Regenerative Therapy 9 (2018) 24e31

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Regenerative Therapy
journal homepage: http://www.elsevier.com/locate/reth

Original Article

Cartilage repair and inhibition of the progression of cartilage
degeneration after transplantation of allogeneic chondrocyte sheets ina nontraumatic early arthritis model
Naoki Takatori 1, Masato Sato*, Eriko Toyoda, Takumi Takahashi, Eri Okada, Miki Maehara,
Masahiko Watanabe
Department of Orthopaedic Surgery, Surgical Science, Tokai University School of Medicine, 143 Shimokasuya, Isehara, Kanagawa 259-1193, Japana r t i c l e i n f o

a b s t r a c t

Article history:
Received 6 July 2018
Received in revised form
19 July 2018
Accepted 22 July 2018

Introduction: Using a rat model of nontraumatic early arthritis induced by intra-articular administrationof low-dose monoiodoacetic acid (MIA), we transplanted allogeneic chondrocyte sheets and examinedthe effects on tissue repair.
Methods: MIA (0.2 mg/50 ml) was injected into the right knee of 20 male Wistar rats. Four weeks later,rats were randomly allocated into three groups: Group A was examined 4 weeks after administration of
MIA; Group B, 8 weeks after MIA injection and chondrocyte sheet transplantation, and Group C, 8 weeks
after MIA injection but without chondrocyte sheet transplantation. Allogeneic chondrocyte sheets weretransplanted into the right knee of Group B rats. Pain was assessed as the weight distribution ratio of thedamaged to undamaged limb. The OARSI score was used for histological scoring.
Results: The limb weight distribution ratio indicated signiﬁcantly less pain in Group B. Histologicalscoring showed signiﬁcant differences in cartilage repair and inhibition of the progression of cartilage
degeneration between Groups B and C, but not between Groups A and B, or Groups A and C.
Conclusions: These ﬁndings suggest that, in this rat model of nontraumatic early arthritis induced by
low-dose MIA injection, allogeneic chondrocyte sheet transplantation induces cartilage repair andsuppresses cartilage degeneration.
© 2018, The Japanese Society for Regenerative Medicine. Production and hosting by Elsevier B.V. This is
an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Keywords:
Osteoarthritis
Chondrocyte sheet
Transplantation
Monoiodoacetic acid (MIA)OARSI score

1. Introduction
Osteoarthritis (OA) is one of the most prevalent joint diseases,
especially in mature women [1]. The condition is characterized byAbbreviations: AB, Antibiotic-antimycotic solution; Acan, Aggrecan; Col1A1,
Collagen type Ⅰalpha 1; Col2A1, Collagen type Ⅱalpha 1; Comp, Cartilage oligomericmatrix protein; DMEM, Dulbecco's modiﬁed Eagle's medium; DPBS, Dulbecco's
phosphate-buffered saline; EDTA, Ethylenediaminetetraacetic acid; FBS, Fetal
bovine serum; IFP, Infrapatellar fat pad; ITGa10, Integrin alpha-10; MIA, Monoiodoacetic acid; Mmp13, Matrix metalloproteinase-13; OA, Osteoarthritis; OARSI,
Osteoarthritis research society international; PVDF, Polyvinylidene diﬂuoride;qPCR, Quantitative real-time polymerase chain reaction.
* Corresponding author. Fax: þ81 463 96 4404.
E-mail addresses: n.tori001@tsc.u-tokai.ac.jp (N. Takatori), sato-m@is.icc.u-tokai.
ac.jp (M. Sato).
Peer review under responsibility of the Japanese Society for Regenerative
Medicine.
1
Fax: þ81 463 96 4404.

degenerative changes in articular cartilage, which cause pain andmalfunctions as it progresses [2,3]. Structural changes include lossof articular cartilage, subchondral bone sclerosis, and formation ofcysts and osteophytes [4]. The low cellularity and avascular natureof articular cartilage means that its capacity to self-regenerate after
injury or degeneration is limited [5]. Mild OA is treated conservatively with oral administration of nonsteroidal anti-inﬂammatory
drugs, intra-articular injection of corticosteroid [6] and/or hyaluronic acid [7], and exercise therapy [8]. However, surgical treatment may be required as the disease progresses. The main
treatments are high tibial osteotomy and total knee arthroplasty.
Various arthritis models, such as surgical intervention and intraarticular drug injection, have been used to study cartilage regeneration. The anterior cruciate ligament resection model is the surgical intervention model used most frequently. In this model,
anterior cruciate ligament resection triggers cartilage degeneration,
subchondral bone sclerosis, and osteophyte formation, whichmimic the pathological changes observed in human OA [9e11].

https://doi.org/10.1016/j.reth.2018.07.003
2352-3204/© 2018, The Japanese Society for Regenerative Medicine. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/).

N. Takatori et al. / Regenerative Therapy 9 (2018) 24e31

Intra-articular injection of monoiodoacetic acid (MIA) is one
method for inducing OA experimentally. MIA inhibits glyceraldehyde-3-phosphate dehydrogenase activity in chondrocytes andcauses cell death and decreased proteoglycan content. The reaction
is simple, rapid, and reproducible [12e15]. Some reports indicatethat the OA caused by MIA-induced chemical induction is different
from human OA [16,17]. On the other hand, Sangeetha et al. andothers have reported histological and morphological changes in the
MIA model similar to those in human OA [18e23]. Udo et al. [24]reported that the changes caused by MIA depend on the dose andtiming, and that low-dose administration is useful for evaluating
the pathology of OA, pain mechanisms, and therapeutic effects.
Mohan et al. [25] reported that the low-dose MIA-induced arthritis
model is suitable for studying nontraumatic OA and the therapeutic
effects on cartilage and bone.
We have transplanted chondrocyte sheets into areas showing
traumatic changes in an articular cartilage damage model in various
animals and have conﬁrmed its effectiveness [26e28]. The purposeof this study was to determine whether the repair or inhibition of
the degeneration of damaged cartilage can be obtained by transplanting allogeneic chondrocyte sheets in a nontraumatic arthritis
model of OA induced by intra-articular injection of low-dose MIA.
2. Materials and methods
All procedures using animals in this study were performed inaccordance with the Guide for the Care and Use of Laboratory
Animals (NIH Publication No. 8023, revised 1978) published by the
National Institutes of Health, USA, and the Guidelines of Tokai
University on Animal Use. This animal experiment was approved by
the Animal Committee of Tokai University (permission number
171059).
2.1. Animals
Twenty male Wistar rats (CLEA Japan Inc., Tokyo, Japan) at 8
weeks of age and three male Lewis rats (Charles River Japan Inc.,
Kanagawa, Japan) at 6 weeks of age were used for the study. The
animals were housed under normal conditions for 2 weeks before
the start of the experiments to acclimate them to the environment.
One to two rats were housed per cage in sterile conditions, androdent chow and water were allowed ad libitum.
2.2. Chondrocyte sheets
2.2.1. Harvest of chondrocytes and fabrication of chondrocyte
sheetsTo prepare allogeneic cell sheets for transplantation, three 6week-old male Lewis rats were used. The rats were sacriﬁced byan overdose of 50% isoﬂurane. A medial parapatellar incision wasmade in both legs, each patella was dislocated laterally, and thecollateral ligaments and the anterior and posterior cruciate ligaments were severed. The joint capsule on the hip joint side wasincised, and the femur was extracted. The articular cartilage tissue
was collected with a scalpel from the bilateral femoral head andbilateral femoral condyle. Cells were isolated from the tissue
sample using an enzymatic procedure and then incubated in Dulbecco's modiﬁed Eagle's medium/F12 (DMEM/F12; Gibco, Grand
Island, NY, USA) supplemented with 20% fetal bovine serum (FBS;
AusGeneX, Molendinar, Australia), 1% antibioticeantimycotic solution (AB; Gibco), and 5 mg/ml collagenase type 1 (Worthington
Biochemical, Mannheim, Germany) for 3 h at 37  C in a humidiﬁed
atmosphere of 5% CO2 and 95% air. The cell suspension was washedand passed through a 100 mm strainer (BD Falcon, Franklin Lakes,
NJ, USA).

25

The collected cells were seeded at a density of 1  104 cells/cm2
in a 150 cm2 culture ﬂask in DMEM/F12 supplemented with 20%
FBS and 1% AB, and incubated at 37  C. After 3 days, 100 mg/ml
ascorbic acid (Nissin Pharmaceutical, Yamagata, Japan) was added
to the medium, and the medium was replaced every 3 or 4 days.
Cells were passaged once when they reached conﬂuence and thencryopreserved.
To fabricate cell sheets, cells were thawed, passaged once, andthen seeded onto UpCell® inserts (CellSeed Inc., Tokyo, Japan) at
1  104 cells/cm2. Ascorbic acid was added, the medium was
replaced every 3 or 4 days, and the cells were cultured for 14 days.
To harvest the cells using the characteristics of the UpCell insert,
the culture temperature was lowered to <25  C, which caused thechondrocytes to detach, after which they were preserved forfurther analysis.
2.2.2. Cell counting
The cell sheets were washed in Dulbecco's phosphate-bufferedsaline (PBS; Gibco). The sheets were then incubated in TripLE Express® (Gibco) at 37  C for 40 min and centrifuged at 1500 rpm for
5 min. The cell sheets were resuspended in 0.25 mg/ml collagenase
P (Roche, Basel, Switzerland) at 37  C for up to 5 min and thencentrifuged at 1500 rpm for 5 min. Finally, the isolated cells were
resuspended in DMEM/F12, and the cells were counted using a
trypan blue exclusion assay.
2.2.3. Histological staining
The collected cell sheets were ﬁxed with 20% formalin (Wako
Pure Chemical, Japan) and embedded in parafﬁn wax. The tissue
was cut into 3 mm sections, which were then deparafﬁnized according to standard procedures, and stained with hematoxylineeosin. The sections were stained with Safranin O in a 0.08%
fast green aqueous solution and 0.1% Safranin O aqueous solution.
The sections were also stained with a 0.05% toluidine blue solution.
2.2.4. Immunohistochemical staining
Parafﬁn-embedded tissue was sectioned at 3 mm and deparafﬁnized using standard procedures. The sections were stained forcollagen type I with goat anti-type I collagen-UNLB (Southern
Biotech, USA). The sections were washed with PBS and processedusing an ImmPRESS Anti-goat Ig Reagent Kit (Vector, USA) and theslides were sealed. Sections for collagen type II staining weredeparafﬁnized and then reacted with Anti-hCL (II) puriﬁed IgG(Daiich Fine Chemical, Japan). The sections were washed with PBS
and processed using an ImmPRESS Anti-mouse Ig Reaction Kit(Vector), and the slides were sealed.
2.2.5. RNA isolation, cDNA synthesis, and quantitative real-time
PCR
Three samples of passage 1 chondrocytes and three samples ofcultured cartilage cell sheets were analyzed. The recovered chondrocyte sheets were crushed at 1500 rpm for 3 min using SHAKE
Master Neo (Bio Medical Science, Japan). Total RNA was extractedusing an RNeasy Mini Kit (Qiagen, Germany) according to the manufacturer's instructions. Next, ﬁrst-strand cDNA was synthesized using 1 mg of total RNA and the enzyme, buffer, and primer of a
QuantiTect Reverse Transcription Kit (Qiagen) and elongated using a
GeneAmp PCR system 9600 (Thermo Fisher Scientiﬁc, USA) at 42  C
for 15 min and 95  C for 3 min. Quantitative real-time polymerase
chain reaction (qPCR) was performed using an Applied Biosystems
7300 Real-Time PCR system (Applied Biosystems, USA). The primers
were as follows for TaqMan® Gene Expression Assays (Applied Biosystems, USA): Col1A1 (Rn01637087_m1), Col2A1 (Rn01463848_m1),
Sox9(Rn01751070_m1),
Acan(Rn00573424_m1),
ITGa10(Rn01533928_m1),
Mmp13(Rn01448194_m1),
Lect1

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N. Takatori et al. / Regenerative Therapy 9 (2018) 24e31(Rn00578277_m1),
Comp(Rn00563255_m1),
and
Gapdh
(Rn01775763_g1). The ﬁnal reaction volume was 20 ml, and thethermocycling conditions were as follows: 50  C for 2 min, 95  C for
10 min, and 95  C for 15 s and 60  C for 1 min for 40 cycles. The
expression level of the internal control GAPDH was used as ahousekeeping gene, and the comparative 2⊿⊿Ct method was used foranalysis.
2.2.6. Evaluation of cell sheets
We measured cell number and viability and cell sheet thickness,and conﬁrmed the existence of the cartilage matrix in tissue sections. We also used qPCR to quantify the gene expression levels inchondrocyte sheets compared with passage 1 chondrocytes.
2.3. Induction of OA by MIA
We used 20 male Wistar rats at 8 weeks of age. With the animal
under anesthesia with isoﬂurane and oxygen inhalation, both knees
were shaved and disinfected. Under sterile conditions, a 1 cm
incision was made in the skin via the medial parapatellar approach
to expose the patellar tendon. The knee joint was ﬂexed 90 , andMIA (0.2 mg dissolved in 50 ml of physiological saline; SigmaeAldrich, St. Louis, MO, USA) was injected into the knee joint
through a 27 G needle. We considered the rise of joint pressure due
to intraarticular administration, so the same volume of saline wasinjected into the left knee joint using the same method. After injection, the skin was sutured.
2.4. Transplantation of chondrocyte sheets
Four weeks after MIA administration, 20 rats were randomly
allocated to three groups. All rats were injected with MIA to induce
OA. Group A rats (n ¼ 6) were sacriﬁced 4 weeks after MIA injection. Group B rats (n ¼ 8) received chondrocyte sheet transplantation 4 weeks after MIA injection and were sacriﬁced 4 weeks
later (i.e., 8 weeks after MIA injection). Group C rats (n ¼ 6) did notreceive chondrocyte sheets and were sacriﬁced 8 weeks after MIA
injection. For chondrocyte sheet transplantation, Group B rats wereanesthetized with isoﬂurane and oxygen. Under sterile conditions,both knees were shaved and disinfected, a ~2 cm skin incision wasmade to the medial side of the right knee using the parapatellar
approach, and the patella was dislocated to the lateral side. Next,
the medial side of the knee joint capsule was dissected and the joint
was exposed. A chondrocyte sheet that had been cultured for 14
days was transplanted into the femoral condylar region, the dislocation of the patella was reduced, and the joint capsule and skinwere sutured.
A sham operation was performed for the joint capsule of the left
knee joint of Group B rats and both knee joints of Group C rats.
2.5. Pain evaluation
An incapacitance meter (BrainScience Idea Co., Ltd, Japan) wasused to detect changes in the distribution ratio of the damaged limb
to the undamaged limb, and the ratio served as the gauge for
evaluating pain. This device is used widely to investigate pain andpain-alleviating effects [27,29,30]. The measurements were madewhen the rat's hind legs were both positioned over the platforms
and the rat was stationary. The weight distribution of both hind legs
was measured 10 times, and the following formula was used to
calculate the limb weight distribution ratio (%).
Limb weight distribution ratio (%) ¼ damaged limb load (g)/(undamaged limb load (g) þ damaged limb load (g))  100

After MIA injection, the weight distribution was measured 14
times on days 1, 7, 14, 21, 28, 29, 31, 35, 38, 42, 45, 49, 52, and 55.
Fig. 1 shows the schedule for MIA administration, transplant
surgery, and load measurement.
2.6. Histological evaluation
Four weeks after MIA injection, six rats in Group A were sacriﬁced, and 4 weeks after transplantation of chondrocyte sheets (8weeks after MIA injection), eight rats in Group B and six rats inGroup C were sacriﬁced. The knee joint was exposed using themedial parapatellar approach and the collateral ligaments andanterior and posterior cruciate ligaments were severed. The femur
and the tibia were then separated.
The femur and tibia were ﬁxed in 20% formalin (Wako Pure
Chemical) for 5e6 days. Each section was degreased and demineralized with acetone solution (Wako Pure Chemical) and ethylenediaminetetraacetic acid (EDTA) solution (Wako Pure Chemical).
It was ﬁxed again with 20% formalin, decalciﬁed with EDTA solution, and embedded in parafﬁn wax. In the rat, the load distribution
on the medial side of the femur is higher than on the lateral side
[23,31,32]; therefore, the section of the medial femoral condyle wascut in the sagittal direction (3 mm sections). The sections were
stained with Safranin O, collagen type I staining, and collagen type
II staining in the same protocol. Microscopic images were captured
under a BZ-9000 Biorevo ﬂuorescence microscope (Keyence,
Japan).
Bone samples from Groups A, B, and C were analyzed histologically. We evaluated the distal part of the femoral articular cartilage
and the intermediate region of the articular cartilage of the tibia
based on the cartilage region determination method of Nomura
et al. [33]. The Osteoarthritis Research Society International (OARSI)OA cartilage index system was used to score the histopathology, asestablished by Pritzker et al. [34]. The scoring system evaluates thegrade according to the depth of cartilage damage and the stage
according to the extent of damage over the joint surface. The score
for grade  stage is calculated and can range from 0 to 24, withhigher scores indicating more advanced cartilage degeneration. We
show the OARSI score (Table 1). Scoring was performed by three
single-blinded examiners.
2.7. Statistical analysis
The results are expressed as the mean ± standard deviation (SD).
Gene expression levels in the cell sheets were analyzed using theManneWhitney U test. A p-value < 0.05 was considered to indicate
a signiﬁcant difference; however, changes in gene expression <2fold are considered to be within the physiological limit and,
therefore, such changes were considered to be unchanged despite
being statistically signiﬁcant. The damaged limb weight distribution ratio and histological assessment as measured by the OARSI
score were evaluated using analysis of variance. The TukeyeKramer
test was used for post hoc testing. A p-value < 0.05 was deemed tobe signiﬁcant.
3. Results
3.1. Sheet thickness, cell count, and tissue evaluation of chondrocyte
sheetsThe macroscopic and microscopic ﬁndings for cell sheets areshown in Fig. 2A and B. Chondrocyte sheets contained cells at a highdensity, and these cells were easily harvested and manipulated
without tearing after 14 days of culture. Chondrocyte sheets contained an average 1.32 ± 0.28  106 cells and had a survival rate of

N. Takatori et al. / Regenerative Therapy 9 (2018) 24e31

27

Fig. 1. Experimental schedule. Four weeks after administration of MIA, Group A was sacriﬁced, and Groups B and C were subjected to cell sheet transplantation surgery. At 8 weeks
after MIA administration, Group B and C were sacriﬁced.

Table 1OARSI osteoarthritis cartilage histopathology scoring.
Grade (key feature)

Stage % Involvement (surface, area, volume)
Stage 1 (<10%)Stage 2 (10e25%)

Stage 3 (25e50%)

Stage 4 (>50%)

Grade 1 (surface intact)
Grade 2 (surface discontinuity)
Grade 3 (vertical ﬁssures, clefts)
Grade 4 (erosion)
Grade 5 (denudation)
Grade 6 (deformation)
Score ¼ grade  stage

1
2
3
4
5
6

2
4
6
8
10
12

3
6
9
12
15
18

4
8
12
16
20
24

Fig. 2. Macroscopic and histological ﬁndings, and gene expression in cell sheets. (A) Representative images of a cell sheet attached to a white polyvinylidene membrane. (B)Hematoxylineeosin, Safranin O, toluidine blue, and collagen type II staining of the cartilage matrix. Collagen type I did not stain (40, scale bar ¼ 50 mm). (C) The expression of
Col1A1 and ITGa10 increased, but there was no signiﬁcant difference (p > 0.05). Expression of Sox9 and Mmp13 increased signiﬁcantly but not more than 2-fold (*p < 0.05, <2-fold).
Expression of Col2A1, Acan, Lect1, and Comp increased signiﬁcantly by >2-fold (*p < 0.05, >2-fold).

81.3% ± 2.4% and an average thickness of 29.53 ± 2.23 mm. The
sheets formed a thick structure with integrated layers, which were
examined histologically and by staining the cartilage matrix with
Safranin O and toluidine blue.

values were 1.5 times for Col1A1, 4.73 times for Col2A1, 1.6 times for
Sox9, 3.10 times for Acan, 1.23 times for ITGa10, 1.83 times for
Mmp13, 58.63 times for Lect1, and 9.57 times for Comp. This analysis
conﬁrmed an increase in the expression of all genes analyzed.

3.2. qPCR of chondrocyte sheets

3.3. Pain evaluation

Gene expression in three chondrocyte sheets was evaluated andcompared with that in passage 1 chondrocytes (Fig. 2C). The relative gene expression of chondrocytes was set at 1.0; the relative

The normal limb weight distribution ratio is 50%. Fig. 3Ashows the changes in the weight distribution ratio with timeafter MIA injection in each group. Up to 4 weeks after MIA

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N. Takatori et al. / Regenerative Therapy 9 (2018) 24e31

injection, the ratio did not differ signiﬁcantly between anygroups. Fig. 3B shows the limb weight distribution ratio on thelast day was 47.4% ± 1.0% in Group A, 49.4% ± 0.2% in Group B,
and 48.4% ± 0.6% in Group C; the value was signiﬁcantly higher inGroup B than in the other two groups.

Group B, the superﬁcial cartilage layer and partial staining wereretained in the femur, and only mild damage was found in thesurface cartilage layer of the tibia. Immunostaining showedcollagen type II staining in all intact cartilage, but collagen type I
staining was observed only in the superﬁcial regions of cartilage innormal, Group A, and Group B.

3.4. Macroscopic evaluation
3.6. Histological scoring
No signs of infection such as swelling or redness of the knee
joint were observed macroscopically throughout the entire study.
Fig. 3C shows the macroscopic ﬁndings for femoral condylar andtibial articular cartilage in each group compared to normal. Normal
femoral condylar articular cartilage did not stain with indigo
carmine and its surface was smooth. In Group A, damage wasobserved in the medial condyle. Minor injury was observed inGroup B, and severe injury was observed in the medial and lateralcondyles in Group C. Tibial articular cartilage damage was observedin all groups as for the femur, but there was less evidence of erosion
in Group B than in Group C.
3.5. Histological evaluation
Fig. 4A shows the histological ﬁndings for femoral condylar
articular cartilage and tibial articular cartilage. Safranin O stainingof the femur and tibia showed loss of chondrocytes and decreased
proteoglycan staining in Group A, but the superﬁcial cartilage layer
was stable. Similar ﬁndings were observed in Group C, but thinning of the articular cartilage was also observed. By contrast, inFig. 4B shows the OARSI scores for the femur and tibia in thethree groups. The score for the femur was signiﬁcantly lower(indicating less damage) in Group B (5.83 ± 3.02 points) than inGroup A (8.28 ± 4.87 points) and Group C (11.5 ± 4.5 points). For thetibia sites, the scores did not differ signiﬁcantly between groups:
9.89 ± 5.09 points in Group A, 11.33 ± 4.57 points in Group B, and13.83 ± 2.26 in Group C.
4. Discussion
We have previously investigated defects created in articularcartilage of various animals such as rats [26], rabbits [27], and mini
pigs [28], and we have transplanted chondrocyte sheets to conﬁrm
the efﬁciency of articular cartilage repair. The purpose of this study
was to investigate the application of allogeneic chondrocyte sheets
in a rat model of nontraumatic early arthritis.
The prepared chondrocyte sheets had a survival rate of 80% or
more, sufﬁcient thickness, abundant extracellular matrix, and signiﬁcant increase in the expression (>2-fold) of Col2A1, Acan, Lect1,

Fig. 3. Time course after administration of MIA, limb weight distribution ratio on the last day of each group, and macroscopic ﬁndings for femoral condylar articular cartilage. (A)The limb weight distribution ratio decreased transiently after administration of MIA and after transplantation surgery. At 28 days after MIA administration, the ratios did not differ
signiﬁcantly (p > 0.05) between any group: 47.4% ± 1.0% in Group A, 47.7% ± 0.3% in Group B, and 47.6% ± 0.6% in Group C. (B) On the last day, the limb weight distribution ratio wassigniﬁcantly higher in Group B than in the other two groups: 47.4% ± 1.0% in Group A, 49.4% ± 0.2% in Group B, and 48.4% ± 0.6% in Group C (**p < 0.01 for Group A vs B; *p < 0.05 forGroup B vs C; p > 0.05 for Group A vs C). (C) The upper panel shows unstained samples, and the lower panel shows samples stained with indigo carmine. Compared with undamaged tissue, Group A showed damage to the medial condyle, and Group B showed less damage. More damage was observed in the lateral condylar region in Group C.

N. Takatori et al. / Regenerative Therapy 9 (2018) 24e31

29

Fig. 4. Histological ﬁndings in the femur and tibia, and OARSI scores. (A) Undamaged femoral condylar Safranin O staining was observed in Group A with loss of chondrocytes andproteoglycans. Group B showed a cartilage layer and some Safranin O staining. Group C showed erosion and thinning of the cartilage layer. No peeling of the transplanted cartilage
sheet or uninitiated parts were observed in Group B. Safranin O staining of the normal tibia and loss of chondrocytes and proteoglycans were observed in Group A, as seen in the
femur. Compared with the femur in Group B, although the cartilage layer remained, slight erosion was observed and this tissue did not stain with Safranin O in Group A. In Group C,
erosion and thinning of the cartilage layer were observed (20, scale bar ¼ 50 mm). Immunostaining of the femur showed collagen type II staining in all intact cartilage, but collagentype I staining was observed only in the superﬁcial regions of cartilage in normal, Group A, and Group B. Immunostaining of the tibia showed both collagen type I and collagen type
II staining (20, scale bar ¼ 50 mm). (B) The OARSI score for the femur differed signiﬁcantly between Groups B and C but not between Groups A and B, or Groups A and C (*p < 0.05,
Group B vs C; p > 0.05, Group A vs B, and Group A vs C). The OARSI score for the tibia did not differ signiﬁcantly between the three groups.

and Comp. Collagen type II and aggrecan are the major components
of cartilage matrix and are essential for the maintenance of normalchondrocytes [35]. Collagen type II is resistant to tensional force[36]. Aggrecan is also involved in providing resistance to joint
loading by forming a complex through interactions with proteinssuch as hyaluronic acid [37]. Lect1 encodes a chondromodulin I
protein, and Miura et al. [38] reported that it has an antiangiogenic
effect in cartilage tissue and promotes cartilage formation. The
STAT signaling pathway regulates transcription of multiple genesthat regulate cell proliferation and differentiation, including osteoblast differentiation, and bone-related factors such as alkaline
phosphatase and collagen type X [39,40]. Chondromodulin I inhibits the STAT signaling pathway [41]. Klinger et al. [42] reported
that chondromodulin I strongly suppresses chondrocyte hypertrophy and endochondral ossiﬁcation and helps to stabilize cartilage
repair tissue.
Cartilage oligomeric matrix protein (COMP) is a noncollagenous
protein that is important for multicellular networks and is known
to stimulate chondrocyte proliferation and cartilage formation[43,44]. Some have reported interactions between COMP andcollagen types I and II, and that these interactions play a role inmaintaining the stability of the collagen network [45,46]. Although
COMP is found in various tissues, it is most abundant in tissues thatbear mechanical loading such as tendons and cartilage [47]. Ourdata suggest that chondrocyte sheets exhibit sufﬁcient expressionof genes important to maintaining a stable cartilage matrix.
Using the limb load distribution ratio as an index of pain, we
observed a signiﬁcant pain-alleviating effect in the rats thatreceived transplanted chondrocyte sheets. Our results are similar to
those reported for transplantation of allogeneic chondrocyte sheets
in a rabbit articular cartilage defect model at 4 weeks after

transplantation reported by Ito et al. [27] and 12 weeks after
transplantation reported by Tani et al. [30].
Macroscopic observations showed no inﬂammatory ﬁndings in
the knee such as swelling or reddening. In addition, the transplanted group (Group B) showed evidence of suppression of cartilage degeneration compared with the nontransplanted groups(Groups A and C), which did not receive transplanted sheets.
Histologically, the adhesion of the margin and bottom of the cellsheet was good, and tissue was signiﬁcantly repaired on the femur
sites. On the tibia sites, there was no signiﬁcant difference between
groups that received or did not receive chondrocyte sheet transplantation. However, there was a tendency toward less progressionof cartilage degeneration in Group B. In this study, the cell sheet
was transplanted into the femoral condylar aspect, and signiﬁcantcartilage repair was obtained on the femur side. These ﬁndings
suggest that degeneration of the tibia sites may have been suppressed by a paracrine effect.
Udo et al. [24] also used an MIA arthritis model, but ourmacroscopic ﬁndings and histological scoring of Groups A and C
were more favorable in our study. As noted earlier, the effects of
MIA depend on the timing and dose. Although there are no reportsof the residual time in joints, Udo et al. reported the progress of
cartilage degeneration for 12 weeks after low-dose MIA injection.
Future studies should extend the observation period and investigate the effectiveness of chondrocyte sheet transplantation. We did
not measure inﬂammatory factors in the synovium or infrapatellar
fat pad (IFP) in this study. Udo et al. observed decreased inﬂammation in the IFP and reversible changes 7 days after low-dose MIA
injection. Synovitis and cartilage degeneration are typical featuresof knee joint OA, and it is necessary to conﬁrm the effectiveness ofchondrocyte sheet transplantation against synovitis.

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N. Takatori et al. / Regenerative Therapy 9 (2018) 24e31

5. Conclusion
In this study, we transplanted allogeneic chondrocyte sheets
into rats with OA induced by low-dose MIA injection. Transplantation of cell sheets alleviated pain, as shown by a higher limb
weight ratio in the group that received transplanted sheets. Histological ﬁndings showed less degeneration in rats that received
transplanted sheets. These data suggest that, in this nontraumatic
early arthritis model, transplantation of allogeneic chondrocyte
sheets reduced the effects of OA, induced cartilage repair, andinhibited the progression of articular cartilage degeneration.
Declarations of interest
M.S. is one of the inventors on the patent (WO2006093151)
submitted by CellSeed Inc. for the manufacturing process ofchondrocyte sheets. M.S. receives research funds from CellSeed Inc.
Acknowledgements
This research was supported by the Project Focused on Developing Key Evaluation Technology: Manufacturing Technology for
Industrialization in the Field of Regenerative Medicine (No.
14525207) and by the Project Focused on Developing Key Evaluation Technology: Evaluation for Industrialization in the Field ofRegenerative Medicine (No. 15667006 to M.S.) from the Japan
Agency for Medical Research and Development.
We are grateful to the Support Center for Medical Research and
Education, Tokai University.
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