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Safety and efﬁcacy of human juvenile chondrocyte-derived cellsheets for osteochondral defect treatment

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Makoto Kondo 1 ✉, Sumako Kameishi1, Kyungsook Kim1, Nicolas F. Metzler1,2, Travis G. Maak3, Douglas T. Hutchinson3,4,Angela A. Wang3,4, Miki Maehara5, Masato Sato 5, David W. Grainger1,2 and Teruo Okano1,6 ✉Knee cartilage does not regenerate spontaneously after injury, and a gold standard regenerative treatment algorithm has not been
established. This study demonstrates preclinical safety and efﬁcacy of scaffold-free, human juvenile cartilage-derived-chondrocyte(JCC) sheets produced from routine surgical discards using thermo-responsive cultureware. JCCs exhibit stable and high growthpotential in vitro over passage 10, supporting possibilities for scale-up to mass production for commercialization. JCC sheetscontain highly viable, densely packed cells, show no anchorage-independent cell growth, express mesenchymal surface markers,
and lack MHC II expression. In nude rat focal osteochondral defect models, stable neocartilage formation was observed at 4 weeks
by JCC sheet transplantation without abnormal tissue growth over 24 weeks in contrast to the nontreatment group showing no
spontaneous cartilage repair. Regenerated cartilage was safranin-O positive, contained type II collagen, aggrecan, and humanvimentin, and lacked type I collagen, indicating that the hyaline-like neocartilage formed originates from transplanted JCC sheets
rather than host-derived cells. This study demonstrates the safety of JCC sheets and stable hyaline cartilage formation withengineered JCC sheets utilizing a sustainable tissue supply. Cost-beneﬁt and scaling issues for sheet fabrication and use support
feasibility of this JCC sheet strategy in clinical cartilage repair.
npj Regenerative Medicine (2021)6:65 ; https://doi.org/10.1038/s41536-021-00173-9

INTRODUCTION
Articular cartilage plays an essential role in reducing friction in
joint motion and mitigating joint load stress. In contrast to bone,
the post-traumatic repair capacity of cartilage is limited1. As such,
focal cartilage defects have been identiﬁed as a potent risk factor
for early osteoarthritic disease2,3, and restoring cartilage integrity
is considered to be a key means of preventing or delaying the
reduction in patient quality of life associated with osteoarthritis
development4,5. Autologous and allogeneic osteochondral grafts
have been applied as mosaicplasty, bulk grafts, and particulated
cartilage6 to replace damaged cartilage. However, allogeneic
grafts have limited supply, treatable lesion location, and area
restricts graft options, and the limited duration of bulk osteochondral allograft (OCA) transplantation viability makes the timing ofsurgical treatment difﬁcult7. Finally, in many cases, ﬁbrocartilage isoften reported following chondral repair techniques.
After a milestone report of in vitro cultured autologous
chondrocyte implantation8, various tissue engineering methods
using chondrocytes and mesenchymal stem/stromal cells (MSCs),
with or without accompanying biomaterials, have been developedwith a myriad of clinical studies ongoing9–12. Juvenile cartilage isnoted as a desirable source for neocartilage regeneration due toits immune tolerance13, proliferative capacity, and maintenance ofcharacteristic matrix proteins in vitro compared to adult cartilagederived chondrocytes14,15. Allogeneic sourcing reduces chondral
defect treatment to a single-stage surgical procedure16,17. However, the adhesion of chondral grafts to cartilage and bone
surfaces is difﬁcult and graft failure is reported18.

Cell sheet technology using poly(N-isopropylacrylamide)grafted temperature-responsive cultureware allows reproducibly
consistent creation of sheets via detachment of conﬂuently
cultured cells19. Harvested sheets readily adhere on tissue
lesions with endogenous extracellular matrix (ECM) and surface
proteins eliminating secondary adhesion techniques, such as
suture or ﬁbrin glue. Cell sheets are applied to treat multiple
diseases in patients using various cell types20–25. The safety andclinical efﬁcacy of autologous chondrocyte sheet transplantation
was demonstrated in a small cohort study with concomitantanterior cruciate ligament reconstruction or high tibial osteotomy (HTO) and microfracture26. Recently, surgically discarded
juvenile tissue obtained from routine polydactyly resection isreported as a source of allogeneic human chondrocytes to form
hyaline cartilage in implanted subcutaneous space of immunodeﬁcient mouse27,28. Polydactyly-derived chondrocyte sheets
were compared to adult chondrocyte sheets fabricated fromadult tissue discards obtained from patients undergoing total
knee arthroplasty and shown to exhibit higher secreted TGFβ1
concentrations, with practical advantages useful to future
cartilage repair29.
The current study reports the preclinical safety and efﬁcacy ofpolydactyly-sourced juvenile cartilage-derived chondrocyte (JCC)
sheets in vitro and in vivo using a rat focal osteochondral defectmodel. Properties of these JCC sheets in sheet production androdent cartilage repair suggest feasibility for possible future
clinical translation of this therapy.

1
Cell Sheet Tissue Engineering Center (CSTEC), Department of Pharmaceutics and Pharmaceutical Chemistry, Health Sciences, University of Utah, 30 South 2000 East, Salt Lake
City, UT 84112, USA. 2Department of Biomedical Engineering, University of Utah, 36 S. Wasatch Drive SMBB 3100, Salt Lake City, UT 84112, USA. 3Department of Orthopaedic
Surgery, University of Utah Orthopaedic Center, University of Utah, 590 Wakara Way, Salt Lake City, UT 84108, USA. 4Pediatric Orthopaedics Surgery, Primary Children’s Hospital
Orthopedics, 100 North Mario Capecchi Dr. Suite 4550, Salt Lake City, UT 84113, USA. 5Department of Orthopaedic Surgery, Surgical Science, Tokai University School of Medicine,
143 Shimokasuya, Isehara, Kanagawa 259-1193, Japan. 6Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University, TWIns, 8-1 Kawada-cho,Shinjuku-ku, Tokyo 162-8666, Japan. ✉email: makoto.kondo@utah.edu; teruo.okano@utah.edu

Published in partnership with the Australian Regenerative Medicine InstituteM. Kondo et al.

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In vivo
experiment
Surfacemarker
CS Histology Transformation
assay
Cell sheet CS
Total cell
Cell
Serial passage
formation detachment test number of CS viability of CS culture
Viability at
end of P1
Donor ID Age
Sex Ethnicity Anatomical(months)site

In vitro safety evaluation of juvenile cartilage-derivedchondrocyte sheets
JCC isolated from sheets prepared from four individual donors
showed no anchorage-independent colony growth in
10,000 seeded cells (<0.01%) (Fig. 3A(a)). Cells from both regular
duration (2-week) cultured cell sheets and extended (3.5-week)
cultured JCC sheets showed no increased signal after 8-day soft
agar culture, suggesting no anchorage-independent cell growth,whereas HepG2 cells (positive control) showed signiﬁcant cell
growth at 8 days after seeding (Fig. 3A(b)). Cell surface markers forleukocytes (CD45 and lineage cocktail) and vascular endothelial
cells (CD31) were negligible (Fig. 3B), indicating that the isolationand culture processes are free of contaminants. MHC-I molecules,
HLA-ABC, were expressed 100% whereas MHC-II molecules, HLADR, -DP, -DQ, were not detected in the cells isolated from JCC
sheets (Fig. 3B), suggesting low allogeneic immunogenicity inlong-term graft retention. Expression of mesenchymal cellmarkers, CD44, CD90, and CD81, were 100%, suggesting pure
populations of cultured chondrocytes as previously reported29. Bycontrast, variability of CD106 expression among donors wasobserved (5.6 ± 4.6%), indicating CD106 cannot be used as a purity
marker. To establish phenotypic stability, JCC were seriallypassaged and morphology and population doubling times at
each passage were evaluated. We observed constant cell growthin serial passage culture up to P13 (Fig. 3C), which suggests thatengineered juvenile chondrocyte sheets are unlikely to undergo amalignant transformation during a production process.

Juvenile chondrocyte donor sample information.

In vitro characterization of juvenile cartilage-derivedchondrocyte sheets
P2 JCCs cultured on temperature-responsive cell culture insert for
2 weeks were conﬂuent (Fig. 2A) and able to be harvested as cellsheets (Fig. 2B). JCC sheets maintain high cell viability (98.0 ±1.3%) and rich cell numbers in each sheet construct (1.90 ± 0.48 ×
106 cells per sheet) (Fig. 2C). After detachment, JCC sheets
undergo a spontaneous, endogenous contraction resulting in a
multicell thick sheet structure without folding (Fig. 2D). The cellsheet stains negatively for safranin-O and toluidine blue (only at
nuclei), but positively for aggrecan and type I collagen withimmunohistochemistry (Fig. 2D). The expression of type II collagenwas not evident in the cell sheets. These in vitro characters weremaintained in the cell sheets prepared at passage 9 (Supplementary Fig. 2). Importantly, cells isolated from JCC sheets exhibit
robust chondrogenic capacity in pellet cultures (Fig. 2E).
Theoretical numbers of JCC sheets prepared from a single
polydactyly donor at a given passage are shown in Fig. 2F.

Table 1.

1234567890():,;RESULTSIn vitro characterization of juvenile cartilage-derived
chondrocytes
To demonstrate the safety of juvenile cartilage-derivedchondrocyte (JCC) sheets, juvenile cartilage tissue, and JCCs inculture were characterized. Surgically discarded polydactyly
cartilage samples from 12 juvenile human donors (Table 1) were
harvested (Fig. 1A) and conﬁrmed to contain hyaline cartilageusing safranin-O staining (Fig. 1B). The morphology of isolatedchondrocytes was observed after initial attachment and during
subsequent culture. The chondrocytes showed stellate shapes
after surface attachment, then spread after one passage (Fig. 1C).
These cells exhibit a constant growth rate for over 10 passages(Fig. 1D). Harvested JCCs at the end of P2 and P9 both formed fully
differentiated hyaline cartilage pellets after 3-week differentiation
culture although the size of P9 pellets were slightly smaller
compared to P2 pellets (Fig. 1E), suggesting decreased growthpotential, but maintained differentiation potential at P9.

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Published in partnership with the Australian Regenerative Medicine InstituteM. Kondo et al.

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Fig. 1 Isolation of chondrocytes from juvenile cartilage surgical discards and in vitro expansion of juvenile chondrocytes. A Juvenile
donor-derived cartilage tissues under stereomicroscope. Scale bar: 5 mm. B Safranin-O staining of cartilage tissue. Scale bar: 500 μm. C Phasecontrast images of cultured chondrocytes. Scale bars: 200 μm. D Average fold-change of in vitro cell expansion. Data shown as mean and SD(n = 13 individual donors). E In vitro differentiated JCC pellets. Photos show Safranin-O staining of P2 JCCs (top) and P9 JCCs (bottom). Bars:
500 µm. Right graph shows pellet size diameter measurements (n = 2). Error bars indicate SD. **p < 0.01 by Student’s t test.

In vivo safety and efﬁcacy evaluation
To demonstrate both safety and efﬁcacy in vivo, JCC sheets weretransplanted at the time of surgical focal osteochondral defectcreation in rats. The two experimental groups, (JCC sheet treatmentgroup and defect-only negative control group) were observed
under stereomicroscopy, and histologically examined and compared post-transplantation. Preliminary JCC sheet transplantation
studies using immunocompetent Sprague Dawley (SD) rats failed
to regenerate cartilage (Supplementary Fig. 3). Therefore, an
immunocompromised athymic rat model was selected to evaluate
sheet-induced neocartilage formation. Seven-week-old nude rats
received focal osteochondral defects in the trochlear groove (2mm diameter, 200–350 µm depth. Depressed knee surface andﬁbrotic pannus indicative of failure to spontaneously regeneratecartilage tissue were observed in the defect-only group at all-time
points (4, 8, 12, and 24 weeks) (Fig. 4B). This is consistent withpreviously reported critical size defects (>1.4 mm diameter) in rat
knee cartilage30. Contrary to the defect-only group, the treatment
Published in partnership with the Australian Regenerative Medicine Institutegroup showed regenerated, white cartilage in defect areas at alltime points (Fig. 4A). Moreover, histological examination revealed
the defect-only group to be safranin-O negative, whereas thetreatment group exhibited thick, safranin-O positive, hyaline
neocartilage at all-time points (4, 8, 12, and 24 weeks) (Fig. 4B).
Importantly, the interface between the regenerated cartilage andhost tissue is stably integrated (Fig. 4B). In addition, samples at alltime points exhibited lacuna formation, suggesting mature
cartilage formation in defect areas by 4 weeks post-transplantation,and maintenance of native tissue architecture. Interestingly, thesize of lacuna was smaller than host native cartilage (Supplementary Fig.. 4), indicating that tissue is not the residue of the original
host cartilage. Moreover, while regenerated cartilage was substantially thicker than rat native cartilage, no tumorigenic tissue
formation was observed in all rats, suggesting the in vivo safety oftransplanted JCC sheets throughout 24 weeks of study.
The presence of aggrecan (ACAN) and type 2 collagen (COL2),hyaline cartilage-speciﬁc matrix proteins, and type 1 collagen
npj Regenerative Medicine (2021) 65

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4

B

Total cell # per sheetC

2.5×10 6

P2 day 14

Cell sheet
Ring-shape support membraneDcell #number
TotalCell/ cell sheetA

2.0×10 6
1.5×10 6
1.0×10 6
5.0×10 5
0E

H&E

Toluidine
blue

AggrecanCollagen I
Collagen II

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Cell sheets possible froma single donor per passage

10 7

Number of
Preparable
CS #cell sheets possible

Safranin-O

10 6
10 5
10 4
10 3
10 2
10 1
10 0
0

1

2

3

4

5

PassageFig. 2 Characterization of engineered JCC sheet. A A phase-contrast image of conﬂuent chondrocytes at day 14 of passage 2. Scale bar:
200 µm. B Macroscopic image of engineered JCC sheet. Scale bar: 5 mm. C Total cell number in one cell sheet (n = 13 individual donors).
D Hematoxylin and eosin staining, Safranin-O staining, Toluidine blue staining, aggrecan, type I collagen, and type II collagen
immunohistochemistry of JCC sheets. Bars: 50 µm. E Safranin-O staining of an in vitro differentiated pellet from P2 JCC sheet. Bar: 500 µm.
F Theoretical cell sheet numbers possibly prepared from cultured JCCs at each passage. Data shown as mean and SD (n = 11 individual
donors).

(COL1), a damaged or arthritic articular cartilage marker wasassessed on harvested knee samples by immunohistochemistry(IHC). The pannus at the defect site in the defect-only group
showed strong COL1 expression, but ACAN and COL2 were notexpressed (Fig. 5A). Regenerated neocartilage in the JCC sheet
treatment group showed a limited expression of COL1 localized toneocartilage surfaces and abundant ACAN and COL2 expressions
in the neocartilage body (Fig. 5B). We also used human-speciﬁcvimentin staining to determine the origin of the regenerated
tissue. Speciﬁcity of human-speciﬁc anti-vimentin antibody was
conﬁrmed on superﬁcial cartilage of ﬁbrotic tissue in the
nontreatment group by comparison to a “pan” vimentin antibody
that cross-reacts to both human and rat cartilage (Supplementary
Fig. 5). Human-vimentin-speciﬁc antibody did not react with rat
cartilage or stromal tissue, whereas it reacted with the neocartilage tissue areas on JCC sheet treatment samples (Fig. 6A).
Interestingly, COL2 was observed at the periphery of humanvimentin positive cells and adjacent interstitial matrix (Fig. 6B).
These data strongly suggest that transplanted human JCC sheets
are responsible for deposition of new cartilage matrix proteins andneocartilage generation.
We assessed functional recovery induced by JCC sheettransplantation on the rat focal osteochondral defect models
by measuring rat hind limb weight bearing. The JCC sheet
treatment group showed rapid recovery in weight distribution
on each leg after 3 weeks, while reduced weight bearing on the
injured leg persisted in the defect-only group for over 6 weeks
npj Regenerative Medicine (2021) 65(Fig. 7 and Supplementary Fig. 6A), indicating that regenerated
neocartilage ameliorates pain caused by the focal defect. JCC
sheet transplantation did not affect total body weight (Supplementary Fig. 6B).
DISCUSSION
The many current clinical demands for treating cartilage defects
are not satisﬁed by current tissue engineering products due tohigh graft costs, tissue availability, and poor adhesion techniques31,32. Osteochondral autograft transplantation is limited by (1)patient chondral integrity in the notch and peritrochlear region,(2) graft availability in large osteochondral defects, and has welldocumented donor site morbidity. Autologous chondrocyte
implantation (ACI) also sacriﬁces native patient cartilage and has
signiﬁcant preparation costs that proportionally increase withincreasing patient treatment numbers. Moreover, ACI requires atwo-stage surgical treatment for tissue harvest and subsequent
processing/handling and re-implantation thereby resulting indelayed patient recovery and increased treatment costs. In
contrast, fresh bulk osteochondral allograft transplantation (OCA)
from deceased donors allows a single-stage treatment but islimited by chondral viability during storage and biological graft
quality control, resulting in difﬁculty in uniform product preparation and limited tissue availability that may result in patient
waiting months to years to obtain treatment. In contrast, JCCs canbe obtained from surgical discards of juvenile donors without
Published in partnership with the Australian Regenerative Medicine InstituteM. Kondo et al.

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Fig. 3 Tumorigenicity assay, population doubling time, and cell surface markers. A (a) Microscopic images of in vitro tumorigenicity assay
in soft agar culture conditions. Images of the left column show seeded cells from 2-week cultured cell sheet. Images of the middle column
show seeded cells from 3.5-week cultured cell sheet. Images of the right column show seeded HepG2 cells as a positive control. The top row
shows the image at day 0 and the bottom shows images at day 8 after seeding. Bars: 200 µm. (b) Semi-quantiﬁcation of cell number by DNAbound ﬂuorescence in soft agar culture at day 0 and day 8. Data shown as mean and SD (n = 4 individual donors). **p < 0.01, *p < 0.05, N.S.
(non-signiﬁcant) by Student’ t test. B Flow cytometry analysis for cell purity and surface marker characterization. (a) Representative histogramsfor CD45, lineage cocktail (mixture of CD3, CD14, CD16, CD19, CD20, CD56), CD31, HLA-ABC, and HLA-DR, -DP, -DQ, CD44, CD90, CD81, andCD106. Column colors represent ﬂuorophores (blue: Paciﬁc blue, green: FITC or Alx488, red: PE, magenta: APC or Alx647) (b) Average
percentages for CD45, lineage cocktail (mixture of CD3, CD14, CD16, CD19, CD20, CD56), CD31, HLA-ABC and HLA-DR, -DP, -DQ, CD44, CD90,
CD81, and CD106. n = 4–6 individual donors. C Population doubling time in the extended subculture in chondrocyte culture medium up to
P13. Data shown as mean and SD (n = 13 individual donors).

donor site morbidity. Moreover, cultured JCCs show high growthpotential (Fig. 1D), low HLA levels (Fig. 3B), and can becryopreserved as a cell bank, enabling the manufacturing of offthe-shelf JCC sheets that allow a single-stage chondral repair
option that is not limited by graft size, availability, or defectlocation architecture. Finally, JCC sheets can be carefully
controlled for product quality surveillance before patient treatment thereby ensuring little to no graft variability.
Published in partnership with the Australian Regenerative Medicine Institute

Clinical data reporting outcomes of ACI at 8–60 months
observed that only 15% of the cohort shows type II collagendominant cartilage33. This low hyaline cartilage level may be
attributed to the variable potency of transplanted autologous cells
from individual adult patients and/or the cell delivery method
using processed cell suspensions covered in situ by periosteal
ﬂaps or collagen membranes that may cause low cell density andvariable defect retention. Autologous cultured chondrocytes on
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Fig. 4 Middle- and long-term in vivo efﬁcacy of focal osteochondral defect treatment in nude rats. A Macroscopic images of surgicallycreated focal defects (left images of each group) and 4, 8, 12, and 24 weeks after treatments (right images of each time point). The top row
shows native and defect only group. The bottom row shows defect and cell sheet group. B Safranin-O staining of each condition.
Representative images of samples at 4 weeks (n = 14), 8 weeks (n = 3), 12 weeks (n = 3), and 24 weeks (n = 3) are shown. Bars: Top and thirdrows: 500 µm, second and bottom rows: 100 µm.

porcine collagen membrane (MACI) employs chondrocytes frompatients’ own native cartilage in collagen I/III matrices fortransplant into the chondral lesion. MACI has had promisingclinical outcomes, but the superiority of MACI over othertechniques remains unclear34. Six-month postoperative histological analysis shows hyaline-like cartilage rather than hyaline
cartilage35. Chondrogenic differentiation is enhanced by cellcondensation36–38. The hyaline-like regeneration in MACI may be
attributable to the adult patients’ own cell origin and lack ofcell–cell communication in the collagen matrix.
Chondrocytes are well-known to de-differentiate in passage
cultures39. However, in contrast to adult chondrocytes, JCCs
maintain higher proliferative capacity and differentiation potential
after subculture14,40. Our results demonstrated that passaged JCCs
exhibit an elongated cell morphology (Fig. 1C), but successfully
redifferentiate to hyaline cartilage both in vitro (Figs. 1E and 2E)
and in vivo (Fig. 4A, B). Interestingly, isolated JCCs also show stable
cell growth over passage 10 (Figs. 1D and 3C). Collectively, theseresults, together with allogeneic cell sheet animal studies and
npj Regenerative Medicine (2021) 65

many allograft studies in the knee, allow allogeneic transplantation of JCCs for cartilage regeneration, probably due to the fact of
scarce accessibility of antigen-presenting cells and cytotoxic T
lymphocytes to the regenerated cartilage tissue. Neocartilage
forming capability and safety of highly passaged JCCs must be
demonstrated in future preclinical studies and clinical focal
cartilage defect treatments.
A single juvenile polydactyly donor can theoretically produce
thousands of JCC cell sheets at P2 (Fig. 2F). In addition to theadvantage of abundant cell sourcing for allogeneic transplantation41,42, we exploit the engineered JCC sheet production using
commercial temperature-responsive cultureware that enablesscalable, reliable harvesting and handling of intact allogeneic cellsheets and direct delivery of the tissue-like structure onto target
tissue sites. Such scaffold-free tissue engineering approaches can
avoid unexpected reaction/rejection of scaffold biomaterials and/or their degradation products43,44, which should enhance cartilage
regeneration and integration in host tissue. Furthermore, JCC
sheets are ﬂexible and adjustable to ﬁt on and into defect areas
Published in partnership with the Australian Regenerative Medicine InstituteM. Kondo et al.

7

Fig. 5 Cartilage-speciﬁc marker expression by transplanted human JCC sheet-derived tissue. Aggrecan staining (top), type II collagenstaining (middle), type I collagen staining (bottom) are shown. All samples shown are 4 weeks after transplantation. Bars: Left columns of eachtreatment group: 500 µm, right columns of each treatment group: 100 µm.

regardless of surface asperity, which can better facilitate graft
integration with host tissue, a major issue in conventional
osteochondral graft implantation, pellet/aggregate culture techniques, or biomaterial-based osteochondral repair45. Highly
proliferative JCCs in the sheet occupy the defect area, show rapid
and mature cartilage formation speciﬁcally at the defect area, andspontaneously close the gap with the host lateral interface of
native cartilage in 4 weeks (Fig. 4A, B). Neocartilage formed by JCC
sheets was substantially thicker than native rat cartilage (Fig. 4B),
suggesting the innate developmental potential of humanchondrocytes and their possible capacity to treat full-thickness
chondral defects in human patients. Notably, the newly formed
cartilage tissue did not display tumorigenic growth over 6 months(Fig. 4A, B), supporting the safety and stability of JCC sheets in situ.
Chondrogenesis in embryonic development is a complex process
initiated by MSC condensation on the bone-forming site, followedby terminal differentiation to mature cartilage46. In addition,
contraction of MSC sheet promotes chondrogenic differentiationin vitro38. The mechanism of the differentiation process oftransplanted JCC sheets may fundamentally follow this differentiation path, but future study must probe the detailed molecularinteractions that compel neocartilage formation in situ.
Allogenic osteochondral grafts have been applied to many
patients47,48 and previous allogeneic adult chondrocyte sheettransplantation models show cell retention in rabbits at 12 weeks49
and pigs at 3 months50. Allograft and allogeneic cell rejection are
regulated by interactions between the donor HLA molecules andrecipient T cells. Although class I MHC was expressed in JCC sheet
cells (Fig. 3B), leukocyte number in synovial ﬂuid is very low (<200cells/μL in healthy and <5000 in osteoarthritis)51. Thus, the knee
joint space is often considered an immune-privileged site. The
graft failure in SD rats and long-term engraftment in nude ratssuggests a critical role of T cells in a xenogeneic reaction to theimplanted chondrocyte sheets in the immunocompetent rat knee.
Possible mechanism in the observed xenogeneic rejection may bemacrophage activation by T cells that recognize xenoantigens aspreviously described52,53. MHC II plays an important role in the
immune rejection of transplanted allogeneic MSC via alloantibody formation demonstrated with a class II transactivator
knock-down model54; therefore, the absence of MHC II expression
in the JCC sheet (Fig. 3B) suggests negligible chronic rejection andpossible long-term engraftment in an allogeneic setting. In
addition, cultured JCCs are reported to inhibit T cell proliferationand express inhibitory cell surface B7 co-stimulatory molecules13.
Published in partnership with the Australian Regenerative Medicine Institute

Therefore, engineered JCC sheets might also be able to promote
long-term cartilage regeneration with low immunogenicity as anallogeneic cell therapy.
In summary, we report that JCCs isolated from juvenile donor
surgical discards can yield thousands of P2 JCC sheets, and wehave conﬁrmed consistent cell viability and quality, total cellnumbers, surface markers, and neocartilage regeneration in vivo
in a rodent focal osteochondral defect model. Beyond reliable
cartilage formation, the method has scaling features amenable toallogeneic off-the-shelf production strategies, including intrinsically low HLA human cell sources, use of mass-handledcommercial temperature-responsive cultureware, cell stability,
expansion, banking and storage, and sheet durability in shipment
or on-site fabrication adoption. These data strongly support thefurther applicability of JCC sheets in possibly addressing current
substantial clinical unmet needs in focal chondral defect patients.
Recent studies have revealed the molecular traits of human JCC
sheets correlated with in vivo efﬁcacy in rabbit osteochondral
defect treatment55. Strategies that can validate regenerative
capacity and minimize variability in new donor tissue sources
without extensive in vitro and in vivo testing are needed before
large-scale studies commence. In addition, better understanding
the regenerative mechanisms of JCC sheets will allow morerational development of this reliable therapy and encourage
future clinical adoption.
METHODS
Cartilage sampling from juvenile human polydactyly donors
Cartilage from the phalanx and metacarpal bones of amputatedpolydactylous ﬁngers and toes from 12 patients (24.8 ± 17.0 months old)
(Table 1) was sharply dissected using a scalpel and maintained in saline
immediately following harvest. All patients were prospectively enrolled at
Intermountain Primary Children’s Hospital (Salt Lake City, USA). Institutional Review Board oversight from University of Utah and Intermountain
Primary Children’s Hospital was waived due to use of deidentiﬁed routine
surgical discards.

Chondrocyte isolation
Cartilage harvested from juvenile donor tissues was transferred into saline,
cut into <4 mm2 pieces by scalpel, and then incubated with 5 mg/mL of
Type 1 collagenase at 37 °C for 1.5–3.0 h (LS004197, Worthington
Biochemical, Lakewood, USA). Resulting cells were ﬁltered through a
100-µm cell strainer, washed with saline and then resuspended inchondrocyte culture medium (DMEM-F12, 11320082, ThermoFisher
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8

Fig. 6 Transplanted human chondrocyte engraftment accompanied by type II collagen deposition. A (a) Human antigen-speciﬁc vimentin
staining of a JCC sheet-treated sample. Red arrowheads indicate regenerated cartilage. Blue arrowheads indicate host cartilage. Bar: 500 µm(b) Magniﬁed area of regenerated cartilage with human antigen-speciﬁc vimentin staining. Bar: 200 µm. B Double staining of human vimentin(red) and type II collagen (green). DAPI + Ph: DAPI and phase-contrast image to show nuclei (blue with white edge). The right panel shows themerged image of human vimentin, type II collagen, and DAPI. Bars: 200 µm. A histology sample of 4 weeks after treatment is shown.
of culture, cell sheets were harvested with forceps after incubation atroom temperature.

Cell viability and total cell number of chondrocyte sheet
Single cells from fabricated JCC sheets were isolated by incubation with
TrypLE Select for 15 min and 0.25 mg/mL collagenase P (11 213 857 001,
Roche, Basel, Switzerland) for 30 min. Cells were counted via hemocytometer and cell viability was demonstrated via trypan blue (T8154,
MilliporeSigma) dye exclusion56.

Chondrogenic differentiation culture

Fig. 7 Rodent weight-bearing test. Weight distribution of rat left
and right hind legs are measured, and percentages of treatment
side weight bearing are shown. Sample numbers at week 2, 3, 4, 5,
and 6 = 13, 12, 12, 8, and 8 (defect only group); = 16, 15, 15, 9, and 9(defect + CS group), respectively. Data shown as mean and SEM.
**p < 0.01, *p < 0.05 by Welch’s t test.
Scientiﬁc, Waltham, USA) containing 1% antibiotic-antimycotic (15240062,
ThermoFisher) and 20% fetal bovine serum (FBS) (16000044,
ThermoFisher).

Cell culture
Isolated chondrocytes were seeded on polystyrene dishes (CELLTREAT,
Pepperell, USA) at 5000–10,000 cells/cm2 in a chondrocyte culture medium(described above). The medium was replaced with chondrocyte medium
supplemented with 100 μg/mL L-ascorbic acid phosphate magnesium saltn-hydrate (013–19641, Fujiﬁlm Wako Pure Chemical, Osaka, Japan) at theﬁrst medium change on day 4. Cells were passaged with this medium
thereafter with the daily observation by phase-contrast microscopy
throughout cell culture. Subconﬂuent cells were collected by TrypLE
Select (12563011, ThermoFisher) dissociation and counted. Expanded cells
were cryopreserved in STEM-CELLBANKER GMP grade (Zenoaq, Fukushima,
Japan) at the end of P0. Serial subculture was performed with the thawed
cells at the initial density of 10,000 cells/cm2 passaged every 3–5 days.

Juvenile chondrocyte sheet preparation
Cell sheets were prepared from passage 1 cells sourced from thawed
cryopreserved cells. Subconﬂuent P1 cells were collected with 1x TrypLE
Select for 5 min, then seeded at a density of 10,000 cells/cm2 ontemperature-responsive cell culture inserts (CellSeed, Tokyo, Japan).
Chondrocyte culture media was changed every 3–4 days. After 2 weeks
npj Regenerative Medicine (2021) 65

Chondrogenic pellet culture was performed based on a previous study38.
JCCs harvested at the end of P2 and P9 cultures or isolated cells from P2
JCC sheets were aliquoted in chondrocyte culture medium at 2.5 × 105 cells
in 15 mL conical tubes for pellet cultures. Tubes were spun at 500 × g for
10 min. Caps were loosened and cells were incubated at 37 °C, 5% CO2 for
3 days to facilitate pellet formation. After the 3-day incubation step,chondrogenic samples were induced with chondrogenic medium, control
samples were replaced with a new chondrocyte culture medium, and all
samples were transferred to a hypoxic incubator (37 °C, 5% CO2, 5% O2).
Chondrogenic medium contained high glucose DMEM supplemented with
10 ng/mL transforming growth factor beta-3 (TGFβ3) (ThermoFisher),
200 ng/mL bone morphogenic protein-6 (BMP6) (PeproTech), 1% InsulinTransferrin-Selenium (ITS-G) (ThermoFisher), 1% PS (Life Technologies), 1%
non-essential amino acids (NEAA) (ThermoFisher), 100 nM dexamethasone(MP Biomedicals, Irvine, USA), 1.25 mg/ml bovine serum albumin (BSA)(MilliporeSigma), 50 μg/mL L-ascorbic acid phosphate magnesium salt nhydrate (Fujiﬁlm Wako Pure Chemical), 40 μg/mL L-proline (MilliporeSigma), and 5.35 μg/mL linoleic acid (MilliporeSigma). Media was changed
twice a week for 3 weeks.

In vitro cell transformation assay
In vitro cell transformation was evaluated by serial passage culture and soft
agar assay. Juvenile chondrocytes were passaged up to 13 passages(57–67 days in total) with a seeding density of 10,000 cells/cm2. Cells wereobserved every day and cell growth rate was calculated as doubling time.
Cell transformation was assessed by detecting anchorage-free proliferation. Cultured cells isolated from P2 chondrocyte sheets of normal culture
periods (2 weeks) and extended culture periods (3.5 weeks) were seeded ina soft agar gel with chondrocyte culture media with ascorbate at a densityof 5,000 cells per well by using CytoSelect Cell Transformation Assay (CBA140, Cell Biolabs, San Diego, USA). Fluorescent signal representing cellnumber was measured with a spectroﬂuorometer (Cytation 3 image
reader, BioTek, Winooski, USA) on day 0 and day 8 according to
manufacturer’s protocol. HepG2 cells (HB-8065, ATCC) in DMEM containing
10% FBS and 1% antibiotic-antimycotic were used as positive control foranchorage-free cell growth. Data are shown as averages of relative
ﬂuorescent units from duplicate or triplicate assays.
Published in partnership with the Australian Regenerative Medicine InstituteM. Kondo et al.

9
Flow cytometry
Isolated cell suspensions from chondrocyte sheets (dissociated as
described before) were aliquoted and incubated in 1 µg/mL Fc block
solution (564220, BD, Franklin Lakes, USA), resuspended in 10% FBScontaining PBS for 5–10 min, then labeled with ﬂuorescent-conjugatedantibodies (Supplementary Table 1) for 15 min with brief vortexing steps.
Cells were washed with 10% FBS-containing PBS, centrifuged, resuspended
with 1:1000 propidium iodide (PI) (556463, BD) in 10% FBS-containing PBS.
Samples were analyzed with a cell analyzer (Canto, BD). Doublets were
excluded with FSC-W and SSC-W gating, then the PI-negative population
was analyzed for speciﬁc antibody staining. The gating strategy is shown in
Supplementary Fig. 1.

Histology
Fabricated cell sheets were ﬁxed with 4% paraformaldehyde for 30 min at
RT. Harvested rat knee tissue was ﬁxed in 4% paraformaldehyde for fourdays and decalciﬁed in RapidCal Immuno (BBC Biochemical, Mount Vernon,
USA) for one day at RT. Samples were embedded in parafﬁn blocks andthen cut into 5-µm transverse sections with a microtome. Slides weredeparafﬁnized by baking in an oven at 65 °C and subsequent washes withxylene and ethanol. Sections were hydrated by gradual ethanol replacement by distilled water. Safranin-O was used for metachromatic staining
for sulfated glycosaminoglycans. Samples were stained for 5 min with
Wiegert’s Iron Hematoxylin (MilliporeSigma), 5 min with 0.5 g/L Fast Green
FCF (MilliporeSigma), and 5 min with 0.1% Safranin-O (MilliporeSigma).
Images were taken with a BX41 microscope (Olympus, Tokyo, Japan) andAmScope Software (USA).

Immunohistochemistry
Sections of histology samples were hydrated, then antigen retrieval was
performed. The retrieval method was chosen to preserve the tissue
integrity of knee samples and cell sheet samples after the staining
optimization: protease K (S3020, Agilent Technologies, Santa Clara, USA)
for COL2 and vimentin staining of knee samples; heat antigen retrieval in
citrate buffer (pH 6.0) (C9999, MilliporeSigma, Burlington, USA) for
COL2 staining of cell sheet samples. Peroxidase blocking was performed
with Hydrogen Peroxide Blocking Reagent (ab64218, Abcam, Cambridge,
UK). After blocking with 5% donkey serum and 0.1% Triton-X in PBS for 1
h. Samples were then incubated overnight with primary antibodies at
4 °C. Polyclonal goat anti-type I collagen (1:200, SouthernBiotech,
Birmingham, USA), monoclonal mouse anti-type II collagen (1:200,
2B1.5, ThermoFisher), polyclonal goat anti-aggrecan (1:100, AF1220,
R&D Systems, Minneapolis, USA), and monoclonal rabbit anti-humanvimentin (1:200, SP20, Abcam)57,58 were used as primary antibodies.
Normal mouse IgG2a (X0943, Agilent), normal goat IgG (NI02, MilliporeSigma), or normal rabbit IgG (X0903, Agilent) were used as isotype
controls at the same concentration as the primary antibodies. Horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody (1:1,000,
115–035–166, Jackson ImmunoResearch, West Grove, USA) was used fortype II collagen. HRP-conjugated donkey anti-goat antibody (1:1,000,
705-035-147, Jackson) was used for type I collagen and aggrecanstaining. HRP-conjugated goat anti-rabbit antibody (1:1,000, 111-035144, Jackson) was used for human vimentin staining. ImmPACT DAB
Peroxidase (HRP) Substrate (SK-4105, Vector Laboratories, Burlingame,USA) was used as a chromogen. Brightﬁeld images were taken with a
BX41 microscope and AmScope Software. Fluorescent images were takenwith Axio Vert.A1 microscope and ZEN software (Zeiss, Oberkochen,
Germany). All primary and secondary antibodies are listed in Supplementary Tables 2 and 3, respectively.

Surgical and transplantation procedure
The animal study plan was evaluated and approved by Institutional
Animal Care & Use Committee (IACUC, University of Utah) (assigned ID:
17-09011). SD rats and nude rats (6-week-old), male and female, werepurchased from Charles River Laboratories (Wilmington, USA). After aweek of acclimatization at the animal facility, animals were anesthetizedusing isoﬂurane and O2 gas. A medial parapatellar incision was made toexpose the knee joint; the patella was laterally dislocated and a focal
osteochondral defect (diameter 2 mm; depth 200–350 μm) was createdon the patellar groove of the femur using an electric grinder without
penetration to the bone marrow. Defect depth was controlled by theprocedure under a surgical stereo zoom microscope (SZX10, Olympus,
Published in partnership with the Australian Regenerative Medicine InstituteJapan) with repeated depth measurement with a needle tip (25 G, BD).
Chondrocyte sheets prepared in temperature-responsive cell culture
inserts in six-well plates (described above) were washed with saline, thencut in halves by a razor and single sheet halves transplanted to eachsurgical knee defect after defect creation. All animals received
buprenorphine for 2 days and carprofen for 3 days in compliance with
the IACUC protocol. Animals were sacriﬁced after 4, 8, 12, and 24 weeks
for further histological evaluations.

Rat hind limb weight-bearing distribution assessment
Weight distribution in rats was demonstrated with Incapacitance Tester(Linton Instrumentation, UK), a device with two separate boards onwhich the rats sit, measuring how the rats distribute their weight. All
animals were acclimatized 1–2 times before and after surgery. No
measurement was done until 2-week time point after surgery to avoid
effects of muscle trauma and analgesia. Weight distribution was
calculated by the following formula:
Weight distributionð%Þ ¼ ðTreated side0s load Þ=ðTreated side0s load þ intact side0s loadÞ  100

Sample numbers at week 2, 3, 4, 5, and 6 = 13, 12, 12, 8, and 8(nontreatment group); = 16, 15, 15, 9, and 9 (defect + CS group),
respectively. Data are shown as mean and SEM. **p < 0.01, *p < 0.05 by
Welch’s t test.

Reporting Summary
Further information on research design is available in the Nature Research
Reporting Summary linked to this article.

DATA AVAILABILITY
Data sets generated and/or analyzed during the current study are available from thecorresponding author upon reasonable request.

Received: 2 September 2020; Accepted: 14 September 2021;

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ACKNOWLEDGEMENTS
This work was supported in part by the University Technology Acceleration Grant(UTAG) from the Utah Science, Technology, and Research (USTAR) program. We thankTakumi Takahashi, Hallie Thorp, and Masatoshi Oka for their scientiﬁc comments. We
thank Manshu Tang, Goro Kuramoto, and Miranda Rogers for consultation aboutanimal experiments. The University of Utah HSC Core Flow Cytometry Facility is
acknowledged as a valued technical resource.

AUTHOR CONTRIBUTIONS
M.K., T.O., and T.G.M. conceptualized the study concepts. M.K. designed, performed,
interpreted all experiments and analysis, and wrote the manuscript. S.K. supported
primary cell culture, assisted weight-bearing experiments, interpreted the data, andedited the manuscript. K.K. participated in animal model development. N.F.M.
supported primary cell culture, provided data interpretation, and edited themanuscript. D.T.H. and A.A.W. harvested juvenile cartilage tissue. M.M. and M.S.
provided technical input and scientiﬁc comments on cell sheet preparation andanimal study. T.G.M. provided clinical input for designing knee defect model andoriginated the rodent surgical model. D.W.G. and T.O. supervised the technical teams,
coordinated resources, and reviewed and edited the manuscript. All authors read andapproved the ﬁnal manuscript.

COMPETING INTERESTS
Teruo Okano holds equity in CellSeed, Inc. (Japan) and is an inventor/developer
designated on the patent for CellSeed’s commercialized temperature-responsive
cultureware. No other competing ﬁnancial interests exist and all authors declare thatthey have no other competing interests.

Published in partnership with the Australian Regenerative Medicine InstituteM. Kondo et al.

11
ADDITIONAL INFORMATION
Supplementary information The online version contains supplementary material
available at https://doi.org/10.1038/s41536-021-00173-9.
Correspondence and requests for materials should be addressed to Makoto Kondo
or Teruo Okano.
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Published in partnership with the Australian Regenerative Medicine Institutenpj Regenerative Medicine (2021) 65