SOX-9 pathway

Accepted Manuscript Hyaluronan size alters chondrogenesis of adipose-derived stem cells via the CD44/ERK/SOX-9 pathway Shun-Cheng Wu, Chung-Hwan Chen, Jyun-Ya Wang, Yi-Shan Lin, Je-Ken Chang, Mei-Ling Ho PII: DOI: Reference:

S1742-7061(17)30709-2 https://doi.org/10.1016/j.actbio.2017.11.025 ACTBIO 5178

To appear in:

Acta Biomaterialia

Received Date: Revised Date: Accepted Date:

29 June 2017 2 November 2017 7 November 2017

Please cite this article as: Wu, S-C., Chen, C-H., Wang, J-Y., Lin, Y-S., Chang, J-K., Ho, M-L., Hyaluronan size alters chondrogenesis of adipose-derived stem cells via the CD44/ERK/SOX-9 pathway, Acta Biomaterialia (2017), doi: https://doi.org/10.1016/j.actbio.2017.11.025

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.

Hyaluronan size alters chondrogenesis of adipose-derived stem cells via the CD44/ERK/SOX-9 pathway Shun-Cheng Wu1, Chung-Hwan Chen1,2,3,4, Jyun-Ya Wang1, Yi-Shan Lin1, Je-Ken Chang1,2,3,4#, Mei-Ling Ho1, 5, 6, 7,8*# 1

Orthopaedic Research Center, Kaohsiung Medical University, Kaohsiung, Taiwan Department of Orthopaedics, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan 2

3

Division of Adult Reconstruction Surgery, Department of Orthopedics, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan 4 Department of Orthopedics, Kaohsiung Municipal Ta-Tung Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan 5

Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan 6 Department of Physiology, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan 7 Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung, Taiwan 8 Department of Medical Research, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan # Mei-Ling Ho and Je-ken Chang contributed equally to the study. Corresponding author: Dr. Mei-Ling Ho, Department of Physiology, College of Medicine, Kaohsiung Medical University, No. 100, Shih-Chuan 1st Road, Kaohsiung 807, Taiwan Tel.: 886-7-3121101 (Ext. 2553); Fax: 886-7-3219452; E-mail: [email protected] Dr. Je-ken Chang, Department of Orthopaedics, College of Medicine, Kaohsiung Medical University, No. 100, Shih-Chuan 1st Road, Kaohsiung 807, Taiwan Tel.: 886-7-3121101 (Ext. 2553); Fax: 886-7-2911590 E-mail: [email protected]

1

Abstract: Hyaluronan (HA) is a natural linear polymer that is one of the main types of extracellular matrix during the early stage of chondrogenesis. We found that the chondrogenesis of adipose-derived stem cells (ADSCs) can be initiated and promoted by the application of HA to mimic the chondrogenic niche. The aim of this study is to investigate the optimal HA molecular weight (Mw) for chondrogenesis of ADSCs and the detailed mechanism. In this study, we investigated the relationships among HA Mw, CD44 clustering, and the extracellular signal-regulated kinase (ERK)/SOX-9 pathway during chondrogenesis of ADSCs. Human ADSCs (hADSCs) and rabbit ADSCs (rADSCs) were isolated and expanded. Chondrogenesis was induced in rADSCs by culturing cells in HA-coated wells (HA Mw: 80 kDa, 600 kDa and 2000 kDa) and evaluated by examining cell aggregation, chondrogenic gene expression (collagen type II and aggrecan) and sulfated glycosaminoglycan (sGAG) deposition in vitro. Cartilaginous tissue formation in vivo was confirmed by implanting HA/rADSCs into joint cavities. CD44 clustering, ERK phosphorylation, SOX-9 expression and SOX-9 phosphorylation in cultured hADSCs were further evaluated. Isolated

and

expanded

rADSCs

showed

multilineage

potential

and

anchorage-independent growth properties. Cell aggregation, chondrogenic gene expression, and sGAG deposition increased with increasing HA Mw in rADSCs. The 2000 kDa HA had the most pronounced chondrogenic effect on rADSCs in vitro, and implanted 2000 kDa HA/rADSCs exhibited marked cartilaginous tissue formation in vivo. CD44 clustering and cell aggregation of hADSCs were enhanced by an increase in HA Mw. In addition, higher HA Mws further enhanced CD44 clustering, ERK phosphorylation, and SOX-9 expression and phosphorylation in hADSCs. Inhibiting CD44 clustering in hADSCs reduced HA-induced chondrogenic gene expression. Inhibiting ERK phosphorylation also simultaneously attenuated HA-induced SOX-9 2

expression and phosphorylation and chondrogenic gene expression in hADSCs. Our results indicate that HA initiates ADSC chondrogenesis and that higher Mw HAs exhibit stronger effects, with 2000 kDa HA having the strongest effect. These effects may be mediated through increased CD44 clustering and the ERK/SOX-9 signaling pathway. Key Words: Hyaluronan (HA), Molecular weight (Mw), Adipose-derived stem cells (ADSCs), Chondrogenesis, CD44 clustering, ERK/SOX-9.

3

Introduction: Damaged articular cartilage has a limited capacity for self-repair [1]. Cell-based tissue engineering provides a novel therapy for cartilage defects [1], and attempts have been made to regenerate cartilage and repair defects using in vitro expanded chondrocytes or undifferentiated mesenchymal stem cells (MSCs). Due to the poor proliferation capacity and de-differentiation of chondrocytes caused by in vitro expansion, using MSCs as the cell source is becoming an important approach for articular cartilage tissue engineering [2-4]. Adipose-derived stem cells (ADSCs) are thought to be beneficial for articular cartilage tissue engineering due to their ease of harvest, high ADSCs yield rates, and chondrogenic differentiation potential [5-7]. Chondrogenesis is a complex process that is initiated by condensation (aggregation) of undifferentiated mesenchymal cells followed by the formation of cartilage in the extracellular matrix [4, 8, 9]. Chondrogenic induction for ADSCs is important in ADSC-based tissue engineering for articular cartilage [1]. The niche interactions for stem cell differentiation include growth factors, cell-cell contacts, and cell-matrix adhesions [10]. An appropriate interplay of the niche components can be utilized to induce chondrogenesis of ADSCs and thus produce cartilage tissue. Hyaluronan (HA) is one of the main extracellular matrices in articular cartilage as well as in mesenchyme during the early stage of chondrogenesis [8, 11-16]. We found 4

that ADSC chondrogenesis can be initiated and promoted with the use of HA substrate to mimic the chondrogenic niche and that this effect is mediated by CD44 [17, 18]. Cell aggregation, chondrogenic gene expression (collagen type II & aggrecan), and cartilaginous matrix formation were increased when ADSCs were cultured on HA substrate compared with a petri dish without HA [17, 18]. HA is a natural linear polymer that is thought to be beneficial for stem cell-based articular cartilage tissue engineering, but the most effective HA molecular weight (Mw) for the HA/CD44 interaction is still unclear. In addition, the mechanism of HA/CD44 signaling is not well-defined. The surface antigen CD44 is the main HA receptor, and the interaction between HA and CD44 on chondrocytes is crucial to maintaining cartilage homeostasis [19-22]. Previous reports have indicated that changing the molecular weight (Mw) of HA alters its biological effects, including extracellular matrix homeostasis, angiogenesis, and immunosuppression [23-27]. The interaction between CD44 and HA has also been shown to be influenced by HA Mw, and clustering of CD44 is a typical characteristic that affects the immune cell response to HA binding [28-31]. Based on these previous reports, we propose that changing HA Mw may alter CD44 clustering and subsequently affect chondrogenesis in ADSCs. Sry-type HMG box-9 (SOX-9) is required for chondrogenic differentiation and 5

cartilage formation [32-34], is one of the earliest markers expressed in cells undergoing precartilaginous condensation, and functions as a transcription factor to directly regulate the expression of cartilaginous-specific matrices including collagen type II and aggrecan [32, 34-38]. In addition, the extracellular signal-related kinase (ERK) pathway is a signaling cascade activated by various extracellular stimuli including HA [39-41]. The ERK pathway has been indicated to be involved in chondrogenesis in MSCs and chondrogenic cells [39, 40]. Another study showed that SOX-9 expression can be regulated by the ERK pathway in pluripotent stem cells and chondrocytes [42]. Therefore, ERK/SOX-9 signaling may be involved in HA/CD44-induced chondrogenesis in ADSCs. Accordingly, in this study, we hypothesize that the chondrogenesis of ADSCs induced by different Mw HA niches may alter CD44 clustering and ERK/SOX-9 signaling and eventually induce ADSC chondrogenesis. To prove the hypothesis, human ADSCs (hADSCs) and rabbit ADSCs (rADSCs) were isolated and characterized. The adherent and expanded rADSCs isolated from rabbit adipose tissue showed multilineage differentiation potential. We first evaluated the effect of different Mws of HA on the chondrogenesis of rADSCs in vitro. Cartilaginous tissue formation of rADSCs induced by HA in a joint cavity was also confirmed. Furthermore, the effects of different HA Mws on CD44 clustering, ERK phosphorylation, and SOX-9 6

levels in hADSC chondrogenesis were examined.

7

Materials and Methods:

Materials

Sodium hyaluronan with Mws of 2000 kDa and 80 kDa (FCH-200, Mw=1800~2200 kDa, average Mw is 2,000 kDa; FCH-SU, Mw=50~110 kDa, average Mw is 80 kDa) was purchased from Kikkoman Co (Japan). Sodium hyaluronan with a Mw of 600 kDa (Hyalgan®, Mw=500~730 kDa, average Mw is 600 kDa) was purchased from Fidia Farmaceutici S. p. A (Italy). All chemicals were purchased from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA) unless otherwise specified.

Isolation and culture of rabbit adipose-derived stem cells (rADSCs) or human adipose-derived stem cells (hADSCs)

The method for the isolation and characterization of rADSCs and hADSCs was based on a previous report [43] with some modifications. For isolation of rADSCs, male New Zealand White rabbits weighing 2.5–3 kg were used. The animal study was approved by the Animal Experiment Committee of Kaohsiung Medical University, Taiwan. The rabbits were first anesthetized with xylazine (5 mg/kg) and ketamine (45 mg/kg) [44]. The anesthesia was supplemented with a subcutaneous injection of 0.5% lidocaine hydrochloride. Then, 3 g of subcutaneous adipose tissue was excised 8

from the groin area of each rabbit. For isolation of ADSCs from humans, hADSCs were isolated from subcutaneous adipose tissue as previously described [45]. Human adipose tissue was obtained from patients with approval from the ethical committee at the Kaohsiung Medical University hospital (KMUH-IRB-20120372). After obtaining informed consent, subcutaneous adipose tissue from the gluteal area was taken from patients during orthopedic surgery. In this study, the human adipose tissues were isolated from six donors and the rabbit adipose tissues were isolated from six rabbits. After 3 g of subcutaneous adipose tissue was isolated from rabbits or humans, the adipose tissues were minced with scissors. The minced adipose tissues from rabbits or humans were digested with 1 mg/ml type IA collagenase (125 units/mg) (catalog number: 17100017; Thermo Scientific) at 37°C under 5% CO2 for 24 h. The digested tissue was centrifuged at 1000 rpm for 5 min, and the pellet was washed twice with PBS. The pellet was then resuspended in K-NAC medium and plated in a 100-mm culture dish. The K-NAC medium, which is a low calcium medium supplemented with a glutathione-enhancing agent and antioxidants, was developed for culturing ADSCs [43]. The K-NAC medium used in this study helps isolate and expand ADSCs according to previous reports [43, 45-47]. The K-NAC medium was composed of Keratinocyte-SFM Basal Medium (Gibco-BRL, Rockville, MD) supplemented with 25 mg Bovine Pituitary Extract (BPE) (Gibco-BRL, Rockville, MD), 2.5 µg human 9

recombinant Epidermal Growth Factor (rEGF) (Gibco-BRL, Rockville, MD), 2 mM N-acetyl-L-cysteine, 0.2 mM L-ascorbic acid 2-phosphate sesquimagnesium salt, and 5% fetal bovine serum (FBS) [43]. The medium was refreshed 24 h after the initial plating and the unattached cells were removed by washing with PBS. The medium was changed every 2 days thereafter, and the cells were allowed to grow until near confluence. After 1 week, sufficient cells were generated for trypsinization and storage in liquid nitrogen or for subculturing.

In vitro differentiation assay of isolated rADSCs

To induce chondrogenic differentiation, rADSCs were suspended in basal medium (Dulbecco's modified Eagle's medium supplemented with 10% FBS, 1% non-essential amino acids, and 100 U/ml penicillin/streptomycin (Gibco-BRL, Grand Island, NY)) at a cell density of 1×107 cells/ml, and then, 10 µl of cell suspension was placed in the center of each well in a 24-well plate. The cell suspension was incubated at 37°C for 2.5 h to form a pelleted micromass and then cultured in 2 ml of chondrogenic medium (basal medium supplemented with insulin (catalog number: I1882) (6.25 μg/mL), transforming growth factor β1 (catalog number: T1654) (TGF-β1; 10 ng/mL), and ascorbic acid-2-phosphate (catalog number: A8960) (50 μM)) for 2 weeks [43]. The medium was changed every 2 days, and cartilage-specific

10

matrix deposition was assessed at the end of 2 weeks post-induction. The pellets were fixed with 2% paraformaldehyde for 15 min and then stained with 1% Alcian blue for 20 min [43].

To induce adipogenic differentiation, 1×104 rADSCs per well were plated onto a 24-well plate, and the medium was exchanged with adipogenic differentiation medium. The rADSCs were incubated for 2 days in IDI-I differentiation medium (basal medium supplemented with indomethacin (catalog number: I8280) (1 μM), dexamethasone (catalog number: D8893) (1 μM), IBMX (catalog number: I7018) (500 μM), and insulin (catalog number: I1882) (10 μg/ml)) and then the medium was replaced with I medium (basal medium supplemented with insulin (10 μg/ml)) for 1 day. This treatment was repeated for 2 weeks. For visualization of lipid vacuole accumulation, the cells were fixed with 2% paraformaldehyde and stained with Oil Red O for 20 min at room temperature [43].

To induce osteogenic differentiation, 1×104 rADSCs per well were plated onto a 24-well-plate, and the medium was exchanged with osteogenic differentiation medium (basal medium supplemented with L-ascorbic acid-2-phosphate (catalog number: A8960) (50 μM), β-glycerophosphate disodium (catalog number: G9891) (10 mM) and dexamethasone (catalog number: D8893) (0.1 μM). The medium was

11

changed every 2 days for 2 weeks. Deposition of calcium in a calcified matrix was stained with von Kossa staining to assess osteogenic differentiation [43].

Anchorage-independent growth of rADSCs

A total of 5×105 rADSCs in 3 ml of K-NAC containing 0.33% agarose were plated on top of 3 ml of prehardened 0.5% agarose in 6-cm culture dishes. Then, 2.5 ml of K-NAC medium was added and renewed once every 3 days. At the end of 2 weeks, the colony developed by each rADSC was observed with microscopy [43].

Cell culture on HA-coated wells

HA dissolved in distilled water (average Mw of HA: 80 kDa, 600 kDa and 2000 kDa) was coated on 6-well plates (0, 0.05 mg/cm2 ) for 48 h at 37°C, followed by two washes with PBS [17]. The HA substrate remaining on the cell culture plates was confirmed using Alcian blue staining (Supplementary Fig. S1). The ADSCs were seeded at a density of 4×105 cells/2 ml of basal medium consisting of Dulbecco's modified Eagle's medium supplemented with 10% FBS, 1% non-essential amino acids, and 100 U/ml penicillin/streptomycin (Gibco-BRL, Grand Island, NY). The ADSCs cultured in HA-coated wells were divided into four groups: the Control group, ADSCs cultured in wells without HA coating; the 80 kDa group, ADSCs cultured in wells coated with HA (Mw: 80 kDa); the 600 kDa group, ADSCs cultured in wells coated 12

with HA (Mw: 600 kDa); and the 2000 kDa group, ADSCs cultured in wells coated with HA (Mw: 2000 kDa). The medium was changed every 2 days until the cells were harvested. At every indicated time interval, cells were collected for further experimental analysis.

RNA isolation and quantitative real-time polymerase chain reaction (real-time PCR)

At the indicated time points, ADSCs were collected from the 6-well plate in each experimental group. TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was used to extract the total RNA from these cells following the manufacturer’s instructions. The RNA quality was confirmed by detecting the absorbance ratio at 260 nm and 280 nm using a Thermo Scientific NanoDropTM 1000 Spectrophotometer (Thermo Fisher Scientific). Subsequently, 0.5-1 μg of total RNA per 20 μl of reaction volume was reverse transcribed into cDNA using a SuperScript® III First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA). Real-time PCR reactions were performed and monitored using iQTM SYBR Green® Supermix (Bio-Rad Laboratories Inc., Hercules, CA) and a quantitative real-time PCR detection system (Bio-Rad Laboratories Inc., Hercules, CA). The cDNA samples (2 µl of samples in a total volume of 25 µl per reaction) were analyzed for the genes of interest [17, 18, 48], including human

13

collagen

type

II

(hCOL-II),

human

aggrecan

(hAGG),

and

human

glyceraldehyde-3-phosphate-dehydrogenase (hGAPDH) (Table 1), rabbit collagen type II

(rCOL-II),

rabbit

aggrecan

(rAGG),

and

rabbit

glyceraldehyde-3-phosphate-dehydrogenase (rGAPDH) (Table 2). After the real-time PCR reaction, a dissociation (melting) curve was generated to determine the specificity of the reaction. The relative mRNA expression levels of each target gene were calculated from the threshold cycle (Ct) value of each PCR product and normalized to the expression of GAPDH using the comparative Ct method [49]. For each gene of interest, the readings of each experimental group at every indicated time point were collected.

Chemical cross-linking for CD44 clustering at the cell surface

A protein chemical cross-linking method can be used to assess CD44 clustering [50, 51]. At the indicated time point, the hADSCs in each group were washed 2 times with ice-cold PBS (20 mM sodium phosphate, 0.15 M NaCl, pH 8.0). CD44 cross-linking was performed by incubation with 2 mM bis(sulfosuccinimidyl) suberate (BS3) (Pierce) for 1 h at 4°C and quenched by incubation with 20 mM Tris, pH 7.5, for 15 min at room temperature [50, 51]. Cells were washed twice with PBS and lysed with cell lysis buffer for further western blot analysis.

14

Western blot analysis

At each indicated time point, the cells from each group were washed twice with ice-cold PBS with 1 mM sodium vanadate and lysed in modified radio immunoprecipitation assay buffer (RIPA; 150 mM NaCl, 1 mM EGTA, 50 mM Tris, pH 7.4, 10% glycerol, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) containing a protease inhibitor cocktail (Complete Protease Inhibitor Cocktail Tablets; Roche Diagnostics Ltd., Taiwan) and 1 mM sodium vanadate. The lysates were cleared by centrifugation at 14,000 rpm for 15 min at 4°C. The expression levels were analyzed by western blot using antibodies against CD44 (catalog number: 5640; Cell Signaling, Danvers, MA, USA), SOX-9 (catalog number: ab76997; Abcam, Cambridge, UK), phospho-SOX9 (phospho-S181) (p-SOX-9) (catalog number: PA5-35636; Thermo Scientific), GAPDH (catalog number: MA5-15738; Thermo Scientific), phospho-p44/42 MAPK (Erk1/2) (p-ERK) (catalog number: 4370; Cell Signaling, Danvers, MA, USA), and p44/42 MAP Kinase (ERK) (catalog number: 4696; Cell Signaling, Danvers, MA, USA), and they were monitored using enhanced chemiluminescence analysis (ECL system; Amersham).

Alcian blue staining for the detection of sulfated glycosaminoglycan (sGAG) deposition

15

To assess the presence of sulfated glycosaminoglycan (sGAG) deposition, the ADSCs from each group were cultured for 5 days. Before the Alcian blue staining, the ADSCs were washed at least three times with PBS to remove the HA residue remaining on the tissue culture plate and were then fixed overnight using 4% paraformaldehyde in PBS. Staining was accomplished by applying a solution of 0.1% Alcian blue 8 GX in 0.1 M HCl to the cells for 2 h at room temperature to assess sGAG deposition. Dimethylmethylene blue (DMMB) assay for quantification of

sulfated

glycosaminoglycan (sGAG) deposition DMMB reacts with the sulfate group of the GAG chain and, therefore, will not react with unsulfated GAGs such as HA [52]. Therefore, the DMMB assay was used to quantify sGAG deposition by ADSCs. At each indicated time point, the DNA content and sGAG deposition in the wells were quantified spectrofluorometrically using 33258 Hoechst dye and dimethylmethylene blue (DMMB), respectively [52, 53]. A standard curve for the DMMB assay was generated using an aqueous solution of chondroitin sulfate C (Sigma-Aldrich, St. Louis, MO, USA), with concentrations ranging from 0 to 25 μg/μl.

Inhibition of the activation of ERK

16

For the ERK inhibition study, the MAP/ERK inhibitor U0126 (Cell Signaling, Danvers, MA, USA) was used at a concentration of 10 μM [54]. The hADSCs were cultured in basal medium with/without U0126 at a concentration of 10 μM for 30 min. After 30 min, the ADSCs were washed with PBS. The hADSCs were resuspended in basal medium with/without U0126 at densities of 4×105 cells/2 ml, and then seeded on HA-coated wells. Control cultures were supplemented with 0.01% dimethyl sulfoxide (DMSO) alone because the inhibitors were dissolved in DMSO.

Inhibition of CD44 clustering

For the inhibition of CD44 clustering study, nystatin was used at a concentration of 50 μg/ml [55]. ADSCs were cultured in basal medium with/without nystatin at a concentration of 50 μg/ml for 30 min. After 30 min, the ADSCs were washed with PBS, resuspended in basal medium with/without nystatin at densities of 4×105 cells/2 ml, and then seeded on HA-coated wells. Control cultures were supplemented with 0.01% DMSO alone because the inhibitors were dissolved in DMSO.

Cartilaginous tissue formation of rabbit ADSCs with HA in joint cavities

To investigate the effect of HA on cartilaginous tissue formation of rADSCs in a joint cavity, rADSCs suspended within HA (Mw of HA: 600 kDa or 2000 kDa) or PBS were first encapsulated in fibrin hydrogel and then implanted in the joint cavity 17

of a rabbit knee. For encapsulation of rADSCs in HA, the rADSCs were first mixed in 1% HA solution or in PBS, and then, every 30 l of rADSCs (5×106 cells/30 l) suspended in HA or PBS was mixed with 120 l of fibrin solution (100 mg/ml in PBS) and placed in a Teflon mold 5.5 mm in depth and 5.5 mm in diameter. To the mold, 40 l of bovine thrombin (300 U/ml) in 40 mM CaCl2 was added and was mixed well with the cell/fibrin solution. This mixture was incubated at room temperature for 15 min to form a hydrogel. The rADSCs with HA or PBS in fibrin hydrogels were divided into three groups: the Control group, rADSCs mixed with PBS then encapsulated in fibrin hydrogel; the 600 kDa group, rADSCs mixed with HA (Mw: 600 kDa); and the 2000 kDa group, rADSCs mixed with HA (Mw: 2000 kDa) and then encapsulated in fibrin hydrogel. After hydrogel formation, the hydrogels from each group were removed from the Teflon molds and implanted into the joint cavity immediately. For joint cavity implantation, 12 male New Zealand White rabbits weighing 2.5–3 kg were used (four rabbits in each group). The rabbits were first anesthetized as previously described, and the joint cavity of the knee was opened. The fibrin hydrogels of the three groups were first washed with sterile PBS twice and then implanted into the joint cavity of the knee. After 3 weeks, the rabbits were sacrificed with an overdose of inhaled CO2 at the same time point. The cartilaginous tissue formation of implanted HA/fibrin hydrogels was collected for histological analysis. 18

For evaluation of cartilaginous tissue formation, the neocartilage from each group was removed, embedded in paraffin and cut into 5-μm sections. The sections were stained with Safranin O to assess sGAG synthesis and were counterstained with Fast Green for background. sGAG was stained red by Safranin O, and the total and red-stained areas in the neocartilage tissues of the 3 groups were measured using Image-Pro Plus software, version 5.0 (Media Cybernetics, Silver Spring, MD, USA). The average relative sGAG staining intensity (red staining intensity) was determined. Compared to the sGAG content in the Control group, the sGAG content in the HA groups was calculated using the following formula: the sGAG content in the neocartilage tissue (relative to the Control group)=average sGAG staining intensity of the neocartilage tissue in the individual group/average sGAG staining intensity of the neocartilage tissue in the Control group (Supplementary Fig. S3).

Statistical analysis

The data are expressed as the means ± standard error of the mean (SEM) of the data from each experiment. Statistical significance was evaluated by one-way analysis of variance (ANOVA), and multiple comparisons were performed using Scheffe’s method. A p<0.05 was considered significant.

19

Results: Isolated rabbit ADSCs show multilineage differentiation potential and anchorage-independent growth capability The multipotent ability of hADSCs to differentiate into osteoblasts, adipocytes and chondrocytes was confirmed in our previous unpublished data (Supplementary Fig. S2). Here, we further tested the isolated rADSCs. To determine the multilineage potential of isolated rADSCs, the multilineage differentiation potential was tested. After osteogenic induction, calcium deposits in the rADSCs were stained with von Kossa (Fig. 1A). After chondrogenic induction, cartilage nodules appeared in the rADSCs and were positively stained with Alcian blue (Fig. 1A). After adipogenic induction, the rADSCs also developed Oil Red O-positive lipid droplets (Fig. 1A). These results showed that the isolated rADSCs had multilineage differentiation potential. The ability of the isolated rADSCs to grow in soft agar was also tested. rADSCs cultured in soft agar showed colony formation after 14 days of culture (Fig. 1B). These results indicated that isolated rADSCs exhibited MSC characteristics. Increase in the Mw of HA enhances chondrogenesis of rADSCs in vitro To test whether altering the Mw of HA affects chondrogenesis of ADSCs in vitro, the rADSCs were cultured in non-coated (Control group) or HA-coated wells 20

(80 kDa, 600 kDa or 2000 kDa groups). The cell aggregation, chondrogenic gene expression and cartilaginous matrix synthesis of rADSCs in these four groups were compared. The results of cell aggregation, chondrogenic gene expression and sGAG deposition of the Control, 80 kDa, 600 kDa and 2000 kDa groups are shown in Figure 2. Obvious cell aggregation was observed 4 h after rADSCs were cultured in HA-coated wells, and no cell aggregation was found in the Control group. More pronounced rADSC cell aggregation was found in the 80 kDa, 600 kDa and 2000 kDa groups in comparison with the Control group (Fig. 2A). Real-time PCR was used to confirm the expression of chondrogenic genes when rADSCs were cultured in non-coated (Control group) or HA-coated wells (80 kDa, 600 kDa or 2000 kDa groups). The real-time PCR results showed that the expression of chondrogenic genes (rCOL-II and rAGG) in rADSCs was increased after culture in HA-coated wells for 24 h. Chondrogenic gene expression (rCOL-II and rAGG) was enhanced in rADSCs in the 80 kDa, 600 kDa and 2000 kDa groups in comparison with the Control group (Fig. 2B). Enhanced chondrogenic gene expression, including rCOL-II and rAGG, was found to accompany the increase in HA Mw. The chondrogenic gene expression of rADSCs in the 2000 kDa group showed the most pronounced chondrogenic gene expression 24 h after culture on HA-coated wells (Fig. 2B). 21

Alcian blue staining and a DMMB assay were used to confirm the sGAG deposition of rADSCs cultured in non-coated (Control group) or HA-coated wells (80 kDa, 600 kDa or 2000 kDa groups). The Alcian blue staining results showed more intense sGAG deposition by rADSCs cultured in the 3 HA-coated groups for 5 days in comparison to the Control group (Fig. 2C). The DMMB assay quantification results showed that the average amount of sGAG (sGAG/DNA) produced by rADSCs was significantly increased in the 3 HA-coated groups (Fig. 2C). Of the HA-coated groups, the sGAG/DNA synthesis of rADSCs in the 2000 kDa group showed the most pronounced sGAG deposition after 5 days of culture (Fig. 2C). Overall, these results suggest that an increase in HA Mw enhances chondrogenesis of rADSCs in vitro. The 2000 kDa HA induces cartilaginous tissue formation of rADSCs in vivo To confirm whether HA induces cartilaginous tissue formation of rADSCs in vivo, the cartilaginous tissue formation of rADSCs with HA was tested in a joint cavity. The rADSCs were mixed with PBS (Control group) or HA (2000 kDa group), encapsulated within fibrin hydrogel and then implanted into the joint cavity of the knee for 3 weeks. After 3 weeks of implantation, the neocartilage formations were harvested and analyzed. The sGAG synthesis of implanted rADSCs with HA were stained with Safranin O staining. The results of neocartilage formation of rADSCs in the Control and 2000 kDa groups are shown in Figure 2D&2E. The results showed 22

that the cartilaginous tissue formation in the 2000 kDa groups was enhanced compared with that in the Control group after 3 weeks of implantation (Fig. 2D). The Safranin O staining results showed more intense sGAG staining in the 2000 kDa group than in the Control group (Fig. 2E). The quantitative results of the sGAG content analysis in the neocartilage tissues among all the groups also showed that the 2000 kDa group had a more pronounced effect on cartilaginous tissue formation than the Control and 600 kDa groups (Supplementary Fig. S3). These results suggest that 2000 kDa HA induces cartilaginous tissue formation of rADSCs in vivo. The hADSC aggregation enhanced by HA Mw is accompanied by CD44 clustering To investigate whether the effects of HA Mw on the chondrogenesis of hADSCs are accompanied by a change in CD44 clustering, hADSCs were cultured in non-coated (Control group) or HA-coated wells (80 kDa, 600 kDa or 2000 kDa groups). Cell aggregation and the protein levels of CD44 clustering in hADSCs were evaluated. The results of the cell aggregation and the protein levels of CD44 clustering analyses in hADSCs of the Control, 80 kDa, 600 kDa and 2000 kDa groups are shown in Figure 3. The onset of cell aggregation 2 h after cell seeded was found to be accompanied by clustering of CD44 in hADSCs (Fig. 3A&B). The CD44 clustering in hADSCs in the HA-coated groups was higher than that in the Control 23

group (Fig. 3B). The results showed that cell aggregation and CD44 clustering of hADSCs were increased along with the increase in HA Mw. Phosphorylation levels of ERK and SOX-9 in hADSCs increased with the increase in HA Mw To investigate whether HA Mw affects the phosphorylation level of ERK or SOX-9, the hADSCs were cultured in non-coated (Control group) or HA-coated wells (80 kDa, 600 kDa or 2000 kDa groups). The protein levels of phosphorylated ERK, SOX-9 and p-SOX-9 of hADSCs were evaluated. Accompanied by the increase in HA Mw, the phosphorylation of ERK in hADSCs was enhanced at 2 h and 24 h after cell seeding (Fig. 4A). An increase in SOX-9 expression and phosphorylation in hADSCs was also detected in the 80 kDa 600 kDa and 2000 kDa groups in comparison to the Control group at 24 h (Fig. 4B). Increased SOX-9 phosphorylation in hADSCs was also detected in the 80 kDa, 600 kDa and 2000 kDa groups in comparison to the Control group at 24 h (Fig. 4C). The results showed that the phosphorylation of ERK, SOX-9 and p-SOX-9 in hADSCs was increased along with the increase in HA Mw. Moreover, p-SOX-9 in hADSCs was also increased upon HA treatment. Inhibition of CD44 clustering by nystatin inhibited ERK phosphorylation and chondrogenic gene expression induced in ADSCs by the increase in HA Mw 24

To investigate whether CD44 clustering contributes to chondrogenesis of ADSCs induced by HA with increased Mw, the hADSCs were pretreated with or without nystatin before being cultured in non-coated (Control group) or HA-coated wells (80 kDa, 600 kDa or 2000 kDa groups), and the ERK phosphorylation and expression of chondrogenic genes in hADSCs were evaluated. The results showed that nystatin pretreatment decreased ERK phosphorylation and chondrogenic gene expression in hADSCs. The increased ERK phosphorylation in hADSCs induced by HA was decreased by nystatin treatment (Fig. 5A). Decreased ERK phosphorylation in the 2000 kDa group of hADSCs was found when cells were pretreated with nystatin (Fig. 5A). Increased chondrogenic gene expression induced by HA with increased Mw in hADSCs was found (Fig. 5B). Decreased collagen type II expression and aggrecan expression was also observed in the 80 kDa, 600 kDa and 2000 kDa groups of hADSCs after pretreatment with nystatin (Fig. 5B). These results suggest that the CD44 clustering in hADSCs contributes to ERK phosphorylation and chondrogenesis induced in hADSCs by HA with increased Mw. Inhibition of ERK phosphorylation inhibited the SOX-9 and chondrogenic gene expression in ADSCs induced by increased Mw HA To test whether phosphorylation of ERK alters SOX-9 levels and chondrogenesis of hADSCs in response to HA, we inhibited phosphorylation of ERK 25

in hADSCs and then measured the SOX-9 levels and chondrogenic gene expression in hADSCs. U0126 was used to inhibit the phosphorylation of ERK. The results showed that inhibition of ERK phosphorylation in hADSCs induced by HA Mw decreased SOX-9 phosphorylation and expression (Fig. 6A). The expression of chondrogenic genes in hADSCs induced by HA Mw also decreased when ERK phosphorylation was inhibited in hADSCs (Fig. 6B). These results indicated that inhibition of the ERK phosphorylation induced by HA decreased SOX-9 expression and phosphorylation as well as the expression of chondrogenic genes in hADSCs.

26

Discussion: HA is a high molecular weight polysaccharide that consists of repeating units of glucuronic acid and N-acetylglucosamine, and it has been studied as a scaffold for carrying stem cells [47, 56-59]. The biological effects of HA are altered with changes in its molecular weight (Mw); however, whether the Mw of HA affects chondrogenesis of ADSCs is still not well understood. The purpose of this study was to investigate the signal transduction mechanism of the influence of HA Mw on chondrogenesis of ADSCs. In this study, we found that with an increase in the Mw of HA (80 kDa~2000 kDa) the chondrogenesis of ADSCs increases, including initial cell aggregation, expression of chondrogenic genes and sGAG deposition in vitro (Fig. 2A-C). The best HA Mw for ADSC chondrogenesis is 2000 kDa, and in vivo implantation of 2000 kDa HA/ADSCs also showed obvious cartilaginous tissue formation (Fig. 2D&E). More importantly, we found that 2000 kDa HA had the best effect on CD44 clustering, ERK phosphorylation and SOX-9 expression and phosphorylation in ADSCs (Fig. 3&4). Suppression of CD44 clustering significantly decreased HA-induced ERK phosphorylation and chondrogenic gene expression in hADSCs (Fig. 5). Furthermore, suppression of ERK phosphorylation also significantly decreased both the HA-induced SOX-9 levels and chondrogenic gene expression in ADSCs (Fig. 6). The HA Mw-dependent changes in CD44 clustering,

27

ERK phosphorylation and SOX-9 expression and phosphorylation were abrogated by both blockade of CD44 clustering and ERK phosphorylation. Based on these results, we suggest that different Mws of HA alter its chondrogenic effect on ADSCs by altering CD44 clustering and the ERK/SOX-9 signaling pathway. Adipose tissue consists of adipocytes, fibroblasts, and vascular smooth muscle cells as well as stem cells. High calcium concentrations are known to cause cell differentiation [60, 61]. The K-NAC medium, which is a low-calcium medium supplemented with a glutathione-enhancing agent and antioxidants, has been used to isolate and expand hADSCs [43]. In this study, we further used K-NAC medium to isolate and expand rADSCs, and the stem cell properties were also tested. The multipotent ability of ADSCs to differentiate into osteoblasts, adipocytes and chondrocytes is one of thecriteria that defines MSCs [43, 62]. To confirm that rADSCs were successfully isolated from rabbit adipose tissues, we evaluated both the multipotent differentiation ability and anchorage-independent growth of rADSCs isolated from rabbit adipose tissues. In this study, adherent and expanded cells isolated from rabbit adipose tissues showed multilineage differentiation potential (Fig. 1A). Expression of specific surface antigens on the cell surface is another important criterion that defines MSCs [62]. Due to the lack of commercially available antibodies for rabbit surface antigens, it is difficult to analyze the surface antigens expressed on 28

isolated rADSCs. The ability to grow in soft agar and anchorage-independent growth are also characteristic of stem cells [43, 63]. Therefore, the anchorage-independent growth of rADSCs was tested to confirm that the isolated rADSCs possess stem cell properties (Fig. 1B). In articular cartilage homeostasis, a report indicated that HA Mw in synovial fluid is changed in association with pathogenic progression of articular cartilage [64]. The Mw of HA in articular cartilage is age-related, HA from young people is approximately 2000 kDa, and this Mw decreases with age [65]. Therefore, we tested the effects of HA Mw on chondrogenesis of ADSCs by culturing cells in HA with Mws ranging from 80 kDa to 2000 kDa. Chondrogenesis is a complex process that is initiated by mesenchymal stem cell condensation (aggregation) and subsequent expression of collagen type II and aggrecan [4, 8, 9, 34, 66, 67]. The normal functional extracellular matrix of articular cartilage is mainly composed of sGAG and collagen type II [68]. To investigate the chondrogenic effect of HA Mw on ADSCs, we tested cell aggregation, chondrogenic gene expression and sGAG deposition of ADSCs in vitro. We found that with an increase in HA Mw cell aggregation increased at 4 h; the mRNA expression of collagen type II and aggrecan in rADSCs increased at 24 h (Fig. 2A & 2B); and sGAG deposition also increased on day 5 (Fig. 2C). Overall, we suggest that HA with a Mw of 2000 kDa showed the most pronounced 29

chondrogenic effect on ADSCs. Limitations of methods to investigate the neo-cartilaginous tissue development of MSCs in vitro have been reported, such as instability of the chondrocyte phenotype and lack of extracellular matrix secretion [69-71]. Synovial fluid in the joint cavity is one of the tissue environmental factors that induces MSC chondrogenesis [72, 73]. To investigate the cartilaginous tissue formation of rADSCs induced by HA, we implanted an HA/ADSC construct into the knee joint cavity of rabbits. The primary constituents of extracellular matrices of articular cartilage are collagen type II and sulfated glycosaminoglycan [68]. However, it is difficult to analyze synthesized collagen type II from implanted rADSCs due to a lack of commercially available antibodies targeting rabbit collagen type II. Therefore, we examined sulfated glycosaminoglycans synthesized by implanted rADSCs with HA by using Safranin O staining. Our results showed that 2000 kDa HA-induced cartilaginous tissue development of rADSCs in the joint cavity (Fig. 2D&E). This confirms that 2000 kDa HA can be used to enhance cartilaginous tissue formation of ADSCs in vivo. Recent studies have shown that CD44 plays an important role in cell differentiation, and HA binding with CD44 mediates cellular activation [74, 75]. We previously also found that the HA microenvironment initiates cell aggregation and promotes chondrogenesis of hADSCs, and blockade of HA binding with CD44 30

reduces the chondrogenic effect on hADSCs [17, 18]. Prechondrogenic condensation of MSCs (cell aggregation) is the initial stage of chondrogenesis [8, 76]. Clustering of CD44 upon HA stimulation is a crucial process, and dimerization of CD44 on the cell membrane is used to evaluate CD44 clustering [28-31, 77]. A protein chemical cross-linking method that can be observed with western blot analysis was used to assess CD44 dimerization [50, 51]. We found that CD44 dimerization in hADSCs increased along with the HA-induced cell aggregation of hADSCs at 2 h (Fig. 2A&B). Inhibition of CD44 clustering by nystatin decreased chondrogenic gene expression in hADSCs (Fig. 5B). In this study, we found that dimerization of CD44 in ADSCs is responsive to HA treatment and contributes to chondrogenic differentiation of ADSCs. CD44 clustering has been shown to lead to ERK phosphorylation and increase SOX-9 protein levels in primary chondrocytes [42, 51]. SOX-9 is a transcription factor that regulates collagen type II and aggrecan expression in human MSCs [40]. Other studies have shown that ERK has either promotive or inhibitory effects on chondrogenic differentiation [40, 78, 79]. In this study, we found that phosphorylated ERK was increased by an increase in HA Mw from 2 h to 24 h (Fig. 4A), and inhibition of CD44 clustering by nystatin reduced the HA Mw-induced ERK phosphorylation and chondrogenesis of ADSCs (Fig. 5 A&B). Our results showed that 31

CD44 clustering up-regulates ERK phosphorylation. Moreover, consistent with previous findings on ERK [40, 42, 51], our results also showed that ERK positively regulates SOX-9 expression and chondrogenic differentiation in ADSCs. The expression level of not only SOX-9 but also phosphorylated SOX-9 can regulate chondrogenesis [80]. Binding of HA with CD44 has been indicated to increase parathyroid hormone-related peptide (PTHrP) expression, which enhances SOX9's ability to transactivate chondrogenic genes by phosphorylating SOX9 [81-83]. To explore whether ERK phosphorylation-mediated SOX-9 expression and phosphorylation is necessary for HA Mw-induced chondrogenesis of hADSCs, we further tested whether the ERK antagonist U0126 blocked the regulatory effect of SOX-9 on chondrogenic gene expression in hADSCs. We found that U0126 pretreatment reduced SOX-9 phosphorylation and expression as well as the chondrogenic gene expression induced by increased HA Mw (Fig. 6). The results suggest that both expression and phosphorylation of SOX-9 contributes to the chondrogenesis of hADSCs induced by increased HA Mw, and the effect is mediated through ERK phosphorylation. The biological effect of HA may be influenced by physical and chemical signals. HA can function as a docking site for sulfated GAGs or proteoglycans through molecular electrostatic-mediated entanglement effect, which may enhance the 32

HA-CD44 interaction [84-86]. The viscosity of the HA solution can be changed by altering the Mw of HA, which then changes the shear stress of liquid flow. Shear stress has been indicated to modulate stem cell differentiation under fluid flow cell culture conditions [87-89]. For excluding these artifacts, we tested the Mw-dependent effect of HA on the chondrogenesis of ADSCs under static culture conditions rather than under fluid flow culture conditions. We also demonstrated that the direct receptor-mediated HA-CD44 interaction is the primary effector on the chondrogenesis of ADSCs in this study. We observed that CD44 clustering and the expression of chondrogenic genes (Collagen type II and Aggrecan) in ADSCs are increased concomitant with the increase of HA Mw (Figure 3B&5B). The inhibition of CD44 clustering by nystatin in hADSCs also reduces the primary chondrogenic effect of HA on hADSCs (Figure 5B). Based on these results, we suggest that the HA-CD44 interaction but not the physical properties of HA, such as viscosity, is the primary mechanism by which HA influences chondrogenesis of ADSCs. One limitation of this study is that the mechanism of HA-CD44 signaling involves multiple pathways. Pathways other than ERK/SOX-9 are also worth investigating in the future. CD44 can also function as a co-receptor in other cells, for example, coupling to transforming growth factor β (TGF-β) receptor or epidermal growth factor receptor [90, 91]. In addition, CD105, a member of the TGF-β receptor 33

family, is one of the surface markers of ADSCs and can mediate TGF-β1 and TGF-β3 signaling [92-94]. However, little is known regarding whether CD44 functions as a co-receptor with CD105 upon HA-CD44-mediated signaling during chondrogenesis of ADSCs. This mechanism needs to be investigated in the future. Conclusion: In this study, we found that increasing HA Mw (80 kDa~2000 kDa) increases the HA-induced chondrogenic effect on ADSCs. Different HA Mws may alter ADSC chondrogenesis by altering CD44 clustering and the ERK/SOX-9 pathway. This finding

provides

new

information

regarding

the

biochemical

control

of

chondrogenesis by HA substrates that may add value to the development of HA-based biomaterials for articular cartilage regeneration.

Acknowledgments:

The authors acknowledge grant support for this study provided by the National Science Council (MOST 103-2314-B-037-017-MY3) and Kaohsiung Medical University Hospital (KMUH103-3R37). This study was also supported in part by Kaohsiung Medical University “Aim for the Top” Universities Grants, grant Nos. KMU-TP105B00, KMU-TP105B02 and KMU-TP105B10.

34

Figure legends: Fig. 1. Characterization of adipose-derived stem cells (ADSCs) isolated from rabbits. (A) Rabbit ADSCs were induced to differentiate into osteocytes, chondrocytes or adipocytes. (a) ADSCs were cultured for 21 days with osteogenic induction medium, and calcium deposition was visualized with von Kossa staining (Scale bar = 200 μm). (b) ADSCs were cultured for 14 days with chondrogenic induction medium, and the cartilage-specific matrix generated was visualized with Alcian blue staining (Scale bar = 200 μm). (c) ADSCs were cultured for 28 days with adipogenic induction medium, and lipid droplets were observed (Scale bar = 100 μm). (B) The ability of rabbit ADSCs to grow in soft agar. Colonies of ADSCs developed in soft agar. (a) ADSCs were cultured in soft agar on day 1 (Scale bar = 200 μm). (b) ADSCs were cultured in soft agar on day 1 (Scale bar = 50 μm). (c) ADSCs were cultured in soft agar on day 14 (Scale bar = 200 μm). (d) ADSCs were cultured in soft agar on day 14 (Scale bar = 50 μm). Fig. 2A-C Effects of changes in HA Mw on chondrogenic differentiation of rADSCs in vitro. rADSCs were cultured in wells coated with different molecular weight HAs. (A) Aggregation of rADSCs in the 6 wells of the Control, 80 kDa, 600 kDa and 2000 kDa groups at 4 h (Scale bar = 200 μm). 35

(B) The mRNA expression levels of chondrogenic genes (collagen type II: rCOL-II and aggrecan: rAGG) in the Control, 600 kDa and 2000 kDa groups at 24 h. The gene expression levels are expressed relative to the Control group, which is defined as 1. (C) Alcian blue staining of sGAG in the Control, 80 kDa, 600 kDa and 2000 kDa groups on day 5. Blue: Alcian blue staining. (Scale bars = 200 μm). The total sGAG synthesis by rADSCs in the Control, 80 kDa, 600 kDa and 2000 kDa groups was quantified by a DMMB assay. The sGAG synthesis normalized to the total DNA in each group is expressed as sGAG/DNA. The sGAG/DNA content is expressed relative to the Control group at day 5, which is defined as 1. The values presented are the means ± SEM (n=6). (*) and (**) indicate p<0.05 and p<0.01, respectively, in comparison with the Control group. (#) and (##) indicate p<0.05 and p<0.01, respectively, between two groups. Fig. 2D&E. Effects of HA on cartilaginous tissue formation of rADSCs in vivo. (D) Gross image of neo-formed cartilage (arrow) in the Control (a), and 2000 kDa (b) groups. (E) Images of Safranin O and Fast Green staining to assess sGAG synthesis in the Control (a) and 2000 kDa (b) groups. Red: Safranin O staining. Blue: Fast Green staining. (Scale bars = 100 μm). Fig. 3. Effect of changes in HA Mw on cell aggregation and CD44 clustering of 36

human ADSCs (hADSCs). hADSCs were cultured in wells coated with different molecular weight HAs. (A) The aggregation of hADSCs in the Control, 80 kDa, 600 kDa and 2000 kDa groups at 2 h after cell seeding before BS3 treatment. The hADSCs in each group were then treated with BS3 for further western blot analysis. (Scale bars = 200 μm). (B) The hADSCs in each group were treated with BS3 and analyzed with western blotting. The protein expressions levels of CD44 clustering, CD44 and GAPDH in the Control, 80 kDa, 600 kDa and 2000 kDa groups at 2 h. The CD44 clustering/CD44 expression level of each group is expressed relative to the Control group, which is defined as 1. The values presented are the means ± SEM (n=6). (*) and (**) indicate p<0.05 and p<0.01, respectively, in comparison with the Control group. (#) indicates p<0.05 between two groups. Fig. 4 ERK phosphorylation, SOX-9 expression and SOX-9 phosphorylation in hADSC induced by HA Mw. hADSCs were cultured in wells coated with different molecular weight HAs. (A) The protein expression levels of ERK, p-ERK and GAPDH in the Control, 80 kDa, 600 kDa and 2000 kDa groups at 2 and 24 h. The protein levels of phosphorylated ERK are expressed relative to that of the Control group at 24 h, which is defined as 1. (B) (C) The protein expression levels of SOX-9, p-SOX-9 and GAPDH in the Control, 80 kDa, 37

600 kDa and 2000 kDa groups at 24 h. The levels of SOX-9 expression (SOX-9) and SOX-9 phosphorylation (p-SOX-9) are expressed relative to those of the Control group at 24 h, which are defined as 1. The values presented are the means ± SEM (n=6). (*) and (**) indicate p<0.05 and p<0.01, respectively, in comparison with the Control group. (#) indicates p<0.05 between two groups. Fig. 5. Effect of CD44 clustering inhibition by nystatin on ERK phosphorylation and chondrogenic gene expression in hADSCs. hADSCs were pretreated with (+Nystatin) or without nystatin (-Nystatin) and then cultured in 6-well plates coated with different molecular weight HAs. (A) The protein expression levels of ERK, p-ERK and GAPDH in the Control and 2000 kDa groups at 24 h. The protein levels of ERK phosphorylation are expressed relative to that of the Control group at 24 h, which is defined as 1. The values presented are the means ± SEM (n=6). (B) The mRNA expression of collagen type II (hCOL-II), and Aggrecan (hAGG) is expressed relative to that of the Control group at 24 h, which is defined as 1. The values presented are the means ± SEM (n=4). (*) and (**) indicate p<0.05 and p<0.01, respectively, in comparison with the Control group. (#) and (##) indicate p<0.05 and p<0.01, respectively, and represent the comparison between +Nystatin and -Nystatin in each group. 38

Fig. 6 The effect of inhibition of ERK phosphorylation by U0126 on SOX-9 expression, SOX-9 phosphorylation and chondrogenic gene expression of hADSCs induced by HA Mw. (A) hADSCs were pretreated with (+U0126) or without U0126 (-U0126) and then cultured in 6-well plates coated with different molecular weight HAs. The protein expression levels of SOX-9, p-SOX-9 and GAPDH in the Control, and 2000 kDa groups at 24 h. (B) The mRNA expression levels of collagen type II (hCOL-II) in the Control, 80 kDa, 600 kDa and 2000 kDa groups at 24 h. (C) The mRNA expression levels of aggrecan (hAGG) in the Control, 80 kDa, 600 kDa and 2000 kDa groups at 24 h. The mRNA expression level of each gene is expressed relative to the Control group, which is defined as 1. The values presented are the means ± SEM (n=4). (*) and (**) indicate p<0.05 and p<0.01, respectively, in comparison with the Control group. (#) and (##) indicate p<0.05 and p<0.01, respectively, and represent the comparison between +U0126 and -U0126 in each group.

39

References:

[1] K.L. Caldwell, J. Wang, Cell-based articular cartilage repair: the link between development and regeneration, Osteoarthritis Cartilage 23(3) (2015) 351-62. [2] S.M. Richardson, G. Kalamegam, P.N. Pushparaj, C. Matta, A. Memic, A. Khademhosseini, R. Mobasheri, F.L. Poletti, J.A. Hoyland, A. Mobasheri, Mesenchymal stem cells in regenerative medicine: Focus on articular cartilage and intervertebral disc regeneration, Methods 99 (2016) 69-80. [3] C.R. Fellows, C. Matta, R. Zakany, I.M. Khan, A. Mobasheri, Adipose, Bone Marrow and Synovial Joint-Derived Mesenchymal Stem Cells for Cartilage Repair, Front Genet 7 (2016) 213. [4] M.J. Zuscik, M.J. Hilton, X. Zhang, D. Chen, R.J. O'Keefe, Regulation of chondrogenesis and chondrocyte differentiation by stress, J Clin Invest 118(2) (2008) 429-38. [5] J.W. Kuhbier, B. Weyand, H. Sorg, C. Radtke, P.M. Vogt, K. Reimers, [Stem cells from fatty tissue : A new resource for regenerative medicine?], Chirurg 81(9) (2010) 826-32. [6] J.W. Kuhbier, B. Weyand, C. Radtke, P.M. Vogt, C. Kasper, K. Reimers, Isolation, characterization, differentiation, and application of adipose-derived stem cells, Adv Biochem Eng Biotechnol 123 (2010) 55-105. [7] G.I. Im, Regeneration of articular cartilage using adipose stem cells, J Biomed Mater Res A 104(7) (2016) 1830-44. [8] M.B. Goldring, K. Tsuchimochi, K. Ijiri, The control of chondrogenesis, J Cell Biochem 97(1) (2006) 33-44. [9] A. Woods, G. Wang, F. Beier, Regulation of chondrocyte differentiation by the actin cytoskeleton and adhesive interactions, J Cell Physiol 213(1) (2007) 1-8. [10] D.E. Discher, D.J. Mooney, P.W. Zandstra, Growth factors, matrices, and forces combine and control stem cells, Science 324(5935) (2009) 1673-7. [11] B.P. Toole, Hyaluronan in morphogenesis, J Intern Med 242(1) (1997) 35-40. [12] P.V. Thorogood, J.R. Hinchliffe, An analysis of the condensation process during chondrogenesis in the embryonic chick hind limb, J Embryol Exp Morphol 33(3) (1975) 581-606. [13] R.A. Kosher, M.P. Savage, K.H. Walker, A gradation of hyaluronate accumulation along the proximodistal axis of the embryonic chick limb bud, J Embryol Exp Morphol 63 (1981) 85-98. [14] C.T. Singley, M. Solursh, The spatial distribution of hyaluronic acid and 40

mesenchymal condensation in the embryonic chick wing, Dev Biol 84(1) (1981) 102-20. [15] E.B. Hunziker, Articular cartilage repair: basic science and clinical progress. A review of the current status and prospects, Osteoarthritis Cartilage 10(6) (2002) 432-63. [16] H.S. Yoo, E.A. Lee, J.J. Yoon, T.G. Park, Hyaluronic acid modified biodegradable scaffolds for cartilage tissue engineering, Biomaterials 26(14) (2005) 1925-33. [17] S.C. Wu, J.K. Chang, C.K. Wang, G.J. Wang, M.L. Ho, Enhancement of chondrogenesis of human adipose derived stem cells in a hyaluronan-enriched microenvironment, Biomaterials 31(4) (2010) 631-640. [18] S.C. Wu, C.H. Chen, J.K. Chang, Y.C. Fu, C.K. Wang, R. Eswaramoorthy, Y.S. Lin, Y.H. Wang, S.Y. Lin, G.J. Wang, M.L. Ho, Hyaluronan initiates chondrogenesis mainly via CD44 in human adipose-derived stem cells, J Appl Physiol (1985) 114(11) (2013) 1610-8. [19] C.B. Knudson, Hyaluronan and CD44: strategic players for cell-matrix interactions during chondrogenesis and matrix assembly, Birth Defects Res C Embryo Today 69(2) (2003) 174-96. [20] C.B. Knudson, W. Knudson, Cartilage proteoglycans, Semin Cell Dev Biol 12(2) (2001) 69-78. [21] D.J. Aguiar, W. Knudson, C.B. Knudson, Internalization of the hyaluronan receptor CD44 by chondrocytes, Exp Cell Res 252(2) (1999) 292-302. [22] A. Aruffo, I. Stamenkovic, M. Melnick, C.B. Underhill, B. Seed, CD44 is the principal cell surface receptor for hyaluronate, Cell 61(7) (1990) 1303-13. [23] D.C. West, S. Kumar, Hyaluronan and angiogenesis, Ciba Found Symp 143 (1989) 187-201; discussion 201-7, 281-5. [24] C. Tempel, A. Gilead, M. Neeman, Hyaluronic acid as an anti-angiogenic shield in the preovulatory rat follicle, Biol Reprod 63(1) (2000) 134-40. [25] A.J. Day, C.A. de la Motte, Hyaluronan cross-linking: a protective mechanism in inflammation?, Trends Immunol 26(12) (2005) 637-43. [26] C.M. Milner, V.A. Higman, A.J. Day, TSG-6: a pluripotent inflammatory mediator?, Biochem Soc Trans 34(Pt 3) (2006) 446-50. [27] S. Ohno, H.J. Im, C.B. Knudson, W. Knudson, Hyaluronan oligosaccharides induce matrix metalloproteinase 13 via transcriptional activation of NFkappaB and p38 MAP kinase in articular chondrocytes, J Biol Chem 281(26) (2006) 17952-60. [28] P.L. Bollyky, B.A. Falk, S.A. Long, A. Preisinger, K.R. Braun, R.P. Wu, S.P. Evanko, J.H. Buckner, T.N. Wight, G.T. Nepom, CD44 costimulation promotes FoxP3+ regulatory T cell persistence and function via production of IL-2, IL-10, and 41

TGF-beta, J Immunol 183(4) (2009) 2232-41. [29] P.L. Bollyky, B.A. Falk, R.P. Wu, J.H. Buckner, T.N. Wight, G.T. Nepom, Intact extracellular matrix and the maintenance of immune tolerance: high molecular weight hyaluronan promotes persistence of induced CD4+CD25+ regulatory T cells, J Leukoc Biol 86(3) (2009) 567-72. [30] P.L. Bollyky, J.D. Lord, S.A. Masewicz, S.P. Evanko, J.H. Buckner, T.N. Wight, G.T. Nepom, Cutting edge: high molecular weight hyaluronan promotes the suppressive effects of CD4+CD25+ regulatory T cells, J Immunol 179(2) (2007) 744-7. [31] J. Sleeman, W. Rudy, M. Hofmann, J. Moll, P. Herrlich, H. Ponta, Regulated clustering of variant CD44 proteins increases their hyaluronate binding capacity, J Cell Biol 135(4) (1996) 1139-50. [32] V. Lefebvre, B. Dumitriu, A. Penzo-Mendez, Y. Han, B. Pallavi, Control of cell fate and differentiation by Sry-related high-mobility-group box (Sox) transcription factors, Int J Biochem Cell Biol 39(12) (2007) 2195-214. [33] G.H. Goodwin, C. Sanders, E.W. Johns, A new group of chromatin-associated proteins with a high content of acidic and basic amino acids, Eur J Biochem 38(1) (1973) 14-9. [34] W. Bi, J.M. Deng, Z. Zhang, R.R. Behringer, B. de Crombrugghe, Sox9 is required for cartilage formation, Nat Genet 22(1) (1999) 85-9. [35] E. Wright, M.R. Hargrave, J. Christiansen, L. Cooper, J. Kun, T. Evans, U. Gangadharan, A. Greenfield, P. Koopman, The Sry-related gene Sox9 is expressed during chondrogenesis in mouse embryos, Nat Genet 9(1) (1995) 15-20. [36] D.M. Bell, K.K. Leung, S.C. Wheatley, L.J. Ng, S. Zhou, K.W. Ling, M.H. Sham, P. Koopman, P.P. Tam, K.S. Cheah, SOX9 directly regulates the type-II collagen gene, Nat Genet 16(2) (1997) 174-8. [37] V. Lefebvre, W. Huang, V.R. Harley, P.N. Goodfellow, B. de Crombrugghe, SOX9 is a potent activator of the chondrocyte-specific enhancer of the pro alpha1(II) collagen gene, Mol Cell Biol 17(4) (1997) 2336-46. [38] I. Sekiya, K. Tsuji, P. Koopman, H. Watanabe, Y. Yamada, K. Shinomiya, A. Nifuji, M. Noda, SOX9 enhances aggrecan gene promoter/enhancer activity and is up-regulated by retinoic acid in a cartilage-derived cell line, TC6, J Biol Chem 275(15) (2000) 10738-44. [39] H. Watanabe, M.P. de Caestecker, Y. Yamada, Transcriptional cross-talk between Smad, ERK1/2, and p38 mitogen-activated protein kinase pathways regulates transforming growth factor-beta-induced aggrecan gene expression in chondrogenic ATDC5 cells, J Biol Chem 276(17) (2001) 14466-73. [40] R. Tuli, S. Tuli, S. Nandi, X. Huang, P.A. Manner, W.J. Hozack, K.G. Danielson, 42

D.J. Hall, R.S. Tuan, Transforming growth factor-beta-mediated chondrogenesis of human mesenchymal progenitor cells involves N-cadherin and mitogen-activated protein kinase and Wnt signaling cross-talk, J Biol Chem 278(42) (2003) 41227-36. [41] M.A. Solis, Y.H. Chen, T.Y. Wong, V.Z. Bittencourt, Y.C. Lin, L.L. Huang, Hyaluronan regulates cell behavior: a potential niche matrix for stem cells, Biochem Res Int 2012 (2012) 346972. [42] S. Murakami, M. Kan, W.L. McKeehan, B. de Crombrugghe, Up-regulation of the chondrogenic Sox9 gene by fibroblast growth factors is mediated by the mitogen-activated protein kinase pathway, Proc Natl Acad Sci U S A 97(3) (2000) 1113-8. [43] T.M. Lin, J.L. Tsai, S.D. Lin, C.S. Lai, C.C. Chang, Accelerated growth and prolonged lifespan of adipose tissue-derived human mesenchymal stem cells in a medium using reduced calcium and antioxidants, Stem Cells Dev 14(1) (2005) 92-102. [44] N.K. Singh, G.R. Singh, Amarpal, P. Kinjavdekar, A.K. Sharma, T.R. Mohanty, S. Kumar, H.S. Chae, Y.M. Yoo, C.N. Ahn, Articular cartilage repair with autografting under the influence of insulin-like growth factor-1 in rabbits, J Vet Med A Physiol Pathol Clin Med 54(4) (2007) 210-8. [45] H.T. Chen, M.J. Lee, C.H. Chen, S.C. Chuang, L.F. Chang, M.L. Ho, S.H. Hung, Y.C. Fu, Y.H. Wang, H.I. Wang, G.J. Wang, L. Kang, J.K. Chang, Proliferation and differentiation potential of human adipose-derived mesenchymal stem cells isolated from elderly patients with osteoporotic fractures, J Cell Mol Med 16(3) (2012) 582-93. [46] C.Z. Wang, S.M. Chen, C.H. Chen, C.K. Wang, G.J. Wang, J.K. Chang, M.L. Ho, The effect of the local delivery of alendronate on human adipose-derived stem cell-based bone regeneration, Biomaterials 31(33) (2010) 8674-83. [47] J. Kim, Y. Park, G. Tae, K.B. Lee, C.M. Hwang, S.J. Hwang, I.S. Kim, I. Noh, K. Sun, Characterization of low-molecular-weight hyaluronic acid-based hydrogel and differential stem cell responses in the hydrogel microenvironments, J Biomed Mater Res A 88(4) (2009) 967-75. [48] C.H. Chen, Y.S. Lin, Y.C. Fu, C.K. Wang, S.C. Wu, G.J. Wang, R. Eswaramoorthy, Y.H. Wang, C.Z. Wang, Y.H. Wang, S.Y. Lin, J.K. Chang, M.L. Ho, Electromagnetic fields enhance chondrogenesis of human adipose-derived stem cells in a chondrogenic microenvironment in vitro, J Appl Physiol (1985) 114(5) (2013) 647-55. [49] K.J. Livak, T.D. Schmittgen, Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method, Methods 25(4) (2001) 402-8. 43

[50] C. Yang, M. Cao, H. Liu, Y. He, J. Xu, Y. Du, Y. Liu, W. Wang, L. Cui, J. Hu, F. Gao, The high and low molecular weight forms of hyaluronan have distinct effects on CD44 clustering, J Biol Chem 287(51) (2012) 43094-107. [51] G. Zhang, H. Zhang, Y. Liu, Y. He, W. Wang, Y. Du, C. Yang, F. Gao, CD44 clustering is involved in monocyte differentiation, Acta Biochim Biophys Sin (Shanghai) 46(7) (2014) 540-7. [52] R.W. Farndale, D.J. Buttle, A.J. Barrett, Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue, Biochim Biophys Acta 883(2) (1986) 173-7. [53] C.G. Williams, T.K. Kim, A. Taboas, A. Malik, P. Manson, J. Elisseeff, In vitro chondrogenesis of bone marrow-derived mesenchymal stem cells in a photopolymerizing hydrogel, Tissue Eng 9(4) (2003) 679-88. [54] M.F. Favata, K.Y. Horiuchi, E.J. Manos, A.J. Daulerio, D.A. Stradley, W.S. Feeser, D.E. Van Dyk, W.J. Pitts, R.A. Earl, F. Hobbs, R.A. Copeland, R.L. Magolda, P.A. Scherle, J.M. Trzaskos, Identification of a novel inhibitor of mitogen-activated protein kinase kinase, J Biol Chem 273(29) (1998) 18623-32. [55] A.C. Midgley, M. Rogers, M.B. Hallett, A. Clayton, T. Bowen, A.O. Phillips, R. Steadman, Transforming growth factor-beta1 (TGF-beta1)-stimulated fibroblast to myofibroblast differentiation is mediated by hyaluronan (HA)-facilitated epidermal growth factor receptor (EGFR) and CD44 co-localization in lipid rafts, J Biol Chem 288(21) (2013) 14824-38. [56] S. Gerecht, J.A. Burdick, L.S. Ferreira, S.A. Townsend, R. Langer, G. Vunjak-Novakovic, Hyaluronic acid hydrogel for controlled self-renewal and differentiation of human embryonic stem cells, Proc Natl Acad Sci U S A 104(27) (2007) 11298-303. [57] C. Chung, J.A. Burdick, Influence of three-dimensional hyaluronic acid microenvironments on mesenchymal stem cell chondrogenesis, Tissue Eng Part A 15(2) (2009) 243-54. [58] L. Pan, Y. Ren, F. Cui, Q. Xu, Viability and differentiation of neural precursors on hyaluronic acid hydrogel scaffold, J Neurosci Res 87(14) (2009) 3207-20. [59] T.C. Laurent, J.R. Fraser, Hyaluronan, FASEB J 6(7) (1992) 2397-404. [60] S.H. Yuspa, D.L. Morgan, Mouse skin cells resistant to terminal differentiation associated with initiation of carcinogenesis, Nature 293(5827) (1981) 72-4. [61] J.G. Rheinwald, H. Green, Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells, Cell 6(3) (1975) 331-43. [62] M. Dominici, K. Le Blanc, I. Mueller, I. Slaper-Cortenbach, F. Marini, D. Krause, R. Deans, A. Keating, D. Prockop, E. Horwitz, Minimal criteria for defining 44

multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement, Cytotherapy 8(4) (2006) 315-7. [63] C.C. Chang, W. Sun, A. Cruz, M. Saitoh, M.H. Tai, J.E. Trosko, A human breast epithelial cell type with stem cell characteristics as target cells for carcinogenesis, Radiat Res 155(1 Pt 2) (2001) 201-207. [64] P.A. Band, J. Heeter, H.G. Wisniewski, V. Liublinska, C.W. Pattanayak, R.J. Karia, T. Stabler, E.A. Balazs, V.B. Kraus, Hyaluronan molecular weight distribution is associated with the risk of knee osteoarthritis progression, Osteoarthritis Cartilage 23(1) (2015) 70-6. [65] M.W. Holmes, M.T. Bayliss, H. Muir, Hyaluronic acid in human articular cartilage. Age-related changes in content and size, Biochem J 250(2) (1988) 435-41. [66] C.J. Liu, Y. Zhang, K. Xu, D. Parsons, D. Alfonso, P.E. Di Cesare, Transcriptional activation of cartilage oligomeric matrix protein by Sox9, Sox5, and Sox6 transcription factors and CBP/p300 coactivators, Front Biosci 12 (2007) 3899-910. [67] Y. Kawakami, J. Rodriguez-Leon, J.C. Belmonte, The role of TGFbetas and Sox9 during limb chondrogenesis, Curr Opin Cell Biol 18(6) (2006) 723-9. [68] I.M. Khan, S.J. Gilbert, S.K. Singhrao, V.C. Duance, C.W. Archer, Cartilage integration: evaluation of the reasons for failure of integration during cartilage repair. A review, Eur Cell Mater 16 (2008) 26-39. [69] D.M. Bradham, A. Passaniti, W.E. Horton, Jr., Mesenchymal cell chondrogenesis is stimulated by basement membrane matrix and inhibited by age-associated factors, Matrix Biol 14(7) (1995) 561-71. [70] B. Johnstone, T.M. Hering, A.I. Caplan, V.M. Goldberg, J.U. Yoo, In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells, Exp Cell Res 238(1) (1998) 265-72. [71] A.A. Worster, B.D. Brower-Toland, L.A. Fortier, S.J. Bent, J. Williams, A.J. Nixon, Chondrocytic differentiation of mesenchymal stem cells sequentially exposed to transforming growth factor-beta1 in monolayer and insulin-like growth factor-I in a three-dimensional matrix, J Orthop Res 19(4) (2001) 738-49. [72] A.I. Caplan, S.P. Bruder, Mesenchymal stem cells: building blocks for molecular medicine in the 21st century, Trends Mol Med 7(6) (2001) 259-64. [73] J. Chen, C. Wang, S. Lu, J. Wu, X. Guo, C. Duan, L. Dong, Y. Song, J. Zhang, D. Jing, L. Wu, J. Ding, D. Li, In vivo chondrogenesis of adult bone-marrow-derived autologous mesenchymal stem cells, Cell Tissue Res 319(3) (2005) 429-38. [74] R.S. Charrad, Y. Li, B. Delpech, N. Balitrand, D. Clay, C. Jasmin, C. Chomienne, F. Smadja-Joffe, Ligation of the CD44 adhesion molecule reverses blockage of differentiation in human acute myeloid leukemia, Nat Med 5(6) (1999) 669-76. 45

[75] S. Bourcier, A. Sansonetti, L. Durand, C. Chomienne, J. Robert-Lezenes, F. Smadja-Joffe, CD44-ligation induces, through ERK1/2 pathway, synthesis of cytokines TNF-alpha and IL-6 required for differentiation of THP-1 monoblastic leukemia cells, Leukemia 24(7) (2010) 1372-5. [76] M. Demoor, D. Ollitrault, T. Gomez-Leduc, M. Bouyoucef, M. Hervieu, H. Fabre, J. Lafont, J.M. Denoix, F. Audigie, F. Mallein-Gerin, F. Legendre, P. Galera, Cartilage tissue engineering: molecular control of chondrocyte differentiation for proper cartilage matrix reconstruction, Biochim Biophys Acta 1840(8) (2014) 2414-40. [77] D. Liu, M.S. Sy, Phorbol myristate acetate stimulates the dimerization of CD44 involving a cysteine in the transmembrane domain, J Immunol 159(6) (1997) 2702-11. [78] C.D. Oh, S.H. Chang, Y.M. Yoon, S.J. Lee, Y.S. Lee, S.S. Kang, J.S. Chun, Opposing role of mitogen-activated protein kinase subtypes, erk-1/2 and p38, in the regulation of chondrogenesis of mesenchymes, J Biol Chem 275(8) (2000) 5613-9. [79] B.E. Bobick, W.M. Kulyk, The MEK-ERK signaling pathway is a negative regulator of cartilage-specific gene expression in embryonic limb mesenchyme, J Biol Chem 279(6) (2004) 4588-95. [80] Y. Kawakami, J. Rodriguez-Leon, J.C. Izpisua Belmonte, The role of TGFbetas and Sox9 during limb chondrogenesis, Curr Opin Cell Biol 18(6) (2006) 723-9. [81] L.Y. Bourguignon, P.A. Singleton, H. Zhu, B. Zhou, Hyaluronan promotes signaling interaction between CD44 and the transforming growth factor beta receptor I in metastatic breast tumor cells, J Biol Chem 277(42) (2002) 39703-12. [82] W. Huang, X. Zhou, V. Lefebvre, B. de Crombrugghe, Phosphorylation of SOX9 by cyclic AMP-dependent protein kinase A enhances SOX9's ability to transactivate a Col2a1 chondrocyte-specific enhancer, Mol Cell Biol 20(11) (2000) 4149-58. [83] W. Huang, U.I. Chung, H.M. Kronenberg, B. de Crombrugghe, The chondrogenic transcription factor Sox9 is a target of signaling by the parathyroid hormone-related peptide in the growth plate of endochondral bones, Proc Natl Acad Sci U S A 98(1) (2001) 160-5. [84] M. Wang, X. Liu, Z. Lyu, H. Gu, D. Li, H. Chen, Glycosaminoglycans (GAGs) and GAG mimetics regulate the behavior of stem cell differentiation, Colloids Surf B Biointerfaces 150 (2017) 175-182. [85] R.L. Jackson, S.J. Busch, A.D. Cardin, Glycosaminoglycans: molecular properties, protein interactions, and role in physiological processes, Physiol Rev 71(2) (1991) 481-539. [86] D.C. Kraushaar, S. Dalton, L. Wang, Heparan sulfate: a key regulator of embryonic stem cell fate, Biol Chem 394(6) (2013) 741-51. [87] M.K. Cowman, T.A. Schmidt, P. Raghavan, A. Stecco, Viscoelastic Properties of 46

Hyaluronan in Physiological Conditions, F1000Res 4 (2015) 622. [88] Y.C. Toh, J. Voldman, Fluid shear stress primes mouse embryonic stem cells for differentiation in a self-renewing environment via heparan sulfate proteoglycans transduction, FASEB J 25(4) (2011) 1208-17. [89] S. Stolberg, K.E. McCloskey, Can shear stress direct stem cell fate?, Biotechnol Prog 25(1) (2009) 10-9. [90] R. Thapa, G.D. Wilson, The Importance of CD44 as a Stem Cell Biomarker and Therapeutic Target in Cancer, Stem Cells Int 2016 (2016) 2087204. [91] B.P. Toole, M.G. Slomiany, Hyaluronan, CD44 and Emmprin: partners in cancer cell chemoresistance, Drug Resist Updat 11(3) (2008) 110-21. [92] K. Warrington, M.C. Hillarby, C. Li, M. Letarte, S. Kumar, Functional role of CD105 in TGF-beta1 signalling in murine and human endothelial cells, Anticancer Res 25(3B) (2005) 1851-64. [93] M. Altomonte, R. Montagner, E. Fonsatti, F. Colizzi, I. Cattarossi, L.I. Brasoveanu, M.R. Nicotra, A. Cattelan, P.G. Natali, M. Maio, Expression and structural features of endoglin (CD105), a transforming growth factor beta1 and beta3 binding protein, in human melanoma, Br J Cancer 74(10) (1996) 1586-91. [94] H. Tapp, E.N. Hanley, Jr., J.C. Patt, H.E. Gruber, Adipose-derived stem cells: characterization and current application in orthopaedic tissue repair, Exp Biol Med (Maywood) 234(1) (2009) 1-9.

47

Figure 1.

A.

a

b

c

Figure 1.

B. a

b

c

d

Figure 2.

A.

4hr

B.

80 kDa

rAGG

rCOL-II

24hr

Normalized Fold Expression

## #

4

# 3

**

2

**

*

1 0 Control

80 kDa

600 kDa

2000 kDa

600 kDa

2000 kDa

24hr

## Normalized Fold Expression

Control

##

4

#

**

3 2

*

**

1 0 Control

80 kDa

600 kDa

2000 kDa

Figure 2.

C.

Day 5

Control

80 kDa

2000 kDa

600 kDa

sGAG/DNA synthesis

sGAG synthesis

Day 5

##

4

# #

3

**

2

** **

1 0 Control

80 kDa

600 kDa

2000 kDa

Figure 2.

D.

a

b

Control group

E.

a

2000kDa group b

Control group

2000kDa group

Figure 3. A.

2hr

Control

2000kDa

600kDa

80kDa

B.

2hr Control 80kDa 600kDa 2000kDa

170 kDa

CD44 Clustering CD44

85 kDa

GAPDH

36 kDa

Integrated band intensity (Ratio to Control)

CD44 Clustering/CD44 #

2

# #

1.5

*

*

*

1 0.5 0

Control

80kDa

600kDa

2000kDa

Figure 4.

A. 2hr Control 80kDa 600kDa 2000kDa

p-ERK

44 kDa 42 kDa

ERK

44 kDa 42 kDa

GAPDH

36 kDa

24hr Control 80kDa 600kDa 2000kDa

p-ERK

44 kDa 42 kDa

ERK

44 kDa 42 kDa

GAPDH

36 kDa

p‐ERK/ERK 4 Integrated band intensity (Ratio to Control)

24hr

# # #

3

**

2

**

*

1 0

Control

80kDa

600kDa

2000kDa

Figure 4.

B.

24hr Control 80kDa 600kDa 2000kDa

SOX-9

38 kDa

p-SOX-9

56 kDa

GAPDH

36 kDa

C. Integrated band intensity (Ratio to Control)

SOX‐9

#

24hr

#

2

#

1.5

*

*

1

**

0.5 0 Control

80kDa

600kDa

2000kDa

24hr

Integrated band intensity (Ratio to Control)

p‐SOX‐9 2 1.5

**

**

80kDa

600kDa

**

1 0.5 0

Control

2000kDa

Figure 5.

A.

Integrated band intensity (Ratio to Control)

p‐ERK/ERK 2

-Nystatin

+Nystatin

**

1.5 1 0.5 0

Control

2000kDa

Control

2000kDa

Figure 5.

B.

24hr

Normalized Fold Expressions

hCOL-II ‐ Nystatin

4

+ Nystatin

**

3

##

**

2

**

## #

1 0 Control

80kDa

600kDa

2000kDa 24hr

Normalized Fold Expressions

hAGG 4

‐ Nystatin

+ Nystatin

**

**

3 *

2

#

##

#

1 0 Control

80kDa

600kDa

2000kDa

Figure 6.

A.

B.

24hr

Normalized Fold Expressions

hCOL-II ‐ U0126

+ U0126

4

**

3

**

*

2

#

##

#

1 0

Control

80kDa

600kDa

2000kDa

Normalized Fold Expressions

hAGG ‐ U0126

4 3

*

2

24hr

+ U0126

**

** #

#

##

1 0

Control

80kDa

600kDa

2000kDa

Table 1

Primer sequences and cycling conditions for real-time PCR Gene

PCR primers Sequence (forward and reverse)

Human Collagen type II (hCOL-II)

Forward : 5’-CAA CAC TGC CAA CGT CCA GAT-3’

Annealing Temperature, 61

Reverse : 5’-TCT TGC AGT GGT AGG TGA TGT TCT-3’ Human Aggrecan (hAGG)

Forward : 5’-ACA GCT GGG GAC ATT AGT GG-3’

61

Reverse : 5’-GTG GAA TGC AGA GGT GGT TT-3’ Human Glyceraldehyde-3-phosphate-dehydrogenase

Forward : 5’-TCT CCT CTG ACT TCA ACA GCG AC-3’

(hGAPDH) Reverse : 5’-CCC TGT TGC TGT AGC CAA ATT C-3’ Denature: 95°C for 30 s, 95°C for 4 min, followed by 35 cycles of Cycling conditions

95°C for 10 s, 61°C(shown in column of Annealing Temperature) for 15 s and 72°C for 15 s

48

61

°C

Table 2

Primer sequences and cycling conditions for real-time PCR Annealing

Gene

PCR primers Sequence (forward and reverse)

Rabbit Collagen type II (rCOL-II)

Forward : 5’-CCA CGC TCA AGT CCC TCA AC -3’

Temperature, 60

Reverse : 5’-TGC TGC TCC ACC AGT TCT TC -3’ Rabbit Aggrecan (rAGG)

Forward : 5’-AAG GCT TAG GGT CTG TGG A

-3’

60

Reverse : 5’-CTG CTT CTT GGG CTG TTG G -3’ Rabbit Glyceraldehyde-3-phosphate-dehydrogenase

Forward : 5’- CCA CTT TGT GAA GCT CAT TTC CT -3’

(rGAPDH) Reverse : 5’-TCG TCC TCC TCT GGT GCT CT -3’ Denature: 95°C for 30 s, 95°C for 4 min, followed by 35 cycles Cycling conditions

of 95°C for 10 s, 60°C(shown in column of Annealing Temperature) for 15 s and 72°C for 15 s

49

60

°C

50