Novel biomimetic tripolymer scaffolds consisting of chitosan, collagen type 1, and hyaluronic acid for bone marrow-derived human mesenchymal stem cells-based bone tissue engineering Smitha Mathews,1 Ramesh Bhonde,2 Pawan Kumar Gupta,2 Satish Totey3 1

Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad-500007, India School of Regenerative Medicine, Manipal University, GKVK Post, Yelahanka, Bangalore-560065, India 3 Kasiak Research Pvt. Ltd., DIL Complex, Ghodbunder Road, Thane (West) 400 610, India 2

Received 8 October 2013; revised 4 March 2014; accepted 13 March 2014 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.33152 Abstract: Human bone marrow-derived mesenchymal stem cells (hMSCs) are an ideal osteogenic cell source for bone tissue engineering (BTE). A scaffold, in the context of BTE, is the extracellular matrix (ECM) that provides the unique microenvironment and play significant role in regulating cell behavior, differentiation, and development in an in vitro culture system. In this study, we have developed novel biomimetic tripolymer scaffolds for BTE using an ECM protein, collagen type 1; an ECM glycosaminoglycan, hyaluronic acid; and a natural osteoconductive polymer, chitosan. The scaffolds were characterized by scanning electron microscopy (SEM) and swelling ratio. The scaffolds were seeded with hMSCs and tested for cytocompatibility and osteogenic potential. The scaffolds supported cell adhesion, enhanced cell proliferation, promoted

cell migration, showed good cell viability, and osteogenic potential. The cells were able to migrate out from the scaffolds in favorable conditions. SEM, alkaline phosphatase assay, and immunofluorescent staining confirmed the differentiation of hMSCs to osteogenic lineage in the scaffolds. In conclusion, we have successfully developed biomimetic scaffolds that supported the proliferation and differentiation of hMSCs. These scaffolds hold great promise as a cell-delivery vehicle for regenerative therapies and as a support system for enhancing C 2014 Wiley Periodicals, Inc. J Biomed Mater bone regeneration. V Res Part B: Appl Biomater 00B: 000–000, 2014.

Key Words: bone tissue engineering, mesenchymal stem cells, biomimetic scaffold, osteoblast differentiation, chitosan

How to cite this article: Mathews S, Bhonde R, Kumar Gupta P, Totey S. 2014. Novel biomimetic tripolymer scaffolds consisting of chitosan, collagen type 1, and hyaluronic acid for bone marrow-derived human mesenchymal stem cells-based bone tissue engineering. J Biomed Mater Res Part B 2014:00B:000–000.

INTRODUCTION

Tissue engineering has emerged as a promising alternative approach in the treatment of malfunctioning or lost organs.1 In this approach, a temporary scaffold is needed to serve as an adhesive substrate for the implanted cells and as a physical support to guide the formation of the new tissue or organ.2 The scaffold should be biocompatible, biodegradable, and highly porous with large surface/volume ratio. An ideal scaffold should support cell growth, cell migration, and retention of the differentiated cell function.3 As scaffolds are considered as the in vitro replacement for the in vivo extracellular matrix (ECM), mimicking the ECM cues for the scaffold preparation and modification should improve their performance.4 This school of thought, mimicking the in vivo scenario, has led to the emergence of the biomimetic approach of tissue engineering. ECM, the acellular material surrounding the cell, mainly consists of proteins and polysaccharides or glycosaminoglycans (GAGs). As ECM plays an important role in cell

adhesion, proliferation, migration, and differentiation,5 its physical and chemical properties are adapted for designing the biomimetic scaffolds. The ECM of bone is composed of organic and inorganic matrices. Collagen type I (COL) is the major component of the organic matrix of bone. Bone tissue has mineralized matrix that can withstand significant compressive loads. Therefore, the scaffolds used for bone tissue engineering (BTE) should be able to provide a framework for mechanical stability along with interconnected large pores for osteointegration.6 Since there is no ideal biomaterial that will accomplish all the required properties necessary for various tissue-engineering applications, composite scaffolds are a better choice. Composite scaffolds have two or more biomaterials combined to improve either their mechanical properties or their functional efficiency or both. The choice of biomaterials, the concentration, the mixing ratio, and the method of preparation are the key factors influencing the structural and functional aspects of composite polymers.7,8 The mixing ratio and the degree of

Additional Supporting Information may be found in the online version of this article. Correspondence to: S. Totey ([email protected]) Contract grant sponsor: Council of Scientific and Industrial Research (CSIR), New Delhi, India.

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TABLE I. Composition of the Tripolymer Scaffolds Scaffold Label S1 S2 S3 S4 S5 S6 S7

CHI 10 (mg/mL)

COL 10 (mg/mL)

HA 10 (mg/mL)

Volumetric Ratio CHI:COL:HA

1 1 1 0.1 1 1 1

1 0.1 1 0.1 0.1 1 0

1 1 0.1 1 0.1 0 1

1:1:1 1:0.1 :1 1:1:0.1 0.1:0.1:1 1:0.1:0.1 1:1:0 1:0:1

cross-linking of the selected polymers are very crucial in determining the biostability, mechanical strength, and microstructure of composite scaffolds.9 Identifying the ideal cell source and the in vivo microenvironment; and understanding the regulatory mechanisms of their growth and differentiation play a significant role in determining the successful performance of a tissueengineered construct. Mesenchymal stem cells with its excellent properties like self-renewal, high expansion, and osteogenic potential10,11 are considered as the most prospective candidate for cell-based tissue engineering for bone regeneration. We have previously demonstrated the beneficial effect of the ECM protein, collagen type 1 (COL),12 ECM-GAG, hyaluronic acid,13 and a natural polymer, chitosan14 in supporting hMSC culture, and enhancing their differentiation to osteogenic lineage. We have also successfully developed a two-dimensional (2D) tripolymer coating demonstrating their synergistic effect on osteoblast differentiation and mineralization.8 Our data showed that when chitosan, COL, and hyaluronic acid were used in a 1:1:1 ratio combination, there was significant enhancement in mineralization with high amount of calcium with respect to the single component treated and untreated plates. In this study, we have developed a novel biomimetic 3D scaffold consisting of chitosan, COL, and hyaluronic acid and evaluated the proliferation and osteoblast differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs) in these scaffolds. We hypothesize that a combination of polymers enhancing osteogenesis is an ideal choice for fabricating composite scaffold with superior properties for bone tissue-engineering applications. MATERIALS AND METHODS

Chitosan and collagen solutions were first mixed thoroughly on a vortex machine. Hyaluronic acid solution was, then, added and vortexed again to get a uniform solution. Different tripolymer combinations were made by mixing 1% solutions of chitosan, COL, and hyaluronic acid in different volumetric ratios (Table I). The samples were poured into a 6-well plate and frozen at 280 C overnight. Porous scaffolds were obtained by freeze drying the samples in a freeze dryer (Alpha 2D Plus, Martin Christ, Osterode am Harz, Germany) until completely dry. The samples that formed relatively strong and stable porous scaffolds were selected for further processing and cell culture. The sides of the samples were removed, and the core was cut into 5-mm diameter cylinders having 0.1mm thickness, using a sharp punch. The freeze-dried scaffolds were cross-linked by immersing it in 40% ethanol containing 50 mM 2morpholinoethanesulphonic acid (MES, Sigma Aldrich; pH 5.5) and 33 mM 1-ethyl-3-(3-dimethyl aminopropyl) carbodimide (EDC, Sigma Aldrich) for 6 h at room temperature (RT). After cross-linking, the samples were washed in 0.1 M Na2HPO4 (pH 9.1), 1.0 M NaCl, and distilled water, respectively. The samples were again freeze dried until dry. The cross-linked samples were sterilized by incubating them in 70% ethanol for 24 h at 4 C followed by incubation in antibiotic-antimycotic solution (GIBCO, Invitrogen, NY) for at least 48 h at 4 C. Prior to the cell culture, the scaffolds were incubated in phenol red containing cell culture medium overnight in a humidified incubator at 37 C and 5% CO2 in the air. Scanning electron microscopy The lyophilized samples were sputter coated with gold in an ion sputtering device (JEOL-JFC- 1100 E, JEOL Technics; Tokyo, Japan) and observed in FEI Quanta 200 environmental scanning electron microscope (ESEM; FEI, Oregon), and the images were captured. Swelling measurements Three dry scaffolds were weighed (Wd) and placed in phosphate-buffered saline (PBS) at RT for 10 h. After removing the unabsorbed solution, the wet weight (Ww) of the scaffold was determined. The swelling ratio of the scaffold was defined as the ratio of the weight increase (Ww 2 Wd) to the initial weight (Wd) and was calculated by the following equation:

Preparation of tripolymer scaffolds Tripolymer scaffolds were prepared using 1% solution of chitosan (>87.61% degree of deacetylation), COL, and hyaluronic acid. Chitosan (CHI, 1%) solution was prepared by dissolving chitosan powder (manufactured from shrimp and crab shells, a kind gift from India Sea Foods, Cochin, India) in 0.1 N sterile acetic acid (Merck, India). One percentage of COL solution was prepared by dissolving collagen powder (Sigma Aldrich, St. Louis, MO) in 0.1 N sterile acetic acid. Hyaluronic acid (HA, 1%) solution was prepared by dissolving potassium salt of hyaluronic acid (Sigma Aldrich) in sterile distilled water.

hMSCs culture on the scaffolds hMSCs were isolated from bone marrow of adult human donors after taking informed written consent by method mentioned elsewhere.14 The isolated cells were characterized by flow cytometry and differentiation potential. The cells were grown in hMSC media consisting of KnockoutDulbecco’s modified Eagle medium (DMEM; GIBCO, Invitrogen) medium supplemented with 10% fetal bovine serum (certified Australian, HyClone, Victoria, Australia), 2

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Swelling ratio ð%Þ5ððWw 2Wd Þ=Wd Þ3100

ORIGINAL RESEARCH REPORT

FIGURE 1. Scanning electron micrographs depicting the microstructure of the scaffolds. (A) S1 scaffold showed highly heterogeneous microstructure and pore distribution. (B) S3 scaffold showed relatively less heterogeneous structure. Inset depicts the macroscopic structure of the scaffolds. SEM images, scale bar—200 mm.

mM L-glutamine (GIBCO, Invitrogen), and 1% Pen Strep (10,000 units/mL Penicillin and 10,000 mg/mL Streptomycin, GIBCO, Invitrogen). The cells were labeled with PKH26 using PKH26 red fluorescent cell linker kit for general cell membrane labeling (Sigma Aldrich) according to the manufacturer’s instructions. The hMSCs stained with PKH26 dye will be referred to as PKH26 labeled cells and the unstained hMSCs will be referred to as unlabeled cells henceforth. The scaffolds were seeded with PKH26 labeled hMSCs at a seeding density of 1 3 105 cells per cylindrical scaffolds of 5 mm of diameter and 0.1-mm thickness. The scaffolds were placed in 2methacryloyloxyethyl phosphorylcholine treated, nonadherent culture plates (Nalge Nunc International, Rochester, NY), and maintained in hMSC media. The plates were incubated in a humidified incubator at 37 C and 5% CO2 in the air. Calcein-propidium iodide live-dead assay The scaffolds seeded with unlabeled cells were stained with cell-permeable calcein acetoxymethyl (calcein AM) and propidium iodide (PI; Sigma Aldrich). For the staining, the culture medium was aspirated and fresh media containing 2 mM cal-

FIGURE 2. Swelling fold (%) of the scaffolds soaked in physiological fluid. S3 scaffold had relatively higher swelling fold than S1 scaffold (*p < 0.05). Error bars represent standard deviation (n 5 3).

cein and 0.01 mg/mL PI was added to the plate and incubated at 37 C for 10 min. The stain was removed and the plates were washed thrice with Dulbecco’s phosphatebuffered saline. As the PKH26 interfered with the fluorescence of PI, cells labeled with PKH26 were stained with calcein alone for tracking the viability of the cells seeded into the scaffolds. The plates were, then, observed under a fluorescent microscope (Nikon Eclipse 80i, Nikon Corporation, Tokyo, Japan) and analyzed by the Q Capture Pro 6 software. Osteoblast differentiation studies on the selected scaffolds After 7 days of culture, the cells were directed to osteogenic lineage by maintaining the cell containing scaffolds in osteogenic induction (OST) media (hMSC media supplemented with 50 mg/mL ascorbic acid, 10 mM b-glycerophosphate and 1028 M dexamethasone [Sigma Aldrich]). The cells were tracked at different time points by fluorescent microscopy for PKH26 staining. Calcein or calcein-PI fluorescent staining were used to evaluate the viability of the cells. As the scaffold material interfered with commonly used confirmatory tests for osteoblast differentiation like Alizarin Red S staining, Kossa staining; and calcium quantification by o-cresolphthalein method, we have used alternative methods like SEM analysis, ALP assay; and expression of osteocalcin and fibronectin for verifying the differentiation of hMSCs to osteogenic lineage. SEM analysis for cell morphology and mineralization The scaffolds, seeded with the cells were processed for SEM to study the cell morphology and distribution. The scaffold was fixed in the fixation buffer containing 2.5% gluteraldehyde and 4% paraformaldehyde (PFA) in PBS for 4 h at RT or at 4 C overnight. The fixed scaffold were washed thoroughly with PBS and dehydrated with serial ethanol washes

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FIGURE 3. Calcein-PI viability assays after 24 h of seeding. hMSCs on (A, B, C) S1 and (D, E, F) S3 scaffolds. Live cells are stained green by calcein and dead cells red by PI. Scale bar—20 mm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

(30% ethanol for 10 min, 50% ethanol for 10 min, 70% ethanol for 10 min or overnight, 85% ethanol for 20 min, 95% ethanol for 20 min, 100% ethanol for 20 min or overnight, 100% acetone for 20 min, and a final wash of 100% acetone for 20 min). The scaffolds were immediately freeze dried. The freeze-dried scaffolds were sputter coated with gold and observed in a FEI Quanta 200 ESEM. The images were captured at different magnifications to analyze the cell morphology and mineralization. Alkaline phosphatase assay Cell lysate was collected on day 14 of differentiation from various scaffolds and alkaline phosphatase (ALP) assay was performed to evaluate the ALP activity according to the method mentioned elsewhere.14 Immunofluorescent staining for fibronectin and osteocalcin Since the scaffold showed autofluorescence when treated with 4% PFA, it was not possible to demonstrate the presence of any protein directly in the cells within in the scaffold. So, an indirect method was used for the immunofluorescent staining to demonstrate the protein of interest. The scaffolds maintained in OST media for 14 days in nonadherent plates were placed in the tissue culture treated chamber slides. After 24 h of incubation, cells migrated from the scaffolds were fixed with 4% PFA and stained for fibronectin and osteocalcin using anti-human antibodies raised in mouse (BD Pharmingen, San Jose). The nuclei were stained with 40 ,6-diamidino-2-phenylindole (Sigma Aldrich). The slides were observed under a fluorescent microscope (Nikon Eclipse 80i) and analyzed by the Q Capture Pro 6 software.

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Statistical analysis The experiments were repeated with three different donor samples. Each set of experiments were conducted in triplicates and results were expressed as mean 6 standard deviation (SD). Statistical analysis was done using the two-tailed, paired Student’s t-test. p-value < 0.05 (*) was considered significant. RESULTS

Characterization of the scaffolds We have prepared seven scaffolds (S1–S7) with varying concentrations of CHI, COL, and HA (Table I). The tripolymer scaffolds, S1 and S3, which formed strong, stable, and porous scaffolds, were selected for further characterization and osteogenic potential evaluation. S1 had equal proportion of CHI, COL, and HA (1:1:1), whereas S3 had less amount of HA (1:1:0.1). The SEM images of the scaffolds showed porous structure with varying pore size and shape (Figure 1). The tripolymer scaffold, S1 showed heterogeneous structure and pore distribution. The S3 scaffold showed significant difference in the microstructure and unlike S1, S3 scaffold had relatively less heterogeneous, sheet-like microstructure. The stability of the scaffold was evaluated by determining the swelling ratio after soaking it in physiological fluid (Figure 2). All the samples could bind 400–500-fold of physiological fluid and still maintain their firm structure and stability. The swelling ratio decreased as the proportion of hyaluronic acid increased in the tripolymer scaffolds. S3 scaffold showed higher swelling fold percentage than S1 scaffold. hMSC culture on tripolymer scaffolds The cells isolated from the bone marrow showed markers and characteristic features of hMSCs (Supplementary

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FIGURE 4. SEM depicting the morphology of hMSCs on the scaffolds. (A, B) S1 and (C, D) S3 scaffolds. The cells are seen in layers and shows spindle-shaped fibroblastic morphology. B, D shows the higher magnification of the area marked with white square in A, C, respectively. Scale bar—(A, C) 100 mm and (B, D) 10 mm.

Information; Figure S1). The calcein-PI staining performed after 24 h of unlabeled hMSC seeding showed good cell viability on both the scaffolds (Figure 3). Calcein also revealed spindle-shaped fibroblast-like cells on S1 and S3 scaffolds. As the bare scaffold did not show any autofluorescence, the strong background fluorescence coming from the scaffolds indicated the presence of cells deep within the scaffolds. The SEM images were taken after 24 h of cell seeding for the evaluation of cell morphology and distribution (Figure 4). S1 and S3 scaffolds showed patches of adherent spindle-shaped cells in layers inside the porous scaffold. Higher magnification (50003) revealed cells with smooth surfaces on both the scaffolds. Evaluation of the osteogenic potential of the scaffolds The PKH26 labeled cells were observed under a fluorescent microscope at different stages of osteoblast differentiation (Figure 5). PKH26 labeled cells were traceable throughout

the differentiation process. S3 scaffold maintained in osteogenic media showed patches of cells with high intensity fluorescence, indicative of clustering of cells, on day 14 of OST [Figure 5(H)]. Good cell viability was observed in both the tested scaffolds even after 14 days of OST (Figure 6). As the culture progressed, the scaffolds showed more number of cells and higher fluorescence (Figures 3 and 6). SEM analysis was done on day 14 of osteoblast differentiation for the evaluation of cell morphology and presence of mineral deposits. Both the scaffolds showed adherent fibroblast-like cells that covered the entire surface of the scaffold in both hMSC and osteogenic media (Figures 7 and 8). Mineral-like deposits were observed on the cells in osteogenic media. Cells maintained in hMSC media had a smooth surface. ALP assay, done on day 14 of osteoblast differentiation showed marked increase (2–3-fold) in the ALP activity by

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osteocalcin and showed a low expression and very hazy fibronectin structure. There was no significant difference in the ostoecalcin and fibronectin expression by the cells of S1 and S3 scaffolds. DISCUSSION

FIGURE 5. Osteogenic differentiation of hMSCs on the tripolymer scaffolds: PKH26-labeled cell tracking. The cells maintained in hMSC media on (A, B) day 7 and (C, D) day 14. The cells maintained in osteogenic (OST) media on (E, F) day 7 and (G, H) day 14. (H) S3 scaffold maintained in OST media showed cluster of cells with high intensity fluorescence. Fluorescent micrographs, scale bar—203 m. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary. com.]

BTE has emerged as a method of combining cells, scaffolds, and bioactive proteins to fabricate functional neotissue to replace the damaged tissue. The advantage of using scaffolds for BTE is that, it will provide initial structural support for the cells and then degrade when the cells secrete their own matrix. Scaffolds play an important role as ECM during engineered tissue development. Biomimetic scaffolds mimic the native ECM and, thus, hold both structural and functional advantages. In this study, our aim was to develop a novel biomimetic scaffold using CHI, COL, and HA, ideal for bone regeneration. We selected CHI for its scaffold forming and osteogenic properties; COL for its cell adhesive and osteogenic properties; and HA for its osteogenic and mineralization properties. We hypothesised that the addition of ECM components such as COL and HA will improve the cell adhesive and osteogenic properties of CHI. We have used PKH26 staining for cell tracking and calcein-PI staining for assessing the morphology and viability of the cells within the scaffolds. Our results show that, two mixing ratios of CHI, COL, and HA; 1:1:1 (S1) and 1:1:0.1(S3), formed stable porous scaffolds. When the tripolymer combination was used, CHI, COL, and HA formed a strong ionic interaction that was further enhanced by the EDC cross-linking. Chitosan with its large number of free amino groups, served as a bridge for cross-linking COL and HA. Yang and Zhang15 reported that chitosan increased the incorporation of GAGs in a CHI-COLGAG composite scaffold. Lin et al.9 hypothesized that, when CHI, COL, and HA were mixed, the ionic interaction will form strong bond that enhances the tensile strength of the

the cells maintained in osteogenic media when compared to the cells maintained in nonosteogenic media (Figure 9). Cells maintained in osteogenic media showed significantly higher ALP activity on S3 scaffold when compared to S1 scaffold. Our experiments demonstrated the migration of differentiated cells from scaffolds maintained in osteogenic medium for 14 days were positive for osteocalcin with prominent expression of fibronectin around the cells. The PKH26 labeled cells migrated out from the scaffolds, when placed on tissue culture-treated adherent chamber slides. The migrated cells grew as adherent fibroblast-like populations. When these cells were stained for fibronectin and osteocalcin, the cells migrated from the scaffolds maintained in the osteogenic media showed presence of osteocalcin and; higher expression and well-defined fibronectin structure (Figure 10). The cells from hMSC media were negative for

FIGURE 6. Viability assay of PKH26 labeled hMSCs in the scaffolds by calcein staining after 14 days of osteogenic induction. Large number of viable, spindle-shaped cells seen in the scaffolds maintained in both (A, B) hMSC and (C, D) OST media. Fluorescent micrographs, scale bar—20 mm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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FIGURE 7. SEM analysis of hMSCs on S1 scaffold on day 14 of osteogenic induction. The cells show spindle-shaped fibroblast-like morphology and covered the entire surface of the scaffold. (A, B) Cells in hMSC media had smooth surface. (C, D) Cells in OST media showed irregular surface and mineral-like deposits in. B, D shows the higher magnification of the area marked with white square in A, C, respectively. Scale bar—(A, C) 100 mm and (B, D) 10 mm.

composite scaffold. They demonstrated that, in a tripolymer mix of CHI, COL, and HA, the thermal transition temperature increased when CHI and HA content increased. Our tripolymer scaffolds, differed from the previously reported, in the unique way of preparation and mixing ratios. Incorporation of chitosan in the scaffold increased the biostability of the scaffold. The EDC cross-linking and relyophilisation altered the microstructure of the scaffolds. As the S1and S3 tripolymer solutions were heterogeneous, the microstructure of the scaffolds had irregular pore shapes and heterogeneous distribution of pores. The proportion of hyaluronic acid in the tripolymer scaffolds significantly changed the microstructure of the scaffolds. SEM analysis showed significant difference in the pore shape, size, and distribution between S1 and S3 scaffolds which differed only in the HA concentration. The S3 scaffold moreover had a sheet-like micro-

structure. Higher HA concentration resulted in a more heterogeneous solution, which formed scaffolds with irregular pore size and decreased swelling ratio. HA–COL interactions might be responsible for this observed difference. The water retaining property of the scaffold can greatly influence their efficacy in tissue-engineering applications. The ability of the scaffold to retain water is dependent on many factors like the hydrophilicity of the material, porosity of the scaffold, and chemical cross-linking.16 The behavior of the scaffold during swelling influences the cell adhesion, distribution, and nutrient-waste exchange. CHI, COL, and HA have good water binding capacity which was reflected in their tripolymer combinations also. Thus, the scaffolds projected a favorable condition for cell adhesion and proliferation within the scaffold. Correia et al.17 showed that incorporation of HA to CHI scaffold influenced the physiological as well as the

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FIGURE 8. SEM analysis of hMSCs on S3 scaffold on day 14 of osteogenic induction. The cells shows spindle-shaped fibroblast-like morphology and covered the entire surface of the scaffold. (A, B) Cells in hMSC media and (C, D) cells in OST media. B, D shows the higher magnification of the area marked with white square in A, C, respectively. Spots of mineralization (black arrows) were visible in OST media. Scale bar—(A, C) 100 m and (B, D) 10 m.

biological properties of the scaffolds. According to their study, as the concentration of HA increased, the pore size and the swelling ratio increased. However, our study has shown that, as the concentration of HA increased, the swelling ratio decreased. During the tripolymer scaffold fabrication, the EDC cross-linking increased the hydrophobicity and the relyophilization, decreased the porosity. The increase in the degree of cross-linking reduced the swelling ratio of the scaffolds.17 Higher degree of cross-linking will decrease the availability of free hydrophilic groups like amino group. Equal amount of chitosan, COL, and hyaluronic acid in S1 scaffold resulted in higher degree of cross-linking than S3. As S3 had lesser amount of hyaluronic acid, more number of free amino groups was available on chitosan, which explains the higher swelling ratio of S3 over S1 scaffold.

Biomimetic scaffolds aim at providing cells with tissue specific biological cues necessary for its proliferation and/or differentiation. Our previous studies14 showed that the coating density of chitosan influences the adhesion of hMSCs to chitosan-treated culture plates. The biomimetic modification of chitosan by the incorporation of COL and HA created favorable surface for hMSC adhesion and proliferation. The composite scaffold of CHI, COL, and HA, showed minimum toxicity, good cell viability, and cell retention. The scaffolds showed adherent spindle-shaped cells, the characteristic feature of hMSCs in an in vitro culture system. There was extensive hMSC proliferation evidenced from the staining and SEM analysis. Calcein staining confirmed good viability of the cells even after 14 days of culture in the scaffolds. As the scaffolds had large surface area, they were able to

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CONCLUSIONS

FIGURE 9. ALP assay on day 14 of osteogenic induction. The dotted line represented the level of ALP in the initial undifferentiated population. Note the significant increase in the ALP activity of the cells maintained in OST media. Cells undergoing osteogenic differentiation in S3 scaffold showed significantly higher (*p < 0.05) ALP level than S1 scaffold. Error bars represent standard deviation (n 5 3).

support the adhesion and growth of more number of cells. The 24-h post seeding SEM images showed only patches of cells on the scaffolds and after 14 days of culture the SEM image showed the entire scaffold surface covered with layers of cells. There was strong cell–cell and cell–material interactions, which hindered the action of trypsin. Thus, cell harvesting by trypsinization gave very poor cell yield. The tripolymer scaffolds, which had three components with known osteogenic property, also favored osteoblast differentiation. Increase in ALP activity and mineral-like deposits by the cells in osteogenic media indicated the osteogenic potential of the scaffolds. Significant increase in ALP activity indicated the differentiation to osteogenic lineage.12–14 S3 scaffold showed clustering of cells when hMSCs were induced to osteogenic lineage. Clustering of cells and matrix synthesis is an indicator of osteogenic differentiation and mineralized nodule formation.18 The ECM secreted by osteoblasts contains fibronectin, which is required for further differentiation and survival of the osteoblasts.19–21 Osteocalcin is a more specific marker of mature osteoblasts.22 This demonstrates the presence of osteoblast in the scaffold and their ability to migrate to the surrounding environment. This will have implication in BTE where scaffolds containing differentiated cells are applied to the site of injury or fracture. Among the tripolymer scaffolds tested here, we consider S3 scaffold with lower concentration of HA as more cost effective for bone tissue-engineering applications. Compared to S1, S3 showed more uniform microstructure, higher swelling ratio, and better osteogenesis. PKH26 cell tracking, calcein staining, and SEM analysis proved the proliferation, migration, differentiation, and survival of cells within the scaffold. We have demonstrated the ability of the undifferentiated and differentiated cells to migrate from the scaffold and grow in favorable environment. This is one of the most desirable characteristic features for in vivo applications like cell delivery to the site of injury or healing bone fractures.

In summary, we have developed stable, biomimetic, chitosan- COL– hyaluronic acid-based scaffolds, S1 (1CHI: 1COL: 1HA), and S3 (1CHI: 1COL: 0.1HA) which supported and promoted hMSC adhesion and osteoblast differentiation. Cells grew and differentiated as adherent fibroblastic population on S1 and S3 scaffolds. Both the tested scaffolds showed excellent cell compatibility, conductivity, and good osteogenic potential. These findings recapitulate our own previous studies showing the synergistic effect of chitosan and selected ECM components in enhancing osteogenesis in a 2D culture system. These scaffolds hold great promise in hMSCs-based BTE. S3 scaffold with lesser amount of hyaluronic acid was considered a better choice as it was more economical and showed optimum performance. Based on our findings, we propose the use of these scaffolds for BTE and therapeutic applications. This biomimetic scaffold that

FIGURE 10. Immunofluorescent staining of cells from S1and S3 scaffolds. Cells migrated from the scaffold maintained in hMSC (A) or osteogenic media (B) for 14 days were stained for fibronectin and osteocalcin after 24 h of migration. FITC stained fibronectin and osteocalcin green and DAPI stained the nuclei blue. More expression of fibronectin was observed around the cells migrated from the scaffolds maintained in osteogenic media (B) when compared to the cells from hMSC media. Only cells migrated from the scaffolds maintained in osteogenic media showed the expression of osteocalcin. Scale bar—20 mm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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support, enhances, and retains hMSC proliferation and differentiation is ideal for cell delivery at the site of injury or damage.

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NOVEL BIOMIMETIC TRIPOLYMER SCAFFOLDS FOR BONE TISSUE ENGINEERING

REFERENCES

MATHEWS ET AL.

Novel biomimetic tripolymer scaffolds consisting of chitosan, collagen type 1, and hyaluronic acid for bone marrow-derived human mesenchymal stem cells-based bone tissue engineering.

Human bone marrow-derived mesenchymal stem cells (hMSCs) are an ideal osteogenic cell source for bone tissue engineering (BTE). A scaffold, in the con...
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