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Artificial Organs
35(1):49–57, Wiley Periodicals, Inc.
© 2010, Copyright the Authors
Artificial Organs © 2010, International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.
Repair of Bone Defect in Caprine Tibia Using a Laminated
Scaffold With Bone Marrow Stromal Cells Loaded Poly
(L-Lactic Acid)/b-Tricalcium Phosphate
Jianyan Huang, Lingmin Zhang, Bin Chu, Xiaohui Peng, and Shunqing Tang
Biomedical Engineering Institute of Jinan University, Guangzhou, China
Abstract: Repair of bone defects of a critical size encoun-
ters many problems, and many efforts aim to build a porous
scaffold loading bone marrow stromal cells (BMSCs) or
bone morphogenetic protein (BMP2) to quickly repair
bone defects. In this paper, a laminated scaffold was
designed and tested for the repair of bone defects in a
caprine tibia. Beta-tricalcium phosphate (b-TCP) and poly
(L-lactic acid) (PLLA) were fabricated to a sandwich struc-
tured composite that was then rolled up to form a cylindri-
cal shaped, porous scaffold. The porosity and bending
strength of the PLLA/b-TCP laminated scaffold were
around 70% and 1.7 MPa, respectively. Results from in
vitro experiments showed that the pH value of the scaffold
in water fluctuated between 4.9 and 7.0 during its
degradation. When exposed to the simulated body fluid, the
The repair process of a bone defect, in particular
with a critical defect (excessive 2 cm gap), is very
slow. In general, over 12 months of recovery are
needed, even though a treatment was applied using a
scaffold with porous degradable bioceramic (1) or
bone cement (2), degradable polymer (3), or their
composite (4). With convenient processing and
shorter degradation time, degradable polymers and
their composites have been developed as scaffolds
for quick bone repair (less than 12 months).
However, the degradation of commonly used poly-
mers makes the mechanical strength of the scaffold
decrease quickly. When the degradation rate is faster
than the formation rate of the new bones, collapse of
the implanted scaffold often takes place during the
doi:10.1111/j.1525-1594.2010.01042.x
Received November 2009; revised January 2010.
Address correspondence and reprint requests to Dr. Shunqing
Tang, Biomedical Engineering Institute of Jinan University,
Guangzhou 510632, China. E-mail: tshunqt@jnu.edu.cn
49
aor_1042 49..57
scaffold lost its strength after 11 weeks of degradation.
After implantation in Chinese caprines’ diaphyseal defects
with loaded allogeneic BMSCs, the scaffold sped up the
bone repair without collapse of the scaffold and the
unwanted inflammatory response, and then rapidly
degraded and finally disappeared at 12 weeks. Gross exami-
nations and pullout tests showed that the experimental
caprines walked normally and the implanted leg could be
heavily loaded. X-ray examinations and histological analy-
ses showed new bone tissues formed with similar structures
to normal ones. It is suggested that the novel PLLA/b-TCP
laminated scaffold with BMSCs loading can regenerate
new bones quickly. Key Words: Poly (L-lactic acid)—b-
Tricalcium phosphate—Bone defect and repair—Bone
marrow stromal cells.
bone repair. In this case, the extracellular matrix
(ECM) expressed quickly by the seeded cells or rapid
osteogenesis in the scaffold is needed to fix or sustain
the debris of the broken scaffold. Therefore, it is
important to find out a way to achieve quick osteo-
genesis in the implant before its collapse. Studies on
the protocol of rapid bone repair will be of significant
benefit to patients in shortening therapy time. Also, it
will be helpful to find a path to achieve in vitro bone
construction that has not yet succeeded (5). Concepts
of tissue engineering (6) and regenerative medicine
(7) have contributed to finding new methods to
achieve rapid repair of bone defects. Unfortunately,
up to now, bone tissue cannot be constructed by in
vitro culture, so it may be that the only way to regen-
erate bone in situ quickly is by some regenerative
medicine process (5). To quickly repair bone defects
of a critical volume is still a challenge for researchers
who focus on bone tissue engineering and bone
regenerative medicine. As usual, the difficulty is to
balance the degradation rate of scaffolds and the

50 J. HUANG ET AL.
speed of osteogenesis before the collapse of the scaf-
fold in a quick repair process. By introducing seeded
cells (BMSCs) or growth factors (bone morphologi-
cal protein type 2 [BMP2], etc.) into scaffolds for
accelerating syntheses of ECMs and osteogenesis, the
bone repair process will be significantly speeded up
(8). A strategy for quick bone repair therefore is to
implant degradable scaffolds with loading the seeded
cells (in general BMSC), growth factors (BMP2), or
both of them into the defect.
To achieve rapid bone regeneration, it is important
to construct a novel scaffold which has high porosity
and mechanical strength for sustaining cells and their
ECMs at the initial stage of the repair and a degrada-
tion rate matching new bone formation during
the repair process. Some biomaterials including poly
(lactic acid) (PLA) (9), collagen (or gelatin) (10),
hydroxyapatite (HA) (11), b-tricalcium phosphate
(b-TCP) (12) and calcium phosphate bone cements
(13), etc., have been used as bone regeneration scaf-
folds, but they have some obvious shortages. For
example, firstly, the pH value of the solution is very
low during the degradation of single PLA scaffolds,
which provokes an inflammatory response to the sur-
rounding tissues in the implanted region. Second, it is
difficult for HA or b-TCP to be processed in a desired
shape or a large volume with pores because of their
brittleness, although they have good osteoinduction,
and are intensively investigated for imitating natural
bone tissue. And thirdly, the bulk scaffold of HA or
b-TCP with a critical volume slowly degrades and
often persists over 12 months or more. Compared
with a slow osteogenesis process of the bone tissue in
the lesion site, collagen, gelatin, or their cross-linked
matrix degrades more quickly than the new bone
formation, which might lead to the collapse of the
scaffold before the achievement of the bone repair.
However, currently no significant facts show that any
new material except these kinds of biomaterials or
similar are more suitable for bone regenerative scaf-
fold.A wise way is to combine these materials as bone
regenerative scaffolds with some new approaches.
As is usual, a composite scaffold made from inor-
ganic components and polymers is processed by
simply mixing two or more components and fabricat-
ing with freeze-drying, hot-pressing, or bio-mimetic
coprecipitation method, etc. Although every compo-
nent homogenously disperses in the composite by
these processes, the mechanical properties of the
composite scaffold commonly depend on the degra-
dation rate of a polymer that acts as an adhesive
agent for inorganic components in the composite. For
common bone regeneration, a slowly degradable
polymeric component should be preferred, but it
Artif Organs, Vol. 35, No. 1, 2011
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leads to slow bone repair. As an alterative, if a slowly
degradable inorganic component is constructed in a
suitable shape instead of a particle form, the compos-
ite scaffold will sustain very well, even though the
polymeric component quickly degrades.
As we have known, natural bone tissue is
assembled by collagen layers and HA slices in a mul-
tilayer laminated way (14), and the composite has a
suitable elasticity and a high modulus. To imitate the
structure of natural bone, the inorganic component
with a relatively slow degradation rate in the scaffold
was fabricated into a slice, a strip, or a relatively large
bulk for independently sustaining its strength, and
the scaffold will keep its shape instead of collapse due
to the degradation and rapid loss of adhesive prop-
erty of the polymeric component.
The objective of the present study is to prepare a
sandwich-structured, PLLA/b-TCP/PLLA composite
scaffold with a critical size, and to evaluate the effec-
tiveness of the designed composite scaffold loading
BMSCs in promoting rapid regeneration of tibia
bone in the Chinese caprine.
MATERIALS AND METHODS
Preparation of scaffolds
Preparation of b-TCP slice or strip
Polystyrene resin, as porogensis, was milled and
sieved with a 500-mm sieve. b-TCP powders were
mixed with the milled polystyrene resin particles in a
6:1 weight ratio, along with 5% polyvinyl alcohol
aqueous solution as an adhesive agent. The mixture
was then pressured in a thin steel model (about 2 mm
depth) with a size of 30 ¥ 10 ¥ 2 mm, dried at 300°C
and sintered at temperatures from 1050 to 1150°C for
3 h, then cooled, and washed with distilled water. For
making the cylinder-shaped scaffolds, the strip of
b-TCP with a size of 30 ¥ 3 ¥ 2 mm was used.
Preparation of PLLA slices
PLLA (Mw = 4 ¥ 104, from Polymer Institute,
Zhongshan University, China) was dissolved in
dichloromethane, and mixed with a certain volume of
NaCl particles (sieved with a 500 mm sieve), then the
mixture was placed in a 2-mm deep dish. After vola-
tilization of dichloromethane, a PLLA slice was
dipped into distilled water multiple times to leach out
NaCl particles. The porosity of the PLLA slice was
controlled by the weight ratio of NaCl and PLLA
(from 4:1 to 10:1).
Multiple solvent-dipped b-TCP strips with a thick-
ness of 2 mm were adhered with two PLLA slices.The
strips were arranged in a parallel manner. The sand-

REPAIR OF BONE DEFECT IN CAPRINE TIBIA
FIG. 1. Schematics of fabrication process for the sandwich-
structured, cylinder-shaped scaffold. Step 1: preparing slices or
strips; Step 2: adhering slices together; Step 3: rolling into hollow
cylinder-shaped scaffold and sticking the interface with solvent.
wich structured composite was rolled up to form a
cylinder-shaped scaffold as was the need at the
repaired lesion site. Figure 1 shows the scheme of the
process (the interface of cylinder wall was adhered by
dichloromethane dissolving process). The cylinder-
shaped scaffold has a length of 3 cm,an outer diameter
of 1.2 cm,and inner diameter of 0.4 cm.The dimension
just matched the size of caprine’s tibia defect.
Morphological evaluation of scaffold
Digital images of the scaffolds were captured by an
Olympus SP-500UZ digital camera (Olympus Corpo-
ration, Japan). The morphology of the cross-section
of the scaffold, PLLA slice, or b-TCP strip was
observed under a JSM-T300 scanning electron
microscopy (JEOL Ltd., Tokyo, Japan) after the
samples were sputter-coated with gold.
Porosity of scaffold
A gravity bottle was filled with absolute alcohol
and weighed (W0) at first. The scaffold (n = 5) sample
with a certain weight (WS) was put into the bottle,
which was then transferred to a three-neck bottle.
The sample was degassed under vacuum, alcohol was
added to fill up the gravity bottle with the sample
through one of three necks, and the gravity bottle
with the sample and filled alcohol was taken out and
weighed (W1) again.After that, the scaffold was taken
out of the gravity bottle and we weighed the bottle
with the remaining alcohol (W2). The porosity of the
scaffold (e) was calculated as follows,
ε = ( W1 − W2 − WS ) (W0 − W2 )
Properties of scaffolds during in vitro degradation
Samples including PLLA/b-TCP laminated scaffold,
two PLLA slices, and one b-TCP slice with different
porosity (n = 5) were dried at 80°C in a vacuum for
6 h, and cooled down in a desiccator and weighed
(w0), then put into a simulated body fluid (SBF) for a
certain time, and taken out and washed with distilled
water, dried at the same conditions as mentioned
earlier, and weighed (w1), the weight loss of the scaf-
fold (wl) was calculated as follows
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51
W1 = ( w1 − w0 ) w0
In different intervals, 1 mL SBF was taken out, and
diluted to 50 mL. Calcium (Ca) ion concentration in
the diluted SBF was determined by a Hitachi 180-80
atomic emission spectroscopy (Hitachi Co. Ltd.,
Tokyo, Japan).
The PLLA/b-TCP laminated scaffold, PLLA slice,
and b-TCP slice with different porosity (n = 5) were
put into three 50-mL beakers with 30 mL distilled
water, respectively. The pH value of the distilled
water was recorded by a PHS-II acidometer
(TAYASAF Sci & Tech Co. Ltd., Beijing, China) in a
certain interval during degradation.
For bending strength tests, all samples (n = 5) were
prepared with a same size of 3 cm (L) ¥ 1 cm
(W) ¥ 6 mm (D). The samples were dipped into
40 mL SBF. After 4, 8, 12, and 16 weeks, they were
taken out, washed and dried, and then their bending
strength was recorded by an AG-1 versatile testing
machine (Shimadzu Autograph, Shimadzu Co.,
Kyoto, Japan).
Repair of caprine tibia bone defects
A 12-month-old caprine was anesthetized and
10 mL of bone marrow was taken out by a syringe
moistened with a heparinized saline solution (1:1000
heparin in 100 mL of 0.9% sodium chloride) after a
16-G bone marrow needle was inserted into the iliac
bone. The bone marrow was mixed with Dulbecco’s
modified Eagle’s medium (DMEM) and 15 mg/mL of
heparin sodium to prevent clotting, and centrifuged
at 1500 rpm for 15 min. The upper solution was
removed and the rest was sieved through a 150-mm
sieve. The harvested allogeneic BMSCs were
expanded in monolayer through three subcultures
(passage 3 [P3] BMSCs). The medium for the mono-
layer expansion was DMEM solution containing
10% fetal bovine serum, 50 mg/mL ascorbic acid
2-phosphate, 10-8 M dexamethasone, and 10-8 M
b-glycerophosphate sodium.
The P3 BMSCs were resuspended at a concentra-
tion of 12 million cells per 1 mL culture medium, 7
million cells were pipetted onto the outer surface of
the cylinder, and 5 million cells onto the inner
surface, then cultured in vitro for 3 days. The prolif-
eration of the BMSCs was assayed with 3-(4,5-
dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium
method. The left leg of a Chinese caprine (total 20
caprines) was fixed by an AO steel plate, and a 3-cm-
length defect of the tibia diaphyseal was made, then
the PLLA/b-TCP laminated scaffold loaded cells
were implanted into the defect (n = 3). At 1 week
after surgery, 8 ¥ 105 U penicillin per caprine was
Artif Organs, Vol. 35, No. 1, 2011

52 J. HUANG ET AL.
injected every day, 2 weeks later sutures were
removed and caprines were allowed to roam free.
After 4, 8, and 12 weeks respectively, the caprines
were sacrificed, and the defects were examined by
X-ray and pullout tests, then dissected, and fixed with
10% formaldehyde, dipped into dilute HCl solution
for dissolving the inorganic component of bone, then
embedded into paraffin and microtomed, and stained
with hematoxylin and eosin (H&E) for histological
analyses. In one group, the steel plate was taken out
at the 12th week, and sacrificed after 24 months, and
gross examinations were carried out. Anatomic
analysis was done to check bone marrow formation
after sacrifice. In a control group, the defects were
placed with nothing (n = 3) or with PLLA/b-TCP
laminated scaffolds without BSMCs (n = 5), and their
histological analyses and X-ray examinations were
run in the same way mentioned above.
All animal experiments were approved by Ani-
mal Care Committee of Medical College, Jinan
University.
Statistical analysis
All results were statistically evaluated by analysis
of variance and post hoc testing with Bonferroni’s
correction on SPSS statistical software (SPSS, Inc.,
Chicago, IL, USA). Significance was set at the 5%
level (P < 0.05).
RESULTS AND DISCUSSION
Structure and mechanical property of scaffolds
Because b-TCP ceramic is brittle, it was difficult for
b-TCP powders to be sintered into a large bulk
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FIG. 2. Morphologies of the PLLA/b-TCP
scaffold. (A) Photograph of the cylinder-
shaped scaffold, (B–D) cross-sectional
images intersections of composite scaf-
fold, b-TCP strip, PLLA slice, respectively.
Arrows indicate good integration of PLLA
and b-TCP at the interface.
ceramic with a high porosity. b-TCP strips with a
porosity of 30–40% could be easily obtained in the
lab. As the porosity was larger than 50%, b-TCP
strips were too weak to be processed. PLLA slices
have a high porosity, which could reach to 90% when
the weight ratio of PLLA/NaCl particle was 10:1, but
was just 76% when the ratio was 4:1. In this study, the
prepared PLLA/b-TCP laminated scaffold for
implantation had a porosity of 35% for the b-TCP
strips and 90% for the PLLA slices. The fabrication
process of the scaffold is shown in Fig. 1. From step 1
to step 3, two PLLA slices and one b-TCP strip were
fabricated in a sandwich-structured, cylinder-shaped
scaffold. The images and morphologies of the scaf-
folds are shown in Fig. 2. The prepared scaffold could
be a different size (Fig. 2A). The b-TCP strip in the
middle layer tightly attached to the PLLA slices (Fig.
2B). No large gaps were found at the interface
between the two components, and they were firmly
adhered. It was also shown that the breakage of the
tested scaffold occurred in phase of the b-TCP strip
instead of at their interface. Results of quantitative
analyses showed that the average porosity of the
PLLA/b-TCP laminated scaffold was about 70%, and
the average diameters of the pores in the scaffold
ranged from 200 mm to 500 mm (Fig. 2C,D).The pores
in the PLLA slices were kept very well. But in the
b-TCP strip (Fig. 2C), there were many globular
pores accompanying smaller ones.
The bending strength is listed Table 1. The PLLA/
b-TCP laminated scaffold showed 1.9 MPa of
bending strength which is much higher than that of
single PLLA scaffold. It indicates that b-TCP strips
could reinforce the single PLLA scaffold. The

REPAIR OF BONE DEFECT IN CAPRINE TIBIA
TABLE 1. Bending strength of the scaffolds with degradation time (MPa)*
Scaffold 0 4
PLLA 1.24 ⫾ 0.03 0.94 ⫾ 0.03
Composite PLLA/b-TCP 1.69 ⫾ 0.04 1.32 ⫾ 0.03
* Expressed as mean ⫾ standard deviation, n = 5.
bending strength of the PLLA/b-TCP laminated scaf-
fold decreased slowly during the in vitro 12-week
degradation. On the contrary, a single PLLA scaffold
almost lost its entire strength at the same time. This
means that b-TCP could sustain the scaffold more
effectively and longer during degradation. Further-
more, with the help of thermal plastic PLLA, the
PLLA/b-TCP laminated scaffold can be easily fabri-
cated to the desired shapes than the single b-TCP. For
example, the PLLA/b-TCP laminated scaffolds could
be conveniently cut into small pieces. These facts
demonstrated that the b-TCP strip in the laminated
composite could reinforce PLLA and retard the
decline of its mechanical strength due to degradation
of PLLA. PLLA synergistically promotes b-TCP
easily to be processed into the desired shape.
Degradation of scaffold
A single PLLA slice quickly degraded in an acidic
environment because of its accelerated degradation
effect by acid catalyst, but in the PLLA/b-TCP lami-
nated scaffold, that acid was partly neutralized
by b-TCP reducing the acid catalysis effect. This sup-
ports the fact that the PLLA/b-TCP laminated scaf-
fold is sustained for a longer time during degradation.
FIG. 3. Weight loss of the scaffolds during in vitro degradation.
Scaffolds were prepared with a porosity of (A) 66%, (B) 74%, (C)
80%, (D) 89% in PLLA, (E) 34% in b-TCP.
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53
Degradation time (weeks)
8 12 16
0.32 ⫾ 0.01 0.09 ⫾ 0.01 —
0.91 ⫾ 0.02 0.58 ⫾ 0.02 —
Results of the in vitro degradation experiments
(Fig. 3) showed that a small amount of weight loss in
the PLLA/b-TCP laminated scaffold was observed at
3 weeks. The weight loss reached 10% in the 11th
week. The higher the porosity, the larger the weight
loss of the scaffold. As compared with the PLLA/b-
TCP laminated scaffold, a single PLLA slice with
high porosity has a larger weight loss; but all of
b-TCP had a relatively smaller weight loss at 17
weeks. It indicates that a high porosity increased the
weight loss of the scaffold containing PLLA. It was
also found that the weight loss of the scaffold was
smaller than that of a single PLLA slice with the
same porosity.This might be due to a low degradation
rate of b-TCP strip hindering degradation of PLLA
in the PLLA/b-TCP laminated scaffold. After the
11th week, the degradation rate of the scaffold sped
up. This might be related to the acidic monomers
from the degraded PLLA accelerating the dissolu-
tion of b-TCP. It was mentioned that the strength of
the single PLLA slice was too weak to be measured
after 13 weeks of degradation.
Figure 4 shows the pH value of single b-TCP strip
around 7.0 with degradation. The pH of PLLA/b-
TCP laminated scaffold changed from 4.9 to 6.5, but a
FIG. 4. pH of the scaffolds over time during in vitro degradation.
(A) scaffolds with a porosity of 66%; (B) scaffolds with a porosity
of 74%; (C) PLLA slices with a porosity of 80%; (D) PLLA slices
with a porosity of 89%; (E)b-TCP with a porosity of 34%.
Artif Organs, Vol. 35, No. 1, 2011

54 J. HUANG ET AL.
single PLLA slice fluctuated around 2.9.This suggests
that the b-TCP strip in the PLLA/b-TCP laminated
scaffold could neutralize the acidity of PLLA slices
during their degradation. This did well in restraining
the inflammatory response caused by high acidity of
PLLA monomer. At about week 11, the pH value of
the scaffold was the lowest, and reached 4.9. This
means that a lot of the PLLA monomers appeared
and the PLLA in the scaffold would break into small
pieces. In fact, after 15 weeks of degradation, most of
the PLLA in the scaffold broke into pieces and only
a small amount was left, and after 17 weeks PLLA
almost disappeared, but the b-TCP strip still kept its
shape. This indicates that b-TCP could sustain the
composite scaffold although most of PLLA slices
degraded.The concentration of calcium ion increased
with degradation time, and reached the maximum
value at about 80 ppm, at about week 11. Coinci-
dently, at the same time, the pH value of the PLLA/
b-TCP laminated scaffold reached a maximum. This
might be explained that in acidic medium, b-TCP
might react with acidic prepolymers or monomers
from the degraded PLLA, which leads to its quick
degradation or dissolution.
X-ray examination and histological analysis
of implantation
Gross examinations showed that no inflammatory
response was observed in these experimental
caprines, and the implanted caprines walked regu-
larly after the fixed plates were taken out. In the case
of the control group, however, the caprines receiving
the laminated scaffold without BMSCs were limited,
and it was found that a significant inflammatory
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FIG. 5. X-ray images of bone defects post
implantation of BMSCs loaded PLLA/b-
TCP scaffolds in caprine tibia. (A–C) with
PLLA/b-TCP scaffolds in week 4, 8, 12,
respectively; (D) control defect without the
scaffold in week 12.
response took place at the lesion site. The caprines
receiving nothing were lame and the defect was
partly filled with some connective tissues instead of
bone.
The X-ray examination (Fig. 5) results show that in
the 4th week, a clear gap was found between the
implanted and autologous tissue. This indicates that
no osteogenesis took place at the early stage of the
repair process, but there were some connective
tissues to wrap the defect by anatomic analysis. The
gap between the implanted scaffold and tibia bone
disappeared after an 8-week implantation. There
were effective bone bridge connections between the
PLLA/b-TCP laminated scaffold and autologous
bone, and some newly formed bone tissues substi-
tuted for the scaffold. However, the heavy X-ray
image for solid bone showed that a lot of b-TCP still
remained. In the 12th week, the density of bone in the
lesion site looks like that from autologous bone. This
suggests that the repair process is totally completed.
As a contrast, for the control group, the 3-cm defect
remained after 12-weeks.
Pullout tests also showed that the gap between the
defect and autologous bone was weakly connected
by connective tissues after 4-weeks and the repaired
defect was easily broken again. After 12-weeks there
was apparently bone connection, and it was not easy
to pull out the defect with certain force.
Anatomic analyses and histological assays as
shown in Fig. 6A also demonstrated that in the 4th
week, there was a thin connective tissue wrapping the
implant, and some cartilage-like or few bone tissues
(woven bone or trabeculae of bone) were dispersed
in the scaffolds and arranged in bone tube direction.

REPAIR OF BONE DEFECT IN CAPRINE TIBIA
It was also found that few blood vessels, macrophages
and bone marrow existed in the implant.
In the 8th week (Fig. 6B), some cartilage-like
tissues in the PLLA/b-TCP laminated scaffold were
ossified, and there were many bone trabecula and
bone islands in the scaffold. The scaffold was divided
into many parts by the newly formed woven bones. In
sections of the histological slides, newly formed bone
covered the two tops of the implant that connected
with autologous tissue where a lot of cells were found.
In the 12th week (Fig. 6C), the formed bones in
implanted area were similar to normal bone, and the
defects disappeared, and the implants were com-
pletely ossified and the two tops of the implants com-
pletely connected with autologous bone. There were
multiple bone marrow tissues and mature bone tra-
becula in the middle of the implant (the channel of
the scaffold), showing that the bone marrow channel
was forming. Very few pieces of scaffold remained,
and the structure regeneration of bone in the defect
seemed to be achieved.
In the control group (Fig. 6D), the gap between the
implant and natural bone was only filled with connec-
tive tissues. Only fibrous collagen tissues were found.
In the PLLA/b-TCP laminated scaffolds without
BMSCs group, four of five defects had severe inflam-
matory responses and formed pus instead of osteo-
genesis, and the scaffolds collapsed at 8 weeks. The
left one had a lot of connective tissues and just minor
osteogenesis at the tops of the defect.
By anatomic examinations and histological analy-
ses, mature bone marrow and channels began to form
in the defect after 12 weeks, and after 24 months the
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55
FIG. 6. Histological examination (H&E
staining) of bone defects after implantation
of BMSCs loaded PLLA/b-TCP scaffolds in
caprine tibia. (A) with PLLA/b-TCP scaf-
folds in week 4, arrow indicate blood
vessel invading into the scaffold; (B) with
PLLA/b-TCP scaffolds in week 8, arrow
indicates the presence of bone islands; (C)
with PLLA/b-TCP scaffolds in week 12,
arrow shows the mature bone tissue along
with implant debris (small circles); (D)
control defect without the scaffold, the for-
mation of scar tissue in control defect
without scaffold in week 12.
structures of caprine’s tibia defect bone were totally
recovered.
Discussion
Because of different degradation mechanisms,
PLLA (breakages of chain units) and b-TCP (dissolv-
ing process) exhibit different features during
degradation. The mechanical strength of PLLA slice
quickly decreases with degradation. At a critical
point, single PLLA scaffolds suddenly collapse. The
b-TCP strip, however, can sustain a longer time even
though part of solid b-TCP has been lost (dissolved).
In this study, in order to quickly repair bone defects,
b-TCP with quick degradation was introduced into
the scaffold which shows many advantages over other
general inorganic biomaterials, such as HA and 45S5
bioglass.
PLLA was chosen as an adhesive agent to improve
fabrication process of b-TCP. In the rolling process,
few b-TCP strips broke, which had no significant
effect on the mechanical strength of the scaffold.
Another advantage of PLLA was its hydrophobicity
that keeps the pores in the scaffold very wet. It was
important for the seeded BMSCs to obtain nutrients
during longer repair processes in the scaffold.
Although some properties, such as low mechanical
strength of the PLLA and acidity of its prepolymers
or monomers, can be improved by composing with
b-TCP at the early stage of degradation, the draw-
backs of PLLA may remain and be present at the
end of degradation. Especially, the release of many
acidic monomers leads to significant inflammatory
responses. Reports (15) showed that after composing
Artif Organs, Vol. 35, No. 1, 2011

56 J. HUANG ET AL.
with a-TCP, PLA in scaffold presented only a mild
inflammatory response at 12 months; however, after
24 months a strong inflammatory reaction was seen
and the newly formed bone was partly osteolytic
because of acidic monomers. It was also found that
the mechanical properties remained constant until
26-week degradation if PLA molecular weight was
high, such as 340 000, and decreased continuously
until they were completely lost at week 52. In this
study, to build the PLLA/b-TCP laminated scaffold
for quick bone repair, PLLA with a low molecula
weight was chosen for shortening degradation time.
Results of the in vitro experiments showed that
PLLA slice degraded and broke at the 11th week, but
in the in vivo experiment, results of X-ray examina-
tions, anatomy and histological experiments showed
that the scaffold broke at the 8th week or earlier.
This means the PLLA/b-TCP-laminated scaffold
degraded quicker in the in vivo experiment than the
in vitro one. At that moment some woven bones
formed. Little PLLA and b-TCP remained after 12
weeks, and there was no inflammatory response to be
found at the lesion site. After the steel plates were
unloaded at week 12, caprines walked normally.After
24 months, no inflammatory response in the lesion
was found. This might be due to neutralization of
acidic PLLA monomer with b-TCP and rapid osteo-
genesis of the scaffold. Normal body fluid or blood
flow in the osteogenic scaffold was helpful to take the
acidic monomers out of the lesion site. In the control
group, the laminated scaffold without seeded cells
collapsed before the 8th week, and a severe inflam-
matory response took place because of the accumu-
lation of acidic prepolymer or monomer of PLLA.
Other concerns in the present study are if the col-
lapse of the scaffold takes place after the breakage of
PLLA, and if the ECMs synthesized by BMSCs are
helpful to sustain the scaffold or not. Results proved
that the PLLA/b-TCP laminated scaffold degraded
more quickly during the in vivo than the in vitro
experiment as mentioned earlier. In vitro, after
11-week degradation, the scaffolds became very
weak and easily collapsed because most of the PLLA
broke into pieces. However, in caprines’ defects, the
scaffolds seeded BMSCs were osteogenic and did not
collapse, suggesting that the BMSCs played a key
role in sustaining the scaffolds. A possible reason is
that the ECMs synthesized by the seeded BMSCs
adhered to the b-TCP phases substituting PLLA in
the scaffold and the BMSCs accelerate osteogenesis.
Although it is difficult to assay the live BMSCs in the
scaffold after proliferation, we assayed the amount
of BMSCs in a single scaffold and found 8.7 ⫾ 0.97
million after 3-day in vitro proliferation. It was diffi-
Artif Organs, Vol. 35, No. 1, 2011
15251594, 2011, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/j.1525-1594.2010.01042.x by Nat Prov Indonesia, Wiley Online Library on [09/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
cult to track the quantitative relationship between
the synthesis of ECMs and the mechanical strength
of scaffold, but some researches have presented a lot
of evidence of this effect. For example, some practices
(16) on small bulk of bone repair demonstrated this
effect. HA/collagen loading BMP-2 did not result in
collapse of the implant because of quick bone
regeneration. In the case of gelatin/b-TCP which was
studied for fundamental research on the in vitro
osteogenesis of BMSCs, it was found that BMSCs
quickly synthesized ECMs to sustain the scaffold. In
the control group, the PLLA/b-TCP laminated scaf-
fold was implanted without BMSCs, and severe
inflammatory responses were found in almost all five
cases, and the scaffold collapsed before the 8th week
of implantation. A suggested explanation is that the
degradation rate of PLLA in the scaffold is too quick
to match the rate of new bone formation, and there
are no other components as adhesion, which leads to
the collapse of the PLLA/b-TCP laminated scaffold.
After the collapse of the scaffold, a lot of debris of
PLLA can lead to an inflammatory reaction at the
lesion site.
Many studies (17) carried out the repair of bone
defect with a small volume, about 10 to a few hundred
cubic millimeters, where few problems were encoun-
tered related to collapse of the scaffold and osteogen-
esis and more over (18), a tiny bone bioreactor could
be developed to produce bone tissue. However, there
are significant differences for a large bone defect,
usually, it takes a long time to achieve the repair, for
example over 12 months, in some cases over 24
months, because of very low ossification rate of the
defect modulated by autologous bone.This means the
scaffold should keep its shape for a long time. As we
know, it is difficult to balance the degradation of the
scaffold and its osteogenesis before the collapse of the
scaffold. Some scaffolds are usually designed with
very slow degradation rates, but normal bone repair is
hindered. On the other hand, if the designed scaffold
rapidly degrades, the scaffold often collapses before
repair achievement. For a large cylinder-shaped
defect with a size of 3 cm in length and 1.2 cm in
diameter investigated in this study, ECMs synthesized
quickly from BMSCs are required to prevent from the
collapse of the scaffold with the degradation of PLLA.
It might be valuable to note that in this study the
bone densities in the defects increased with the repair
process, and after 12 weeks, mature bone marrow in
the channel began to be formed instead of bone
tissues or connective tissues filling the channel. That
is one of the reasons why the scaffold was designed
into a hollow cylinder-shaped scaffold for the regen-
eration of the structures of the bone.

REPAIR OF BONE DEFECT IN CAPRINE TIBIA
CONCLUSIONS
In conclusion, implantation of allogeneic BMSCs
loaded PLLA/b-TCP laminated scaffold facilitated
bone repair of large defects with a volume of up to
3 cm3 in caprine tibia. In vitro findings showed that
the PLLA/b-TCP laminated scaffold is neutral in
water during its degradation and loses its strength
after 11 weeks in the simulated body fluid. In vivo
results also disclose that allogeneic BMSCs in the
scaffold play a certain role and caprine tibia were
repaired in 12 weeks. Results suggested that BMSCs
played an important role in the repair process, which
warrants further investigation.
Acknowledgments: The authors thank Prof. Z. Lu
for his generosity in providing PLLA material. We
appreciate the valuable help from Drs. B. Chen,
G. Pei, and H. Shi. The article was revised by H. Chen
from McGill University. This work was supported by
the National Fundamental Research “973” Project
(G1999504305), the Guangdong Province Team
Group Project, the National 211 project, and research
fund from Guangdong Key Laboratory of
Biomaterials.
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