Effects of a cell-free method using collagen vitrigel incorporating
TGF-b1 on articular cartilage repair in a rabbit osteochondral
defect model
Hideyuki Maruki,1 Masato Sato,2 Toshiaki Takezawa,3 Yoshiki Tani,2 Munetaka Yokoyama,2Takumi Takahashi,2 Eriko Toyoda,2 Eri Okada,2 Shigehisa Aoki,4 Joji Mochida,2 Yoshiharu Kato1
1

Department of Orthopaedic Surgery, Tokyo Women’s Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666,
Japan
2
Department of Orthopaedic Surgery, Surgical Science, Tokai University School of Medicine, 143 Shimokasuya, Isehara,
Kanagawa 259-1193, Japan
3
Transgenic Animal Research Center, National Institute of Agrobiological Sciences, 2 Ikenodai, Tsukuba, Ibaraki 305-0901,
Japan
4
Department of Pathology and Microbiology Faculty of Medicine, Saga University, 5-1-1 Nabeshima, Saga 849-8501, Japan
Received 26 April 2016; revised 31 August 2016; accepted 12 September 2016
Published online 12 October 2016 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.33792
Abstract: We studied the ability of collagen vitrigel material torepair cartilage in vivo when used alone or with transforming
growth factor-b (TGF-b). We measured the time course andquantity of TGF-b1 released from the collagen vitrigel in vitro toquantify the controlled release of TGF-b1. Over 14 days, 0.91 ng
of TGF-b was released from the collagen vitrigel. Osteochondral
defects were made in the femoral trochlear groove in 36 Japanese white rabbits, which were divided into three groups:
untreated group (group A), collagen vitrigel-implanted group(group B), and TGF-b1-incorporated collagen vitrigel-implantedgroup (group C). The weight distribution ratio between theaffected and unaffected limbs served as an indicator of pain.
Animals were sacrificed at 4 and 12 weeks after surgery, andtheir tissues were assessed histologically. The weight distribution ratio increased in all groups and did not differ significantly
between groups at 12 weeks. Group A needed 6 weeks to attainmaximum improvement, and groups B and C showed nearmaximum improvement at 4 and 2 weeks, respectively. The
International Cartilage Repair Society II score improved significantly in group C relative to the other groups. These findings
suggest that sustained, slow release of TGF-b caused early pain
C 2016 Wiley Periodicals, Inc. J
mitigation and cartilage repair. V
Biomed Mater Res Part B: Appl Biomater, 105B: 2592–2602, 2017.

Key Words: TGF-b, collagen vitrigel, controlled release, cartilage repair, articular cartilage

How to cite this article: Maruki H, Sato M, Takezawa T, Tani Y, Yokoyama M, Takahashi T, Toyoda E, Okada E, Aoki S,
Mochida J, Kato Y. 2017. Effects of a cell-free method using collagen vitrigel incorporating TGF-b1 on articular cartilage repair
in a rabbit osteochondral defect model. J Biomed Mater Res Part B 2017:105B:2592–2602.

INTRODUCTION

Since the report by Brittberg et al.1 describing the implantationof cultured autologous chondrocytes, various cartilage regeneration treatments have been studied.2–4 Treatments such asbone drilling and mosaicplasty are mainstays in the ﬁeld of
orthopedics. However, these methods have some problems,
including narrowness of the treatment range, replacement of
repair tissue with ﬁbrous cartilage and not hyaline cartilage,
and the need to harvest a graft.5–7 We have developed a technique that uses cell sheets produced in temperatureresponsive culture dishes developed by Okano et al.8,9 toimprove the repair of articular cartilage tissue and to treat abroad range of articular cartilage defects such as osteoarthritisof the knee. Chondrocyte sheets can help to repair articularcartilage defects by stimulating growth of hyaline-like cartilage.10–17 A study of the mechanism of action of these cellsheets has conﬁrmed that transforming growth factor-b (TGFb), prostaglandin E2, and other humoral factors also play
important roles in the repair of articular cartilage tissue andthat TGF-b plays a particularly important role.17 TGF-b inhibitsinterleukin-1 (IL-1) and other catabolic cytokines, reportedly
acts to promote cell proliferation and extracellular matrix production by articular chondrocytes, and promotes differentiation of mesenchymal stem cells into chondrocytes.17,18
There are ﬁve TGF-b isoforms, which have 70–80%
homology in their amino acid sequences. Three isoforms

Correspondence to: Masato Sato; e-mail: sato-m@is.icc.u-tokai.ac.jp
Contract grant sponsor: Supported by a Health Labour Sciences Research Grant from the Ministry of Health, Labour, and Welfare of Japan; contract grant number: No. 12103253
Contract grant sponsor: Agri-Health Translational Research Project Grants from the Ministry of Agriculture, Forestry and Fisheries of Japan; contract grant numbers: No. 6240 to M. Sato and No. 6110 to T. Takezawa

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C 2016 WILEY PERIODICALS, INC.
VORIGINAL RESEARCH REPORTFIGURE 1. Collagen vitrigel. Photographs were obtained after the collagen vitrigel was rehydrated in PBS at room temperature 308C for 30
min. (a) The collagen vitrigel used for this study was formed into a
thin, round sheet 15 mm in diameter. The quantity of collagen in thevitrigel was 5.0 mg/cm2. The collagen vitrigel was irradiated by anelectron beam, stored in the dark at 258C, and rehydrated in PBS justbefore use. (b) Scanning electron microscopy images of the collagen
vitrigel membrane were obtained using a JSM 6510 instrument(JEOL, Tokyo, Japan) at an accelerating voltage of 20 kV. Original
magnification is 310,000. Bars 5 1 lm. (c) and (d) Transmission electron microscopy images of the collagen vitrigel membrane obtainedusing a JEM-1210 instrument (JEOL). (c) Bars 5 1 lm and (d)bars 5 500 nm.

(TGF-b1, TGF-b2, and TGF-b3) exist in humans.19 TGF-b1 isparticularly useful for regenerating cartilage tissue.20–22
TGF-b is most effective when a certain concentration is
maintained in the lesion, although the brevity of its durationof activity has been reported as a disadvantage.19,23,24
Although the short-term action of injected TGF-b alone has
been studied extensively in animal models, such injection
leads to the deposition of cartilage ﬁbrocartilage, and a better method is needed to stimulate defect repair by hyaline
cartilage.20
Conventional collagen gel comprises low-density collagen ﬁbrils, which make it soft and difﬁcult to handle.
Takushi et al.25 reported that drying a thermally denatured
protein gel to remove gradually both free and bound water
allows the gel to be converted into a hard transparent material similar to glass. Takezawa et al.26 applied this vitriﬁcation process to conventional collagen gel and extended thiswork by rehydrating the vitriﬁed collagen gel to produce ahigh-density gel comprising collagen ﬁbrils—a novel biomaterial named “collagen vitrigel.” Using this technology, thedisk-shaped conventional gel of 0.25% (w/v) collagen ﬁbrils
can be concentrated up to 100–133 times and converted
into a round collagen vitrigel membrane of 25–33% (w/v)

collagen ﬁbrils, which is equivalent to the composition ofconnective tissue found in the body.27 Relative to conventional collagen gels, this collagen vitrigel membrane offers
superior handling, strength, transparency, and permeability
to proteins with a molecular weight of 100 kDa or
higher.26–28 The collagen vitrigel membrane comprises
mainly collagen type I and has been reported to be an effective carrier for paracrine assays in three-dimensional in
vitro culture and in vivo drug-delivery systems (DDSs).29
In the present study, we made use of the properties ofcollagen vitrigel as a DDS. By incorporating TGF-b into thecollagen vitrigel, we prepared a carrier for the sustained,
slow release of TGF-b without the need for cells. The purpose of this study was to examine the effects of the sustained, slow release of TGF-b on the repair of articularcartilage defects and the tissue-repairing effects of collagen
vitrigel alone and as a sustained-release carrier. We
implanted the sustained-release carrier into full-thickness
articular cartilage defects in Japanese white rabbits, and we
conﬁrmed the therapeutic effects. We also conducted invitro research to measure the quantity and duration of thesustained release of TGF-b from impregnated collagen
vitrigel.
MATERIALS AND METHODS

We used 36 female Japanese white rabbits, aged 18 weeks
and weighing 3 kg. All animal experimentation conformed
to the Care and Use of Laboratory Animals (NIH publication
No. 85–23, revised 1996) and Tokai University guidelines
on animal experimentation (animal experimentation approval number: 151075). In this study, one rabbit withimplanted collagen vitrigel suffered a lower-leg fracture during rearing and was euthanized and excluded from theexperimental results. No adverse events were observed inany of the other 35 rabbits.
Collagen vitrigel
Various studies of tissue regeneration have made use of theadvantages of collagen vitrigel membrane.30–36 Collagen
xerogel membranes, deﬁned as dried collagen vitrigel membranes without free water, can be prepared by simply revitrifying collagen vitrigel membranes on a separable sheet.
Such collagen xerogel membranes are appropriate for longterm preservation because they can be easily converted into
collagen vitrigel membranes by rehydration, as reported
previously.34,36,37 The thin, round membranes of collagen
vitrigel with a collagen content of 5.0 mg/cm2 and a diameter of 15 mm (Fig. 1) were prepared as reported previously.36 Brieﬂy, 1.8 mL of 0.5% porcine-derived atelocollagensolution was poured into a separable mold with an innerdiameter of 15 mm, and the collagen vitrigel membrane wasfabricated by gelation, vitriﬁcation, and rehydration withphosphate-buffered saline (PBS; Gibco, Grand Island, NY,
USA). The membrane was then converted to a collagen xerogel membrane by revitriﬁcation on a separable sheet. This
collagen xerogel membrane was irradiated with electron

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beams and then stored in the dark at 258C. Immediately
before use, the membrane was rehydrated with PBS.
Incorporation of TGF-b1 into the collagen vitrigel
Human TGF-b1 (R&D Systems, Minneapolis, MN, USA) was
dissolved to a ﬁnal concentration of 10 ng/mL in PBS in acontainer precoated with 1% bovine serum albumin (BSA;
Merck, Darmstadt, Germany) solution. Research on themechanism of action of TGF-b in cultures of chondrocytes
derived from mesenchymal stem cells shows that 10 ng/mL
is the optimum concentration of TGF-b during culture.19,22,38–42 Therefore, we used this concentration in our
experiments. A thin, circular sheet of collagen vitrigel with
5.0 mg/cm2 of collagen and a diameter of 15 mm was incubated in this solution for 24 h at 48C before implantation.
Assessment of the quantity and duration of TGF-b1
released from TGF-b1-incorporated collagen vitrigel
After incubating the collagen vitrigel for 24 h at 48C in a 10
ng/mL TGF-b1 solution, the collagen vitrigel was placed in
3 mL of 1% BSA solution and incubated at 378C. The solution was collected and replaced every 2 days for 14 days(n 5 3), and the collected samples were stored at 2308C. A
human TGF-b1 quantikine ELISA Kit (R&D Systems) wasused to measure the quantity of TGF-b1 in the samples.
Implantation
A drill was used to make an osteochondral defect with adiameter of 5 mm and depth of 3 mm in the articular cartilage of the right femoral trochlear groove in 36 Japanesewhite rabbits (Fig. 2). We irrigated the defect with physiological saline (Nipro, Osaka, Japan) during the drilling toprevent infection and thermal damage. The 36 rabbits weredivided into three groups (n 5 12 in each group): an
untreated group (group A), a collagen vitrigel-implantedgroup (group B), and a TGF-b1-incorporated collagen
vitrigel-implanted group (group C). After covering the defectwith collagen vitrigel, we pressed the collagen vitrigel into
the femur by reduction of the patella, which we had dislocated to create the defect. We then sutured the quadriceps
femoris muscle and tendon to prevent dislocation. Six rabbits from each group were sacriﬁced at 4 weeks and another six rabbits at 12 weeks after surgery, and their tissues
were examined histologically to assess the cartilage repair.
Pain assessment
We used the change in the weight distribution ratio
between the affected limb and the unaffected limb as anindicator of pain. This ratio is used widely to assess pain inexperimental animals.15,43 The weight distribution ratio wasmeasured with an incapacitance tester (Linton Instrumentation, Norfolk, UK). Each rabbit was placed on the tester andallowed to stand on its hind limbs, which were adjusted torest on two separate sensors. The rabbit was then surrounded by a box to restrict its movement and was allowed
to rest in this position for 5 min, after which the weight
distribution was measured. The mean weight distribution
ratios were calculated from ﬁve measurements taken for

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FIGURE 2. Osteochondral defect model and gross appearance of thedefects 4 and 12 weeks after surgery. (a) Osteochondral defect model.
We created an osteochondral defect (diameter, 5 mm; depth, 3 mm)by drilling into the articular cartilage of the right femoral trochlear
groove. (b) Model with transplanted collagen vitrigel. Collagen vitrigel
was transplanted to cover the defect in groups B and C. (c) Grossappearance of the defects 4 and 12 weeks after surgery. Filling in of
the defect in groups A and B was insufficient at 4 and 12 weeks after
surgery. In group C, the defect was filled with cartilaginous tissue at 4
and 12 weeks after surgery.

each rabbit in all groups, and group mean values were calculated from those mean ratios. Measurements were taken
at 1, 2, 3, 4, 6, 8, 10, and 12 weeks after surgery. The
weight distribution ratio (expressed here as the affected
limb weight as a percentage of the total weight) was
calculated using the following formula: weight distribution
ratio (%) 5 (affected limb weight/[unaffected limb
weight 1 affected limb weight]) 3 100.
Tissue assessment
Animals were sacriﬁced at 4 and 12 weeks after surgery,and their tissues were examined histologically to conﬁrm
the repair of the osteochondral defect at the right femoral
trochlear groove of the right knee joint. After sacriﬁce, the

EFFECTS OF COLLAGEN VITRIGEL WITH TGF-b1 ON CARTILAGE REPAIRORIGINAL RESEARCH REPORTTABLE I. ICRS II Histological Grading System
Ti:
Tissue morphology (viewed
under polarized light)
0%: Full-thickness collagenfibers100%: Normal cartilagebirefringence
Matx:
Matrix staining(metachromasia)
0%: No staining
100%: Full metachromasia
Cmorph:Cell morphology
0%: No round/oval cells
100%: Mostly round/oval cells
Cclus:
Chondrocyte clustering (4 ormore grouped cells)
0%: Present
100%: Absent
SurfA:
Surface architecture
0%: Delamination, or major
irregularity
100%: Smooth surface

Basl:
Basal integration

Vasc:
Vascularization (within the repaired tissue)

0%: No integration

0%: Present

100%: Complete integration

100%: Absent

Ftide:
Formation of a tidemark

SurfSp:Surface/superficial assessment

0%: No calcification front
100%: Tidemark
SubBA:
Subchondral boneabnormalities/marrow fibrosis
0%: Abnormal
100%: Normal marrow
Infl:
Inflammation

0%: Total loss or complete disruption
100%: Resembles intact articular cartilage
MDzone:Mid/deep zone assessment

0%: Present
100%: Absent
AbnCO:
Abnormal calcification/ossification
0%: Present
100%: Absent

0%: Fibrous tissue
100%: Normal hyaline cartilage
Hgtot
1: Tissue morphology (Ti).
2: Matrix staining (Matx)
3: Cell morphology (Cmorph).
4: Chondrocyte clustering (Cclus).
5: Surface architecture (SurfA).
6: Basal integration (Basl).
7: Formation of a tidemark (Ftide).
8: Subchondral bone abnormalities/marrow
fibrosis (SubBA).
9: Inflammation (Infl).
10: Abnormal calcification/ossification(AbnCO).
11: Vascularization (Vasc).
12: Surface/superficial assessment (SurfSp).
13: Mid/deep zone assessment (MDzone).

This system evaluates repair tissue based on 14 items: 1: Tissue morphology (Ti). 2: Matrix staining (Matx) 3: Cell morphology (Cmorph). 4:
Chondrocyte clustering Cclus). 5: Surface architecture (SurfA). 6: Basal integration (Basl). 7: Formation of a tidemark (Ftide). 8: Subchondralbone abnormalities/marrow fibrosis (SubBA). 9: Inflammation (Infl). 10: Abnormal calcification/ossification (AbnCO). 11: Vascularization (Vasc).
12: Surface/superficial assessment (SurfSp). 13: Mid/deep zone assessment (MDzone). The maximum score is 100 points and the minimum scoreis 0 points for each item. The total score range is from 0 (no repair) to 1300 (normal articular cartilage).

right femur was collected and immobilized in 10% formalinsolution, delipidated for 2–3 days with acetone solution,
washed with water, and decalciﬁed with 10% ethylenediaminetetraacetic acid for 5–7 days. This process was repeated four times. After trimming, samples were embedded inparafﬁn. With the defect placed at the center, sections wereprepared at 3 lm thickness at right angles and were stainedwith safranin O and hematoxylin and eosin. And we performed immunohistochemical staining using mouse primarymonoclonal antibodies which react with human collagentype II (Daiichi Fine chemical, Toyama, Japan). To immunostain for collagens type II, deparafﬁnized sections weretreated with 0.4% pepsin (DAKO) for 30 min. at 378C. Afterwashing in distilled water, sections were treated with 0.3%hydrogen peroxide/methanol solution at RT for 15 min.
After washing in PBS, sections were blocked with 2.5% normal goat serum (MP-7402, Vector Laboratories, Burlingame,
CA, USA) for 10 min. at RT. They were then treated for 3 h
at RT with mouse monoclonal antibodies for human type II
collagen diluted at 1:100 with 1% BSA in PBS. After washing in PBS, they were treated for 1 h at RT with ImmPRESS

polymer anti-mouse IgG reagent (MP-7402, Vector Laboratories, Burlingame, CA, USA). Finally, they were immersed for
2–8 min. in Tris–HCl buffer (pH 7.6) containing 0.02% diaminobenzidine and 0.005% hydrogen peroxide, and thencounterstained with Mayer’s hematoxylin.
The International Cartilage Repair Society (ICRS) II histological grading system was used to assign scores. Twoblinded board-certiﬁed orthopedic surgeons scored thesamples, and a mean score was calculated. The ICRS II histological grading system is an assessment method developed by Mainil-Varlet et al.44 This assessment methodassigns scores for 13 items: 1: Tissue morphology (Ti). 2:
Matrix staining (Matx), 3: Cell morphology (Cmorph), 4:
Chondrocyte clustering (Cclus), 5: Surface architecture(SurfA), 6: Basal integration (Basl), 7: Formation of a tidemark (Ftide), 8: Subchondral bone abnormalities/marrowﬁbrosis (SubBA), 9: Inﬂammation (Inﬂ), 10: Abnormal calciﬁcation/ossiﬁcation (AbnCO), 11: Vascularization (Vasc),
12: Surface/superﬁcial assessment (SurfSp), 13: Mid/deepzone assessment (MDzone). The maximum score is 100
points and the minimum score is 0 points for each item

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2595

FIGURE 3. Quantity of released TGF-b1 measured every 2 days and total quantity released. (a) Quantity of TGF-b1 released measured every 2days up to 14 days after surgery. (b) Total quantity of released TGF-b1 measured up to 8 and 14 days after surgery.

(Table I). We did not do a single score overall assessmentin this study.
Statistical analysis
All data except the weight distribution ratio are expressed
as mean 6 standard deviation. The weight distribution ratio
is expressed as mean 6 standard error (SE). Analysis of variance was used to analyze all data. The Holm–Bonferroni
method was used for post hoc testing. Statistical signiﬁcancewas set at p < 0.05.

improved from 35.6% 6 2.1% to 43.6% 6 1.2% in Group A,
from 37.0% 6 1.4% to 43.6 6 1.4% in group B, and from
38.8% 6 2.1% to 44.5% 6 1.5% in group C. At 1 week after
surgery, the difference between groups A and C was signiﬁcant. At 2 weeks after surgery, the ratio was 36.7% 6 0.7%
in group A, 39.0% 6 0.9% in group B, and 45.5% 6 0.3% ingroup C; the differences between groups A, B, and C weresigniﬁcant. At 3 weeks after surgery, the ratio was 41.4% 6
1.5% in group A, 43.7% 6 0.4% in group B, and 45.4% 6
0.4% in group C; the differences between groups A, B, and

RESULTS

Quantity of TGF-b1 released in vitro from TGF-b1incorporated collagen vitrigel over 2 weeks
The duration and quantity of TGF-b1 released from the
TGF-b1-incorporated collagen vitrigel were measured. The
quantity of TGF-b1 released at different times after incorporation was 0.37 6 0.10 ng at 0–2 days, 0.17 6 0.02 ng at
2–4 days, 0.09 6 0.02 ng at 4–6 days, 0.10 6 0.01 ng at 6–8
days, 0.07 6 0.01 ng at 8–10 days, 0.06 6 0.01 ng at 10–12
days, and 0.05 6 0.01 ng at 12–14 days. We next measured
the sustained release of TGF-b1 for 2 weeks (Fig. 3). The
total quantity of TGF-b released was 0.73 6 0.13 ng overthe ﬁrst 8 days and 0.91 6 0.12 ng over the entire 14 days
after incorporation (Fig. 3).
Pain assessment as measured by the weight
distribution ratio between the affected andunaffected limbs
The weight distribution ratio was compared from 1 to 12
weeks after surgery (Fig. 4). From 1 to 12 weeks, the ratio

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FIGURE 4. Damaged limb weight distribution ratio (%) after surgery.
p < 0.05 was considered significant. (*) versus group A, (§) versus
group B. Damaged limb weight distribution ratio (%) 5 damaged limb
load (g)/[undamaged limb load (g) 1 damaged limb load (g)] 3 100.
The damaged limb weight distribution ratios (mean 6 SE) in weeks 1,
2, 3, 4, 6, 8, 10, and 12 after surgery are shown.

EFFECTS OF COLLAGEN VITRIGEL WITH TGF-b1 ON CARTILAGE REPAIRORIGINAL RESEARCH REPORTFIGURE 5. Histological observations of repair tissue. Samples were stained with Safranin O at 4 and 12 weeks after surgery (bar 5 1000 lm).
Higher magnification images for each sample are shown to the right of each image (bar 5 100 lm). The higher-magnification images represent
the tissue enclosed by black squares in the lower magnification images. Group A: untreated group. Group B: collagen vitrigel-implanted group.
Group C: TGF-b1-incorporated collagen vitrigel-implanted group.

C were signiﬁcant. At 4 weeks after surgery, the ratio was
41.9% 6 0.8% in group A, 44.9% 6 0.1% in group B, and44.3% 6 0.5% in group C; the differences were signiﬁcantbetween groups A and B, and between groups A and C.
However, no signiﬁcant differences were observed between
the groups at 6, 8, 10, and 12 weeks after surgery.
The time needed for signiﬁcant recovery differed
between groups: group A took 6 weeks, group B took 4
weeks, and group C took 2 weeks. Group A showed signiﬁcant improvement of the weight distribution ratio between
1 and 6 weeks after surgery, and group B showed signiﬁcant improvement between 1 and 4 weeks after surgery but
no further signiﬁcant improvement both groups. Group C
showed signiﬁcant improvement between 1 and 2 weeks
after surgery but no further signiﬁcant improvement. At 2
and 3 weeks after surgery, the differences of the weight distribution ratio between groups B and C were signiﬁcant. At
2, 3, and 4 weeks after surgery, the differences between
group A and the other groups were signiﬁcant.
Tissue assessment (characteristic of the tissue of eachgroup and ICRS II scores)
We divided the 36 Japanese white rabbits into threegroups (n 5 12) and sacriﬁced six animals from eachgroup at 4 and 12 weeks after surgery. One rabbit ingroup B, which was to be sacriﬁced at 12 weeks after
surgery, experienced a lower-leg fracture and waseuthanized. No other adverse events, such as infections,were observed during the experiment. After sacriﬁce, theright femur was collected and the osteochondral defects
were assessed using the ICRS II histological grading system (Table I, Fig. 5). We used collagen type II staining as
an indicator of the restoration of cartilage tissue, andhematoxylin and eosin staining as an indicator of inﬂammation. We visually conﬁrmed that collagen vitrigel wasabsorbed by 4 weeks after surgery.
The histology of regenerated cartilage representing the
average score for group A 4 weeks after surgery indicatesdefect ﬁlling with ﬁbrous tissue and poor defect ﬁlling rates.
At best, the histology indicates defect ﬁlling with hyaline
cartilage-like repair tissue. At worst, the defect was not
repaired at all. The histology of regenerated cartilage representing the average score for group A 12 weeks after surgery indicates defect ﬁlling with ﬁbrous tissue andcartilage-like repair tissue. At best, the histology indicatesdefect ﬁlling with hyaline cartilage-like repair tissue. At
worst, the defect was not repaired at all.
The histology of regenerated cartilage representing the
average score for group B 4 weeks after surgery indicatesdefect ﬁlling with ﬁbrous tissue and ﬁbrocartilage-like
repair tissue. At best, the histology indicates defect ﬁllingwith hyaline cartilage-like repair tissue. At worst, the defectwas not repaired at all. The histology the average and bestscore for group A 12 weeks after surgery indicates histology

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2597

FIGURE 6. Histological observations of repair tissue. Samples were stained with hematoxylin and eosin and antibody to collagen type II at 4 and
12 weeks after surgery (bar 5 1000 lm). Group A: untreated group. Group B: collagen vitrigel-implanted group. Group C: TGF-b1-incorporated
collagen vitrigel-implanted group.

almost similar to group B 4 weeks after surgery. At worst,
the histology indicates defect ﬁlling with ﬁbrous tissue andﬁbrocartilage-like repair tissue but defect-ﬁlling rate wasabout 60%.
The histology of regenerated cartilage representing the
average score for group C 4 weeks after surgery indicatesdefect ﬁlling with tissue staining for safranin O and collagen
II. At best, the histology indicates repair with cartilage like
properties. At worst, the histology indicates defect ﬁllingwith cartilage tissue but with poor lateral integration and alack of formation of a tidemark. The average, best, andworst scores of group A 12 weeks after surgery indicateshistology almost similar to group C 4 weeks after surgery.
We evaluated collagen type II staining (Fig. 6). We
observed pale weak staining in groups A and B at 4 and 12
weeks but did not observe staining in most of the repair tissue. We observed staining in most of the repair tissue ingroup C at 4 and 12 weeks. The defect in group C appeared tocontain more collagen type II compared with that in withgroups A and B. We used hematoxylin and eosin staining to
identify inﬂammation (Fig. 6). At 4 weeks, only a few sectionsof tissue from groups A and B showed mild invasion and accumulation of lymphocytes, which conﬁrmed that there was noinﬂammatory reaction in almost all of the repair tissue.
In the groups sacriﬁced at 4 weeks after surgery, the
ICRS II scores were 701.2 6 116.5 in group A, 744.2 6 97.6

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FIGURE 7. ICRS II histological grading system scores. p < 0.05 wasconsidered significant. The total score ranges from 0 (no repair) to
1300 (normal articular cartilage). Group A received an osteochondral
defect only, group B received the defect and collagen vitrigel transplantation, and group C received the defect and TGF-b1-incorporated
collagen vitrigel transplantation. The animals were sacrificed and thetissues were examined 4 and 12 weeks after surgery. (note: one rabbit
in group B was injured and euthanized during the experiment, andwas excluded from the data analysis.)

EFFECTS OF COLLAGEN VITRIGEL WITH TGF-b1 ON CARTILAGE REPAIRORIGINAL RESEARCH REPORTTABLE II. ICRS II Histological Grading System Results at 4
Weeks (Values are the Mean 6 SE)

Ti
Matx
Cmorph
Cclus
SurfA
Basl
Ftide
SubBA
Infl
AbnCO
Vasc
SurfSpMDzone
Total

A

B

C

53.7 6 11.0
42.2 6 13.7
44.2 6 13.6
28.4 6 13.5
68.7 6 10.4
40.8 6 7.9
25.6 6 8.8
43.9 6 10.7
100.0 6 0.0
65.9 6 12.0
100.0 6 0.0
38.7 6 11.9
49.2 6 14.3
701.2 6 116.5

49.8 6 14.5
45.9 6 18.2
48.1 6 16.3
25.3 6 9.6
53.0 6 13.5
53.6 6 12.5
37.0 6 9.7
52.1 6 15.4
100.0 6 0.0
79.8 6 6.8
100.0 6 0.0
42.3 6 13.0
57.3 6 14.6
744.2 6 97.6

82.0 6 5.8
75.2 6 12.7
76.3 6 9.1
64.6 6 12.6
88.3 6 3.9
73.1 6 8.6
69.4 6 9.8
80.1 6 4.7
100.0 6 0.0
85.7 6 6.31
100.0 6 0.0
75.2 6 6.9
73.9 6 12.4
1043.8 6 60.4

The maximum score is 100 points and the minimum score is 0
points for each item. The total score range is from 0 (no repair) to
1300 (normal articular cartilage). The results were as follows group(A): 701.2 6 116.5; group (B): 744.2 6 97.6; group (C): 1043.8 6 60.4.
Group (C) exhibited higher Ti, Matx, Cclus, SurfA, Basl, Ftide, SubBA,
SurfSp, and MDzone than groups (A) and (B). Groups (A) and (B) did
not have major differences.

in group B, and 1043 6 60.4 in group C (Fig. 7). The differences were signiﬁcant between groups A and C, andbetween groups B and C. For the individual ICRS II items
assessed, Matx, Cmorph, and Ftide differed signiﬁcantly
between groups C and A, and between groups B and C. The
values for all ICRS II items excluding Inﬂ and Vasc werehigher in group C than in groups A and B. For the individual
ICRS II items assessed, groups A and B did not have majordifferences (Table II).
In the groups sacriﬁced at 12 weeks after surgery, the
ICRS II score was 740.9 6 79.8 in group A, 809.9 6 68.9 ingroup B, and 1059.5 6 58.2 in group C (Fig. 7). The difference was signiﬁcant between groups A and C, and between
groups B and C. For the individual ICRS II items assessed,
Ti, Matx, Cmorph, and Ftide differed signiﬁcantly between
groups C and A, and between groups C and B. The values
for all ICRS II items excluding Inﬂ and Vasc were higher ingroup C than in groups A and B. For the individual ICRS II
items assessed, groups A and B did not have major differences (Table III). In group A, no signiﬁcant differences wereobserved for total ICRS II scores between 4 and 12 weeks
after surgery. Similarly, no signiﬁcant differences wereobserved between 4 and 12 weeks for either groups B or C.
DISCUSSION

Several in vitro studies of carriers for the sustained releaseof TGF-b have been reported.45–47 This study is the ﬁrst to
perform in vitro experiments to assess collagen vitrigel as asustained-release carrier for TGF-b1. Measurement of theduration and quantity of TGF-b1 released showed a sustained release of 0.37 ng over the ﬁrst 2 days after impregnation, which accounted for 40% of the total quantity
released over the 14 days of measurement. Thereafter, thequantity released decreased gradually. During the second
week following incorporation, sustained release of 0.2 ng
was observed, which conﬁrmed the sustained, slow releaseof TGF-b1. Nicoll et al.45 reported a sustained release of0.5% TGF-b1 injected into a gel during the ﬁrst week. We
measured the sustained release of 3% of the injected TGFb1 during the ﬁrst week after incorporation. This conﬁrmed
the sustained release of TGF-b from the collagen vitrigel.
Research on the mechanism of action of TGF-b in cultures of chondrocytes derived from mesenchymal stem cells
has shown that 10 ng/mL is the optimum concentration of
TGF-b during culture and that 1 week is the optimum duration of action after administration.19,22,38–42 Continued sustained release of a large quantity of TGF-b inside a joint
should be prevented because this can increase the formation of cartilaginous tissue.48,49 Thus, it is believed that boththe environment and carrier must allow for early sustained
release of an appropriate quantity of TGF-b to allow it toact effectively on the cartilage defect. In this regard, becauseof the conﬁrmed sustained-release duration of 2 weeks, wefeel that the collagen vitrigel satisﬁes this requirement.
Analysis of the affected parts of rabbits sacriﬁced at 4
weeks after surgery also showed that no collagen vitrigel
remained; therefore, the collagen vitrigel is believed to havebeen absorbed within 4 weeks.
The weight distribution ratio between the affected andunaffected limbs was measured as an indication of postoperative pain. This ratio improved at 12 weeks after surgery
and did not differ signiﬁcantly between groups. However,group A required 6 weeks to reach maximum improvement,
whereas groups B and C showed near-maximum improvement at 4 and 2 weeks after surgery, respectively. Itoet al.15 used a similar method to assess the weight

TABLE III. ICRS Histological Grading System Results at 12
Weeks (Values are the Mean 6 SE)

Ti
Matx
Cmorph
Cclus
SurfA
Basl
Ftide
SubBA
Infl
AbnCO
Vasc
SurfSpMDzone
Total

A

B

C

52.6 6 10.6
35.2 6 11.9
38.9 6 13.6
24.3 6 10.5
67.1 6 6.1
58.8 6 12.3
30.3 6 8.2
65.1 6 12.3
100.0 6 0.0
69.8 6 11.0
100.0 6 0.0
54.4 6 8.8
44.3 6 11.7
740.9 6 79.8

63.6 6 9.6
39.9 6 10.3
37.1 6 9.6
24.5 6 6.0
63.8 6 13.8
72.8 6 7.0
47.6 6 8.0
70.1 6 7.9
100.0 6 0.0
78.3 6 6.14
100.0 6 0.0
55.6 6 11.8
56.7 6 9.9
809.9 6 68.9

91.4 6 2.4
85.0 6 7.5
79.6 6 7.9
70.7 6 12.9
82.3 6 4.3
73.2 6 5.9
71.0 6 7.4
70.0 6 6.8
100.0 6 0.0
80.8 6 6.3
100.0 6 0.0
75.1 6 6.5
80.6 6 7.9
1059.5 6 58.2

The maximum score is 100 points and the minimum score is 0
points for each item. The total score range is from 0 (no repair) to
1300 (normal articular cartilage). The results were as follows group(A): 740.9 6 79.8; group (B): 809.9 6 68.9; group (C): 1059.5 6 58.2.
Group (C) exhibited higher Ti, Matx, Cclus, Ftide, SurfSp, and MDzone
than groups (A) and (B). Groups (A) and (B) did not have majordifferences.

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distribution ratio in their research on the repair of articularcartilage defects using cell sheets and reported changes up
to 4 weeks. Ito et al.15 reported that the weight distribution ratio improved from 35.6% 6 1.1% to 49.6% 6 0.1%
in the ﬁrst 4 weeks in articular cartilage treated with cartilage cell sheets. Group C in our study exhibited improvement by 3 weeks after surgery similar to that in the
animals in the study by Ito et al.15 However, we found no
improvement in the weight distribution ratio after thispoint. We believe that the cell sheets provide sustained
release of various cytokines in addition to TGF-b1 andthat these also contribute to articular cartilage repair.17
Regardless of whether other cytokines are involved, thesustained release of TGF-b1 alone seems to be related tothe similar improvements in early pain assessment ingroup C in this study and the cell sheet-treated group in
the study by Ito et al.
The histological assessment showed that group C exhibited signiﬁcant improvement relative to groups A and B at 4
and 12 weeks after surgery. No signiﬁcant differences wereobserved between 4 and 12 weeks within each group. These
results suggest that the early, sustained, slow release of
TGF-b1 into the cartilage defect contributed to improved
tissue repair starting immediately after surgery. For theitems of the ICRS II score, improvements were observed inmany items, and these improvements are considered to be acharacteristic effect of TGF-b1. We conﬁrmed that subchondral bone intruded into the repair cartilage area with somesamples of the group for 12 weeks. We believe that subchondral bone intruded into the space that was not ﬁlled
because of slow or poor repair. In a study in which liposomal TGF-b1 was injected to treat articular cartilage defects,
Abe et al.20 found that the extent of improvement did not
differ between the group injected with TGF-b1 alone andthe control group but that the tissue was repaired withﬁbrous cartilage tissue. In our study, we observed that the
repair tissue contained hyaline cartilage, which suggeststhat the sustained, slow release of TGF-b1 stimulated regeneration by a better quality of cartilage. By contrast, in astudy using a hydrogel scaffold and TGF-b used to treat
articular cartilage defects, Holland et al.46 reported that thesustained, slow release of TGF-b alone failed to produce amore favorable improvement in tissue compared with anuntreated group. The possible reasons for the different ﬁndings include differences in the environment between in vivo
and in vitro experimentation or differences in the defectposition and size. Collagen vitrigel is a carrier with good
tissue-engraftment capacity and whose implantation has
been reported to induce tissue repair.32,33,35,36 This implies
that, compared with other carriers, the sustained, slow
release of TGF-b into articular cartilage produces more
effective results.
The ICRS II score did not show signiﬁcant differences
between groups A and B for the items. And there were no
signiﬁcant differences in the total score between thesegroups, which suggests that there was no effective cartilage
repair in the vitrigel-only-treated group (group B).

2600

MARUKI ET AL.

The present study suggests that sustained, slow releaseof TGF-b1 over 2 weeks contributes to early improvementin pain and tissue repair of articular cartilage. The results
also conﬁrm that collagen vitrigel is useful as a sustainedrelease carrier. However, although we identiﬁed areas wherecollagen vitrigel improved the quality of the repaired cartilage tissue, treatment with collagen vitrigel alone did not
produce the same effects as those of the TGF-bincorporated collagen vitrigel. Our study conﬁrmed that sustained, slow release of TGF-b1 occurred for 2 weeks andthat 40% of the total quantity was released in the ﬁrst 2
days; however, the mean sustained release could not be conﬁrmed. The quantity of TGF-b1 released (0.91 ng) was alsoless than the intended 10 ng. Further improvement to thecarrier to achieve the sustained release of a greater quantityof TGF-b1 into articular cartilage defects is a futurechallenge.
Further evaluation of the repair tissue is needed before
this technology can be applied to humans. Collagen vitrigel
offers the advantages of easy manufacturing and handling ofvarious sizes. In this study, we conﬁrmed that collagen vitrigel was effective as a controlled-release carrier in a rabbit
osteochondral defect model. We plan to evaluate further theduration of the controlled-release effect and optimum quantity of TGF-b incorporated into the gel using both in vitro
and larger animal studies.
CONCLUSION

Collagen vitrigel is an effective carrier for the sustained
release of TGF-b1. Implantation of TGF-b1-incorporated collagen vitrigel increased the early mitigation of pain andarticular cartilage repair.
ACKNOWLEDGMENTS

We are grateful to the Education and Research Support Center,
Tokai University.
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EFFECTS OF COLLAGEN VITRIGEL WITH TGF-b1 ON CARTILAGE REPAIR