Biomaterials 30 (2009) 4967–4977

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Biomaterials
journal homepage: www.elsevier.com/locate/biomaterials

Anterior cruciate ligament regeneration using mesenchymal stem cells

and silk scaffold in large animal model
Hongbin Fan a, Haifeng Liu a, Siew L. Toh b, c, James C.H. Goh a, b, *
a
Department of Orthopedic Surgery, National University of Singapore, Singapore
b
Division of Bioengineering, National University of Singapore, Singapore
c
Department of Mechanical Engineering, National University of Singapore, Singapore

a r t i c l e i n f o a b s t r a c t

Article history: Although in vivo studies in small animal model show the ligament regeneration by implanting
Received 3 May 2009 mesenchymal stem cells (MSCs) and silk scaffold, large animal studies are still needed to evaluate the silk
Accepted 19 May 2009 scaffold before starting a clinical trial. The aim of this study is to regenerate anterior cruciate ligament
Available online 18 June 2009
(ACL) in pig model. The micro-porous silk mesh was fabricated by incorporating silk sponges into knitted
silk mesh with lyophilization. Then the scaffold was prepared by rolling the micro-porous silk mesh
Keywords:
around a braided silk cord to produce a tightly wound shaft. In vitro study indicated that MSCs prolif-
Silk
erated profusely on scaffold and differentiated into fibroblast-like cells by expressing collagen I, collagen
Mesenchymal stem cell
Tissue engineering III and tenascin-C genes in mRNA level. Then the MSCs-seeded scaffold was implanted in pig model to
Ligament regenerate ACL. At 24 weeks postoperatively, the MSCs in regenerated ligament exhibited fibroblast
morphology. The key ligament-specific extracellular matrix components were produced prominently and
indirect ligament–bone insertion with three zones (bone, Sharpey’s fibers and ligament) was observed.
Although there was remarkable scaffold degradation, the maximum tensile load of regenerated ligament
could be maintained after 24 weeks of implantation. In conclusion, the results imply that silk-based
material has great potentials for clinical applications.
Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction
Anterior cruciate ligament (ACL) plays an essential role in
keeping the normal motion and stability of knee joint. It connects
a posteriolateral part of the femur to an anteriomedial part of the
tibia. This structure allows it to limit forward motion of the tibia. It
is one of the most commonly injured ligaments of knee joint. In
United States, the incidence of ACL disruption is estimated to be one
in 3000 persons [1]. The injury is exacerbated by the fact that ACL
has a limited capacity to regenerate. Therefore, grafts are needed
for ACL reconstruction. Now, over 100,000 ACL reconstructions are
performed every year in the United States [2]. The grafts (autograft
or allograft) used in ACL reconstruction include the hamstring
tendon and patella tendon. Despite great improvements in surgical
therapies, there still remain some limitations. The autograft or
allograft transplantation has drawbacks such as ligament laxity,
donor site morbidity, long recovery period and pathogen transfer
* Corresponding author. Department of Orthopedic Surgery, National University
of Singapore, 10 Kent Ridge Crescent, Singapore. Tel.: þ65 65165259; fax: þ65
67765322.
E-mail address: dosgohj@nus.edu.sg (J.C.H. Goh).
[3–6]. In recent years, tissue engineering has offered the possibility
of creating functional engineered tissues to treat ACL injuries
without many of the undesirable side effects associated with
current reconstructive options [7,8].
The scaffold selection is a major issue in constructing the tissue-
engineered ligament. The ideal scaffold should mimic the extra
cellular matrix (ECM) to provide appropriate mechanical support
and biochemical stimulation. It can manipulate cell behaviors to
achieve the desired functions such as proliferation, differentiation
and matrix remodeling [9,10]. Recently silk has been increasingly
studied as the scaffold for ligament tissue engineering due to its
good biocompatibility, slow degradability and remarkable
mechanical capability [11,12]. Silk fibers woven into a ‘‘wire-rope’’
scaffold have been shown to possess mechanical properties similar
to those of native ACL. Bone marrow stromal cells can attach,
proliferate and differentiate on it [13]. Moreover, braided silk
scaffold modified with short polypeptide can significantly increase
collagen production [14]. In our previous study, micro-porous silk
sponges were incorporated into the knitted silk mesh to form
a combined scaffold. In vitro cultured mesenchymal stem cells
(MSCs) could differentiate and produce ligament-specific ECM
components on it [15]. The MSCs-seeded scaffold was rolled into
a tightly wound shaft and implanted in rabbit model to reconstruct

0142-9612/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biomaterials.2009.05.048
4968 H. Fan et al. / Biomaterials 30 (2009) 4967–4977
ACL. The regenerated ligament exhibited abundant ECM production
and direct ligament–bone insertion. Its tensile strength could meet
the mechanical requirements for daily activities [16].
However, it should be noted that rabbit spends a larger part of
its time in deeper knee flexion when compared with large
animals (pig, goat and sheep). Although ACL has been regenerated
in rabbits, large animal studies are still needed to extrapolate the
findings from animal data to the humans [16]. The ACL is a two-
bundle ligament consisting of anteromedial (AM) and postero-
lateral (PL) bundles. The differences between ACLs of various
species may lie not in PL bundles variation but in AM bundles
variation. Studies indicate that pig knee differs from human knee
only in the magnitude and direction of force in PL bundle. In
contrast, sheep and goat knees show different magnitude and
direction force in both ACL and AM bundles when compared with
human knee. Therefore, goat and sheep knees may have limita-
tions in modeling the human ACL, while pig knee may be the
preferred model for experimental studies [17,18]. To further
investigate the clinical potential of silk material, we propose to
regenerate ACL in pig model using MSCs and silk scaffold. This
can provide us new insights into the histological changes and
biomechanical properties of tissue-engineered ligament. We
hypothesize that MSCs-seeded silk scaffold can regenerate ACL in
large animal model and provide reliable tensile strength to keep
the stability of knee joint.
2. Materials and methods
2.1. Preparation and characterization of scaffold
The knitted silk mesh of 30 mm  45 mm in dimension was fabricated using raw
Bombyx mori silk fibers (Silk Innovation Center, Thailand) with 24 needles on
a knitting machine (Silver-reed SK270, Suzhou, China) [19]. Then, the knitted raw
silk mesh was degummed in 0.02 M NaHCO3 solution at w100  C for 1.5 h to remove
Fig. 1. (A) Gross observation of a braided silk cord. (B) Gross observation of a knitted silk mesh. Inset of (B) shows micro-porous structure of silk mesh observed by SEM (100). (C)
The scaffold is fabricated by rolling up the silk mesh around a silk cord. (D) Cross-sectional morphology of scaffold. Note: the arrow indicates braided silk cord.
sericin thoroughly. The silk solution was obtained by dissolving sericin-free silk
fibers into a ternary solvent system of CaCl2/CH3CH2OH/H2O (1:2:8 in molar ratio)
followed by dialysis [20]. Then, the knitted sericin-free mesh was immersed in silk
solution (2.6 wt%), frozen at 20  C for 12 h and freeze-dried for 24 h to form the
micro-porous silk sponges in the openings of knitted mesh. Thereafter, the micro-
porous mesh was immersed in a methanol–water (90:10v/v) solution for 10 min to
induce b-sheet structural transition and then washed several times to remove the
residual methanol. The micro-porous silk mesh was then dried overnight in a fume
hood and the morphology was observed by scanning electron microscope (SEM,
JEOL JSM-5600LV, Japan). To get braided silk cord, four degummed silk fibers were
braided in one bundle and six bundles were braided in one cord. Then the cord was
cut into short pieces with 45 mm in length. Finally, the micro-porous mesh and
braided cord were sterilized by autoclave. The scaffold was prepared by rolling the
silk mesh around a braided silk cord to produce a tightly wound shaft with 45 mm in
length and 5.0 mm in diameter. The mechanical properties of scaffolds (n ¼ 4) were
tested using an Instron mechanical testing system (Model 5543, Instron Inc., MA)
with a maximum loading capacity of 1000 N. The tests were performed with a gauge
length of 20 mm at a loading speed of 10 mm/min. During the test, the scaffolds
were always kept moist with phosphate buffered saline (PBS) solution. The
maximum tensile load and stiffness were evaluated.
2.2. Isolation and expansion of MSCs
MSCs were generated from bone marrow aspirates of twelve male Yorkshire pigs
(3 months old, 30–35 kg). The bone marrow was aspirated from the posterior iliac
crest and washed three times with Hanks’ balanced salt solution (HBSS). Cells were
resuspended in 20 ml of Dulbecco’s Modified Eagle’s Medium (DMEM) supple-
mented with 10% fetal bovine serum (FBS) (HyClone Logan, Utah), L-glutamine
(580 mg/L) and penicillin–streptomycin (100 U/mL). Cultures were incubated at
37  C and 5% CO2. After 72 h, non-adherent cells were removed by changing
medium. When reaching 70–80% confluence, adherent cells were freed from the
flask with 0.05% trypsin and subcultured. A homogenous MSCs population was
obtained after 2 weeks of culture and MSCs (passage 2) were harvested for further
study.
2.3. Cell proliferation, differentiation and viability
The MSCs (4.0  106) were loaded on micro-porous silk mesh (dimension:
30 mm  45 mm) and braided silk cord (length: 45 mm) and then cultured
 H. Fan et al. / Biomaterials 30 (2009) 4967–4977
Table 1
Mechanical properties of scaffold, regenerated ligament and native ACL (n ¼ 4; data:
mean  SD).
Sample Maximum load (N) Stiffness (N/mm)
Scaffold 398.8  69.5 58.5  16.8
Regenerated ligament (control group) 120.3  14.2 10.9  1.8
Regenerated ligament (experiment group) 404.9  49.9 57.5  9.4
Native ACL 770.1  105.2 93.9  15.9
overnight for cell adhesion. Thereafter, the mesh was rolled around the silk cord
to produce a tightly wound shaft. The MSCs/scaffolds were cultured over 2 weeks
and the cell proliferation was evaluated at 1d, 3d, 5d, 7d, 9d, 11d, 13d and 15d
time points by alamar blue assay following vender’s instructions (Biosource,
Camarillo, CA). Briefly, the MSCs/scaffold (n ¼ 6) was incubated in culture medium
supplemented with 10% (v/v) alamar blue fluorescent dye for 2 h. Then, 100 ml of
medium was extracted from each sample and measured at 570/600 nm in
a microplate reader (Sunnyvale, CA). The culture medium supplemented with 10%
alamar blue was used as a negative control. The total RNA was extracted from
MSCs/scaffolds (n ¼ 6) using RNeasy Mini Kit (Qiagen, Valencia, CA, USA) after 1
week and 2 weeks of culture. For each sample 200 ng RNA was used to synthesize
cDNA with Iscript cDNA synthesis kit (Biorad laboratories, Hercules, CA). Quan-
titative real-time PCRs were carried out and monitored with a Stratagene
Mx3000P system (Stratagene, La Jolla, CA, USA). QuantiTect SYBR Green PCR kit
(Qiagen, Valencia, CA, USA) was used to quantify the transcription level of liga-
ment-specific genes including collagen I, collagen III and tenascin-C. The tran-
scription level normalized to GAPDH was calculated using the 2DCt formula with
reference to the undifferentiated MSCs. Fluorescein diacetate (FDA) molecule
probe was used to investigate the viability of MSCs on scaffolds (n ¼ 4). After 2
weeks of culture, the MSCs/scaffolds were thoroughly rinsed with PBS and
incubated with 15 mg/mL FDA solution for 15 min. Then, the samples were
observed by confocal microscopy (Zeiss LSM 510 Meta, Germany). The
morphology of MSCs on scaffold was also examined with SEM.
Fig. 2. (A) Proliferation of MSCs on scaffold evaluated by alamar blue assay on days 1, 3, 5, 7, 9, 11, 13, and 15 (Data: mean  SD, n ¼ 6). (B) Collagen I, collagen III and tenascin-C genes
transcription at 1 week and 2 weeks. Undifferentiated MSCs were used as reference sample and all results were normalized with respect to the expression of GAPDH levels (Data:
mean  SD, n ¼ 6, *p < 0.05). (C) Morphology of MSCs on scaffold observed by SEM at 2 weeks (100). (D) Viability of MSCs on scaffold evaluated by confocal microscope (FDA
staining; green: live cells).
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2.4. ACL reconstruction
Institutional Animal Care and Use Committee of National University of
Singapore approved all animal experiments. After two weeks of MSCs expansion,
twelve Yorkshire pigs (3.5 months old, 40–45 kg) mentioned above were divided
into experiment group (MSCs/scaffold implantation, n ¼ 6) and control group
(scaffold implantation, n ¼ 6) for ACL reconstruction. In experiment group, the
autologous MSCs (4.0  106) were loaded on silk mesh (dimension:
30 mm  45 mm) and braided silk cord (length: 45 mm) and then cultured over-
night for cell adhesion. Thereafter, the mesh was rolled up around the silk cord to
produce a tightly wound shaft (length: 45 mm, diameter: 5 mm). Both ends of shaft
were sutured with 1–0 polyester suture (Ethibond, Johnson & Johnson, USA) in
a whip-stitch style. The lateral parapatellar arthrotomy was used to expose the right
hind knee joint. After the ACL was excised, the tibial and femoral bone tunnels were
created with a 5.0 mm diameter drill bit. The shaft was passed through the bone
tunnel and joint cavity. Then, both ends were fixed by sutures tied over screws in
femur and tibia. This procedure was done with the knee flexed at 30 and the
implant was in slight tension. In control group, the tightly wound silk shaft without
MSCs was implanted in knee joint using same method. All animals were allowed to
move freely without immobilization after surgery. The pigs were sacrificed at 24
weeks postoperatively. Then the hind knee joints were collected immediately and
kept at 80  C.
2.5. Observation of regenerated ligament–bone junction
Before biomechanical test and histology staining, the specimens (n ¼ 12) from
experiment group and control group were firstly scanned with micro-CT (SMX-
100CT, SHIMAZDZU, Japan) to determine whether there was mineralized tissue
formation in bone tunnels. After slowly thawed at 4  C, the specimens were fixed
vertically in micro-CT specimen chamber. The optimal threshold value of 300 was
set for scan in this study. The parameters were set with the energy of 50 kV, the
intensity of 80 mA and the isotropic resolution of 24 mm. Scanning area included the
whole length of implants in bone tunnels and joint cavity. For each sample, a total of
1200 cross-sectional images were acquired to assess regenerated tissue in bone
tunnels. To further study the regenerated ligament–bone junction, the femurs and
4970 H. Fan et al. / Biomaterials 30 (2009) 4967–4977

tibias (n ¼ 2) from each group were decalcified in 30% formic acid and embedded in
paraffin. The sections of 5 mm were cut and stained with hematoxylin and eosin
(H.E.) for histology evaluation.
2.6. Biomechanical test
5543 material testing system (Instron, Canton, MA). The load to failure test was
performed by increasing the tensile load continuously with a speed of 10 mm/min.
The tensile load and elongation were recorded and stiffness was determined by the
slope of the load–deformation curve. During the test, the specimens were always
kept moist with PBS solution.

Four samples from each group were used for biomechanical test. Each one was 2.7. Histological and immunohistological assessment
dissected of all soft tissue and ligaments except for the regenerated ligament to
create a femur-regenerated ligament–tibia complex. Four femur-native ACL–tibia The regenerated ligaments (n ¼ 4) from experiment group and control group
complexes were used as normal control. The femur and tibia were fixed in an Instron were fixed in 10% formalin and embedded in paraffin. The sections of 5 mm were cut

Fig. 3. (A) Macroscopic view of native ACL in pig knee joint. (B) Macroscopic view of ACL reconstruction in pig knee joint (the arrow points to implant). Inset of (B) shows MSCs-
seeded scaffold. (C) Macroscopic view of regenerated ACL in experiment group at 24 weeks postoperatively. (D) Macroscopic view of regenerated ACL in control group at 24 weeks
postoperatively.
 H. Fan et al. / Biomaterials 30 (2009) 4967–4977
and stained with hematoxylin and eosin (H.E.) for histological observation. For
immunohistochemistry staining, endogenous peroxidase was firstly blocked with
hydrogen peroxide before pepsin treatment for 20 min. Monoclonal antibodies for
collagen I (Sigma, USA), collagen III (Chemicon, CA) and tenascin-C (Chemicon, CA)
were applied for 1 h followed by incubation with biotinylated goat anti-mouse
antibody (Lab Vision Corporation, CA) for 30 min. Streptavidin peroxidase was
added for 45 min and 3,30 -diaminobenzidine was used as a chromogenic agent.
Counterstaining was done with hematoxylin. The results were evaluated by three
individuals who were blinded to treatments. They selected the most typical sections
from each specimen to assess ligament regeneration.
2.8. Statistical analysis
All data were analyzed using SPSS 13.0 software and statistically significant
values were defined as p < 0.05. The data were expressed as mean  standard
deviation (SD) in quantification of alamar blue reduction, gene transcription level
and mechanical properties. The difference was detected using one-way analysis of
variance (ANOVA) test.
3. Results
3.1. Characterization of scaffold
The braided silk cord exhibited wire-rope morphology and silk
fibers were found to have smooth surface due to complete removal
Fig. 4. Micro-CT images of implant-bone junction in femoral bone tunnels (A,C) and tibial bone tunnels (B) in experiment group (A,B) and control group (C,D) at 24 weeks
postoperatively. The arrows indicate the bone tunnels.
4971
of sericin (Fig. 1A). The micro-porous silk mesh showed high
porosity and interconnected pores. SEM observation indicated that
web-like silk sponges were spread evenly over the surface and in
the openings of knitted silk fibers. The micro pore size ranged from
20 mm to 100 mm (Fig. 1B). The scaffold was prepared by rolling up
the silk mesh along its short axis with braided silk cord inside to
produce a tightly wound shaft. The length was 45.0 mm and the
diameter was 5.0 mm (Fig. 1C). The cross-sectional image revealed
the dense cord surrounded by loose, porous silk mesh (Fig. 1D).
Results of biomechanical test showed the maximum load and
stiffness of scaffold were 398.8  69.5 N and 58.5  16.8 N/mm,
respectively (Table 1).
3.2. Cell proliferation, differentiation and viability
Alamar blue is an indicator dye, formulated to quantitatively
measure the proliferation of cells. It consists of an oxidation–
reduction (REDOX) indicator that yields a colorimetric change and
a fluorescent signal in a response to a metabolic activity. The
proliferation of MSCs on scaffold increased rapidly with culture
time at early stage. The value of alamar blue reduction increased
from 35.2  5.2% to 83.5  4.9% after 9 days of culture. Subsequently
4972 H. Fan et al. / Biomaterials 30 (2009) 4967–4977

it increased gradually and reached a plateau. After 15 days the value
was 86.9  3.2% (Fig. 2A). The transcription level of collagen I gene
was significantly up-regulated with 0.29  0.05 fold at 1 week and
0.70  0.05 fold at 2 weeks (p < 0.05). After 2 weeks of culture, the
transcription level increased by 2.41 folds. The transcription levels
of collagen III and tenascin-C genes increased slowly with culture
time. The levels of collagen III gene were 0.75  0.08 fold and
0.83  0.09 fold at 1 week and 2 weeks, respectively. There was no
significant difference (p < 0.05). The transcription levels of tenas-
cin-C gene also showed the similar trend with 0.87  0.08 fold at 1
week and 0.94  0.07 fold at 2 weeks (Fig. 2B). After 2 weeks of
culture, the MSCs proliferated and spread on scaffolds to bridge
neighboring micro-pores. The cells grew along the pore walls or silk
fibers and exhibited semi-ovoid or spindle-shape morphology
(Fig. 2C). Confocal microscopy observation showed robust cell
proliferation and good cell viability on scaffolds. The cells were
distributed throughout scaffolds and there was no significant cell
death (Fig. 2D).
3.3. Gross observation of regenerated ligament
The swine ACL was exposed and dissected (Fig. 3A). The MSCs-
seeded silk mesh was carefully rolled up around the braided silk
cord to form a tightly wound shaft (length: 45.0 mm, diameter:
5.0 mm). Then the in vitro cultured MSCs/scaffold was implanted to
reconstruct ACL in right hind knee joint (Fig. 3B). At 24 weeks
postoperatively, gross observation revealed that the regenerated
ligament resembled the native ACL. The scaffold was filled with
abundant newly formed fibrous tissue and silk fibers couldn’t be
discerned. In addition, the surface was covered by a thin layer of
synovium-like tissue. The color and luster of regenerated ligament
were similar to those of native ACL. Arthrosis was found in one
sample of experiment group. Anterior drawer test by hand indi-
cated that the knee joints were stable with regenerated ligaments
in proper tension. The regenerated ligament–bone junction also
healed well with in-growth of connective tissue (Fig. 3C and D).
3.4. Observation of regenerated ligament–bone junction
The cross-sectional images of bone tunnels were acquired with
micro-CT. The results showed there was no obvious high density
signal detected in femoral and tibial bone tunnels of experiment
group and control group. It indicated that the mineralized tissue
was scarce in the intra-tunnel portion of implants (Fig. 4). After
micro-CT scan, the H.E. staining was performed to further examine
the histological changes of regenerated ligament–bone junction.
After 24 weeks, the interface was filled with abundant connective
tissue with perpendicular collagen fibers projecting from regen-
erated ligament into bone tissue in experiment group. These fibers
resembled Sharpey’s fibers to bridge the gap between the regen-
erated ligament and bone. Indirect ligament–bone insertion with
typical three zones (bone, Sharpey’s fibers and ligament) was
observed (Fig. 5A and B). In contrast, there was no typical insertion
formed in control group (Fig. 5C and D).

Fig. 5. (A) Histological observation of regenerated ligament–bone junction in experiment group at 24 weeks postoperatively (H.E. 100). (B) Magnified view of the black rectangle
frame from (A). The arrows and asterisks indicate the Sharpey-like fibers and degraded silk fibers, respectively (H.E. 400). (C) Histological observation of regenerated ligament–
bone junction in control group at 24 weeks postoperatively (H.E. 100). (D) Magnified view of the black rectangle frame from (C) (H.E. 400).
 H. Fan et al. / Biomaterials 30 (2009) 4967–4977
3.5. Biomechanical analysis
The tensile properties of regenerated ligament and native ACL
were compared. All samples were broken within the intra-articular
zone during biomechanical test although the exact rupture sites
were inconsistent (Fig. 6A–C). The load–deformation curves in
experiment group and control group both comprised toe region,
linear region, microfailure region and failure region. In experiment
group, the curves changed smoothly from a flat slope to a steep one
with extension of regenerated ligaments. The maximal load was
404.9  49.9 N, which was 52.6% of native ACL maximal load at 24
weeks postoperatively (Fig. 6D and E). Due to the degradation of
silk scaffold, the maximal load and stiffness of regenerated liga-
ment in control group were 120.3  14.2 N and 10.9  1.8 N/mm,
respectively (Fig. 6F). Although there was remarkable scaffold
degeneration, the tensile properties of regenerated ligament could
be maintained. This is an obvious sign of ECM production (Table 1).
3.6. Histological and immunohistochemical analysis
H.E. staining and immunohistochemistry staining were used to
assess cell proliferation and ECM production in regenerated liga-
ment. Results indicated that MSCs were distributed throughout the
scaffold and proliferated robustly in experiment group. These cells
exhibited a flat and elongated morphology, which was similar to
that of fibroblasts in native ligament. Abundant ECM was synthe-
sized and the organized collagen fibers were arranged in the
direction of tensile load. After 24 weeks, the micro-porous silk
Fig. 6. The femur–ACL–tibia (A) and femur–regenerated ligament–tibia complexes from experiment group (B) and control group (C) were firmly fixed on Instron machine to
perform mechanical test. The load–deformation curves of native ACL (D) and regenerated ligaments from experiment group (E) and control group (F) were recorded at 24 weeks
postoperatively.
4973
sponges were completely degraded. In contrast, the silk fibers were
degraded partially and still detectable. The remnants of degraded
silk fibers were thoroughly integrated into regenerated ligamen-
tous tissue. On the contrary, the implants of control group appeared
hypocellular. It was mainly composed of silk fibers and only a layer
of loose connective tissue was distributed in the out surface. Cells
were scarcely observed in the inner part of implants and less ECM
production was observed (Fig. 7). The immunohistochemistry
staining for collagen I was strongly positive in newly regenerated
tissue of experiment group. On the other hand, the staining for
collagen III and tenascin-C was weak positive. The normal ligament
consists of 90% collagen I, 9% collagen III and other proteoglycans
[21]. It was interesting to note that collagen I was more prominent
in comparison with collagen III and tenascin-C, which was consis-
tent with the composition of native ligament ECM (Fig. 8).
4. Discussion
ACL reconstruction is still a challenging operation for ortho-
paedic surgeons due to the absence of optimal graft [22,23].
Recently, tissue-engineered ligament has proved to be one of the
most promising alternative therapies. In this study, ACL was
regenerated by implanting silk scaffold and MSCs in pig model. The
regenerated ligament showed similarities to the native ACL in ECM
composition and mechanical properties.
In vitro studies are important to investigate the structure and
biocompatibility of scaffold. However, further progress can only be
made through critical assessment of tissue-engineered ligament in
4974 H. Fan et al. / Biomaterials 30 (2009) 4967–4977

Fig. 7. Histological observation of native ACL (A: 100; B: 200) and regenerated ligaments in control group (C: 100; D: 200) and experiment group (E: 100; D: 200) by H.E.
staining at 24 weeks postoperatively. Note: The arrows and asterisks indicate the sutures and degraded silk fibers, respectively.

appropriate animal models. Tissue-engineered devices are generally
classified as class III medical devices requiring FDA approval for use.
There are many hurdles in developing and testing proposed mate-
rials for the treatment of tendon and ligament injuries in humans.
The general progression goes from success in cell culture or a small
animal to a larger animal in an anatomically correct location and
then to human trials. So we reconstructed ACL in pig model with
tissue-engineered ligament in this study after successful regenera-
tion of ACL in rabbit model [16]. The direction of in situ force in the
human ACL was reported to differ significantly from those of goat
and sheep ACL. In contrast, it was approximately similar to that of pig
ACL [17]. Although primates are appealing models due to their
anatomic and genetic similarities, the costs and management issues
related to large scale testing are prohibitive [24].
In our previous studies, fibroblasts isolated from rabbit ACL,
medical collateral ligament (MCL) and MSCs were regarded as
attractive candidates to compare. It was found that MSCs had the best
proliferation and collagen excretion rate among three cell types
[8,15,25]. Therefore, MSCs were seeded on scaffold to reconstruct ACL
in this study. The proliferation of MSCs on scaffold increased rapidly
at initial stage. Then it increased slowly and reached a plateau on day
15. The results indicated the excellent biocompatibility of scaffold
(Fig. 2). The ECM of ACL is composed primarily of collagen I, collagen
III and small amounts of elastin, proteoglycans and glycoproteins
[21]. The collagens mainly provide mechanical strength to ligament
tissue. As one of glycoproteins, tenascin-C contributes to the visco-
elasticity of the ECM by interacting with collagen fibrils and
proteoglycan decorin [26]. It was noted that during two weeks of
culture MSCs modulated the transcription levels of collagen I,
collagen III and tenascin-C genes. After 2 weeks of culture, the
transcription level of collagen I gene was significantly up-regulated
(p < 0.05), while those of collagen III and tenascin-C genes had no
significant differences (p > 0.05) (Fig. 2). Since cell responses are
highly related to the topography of scaffolds surface, it is reasonable
 H. Fan et al. / Biomaterials 30 (2009) 4967–4977 4975

Fig. 8. Histological observation of regenerated ligament by immunohistochemistry staining specific for collagen I (A,B), collagen III (C,D) and tenascin-C (E,F) at 24 weeks post-
operatively (100). (B), (D) and (F) were magnified view of the black rectangle frames from (A), (C) and (E) respectively (200). Note: The arrows and asterisks indicate the sutures
and degraded silk fibers, respectively.

to assume that the structures of scaffold may trigger cell-surface
receptors and adhesion sites that regulate the genes responsible for
the synthesis and secretion of ligament ECM components.
The methodology of in situ tissue engineering has been applied in
this study to reconstruct ACL. The MSCs-seeded scaffold was
implanted in pig model and the host knee joint served as a functional
bioreactor, which could provide various cytokines and physiological
cyclic loading on implants to induce the differentiation of MSCs. The
cells of regenerated tissue exhibited fibroblast morphology and the
immunohistochemistry staining indicated the production of liga-
ment ECM components including collagen I, collagen III and tenas-
cin-C (Figs. 7 and 8). In previous study, we regenerated ACL using
MSCs and micro-porous knitted silk scaffold in rabbit model [16]. The
scaffold was modified in present research due to the increased
mechanical requirement in pig model. The micro-porous knitted silk
mesh was rolled around a braided silk cord to increase tensile
strength (Fig. 1). The braided silk cord was reported to have excellent
tensile strength similar to that of ACL [13,14,27]. However, MSCs are
usually held up on the surface of braided silk cord due to small gaps
(5–60 mm) between individual fibroin filaments after cell loading
[13]. In this study, the micro-porous silk mesh had proper pore size
(20–100 mm) and larger surface area, which facilitate cell adhesion,
proliferation and ECM production [19]. The pre-seeded silk mesh was
rolled around silk cord to produce a tightly wound shaft for
implantation. This cell seeding methodology and scaffold geometry
ensures the excellent tissue infiltration.
The native ACL attaches to the bone via a direct insertion, which
contains four distinct histologic zones: (1) ligament, (2) uncalcified
fibrocartilage, (3) calcified fibrocartilage and (4) bone. The normal
insertion site is a highly specialized zone of tissue that functions to
transmit stress from hard tissue to soft tissue [28]. In contrast,
indirect insertion comprises three zones including ligament,
4976 H. Fan et al. / Biomaterials 30 (2009) 4967–4977
Sharpey’s fibers and bone. The Sharpey’s fibers extend from ligament
tissue deep into bone [29]. Previously we used a rabbit model for ACL
reconstruction in which the MSCs-seeded scaffold was implanted to
regenerate ACL. A mature direct insertion with four typical zones was
observed after 24 weeks, which was consistent with other studies
[30,31]. However, the healing proceeded by means of fibrous tissue
at the regenerated ligament–bone junction in pig model. The
collagen fibers resembling Sharpey’s fibers projected from regen-
erated ligament into bone tissue. The indirect insertion was observed
after 24 weeks. The discrepancy between these two models may be
due to differences in the species and mechanical environment.
For ligament tissue engineering, the maintenance of mechanical
properties of the scaffold until the newly regenerated tissue
becomes mechanically competent is of great importance. The
scaffold’s mechanical properties and physical signals can direct
cellular activity and phenotype toward functional tissue assembly
and mechanical competent tissue repair [32]. In this study the
scaffold is designed to withstand the forces associated with daily
activities rather than failure forces occurred in trauma. During daily
activities, the ACL is loaded only about 20% of its failure capacity
[33]. The scaffold has the original maximum load of 398.8  69.5 N,
which was 51.8% of native ACL maximum load. It was comparable
with the values of native ACL during normal physical activity and
provided enough tensile strength at initial stage after implantation.
Studies demonstrate that the tensile strength loss of silk scaffolds
ranges from 55% at 6 weeks to 73% at 30 days after implantation
[12,34]. The rate is mainly dependent upon the implantation site,
mechanical environment and scaffold structure. This study evalu-
ates the silk scaffold implanted in swine knee joint and the results
can provide more useful information for future clinical applications.
Although there was remarkable scaffold degeneration at 24 weeks
postoperatively, the tensile properties of regenerated ligament
could be maintained. The newly formed tissue can compensate the
tensile loss caused by the degradation of silk scaffold. There was no
obvious ligament laxity and knee joints were stable. Degenerative
cartilage changes were observed around intra-condylar notch in
one sample of experiment group, which resulted from the place-
ment of the femoral tunnel too anterior in the intercondylar notch.
There are two limitations of this study. Firstly, we didn’t label
and track MSCs on scaffolds after implantation. In our previous
report, green fluorescence protein (GFP) labeled MSCs remained
alive on the similar scaffold implanted in rabbit knee joint at 4
weeks postoperatively [15]. The knee joint is covered with syno-
vium layer and provides a less vascular microenvironment. So there
will be few cells attracted into the scaffold. Furthermore, the
control group (scaffold implantation) showed fewer infiltrated cells
and less ECM production compared to experiment group (scaffold/
MSCs implantation). It partially indicated that few cells migrated
into the scaffold from surrounding tissues. So we can infer that the
regenerated fibrous tissue and proliferated cells on scaffold are
mostly attributed to the MSCs implanted. Secondly, all samples
were harvested from only one time point (24 weeks). The advan-
tage of pig model is that it can more closely mimic human knee
joint compared with rabbit model. However, large animal models
are more costly, more time-consuming, more demanding and more
scrutinized by animal rights activists. In addition, 24-week time
point is appropriate to analyze regenerated ligament with our
previous experience [16]. Therefore, 24-week time point was set in
this research. This study is just a preliminary study. More data will
be needed in future to precisely describe the regenerated ligament.
5. Conclusions
In this study, a pig model which closely mimics the human knee
joint was used to investigate the ACL regeneration. The scaffold was
fabricated by rolling the micro-porous silk mesh around braided
silk cord to increase mechanical properties for large animal model.
In vitro study demonstrated that MSCs on scaffold showed robust
proliferation and fibroblast differentiation. After implantation of
MSCs/scaffold, the regenerated ligament exhibited abundant ECM
and proliferating fibroblast-like cells at 24 weeks postoperatively.
The tensile strength could meet the mechanical requirements for
daily activities. It implies that silk scaffold has great potentials in
clinical applications.
Acknowledgement
This study was supported by grant (R-175-000-054-305) from
Biomedical Research Council, Singapore.
Appendix
Figures with essential colour discrimination. The majority of
figures in this article have parts that are difficult to interpret in
black and white. The full colour images can be found in the on-line
version, at doi:10.1016/j.biomaterials.2009.05.048.
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