International Journal of

Molecular Sciences
ArticleDevelopment of Injectable Polydactyly-Derived
Chondrocyte Sheets
Shiho Wasai 1,2 , Eriko Toyoda 1,2 , Takumi Takahashi 1,2 , Miki Maehara 1,2 , Eri Okada 1,2 , Ryoka Uchiyama 1,2 ,Tadashi Akamatsu 3 , Masahiko Watanabe 1,2 and Masato Sato 1,2, *
1

2

3

*

Department of Orthopaedic Surgery, Surgical Science, Tokai University School of Medicine, 143 Shimokasuya,
Isehara, Kanagawa 259-1193, Japan; sw12345@tsc.u-tokai.ac.jp (S.W.); etoyoda@tokai-u.jp (E.T.);
takumi.takahashi22@gmail.com (T.T.); m-maehara@tsc.u-tokai.ac.jp (M.M.); okada-eri@tokai-u.jp (E.O.);
9bmrm002@mail.u-tokai.ac.jp (R.U.); masahiko@is.icc.u-tokai.ac.jp (M.W.)
Center for Musculoskeletal Innovative Research and Advancement (C-MiRA), Graduate School of Medicine,
Tokai University, 143 Shimokasuya, Isehara, Kanagawa 259-1193, Japan
Department of Plastic Surgery, Surgical Science, Tokai University School of Medicine, 143 Shimokasuya,
Isehara, Kanagawa 259-1193, Japan; akamatu@is.icc.u-tokai.ac.jp
Correspondence: sato-m@is.icc.u-tokai.ac.jp; Tel.: +81-46-393-1121; Fax: +81-46-396-4404

Academic Editor: Bernd Rolauffs

Abstract: We are conducting a clinical study of the use of allogeneic polydactyly-derived chondrocyte
sheets (PD sheets) for the repair of articular cartilage damage caused by osteoarthritis. However, thetransplantation of PD sheets requires highly invasive surgery. To establish a less invasive treatment,
we are currently developing injectable fragments of PD sheets (PD sheets-mini). Polydactyly-derived
chondrocytes were seeded in RepCell™ or conventional temperature-responsive inserts and cultured.
Cell counts and viability, histology, enzyme-linked immunosorbent assay (ELISA), quantitative
real-time polymerase chain reaction (qPCR), and flow cytometry were used to characterize PD sheetsmini and PD sheets collected from each culture. To examine the effects of injection on cell viability,
PD sheets-mini were tested in four experimental conditions: non-injection control, 18 gauge (G)needle, 23G needle, and syringe only. PD sheets-mini produced similar amounts of humoral factors
as PD sheets. No histological differences were observed between PD sheets and PD sheets-mini.
Except for COL2A1, expression of cartilage-related genes did not differ between the two types of PD
sheet. No significant differences were observed between injection conditions. PD sheets-mini havecharacteristics that resemble PD sheets. The cell viability of PD sheets-mini was not significantly
affected by needle gauge size. Intra-articular injection may be a feasible, less invasive method totransplant PD sheets-mini.

Received: 18 February 2021

Keywords: cartilage regeneration; cell sheet; osteoarthritis; minimally invasive treatment

Citation: Wasai, S.; Toyoda, E.;
Takahashi, T.; Maehara, M.; Okada, E.;
Uchiyama, R.; Akamatsu, T.;
Watanabe, M.; Sato, M. Developmentof Injectable Polydactyly-Derived
Chondrocyte Sheets. Int. J. Mol. Sci.
2021, 22, 3198. https://doi.org/
10.3390/ijms22063198

Accepted: 17 March 2021
Published: 21 March 2021

Publisher’s Note: MDPI stays neutral

1. Introduction

with regard to jurisdictional claims in

Articular cartilage is a unique tissue that comprises mainly extracellular matrix (ECM),
in the form of collagens and proteoglycans, and has a hypocellular and avascular structure [1]. Given its tissue properties, articular cartilage can tolerate intensive and repetitive
physical stress but, once damaged, is difficult to repair [2]. Diffuse damage to articularcartilage gradually promotes joint degeneration, which eventually leads to osteoarthritis(OA), especially in the knee joint.
OA of the knee (OAK) is the most common joint disease, involves cartilage defects ofvaried depth and breadth, and is the most frequent cause of knee pain and dysfunction. OA
progresses slowly with age, reduces the quality of life and work productivity, and ultimately
imposes socioeconomic costs worldwide [3]. The Framingham osteoarthritis study foundthat the age-standardized prevalence of OAK reached 19.2% (18.6% for men and 19.3% forwomen) in people older than 45 years. In Japan, where the aging population is increasing,
the number of OAK patients is expected to continue increasing in the future [4–6].

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Int. J. Mol. Sci. 2021, 22, 3198. https://doi.org/10.3390/ijms22063198

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Int. J. Mol. Sci. 2021, 22, 3198

2 of 12Current methods of treatment for OAK include patient education, exercise, oral
treatments, conservative therapy by intra-articular injection of hyaluronic acid or steroids,
and surgical treatments, including arthroscopic debridement of the knee joint, osteotomy
around the knee such as a high tibial osteotomy, unicompartmental knee arthroplasty, andtotal knee arthroplasty [7,8].
Autologous chondrocyte implantation (ACI) is performed as a regenerative treatment
for cartilage defects. However, most of the indications for ACI are cartilage defects causedby trauma and osteochondritis dissecans, and ACI does not have a sufficient therapeutic
effect on OAK. Moreover, because OAK is characterized by heterogeneous cartilage defects,
no fully effective regenerative treatment has been developed to stop the progression of
OAK or to induce cartilage regeneration [9–11].
Cell sheet engineering is a technique that enables the collection of cells in sheet form
simply by lowering the temperature from 37 ◦ C to below 32 ◦ C without using enzymes
such as trypsin and growing the cells in a temperature-responsive culture dish with thetemperature-responsive polymer poly (N-isopropyl acrylamide) [12]. Cell sheets havebeen applied as a regenerative treatment of various tissues including the cornea [13],esophagus [14], myocardium [15], and periodontium [16]. We have been engaged inresearch on cartilage regenerative medicine using this cell sheet engineering since 2004. At
present, autologous chondrocyte sheet transplantation is available as an advanced medicalcare B for the treatment of OAK in Japan. However, two surgical procedures are required
to fabricate and transplant an autologous chondrocyte sheet. In addition, the proliferative
capacity of adult chondrocytes varies greatly among individuals. To overcome these issues,
we explored the possibility of using allogeneic cell sources [17].
By focusing on the immunotolerant properties of cartilage that has already beenreported [18], particulated juvenile cartilage implants (De Novo ® NT Graft; Zimmer,
Warsaw, IN, USA) are used clinically in the United States [19]. However, the use ofthis product in Japan is difficult, due to the difficulty in ensuring traceability. Therefore,
we focused on surgical remains obtained from patients with polydactyly as a source of
allogeneic chondrocytes.
We are currently conducting a clinical study of the use of allogeneic polydactylyderived chondrocyte sheets (PD sheets) for articular cartilage repair [20]. However, thetransplantation of PD sheets requires open-knee surgery and is currently performed inconjunction with open-wedge high tibial osteotomy, which is highly invasive.
Injection is a promising approach to deliver substances for less-invasive types ofregenerative medicine. To establish a less-invasive treatment for OAK, we are currently
developing injectable PD sheets fabricated in RepCell™ plates (CellSeed Inc., Tokyo, Japan),a temperature-responsive culture dish with 3 mm × 3 mm grid walls etched into the culture
surface. Our aim is to develop sheet transplantation treatments that can be delivered
through needle injection into the knee joint (Figure 1).
The purposes of this study were to examine the potential clinical applications ofinjectable fragments of PD sheets (PD sheets-mini) by comparing their sheet characteristics
with those of conventional PD sheets and to examine the effects of PD sheets-mini injectedusing needles of different gauge size on cell viability.

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Figure 1. Fabrication of polydactyly-derived chondrocyte (PD) sheets and mini PD sheets. PD
sheets and mini PD sheets were fabricated in temperature-responsive culture inserts called UpCell® and temperature-responsive culture dishes called RepCell™ (CellSeed Inc.), respectively.
RepCell™ isofapolydactyly-derived
unique culture dish with grid walls
etched onto
the sheets-mini.
culture surface creating
3 mm ×Figure 1.
1. Fabrication
Fabrication
Figureof polydactyly-derived chondrocytechondrocyte(PD)(PD)sheets
sheetsandandPD
mini PD sheets.PD
PDsheets
3 mm grids.
sheetsandmini PD sheets
were fabricatedin temperature-responsive
cultureinserts
Upand PD
sheets-mini
were fabricatedin temperature-responsive
cultureinserts
calledcalled
UpCell®andCell®
and temperature-responsive
culturecalled(CellSeed
RepCell™
(CellSeed
Inc.), respectively.
temperature-responsive
culture dishes
calleddishes
RepCell™
Inc.),
respectively.
RepCell™ is a2.
RepCell™
is Resultsa uniqueculturegridonto
walls
onto
the culture
surfacemm ×
unique culture
dish withgrid dishwallswithetchedtheetchedculturesurfacecreating
3 mm creating× 3 mm 3grids.
3 mm grids.
2.1. Comparison of Sheet Characteristics of Polydactyly-Derived Chondrocyte (PD) Sheets and2. Results
Mini PD Sheets2.
Results
2.1.
Comparisonof Sheet
Characteristics
of Polydactyly-Derived Chondrocyte (PD) Sheets and PD
2.1.1. CellCount
and Viability
Sheets-Mini
2.1.
Comparison of Sheet Characteristics of Polydactyly-Derived Chondrocyte (PD) Sheets andAfterculturing
2.1.1.PD
Cell
Count
and
Viabilityfor 2 weeks, both mini PD sheets and PD sheets were collected, andMini
Sheetsthe cell count corrected by the area of the culture surface was calculated. The cell counculturing
2 weeks, both PD sheets-mini and PD sheets were collected, and2.1.1. After
CellandCount
and forViability
viability
did not differ significantly between mini PD sheets and PD sheets (Figure
the cell count corrected by the area of the culture surface was calculated. The cell count andAfter2a,b).
culturing for 2 weeks, both mini PD sheets and PD sheets were collected, andviability did not differ significantly between PD sheets-mini and PD sheets (Figure 2a,b).
the cell count corrected by the area of the culture surface was calculated. The cell countand viability did not differ significantly between mini PD sheets and PD sheets (Figure2a,b).

(a)(b)

Figure 2. Comparisonof2.cellcount and of
cellcellviability
for PD
andformini
sheets.
are expressed
as aremean ±
FigureComparison
count andcellsheets
viability
PD PD
sheetsand Values
PD sheets-mini.
Valuesstandard deviation. (a) Cell count of PD sheets and mini PD sheets. (b) Cell viability of PD sheets and mini PD sheets. The
expressed as mean ± standard deviation. (a) Cell count of PD sheets and PD sheets-mini. (b) Cell(a) did not differ between the two types of cell(b)cell counts and cell viability
sheets.
viability of PD sheets and PD sheets-mini. The cell counts and cell viability did not differ between
Figure 2. Comparison of cellthe
countviability
for PD sheets and mini PD sheets. Values are expressed as mean ±two andtypescellof cellsheets.
2.1.2.
Flow
Analysisstandard deviation. (a) Cell count of PD
sheetsandCytometricmini PD sheets.
(b) Cell viability of PD sheets and mini PD sheets. The
Flow
CytometricAnalysiscell counts and cell viability 2.1.2.
did not
differbetweentwo typesof cellsheets.
mini PDthe
sheetsand PD
sheetsshowed similar surface markers (Figure 3). Both sheets

were negative
vascularsimilar
endothelium
marker, andCD45,
a bloodcell marker
PD sheets-mini
andforPDCD31,sheetsashowedsurface markers(Figure3). Bothsheets
2.1.2.
Flow
CytometricAnalysismini PD
0.7%;endothelium
CD45, 0.8%; PD
sheets
CD31,
0.8%;
CD45,
1.4%.
Both types owere negative
for sheets
CD31,CD31
a vascularmarker,andCD45,
a bloodcellmarker:were
positivefor themesenchymal
stem
(MSC)
markers
CD29,
CD44, CD73
minisheet
PD sheetsandPD sheets
showedsimilarsurfacemarkers(Figure3). Both
PD sheets-mini
CD31
0.7%;
CD45,
0.8%;
PD
sheets
CD31,cell
0.8%;
CD45,
1.4%.
Bothsheets
types
CD81,
CD90,
and
CD105:mini
PD sheets
CD29,
98.4%;
CD44,
99.9%;
CD73,
99.9%; CD81of sheetwerepositiveformesenchymal
stemmarker,
cell
(MSC)
CD44,
CD73,
were
negative
for
CD31,
a the
vascularendothelium
andmarkers
CD45,
aCD29,
bloodcellmarker:
CD81,
CD90,
CD105:
sheets-mini
CD29,
98.4%;
99.9%;
CD81,
mini
PD
sheetsandCD31
0.7%;PD
CD45,
0.8%; PD
sheets
CD31,CD44,
0.8%;99.9%;
CD45, CD73,
1.4%. Both
types
of
100%;
CD90,
99.9%;
CD105,
88.1%;
PD
sheetsCD29,
98.9%;
CD44,
99.9%;
CD73,
100%;
sheet were positive for the mesenchymal stem cell (MSC) markers CD29, CD44, CD73,
CD81, CD90,
100%; andCD90,
99.9%;mini
CD105,
90.0%.CD29, 98.4%; CD44, 99.9%; CD73, 99.9%; CD81,
CD81,
CD105:PD sheetsInt. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW

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Int. J. Mol. Sci. 2021, 22, 3198

4 of 12100%; CD90, 99.9%; CD105, 88.1%; PD sheets CD29, 98.9%; CD44, 99.9%; CD73, 100%;
CD81, 100%; CD90, 99.9%; CD105, 90.0%.

Figure3. Flowcytometricanalysiscomparing
PD sheetsPD
andsheets
mini PD
sheets.
sheets were
Figure3. Flowcytometricanalysiscomparingand
PD Bothsheets-mini.
Both sheets werepositiveformesenchymal
stem
cell
markers
such
as
CD29,
CD44,
CD73,
CD81,
CD90,
and CD105,
positive for mesenchymal stem cell markers such as CD29, CD44, CD73, CD81,
CD90, and CD105,
and negative for vascular endothelium and blood cell markers such as CD31 and CD45.

and negative for vascular endothelium and blood cell markers such as CD31 and CD45.

2.1.3.
Measurementof theof Humoral
FactorsFactors
2.1.3.
MeasurementofAmounts
the Amountsof Humoral
The amounts of humoral factors were corrected by the culture surface area (Figure
The amounts of humoral factors were corrected by the culture surface area (Figure 4).
4). There was no significant difference between mini PD sheets and PD sheets in the
There was no significant difference between PD sheets-mini and PD sheets in theamounts
amounts of transforming growth factor beta-1 (TGF-β1): mini PD sheets 0.87 ± 0.09 ng/cm2 2oftransforming
growthfactorbeta-1(TGF-β1):PDsheets-mini
0.87
±
0.09
ng/cm
and PDand PD sheets 0.68 ± 0.36 ng/cm22. mini PD sheets produced similar amounts of melanoma
sheets
0.68
±
0.36
ng/cm
.
PD
sheets-mini
producedsimilaramountsofmelanomainhibitory activity (MIA) as PD sheets: mini PD sheets 5.52 ± 3.69 ng/cm2 and PD sheets
2
inhibitory
activity(MIA)
PD sheets:
PD sheets-mini
5.52did± not
3.69differ
ng/cm
2. Similarly,
5.93± 5.07 ng/cm
theasamountsof humoral
factors produced
be- and PD
2sheets± and5.07mini
ng/cm. Similarly,
the amountsof humoral
produced did
tween
PD5.93sheetsPD sheets
for the following
markers:
endothelialfactors
cell-specificnot differ
betweensheets 0.31
and±PDfor sheets,the following
marker1 (ESM-1),
mini PD
PD sheets,
0.11sheets-mini
ng/cm2 and PD
0.42 ± 0.24markers:
ng/cm2; endothe2 and PD sheets 1.062 ± 0.52
Dickkopf-1
protein (DKK-1)
PD sheets,± 0.30 ng/cm
lial cell-specificmarker mini
1 (ESM-1),
PD1.19sheets-mini,
0.31± 0.11 ng/cm and PD sheets,
22
protein(DKK-1)
1 (MCP-1),
PD sheets, 1.19
0.40 ±±0.09
ng/cm
0.42 ;±and0.24monocyte
ng/cm ;chemoattractant
Dickkopf-1 protein
PDminisheets-mini,
0.30 ng/cm2
2
2
2
ng/cm
andsheets
PD sheets± 0.06
ng/cm; and. Themonocyte
amounts ofenzymes that decompose
and PD
1.06 0.34± 0.52ng/cm
chemoattractant
protein 1the
(MCP-1), PD
2 . Theextracellularmatrix,and ±MMP-13,also did
not differ
sheets-mini,
0.40 matrix± 0.09metalloproteinaseng/cm2 and PD(MMP)-3
sheets 0.34
0.06 ng/cm
amounts of enbetween PD sheets and mini PD sheets. The respective values were mini PD sheets, 3.79 ±zymes that decompose the extracellular matrix, matrix metalloproteinase (MMP)-3 and2.53 ng/cm2 and PD sheets 2.73 ± 1.91 ng/cm2 and mini PD sheets, 0.56 ± 0.12 ng/cm2 and
MMP-13, also did not differ between PD sheets and PD sheets-mini. The respective valPD sheets 0.48 ± 0.16 ng/cm2.
2
2

ues were PD sheets-mini, 3.79 ± 2.53 ng/cm and PD sheets
2.73 ± 1.91 ng/cm and PD
5 of 13sheets-mini, 0.56 ± 0.12 ng/cm2 and PD sheets 0.48 ± 0.16 ng/cm2 .

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW

Figure4. Amountsof secreted
factors.
The amountsof transforming
growth factor
Figure4. Amounts
ofhumoral
secretedhumoralfactors.
The amountsof beta-1transforming growth factor beta-1(TGF-β1), melanoma inhibitory activity (MIA), endothelial cell-specific marker 1 (ESM-1), Dick(TGF-β1),inhibitory
activity(MIA),
endothelial
cell-specific marker 1 (ESM-1), Dickkopf-1
kopf-1protein melanoma(DKK-1), monocyte
chemoattractant
protein
1 (MCP-1),
matrix metalloproteinase(MMP)-3, and MMP-13, humoral factors related to cartilage, did not differ significantly between
protein (DKK-1), monocyte chemoattractant protein 1 (MCP-1), matrix metalloproteinase (MMP)-3,
mini PD sheets and PD sheets.

and MMP-13, humoral factors related to cartilage, did not differ significantly between PD sheets-mini
and PD sheets.

2.1.4. Gene Expression Analysis

The relative gene expression of PD sheets was set at 1.0. The relative ratios of gene
expression of mini PD sheets were 0.68 times for COL1A1, 0.19 times for COL2A1, 0.73
times for SOX9, 0.63 times for ACAN, 1.23 times for RUNX2, and 1.30 times for MMP3.
Except for COL2A1, the expression of cartilage-related genes did not differ between PD
sheets and mini PD sheets. The expression of COL10A1 was not detected (Figure 5).

Int. J. Mol. Sci. 2021, 22, 3198

Figure 4. Amounts of secreted humoral factors. The amounts of transforming growth factor beta-1(TGF-β1), melanoma inhibitory activity (MIA), endothelial cell-specific marker 1 (ESM-1), Dickkopf-1 protein (DKK-1), monocyte chemoattractant protein 1 (MCP-1), matrix metalloproteinase
5 of 12(MMP)-3, and MMP-13, humoral factors related to cartilage, did not differ significantly between
mini PD sheets and PD sheets.

2.1.4.
Gene
Expression
Analysis2.1.4.
Gene
Expression
AnalysisThe
relative
gene
expression
The relative gene expressionofofPD
PDsheets
sheetswas
wassetsetatat1.0.
1.0.The
Therelative
relativeratios
ratiosofofgene
gene
expression
of
mini
PD
sheets
were
0.68
times
for
COL1A1,
0.19
times
for
COL2A1,expression of PD sheets-mini were 0.68 times for COL1A1, 0.19 times for COL2A1,0.73
0.73
times
forforRUNX2,
and
1.30
times
forforMMP3.
timesfor
forSOX9,
SOX9,0.63
0.63times
timesfor
forACAN,
ACAN,1.23
1.23times
times
RUNX2,
and
1.30
times
MMP3.
ExceptExceptfor
forCOL2A1,
COL2A1,thetheexpression
expressionofofcartilage-related
cartilage-relatedgenes
genesdiddidnotnotdifferdifferbetween
betweenPD
PD
sheetsandsheets. The
expression
ofof
COL10A1
wasnotdetected(Figuresheetsandmini
PD PD
sheets-mini.
The
expression
COL10A1
wasnotdetected(Figure5).5).

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6 of 13

FigureCartilage-related
gene
expression
analysis
sheetsand
PDPD
sheets-mini.
relative
Figure5. 5.
Cartilage-related
gene
expression
analysisin in
PDPD
sheetsandmini
sheets. TheThe
relative
gene
expressionofofPD
PDsheets
sheetswas
wassetsetatat1.0.
1.0.The
Thevalues
valuesareareexpressed
expressedasasmean
mean±±standard
standarddeviadeviation.
gene
expression** p**< p0.01;
ND,
notnotdetected.
Only
thetheexpression
ofof
COL2A1
differed
betweention.
< 0.01;ND,
detected.
Only
expression
COL2A1
differed
betweenPD
PDsheets
sheetsand
andPD
mini
PD sheets.
sheets-mini.

2.1.5.HistologicalHistologicalAnalysis
Analysis2.1.5.
Sectionsofofboth
bothPD
PDsheets
sheetsand
andPD
mini
PD sheetsshowed
showedsimilar
similarmultiple
multiplestratification
stratification
Sections
sheets-minichondrocytesderived
derivedfrom
frompolydactyly
polydactylytissues.
tissues.Both
BothPDmini
PD sheetsand
andPD
PDsheets
sheetsofofchondrocytes
sheets-mini
werestained
stainedweakly
weaklyorornot
notatatallallwith
withSafranin
SafraninOOand
andtoluidine
toluidineblue.
blue.HistologicalHistologicalanalysis
analysis
wererevealedno
nodifferences
differencesbetween
betweenPD
PDsheets
sheetsand
andPD
mini
PD sheets(Figure(Figure6).6).
revealed
sheets-mini

Figuresheets-mini.
sheets-mini
Figure6.6.HistologicalHistologicalanalysis
analysisofofPD
PDsheets
sheetsand
andPD
mini
PD sheets.Sections
Sectionsofofboth
bothPD
PDsheets
sheetsand
andPD
mini
PD sheetswere
werestainedwith
withhematoxylin–eosin,
hematoxylin–eosin,
safraninandtoluidineblue
scalebar
bar==100
100µm).
μm).
stainedsafraninO,O,andtoluidineblue(×(×10,10, scale

2.2. Effects of Injection of Mini PD Sheets on Cell Viability
Cell viability decreased with time after injection but did not differ significantly between the four injection conditions immediately after (p = 0.748), 4 h (p = 0.987), and 24 h
(p = 0.994) after injection. Cell viability of the conventional PD sheets after detachment did
not differ between the four conditions (Figure 7a,b).

Figure6. Histologicalanalysis of PD sheets and mini PD sheets. Sections of both PD sheets and mini PD sheets were6 of 12
Int. J. Mol.
Sci. 2021,
22, 3198
stained with hematoxylin–eosin, safranin O, and toluidine blue (×10, scale bar = 100 μm).

2.2. Effects of Injection of Mini PD Sheets on Cell Viability
2.2. Effects
of Injectionof PD Sheets-Mini
Cellinjection
Viability but did not differ significantly beCell viability
decreasedwith timeon
after
Cell
decreasedwith timeafter injection
but= did
not4differ
betweentheviability
four injection
conditions
immediately
after (p0.748),h (p =significantly
0.987), and 24htween
the fourimmediately
after (p = PD
0.748),
4 h after(p = 0.987),
and 24h(p = 0.994)
after injection
injection.conditions
Cell viabilityof the conventional
sheetsdetachmentdid(p= differ
0.994) between
after injection.
Cellviability of
the conventional
PD sheets after detachment did
notthe fourconditions(Figure7a,b).
not differ between the four conditions (Figure 7a,b).

(a)

(b)

Figureonon
cellviability
of ofPDminisheets-mini.
(a) (a)To To
examinethe theeffectof injection
on cellFigure 7.7. Evaluation
Evaluationofofthe
theeffecteffectofofinjection
injection
cellviability
PD sheets.
examineeffectof injectiononviability,thePDsheets-mini
suspension
was
examinedunder
four
conditions:non-injection
control,
18
gauge
(G)
needlecell viability, the mini PD sheets suspension was examined under four conditions: non-injection control, 18 gauge (G)injection,
23G needle
injection,and syringe
injection,and cellviability
was measured
at 0, 4,after
injection.
Atneedle injection,
23G needle
injection,and syringe
injection,andcell viability
was measured
at and0, 4, 24
andh 24h after
injection.
At
0
and
4
h
after
injection,
cellviabilityofconventionalPDsheetswascompared.
(b)Comparisonofcellviabilityofmini
0 and 4 h after injection, cell viability of conventional PD sheets was compared.
of cell viability of PD
sheets-mini between the four conditions immediately after, 4 h, and 24 h after injection. Cell viability decreased with time
but did not differ significantly between the four conditions at any time.

3. Discussion
Cartilage regeneration is difficult when induced only with concentrated humoral
factors without cells, and the presence of cells producing cartilage-related humoral factors
is considered to be important. Cell transplantation therapy by injection is a promising
approach for less-invasive regenerative medicine, but the retention of transplanted cells at
the injected sites is an important issue [21].
In cell sheet engineering, cells can be collected in sheets without the need for enzymes
such as trypsin. Cell sheets of most types of cells can be transplanted while retaining theextracellular matrix and cell adhesion factors.
Wang et al. reported that the ability for cell attachment and proliferation was maintained even after MSC sheets were fragmented and injected through a needle, and thismethod may be superior to that using dissociated MSCs [21]. We believe that transplanted
cells engraft at the cartilage defect sites or in the vicinity, even temporarily, and that humoral factors secreted from the cells are important for cartilage regeneration. The results ofgene expression and histological evaluation suggest that dedifferentiation of chondrocytes
occurs in both PD sheet and PD sheet-mini. We consider that cartilage repair is promotedby the recipient’s chondrocytes, which are influenced by the humoral factors secreted byboth sheets, rather than replacing both of the transplanted sheets with cartilage tissue.
In conducting clinical studies on the transplantation of autologous chondrocyte
sheets [20] and polydactyly-derived chondrocyte sheets for articular cartilage regeneration, we have reported the characteristics and therapeutic effects of both types of sheet inanimal experiments using mini-pigs, rabbits, and rats [22–25]. Our previous flow cytometric analysis studies have shown that PD sheets express MSC markers and our histologicalInt. J. Mol. Sci. 2021, 22, 3198

7 of 12analyses have shown that PD sheets are multiply stratified without stacking, as in autologous chondrocyte sheets [17]. Similar results to these studies were obtained for both PD
sheets and PD sheets-mini in this study.
MIA and TGF-β1 secreted from autologous chondrocyte sheets and PD sheets play animportant role in cartilage differentiation and repair. MIA, which is a cartilage differentiation marker and anabolic factor, modulates chondrogenic differentiation via MIA-regulated
extracellular signal-regulated kinase (ERK)-signaling [26]. TGF-b is known to be very
beneficial for cartilage as it stimulates chondrocytes to induce elevation of proteoglycan
and collagen type II production and also counteracts the main catabolic factors in OA [27].
Importantly, more TGF-β1 is secreted from PD sheets than from autologous chondrocyte
sheets [17,28]. We also measured the production of ESM-1 and DKK-1, which we previously
reported as humoral factors whose production correlates positively with the effectivenessof PD sheets for articular cartilage repair. ESM-1 is a proteoglycan involved in angiogenesis
and is expressed in the vascular endothelium and adipocytes. DKK-1 is a protein that has
an inhibitory effect on the Wnt signaling pathway and is involved in bone metabolism
and differentiation [29]. In this study, the secretion of these humoral factors did not differ
significantly between PD sheets and PD sheets-mini and, therefore, we consider that both
sheets would have an equivalent effect of cartilage regeneration.
MCP-1, a chemokine with monocyte/macrophage migration activity, is also called
C–C chemokine ligand 2 (CCL2). CCL2 provokes an inflammatory response through the
C–C chemokine receptor 2 (CCR2), and the CCL2–CCR2 signal plays a role in cartilage
degeneration. However, Jablonski et al. reported that, in the absence of CCR2, CCL2 may
be required for cartilage regeneration [30]. Based on the report by Jablonski et al., wemeasured MCP-1 production in this study, but found no significant difference in MCP-1
production between PD sheet and PD sheet-mini.
MMP-3 and MMP-13 are known as catabolic factors that lead to cartilage degeneration
by degrading ECM [31,32]. We have reported that PD sheets produce less MMP-3 thanadult autologous chondrocytes, suggesting that polydactyly-derived chondrocytes havean advantage as a cell source [17]. In this study, there was no significant difference in the
production of MMP-3 and MMP-13 by PD sheets-mini compared with the conventional PD
sheets.
For all humoral factors measured in this study, we found no significant differencesin the amounts secreted between both types of cell sheet. The new injection form of thechondrocyte sheets used in this study (PD sheets-mini) had similar characteristics as the
PD sheets. However, the expression of COL2A1 was lower in the PD sheets-mini, andthis may reflect the culture of sheets in a flat culture dish instead of an insert, unlike inconventional PD sheets. From the results of clinical studies and accompanying animal
experiments that we have conducted so far, we consider that the humoral factors secretedby the transplanted chondrocyte sheets play a more important role in the regeneration ofarticular cartilage than the genes expressed by the sheets [20,28,29].
Injection of MSCs has been reported to have no effects on the trilineage differentiationof osteogenic, adipogenic and chondrogenic cell lines, or on cell viability [33], although it
has been suggested that apoptosis may occur early after injection through a 21 gauge (G)or 23G needle [34]. However, those studies examined this issue at the cellular level, and noreports have examined the effects of injection of mini-tissues such as cell sheets.
Our findings that the injection condition had almost no effect on the cell viability of
PD sheets-mini suggest that injection of PD sheets-mini is feasible. Even if cell apoptosis
occurs after injection through a 21G or 23G needle, the size of the needle usually used forarthrocentesis of knee is 18G, which should have less effect on cells.
PD sheets-mini have sheet characteristics that closely resemble those of PD sheets.
Our results suggest that the cell viability of PD sheets-mini is not significantly affected by
the clinically relevant needle gauge size. Intra-articular injection may be a feasible method
for the administration of PD sheets-mini as a less-invasive method. In the future, it isnecessary to examine the effect of cartilage treatment of PD sheets-mini in vivo.

Int. J. Mol. Sci. 2021, 22, 3198

8 of 124. Materials and Methods
4.1. Collection of Polydactyly-Derived Chondrocytes
Cartilage tissue was obtained from six polydactyly patients (average age, 15.5 months;
range, 12–23 months; three boys and three girls) who underwent surgery at Tokai University
Hospital.
Cartilage tissue was finely minced with scissors and treated with the following procedure to isolate chondrocytes from cartilage tissue. The minced cartilage tissue wasincubated in Dulbecco’s modified Eagle’s medium/F12 (DMEM/F12; Gibco, Grand Island,
NY, USA) supplemented with 20% fetal bovine serum (FBS; SAFC Biosciences, Lenexa, KS,
USA), 1% antibiotic–antimycotic solution (AB; Gibco), and 5 mg/mL collagenase type 1(CLS1; Worthington, Mannheim, Germany) for 1.5 h at 37 ◦ C in a humidified atmosphereof 5% CO2 and 95% air. The collected cell suspension was washed and passed through a
100 µm strainer (BD Falcon, Franklin Lakes, NJ, USA).
The isolated chondrocytes were seeded at a density of 1 × 104 cells/cm2 in six-wellculture plates (Corning, Corning, NY, USA) in DMEM/F12 supplemented with 20% FBS
and 1% AB, and incubated at 37 ◦ C. After 4 days, 100 µg/mL ascorbic acid (Nissin Pharmaceutical, Yamagata, Japan) was added to the medium, and the medium was replaced every
3 or 4 days thereafter. When the chondrocytes reached confluency, they were passaged
once or twice, and passaged chondrocytes were cryopreserved at −180 ◦ C.
4.2. Fabrication of PD Sheets and PD Sheets-Mini
PD sheets and PD sheets-mini were fabricated using a temperature-responsive culture
insert and RepCell™ plate (CellSeed Inc.). The latter is a unique temperature-responsive
culture plate with 3 mm × 3 mm grid walls etched into the culture surface. The areas of
the culture dishes were 4.2 cm2 and 8.8 cm2 , respectively (Figure 1).
To fabricate PD sheets, cryopreserved chondrocytes isolated from polydactyly patientswere thawed, passaged once, and then seeded in temperature-responsive culture inserts(CellSeed Inc.) at 1 × 104 cells/cm2 . After culturing for 2 weeks with the medium changed
every 3 or 4 days, the culture plates were allowed to stand at 25 ◦ C for 30 min to detach PD
sheets from the inserts, and each sheet was collected by hooking it onto a paper ring. PD
sheets were checked visually to confirm the strength and to identify any tearing.
PD sheets-mini were fabricated using a method similar to that for PD sheets. The lysedchondrocytes were passaged and then seeded at 1 × 104 cells/cm2 in RepCell™ plates.
After culturing for 2 weeks with the medium changed every other day, each RepCell™
plate was kept at 25 ◦ C for 30 min, and multiple 3 mm × 3 mm-sized fragments of PD
sheets were collected (Figure 1).
4.3. Comparison of Sheet Characteristics between PD Sheets and PD Sheets-Mini
4.3.1. Cell Count and Viability
PD sheets and PD sheets-mini were washed in Dulbecco’s phosphate-buffered saline(DPBS; Gibco). Each type of sheet was then incubated in TripLE Express® (Gibco) at
37 ◦ C for 15 min and centrifuged at 1500 rpm for 5 min. The sheets were resuspended
in 0.25 mg/mL Collagenase P (Roche, Basel, Switzerland) at 37 ◦ C for up to 30 min andthen centrifuged at 1500 rpm for 5 min. Cells isolated from both types of sheets were
resuspended in DMEM/F12, and cell count and viability were determined using trypan
blue exclusion.
4.3.2. Flow Cytometric Analysis
After the cell count and viability were measured, isolated cells were washed with DPBS
containing 0.2% bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO, USA) and1 mM ethylenediaminetetraacetic acid (EDTA; Gibco). About 1.5 × 105 cells were mixed ineach tube with the following antibodies: hCD31–fluorescein isothiocyanate (FITC) (clone:
5.6E; Beckman Coulter, Brea, CA, USA), hCD44–FITC (clone: G44-26; BD Biosciences,Franklin, NJ, USA), hCD45–FITC (clone: J33; Beckman Coulter), hCD73–FITC (clone: AD2;

Int. J. Mol. Sci. 2021, 22, 3198

9 of 12BD Biosciences), hCD81–FITC (clone: JS-81; BD Biosciences), hCD90–allophycocyanin(clone: 5E10; BD Biosciences), and CD105–phycoerythrin (clone: 266; 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. Fluoroprobe-labeled mouse IgG1 antibody (Beckman Coulter)and rat IgG2b antibody (BioLegend, San Diego, CA, USA) were used as negative controls.
Stained cells were analyzed using a FACS Verse TM cell sorter (BD Biosciences).
4.3.3. Measurement of Humoral Factors
PD sheets and PD sheets-mini fabricated as described above were used. PD sheetscollected by hooking on paper rings were cultured for 24 h in 3 mL of DMEM/F12 supplemented with 5% FBS and 1% AB in an adherent culture dish. PD sheets-mini were
harvested from RepCell™ plates after the plate was left at room temperature for more than
30 min. PD sheets-mini were suspended in 3 mL of DMEM/F12 supplemented with 5%
FBS and 1% AB, and cultured for 24 h in a culture dish. Each supernatant was collected
and centrifuged at 15,000× g for 10 min to remove cell debris. The concentrations of
TGF-β1, MIA, ESM-1, DKK-1, MCP-1, MMP-3, and MMP-13 were measured using the
enzyme-linked immunosorbent assays (ELISAs) described below.
TGF-β1, MIA, and ESM-1 concentrations were measured using their respective singlesample ELISA kits: Human TGF-β1 Quantikine ELISA Kit (R&D Systems, Minneapolis,
MN, USA), MIA ELISA (Roche), and Human ESM-1 ELISA Kit (Abcam, Cambridge, UK).
The other humoral factors were measured using Quantibody Multiplex ELISA Array(RayBiotech Life, Inc., Norcross, GA, USA).
The signal detected for blank medium containing 5% FBS was subtracted to adjust
for proteins contained in FBS. Measurements were repeated at least twice for each donor,
and averages were used. The amount of humoral factor per area of the culture surface wascalculated.
4.3.4. Gene Expression Analysis
Each of the PD sheets and PD sheets-mini fabricated by culturing for 2 weeks was
disrupted in TRIzol Reagent (Life Technologies, Carlsbad, CA, USA) and crushed at
1500 rpm for 3 min using a SHAKE Master Neo instrument (Bio Medical Science, Tokyo,
Japan). Total RNA was extracted using an RNeasy Mini Kit (Qiagen, Hilden, Germany).
Next, first-strand cDNA was reverse transcribed from 1 mg of total RNA and treated witha QuantiTect Reverse Transcription Kit (Qiagen) in a SimpliAmp Thermal Cycler (Thermo
Fisher Scientific, Waltham, MA, USA) at 42 ◦ C for 15 min and 95 ◦ C for 3 min. Quantitative
real-time polymerase chain reaction (qPCR) was performed using an Applied Biosystems
7300 Real-Time PCR system (Applied Biosystems, Waltham, MA, USA). The SYBR™ Green
primers used are shown in Table 1. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
primers were used to normalize the samples.
Table 1. Genes and their primers used for quantitative real-time polymerase chain reaction (qPCR).
Genes

Forward Primer Sequence

Reverse Primer Sequence

ACAN
COL1A1COL2A1
COL10A1
GAPDH
MMP3
RUNX2
SOX9

AGGAGACAGAGGGACACGTCGTCGAGGGCCAAGACGAAGGTGGAGCAGCAAGAGCAAATGCTGCCACAAATACCCTTTAGAAGGCTGGGGCTCATTTG
ATGATGAACAATGGACAAAGGAACCATGGTGGAGATCATCGAACGCCGAGCTCAGCAAGA

TCCACTGGTAGTCTTGGGCATCAGATCACGTCATCGCACAAC
TGTTGGGAGCCAGATTGTGGTAGTGGGCCTTTTATGCCTAGGGGCCATCCACAGTCTTCGAGTGAAAGAGACCCAGGGACGCCATGACAGTAACCACAG
CCGCGGCTGGTACTTGTAATC

4.3.5. Histological Analysis
The harvested PD sheets and PD sheets-mini were fixed with 20% formalin (Wako
Pure Chemical, Osaka, Japan) and embedded in paraffin wax. The embedded tissue was cut

Int. J. Mol. Sci. 2021, 22, 3198

10 of 12

into 20-µm-thick sections, which were deparaffinized and stained with hematoxylin–eosin,
Safranin O, and toluidine blue according to standard methods.
4.4. Analysis of the Effect of Injection of PD Sheets-Mini on Cell Viability
PD sheets-mini harvested from one RepCell™ plate were suspended in 3 mL of
medium. To analyze the effect of injection on cell viability, suspensions of PD sheets-mini
were tested for the following four injection conditions: non-injection control, 18G needle,
23G needle, and syringe only. For the injection condition, a 3 mL suspension was aspirated
slowly into a 5 mL syringe and then injected through a needle or the syringe itself.
Cell viability was examined using the trypan blue exclusion assay at 0, 4, and 24
h after injection. Cell viability was compared among the four injection conditions, andalso between PD sheets-mini and after detachment of cells from PD sheets at 0 and 24 h
(Figure 7a).
4.5. Statistical Analysis
Significant differences between groups were identified using the Mann–Whitney U
test for non-normally distributed quantitative data. The Kruskal–Wallis test was used todetect significant differences between three groups or more. All statistical analyses were
performed using IBM SPSS Statistics (v. 25.0; IBM, Armonk, NY, USA). Probability valuesof <0.05 were considered to be significant.
5. Conclusions
PD sheets-mini have sheet characteristics that closely resemble those of PD sheets.
Our results suggest that the cell viability of PD sheets-mini is not significantly affectedby injection using a clinically relevant needle gauge size. Intra-articular injection may be
a feasible way to administer PD sheets-mini in a less-invasive method as a point-of-caretreatment for OAK.
6. Patents
The work reported in this study is patent pending; the application number is 2020148200 in Japan, and the application date was 3 September 2020.
Author Contributions: Conceptualization, M.S. and S.W.; formal analysis, S.W.; funding acquisition,
M.S.; investigation, S.W., T.T., M.M., E.O., E.T. and R.U.; resources, S.W., T.A. and M.S.; data curation,
S.W.; writing—original draft preparation, S.W.; writing—review and editing, M.S.; supervision, M.S.
and M.W. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: This study was reviewed and approved by the Provision
Plan of Class 1 Regenerative Medicine (jRCTa030190242) in accordance with the Act on the Safety ofRegenerative Medicine and conducted in compliance with relevant guidelines.
Informed Consent Statement: Written informed consent was obtained from all subjects involved in
the study.
Data Availability Statement: The data presented in this study are available on request from thecorresponding author. The data are not publicly available because of confidentiality issues.
Acknowledgments: We would like to thank Saori Shirasuna, Ayako Watanabe (Tokai University)
and Yuka Kawaguchi (CellSeed Inc.) for their clinical and technical contributions to this study. We
also thank all members of the Department of Plastic Surgery, Surgical Science, Tokai University
School of Medicine, for assistance in the collection of chondrocytes. Finally, we are grateful to the
Support Center for Medical Research and Education at Tokai University for technical support in thisstudy.
Conflicts of Interest: M.S. receives research funding from CellSeed Inc.

Int. J. Mol. Sci. 2021, 22, 3198

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