Received: 25 April 2016

Revised: 30 July 2017

Accepted: 17 July 2018

DOI: 10.1002/term.2748

RESEARCH ARTICLE

Intra‐articular administration of EP2 enhances the articularcartilage repair in a rabbit model
Yoshiki Tani1 | Masato Sato1| Munetaka Yokoyama1 | Miyuki Yokoyama1 |Takumi Takahashi1 | Eriko Toyoda1 | Eri Okada1 | Shinsei Fujimura2 | Hideyuki Maruki3 || Teruo Okano4 | Joji Mochida1
Yoshiharu Kato3 | Masayuki Yamato4
1

Department of Orthopaedic Surgery, Surgical
Science, Tokai University School of Medicine,
Isehara, Japan

2

Abstract
We have reported the usefulness of chondrocyte sheets on articular cartilage repair in

Minase Research Institute, Ono
Pharmaceutical Co., Ltd., Osaka, Japan

animal experiments. Here, we investigated the regenerative effects of EP2 signalling3

with or without chondrocyte sheets. Forty‐five rabbits were used, with six rabbits

Department of Orthopaedic Surgery, Tokyo
Women's Medical University, Tokyo, Japan4

in each of the six groups and nine rabbits for chondrocytes and synovial cells harvest-

Institute of Advanced Biomedical
Engineering and Science, Tokyo Women's
Medical University, Tokyo, Japaning to fabricate triple‐layered chondrocyte sheets: osteochondral defect only (control,

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(Group D), EP2 agonist and chondrocyte sheets (Group E), and EP2 antagonist andFunding information
Japan Agency for Medical Research andDevelopment: Research Project for Practical
Applications of Regenerative Medicine

Group A), EP2 agonist (Group B), EP2 antagonist (Group C), chondrocyte sheetschondrocyte sheets (Group F). After surgery, the weight distribution ratio was measured as an indicator of pain alleviation. Injections of the EP2 agonist or EP2 antagonist were given from 4 weeks after surgery. The rabbits were sacrificed at 12 weeks,
and the repaired tissues were evaluated for histology. The weight distribution ratio
and International Cartilage Repair Society grading were as follows: Group A: 40.5%± 0.2%, 14.8 ± 0.5; Group B: 43.4% ± 0.7%, 25.4 ± 0.8; Group C: 38.7% ± 0.7%,
13.7 ± 0.3; Group D: 48.6% ± 0.6%, 40.2 ± 0.5; Group E: 49.1% ± 0.3%, 40.5 ± 0.4;
and Group F; 46.8% ± 0.4%, 38.7 ± 0.5. Significant differences in histology and pain
alleviation were observed between groups except between Groups A and C, between
Groups D and E, and between Groups D and F. These findings show that the intra‐
articular administration of an EP2 agonist achieved pain alleviation and tissue repair.
However, no synergistic effect with chondrocyte sheets was observed.
KEY W ORDS

cartilage regeneration, chondrocyte sheet, transplantation

1|

I N T RO D U CT I O N& Szerb, 1997; Szerb, Hangody, Duska, & Kaposi, 2005), or autologous
chondrocyte implantation (Ochi, Uchio, Kawasaki, & Iwasa, 2002).

Osteoarthritis (OA) is the most common disease in older adults, is often

There are other treatments for OA caused by articular cartilage degen-refractory to treatment, and can lead to articular cartilage degeneration

eration and damage, such as high tibial osteotomy, unicompartmental(Peat, McCarney, & Croft, 2001). With progression, cartilage degenera-

knee arthroplasty, and total knee arthroplasty.

tion causes dysfunction of the knee joint and makes activities of daily

The process of OA development involves various factors, includ-living difficult. Post‐traumatic articular cartilage defects are treateding cartilage degeneration, repair, and inflammation (Buckwalter,

mainly by microfracture (Mithoefer et al., 2006; Steadman, Rodkey, &Saltzman, & Brown, 2004), but there is no intention to prevent human

Briggs, 2002; Steadman, Rodkey, & Rodrigo, 2001), mosaicplasty

articular cartilage degeneration and damage by these treatments(Hangody, Feczko, Bartha, Bodó, & Kish, 2001; Hangody, Kish, Karpati,(Hunziker & Rosenberg, 1996; Kaneshiro et al., 2006). As treatments

J Tissue Eng Regen Med. 2018;1–9.

wileyonlinelibrary.com/journal/term© 2018 John Wiley & Sons, Ltd.

1

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TANI ET AL.

for damaged articular cartilage, a variety of autologous chondrocyte

activities are in dynamic equilibrium (Mitsui et al., 2011). In chondro-

implantation methods are used throughout the world using

cyte sheet transplantation, articular cartilage repair is achieved in achondrocytes recovered from patients and then cultured in vitro andmore normal environment, which shows the importance of the extra-

transplanted to articular cartilage defects. Post‐traumatic articular

cellular environment to articular cartilage repair.

cartilage defects are the main indications for this treatment but OA

Hamahashi et al. have reported that the abundant secretion ofis not. Such untreated cartilage defects progress to OA because

factors such as transforming growth factor‐β (TGFβ) and prostaglandinchondrocytes have poor regenerative ability and metabolic activity

E2 (PGE2) accelerates the effects of chondrocyte sheets in cartilage(Anderson et al., 2011; Otsuka et al., 2009). The possibility of exacer-

repair (Hamahashi et al., 2015). PGE2 works through four isoforms

bating OA by using conventional cell therapies is a concern becauseof the EP receptor, EP1 to EP4, and then EP2 receptor‐mediatedfibrous cartilage is mixed in with the regenerated tissue.

PGE2 signalling stimulates the growth of chondrocytes and promotes

We have previously reported the usefulness of layered chondro-

the regeneration of articular cartilage in rabbits (Aoyama et al., 2005;

cyte sheets as a treatment for degenerated articular cartilage in animal

Mitsui et al., 2011; Otsuka et al., 2009). Signals through EP1 andexperiments. Kaneshiro et al. reported the effects of layered chondro-

EP3, coupled by Gi protein, increase the intracellular Ca2+ concentra-

cyte sheets obtained from temperature‐responsive culture dishes as ation, and those through EP2 and EP4, coupled by Gs protein, increase

treatment for partial‐thickness defects, which are considered difficult

cyclic adenosine monophosphate (cAMP). Although they have a com-to treat conventionally (Kaneshiro et al., 2006). The characteristics of

mon second messenger, the amide acid identity between EP2 (362the humoral factors produced by layered chondrocyte sheets have alsoamino acids) and EP4 (513 amino acids) is only 31% (Sugimoto &been clarified (Hamahashi et al., 2015). Experiments in humans have

Narumiya, 2007). In addition, a specific agonist for EP2 but not EP4

shown that synovial cell cocultures provide an optimal environment

accelerated the growth of mouse and human chondrocytes by upreg-for the preparation of such sheets for tissue transplantation and are

ulating the expression of growth‐accelerating genes such as the cyclin

particularly beneficial for shortening the required culture period

D gene, thereby stimulating an increase in cAMP (Aoyama et al.,(Hamahashi et al., 2015; Kokubo et al., 2013; Mitani et al., 2009). More-

2005). Sato et al. demonstrated that EP2 signalling was responsible

over, using experiments in minipigs (Ebihara et al., 2012), rabbits (Itofor the down‐regulation of metalloproteinase‐13 (MMP13) in protein

et al., 2012), and rats (Takaku et al., 2014), we achieved good treatment

level (Sato et al., 2010). It has been reported that MMP13 causes

outcomes by transplanting chondrocyte sheets into models of full‐

destruction of the matrix network (Goldring & Goldring, 2004;

thickness defects. These approaches are effective in treating both par-Goldring & Goldring, 2007), that inhibitors of MMP13 prevent thetial‐thickness defects (damage confined to cartilage) and full‐thickness

degradation of articular cartilage (Billinghurst et al., 1997). Hamahashi

defects (damage reaching the subchondral bone). Studies have sug-

et al. measured the amount of humoral factors produced by layered

gested that chondrocyte sheets have therapeutic effects on both types

chondrocyte sheets, and they confirmed that COL II and PGE2 secre-of cartilage damage. Because there is always a mix of partial‐ and full‐tion increased, and MMP13 secretion was inhibited, suggesting thatthickness defects in OA, chondrocyte sheets provide a promisinglayered chondrocyte sheets maintain an intact Extracellular matrix

option for inducing cartilage regeneration.

(ECM) and exert a chondroprotective action (Hamahashi et al.,In the case of autologous chondrocyte sheet transplantation, we2015). Using a rabbit model in which the animals received transection

confirmed the improvement of symptoms and magnetic resonanceof the anterior cruciate ligament, partial resection of the medial menis-

imaging findings for cartilage at 1 year after surgery according tocus, and intra‐articular administration of a gelatin hydrogel containing

our previous studies. By contrast, the problems are that results at

an EP2 agonist, Mitsui et al. reported that EP2 agonists may exert a

1 month after surgery are inconsistent in autologous chondrocyte

protective effect on articular cartilage by inhibiting MMP13 (Mitsui

sheet transplantation. Furthermore, the structural and temporal dif-

et al., 2011). Using a rabbit model of femoral chondral andferences in the formation of chondrocyte sheets between animal spe-osteochondral defects and a similar gelatin hydrogel, Otsuka et al.

cies (Kokubo et al., 2013) remain to be studied. To address thesefound that an EP2 agonist promoted regeneration of cartilage tissues

problems, new treatments that can be used in combination with

(Otsuka et al., 2009). However, there are concerns about the biocom-chondrocyte sheet transplantation are needed to provide stablepatibility of the scaffolds used in some studies (Hunziker, 2001;

results from a very early stage after chondrocyte sheet transplanta-

Mainil‐Varlet et al., 2001) and the repeated administration and con-tion. Chondrocyte sheets possess barrier functions that defend

comitant use of other treatments such as intra‐articular injection ofagainst invasion by catabolic factors in synovial fluid, prevent leakage

EP2 agonists. On the other hand, novel treatments that can be usedof proteoglycans from the extracellular matrix, and preserve bonein concomitant with chondrocyte sheet transplantation are necessary

marrow cells in the damaged cartilage. These sheets also release ana-

to provide stable results from a very early stage after chondrocyte

bolic factors to the defect, physically block catabolic factors in syno-

sheet transplantation. The effects of a selective EP2 receptor antago-

vial fluid, promote the differentiation of bone marrow cells while

nist were reported (Af Forselles et al., 2011), and it may be useful toprotecting the damaged part, and eventually enable cartilage regener-

elucidate the mechanisms underlying these effects by comparing

ation (Ebihara et al., 2012; Hamahashi et al., 2015; Ito et al., 2012;

EP2 receptor antagonists and EP2 agonists.

Kaneshiro et al., 2006; Mitani et al., 2009).

Here, we examined the efficacy of intra‐articular administration

In OA, catabolic activity is increased in chondrocytes, the prolifer-of an EP2 agonist and EP2 antagonist after chondrocyte sheet trans-

ation of chondrocytes is decreased, and the subchondral structure isplantation in treating full‐thickness articular defects in the knees of

weak (Otsuka et al., 2009). In normal cartilage, catabolic and anabolic

rabbits.

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TANI ET AL.

2|

MATERIALS AND METHODS

sectioned and stained for histological evaluation using the International Cartilage Repair Society (ICRS) grading system.

All procedures using animals in this study were performed in accordance with the Guide for the Care and Use of Laboratory Animals(NIH Publication No. 85‐23, revised 2010) published by the National

2.1|

Temperature‐responsive culture dishes

Institutes of Health, USA, and the Guidelines of Tokai University on

The temperature‐responsive culture dishes (UpCell; provided by

Animal Use (Authorization No. 141013).

CellSeed, Tokyo, Japan) were coated with poly (N‐isopropylacrylamide),

Forty‐five Japanese white rabbits, aged 16 to 18 weeks andwhich can change between hydrophilic and hydrophobic properties

weighing about 3 kg, were used in this study. These rabbits weredepending on the temperature and were developed by Okano et al.

purchased from a professional breeder (Tokyo Jiken Dobutsu, Tokyo,(Okano, Yamada, Okuhara, Sakai, & Sakurai, 1995; Okano, Yamada,Japan). Nine Japanese white rabbits were used as the source of artic-

Sakai, & Sakurai, 1993). This polymer facilitates cell adhesion andular chondrocytes and synovial cells. After the chondrocytes andgrowth in normal culture conditions at 37 °C. Reducing the tempera-

synovial cells had been harvested and isolated enzymatically, theture of the culture to <30 °C causes the surface to hydrate and swellchondrocytes were seeded onto temperature‐responsive inserts andrapidly, which prompts the complete detachment of adherent cells

the synovial cells into temperature‐responsive culture dishes, andwithout the need for typical treatment using proteolytic enzymes orthe cells were cocultured for 2 weeks. Each retrieved cell sheet

trypsin. The culture dishes were sterilized using ethylene oxide gas

was then layered to prepare triple‐layered chondrocyte sheets, which(Sekiya, Shimizu, Yamato, Kikuchi, & Okano, 2006).

were cultured for 1 week (Ebihara et al., 2012; Ito et al., 2012;
Kokubo et al., 2013).
The rabbits were anaesthetized by exposure to isoflurane, N2O,
and O2. Under sterile conditions, a medial parapatellar approach on

2.2 | Cell culture using temperature‐responsive
culture dishes

the right side was used to release the joint capsule and to create

The harvested cartilaginous and synovial tissues were finely sliced withan osteochondral defect (diameter 5 mm; depth 3 mm) on thescissors and incubated in Petri dishes in Dulbecco's modified Eagle's

patellar groove of the femur using a drill and biopsy punch (REF‐

medium/F12 (DMEM/F12; Gibco, Grand Island, NY, USA) containing

BP‐50F; Kai Industries, Seki, Japan). Bleeding from the bone was

0.5% (w/v) collagenase type 1 (Worthington, Lakewood, NJ, USA) atobserved, confirming that the osteochondral defects had been

37 °C and 5% CO2 in humidified air for 4 hr while being stirred with aproduced.

stirrer to degrade the proteins. The isolated chondrocytes and synovial

The rabbits underwent the following six treatments, with six ani-cells were passed through a cell strainer (BD Falcon Labware, Franklin

mals in each group: Group A had an osteochondral defect only

Lakes, NJ, USA) with a pore size of 100 μm, and the cells were retrieved(defect/no treatment); Group B received an EP2 agonist (EP2 Ag);by centrifugation at 1,500 rpm for 10 min. The chondrocytes were incu-

Group C received an EP2 antagonist (EP2 An); Group D received

bated in a culture medium of DMEM/F12 supplemented with 20% fetalchondrocyte sheets (ChSh); Group E received an EP2 agonist andbovine serum (Gibco) and 1% antibiotics–antimycotics (Gibco). Fromchondrocyte sheets (EP2 Ag + ChSh); and Group F received an EP2

Day 7 onward, the culture was maintained by adding a further 50‐μg/

antagonist and chondrocyte sheets (EP2 An + ChSh). These operations

ml ascorbic acid (Wako Junyaku Kogyo Corp, Osaka, Japan), and thewere performed in six unilateral knees of six rabbits in each group.

synovial cells were maintained in a culture medium of DMEM/F12 sup-After the surgery, all of the rabbits were returned to the cage without

plemented with 10% fetal bovine serum and 1% antibiotics–

splinting or immobilization.

antimycotics. From Day 4 onward, the culture was maintained by

The first injection of the EP2 agonist or EP2 antagonist was given

adding a further 50‐μg/ml ascorbic acid. All culturing was performed

4 weeks after the surgery; the second and subsequent injections wereat 37 °C in 5% CO2 and 95% humidified air. The chondrocytes wereadministered once every 2 weeks up to 10 weeks (Figure 1). The

seeded on temperature‐responsive inserts (4.2 cm2; CellSeed), andweight distribution ratio of the damaged limb was observed as a mea-

the synovial cells were seeded in temperature‐responsive culture

sure of pain evaluation. The rabbits were sacrificed at 12 weeks by andishes (9.6 cm2; CellSeed) and cocultured for 14 days. The

intravenous pentobarbital overdose. The transplanted tissues werechondrocytes were seeded at a density of 50,000 cells/cm2, and the

FIGURE 1 Timing of chondrocyte sheettransplantation and intra‐articularadministration. The first injection of the EP2
agonist or EP2 antagonist was given 4 weeks
after surgery; the second and subsequent
injections were administered once every
2 weeks up to 10 weeks. The rabbits weresacrificed at 12 weeks. Both of theconcentrations were 100 nM and both of thevolumes were 0.5 ml

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TANI ET AL.

synovial cells were seeded at a density of 10,000 cells/cm2 (Hamahashi

subsequent injections were administered once every 2 weeks up to

et al., 2015; Ito et al., 2012).

10 weeks (Figure 1).

2.3|

Chondrocyte sheet retrieval

After the cells had been cultured for 2 weeks, they reached a confluent
state and the temperature‐responsive inserts were removed from the
incubator and left for 30 min at 25 °C. After removal of the culture
medium, a polyvinylidene fluoride (PVDF) membrane was used toretrieve a chondrocyte sheet, as described (Yamato, Utsumi, Kushida
Konno, Kikuchi, & Okano, 2001). Briefly, the chondrocyte sheet wascovered with a PVDF membrane, and both were harvested carefully
as one unit. This method facilitated good retrieval of the cultured chondrocyte sheets. Next, the PVDF membrane covering the chondrocyte
sheet was harvested by rolling the membrane up at the edge of the culture dish and then overlaying the membrane on top of another chondrocyte sheet. This operation was performed three times to fabricate
a triple‐layered chondrocyte sheet. Because the layered chondrocyte
sheet floated in the culture fluid, a cell strainer (BD Falcon Labware)
was placed on top. The layered chondrocyte sheets were cultured for
1 week further in temperature‐responsive culture dishes.

2.5|

Pain evaluation

One week after surgery, an incapacitance tester (Linton Instrumentation, Norfolk, UK) was used to determine any trends in differences in
the ratio of weight distribution between the undamaged and damaged
limbs, and these trends served as the gauge for evaluating pain. This
device facilitates automatic and reproducible pain evaluation bymeasuring the distribution of load between the hind limbs using adual‐channel weight‐averaging technique, which is used widely toinvestigate pain amelioration in animal models (Ito et al., 2012; Mihara,
Higo, Uchiyama, Tanabe, & Saito, 2007). To acclimatize the animals to
the incapacitance tester, each day for 7 days after they were delivered, each animal was placed in the main container (holder) of thedevice and held still for 5 s. The measurements were performed when
the animals were immobile, after they had been transferred into therabbit holder, and when they were immobile after being removed
from, and then returned to, the holder. This operation was performed
five times, and the weight distribution ratio between the hind legs wascalculated using the following formula and is expressed here as apercentage:
Damaged limb weight distribution ratio (%) = damaged limb

2.4 | Intra‐articular administration of the EP2 agonist
and EP2 antagonist

load (g)/(undamaged limb load [g] + damaged limb load [g]) × 100.

ONO‐2750481 (Ono Pharmaceutical Co., Ltd., Tokyo, Japan) was

After surgery, the measurements were performed eight times atadministered as the EP2 agonist. PF‐04418948 (Cayman Chemical

Weeks 1, 2, 3, 4, 6, 8, 10, and 12.

This measurement was recorded as the mean of five repeats.

Co., Ltd., Ann Arbor, MI, USA) was used as the EP2 antagonist. Bothwere used at a concentration of 100 nM in reference to the past
papers (Af Forselles et al., 2011; Aoyama et al., 2005; Mitsui et al.,

2.6|

Histological evaluation of cartilage repair

2011; Otsuka et al., 2009). Briefly, 0.4‐mg ONO‐2750481 was dis-

The rabbits were sacrificed at 12 weeks by an intravenous pentobar-

solved in 0.5 ml of distilled water, and 0.02 μg of PF‐04418948 was

bital overdose. The transplanted tissue was removed from the distal

dissolved in 0.025 ml of ethanol and 0.475 ml of phosphate‐bufferedportions of the unilateral femur, fixed in 4% paraformaldehyde forsaline (PBS). The solution was administered intra‐articularly with a 27‐

1 week, and then decalcified for 2–3 weeks using distilled water

gauge needle on a 1.0‐ml syringe through the lateral infrapatellar area

(pH 7.4) containing 10% ethylenediaminetetraacetic acid. Next, thetoward the intercondylar space of the femur with each animal in atissues were embedded in paraffin wax and sectioned perpendicularly

deep knee‐flexed position. The first injection of the EP2 agonist or(8‐mm sections) through the centre of the defect. Each section was

EP2 antagonist was given 4 weeks after the surgery; the second andstained with Safranin O for glycosaminoglycans. Immunostaining

FIGURE 2 Damaged limb weight
distribution ratios at various weeks (w) after
surgeryGroup A had an osteochondral defectonly (defect/no treatment); Group B received
an EP2 agonist (EP2 Ag); Group C received an
EP2 antagonist (EP2 An); Group D received
chondrocyte sheets (ChSh); Group E received
an EP2 agonist and chondrocyte sheets(ChSh + EP2 Ag); and Group F received an
EP2 antagonist and chondrocyte sheets(ChSh + EP2 An). The weight distribution ratio(%) was used to evaluate pain and was
calculated as damaged limb load (g)/(undamaged limb load [g] + damaged‐limb load[g]) × 100. Groups D, E, and F exhibited asignificantly higher ratio than Groups A, B, andC at 1 week after surgery (*p < 0.05)

TANI ET AL.

was performed as described (Ito et al., 2012; Nagai et al., 2008).

5

Co., Toyama, Japan) were diluted 1:200 with PBS plus 1% bovine

Briefly, the sections were deparaffinized using standard procedures,

serum albumin (Sigma‐Aldrich). The sections were left in the solution

immunostained, and treated with 0.005% proteinase (type XXIV;

at 4 °C for one night, washed 10 times with PBS, and reacted at

Sigma‐Aldrich, St. Louis, MO, USA) at 37 °C for 30 min. After wash-room temperature for 1 hr with goat antimouse biotin‐conjugateding in PBS, the sections were treated with 0.3% hydrogen peroxide/

secondary antibodies that had been diluted 1:100 with 1% bovine

methanol solution at room temperature for 15–20 min to activate

serum albumin in PBS. The sections were then treated for 1 hr withendogenous peroxidase. The sections were then reacted for 30 min

horseradish peroxidase, stained with streptavidin–horseradish perox-

in normal goat serum diluted 1:20 in PBS. Mouse primary monoclonal

idase, and immersed for 2–4 min in Tris–HCl buffer (pH 7.6) contain-

antibodies to human types I and II collagen (Daiichi Fine Chemical

ing 0.05% diaminobenzidine and 0.005% hydrogen peroxide. After

FIGURE 3 Weight distribution ratio (%) at
12 weeks after surgery. Group A,
osteochondral defect only (defect/notreatment); Group B, EP2 agonist (EP2 Ag);Group C, EP2 antagonist (EP2 An); Group D,
chondrocyte sheets (ChSh); Group E,
chondrocyte sheets and an EP2 agonist(ChSh + EP2 Ag); and Group F, chondrocyte
sheets and an EP2 antagonist (ChSh + EP2
An). Significantly different weight distribution
ratios were found in Group A compared withGroups B, D, E, and F; in Group B comparedwith Groups C, D, E, and F; in Group C
compared with Groups D, E, and F; and inGroup E compared with Group F (all p < 0.05)

FIGURE 4 Histology of repaired tissues at 12 weeks after surgery after immunostaining for collagen I (Col I) and II (Col II; middle and lower rows)
and staining with Safranin O (upper row; bars = 1,000 μm). Group A, osteochondral defect only (defect/no treatment); Group B, EP2 agonist (EP2Ag); Group C, EP2 antagonist (EP2 An); Group D, chondrocyte sheets (ChSh); Group E, chondrocyte sheets and an EP2 agonist (ChSh + EP2 Ag);and Group F, chondrocyte sheets and an EP2 antagonist (ChSh + EP2 An). In Group A, stainability with Safranin O was minimal, and the defects
were repaired with fibrous tissue. In Group B, the implants were partially replaced with fibrous tissue and bone. Group C shows no staining andfibrous tissue repair. In Groups D and E, repair with hyaline cartilage was achieved, and the condition of the graft tissue, structural consistency,
defect filling rate, and subchondral bone formation were all good. In Group F, the formation of convex repaired tissue was inferior to that ofGroups D and E, but it was considered good repair. In Groups D, E, and F, type II collagen expression (brown) was observed in tissues that also
stained with Safranin O. Type II collagen was expressed uniformly in the surrounding cells (images in the lower rows), which made the cartilaginous
repair tissue borders clearer. By contrast, in Groups A, B, and C, no type II collagen expression was observed in the part of the defect that had
been replaced with fibrous tissue. Significant differences were found in Group A compared with Groups B, D, E and F; in Group B compared withGroups C, D, E, and F; and in Group C compared with Groups D, E, and F (all p < 0.05) [Colour figure can be viewed at wileyonlinelibrary.com]

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TANI ET AL.

immunostaining, the slides were counterstained with Mayer's hema-

TABLE 1

toxylin to increase cell visibility.

Item

Description

Ti: Tissue morphology

1: Exclusively not cartilage
2: Mostly not cartilage
3: Mostly fibrocartilage
4: Mostly hyaline cartilage

Matx: Matrix staining

1: None
2: Slight
3: Moderate
4: Strong

Stru: Structural integrity

1: Severe disintegration
2: Cysts or disruptions
3: No organization of chondrocytes
4: Beginning of columnarorganization of chondrocytes
5: Normal, similar to healthy mature
cartilage

Clus: Chondrocyte clustering in
implant

1: 25–100% of the cells clustered
2: <25% of the cells clustered
3: No clusters

Tide: Intactness of the calcified
cartilage layer, formation oftidemark

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

Bform: Subchondral boneformation

1: No formation
2: Slight
3: Strong

SurfH: Histological appraisalof surface architecture

1: Severe fibrillation or disruption
2: Moderate fibrillation or irregularity
3: Slight fibrillation or irregularity
4: Normal

FilH: Histological appraisal
defect filling

1: <25%
2: 26–50%
3: 51–75%
4: 76–90%
5: 91–110%

Latl: Lateral integration of
implanted material

1: Not bonded
2: Bonded at one end or partiallyboth ends
3: Bonded at both sides

Basl: Basal integration of
implanted material

1: <50%
2: 50–70%
3: 70–90%
4: 91–100%

InfH: Inflammation

1: No inflammation
3: Slight inflammation
5: Strong inflammation

Maximum total

45 points

In the histological evaluation, scoring was performed by three
independent blinded examiners using a modified form for Safranin

ICRS Histological grading system

O staining outcome as reported (O'Driscoll, Keeley, & Salter, 1998)
and the ICRS grading system (Brehm et al., 2006; Mainil‐Varlet,
Aigner, Brittberg, et al., 2003). This system can be used to evaluate
repaired tissue based on 11 items: tissue morphology, matrix staining, structural integrity, cluster formation, tidemark opening, boneformation, histological appraisal of surface architecture, histologicalappraisal of the degree of defect filling, lateral integration of
defect‐filling tissue, basal integration of defect‐filling tissue, andhistological signs of inflammation. The total score range is from 11(no repair) to 45 (normal articular cartilage).

2.7|

Statistical analysis

Analysis of variance was used to analyse the weight distribution ratio
12 weeks after surgery and the histological appraisal scores. The
Tukey–Kramer method was used for post hoc testing. The results
are expressed as the mean ± standard error, and p < 0.05 was considered significant.

3
3.1

RESULTS||

Rate of loading

Figure 2 shows the weight distribution ratio (mean ± standard error)
at Weeks 1, 2, 3, 4, 6, 8, 10, and 12 after surgery. Pain‐alleviating
effects were observed from the onset in the chondrocyte sheettransplantation groups (Groups D, E, and F). Groups A and C showed
little improvement: for Group A, from 36.2% ± 0.4% to 40.5% ± 0.2%
and for Group C, from 36.3% ± 0.4% to 38.7% ± 0.7% in Week 12
compared with Week 1 after surgery. Group B exhibited some
improvement: 35.7% ± 0.6% to 43.4% ± 0.7%. Groups D, E, and F
showed greater improvement in the ratios: Group D, 39.5% ± 0.5%
to 48.6% ± 0.6%; Group E, 40.5% ± 0.4% to 49.1% ± 0.3%; andGroup F, 40.4% ± 0.3% to 46.8% ± 0.4%. Groups D, E, and F exhibited a significantly higher ratio than Groups A, B, and C at 1 week
after surgery. Figure 3 shows the damaged limb weight distribution
ratios at Week 12 after surgery. At 12 weeks after surgery, pain‐alleviating effects were also observed in the chondrocyte sheet transplantation groups.

3.2|

Histology of repaired tissues

The operations were uneventful and all of the rabbits were returnedto the cage and allowed to act freely. There were no signs of infection.
Twelve weeks after surgery, six knees from each group were evalu-

Note. This system evaluates repaired tissue based on 11 items: tissue morphology (Ti); matrix staining (Matx); structural integrity (Stru); cluster formation (Clus); tidemark opening (Tide); bone formation (Bform);
histological appraisal of surface architecture (SurfH); histological appraisalof the degree of defect filling (FilH); lateral integration of defect‐filling tissue (Latl); basal integration of defect‐filling tissue (Basl); and histologicalsigns of inflammation (InfH). The total score range was 11–45. ICRS: International Cartilage Repair Society.

ated after the animals had been sacrificed. Figure 4 shows a histological image of repaired tissue stained with Safranin O.
In these rabbits, the total score range using the ICRS histological40.2 ± 0.5; Group E, 40.5 ± 0.4; and Group F, 38.7 ± 0.5. The chondro-

grading system was 13–41 (Table 1). Table 2 shows the ICRS grading

cyte sheet transplantation groups exhibited significantly higher scoressystem results 12 weeks after surgery. The results were Group A,

than Groups A, B, and C for every item except the histological signs of

14.8 ± 0.5; Group B, 25.4 ± 0.8; Group C, 13.7 ± 0.3; Group D,

inflammation.

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TANI ET AL.

TABLE 2ICRS grades 12 weeks after surgery
A
Defect/notreatment

B

C

D

E

F

EP2 AgEP2 An

ChSh

ChSh + P2 Ag

ChSh + EP2 An

Tissue morphology (Ti)

1.11 ± 0.07

2.39 ± 0.18

1.06 ± 0.06

3.95 ± 0.06

3.84 ± 0.09

3.78 ± 0.10

Matrix staining (Matx)

1.06 ± 0.05

2.22 ± 0.17

1.00 ± 0.00

3.89 ± 0.08

3.84 ± 0.09

3.78 ± 0.10

Structural integrity (Stru)

1.33 ± 0.16

2.61 ± 0.18

1.17 ± 0.09

4.44 ± 0.22

4.56 ± 0.19

4.11 ± 0.10

Cluster formation (Clus)

1.06 ± 0.05

2.00 ± 0.00

1.00 ± 0.00

3.00 ± 0.00

3.00 ± 0.00

3.00 ± 0.00

Tidemark opening (Tide)

1.28 ± 0.10

2.61 ± 0.19

1.22 ± 0.10

4.22 ± 0.21

4.11 ± 0.14

4.00 ± 0.00

Bone formation (Bform)

1.22 ± 0.10

2.00 ± 0.00

1.00 ± 0.00

3.00 ± 0.00

3.00 ± 0.00

3.00 ± 0.00

Histologic appraisal of surface architecture (SurfH)

1.11 ± 0.07

2.06 ± 0.06

1.00 ± 0.00

3.78 ± 0.10

3.84 ± 0.11

3.22 ± 0.10

Histologic appraisal of the degree of defect filling (FilH)

1.44 ± 0.18

2.44 ± 0.17

1.22 ± 0.18

3.95 ± 0.10

4.28 ± 0.09

3.95 ± 0.10

Lateral integration of defect‐filling tissue (Latl)

1.11 ± 0.09

2.11 ± 0.07

1.00 ± 0.00

3.33 ± 0.20

3.50 ± 0.20

3.56 ± 0.12

Basal integration of defect‐filling tissue (Basl)

1.11 ± 0.08

2.00 ± 0.00

1.00 ± 0.00

3.67 ± 0.21

3.61 ± 0.21

3.11 ± 0.08

Histologic signs of inflammation (InfH)

3.00 ± 0.00

3.00 ± 0.00

3.00 ± 0.00

3.00 ± 0.00

3.00 ± 0.00

3.00 ± 0.00

Total scores (Hgtot)

14.8 ± 0.54

25.4 ± 0.82

13.7 ± 0.29

40.2 ± 0.51

40.5 ± 0.42

38.7 ± 0.50

Note. Group A, osteochondral defect only (defect/no treatment); Group B, EP2 agonist (EP2 Ag); Group C, EP2 antagonist (EP2 An); Group D, chondrocyte
sheets (ChSh); Group E, chondrocyte sheets and an EP2 agonist (ChSh + EP2 Ag); and Group F, chondrocyte sheets and an EP2 antagonist (ChSh + EP2 An).
ICRS: International Cartilage Repair Society.

3.3|

Immunohistochemistry

Figure 4 shows immunostained repaired tissue 12 weeks after surgery.
In Groups D, E, and F, type II collagen expression (brown) was
observed in tissue that also stained with Safranin O. No type II collagen expression was observed in the defect in Groups A, B, and C. TypeI collagen expression was observed in the superficial portion offibrocartilage and all layers of subchondral bone.

sheet transplantation (Groups D, E, and F) produced significantly better pain alleviation and tissue repair compared with no treatment(Group A). The chondrocyte sheet transplantation groups (Groups
D, E, and F) obtained pain relief from 1 week after surgery, whichsuggests that the early pain‐alleviating effects resulted from thechondrocyte sheet transplantation.
One limitation of this study is that the results from the pain andhistological evaluations used were inconclusive about whether theEP2 agonist had a synergistic effect with chondrocyte sheets in

4|

DISCUSSION

repairing articular cartilage. No significant differences were observed
between Groups D and E in this study, and further studies are

In this study, we confirmed that intra‐articular injection of the EP2

necessary to clarify the mechanism of EP2 agonists and whether aagonist achieved better pain alleviation and tissue repair, as shown

synergistic effect exists.

by the significant differences in these variables between Groups A

We have performed clinical studies of joint repair using autolo-

and B, and between Groups B and C, and the lack of significant differ-gous chondrocyte sheets and have achieved excellent results. How-

ences between Groups D and E. These findings suggest that intra‐

ever, we still do not understand fully the mechanisms responsible forarticular administration of the EP2 agonist was inferior to chondrocyte

the effects of chondrocyte sheets and the factors that reinforce or

sheet transplantation for both pain alleviation and tissue repair.

weaken these effects on pain alleviation and tissue repair. Hamahashi

Although the differences between Groups A and C were not signifi-

et al. have reported significantly greater secretory capacities of TGFβcant, there were trends toward better pain alleviation and tissue repair

and PGE2 in layered chondrocyte sheets fabricated using a coculture

for intra‐articular injection of the EP2 antagonist.

method. They found that the chondrocyte sheets provide a barrier

PGE2 works through four isoforms of the EP receptor, EP1 tofunction and that the abundant secretion of humoral factors produced

EP4, and EP2 receptor‐mediated PGE2 signalling stimulates the prolif-

by the sheets supports the efficacy of chondrocyte sheets in cartilage

eration of chondrocytes and promotes the regeneration of articular

repair (Hamahashi et al., 2015). Signs of improved symptoms andcartilage in rabbits using a gelatin hydrogel containing an EP2 agonist

magnetic resonance imaging findings indicating cartilage repair were(Mitsui et al., 2011; Otsuka et al., 2009). Aoyama et al. have previously

evident from 3 months after surgery for autologous chondrocyte

demonstrated that EP2 is the major PGE2 receptor in articular

sheet transplantation in our previous studies (data not shown).

chondrocytes (Aoyama et al., 2005). In addition, EP2 agonists prevent

There are structural and temporal differences in the formation of

the degeneration of articular cartilage by inhibiting MMP13 in rabbits

chondrocyte sheets between animal species (Kokubo et al., 2013).

(Mitsui et al., 2011; Otsuka et al., 2009). However, the use of such

New treatments that can be used in combination with chondrocyte

scaffolds has problems in terms of the long‐term results and complica-

sheet transplantation are needed to provide stable results from a verytions and involves many biocompatibility issues (Hunziker, 2001;

early stage after chondrocyte sheet transplantation. Our data suggest

Mainil‐Varlet et al., 2001).

that intra‐articular administration of an EP2 agonist may be effective

In this study, the repeated intra‐articular injection of the EP2 ago-

in repairing articular cartilage while synergy with chondrocyte

nist and concomitant use of the EP2 agonist (Group B) and chondrocyte

sheet transplantation needs to be further investigated. In the early

8

TANI ET AL.

post‐operative period, the barrier function of the chondrocyte sheets
may provide some pain relief, but this needs to be confirmed byfurther studies. PGE2 and TGFβ also have an inhibitory effect on T cellproliferation (Aggarwal & Pittenger, 2005; Di Nicola et al., 2002). By
counteracting immune rejection, such as graft‐versus‐host disease,this inhibitory effect may be an advantage and provide a clinicalapplication. We plan to investigate further the protocols and safetyof administration of EP2 agonists.

5|

C O N CL U S I O N S

We have confirmed the effectiveness of intra‐articular administrationof an EP2 agonist in articular cartilage repair. No synergistic effectBrehm, W., Aklin, B., Yamashita, T., Rieser, F., Trüb, T., Jakob, R. P., &Mainil‐Varlet, P. (2006). Repair of superficial osteochondral defects
with an autologous scaffold‐free cartilage construct in a caprine model:
Implantation method and short‐term results. Osteoarth Cart, 14,1214–1226.
Buckwalter, J. A., Saltzman, C., & Brown, T. (2004). The impact of osteoarthritis: Implications for research. Clinical Orthopaedics and Related
Research, 427, S6–S15.
Di Nicola, M., Carlo‐Stella, C., Magni, M., Milanesi, M., Longoni, P. D.,
Matteucci, P., … Gianni, A. M. (2002). Human bone marrow stromalcells suppress T‐lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli. Blood, 99, 3838–3843.
Ebihara, G., Sato, M., Yamato, M., Mitani, G., Kutsuna, T., Nagai, T., …
Mochida, J. (2012). Cartilage repair in transplanted scaffold‐free chondrocyte sheets using a minipig model. Biomaterials, 33, 3846–3851.

with chondrocyte sheets was observed.

Goldring, M. B., & Goldring, S. R. (2007). Osteoarthritis. Journal of Cellular
Physiology, 213, 626–634.

ACKNOWLEDGEMEN TS

Goldring, S. R., & Goldring, M. B. (2004). The role of cytokines in cartilage
matrix degeneration in osteoarthritis. Clinical Orthopaedics and Related
Research, 427, S27–S36.

This work was supported by a Health Labour Sciences Research Grant
from the Ministry of Health, Labour, and Welfare of Japan, and theJapan Agency for Medical Research and Development: Research Project for Practical Applications of Regenerative Medicine. We thank
CellSeed Inc. for supplying the temperature‐responsive culture insertsthat were used in this study. We are grateful to the Education andResearch Support Center, Tokai University. Masato Sato, M.D., Ph.D.
is receiving research funding from CellSeed Inc. Teruo Okano, Ph.D.
is a founder and a member of the board of CellSeed Inc., Tokyo, Japan,
which has licences for certain cell sheet‐related technologies andpatents from Tokyo Women's Medical University. Masayuki Yamato,
Ph.D. is a shareholder of CellSeed Inc. Tokyo Women's Medical
University is receiving research funding from CellSeed Inc.
CONF LICT OF INT E RE ST
The authors have declared that there is no conflict of interest.
ORCID
Masato Sato

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

Masayuki Yamato

http://orcid.org/0000-0002-3146-1024

RE FE R ENC E S
Af Forselles, K. J., Root, J., Clarke, T., Davey, D., Aughton, K., Dack, K., &Pullen, N. (2011). In vitro and in vivo characterization of PF‐
04418948, a novel, potent and selective prostaglandin EP2 receptor
antagonist. British Journal of Pharmacology, 164, 1847–1856.
Aggarwal, S., & Pittenger, M. F. (2005). Human mesenchymal stem cells
modulate allogeneic immune cell responses. Blood, 105, 1815–1822.
Anderson, D. D., Chubinskaya, S., Guilak, F., Martin, J. A., Oegema, T. R.,
Olson, S. A., & Buckwalter, J. A. (2011). Post‐traumatic osteoarthritis:
Improved understanding and opportunities for early intervention. Journal of Orthopaedic Research, 29, 802–809.
Aoyama, T., Liang, B., Okamoto, T., Matsusaki, T., Nishijo, K., Ishibe, T., …
Toguchida, J. (2005). PGE2 signal through EP2 promotes the growthof articular chondrocytes. Journal of Bone and Mineral Research, 20,
377–389.
Billinghurst, R. C., Dahlberg, L., Ionescu, M., Reiner, A., Bourne, R.,
Rorabeck, C., … Poole, A. R. (1997). Enhanced cleavage of type II collagen by collagenases in osteoarthritic articular cartilage. The Journal of
Clinical Investigation, 99, 1534–1545.

Hamahashi, K., Sato, M., Yamato, M., Kokubo, M., Mitani, G., Ito, S., …
Mochida, J. (2015). Studies of the humoral factors produced by layered
chondrocyte sheets. Journal of Tissue Engineering and Regenerative Medicine, 9, 24–30.
Hangody, L., Feczko, P., Bartha, L., Bodó, G., & Kish, G. (2001).
Mosaicplasty for the treatment of articular defects of the knee andankle. Clinical Orthopaedics and Related Research, 391(Suppl), 328–336.
Hangody, L., Kish, G., Karpati, Z., & Szerb, I. (1997). Autogenous
osteochondral graft technique for replacing knee cartilage defects indogs. Orthopedics, 5, 175–181.
Hunziker, E. B. (2001). Articular cartilage repair: Basic science and clinicalprogress. A review of the current status and prospects. Osteoarthritis
Cartilage, 10, 432–463.
Hunziker, E. B., & Rosenberg, L. C. (1996). Repair of partial‐thickness
defects in articular cartilage: Cell recruitment from the synovial membrane. The Journal of Bone and Joint Surgery. American Volume, 78,
721–733.
Ito, S., Sato, M., Yamato, M., Mitani, G., Kutsuna, T., Nagai, T., … Mochida,J. (2012). Repair of articular cartilage defect with layered chondrocyte
sheets and cultured synovial cells. Biomaterials, 33, 5278–5286.
Kaneshiro, N., Sato, M., Ishihara, M., Mitani, G., Sakai, H., & Mochida, J.
(2006). Bioengineered chondrocyte sheets may be potentially usefulfor the treatment of partial thickness defects of articular cartilage. Biochemical and Biophysical Research Communications, 349, 723–731.
Kokubo, M., Sato, M., Yamato, M., Mitani, G., Kutsuna, T., Ebihara, G., …
Mochida, J. (2013). Characterization of chondrocyte sheets preparedusing a co‐culture method with temperature‐responsive culture inserts.
Journal of Tissue Engineering and Regenerative Medicine, 10, 486–495.
https://doi.org/10.1002/term.1764
Mainil‐Varlet, P., Aigner, T., Brittberg, M., Bullough, P., Hollander, A.,
Hunziker, E., … International Cartilage Repair Society (2003). Histological assessment of cartilage repair: A report by the histology endpointcommittee of the international cartilage repair society (ICRS). The Journal of Bone and Joint Surgery. American Volume, 85‐A(Suppl 2), 45–57.
Mainil‐Varlet, P., Rieser, F., Grogan, S., Mueller, W., Saager, C., & Jakob, R.
P. (2001). Articular cartilage repair using a tissue‐engineered cartilage‐
like implant: An animal study. Osteoarthritis and Cartilage, 9, S6–S15.
Mihara, M., Higo, S., Uchiyama, Y., Tanabe, K., & Saito, K. (2007). Different
effects of high molecular weight sodium hyaluronate and NSAID on the
progression of the cartilage degeneration in rabbit OA model.
Osteoarth Cart, 15, 543–549.
Mitani, G., Sato, M., Lee, J. I., Kaneshiro, N., Ishihara, M., Ota, N., …
Mochida, J. (2009). The properties of bioengineered chondrocyte
sheets for cartilage regeneration. BMC Biotechnology, 9, 17.

9

TANI ET AL.

Mithoefer, K., Williams, R. J. 3rd, Warren, R. F., Potter, H. G., Spock, C. R.,
Jones, E. C., … Marx, R. G. (2006). Chondral resurfacing of articular cartilage defects in the knee with the microfracture technique. Surgical
technique. Journal of Bone and Joint Surgery, 88, 294–304.

Sato, T., Konomi, K., Fujii, R., Aono, H., Aratani, S., Yagishita, N., …
Nakajima, T. (2010). Prostaglandin EP2 receptor signalling inhibits theexpression of matrix metalloproteinase 13 in human osteoarthritic
chondrocytes. Annals of the Rheumatic Diseases, 70, 221–226.

Mitsui, H., Aoyama, T., Furu, M., Ito, K., Jin, Y., Maruyama, T., … Toguchida,J. (2011). Prostaglandin E2 receptor type 2‐selective agonist prevents
the degeneration of articular cartilage in rabbit knees with traumatic
instability. Arthritis Research & Therapy, 13, R146.

Sekiya, S., Shimizu, T., Yamato, M., Kikuchi, A., & Okano, T. (2006).
Bioengineered cardiac cell sheet grafts have intrinsic angiogenic potential. Biochemical and Biophysical Research Communications, 341,
573–582.

Nagai, T., Sato, M., Furukawa, K. S., Kutsuna, T., Ohta, N., Ushida, T., & Mochida,J. (2008). Optimization of allograft implantation using scaffold‐freechondrocyte plates. Tissue Engineering. Part a, 14, 1225–1235.

Steadman, J., Rodkey, W., & Briggs, K. (2002). Microfracture to treat full‐thickness chondral defects: Surgical technique, rehabilitation, and outcomes. The Journal of Knee Surgery, 15, 170–176.

Ochi, M., Uchio, Y., Kawasaki, K., & Iwasa, J. (2002). Transplantation of cartilage‐like tissue made by tissue engineering in the treatment ofcartilage defects of the knee. Journal of Bone and Joint Surgery. British
Volume (London), 84, 571–578.

Steadman, J., Rodkey, W., & Rodrigo, J. (2001). Microfracture: Surgical
technique and rehabilitation to treat chondral defects. Clinical Orthopaedics and Related Research, 391(Suppl), S362–S369.

O'Driscoll, S. W., Keeley, F. W., & Salter, R. B. (1998). Durability of regenerated articular cartilage produced by free autogenous periosteal grafts
in major full‐thickness defects in joint surfaces under the influence ofcontinuous passive motion. A follow‐up report at one year. Journal of
Bone and Joint Surgery, 70, 595–606.
Okano, T., Yamada, N., Okuhara, M., Sakai, H., & Sakurai, Y. (1995). Mechanism of cell detachment from temperature‐modulated, hydrophilic‐hydrophobic polymer surfaces. Biomaterials, 16, 297–303.
Okano, T., Yamada, N., Sakai, H., & Sakurai, Y. (1993). A novel recovery
system for cultured cells using plasma‐treated polystyrene dishes
grafted with poly (N‐isopropylacrylamide). Journal of Biomedical Materials Research, 27, 1243–1251.
Otsuka, S., Aoyama, T., Furu, M., Ito, K., Jin, Y., Nasu, A., … Toguchida, J.
(2009). PGE2 signal via EP2 receptors evoked by a selective agonist
enhances regeneration of injured articular cartilage. Osteoarthritis andCartilage, 17, 529–538.
Peat, G., McCarney, R., & Croft, P. (2001). Knee pain and osteoarthritis inolder adults: A review of community burden and current use of primary
health care. Annals of the Rheumatic Diseases, 60, 91–97.

Sugimoto, Y., & Narumiya, S. (2007). Prostaglandin E receptors. Journal of
Biological Chemistry, 282, 11613–11617.
Szerb, I., Hangody, L., Duska, Z., & Kaposi, N. P. (2005). Mosaicplasty:
Long‐term follow‐up. Bulletin/Hospital for Joint Diseases, 63, 54–62.
Takaku, Y., Murai, K., Ukai, T., Ito, S., Kokubo, M., Satoh, M., … Sato, M.
(2014). In vivo cell tracking by bioluminescence imaging after transplantation of bioengineered cell sheets to the knee joint. Biomaterials,
35, 2199–2206.
Yamato, M., Utsumi, M., Kushida Konno, A. C., Kikuchi, A., & Okano, T.
(2001). Thermoresponsive culture dishes allow the intact harvest ofmultilayered keratinocyte sheets without dispase by reducing temperature. Tissue Engineering, 7, 473–480.

How to cite this article: Tani Y, Sato M, Yokoyama M, et al.
Intra‐articular administration of EP2 enhances the articular cartilage repair in a rabbit model. J Tissue Eng Regen Med.
2018;1–9. https://doi.org/10.1002/term.2748