International Journal of

Molecular Sciences
ArticleTranscriptomic and Proteomic Analyses Reveal the
Potential Mode of Action of Chondrocyte Sheets in
Hyaline Cartilage Regeneration
Eriko Toyoda 1 , Masato Sato 1, *, Takumi Takahashi 1 , Miki Maehara 1 , Eri Okada 1 , Shiho Wasai 1 ,Hiroshi Iijima 2 , Ken Nonaka 2 , Yuka Kawaguchi 3 and Masahiko Watanabe 1
1
2
3

*

Department of Orthopaedic Surgery, Surgical Science, Tokai University School of Medicine,
143 Shimokasuya, Isehara, Kanagawa 259-1193, JapanDNA Chip Research Inc., 1-15-1, Minato-ku, Tokyo 105-0022, Japan
CellSeed Inc., 2-5-10, Aomi, Koto-ku, Tokyo 135-0064, Japan
Correspondence: sato-m@is.icc.u-tokai.ac.jp; Tel.: +81-463-93-1121; Fax: +81-463-96-4404

Received: 11 November 2019; Accepted: 22 December 2019; Published: 24 December 2019


Abstract: Chondrocyte sheet transplantation is a novel and promising approach to treating patients
who have cartilage defects associated with osteoarthritis. Hyaline cartilage regeneration by autologous
chondrocyte sheets has already been demonstrated in clinical research. In this study, the efficacy
of polydactyly-derived chondrocyte sheets (PD sheets) as an allogeneic alternative to standard
chondrocyte sheets was examined using an orthotopic xenogeneic transplantation model. In addition,
the expression of genes and the secreted proteins in the PD sheets was analyzed using a microarray
and a DNA aptamer array. The efficacy of PD sheets with respect to cartilage defects was assessedusing histological scores, after which the expressions of genes and proteins exhibiting a correlation
to efficacy were identified. Enrichment analysis of efficacy-correlated genes and proteins showedthat they were associated with extracellular matrices, skeletal development, and angiogenesis. Eightgenes (ESM1, GREM1, SERPINA3, DKK1, MIA, NTN4, FABP3, and PDGFA) exhibited a positive
correlation with the efficacy of PD sheets, and three genes (RARRES2, APOE, and PGF) showed anegative correlation for both transcriptomic and proteomic analyses. Among these, MIA, DKK1, andGREM1 involved in skeletal development pathways and ESM1 involved in the angiogenesis pathway
exhibited a correlation between the amount of secretion and efficacy. These results suggest that these
secreted factors may prove useful for predicting PD sheet efficacy and may therefore contribute tohyaline cartilage regeneration via PD sheets.
Keywords: cartilage regeneration; cell sheet; xenogeneic transplantation model; efficacy

1. Introduction
It is well known that articular cartilage does not regenerate easily. Spontaneous repair of cartilage
defects cannot be expected following damage by trauma or osteoarthritis (OA). In fact, OA is designatedas a degenerative cartilage disease, with about half of those over 40 years old being estimated as
potential patients [1]. Although OA accounts for the majority of cartilage injuries that require treatment,
there is no established definitive therapy for OA.
Chondrocyte sheets can be created without using material such as animal-derived collagen byusing temperature-responsive culture devices such as thermo-responsive polymer grafted culture
dishes [2,3], where cells can adhere, proliferate, and form into a sheet. The sheet can be harvested
without enzymatic digestion by lowering the temperature. By using this technology, chondrocyte
sheets can be collected and triple-layered and then transplanted onto cartilage lesions while retaining

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the extracellular matrix and adhesion molecules that are produced by the chondrocytes themselves [4].
Because chondrocyte sheets can adhere to the recipient site without sutures, the chondrocytes willremain at the site of implantation.
Chondrocyte sheet transplantation is therefore a promising approach to treating articular cartilage
lesions. Articular cartilage regeneration with hyaline cartilage, which is important for joint function,
has been demonstrated using partial-thickness and osteochondral defect models of rats, rabbits, andpigs [4–8].
Treatment with chondrocyte sheets using the patient’s own cells has been performed on 20- to
60-year-old patients who had knee joint cartilage damage caused by traumatic or OA degeneration.
The articular cartilage was repaired to hyaline cartilage, and the treatment improved the clinical joint
condition score [9]. These results suggest that cell sheet transplantation could become the definitive
treatment for OA-associated articular cartilage lesions.
Based on years of experience with allograft transplantation, it is well known that cartilage tissue
is immune tolerant. Allogeneic cartilage fragments are commercially available and have been widelytransplanted in the United States [10]. It is therefore considered that the use of allogeneic chondrocyte
sheets can be an applicable treatment. In considering the application of allogeneic chondrocyte sheets tothe treatment of joint disease, we have investigated the application of chondrocytes obtained from thesurgical remains of juvenile polydactyly patients. To date, we have reported that polydactyly-derived
chondrocytes have excellent proliferative capacities that allow them to establish a layered structure andto form sheets that have similar characteristics to autologous chondrocyte sheets, such as the expressionof mesenchymal cell surface markers and the production of various cartilage anabolic factors [11].
Because most polydactyly surgery is performed in infancy, highly proliferative chondrocytes canbe obtained. If the number of chondrocytes obtained from a single donor is insufficient to fabricate
many sheets, the chondrocytes are culture expanded and cryopreserved as a material for future
polydactyly-derived chondrocyte sheet (PD sheet) fabrication.
However, the cartilaginous tissues obtained from polydactyly surgery include the parts that may
differentiate into secondary ossification center, growth plate, articular cartilage, or bone. It is also
well known that chondrocytes are easily dedifferentiated on expansion culture on dishes, and we
can assume that the PD sheet characteristics will be affected by variations in the culture period andpassage number. For these reasons, it is necessary to clarify the effects of the diversity associated withallogeneic cell sheets on the effectiveness of cell sheet transplantation.
We have previously reported that the efficacy of human adult knee chondrocyte sheets on
osteochondral defects can be directly evaluated via a xenogeneic transplantation model involving
immunosuppressant-treated rabbits [12]. In this study, we evaluated the efficacy of PD sheets forosteochondral defects in terms of this xenogeneic model, with gene expression and the secreted proteinsof transplanted PD sheets also being evaluated. Transcriptomic and proteomic analyses revealed thegene expression profiles and production factors that are characteristic of effective or less effective PD
sheets. In addition, the biological functions of the molecules that contributed to PD sheet effectivenesswere estimated.
2. Results
2.1. Effect of PD Sheets on Hyaline Cartilage Regeneration
Our overall experimental design is shown in Figure 1a. To assess the impact of diversity of PD
sheets on the effectiveness and transcriptomic and proteomic profiles, PD sheets were created for 12
lots of PD chondrocytes obtained from 10 donors. These lots involved different passage numbers,
expansion under different culture conditions, and different cryopreservation media (see Supplementary
Table S1). Figure 1b,c shows the results of the PD sheet evaluation for osteochondral defects in the rabbit
orthotopic xenograft model. Column A in Figure 1b shows representative results of the no-treatment
control group. Osteochondral defects were mainly filled by bone-like and fibroblastic cartilage-like

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tissue that stained for type I collagen (COL1) and absent for type II collagen (COL2). Columns B and
C are representative results of PD sheet transplantation groups of different lots that had the lowest
and highest International Cartilage Repair Society (ICRS) scores, indicating poor cartilage repair andexcellent cartilage repair, respectively. Column B showed incomplete filling with fibrous cartilage.
Column C exhibited hyaline cartilage regeneration in which the tissue clearly stained for safranin-O,
indicating proteoglycan deposition, and for COL2, indicating hyaline cartilage regeneration at 4 weeks
after transplantation. These results indicated the replacement of the original osteochondral defectarea by hyaline cartilage at this time point (4 weeks). We expect the eventual replacement of thehyaline cartilage by subchondral bone in the areas which are typically filled with bone in healthycartilage at longer periods (12 weeks) as we have observed in a previous study [6]. The ICRS score atfour weeks in the untreated group, indicated as D, was 22.13 ± 3.09 (n = 5), and ICRS scores for thetransplantation groups indicated as #1 to #12 varied between 18.1 and 35.2 (see Figure 1c). Among
PD sheet transplantation groups, four lots demonstrated statistically significant efficacy in repairing
osteochondral defects and several lots clearly failed in doing so.
In both column B and C, the presence of human vimentin positive cells in regenerated tissue wasobserved. These results suggest that the regenerated tissue was derived from the transplanted PD
sheet in both cases.
Figure 1d shows the property distributions for the PD sheets with respect to thickness, cell number,and viability of the cells contained in the PD sheets. The PD sheets formed piled-cell structures,and their thicknesses were 7.7–18.3 µm (average 11.52 ± 3.23 µm). Cell numbers also varied acrossthe range 1.7–4.2 × 106 cells per PD sheet (average 2.65 ± 0.67 × 106 ). The PD sheets were digested
enzymatically, and their surface marker expressions were analyzed. Flow cytometric analysis of thedispersed cells showed that the markers that are generally expressed by blood lineage cells, CD31
(platelet endothelial cell adhesion molecule-1), and CD45 (leukocyte common antigen) were almost
always negative. For CD81 (cluster of differentiation 81) and Mesenchymal stem cell (MSC) markers,
CD90 (also known as Thy-1) and CD44 (a receptor for hyaluronic acid) were more than 95% positive
for all lots. We have reported that these markers are expressed in both adult chondrocyte sheets, which
exhibited effectiveness in a previous clinical study and PD sheets [10]. There was almost no significant
difference in the expression of these cell surface markers between the lots (see Figure 1e).

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Figure1. Comparisonof polydactyly-derived
12 polydactyly-derived
chondrocyte
sheetsrespect
to efficacy
their
Figure1. Comparisonof 12(PD)(PD)
chondrocyte
sheetswith withrespect
to their
efficacyonhyalinecartilage
regenerationinarabbitxenogeneictransplantation
model:(a)
Overview
on hyaline cartilage regeneration in a rabbit xenogeneic transplantation model: (a) Overview of theof thestudy. (b) Representative
images, using
histologicalstainingand
withsafranin-O andstudy.
(b) Representative
images, using histologicalstainingwith safranin-O
immunohistochemical
immunohistochemical staining of COL2, COL1, and vimentin for rabbit knee cartilage defects withstaining of COL2, COL1, and vimentin for rabbit knee cartilage defects with and without treatment
and without treatment with cell sheet transplantation. A: no sheet transplantation, B: poor cartilage
with cell sheet transplantation. A: no sheet transplantation, B: poor cartilage repair (#1 in (c)), C:
repair (#1 in (c)), C: excellent cartilage repair (#12 in (c)). Scale bar = 1 mm. (c) ICRS scores for the PD
excellent cartilage repair (#12 in (c)). Scale bar = 1 mm. (c) ICRS scores for the PD sheets: Each
sheets: Each circle indicates the average ICRS score for rabbits in a group. D represents a groupcircle indicates the average ICRS score for rabbits in a group. D represents a group without PD sheet
without PD sheet transplantation. The number indicates each lot of PD sheets in Supplementarytransplantation. The number indicates each lot of PD sheets in Supplementary Table S1. * p < 0.05.
Table S1. * p < 0.05. (d) Distribution of PD sheet characteristics: Cell sheets were dispersed by(d) Distribution of PD sheet characteristics: Cell sheets were dispersed by enzymatic digestion, withenzymatic digestion, with cell numbers and the viability of cell suspension being determined. (e) PD
cell numbers and the viability of cell suspension being determined. (e) PD sheets showing similar
sheets showing similar surface marker expressions.
surface marker expressions.

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5 of 202.2. Gene Expression Profiles Associated with the Effectiveness of PD Sheets
2.2. Gene Expression Profiles Associated with the Effectiveness of PD Sheets
To identify transcriptomic characteristics related to PD sheet effectiveness, the gene expression of
To identify
transcriptomic
characteristics
relatedwith
to PD
sheet effectiveness,the via
gene
expression
the 12 lotsof PD sheets,
which hadbeen examined
respect
to their efficacy
theorthotopic
of
the
12
lots
of
PD
sheets,
which
had
been
examinedwithrespect
totheirefficacyvia
theorthotopic
xenograft model, was analyzed using microarrays. We extracted genes based on the correlation
xenograftmodel,
was analyzed
usingscores,
microarrays.
We205
extracted
genes basedon thecorrelation
betweenthe arraysignals
and the ICRS
obtaining
genes showing
a positive
correlation
withbetweenthearraysignals
and
the
ICRS
scores,obtaining205
genes
showingapositivecorrelation
the ICRS score and 238 genes showing a negative correlation. The genes for the 20 highest
positivewith the ICRS score and 238 genes showing a negative correlation. The genes for the 20 highest
and negative correlations are listed in Supplementary Tables S2 and S3, respectively. Figure 2a shows
positive and negative correlations are listed in Supplementary Tables S2 and S3, respectively. Figure
the results of a cluster analysis of 443 extracted genes via a heat map of gene expression. We can
2a shows the results of a cluster analysis of 443 extracted genes via a heat map of gene expression.
consider these as candidate marker genes for efficacy-related characteristics. For the five lots of PD
We can consider these as candidate marker genes for efficacy-related characteristics. For the five lots
sheets with the lowest ICRS scores, four lots are classified into the same cluster, suggesting that geneof PD sheets with the lowest ICRS scores, four lots are classified into the same cluster, suggestinggroupsthat contribute
this clustering
be an index
to anexcludethat arelessthateffective.
that genegroups thattocontributeto thismay
clustering
may beindex those
to exclude
those
are lessWe
further
identified
171 genes
with171changesin expression
greater than
2-fold thanbetweentheeffective
effective.
We further
identified
genes withchanges inofexpressionof greater
2-foldbetween4 lotsthat
exhibited
efficacyin
Figure1candlesseffective
lots
that
fall
into
the
cluster.
Inthe less
the effective 4 lots that exhibited efficacy in Figure 1c and less effective lots that fall into the cluster.
effective
lots,
149
genes
were
upregulated
and
22
genes
were
downregulated(absolutefoldchangeIn the less effective lots, 149 genes were upregulated and 22 genes were downregulated (absolute> 2;
adjusted
p-value
Figure2b shows
clear
separationof PDclear
lot groups,
suggesting
thatgroups,
the genefold change><
2; 0.05).
adjusted
p-value< 0.05).
Figure2b shows
separationof PD lotsetsuggesting
could be candidate
markers
thatbeidentify
lessmarkers
effectivethat
PDidentify
sheets. less effective PD sheets.
that the geneset could
candidate

FigureHeatmapmapandandhierarchicalhierarchicalclustering
clustering ofof efficacy-correlated
efficacy-correlated gene
sheetsFigure2. 2.Heatgeneexpressions:expressions:PDPD
sheetsexhibitedadistinctgene
expression
profileaccording
to
their
in
vivo
efficacy.
Red
indicates
highexhibited a distinct gene expression profile according to their in vivo efficacy. Red indicates highexpression
levels,andgreengreenrepresents
representslow
lowexpression
expression levels.
correlated
expression
levels,andlevels. (a)(a)Analyzed
Analyzedusing
usingefficacy
efficacy
correlated
443
gene
set.
(b)
Differentiallyexpressed
171
genes
in
effectiveandlesseffective
lots
of
PD
443 gene set. (b) Differentially expressed 171 genes in effective and less effective lots of PDsheets.
sheets.

2.3.2.3.
Function
Prediction
Function
PredictionbybyEfficacy-Related
Efficacy-RelatedGenes
Genes
selectmarker
genes
that
have
reasonablebiologicalrelevance
relevancefor
forefficacy,
efficacy,weweclarified
clarifiedthe
ToToselectthethemarker
genes
that
have
reasonablebiologicalthe biological
functionsof efficacy-related
by performing
an enrichment
analysisof 443genes
biological
functionsof efficacy-related
gene geneby performing
an enrichment
analysisof 443
genes
using
using
Metascape[13].
The
resultssuggestthat
thepositively
correlated
genes
were
involvedinMetascape [13]. The results suggest that the positively correlated genes were involved in cartilage

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6 of 20development and mesenchymal stem cell differentiation (Figure 3). In addition, some of the genes
cartilage development and mesenchymal stem cell differentiation (Figure 3). In addition, some of thewereinvolvedin angiogenesis
and extracellularmatrix matrixorganization.
genes
were involvedin angiogenesis
and extracellularorganization.

Figure3. Meta-enrichment
analysisof thegenes:(a) Heatmap ofthe oftopthe20 enriched
Figure3. Meta-enrichment
analysisof efficacy-associatedthe efficacy-associatedgenes:(a) Heatmap
top 20clusters
basedon positive
negativeandcorrelation
lists. Colors
representenrichedclusters
based andon positive
negative genecorrelation
gene lists.
Colorsstatisticalrepresentsignificance,
statisticalwithgray indicatinga lackof significance.
extracellular
IGF: insulin-likefactor,significance,with grayindicatinga lack ofECM:
significance.
ECM:matrix,
extracellularmatrix, IGF:growth
insulin-like
IGFBPs:growthinsulin-likefactor binding
proteins.
Enrichment
networkvisualization:
Clustergrowthinsulin-likefactor, IGFBPs:
growthfactor(b)binding
proteins.
(b) Enrichment
network
annotations
are shownsize ofcolor-coded.
the node indicatesthegenes includedin thevisualization:
Clustercolor-coded.
annotations The
are shown
The size
ofnumberthe nodeofindicatesthe number
ontology
terms
or pathways.
(c) Theterms
colororscale
for statisticalwasappliedsignificanceto the sameof genes
includedin the ontology
pathways.
(c) The significancecolor scale forstatistical
was network.
applied to the same cluster network.
cluster

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Table
Table11shows
showsthetheefficacy-related
efficacy-relatedgenes
genesdescribed
described andand annotatedannotated inin terms
terms ofof enrichedenriched gene
gene
ontology
(GO).
Some
genes
associatedwith
GO:0030198
extracellularmatrixorganization
ontology (GO). Some genes associated with GO:0030198 extracellular matrix organizationwere
werealso
also
associatedwith
GO:0061035
regulationofcartilage
development.
We
selected
five
GO-annotatedgenes
associated with GO:0061035 regulation of cartilage development. We selected five GO-annotatedin
Figurefor reverse
polymerasepolymerase
chain reaction(RT-PCR)
The expressions
genes
in 4a
Figure4a fortranscription
reverse transcription
chain
reactionassay.
(RT-PCR)
assay. Theof
aggrecan(ACAN),
integrin
alpha
10(ITGA10),
growthdifferentiation
factor
5(GDF5),
expressions of aggrecan (ACAN), integrin alpha 10 (ITGA10), growth differentiation SRY-box
factor 5transcription
factortranscription
9 (SOX9), andfactor
SRY-boxtranscription
factor 5 (SOX5)
were validated
via RT-PCR,(GDF5), SRY-box
9 (SOX9),and SRY-box
transcription
factor 5 (SOX5)
were
andthe correlation
withandthe theICRS
score waswith
analyzed.
Thescore
results
showthat therea correlation
validated
via RT-PCR,
correlation
the ICRS
wasanalyzed.
The wasresults
show thatbetweenthea ∆Ct
with respect
to actin
and thefor ACAN,
there wascorrelation
betweenthe beta
ΔCt (ACTB)
with respect
to ICRS
actin scoresbeta (ACTB)
and ITGA10,
the ICRSGDF5,scoresand
for
SOX9
but
not
SOX5
(Figure4b).
ACAN,
ITGA10,
GDF5,
and
SOX9
were
selectedastheefficacy-related
ACAN, ITGA10, GDF5, and SOX9 but not SOX5 (Figure 4b). ACAN, ITGA10, GDF5, and SOX9 weremarkerAfter Z-score marker
normalization,
the gene
expression(GES) was calculated
selectedcomponents.
as the efficacy-related
components.
After
Z-scorescorenormalization,
the geneby
each
predictive
marker
gene
based
on
weighted
linearfunctions
(GES
=
(∆Ct
(ACAN)
×3 +linear∆Ct
expression score (GES) was calculated by each predictive marker gene based on weighted(ITGA10)
×2+∆Ct(GDF5)×0.4+∆Ct(SOX9)
×1).
The
correlation
coefficient
of
these
data
sets
was
functions (GES = (ΔCt (ACAN) × 3 + ΔCt (ITGA10) × 2 + ΔCt (GDF5) × 0.4 + ΔCt (SOX9) × 1). The
0.44
for ICRScoefficientscores (Figure4c).data sets was 0.44 for ICRS scores (Figure 4c).
correlationof these

Figureofof
selected
genes
show
a
Figure4.4.Potential
Potentialefficacy-related
efficacy-relatedmarkers
markersfor
forPD
PDsheets:
sheets:(a)The(a)Thescatterplots
scatterplots
selected
genes
show
correlation
betweennormalized
microarraysignaland
ICRS
scoresofPDsheets
(n
=
12).
(b)
A
weak
a correlation between normalized microarray signal and ICRS scores of PD sheets (n = 12). (b) A weakcorrelation
in in
PDPD
sheetsthatthatis consistent
withwith(a) andwere
correlationbetween
betweengene
geneexpression
expression
sheetsis consistent(a) their
and ICRS
their scores
ICRS scoresvalidatedby RT-PCR.
The horizontal
axis showstheshows
gene expression
level in terms
of in
∆Ct
valueswere validatedby RT-PCR.
The horizontal
axis
the gene expression
level
terms
of thatΔCt
were
calculated
bycalculated
subtracting
Ct value ofgenes
Ct valuebeta (ACTB)
avaluesthat were
bythe
subtracting
thetarget
Ct valueof fromtargetthe
genesfrom oftheactinCt valueof actin as
beta
referencegene.
(c)Scatterplot
ofgene
expression
score
(GES)
calculated
from
gene
expression
levelsof(ACTB) as a reference gene. (c) Scatterplot of gene expression score (GES) calculated from genemarkergenes.
expression
levels of marker genes.

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Table 1. Enriched gene ontology (GO) terms and genes fall into clusters.
Term

GO:0030198

Description

extracellular matrixorganization

LogP−14.18Log (q-Value)

Symbols−9.87

ACAN, COL4A2, COL9A3, HAPLN1, DAG1, FOXC1,
HSPG2, CCN1, ITGA6, ITGA3, LAMB3, MELTF, PDGFA,
SOX9, TNXB, ITGA10, ADAMTSL2, SPINT2, ABI3BP,
GREM1, NTN4, THSD4, SCUBE1, OLFML2A, DMD,
GDF5, ADAMTS1, P3H2, COL10A1, MMP17, CAPNS2,
CTRB2

GO:0001525

angiogenesis

−9.15

−5.53

COL4A2, CSPG4, DAG1, FGF1, FOXC1, HGF, HOXA3,
HSPG2, CCN1, LEPR, MFGE8, NOTCH3, PDGFA,PTGIS, FZD8, APLN, ADGRG1, RAMP1, ESM1, GREM1,
HIPK2, RASIP1, ISM1, WNT11, ERRFI1, ADRB2, DOK1,
EFNB1, GALNT3, IGFBP2, PTGIR, SOX9, MTSS1,
PLCB1, NGEF, RHBDF2, SPRY4

GO:0070848

response to growth
factor

−7.29

−4.01

RUNX3, COL4A2, FGF1, FOXC1, GALNT3, GDF10,
HGF, CCN1, ITGA3, PENK, SOX5, SOX9, GDF5, APLN,ADAMTSL2, SPINT2, DKK1, BAMBI, GREM1, DKK3,
HIPK2, ERRFI1, SPRY4, INHBB

GO:0001503

ossification

−6.64

−3.50

ACP5, ADRB2, FOXC1, GDF10, HGF, CCN1, P2RX7,
PENK, SRGN, SOX9, WNT3, WNT11, GDF5, DKK1,
GREM1, SMOC1, ACAN, RUNX3, HAPLN1, HOXA3,
HOXD10, SOX5, SPNS2, CSPG4, LEPR

GO:0003002

regionalization

−6.33

−2.93

EMX1, EVX1, FGF1, FOXC1, HOXA3, HOXA10,
HOXD10, LFNG, WNT3, LHX3, ADGRG1, LHX2, ZEB2,SPRY1, DKK1, GREM1, FOXB1, HIPK2, HES2, GSC, SP8,
EFNB1, SHROOM3

GO:0048598

embryonic
morphogenesis

−5.72

−2.71

COL4A2, DAG1, FOXC1, HOXA3, HOXD10, CCN1,
ITGA3, LAMB3, SOX9, WNT3, WNT11, GDF5, APLN,
LHX2, SPINT2, DKK1, HIPK2, SHROOM3, ACAN,ADAMTS1, GREM1, HSPB7, ALPK3, SCUBE1, PDGFA

GO:0035272

exocrine systemdevelopment

−5.57

−2.58

DAG1, FOXC1, HGF, PDGFA, SOX9, NTN4, FGF1,
SPINT2, RASIP1, WNT11, DKK1, GREM1, CYP19A1,
HOXA3, WNT3, APLN, DKK3, PLCB1, PRR5L, PEAR1,
LHX2

GO:0072006

nephrondevelopment

−5.52

−2.55

FGF1, FOXC1, ITGA3, NOTCH3, PDGFA, PODXL,SOX9, WNT11, MTSS1, DAG1, HGF, CCN1, WNT3,
LHX2, SPINT2, DKK1, RIPK4, RASIP1, SHROOM3,
NTN4, CYP19A1, ITGA6, CRLF1, ADAMTS1,
ADAMTSL2, ERRFI1, PGF, SPRY1

GO:2000272

negative regulationof signaling
receptor activity

−5.38

−2.19

DKK1, DKK3, ERRFI1, LYNX1, MTRNR2L7,
MTRNR2L3, MTRNR2L8, MTRNR2L9, ADRB2, MUC4,SHANK2, GREM1

GO:0043408

regulation of
mitogen-activatedprotein kinasecascade

−5.17

−2.35

ADRB2, CSPG4, DAG1, FGF1, GDF10, HGF, CCN1,
INHBB, IRAK2, P2RX7, PDGFA, GDF5, FZD8, DKK1,
PLCB1, GREM1, HIPK2, ERRFI1, NDRG2, SPRY4, DMD,RASIP1, PRR5L, CAMK2N2, WNT11

GO:0031128

developmental
induction

−5.08

−2.30

FGF1, SOX9, WNT3, DKK1, HIPK2, COL4A2, DAG1,
FOXC1, ITGA3, LAMB3, WNT11, APLN, ADRB2,
IGFBP2, P2RX7, FZD8, SHANK2, PLCB1, BAMBI,
GREM1, DKK3, NDRG2, LHX2, ANK1, HGF, HOXD10,
HSPG2, NOTCH3, SOX5, RAMP1, GDF5

GO:0061035

regulation of
cartilage
development

−4.54

−1.84

CCN1, SOX5, SOX9, WNT11, GDF5, GREM1, ACAN,RUNX3, HOXA3, PDGFA, ANXA2, GDF10, SRGN,
DKK1

GO:0072497

mesenchymal stem
cell differentiation

−4.52

−1.83

SOX5, SOX9, WNT3, FOXC1, TNXB, THSD4,ADAMTSL2, ERRFI1, DKK1

2.4. Analysis of Secretory Factors Related to PD Sheet EffectivenessIt might be expected that the humoral factors continuously supplied from a PD sheet contribute to
the promotion of hyaline cartilage regeneration. Therefore, we analyzed the relative amounts of secreted
proteins from PD sheets using the slow off-rate modified aptamers (SOMA) scan platform [14,15] andtheir correlation with ICRS scores. Among the protein factors that can be analyzed with SOMAscan,
49 proteins having a positive correlation with efficacy score and 63 proteins having a negative
correlation were extracted. The factors for the 20 highest positive and negative correlations are listed.
(See Supplementary Tables S4 and S5.) Figure 5a shows the results of the enrichment analysis of

Int. J. Mol. Sci. 2020, 21, 149
Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW

9 of 20
9 of 20

the efficacy-correlated
factors. Negatively
factors5awere
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eight proteins were identified in the supernatant that correlated with effectiveness: ESM1, GREM1,
expressionof genes,eight proteins
werePDGFA
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the supernatantthat showedcorrelated
with
SERPINA3,
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effectiveness: ESM1, GREM1, SERPINA3, DKK1, MIA, NTN4, FABP3, and PDGFA (Figure 5b–d).
correlation: retinoic acid receptor responder 2 (RARRES2), apolipoprotein E (APOE), and placental
Three proteins showed a negative correlation: retinoic acid receptor responder 2 (RARRES2),
growth factor (PGF).
apolipoprotein E (APOE), and placental growth factor (PGF).

FigureAnalysisofofproteinsproteinssecreted
secreted byby PD
PD sheets:
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Figure5. 5.
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sheetsexaminedby
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logRFU).
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ICRS
examined by SOMAscan (log10 relative fluorescence unit: logRFU). (e) Scatterplots between ICRS scoreand protein concentration as determined by ELISA.
andscore
proteinconcentration as determined by ELISA.

Int. J. Mol. Sci. 2020, 21, 149

10 of 20Of these, we focused on DKK1, which is expected to demonstrate OA-preventing effects [16];GREM1, known to be involved in chondrocyte differentiation [17]; MIA, which is a cartilage
differentiation marker and anabolic factor [18–20]; and NTN4 and ESM1, which are both angiogenesis
related factors. The amounts of these factors were quantified via enzyme-linked immunosorbent assay(ELISA), and the results showed that the amount of protein varied between PD sheets, with the ranges
being 2.6 to 26.6 ng/mL for DKK1, 58.2 to 138 ng/mL for GREM1, 0 to 38.4 ng/mL for MIA, 153 to
714 pg/mL for NTN4, and 128 to 849 pg/mL for ESM1. Among them, NTN4 failed to exhibit correlation
with ICRS scores in ELISA. For the concentration of MIA and DKK1, a weak correlation with ICRS
scores was observed. For GREM1 and ESM1, a moderate correlation was demonstrated (Figure 5e).
3. Discussion
In this study, we have confirmed that using PD sheets is effective in the orthotopic xenograftmodel, similar to using adult knee chondrocyte sheets. In the fabrication of human adult chondrocyte
sheets, chondrocytes proliferate in the 2D-culture environment, causing the expression of Col2A1 to be
reduced and the chondrocyte sheets to be considered dedifferentiated. However, it has been foundthat they redifferentiate into hyaline cartilage in the transplanted joint for both animal model [12] andhuman clinical research [9]. Using PD sheets, we have found hyaline cartilage-like tissue in defect areas
derived from human cells, indicating that PD sheets also redifferentiate to hyaline cartilage in vivo.
The results of using an in vivo imaging system to track chondrocyte-sheet orthotopic transplants inrats indicate that most of the luciferase signal of the allogeneic chondrocytes in the joint will disappear
within a few months [21], suggesting that allograft-derived tissue is replaced by recipient-derived
tissue in the long term. Our results suggest that, at least in the short term, PD sheets may act as
cartilage progenitor cells.
It has been suggested that the chondrocyte sheets that adhere to the cartilage lesion act as a barrier
against catalytic factors while prevent the outflow of cartilage-tissue matrices [4,22]. Moreover, there
is an implication that anabolic factors produced from chondrocyte sheets act as paracrine factors forpromoting cartilage regeneration [11,23,24]. Our study shows that the expression of genes and thesecretion of protein factors associated with the extracellular matrix, cartilage development, ossification,
and angiogenesis are both involved in the efficacy of PD sheets. We previously reported that PD sheets
secrete MIA as an anabolic factor [11], and it is confirmed again in this study. In addition, we haveidentified other candidates for anabolic factors (GREM1, DKK1, and PDGF) that contribute to theefficacy of PD sheets.
GREM1 and DKK1, of which secretion was confirmed by PD sheets and exhibited a correlation toefficacy, are known to be involved in Wnt signal regulation, and Wnt signal disruption in articularcartilage is thought to cause cartilage homeostasis failure [25–28]. They are reported to suppress
hypertrophic chondrocyte differentiation and to increase cartilage surface and quiescent chondrocyte
layers [17]. Suppression of the onset of OA in DKK1-transgenic mice has also been reported [16].
PDGF-AA is indicated as a cartilage trophic factor that promotes chondrocyte proteoglycan production
and chondrocyte proliferation [29,30] and a decrease in PDGF-AA promotes cartilage degeneration [31].
Our results suggest that PD sheets promote the regeneration of hyaline cartilage by producing
these factors. Because PD sheets are living tissues, they may be able to tune their factor production
during the process of redifferentiating into hyaline cartilage according to the microenvironment: thecartilage surface layer, the deeper layers, and the subchondral bone. Production of these factors may
be a useful marker for predicting the effectiveness of the PD sheets when selecting donors for cell sheet
production or improving culture methods.
In addition, our study shows that the production of NTN4 and ESM1, which have not beenreported as having any specific role in cartilage tissue, and ESM1 was correlated with the efficacy of PD
sheets. NTN4 is a netrin family factor that has been reported to promote osteoblast differentiation [32]and to modify SMAD signaling in chondrocytes [33], suggesting that NTN4 may have some effect on
the chondrocytes themselves and the subchondral bone. NTN4 has also been reported to suppress

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angiogenesis [34,35]. ESM1 is a dermatan sulfate proteoglycan of which the expression is enhanced by
vascular endothelial growth factor (VEGF) [36,37]. It has been reported that ESM1 expresses higher inthe deep cartilage layer than in other parts of the cartilage [38], implying a biological role related tochondrocyte differentiation. Rocha et al. report that ESM1 increases the bioavailability of VEGF-165
by competitively binding to fibronectin [39]. Because PD sheets express abundant fibronectin, it isassumed that the ESM1 secreted from the PD sheets binds and saturates the fibronectin in the PD
sheets themselves or in the recipient tissue. ESM1 may prevent binding of VEGF and invagination of
new blood vessels.
Angiogenesis in articular cartilage is thought to cause cartilage degeneration and osteophyte
formation. We previously reported that osteophyte formation is suppressed by anti-VEGF antibody
administration in an anterior-cruciate-ligament transection rabbit model [40,41]. These results suggestthat the suppression of angiogenesis may be a promising approach to OA prevention. The NTN4 andESM1 produced by PD sheets may contribute to the regeneration of hyaline cartilage by suppressing
angiogenesis and subsequent calcification during the repair of a cartilage defect.
SERPINA3 is a member of the serine protease family and has been reported as a gene of which theexpression level changes when MSC differentiate into chondrocytes [42]. FABP3 is a fatty acid binding
protein which is required for the accumulation of intracellular lipid droplets and has been reported toincrease MSC survival [43,44]. However, because FABP3 is a protein that is normally localized in the
Golgi body, the increased amount in the supernatant may be related to the cell death of cells expressing
FABP3. The contribution of these factors to effectiveness is currently unknown, and further analysis
is required.
One limitation of our study was that the results were obtained from a xenograft model using
immunosuppressants. There could therefore be some differences in the engraftment and rejection
processes between xenogeneic and allogeneic transplantation, which should be considered in humanapplications. The engraftment of PD sheets is considered to be very important for effectiveness,and some factors could promote engraftment in xenogeneic transplantation rather than promote theregeneration of cartilage.
In regenerative medicine using allogeneic cells, which may well vary in their properties becauseof donor differences, stabilizing the properties of the tissue engineering products is an inevitable issue
for clinical use. To address this issue, it is essential to identify biological characteristics that contributeto in vivo efficacy. This would then enable validation of donor cells and culture conditions based on
identified biological characteristics.
PD sheets are transplanted to histoincompatibile patients in parallel with microfracture treatment
in our ongoing clinical study, so the contact between bone marrow and PD sheets should be considered.
The potential immune modulatory function of PD sheets may affect their efficacy. We have demonstrated
the efficacy of chondrocyte sheet transplantation in allogeneic settings in rats, rabbits, and minipigs
without immunosuppression [5–8]. Additionally, many allogeneic osteochondral transplantations
in histoincompatibile patients have been conducted safely [10,45,46]. However, the influence ofhistoincompatiblility on efficacy of PD sheet transplantation is still unknown, as we are currently
evaluating the immune response in the PD sheet transplant recipients. To reduce the immune response
through the contact between blood and PD sheets, PD sheet transplantation without microfracture or
its application in early OA patients could be considered, as chondrocyte sheets have demonstrated
their effectiveness on partial thickness defects of articular cartilage [4,8].
In this study, we have identified biological pathways and associated genes that may contributeto hyaline cartilage regeneration by PD sheet transplantation. Although further improvements areneeded, we have proposed gene expression scores and relevant secreted factors that could be used as
markers to predict the in vivo efficacy of PD sheets. To use these markers for efficacy prediction, thecorrelation with efficacy needs to be further analyzed via independent data and could be replaced bybetter genes and the thresholds for the markers need to be determined. Eventually, the contribution of

Int. J. Mol. Sci. 2020, 21, 149

12 of 20these factors to the efficacy of PD sheets will need to be confirmed in human clinical studies before
they can be used as quality markers.
4. Materials and Methods
4.1. Ethics Statement
The experiments were performed under the approval and guidance of the Clinical Research
Review Committee of Tokai University School of Medicine. Informed consent was obtained from theparents 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 Regulations
for Animal Experiments and the Fundamental Guidelines for Proper Conduct of Animal Experiments
and Related Activities in Academic Research Institutions under the jurisdiction of the Japanese Ministryof Education, Culture, Sports, Science, and Technology for animal handling and care.
4.2. Fabrication of PD Sheets
4.2.1. Preparation of Chondrocytes
Chondrocytes were isolated according to the preparation procedure described previously [11].
Briefly, cartilaginous tissue obtained from juvenile polydactyly patients (11 patients, aged 8–23 months,
five girls, four boys, and two de-identified donors) was digested with 5 mg/mL CLS1 (Worthington
Biochemical Corp., Lakewood, NJ, USA) in a preparation medium (DMEM/F12, Gibco, Waltham, MA,
USA) supplemented with 20% fetal bovine serum and 1% antibiotic–antimycotic solution (Gibco)for 2–3 h at 37 ◦ C in a humidified atmosphere of 5% CO2 and 95% air. The isolated chondrocytes
were washed with the preparation medium and seeded on culture dishes. After the cells adheredand started to proliferate, the medium was replaced with a culture medium (the preparation medium
with 100 µg/mL ascorbic acid (Nissin Pharmaceutical, Yamagata, Japan)). In two cases, tissues wereminced into pieces of 1 mm3 or less, placed on culture dishes in the preparation medium, and cultured
until the cells became subconfluent. Chondrocytes were harvested using TrypLE Express (Thermo
Fisher Scientific, Tokyo, Japan) and cryopreserved either with STEM-CELLBANKER™ (ZENOAQ,
Fukushima, Japan) or TC-Protector cell freezing media (KAC Co., Ltd., Kyoto, Japan). Some donors
were passaged up to five times before being cryopreserved.
4.2.2. Fabrication of PD Sheets
Twelve lots of cryopreserved chondrocytes were thawed, passaged once in the culture medium,
seeded on temperature-responsive culture inserts (CellSeed Inc., Tokyo, Japan) at 1 × 104 cells/cm2 , andcultured for two weeks. The PD sheets were divided into four groups for use in efficacy evaluation,
evaluation of characteristics, analysis of gene expression, and analysis of protein secretion.
4.2.3. Evaluation of Characteristics of PD Sheets
To determine the cell number, PD sheets were digested with TrypLE Express (Thermo Fisher
Scientific) for 30 min at 37 ◦ C followed by incubation with 0.25 mg/mL Collagenase-P (Roche, Basel,
Switzerland) for 30 min at 37 ◦ C. The dispersed cells were washed with culture medium, and cellnumber and viability were determined by trypan blue exclusion assay. To determine the thicknessof the PD sheets, each PD sheet was embedded in Tissue-Tek O.C.T. Compound (Sakura Finetech,
Tokyo, Japan) and frozen at −80 ◦ C. Cross sections of the PD sheet at 10 µm thicknesses were cut andmounted onto glass slides, air-dried, and fixed with 4% paraformaldehyde in 0.01 M phosphate buffer
for 30 min at RT. The sections were stained with H&E according to standard protocols. Microscopic

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images were captured, and the distances between pairs of points were measured using a BZ-8000
microscope (Keyence, Osaka, Japan). For the flow cytometry, dispersed cells were suspended in
Ca/Mg-free phosphate buffer saline (PBS) containing 0.2% human serum albumin (Sigma-Aldrich,Tokyo, Japan) and 1 mM ethylenediaminetetraacetic acid (EDTA: Wako Chemical Co. Ltd., Tokyo,
Japan). They were then incubated with five antibodies: CD31/FITC (clone: 5.6E) and CD45/FITC (clone:J.33) were supplied by Beckman Coulter; CD81/APC (clone: JS-81), CD90/APC (clone: 5E10), andCD44/FITC (clone: G44-26) were supplied by BD Biosciences (San Diego, CA, USA). The stained cells
were washed once and analyzed using a FACSVerse flow cytometer (BD Biosciences).
4.3. Evaluation of the Efficacy of PD Sheets for Osteochondral Defects
4.3.1. Immunosuppressed Rabbit Osteochondral Defect Model
Efficacy evaluation of PD sheets in rabbits was performed as described previously [12]. A total of 69
female Japanese white rabbits (average weight = 3.0 kg: Tokyo Laboratory Animals Science Co., Tokyo,
Japan) was used. Before surgery, the rabbits were randomly assigned by weight to either the defect-onlygroup or to PD sheet transplantation groups. For immunosuppression, tacrolimus (1.6 mg/kg/day)
was administered intramuscularly daily for 10 days, starting two days before transplantation andthen every other day until four weeks after surgery. The rabbits were anesthetized with 2 L/min
nitrous oxide, 1 L/min oxygen, and 2.5–3.0% isoflurane (Pfizer, New York, NY, USA) before surgery
and transplantation. An osteochondral defect (diameter = 5 mm; depth = 3 mm) was created in thepatellar groove of the femur using a 5-mm biopsy punch (Kai Industries, Gifu, Japan) as a marking
guide and a 5-mm drill. Slight bleeding from the subchondral bone was confirmed, and physiologicalsaline (Nipro, Osaka, Japan) was used to clean the defect and to prevent thermal damage.
4.3.2. PD Sheet Transplantation
For PD sheet transplantation, the culture plates containing the PD sheets were kept at 25 ◦ C for
30 min to allow their detachment. One PD sheet per knee was transplanted on the osteochondral defectusing a polyvinylidene difluoride membrane. After restoration of the patella, the quadriceps femorismuscle and tendon were sutured to prevent dislocation.
4.3.3. Histological Scoring for Regenerated Cartilage
Four weeks after PD sheet transplantation, the rabbits were euthanized by an intravenous
administration of 50 mg/mL pentobarbital (Tokyo Chemical Industry, Tokyo, Japan). The operated-on
femur was collected and fixed in 20% formalin (Wako Pure Chemical Industries) for 3–5 days.
The specimen was decalcified in 10% EDTA (Wako Pure Chemical Industries) for 3–4 weeks andparaffin embedded. The serial sections (3 µm) were cut near the center of the defect area, parallel tothe long axis of the femur. For histological examination, sections were stained with hematoxylin andeosin (H&E), safranin-O, and fast green according to standard protocols. The stained sections wererandomized and scored separately by two trained orthopedic surgeons, using a modified version of
the O’Driscoll score and the International cartilage repair society (ICRS) score [47,48] (Details of thescoring method are shown in Supplementary Table S6).
4.3.4. Immunostaining
For type I collagen (COL1) and type II collagen (COL2) immunostaining, deparaffinized 3-µm
sections were treated with 0.4% pepsin (Agilent Technologies, Santa Clara, CA, USA) for 30 min at
37 ◦ C for antigen retrieval. The sections were then incubated with 0.4% pepsin (DAKO, Glostrup,
Denmark) at 37 ◦ C for 30 min and washed in distilled water, followed by incubation in 0.3% hydrogen
peroxide/methanol solution for 30 min at room temperature (RT) to block endogenous peroxidase
activity. Next, the sections were washed in PBS, blocked with 2.5% normal goat serum (NGS) for 10 min
at RT, and incubated for 3 h at RT with mouse monoclonal antibody in either human COL1 or humanInt. J. Mol. Sci. 2020, 21, 149

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COL2 (Kyowa Pharma Chemical Co., Toyama, Japan) diluted at 1:100 with 1% bovine serum albumin(Sigma-Aldrich) in PBS. Finally, the stained sections were washed in PBS, followed by incubation with
the ImmPRESS Reagent Anti Mouse Ig (Vector Laboratories, Burlingame, CA, USA) at RT, immersed
for 2–8 min in Tris–HCl buffer (pH 7.6) containing 0.02% diaminobenzidine and 0.005% hydrogen
peroxide, and then counterstained with H&E.
For human vimentin detection, deparaffinized sections were treated with 10-mM sodium citratebuffer (pH 6.0) for 10 min at 98 ◦ C in a microwave for antigen retrieval. The sections were incubatedwith 5% NGS containing PBS for blocking, followed by incubation with Alexa Fluor 647-conjugatedrabbit monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA) diluted at 1:100 with
1% bovine serum albumin in PBS overnight at 4 ◦ C to detect human vimentin. The sections were
washed in distilled water and then mounted and cured with 40 ,6-diamidino-2-phenylindole (Vector
Laboratories) according to the manufacturer’s instructions. All microscopic images were obtainedusing a BZ-9000 Generation II fluorescence microscope (Keyence Corp., Osaka, Japan).
4.4. Gene Expression Analysis of PD Sheets
4.4.1. RNA Isolation
The PD sheets were disrupted in TRIzol Reagent (Life Technologies, Carlsbad, CA, USA) using
SHAKE Master Neo (Bio Medical Science, Japan), and the total RNA was further purified using the
Qiagen RNeasy Mini Kit (QIAGEN, Valencia, CA, USA) according to the manufacturer’s instructions.
RNA quantity and quality were determined using a Nanodrop One spectrophotometer (Thermo Fisher
Scientific Inc.) and an Agilent Bioanalyzer (Agilent Technologies), as recommended.
4.4.2. cRNA Amplification and Labeling
Total RNA was amplified and labeled with cyanine 3 (Cy3) using the Agilent Low Input Quick
Amp Labeling Kit, one-color (Agilent Technologies) following the manufacturer’s instructions. Briefly,
the total RNA was reversed transcribed to double-strand cDNA using a poly dT-T7 promoter primer.
The primer, template RNA, and quality-control transcripts of known concentration and quality werefirst denatured at 65 ◦ C for 10 min and incubated for 2 h at 40 ◦ C with 5× First-Strand Buffer, 0.1 M
dithiothreitol, 10 mM deoxyribonucleotide triphosphate mix, and AffinityScript RNase Block Mix.
The AffinityScript enzyme was inactivated at 70 ◦ C for 15 min. cDNA products were then used as
templates for in vitro transcription to generate fluorescent cRNA. The cDNA products were mixed
with a transcription master mix in the presence of T7 RNA polymerase and Cy3-labeled cytidine
triphosphate and incubated at 40 ◦ C for 2 h. Labeled cRNAs were purified using QIAGEN’s RNeasy
mini spin columns and eluted in 30 µL of nuclease-free water. After amplification and labeling, thecRNA quantity and degree of cyanine incorporation were determined using a Nanodrop ND-1000
spectrophotometer and an Agilent Bioanalyzer.
4.4.3. Sample Hybridization
For each hybridization, 0.60 µg of Cy3-labeled cRNA were fragmented and hybridized at 65 ◦ C
for 17 h using an Agilent SurePrint G3 Human GE v3 8 × 60K Microarray (Design ID: 072363). Afterwashing, the microarrays were scanned using an Agilent DNA microarray scanner.
4.4.4. Identification of Efficacy Correlation Genes
The intensities of each scanned feature were quantified using Agilent feature extraction software
version 11.5.1.1, which performs background subtraction. We used only those features that were
flagged as error-free (“Detected” flags) and excluded any feature that was flagged as not positive, notsignificant, not uniform, not above background, saturated, or a population outlier (“Not Detected” and“Compromised” flags). Quantile normalization was performed using Agilent GeneSpring software
version 14.9.1. After quantile normalization of the raw signal data, the probes were filtered based

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on preexisting annotations with a gene symbol and average signal intensity for all samples (higher
than 5 coveted log2 intensity). For extraction of the marker genes, Pearson’s correlation coefficientswere calculated between the expression level of each of those probes and the individual ICRS scores,
then probes with those having more than 0.4 or less than −0.4 were selected. Finally, 443 genes
were identified using these criteria with the large variance being more than 0.5 in log2 intensity for
all samples. Welch’s t-tests were used between the 2 groups that exhibited efficacy and inefficacy:
171 genes were selected with absolute fold change > 2 and corrected p-value < 0.05. Hierarchicalclustering analysis was performed using Agilent GeneSpring software version 14.9.1. (Similarity
Measure: Euclidian, Linkage Rule: Average).
4.4.5. Enrichment Analysis
Efficacy-correlated genes were analyzed using online tools in Metascape (http://metascape.or./) [13].
Functional enrichment was performed in a default setting. Multiple input gene lists containing positively
correlated genes and negatively correlated genes were analyzed.
4.4.6. RT-PCR
The total RNA of the PD sheets was converted to cDNA using QuantiTect Reverse Transcription
Kit (Qiagen). The TaqMan PreAmp Master Mix Kit (Applied Biosystems, Waltham, MA, USA) was used
to preamplify the cDNA. TaqMan Gene Expression Assays (Applied Biosystems) (see Supplementary
Table S6) including fluorescent probes and forward/reverse primers were diluted with Tris and EDTA
(TE) buffer (1×) according to the manufacturer’s instructions to obtain a final concentration of 0.2×
pooled assay mix. The reactions were performed at 50 µL and prepared as follows: 25 µL of TaqMan
PreAmp Master Mix (2×), 12.5 µL of pooled assay mix (0.2×), 1 µL of cDNA sample, and nuclease-freewater to adjust to the 50 µL total. The thermal cycler GeneAmp PCR System 9700 (Applied Biosystems)
was run at 95 ◦ C for 10 min for denaturing, at 95 ◦ C for 14 15-s cycles, and at 60 ◦ C for 4 min forpreamplification PCR. The preamplified cDNA products were diluted with TE buffer (1×) at 1:20 for
use as templates for real-time RT-PCR analysis. TaqMan real-time PCR was performed using the 7500
Real-Time PCR System (Applied Biosystems) and the probes in Supplementary Table S7. The PCR
reaction mixture comprised 10 µL of TaqMan Gene Expression Master Mix (2×), 1 µL of TaqMan Gene
Expression Assay (20×), 5 µL of preamplified cDNA, and nuclease-free water to adjust to the 20 µL
total. The thermal cycler 7500 Real-Time PCR System was run at 50 ◦ C for 2 min, 95 ◦ C for 10 min, at
95 ◦ C for 40 15-s cycles, and at 60 ◦ C for 1 min for amplification. The cycle threshold (Ct) values were
determined by 7500 Real-Time PCR System software v. 2.0.6 (Thermo Fisher Scientific). The relative
expression value of each gene (∆Ct value) was derived against the Ct of the internal control ACTB.
4.5. Analysis of Proteins Secreted by PD Sheets
4.5.1. Preparation of the Supernatant of the PD sheets
Each PD sheet was cultured in 3 mL of DMEM/F12 (Gibco) supplemented with 1% fetal bovine
serum and 1× antibiotic–antimycotic solution (Gibco) for 72 h. The supernatant was collected, andany debris was removed by centrifugation at 13,000 g for 10 min, divided into aliquots, and stored at−80 ◦ C until required.
4.5.2. Proteomic Analysis Using SOMAscan
The supernatant was analyzed using the SOMAscan assay platform (SomaLogic Inc., Boulder,
CO, USA), a multiplexed aptamer-based assay detecting 1129 proteins by slow off-rate modified
aptamers (SOMAmers) [14]. The assay uses chemically modified nucleotides to transform a protein
signal into a nucleotide signal that can be quantified using relative fluorescence on microarrays.
We applied rank-based quantile normalization to the raw signal data. Next, probes were filteredbased on signal intensity (higher than 6 coveted log2 intensity) to obtain the average intensity for all

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samples. For extraction of marker proteins, Pearson’s correlation coefficients were calculated between
the protein expression level of each of those probes and the individual ICRS scores, with those having
more than 0.4 or less than −0.4 being selected. Finally, 112 genes were identified using these criteria,with the large variance being more than 0.5 in log2 intensity for all samples.
4.5.3. Enzyme-Linked Immunosorbent Assay
The supernatants were defrosted, and the concentrations of proteins were determined using
ELISA kits for MIA (Roche), DKK1 (R&D Systems, Minneapolis, MN, USA), ESM1 (abcam, Cambridge,UK), GREM1, and NTN4 (Cloud-Clone Corp., Katy, TX, USA).
4.6. Statistical Analysis
Numerical result summaries were expressed as a mean and a standard deviation. Analysis ofvariance was used to analyze ICRS scores, and Dunnett’s multiple comparison was used for post
hoc analysis.
5. Conclusions
In this study, we have identified efficacy-associated genes that may contribute to hyaline cartilage
regeneration via PD sheet transplantation. The identified characteristics could act as markers to predict
the in vivo efficacy of using PD sheets. In addition, if the mechanism by which these factors promote
cartilage regeneration is further clarified, this will offer a new approach to OA treatment.
Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/21/1/149/s1.
Author Contributions: Conceptualization, M.S. and E.T.; data curation E.T.; formal analysis, H.I.; funding
acquisition, M.S.; investigation, T.T., M.M., S.W., E.O., H.I., K.N., and Y.K.; resources, S.W. and M.W.; visualization,
K.N.; writing—original draft preparation, E.T.; 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 was supported by AMED under grant numbers JP18be0104013, JP19bk0104063,
and JP19bm0404035.
Acknowledgments: We would like to express our gratitude to Hidenori Akutsu and Akihiro Umezawa, National
Center for Child Health and Development, for assistance in the collection of chondrocytes. We are grateful to
the Education and Research Support Center, Tokai University, for providing expertise that greatly assisted theresearch. The authors used the rabbit images in Figure 1 with permission from the Database Center for Life Science(DBCLS) in Japan (2016 DBCLS TogoTV/CC-BY-4.0).
Conflicts of Interest: M.S. receives research funding from CellSeed Inc.

Abbreviations
ACAN
ACTB
APOE
CLS1
COL1
COL2
Ct
Cy3
∆Ct
DKK1
DMEM/F12
EDTA
ELISA
ESM1
FABP3
GDF5

AggrecanActin beta
Apolipoprotein E
Collagenase Type I
Type I collagenType II collagen
Cycle threshold
Cyanine 3
Delta Ct
Dickkopf WNT signaling pathway inhibitor 1
Dulbecco’s modified Eagle’s medium–Nutrient Mixture F-12
Ethylenediaminetetraacetic acid
Enzyme-linked immunosorbent assay
Endothelial cell-specific molecule 1
Fatty acid-binding protein 3
Growth differentiation factor 5

Int. J. Mol. Sci. 2020, 21, 149

GES
GO
GREM1
H&E
ICRS
ITGA10
MIA
MSC
NGS
NTN4
OA
PBS
PCR
PD
PD sheetPDGFA
PGF
RARRES2
RT
RT-PCRSERPINA3
SOMA
SOX5
SOX9
SRY
TE

17 of 20

Gene expression score
Gene ontology
Gremlin 1
Hematoxylin and eosinInternational Cartilage Repair Society
Integrin alpha 10
Melanoma inhibitory activity
Mesenchymal stem cells
Normal goat serum
Netrin 4
Osteoarthritis
Phosphate buffer saline
Polymerase chain reaction
Polydactyly-derived
PD chondrocyte sheet
Platelet-derived growth factor subunit A
Placental growth factor
Retinoic acid receptor responder 2
Room temperature
Reverse transcription PCR
Serpin family A member 3
Slow off-rate modified aptamers
SRY-box transcription factor 5
SRY-box transcription factor 9
Sex-determining region Y
Tris and EDTA

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