Osteoarthritis-derived chondrocytes are a potential source of multipotent progenitor cells for cartilage tissue engineering

Accepted Manuscript Osteoarthritis-derived chondrocytes are a potential source of multipotent progenitor cells for cartilage tissue engineering Tomoyuki Oda, Tadahiro Sakai, Hideki Hiraiwa, Takashi Hamada, Yohei Ono, Motoshige Nakashima, Shinya Ishizuka, Tetsuya Matsukawa, Satoshi Yamashita, Saho Tsuchiya, Naoki Ishiguro PII:

S0006-291X(16)31545-5

DOI:

10.1016/j.bbrc.2016.09.085

Reference:

YBBRC 36464

To appear in:

Biochemical and Biophysical Research Communications

Received Date: 12 September 2016 Accepted Date: 16 September 2016

Please cite this article as: T. Oda, T. Sakai, H. Hiraiwa, T. Hamada, Y. Ono, M. Nakashima, S. Ishizuka, T. Matsukawa, S. Yamashita, S. Tsuchiya, N. Ishiguro, Osteoarthritis-derived chondrocytes are a potential source of multipotent progenitor cells for cartilage tissue engineering, Biochemical and Biophysical Research Communications (2016), doi: 10.1016/j.bbrc.2016.09.085. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT 1

Osteoarthritis-derived chondrocytes are a potential source of

2

multipotent progenitor cells for cartilage tissue engineering

3

Tomoyuki Oda, Tadahiro Sakai, Hideki Hiraiwa, Takashi Hamada, Yohei Ono, Motoshige

5

Nakashima, Shinya Ishizuka, Tetsuya Matsukawa, Satoshi Yamashita, Saho Tsuchiya and

6

Naoki Ishiguro

7

SC

RI PT

4

Department of Orthopaedic Surgery, Nagoya University Graduate School of Medicine

9

65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan

10

M AN U

8

*Corresponding author: Tadahiro Sakai

12

Department of Orthopaedic Surgery, Nagoya University Graduate School of Medicine

13

65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan

14

Telephone: +81-52-741-2111; Fax: +81-52-744-2260

15

E-mail: [email protected]

EP

17

Word counts: 4563/4600 words

AC C

16

TE D

11

1

ACCEPTED MANUSCRIPT 18

ABSTRACT

The natural healing capacity of damaged articular cartilage is poor, rendering joint

20

surface injuries a prime target for regenerative medicine. While autologous chondrocyte or

21

mesenchymal stem cell (MSC) implantation can be applied to repair cartilage defects in

22

young patients, no appropriate long-lasting treatment alternative is available for elderly

23

patients with osteoarthritis (OA). Multipotent progenitor cells are reported to present in adult

24

human articular cartilage, with a preponderance in OA cartilage. These facts led us to

25

hypothesize the possible use of osteoarthritis-derived chondrocytes as a cell source for

26

cartilage tissue engineering. We therefore analyzed chondrocyte- and stem cell-related

27

markers, cell growth rate, and multipotency in OA chondrocytes (OACs) and bone

28

marrow-derived MSCs, along with normal articular chondrocytes (ACs) as a control. OACs

29

demonstrated similar phenotype and proliferation rate to MSCs. Furthermore, OACs

30

exhibited multilineage differentiation ability with a greater chondrogenic differentiation

31

ability than MSCs, which was equivalent to ACs. We conclude that chondrogenic capacity is

32

not significantly affected by OA, and OACs could be a potential source of multipotent

33

progenitor cells for cartilage tissue engineering.

34

Keywords: autologous chondrocyte implantation, cartilage tissue engineering, chondrocytes,

35

multipotency, osteoarthritis

AC C

EP

TE D

M AN U

SC

RI PT

19

2

ACCEPTED MANUSCRIPT 36

1. Introduction

There have been numerous attempts to develop tissue-engineered grafts or patches to

38

repair focal chondral and osteochondral defects; however, the clinical application of

39

cell-based therapies for cartilage repair remains challenging.

RI PT

37

Autologous chondrocyte implantation (ACI), first introduced by Brittberg et al.[1] in

41

1994, is based on the implantation of cultured chondrocytes onto the defect. However, ACI

42

has major inherent limitations, including patient age and cell culture[1,2]. At present,

43

International Cartilage Repair Society criteria do not recommend ACI as a therapeutic option

44

for elderly patients or OA patients, while chondrocytes from OA patients may have the

45

capacity to form cartilage tissue and fulfill the prerequisites for use in ACI[3]. Furthermore,

46

ACI requires cell expansion on monolayer cultures for weeks, leading chondrocytes to

47

dedifferentiate, which involves a decrease in the expression of type II collagen (COL2)[4].

48

The cells develop a fibroblastic morphology, preventing chondrocytes in prolonged culture

49

from being able to produce long-lasting cartilage[4]. However, our previous study

50

successfully demonstrated re-differentiation capacity of OACs even after multiple monolayer

51

passages[5].

EP

TE D

M AN U

SC

40

The cell source for cartilage tissue engineering is not limited to autologous

53

chondrocytes. Possible use of mesenchymal stem cells (MSCs) from various tissues (e.g.

54

synovium, bone marrow) has been reported by authors, demonstrating their sufficient

55

chondrogenic

56

dedifferentiation-resistant

57

chondrogenesis is known to vary among different MSC populations or source tissues[6,9].

AC C

52

differentiation

ability

alternative to

in

vitro[6,7].

chondrocytes[8].

MSCs

may

However,

provide

a

the ability of

58

Interestingly, multipotent progenitor cells (MPCs) have been reported to be present

59

in adult human articular cartilage, and they are particularly abundant in OA cartilage[10].

60

Therefore, while MSCs have attracted attention as a cell source for cartilage tissue 3

ACCEPTED MANUSCRIPT engineering[8,11], we hypothesized that OACs, which consist of MPCs, could be a more

62

promising cell source with excellent chondrogenic capacity. The purpose of our study was to

63

investigate the multilineage differentiation ability of OACs in comparison to MSCs, with

64

particular focus on their chondrogenic differentiation potential after monolayer expansion.

AC C

EP

TE D

M AN U

SC

RI PT

61

4

ACCEPTED MANUSCRIPT 65

2. Materials and methods

66

2.1. Tissue harvesting Human bone marrow and articular cartilage were harvested from the knees of 29

68

patients (6 men, 23 women; mean age, 71.0 years) undergoing knee joint replacement for OA,

69

who fulfilled the American College of Rheumatology criteria for this disease, at Nagoya

70

University Hospital (Nagoya, Japan). Non-OA cartilage was harvested from 9 patients who

71

underwent hemiarthroplasty for hip fracture and arthroscopic Bankart repair for shoulder

72

instability (2 men, 7 women; mean age, 68.2 years). The acquisition of tissues was approved

73

by the Ethics Committee of Nagoya University. All patients gave written consent for this

74

research.

M AN U

SC

RI PT

67

75 76

2.2. Cell culturing

Cartilage slices were digested with 3 mg/mL collagenase XI (Sigma, St. Louis, MO)

78

in Dulbecco’s Modified Eagle’s Medium (DMEM; Sigma) for 6 h at 37°C, filtered, and

79

washed thoroughly. Freshly isolated cells were defined as ‘‘P0’’ cells. Chondrocytes were

80

resuspended in DMEM supplemented with 10% fetal bovine serum (FBS) and plated in tissue

81

culture flasks at a density of 1×104 cells/cm2. The medium was changed twice weekly. When

82

the monolayer culture reached confluence, 1 plate was lysed directly for RNA extraction and

83

another detached by treatment with trypsin–EDTA (0.25% trypsin/2.21 mM EDTA; Sigma).

84

The released cells were replated as a monolayer at 1×104 cells/cm2 or cultured as a pellet.

85

Passage number was defined as the number of times cells were trypsinized and replated as

86

monolayers (P1–P3).

AC C

EP

TE D

77

87

Bone marrow (approximately 2 mL from each patient) was collected from distal

88

femur. Primary culture of bone marrow-derived MSCs was performed as previously

89

described[12]. Cells were isolated and resuspended in MSC growth medium containing 5

ACCEPTED MANUSCRIPT MSCGM BulletKit with SingleQuots (10% FBS, L-glutamine; Lonza, Allendale, NJ). Cells

91

were filtered and seeded at a density of 2x105/cm2 in fresh medium. After 6 days,

92

nonadherent hematopoietic cells were removed, and the MSCs on the culture plate were

93

replenished with fresh medium. The medium was changed twice weekly thereafter. The cells

94

were expanded in a monolayer culture until P3, with an interval of ~1–2 weeks for each

95

passage.

RI PT

90

96

2.3. Cell phenotypes

SC

97

The phenotypes of cells isolated from 11 donors (ACs, n=4; OACs, n=7; MSCs,

99

n=7) was investigated by flow cytometry. For immunofluorescence analysis, cells were

100

incubated on ice for 15 min with phycoerythrin-conjugated monoclonal antibodies against the

101

indicated CD molecules. Antibody conjugates against CD45 and HLA-DR (not expressed in

102

MSCs) and CD29, CD44, CD73, CD90, and CD105 (expressed in MSCs) were from BD

103

Pharmingen (San Diego, CA). In an additional set of measurements, surface expression of

104

CD29, CD44, CD73, CD90, and CD105 was continuously quantified (P0–P2). After staining,

105

cells were analyzed on a FACSCanto™ II instrument (BD Biosciences, Heidelberg,

106

Germany); data were analyzed with CellQuest software (BD Biosciences).

EP

TE D

M AN U

98

107

109

2.4. Proliferation assays

AC C

108

Cell

proliferation

was

measured

by

the

110

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay

111

using the Cell Counting Kit (Dojindo, Kumamoto, Japan) according to the following scheme:

112

OACs and MSCs at 2 weeks after isolation were trypsinized and seeded in triplicate in a

113

96-well plate at a density of 3000 cells/well and incubated at 37°C in a CO2 incubator. On

114

days 1, 3, 4, and 7, 10 µL of MTT was added, and the plate was incubated for 30 min at 37°C. 6

ACCEPTED MANUSCRIPT 115

The absorbance value was measured at 450 nm using a microplate reader.

116 117

2.5. Cell differentiation assays

118

2.5.1. Chondrogenic differentiation and redifferentiation Chondrogenic differentiation potential of the MSCs and redifferentiation potential of

120

the OACs were investigated by pellet culture[11]. A pellet containing 2.5×105 cells was

121

cultured in 500 µL of chondrogenic induction medium containing hMSC chondrogenic

122

differentiation BulletKit with SingleQuots (dexamethasone,

123

proline, pyruvate, and ITS+ Premix; Lonza) supplemented with 10 ng/mL transforming

124

growth factor-β3 (TGF-β3; Peprotech, Rocky Hill, NJ). Pellets were cultured for 14 or 21

125

days, with the medium being changed twice weekly.

SC

L-ascorbic

acid-2-phosphate,

M AN U

126 127

RI PT

119

2.5.2. Osteogenic differentiation

Osteogenic differentiation was induced in monolayer cultures using well-established

129

medium supplements[13]. Cells were seeded in 6-well plates at 3×103 cells/cm2 in 2 mL of

130

osteogenic induction medium containing hMSC osteogenic differentiation BulletKit with

131

SingleQuots

132

acid-2-phosphate; Lonza) and cultured for 3 weeks, with the medium being changed twice

133

weekly.

135

β-glycerophosphate,

EP

FBS,

dexamethasone,

and

L-ascorbic

AC C

134

(10%

TE D

128

2.5.3. Adipogenic differentiation assay

136

Adipogenic differentiation was induced in monolayer cultures using cycles of

137

treatment with different media[7]. Cells were seeded in 6-well plates at 5×103 cells/cm2 and

138

cultured in basic medium with 10% FBS until confluent. Cells were then treated with

139

adipogenic induction medium containing hMSC adipogenic differentiation BulletKit with 7

ACCEPTED MANUSCRIPT SingleQuots (10% FBS, insulin, dexamethasone, 3-isobutyl-1-methyl xanthine; Lonza) for 72

141

h and subsequently with adipogenic maintenance medium containing hMSC adipogenic

142

maintenance BulletKit with SingleQuots (10% FBS, insulin; Lonza) for 24 h. After the 96-h

143

treatment cycle was repeated 4 times, cells were cultured for an additional week in

144

adipogenic maintenance medium

145 146

2.6. Reverse transcription-polymerase chain reaction

RI PT

140

Total RNA was extracted from monolayers and pellets with RNeasy Mini kit

148

(Qiagen, Hilden, Germany), and reverse transcription was performed using the

149

High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA).

150

Relative quantification of mRNA expression was performed using the LightCycler480 SYBR

151

Green I Master (Roche Diagnostics, Mannheim, Germany). Expression levels were measured

152

in triplicate and normalized to GAPDH expression. Details of the primers used are listed in

153

Table 1. Total RNA containing microRNA (miRNA) was extracted from monolayers and

154

pellets using the mirVana miRNA isolation kit (Applied Biosystems, Life Technologies,

155

Carlsbad, CA). Expression of mature miR-27b and miR-140 or U6 small nuclear RNA

156

(RNU6B) as the endogenous control was quantified using TaqMan Micro-RNA Assays

157

(Applied Biosystems, Life Technologies). Purified microRNA was reverse transcribed, and

158

quantitative PCR was performed[14].

M AN U

TE D

EP

AC C

159

SC

147

Reaction mixtures were incubated at 95°C for 10 min, followed by 40 cycles of 95°C

160

for 30 s and 60°C for 1 min. The threshold cycle was determined in the exponential phase of

161

amplification, and relative expression levels were quantified by the ∆∆Ct method.

162 163 164

2.7. Histology and immunohistochemistry Cell pellets cultured in chondrogenic medium were fixed in 4% formalin, embedded 8

ACCEPTED MANUSCRIPT in paraffin, cross-sectioned (5-µm thick), and stained with Safranin O to determine the

166

presence of sulfated glycosaminoglycans. Cell layers cultured in osteogenic medium were

167

fixed in 4% paraformaldehyde (PFA) for 10 min and stained with 1% Alizarin Red S

168

(Sigma-Aldrich Co., Tokyo, Japan) for 10 min. Cell layers cultured in adipogenic medium

169

were fixed in 4% PFA for 10 min and incubated in Oil Red O solution (Sigma-Aldrich Co.)

170

for 20 min. For immunohistochemistry, sections were deparaffinized and treated with

171

testicular hyaluronidase (0.05%; type I-S, Sigma-Aldrich Co.) for 1 h at 37°C. Sections were

172

then blocked with endogenous peroxidase in 3% H2O2/MeOH, followed by blocking with

173

protein blocking agent (PBA). Mouse anti-human monoclonal antibody for COL2 (Daiichi

174

Fine Chemical Co. Ltd., Toyama, Japan) diluted to 1:500 (1 µg/mL protein concentration)

175

was then applied for 18 h at 4°C. The primary antibody was detected by the avidin–biotin

176

conjugate method, which was applied using the Histofine MOUSESTAIN Kit (Nichirei

177

Biosciences, Tokyo, Japan). Peroxidase activity was detected using the Histofine DAB kit

178

(Nichirei Biosciences) and counterstained with hematoxylin. Sections for negative controls

179

were incubated with PBA instead of the primary antibody.

180

183

EP

182

2.8. Statistical analysis

All data are presented as mean ± standard deviation. The Mann–Whitney U-test was used to determine statistical significance with a p-value set less than 0.05.

AC C

181

TE D

M AN U

SC

RI PT

165

9

ACCEPTED MANUSCRIPT 184

3. Results

185

3.1. Cell surface phenotypes

Flow cytometry analysis revealed that OACs, MSCs, and ACs were positive for

187

CD29, CD44, CD73, CD90, and CD105 and negative for CD45 and HLA-DR (Fig. 1A). The

188

percentage of MSC marker expression of OACs was high (70%~92%) at P0 (Fig. 1B).

189

Throughout all passages, OACs maintained MSC marker expression in more than 95% of

190

cells.

RI PT

186

192

SC

191

3.2. Proliferation rate

The cell growth rate over a 7-day period was greater in OACs compared to MSCs

194

(Fig. 1C). OACs also had a shorter population doubling time (Fig. 1D), although this was not

195

statistically significant.

M AN U

193

196

3.3. Differentiation into mesenchymal lineages

198

3.3.1. Chondrogenic differentiation

TE D

197

To evaluate the chondrogenic potential of MSCs and OACs at P2, cell pellets were

200

treated with chondrogenic medium for 21 days. After chondrogenic induction, the pellets

201

were morphologically smooth and large owing to the production of extracellular matrix. The

202

expression of chondrogenesis-specific genes (i.e. SOX9, COL2, ACAN) in OACs were all

203

significantly elevated in the induced groups compared with the control monolayer cultures

204

(Fig. 2A). MSCs did not respond to chondrogenic induction as well as OACs and these

205

chondrogenic markers were expressed significantly higher in OACs than MSCs (Fig. 2A).

206

OACs formed pellets that stained more intensely with COL2 and Safranin O compared to

207

MSCs (Fig. 2B).

AC C

EP

199

208

10

ACCEPTED MANUSCRIPT 209

3.3.2 Osteogenic differentiation To evaluate the osteogenic potential of the MSCs (P2) and OACs (P2), cells were

211

cultured in osteogenic medium for 21 days. Osteogenesis-specific gene expression in OACs

212

was elevated in the induced group compared with the control monolayer cultures, while

213

significance was obtained only for RUNX2 (Fig. 2C). There was no significant difference in

214

mRNA expression of Runx2 and ALP between OACs and MSCs (Fig. 2C). After 3-week

215

culture in osteogenic medium, both OACs and MSCs generated a mineralized matrix that

216

stained positive for Alizarin Red S (Fig. 2D). Cells grown in the control medium did not form

217

a mineralized matrix.

218 219

3.3.3. Adipogenic differentiation

M AN U

SC

RI PT

210

To evaluate the adipogenic potential of the MSCs (P2) and OACs (P2), cells were

221

cultured in adipogenic medium for 21 days. The expression of adipogenesis-specific genes

222

(FABP4, LPL) in OACs and MSCs was elevated in the induced groups compared with the

223

control monolayer cultures(Fig. 2E). There was no significant difference in mRNA

224

expression of FABP4 and LPL between OACs and MSCs (Fig. 2E). After 3-week culture in

225

adipogenic medium, both OACs and MSCs generated cells that stained positive with Oil red

226

O (Fig. 2F). Lipid droplets were observed as early as after the second induction treatment,

227

increasing in both size and number over time. Lipid vacuoles were not formed in the control

228

medium.

EP

AC C

229

TE D

220

230

3.4. Comparison of gene expression between ACs and OACs

231

3.4.1. Chondrogenic gene expression

232

To evaluate the phenotypic difference between OACs and ACs, we determined

233

chondrogenic gene expression in ACs at P0 and in OACs at P0 followed by monolayer 11

ACCEPTED MANUSCRIPT 234

expansion and chondrogenic induction. Mean mRNA expression level of the chondrogenic

235

markers (i.e. SOX9, COL2, ACAN) did not differ between OACs and ACs (Fig. 3A).

236

Monolayer passaging of chondrocytes results in dramatic changes in cell shape and loss of

237

the chondrocyte phenotype (Fig. 3A). However, after chondrogenic induction, OACs

238

remarkably recovered all the chnodrogenic markers

239

(Figu. 3A). Positive staining for both COL2 and Safranin O was observed, principally within

240

the pericellular matrix (Fig. 3B). Negative controls did not show significant staining.

RI PT

to the same level as those in ACs (P0)

242

SC

241

3.4.2. Catabolic gene expression

To further evaluate the phenotypic difference between OACs and ACs, we

244

determined catabolic marker expression in ACs at P0 and in OACs at P0 followed by

245

monolayer expansion and chondrogenic induction. The expression levels of MMP13 and

246

ADAMTS5 at P0 were significantly higher in OACs than in ACs (Fig. 4A). The expression

247

of these genes in OACs decreased over time, during monolayer culture followed by

248

chondrogenic induction, to the level of ACs or even below (Fig. 4A). The expression of

249

miR-27b and miR-140, regulating MMP-13 [19] and ADAMTS5 [20], respectively,

250

significantly increased after chondrogenic induction (Fig. 4B).

AC C

EP

TE D

M AN U

243

12

ACCEPTED MANUSCRIPT 251

4. Discussion OACs demonstrated multilineage differentiation with superior chondrogenic

253

capacity to MSCs. The expression of chondrogenesis-specific genes (i.e. SOX9, COL2,

254

ACAN) after chondrogenic induction was similar to the expression in normal articular

255

chondrocytes, with equivalent extracellular matrix production. In addition, cartilage related

256

catabolic markers (i.e. MMP13, ADAMTS5) were both downregulated to the same or even

257

lower level of normal articular chondrocytes.

RI PT

252

When cell surface marker expression profiles of ACs, OACs, and MSCs were

259

compared, little difference was observed between the 3 groups. The expression of CD44,

260

CD73, CD90, and CD105, regarded as distinctive of MSCs[7,15], was upregulated in ACs

261

and OACs. A previous study reported that the cell surface marker expression of

262

dedifferentiated ACs resembles that of MSCs, and the possibility of reversion of

263

differentiated ACs to a more primitive, undifferentiated state has been suggested[15]. Adult

264

ACs dedifferentiated by monolayer expansion share a major functional feature of MSCs: the

265

ability to differentiate into diverse mesenchymal lineages[16]. In the current study, we

266

demonstrated that OACs and MSCs isolated from progressive OA—especially OACs with a

267

chondrogenic potential higher than that of MSCs—could differentiate toward chondrocytic,

268

osteoblastic, and adipocytic lineages.

M AN U

TE D

EP

A recent review of cell therapy in cardiac medicine suggested that the efficacy of cell

AC C

269

SC

258

270

therapy may depend on cell characterization, and that cell therapy using heterogeneous

271

populations of uncharacterized cells may also account for the disparate results in various cell

272

therapy studies[17]. Similarly, even among MSCs, different cell populations or cells from

273

different tissue sources present various level of chondrogenic differentiation[6,18]. However,

274

we speculate that OACs themselves have multidifferentiation potential since MSC marker

275

expression was elevated to more than 60% from early stages of isolation and were all positive 13

ACCEPTED MANUSCRIPT 276

at P1. Furthermore, OACs could successfully demonstrated excellent induced chondrogenesis

277

even after monolayer expansion to the level of normal chondrocytes. Several studies have demonstrated in vivo phenotypic alterations in OACs vis-a-vis

279

ACs. The expression of genes belonging to hypertrophic cartilage (e.g. COL10, VEGF,

280

MMP13) increased, while the expression of genes characteristic for a mature articular

281

cartilage phenotype decreased considerably (SOX9, COL2, ACAN) in comparison with

282

normal cartilage[19,20]. These OA-related alterations might influence bioactivity and matrix

283

gene expression negatively in vitro[21,22]. However, OACs may possibly display a good

284

proliferation potential and redifferentiate, resulting in a matrix rich in proteoglycans and

285

COL2[22]. We previously demonstrated that OACs could successfully re-activate their

286

chondrocyte phenotype in vitro even after dedifferentiation during monolayer expansion[5].

287

In the present study, MMP13 and ADAMTS5 were highly expressed in OA cartilage

288

compared with normal cartilage, while COL10 and VEGF expression did not differ (data not

289

shown). Interestingly, the upregulated expression of MMP13 and ADAMTS5 was reversed

290

when cultured in monolayer, and further decreased to the level below normal chondrocytes

291

after chondrogenic induction.

TE D

M AN U

SC

RI PT

278

Micro RNAs (miRNAs) are small noncoding RNAs, which are important regulators

293

of gene expression in human cells. A number of miRNAs are regulated in chondrogenesis,

294

and their functions are beginning to be delineated. MicroRNA-140 (miR-140) is highly and

295

selectively expressed in cartilage, and transfection of chondrocytes with miR-140 has been

296

found to downregulate ADAMTS5 expression and rescue ACAN expression[23]. Similarly,

297

the expression of miR-27b is inversely correlated with the expression of MMP13, a direct

298

target, in OA cartilage[24]. MiR-140 and miR-27b expression in the cartilage of OA patients

299

is significantly lower than that in normal cartilage[23,25]. Here, we showed that miR-140 and

300

miR-27b expression increased during pellet culture for redifferentiation (2.8-fold and 30-fold

AC C

EP

292

14

ACCEPTED MANUSCRIPT increase, respectively), indicating that the epigenetic status of OACs changed under in vitro

302

cultivation for redifferentiation. While OACs are exposed to prolonged inflammation and

303

mechanical stress, we speculated that epigenetic changes might occur after release from

304

strong stimuli, leading the cells to fully recover their normal status as articular chondrocytes

305

in vitro.

RI PT

301

There are several limitations to the current study mainly due to the nature of basic

307

research in vitro. First, all the data shown were at cellular or molecular level, therefore further

308

investigations including in vivo and clinical researches are necessary to apply the use of

309

OACs in clinical settings. Second, while we demonstrated promising differentiation potential

310

of OACs at mRNA and protein levels, whole sequence of chondrogenic differentiation and

311

the effects of osteoarthritis, are still unknown. For example, DNA methylation, protein-DNA

312

interaction, or activity levels of catabolic enzymes have not been investigated. Further

313

analyses, such as bisulfate sequencing, ChIP assay, and zymography may be needed. Third,

314

MSCs were only obtained from bone marrows and other source tissues were not tested.

315

However, considering the excellent chondrogenic capacity of OACs, equivalent to normal

316

articular chondrocytes, we do not believe it is necessary to compare OACs to various types of

317

MSCs. Finally, we could not selectively isolate chondrogenic OAC populations with a higher

318

ability to produce cartilage matrix. However, considering the limited number of autologous

319

chondrocytes obtainable from donor cartilage, extended monolayer expansion to achieve

320

isolation of selective populations might not be ideal, even though we have shown a great

321

re-differentiation potential of monolayer cultured OACs.

AC C

EP

TE D

M AN U

SC

306

322

Nevertheless, our findings indicate that (1) ACs, OACs, and MSCs did not differ

323

significantly in cell surface marker expression profiles of MSC markers; (2) expanded OACs

324

could differentiate into chondrocytic, osteoblastic, and adipocytic lineages; (3) ACs and

325

OACs did not differ significantly in gene expression profiles; and (4) OACs could produce 15

ACCEPTED MANUSCRIPT cartilage matrix akin to ACs in chondrogenic pellet culture. Irrespective of age and OA

327

etiology, OACs have multilineage differentiation capacity and possess adequate

328

redifferentiation potential. Therefore, a therapeutic application of OACs as multipotent

329

progenitors for cartilage-tissue engineering seems feasible.

AC C

EP

TE D

M AN U

SC

RI PT

326

16

ACCEPTED MANUSCRIPT Figure and Table legends

Figure 1. Cell surface marker analyses and proliferation assays of OACs, MSCs, and ACs. (A) Cell surface marker analysis for CD29, CD44, CD73, CD90, CD105, CD45, and

RI PT

HLA-DR by flow cytometry of OACs (n=7), MSCs (n=7), and ACs (n=5). (B) MSC marker expression of OACs (n=4) at different passages (P0–P2). (C) Cell proliferation rate assayed by MTT over a 7 day period. Results compared to day 1 as control (OACs, n=4; MSC, n=4)

SC

(D) Population doubling time calculated from total cell numbers at days 3 and 7. Data shown

M AN U

as mean ± SD. n.s, not significant.

Figure 2. Mesenchymal differentiation of MSCs and OACs. (A) mRNA expression of SOX9, COL2, and ACAN and (B) Safranin O and immunohistochemical staining for COL2 in MSCs and OACs after 3 weeks of chondrogenic induction (n=5). (C) mRNA expression of

TE D

RUNX2 and ALP and (D) Alizarin Red S staining in MSCs and OACs after 3 weeks of osteogenic induction (n=5). (E) mRNA expression of FABP4 and LPL and (F) Oil red O staining in MSCs and OACs after 3 weeks of adipogenic induction (n=5). Data shown as

EP

mean ± SD. * p<0.05; ** p<0.01; n.s., not significant; M, monolayer culture; P, pellet culture;

AC C

O, osteogenic induction; A, Adipogenic differentiation.

Figure 3. Chondrogenic maker expression and redifferentiation potential of ACs and OACs. (A) mRNA expression of SOX9, COL2, and ACAN in ACs (n=5) at P0 compared to OACs (n=5) at P0 with following monolayer culture and pellet culture. (B) Safranin O and immunohistochemical staining for COL2 in pellets formed by ACs and OACs at P2. Data shown as mean ± SD. * p<0.05; ** p<0.01; n.s, not significant; M, monolayer culture; P, 17

ACCEPTED MANUSCRIPT pellet culture.

Figure 4. Catabolic marker expression in ACs and OACs, and alteration of miRNA in OACs. (A) mRNA expression of MMP13 and ADAMTS5 in ACs (n=5) at P0 compared to

RI PT

OACs (n=4) at P0 with following monolayer culture and pellet culture. (B) Relative

expression of miR-27b and miR-140 in OACs (n = 4). The fold increase of each miR

not significant; M, monolayer culture; P, pellet culture.

M AN U

Table 1. Primer sequences for RT-PCR.

SC

expression was compared with the control at P1. Data shown as mean ± SD; * p<0.05; n.s.,

MMP13, matrix metalloproteinase 13; ADAMTS5, a disintegrin and metalloproteinase with thrombospondin motifs 5; SOX9, SRY (sex determining region Y)-box 9; COL2, collagen type II, ACAN: aggrecan; RUNX2: runt-related transcription factor 2; ALP: alkaline

TE D

phosphatase; FABP4: fatty acid binding protein 4; LPL: lipoprotein lipase; GAPDH:

AC C

EP

glyceraldehyde-3-phosphate dehydrogenase

18

ACCEPTED MANUSCRIPT References

[1] M. Brittberg, A. Lindahl, A. Nilsson, C. Ohlsson, O. Isaksson, L. Peterson, Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation, N Engl J Med 331 (1994) 889-895.

RI PT

[2] S.P. Krishnan, J.A. Skinner, W. Bartlett, R.W.J. Carrington, A.M. Flanagan, T.W.R. Briggs, G. Bentley, Who is the ideal candidate for autologous chondrocyte implantation?, J Bone Joint Surg Br 88 (2006) 61-64. [3] T. Dehne, C. Karlsson, J. Ringe, M. Sittinger, A. Lindahl, Chondrogenic differentiation

M AN U

SC

potential of osteoarthritic chondrocytes and their possible use in matrix-associated autologous chondrocyte transplantation, Arthritis Res Ther 11 (2009) R133. [4] K. von der Mark, V. Gauss, H. von der Mark, P. Müller, Relationship between cell shape and type of collagen synthesised as chondrocytes lose their cartilage phenotype in culture., Nature 267 (1977) 531-532. [5] Y. Ono, T. Sakai, H. Hiraiwa, T. Hamada, T. Omachi, M. Nakashima, S. Ishizuka, T. Matsukawa, W. Knudson, C.B. Knudson, N. Ishiguro, Chondrogenic capacity and alterations in hyaluronan synthesis of cultured human osteoarthritic chondrocytes, Biochem Biophys Res Commun 435 (2013) 733-739.

TE D

[6] Y. Ogata, Y. Mabuchi, M. Yoshida, E.G. Suto, N. Suzuki, T. Muneta, I. Sekiya, C. Akazawa, Purified Human Synovium Mesenchymal Stem Cells as a Good Resource for Cartilage Regeneration, PLoS One 10 (2015) e0129096. [7] M.F. Pittenger, A.M. Mackay, S.C. Beck, R.K. Jaiswal, R. Douglas, J.D. Mosca, M.A.

EP

Moorman, D.W. Simonetti, S. Craig, D.R. Marshak, Multilineage potential of adult human mesenchymal stem cells, Science 284 (1999) 143-147. [8] M.K. Majumdar, V. Banks, D.P. Peluso, E.A. Morris, Isolation, characterization, and chondrogenic potential of human bone marrow-derived multipotential stromal cells, J

AC C

Cell Physiol 185 (2000) 98-106. [9] H. Yoshimura, T. Muneta, A. Nimura, A. Yokoyama, H. Koga, I. Sekiya, Comparison of rat mesenchymal stem cells derived from bone marrow, synovium, periosteum, adipose tissue, and muscle, Cell Tissue Res 327 (2007) 449-462. [10] S. Alsalameh, R. Amin, T. Gemba, M. Lotz, Identification of mesenchymal progenitor cells in normal and osteoarthritic human articular cartilage, Arthritis Rheum 50 (2004) 1522-1532. [11] I. Sekiya, J.T. Vuoristo, B.L. Larson, D.J. Prockop, In vitro cartilage formation by human adult stem cells from bone marrow stroma defines the sequence of cellular and molecular events during chondrogenesis, Proc Natl Acad Sci U S A 99 (2002) 4397-4402. 19

ACCEPTED MANUSCRIPT [12] H.J. Lee, B.H. Choi, B.H. Min, S.R. Park, Changes in surface markers of human mesenchymal stem cells during the chondrogenic differentiation and dedifferentiation processes in vitro, Arthritis Rheum 60 (2009) 2325-2332. [13] N. Jaiswal, S.E. Haynesworth, A.I. Caplan, S.P. Bruder, Osteogenic differentiation of purified, culture-expanded human mesenchymal stem cells in vitro, J Cell Biochem 64 (1997) 295-312.

RI PT

[14] T. Matsukawa, T. Sakai, T. Yonezawa, H. Hiraiwa, T. Hamada, M. Nakashima, Y. Ono, S. Ishizuka, H. Nakahara, M.K. Lotz, H. Asahara, N. Ishiguro, MicroRNA-125b regulates the expression of aggrecanase-1 (ADAMTS-4) in human osteoarthritic chondrocytes, Arthritis Res Ther 15 (2013) R28.

M AN U

SC

[15] J. Diaz-Romero, J.P. Gaillard, S.P. Grogan, D. Nesic, T. Trub, P. Mainil-Varlet, Immunophenotypic analysis of human articular chondrocytes: changes in surface markers associated with cell expansion in monolayer culture, J Cell Physiol 202 (2005) 731-742. [16] A. Barbero, S. Ploegert, M. Heberer, I. Martin, Plasticity of clonal populations of dedifferentiated adult human articular chondrocytes, Arthritis Rheum 48 (2003) 1315-1325.

TE D

[17] A. Rosenzweig, Cardiac cell therapy--mixed results from mixed cells, N Engl J Med 355 (2006) 1274-1277. [18] Y. Sakaguchi, I. Sekiya, K. Yagishita, T. Muneta, Comparison of human stem cells derived from various mesenchymal tissues: superiority of synovium as a cell source,

EP

Arthritis Rheum 52 (2005) 2521-2529. [19] H. Kawaguchi, Endochondral ossification signals in cartilage degradation during osteoarthritis progression in experimental mouse models, Mol Cells 25 (2008) 1-6. [20] N. Takahashi, C.B. Knudson, S. Thankamony, W. Ariyoshi, L. Mellor, H.J. Im, W.

AC C

Knudson, Induction of CD44 cleavage in articular chondrocytes, Arthritis Rheum 62 (2010) 1338-1348. [21] R. Dorotka, U. Bindreiter, P. Vavken, S. Nehrer, Behavior of human articular chondrocytes derived from nonarthritic and osteoarthritic cartilage in a collagen matrix, Tissue Eng 11 (2005) 877-886. [22] T. Tallheden, C. Bengtsson, C. Brantsing, E. Sjogren-Jansson, L. Carlsson, L. Peterson, M. Brittberg, A. Lindahl, Proliferation and differentiation potential of chondrocytes from osteoarthritic patients, Arthritis Res Ther 7 (2005) R560-568. [23] S. Miyaki, T. Nakasa, S. Otsuki, S.P. Grogan, R. Higashiyama, A. Inoue, Y. Kato, T. Sato, M.K. Lotz, H. Asahara, MicroRNA-140 is expressed in differentiated human articular chondrocytes and modulates interleukin-1 responses, Arthritis Rheum 60 (2009) 2723-2730. [24] N. Akhtar, Z. Rasheed, S. Ramamurthy, A.N. Anbazhagan, F.R. Voss, T.M. Haqqi, 20

ACCEPTED MANUSCRIPT MicroRNA-27b regulates the expression of matrix metalloproteinase 13 in human osteoarthritis chondrocytes, Arthritis Rheum 62 (2010) 1361-1371.

AC C

EP

TE D

M AN U

SC

RI PT

[25] G. Tardif, D. Hum, J.P. Pelletier, N. Duval, J. Martel-Pelletier, Regulation of the IGFBP-5 and MMP-13 genes by the microRNAs miR-140 and miR-27a in human osteoarthritic chondrocytes, BMC Musculoskelet Disord 10 (2009) 148.

21

ACCEPTED MANUSCRIPT

MMP13 ADAMTS5 SOX9 COL2 ACAN RUNX2 ALP FABP4 LPL GAPDH

Oligonucleotide sequence Forward Reverse 5' GGTGGTGATGAAGATGATT 3' 5' TCAGTCATGGAGCTTGCT 3' 5‘ TGACAAGTGCGGAGTATG 3' 5' AGGCAGTGAATCTAGTCTGG 3' 5' CTGGGCAAGCTCTGGAGA 3' 5' ATGTGCGTCTGCTCCGTG 3' 5' GAAGGATGGCTGCACGAAACA 3‘ 5' GCAATGTCAATGATGGGGAGGC 3‘ 5' AGGAGCAGGAGTTTGTCAACAAC 3' 5' AGTTCTCAAATTGCATGGGGTGT 3' 5' GGACCTCGGGAACCCAGAAG 3' 5' ACTTGGTGCAGAGTTCAGGGA 3' 5' TAACATCAGGGACATTGACGTGATC 3‘ 5‘TCCAGATGAAGTGGGAGTGCTT 3‘ 5' GCAGCTTCCTTCTCACCTTGAA 3' 5' CCATGCCAGCCACTTTCCTG 3' 5' CGTTCTCAGATGCCCTACAAAGT 3‘ 5' CACGGTGCCATACAGAGAAATCT 3‘ 5' TGCACCACCAACTGCTTAGC 3‘ 5' GGCATGGACTGTGGTCATGAG 3‘

Product size 125 bp 167 bp 179bp 96 bp 117 bp 159 bp 182 bp 155 bp 114 bp 87 bp

RI PT

Gene

AC C

EP

TE D

M AN U

SC

Table 1, oda et al

120

CD29

ACs OACs MSCs

100 80

CD44 60 40

CD73

20 0 CD45 HLA-DR CD29

CD44

CD73

CD90

CD105

CD90

C 4

OACs OACs

2

D

1 0 0

0

1

1

2

3

3

4

5

5

Cultivation time (day)

7

7

AC C

EP

TE D

Figure 1, oda et al

6

8

P0 P0

P1 P1

P2 P3

P0 P0

P1 P1

P2 P3

P0 P0

P1 P1

P2 P3

P0 P0

P1 P1

P2 P3

P1 P1

P3 P2

100 100 90 90 80 80 70 70 60

60

100 100 90 90 80 80 70 70

100 100 90 90 80 80 70 70

SC

MSCs MSCs

Population doubling time (days)

33

100 100 90 90 80 80 70 70

M AN U

Relative cell number

CD105

100 100 90 90 80 80 70 70

RI PT

B percentage of cells (Mean + SEM)

A

percentage of cells percentage of cells percentage of cells percentage of cells percentage of cells (Mean + SEM) (Mean + SEM) (Mean + SEM) (Mean + SEM) (Mean + SEM)

ACCEPTED MANUSCRIPT

P0 P0

5

n.s.

4 3 2 1 0

OACs

MSCs

ACCEPTED MANUSCRIPT

P2 (M)

0.0008 0.0008

0.01 0.01

0.0004 0.0004

0.005 0.005

00

P2 (P)

Relative expression

Mono COL2 Pellet

**

0.3

0.3

0.2

0.2

OACs MSCs

D

MSCs

E

FABP4

0

0

P2 (P)

Pellet

ACAN

0

MSCs

Mono

P2 (P)

Pellet OACs

0.016 0.016 0.012 0.012 0.008 0.008 0.004 0.004 00

COL2

Safranin O

Safranin O

AC C

EP

TE D

Figure 2, oda et al

LPL

0.03 0.03

**

OACs MSCs

0.01 0.01

00

P2 (A) Adip

M AN U

COL2

Osteo

OACs

x 100

0.02 0.02

P2 (M) Mono

F

P2 (O)

Mono

SC

0

P2 (M)

B

Relative expression

0.02 0.02

*

0.04 0.04

*

Relative expression

*

0.06 0.06

P2 (M)

P2 (O) Osteo

0.1 P2 (M)

OACs MSCs

0 P2 (M) Mono

0.1

Mono

ALP 0.015 0.015

RI PT

Relative expression

**

RUNX2 0.0012 0.0012

Relative expression

C

SOX9 0.2 0.2 0.15 0.15 0.1 0.1 0.05 0.05 00

*

A

MSCs

x 100

P2 (M) Mono

P2 (A) Adipo

OACs

ACCEPTED MANUSCRIPT

A

SOX9

COL2 COL2

SOX9

n.s.

n.s.

2000

n.s.

*

2

ACs OACs

1.5

n.s.

**

1500 1000

1 500

0.5

0

0 ACs (P0) Acs P0

OACs (P0) OACsP0

P1 P1(M)

ACs (P0) Acs P0

P2 (P) pellet

B

ACs

ACAN AGC n.s.

0.06

**

COL2

0.02 0 OACs (P0) OACsP0

P1 P1(M)

M AN U

0.04

ACs (P0) Acs P0

P2 (P) pellet

EP

TE D

Figure 3, oda et al

AC C

OACs (P0) OACsP0

SC

n.s.

0.08

RI PT

Relative expression

2.5

Safranin O

P1 P1(M)

P2 (P) pellet

OACs

COL2

Safranin O

ACCEPTED MANUSCRIPT

A

MMP13

*

*

0.08

* 0.4 ACs

0

0.2

0 P2(P) P2 (P)

ACｓP0 ACs (P0)

* *

5

40

4

30

3

M AN U

20

2

P2(P) P2 (P)

miR-140/RNU6B

50

SC

miR-27b/RNU6B 6

P1(M) P1

OACsP０ OACs (P0)

*

B

P1P1 (M)

OACsP０ OACs (P0)

*

ACｓP0 ACs (P0)

RI PT

OACs

0.04

*

Relative expression

0.6

n.s.

*

0.12

ADAMTS5

10

1

0

0 P1(M) P1 (M)

P2(M) P2 (M)

AC C

EP

TE D

Figure 4, oda et al

P2(P) P2 (P)

P1(M) P1 (M)

P2(M) P2 (M)

P2(P) P2 (P)

ACCEPTED MANUSCRIPT Highlights Osteoarthritis chondrocytes (OACs) have multilineage differentiation capacity

•

Articular chondrocytes (ACs) and OACs have similar gene expression profiles

•

OACs have high chondrogenic potential

•

OACs could be a cell resource for cartilage tissue engineering

AC C

EP

TE D

M AN U

SC

RI PT

•