Received: 8 September 2019

Revised: 5 June 2020

Accepted: 2 July 2020

DOI: 10.1002/term.3101

RESEARCH ARTICLE

Regenerative effects of human chondrocyte sheets in a
xenogeneic transplantation model using immune-deficient rats
Daichi Takizawa1,2 | Masato Sato1,2| Eri Okada1,2 | Takumi Takahashi1,2Miki Maehara1,2 | Ayako Tominaga3 | Yasuyuki Sogo1,2 | Eriko Toyoda1,2 ||Masahiko Watanabe1,2
1
Department of Orthopaedic Surgery, Surgical
Science, Tokai University School of Medicine,
Isehara, Japan

Abstract
Although cell transplantation has attracted much attention in regenerative medicine,

2Center for Musculoskeletal innovative
Research and Advancement (C-MiRA), Tokai
University Graduate School, Isehara, Japan3

Department of Orthopaedic Surgery, Tokyo
Women's Medical University, Shinjuku-ku,
Japananimal models continue to be used in translational research to evaluate safety andefficacy because cell sources and transplantation modalities are so diverse. In the present study, we investigated the regenerative effects of human chondrocyte sheets onarticular cartilage in a xenogeneic transplantation model using immune-deficient rats.
Osteochondral defects were created in the knee joints of immune-deficient rats that

Correspondence
Masato Sato, Department of Orthopaedic
Surgery, Surgical Science, Tokai University
School of Medicine, 143 Shimokasuya, Isehara,
Kanagawa 259-1193, Japan.
Email: sato-m@is.icc.u-tokai.ac.jp
Funding information
Japan Agency for Medical Research andDevelopment, Grant/Award Numbers:
JP19bk010406, 20bk0104101h0001,
20bm0404035h0003

were treated as Group A, untreated (without transplantation); Group B, transplantation of a layered chondrocyte sheet containing 5.0 × 105 cells (layered chondrocyte
sheet transplantation); Group C, transplantation of a synoviocyte sheet containing
5.0 × 105 cells (synoviocyte sheet transplantation); or Group D, transplantation ofboth a synoviocyte sheet plus a layered chondrocyte sheet, each containing 5.0 × 105
cells (synoviocyte sheet plus layered chondrocyte sheet transplantation). Histologicalevaluation demonstrated that Group B showed cartilage regeneration with hyaline
cartilage and fibrocartilage. In Groups C and D, the defect was filled with fibrous tissue but no hyaline cartilage. Transplanted cells were detected at 4 and 12 weeks after
transplantation, but the number of cells had decreased at 12 weeks. Our results indicate that layered chondrocyte sheet transplantation contributes to articular cartilage
regeneration; this model proved useful for evaluating these regenerative effects.
KEYWORDS

allogeneic, transplantation, articular, cartilage, chondrocytes, preclinical, transplantation,
xenogeneic transplantation

1 | I N T RO D UC TI O NMankin, & Grodzinsky, 2005). Because articular cartilage is an avascular tissue, it has little potential for self-repair after injuries or lesions,

Articular cartilage comprises sparsely distributed chondrocytes in awhich often worsen over time, leading to knee osteoarthritis (OA;

rich extracellular matrix consisting of type II collagen and proteogly-

Brittberg et al., 2016).

cans. It primarily functions to absorb shock and facilitate smoothCurrent surgical approaches for small cartilage defect include

articular movements (Buckwalter & Mankin, 1998; Buckwalter,bone marrow stimulation (Steadman, Rodkey, & Briggs, 2002),

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,provided the original work is properly cited.
© 2020 The Authors. Journal of Tissue Engineering and Regenerative Medicine published by John Wiley & Sons Ltd
J Tissue Eng Regen Med. 2020;1–11.

wileyonlinelibrary.com/journal/term

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TAKIZAWA ET AL.

osteochondral2004),

Animal Use (Authorization No. 173035). Articular chondrocytes andosteochondral allograft transplantation (Bugbee, Pallante-Kichura,

autografttransfer(Hangodyetal.,synoviocytes were collected and used after obtaining patients'

Görtz, Amiel, & Sah, 2016), and autologous chondrocyte implantation

informed consent and with the approval of our Hospital's Clinical(Brittberg et al., 1994; Saris et al., 2009). Although clinical outcomes

Research Review Committee (Authorization No. 09R-070).

suggest some success, those approaches are insufficient for evidence,tissue engineer approaches are emerging as alternatives to current

2.1 | Fabrication of LC sheets and SY sheets

surgical approaches for cartilage repair (Kwon et al., 2019; Makris,
Gomoll, Malizos, Hu, & Athanasiou, 2015). Especially using scaffold-

Articular cartilage and synovial tissues were collected from thefree approach, we produced chondrocyte sheets fabricated on

unwanted postoperative tissues of six patients (age 71–78 years;

temperature-responsive cell culture inserts, which allow the transplan-

average age 74 years) who underwent total knee arthroplasty at Tokaitation of chondrocytes with their extracellular matrix intact

University Hospital. Articular cartilage and synovial tissues were enzy-(Kaneshiro et al., 2007; Kokubo et al., 2016; Mitani et al., 2009).

matically

Translational models including rats (Takaku et al., 2014; Takatori

synoviocytes, respectively. Layered chondrocyte sheets (LC sheets)

et al., 2018), rabbits (Ito et al., 2012; Kaneshiro et al., 2006; Taniand SY sheets were made according to the methods described by

et al., 2017), and minipigs (Ebihara et al., 2012) were used previously

Kokubo et al. (2016) and Takahashi et al. (2018), as summarized

to confirm the safety and efficacy of chondrocyte sheet transplanta-

below. Briefly, articular cartilage or synovial tissues were minced in ation. Next, we conducted a clinical study using autologous transplan-

petri dish using scissors, and then digested in Dulbecco's modified

tation of human chondrocyte sheets to treat patients with knee OA,

Eagle's medium-Nutrient Mixture F-12 (DMEM/F12; Gibco, Waltham,

after which we detected no severe adverse events after more thanMA, USA) containing 5 mg/ml collagenase type I (Worthington, NJ,

3 years of follow-up. In addition, histological analysis of theUSA), 20% fetal bovine serum (FBS; Ausgenex, Molendinar, Australia),

regenerated cartilage demonstrated that chondrocyte sheet transplan-and 1% antibiotic–antimycotic solution (AA; Gibco). The cell suspen-tation could lead to regeneration of hyaline cartilage (Satosion was passed through a 100-μm cell strainer (Becton Dickinson and

et al., 2019). Recently, we reported the mode of action of chondro-

Company [BD], Franklin Lakes, NJ) and washed in 1 × Dulbecco's

cyte sheets in hyaline cartilage regeneration by transcriptomic andphosphate-buffered saline (PBS; Gibco). Primary chondrocytes were

proteomic analyses (Toyoda et al., 2019). We are currently conducting

suspended in Cellbanker 1 cryopreservation medium (Zenoaq,

our second clinical study using allogeneic transplantation of chondro-

Fukushima, Japan) and cryopreserved at −80 C. Synoviocytes werecyte sheets fabricated from unwanted surgical tissue derived from

cultured to Passage 1 and preserved in the same manner as the cho-patients with polydactyly (Maehara et al., 2017).

ndrocytes. The frozen chondrocytes and synoviocytes were thawed,

digested

separately

toisolatechondrocytes

andTo evaluate new human cell sources of chondrocyte sheets, it isand the chondrocytes were seeded on temperature-responsive cul-preferable to use translational models that can directly evaluate theture inserts (UpCell® 4.2 cm2, CellSeed, Tokyo, Japan) at 50,000 cel-

final product. In other words, xenogeneic orthotopic transplantationls/cm2, whereas the synoviocytes were seeded on six-well companion

models that approximate the in vivo efficacy of clinical transplantation

plates (Corning, Corning, NY, USA) at 10,000 cells/cm2, followed by

are required. We previously reported the use of an immuno-

coculturing.

suppressed rabbit xenogeneic transplantation model to directly evalu-

DMEM/F12, 20% FBS, and 1% AA. The medium was changed every

ate human chondrocyte sheets for articular cartilage repair (Takahashi

3 or 4 days thereafter, with the addition of 100 μg/ml ascorbic acidet al., 2018), but the duration for which immunosuppression was(Wako Pure Chemical Industries, Osaka, Japan). After coculturing foreffective limited long-term studies. Ito et al. (2012) and Shimizu

2 weeks, three chondrocyte sheets were layered using a poly-

et al. (2015) reported conflicting results concerning the effectivenessvinylidene difluoride membrane and were cultured for another 7 days.

of chondrocyte sheets and synoviocyte (SY) sheets using allogeneic

The synoviocytes were also collected in sheet forms and were cul-transplantation in rabbits and rats, respectively. In the present study,

tured for another 7 days. To count the cells, fabricated LC sheets and

we investigated the regenerative effects of human chondrocyte

SY sheets were digested enzymatically, and trypan blue exclusion

sheets and SY sheets for articular cartilage regeneration in a xenoge-

assays were performed. LC sheets and SY sheets were cut appropri-

neic model using immune-deficient rats to establish a translational

ately just prior to transplantation so that they contained approxi-

model that can approximate the efficacy of allogeneic transplantation

mately 5.0 × 105 cells.

The culture medium was initiallycomposedofof human chondrocyte sheets.

2.2 | Histological and immunohistochemical analysis
of LC sheets and SY sheets
2 | MATERIALS AND METHODSFor histological analysis, LC sheets and SY sheets were fixed in 4%
All procedures using animals in this study were performed in accor-

paraformaldehyde in 0.01 M PBS and embedded in optimum cutting

dance with the Guide for the Care and Use of Laboratory Animals

temperature compound (Sakura Finetek, Tokyo, Japan). Frozen 10 μm(NIH Publication No. 85-23, revised 2010) published by the National

sections were stained with 0.1% Safranin O aqueous solution

Institutes of Health, USA, and the Guidelines of Tokai University on(Chroma Gesellschaft Schmid & Co., Munster, Germany) and 0.08%

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TAKIZAWA ET AL.

fast green aqueous solution (Chroma Gesellschaft Schmid & Co.,

2.4 | Measurement of humoral factors

Munster, Germany). The sections were also stained with hematoxylin(Merck Millipore, Darmstadt, Germany) and eosin (Muto pure

A random selection of LC sheets and SY sheets were further cultured

chemicals Co., Ltd., Tokyo, Japan; HE). For immunohistochemical anal-

for 72 h in 3 ml of DMEM/F12, supplemented with 1% FBS and 1%

ysis, 20 μm frozen sections were blocked with 5% normal goat serum

AA incubated at 37 C under appropriate cell culture condition.

(NGS; Rockland Immunochemicals, Limerick, PA, USA) and 0.3%

Supernatants were collected and centrifuged at 15,000 g for 10 min

TritonX-100 (Sigma-Aldrich, St. Louis, MO, USA) for 30 min, thento remove cell debris. The concentrations of transforming growthincubated at 4 C overnight with primary antibodies to type I collagenfactor-β-1 (TGF-β1; R&D Systems), and melanoma inhibitory activity(COL I; Southern Biotech, Birmingham, AL, USA; dilution, 1:200); type

(MIA; Roche, Basel, Switzerland) were measured using enzyme-linked

II collagen (COL II; Kyowa Pharma Chemical, Toyama, Japan; dilution

immunosorbent assay (ELISA) kits. The signal detected for blank

1:200), or fibronectin (Merck Millipore, Darmstadt, Germany; dilution

medium containing 1% FBS was subtracted to adjust for the protein

1:500). The sections were washed and incubated at room temperature

content of the FBS. Measurements were repeated at least twice for(RT) for 1 h with secondary antibody goat anti-mouse immunoglobulineach donor, and averages are reported. The concentrations of TGF-β1(Ig) Alexa Fluor 488 (Thermo Fisher Scientific, Waltham, MA, USA) foror MIA were normalized by the number of cells (1.0 × 106 cells).

COL II and fibronectin, and donkey anti-goat Ig Alexa Fluor
546 (Thermo Fisher Scientific) for COL I. The slides were thenstained with 40 ,6-diamidino-2-phenylindole (DAPI; Vector Laborato-

2.5 | Transplantation of LC sheets and SY sheets

ries, Burlingame, CA, USA), and observed under a fluorescencemicroscope (BZ-9000 Generation II, Keyence Corp., Osaka, Japan).

Forty-eight 12-week-old rats (F334/NJcl-rnu/rnu; Clea Japan, Tokyo,
Japan), weighing approximately 270 g, were used in the transplanta-

2.3 | RNA extraction and reverse transcription–PCRtion experiments. The animals were housed two animals per cage andwere given daily standard chow and access to water ad libitum. LC

LC sheets and SY sheets were incubated with TRIzol (Thermo Fisher

sheets and SY sheets fabricated from each of the donors were allo-

Scientific) for RNA extraction. Total RNA was extracted using thecated equally to each transplantation group. The rats were anesthe-

RNeasy Mini Kit (Qiagen Inc., Valencia, CA, USA), according to the man-tized using isoflurane, N2O, and O2. An osteochondral defect was

ufacturer's instructions. cDNA was synthesized using the QuantiTect

created using the methods described by Itokazu et al. (2016). Briefly,



Reverse Transcription Kit (Qiagen Inc., Hilden, Germany) at 42 C fora medial parapatellar incision was made on the right knee; the patella

15 min and 95 C for 3 min. Table 1 lists the oligonucleotide primers

was moved laterally and an osteochondral defect (diameter 2 mm;

used. Polymerase chain reaction (PCR) was performed using SYBR

depth 1 mm) was created on the patellar groove of the femur using a

Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) andbiopsy punch (Kai Industries, Seki, Japan) and hand drill. Marrow

the cDNA template (2.5 ng) in a total reaction volume of 25 μl using a

bleeding during creation of the osteochondral defect was confirmed.

7300 Real-time PCR system (Applied Biosystems) at 95 C for 15 s,

Next, LC sheets and SY sheets were transplanted as: Group A,

60 C for 60 s, for 35–45 cycles. The results were analyzed using Smartuntreated (without transplantation); Group B, LC sheet containing

Cycler II software (Applied Biosystems). The internal control

5.0 × 105 cells; Group C, SY sheet containing 5.0 × 105 cells, andglyceraldehyde-3-phosphate dehydrogenase was used as a referenceGroup D: both SY and LC sheets, each containing 5.0 × 105 cells. For–ΔΔCtgene, and the comparative 2
TABLE 1

method was used for analysis.

Group D, the SY sheet was transplanted first and then covered with

Primers used for reverse-transcriptase–polymerase chain reaction

Target genes

Primer sequence: forward

Primer sequence: reverse

0

GAPDH

5 -ACCCAGAAGACTGTGGATGG-3

COL1A1

50 -GTCGAGGGCCAAGACGAAG-30

050 -TTCTAGACGGCAGGTCAGGT-30
50 -CAGATCACGTCATCGCACAAC-30

0

COL1A2

5 -AATTGGAGCTGTTGGTAACGC-3

COL2A1

50 -GTGGAGCAGCAAGAGCAA-30

0

50 -CACCAGTAAGGCCGTTTGC-30
50 -TGTTGGGAGCCAGATTGT-30

0

COL10A1

5 -ATGCTGCCACAAATACCCTTT-3

SOX9

50 -AACGCCGAGCTCAGCAAGA-30

050 -CCGCGGCTGGTACTTGTAATC-30

MMP13

5 -ACTGAGAGGCTCCGAGAAATG-3

50 -GAACCCCGCATCTTGGCTT-30

ACAN

50 -AGGAGACAGAGGGACACGTC-30

50 -TCCACTGGTAGTCTTGGGCAT-30

RUNX2

0

50 -GGTAGTGGGCCTTTTATGCCT-30

0

0

0

5 -ACCATGGTGGAGATCATCG-3

50 -CGCCATGACAGTAACCACAG-30

Note: Primer sequences for COL1A1, COL1A2, and COL10A1 from Yamashita et al. (2015).
Abbreviations: ACAN, aggrecan; COL10A1, collagen, type X, alpha 1; COL1A1, collagen, type I, alpha 1; COL1A2, collagen, type I, alpha 2; COL2A1, collagen, type II, alpha 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MMP13, matrix metalloproteinase 13; RUNX2, Runt-related transcription factor 2; SOX9, SRY-Box 9.

4

TAKIZAWA ET AL.

the LC sheet. The patella was anatomically repositioned and the

TABLE 2

quadriceps femoris muscle, tendon, and skin were sutured. After the

Item

Description

surgery, all the rats were returned to their cages without splinting orTi

1: Exclusively noncartilage

immobilization.

ICRS histological grading system

2: Mostly noncartilage
3: Mostly fibrocartilage
4: Mostly hyaline cartilage

2.6 | Pain evaluationMatx

1: None
2: Slight

An incapacitance tester (Linton Instrumentation, Norfolk, UK) was

3: Moderateused to evaluate the degree of pain, inflammation, or discomfort aspreviously reported (Ito et al., 2012; Mihara, Higo, Uchiyama,

4: StrongStru

1: Severe disintegration

Tanabe, & Saito, 2007; Takahashi et al., 2018; Tani et al., 2017). All

2: Cysts or disruptions

animals were acclimated to the incapacitance tester for 7 days prior

3: No organization of chondrocytes

to transplantation. Nine measurements were taken, on days 1, 5,

4: Beginning of columnar organization of chondrocytes

8, 12, 15, 19, 22, 26, and 28 after transplantation. The averagedamaged-limb weight distribution ratio (%) of the hind limbs was

5: Normal, similar to healthy mature cartilage
Clus

calculated from 10 repeated measurements for each animal and2: <25% of the cells clusteredaveraged for each group as: Damaged-limb weight distribution ratio(%) = damaged-limb load (g)/total limb load (g) × 100.

1: 25–100% of the cells clustered

3: No clusters
Tide

1: <25% of the calcified cartilage layer intact
2: 25–49% of the calcified cartilage layer intact
3: 50–75% of the calcified cartilage layer intact

2.7 | Histological evaluation of repaired cartilage

4: 76–90% of the calcified cartilage layer intact
5: Complete intactness of the calcified cartilage layer

The animals were euthanized by administration of high-dose anes-

Bform

thesia at 4 or 12 weeks after transplantation. The operated knee

2: Slight

was opened, and the distal portion of the femur was excised andfixed for 1 week in 20% formalin (Wako Pure Chemical Industries).

1: No formation

3: Strong
SurfH

1: Severe fibrillation or disruption

The sample was decalcified in 10% EDTA (Wako Pure Chemical

2: Moderate fibrillation or irregularity

Industries) for 2 and 3 weeks and embedded in paraffin wax, and

3: Slight fibrillation or irregularity

8 μm sections were cut near the center of the defect area, parallelto the long axis of the femur. Standard protocols were used for his-

4: Normal
FilH

1: <25%

tological staining. Deparaffinized sections were stained with 0.1%

2: 26–50%

Safranin O aqueous solution (Chroma Gesellschaft Schmidt & Co.,

3: 51–75%

Munster, Germany) and 0.08% fast green aqueous solution (Chroma

4: 76–90%

Gesellschaft Schmid & Co., Munster, Germany). These were randomized and scored separately by two trained orthopedic surgeons (A.T.

5: 91–110%
Latl

and Y.S.), who were blinded to their identities, using the Interna-

2: Bonded at one end/partially both ends

tional Cartilage Repair Society (ICRS) histological grading system(Brehm et al., 2006; Mainil-Varlet et al., 2003; O'Driscoll, Keeley, &1: Not bonded

3: Bonded at both sidesBasl

1: <50%

Salter, 1988), which is a modification of that reported by O'Driscoll

2: 50–70%

et al. (1988). This system evaluates repaired tissue based on

3: 70–90%

11 items: tissue morphology, matrix staining, structural integrity,

4: 91–100%

cluster formation, tidemark opening, bone formation, histological

InfH

5: No inflammationof defect filling, lateral integration of defect-filling tissue, basal
integration of defect-filling tissue, and histological signs of inflammation. Eleven items were evaluated by scoring as shown in Table 2.
The total score ranged from 11 points (no repair) to 45 points(normal articular cartilage).
To immunostain for COL I, deparaffinized sections were treatedwith 0.01 M citric acid buffer for 10 min at 98 C; to immunostain for
COL II, deparaffinized sections were treated with 0.4% pepsin for
30 min at 37 C. The sections were washed in distilled water, blocked

1: Strong inflammation
3: Slight inflammation

appraisal of surface architecture, histological appraisal of the degree
Hgtot

45 points

Note: This system evaluates repaired tissue based on 11 items: Ti: tissuemorphology; Matx: matrix staining; Stru: structural integrity; Clus: cluster
formation; Tide: tidemark opening; Bform: bone formation; SurfH:
histological appraisal of surface architecture; FilH: histological appraisal ofthe degree of defect filling; Latl: lateral integration of defect-filling tissue;
Basl: basal integration of defect-filling tissue; InfH: histological signs ofinflammation. The total score (Hgtot) range is 11–45. ICRS: International
Cartilage Regeneration and Joint Preservation Society.

5

TAKIZAWA ET AL.

with normal equine serum for 10 min at RT, and then treated withimmersed for 2–4 min in Tris–HCl buffer (pH 7.6) containing 0.05%

primary antibodies to COL I (Southern Biotech; dilution 1:100) or COL

diaminobenzidine and 0.005% hydrogen peroxide, and then counter-

II (Kyowa Pharma Chemical; dilution 1:100) for 3 h at RT. The stained

stained with HE.

sections were washed in 0.01 M PBS, treated with ImmPRESS

To immunostain for human vimentin (hVIM), deparaffinizedpolymer anti-mouse IgG reagent (Vector Laboratories) for 1 h at RT,

sections were treated with target retrieval solution (S1699; Dako,F I G U R E 1 Properties of LC sheets and SY sheets. For each sheet, n = 6. (a) Histological analysis of LC sheets and SY sheets revealed negative
staining for Safranin O. Immunohistochemical analysis revealed positive staining for COL I and fibronectin, but negative staining for COL II for
both sheet types. Scale bar = 50 μm. (b) Gene expression profile of LC sheets and SY sheets. mRNA expression for COL2A1, SOX9, and MMP13
was significantly higher in LC sheets than in SY sheets (p < 0.05). (c) The concentrations of humoral factors secreted by LC sheets and SY sheets.
The secretion of TGF-β1 was 1.6 ± 0.2 ng/1.0 × 106 cells by LC sheets and 0.40 ± 0.02 ng/1.0 × 106 cells by SY sheets. The secretion of MIA by
LC sheets was 16.5 ± 2.1 ng/1.0 × 106 cells, compared with undetectable levels secreted by SY sheets. The secretion of MIA and TGF-β1 by LC
sheets was significantly higher than that by SY sheets (p < 0.05). The results are presented as mean ± standard deviation. LC: layered
chondrocyte; SY: synoviocyte; COL I: type I collagen; COL II: type II collagen; COL1A1: collagen, type I, alpha 1; COL1A2: collagen, type I, alpha2; COL2A1: collagen, type II, alpha 1; COL10A1: collagen, type X, alpha 1; SOX9: SRY-Box 9; MMP13: matrix metalloproteinase 13; ACAN:
aggrecan; RUNX2: Runt-related transcription factor 2; TGF-β1: transforming growth factor-β-1; MIA: melanoma inhibitory activity; L: LC sheets;
S: SY sheets [Colour figure can be viewed at wileyonlinelibrary.com]

6

TAKIZAWA ET AL.

Glostrup, Denmark) for 30 min at 98 C and then cooled for 30 min.
The sections were washed in distilled water, blocked with 5% NGS for
10 min at RT, and then treated with primary antibody to hVIM
(ab16700; Abcam, Cambridge, MA, USA; dilution 1:100) overnight at
4 C. The stained sections were washed in 0.01 M PBS, treated withsecondary antibody gout anti-rabbit Ig Alexa Fluor 488 (Thermo
Fisher Scientific) for 1 h at RT, and then stained with DAPI. All microscopic images were obtained using a fluorescence microscope (BZ9000 Generation II, Keyence Corp., Osaka, Japan).

2.8 | Statistical analysis
The results are presented as mean ± standard deviation, and p < 0.05
was deemed significant. Statistical analysis was performed using SPSS
v23.0 software (IBM Corp., Armonk, NY, USA). The reverse transcription PCR and ELISA results were analyzed using the Mann–Whitney
U test. Analysis of variance was used to analyze the weight distribution ratios and ICRS scores, and the Tukey honest significant difference (HSD) method was used for post hoc tests.

3 | RESULTS
3.1 | Properties of LC sheets and SY sheets
An average LC sheet and SY sheet contained 1.3 ± 0.3 × 106 cells and
1.4 ± 0.2 × 106 cells, respectively. LC sheets and SY sheets stained
negative for Safranin O and COL II and positive for COL I and fibronectin; no differences in staining patterns were detected between thetwo sheet types (Figure 1a). The expression of mRNA for collagen,type II, alpha 1 (COL2A1), SRY-Box 9 (SOX9), and matrix
metalloproteinase 13 (MMP13) was significantly higher in LC sheets
than in SY sheets (Figure 1b). The concentrations of humoral factors
secreted by LC sheets and SY sheets are summarized in Figure 1c.
Compared with SY sheets, LC sheets secreted higher concentrationsof TGF-β1 (LC sheets 1.6 ± 0.2 ng/1.0 × 106 cells; SY sheets
0.40

±

0.02

ng/1.0×106

cells)

and

MIA

(LC

sheetsF I G U R E 2 Pain evaluation. (a) Weight distribution ratios of thedamaged limb after transplantation. Group A, untreated; Group B, LC
sheet containing 5.0 × 105 cells alone; Group C, SY sheet containing
5.0 × 105 cells alone; and Group D, SY sheet plus LC sheet, eachcontaining 5.0 × 105 cells. For each group, n = 6. Group A showed
minimal improvement. Groups B, C, and D all showed improvement.
(b) Weight distribution ratios on Day 28 after transplantation: GroupA = 41.9 ± 5.0%, Group B = 47.9 ± 3.9%, Group C = 45.3 ± 4.0%, andGroup D = 46.3 ± 3.6%. The weight distribution ratios of Groups B
and D were significantly higher than that of Group A (p < 0.05). The
results are presented as mean ± standard deviation. LC: layered
chondrocyte; SY: synoviocyte

16.5 ± 2.1 ng/1.0 × 10 cells; SY sheets were below the detection
6

limit).

3.3 | Microscopic analysis of repaired tissue
3.2 | Pain evaluation

No signs of infection or rejection were observed in any group during
the experiment. Safranin O staining was performed to evaluate the

The weight distribution ratio was used as a measure of pain and was

repaired tissue, as shown in Figure 3. Four weeks after transplanta-

tested daily for 28 days after transplantation (Figure 2a). Group A

tion, the defects in Groups B, C, and D were filled with repaired tissue.

showed minimal improvement from Day 1 to Day 28: 30.9 ± 6.8% to

In Group A, the defects were not filled with a chondral layer, but with

41.9 ± 5.0%. Groups B, C, and D all showed improvement from Day

either bone-like tissue or fibrous tissue that did not stain for Safranin

1 to Day 28: 30.1 ± 5.3% to 47.9 ± 3.9% in Group B, 31.8 ± 4.5% toO or COL II staining but did stain for COL I. In Group B, the repaired

45.3 ± 4.0% in Group C, and 32.2 ± 6.4% to 46.3 ± 3.6% in Grouptissue showed Safranin O and COL II staining and no COL I staining,

D. The weight distribution ratios for Day 28 were significantly higher

good integration with the surrounding cartilage, a high defect-fillingfor Groups B and D than for Group A, as shown in Figure 2brate, and a smooth superficial layer, although the subchondral bone(p < 0.05).

formation was inadequate. In Groups C and D, the repaired tissue

TAKIZAWA ET AL.

7

F I G U R E 3 Representative microscopic images from the histological and immunohistochemical analyses. Group A, untreated; Group B, LC
sheet containing 5.0 × 105 cells alone; Group C, SY sheet containing 5.0 × 105 cells alone; and Group D, SY sheet plus LC sheet, each containing
5.0 × 105 cells. For each group, n = 6. Four weeks after transplantation in Groups B, C, and D, the defects were filled with repaired tissue.
Histological analysis of Group A revealed no Safranin O staining or formation of a chondral layer but revealed bone-like tissue or fibrous tissue. In
Group B, strong Safranin O staining was observed. In Groups C and D, no Safranin O staining was observed. Immunohistochemical analysis
revealed negative staining for COL II and hVIM in Group A, positive staining for COL II and hVIM in Group B, and positive staining for COL I andhVIM in Groups C and D. At 12 weeks after transplantation, the defects in Groups B, C, and D, in which transplantation was performed, werefilled with repaired tissue. Histological analysis of Group A revealed no Safranin O staining or formation of a chondral layer but revealed bone-liketissue. In Group B, strong Safranin O staining was observed. In Groups C and D, no Safranin O staining was observed. Immunohistochemical
analysis revealed negative staining for COL II and hVIM in Group A, positive staining for COL II and hVIM and partial staining for COL I in
Group B, and positive staining for COL I and hVIM in Groups C and D. Low-power images of Safranin O, COL I, COL II, and hVIM staining areshown in the upper rows (scale bar = 500 μm). High-power images of Safranin O and hVIM are shown in the lower rows (scale bar of Safranin
O = 200 μm, scale bar of hVIM = 50 μm). LC: layered chondrocyte; SY: synoviocyte; COL I: type I collagen; COL II: type II collagen; hVIM: humanvimentin [Colour figure can be viewed at wileyonlinelibrary.com]

showed good integration with the surrounding cartilage, but thetransplantation, the defects in Groups B, C, and D were filled withrepaired tissue was mostly fibrous tissue, with no Safranin O or COL II

repaired tissue. In Group A, the defects were filled with bone-likestaining and with COL I staining, and the superficial layer andtissue. In Group B, the repaired tissue showed Safranin O and COL II

subchondral bone formation were inadequate. At 12 weeks afterstaining and partial COL I staining, integration with the surrounding

8

tissue, a high defect-filling rate, a smooth superficial layer, and acolumnar arrangement of the chondrocytes. There was moderate

TAKIZAWA ET AL.

3.4 | Localization of human-derived cells in repaired
tissueformation of subchondral bone. In Groups C and D, the repaired tissue
had not changed significantly from that which was present 4 weeks

Figure 3 shows the immunostaining for hVIM. Groups B, C, and D

after transplantation. A modified version of the ICRS grading systemshowed positive hVIM staining at 4 weeks after transplantation, but

was used to evaluate cartilage repair (Table 2). Figure 4 and Table 3

the overall number of positive cells decreased at 12 weeks aftershow the ICRS grading system results 4 and 12 weeks after transplan-transplantation.

tation. At 4 weeks after transplantation, the scores for Groups A, B, C,
and D were 24.6 ± 4.1, 32.3 ± 5.4, 24.9 ± 3.2, and 25.2 ± 5.9,
respectively. The score for Group B was significantly higher than that

4 | DISCUSSION

for Group A. At 12 weeks after transplantation, the scores forGroups A, B, C, and D were 22.8 ± 4.6, 33.5 ± 4.4, 23.3 ± 2.1, andAppropriate translational models that can directly evaluate the final

25.5 ± 2.1, respectively. The score for Group B was significantly

outcomes are necessary to confirm the safety and efficacy of humanhigher than those for Groups A, C, and D.

cell-derived products. In the present study, LC sheets, similar to thoseused in our previous clinical study, and SY sheets fabricated fromsynovium-derived cells, were evaluated in vitro and in vivo.
LC sheets and SY sheets were both negative for Safranin O
staining and positive for fibronectin and COL I immunostaining.
Compared with the chondrocyte sheets fabricated from the surgicalwaste material of younger patients (Kokubo et al., 2016), the LC
sheets used in this study were fabricated from older patients, which
may explain why these LC sheets did not stain for Safranin O or COL
II. Gene expression analysis revealed that compared with SY sheets,
LC sheets had significantly higher expression of COL2A1 and SOX9
suggesting that they had better chondrogenic properties. MMP13
expression was significantly higher in LC sheets than in SY sheets. In
previous study, the LC sheets restrain the catabolic factors matrix
metalloproteinase 3, MMP13, and ADAMTS5 in allogenic transplantation on rabbit model (Kaneshiro et al., 2006). Also, MMP13 expression
did not differ significantly between LC sheets and layered SY sheets,
and that ADAMTS4 and 5 were significantly higher in the layered SY
sheets than the LC sheets in allogenic transplantation on rat model(Shimizu et al., 2015). The difference is that the cells for the current
study were collected from older patients with knee OA. Furthermore,collagen, type X, alpha 1 expression in this study was higher in the LC
sheets than in SY sheets. Thus, it is possible that LC sheets may beparticularly affected by OA.
ELISA analysis revealed that LC sheets produced significantly
higher levels of TGF-β1 and MIA, which have, respectively, beensuggested to make important contributions to cartilage repair and to

F I G U R E 4 Histological evaluation of repaired tissue at(a) 4 weeks and (b) 12 weeks after transplantation. Group A,
untreated; Group B, LC sheet containing 5.0 × 105 cells alone;Group C, SY sheet containing 5.0 × 105 cells alone; and Group D, SY
sheet plus LC sheet, each containing 5.0 × 105 cells. For each group,n = 6. At 4 weeks after transplantation, the scores for Groups A, B, C,
and D were 24.6 ± 4.1, 32.3 ± 5.4, 24.9 ± 3.2, and 25.2 ± 5.9,
respectively. ICRS scores in Group B were significantly higher thanthose in Group A (p < 0.05). At 12 weeks after transplantation, thescores for Groups A, B, C, and D were 22.8 ± 4.6, 33.5 ± 4.4,
23.3 ± 2.1, and 25.5 ± 2.1, respectively. ICRS scores in Group B weresignificantly higher than those in Groups A, C, and D (p < 0.05). The
results are presented as mean ± standard deviation. LC: layered
chondrocyte; SY: synoviocyte; ICRS: International Cartilage
Regeneration and Joint Preservation Society

be a marker for the cartilage phenotype.
Cartilage repair was observed at 4 and 12 weeks after transplantation of LC sheets, with effects including alleviation of pain and an
improvement in histological scores. To our knowledge, this is the firststudy in which human chondrocyte sheets were transplanted toimmune-deficient rats and cartilage repair was observed. Consistent
with the reported by Takahashi et al. (2018), the repaired tissue inGroup B contained a mixture of hyaline cartilage and fibrocartilage,
possibly because the cells were collected from older patients withknee OA. We previously reported the high gene expression of fibronectin, integrin α10, other cell adhesion factors, SOX9 (a key regulatorof chondrocyte differentiation), and COL2A1 and collagen, type
XXVII, alpha 1 in LC sheets, indicating that these sheets have excellent

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TAKIZAWA ET AL.

TABLE 3ICRS grades 4 and 12 weeks after transplantation
Item

4W

12W

A

B

C

D

Ti

2.1 ± 0.7

3.5 ± 0.5

1.8 ± 0.2

2.1 ± 0.6Matx

1.3 ± 0.4

2.4 ± 0.8

1.8 ± 0.3

1.6 ± 0.5Stru

2.3 ± 0.6

3.7 ± 0.5

1.9 ± 0.1

2.4 ± 1.1Clus

1.0 ± 0.0

1.1 ± 0.1

1.0 ± 0.0

1.0 ± 0.0

Tide

1.0 ± 0.0

2.2 ± 1.1

1.0 ± 0.0

1.2 ± 0.4Bform

2.4 ± 0.4

2.3 ± 0.4

1.0 ± 0.0

1.4 ± 0.6

SurfH

2.4 ± 0.6

2.8 ± 0.8

2.0 ± 0.3

2.0 ± 0.6FilH

3.5 ± 0.9

3.9 ± 0.9

3.6 ± 0.9

3.3 ± 1.3Latl

1.7 ± 0.8

2.4 ± 0.5

2.4 ± 0.6

1.9 ± 0.7Basl

1.9 ± 0.8

3.1 ± 1.0

3.4 ± 1.1

2.7 ± 0.9InfH

5.0 ± 0.0

5.0 ± 0.0

5.0 ± 0.0

5.0 ± 0.0

Hgtot

24.6 ± 4.1

32.3 ± 5.4

24.9 ± 3.2

25.2 ± 5.9

Item

A

B

C

D

Ti

1.7 ± 0.4

3.0 ± 0.9

1.9 ± 0.1

1.9 ± 0.1Matx

1.2 ± 0.4

2.4 ± 1.3

1.3 ± 0.5

1.5 ± 0.5Stru

2.2 ± 0.7

2.9 ± 0.8

1.9 ± 0.5

2.2 ± 0.3

Clus

1.1 ± 0.1

1.1 ± 0.1

1.0 ± 0.0

1.0 ± 0.0

Tide

1.0 ± 0.0

2.2 ± 1.2

1.0 ± 0.0

1.0 ± 0.0Bform

2.8 ± 0.2

2.6 ± 0.5

1.3 ± 0.6

1.4 ± 0.5

SurfH

2.1 ± 0.6

3.1 ± 0.5

1.9 ± 0.5

2.4 ± 0.6FilH

3.4 ± 0.8

5.0 ± 0.0

3.1 ± 0.6

3.6 ± 0.7Latl

1.5 ± 0.8

2.6 ± 0.4

1.7 ± 0.5

1.9 ± 0.6Basl

1.5 ± 0.6

3.6 ± 0.6

3.1 ± 0.6

3.3 ± 0.6InfH

5.0 ± 0.0

5.0 ± 0.0

5.0 ± 0.0

5.0 ± 0.0

Hgtot

22.8 ± 4.6

33.5 ± 4.4

23.3 ± 2.1

25.5 ± 2.1

Note: Group A, untreated; Group B, LC sheet containing 5.0 × 10 cells alone; Group C, SY sheet containing 5.0 × 10 cells alone; and Group D, SY sheet
plus LC sheet, each containing 5.0 × 105 cells. LC: layered chondrocyte; SY: synoviocyte. For each group, n = 6. ICRS: International Cartilage Regeneration
and Joint Preservation Society; Ti: tissue morphology; Matx: matrix staining; Stru: structural integrity; Clus: cluster formation; Tide: tidemark opening;
Bform: bone formation; SurfH: histological appraisal of surface architecture; FilH: histological appraisal of the degree of defect filling; Latl: lateral integration of defect-filling tissue; Basl: basal integration of defect-filling tissue; InfH: histological signs of inflammation. The total score (Hgtot) range is 11–45.
5

5adhesiveness and maintenance of the cartilage phenotype (Mitani

At 4 and 12 weeks after transplantation, immunostaining withet al., 2009). In the previous study, we confirmed that LC sheets

hVIM showed that transplanted cells were still present in thesecrete MIA, TGF-β1, and prostaglandin E2, which are anabolic factors

osteochondral defects. However, at 12 weeks after transplantation,

involved in cartilage repair (Hamahashi et al., 2015).

the number of transplanted cells had decreased. This finding is consis-

Cartilage repair was not observed in Groups C and D at 4 ortent with those of Itokazu et al. (2016), who reported that the number

12 weeks after transplantation, and the osteochondral defects wereof transplanted cells had decreased 12 weeks after transplantation of

filled with fibrous tissue. Ito et al. (2012) reported that, in rabbits,

xenogeneic cells to immune-deficient rats, and with those of Takaku

better cartilage regeneration was achieved by transplanting a combi-

et al. (2014), who reported that after transplantation of allogeneic

nation of LC sheets and SY sheets than by LC sheets or SY sheetschondrocyte sheets in rats, cells derived from the transplants mostly

alone. Shimizu et al. (2015) reported that in rats, the transplantation ofdisappeared within the joints at 4–8 weeks after transplantation,

SY sheets as a monolayer or a triple layer did not promote hyaline

although cells were still detected 21 months after transplantation.

cartilage repair. Our results show that SY sheets, alone or in combina-

Although cartilage is a tissue with relatively high immunological toler-tion with LC sheets at a ratio of 1:1, did not promote hyaline cartilage

ance, it is likely that immunosuppression is required for xenogeneic

repair. These differences may relate to the differing characteristics oftransplantation. Takahashi et al. (2018) performed xenogeneic trans-

SY sheets from different animal species (rabbits, rats, or humans) andplantation of human chondrocyte sheets into an immune-deficientthe fact that, in our study, the cells were collected from older patients

rabbit under immunosuppression. Although they confirmed thatwith knee OA.

transplanted cells were present at 4 weeks after transplantation, they

10

TAKIZAWA ET AL.

reported immune rejection at 12 weeks after transplantation, whenthe effects of the immunosuppression wore off. In the present study,
we used F334/NJcl-rnu/rnu rats for xenogeneic transplantation
experiments. These rats have an immunological deficit in T-cellfunction and are not likely to reject transplanted tissues; therefore,
replacement of transplanted cells by host-derived cells secondary tochondrogenesis and time is believed to be the reason for thedecreased number of cells detected.
There were two limitations to this study. First, 12 weeks posttransplantation is a short period for evaluating cartilage repair.
Therefore, evaluation over a longer term is warranted. Second, thisstudy was conducted on small animals. The rat model is low cost andsuitable for the selection of cell sources; however, validation of theefficacy of transplantation is required in larger animals that moreclosely resemble humans.
Evaluation of human chondrocyte sheets using immune-deficient
rats was possible for up to 12 weeks after transplantation. The in vitro
evaluation revealed that human chondrocyte sheets with a cartilagespecific phenotype contributed to cartilage regeneration in vivo. We
plan to verify the efficacy of various cell sources for chondrocyte
sheets using this translational model.
ACKNOWLEDGMENTS
We are grateful to the Support Center for Medical Research and
Education at Tokai University for histological analysis technicalsupport. This research was supported by the Japan Agency for Medical Research and Development under grant number JP19bk0104063,
20bk0104101h0001, and 20bm0404035h0003.
CONF LICT OF IN TE RE ST
M.S. receives research funds from CellSeed Inc.
AUTHOR CONTRIBUTIONS
D.T.: Lead for all in vivo and in vitro elements of experimental workand manuscript preparation. M.S.: Guarantor, contributions to experimental design, lead for all elements of experimental work, provision offunding, and manuscript proofing. E.O.: Contributions to experimental
design and in vivo elements of experimental work. T.T.: Contributions
to experimental design, in vivo elements of experimental work,
statistical analysis, and manuscript proofing. M.M.: Contributions toin vitro elements of experimental work. A.T.: Contributions to in vivo
elements of experimental work. Y.S.: Contributions to in vivo elements of experimental work. E.T.: Contributions to experimental
design and in vitro elements of experimental work. M.W.: Contributions to experimental design and manuscript proofing.
ORCID
Masato Satohttps://orcid.org/0000-0003-4049-7325

Takumi Takahashi

https://orcid.org/0000-0003-0217-1324

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How to cite this article: Takizawa D, Sato M, Okada E, et al.
Regenerative effects of human chondrocyte sheets in a
xenogeneic transplantation model using immune-deficientrats. J Tissue Eng Regen Med. 2020;1–11. https://doi.org/10.
1002/term.3101