Materials Science and Engineering C 52 (2015) 90–96

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Silk fibroin/gelatin–chondroitin sulfate–hyaluronic acid effectively enhances in vitro chondrogenesis of bone marrow mesenchymal stem cells Nopporn Sawatjui a,e, Teerasak Damrongrungruang b, Wilairat Leeanansaksiri c,d, Patcharee Jearanaikoon e, Suradej Hongeng f, Temduang Limpaiboon e,⁎ a

Biomedical Sciences, Graduate School, Khon Kaen University, Khon Kaen 40002, Thailand Department of Oral Diagnosis, Faculty of Dentistry, Khon Kaen University, Khon Kaen 40002, Thailand c Stem Cell Therapy and Transplantation Research Group, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand d School of Microbiology, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand e Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand f Department of Pediatrics, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok 10400, Thailand b

a r t i c l e

i n f o

Article history: Received 15 October 2014 Received in revised form 4 February 2015 Accepted 22 March 2015 Available online 24 March 2015 Keywords: Cartilage tissue engineering Scaffold Chondrogenesis Mesenchymal stem cells Biomaterials Biomimetic

a b s t r a c t Tissue engineering is becoming promising for cartilage repair due to the limited self-repair capacity of cartilage tissue. We previously fabricated and characterized a three-dimensional silk fibroin/gelatin–chondroitin sulfate–hyaluronic acid (SF–GCH) scaffold and showed that it could promote proliferation of human bone marrow mesenchymal stem cells (BM-MSCs). This study aimed to evaluate its biological performance as a new biomimetic material for chondrogenic induction of BM-MSCs in comparison to an SF scaffold and conventional pellet culture. We found that the SF–GCH scaffold significantly enhanced the proliferation and chondrogenic differentiation of BM-MSCs compared to the SF scaffold and pellet culture in which the production of sulfated glycoaminoglycan was increased in concordance with the up-regulation of chondrogenic-specific gene markers. Our findings indicate the significant role of SF–GCH by providing a supportive structure and the mimetic cartilage environment for chondrogenesis which enables cartilage regeneration. Thus, our fabricated SF–GCH scaffold may serve as a potential biomimetic material for cartilage tissue engineering. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Cartilage tissue has a poor self-repair capacity due to the sparsely embedded chondrocyte, slow matrix turnover, lack of vascular supply and low supply of progenitor cells. Autologous chondrocyte transplantation (ACT) is a cell-based clinical treatment for articular cartilage defects [1]. However, poor chondrocyte proliferation in vitro, loss of chondrocyte phenotype upon ex vivo expansion, and inferior fibrocartilage formation at the defect site and donor site have limited its clinical application [2, 3]. Mesenchymal stem cells (MSCs) have shown to have the potential to differentiate into several cell types including chondrocytes and to be present in various tissues such as bone marrow, adipose tissue, cord blood, and trabecular bone. Due to their high proliferation capacity, MSCs are attractive as a distinguished cell substitute for chondrocytes in cartilage regeneration [4]. The scaffold is an important key component in cartilage tissue engineering, which must support cell proliferation and/ or differentiation as well as bear mechanical stress associated with body movement prior to new tissue formation. Although synthetic materials ⁎ Corresponding author. E-mail address: [email protected] (T. Limpaiboon).

http://dx.doi.org/10.1016/j.msec.2015.03.043 0928-4931/© 2015 Elsevier B.V. All rights reserved.

allow better control of mechanical, morphological and physicochemical properties than natural materials, most synthetic materials such as poly(lactic-co-glycolic acid) (PLGA), poly(lactic acid) (PLA) or glycolic acid (PGA) induce some inflammation in vivo [5]. Many natural composite materials such as silk fibroin (SF), collagen, gelatin, alginate, chondroitin sulfate and hyaluronic acid have been widely used to fabricate scaffolds for cartilage tissue engineering in recent years [6–9]. SF produced by Bombyx mori silkworm has been used commercially as biomedical sutures for decades. SF is considered to be a suitable material for cartilage tissue engineering because of its good oxygen and water-vapor permeabilities, minimal inflammatory reaction, relatively slow degradability and high mechanical strength [10,11]. However, single component SF scaffolds may not be sufficient for cartilage tissue engineering as there is a deficiency of cell specificbinding sites and limited options for growth factor anchorage capacity. Many studies have reported on SF combining with other biomaterials including hyaluronic acid [12] and chitosan [13] to produce scaffolds for supporting MSCs in tissue engineering. Gelatin, a denatured form of collagen, is less immunogenic compared to collagen. It contains informational signals such as the ArgGly-Asp (RGD) motif that can promote cell adhesion and proliferation

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[14]. Chondroitin sulfate, a sulfated glycoaminoglycan (GAG), is the main GAG in the native articular cartilage. It is composed of repeating disaccharide units containing sulfate ester and carboxylic groups in which the positively charged growth factors can be attached [15,16]. Some investigators have reported that chondroitin sulfate stimulates proliferation and extracellular matrix production of seeded chondrocytes in collagen–GAG matrices in vitro [17,18]. Hyaluronic acid, a water-soluble polysaccharide, is a key component of the extracellular matrix in cartilage. The specific interaction of hyaluronic acid with a CD44 cell surface receptor plays an important role in cell migration and also interacts with other molecules to maintain articular chondrocyte proliferation and morphology [19]. Our previous study showed that a biocomposite scaffold of silk fibroin, gelatin, chondroitin sulfate and hyaluronic acid (SF–GCH) could promote MSC proliferation compared to an SF scaffold [20]. The aim of this study was to evaluate the effective enhancement of in vitro chondrogenesis of bone marrow derived MSCs in an SF–GCH scaffold compared to SF and conventional pellet culture. We hypothesized that SF acts as a main structure which provides a mechanically stable structure and the GCH composite provides a biomimetic surface like the extracellular matrix for MSC proliferation and chondrogenic differentiation. Thus, SF– GCH might be a potential blending scaffold for cartilage tissue engineering.

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recombinant human bone morphogenetic protein-6 (BMP-6, R&D Systems, Minneapolis, MN) and 10 ng/mL recombinant human transforming growth factor-β3 (TGF-β3, R&D Systems). For the scaffold culture, the sterile scaffold was pre-wetted with culture media for 4 h in 5% CO2 at 37 °C before 3 × 105 BM-MSCs were seeded and maintained at 37 °C with 5% CO2 for 4 h then chondrogenic medium was added. The medium in both the pellet and scaffold culture was changed every 3 to 4 days during the culture period of 21 days. 2.3. Scanning electron microscopy (SEM) After culture for 21 days, constructs were rinsed with PBS, fixed with Karnovsky's solution at 4 °C, post fixed with 1% osmium tetroxide, and sequentially treated with series of ethanol and isoamyl acetate. Finally, critical point drying was performed and the constructs were observed by SEM (Hitachi S-3000N) after being sputter-coated with gold. 2.4. Cellular proliferation

2. Materials and methods

The proliferation of chondrogenic BM-MSCs was evaluated by quantification of genomic DNA [24]. After culture for 7, 14, and 21 days, the genomic DNA was extracted from each pellet and seeded scaffold using DNeasy tissue kit (Qiagen, Chatsworth, CA) according to the manufacturer's protocols. The absorbance was measured at 260 nm by a UV/visible spectrophotometer (NanoVue, Buckinghamshire, UK).

2.1. Scaffold fabrication

2.5. Quantitation of sulfated glycosaminoglycan (sGAG) production

B. mori silk cocoons (Nangnoi-Srisaket strain) were obtained from the Queen Sirikit Sericulture Center, Khon Kaen, Thailand. Silk fibroin (SF) aqueous solution (3%, w/v) was prepared as described previously [21]. The mixture of gelatin (G), chondroitin sulfate (C) and hyaluronic acid (H) (3:1:0.05 w/w) (Sigma-Aldrich, St. Louis, MO) was used to prepare 3% (w/v) GCH solution at 40 °C for 30 min. The three-dimensional SF and SF–GCH scaffolds were fabricated by a freeze-drying technique. The blended SF and GCH solution (2:1 w/w) was cross-linked by adding 1% N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC, Sigma-Aldrich) and 0.5% N-hydroxysuccinimide (NHS, SigmaAldrich) at room temperature for 15 min prior to injecting into the cylindrical Teflon molds, frozen at − 20 °C for 4 h, frozen at − 80 °C for 4 h and lyophilized for 48 h. The freeze-dried scaffolds were immersed in methanol for 1 h, then repeatedly cross-linked in 0.2% EDC/0.1% NHS solution and lyophilized under the same condition. The scaffolds were cut into a cylindrical shape (5 mm diameter × 2 mm height) and sterilized with 70% alcohol under UV light for 1 h followed by washing with sterile phosphate buffer saline (PBS, pH 7.4).

The sGAG content was determined by the 1,9-dimethylmethylene blue (DMMB, Sigma-Aldrich) colorimetric method as previously described [25]. After 7, 14, and 21 days of cultivation, samples were digested with papain (BDH Laboratory Supplies, Poole, UK) for 24 h at 60 °C. The digested sample was reacted with DMMB solution and absorbance was measured at 525 nm using a microplate reader (Sunrise, TECAN). The amount of sGAG in each sample was extrapolated using a standard plot of chondroitin sulfate ranging from 0 to 100 μg/mL.

2.2. Cell culture Human mesenchymal stem cells were isolated from bone marrow aspirate and characterized as described previously [22]. This study received an approval from the Ethics Committee on Research Involving Human Subjects, Faculty of Medicine, Ramathibodi Hospital, Mahidol University. Written informed consent was obtained from all participants involved in this study (MURA 2011/404). The BM-MSCs were expanded to passage 3 in DMEM-low glucose containing 10% fetal bovine serum, 100 U penicillin and 100 μg/mL streptomycin (all were obtained from Invitrogen, Carlsbad, CA). The pellet culture was performed as previously described [23]. In brief, 3 × 105 BM-MSCs were seeded in a sterile 15 mL polypropylene tube and centrifuged at 500 ×g for 5 min. The BM-MSC pellet was maintained at 37 °C with 5% CO2 in chondrogenic medium containing DMEM-high glucose with L-glutamine, sodium pyruvate, pyridoxine hydrochloride (Invitrogen), 100 U penicillin, 100 μg/mL streptomycin, 50 μg/mL L-ascorbic acid-2-phosphate (Sigma-Aldrich), 0.4 mM L-proline (Sigma-Aldrich), 10−7 M dexamethasone (Sigma-Aldrich), 1% ITS + 1 (Sigma-Aldrich), 250 ng/mL

2.6. RNA isolation, cDNA synthesis and semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) Total RNA was isolated from samples using a TRIzol® reagent (Invitrogen) and was transcribed into cDNA using the ImProm-II™ Reverse Transcription System (Promega, Madison, WI) according to the manufacturer's protocols. The specific gene expression for collagen type II (COL II), SRY (sex determining region Y)-box9 (SOX-9), aggrecan (AGC) and collagen type X (COL X) was evaluated to verify the induction of chondrogenesis by semi-quantitative RT-PCR in which the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was used as an internal control. The primer sequences of these genes are shown in Table 1. The PCR reaction was performed using a GStorm GS482 thermal cycler (GRI, Rayne, UK). PCR products were then

Table 1 Primers and their sequences for RT-PCR. Gene

Primer sequence

Product size (bp)

Access no.

GAPDH

F: 5′-CAACAGCCTCAAAGATCATCA-3′ R: 5′-AGGTCCACCACTGACACGTT-3′ F: 5′-CAGAAGACCTCACGCCTC-3′ R: 5′-TAGTTTCCTGCCTCTGCCTTGAC-3′ F: 5′-TGAAGAAGGAGAGCGAGGAG-3′ R: 5′-GCGGCTGGTACTTGTAATCC-3′ F: 5′-CAGGTGAAGACTTTGTGGACATCC-3′ R: 5′-CCTCCTCAAAGGTCAGCGAGTAGC-3′ F: 5′-ACTCCCAGCACGCAGAATCCA-3′ R: 5′-ACAGCTGATGGTCCCGGTGGT-3′

313

NM_002046.4

362

NM_001844.4

351

NM_000346.3

438

NM_001135.3

344

NM_000493.3

COL II SOX-9 AGC COL X

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Fig. 1. SEM images of SF (top panel) and SF–GCH (bottom panel) scaffolds 21 days after seeding with BM-MSCs and cultured in chondrogenic medium. Cells showed a spherical shape with a phenotypic appearance of chondrocyte-like cells.

separated on 2% agarose gel electrophoresis, stained with ethidium bromide and visualized under an UV illuminator. The intensities of PCR bands were measured using the Gel Analyzer 2010a software program. The band intensity of each target gene was then normalized to GAPDH.

2.7. Immunohistological analysis The samples were harvested, washed in PBS and fixed in 4% phosphate-buffered paraformaldehyde at room temperature for 18–24 h, then dehydrated through a graded series of ethanol, embedded in paraffin and sectioned to a 4 μm-thickness. The sections were then deparaffinized in xylene and rehydrated using a graded series of ethanol/water mixtures. For immunohistological analysis, collagen type II and proteoglycan were analyzed using mouse monoclonal anti-human collagen type II (Neomarkers, Fremont, CA) and mouse monoclonal anti-human cartilage proteoglycan (Chemicon International, Temecula, CA), respectively. The sections were digested with proteinase K (Invitrogen) for 10 min to retrieve antigens and the endogenous peroxidase activity was blocked by 3% H2O2 in distilled water for 10 min, then 10% fetal bovine serum was used to prevent non-specific background staining. All reactions were conducted at room temperature. The sections were then incubated at room temperature with primary antibodies; 1:400 anti-proteoglycan for 30 min and 1:200 anti-collagen type II overnight. The target proteins were detected by incubation at room temperature with goat anti-mouse secondary antibody conjugated to horseradish peroxidase (DAKO Corporation, Carpinteria, CA) for 60 min, followed by 3,3′ diaminobenzidine (DAB, DAKO Corporation) substrate for 15 min. The nuclei were counterstained with Meyer's hematoxylin.

2.8. Statistical analysis Data are presented as mean ± standard deviation. All statistical analyses were performed using SPSS version 17.0 software and statistical comparisons were performed using one-way ANOVA with Tukey's post hoc test. P b 0.05 was considered statistically significant.

3. Results 3.1. SEM observation The morphology of chondrogenic differentiation of BM-MSCs on the SF and SF–GCH scaffolds were observed by SEM after culture for 21 days. Cells were well attached on each scaffold which appeared to have a spreading and spherical chondrocyte-like morphology (Fig. 1). No major difference in cell morphology was observed between these scaffold types. 3.2. Cellular proliferation and sGAG production Cell proliferation of each culture system was determined for its DNA content. Both SF and SF–GCH scaffolds showed a continuously increasing DNA content along with cultured time. By contrast, the DNA content in the pellet culture was maintained in a similar level until day 14 and seemed to decrease at day 21 but was not statistically significant (p = 0.124; D14 vs D21) (Fig. 2a). The DNA content of the pellet culture, and SF and SF–GCH scaffolds after culture for 21 days was 3.93 ± 0.20 μg, 5.6 ± 0.27 μg and 5.8 ± 0.56 μg, respectively. It was found that the DNA content produced by cells cultured on SF–GCH was significantly high compared to the pellet culture on day 14 (p b 0.05) until day 21 (p b 0.01) suggesting that the SF–GCH content enhanced cell proliferation better than cell aggregation per se. However, no significant difference of DNA content was observed between SF and SF–GCH scaffolds at any time point. This result suggested that a scaffold is essential for the enhancement of proliferation of BM-MSCs. The cartilage-specific extracellular matrix, sGAG, was evaluated to indicate the chondrogenesis of BM-MSCs. No significant increase in the sGAG level was observed in the pellet culture during the culture period (Fig. 2b). The sGAG production of SF and SF–GCH scaffolds was dramatically increased throughout the culture time. Interestingly, the sGAG content of SF–GCH was significantly higher than that of the pellet culture and SF scaffold (p b 0.01) (Fig. 2b). However, no significant difference in the sGAG content was observed at the early stage of chondrogenesis (day 7) between SF and SF–GCH scaffolds. When normalized to DNA content, the sGAG/DNA ratio of the pellet culture, and SF and SF–GCH scaffolds of day 21 was 1.45-, 1.76-, and 2.07-fold, respectively, higher than the ratio of day 7 (Fig. 2c). Our finding

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Fig. 2. (a) DNA content, (b) sGAG content, and (c) sGAG/DNA ratio of the BM-MSCs cultured in chondrogenic medium under pellet culture, SF scaffold and SF–GCH scaffold after 7, 14 and 21 days. Data represent mean ± SD (n = 4). * and ** denote statistically significant differences at p b 0.05 and p b 0.01, respectively.

demonstrated that the presence of a GCH component significantly enhanced sGAG production in comparison to the pellet culture and SF scaffold suggesting the significant role of GCH as an extracellular matrix composite for chondrogenesis. 3.3. Chondrogenic gene expression signature A chondrogenic gene expression signature such as COL II, SOX-9, AGC and COL X was evaluated by RT-PCR which was up-regulated in all kinds of culture systems (Fig. 3). Band intensity of COL II in the SF– GCH scaffold was 1.8-, 1.6- and 2.8-fold higher than the pellet culture (p b 0.05) at days 7, 14, and 21 respectively, and was 2.2- and 1.8-fold higher than the SF scaffold at days 14 and 21 (p b 0.01) (Fig. 4a). Interestingly, AGC expression in SF–GCH was 2.1-, 1.7- and 1.9-fold higher than the pellet culture (p b 0.01), and was 2.0-, 1.9- and 1.9-fold higher than the SF scaffold at every time point (p b 0.01) (Fig. 4c). The significantly increased levels of COLII and AGC in the SF–GCH scaffold at the early phase of chondrogenesis (day 7) suggested that GCH effectively

accelerated chondrogenic differentiation of BM-MSCs. The level of transcription factor SOX-9 seemed to be constant in the pellet culture but was gradually increased in SF and SF–GCH scaffolds (Fig. 4b). However, no significant difference was found in SOX-9 and COL X expression throughout the culture period in these culture systems (Fig. 4b, d). Our finding supported our hypothesis in which extracellular matrix is essential for the improvement of chondrogenesis of BM-MSCs. 3.4. Immunohistochemistry Chondrogenic differentiation in the pellet and scaffold culture system was also characterized for the presence of chondrocyte-specific proteins such as collagen type II and proteoglycan by immunohistochemistry. The results showed that collagen type II and proteoglycan were both positively stained in all culture groups at day 21 (Fig. 5) suggesting successful chondrogenic differentiation of BM-MSCs. The cells grown on SF and SF–GCH scaffolds showed homogeneous distribution throughout the scaffold.

Fig. 3. RT-PCR analysis of genes encoding COL II, SOX-9, AGC and COL X for chondrogenic induction of BM-MSCs under pellet culture, SF scaffold and SF–GCH scaffold after 7, 14 and 21 days. The housekeeping gene (GAPDH) was used as a control.

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Fig. 4. Chondrogenic-specific gene expression normalized to GAPDH after 7, 14 and 21 days of culture, (a) COL II, (b) SOX-9, (c) AGC and (d) COL X. Data represent mean ± SD (n = 4). * and ** denote statistically significant differences at p b 0.05 and p b 0.01, respectively.

4. Discussion The ideal scaffold required for cartilage tissue engineering should be biocompatible, biodegradable, mechanically flexible and highly porous. It should have a three-dimensional structure which provides a supportive microenvironment for cell migration, proliferation and differentiation. Furthermore, the biomaterials with the mimetic environment of cartilage are a promising scaffold for cartilage repair in regenerative medicine. Recently, the SF–GCH scaffold was fabricated by the freezedrying method [20]. This three-dimensional scaffold containing key components of cartilage extracellular matrix showed mechanical strength as well as high porosity which could be easy for cell seeding and cultivation. The SF–GCH scaffold with a pore size of 211.2 ± 30.0 μm and 73.5 ± 3.9% porosity promoted BM-MSCs proliferation more effectively than the SF scaffold [20]. The present work aimed to evaluate the biological performance of the SF–GCH scaffold as a potential biomimetic material for in vitro chondrogenesis in comparison to a conventional pellet culture system and SF scaffold. The pellet culture which has been widely used to induce chondrogenic differentiation of MSCs was employed as a gold standard method [26]. The porous structure of a scaffold with high porosity is a crucial feature in three-dimensional cell cultivation as a main transport of nutrients, oxygen and signal molecules which might increase cell proliferation and contribute to homogenous cell distribution [24]. Our finding indicated the advantage of the three-dimensional scaffold over the pellet culture for chondrogenic differentiation as shown by an increase in

sGAG production, a main matrix composition of cartilage. The high level of sGAG in the SF–GCH scaffold after 21 day culture was found to be consistent with the extreme up-regulation of the chondrogenic gene markers, collagen type II and aggrecan, whereas the expression levels of these markers were low in the pellet culture and SF scaffold suggesting that GCH are the key components for enhancing chondrogenic differentiation in the SF–GCH scaffold. The presence of GCH in the scaffolds might stimulate the growth of MSCs because growth factors interact with GCH to provide appropriate signals with respect to differentiation. It has been reported that an RGD motif presented in gelatin interacted with αVβ6 integrin subunits which activated TGF-β3 actions [27]. Salinas et al. [28] demonstrated that the RGD peptide could promote chondrogenesis of MSCs. Chondroitin sulfate is a high negative polarity molecule resulting from the carboxylic and sulfate ester groups which allow chondroitin sulfate to be used as a polyanion, onto which positively charged growth factors can be adsorbed and enriched to induce cell adhesion and differentiation [15,29]. It has been demonstrated that the presence of chondroitin sulfate in the scaffold promoted the secretion of proteoglycan and type II collagen [30]. Moreover, addition of chondroitin sulfate in the polyethylene glycol (PEG)-based hydrogel also improved chondrogenic differentiation of goat MSCs [31]. Hyaluronic acid or hyaluronan is an important component of articular cartilage. It has been reported that a hyaluronan enriched microenvironment initiates and enhances chondrogenesis of adipose derived stem cells via CD44, a cell receptor for hyaluronic acid, which

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Fig. 5. Immunohistochemical staining for collagen type II and proteoglycan of pellet culture, SF scaffold and SF–GCH scaffold after culture for 21 days. Scale bar: 200 μm.

subsequently contributes to cartilaginous matrix formation [32]. The interaction between hyaluronan and a CD44 cell surface receptor plays an important role in maintaining the differentiated characteristic of chondrocytes [33]. Matsiko et al. [34] reported that the presence of hyaluronic acid in a collagen-based scaffold improves cellular infiltration and promotes early-stage chondrogenesis for cartilage tissue engineering. They suggested that the presence of hyaluronic acid in the scaffold may facilitate the interaction of soluble growth factors available through immobilization. Thus, hyaluronic acid may provide coreceptors for the presentation of the necessary biochemical factors for commitment towards a chondrocytic lineage leading to gene expression and protein synthesis. In this study, TGF-β3 and BMP-6 were selected due to their being a strong inducer of the chondrogenesis of MSCs based on a previous study [35]. The SF–GCH scaffold showed no significant change in the DNA content but a significant increase in the expression of chondrogenic-specific gene markers in comparison to an SF scaffold. Collagen type II and aggrecan are the main extracellular matrix of cartilage which were increased during chondrogenic induction. Our finding indicated the significant role of SF–GCH on the enhancement of chondrogenesis of BM-MSCs. No significant difference in SOX-9 expression throughout the culture period was observed indicating that the transcription factor Sox-9 is a key regulator of early chondrogenic differentiation and maintenance of the chondrocyte phenotype [36]. The expression level of Sox-9 was high in all culture systems suggesting that chondrogenic differentiation was successfully induced by TGF-β3 and BMP-6. The up-regulation of COL II, AGC, SOX-9 and COL X is believed to represent the physiological adaptation of MSCs to a specialized environment of the articular matrix. Collagen type X is well known to be a maker of hypertrophic chondrocytes. In this study, there was no significant difference in COL X mRNA expression of all culture groups indicating that all culture systems could induce the differentiation of BM-

MSCs to hypertrophic chondrocytes. This effect might be explained by the presence of BMP-6 which was consistent with the previous report of the effect of BMP-6 on chondrogenic induction of BM-MSCs in pellet culture [37,38]. Other investigators demonstrated the maintenance of chondrocyte phenotype in the mimetic cartilage environment scaffold. Chang et al. [39] studied the porcine chondrocytes in the GCH tricopolymer scaffold and Ko et al. [40] cultivated human chondrocytes in type II collagen–chondroitin sulfate–hyaluronan porous scaffolds. Both research groups reported that chondrocytes are evenly distributed in the scaffold, retain their phenotype, and secrete an extracellular matrix including collagen type II and proteoglycan. 5. Conclusions The aim of this study was to evaluate the potential role of the SF– GCH scaffold as a biomimetic material for enhancing proliferation and chondrogenic differentiation of BM-MSCs. Our study showed the advantage of SF–GCH over the SF scaffold by which a blending scaffold could effectively enhance chondrogenesis of BM-MSCs under the mimetic cartilage environment served by GCH. The SF–GCH blending scaffold could promote proliferation and chondrogenic differentiation of seeded BMMSCs. These findings suggested that the SF–GCH hybrid scaffold may be suitable to serve as a new biomaterial for promoting and maintaining the chondrogenic differentiation of MSCs which leads to the enhancement of cartilage repair and applicability for cartilage tissue engineering. Acknowledgements This work was supported by the Royal Golden Jubilee Ph.D. Program (grant no. PHD/0149/2550 to N. Sawatjui); the Research and Technology Transfer Affairs Division (grant no. 2553/15309), Khon Kaen

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University; and the Centre for Research and Development of Medical Diagnostic Laboratories (CMDL) (grant no. CMDL 2555), Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen, Thailand.

[21] [22]

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