Received: 20 December 2017

Revised: 27 May 2018

Accepted: 9 July 2018

DOI: 10.1002/term.2741

RESEARCH ARTICLE

Rabbit xenogeneic transplantation model for evaluating humanchondrocyte sheets used in articular cartilage repair
Takumi Takahashi1| Masato Sato1| Eriko Toyoda1 | Miki Maehara1 |Daichi Takizawa1 | Hideyuki Maruki2 | Ayako Tominaga2 | Eri Okada1 | Ken Okazaki2 |Masahiko Watanabe1
1Department of Orthopaedic Surgery, Surgical
Science, Tokai University School of Medicine,
Isehara, Japan2

Department of Orthopaedic Surgery, Tokyo
Women's University, Tokyo, Japan

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 Number:
14525207 (Project Focused on Developing
Key Evaluation Technology: Manufacturing
Technology for Industrialization in the Field ofRegenerative Medicine) and 15667006(Research Project for Practical Applications ofRegenerative Medicine)

Abstract
Research on cartilage regeneration has developed novel sources for humanchondrocytes and new regenerative therapies, but appropriate animal models fortranslational research are needed. Although rabbit models are frequently used in suchstudies, the availability of immunocompromised rabbits is limited. Here, we investigated the usefulness of an immunosuppressed rabbit model to evaluate directly theefficacy of human chondrocyte sheets through xenogeneic transplantation. Humanchondrocyte sheets were transplanted into knee osteochondral defects in Japanesewhite rabbits administered with immunosuppressant tacrolimus at a dosage of 0.8
or 1.6 mg/kg/day for 4 weeks. Histological evaluation at 4 weeks after transplantationin rabbits administered 1.6 mg/kg/day showed successful engraftment of humanchondrocytes and cartilage regeneration involving a mixture of hyaline cartilage andfibrocartilage. No human chondrocytes were detected in rabbits administered
0.8 mg/kg/day, although regeneration of hyaline cartilage was confirmed. Histologicalevaluation at 12 weeks after transplantation (i.e., 8 weeks after termination of
immunosuppression) showed strong immune rejection of human chondrocytes, whichindicated that, even after engraftment, articular cartilage is not particularly immune
privileged in xenogeneic transplantation. Our results suggest that Japanese whiterabbits administered tacrolimus at 1.6 mg/kg/day and evaluated at 4 weeks may be
useful as a preclinical model for the direct evaluation of human cell‐based therapies.
KEY W ORDS

allogeneic transplantation, articular, cartilage, chondrocytes, preclinical, transplantation, xenogeneictransplantation

1|

I N T RO D U CT I O Nnature, articular cartilage is prone to irreversible damage from trauma
or aging that often progresses to degenerative conditions such as oste-

Articular cartilage is a complex tissue capable of withstanding repetitive

oarthritis (OA) (Brittberg et al., 2016). OA is a debilitating disease thatforces experienced by the knee joint through daily movements such as

limits mobility and imposes a major social and healthcare burden,

walking and stair climbing. Because of its complexity and avascularespecially in ageing populations. The increases in longevity and size--------------------------------------------------------------------------------------------------------------------------------

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.
© 2018 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons Ltd

J Tissue Eng Regen Med. 2018;12:2067–2076.

wileyonlinelibrary.com/journal/term

2067

2068

TAKAHASHI ET AL.

of the elderly population mean that more people are living longer

tacrolimus, a potent immunosuppressant used in organ transplanta-

with disability and a reduced quality of life (Ondrésik et al., 2017).

tion (Ikebe et al., 1996; Kino et al., 1987). We hypothesized that a

Therapies for restoring normal knee function through the regenera-

xenogeneic transplantation rabbit model would be a cost‐effectivetion of articular cartilage have been investigated (Mardones, Jofré, &method for the direct evaluation of the in vivo efficacy of human

Minguell, 2015), but the development of an effective treatment forchondrocyte sheets.

OA is still a work in progress. To date, bone marrow‐stimulation
techniques (Erggelet & Vavken, 2016); autologous and allogeneic
osteochondral grafts (Bugbee, Pallante‐Kichura, Görtz, Amiel, &2

MATERIALS AND METHODS|

Sah, 2016; Hangody & Füles, 2003); and autologous chondrocyte
implantation (Brittberg et al., 1994; Clavé et al., 2016; Tohyama et al.,The animal experiments were approved by the Institutional Animal2009) have been used for the treatment of relatively small cartilage

Experiment Committee at Tokai University and were performed in

defects. The clinical outcomes so far suggest some success, but evidence

accordance with the guidelines of the Institutional Regulation for Ani-

is inconclusive, and there are concerns about repair with fibrocartilage

mal Experiments and the Fundamental Guideline for Proper Conduct

and donor site morbidity (Matricali, Dereymaeker, & Luyten, 2010).

of Animal Experiment and Related Activities in Academic Research

Novel therapies being developed for articular cartilage repair can be
categorized as scaffold only, cells with a scaffold, or cells without a scaf-

Institutions under the jurisdiction of the Ministry of Education, Culture, Sports, Science, and Technology for animal handling and care.

fold (scaffoldless; Kon, Roffi, Filardo, Tesei, & Marcacci, 2015). Our
approach has been to use cell sheet technology developed in the
1990s (Kikuchi, Okuhara, Karikusa, Sakurai, & Okano, 1998; Okano,

2.1|

Fabrication and analysis of TKA sheets

Yamada, Okuhara, Sakai, & Sakurai, 1995; Yamada et al., 1990) to cul-

Under the approval and guidance of the Tokai University Ethics Com-

ture chondrocytes on temperature‐responsive culture inserts coatedmittee and with patients' informed consent, cartilage and synovial tis-with poly(N‐isopropylacrylamide), which allows the collection ofsues were obtained from five patients (age 61–80 years; average age

chondrocytes as cell sheets without disrupting the extracellular matrix.

71.4 years) who underwent TKA at Tokai University Hospital.

Using this scaffoldless technology, we have reported the usefulness of

Chondrocytes and synovial cells were enzymatically isolated, andlayered chondrocyte sheets (Kokubo et al., 2016) in the treatment ofchondrocyte sheets made from cells obtained from TKA (TKA sheets)

partial‐thickness defects in rabbits (Kaneshiro et al., 2006) and full‐thick-were prepared separately from each donor using the coculture

ness defects in rats (Takaku et al., 2014); rabbits (Ito et al., 2012); andmethod as previously described (Kokubo et al., 2016). Briefly, cartilage

minipigs (Ebihara et al., 2012). In a clinical study from 2011 to 2014,

tissue and synovial tissue were separately minced and digested with

we treated eight patients with autologous chondrocyte sheets and con-

5 mg/ml collagenase Type I (Worthington Biochemical Corp., Lake-firmed the safety and effectiveness of this method in treating patientswood, NJ, USA) in Dulbecco's modified Eagle's medium–Nutrient Mix-

with OA (Sato, Yamato, Hamahashi, Okano, & Mochida, 2014).

ture F‐12 (DMEM/F12; Gibco, Waltham, MA, USA) supplemented

However, autologous transplantation requires two surgeries, andwith 20% fetal bovine serum (FBS; Ausgenex, Molendinar, Australia)

the cell source is often limited. Thus, we are currently investigating theand 1% antibiotic–antimycotic solution (AA; Gibco). The cells were fil-possibility of creating allogeneic chondrocyte sheets. For cartilage regen-tered through a 100‐μm cell strainer (Becton, Dickinson, and Company

eration, both differentiated (Ham et al., 2015) and undifferentiated mes-[BD], Franklin Lakes, NJ, USA) and washed in phosphate‐bufferedenchymal stem cells (Richardson et al., 2016) from various sources suchsaline (PBS; Gibco). Primary chondrocytes were stored at −80°C in

as bone marrow, adipose tissue, and synovial tissue have been investi-

Cellbanker 1 cryopreservation medium (Zenoaq, Fukushima, Japan),gated as potential sources. Hyaline cartilaginous tissue generated fromand synovial cells were cultured to Passage 1 and then stored similarly.

embryonic stem cells (Oldershaw et al., 2010) and, more recently, from

To fabricate TKA sheets, synovial cells at Passage 1 were firstinduced pluripotent stem cells (Yamashita et al., 2015) shows great

plated as feeder cells in six‐well dishes (BD) at a density of 1 × 104 cells

potential. To address the feasibility and traceability issues, we have been

per cm2 with 3 ml of medium. After 1 hr, temperature‐responsive cul-investigating the use of chondrocytes obtained from young polydactyly

ture inserts (Cellseed, Tokyo, Japan) were placed in the wells, and pri-patients (Nasu, Takayama, & Umezawa, 2016) in preparation for amary chondrocytes were seeded without direct contact to synovial

first‐in‐human clinical study for allogeneic chondrocyte sheets that has

cells at a density of 5 × 104 cells per cm2 with 2 ml of medium. The

been approved by the Ministry of Health, Labour and Welfare of Japan.

medium was changed every 3 or 4 days thereafter with the addition

The main objective of this study was to establish a preclinicalof 100 μg/ml ascorbic acid (Wako Pure Chemical Industries, Osaka,

animal model that would allow the direct evaluation of humanJapan). At 2 weeks, three sheets of only chondrocytes were layered

chondrocyte sheets for translational research purposes. Rabbits areusing a support membrane of polyvinylidene difluoride, and the lay-

phylogenetically and anatomically more closely related to humans

ered sheets were cultured for another 7 days.

than rodents (Schnupf & Sansonetti, 2012), and the size of defects

To count the cells, fabricated TKA sheets were digested enzymat-that can be created allows for the transplantation of a single

ically and stained with trypan blue. For histological analysis, TKA

chondrocyte sheet without overpopulating the defect. Human

sheets were fixed in 4% paraformaldehyde in phosphate buffer andadult chondrocyte sheets were fabricated from surgical remains ofembedded in optimal cutting temperature compound (Sakura Finetek

total knee arthroplasty (TKA) and transplanted into osteochondral

Japan, Tokyo, Japan). Twenty‐micrometre‐thick sections were stained

defects in Japanese white (JW) rabbits immunosuppressed bywith haematoxylin and eosin (HE) or with Safranin O, Fast Green, and2069

TAKAHASHI ET AL.

HE. For immunohistochemical analysis, 10‐μm sections were blocked

after surgery. The intramuscular injections alternated between thewith 5% normal goat serum (NGS; Rockland Immunochemicals, Limerick,right and left hind legs.

PA, USA) and 0.3% Triton X‐100 (Sigma‐Aldrich, St. Louis, MO, USA) in

For surgery and transplantation, the rabbits were anaesthetized

phosphate buffer for 30 min. The sections were then incubated with pri-with 2 L/min nitrous oxide, 1 L/min oxygen, and 2.5–3.0% isofluranemary antibodies to human Type I collagen (COL1; SouthernBiotech, Bir-

(Pfizer, New York City, NY, USA). A medial parapatellar incision wasmingham, AL, USA; dilution 1:200); Type II collagen (COL2; Kyowa

made to the right knee, and the patella was dislocated to access the

Pharma Chemical, Toyama, Japan; dilution 1:200); aggrecan (R&D Sys-

patellar groove of the femur. A 5‐mm biopsy punch (Kai Industries, Gifu,

tems, Minneapolis, MN, USA; dilution 1:10); or fibronectin (Merck

Japan) was used as a marking guide, and a 5‐mm drill was used to create

Millipore, Darmstadt, Germany; dilution 1:500) at 4°C overnight. The

an osteochondral defect (diameter = 5 mm; depth = 3 mm). Slight bleed-sections were washed and incubated at room temperature (RT) foring from the subchondral bone was confirmed, and physiological saline

1 hr with the secondary antibody Alexa Fluor 488‐conjugated goat

(Nipro, Osaka, Japan) was used to clean the defect and prevent thermalanti‐mouse immunoglobulin (Ig; Thermo Fisher Scientific, Waltham,damage. For transplantation groups B, C, and E, one TKA sheet was

MA, USA) for COL2 and fibronectin, and Alexa Fluor 488‐conjugatedtransplanted into each defect without suturing. After restoration ofanti‐goat Ig (Thermo Fisher Scientific) for COL1 and aggrecan. The

the patella, the quadriceps femoris muscle and tendon were sutured

slides were then stained with 4′,6‐diamidino‐2‐phenylindole (Vector

to prevent dislocation.

Laboratories, Burlingame, CA, USA) and observed under a BZ‐9000
Generation II fluorescence microscope (Keyence Corp., Osaka, Japan).

2.4|

Monitoring of biochemical markers in blood

To measure the concentrations of humoral factors produced by
TKA sheets, fabricated sheets were placed in DMEM/F12 supplemented with 1% FBS and 1% AA, and the culture supernatants werecollected after 72 hr, centrifuged at 15,885 g for 5 min to remove
debris, and stored at −80°C. Commercial enzyme‐linked immunosorbent assays were used to quantify the concentrations of transforming
growth factor‐β1 (R&D Systems) and melanoma‐inhibitory activity(MIA; Roche, Basel, Switzerland).

Blood monitoring was performed weekly for selected rabbits (n = 3 foreach group) in defect group A and transplantation group C from Day 0(before surgery) to Day 28 (before euthanasia). Blood samples werecollected from the ear, placed in EDTA 2 K tubes and BD Vacutainer
SST II Advance tubes, and frozen at −30°C. Samples were sent toFujifilm Monolith Co. (Tokyo, Japan) for analysis. Abnormalities inblood chemistry were monitored, especially to detect changes in kidney and liver function.

2.2 | Measurement of blood tacrolimus
concentration in JW rabbits

2.5

All JW rabbits were purchased from Tokyo Laboratory Animals Sci-folk, England) was used to evaluate the degree of pain, inflammation,ence Co. (Tokyo, Japan). The blood concentration of tacrolimus wasor discomfort, as previously reported (Ito et al., 2012). Measurements

monitored in three female JW rabbits (average weight = 3.0 kg) inde-

were made before surgery, on Days 1, 4, 7, 10, 14, 17, 21, 24, and 28

pendent from the transplantation experiment. Tacrolimus (Astellas

for the first 4 weeks, and on Days 35, 42, 49, 56, 70, and 84 for the

Pharma, Tokyo, Japan) was administered daily for 14 days intramuscu-

following 8 weeks. The average damaged limb weight distribution

larly at a dosage of 1.6 mg/kg/day. Blood samples from the ear wereratio (%) of the hind limbs was calculated from 10 repeated measure-collected into EDTA 2 K tubes (Tokuyama Sekisui Co., Yamaguchi,ments for each animal and averaged for all groups as follows.

|

Pain evaluation

The Linton Incapacitance Tester (Linton Instrumentation, Diss, Nor-Japan); frozen at −30°C; and sent to SRL Inc. (Tokyo, Japan) for analDamaged limb weight distribution ratio ð%Þ
Damaged limb load ðgÞ¼
×100
Total limb load ðgÞ

ysis. Tacrolimus concentration in blood was measured using anelectrochemiluminescence immunoassay on a Cobas e 411 immunoassay analyser (Hitachi High Technologies Co., Tokyo, Japan) with a minimum detection level of 0.5 ng/ml.

2.6
2.3

|

Transplantation of TKA sheets|

Histological evaluation of regenerated cartilage

Rabbits were euthanized by an intravenous administration of 50 mg/

Thirty female JW rabbits (average weight = 3.0 kg) were used in theml pentobarbital (Tokyo Chemical Industry, Tokyo, Japan) at 4 weeks

transplantation experiment. The animals were housed one animal per

or 12 weeks. The operated knee was opened, and the distal portion

cage and were given daily standard chow and access to water adof the femur was excised and fixed in 20% formalin (Wako Pure

libitum. Before surgery, rabbits were randomly assigned by weight to

Chemical Industries) for 3–5 days. The sample was decalcified in

one of five groups: A (defect only, 4 weeks, 1.6 mg/kg/day of tacroli-

10% EDTA (Wako Pure Chemical Industries) for 3–4 weeks andmus); B (TKA sheet, 4 weeks, 0.8 mg/kg/day); C (TKA sheet, 4 weeks,

embedded in paraffin wax, and 3‐μm sections were cut near the cen-

1.6 mg/kg/day); D (defect only, 12 weeks, 1.6 mg/kg/day); and E (TKA

tre of the defect area, parallel to the long axis of the femur.

sheet, 12 weeks, 1.6 mg/kg/day). TKA sheets fabricated from each ofStandard

protocols

were

used

for

histologicalstaining.

the donors were equally allocated to each transplantation group.

Deparaffinized sections were stained with HE only or with Safranin O,

Tacrolimus was administered intramuscularly daily for 10 days starting

Fast Green, and HE. Safranin O‐stained sections were randomized and2 days before transplantation and then every other day until 4 weeks

scored separately by two trained orthopaedic surgeons (H. M. and2070

TAKAHASHI ET AL.

D. T.), who were blinded to their identity, using a modified version ofsignificance test was used for post hoc analysis. The weight distribution

the O'Driscoll score and International Cartilage Repair Society (ICRS)

ratios were compared with values before surgery using the paired t test.

score (Mainil‐Varlet et al., 2003; O'Driscoll, Keeley, & Salter, 1986).
To immunostain for COL1 and COL2, deparaffinized sections weretreated with 0.4% pepsin (Agilent, Santa Clara, CA, USA) for 30 min at

3

RESULTS|

37°C. The sections were washed in distilled water, treated with 0.3%

Properties of TKA sheetshydrogen peroxide–methanol solution at RT for 15 min, washed in

3.1

PBS, blocked with 2.5% NGS for 10 min at RT, and then treated for

An average TKA sheet contained 1.6 ± 0.2 × 106 cells and had a thick-3 hr at RT with mouse monoclonal antibody to either human COL1 orness of 50.0 ± 6.5 μm. The sheets were layered and manipulated using

human COL2 (Kyowa Pharma Chemical Co., Toyama, Japan) diluted ata polyvinylidene difluoride support membrane, which was removed

1:100 with 1% bovine serum albumin (Sigma‐Aldrich) in PBS. The stained

upon transplantation (Figure 1a,b). HE staining of TKA sheets showed

sections were washed in PBS, treated for 1 hr at RT with ImmPRESS

the integration of the three chondrocyte sheet layers and the multi-

polymer anti‐mouse IgG reagent (Vector Laboratories), immersed forlayer of chondrocytes 1 week after layering (Figure 1c). TKA sheets

2–8 min in Tris–HCl buffer (pH 7.6) containing 0.02% diaminobenzidine

stained negative for Safranin O (Figure 1d), positive for COL1

and 0.005% hydrogen peroxide, and then counterstained with HE.

(Figure 1e), slightly positive for COL2 (Figure 1f), positive for aggrecan|

To immunostain for human vimentin, deparaffinized sections were

(Figure 1g), and positive for fibronectin (Figure 1h). Enzyme‐linked

treated with 10‐mM sodium citrate buffer (pH 6.0) for 10 min at 98°C

immunoassays showed that an average TKA sheet produced

in a microwave. The sections were cooled for 30 min, washed in PBS,

1.8 ± 0.2 ng/ml of transforming growth factor‐β1 and 14.3 ± 2.1 ng/

and then treated with 5% NGS, followed by Alexa Fluor 647‐conjugated

ml of MIA in 3 ml of culture media in 72 hr.

rabbit monoclonal antibody to human vimentin (Cell Signaling Technology, Danvers, MA, USA) diluted at 1:100 with 1% bovine serum albumin
in PBS overnight at 4°C. Sections were washed in distilled water and

3.2|

Blood tacrolimus concentration in JW rabbits

then mounted and cured with 4′,6‐diamidino‐2‐phenylindole (Vector

The blood tacrolimus concentration (ng/ml) in three JW rabbits admin-

Laboratories) according to the manufacturer's instructions.

istered 1.6 mg/kg/day for 14 days was monitored for 17 days. PeakAll microscopic images were obtained using a BZ‐9000 Generation II fluorescence microscope (Keyence Corp.).

concentration measured 2 hr after administration on Days 1, 2, and3 was 171.5 ± 36.5, 188.5 ± 16.5, and 196.0 ± 5.0, respectively(Figure 2a). The trough concentration was measured at 24 hr after

2.7|

Statistical analysis

injection and was highest on Day 1 (75.3 ± 28.8) and lowest on Days 5(31.0 ± 1.4) and 7 (31.2 ± 2.4). The blood tacrolimus concentration

Numerical results are expressed as mean and standard deviation unless oth-increased on Days 10 (45.5 ± 11.3) and 14 (57.6 ± 11.3). After the final

erwise noted. ICRS scores are expressed as mean and standard error of theinjection on Day 14, the concentration continued to decrease tomean. Repeated measures analysis of variance was used to analyse mea-

Day 17 (4.0 ± 0.9; Figure 2b). After the immunosuppression was termi-

surements from the monitoring of biochemical makers in blood. Analysis

nated, the animals' appetite returned to normal, and body weightof variance was used to analyse ICRS scores, and Tukey's honest

increased gradually.

FIGURE 1 Representative macrographs andmicrographs of TKA sheets. (a) Macrograph of
a TKA sheet attached to a PVDF support
membrane and (b) the same thin sheet seenfrom an angle. Scale bar = 1 cm. Histologicalanalysis of sections of layered chondrocyte
sheets stained with (c) HE and (d) Safranin O.
Immunohistochemical analysis revealed (e)positive staining for COL1, (f) slight staining
for COL2, (g) positive staining for ACAN, and(h) positive staining for FN. Scale bar = 50 μm.
ACAN: aggrecan; COL1: Type I collagen;
COL2: Type II collagen; FN: fibronectin; HE:haematoxylin and eosin; TKA: total knee
arthroplasty; PVDF: polyvinylidene difluoride

2071

TAKAHASHI ET AL.

decrease in average weight for each group was as follows: 0.16 kg at
Day 14 in Group A; 0.32 kg in Group B at Day 21; 0.09 kg in Group C
at Day 21; 0.24 kg in Group D at Day 10; and 0.13 kg in Group E at
Day 4. Adverse events were detected near the end of the 4 weeks
in two rabbits from Group C and in four rabbits from Group B. Self‐
inflicted wounds to the end of the hind limbs were observed, but noabnormalities to the surgical areas were detected. Adverse eventswere detected in two rabbits from Group E. Self‐inflicted wounds tothe end of the hind limbs were observed in one rabbit, and swellingin the surgical knee joint was detected in the other. In all rabbits, muscle stiffness and muscle loss were observed in areas where tacrolimus
had been administered.

3.4|

Monitoring of biochemical markers in blood

Blood monitoring was performed weekly for selected rabbits (n = 3) in
defect group A and transplantation group C (Table 1) starting from
Day 0 before transplantation. Creatinine concentration remained
within the standard physiological level in the defect group (0.7–
1.0 mg/dl) and transplantation group (0.7–0.9 mg/dl). Blood urea
nitrogen level also remained within the standard physiological levelin the defect group (11.7–18.0 mg/dl) and transplantation group
FIGURE 2 Blood tacrolimus concentration (ng/ml) in Japanese whiterabbits (n = 3) administered 1.6 mg/kg/day intramuscularly from Days
0 to 13. (a) Measurements were made at 2, 8, and 24 hr after injection
for the first 3 days. (b) Trough levels were measured at 24 hr after
injection just before the next dose for 14 days. Additional
measurements on Days 15, 16, and 17 indicated the metabolism oftacrolimus after the end of the injections(12.5–19.0 mg/dl). No indications of liver or kidney failure were

3.3 | Xenogeneic transplantation of TKA sheets inimmunosuppressed JW rabbits

3.5

The surgeries were uneventful, and the TKA sheets fully covered thefollowed for 4 weeks (Figure 3a) and 12 weeks (Figure 3b). This ratio

defect areas. Loss of appetite and diarrhoea were observed after sur-recovered to the value before surgery by Day 21 in transplantation

gery, and a subsequent decrease in body weight was observed; largest

groups B (p = 0.972) and C (p = 0.214) but did not return fully to the

TABLE 1

observed. Creatine phosphokinase level increased on Day 7 in thetransplantation group, and C‐reactive protein level increased on Days 7
and 14 in both groups, but these decreases may have reflected themuscle damage caused by the surgery and intramuscular administration. No significant differences were detected between the twogroups for the measured biochemical markers.

|

Pain evaluation

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

Monitoring of biochemical markers in blood
p valueDay

Defect group A

Transplantation group C

0

7

14

21

28

0

7

14

21

Albumin (g/dl)

0.141

4.5

4.0

4.3

4.5

4.4

4.6

4.3

4.4

4.7

28
4.4

Total bilirubin (mg/dl)

1.000

0.1

0.1

0.1

0.1

0.1

0.1

0.1

0.1

0.1

0.1

AST (IU/l)

0.836

23.7

21.7

19.7

17.7

17.7

22.0

23.0

18.0

13.5

20.5

ALT (IU/l)

0.701

50.0

59.0

52.3

38.3

36.3

63.0

53.5

55.5

40.5

39.0

CPK (IU/l)

0.117

1470

1740

1294

842

893

708

4159

1836

895

1688

BUN (mg/dl)

0.238

14.3

18.7

11.7

13.7

18.0

17.0

19.0

12.5

15.5

16.5

Creatinine (mg/dl)

0.687

1.0

0.8

0.7

0.8

0.8

0.9

0.8

0.7

0.8

0.8

Uric acid (mg/dl)

0.163

0.1

0.1

0.1

0.1

0.1

0.3

0.2

0.1

0.2

0.2

Sodium (mEq/l)

0.504

143

141

142

143

144

139

142

141

141

142

Chloride (mEq/l)

0.354

103

104

102

104

107

105

102

104

105

106

Potassium (mEq/l)

0.508

3.4

4.3

4.2

4.9

5.1

3.9

4.4

4.2

4.4

4.8

Blood glucose (mg/dl)

0.760

111

151

123

125

113

112

147

127

128

122

CRP (mg/dl)

0.923

0.4

10.5

5.3

1.3

0.4

0.4

6.5

6.3

0.7

0.6

Note. ALT: alanine transaminase; AST: aspartate aminotransferase; BUN: blood urea nitrogen; CPK: creatine phosphokinase; CRP: C‐reactive protein.

2072

TAKAHASHI ET AL.

O staining or an increase in subchondral bone filling in the defect area
(Figure 4a). Group B showed strong Safranin O staining (Figure 4b),whereas Group C showed weak Safranin O staining (Figure 4c).
Group D showed weak Safranin O staining for surface areas surrounding the defect area. Group E showed signs of inflammatory cells within
the defect area and subchondral bone, and no regeneration of articularcartilage (Figure 4e).
Immunohistochemical analysis was used to evaluate the regenerated cartilage (Figure 5). Samples from transplantation group B stained
strongly for COL2 and minimally for COL1 (Figure 5b), which indicatedrepair by hyaline cartilage. Samples from transplantation group C
stained weakly for COL2 and strongly for COL1 (Figure 5c), whichindicated repair by both hyaline cartilage and fibrocartilage. Immunostaining with human‐specific vimentin antibody showed successful
engraftment of human cells only in Group C (Figure 5c). No regenerated cartilage was detected in Groups D or E.
A modified version of the ICRS grading system was used toevaluate cartilage repair (Figure 6). At 4 weeks, the scores weresignificantly higher in transplantation groups B (30.4 ± 2.8,
p = 0.020) and C (31.0 ± 2.2, p = 0.014) than in defect group A(20.1 ± 2.0). At 12 weeks, the scores did not differ significantly(p = 0.07) between transplantation group E (18.2 ± 2.8) and defect
FIGURE 3 Change in weight distribution ratio determined as theratio between the operated hind limb load and the total hind limb
load. (a) Group A: defect only, 4 weeks, 1.6 mg/kg/day of tacrolimus;(b) Group B: total knee arthroplasty (TKA) sheet, 4 weeks, 0.8 mg/kg/
day; (c) Group C: TKA sheet, 4 weeks, 1.6 mg/kg/day; (d) Group D:
defect only, 12 weeks, 1.6 mg/kg/day; and (e) Group E: TKA sheet,
12 weeks, 1.6 mg/kg/day. (a) The ratio recovered to the value before
surgery in transplantation groups B (p = 0.972) and C (p = 0.214) byDay 21 but never fully recovered by Day 28 in defect group A(p = 0.008). (b) The ratio recovered to the value before surgery by
Day 21 in transplantation group E (p = 0.593) but worsened at Day 42(p = 0.010) and never recovered fully by Day 84 in Group D (p = 0.015)

group D (25.8 ± 1.6).

4|

DISCUSSION

Hyaline cartilage regeneration using chondrocyte sheets may provide
an effective and long‐term treatment for OA. To ensure the safety
and efficacy of this treatment using different cell sources, preclinicalmodels are needed for evaluating human chondrocyte sheets directly.
Such models will also be critical for evaluating differences associatedwith donor age, gender, health status, and other factors yet to be identified. In this study, we have shown the usefulness of a rabbit xenogeneic transplantation model for the direct evaluation of humanvalue before surgery by Day 28 in defect group A (p = 0.008; Figure 3a). The results of the 12‐week evaluation are shown in Figure 3b. The
weight distribution ratio recovered to the value before surgery by
Day 21 in transplantation group E (p = 0.593) but worsened at Day 42(p = 0.010) and did not recover thereafter. This ratio did not recover
fully by Day 84 in defect group D (p = 0.015).

chondrocyte sheets.
JW rabbits were immunosuppressed by tacrolimus at two different concentrations, 0.8 and 1.6 mg/kg/day, and human TKA sheets
were transplanted into osteochondral defects. We verified that TKA
sheets expressed fibronectin important to the adhesive properties ofchondrocyte sheets and that they also produced TGFβ‐1 and MIA,
which are known anabolic factors that may contribute to the regener-

3.6 | Macroscopic and microscopic analysis ofregenerated cartilage

ative effects. At 4 weeks and under immunosuppression of 1.6 mg/kg/
day, successful engraftment of human chondrocytes, pain alleviation,and improvement in histological scores were observed. To our knowl-

Representative macroscopic images of the defect area for each groupedge, this is the first study to provide clear evidence of the successful

are shown in Figure 4. The images show filling of the defect by smoothengraftment of human chondrocytes in the injured rabbit knee and towhite tissue in transplantation groups B (Figure 4b) and C (Figure 4c).

characterize the cartilage matrix produced by the transplanted

By contrast, the defect areas were either unfilled or partially filled withchondrocytes. Immunostaining indicated repair by both hyaline

irregular tissue in defect groups A (Figure 4a) and D (Figure 4d). In

cartilage and fibrocartilage, which may be indicative of the advanced

transplantation group E, the defect areas were filled with synovial fluid

age and OA nature of the adult chondrocytes used in this study. Mus-and showed attachment of surrounding tissue to the synovium andcle stiffness and muscle atrophy at the sites of tacrolimus injections

indications of inflammation (Figure 4e).

were observed on both hind legs, but the results of the weight distri-

HE and Safranin O staining was performed to evaluate the regenerated cartilage, as shown in Figure 4. Group A showed little Safraninbution ratios were comparable with those reported in our previous
study (Ito et al., 2012).

TAKAHASHI ET AL.

2073

FIGURE 4 Representative macroscopic images of the defect areas in the patellar groove of the femur and representative microscopic images
from the histological analysis of paraffin sections of the defect area. (a) Group A: defect only, 4 weeks, 1.6 mg/kg/day of tacrolimus; (b) Group
B: total knee arthroplasty (TKA) sheet, 4 weeks, 0.8 mg/kg/day; (c) Group C: TKA sheet, 4 weeks, 1.6 mg/kg/day; (d) Group D: defect only,
12 weeks, 1.6 mg/kg/day; and (e) Group E: TKA sheet, 12 weeks, 1.6 mg/kg/day. Macroscopically, at 4 weeks, the defect was (a) filled withirregular tissue and (b, c) filled with white smooth material. At 12 weeks, the defect was (d) unfilled and (e) unfilled and showing signs of severe
synovial fluid accumulation and inflammation. Left scale bars = 1 cm. Histological analysis revealed (a) subchondral bone filling in part of the defectarea. (b) Strong staining for Safranin O was observed. (c) Slight staining for Safranin O was observed. (d) The defect area was unfilled, and Safranin
O staining was weak for surface areas surrounding the defect area. (e) Inflammatory cells filled the defect area including parts of the subchondralbone, and no staining for Safranin O was observed in the defect area. Middle and right scale bars = 1 mm

At 4 weeks and under immunosuppression of 0.8 mg/kg/day,We also examined whether this model could be used to evaluatealmost no engraftment of human chondrocytes was observed, but

the remodelling of articular cartilage over the long term. We hypothe-

there was strong regeneration with hyaline cartilage as well as pain

sized that, after successful engraftment and matrix production, immu-

alleviation and improvement in histological scores. The observed

nosuppression may be unnecessary. However, histological evaluation

regenerative effect may be attributed to the paracrine effect of

at 12 weeks after transplantation (i.e., 8 weeks after termination ofhumoral factors produced by chondrocyte sheets, as we reported pre-immunosuppression) showed that immune rejection had occurred.

viously (Hamahashi et al., 2015). The paracrine effect was also

The articular cartilage has long been considered a relatively immune‐

reported to be the major mode of action of cell sheet treatment ofprivileged site, but recent findings have been inconsistent. For

ischaemic cardiomyopathy in a porcine xenogeneic transplantation

example, using porcine chondrocytes in a rabbit model, Ramallal

model (Kawamura et al., 2012; Kawamura et al., 2015). The rejection

et al. (2004) reported no immune rejection at 24 weeks in a xenoge-of transplanted cells may occur in parallel with the paracrine effectneic transplantation study. However, delayed immune rejection wasand may result in regeneration of hyaline cartilage by activated hostsuggested in a similar study by another laboratory (Pei, Yan, Shoukry,

cells even when no donor cells remain.

& Boyce, 2010). Xenogeneic transplantation studies using human

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TAKAHASHI ET AL.

FIGURE 5 Representative microscopic images from the immunohistochemical analysis of paraffin sections of the defect area. (a) Group A: defectonly, 4 weeks, 1.6 mg/kg/day of tacrolimus; (b) Group B: total knee arthroplasty (TKA) sheet, 4 weeks, 0.8 mg/kg/day; (c) Group C: TKA sheet,
4 weeks, 1.6 mg/kg/day; (d) Group D: defect only, 12 weeks, 1.6 mg/kg/day; and (e) Group E: TKA sheet, 12 weeks, 1.6 mg/kg/day. Sections were
stained for Types I and II collagens and for human‐specific vimentin (hVimentin). (a) Positive for Type I collagen but negative for Type II collagen.
(b) Strong staining for Type II collagen but negative for Type I collagen and hVimentin. (c) Staining for both Types I and II collagens and for
hVimentin in the entire defect area. (d) The defect area was unfilled. (e) Inflammatory cells filled the defect area, which was negative for Types I
and II collagens and hVimentin. Scale bars = 1 mmchondrocytes in minipigs (Niemietz et al., 2014) and humanadministration to 4 weeks in this study and would limit its use in lon-

osteochondral biphasic composite constructs in rabbits (Jang, Lee,ger studies. Differences in tacrolimus toxicity between rabbit species

Park, Song, & Wang, 2013) have also reported immune rejection. Thus,

must also be considered in order to translate our results to othermost of the evidence suggests that immunosuppression is necessary

rabbit species. Severe tacrolimus toxicity was reported in the Dutch‐

for long‐term studies and that articular cartilage is not necessarily

Belted rabbit, and a much lower dosage of 0.08 mg/kg/day has been

immune‐privileged in xenogeneic transplantation.

suggested as feasible (Giessler, Gades, Friedrich, & Bishop, 2007).

A key limitation of our study is that tacrolimus has been shown to

JW rabbits can tolerate 1.6 mg/kg/day, as first described by Ikebe et al.

reduce OA‐like responses and to protect cartilage matrix integrity(1996) in bone xenogeneic transplantation, but the optimal concentra-

in vitro and in vivo (Siebelt et al., 2014). These effects may complicate

tions need to be determined in further studies.

the interpretation of our results. Intramuscular administration of tacro-

Chondrocyte sheets are unique in that the transplanted

limus alone was insufficient for allowing the regeneration of articularchondrocytes may survive over the long term in the recipient in addi-

cartilage in this rabbit model. However, tacrolimus may stimulate ortion to contributing to the regeneration of cartilage through a para-

modify the cartilage‐regenerating effect resulting from the transplan-crine effect. Few clinical studies have tracked the fate of donor

tation of chondrocyte sheets. Further studies are needed to determine

chondrocytes in humans. In several studies with fresh osteochondral

the extent to which transplanted cells may be affected.

allografts, donor chondrocytes were reported to be alive and active

Another limitation is that tacrolimus administration was accompa-

in the patients after 29 years (Jamali, Hatcher, & You, 2007). The same

nied by adverse events such as weight loss and self‐inflicted wounds.

research group published another case report identifying, without

Self‐inflicted wounds and muscle loss increased the variability in

exception, the engraftment of donor allograft cells in the location of

the weight distribution ratio. Blood monitoring did not indicate kidney

the allografts after 3 years (Haudenschild, Hong, Hatcher, & Jamali,

or liver failure, but these adverse events limited tacrolimus2012). Although xenogeneic transplantation may not completely

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TAKAHASHI ET AL.

RE FE RE NC ES
Brittberg, M., Gomoll, A. H., Canseco, J. A., Far, J., Lind, M., & Hui, J. (2016).
Cartilage repair in the degenerative ageing knee. Acta
Orthopaedica, 87(Suppl 363), 26–38. https://doi.org/10.1080/
17453674.2016.1265877
Brittberg, M., Lindahl, A., Nilsson, A., Ohlsson, C., Isaksson, O., & Peterson,
L. (1994). Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. New England Journal of Medicine,
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Bugbee, W. D., Pallante‐Kichura, A. L., Görtz, S., Amiel, D., & Sah, R. (2016).
Osteochondral allograft transplantation in cartilage repair: Graftstorage paradigm, translational models, and clinical applications. Journalof Orthopaedic Research, 34, 31–38. https://doi.org/10.1002/
jor.22998

FIGURE 6 International Cartilage Repair Society (ICRS) scores in thetreatment groups. (a) Group A: defect only, 4 weeks, 1.6 mg/kg/day oftacrolimus; (b) Group B: total knee arthroplasty (TKA) sheet, 4 weeks,
0.8 mg/kg/day; (c) Group C: TKA sheet, 4 weeks, 1.6 mg/kg/day; (d)Group D: defect only, 12 weeks, 1.6 mg/kg/day; and (e) Group E: TKA
sheet, 12 weeks, 1.6 mg/kg/day. At 4 weeks, ICRS scores weresignificantly higher for groups B (*p = 0.020) and C (*p = 0.014) thanfor Group A. At 12 weeks, the scores did not differ significantly
between Groups D and E (p = 0.07)

reproduce the results of allogeneic transplantation, a xenogeneictransplantation model that assesses both the paracrine effect andthe characteristics of the engrafted chondrocytes is essential.
A rabbit xenogeneic transplantation model using JW rabbits withintramuscular administration of tacrolimus was feasible over a shortspan of 4 weeks. Ascertaining the efficacy of human chondrocyte
sheets and other regenerative therapies for articular cartilage repair
through xenogeneic transplantation of human cells is important for
determining the in vivo characteristics of donor cells. We will use thispreclinical model in the future to evaluate different cell sources anddonor differences to ensure in vivo efficacy.
ACKNOWLEDGEMEN TS
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 to
M. S.) and by the Research Project for Practical Applications ofRegenerative Medicine (No. 15667006 to M. S.) from the Japan
Agency for Medical Research and Development. We also acknowledge the Support Center for Medical Research and Education at
Tokai University for technical support in histological analysis andanimal care.
CONF LICT OF INT E RE ST
M. S. is one of inventors on the patent (WO2006093151) submitted
by the main applicant CellSeed Inc. for the manufacturing process ofchondrocyte sheets. M. S. receives research funds from CellSeed Inc.
ORCID
Takumi Takahashi
Masato Sato

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

http://orcid.org/0000-0003-4049-7325

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How to cite this article: Takahashi T, Sato M, Toyoda E, et al.
Rabbit xenogeneic transplantation model for evaluating humanchondrocyte sheets used in articular cartilage repair. J Tissue
Eng

Regen

Med.

10.1002/term.2741

2018;12:2067–2076.

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