
scaffolds for tissue engineering.[32,41] Printed 3D scaffolds com-
Natural or synthetic biomaterials used for 3D printing of prising BM have demonstrated promising in vitro results. In
scaffolds can vary greatly depending on the type of printing vitro studies have shown that an iron-manganese (Fe-Mn) alloy
technology used. These materials include metals, ceramics, was suitable for bone scaffold fabrication.[32] Scaffolds were fab-
polymers, and composites.[28–30] Metals with the potential for ricated using an inkjet 3D printing technique, where Fe-Mn
use in 3D printing of scaffolds include iron, cobalt, chromium, constituted the powder bed and an unspecified water-based
stainless steel and titanium alloy. Metals are attractive mate- organic solvent was used as the binder solution. The Fe-Mn
rials to use in the 3D printing of scaffolds because of their high scaffold showed strong tensile mechanical properties similar
mechanical strengths which have been shown to be similar to bone, as well as biodegradability, which allowed the scaffold
to that of the bone, hence their common application to bone to adequately support cell proliferation and differentiation.[32]
tissue regeneration.[31,32] Aside from displaying high mechan- A similar method was implemented in a separate study using
ical stabilities, metals are promising materials to explore since, an inkjet 3D printer involving Mg3(PO4)2 powder with a binder
to a limited degree, they are safe to use in vivo. To date, many solution containing 2 M K2HPO4, 0.5 M (NH4)2HPO4 or 20%
of the aforementioned metals have been used to design con- H3PO4 to form a matrix of either struvite-(K) (MgKPO4.6H2O),
ventional scaffolds, but only a few metals are actually used in struvite (MgNH4PO4.6H2O) or newberyite (MgHPO4.3H2O) by
3D printing.[31] These metals include stainless steel, cobalt- a hydraulic setting reaction.[41] It was demonstrated that hard-
chromium alloys, titanium and its alloys, nitinol, and others ening of the scaffold post-printing increased the compressive
that are used for biomedical implants.[33] Further work would strength to 10 MPa for struvite and 35 MPa for newberyite scaf-
need to be performed to establish the feasibility of using certain folds compared to the initial strengths of the printed samples
metal materials as components of 3D printed scaffolds despite (1.3–2.8 MPa).[41] These compressive strengths are higher than
having been successfully used in conventional scaffolding. The the minimum postulated strength required for scaffolds used
current limitations include: 1) the 3D printing technology avail- for bone regeneration (2 MPa).[42] However, the potential of
able, thus limiting the type of metals that can be used, and these materials to be used for tissue engineering is currently
2) the toxicity of metal ions caused by metal corrosion and deg- uncertain due to a lack of data on the impact of these materials
radation inside the body. This corrosion produces metal ions on cell viability as well as uncertainty as to their biocompati-
that may be toxic to the body at high concentrations and with bility. Printing of 3D scaffolds using BMs for bone regeneration
the lack of a clearance pathway, a system designed to capture shows promise and should be further researched using a variety
the metal ions or avoid systemic toxicity is necessary in addi- of metal materials in order to increase the availability of mate-
tion to scaffold fabrication.[34,35] Another parameter that hinders rials for 3D printing. Although metallic materials and scaffolds
the use of metals is long degradation times, which results in designed to date have been developed for bone replacement,
functional tissue forming around the scaffold rather than ulti- they may have many other applications for other tissues since
mately replacing the scaffold. However, the use of minute or non-metallic scaffolds are less robust and can collapse under
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REVIEW
the contractile force applied by cells during cellular attachment femur defects in rats. Other ceramic materials commonly used
and proliferation.[43] for 3D printed bone scaffolds include calcium phosphate and
As the availability of BM steadily increases, the need for, and TCP,[50–52] which, when combined with other ceramics, such as
complications brought on by, permanent prosthetics and metal HA, yields scaffolds with pore sizes of 300 µm that are large
replacement parts can be reduced, thereby allowing patients to enough to allow nutrient transfer for cells. These scaffolds were
regenerate their own bone in the ultimate absence of non-phys- also fabricated with >96.5% accuracy compared to the com-
iological materials. The paucity of research involving metals puter-aided design, allowing for future scaffolds to be intricately
as biomaterials for 3D printed scaffolds is primarily due to the designed to mimic the ECM structure necessary for optimal cell
traditional notion of non-biodegradability and limited processa- attachment and proliferation.[52] This acquired pore size and
bility (high temperatures are needed to form desired structures) the high resolution design makes calcium phosphate a suit-
attributed to metal materials.[28,44] However, the implementa- able material for scaffold fabrication and cellular growth. Other
tion of BM aims to counter this notion and increase the prac- studies have also shown the printability of calcium phosphate
ticality of using metals in 3D printing of more innovative and with other blended compounds, such as calcium sulfate, to
effective scaffolds. BMs are an untapped source for 3D printing create a powder composite with a water-based binder.[51] Using
of scaffolds for tissue engineering, as they could provide addi- a combination of ceramic materials for 3D printing of scaffolds
tional mechanical strength to current scaffolds to withstand should be further investigated to create materials with high pre-
most compressive and tensile forces. cision design, possessing adequate compressive strength, and
the ability to promote cell proliferation and differentiation that
can be applicable to both non-load bearing and load bearing
2.2. Ceramics orthopedic applications.
Ceramics contain both metallic and nonmetallic elements and

have been used as materials for 3D printed scaffolds due to their 2.3. Polymers
high mechanical strength and biocompatibility.[45] Ceramics are
capable of scaffold fabrication for bone regeneration mainly Polymers represent a major category of materials with potential
due to their apatite-mineralization ability.[46] Hydroxyapatite for use in the 3D printing of scaffolds for tissue engineering.
(HA), itself a ceramic, is commonly found in human teeth Such polymers include synthetic poly(ethylene glycol) diacrylate
and bones,[47] thus making the use of HA, or similar ceramics, (PEGDA) and natural gelatin methacrylate (GelMA),[53] which
attractive materials for creating scaffolds with strong mechan- are both used in the formation of hydrogels.[16] Hydrogels are
ical properties similar to that of natural bone. HA has garnered attractive biomaterials for tissue engineering since they possess
much attention in the field of regenerative medicine, and as adjustable mechanical properties, are biocompatible and have
such, is a commonly used material for 3D printed scaffolds. the ability to be hydrated while remaining insoluble and main-
In one study, a rapid prototyping technique was used to create taining their 3D structure. In addition, the hydrating properties
3D printed scaffolds from HA with complex internal structures of hydrogels allow them to mimic those of biological tissue.[54,55]
with slopes of 45° to allow for cell proliferation inside of the Both poly(ε-caprolactone) (PCL) and poly(D,L-lactic-co-glycolic
structure.[48] The interconnecting channels with pore sizes of acid) (PLGA) scaffolds have been manufactured using rapid
500 µm in the designed scaffolds displayed the ability to facili- prototyping. The use of these scaffolds in the defects of rabbit
tate mouse MC3T3-E1 cell proliferation, illustrating the poten- tibias demonstrated their safety as well as their capacity to pro-
tial of HA scaffolds to regenerate bone. Another study used mote the generation of bone tissue.[56] An advantage of using
computer-assisted 3D printing in a rapid prototyping technique synthetic polymer materials such as PLGA, and PCL is that
to fabricate HA and tricalcium phosphate (TCP) scaffolds. The both these synthetic polymers have been approved by the FDA
scaffolds were created using HA and tricalcium phosphate in for clinical use.[57] Another advantage of using PCL polymers
a layer-by-layer process followed by sintering.[49] These scaf- and PLGA copolymers is the low toxicity of their degradation
folds were seeded with human osteoblasts that were isolated products, which feeds into metabolic pathways.[13] A disadvan-
from human iliac crest cancellous bone, and showed high bio- tage of using PLGA is that it can cause inflammatory responses
compatibility and low cytotoxicity. The results provide further when there is a build-up of acidic oligomers.[58] Inflammation
evidence that HA materials demonstrate biocompatibility and plays a key role in tissue regeneration,[59] but it is important to
the ability to assist in cell growth and viability. More recently, control or limit this response, as high levels of inflammation
the ceramic, calcium silicate (CaSiO3) was used to make scaf- can lead to fibrosis resulting in poor tissue function, or even
folds with higher bone healing capacity than that of tricalcium rejection of the implanted scaffold. Thus, it is crucial to under-
phosphate scaffolds.[46] The 3D printed scaffolds were printed stand the inflammatory effect of the particular biomaterial(s)
through a device developed by the Fraunhofer Institute for used and the scaffold structure used to generate the desired
Materials Research and Beam Technology. The device was able tissue. The inflammatory response, generally mounted by the
to print the CaSiO3 solution (in polyvinyl alcohol) in a controlled innate immune system, promotes the recruitment of cells (pri-
layer-by-layer plotting. Using this method it was demonstrated marily neutrophils and monocytes) to the area of tissue damage
that pore morphology, pore size and porosity of the scaffold in order to assist with tissue repair and regeneration.[60] In
could be controlled.[46] It was also found that the CaSiO3 scaf- one study, 3D printed polylactic acid (PLA) and chitosan scaf-
folds could significantly improve bone regeneration compared folds were compared for their ability to induce inflammation
to tricalcium phosphate scaffolds when implanted in vivo into and consequently impact on tissue regeneration.[61] Through
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REVIEW
the analysis of macrophage morphology and

human monocyte cytokine profiles (tumor
necrosis factor (TNF)-α, interleukin (IL)-6,
IL-10, IL-12/23(p40) and transforming
growth factor (TGF)-β1) it was concluded that
the inflammatory properties of the scaffolds
were determined by both scaffold geometry
and composition. PLA-based scaffolds pro-
moted a higher production of Il-6, IL-12/23,
and IL-10 compared to chitosan scaffolds.
Whilst chitosan scaffolds only promoted a
higher secretion of TNF-α when compared
to PLA-based scaffolds across the range
of cytokines tested. It was also concluded
that orthogonal scaffolds induced stronger
inflammatory reactions compared to that of
diagonal scaffolds due to an increased pres-
ence of multinucleated giant cells in the
orthogonal scaffolds.[61] However, the ideal
levels of inflammation to generate functional Figure 1. In vivo bone formation in rat calvarial defects implanted with collagen scaffolds:
tissue were not tested and further studies representative microCT scans showing the level of regenerated bone tissue in calvarial defects 4
weeks after implantation with A,D) empty defects (no collagen scaffolds), B,E) empty scaffolds
involving other cells types and the formation (collagen scaffolds alone), and C,F) PEI-pPDGF-B complex-loaded scaffolds(collagen scaffold
of functional tissue are required. with complex). Images (D–F) are close up images of the defects and shows the rate of bone
Another interesting characteristic of these regeneration. Reproduced with permission.[69] Copyright, 2014, Elsevier.
polymers is their rate of biodegradation,
which is often too fast when PLGA is used and too slow when 3D ink-jet printing technique to yield scaffolds with improved
PCL is used. In a polymer degradation assay performed using mechanical strengths, cytocompatibility and bone regeneration
scaffolds, it was shown that with comparable concentrations of capacities.[68] Other natural polymers that have been tested for
PCL and PLGA, PLGA degraded by 18% at 14 days and 56% their potential use in 3D printing are corn starch and dextran.
by 28 days compared to PCL whose degradation was 33% at 21 These materials were used in a binder printing technique that
days, and 39% at 28 days.[62] Nevertheless both PLGA and PCL utilized a water-based binder to form porous scaffolds. The
may still have beneficial regenerative traits depending on the scaffolds were not tested on cells for biocompatibility, however,
type of injury.[56,63] Long term healing may be necessary in open with post-processing and additional fabrication methodologies,
bone fractures.[64,65] PCL is possibly a preferable choice for open these materials could yield interconnected, high resolution
fractures due to its slower degradation rate. A longer healing scaffolds.[70]
period is often required in open fractures because the bone has
penetrated the skin, which can oftentimes lead to infections
that increase the time for healing. The slow degradation rate of 2.4. Composites
PCL allows the scaffold to provide support for growing cells for
a longer period of time enabling more dense tissue to form.[56] Composite materials, or composites, are important in the field
For cases such as closed fractures (broken bone that has not of tissue engineering as they enable commonly used mate-
penetrated the skin), PLGA could be a potential candidate for rials for 3D printing to have increased mechanical strength
bone regeneration. and more intricately designed scaffolds. One group made 3D
PCL-based copolymers, such as PLGA-PCL-PLGA[66] and printed scaffolds for bone regeneration comprising a composite
PCL-PEG-PCL,[67] have been synthesized to control the degrada- of calcium phosphate and type I collagen using a ZPrinter
tion of PCL for controlled drug release applications. However, 450 printer to demonstrate the feasibility of the process and
these copolymers have the potential to be used for tissue engi- to enhance mechanical and cellular benefits in vitro.[68] In a
neering applications as well. Other synthetic polymers used separate study, a nozzle extrusion-based 3D printer was used
for scaffolding include polyglycolic acid (PGA), poly(propylene to generate PLA-based composite scaffolds consisting of PLA
fumarate) (PPF) and poly(hydroxy butyrate) (PHB). Natural and bioactive CaP glass. These scaffolds were made with two
polymers, such as proteins and polysaccharides, have also been different layer designs: orthogonal layer configuration (ORTH)
used for scaffold fabrication, and among these polymers, the (Figure 2) and displaced double-layer design (DISPL) (Figure 3)
most popular candidate for tissue engineering has been col- to test the ability of nozzle-based rapid prototyping printing
lagen type 1.[13,68] Collagen scaffolds loaded with cationic PEI- to print biodegradable scaffolds with different porosity and
pDNA complexes were recently used to create a patch capable high mechanical properties.[71] The printed scaffolds were
of bone regeneration in calvarial defects in rats (Figure 1).[69] highly porous and possessed mechanical properties that were
This research illustrates the great potential for collagen to be dependent on the layer design, where ORTH scaffolds yielded
used in 3D printing of implantable scaffolds. In another study, three times higher compression modulus strength (90k MPa)
collagen was used with a phosphoric acid binder solution in a compared to DISPL scaffolds (∼30k MPa). Composites also play
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REVIEW
searching for more effective scaffolds capable of mimicking
the ECM for cellular attachment, proliferation, and differen-
tiation resulting in the formation of functional tissue. To date,
the majority of research on 3D printed scaffolds has been con-
cerned with bone tissues, and therefore more research is nec-
essary in the field of tissue engineering with respect to other
tissues such as cardiac tissue. Newly designed composites and
synthesized biomaterials may pave the way for 3D printed scaf-
folds with >99% precision, 100% interconnectivity, versatile
pore size manipulation, and high mechanical strengths for a
range of load-bearing and tissue formation applications.
3. Three-Dimensional Printing Techniques for

Scaffold Fabrication
In the last decade, many different techniques have been used
Figure 2. 3-D printed scaffolds with orthogonal layer configuration: to form porous 3D biomimetic scaffolds, and have included
SEM images of scaffolds made from A,B) PLA/PEG; C,D) PLA/PEG/G5; phase-separation, self-assembly, electrospinning, freeze drying,
A,C) top view; B,D) cross-section view. Reproduced with permission.[71] solvent casting/particulate leaching, gas foaming, and melt
Copyright 2013, Elsevier. molding. Using scaffolds, the architecture of native extracellular
matrices can be mimicked at the nanoscale level and therefore
a major role in increasing the mechanical properties of hydro- provide the primary base for the regeneration of new tissue.[72]
gels. Composite hydrogels comprising ceramics, for example, Originally a “top-down” approach was used as a tissue engi-
can maintain their hydrophilic polymeric network to mimic that neering method for scaffold fabrication. In this method, cells
of innate tissue, whilst increasing their mechanical strength to are seeded onto a biodegradable and biocompatible scaffold,
withstand the compression forces caused by cell proliferation and are predicted to migrate and fill the scaffold hence creating
and differentiation.[54] Composites may be the key biomaterials their own matrix. By using this technique, several avascular tis-
for 3D printing of ECM-like scaffolds. sues such as bladder[73] and skin[74] have been engineered effec-
A more comprehensive review of 3D printed materials for tively. However, due to the limited diffusion properties of these
bone tissue engineering has been published elsewhere.[28] The scaffolds, this technique faces several challenges for fabrication
search for the optimal material or material blend for 3D printed of more complex tissues such as heart and liver.[75] Therefore,
scaffold fabrication is an ongoing challenge. This research is “bottom-up” methods have been developed to overcome this
necessary, as different types of tissue replacements require dif- problem.[76] Bottom-up approaches include cell-encapsulation
ferent specifications such as specific pore sizes, scaffold mor- with microscale hydrogels, cell aggregation by self-assembly,
phologies, or mechanical strengths. Scientists are continually generation of cell sheets, and direct printing of cells.[77] These
complex tissue blocks can be assembled using various methods
including microfluidics,[78] magnetic fields,[79] acoustic fields,[80]
and surface tension.[81] These methods are relatively easy and
have provided a solid foundation for the fabrication of scaffolds.
However, as mentioned previously, these conventional methods
suffer from several limitations including inadequate control
over scaffold properties such as pore size, pore geometry, dis-
tribution of high levels of interconnectivity, and mechanical
strength. As such, it is necessary to develop technologies with
sufficient control so as to design more intricate tissue-spe-
cific scaffolds. In addition, scaffolds can be coated using sur-
face modification techniques (such as introducing functional
groups) to enhance cell migration, attachment and prolifera-
tion. Three-dimensional printing allows scaffolds to become
more precisely fabricated (similar to that of the computer-aided
design (CAD)) with higher flexibility in the type of materials
used to make such scaffolds. Three-dimensional printing uses
an additive manufacturing process where a structure is fabri-
cated using a layer-by-layer process. Materials deposited for the
formation of the scaffold may be cross-linked or polymerized
Figure 3. 3D printed scaffolds with displaced double-layer design:
SEM images of scaffolds made from A,B) PLA/PEG; C,D) PLA/PEG/G5; through heat, ultraviolet light, or binder solutions. Using this
A,C) top view; B,D) cross-section view. Reproduced with permission.[71] technology, 3D printed scaffolds can be prepared for optimized
Copyright 2013, Elsevier. tissue engineering.
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REVIEW
For appropriate formation of tissue architecture, the seeding producing signals that are taken up by digital X-ray detectors
cells (often stem cells) require a 3D environment/matrix sim- and sent to be processed by a computer to generate cross-sec-
ilar to that of the ECM. The ECM acts as a medium to provide tional images of the body. These cross-sections are then stacked
proteins and proteoglycans among other nutrients for cellular to create a 3D image of the scanned organ(s). CT scans are gen-
growth. The ECM also provides structural support to allow for erally used for imaging bone due to its density while soft tissues
cellular functionality such as regulating cellular communica- can be problematic as they have varying abilities to inhibit X-ray
tion, growth, and assembly.[82] With this in mind, scientists and penetration resulting in faint or undefined images. In order to
engineers originally attempted to replicate the ECM through image these soft tissues, contrasting agents such as iodine or
conventional techniques, which consequently established a barium-based compounds may be used to facilitate contrast and
framework for using more advanced techniques, such as 3D increase visibility.[91]
printing, to yield higher quality scaffolds. The 3D printing tech- MRI is preferable over CT when attempting to image soft
nique can create defined scaffold structures with controlled tissue and other organs besides bones as the contrast of tightly
pore size and interconnectivity and the ability to support cell placed organs can more readily seen when changes to radio
growth and tissue formation.[28,83,84] The current methods waves and magnetic fields are applied. The radio waves and
for 3D printing involve a CAD, which is then relayed to each magnetic field enables the ability of the instrument to highlight
3D printing system to “print” the desired scaffold structure. the desired tissue in tightly knitted areas. However, CT scans
Through various 3D printing technologies, discussed below, create better quality images of bone structures than MRI due
researchers are trying to fabricate biocompatible scaffolds that to the low concentration of water in bones resulting in less
efficiently support tissue formation. hydrogen atoms emitting energy to succinctly create a cross-
sectional image. Creating a scaffold directly from the images is
not always feasible due to the possibility of scanning diseased
3.1. Computer Aided Design and Digital Imaging or damaged organs.[85] In this case, computer modeling may be
necessary to recreate the missing parts of the organ or tissue.
The start of many 3D printing processes involves a CAD that With MRI and CT imaging techniques, the reconstruction of
must be drawn or taken from known organ structures. Gener- both 2D and 3D images is a powerful tool to recreate the com-
ally 2D slices acquired from imaging instruments are compiled plexity of tissue structures. These tools allow researchers to be
and stacked on top of one another to form a 3D structure.[85,86] one step closer to fabricating a precise replica of the needed
In tissue engineering, it is imperative to grow tissue similar to extracellular matrix to enhance functional tissue formation.
that of the native tissue and in order to accomplish this, imaging
techniques can be used to produce scaffolds that closely mimic
the structure of native tissues.[87] These images inform scaffold 3.2. Direct 3D Printing
designs by providing morphology and size parameters to which
scaffolds need to conform in order to fit into irregularly shaped Three-D printing involves the fabrication of structures through
defects/fractures where tissue formation is desired. The scaf- successive layer deposition using a computerized process. The
fold shape also helps to direct the growth of cells and provide first “3D printer” was developed by investigators at the Massa-
shape for the final tissue.[88] It is also worth noting that scaf- chusetts Institute of Technology (MIT) in the 1990s, and was
fold shape can affect the type of tissue regenerated as can be based on the technology of an ordinary inkjet printer. This
seen in dentin tissue regeneration with differentially shaped printing technique can sometimes be referred to as “binder jet-
scaffolds using dental pulp-derived cells.[89] The complexities in ting” or “drop-on-powder”.[92] In an ordinary 2D inkjet printer,
morphology and architecture of tissues can be delineated with the ink nozzle moves in a side to side motion incrementally
imaging technologies such as magnetic resonance imaging along one plane such that the printed material has the 2 dimen-
(MRI) and computer tomography (CT). These imaging technol- sions of length and width. A 3D printer uses the same tech-
ogies help to take cross-sectional slices of organs and compile nology, but as well as moving side to side along one plane,
them into a 3D image, thus allowing the design of scaffolds to the printer has a platform capable of moving up and down
be a close representation of native organs.[85] (90 degrees to the side to side motion), hence adding the
MRI functions by using magnetic fields and pulsating radio dimension of height and thereby printing in 3 dimensions. The
waves to yield detailed pictures of organs and soft tissues. 3D printer designed by MIT has similar characteristics to the
Using gradient coils to interpret energy signals produced by 2D inkjet printer, however, instead of ink, the 3D printer uses a
water molecules within the tissue, 2D images are generated.[90] liquid binder solution that is selectively deposited on a powder
These 2D images are then stacked to create a 3D image of the bed instead of paper. The process begins with a powder bed,
scanned area. Because MRI requires hydrogen molecules gen- which could vary depending on materials used, that is spread
erally in water, they are best used for soft tissue imaging such onto the build platform and leveled using a roller system. The
as ligaments and tendons and organs of the chest and abdomen printer nozzle then dispenses binder solution in the designated
(heart, spleen, pancreas, liver, kidneys). They are also used powdered areas directed by the CAD. Once the binder solution
to image pelvic organs such as the bladder and reproductive and powder are combined, the excess powder is removed (blown
organs. off). The build platform is then lowered, and a new powder
CT, also known as computerized axial tomography, is a tech- layer is deposited and leveled. This process is then repeated
nology that uses X-rays to produce images from a scanned area. until the final structure is created (Figure 4). This technique
In a CT scanner, X-ray tubes are rotated around a patient’s body also has the versatility to change the composition of binder and
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REVIEW
powder if it is deemed necessary where certain parts of the scaf-
fold may require a material with higher mechanical strengths
and/or smaller pore sizes. An example of this may be building
a scaffold with larger pore sizes deep within the scaffold, while
having smaller surface pores. With the increase in pore sizes,
cells deep within the scaffold will be able to maintain their cel-
lular processes as vital resources such as nutrients, oxygen, and
waste are able to diffuse without difficulty compared to small
pore sizes that may result in the nutrient deprivation of cells
leading to cell death. Cell death on a large scale ultimately
leads to the collapse of the scaffold and the inability to form
functional tissue. The resulting desired effect of the fabricated
scaffold will be to provide a medium that guarantees high pro-
liferation and differentiation of cells to generate functional tis-
sues. The advantages of this method are the expansive list of
powder-binder solutions available to yield the desired scaffold.
The use of binders, however, can lead to toxicity if they are not
completely removed once the scaffold is ready to be implanted,
as in the case of organic solvents that are used as binders for
some powdered polymer materials. Another disadvantage for
this printing technique is the post processing required, where
heat treatment may be necessary to ensure durability.[93]
Three-D printed scaffolds for tissue engineering are showing
great promise as many new 3D printers become available
through various companies. Using a Palmetto 3D printer, devel-
oped by investigators at the Medical University of South Caro-
lina and Clemson University (South Carolina), the feasibility of
fabricating alginate scaffolds using CaCl2 as the liquid binder
was established[95] (Figure 5).
Figure 4. Schematic of direct 3D printing of a CAD scaffold. Adapted Biocomposite scaffolds comprising of chitosan and
with permission.[94] Copyright 2005, Nature Publishing Group.
hydroxyapatite were 3D printed using a Spectrum 510 3D
Figure 5. Fabrication of alginate scaffolds using 3D printing: Formation of scaffolds facilitated by 3D printed alginate hydrogel microdroplets with
different geometries (cube (A), square frame (B) and pyramid (C)). The scale bar for (A–C) is 1 mm. The scale bar for (D) is 2 mm. The blue, green,
yellow, gray and red represent the first, second, third, fourth and fifth layers of bioprinted alginate microdroplets, respectively. Reproduced with per-
mission.[95] Copyright 2014, IOP Publishing.
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REVIEW
printer made by Z-Corp to yield dense (solid, nonporous) and Three-D printing is also attractive because it can allow for
porous cylindrical scaffolds.[96] These scaffolds were created by the introduction of growth factors such as vascular endothelial
applying a binder solution consisting of 40 wt% lactic acid to growth factor (VEGF) or platelet-derived growth factor (PDGF)
different chitosan/hydroxyapatite composites (20 wt%, 25 wt%, in the bioink to enhance cell proliferation and differentiation
and 30 wt% chitosan) followed by a post-hardening process. and, in particular, to promote angiogenesis. The addition of
Optimal mechanical properties were observed with solid scaf- these growth factors can increase the rate of tissue formation in
folds printed using 25 wt% chitosan as demonstrated by their scaffolds as well as generating a resilient tissue due to enhanced
high compression strength of 16.32 MPa and Young’s modulus differentiation. In one study, in order to promote vasculariza-
of 4.4 GPa.[96] However, the researchers were only capable of tion, a bioscaffolder pneumatic dispensing system was used
fabricating nonporous scaffolds with the desired high mechan- to incorporate VEGF in a 3D printed matrigel-alginate scaf-
ical strength, which is problematic as porosity is an important fold.[99] The use of gelatin microparticles to control the release
property since it allows for diffusion of oxygen, nutrients, and of VEGF in a sustained manner resulted in higher vasculariza-
cellular waste. The reason for the ineffectiveness of the porous tion compared to scaffolds with no growth factors and scaffolds
scaffold is due to the post hardening process, which involves with fast release of VEGF when applied in murine models. The
high concentrations of solvent immersion that ultimately led to use of growth factors is still somewhat in its infancy in regards
the collapse of the porous scaffolds.[96] Therefore the fabrication to 3D printing, as there have been fewer than 10 studies per-
of 3D printed chitosan/hydroxyapatite scaffolds for tissue engi- formed in the last 5 years in regards to fabricating scaffolds
neering, whilst promising, still requires further optimization. with embedded growth factors, however, with their increased
use, researchers will be able to create complex shaped scaffolds
to more closely mimic the in situ ECM conditions required for
3.3. Bioplotter Printing optimal organogenesis.
In order to develop a more efficient all-in-one system, one
Bioplotter printing is a rapid manufacturing technique that group used a NovoGen MMX Bioprinter from Organovo which
uses a nozzle extrusion system of thermally or chemically comprised two pumps and two nozzles.[100] This system was
treated materials (Figure 6). As with all 3D printing methods, a capable of dispensing gelatin methacrylate (GelMA) hydrogels,
CAD is first created and then sent to the 3D printer. The mate- whilst simultaneously dispensing cells to seed the scaffold.
rials are deposited in a layer-by-layer fashion, where each layer This system enables the direct addition of cells into the scaffold
may contain a combination of different materials. Similar to rather than waiting to seed the cells after scaffold fabrication.
that of ink cartridges in an inkjet printer, the Bioplotter printer This direct seeding has the advantage of homogenously distrib-
is capable of using and changing “bioink” to develop the final uting cells throughout the scaffold, as well as being less time
scaffold structure. A key feature of Bioplotter systems is that consuming. A UV light guide was also added to the printer to
they print cell-laden gels, often with other polymeric materials allow for photopolymerization of the GelMA. This system gen-
such as PCL, to yield viable and functional scaffolds.[15,97] The erated HepG2 cell-laden scaffolds capable of retaining high cell
printer utilizes a pneumatic pressurized system to extrude the viability for at least eight days in vitro. This study illustrates
material from the bioink cartridges. The disadvantage of using the viability of using a 3D printer to print scaffolds for com-
this system is the shear stress from the variously sized noz- plex tissue engineering processes. In a separate study, similar
zles, which may impact negatively on cell viability during the materials were used in a projection stereolithography system to
printing process.[98] 3D print GelMA scaffolds in a layer-by-layer process.[101] This
technique allowed scaffolds to be fabricated down to microscale
sizes while still being a viable option for supporting cell growth.
The emergence of projection printing as a potential tool for 3D
printing of scaffolds will allow researchers to design micro-
sized scaffolds with high precision and be implanted directly
into certain parts of the body without being overly intrusive.
Researchers have seen success in the use of a 3D-Bioplotter
from EnvisionTEC to produce cell-laden 3D printed scaffolds.
The 3D-Bioplotter has been used for bone regeneration[102,103]
and soft tissue biofabrication.[104]
Another Bioplotter printer is a nozzle-deposition tool used
to fabricate 3D scaffolds called the Tissue Engineering 3Dn-
300 that was designed by Sciperio/nScrypt Incorporated. This
printer was used to yield PLA/PEG blends with 5, 10, and 20%
(w/w) of PEG and PLA/PEG/bioactive calcium phosphate (CaP)
glass composite scaffolds. The blend incorporated the use of
PEG as a plasticizer to decrease the glass transition tempera-
ture of the blend and enable processing at low temperatures.[105]
The addition of PEG improved scaffold processing, however,
Figure 6. Schematic of Bioplotter Printer set-up. Adapted with permis- the ability of these scaffolds to support cell growth both in vitro
sion.[94] Copyright 2005, Nature Publishing Group. and in vivo is yet to be explored. Using a modified Bioplotter
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differentiation into a chondrogenic lineage was observed.[107]
REVIEW
The researchers demonstrated for the first time, the ability of
dECM bioink to be incorporated into 3D printed cell-laden scaf-
fold constructs that were capable of harboring cells with high
cell viability, cell differentiation, and generating functional
tissue formation. The potential of using dECM increases the
versatility of 3D scaffolds used for tissue engineering.
3.4. Fused Deposition Modeling
Figure 7. Ear-shaped scaffolds: Photographs of ear-shaped structures Fused deposition modeling (FDM) is a 3D printing technique
made from PCL using a PEG sacrificial layer (not shown) through a hybrid that utilizes thermoresponsive polymers that have been heated
3D printing system combining both inkjet printing and fused deposition
beyond their glass transition temperatures and deposited onto
modeling. A) back of ear scaffold, B) front of ear scaffold. Reproduced
with permission.[27] Copyright 2014, IOP Publishing. a solid medium. This process works using a coiled thermo-
plastic polymer filament that is unwound and extruded through
a heated nozzle onto a fabrication platform. Upon contact with
printer, a multihead tissue/organ building system was used the base (platform), the polymer hardens and sets. After a layer
to print PCL and cell laden hydrogels, while using PEG as a has been deposited, the process repeats itself in a layer-by-layer
sacrificial layer. This system utilized a hybrid system of inkjet process until the CAD structure has been completed (Figure 8).
printing (hydrogels) and fused deposition modeling (thermo- An advantage of using FDM is that it eliminates any poten-
plastics) to enhance the mechanical stability of the scaffold, as tial toxicity caused by organic solvents that are often neces-
some hydrogels demonstrate poor mechanical properties.[106] sary to solubilize certain polymers, such as dichloromethane
Using this system, a complex-shaped scaffold for ear regenera- being used to solubilize PLGA.[108] The requirement of a
tion was fabricated.[27] The process involved the creation of a
sacrificial layer as a base for the formulation of complex struc-
tures that was easily dissolved away. Similar to trying to build
an inverse pyramid or bowl-shaped structures, there needs to
be a support layer to allow the complex structure to take shape.
Using these complex ear-shaped scaffolds (Figure 7), positive
in vitro results of chondrogenesis and adipogenesis from the
co-printed chondrocytes and adipocytes were obtained. The use
of this hybrid system will allow direct 3D printing to increase
its flexibility in designing scaffolds with even the most complex
shapes.
In a more recent study, a multihead tissue/organ building
system was used to bioprint cell-laden scaffolds with decellu-
larized extracellular matrix (dECM).[107] The use of dECM is
an attempt to closely mimic the complexities of the ECM by
providing infiltrating or embedded cells with an environment
similar to native tissue. Adipose ECM, cartilage ECM, and heart
ECM were decellularized and used to create matrices capable of
yielding high cell viability and functionality. The ability of scaf-
folds to yield tissue-specific gene expression in cell-laden gels
and PCL frameworks was investigated. In this part of the study,
human adipose-derived stem cells (hASCs) and human inferior
turbinate-tissue derived mesenchymal stromal cells (hTMSCs)
were used in scaffolds containing adipose ECM or cartilage
ECM respectively to assess the ability of the scaffolds to pro-
vide an environment with the ability to effectively increase adi-
pogenic or chondrogenic differentiation of adult stem cells.[107]
Depending on the dECM used in the printing process, it can
be specifically related to the increase in cellular differentiation
of that specific type of tissue formation, as can be seen when
the researchers cultured hASCs in decellularized adipose or
hTMSCS in decellularized cartilage to assess tissue-specific Figure 8. Schematic of Fused Deposition Modeling: a coiled filament
gene expression. The hASC culturing yielded increased expres- is printed through a heated extrusion nozzle and solidifies upon con-
sion of adipogenic markers over time and the adECM demon- tact with the fabrication platform. Adapted with permission.[94] Copyright
strated an “adipoconductive” environment, while the hTMSC 2005, Nature Publishing Group.
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REVIEW
(HEMA–MMA–MAA). This hybrid scaffold,

consisting of synthetic and natural polymers,
allowed cells to penetrate through the entire
structure. In vitro experiments were per-
formed where cell suspensions were seeded
into the scaffold during the addition of meth-
ylated collagen to the freeze-dried PCL-HA
scaffolds. The researchers were able to sup-
port their hypothesis that self-assembled
composite matrix and dynamic culture could
enhance mesenchymal stem cell attachment,
differentiation, and distribution compared
to static cultures. In the dynamic cultures
with the PCL composite scaffold, the scaffold
promoted synergistic enhancement of osteo-
genesis in a telomerase reverse transcriptase
gene-transduced cell population of human
bone marrow-derived mesenchymal stem
cells (hMSC-TERT) when compared to naked
Figure 9. SFF-based 3D PCL/PLGA scaffold. SEM images of A) whole structure of the SFF- (non-coated PCL scaffolds) dynamic cultures
based 3D PCL/PLGA scaffold. B) Surface morphologies of (left) control, (middle) fp-151-, and and naked and embedded (coated) static cul-
(right) fp-151-RGD-coated scaffolds. Reproduced with permission.[108] Copyright 2012, Elsevier. tures.[19] The enhancement of osteogenesis
was defined by calcium deposition levels and
thermoplastic material for this technology limits its application gene expression of osteogenic markers (alkaline phosphatase,
and versatility with respect to scaffold fabrication where the osteocalcin, RunX2, Collagen, and BSP-1) which were notice-
most commonly used material is acrylonitrile butadiene styrene ably higher in embedded dynamic cultures, indicating higher
(ABS). Other polymers, such as polycarbonate (PC), polyether- levels of mineralization.[19]
imide (PEI), and polyphenylsulfone (PPSF) have been used in A new approach that may warrant the increased use of FDM is
FDM.[109] However, these materials are generally not used for the addition of electrospinning. One group was able to use an in-
tissue engineering purposes but rather for printing everyday house developed melt electrospinning device to create a biphasic
objects such as most modern LEGOs. Further research is nec- scaffold with a bone and periodontal compartment. Medical
essary to discover other thermoplastics, such as polyesters, grade PCL-TCP membrane scaffolds, acting as the bone com-
that are viable options for scaffolding for tissue engineering. partment, were fabricated using FDM and then coated with cal-
Even with this limitation, FDM has proven to be an impor- cium phosphate, while the periodontal compartment was electro-
tant technique in fabricating scaffolds for tissue engineering. spun through a melt electrospinning device. A biphasic scaffold
The primary option for FDM printed scaffold for tissue engi- was then assembled by compressing a partially fused CaP-coated
neering is the polyester, PCL[110] as well as composites of PCL bone compartment (FDM scaffold) onto a periodontal compart-
such as PCL-TCP,[111,112] and HA/PCL.[113] In a recent study, a ment (melt electrospun mesh).[114] The newly designed scaf-
multihead deposition system was used to mix PCL and PLGA fold had pore sizes ranging from 100–400 µm, which was large
to yield a blended scaffold with pore sizes of ∼300 µm, and a enough for cellular diffusion. In vivo testing of the scaffold con-
high compressive strength of 3.2 MPa (Figure 9).[108] The fab- firmed tissue integration between both compartments, forming
ricated scaffolds, combined with mussel adhesive proteins as a tissue structurally resembling native periodontal tissues and
a functionalization material, yielded high cell attachment and establishing high levels of vascularization and tissue orientation
proliferation of human adipose tissue-derived stem cells. This in both bone and periodontal compartments.[114] The addition of
scaffold also yielded positive results in in vivo studies, where multiple printing techniques and novel scaffold designs may give
enhanced bone regeneration was seen in a calvarial defect rat rise to advanced 3D printing technologies capable of fabricating
model (Figure 10).[108] higher quality scaffolds for tissue engineering.
Another group further utilized FDM in a hybrid system to
generate a scaffold with both natural and synthetic polymers in
order to take advantage of the ability of the natural polymers 3.5. Selective Laser Sintering (SLS)
to mimic the structure and biological function of the ECM.[19]
In this study, PCL was processed by FDM as the base of the In the fabrication of 3D scaffolds using the selective laser sin-
scaffold, which was then immersed into hyaluronic acid solu- tering (SLS) method, a layer of powder is smeared onto a sur-
tion for 24 hours, followed by freeze drying. After the scaffold face, followed by the sintering of powdered particles together in
was dried, a coacervation reaction was completed by pipetting a a desired pattern using a laser beam (usually a CO2 laser). After
methylated collagen solution into the dried scaffold. The newly deposition of the first layer, the process is repeated and another
coated PCL-HA-collagen scaffold was completed with a final layer is applied to the pre-existing layer (Figure 11).
coating of a 3% terpolymer solution consisting of hydroxyle- The SLS process has been used to prepare scaffolds
thyl methacrylate, methyl methacrylate, and methacrylic acid from biocompatible and biodegradable polymers such as
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REVIEW
requirement for powdered material to be
capable of withstanding laser heat, as well
as resisting shrinkage of the scaffold during
the sintering process. Another potential dis-
advantage of using SLS is the pre- and post-
heating treatments of the powdered material
between the crystallization and glass transi-
tion or the melting temperatures to reduce
the shrinkage caused by the laser to the scaf-
folds.[116] Another problem is that the mate-
rial formulation of the scaffold must be able
to withstand high temperatures reaching
up to 1400°C[115] depending on the material
being used.
SLS utilizes many parameters, such
as powdered material, laser power, scan
spacing, layer thickness, part bed tempera-
ture, scan speed, and roller speed, all of
which can be optimized in order to fabri-
cate complex and intricate scaffolds.[117,118]
Three of these parameters, laser power,
scan spacing, and laser thickness have been
optimized in the production of quality SLS
scaffolds.[118] By optimizing these para-
meters the researchers were able to fabri-
cate a nanocomposite scaffold made from
poly(hydroxybutyrate-co-hydroxyvalerate)
(PHBV) microparticles (oil-in-water method)
and calcium phosphate/PHBV nanocom-
posite microspheres (solid-in-oil-in water
method). It was determined that the param-
eters investigated had a significant effect
on mechanical properties (compressive
properties), accuracy, and stability of the
scaffolds.[118] Yielding higher quality scaf-
folds with predetermined properties can be
achieved by optimizing the parameters of
SLS, however, these scaffolds will need to be
further investigated in vitro and in vivo to
assess their functionality and efficacy.
Figure 10. Effect of scaffolds on bone regeneration: Calvarial defects in rats were implanted Researchers have also shown the ability
with various (indicated) PCL/PLGA blended scaffolds made using fused deposition modeling. to use SLS printing to print scaffolds using
A) µCT scan 3-D and B) X-ray 3-D axis images of rat calvaria and C) quantified new bone PCL[119] or PCL/HA[120] powdered mixtures
volume after 8 weeks of implantation. (A) represents the calvarial defect margin. Values and with precision (Figure 13). The scaffolds fab-
error bars represent the means of quintuple samples and standard deviations with statistical
ricated had high compression moduli ranging
significance (*p < 0.05 and **p < 0.01). D) Histological analysis of in vivo bone regenera-
tion 8 weeks after implantation. hADSC = human adipocyte-derived stem cells; fp-151-RGD from 52-67 MPa and with the seeding of bone
= a genetically redesigned hybrid mussel adhesive proteins. Reproduced with permission.[108] morphogenetic protein-7-transduced primary
Copyright 2012, Elsevier. human gingival fibroblasts, demonstrated the
generation of bone in vivo as shown by histo-
polyetheretherketone, polycaprolactone, polyvinyl alcohol, and logical staining and µCT data.[119] The studies show the poten-
poly(lactic acid). In one report, a biocomposite slurry consisting tial use of SLS printing for bone tissue engineering.
of hydroxyapatite (HA), silica sol, and sodium tripolyphosphate
was used to generate scaffolds using SLS (Figure 12).[115] These
scaffolds demonstrated high mechanical strengths of up to 3.6. Stereolithography (SLA)
43.26 MPa but possessed low porosity. However, the in vitro
studies indicated the feasibility of using these scaffolds for the Stereolithography (SLA) is a process where 3D scaffolds are
growth of osteoblast-like cells.[115] Using SLS techniques can formed from a liquid polymer via a light-mediated chemical
be beneficial when high mechanical strength and low porosity reaction. In this process, a photosensitive (photocurable)
are required; however, a limitation of this technique is the polymer is deposited onto a surface medium which is then
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REVIEW
Figure 13. SLS design file and actual fabricated scaffold. A) STL design file
for the 1.75 mm x=y=z porous scaffold. B) 1.75 mm x=y=z PCL scaffold fab-
ricated by SLS. Reproduced with permission.[119] Copyright 2005, Elsevier.
exposed to light (UV range of 300–400 nm).[121–124] After the first

layer is cured, this process is repeated, overlaying the previous
layer, until the scaffold has been fully designed (Figure 14).
Examples of biomaterials used for SLA are polypropylene
fumarate (PPF) with photocrosslinkable bonds and polyeth-
ylene glycol acrylate. An advantage of using SLA is the ability of
the user to control and create defined geometries of the scaffold
with high resolution, which almost mimics that of the CAD
design.[125] A disadvantage of using SLA is that the materials
are required to be photopolymers that utilize photoinitiators.
The major categories of photoinitiators include radical photo-
polymerization through photocleavage, hydrogen abstraction,
and cationic photopolymerization where cationic photoinitiators
Figure 11. Schematic of Selective Laser Sintering technique. Adapted

with permission.[94] Copyright 2005, Nature Publishing Group.
Figure 12. Bone scaffolds generated by SLS: A) Image of the scaffold,

B) front view, C) top view, and D) back view of bone scaffold parts. Size is Figure 14. Schematic of Stereolithographical technique for manufac-
based on a centimeter ruler, with hatch marks indicating per millimeter. turing scaffolds. Adapted with permission.[94] Copyright 2005, Nature
Reproduced with permission.[115] Copyright 2014, Elsevier. Publishing Group.
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REVIEW
Figure 15. CLIP schematic and printed objects. A) Schematic of CLIP printer displaying an oxygen permeable window and dead zone space. B) Scaf-
fold design using CLIP with print speeds of 500 mm/hour (movies S1 and S2). C) Ramp test analysis, illustrating that the same speed is achievable
regardless of 3D model slicing thickness (100 µm, 25 µm, and 1 µm). Reproduced with permission.[129] Copyright 2015, AAAS.
are not used for tissue engineering due to the generation of 20 µm, and optical absorption height of the resin of 100 µm the
toxic byproducts.[126,127] Researchers recently used a SLA 250/40 speeds of fabrication were in excess of 300 mm/hour. When the
stereolithograph machine to print scaffolds made from PPF optical absorption height was changed to 300 µm, thereby sac-
and using the photoinitiator, Irgacure 819.[124] The fabricated rificing resolution, speeds of greater than 1000 mm/hour were
scaffolds had a pore size range of 150–800 µm and a porosity of achieved. This is compared to layer-by-layer processing that typ-
90%. Their study showed the feasibility of using PPF material ically produces only a few millimeters per hour. The CLIP tech-
with SLA for scaffolding purposes. Future in vitro and in vivo nique operates by projecting UV images through a UV trans-
studies would need to be conducted to establish cytotoxicity and parent and oxygen-permeable window stationed underneath a
biocompatibility of the fabricated scaffolds. liquid resin bath. A dead zone is created between the window
Using an EnvisionTEC Per-factory Mini Multilens SLA and the continuously elevating curing part such that reactive
machine, photocrosslinkable PCL macromers were printed into a liquid resin is always available for curing (Figure 15).[129] There
3D scaffold using Irgracure 369 as the photoinitiator. These scaf- is a tradeoff in this system where the increase in speed of pro-
folds showed no material shrinkage during the fabrication production yields poorer resolution 3D objects (Figure 16). How-
cess, as well as possessing porosity of ∼70.5%, with an average ever, the novel implementation surpasses conventional stereo-
pore size of 465 µm. The method in which the scaffold was lithography techniques in terms of production time and is only
printed required additional solvents and showed the potential to limited in terms of fabrication time based on resin cure rates
be used for tissue engineering.[128] However, the researchers did and viscosity, rather than step by step layer formation seen in
not test the functionality of the scaffolds. Although the ability to typical stereolithography.[129]
print scaffolds for tissue engineering using stereolithography has
been demonstrated, future in vitro and in vivo experiments are
needed to establish the potential of this method and to increase 4. Nanofibrous Scaffolds
adoption of stereolithography for this purpose.
Using a continuous liquid interface production (CLIP) tech- 4.1. Electrospinning
nique, Tumbleston et al. was able to utilize stereolithography in
a manner that surpasses any 3D printing technique in terms Electrospinning differs from SFF technologies by forming scaf-
of production time for object fabrication.[129] Current layer-by- folds through random orientations of nanofibers compared
layer printing takes hours to construct, and even longer for to a strategic layer-by-layer process. Electrospinning works by
finer resolution constructs. With a “dead zone” thickness of using electrostatic forces to produce fine fibers from polymer
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were seen to beat spontaneously for a period of 5 weeks.[135]

REVIEW
Further details on bio-electrospraying are available in a compre-

hensive review by Suwan Jayasinghe.[136] Cell electrospinning is
another technique that uses electrospinning, but uses a coaxial
system to generate cell laden scaffolds.[137] This technology has
seen success in printing polydimethylsiloxane polymers with
the immortalized human astrocytoma cell line, 1321N1. The cell
laden fiber mesh/scaffold created using this method displayed
no cellular damage during the nanofabrication process.[138]
These results demonstrate the potential for this technique to
provide functionalized scaffolds harboring living cells.
Figure 16. Resolution patterned test. Reproduced with permission.[129] 4.3. Self-Assembling Scaffolds
Copyright 2015, AAAS.
A novel approach to designing 3D printed scaffolds is through

solutions or melts. The electrospinning process applies an the use of self-assembled scaffolds. This method relies on non-
electric field (high voltage) to a liquid polymer solution that is covalent spontaneous interactions between hydrophilic and
held by its surface tension to induce an electric charge on the hydrophobic amino acid motifs to drive scaffold assembly.[139]
liquid surface. Once this applied force reaches a point where Different environmental cues can trigger self-assembly of
the repulsive electrical forces overcome the surface tension, a peptides or nucleic acids. One research team has developed
jet of solution is ejected from the tip of the Taylor cone (point three new threonine-based nanofibrous, self-assembling
of eruption). The unstable and rapid whipping, caused by elec- multidomain peptides (MDP) capable of forming porous
trostatic repulsion, elongates the fibers which are then collected hydrogels (Figure 17).[139] The researchers formulated pep-
by a grounded collector. The jet of solution continually under- tides with the ability to self-assemble into biomimetic β-sheet
goes elongation and solvent evaporation, which eventually nanofibers.[139–141] Stem cells from human exfoliated deciduous
leads to the production of randomly oriented nanofiber con- teeth were encapsulated in hydrogels formed by the MDPs and
structs.[130–132] Electrospinning is capable of producing inter- cytocompatibility was observed. In contrast to serine-based
connected nanofibers ranging from 5 nm to more than 1 µm in MDPs, the threonine-based MDPs were dependent on bioactive
diameter. This method has been shown to form randomly ori- functionalization with the RGDS sequence for cell attachment
ented nanofibers possessing a high surface area with low den-
sity as well as high porosity with small pore size (≈100 µm).[130]
Recently, production of aligned nanofibers has been reported
using synthetic and natural biomaterials that include collagen,
chitosan, and gelatin. In a proof of concept experiment, micro-
contact printing was used in combination with biocompatible
electrospun scaffolds.[133] Poly(ε-caprolactone) matrices were
obtained by electrospinning, which was followed by the transfer
of poly-L-lysine protein patterns from polydimethylsiloxane
stamps onto the electrospun substrates. This scaffold was
then tested using human bone-marrow-derived mesenchymal
stromal cells and displayed the ability to be an effective sub-
strate for cell adhesion. The use of electrospinning technologies
to fabricate scaffolds for tissue engineering has attracted the
interest of researchers due to the similarity in morphology to
that of native ECM.[133]
4.2. Bio-Electrospraying and Cell Electrospinning
Another interesting approach to the development of a novel 3D

printing method for the fabrication of scaffolds is through bio-
electrospraying.[134] Bio-electrospraying works similarly to elec-
trospinning, except that small droplets are formed instead of Figure 17. Self-assembling scaffolds: AFM of a 0.01% by weight peptide
fine fibers. Keat-Eng Ng et al. were able to use this technology solution under A) nongelation conditions and B) gelation conditions;
C) TEM of a 0.01% by weight peptide solution under non-gelation condi-
to process living cells and overcome the disadvantages of ink-jet
tions; and D) SEM of 1% by weight hydrogel of K(TL)2SLRG(TL)3KGRGDS
printing due to the shear stress of the needle gauge affecting cell nanofibers illustrate the different morphology observed with the threo-
mortality. Using bio-electrospraying, primary cardiac cells could nine-leucine (TL)-based nanofibers. Reproduced with permission.[139]
be printed to yield functional cardiac tissue, where myocytes Copyright 2014, American Chemical Society.
www.advhealthmat.de
REVIEW
to occur. The most popular polymers to have been used in self- showed higher proliferation of BMSCs compared to non-coated
assembled scaffolds include rationally designed nucleic acids PCL scaffolds. In another study involving indirect 3D printing,
and peptides.[142] The ability to create peptide-based assembled Park, Jung, and other colleagues created an advanced indirect
scaffolds that can mimic the structure and function of the ECM 3D printing technique with projection-based micro-stereolithog-
can be a huge advantage to allow cells to attach and proliferate raphy and an injection molding system.[145] Using an alkali-sol-
at a much faster rate. This technology can improve current 3D uble photopolymer, a sacrificial mold was initially made using
printed scaffolds by increasing the recruitment of cells to the projection-based micro-stereolithography. From this mold, PCL
desired site. scaffolds were fabricated by either solvent-based or thermal
molding using the injection molding system. This advanced
indirect 3D printing method showed that the thermal molding
5. Indirect 3D Printing process achieved a substantial reduction in scaffold fabrication
time and stronger mechanical properties compared to scaffolds
With indirect 3D printing, a 3D printed scaffold is made by generated by conventional indirect 3D printing. Thus, with
using a negative mold based on a scaffold design. Once the combinatorial techniques, indirect 3D printing disadvantages
mold is made, the biomaterial is cast into the negative mold. can be negated. Indirect 3D printing has also been use to fab-
After casting is complete, the negative mold is dissolved to ricate scaffolds with natural polymer such as collagen. These
obtain the desired scaffold. This technique is more conven- novel collagen scaffolds demonstrated the ability to support the
tional compared to the techniques discussed thus far for 3D growth of hMSCs for up to 4 weeks in vitro and have little to no
printing of scaffolds and requires a solvent-based system to cytotoxicity on the seeded cells.[146] Indirect 3D printing has the
dissolve away support layers leading to longer scaffold fabrica- ability to print viable scaffolds and may be a useful and cheaper
tion times.[143] However, an advantage to this technique is that alternative to fabricate scaffolds at a laboratory scale when com-
it is capable of creating customized scaffolds that precisely fit pared to purchasing a 3D printer.
the patient’s needs, for example, researchers at the Univer-
sity of California were able to print a scaffold that mimicked
a human mandibular condyle.[144] In this study, the research 5.1. Wax Printing
group used indirect 3D printing to make biomimetic scaffolds
composed of PCL and chitosan. The scaffold was prepared by Wax printing is a form of indirect 3D printing, as the wax
adding PCL dissolved in chloroform to a gelatin mold made printer prints a negative mold from which the scaffold material
from 3D printing. Once the scaffold was dried, the gelatin solution is cast. The formation of a 3D printed scaffold from a
mold was then removed by placing the PCL-gelatin scaffold in wax printer starts with droplets of build wax and support wax
dH2O at 50 °C for 6 h to obtain the PCL scaffold. After the PCL being dispensed from a nozzle to build a layer on the surface.
scaffold was acquired, an apatite coating was added to the scaf- Upon completion of the printed layer, the printed surface is flat-
fold to help support the cell growth of bone marrow stromal tened and another layer is printed. Each layer is allowed a cool
cells (BMSCs).[144] The fabricated PCL apatite-coated scaffolds down period so that the wax will harden. After the structure is
Figure 18. Schematic of Wax Printing process. Adapted with permission.[94] Copyright 2005, Nature Publishing Group.
www.advhealthmat.de
REVIEW
Table 1. Comparison table and preclinical progress of various 3D printing techniques used to print scaffolds for tissue engineering.
Printing Method Advantages Disadvantages Preclinical progress

Direct 3D printing/Inkjet • Versatile in terms of usable materials • Potential toxicity (incompletely removed • (Rat/Bone)[152–156]
• No support is necessary for overhang or binders) • (Rabbit/Bone)[157,158]
complex structures • Low mechanical strength prints compared • (Mouse/Bone)[99,158
to laser sintering
• Time Consuming (Post-processing)[93]
w/electrospinning • (Mouse/Cartilage)[159]
Bioplotting • Prints viable cells[15,97] • Limitation on nozzle size*[98] • (Rabbit/Trachea)[160]

• Soft tissue applications[107] (*Must not be cytotoxic during processing) • (Rabbit/Cartilage)[161]
• Requires support structure for printing • (Rat/Cartilage)[162]
complex shapes • (Mouse/Cartilage)[163]
• (Mouse/Tooth regeneration)[164]
• (Mouse/Skin)[165]
Fused Deposition Modeling • Low cytotoxicity vs direct • Limitation on materials (often requires • (Swine/Bone)[167
3D printing[108] thermoplastics)[109] • (Rat/Bone)[108,114]
• Relatively inexpensive • Materials used are non-biodegradable
(printers and materials)[166] • Requires support structure for overhangs
and complex shapes
• Post-processing may be necessary
• Low Resolution[166]
Selective Laser Sintering • Provides scaffolds with high mechanical • Limitation on materials • (Mouse/Bone)[168]
strength (must be shrinkage and heat resistant)[116] • (Rat/Heart)[169]
• Powder bed provides support • Very high temp required ( up to 1400 °C)[115] • (Rat/Bone)[170,171]
for complex structures • Expensive and time consuming • (Mouse/Skin)[171,172]
• Fine resolution[117,118] (processing and post processing) • (Mouse/Heart)[172]
Stereolithography • Very high resolution[166] • Materials must be photopolymers[173] • (Rat/Bone)[174]

• Speed of fabrication[129] • Expensive (two photon printers)[166] • (Rabbit/Trachea)[175]
• Smooth surface finish • Support system is necessary for overhang • (Pig/Tendon)[176]
and intricate objects
Electrospinning • Speed of fabrication • Random orientation of fibers[177] • (Mouse/biocompatibility)[181,182]

• Cell printing[137] • Non-uniform pore sizes[178] • (Rat/Bone)[183,184]
• Soft tissue engineering[135] • High voltage (1–30 kV) requirements[179,180] • (Rabbit/Vascular tissue)[185]
• Low shear stress (bioelectrospraying)[136]
Indirect 3D Printing • Good for prototyping/preproduction • Requires proprietary waxes for • (Rat/Bone)[189]
• Material versatility casting once biocompatibility(Wax Printing)[148] • (Mouse/Tooth regeneration)[190]
mold is obtained[186] • Low accuracies/resolution[187]
• Mold required for casting[188]
• Long production times
(mold → cast → processing → product)
complete, it can be immersed in selective solvents to dissolve More recently, wax printing using paraffin spheres and wax was
the support wax to form a porous structure which is the nega- used to fabricate poly(L-lactide) (PLLA) nanofibrous scaffolds
tive mold. The casting solution made from the scaffold material with controlled shape, pore size, and poor connectivity.[150] The
is then added to the negative mold and hardened. The negative wax support mold was printed with a Modelmaker II (Solid-
mold is then either dissolved or melted to leave behind the scaf- scape Inc.) in a layer-by-layer process and was then filled with
fold only (Figure 18).[147] paraffin spheres (build wax layer). The wax layer was then dis-
The disadvantage of using wax is that it was not originally solved away with cyclohexane, and PLLA casting solution was
designed to be biocompatible. This is problematic as the cast in the mold filled with paraffin spheres. After the PLLA
non-biodegradable wax residuals may contaminate the scaf- scaffold was formed, the paraffin spheres were also dissolved
fold resulting in unsolicited effects.[148] However, the com- away with cyclohexane. In vitro studies for the fabricated
pany TEOX Ltd. has developed new biocompatible waxes, nanofibrous scaffolds indicated strong osteoblast cell growth
BioBuild and BioSupport, to be used as mold materials for wax and differentiation, illustrating the potential for wax printing
printing.[149] These proprietary waxes can be dissolved in either to be used to fabricate scaffolds with the capacity to regenerate
water heated to 35 °C (BioSupport) or ethanol (BioBuild).[147,149] bone tissues.[150]
www.advhealthmat.de
6. Preclinical to Clinical Translation of 3D Printing
REVIEW
ever, alternative approaches also being investigated include the
Technologies printing of functional tissues in vitro. Such 3D printed tissues
can be formed using a patient’s own cells thereby potentially
Although the advances in 3D printing have led to the fabrica- overcoming issues of rejection.[191,192] Ultimately, 3D printing of
tion of scaffolds with fine resolutions (down to 10 µm) suitable scaffolds for tissue engineering may be the key to giving those
for a range of tissues, the translation of 3D printed scaffolds to suffering from organ failure and dysfunction caused by dam-
the clinic has been slow. Current obstacles to using 3D printed aged or diseased tissue a chance at an improved quality of life.
scaffolds in clinical applications reside in issues of biologics,
engineering, cost, and regulation/safety.[151] For biologics, it is
necessary to take into consideration the survival requirements Acknowledgements
of cells such as oxygen diffusion, cell migration, and levels of
vascularization which have often been suboptimal. In terms We acknowledge financial support from a Deans Graduate Fellowship
of engineering, the processability and reproducibility of the from the University of Iowa, the ITI Foundation for the Promotion
of Implantology, Switzerland (ITI Research Grant No. 855 2012), the
scaffolds is necessary to ensure consistency and homogenous Osseointegration Foundation, the Osteology Foundation, the National
application. As 3D printing is a novel approach to tissue engi- Institutes of Health National Institute of Dental and Craniofacial
neering, the procurement of a 3D printer with the ability to Research (1R21DE02420601A1), the National Cancer Institute at the
print scaffolds with fine resolution can be an expensive invest- National Institutes of Health (5P30CA086862) and the Lyle and Sharon
ment (up to $750,000 for a single printer). In addition, the Bighley Professorship. We have no conflicts of interest to declare.
supply of approved and appropriate materials, cell culture facil-
Received: March 10, 2015
ities and, in many cases, recombinant factors will add to the
Revised: April 26, 2015
financial burden. Finally, regulation and safety guidelines must Published online: June 10, 2015
be established and a standard must be set in order to ensure
that scaffolds meet a certain criteria before being used.[151] Nev-
ertheless, the abundance of preclinical in vivo studies using
various 3D printing techniques demonstrates the feasibility
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3D Printing of Scaffolds For Tissue Regeneration

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3D Printing of Scaffolds for Tissue Regeneration

Anh-Vu Do, Behnoush Khorsand, Sean M. Geary, and Aliasger K. Salem*

nonsteroidal anti-inflammatory drugs

Ceramics contain both metallic and nonmetallic elements and

the analysis of macrophage morphology and

3. Three-Dimensional Printing Techniques for

differentiation into a chondrogenic lineage was observed.[107]

3.4. Fused Deposition Modeling

(HEMA–MMA–MAA). This hybrid scaffold,

exposed to light (UV range of 300–400 nm).[121–124] After the first

Figure 11. Schematic of Selective Laser Sintering technique. Adapted

Figure 12. Bone scaffolds generated by SLS: A) Image of the scaffold,

were seen to beat spontaneously for a period of 5 weeks.[135]

Further details on bio-electrospraying are available in a compre-

A novel approach to designing 3D printed scaffolds is through

4.2. Bio-Electrospraying and Cell Electrospinning

Another interesting approach to the development of a novel 3D

Printing Method Advantages Disadvantages Preclinical progress

Bioplotting • Prints viable cells[15,97] • Limitation on nozzle size*[98] • (Rabbit/Trachea)[160]

Stereolithography • Very high resolution[166] • Materials must be photopolymers[173] • (Rat/Bone)[174]

Electrospinning • Speed of fabrication • Random orientation of fibers[177] • (Mouse/biocompatibility)[181,182]

6. Preclinical to Clinical Translation of 3D Printing

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