Research

Engineering 2015, 1(2): 261268

DOI 10.15302/J-ENG-2015061

3D PrintingReview
Design and 3D Printing of Scaffolds and Tissues
Jia An, Joanne Ee Mei Teoh, Ratima Suntornnond, Chee Kai Chua*

ABSTRACT A growing number ofthree-dimensional (3D)-printing processes have been applied to tissue engineering.
This paper presents a state-of-the-art study of 3D-printing
technologiesfor tissue-engineering applications, with
particular focus on the development of a computer-aided
scaffold design system; the direct 3D printing of functionally
graded scaffolds; the modeling of selective laser sintering
(SLS) and fused deposition modeling (FDM) processes; the
indirect additive manufacturing of scaffolds, with both micro
and macro features; the development of a bioreactor; and
3D/4D bioprinting. Technological limitations will be discussed
so as to highlight the possibility of future improvements for
new 3D-printing methodologies for tissueengineering.
KEYWORDS rapid prototyping, 3D printing, additive manu
facturing, tissue engineering, bioprinting

1Introduction
The concept of tissue engineering was formalized in 1993
when Langer and Vacanti published a historical milestone
paper in Science, in which the characteristics and applications
of biodegradable three-dimensional (3D) scaffolds were first
detailed [1]. Ideally, 3D scaffolds should be highly porous,
have well-interconnected pore networks, and have consistent
and adequate pore size for cell migration and infiltration [2].
In the decade following the publication of this paper (1993
2002), a number of conventional manufacturing techniques
were applied to fabricating porous 3D scaffolds, such as fiber
bonding, phase separation, solvent casting, particulate leaching, membrane lamination, molding, and foaming [3]. However, all these methods share a major drawback: They do not
permit enough control of scaffold architecture, pore network,
and pore size, giving rise to inconsistent and less-than-ideal
3D scaffolds. To overcome this problem, researchers proposed the use of 3D-printing methods (also known as rapid
prototyping, solid free-form fabrication, or additive manufacturing) to fabricate customized scaffolds with controlled pore
size and pore structure [46]. Out of more than 40 different
3D-printing techniques in development, fused deposition

modeling (FDM), stereolithography, inkjet printing, selective

laser sintering (SLS), and colorjet printing appeared to be the
most popular, due to their ability to process plastics [7, 8].
As a result, in the second decade of this field (20032012), the
number of studies in the arena of 3D printing for tissue engineering rapidly multiplied. These studies covered scaffold
design, process modeling and optimization, comparisons of
3D-printing methods, post-processing and characterization
of 3D printed scaffolds, in vitro and in vivo applications of 3D
printed scaffolds, new scaffold materials for 3D printing, new
3D-printing methods for scaffold fabrication, and even the
branching out of an entirely new field3D bioprinting, or
organ printing. Our research group has been extensively involved in this vast wave of research. In this paper, we present
our past and current work in this field, and give our perspective on the future of this area as it moves into its third decade
(20132022).

2 Scaffold architecture design

2.1 Scaffold library
Scaffold architecture design can significantly influence
both mechanical property and cell behaviors [9]. We have
adopted a bottom-up approach when constructing a 3D
scaffold; that is, first making unit cells, and then assembling them into a 3D scaffold. Using this approach, we can
fine-tune the mechanical property, based on the porous
structure design. We have developed in-house a computeraided system for tissue scaffolds (CASTS) that can automatically create a highly porous 3D scaffold model with
controlled architecture, and precisely match the external
surface profile of a native anatomic structure such as
bone [1012]. In this system, nearly 20 polyhedral shapes
are selected to form the basic geometry of a unit cell.
The scaffold library and the parameters of each unit cell,
such as pore size and strut size, can be adjusted, and each
polyhedral unit can be repeated automatically in a spatial arrangement and sized to form a block that suits the
intended scaffold application (Figure 1). An anatomically
shaped porous scaffold can then be created through Bool-

Singapore Centre for 3D Printing, School of Mechanical and Aerospace Engineering, Nanyang Technological University, Singapore 639798, Singapore
* Correspondence author. E-mail: [email protected]
Received 30 January 2015; received in revised form 23 March 2015; accepted 30 June 2015
The Author(s) 2015. PublishedbyEngineering Sciences Press. ThisisanopenaccessarticleundertheCCBY license (http://creativecommons.org/licenses/by/4.0/)

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ean operation between the scaffold
block and the actual surface model of
the defect tissue. A detailed derivation of the mathematical formulae of
the CASTS system for designing and
fabricating tissue engineering scaffolds is contained in Ref. [13].

fold is obtained by performing a difference operation between the contour model

and the pore model. Using this method, a well-defined pore size distribution can
be achieved for gradient bone-scaffold design. Recently, a new method based on
sigmoid functionand Gaussian radial basis function has been developed to gene
rate functionally graded structures, and the resulting models can be exported as
STL-files and be 3D printed [19].

Figure 1. An example of five polyhedral units

and their resultant blocks, generated in the
CASTS scaffold library.

Figure 2. Virtual and physical prototypes of the functionally graded porous scaffold of a femur bone
segment.

2.2 Functionally graded scaffold

Natural tissues such as bone usually
have a gradient porous structure, so
matching mechanical strength and
stiffness between porous scaffold design and the target tissue structure is
important [14]. There are two types of
stiffness gradient in bones: radial gradients in long bones, and linear gradients in short and irregular bones. We
have achieved radial gradient design
by arranging cylindrical unit cells in
a concentric manner so that the porosity decreases linearly from the center
to the periphery. This linear gradient
occurs as a result of varying the strut
diameter along the gradient direction.
Therefore, we can tailor the stiffness
variation for CASTS scaffolds by adjusting the porosity-stiffness relationship
[15]. After modifying and improving
the CASTS system, our group successfully fabricated a human mandibular
cancellous bone scaffold and a femur
bone segment, both with functional
gradients [16, 17]. An example of a functionally graded femur bone segment is
shown in Figure 2. This process is highly accurate and reproducible. Another
method of designing gradient structure
is based on shape function and on an
all-hexahedral mesh refinement [18]. In
this method, a truncated bone is subdivided and represented using various irregular hexahedral elements, which are
then converted into various irregular
pore elements based on shape function.
The entire pore model is obtained after
a union operation among the irregular
pores, and then the resulting bone scaf262

2.3 Design for vascularization

In addition to mechanical performance, vascularization is a major limitation in tissue engineering, especially when engineering thick or bulk tissues. Researchers
have proposed various strategies to enhance or accelerate vascularization, in which
scaffold design plays a crucial role [20]. Results show that a designed pore size of
250 m or above favors the growth of blood vessels more than smaller pore sizes [21].
Also, a high porosity does not necessarily lead to more vascularization, because
cell migration and vascularization could be inhibited if there is little interconnectivity between pores [22]. Recently, researchers have developed a tool box for evaluating 3D porous scaffolds [23]. This tool box is based on modular scaffold design,
and allows the fine-tuning of scaffold pore size and porosity for vascularization
study. Our group is exploring a new concept of hybrid scaffold design to address
the vascularization issue. This new approach involves thin porous membranes and
filament meshes that alternate in layers to form a 3D scaffold (Figure 3) [24].

Figure 3. A proposed hybrid scaffold design for vascularization.

3 Direct 3D printing
3.1 Specific forms of materials
At room temperature, the primary forms of materials used for 3D printing are
solidifiable fluid, non-brittle filament, laminated thin sheet, and fine powder (see
Table 1) [25]. Each form is specific for certain 3D-printing processes. If a material

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3D PrintingReview
is deemed suitable for a particular application but cannot be
easily prepared in the specific form required by the desired
3D-printing process, printing this material would be a challenge. Even if a material can be prepared in a specific form,
this does not guarantee that the material is 3D printable, because successful printing in the vertical dimension also relies
on the bonding strength between layers. Therefore, when
exploring a material for 3D scaffold applications, it is important to consider the available forms of the material at the
very first stage. Furthermore, in order to increase the range

Research

of 3D printable biomaterials, future development should

include the invention of new methods to transform existing
biomaterials into suitable forms for 3D printing. For example,
gelatin gel is solidifiable upon a decrease in temperature,
but this low-temperature environment conflicts with what is
favorable for cell survival. Therefore, future research may involve the development of a new mechanism of solidification
for gelatin, such as solidification by enzymatic crosslinking
[26], or the development of a new hybrid mechanism for lowtemperature deposition of hydrogels and cells [27].

Table 1. Specific forms of materials and suitable 3D-printing processes.

Form

Examples

Suitable 3D-printing processes

Refs.

Solidifiable fluid

Photopolymer resins, temperature sensitive

polymers, ion cross-linkable hydrogels, ceramic
paste, etc.

Stereolithography (SLA)

[28]

Polyjet

[29, 30]

Digital light processing (DLP)

[31]

Micro-extrusion

[32]

Non-brittle filament

Thermoplastics, e.g., ABS, PLA, and PCL

Fused deposition modeling (FDM)

[33]

Laminated thin sheet

Paper, plastic sheet, metal foil

Paper lamination technology (PLT)

[34]

Laminated object manufacturing (LOM)

[35]

Ultrasonic consolidation (UC)

[36]

Selective laser sintering/melting (SLS/SLM)

[17, 37, 38]

Electron beam melting (EBM)

[39]

Laser engineered net shaping (LENS)

[40]

Direct metal deposition (DMD)

[41]

Colorjet printing (CJP)

[4244]

Fine powder

Plastic fine powder, ceramic powder, metal

powder

Notes: ABSacrylonitrile-butadiene-styrene; PLApolylactic acid; PCLpolycaprolactone.

3.2 Process parameters and limitations

Our group investigated a range of materials using the SLS
process for fabricating tissue-engineering scaffolds. Table 2
summarizes the main process parameters for SLS, namely
part-bed temperature, laser power, and scan speed. In particular, in the polyetheretherketone/hydroxyapatite (PEEK/
HA) system, results show that HA should be kept at 40wt.%
or below in order to ensure structural integrity. In the polyvinyl alcohol/hydroxyapatite (PVA/HA) and polycaprolactone/

hydroxyapatite (PCL/HA) systems, HA should be kept at 30

wt.% or below in order to yield successful scaffold specimens
with well-defined pore interconnectivity and good structural
integrity. When developing composite material systems,
although the addition of HA initially improved mechanical
properties and bioactivity, it compromised material properties during the hydrolytic degradation process [45]. In addition to scaffolds, we investigated laser sintering of drug
delivery devices and their microfeatures [4648].

Table 2. SLS process parameters for different types of polymers.

Polymer type

Composition (wt.%)

Part-bed temperature (C)

Laser powder (W)

Scan speed (mms1)

Refs.

PCL

100

3055

17

38105080

[49]

PLLA

100

60

1015

1270

[49]

PVA

100

6065

1015

12705080

[49]

PLGA

100

70

10

1651

[50]

100

110140

928

5080

[49]

PEEK/HA

> 60/40

140

16

5080

[51, 52]

PVA/HA

> 70/30

6580

1315

12701778

[53, 54]

PCL/HA

> 70/30

40

12702540

[55]

PEEK

Notes: PLLApoly-L-lactic acid; PLGApoly(lactic-co-glycolic acid).

One limitation in the SLS process is material wastage

when building small prototypes such as tissue-engineering
scaffolds. However, this problem can be overcome by incorporating a compact adaptation system into the SLS part
bed, allowing the adaptor to transfer the motion of the SLS

part bed onto its own small part bed [56]. Up to 6.5 times
the amount of powder can be saved by using this device. A
second limitation of SLS-made scaffolds is the low retention
of cells during cell seeding. One reason is that the materials
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tachment. The other reason is that the pores are much larger
than cells due to SLS resolution issue, as a result, cells fall
through the pores during the seeding process. However, the
use of a hybrid 3D scaffold that consists of alternate electrospun nanofibers and 3D printed scaffold layers will prevent cells from falling through, due to the small size of the
nanofiber pores [57, 58]. An alternative solution is to inject
cell-laden collagen hydrogel into the porous structure [59].
An unresolved limitation in SLS scaffolds does exist, for
example, the entrapment of powder in the interior region of
the porous scaffold. It is difficult to remove the entrapped
powder manually, especially for pore sizes below 500 m.
Researchers have explored ultrasonic cleaning with only
limited success [60].
3.3 Modeling for FDM and SLS
In 3D printing, it is important to understand the process itself, as well as the science behind it, in order to improve the
process further (Figure 4). PCL is a representative biomaterial
for the FDM process. The results from modeling and finite
element analysis indicate that the pressure drop and the
velocity of the PCL melt flow depend on the flow channel
parameters [61]. The temperature gradient of the PCL melt
shows that it liquefies within 35% of the channel length [61].

Similarly, we modeled the heat-transfer phenomena during

the SLS process. By incorporating material properties such as
thermal conductivity, thermal diffusivity, surface reflectivity, and absorption coefficient, our model helped to identify
the biomaterial and laser-beam properties that are critical
to the sintering result [62]. It is also important to understand
the relationship between mechanical properties and scaffold
porosity in 3D printing (Figure 5). We obtained such a plot
for FDM, based on experimental data from ABS samples [63,
64]. In addition, we found a feasible porosity and compressive
stiffness range for our CASTS-designed PCL scaffolds via
the testing of physical prototypes [65]. This stiffness range
fairly matches the stiffness gradient of cancellous bone in the
maxillofacial region, which varies gradually from 35.55MPa
in the molar region to 67.48MPa in the incisor and canine region [66].

4 Indirect 3D printing
Natural polymers usually have very good biocompatibility,
and can provide a favorable micro-environment for cells as
compared to synthetic polymers. However, the 3D printability of natural polymers is generally poor. Indirect 3D printing was developed in order to produce a 3D porous scaffold

Figure 4. Process modelling. (a) Temperature distribution and Gaussian contour in the laser sintering process: 1), 3) are temperature distributions; 2), 4) are
Gaussian contours. (b) Velocity profiles at different zones along the melt flow channel.

Figure 5. Scaffold porosity and mechanical property. (a) Relationship between porosity and modulus
in FDM scaffolds; (b) the feasible porosity and compressive stiffness range (gray band) in SLS-made PCL
scaffolds.

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using natural polymers such as collagen or gelatin. In contrast to direct 3D

printing, which produces a scaffold directly from the model material, indirect
3D printing creates a negative mold,
usually from a support material, and
then casts the desired polymer scaffold
out of the mold via a drying method [6,
67, 68]. Collagen scaffolds with 3D networks of internal channels can be produced using this approach [69]. Moreover, freeze-drying was found to be the
most suitable drying method in indirect

3D PrintingReview
3D printing, as it induced less shrinkage than critical-point drying, and accurately
reproduced the design morphology of the channels [69]. Furthermore, indirect fabrication can be combined with a foaming process to produce highly and uniformly
porous gelatin scaffolds with complex channel architectures [29, 70], as shown in
Figure 6(ad). The order of this structure can be improved further by incorporating
monodispersed microspheres into the casting process [71], as shown in Figure 6(e, f).
In addition to collagen and gelatin, our group has successfully produced porous
scaffolds from silk fibroin protein with both macro- and micro-morphological features [30, 72].

Figure 6. Highly and uniformly porous interconnected network via the combination of indirect 3D
printing and foaming processes

5Bioreactor
A bioreactor is an important post-processing tool in tissue engineering, as it provides a dynamic environment for cell-scaffold construct, and facilitates the maturation of the construct. More importantly, a bioreactor is a part of the automation
line in industry-scale tissue engineering [73]. A recent study reports that scaffold
architecture could influence cell differentiation in a bioreactor, but not in static
culture [74], further evidencing the importance of a bioreactors role. Surprisingly,
by taking the advantage of the air-liquid interface commonly found in a rotating
bioreactor, induced pluripotent stem (iPS) cells can be induced toward the differentiation of alveolar epithelium, offering a new role for a bioreactor in resolving a
cell-source problem [75]. The most recent bioreactor design is a dual-flow bioreactor coupled with mechanical stimulation [76]. This novel design allows nutrients,
anabolic or catabolic factors to diffuse from one side of the construct, enabling the
creation of gradients; as a result, this bioreactor design is well suited for engineering interface tissues. Our group has focused particularly on the effect of interstitial
flow on fibroblast responses [77]. Through a computational fluid dynamics study,
we found that dynamic flow, even a flow rate as low as 0.002 cm.s1, can support
much better mass exchange, higher cell number, and more even cell and nutrient
distribution than static culture [78]. We have also developed a dual-window dualbandwidthspectroscopic optical-coherence tomography (DWDB-SOCT) technique
to monitor fibroblast cell proliferation in scaffolds. Fibroblasts and their distribution in scaffolds are clearly differentiable in the spectroscopic images [79]. In future, we expect that bioreactors will play more biological roles and take on a more
integrated design by combining various types of stimulation and non-invasive
monitoring techniques.

6 3D bioprinting and beyond

One of the major advances in tissue engineering is the emergence of a new

Research

field: 3D bioprinting [80]. Mironov

etal. [81] originally proposed the concept
of 3D bioprinting as organ printing,
and defined it as the computer-aided,
jet-based 3D tissue-engineering of living
human organs. This organ printing process exactly follows the typical process
chain of 3D printing; that is, starting with
a computer-aided design (CAD) model,
converting to a STL file, slicing and then
printing. The major advantages of organ
printing are automation and high cell
density, compared to a traditional scaffold-based approach [82]. The materials
used for organ printing are microtissues,
usually in the form of spheroids. These
closely placed spheroids can undergo
self-assembly and fuse together [83],
forming the foundation of 3D printability
in organ printing. Organ printing carries
the imminent potential to address organization and complexity issues in engineered tissues [84]. It also carries great
potential to foster the establishment of an
industry-scale robotic tissue-fabrication
line [73]. Nevertheless, current application of organ printing is limited to in vitro
drug testing. According to Mironov, the
first bioprinted organ transplant in the
world is likely to be the thyroid gland of
a mouse, in 2015 [85].
Besides organ printing, other elegant
bioprinting approaches and methodologies exist, such as inkjet printing,
micro-extrusion and laser-assisted
forward transfer, which are comprehensively reviewed in Ref. [86]. It is a
major challenge in these approaches
to position and culture multiple types
of cells in a single process at a defined
location. Although researchers have
obtained initial success in printing heterogeneous tissues [87, 88], these were
printed in separate compartments and
did not replicate the microstructure of
the native tissue. To address this challenge, our group plans to focus on a
fundamental study of the bioprinting
process, such as developing a timepressure model to precisely control extruded material [89].
One interesting derivation of 3D bioprinting is the integration of microelectronic and mechatronic components.
For example, a bio-bot is a walking robot
powered by the contraction of a strip of
mammalian skeletal muscle cells [90].
Another interesting derivation of 3D
bioprinting is the concept of 4D bio-

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printing. 4D printing refers to the 3D printing of programmable materials; since the printed part gradually transforms
in shape over the post-printing period, the fourth dimension
refers to time [91]. One physical demonstration of 4D printing involved intelligent active hinges that enabled origami
folding [92], and this concept has been extended further to
make light-responsive windows that open and close automatically in response to the amount of sunshine. Therefore,
the research community considers 4D printing to be a new
and emerging field [93]. Our group is currently collaborating
with Stratasys (www.stratasys.com) to work on 4D-printable
shape-memory polymers [94]. In terms of 4D bioprinting,
the development of programmable biomaterials appears to
be crucial in realizing time-dependent shape change. The
term 4D bioprinting is currently less defined than that of
4D printing, and the shape it will take in the future is largely
unknown, but well worth watching.

in tissue engineering. Part I. Traditional factors. Tissue Eng., 2001, 7(6):

679689
4. S. Yang, K. F. Leong, Z. Du, C. K. Chua. The design of scaffolds for use
in tissue engineering. Part II. Rapid prototyping techniques. Tissue Eng.,
2002, 8(1): 111
5. K. F. Leong, C. M. Cheah, C. K. Chua. Solid freeform fabrication of threedimensional scaffolds for engineering replacement tissues and organs.
Biomaterials, 2003, 24(13): 23632378
6. W. Y. Yeong, C. K. Chua, K. F. Leong, M. Chandrasekaran. Rapid prototyping in tissue engineering: Challenges and potential. Trends Biotechnol.,
2004, 22(12): 643652
7. T. Boland, et al. Rapid, prototyping of artificial tissues and medical devices. Adv. Mater. Process., 2007, 165(4): 5153
8. P. J. Brtolo, C. K. Chua, H. A. Almeida, S. M. Chou, A. S. C. Lim. Biomanufacturing for tissue engineering: Present and future trends. Virtual and
Physical Prototyping, 2009, 4(4): 203216

7 Future prospects

9. S. J. Hollister. Porous scaffold design for tissue engineering. Nat. Mater.,

In the second decade after the birth of tissue engineering, 3D

printing gradually became a definite part of this field, due
to its controllability and manufacturing capability. Looking
into the future, even once the technical challenges described
above are overcome, it will still be a long way from transforming academic know-how into clinical products that
benefit society. Researchers current tasks in the field are to
accelerate the standardization and certification of 3D printed
medical devices. A prolonged delay in this standardization
would make regulatory work even more complicated, especially with the currently trending and transforming 3D bioprinting technologies, because the definition of medical device may soon be redefined. Another future trend may come
in the legal landscape [95], as the infringement and protection of intellectual properties around 3D printing interweave
more intensely. Thus, an early and informed exploration of
various legal approaches could be the best preparation to
cope with tomorrows changes.

10. C. M. Cheah, C. K. Chua, K. F. Leong, S. W. Chua. Development of a tissue

Acknowledgements
The authors would like to thank Singapore National Research
Foundation (NRF) for funding the Singapore Centre for 3D
Printing (SC3DP). The authors would also like to thank Professor Li Lin at the University of Manchester for his kind invitation for submission.

Compliance with ethics guidelines

Jia An, Joanne Ee Mei Teoh, Ratima Suntornnond, and Chee
Kai Chua declare that they have no conflict of interest or financial conflicts to disclose.

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