Biocompatibility and biodegradation studies of PCL/b-TCP bone

tissue scaffold fabricated by structural porogen method

Lin Lu

Qingwei Zhang

David Wootton

Richard Chiou

Dichen Li

Bingheng Lu

Peter Lelkes

Jack Zhou
Received: 2 December 2011 / Accepted: 23 May 2012 / Published online: 6 June 2012
Springer Science+Business Media, LLC 2012
Abstract Three-dimensional printer (3DP) (Z-Corp) is a
solid freeform fabrication system capable of generating
sub-millimeter physical features required for tissue engi-
neering scaffolds. By using plaster composite materials,
3DP can fabricate a universal porogen which can be
injected with a wide range of high melting temperature
biomaterials. Here we report results toward the manufac-
ture of either pure polycaprolactone (PCL) or homoge-
neous composites of 90/10 or 80/20 (w/w) PCL/
beta-tricalcium phosphate (b-TCP) by injection molding
into plaster composite porogens fabricated by 3DP. The
resolution of printed plaster porogens and produced scaf-
folds was studied by scanning electron microscopy. Cyto-
toxicity test on scaffold extracts and biocompatibility
test on the scaffolds as a matrix supporting murine osteo-
blast (7F2) and endothelial hybridoma (EAhy 926) cells
growth for up to 4 days showed that the porogens removal
process had only negligible effects on cell proliferation. The
biodegradation tests of pure PCL and PCL/b-TCP compos-
ites were performed in DMEMwith 10 %(v/v) FBS for up to
6 weeks. The PCL/b-TCP composites show faster degrada-
tion rate than that of pure PCL due to the addition of b-TCP,
and the strength of 80/20 PCL/b-TCP composite is still
suitable for human cancellous bone healing support after
6 weeks degradation. Combining precisely controlled
porogen fabrication structure, good biocompatibility, and
suitable mechanical properties after biodegradation, PCL/
b-TCP scaffolds fabricated by 3DP porogen method provide
essential capability for bone tissue engineering.
1 Introduction
Tissue engineering is a rapidly growing interdisciplinary
eld which offers a promising new approach for bone
repair [1, 2]. One promising method of bone tissue engi-
neering is using biomaterials to build scaffolds serving as
an articial extracellular matrix to support new tissue
growth [25].
Composites comprising a biodegradable polymeric
matrix with inorganic llers show considerable promise in
the eld of orthopedic regenerative medicine since they can
provide enhanced mechanical properties and improved
interaction with host tissue [57]. Poly-e-caprolactone
(PCL), a semicrystalline aliphatic polyester, has been
investigated as a potential biomaterial for orthopedic
applications due to its good biocompatibility and biode-
gradability [8, 9]. However, PCL has insufcient
mechanical strength and its surface chemistry does not
promote cell adhesion. Owing to its osteoconductivity and
osteoinductivity, calcium phosphate is frequently used with
PCL as a scaffold material for bone tissue engineering [10
12]. Incorporation of calcium phosphate with PCL would
overcome the shortcoming of hydrophobicity of PCL and
the brittleness of the ceramic.
L. Lu Q. Zhang R. Chiou J. Zhou (&)
Department of Mechanical Engineering and Mechanics, Drexel
University, 3121 Chestnut St., Philadelphia, PA 19104, USA
e-mail: zhoug@drexel.edu
D. Wootton
Mechanical Engineering, Cooper Union, 41 Cooper Square,
New York, NY 10003, USA
D. Li B. Lu
School of Mechanical Engineering, Xian Jiaotong University,
Xian, China
P. Lelkes
Department of Bioengineering, Temple University, Philadelphia,
PA 19122, USA
1 3
J Mater Sci: Mater Med (2012) 23:22172226
DOI 10.1007/s10856-012-4695-2
An ideal bone tissue engineering scaffold should have
both sufcient mechanical strength similar to natural bone
and interconnected three-dimensional (3D) porous struc-
ture with favorable surface chemistry to support nutrient
transfer, bone cells adhesion, proliferation, and differenti-
ation [35]. Many techniques, including salt leaching [13],
gas foaming [14], solvent casting [15], phase inversion
processes [16], ber meshes [17], and sacricial com-
pression-molding [18] have been developed to fabricate 3D
porous architectures to meet the requirements of bone
scaffolds preparation. However, many of these methods
cannot produce scaffolds with controllable pore size and
precise structure. Solid freeform fabrication (SFF) is one of
the manufacturing technologies for fabricating 3D scaf-
folds with predetermined porosity and structure [1923].
Recent developed porogen method has attracted a lot of
attention since this method can build a scaffold with more
precise dimension than direct SFF [24]. In our previous
work, we have fabricated injectable thermoplastic (wax)
porogens for 3D bone scaffolds preparation [25]. However,
the melting point of the wax used for porogen fabrication is
relatively low (\75 C), which limits the injection of most
of the biopolymers for scaffolds fabrication. A three-
dimensional printer (3DP) is one of the choices to achieve
high melting point biopolymer fabrication since 3DP uses
plaster as the porogen building material. Plaster has very
high melting point (1,4001,500 C), which can support
the molding of almost all the biopolymers. However, the
grain size of plaster is much bigger than wax, and thus may
inuence the precision of the nal 3D scaffolds structure.
In order to test the feasibility of 3DP method for bone
tissue engineering scaffolds fabrication, we incorporated
PCL with beta-tricalcium phosphate (b-TCP) micro-parti-
cles to generate a series of bone scaffolds by injection
molding of the composites into plaster porogens. The reso-
lution of the produced scaffolds was studied. The possible
cytotoxicity introduced by porogen and the biocompatibility
of all the fabricated porous scaffolds were tested. In addition,
the mechanical properties of PCL/b-TCP composites inu-
enced by materials degradation were investigated in order to
mimic the condition in human body.
2 Materials and methods
2.1 Structured porogen design and fabrication
Porogens with strut size of 200400 lm and space size of
800 lm were designed and constructed using CAD soft-
ware (Pro/Engineer, Parametric Technology Corporation).
An STL le of the CAD model was then imported into a
commercially available 3D printer (Z310 plus, Z-Corp) to
print the rectangular honeycomb-like construct layer by
layer following a perpendicular lay-down pattern (0/90)
shown schematically in Fig. 1a and b. The porogens were
printed by selectively gluing layers of 89 lm thick plaster
powder (ZP 130, Z-Corp) using a liquid binder (ZB 58,
clear). The powder is a proprietary blend of calcium sulfate
hemihydrate plaster, polyvinyl alcohol and potassium sul-
fate. The binder is a proprietary water solution of glycerol,
potassium sorbate and surfactant.
After completely printed, the individual porogen was
removed and the excess powders lled inside the pores
were cleared by pressurized air. The porogens were then
sintered at 135 C for 30 min and inltrated with 40 % (w/
v) polyethylene glycol (PEG, Sigma) for 2 s for strength-
ening. The coated porogens were air-dried at room tem-
perature for further use.
2.2 Scaffolds fabrication
Figure 2 shows a simplied illustration of scaffold fabrication
process. PCL pellets (average molecular weight = 65,000,
Sigma, viscosity 385 Pa s at 75 C) or PCL/b-TCP (Fisher
Fig. 1 The designed porogens:
a square porogen with 200 lm
struts spaced 800 lm apart and
b square long porogen with
400 lm struts spaced 800 lm
apart
2218 J Mater Sci: Mater Med (2012) 23:22172226
1 3
Scientic) composites with 90/10 and 80/20 weight ratios
were melted in a vacuum oven (VWR 1410) at 75 C under
vacuum to remove air bubbles. The molten materials were
then injected into the inltrated porogens using disposable
1 ml syringes. After cooling down to room temperature, the
porogen material was dissolved by a commercial proprietary
dental alginate/plaster remover solution (Cavex GreenClean,
Cavex Holland BV, The Netherlands) at 40 C for 5 days
under rotation. The solution was changed every 24 h. After
removing the porogen, the produced scaffolds were washed
with DI water three times, air-dried at room temperature, and
stored in capped tubes for future tests.
2.3 Cytotoxicity and biocompatibility tests
The cell cultures were performed with either murine bone
marrow derived osteoblast cell line (7F2, from ATCC,
CRL-12557) or human umbilical vein-derived EAhy 926
endothelial cells which were kindly provided by Professor
Cora-Jean Edgell at the University of North Carolina,
Chapel Hill. 7F2 cells were routinely maintained in alpha
minimum essential medium (a-MEM) without ribonu-
cleosides and dexoyribonucleosides, with 10 % (v/v) fetal
bovine serum (FBS, Hyclone), 2 mM L-glutamine and
0.5 % (v/v) penicillinstreptomycin in a 5 % CO
2
incu-
bator at 37 C. EAhy926 cells were routinely maintained in
Dulbeccos modication of Eagles medium (DMEM) with
10 % (v/v) FBS and 0.5 % (v/v) penicillinstreptomycin in
a 5 % CO
2
incubator at 37 C.
2.3.1 Cytotoxicity test
In order to evaluate the possible toxicity introduced by the
3DP porogens or plaster remover, the in vitro cytotoxicity
of the porous scaffolds was tested. Briey, ten sterilized
PCL scaffolds were soaked in culture medium without
changing the medium for 5 days to obtain the scaffolds
extracts. 7F2 and EAhy 926 were cultured in 24-well plates
in the medium with and without the scaffolds extracts for
up to 4 days. Cell viability and proliferation were assessed
with Alamar Blue
TM
assay (AB, Biosource, Alameda, CA)
every 2 days.
2.3.2 Biocompatibility
(1) Osteoblast cell line. The PCL, 90/10 and 80/20 PCL/
b-TCP scaffolds were incubated overnight with 20 lg/ml
collagen type I (BD Biosciences) at 37 C to enhance the
cellular attachment. Then the scaffolds were seeded with
a suspension of 1 million 7F2 cells/ml and the initial
seeding was enhanced on the orbital shaker (Belly dan-
cer, Stovall Life Science) for 3 h. Cell viability and
proliferation were assessed with AB
TM
assay at day 4
post-seeding. The samples were xed with 10 % buffered
formalin after 1 and 4 days culturing and stored in PBS
at 4 C until cytological staining. (2) Endothelial cell
line. The sterilized scaffolds were incubated overnight
with 0.1 mg/ml bronectin (BD science) at 37 C on an
orbital shaker to facilitate extracellular matrix (ECM)
protein adsorption and enhance cell attachment. Half
million EAhy 926 cells were seeded on each scaffold and
the initial seeding was enhanced on the orbital shaker for
3 h. The cells were xed with 10 % buffered formalin
(Fisher) after 24 and 48 h culturing. The xed samples
were washed three times with PBS and stored in PBS at
4 C until cytological staining.
For staining, the samples with both cell lines were
washed once more with PBS and incubated with PBS
containing 2 lg/ml Hoechst 33258 (bisbenzimide, a
nuclear stain, Sigma) and 2 lg/ml rhodamine phalloidin
(Phalloidin, TRITC-labeled, a cytoskeleton stain, Sigma).
Samples were visualized with a Leica DMRX microscope
equipped with the appropriate uorescence lters. Digital
images were acquired using a Leica 300F camera.
Fig. 2 A simplied illustration of bone scaffold fabrication processes: a plaster structured porogen, b biopolymer composite injected into the
skeleton cavity and c resulting scaffold
J Mater Sci: Mater Med (2012) 23:22172226 2219
1 3
2.4 In vitro degradation study
The PCL, 90/10 and 80/20 PCL/b-TCP samples were
compression molded into cylinders with 5 mm diameter at
75 C. The cylinders were then cut into 12 mm length for
in vitro degradation test.
The degradation test was performed in DMEMwith 10 %
(v/v) FBS on an orbital shaker in a 5 % CO
2
incubator at
37 C for periods of 2, 4, and 6 weeks. The culture medium
was changed every 2 days. At each time point, six samples of
each specimen were removed, rinsed with DI water three
times and vacuum dried for 4 days before analysis.
2.5 Mechanical test
Compression tests of PCL, 90/10 and 80/20 PCL/b-TCP
cylinders after degradation were performed on a Tinius
Olsen H25KT machine at 1 mm/min speed rate until fail-
ure. Six specimens were tested for each sample.
2.6 Molecular weight test
The molecular weight was measured by gel permeation
chromatography (GPC-HPLC with refractive index detec-
tion, Waters, USA), carried out at a ow rate of 0.8 ml/min
at 25 C. Briey, predetermined amount of samples were
dissolved in THF (Tetrahydrofuran, Sigma; ratio: 2 mg
samples/1 ml THF) and then ltered using 0.45 lm PTFE
(Hydrophobic uoropore) syringe driven lter unit (Mel-
lex). The ltered samples were injected into the GPC. The
molecular weight was determined using an exponential
calibration curve (MW = k 9 exp(-ct)) t to data from
polystyrene standards (5400 kDa, Polyscience Co.), and
t in MS Excel (R
2
= 0.9976).
2.7 Scanning electron microscopy (SEM)
Porogen, inltrated porogen and resultant scaffold were air-
dried and sputter coated with Pt/Pd for 40 s. The scaffolds with
cells weredriedwithacritical point dryer (CPD, SPI CPD7501,
West Chester, PA) prior to sputter coating. All samples were
characterized with an XL-30 Environmental SEM-FEG (Phi-
lips) using an acceleration voltage of 10 kV. A total of 34
images from independent preparations were evaluated.
2.8 Statistical analysis
All the data are expressed as mean value standard
deviation (SD). All biological experiments were performed
at least three times. Statistical analysis of AB
TM
measurements was performed using ANOVA one-way
analysis, dening p \0.05 as signicant.
3 Results
The micro-porous architecture of porogens with and
without PEG inltration, the produced 80/20 PCL/b-TCP
scaffold, as well as the dimensions of porogen and resultant
scaffold were visualized and measured under SEM (Fig. 3).
The average strut width of the porogen (Fig. 3c) and the
pore size of the scaffold (Fig. 3d) were measured as
605.8 40.74 and 421.5 29.7 lm, respectively.
In the cytotoxicity test, AB assay results show that there
is no signicant difference (p [0.05, n = 6) in the cell
viability for both the 7F2 and EAhy926 cell lines between
in the scaffolds extracts and control medium at each time
point (Fig. 4).
In the biocompatibility test, the AB results reveal that
the osteoblasts proliferate on all scaffolds during the
4 days culturing (Fig. 5). The normalized cell prolifera-
tion data shows a similar cell proliferation on pure PCL and
90/10 PCL/b-TCP scaffolds (p [0.05, n = 6). The cell
growth on 80/20 PCL/b-TCP scaffolds presents signicant
difference (p \0.05, n = 6) with the other two scaffolds
(pure PCL and 90/10 PCL/b-TCP scaffolds).
The uorescent staining images show that the 7F2 cells
attach and spread on both 90/10 (Fig. 6b) and 80/20
(Fig. 6c) PCL/b-TCP scaffolds at day 1 post-seeding while
the osteoblasts can only attach on PCL scaffolds in the pore
area (Fig. 6a). Osteoblasts were conuent after 4 days
seeding on all the scaffolds (Fig. 6df). SEM images reveal
that the osteoblasts attached, spread, migrated, proliferated
to conuence within 4 days and the cells were visibly
growing into the interior pore structures (Fig. 7). Besides
osteoblasts, EAhy926 endothelial hybridoma cells were
also used in this study since bone is a highly vascularized
tissue. The images of bisbenzimide and rhodamine phal-
loidin staining of EAhy926 cells cultured on PCL, 90/10
and 80/20 PCL/b-TCP scaffolds after 24 and 48 h seeding
show that the cells attached and proliferated on all the
scaffolds (Fig. 8). It was found that the cell density on pure
PCL scaffolds was lower than that on 90/10 and 80/20
PCL/b-TCP scaffolds.
The degradation of the porous scaffolds in DMEM with
10 % FBS was monitored by the molecular weight changes
with GPC (Table 1). The molecular weight of PCL chan-
ged from 27.05 kDa at week 2 to 26.70 kDa at week 4, and
then degraded to 26.53 kDa at week 6. The molecular
weight changes of 90/10 and 80/20 PCL/b-TCP scaffolds
were not presented here since b-TCP is not soluble in THF
and the data tested by GPC may be inconclusive.
2220 J Mater Sci: Mater Med (2012) 23:22172226
1 3
The total weight changes of all samples over 6 weeks
degradation show a steady weight decrease with the incu-
bation time (Table 2). The degradation proles exhibit
4.05 % mass loss for PCL sample after 6 weeks degradation
while 90/10 and 80/20 PCL/b-TCP samples present 4.73 and
5.43 % mass loss, respectively. The degradation rate of
80/20 PCL/b-TCP samples is signicantly faster than that of
90/10 PCL/b-TCP or pure PCL samples (p \0.05).
The compression test results of PLC, 90/10 and 80/20
PCL/b-TCP scaffolds after 6 weeks degradation test in
DMEM with 10 % FBS are shown in Fig. 9. PCL rods lost
only about 7.0 % compressive strength after 6 weeks
degradation while 90/10 and 80/20 PCL/b-TCP rods lost
about 37.5 and 53.7 % of compressive strength,
respectively.
A B
1mm 1mm
C D
1mm 1mm
Fig. 3 SEM images of plaster
porogen designed with 400 lm
pore size (a), PEG inltrated
porogen (b), size measurement
of inltrated porogen (c), and
size measurement of 80/20
PCL/b-TCP scaffold made from
injection molding of 3D
structured plaster porogen (d)
Alamar Blue Assay for Toxicity Test
3.50
4.00
4.50
7F2
7F2-CONTROL
EAhy 926
2.50
3.00
Eahy 926 CONTROL
1.00
1.50
2.00
N
o
r
m
a
l
i
z
e
d

F
l
u
o
r
e
s
e
c
e
n
c
e
0.00
0.50
0 1 2 3 4 5 0 1 2 3 4 5
Culture Time (Days)
Fig. 4 Cytotoxicity test on scaffolds fabricated from injection
molding of 3D structured plaster porogen. Normalized Alamar
Blue
TM
(AB) readings over the 4 days cell culture in the medium
with and without scaffolds extract. Metabolic activities measured by
AB assay every 2 days following the 24 h seeding period. The data
were normalized to the AB uorescence reading at a day 0. Y-error
bars represent the standard deviation from the mean for each sample
(n = 6)
Alamar Blue Assay for 7F2 on Scaffolds
2.00
2.50
100% PCL
90% PCL-10% CaP
80% PCL-20% CaP
*
1.50
0.50
1.00
N
o
r
m
a
l
i
z
e
d

A
B
0.00
4 days 0 day
Culture time
Fig. 5 Normalized increase in AB readings over the 4 day in vitro
7F2 cell culture on pure PCL, 90/10 and 80/20 PCL/b-TCP scaffolds
with 400 lm pore size. Metabolic activities were measured by AB
assay after 4 days post-seeding following the 24 h seeding period.
The data were normalized to the AB uorescence reading data day 0.
Y-error bars represent the standard deviation from the mean for each
sample (n = 6)
J Mater Sci: Mater Med (2012) 23:22172226 2221
1 3
4 Discussion
Porogen method uses SFF techniques to fabricate a struc-
tured porogen with complicated external shape and
interconnected internal structure precisely designed by
directly reconstructing CT/MRI images or CAD model [23,
25]. Following the porogen fabrication, the biomaterials
can be injected in and the designed scaffolds can be
Fig. 6 Morphology of 7F2 osteoblasts at 1 day (ac) and 4 days (d
f) of post-seeding on PCL, 90/10 and 80/20 PCL/b-TCP composite
scaffolds: at 1 day post-seeding, cells attached on PCL (a), 90/10
PCL/b-TCP (b) and 80/20 PCL/b-TCP (c) scaffolds; at 4 days post-
seeding, cells proliferated on PCL (d), 90/10 PCL/b-TCP (e) and
80/20 PCL/b-TCP (f) scaffolds. Staining for nuclei is bisbenzimide
(blue) and for actin cytoskeletonphalloidin (red). Original magni-
cation is 950 (Color gure online)
A B C
1mm 1mm 1mm
D
E F
50 m 20 m
20 m
Fig. 7 SEM images of 7F2 grown on the different scaffolds after
4 days post-seeding: osteoblast spread on the surface and grow into
the pores of pure PCL (a, d), 90/10 PCL/b-TCP (b, e) and 80/20 PCL/
b-TCP (c, f) scaffolds. (ac) are in low magnication to show overall
scaffolds and (df) are in high magnication to show the detail of cell
morphology
2222 J Mater Sci: Mater Med (2012) 23:22172226
1 3
obtained after removing the porogen. This method over-
comes several existing manufacturing limitations to
improve bone scaffold fabrication with controllable pore
sizes and precise structure dimension [24].
In this study, the 3DP porogen system was used to
fabricate the desired bone tissue engineering scaffolds.
3DP porogen method uses plaster as the building material,
which can resist higher injection temperature and provide
more possibilities for preparing scaffolds with most of the
biopolymers.
It was reported that the optimal porosity for bone
ingrowth is 200400 lm [26], thus porogens with 200, 300
and 400 lm struts spaced 800 lm apart and overall
dimension of 10.4 9 10.4 9 6.2 mm
3
were designed and
fabricated in order to test their feasibility for bone scaffolds
preparation. The attempt to fabricate a porogen with
200 lm struts and 800 lm voids was failed due to the
resolution limit of the 3D printer. Porogen with 300 lm
struts has better resolution, but the weak mechanical
strength makes it impossible to be injected with biomate-
rials. The porogens with 400 lm strut have good structural
integrity comparing with the other two, and their
mechanical strength is suitable for biomaterial injection.
Therefore, we have mainly focused on the scaffolds prep-
aration based on 400 lm strut porogens.
The SEM images (Fig. 3) show that the porogen with
PGE inltration has more regular structure and pores than
that without any treatment, demonstrating that the PEG
treatment does help to improve the integrity of the plaster
porogen and this procedure is necessary for scaffolds fab-
rication with better quality. The average strut width of the
porogen (605 40.74 lm) is different from the designed
width (400 lm), which is probably due to the binder
migration in the loose powder during the printing. After
biomaterials injection and porogen removing, the 80/20
PCL/b-TCP scaffold shows well interconnected and highly
Fig. 8 Morphology of EAhy 926 cells cultured on PCL, 90/10 and
80/20 PCL/b-TCP composite scaffolds for 24 h (ac) and 48 h (df):
at 24 h post-seeding, cells attached on PCL (a), 90/10 PCL-CaP
(b) and 80/20 PCL/b-TCP (c) scaffolds; at 48 h post-seeding, cells
proliferated on PCL (d), 90/10 PCL/b-TCP (e) and 80/20 PCL/b-TCP
(f) scaffolds. Staining for nuclei is bisbenzimide (blue) and for actin
cytoskeletonphalloidin (red). Original magnication is 950 (Color
gure online)
Table 1 Molecular weight changes of PCL scaffolds during
6 weeks degradation in DMEM with 10 % FBS
Degradation time
(weeks)
Mn
(g/mol)
Molecular weight
(kDa)
Week 2 25347 27.05
Week 4 25347 26.70
Week 6 23339 26.53
Table 2 Mass changes of scaffolds during 6 weeks degradation in
DMEM with 10 % FBS
Sample PCL
(g)
90/10 PCL/b-TCP
(g)
80/20 PCL/b-TCP
(g)
Week 2 0.35 0.03 2.42 0.20 2.82 0.43
Week 4 3.01 0.33 3.99 0.15 4.36 0.35
Week 6 4.05 0.15 4.73 0.15 5.43 0.30
J Mater Sci: Mater Med (2012) 23:22172226 2223
1 3
opened structure with the pore size of 421.5 29.7 lm
(Fig. 3d), which is close to the designed 400 lm. The pore
size of our previously prepared PCL scaffolds via injection
molding of 400 lm thermoplastic (wax) porogens was
396 40 lm [25], which is more precise than the scaf-
folds produced by plaster porogens. The result indicate that
in order to use plaster porogen as a mold for most of the
biomaterials with higher melting point, the printing reso-
lution of the machine and the inltration procedure of the
porogen could be used to improve and regulate the scaf-
folds for different requirements.
During the porogen material removal process, the
reaction between the Cavex plaster remover and the plaster
porogen might bring some unknown reactant into the
scaffolds, which may further inuence the application of
produced scaffolds in tissue engineering. In order to assess
the feasibility of the 3DP system for biomedical applica-
tions, indirect cytotoxicity test was performed in the extract
of the scaffolds prepared by porogen method. The results
suggest that the porogen removal procedure does not
introduce any cytotoxicity to the scaffolds.
In addition to cytotoxicity test, the biocompatibility of
the scaffolds was assessed. In this study, 7F2 osteoblasts
were seeded on PCL, 90/10, and 80/20 PCL/b-TCP scaf-
folds. Since the initial contact of osteoblasts with implant
surfaces is very important for osseointegration of implants
[27, 28], the scaffolds were incubated with collagen type I
to improve the initial adhesion before cell seeding. The cell
metabolic activity/cell proliferation was measured by AB
assay. The result indicates that introducing 20 % wt of
b-TCP into PCL matrix can enhance the cell proliferation
dramatically. The uorescent staining images (Fig. 6)
exhibit that the initial seeding of osteoblasts was improved
with increasing the b-TCP concentration in the scaffolds.
After 4 days culturing, the scaffolds with b-TCP present
enhanced cell growth compared with neat PCL scaffolds.
SEM images conrm these ndings, that after 4 days post-
seeding, cells have better proliferation on the scaffolds with
b-TCP than that on neat PCL scaffolds. The uorescent
staining images of the EAhy926 endothelial cells in addi-
tion conrmed that the introducing of b-TCP into PCL
matrix can enhance the cell viability and functional activity
(Fig. 8). The biocompatibility testing results indicate that
the interconnected porous structure produced via 3DP
structured porogens could benet cell growth and be used
as temporary bone tissue regenerating support.
It was reported that the degradation of PCL was rst
caused by the autocatalysis of non-enzymatic random
hydrolytic chain scission in ester linkages, then the
mechanical strength and weight of PCL lost gradually with
the surface area increases facilitating greater bioerosion
[29] to produce the nal degradation products [30]. For a
bone scaffold fabricated by biodegradable materials, it is
necessary to control the degradation at a rate that will
slowly transfer load to the healing bone. Since completely
absorbing PCL takes a few years in vivo [31], an implant
fabricated from these materials may support the bone for
the entire duration of normal healing. In vitro experiments
can simulate the degradation of degradable implants within
the body. In this work, the degradation tests were per-
formed for up to 6 weeks to assess the degradation trends
of designed scaffolds after adding b-TCP. The samples
containing higher b-TCP concentration show higher weight
loss, which is consistent with other reports [32]. Through
varying the concentration of b-TCP in PCL, the degrada-
tion rate can be regulated.
The compression strength as a function of in vitro
degradation shows increased compressive modulus (CM)
of neat PCL samples during the initial stages (2 weeks) of
the degradation study. The CM of neat PCL was then
slowly decreased with degradation until 6 weeks. A similar
prole has been observed in degradation tests of
poly(propylene fumarate) (PPF)-TCP systems [33, 34].
Looney and Park [35] also demonstrated that during in
vitro degradation experiment PMMA bone cements show
an increase in mechanical properties in the initial stage. It
is know that the degradation of biopolymer is initiated in
the amorphous area, since the crystallized area is more
Comparison of CM after 6 weeks of degradation test
(in DMEM with 10% FBS)
700
Comparison of UCS after 6 weeks of degradation test
(in DMEM with 10% FBS)
30
A B
300
400
500
600
Week 0 Week 2 Week 4 Week 6
15
20
25
Week 0 Week 2 Week 4 Week 6
0
100
200
PCL 90% PCL 80% PCL
C
M

(
M
P
a
)
0
5
10
PCL 90% PCL 80% PCL
U
C
S

(
M
P
a
)
Fig. 9 Compressive modulus (CM) and ultimate compressive strength (UCS) comparison of PLC, 90/10 and 80/20 PCL/b-TCP scaffolds after
6 weeks degradation test in DMEM with 10 % FBS
2224 J Mater Sci: Mater Med (2012) 23:22172226
1 3
difcult to be attacked by the surrounding medium. The
increased mechanical properties of PCL could be due to the
increased crystallized area in the biopolymer. However, the
exact mechanism of this phenomenon will need further
studies.
The initial compressive strength of 80/20 PCL/b-TCP
rods is around 26 MPa, which is similar to the reported
24.8 1.3 MPa [25]. The UCS of 80/20 PCL/b-TCP
scaffolds dropped rapidly to around 10 MPa after 6 weeks
degradation, which still closely matches to that of human
cancellous bone (0.2210.44 MPa) [36]. Since the normal
time line for bone healing is around 612 weeks, our
results show a possibility to use 80/20 PCL/b-TCP com-
posites as bone tissue engineering scaffolds.
The mechanical test after degradation may provide a
chance to predict the inuence of the mechanical properties
of scaffolds with different porosities and structures after
degradation. Besides, the objective of the current study was
to monitor the in vitro degradation prole of PCL/b-TCP
composites when immersed in standard culture medium.
Thus the results obtained here would be clinically relevant
and used as a reference for further in vivo study.
5 Conclusion
Interconnected PCL or PCL/b-TCP bone scaffolds with
different pore sizes were fabricated by injection molding of
the biomaterials into 3DP-built porogens followed by
porogen removal. Due to the resolution limitation of 3DP
system, porogens designed for scaffolds with less than
400 lm pores is very difcult, which suggests improving
the resolution of 3DP system is necessary in order to
have ner scaffolds structure. Inltration by PEG shows
enhanced integrity of the plaster porogens. This technique
can overcome several existing manufacturing limitations to
fabricate bone scaffolds with pre-determined structure by
combining the advantages of SFF method, structured
porogen design, and injection molding of bioactive com-
posite materials. The porogens use plaster as building
material, which can support the injection molding of most
biomaterials with wide range of melting temperature. With
incorporation of bioactive b-TCP into the PCL matrix, the
bioactivity and degradability of the scaffolds can be
improved signicantly, while maintaining compressive
strength similar to human cancellous bone. With more
material choices enabled by plaster porogens and good
biocompatibility, 3DP printed porogen based technique
promises improved exibility to make more kinds of
scaffolds with sufcient mechanical integrity.
Acknowledgments We gratefully thank National Science Founda-
tion (NSF) for its nancial support (DMI0300405, CMMI-0700139
and CMMI-0925348). Additionally, the authors are grateful to Dr.
Wei Sun for providing access to 3D printer for this study. We also
would like to thank the laboratory of Dr. Giuseppe Palmese for
assistance with GPC degradation tests and the laboratory of Dr. Boris
Polyak for providing the access to plate reader. The Centralized
Research Facility (CRF) of the College of Engineering, Drexel Uni-
versity provided access to electron microscopes used in this work.
References
1. Williams D. Benet and risk in tissue engineering. Mater Today.
2004;7:249.
2. Kneser U, Schaefer DJ, Polykandriotis E, Horch RE. Tissue
engineering of bone: the reconstructive surgeons point of view.
J Cell Mol Med. 2006;10:719.
3. Burg KJL, Porter S, Kellam JF. Biomaterial developments for
bone tissue engineering. Biomaterials. 2000;21:234759.
4. Thomson RC, Mikos AG, Beahm E, Lemon JC, Sattereld WC,
Aufdemorte TB, et al. Guided tissue fabrication from periosteum
using preformed biodegradable polymer scaffolds. Biomaterials.
1999;20:200718.
5. Ma PX. Biomimetic materials for tissue engineering. Adv Drug
Deliv Rev. 2008;60:18498.
6. Zhang QW, Mochalin VN, Neitzel I, Knoke IY, Han JJ, Klug CA,
et al. Fluorescent PLLA-nanodiamond composites for bone tissue
engineering. Biomaterials. 2011;32:8794.
7. Neumann M, Epple M. Composites of calcium phosphate and
polymers as bone substitution materials. Eur J Trauma. 2006;
32:12531.
8. Shin M, Yoshimoto H, Vacanti JP. In vivo bone tissue engi-
neering using mesenchymal stem cells on a novel electrospun
nanobrous scaffold. Tissue Eng. 2004;10:3341.
9. Rohner D, Hutmacher DW, Cheng TK, Oberholzer M, Hammer
B. In vivo efcacy of bone-marrow-coated polycaprolactone
scaffolds for the reconstruction of orbital defects in the pig.
J Biomed Mater Res B Appl Biomater. 2003;66B:57480.
10. Ruhe PQ, Hedberg EL, Padron NT, Spauwen PHM, Jansen JA,
Mikos AG. rhBMP-2 release from injectable poly(DL-lactic-co-
glycolic acid)/calcium-phosphate cement composites. J Bone
Joint Surg Am. 2003;85A:7581.
11. Xu HHK, Quinn JB, Takagi S, Chow LC. Synergistic rein-
forcement of in situ hardening calcium phosphate composite
scaffold for bone tissue engineering. Biomaterials. 2004;25:
102937.
12. Barralet JE, Grover L, Gaunt T, Wright AJ, Gibson IR. Prepa-
ration of macroporous calcium phosphate cement tissue engi-
neering scaffold. Biomaterials. 2002;23:306372.
13. Yang Q, Chen L, Shen X, Tan Z. Preparation of polycaprolactone
tissue engineering scaffolds by improved solvent casting/partic-
ulate leaching method. J Macromol Sci Part B Phys. 2006;45:
117181.
14. Heijkants R, Van Tienen TG, De Groot JH, Pennings AJ, Buma
P, Veth RPH, et al. Preparation of a polyurethane scaffold for
tissue engineering made by a combination of salt leaching and
freeze-drying of dioxane. J Mater Sci. 2006;41:24238.
15. Mikos (AGH, TX), Sarakinos, Georgios (Boston, MA), Vacanti,
Joseph P. (Winchester, MA), Langer, Robert S. (Newton, MA),
Cima, Linda G. (Lexington, MA). Biocompatible polymer
membranes and methods of preparation of three-dimensional
membrane structures. United States: Massachusetts Institute of
Technology (Cambridge, MA), Childrens Medical Center Cor-
poration (Boston, MA); 1996.
16. Fukuhira Y, Kitazono E, Hayashi T, Kaneko H, Tanaka M,
Shimomura M, et al. Biodegradable honeycomb-patterned
J Mater Sci: Mater Med (2012) 23:22172226 2225
1 3
lm composed of poly(lactic acid) and dioleoylpho-
sphatidylethanolamine. Biomaterials. 2006;27:1797802.
17. Cima LG, Vacanti JP, Vacanti C, Ingber D, Mooney D, Langer R.
Tissue engineering by cell transplantation using degradable
polymer substrates. J Biomech Eng Trans ASME. 1991;113:
14351.
18. Zhang W, Yao D, Zhang Q, Zhou JG, Lelkes PI. Fabrication of
interconnected microporous biomaterials with high hydroxyapa-
tite nanoparticle loading. Biofabrication. 2010;2.
19. Manjubala I, Woesz A, Pilz C, Rumpler M, Fratzl-Zelman N,
Roschger P, et al. Biomimetic mineral-organic composite scaf-
folds with controlled internal architecture. J Mater Sci Mater
Med. 2005;16:11119.
20. Hollister SJ, Maddox RD, Taboas JM. Optimal design and fab-
rication of scaffolds to mimic tissue properties and satisfy bio-
logical constraints. Biomaterials. 2002;23:4095103.
21. Roy TD, Simon JL, Ricci JL, Rekow ED, Thompson VP, Parsons
JR. Performance of degradable composite bone repair products
made via three-dimensional fabrication techniques. J Biomed
Mater Res A. 2003;66A:28391.
22. Taboas JM, Maddox RD, Krebsbach PH, Hollister SJ. Indirect
solid free form fabrication of local and global porous, biomimetic
and composite 3D polymerceramic scaffolds. Biomaterials.
2003;24:18194.
23. Lu L, Zhang Q, Wootton D, Lelkes PI, Zhou J. A novel sucrose
porogen-based solid freeform fabrication system for bone scaf-
fold manufacturing. Rapid Prototyping J. 2010;16:36576.
24. Mooney DJ, Baldwin DF, Suh NP, Vacanti LP, Langer R. Novel
approach to fabricate porous sponges of poly(D,L-lactic-co-gly-
colic acid) without the use of organic solvents. Biomaterials.
1996;17:141722.
25. Mondrinos MJ, Dembzynski R, Lu L, Byrapogu VKC, Wootton
DM, Lelkes PI, et al. Porogen-based solid freeform fabrication of
polycaprolactone-calcium phosphate scaffolds for tissue engi-
neering. Biomaterials. 2006;27:4399408.
26. Boyan BD, Hummert TW, Dean DD, Schwartz Z. Role of
material surfaces in regulating bone and cartilage cell response.
Biomaterials. 1996;17:13746.
27. Geissler U, Hempel U, Wolf C, Scharnweber D, Worch H,
Wenzel KW. Collagen type I-coating of Ti6Al4V promotes
adhesion of osteoblasts. J Biomed Mater Res. 2000;51:75260.
28. Andrianarivo AG, Robinson JA, Mann KG, Tracy RP. Growth on
type-I collagen promotes expression of the osteoblastic pheno-
type in human osteosarcoma MG-63 cells. J Cell Physiol.
1992;153:25665.
29. Schantz JT, Hutmacher DW, Ng KW, Khor HL, Lim TC, Teoh
SH. Evaluation of a tissue-engineered membrane-cell construct
for guided bone regeneration. Int J Oral Maxillofac Implants.
2002;17:16174.
30. Woodward SC, Brewer PS, Moatamed F, Schindler A, Pitt CG.
The Intracellular degradation of poly(epsilon-caprolactone).
J Biomed Mater Res. 1985;19:43744.
31. Ali SAM, Zhong SP, Doherty PJ, Williams DF. Mechanisms of
polymer degradation in implantable devices. 1. Poly(caprolac-
tone). Biomaterials. 1993;14:64856.
32. Kim HW, Knowles JC, Kim HE. Development of hydroxyapatite
bone scaffold for controlled drug release via poly(epsilon-cap-
rolactone) and hydroxyapatite hybrid coatings. J Biomed Mater
Res B Appl Biomater. 2004;70B:2409.
33. Timmer MD, Ambrose CG, Mikos AG. In vitro degradation of
polymeric networks of poly(propylene fumarate) and the cross-
linking macromer poly(propylene fumarate)-diacrylate. Bioma-
terials. 2003;24:5717.
34. Peter SJ, Nolley JA, Widmer MS, Merwin JE, Yaszemski MJ,
Yasko AW, et al. In vitro degradation of a poly(propylene
fumarate)/beta-tricalcium phosphate composite orthopaedic
scaffold. Tissue Eng. 1997;3:20715.
35. Looney MA, Park JB. Molecular and mechanical property
changes during aging of bone cement in vitro and in vivo.
J Biomed Mater Res. 1986;20:55563.
36. Misch CE, Qu ZM, Bidez MW. Mechanical properties of tra-
becular bone in the human mandible: implications for dental
implant treatment planning and. Surgical placement. J Oral
Maxillofac Surg. 1999;57:7006.
2226 J Mater Sci: Mater Med (2012) 23:22172226
1 3