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Polydactyly-derived allogeneic chondrocyte cell-sheettransplantation with high tibial osteotomy as regenerative
therapy for knee osteoarthritis

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Kosuke Hamahashi1,2, Eriko Toyoda 1,2, Miya Ishihara3, Genya Mitani1,2, Tomonori Takagaki1,2, Nagatoshi Kaneshiro1,2,
Miki Maehara1,2, Takumi Takahashi 1,2, Eri Okada1,2, Ayako Watanabe1,2, Yoshihiko Nakamura4, Reiko Kato5, Ryo Matoba6,
Takehiko Takagi7, Hidenori Akutsu7, Akihiro Umezawa7, Hiroyuki Kobayashi8, Tadashi Akamatsu9, Masayuki Yamato 10,
Teruo Okano11, Masahiko Watanabe 1,2 and Masato Sato 1,2 ✉Allogeneic cell therapies are not fully effective in treating osteoarthritis of the knee (OAK). We recently reported that transplantationof autologous chondrocyte cell-sheets along with open-wedge high tibial osteotomy promoted hyaline cartilage repair in humans.
Here we describe our regenerative therapy for OAK using polydactyly-derived allogeneic chondrocyte cell-sheets (PD sheets) andtemperature-responsive culture inserts. Ten patients with OAK and cartilage defects categorized arthroscopically as Outerbridge
grade III or IV received the therapy. Cartilage viscoelasticity and thickness were assessed before and after transplantation.
Arthroscopic biopsies obtained 12 months after transplantation were analyzed histologically. Gene expression was analyzed toevaluate the PD sheets. In this small initial longitudinal series, PD sheet transplantation was effective in treating OAK, as indicatedby changes in cartilage properties. Gene marker sets in PD sheets may predict outcomes after therapy and provide markers for theselection of donor cells. This combined surgery may be an ideal regenerative therapy with disease-modifying effects in OAK
patients.
npj Regenerative Medicine (2022)7:71 ; https://doi.org/10.1038/s41536-022-00272-1

INTRODUCTION
Osteoarthritis (OA) is the most common cause of loss of mobility
and is the most prevalent form of musculoskeletal disease
worldwide1,2. OA adversely affects the quality of life, work
productivity, and cost of health care. We recently reported on
our clinical research on the effects of combined surgery andautologous chondrocyte cell-sheet transplantation in OA3. No
serious adverse events related to the combined surgery wereobserved during the treatment and follow-up period, andexcellent regeneration of the articular surface with hyaline
cartilage was conﬁrmed in second-look arthroscopic surgery. Allbiopsy samples of regenerated cartilage revealed strong staining
for Safranin O and expression of type II collagen (COL2). The Knee
Injury and Osteoarthritis Outcome Score (KOOS) and Lysholm
Knee Score (LKS) improved signiﬁcantly after surgery, and these
improvements were maintained for more than 3 years. However,autologous chondrocyte cell-sheet transplantation has several
limitations. For example, arthroscopic surgery, which requires theharvesting of cartilage and synovial tissues, is needed before
transplantation; the harvested cells and their properties differ
between individuals and are occasionally harvested in insufﬁcientnumbers to fabricate cell sheets, and chromosomal abnormalities
are often found in cells obtained from elderly patients withosteoarthritis of the knee (OAK). Given these limitations and thefact that articular cartilage is immune tolerant, we consider the useof allogeneic cell sheets as a viable option for avoiding the needto harvest tissue.
Allogeneic mesenchymal stem cells (MSCs), adipose-derived stem
cells, and umbilical cord stem cells are often used in clinical trials oftreatments for OAK. However, most of these methods involve celltherapies using intra-articular injection. These therapies tend toachieve temporary improvement of symptoms not but structural
reconstruction or the production of native hyaline cartilage. The
recent increase in the use of cell-based strategies for cartilage repair
has generated an urgent need for potent and safe off-the-shelf cellsources. We have focused on polydactyly surgery as a way ofobtaining tissue samples as allogeneic cell sources and havesucceeded in fabricating polydactyly-derived allogeneic chondrocyte cell-sheets (PD sheets) using temperature-responsive culture
inserts4,5. Recently, Kondo et al. reported the preclinical safety andefﬁcacy of polydactyly-sourced juvenile cartilage derivedchondrocyte sheets in vitro and in vivo using a rat focal
osteochondral defect model6. PD chondrocytes proliferate rapidly,
establish a layered structure with a sufﬁcient extracellular matrix,
and form sheets that can be easily manipulated. Although thenumber of autologous chondrocyte sheets that can be fabricated is

1
Department of Orthopaedic Surgery, Surgical Science, Tokai University School of Medicine, 143 Shimokasuya, Isehara, Kanagawa 259-1193, Japan. 2Center for Musculoskeletal
innovative Research and Advancement (C-MiRA), Tokai University Graduate School, Isehara, Kanagawa 259-1193, Japan. 3Department of Medical Engineering, National Defense
Medical College, 3-2 Namiki, Tokorozawa, Saitama 359-8513, Japan. 4Cell Processing Center, Tokai University Hospital, 143 Shimokasuya, Isehara, Kanagawa 259-1193, Japan.
5
National Institute of Health Sciences, 3-25-26 Tonomachi, Kawasaki-ku, Kawasaki, Kanagawa 210-9501, Japan. 6DNA Chip Research Inc., 1-15-1 Kaigan, Suzue Baydium 5F Minatoku, Tokyo 105-0022, Japan. 7National Center for Child Health and Development, 2-10-1 Okura,Setagaya-ku, Tokyo 157-8535, Japan. 8Department of Clinical Pharmacology, Tokai
University School of Medicine, 143 Shimokasuya, Isehara, Kanagawa 259-1193, Japan. 9Department of Plastic Surgery, Surgical Science, Tokai University School of Medicine, 143
Shimokasuya, Isehara, Kanagawa 259-1193, Japan. 10Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University, 8-1 Kawada-cho, Shinjuku,
Tokyo 162-8666, Japan. 11Cell Sheet Tissue Engineering Center, Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, 30 South 2000, East Salt Lake, UT
84112, USA. ✉email: sato-m@is.icc.u-tokai.ac.jp

Published in partnership with the Australian Regenerative Medicine InstituteK. Hamahashi et al.

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limited4, theoretically, more than 600 PD sheets can be fabricatedfrom passage 2 (P2) cells and more than 3000 PD sheets can be
fabricated from P3 cells. Here, we describe our use of PD sheettransplantation combined with a high tibial osteotomy (HTO) andsuggest that it is an ideal form of regenerative therapy for patientswith OAK.
npj Regenerative Medicine (2022) 71

We have designed a combination therapy (Fig. 1a) in whichconventional surgical treatment for OAK, which is covered by
National Health Insurance in our country, was followed by theremoval of unhealthy tissue, bone marrow stimulation, andchondrocyte sheet transplantation (the “RMSC method”) to treat
cartilage defects3. This study was designed to evaluate the
Published in partnership with the Australian Regenerative Medicine InstituteK. Hamahashi et al.

Fig. 1 Surgical interventions and transplantation of chondrocyte sheets. a Combination of conventional surgical intervention andimplantation of chondrocyte sheets. Polydactyly-derived allogenic chondrocyte sheets (PD sheets) were fabricated from cartilage tissue
obtained from polydactyly surgery. In the cell-processing center, isolated chondrocytes were passaged once and stocked at −180 °C. To
fabricate PD sheets, cells were thawed and passaged once and then seeded on temperature-responsive culture inserts. The PD sheets were
further cultured for 14–16 days and transplanted. OAK patients were ﬁrst treated with conventional surgical treatments OWHTO, followed by
the removal of unhealthy tissue, marrow stimulation, and chondrocyte sheet transplantation (RMSC method). b The overall protocol for theclinical study. The ﬁnal decision about entry into the clinical study was made during the arthroscopic evaluation. For patients meeting theinclusion criteria, LIPA was used to assess their cartilage defects. RMSC removal of unhealthy tissue, marrow stimulation, and chondrocyte
sheet transplantation, OWHTO open-wedge high tibial osteotomy, LIPA laser-induced photoacoustic, KOOS Knee Injury and Osteoarthritis
Outcome Score, LKS Lysholm Knee Score, MRI magnetic resonance imaging. c The CONSORT ﬂow diagram. CONSORT diagram for a singlearm, open-label, uncontrolled, comparative study (that is, enrolment, intervention allocation, follow-up, and data analysis).

effectiveness of the total treatment but not the add-on effects ofeach treatment. HTO for patients with OAK is a joint-preserving
procedure that has garnered attention throughout the world.
Articular cartilage is sometimes regenerated after the improvement of whole leg alignment by HTO, but this is mainly ﬁbrous
cartilage, which has different properties from hyaline cartilage7,8.
We hypothesized that PD sheet transplantation in conjunction
with HTO may accelerate the regeneration of native cartilage, suchas hyaline cartilage, reduce the symptoms of OAK, and help tomaintain the biological qualities of the knee joint.
Temperature-responsive culture dishes or inserts (CellSeed Inc.,Tokyo, Japan), whose use was ﬁrst reported by Okano et al.9,10,
have a special intelligent surface. Using these inserts, chondrocyte
sheets can be produced by maintaining the extracellular matrix
and then reducing the temperature to release the sheets, with noneed for enzymatic digestion.
We were the ﬁrst to report the application of layered
chondrocyte sheets for articular cartilage repair11,12. We havereported evidence from animal studies indicating the potential ofthese layered chondrocyte sheets in the treatment of nontraumatic arthritis in rat13, a partial defect in rabbit articular cartilage14,
and osteochondral defects in rat15,16, rabbit17–19, and minipig20
cartilage. These types of defects are usually present in OAK. We
have also investigated the mode of action of the layered
chondrocyte sheets, and we have suggested that chondrocyte
sheets exert their ongoing regeneration effects in articularcartilage through the continuous secretion of humoral factors
such as melanoma inhibitory activity (MIA), transforming growth
factor β (TGF-β), and prostaglandin E2, which are important in
cartilage regeneration21,22.
Ten patients with OAK and cartilage defects were enrolled inthis study and received therapy with PD sheets. A rigorous
evaluation protocol (Fig. 1b) was designed to assess the endpoints
of safety and efﬁcacy of the therapy and to evaluate the clinicaland structural outcomes. The properties of the transplanted
allogeneic PD sheets were also evaluated thoroughly using gene
expression analysis to investigate their potential use for predicting
the clinical and structural outcomes of the therapy. In this small
initial longitudinal case series, we wanted to determine whether
the expression of speciﬁc marker gene sets in PD sheets could
provide potential markers for predicting the outcomes of this newregenerative therapy for OAK.
RESULTS
Predicting clinical and structural outcomes by analyzing PD
cell sheets
Figure 2 shows the properties of PD sheets transplanted in thisstudy. The PD sheets can be handled using a circular white
support membrane of polyvinylidene diﬂuoride (Fig. 2a). The
multilayered structures were conﬁrmed by histological staining(Fig. 2b). Immunostaining showed that the sheets strongly
expressed ﬁbronectin (FN), COL1, and aggrecan (ACAN). PD sheets
exhibited almost no COL2 expression (Fig. 2b). The cells in the PD
sheets were dedifferentiated, and the properties of chondrocyte
Published in partnership with the Australian Regenerative Medicine Institutesheets were different from those of hyaline cartilage. Figure 2c–eshows the distribution of properties of the transplanted PD sheetswith respect to cell number, viability, and thickness, respectively.
Figure 2f shows the results of the ﬂow cytometric analysis of thedispersed PD sheets. Almost all cells from PD sheets were positive
for cluster of differentiation 81 (CD81) and mesenchymal stem cellmarkers, CD90 (Thy-1), CD44 (receptor for hyaluronic acid), andCD105 (endoglin). CD146 (melanoma cell adhesion molecule)positive and GD2 (disialoganglioside GD2)-positive cells were
present at a frequency of 20–70% and CD49a (integrin α1)-positive
cells at a frequency of 5–25%. Almost all cells were negative forthe blood lineage markers CD31 (platelet endothelial cell adhesion
molecule-1) and CD45 (leukocyte common antigen). The marker
expression was similar to that of adult knee chondrocyte sheetsthat were transplanted in a previous clinical study that usedautologous cells. The production of transforming growth factor β1(TGF-β1) and previously identiﬁed efﬁcacy-associated factors5,
melanoma inhibitory activity (MIA), Dickkopf WNT signaling
pathway inhibitor 1 (DKK1), endothelial cell-speciﬁc molecule 1(ESM1), and gremlin 1 (GREM1) conﬁrmed by enzyme-linked
immunosorbent assay (ELISA) (Fig. 2g). Quantitative polymerase
chain reaction (qPCR) analysis of the genes expressed by the PD
sheets revealed widespread expression of cartilage-related genes
of interest (Fig. 2h).
Clinical outcomes
No serious adverse events related to the combined surgeries, suchas deep infection or pseudoarthrosis, were observed during thefollow-up period. Other adverse events were reported andincluded deep vein thrombosis in one patient, pain in 10 patients,
leukocytosis in 10 patients, and increased C-reactive protein levels
in 10 patients. Deep vein thrombosis was healed by conservative
therapy. These adverse events were considered to have been
related to the open-wedge high tibial osteotomy (OWHTO). We
believe that these were unlikely to have been related to thetransplantation of PD sheets.
The LKS improved signiﬁcantly from 40.1 ± 13.9 points preoperatively to 80.5 ± 15.7 points at the ﬁnal follow-up. All of the
KOOS subscales (symptom, pain, function in daily living, functionin sport and recreation, and quality of life) also improved
signiﬁcantly (Fig. 3a, b and Supplementary Table 1, 2).
Structural outcomes
The cartilage defects and locations are described in Table 1.
Brieﬂy, the mean total defect area was 15.9 cm2 for one patientand >20 cm2 in four patients. Ten patients had medial femoral
condyle (MFC) defects (mean area 10.7 cm2), six patients had
trochlear (Tr) defects (mean area 4.6 cm2), and ﬁve patients had
kissing defects of the tibia (mean area 4.0 cm2).
The femorotibial angle and percentage of mechanical axis (%MA) changed signiﬁcantly from 179.4° ± 2.8° to 168.9° ± 2.8° andfrom 24.1 ± 10.8 to 67.5 ± 9.2 from before to after transplantation,
respectively.
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4

a

c

d

e

b

COL1

f 120
100

Posive cells (%)

HE

80
60
40
20

COL2

0
CD31

g

FN

CD90

CD44

CD105 CD146 CD49a

GD2

ng/mL

ng/mL

ng/mL

ng/mL

3.5

70

250

1400

140

3.0

60

1200

120

2.5

50

1000

100

2.0

40

150

800

80

1.5

30

100

600

60

1.0

20

400

40

0.5

10

200

20

0.0

0

h

Magnetic resonance imaging (MRI) conﬁrmed the regenerationof cartilage in areas that lacked cartilage preoperatively (Supplementary Figure 1). The results of the Magnetic ResonanceObservation of Cartilage Repair Tissue 2.0 (MOCART 2.0) score
improved signiﬁcantly (Table 1).

npj Regenerative Medicine (2022) 71

CD81

ng/mL

Concentraon

ACAN

CD45

TGFβ1

200

50
0

MIA

0

DKK1

0

ESM1

GREM1

The results of the laser-induced photoacoustic (LIPA) evaluation are
shown in Table 1. The viscoelastic characteristics of the regenerated
cartilage are expressed as the ratio relative to that of normal cartilage
in the same joint. The characteristics improved after transplantation,and the mean thickness of the regenerated cartilage was 3.54 mm.

Published in partnership with the Australian Regenerative Medicine InstituteK. Hamahashi et al.

Fig. 2 Properties of PD sheets. a PD sheets were handled using a circular PVDF support membrane. b Representative images of cross-sectionof PD sheet. Histological staining with hematoxylin and eosin (HE), immunohistochemical staining for type I collagen (COL1), type II collagen(COL2), aggrecan (ACAN), and ﬁbronectin (FN). scale bar; 50 μm. c–e Distribution of transplanted PD sheet characteristics of nine lots: The box
represents the interquartile range of the values. The top and bottom of the whiskers represent the maximum and minimum values. The line
inside the box represents the median values. Cell number (c) and cell viability (d) were determined after enzymatic digestion of a cell sheet.
e Thickness of PD sheet was measured using microscopic images of cross sections. f Flow cytometric analysis of surface markers for the cells in
the PD sheets. The PD sheets were digested enzymatically and analyzed by single-color staining. Data are expressed as the mean ± SD of thepercentage of surface marker-expressing cells from nine lot of sheets (Supplementary Fig. 2). Cells were positive for CD81, CD90, CD44, andCD105 and negative for CD31 and CD45. Staining for CD146, CD49a, and GD2 differed between the lots. g Concentrations of humoral factors
secreted by PD sheets. Each mark indicates a lot of sheets. Some marks overlap. h The gene expression proﬁle of transplanted sheets wasanalyzed using qPCR for cartilage-related genes, and the results are reported relative to the expression of GAPDH. A total of nine lots of PD
sheets were fabricated using cryopreserved cells from three polydactyly patient donors. Three lots were from donor 1 and were used to create
sheets 1-1, 1-2, and 1–3 in h. Four lots were from donor 2 and were used to create sheets 2-1, 2-2, 2-3, and 2–4. Two lots were from donor 3
and were used to create sheets 3-1 and 3-2. Each lot was used for one patient, except for sheet 3-2, which was transplanted into two patients(patients 9 and 10). Data with the same color were from the same lot of PD sheets in g, h.

Histological outcomes
All biopsies revealed strong staining for Safranin O and expressionof COL2 (Fig. 3g). Although the degree to which regeneration
occurred varied between patients and some cases represent a signof degeneration, these results nevertheless suggest that regeneration of hyaline cartilage had occurred. The results of the
Osteoarthritis Research Society International (OARSI) histologicalscore, the Mankin score, and the International Cartilage Repair
Society (ICRS II) overall assessment are shown in Table 1. The
quality of regenerated cartilage was very high, with an ICRS IIaverage score of 80.8 (64–96, 0: ﬁbrous tissue, 100: hyaline
cartilage), as shown in Table 1 although ﬁbrocartilage was alsopresent.
Potential markers for predicting the outcomes
To identify PD sheets that promoted superior outcomes, genemarker sets predictive of the postoperative clinical and structural
outcomes were identiﬁed based on the correlational analysis
between the gene expression proﬁle and outcomes. The analyses
were described previously in the study on autologous cell sheets3.
Potential marker sets were selected from cartilage-related genes(list in Supplementary Table 3) with the highest probability of
appearance in a matrix of 100 sets of Pearson correlation
coefﬁcients using the leave-ﬁve-out process.
The gene score was calculated using the gene expression dataof the marker set as described in Methods (Supplementary Tables
4–7). Figure 3c–f shows the scatter plots indicating the genescores of the transplanted PD sheets and clinical outcomes foreach individual case. The marker sets for each outcome score wereas follows: for KOOS (Fig. 3c) PTGS2, TGFB1, MIA, and G0S2; for LKS(Fig. 3d) COL1A2, MATN2, SOX9, and TGFB1; and for OARSI
histological score (Fig. 3e) CCN2, COMP, ACKR4, ESM1, and GREM1;
for ICRS II overall assessment (Fig. 3f) COL2A1, COL27A1, ACKR4,and ESM1.
Case report (Patient 5)
A 51-year-old woman underwent PD-sheet transplantation combined with OWHTO for OAK of the left knee. The femorotibial
angle and %MA changed from 183° to 167° and 11.4 to 68.9,
respectively (Fig. 4a). Subchondral bone was exposed across awide area in the MFC (Fig. 4b) and medial tibial plateau (Fig. 4c).
After using LIPA to evaluate the viscoelastic properties in theresidual cartilage and subchondral bone, we performed microfracture (Fig. 4d) and transplantation of PD sheets (Fig. 4e). One
year after transplantation, regenerated cartilage that fully covered
the defect area was observed in the MFC (Fig. 4f) and medial tibial
plateau (Fig. 4g). Preoperative MRI showed a full-thickness
cartilage defect at the MFC. This defect area appeared to becoated with PD sheets and was ﬁlled with regenerative tissue after
Published in partnership with the Australian Regenerative Medicine Institute3 months, and the regenerative tissue was clearly visible andmaintained at 12 months after transplantation (Fig. 4h).
DISCUSSION
We have conﬁrmed the mode of action, safety, and efﬁcacy of PD
sheets in experiments using the xenogeneic model4 and in otherstudies19,21,23 before undertaking the clinical study.
In the current study, we speculate that the mode of action ofhumoral factors of PD sheets was the most likely mechanism
because we did not use immunosuppressants. In our earlier
research, we found variability in the therapeutic potential of PD
sheets after xenogeneic orthotopic transplantation and recognized the importance of the selection of donors for future studies5.
Cavalli et al.24 reported that polydactyly chondrocytes can be
expanded in vitro and maintain a fast and steady proliferative rate
for up to ﬁve passages, which is an important characteristic of an
off-the-shelf cell source. The proper passage of PD cells is also
critical in clinical trials. To address this issue, we identiﬁed
potential gene marker sets for predicting the outcome of PD sheettransplantation (Fig. 3c–f). The marker genes for each outcome
score are different. TGFB1 appeared in common in KOOS and LKS,
both of which are in the self-report symptom questionnaire. ESM1
and ACKR4 appeared in the OARSI histological score and ICRS II
overall assessment, both scores being related to biopsy tissue
evaluation. The marker set of genes can be useful for selecting theallogeneic cell source for cell sheet fabrication prior to clinical use.
Because we analyzed the expression of cartilage-related genes
in only a small initial longitudinal series, other genes may havebeen extracted if a different gene set had been used for analysis.
The current marker set of genes may be improved by replacing
the genes with newly identiﬁed, more predictive ones.
Articular cartilage sometimes regenerates after bone marrow
stimulation or abrasion arthroplasty combined with HTO. However, these regenerated tissues are mainly ﬁbrous cartilage, which
differs from native hyaline cartilage7,8,25–27 and does not provide
the same long-term load-bearing effects as hyaline cartilage.
Previously, we compared clinical outcomes between patients
undergoing OWHTO and total knee arthroplasty (TKA). In thesepatients, the KOOS was lower in patients who underwent OWHTO
than in those who received TKA, and residual symptoms such as
knee grinding, clicking, and stiffness were worse after OWHTO
than after TKA28. On this point, in the current study, we conﬁrmedthat regeneration of hyaline cartilage had occurred after PD sheettransplantation combined with OWHTO. Our ﬁndings suggest that
long-term therapeutic effects along with improvement of lower
limb alignment can be expected after the combination treatment.
We have previously reported on our use of autologous
chondrocyte sheet transplantation and have continued to observe
the long-term clinical outcomes3. For the previous study, we had
been directed by the Japan Ministry of Health, Labour and Welfare
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K. Hamahashi et al.

6(MHLW) to transplant cell sheets only into knees with defects
measuring <4.2 cm2, which is the size of one cell sheet. However,for the current study, we were able to prepare enough PD sheets
to cover larger defects and we did not limit patient enrollment
because of defect size. In this clinical study, some patients had
npj Regenerative Medicine (2022) 71

defects measuring >20 cm2. Generally, the common exclusion
criteria that deﬁne the “red knee” are lesions measuring >8 cm2,
malalignment, age >55 years, kissing lesions, and diffuse cartilage
thinning29, and some patients with red knee were enrolled in thiscurrent study. Although there were no serious adverse events
Published in partnership with the Australian Regenerative Medicine InstituteK. Hamahashi et al.

Fig. 3 Clinical and histological outcomes. a KOOS was used to assess the effects on knee-related parameters. The patient averages for the
KOOS subscales, symptom, pain, ADL, and QOL improved signiﬁcantly from the baseline. *p < 0.05, **p < 0.01. ADL activities of daily living,
QOL quality of life. b Patient average for LKS improved signiﬁcantly from the baseline. The box represents the interquartile range of the scores.
The top and bottom of the whiskers represent the maximum and minimum scores, except for the data at 12 months that contains the outlier.
The line inside the box represents the median score. *p < 0.05, **p < 0.01. c–f Correlational analysis was used to select a gene marker set thatwas predictive of each of the outcome measures. The scatter plot shows the correlations between the gene scores and KOOS pain at
12 months (c), LKS at 12 months (d), and OARSI (e) and ICRS II (f). The gene score was calculated from the gene expression of the predictive
gene marker set for KOOS pain (PTGS2, TGFB1, MIA, and G0S2), the LKS (COL1A2, MATN2, SOX9, and TGFB1), and the OARSI histological score(CCN2, COMP, ACKR4, ESM1, and GREM1), and ICRS II overall assessment (COL2A1, COL27A1, ACKR4, and ESM1), as described in the Methods
section (Supplementary Tables 4–7). The number assigned to each symbol indicates the corresponding patient. g Histological results ofpatient biopsies and results from clinical evaluations. C = articular cartilage, B = subchondral bone, Dashed line = boundary between articularcartilage region and subchondral bone region. Biopsies of femoral medial condyle were taken from areas of regenerated cartilage 12 months
postoperatively. scale bar = 1 mm. Histological sections from all patients stained for Safranin O. Immunostaining showed the expression oftype II collagen (COL2) in all patients.

Table 1.

Clinical examinations and outcomes.

Patient number

1

2

3

4

5

6

7

8

9

10

Age (years)

56

45

57

52

51

52

53

58

58

57

BMI (kg/m2)

27.3

34.6

31.2

29.0

25.8

35.5

25.9

23.5

23.5

27.3

Duration ofsymptoms (years)

3

8

1

2.5

2

1

1

3

1

6

KL grade
Outerbridgeclassiﬁcation

2
MFC 3
Tr 3

4
MFC 4
Kiss 4
Tr 3

3
MFC 3-4

3
MFC 4

3
MFC 4
Kiss 42
MFC 3-4
Tr 3-43
MFC 3-4

4
MFC 4
Kiss 4
Tr 3

3
MFC 3-4
Kiss 4Tr 3-43
MFC 3-4
Kiss 4Tr 3-4

MFC5.1
Location and Sizeof the defect (cm2) Tr 4.5

MFC 8.8
Kiss 4.2
Tr 4.6

MFC 9.0

MFC 15.0

MFC 15.0
Kiss 5.0

MFC 9.0
Tr 4.0

MFC 12.0

MFC 10.5
Kiss 4.5
Tr 5.3

MFC 11.0
Kiss 6.0
Tr 5.0

MFC 12.0
Kiss 4.5
Tr 4.0

Number oftransplanted cellsheet (total)

MFC 4
Kiss 4
Tr 4 (12)

MFC 9
(9)

MFC
13 (13)

MFC 12Kiss 3 (15)MFC 9
Tr 6 (15)MFC
14 (14)

MFC 6
Kiss 3
Tr 3 (12)MFC 6
Kiss 5
Tr 4 (15)MFC 6
Kiss 5
Tr 4 (15)Thickness of RC
MFC(preop. value) (mm) 2.87 (0.90)

MFC
3.30 (NA)Tr
3.25 (2.45)

MFC
2.90 (1.28)

MFC
3.64 (1.51)

MFC
3.94 (1.86)

MFC
2.64 (1.75)
Tr
2.54 (2.07)

MFC
3.47 (1.66)

MFC
4.44 (0.77)
Tr
3.89 (1.56)

MFC
4.10 (1.03)

MFC
4.37 (1.17)
Tr
4.02 (1.17)

Viscoelasticity ratio MFC
by LIPA
0.86 (0.53)(preop. value)

MFC
0.83 (NA)Tr
0.87 (0.66)

MFC
0.91 (0.53)

MFC
0.90 (NA)MFC
1.00 (NA)MFC
0.94 (0.84)
Tr
0.89 (0.86)

MFC
0.96 (0.71)

MFC
0.93 (NA)Tr
0.90 (0.71)

MFC
0.96 (0.82)

MFC
0.98 (NA)Tr
0.86 (0.76)

ICRS II (Overall
Assessment)

79

64

68

85

86

72

85

96

96

77

OARSI
histological score

7

10

13

3

6

8

6

5

6

6

Mankin score

4

8

5

2

5

5

3

5

4

4

60 (20)50 (25)75 (25)80 (30)75 (55)35 (20)50 (20)60 (40)55 (30)

MFC 6
Tr 4 (10)MOCART 2.0 Knee 50 (10)
Score (preop. value)

BMI body mass index, KL grade Kellgren–Lawrence grade, MFC medial femoral condyle, Kiss Kissing lesion of tibia of tibial plateau, Tr trochlea, RC regenerated
cartilage, LIPA laser-induced photoacoustic measurement, NA not applicable, ICRS International Cartilage Repair Society.

after transplantation of the PD sheets, we must still complete
phase III clinical trials with a well-controlled comparative study by
restricting enrollment to patients without red knee.
The number of transplanted PD sheets, locations of defects, andtotal size of the defects were not related to the clinical outcomes
and histological scores. However, these results may have been
inﬂuenced by the fact that all patients received a sufﬁcientnumber of PD sheets to cover the defects completely.
Our study has three limitations. First, the study included only 10
patients with general OA who underwent OWHTO. This associatedsurgery may have affected the outcomes of the therapy. Second,
we observed promising outcomes for the use of PD sheets for at
Published in partnership with the Australian Regenerative Medicine Instituteleast 1.5 years, but longer-term observations are needed toevaluate this new combination therapy fully. Finally, our clinicalstudy was designed as a single-arm, nonrandomized, anduncontrolled study, and further studies with more patients andconditions are needed.
We have been continuing to develop the techniques forpreserving and transporting cell sheets in preparation for futurecommercialization. We continue to research the vitriﬁcation
method to facilitate the cryopreservation of cell sheets while
maintaining their macro- and microstructures to ensure a highrate of the viability of the constituent cells30. The circulating
vitriﬁcation bag and vitriﬁcation storage box are useful for the
npj Regenerative Medicine (2022) 71

7

K. Hamahashi et al.

8samples indicated hyaline-like cartilage. This combination surgery
may provide an ideal regenerative therapy with disease-modifying
effects in patients with OAK.

a

Preop

Postop.12 mo.

Postop.24 mo.

b

d

f

c

e

g

h

Preop

Postop.3 mo.

Postop.6 mo.

Postop.12 mo.

Fig. 4 Case presentation of regenerative therapy (patient 5). X-ray
photographs (a), arthroscopic images (b–g), and magnetic resonance imaging (h). a X-ray preoperative photograph of the left knee
conﬁrmed a varus deformity and narrowing of the medial joint
space. X-ray photograph 12 months postoperatively showed thatthe alignment was maintained after OWHTO. At 24 months postoperatively (12 months after implants were removed), the alignment
was maintained and beta-tricarcium phosphate graft has beenabsorbed. b, c Preoperative cartilage defect of the medial femoral
condyle and kissing lesion of tibial plateau caused exposure of thesubchondral bone. d Intraoperative pictures of medial femoral
condyle after microfracture. e PD sheets were transplanted just as
putting on it without suturing. f, g The presence of regenerated
cartilage was conﬁrmed at the second look arthroscopic view
(12 months postoperatively). h Sagittal view of the medial
femorotibial joint. T2-weighted images were obtained using a 3.0Tesla MRI. Time course from the preoperative cartilage defect toregenerated cartilage after surgery. The preoperative MR image
shows a broad area of the cartilage defect in the medial condyle ofthe femur, a kissing lesion in the tibia, and the collection of synovial
ﬂuid. The regenerated cartilage was detected 3 months after surgery
and was maintained with time. The irregular subchondral bonecaused by marrow stimulation was initially detected and becamesmooth by 12 months.

long-term preservation of vitriﬁed cell sheets, which will make theclinical application of cryopreserved cell sheets even morefeasible31.
We conﬁrmed the safety and efﬁcacy of the transplantation of PD
sheets. Ten patients underwent PD sheet transplantation combinedwith OWHTO. Second-look arthroscopy 1 year after transplantation
showed regenerated cartilage that covered the defect areas fully.
LIPA evaluation suggested that the regenerated cartilage had
normal mechanical properties, and histological analysis of biopsy
npj Regenerative Medicine (2022) 71

METHODS
The experiments were performed under the approval andguidance of the Clinical Research Review Committee of Tokai
University School of Medicine. We presented these preclinicalresearch data to the MHLW in Japan and applied for a Class I
regenerative medicine provision plan under the law for the safetyof regenerative medicine. The permit was issued on April 27, 2016,
and clinical research using PD sheets was allowed to begin.
Informed consent was obtained from the parents or guardians of
the donors in all cases. Some surgical specimens were irreversibly
de-identiﬁed. All experiments handling human cells and tissues
were performed in line with the tenets of the Declaration of
Helsinki. The animal experiments were approved by the Institutional Animal Experiment Committee at Tokai University and were
performed in accordance with the guidelines of the Institutional
Regulation for Animal Experiments and the Fundamental Guideline for Proper Conduct of Animal Experiment and Related
Activities in Academic Research Institutions under the jurisdictionof the Ministry of Education, Culture, Sports, Science, andTechnology for animal handling and care.
Study design and setting
Our clinical study was designed as a single-arm, nonrandomized,
uncontrolled comparative study. This study (UMIN Clinical Trials
Registry, UMIN000015205, and Japan Registry of Clinical Trials,
jRCTa030190242) was approved by the Institutional Review Boardof the Tokai University School of Medicine and by the MHLW of
Japan. Written informed consent was obtained from all participants. The CONSORT ﬂow diagram is shown in Fig. 1c.
Donor selection
The cell sheets for transplantation were fabricated from donor
cells that exhibited an ability to initiate hyaline cartilage
regeneration after xenogeneic orthotopic transplantation into aJapanese white rabbit model of cartilage full-thickness defect5,19.
We also performed array comparative genomic hybridization(array CGH) and karyotype analysis of the P2 PD cells used tofabricate the cell sheets and as long-term cultured cells (P12 cells)
to assess the genetic stability of the PD cells during in vitro
fabrication. No copy number alterations were detected by array
CGH, and no karyotype abnormalities, as judged by theInternational System for Human Cytogenetic Nomenclature
guidelines, were found in the cells used as the cell source.
Fabrication of PD sheets
Cartilage tissue was obtained from three patients (average age:
12.3 months, range: 10–15 months, one boy and two girls) who
underwent polydactyly surgery at Tokai University Hospital. A
summary of the PD sheet fabrication process is shown in Fig. 1a.
Cartilage tissue was minced with scissors and then incubated in
Dulbecco’s modiﬁed Eagle’s medium/F12 (DMEM/F12; Gibco,
Grand Island, NY, USA) supplemented with 20% fetal bovine
serum (FBS; AusGeneX, Molendinar, Australia, or SAFC Biosciences,
Lenexa, KS, USA), 1% antibiotic–antimycotic solution (AB; Gibco),and 5 mg/mL collagenase type 1 (Worthington, Mannheim,
Germany) for 1.5 h at 37 °C in a humidiﬁed atmosphere of 5%
CO2 and 95% air. The cell suspension was washed and passed
through a 100 μm strainer (BD Falcon, Franklin Lakes, NJ, USA).
The collected cells were seeded at a density of 0.5 to 1 × 104
cells/cm2 into six-well culture plates (Corning Inc., Corning, NY,
USA) in DMEM/F12 supplemented with 20% FBS and 1% AB, andPublished in partnership with the Australian Regenerative Medicine InstituteK. Hamahashi et al.

incubated at 37 °C. After 4 days, 100 μg/mL ascorbic acid (AA;Nissin Pharmaceutical, Yamagata, Japan) was added to themedium, and the medium was replaced every 3 or 4 days. Cells
were cryopreserved using STEM-CELLBANKER™ (ZENOAQ, Fukushima, Japan) when they reached subconﬂuence. Cells were thawed
and seeded once at a density of 0.5 to 1 × 104 cells/cm2 in DMEM/
F12 supplemented with 20% FBS, 1% AB, and 100 μg/mL AA.
When the cells reached subconﬂuence, they were detached using
TrypLE Express (Thermo Fisher Scientiﬁc, Tokyo, Japan) andseeded onto temperature-responsive culture inserts (CellSeed
Inc., Tokyo, Japan) at 1 × 104 cells/cm2. The medium was replaced
every 3 or 4 days. After 2 weeks, the culture plates were kept at
25 °C for 30 min to promote detachment of PD sheets from theinserts, and the PD sheets were harvested. To analyze theproperties of PD sheets using real-time qPCR, ﬂow cytometricanalysis, and histological and immunohistochemical staining, PD
sheets were harvested on day 13 of culture.
Cell count and viability
PD sheets were washed in Dulbecco’s phosphate-buffered saline(DPBS; Gibco). The sheets were then incubated in TripLE Express®
(Gibco) at 37 °C for 15 min and centrifuged at 1500 rpm for 5 min.
The cell sheets were resuspended in 0.25 mg/mL collagenase P(Roche, Basel, Switzerland) at 37 °C for up to 30 min and thencentrifuged at 1500 rpm for 5 min. The isolated cells were ﬁnallyresuspended in DMEM/F12, and the cell count and viability were
determined using the trypan blue exclusion assay.
Flow cytometric analysis
After the cell count was obtained, isolated cells were washed withDPBS containing 0.2% bovine serum albumin (BSA; Sigma-Aldrich,
St. Louis, MO, USA) and 1 mM ethylenediaminetetraacetic acid
(EDTA; Gibco). About 1.5 × 105 cells were mixed in each tube withthe following antibodies: hCD31–ﬂuorescein isothiocyanate (FITC)(1:10; IM1431U, Beckman & Coulter, Brea, CA, USA), CD44–FITC(1:10; 555478, BD Biosciences, Franklin, NJ, USA), hCD45–FITC(1:10; A07782, Beckman & Coulter), hCD81–allophycocyanin (APC)(1:10; 551112, BD Biosciences), hCD90–APC (1:100; 559869, BD
Biosciences), CD105–phycoerythrin (PE) (1:5; A07414, B76299,
Beckman & Coulter), CD146–PE (1:5; 5050-PE100T, BioCytex,
Marseilles, France), CD49-PE (1:5; 559596, BD Biosciences), and
GD2 (1:10; 554272, BD Biosciences). The cells were incubated for
90 min at 4 °C and then washed with DPBS containing 0.2% BSA
and 1 mM EDTA. GD2 was recognized by incubating the cells withthe secondary antibody FITC-conjugated anti-mouse IgG (1:20;
349031, BD Biosciences) for 10 min at 4 °C. Fluoroprobe-labeledmouse IgG1 antibody (1:10; Beckman & Coulter) was used as anegative control. Stained cells were analyzed using a FACSVerse™
cell sorter (BD Biosciences) (Supplementary Fig. 2).
Histological and immunohistochemical staining
PD sheets were harvested after culture and then embedded andfrozen in optimal cutting temperature compound (Sakura Finetek
Japan, Tokyo, Japan). Sections 10 μm thick were stained withhematoxylin and eosin using standard methods (Supplementary
Figure 3). Sections 20 μm thick were immunostained with antihuman COL1 (1:200; 1310-01, Southern Biotech, Birmingham, AL,
USA), COL2 (1:200; F-57, Kyowa Pharma Chemical, Toyama, Japan),FN (1:1000; MA5-11981, Thermo Fisher Scientiﬁc, Waltham, MA,
USA), and ACAN (1:20; 967800, R&D Systems, Minneapolis, MN,
USA) at 4 °C overnight. The sections were washed and incubatedat room temperature for 1 h with the secondary antibody Alexa
Fluor 488-conjugated goat anti-mouse Ig (1:200; A-11017, Thermo
Fisher Scientiﬁc) for COL2 and FN or Alexa Fluor 546-conjugateddonkey anti-goat Ig (1:400, A-11056, Thermo Fisher Scientiﬁc) for
COL1 and ACAN. After immunostaining, the sections were washed
Published in partnership with the Australian Regenerative Medicine Institute9and mounted with VECTASHIELD Antifade Mounting Medium with
4′,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame,
CA, USA). Microscopic images were captured under a BZ-X810
microscope (Keyence, Osaka, Japan).
RNA isolation
The PD sheets were disrupted in TRIzol Reagent (Thermo Fisher
Scientiﬁc) using a SHAKE Master Neo device (Bio Medical Science,
Tokyo, Japan), and the total RNA was isolated using an RNeasy
Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. RNA quantity and quality were determinedusing a Nanodrop One spectrophotometer (Thermo Fisher
Scientiﬁc) and an Agilent Bioanalyzer (Agilent Technologies, Santa
Clara, CA, USA).
Real-time qPCR
Total RNA was converted to cDNA using QuantiTect Reverse
Transcription Kit (Qiagen). A TaqMan PreAmp Master Mix Kit andTaqMan Gene Expression Assays (both from Thermo Fisher
Scientiﬁc) (Supplementary Table 3) were used to preamplify the
cDNA according to the manufacturer’s instructions. TaqMan qPCR
was performed using a 7500 qPCR System or QuantiStudio 3 (bothfrom Thermo Fisher Scientiﬁc). The relative expression values foreach gene (–ΔCt values) were calculated using GAPDH as theinternal control.
Measurement of humoral factors
To collect the supernatants of the fabricated PD sheets, the sheets
were transferred to 3 mL of DMEM/F12 supplemented with 1%
FBS and 1% AB on day 14 of culture and cultured for another 72 h.
Supernatants were collected and centrifuged at 15,000 g for
10 min to remove cell debris. The concentrations of TGF-β1 (R&D
Systems), MIA (Roche), Dickkopf WNT signaling pathway inhibitor
1 (Thermo Fisher Scientiﬁc), endothelial cell-speciﬁc molecule 1(ESM1; Abcam, Cambridge, UK), and gremlin 1 (GREM1; CloudClone Corp., Katy, TX, USA) were measured using ELISA kits. The
signal detected for the blank medium containing 1% FBS was
subtracted to adjust for the proteins contained in FBS.
Clinical research study of PD sheet transplantation
Ten patients (four men and six women) who had cartilage defects
categorized arthroscopically as Outerbridge grade III or IV wereenrolled and received the therapy. Their mean age at the time ofthe operation was 54 years (range 45–58), and their mean bodymass index was 28.3 kg/m2 (range 23.5–35.5). The Kellgren and
Lawrence grades from knee roentgenograms were grade 2 in twopatients, grade 3 in six, and grade 4 in two (Table 1). We
performed PD sheet transplantation in the ﬁrst patient in February
2017 and the last in December 2019.
The inclusion criteria for this clinical research study were having
medial compartment OAK, which is usually indicated for OWHTO(Table 2). In other clinical research studies of autologous
chondrocyte sheet transplantation, the size of the damaged
cartilage lesion was regulated as <4.2 cm2,3. However, there wasno regulation of size for this clinical research study of PD sheettransplantation because more than ten PD sheets could be
prepared in advance. The total number of defects to betransplanted with PD sheets was 21 (10 for MFC, 6 for Tr, 5 forkissing lesions); the average defect sizes were 10.8, 4.6, and 4.0 for
MFC, Tr, and kissing, respectively. The maximum total defect size
was 22.0 cm2 in one patient (Table 1).
Surgery and rehabilitation
We designed a combination therapy in which conventional
surgical treatment for OAK, which is covered by National Healthnpj Regenerative Medicine (2022) 71

K. Hamahashi et al.

10
Table 2.

Inclusion and exclusion criteria of the clinical study.

Eligibility inclusion criteria

Eligibility exclusion criteria

Patients who meet all of the following inclusion criteria and have the ability to consent
will be included in the study.
1 20–60 years of age and any sex
2 Have a knee cartilage lesions
3 Outerbridge classiﬁcation Grade III or IV
4 Cartilage defect in the patellofemoral joint or condyle of the femur that can becovered by a fabricated cell sheet and is indicated for conventional marrow
stimulation, osteochondral autografts, etc.

1 Patients who have difﬁculty providing informed consent
2 Complications that interfere with surgery under generalanesthesia or that affect knee surgery
3 Patients with infectious diseases (including testing positivefor HBV, HCV, HIV, HTLV-1, syphilis)
4 Patients with systemic inﬂammatory diseases, such as
rheumatoid arthritis

HBV hepatitis B virus, HCV hepatitis C virus, HIV human immunodeﬁciency virus, HTLV-1 human T cell leukemia virus type 1.

Insurance in Japan, was followed by the RMSC method (Fig. 1a).
Cryopreserved allogeneic chondrocytes were thawed and cultured
for 3 weeks to fabricate PD sheets before the transplantation
surgery. A total of nine lots were fabricated: three lots from donor
1, four lots from donor 2, and two lots from donor 3. Each lot wasused for one patient, except for the ninth lot, which wastransplanted into two patients. We performed OWHTO as
described by Takeuchi et al.32 and aimed to achieve a postoperative %MA of 62.5. After biplane (oblique and proximal
tuberosity) osteotomy, artiﬁcial bone was transplanted into theopen gap and ﬁxed at the osteotomy site using TomoFix® (DePuy
Synthes, Bettlach, Switzerland) or TriS® (Olympus Terumo Biomaterials, Tokyo, Japan).
After OWHTO, the knee joint was opened about 5 cm using themedial parapatellar approach. The MFC, medial tibial plateau, andpatellofemoral joint were observed, and RMSC was performed(Supplementary Movie). Brieﬂy, after the removal of unhealthy
tissue and bone marrow stimulation (microfracture in sevenpatients and abrasion arthroplasty in three), PD sheets weretransplanted to cover all of the cartilage defect surface. PD sheets
can be transplanted without any suturing or periosteum patch
because of their excellent adhesive properties. This RMSC
method3 is an efﬁcient way to regenerate hyaline cartilage
because it inserts bone marrow-derived MSCs and various
humoral factors secreted by PD sheets into the cartilage defectspace. PD sheets were transplanted into the MFC in 10 patients,
medial tibial plateau in ﬁve, and patellofemoral joint in six. The
mean number of transplanted sheets was 13 (9–15). The Outerbridge grades and location and size of the cartilage defects areshown in Table 1.
All patients were immobilized immediately after surgery using aplaster splint that was maintained for 2 weeks at 20° of knee
ﬂexion to avoid disturbing the transplanted sheets. Patients thenstarted range-of-motion exercise and partial weight bearing at
2 weeks, and full weight bearing at 4 weeks after surgery. In
general, low-impact activities were started at 6 months, and highimpact activities were allowed at 8 months.
Clinical outcomesWe evaluated the clinical outcomes using the patient-oriented
KOOS and LKS preoperatively and at 1, 3, 6, and 12 months
postoperatively33,34.
Structural outcomes
Imaging assessment was performed using X-ray photographs and
MRI. X-ray photographs were examined for knee alignment,
condition of the subchondral bone, and progression of OAK.
Progress was evaluated using the Kellgren–Lawrence grading
scale preoperatively and postoperatively. MRI examinations were
performed using an Achieva 3.0-T TX scanner (Philips Healthcare,Best, The Netherlands), within a TX SENSE Knee eight-channel coil
npj Regenerative Medicine (2022) 71(Philips Healthcare). The images were taken with 10° of knee
ﬂexion. Sagittal and coronal MR images were used to evaluate thecartilage defect areas preoperatively and postoperatively.
For evaluation of the therapy, we used the MOCART 2.0 method
described by Schreiner et al.35 (Table 1). Arthroscopic evaluation of
the cartilage defects included their condition, size, and Outerbridge grade, and was performed preoperatively and postoperatively. The LIPA method was used to evaluate the viscoelastic
properties of cartilage preoperatively and postoperatively (Supplementary Movie). The use of LIPA was approved by theInstitutional Review Board for Clinical Research at Tokai University,and the method has been applied clinically to evaluate themechanical properties of cartilage in patients36. The LIPA method
was used arthroscopically during the ﬁrst surgery (PD sheettransplantation and OWHTO) and 12 months postoperatively(second-look and removal of implants).
Histological outcomes
Histological outcomes were evaluated 12 months postoperatively
using an arthroscopically performed biopsy taken from near thecenter of the regenerated cartilage. Samples were decalciﬁcation
by 10% EDTA Solution B (Wako Pure Chemical Industries, Osaka,
Japan) for 9 days at 4 °C. And then, samples were embedded inparafﬁn wax, and 3 μm sections were cut, deparafﬁnized, stainedwith Safranin O, and immunostained for COL1 and COL2 using
previously reported methods15. The ICRS II overall assessment37
and the OARSI histological score were assessed independently by
three trained orthopedic surgeons. Microscopic scoring of the
cartilage was evaluated using the method described by Little
et al.38, who provided representative images for each score. There
were minimal variations in scoring between the three scorers. The
Mankin score was obtained similarly39,40 Structural compromise(0–6 points), loss of matrix staining (0–4 points), cellularity
anomalies (0–3 points), and violation of tidemark integrity (0 or
1 point) were graded using this scale, with normal cartilage givena score of 0 out of 14 points.
Predictive gene marker sets
Predictive gene marker sets were selected from the 43 genes andcontrol genes examined (GAPDH and ACTB) (Supplementary Table 3)as those whose expression correlated with the KOOS pain score and
LKS, OARSI histological score and ICRS overall assessment score at
12 months postoperatively. First, 36 genes (Supplementary Tables
4–7) with normalized ΔCt values greater than –10 cycles wereidentiﬁed as having detectable gene expression. For each outcome
assessment, Pearson correlation coefﬁcients were calculated
between the expression level of each of the genes and theindividual scores (KOOS, LKS, OARSI histological score, and ICRS
overall assessment score) using the leave-ﬁve-out process, and thegenes were ranked from highest to lowest for the absolute values of
the coefﬁcients. From this, a matrix of 100 sets of Pearson correlation
Published in partnership with the Australian Regenerative Medicine InstituteK. Hamahashi et al.

11
coefﬁcients was calculated. The representative genes were selectedindependently as predictive markers for each of the outcome
measures by selecting >0.5 of absolute value for the Pearson
correlation coefﬁcients for the genes listed according to theirprobability of appearance. Finally, four or ﬁve gene sets with thehighest probability of appearance were selected as the predictive
markers. The gene score was calculated as the normalized value ofthe expression of each gene based on linear functions: Genescore = Σ(–ΔCt value) (Supplementary Tables 4–7).
Statistical analysis
Data were analyzed using repeated-measures analysis of varianceand the post hoc Bonferroni test to identify differences between
the pre-operative and postoperative scores for clinical outcomes(n = 10) for KOOS scores and overall LKS. p values < 0.05 weresigniﬁcant.

DATA AVAILABILITY
The data sets generated and/or analyzed during the current study are available fromthe corresponding author on reasonable request.

Received: 4 November 2021; Accepted: 5 December 2022;

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ACKNOWLEDGEMENTS
We thank Professor Rumiko Shimazawa (Tokai University School of Medicine) and Dr.
Setsuko Hashimoto (CellSeed, Inc.) for their contributions to this project. We also
thank the Support Center for Medical Research and Education at Tokai University fortechnical support in the histological analysis. One of the authors has received funding
from the following sources. This research was supported by the Research Project for
Practical Applications of Regenerative Medicine (Grant numbers JP15bk0104001,
JP17bk0104063, and JP20bk0104101 to M.S.) from the Japan Agency for Medical
Research and Development.

npj Regenerative Medicine (2022) 71

K. Hamahashi et al.

12
AUTHOR CONTRIBUTIONS
K.H.: Contributions to clinical study and manuscript writing. E.T.: Contributions toclinical and basic study and manufacturing cell sheets. M.I.: Contributions to clinicalstudy, especially performing the laser-induced photoacoustic arthroscopy method,and manuscript prooﬁng. G.M.: Contributions to clinical study and manuscript
prooﬁng. T. Takagaki: Contributions to clinical study and manuscript prooﬁng. N.K.:
Contributions to clinical study and manuscript prooﬁng. M.M.: Contributions toclinical and basic study and manufacturing cell sheets. T. Takahashi: Contributions toclinical and basic study and manufacturing cell sheets. E.O.: Contributions to clinicaland basic study and manufacturing cell sheets. A.W.: Contributions to clinical andbasic study and manufacturing cell sheets. Y.N.: Contributions to clinical and basic
study and manufacturing cell sheets. R.K.: Contributions to clinical and basic study
and manufacturing cell sheets. R.M.: Contributions to statistical analyses andmanuscript prooﬁng. T. Takagi: Contributions to clinical study and manuscript
prooﬁng. H.A.: Providing allogeneic cells and contributions to basic study andmanufacturing cell sheets. A.U.: Providing allogeneic cells and contributions to basic
study and manufacturing cell sheets. H.K.: Contributions to statistical analyses andmanuscript prooﬁng. T.A.: Contributions to clinical study and manuscript prooﬁng.
M.Y.: Contributions to experimental design, lead for cell-sheet technology, andmanuscript prooﬁng. T.O.: Contributions to experimental design, lead for cell-sheet
technology, and manuscript prooﬁng. M.W.: Contributions to clinical study andmanuscript prooﬁng. M.S: Guarantor, contributions to clinical study design,experimental design, lead for all in vitro elements of experimental work, provisionof funding, and manuscript preparation.

COMPETING INTERESTS
Each author certiﬁes that M.S. has been involved in the following relevant ﬁnancial
activities outside of this work and/or other relationships or activities that readers
could perceive to have inﬂuenced, or that give the appearance of potentiallyinﬂuencing, this manuscript: funding from CellSeed Inc. M.S. is one of the inventors
on the patents (PCT/JP2006/303759, PCT/JP2017/031136) held by the applicants

npj Regenerative Medicine (2022) 71

Tokai University and CellSeed Inc. for the manufacturing process of chondrocyte
sheets. The remaining authors declare no competing interests.

ADDITIONAL INFORMATION
Supplementary information The online version contains supplementary material
available at https://doi.org/10.1038/s41536-022-00272-1.
Correspondence and requests for materials should be addressed to Masato Sato.
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Published in partnership with the Australian Regenerative Medicine Institute