for peer review onlyousar.lib.okayama-u.ac.jp/files/public/5/55012/...63 migration outside the...

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Hyaluronan stimulates chondrogenic gene expression in

human meniscus cells

Journal: Connective Tissue Research

Manuscript ID GCTS-2016-0072.R3

Manuscript Type: Original Research

Date Submitted by the Author: n/a

Complete List of Authors: Tanaka, Takaaki; Okayama University Graduate School, Furumatsu, Takayuki; Okayama University Graduate School, Orthopaedic Surgery Miyazawa, Shinichi; Okayama University Graduate School, Fujii, Masataka; Okayama University Graduate School, Inoue, Hiroto; Okayama University Graduate School, Orthopaedic Surgery

Kodama, Yuya; Okayama University Graduate School, Ozaki, Toshifumi; Okayama University Graduate School,

Keywords: Meniscus, Hyaluronan, Type II collagen, Chondrocytic gene expression, Inner meniscus, Outer meniscus

URL: http:/mc.manuscriptcentral.com/gcts

Connective Tissue Research

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Hyaluronan stimulates chondrogenic gene expression in human meniscus cells 1

2

Takaaki Tanaka, Takayuki Furumatsu*, Shinichi Miyazawa, Masataka Fujii, Hiroto Inoue, Yuya 3

Kodama, Toshifumi Ozaki 4

5

Department of Orthopaedic Surgery, Okayama University Graduate School, 2-5-1 Shikata-cho, 6

Kitaku, Okayama 700-8558, Japan 7

8

* Correspondence to: Takayuki Furumatsu, MD, PhD 9

Department of Orthopaedic Surgery, Okayama University Graduate School, Okayama, Japan 10

Tel: +81-86-235-7273; Fax: +81-223-9727; E-mail: [email protected] 11

12

Author Contribution Statement: 13

Takaaki Tanaka: Experiments, data collection and analysis, manuscript writing. 14

Takayuki Furumatsu: Study design, manuscript preparation, laboratory organization. 15

Shinichi Miyazawa: Meniscal sample preparation. Masataka Fujii: Data analysis. 16

Hiroto Inoue: Experiments, cell cultures. Yuya Kodama: Data analysis. 17

Toshifumi Ozaki: Laboratory organization. 18

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ABSTRACT 19

Purpose/Aim of the Study: Inner meniscus cells have a chondrocytic phenotype, whereas outer 20

meniscus cells have a fibroblastic phenotype. In this study, we examined the effect of hyaluronan 21

on chondrocytic gene expression in human meniscus cells. 22

Materials and Methods: Human meniscus cells were prepared from macroscopically intact lateral 23

meniscus. Inner and outer meniscus cells were obtained from the inner and outer halves of the 24

meniscus. The cells were stimulated with hyaluronan diluted in Dulbecco’s modified Eagle’s 25

medium without serum to the desired concentration (0, 10, 100, and 1000 µg/mL) for 2–7 days. 26

Cellular proliferation, migration, and polymerase chain reaction analyses were performed for the 27

inner and outer cells separately. Meniscal samples perforated by a 2-mm-diameter punch were 28

maintained for 3 weeks in hyaluronan-supplemented medium and evaluated by histological 29

analyses. 30

Results: Hyaluronan increased the proliferation and migration of both meniscus cell types. 31

Moreover, cellular counts at the surface of both meniscal tissue perforations were increased by 32

hyaluronan treatments. In addition, hyaluronan stimulated α1(II) collagen expression in inner 33

meniscus cells. Accumulation of type II collagen at the perforated surface of both meniscal 34

samples was induced by hyaluronan treatment. Hyaluronan did not induce type I collagen 35

accumulation around the injured site of the meniscus. 36

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Conclusion: Hyaluronan stimulated the proliferation and migration of meniscus cells. Our results 37

suggest that hyaluronan may promote the healing potential of meniscus cells in damaged 38

meniscal tissues. 39

40

Keywords: Meniscus, Hyaluronan, Type II collagen, Inner meniscus, Outer meniscus, 41

Chondrocytic gene expression 42

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Introduction 43

Hyaluronan (HA) is a linear macromolecular polymer composed of a repeated structure 44

of the disaccharide N-acetyl-D-glucosamine and D-glucuronic acid.1 The number of disaccharide 45

repeats is over 10,000, and the molecular weight ranges from 103 to 10

4 kDa. HA is a component 46

of the extracellular matrix (ECM) of various structures and plays a vital role in structural control 47

and homeostasis, including neovascularization, branching morphogenesis, cell migration, and 48

differentiation.2-4

HA has a high water-retention capacity and forms an elastic network structure 49

that HA promotes cell differentiation and migration.5 Some HA functions are thought to derive 50

from its physiochemical characteristics, while HA controls cell behavior at the same time by 51

binding to specific cell surface receptors and binding proteins.6, 7

Normal human meniscus is 52

composed of 72% water, 22% collagen, 0%–8% glycosaminoglycan, and 0%–12% DNA.8 In the 53

bovine meniscus, HA occupies 4%–5% and 10% of the total glycosaminoglycan in the inner and 54

outer regions, respectively.9 HA is a component of proteoglycan aggregaten,

10 which has the 55

ability to increase proteoglycan synthesis, prevent glycosaminoglycan release from the cartilage 56

matrix, and stimulate tissue inhibitor of metalloproteinase-1 in articular chondrocytes.11

However, 57

most studies used animal meniscus and cells, and the effects of HA on human meniscus cells 58

remain unclear. Thus, the effects of HA treatment on the proliferation and migratory ability of 59

human meniscus cells were considered in this study. 60

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HA is currently used as a treatment for gonarthrosis (knee osteoarthritis). The known 61

effects of HA on gonarthrosis include suppression of cartilage degeneration and proteoglycan 62

migration outside the cartilage matrix, cartilage outer layer protection,12, 13

synovial fluid 63

normalization,14

improvement in range of motion, and relief of knee joint pain.15, 16

In vitro, HA 64

has been observed to stimulate the production of tissue inhibitor of metalloproteinase-1 in 65

chondrocytes, inhibit neutrophil-mediated cartilage degradation, and attenuate 66

interleukin-1-induced matrix degeneration and chondrocyte cytotoxicity.17

Articular chondrocytes 67

cultured in the presence of HA have a significantly greater rate of DNA proliferation and ECM 68

production, with increased matrix deposition of chondroitin-6-sulfate and type II collagen, 69

compared with chondrocytes cultured without HA.18

We previously conducted a comparative 70

study on the biological characteristics of meniscus cells. In the primary culture, the inner cells 71

have a small triangular structure, and outer cells are long and narrow.19

The gene expression of 72

α1(II) collagen (COL2A1), chondromodulin-I, and SRY-type HMG box-9 in inner cells was 73

distinctive.19-22

In addition, lipid droplet accumulation was observed by inducing differentiation 74

of both the inner and outer cells to the adipocyte lineage; however, increase in the production of 75

the chondrocytic matrix was only observed in the inner cells.19

From these findings, the cells in 76

the parenchyma of the inner meniscus region seem to maintain chondrocyte-like characteristics in 77

both structure and gene expression, whereas cells in the outer region have fibroblast-like 78

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characteristics within a dense connective tissue. In animal models, intra-articular HA injections 79

improve the healing process,23

stimulate collagen remodeling, and inhibit meniscal swelling after 80

partial menisectomy.11

Because the inner cells maintain chondrocyte-like characteristics, they 81

might respond to HA treatment similarly to articular chondrocytes. 82

The meniscus is a relatively avascular structure with limited peripheral blood supply, and 83

the following three zones determined healing prognosis for meniscal lesions: red, red/white, and 84

white zone. Blood vessels are abundant in the outer region, the “red-zone”, defined as the area 85

beyond the inner two-thirds of the meniscus. Within the inner region, the “white zone” and 86

“red/white zone”, no blood vessels are present or avascular, wherein poor results of the meniscus 87

treatment have been attributed.24

In the clinical setting, a cut in the "red zone” may heal by itself, 88

or can often be repaired with surgery. In contrast, tears in this "white zone” cannot heal. These 89

tears are often in thin, and worn cartilage. Histologically, the meniscus, a fibrocartilaginous tissue, 90

contains multiple cell types, with fibrochondrocytic cells in the parenchyma of the inner meniscus 91

and fibroblast-like cells in the outer region, reportedly leading to differing reactions to 92

mechanical stress.25

93

Based on these findings, we hypothesized that the effects of HA may differ between 94

injuries to the inner region and outer region of the meniscus. In the present study, we used an ex 95

vivo organ-culture model to evaluate the effects of HA treatment on injuries to human inner and 96

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outer meniscus cells. Furthermore, the expression change in COL2A1, which is an articular 97

cartilaginous tissue gene, and the effects of HA treatment on injuries to the inner and outer 98

regions were examined. 99

100

Materials and methods 101

Cells and cell culture: Institutional Review Board approval and informed consent were obtained 102

before all experimental studies. Macroscopically intact lateral meniscus was obtained during total 103

knee arthroplasty in patients suffering from medial osteoarthritis of the knee. Patients with 104

sufficient articular cartilage of the lateral femoral condyle and undegenerated lateral meniscus on 105

magnetic resonance imaging were selected. The selected patients were 58, 61, 63, 69, 71, and 76 106

years old (n = 6). Inner and outer meniscus cells were prepared from the meniscal samples.19, 26

In 107

brief, synovial/capsular tissues and the superficial zones of the meniscus were removed carefully. 108

The width of the obtained lateral meniscus was 10–14 mm. The center line of the meniscal width 109

(5–7 mm from the inner edge) was marked. Inner and outer meniscus tissues were prepared by 110

careful cutting along the center line. Inner and outer meniscus cells were prepared by collagenase 111

(Sigma, St. Louis, MO, USA) treatment.19, 26

After collagenase digestion, attached cells (passage 112

0) were maintained with Dulbecco’s modified Eagle’s medium (DMEM, Wako, Osaka, Japan) 113

containing 10% fetal bovine serum (FBS; HyClone, South Logan, UT), and 1% 114

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penicillin/streptomycin (Sigma). Meniscus cells were used between passages 1 and 2. The cell 115

seeding density was 20% -30% (10,000 cells/cm2) on the dish. 116

HA stimulation: Sodium HA [MW, 500-1200 kDa; kindly provided by Seikagaku Kogyo (Tokyo, 117

Japan) and Kaken Pharmaceutical (Tokyo, Japan)], was diluted in DMEM without serum to the 118

desired concentration (0, 10, 100, and 1000 µg/mL). 119

Cell proliferation assay: Cells were cultured for 2–7 days in serum-free DMEM with HA (0, 10, 120

100, or 1000 µg/mL). Cell proliferation assays were performed as described.20, 27, 28

Cells (5,000 121

cells/well) were incubated on 96-well plates for 12 h prior to the addition of the cell proliferation 122

reagent water-soluble tetrazolium (WST)-1 (Roche Diagnostics, Basel, Switzerland). Optical 123

density (OD) was measured at evaluation and control wavelengths of 450 and 630 nm, 124

respectively. Data obtained by subtracting 630-nm readings from 450-nm readings were used for 125

evaluation (5 wells each). 126

Cell migration assay: Chemotaxis assays were performed using a modified Boyden chamber 127

(48-well chemotaxis chamber AP48, Neuro Probe, Gaithersburg, MD, USA).23, 27, 28

128

Collagen-coated polycarbonate membrane (5 µm pore, Neuro Probe) was placed over the bottom 129

chamber filled with DMEM containing 5% FBS and 0 to 1000 µg/mL HA. Fifty microliters of 130

cell suspension per well (5.0 × 103 cells/well in DMEM) was seeded in the upper wells. The 131

upper and lower wells were separated by a filter coated with type I collagen. The chambers were 132

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incubated for 4 h at a temperature of 37 °C. At the end of the incubation, filters were removed 133

and stained with Diff-Quik (Sysmex International Reagents Co., Hyogo, Japan). Migrated cells 134

were counted and expressed as migrated cells per high-power field. 135

Quantitative real-time polymerase chain reaction (PCR) analysis: RNA samples were obtained 136

from cultured meniscus cells. Total RNAs were isolated using Isogen reagent (Nippon Gene, 137

Toyama, Japan). RNA samples (500 ng) were reverse-transcribed with ReverTra Ace (Toyobo, 138

Osaka, Japan). The cDNAs underwent PCR amplification in the presence of specific primers 139

using rTaq DNA polymerase (TaKaRa, Ohtsu, Japan). For all RT-PCR fragments, the reaction 140

was allowed to proceed for 28–37 cycles. The following specific primer sets were used: α1 (I) 141

and α1 (II) collagen (COL1A1 and COL2A1) and glyceraldehyde-3-phosphate dehydrogenase 142

(G3PDH).19, 20

Quantitative real-time PCR analyses were performed using FastStart DNA Master 143

SYBR Green I kit (Roche Diagnostics). The cycle number crossing the signal threshold was 144

selected in the linear part of the amplification curve. Amplification data of G3PDH were used for 145

normalization. Relative mRNA levels were normalized with the level of meniscus cells with no 146

HA treatment for every sample. 147

Histological analysis: The lateral meniscus was cut into fragments having 1-cm width. Meniscal 148

samples were perforated with a 2-mm diameter punch in the inner 1/4 and outer 1/4 regions to 149

create full-thickness defects as a meniscal injury model (Supplemental Fig. A-E). Although the 150

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circular defect of the meniscus would not occur clinically, we investigated the perforated surface 151

of the defect in our meniscal injury model. Meniscal samples were cultured for 3 weeks in a 152

medium containing 0 or 1000 µg/mL of HA. Meniscal surfaces of the inner and outer regions 153

were evaluated as the superficial layer. The cell numbers for the meniscus superficial layer, 154

perforated surface, were calculated at a length of 200 µm and compared (at 6 locations). The 155

adjacent areas were calculated at an area of 200 × 200 µm2 and compared (at 5 areas). The 156

superficial layer and perforated surface of the meniscus were assessed by immunohistochemical 157

analyses using anti-type I (Abcam, Cambridge, UK) and anti-type II (MP Biomedical, Solon, OH, 158

USA) collagen antibodies as described.29

Anti-type I and anti-type II collagen were used at 1:500 159

and 1:100 dilutions, respectively. Images were analyzed to quantify signal density using Image J 160

software version 1.45 as previously described.30

The mean value derived from 5 different images 161

was evaluated by 2 observers. Intra- and interobserver reliabilities were excellent (ICC > 0.94). 162

Statistical analysis: Quantitative real-time PCR (four reactions for each cDNA sample), 163

immunohistochemistry (3 chambers for each detection), migration assay (4 wells for each 164

treatment) and proliferation assay (5 wells for each treatment) were repeated at least 3 times 165

independently, and similar results were obtained. Data were expressed as means with standard 166

deviations. Mean values were compared with one-way analysis of variance. Post-hoc 167

comparisons were performed using the Holm–Sidak test. Significance was set at P < 0.05. 168

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169

Results 170

HA treatment stimulates the proliferation of meniscus cells 171

Cell proliferation of both inner and outer cells was enhanced in a HA concentration-dependent 172

manner. With HA concentration of 1000 µg/mL, inner cell proliferation was 1.16 times greater 173

than that with 0 µg/mL HA. , and that with 100 µg/mL HA was 1.11 times than 0 µg/mL control 174

(Fig. 1A). In contrast, outer cell proliferation with a HA concentration of 1000 µg/mL was 1.27 175

times than that with 0 µg/mL HA (Fig. 1B). 176

177

HA increases the migration of meniscus cells 178

The migration of inner meniscus cells increased with the addition of HA. Compared with HA 179

concentration of 0 µg/mL, the migration of inner cells increased significantly in the 10, 100, and 180

1000 µg/mL groups (Fig. 2A), and the migration of outer meniscus cells significantly increased in 181

the 100 and 1000 µg/mL groups (Fig. 2B). 182

183

HA enhances the expression of COL2A1 in inner meniscus cells 184

HA did not have any effect on COL1A1 gene expression in either the inner or outer cells (Fig. 185

3A). By contrast, although HA concentration-dependent increase in COL2A1 expression was 186

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observed in the inner cells, no gene expression of COL2A1 was observed in the outer cells (Fig. 187

3A). Quantitative real-time PCR analyses revealed that COL2A1 gene expression significantly 188

increased by HA treatments (100 and 1000 µg/mL) compared with HA-free condition (P < 0.05) 189

(Fig. 3B). COL2A1 gene expression in the outer cells was not affected by HA supplementation 190

(Fig. 3C). 191

192

HA increases the cell number on the perforated surface of organ-cultured meniscus 193

HA-treated groups showed no healing responses in macroscopic observations (Fig. 4A and B). 194

On the perforated surface of the organ-cultured meniscal tissue, an increase in cell number was 195

observed in the HA-treated group in both the inner and outer regions (Fig. 4C, D, G, and H). In 196

the superficial layer, neither the inner nor outer region showed any change in cell number even 197

with HA treatment (Fig. 4E, F, I, and J). Fibroblastic cells were observed in the perforated 198

surfaces of both the inner and outer regions (Fig. 4C, D, G, and H). In both the inner and outer 199

regions, HA treatments (1000 µg/mL) increased cellular counts on the perforated surfaces (Fig. 200

5A and B). The inner and outer regions of the superficial layer showed no change in cell counts 201

(Fig. 5). In the adjacent area of the perforated surface of the inner region, HA treatment resulted 202

in significantly higher reduction in the number of cells than those without HA treatment (Fig. 5C). 203

Although no significant difference was found in the adjacent area of perforated surface of the 204

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outer region, the number of cells was lesser in the HA treatment and without HA treatment (Fig. 205

5D). No significant difference was found in the adjacent area of the perforated surface of both the 206

inner and outer regions (Fig. 5C and D). 207

208

HA induces the accumulation of type II but not type I collagen around the injured site of the 209

meniscus 210

Type I collagen deposition was not influenced by HA treatment in either the perforated surface or 211

the superficial layer of the meniscus (Fig. 6, A-H). By contrast, the perforated surface 212

demonstrated strong staining pattern with anti-type II collagen antibody after HA treatment (Fig. 213

7, A-H). According to image analysis, the staining density with anti-type I collagen antibody 214

showed no significant difference between with and without HA treatment (Fig. 8A and B), 215

whereas the staining density with anti-type II collagen antibody in the perforated surface of both 216

the inner and outer regions was significantly enhanced with HA treatment (Fig. 8C and D). 217

218

Discussion 219

In this study, the meniscus was separated into the inner and outer regions to examine the changes 220

caused by HA treatment. The proliferative and migratory abilities of both the inner and outer cells 221

were similarly enhanced by HA. HA also enhanced COL2A1 gene expression in the inner 222

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meniscus cells. Nakata et al. examined meniscus cell proliferation at several concentrations of 223

HA and observed concentration-dependent effects on cell proliferation.31

Ito et al. reported that 224

HA treatment enhanced the migratory ability of proximal tubule cells.5 In our study, cell 225

proliferation and migratory ability were activated in a HA concentration-dependent manner in 226

both the inner and outer cells. HA promotes cell proliferation, increases volume and surface area 227

for cell migration and cellular activities, and stimulates receptor-mediated events involving CD44, 228

RHAMM, and ICAM-1.32

These receptors forms a link with extracellular signal-regulated protein 229

kinase (ERK), P125fak

, and pp60c-src

. The present study did not investigate these receptors. 230

However, our results indicate the possibility that the healing process of meniscus injuries could 231

be accelerated by HA treatment. 232

In the organ-culture model, no cell proliferation was induced in the superficial layers of 233

either the inner region or the outer region. However, the increase of cell counts was observed on 234

the perforated surface after HA treatment, and cell counts decreased in the adjacent area of the 235

perforated surface in the inner region after HA treatment. Whether the cells on the perforated 236

surfaces increased in number or the meniscus cells migrated remains unclear. Extracellular HA 237

leads to its intracellular degradation via CD44 receptors.33

HA accumulation coincides with cell 238

migration and stimulates cell proliferation in vitro.32

In our study, the superficial and perforated 239

layers were both exposed to high HA concentration, but increased cell counts were observed only 240

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on the perforated surface after HA treatment. We hypothesized that unless cells are exposed from 241

the ECM due to events such as injury, the effects of HA may not be elicited. 242

No effects of HA on type I collagen syntheses were detected in both the superficial layer 243

and perforated surface of organ-cultured meniscal samples. HA was directly linked to the 244

cytokine-transforming growth factor-β1-dependent response.34, 35

Transforming growth factor-β1 245

promotes the synthesis of type II collagen and aggrecan in human meniscus cells.39

In addition, 246

the majority of meniscal ECM consists of type I collagen. The ratio of type II collagen to type I 247

collagen is 1:918

, which may have contributed to the finding that HA had no effect on the increase 248

of type I collagen production. Based on these findings, HA treatment did not lead to a detectable 249

significant increase in its production, whereas HA enhanced type II collagen deposition. In a 250

previous study, HA enhanced the expression of chondrogenic genes and blocked the expression of 251

fibrogenic genes in hyaline cartilage.36

Because the inner cells have a chondrocyte-like 252

morphology20

, we presumed that the effect of HA on the meniscus is similar to that on 253

chondrocytes. 254

This study has several limitations. HA with a mean molecular weight of 900,000 (630–255

1,170 kDa) was used in this study. The effects of NaHA have been reported to depend on the 256

molecular size in vitro.37

However, other references have indicated opposing findings in vivo.37, 38

257

Because a low-molecular-weight HA was used in the present study, comparison with a 258

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high-molecular-weight HA remains to be performed. Furthermore, changes in intracellular signals 259

by means of HA treatment were not examined. Although we observed that cell proliferative and 260

migration abilities of the inner and outer cells and COL2A1 gene expression of inner cells were 261

enhanced by HA, we did not examine the underlying mechanism of these phenomena, which 262

remains a question of future research. Finally, the study samples were older adults, showing 263

osteoarthritis effects. However, we selected the patients with sufficient articular cartilage of the 264

lateral femoral condyle and undegenerated lateral meniscus on magnetic resonance imaging. In 265

addition, the lateral meniscus was macroscopically intact. Further investigations using healthy 266

menisci or animal models will be required to understand the behavior of meniscus cells at the 267

surface of injured menisci. 268

In conclusion, this study demonstrated that HA enhanced the proliferation and migration 269

abilities of both inner and outer meniscus cells, whereas an increase in type II collagen expression 270

could be observed in inner meniscus cells. HA supplementation may possibly promote the healing 271

of the inner meniscus by inducing the proliferation, migration, and type II collagen synthesis of 272

inner meniscus cells. 273

274

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Acknowledgments 275

We thank Ms. Ami Maehara for her technical support. This study was supported by the JSPS 276

KAKENHI Grant Number 16K10904 and the Japanese Foundation for Research and Promotion 277

of Endoscopy. 278

279

Declaration of interest: 280

The authors report no conflicts of interest. The authors alone are responsible for the 281

content and writing of the paper. 282

283

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higher chondrogenic phenotype compared with outer meniscus cells. Connect Tissue Res. 325

2011;52:459–465. 326

20. Kanazawa T, Furumatsu T, Hachioji M, Oohashi T, Ninomiya Y, Ozaki T. Mechanical stretch 327

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inner meniscus cells. J Orthop Res. 2012;30:468–474. 329

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increases Smad3-dependent CCN2 expression in inner meniscus cells. J Orthop Res. 331

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2002;97:313–322. 336

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Amiel D. The effects of hyaluronan on tissue healing after meniscus injury and repair in a 338

rabbit model. Am J Sports Med. 2000;28:90–97. 339

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meniscus. Arthroscopy. 1985;1:58–62. 341

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using Photoshop and Image J. J Neurosci Methods. 2010;190:240–243. 357

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tissue engineering. Clin Orthop Relat Res. 2001;391:S208–218. 360

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matrix assembly. J Biol Chem. 1999;274:21893–21899. 365

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Figure legends 383

384

Figure 1. HA treatment stimulated the proliferation of both the inner and outer meniscus cells. 385

Compared with HA concentration of 0 µg/mL, a maximum 1.16-fold increase in the proliferation 386

of inner meniscus cells was observed with the concentrations (10 to 1000 µg/mL) of HA 387

treatment (A). Compared with HA concentration of 0 µg/mL, a maximum 1.27-fold increase in 388

the proliferation of outer meniscus cells was observed with the concentration (1000 µg/mL) of 389

HA treatment (B). *P < 0.05 compared with 0 µg/mL control. 390

391

Figure 2. HA increased the migration of the inner and outer meniscus cells. Compared with HA 392

concentration of 0 µg/mL, a maximum 2.0-fold increase in the migration of inner meniscus cells 393

was observed with the concentrations (10 to 1000 µg/mL) of HA treatment (A). Compared with 394

HA concentration of 0 µg/mL, a maximum 3.1-fold increase in the migration of outer meniscus 395

cells was observed with the concentration (1000 µg/mL) of HA treatment (B). *P < 0.05 396

compared with 0 µg/mL control. 397

398

Figure 3. HA treatment did not affect COL1A1 gene expression in cultured meniscus cells. HA 399

enhanced COL2A1 expression in the inner meniscus cells but not outer meniscus cells (A). 400

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Compared with HA concentration of 0 µg/mL, a maximum 2.4-fold increase in COL2A1 gene 401

expression was observed with HA concentration of 1000 µg/mL (B). By contrast, no significant 402

difference was observed in the outer meniscus cells (C). *P < 0.05 compared with 0 µg/mL 403

control. 404

405

Figure 4. Meniscal fragments were fully penetrated with a 2-mm diameter punch at the 1/4th of 406

the inner and outer regions (A and B). Meniscal samples were cultured for 3 weeks in a medium 407

containing HA (0 and 1000 µg/mL). White arrowheads, perforated surfaces. Black arrowheads, 408

superficial layers. Bars, 1 cm (A and B). HA increased the cell number on the perforated surface 409

of the inner and outer meniscus cells (C, D, G, and H). By contrast, the cell number in the 410

superficial layers did not change with the HA treatment (E, F, I, and J). Fibroblastic cells were 411

observed in the perforated surfaces of both the inner and outer regions (C, D, G, and H). Open 412

arrowheads indicate the meniscus cells. Bars, 50 µm (C-J). 413

414

Figure 5. HA treatment increased cell counts on the perforated surface in both the inner and outer 415

region (A and B). No change in the number of meniscus cells on the superficial layer was 416

observed with HA supplementation (A and B). In the adjacent area (200 × 200 µm2) of perforated 417

surface of inner region, HA treatment resulted in significantly higher reduction in the number of 418

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cells than without HA treatment (C). No significant difference was found in the adjacent area of 419

the perforated surface of the outer region (D). No significant difference was found in the adjacent 420

area of the superficial layer (C and D). *P < 0.05 compared with 0 µg/mL control. 421

422

Figure 6. Type I collagen deposition in the perforated meniscal samples. HA treatment did not 423

affect the distribution of type I collagen at perforated surfaces and superficial layers. 424

Immunohistological staining of the inner (A-D) and outer (E-H) regions using anti-type I 425

collagen antibody. HA-free (A, C, E, and G) and HA-supplemented (B, D, F, and H) conditions. 426

Inserted small panels indicate negative controls in the absence of the primary antibody (A, B, E, 427

and F). Open arrowheads indicate the meniscus cells. Bars, 50 µm. 428

429

Figure 7. Type II collagen deposition in the perforated meniscal samples. HA induced the 430

accumulation of type II collagen around the injured site of the inner and outer meniscus regions. 431

Inner (A-D) and outer (E-H) regions stained with anti-type II collagen antibody. Inserted small 432

panels indicate negative controls in the absence of the primary antibody (A, B, E, and F). Open 433

arrowheads indicate the meniscus cells. Bars, 50 µm. 434

435

Figure 8. Relative density of collagen deposition in perforated meniscal samples. HA treatments 436

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showed no difference in staining density of anti-type I collagen antibody between the inner and 437

outer regions (A and B). However, a significant increase in the staining density of anti-type II 438

collagen antibody on the perforated surface was observed (C and D). *P < 0.05 compared with 0 439

µg/mL control. 440

441

Supplemental Figure. Meniscal fragments (B) were obtained from the lateral meniscus (A). 442

Perforated holes (2-mm diameter) were created in both the inner and outer regions (C). Coronal 443

sections of perforated holes in the inner (D) and outer region (E). Bars, 1 cm. 444

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Fig. 1 Takaaki Tanaka

A

OD

(45

0−63

0 nm

)

0 10 100 1000

*

0

0.3

0.6

0.9

HA （μg/mL)

0

0.3

0.6

0.9

B

OD

(45

0−63

0 nm

)

0 10 100 1000HA (μg/mL)

**

Inner cells Outer cells

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HA (μg/mL)

Cel

l cou

nts

0 10 100 1000

0

100

200

300

0

50

100

150

HA (μg/mL)

Cel

l cou

nts

0 10 100 1000

A B

* * * * *


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HA (μg/mL) 0 10 100

Inner cells

1000 0 10 100

Outer cells

1000

COL1A1

COL2A1

Rel

ativ

e C

OL2

A1

leve

l(C

OL2

A1/

G3P

DH

)

0

1

2

3

4

HA (μg/mL) 0 10 100 1000

0

1

2

Rel

ativ

e C

OL2

A1

leve

l(C

OL2

A1/

G3P

DH

)

HA (μg/mL) 0 10 100 1000

G3PDH

A

B C* *


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Superficial layerPerforated surface

Inne

r re

gion

Out

er r

egio

n

C D E F

G H I J

HA 0 μg/mL HA 1000 μg/mL HA 0 μg/mL HA 1000 μg/mL

F

Outer regionBInner regionA

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Cel

l cou

nts

/ 200

μm

HA(1000 μg/mL) Superficial

layerPerforated

surface

*

Cel

l cou

nts

/ 200

μm

HA(1000 μg/mL)

0

2

4

6

8

0

2

4

6

8

A

－＋－＋－＋－＋

*

Superficiallayer

Perforatedsurface

BInner region Outer regionC

ell c

ount

s / 4

x104

μm

2


layerPerforated

surface

*

HA(1000 μg/mL)

C

－＋－＋－＋－＋

Superficiallayer

Perforatedsurface

DInner region Outer region

0

2

4

6

8

0

2

4

6

8

Cel

l cou

nts

/ 4x1

04μ

m2

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Inne

r re

gion

Out

er r

egio

n

A B C D

E F G H


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Inne

r re

gion

Out

er r

egio

n

A B C D

E F G H


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0

0.5

1

1.5

Rel

ativ

eC

OL1

den

sity


layerPerforated

surface

－＋－＋0

0.5

1

1.5

0

0.5

1

1.5

2

0

0.5

1

1.5

2

Rel

ativ

eC

OL1

den

sity


layerPerforated

surface

－＋－＋


layerPerforated

surface

－＋－＋HA(1000 μg/mL) Superficial

layerPerforated

surface

－＋－＋

Rel

ativ

eC

OL2

den

sity

Rel

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eC

OL2

den

sity

*

*

A B

C D

Inner region Outer region

Inner region Outer region

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A B

B

CE

D

D E

InnerOuter

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