Study the effect of chitosan on adhesion, proliferation and osteoblast differentiation of bone marrow derived human mesenchymal stem cells

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1 CHAPTER 7 Study the effect of chitosan on adhesion, proliferation and osteoblast differentiation of bone marrow derived human mesenchymal stem cells 7.1 Introduction Every year an estimated 9 million fracture cases occurred globally (Johnell and Kanis, 2006). Currently, autologous bone graft is considered as the gold standard for augmenting bone regeneration. However, limited source and volume of bone graft as well as donor site morbidity and related complications have led the scientists to develop an alternative treatment to the autologous bone graft that can maintain its effectiveness, minimize its disadvantages, and be made affordable for the global population (Salgado et al., 2004). Tissue engineering and cell-based therapies have opened up a new era of regenerative medicine, promising the replacement or regeneration of any non-functional or damaged body parts (Kneser et al., 2006; Bajada et al., 2008). One of the most exciting approaches is cell-based bone tissue engineering (BTE), which combines living osteogenic cells with biomaterial scaffolds ex vivo to allow the development of a three-dimensional tissue construct (Meijer et al., 2007). With the advancement in the field of tissue engineering, significant research was done in identifying a suitable scaffold material and cell source for reconstructing or repairing the tissue of interest (Corona et al., 2010). The identification of the multi-potential mesenchymal stem cells (MSCs) derived from various adult human tissues has provided exciting prospects for the cell-based tissue engineering and regeneration (Bianco and Robey 2001; Chatterjea et al., 2010) 8). Since MSCs have the potential to differentiate into osteoblasts, they can be explored for the development of a bioengineered bone 107

2 construct using various biomaterial scaffolds (Mauney et al., 2005). The goal is to get the cells attached to the scaffold, then replicate, differentiate and organize into the normal healthy tissue as the scaffold degrades. While selecting a biomaterial for tissue engineering applications, apart from the basic qualities like biocompatibility and biodegradability, it is important to understand the role of the particular biomaterial in controlling the behaviour and fate of the cells of interest (Hernandez and Mikos, 2008). The cell-material interactions should be understood in terms of the chemical nature of the material; protein adsorption; cell adhesion and differentiation; and gene expression patterns (Meinel et al., 2004). This knowledge will help us to prepare tailor-made scaffolds of our interest for various tissue engineering applications. Chitosan is a natural biomaterial with a structural similarity to hyaluronic acid of extra-cellular matrix (ECM) (Nair and Laurencin, 2006; Peniche et al., 2007). Though the osteogenic property of chitosan is well established in tissue engineering applications, the exact mechanism is not yet well understood (Venkatesan and Kim, 2010; Yang et al., 2009). The specific details of the osteogenic differentiation of human mesenchymal stem cells on two dimensional chitosan matrices have not been studied in depth so far. In the present investigation, we have utilised chitosan to modify tissue culture plate to understand its effect on adhesion and osteoblast differentiation of bone marrow derived human mesenchymal stem cells (hmscs). Two of our major objectives are to evaluate the potential of chitosan for tissue culture plate modification and to understand the mechanisms of osteogenesis by chitosan in terms of gene expression and mineral matrix deposition. These results will pave way to develop chitosan based culture plates and biomimetic scaffolds for various cell culture and bone tissue engineering applications. 108

3 7.2 Materials and methods Preparation of chitosan coated tissue culture plates The Chitosan powder (>87.61% degree of deacetylation, DD) which was a kind gift from Indian Sea Foods (Cochin, India) was purified by repeated precipitation with 10% NaOH (Merck, India). One percentage chitosan solution was prepared by autoclaving purified chitosan powder in distilled water and then dissolving it by adding 1M sterile glacial acetic acid (Merck, India). Chitosan coated culture plates were prepared by covering the culture plate surface with sufficient volume of sterile chitosan solution at different concentrations. The solution was then evaporated to dryness at room temperature in a biological laminar flow hood. This resulted in the formation of a thin chitosan acetate film on the plate surface. The acidity of the surface was neutralised with 0.1M NaOH solution. The plates were washed several times with distilled water until the ph became neutral, dried and stored at 4 ºC until use. The plates were equilibrated with Dulbecco s phosphate buffered saline (DPBS) (GIBCO, Invitrogen, NY, USA) before using for cell culture experiments and these plates will be referred to as chitosan coated plates here after. The normal tissue culture plates without any coating will be referred to as untreated plates Culture of human mesenchymal stem cells on chitosan coated plates Human MSCs were plated at a seeding density of 5000 cells per sq. cm on the chitosan coated plates prepared at varying concentrations in hmsc media. Untreated plates served as the control for these experiments. The plates were observed under an inverted phase contrast microscope for morphological changes and adherence of the cells. The cell viability on different chitosan coated plates were determined by trypan blue exclusion test (Section 4.2.7) and the doubling time of the hmscs on these plates were calculated (Section ). 109

4 7.2.3 Osteoblast differentiation studies on the chitosan coated plates Human MSCs grown on the selected chitosan coated plate was directed to osteoblast lineage by growing them in osteogenic induction media. The osteogenic potential of the chitosan coated plate was evaluated at different time points by methods like alkaline phosphate (ALP) staining (Section ), ALP assay (Section ), histochemical staining for secreted mineral matrix; Alizarin Red S (Section ) and von Kossa staining (Section ), calcium assay (Section ) and osteoblast differentiation associated gene expression profile (Section ) Statistical analysis The experiments were repeated with three different donor samples. Measures for each sample were performed in triplicates or duplicates and the results were expressed as mean ± standard deviation (SD). The statistical analysis was done by the two tailed, paired Student s t test. In all the analysis a p-value less than 0.05 was considered significant and asterisks were given accordingly to indicate the level of significance as *p<0.05, **p<0.01 and ***p< Results Culture of hmscs on chitosan coated plates We used 5, 10, 25, 50 and 100µg/cm 2 coating density of chitosan in this experiment. On solvent evaporation, chitosan formed a thin film on the polystyrene tissue culture plates. Attached hmscs showed fibroblast like morphology on chitosan coated plates with the coating density of 5, 10 and 25µg/cm 2 (Fig. 7.1A, B, C). 110

5 Figure 7.1 Human MSCs culture on tissue culture plates coated with different concentrations of chitosan. Human MSCs attached and showed fibroblast like morphology on chitosan plates of coating density 5 (B), 10 (C) and 25(D) µg per sq. cm. There was uniform cell distribution on these coated plates. Chitosan plate with a coating density of 50µg per sq. cm (E) also showed adherent hmscs, but there was non-uniform cell distribution. Chitosan plate with a coating density of 100µg per sq. cm (F) did not show any adherent hmscs but there was formation of non-adherent cell aggregates (black arrow) on this plate. Untreated tissue culture plate (A) served as the control. A coating density of 25µg per sq. cm, which supported hmscs adhesion, was selected for further osteoblast differentiation experiments. Phase contrast micrographs, scale bar - 50µm. The cells were uniformly distributed onto these plates. Although chitosan plate with a coating density of 50µg/cm 2 showed adherent hmscs, the growth and cell distributions were not uniform (Fig. 7.1E). The cell viability assay (Table 7.1) revealed better cell viability (>93%) on chitosan coated plates having the coating density of 5, 10 and 25µg/cm 2 when compared to the untreated plate (83.1 ± 4.4%). Chitosan coated plates with the coating density of 50 µg/cm 2 also showed good cell viability (91.9 ± 1.0%). We observed that coating density of 25ug/cm 2 of chitosan was most suitable that favoured optimum growth, morphology and uniform distribution of hmsc and hence selected for subsequent experiments. 111

6 Chitosan plate with a coating density of 100µg/cm 2 did not show any adherent hmscs but there was formation of non-adherent cell aggregates on this plate (Fig. 7.1F). Re-plating of these cell aggregates to normal tissue culture plates showed viable cells, which not only adhered to normal tissue culture plate but also proliferated (Fig. 7.2A, B). Figure 7.2 Human MSC aggregates on chitosan coated plates. 6 days old cell aggregates on chitosan coated plates (A, 100µg per sq. cm). The aggregates re-plated into normal tissue culture plates showed attached spindle shaped cells spreading from the aggregates (B).Scale bar -10µm. When the cell aggregates on the chitosan coated plate was maintained in osteogenic induction media for 21 days, the cells underwent differentiation giving a positive Alizarin Red S staining for calcium (Fig. 7.3). Figure 7.3 Osteogenic differentiation experiments with the cell aggregates on chitosan coated plates. Cell aggregates grown in osteogenic media for 21 days gave a positive staining with Alizarin Red S (A), indicating osteoblast differentiation and mineralization. Alizarin Red S staining for the aggregates in non osteogenic medium for 21 days showed no calcium deposition (B). Phase contrast micrographs, scale bar - 50µm. 112

7 Human MSCs showed good cell viability (>85%) on all the tested chitosan coated plates except the plate with a coating density of 100µg /cm 2 (Table 7.1). Since hmscs did not adhere but formed cell aggregates, the cells did not dissociate effectively on trypsinization. Therefore, we were not able to do a proper cell counting for the viability or doubling time assay on 100µg /cm 2 chitosan coated plates. All the chitosan coated plates which supported hmscs adhesion, showed more percentage of viable cells than the untreated plate. Among the chitosan coated plates, more than 94% hmsc viability was observed on 10µg /cm 2 and 25µg /cm 2 chitosan coated plates, which was remarkably higher than the viability on the untreated plates (83.1 ± 4.4). Plate Viability, % ± SD Untreated 83.1 ± 4.4 Chitosan (1µg /cm 2 ) 86.4 ± 9.2 Chitosan (5µg /cm 2 ) 93.7 ± 0.4 Chitosan (10µg /cm 2 ) 94.1 ± 6.0 Chitosan (25µg /cm 2 ) 94.0 ± 0.9 Chitosan (50µg /cm 2 ) 91.9 ± 1.0 Table 7.1 Viability assay by trypan blue exclusion test (n=4). The doubling time assay of the hmscs on various chitosan coated plates showed interesting results. No general trend in the doubling time with respect to the coating density was observed here. There was marked variation in the hmsc doubling time on different chitosan coated plates (Table 7.2). Only chitosan coated plate with a coating density of 25µg /cm 2 showed a noticeable reduction the doubling time (33.8 ± 9.6h) with respect to the untreated (43.5 ± 19.1h) or other chitosan coated plates. Chitosan coated plate with a lower coating density (1µg /cm 2 ) showed a doubling time for the hmscs 113

8 (42.6 ± 17.8h) comparable to that on the untreated plate. Whereas chitosan coated plate with a higher coating density (50µg /cm 2 ) showed a higher doubling time for the hmscs (51.7 ± 10.3h) when compared to that on the untreated plate. Plate Doubling time in h ± SD Untreated 43.5 ± 19.1 Chitosan (1µg /cm 2 ) 42.6 ± 17.8 Chitosan (5µg /cm 2 ) 50.8 ± 10.9 Chitosan (10µg /cm 2 ) 41.7 ± 14.7 Chitosan (25µg /cm 2 ) 33.8 ± 9.6 Chitosan (50µg /cm 2 ) 51.7 ± 10.3 Table 7.2 Doubling time (DT) of hmscs on various chitosan coated plates (n=4) Evaluation of the osteogenic potential of chitosan coated plates The hmscs were maintained in the osteogenic induction media for a total of 21days. The daily observation of the plates under an inverted phase contrast microscope showed the presence of mineral deposits after 7 to 10 days of osteogenic induction. There was no marked difference in the onset of mineralization for both the cells on chitosan coated and untreated plates. However, there was difference in the amount and pattern of mineral deposition on both the plates. Mineralization was higher on chitosan coated plates, which also gave rise to the formation of bone nodules (Fig. 7.4). The differentiation was confirmed and compared by histochemical staining, quantified using ALP, and calcium assay. The cells maintained in non osteogenic media did not show any mineralization on both the plates. 114

9 Figure 7.4 Phase contrast micrographs of cells undergoing osteoblast differentiation on chitosan coated plates. The upper panel (A-D) shows the osteoblast differentiation on the untreated plates and lower panel (E-H) shows the osteoblast differentiation on the chitosan coated plates. There were more amounts of mineral deposits by the cells differentiated on chitosan coated plates. (F-H) Bone nodule formation was observed on chitosan coated plates on day 14 of differentiation (G) which increased in size by day 21 (H). Scale bar - 10µm ALP staining and assay Alkaline phosphatase is a well-known marker of osteoblast differentiation and staining for its presence using NBT-BCIP gave us the indication of differentiation at an early stage. ALP staining done after 7 days of osteogenic induction was used here to compare and analyse the expression pattern on chitosan coated plates with respect to the untreated plates (Fig. 7.5A, B). The intensity of staining was higher on chitosan coated plates. In the ALP assay, alkaline phosphatase hydrolyzed the substrate p-nitrophenyl phosphate to give a yellow colored product p-nitrophenol, which was quantified by photometry. The ALP assay showed similar pattern of expression by the cells on both untreated and chitosan coated plates (Fig.7.5C). The ALP level increased during the course of differentiation reaching a peak by the second week, which gradually decreased 115

10 towards the final phase of mineralization. The level of ALP expression by the cells in non-osteogenic medium was very low and there was no significant change in their expression level during the entire course of the differentiation experiment Figure 7.5 ALP staining and assay. Cells undergoing osteoblast differentiation stained for ALP by BCIP - NBT method after 7 days of induction (A, B). The upper panel (A) shows the microscopic view and lower panel (B) shows the macroscopic view. Chitosan coated plates (Chitosan) showed more intensely stained cells than the untreated plate. ALP positive cells were mainly present in the periphery of both the plates (B). Phase contrast micrographs, scale bar - 50µm. ALP assay (C) showed similar ALP expression pattern on both the untreated (UT) and chitosan coated plates (chitosan) plates. The peak in ALP activity was observed in the mid-stage (day 14) of differentiation Alizarin Red S and von Kossa staining for calcium Onset of mineralization was determined by microscopic observation of the cells for the mineral deposits and confirmed by Alizarin Red S staining for calcium (Fig. 7.6A-D). Since Alizarin Red S stained intra-cellular calcium, calcium binding proteins and proteoglycans also, it was useful in evaluating the differentiation at an early phase of differentiation. The area and intensity of staining was more on the chitosan coated plates and the intensity increased as the differentiation progressed. Intensely stained patches were observed on chitosan coated plates compared to the untreated plate that showed almost a uniform staining pattern. 116

11 The von Kossa staining, which detected the phosphate in the calcium phosphate of the secreted matrix confirmed the extra-cellular calcium deposition by the mature osteoblasts. The cells undergoing osteoblast differentiation and mineralization gave a positive staining enabling comparison in the late stage of differentiation. Here also, the intensity of staining was more for mineral deposits on chitosan coated plates and there were many patches of intense staining (Fig. 7.6E-H). Figure 7.6 Histochemical staining for calcium. The upper panel shows the staining on the untreated plates and the lower panel shows the staining on chitosan coated plates (Chitosan). Alizarin Red S staining on day 14 of osteogenic differentiation (A-D).The staining on chitosan coated plates (C,D) showed more intense staining then the untreated plates (C, D) indicating more calcium deposits or mineralization. Von Kossa staining on day 21 of osteogenic differentiation (E-H). The von Kossa staining confirmed the enhancement of mineral matrix deposition on the chitosan coated plates (G, H) when compared to the untreated plate (E, F). Phase contrast micrographs, scale bar -10µm Calcium assay Quantification of calcium in the secreted mineral matrix showed a 30% increase in the calcium deposition when the osteogenic induction was carried out on chitosan coated plates (Fig. 7.7). This indicated the potential of chitosan to induce ideal bone formation. 117

12 Figure 7.7 Calcium assay. The amount of calcium in the deposited mineral matrix was quantified on day 21 of osteoblast differentiation and was expressed per mg of total protein. Cells on chitosan coated plate (chitosan) showed significantly high (* p<0.05) calcium deposits compared to the untreated plate Osteoblast differentiation associated gene expression analysis To analyse the expression of osteoblast differentiation associated genes, we quantified the gene expression of the starting undifferentiated population and after 7, 14 and 21 days of osteogenic induction. Eight osteoblast differentiation associated genes were evaluated for their level of expression during osteogenic differentiation. There was several fold increase in the level of expression of most of the genes on chitosan coated plates compared to the untreated plate at different stages of differentiation (Fig. 7.8). Among the osteogenic differentiation associated genes evaluated, OPN showed 100 fold higher expressions at early stage of osteogenic differentiation on chitosan coated plates as compared to that of untreated plates. Similarly, IBSP gene also showed 35 fold higher expression on chitosan coated plate at the early stage of differentiation. At mid-stage differentiation, there were two specific genes COL1A1 and ON showed significant upregulation in chitosan coated plate. COL1A1 showed 30 fold and ON showed six fold 118

13 higher expressions at mid-stage differentiation. These changes were not seen on the untreated plates. On the contrary, OSX gene showed 10 fold higher expressions at early stage of differentiation, which was later down-regulated at mid-stage differentiation and again up-regulated by18 fold at late stage of differentiation. Other important gene that upregulated at late stage of differentiation was ALPL (seven fold) and OCN (six fold) on chitosan coated plate. RUNX2 however, did not show any change in expression pattern in chitosan coated and untreated plates. Figure 7.8 Osteoblast differentiation associated gene expression profile. The graphs represent the relative fold difference in the gene expression on chitosan coated (chitosan) plates with respect to the untreated (UT) plates. The fold difference was calculated by the 2 -ΔΔCt method. Cells on chitosan coated plates showed significantly high fold difference in the expression of OPN, IBSP and COL1A1, the three genes regulating respective proteins associated with calcium binding and bone matrix formation. ALPL showed a marked increase and a significantly high expression on day 14 and 21 of differentiation respectively. ON, a later stage and OCN, a final stage differentiation marker also showed significant fold difference on chitosan coated plates. Another key transcription factor OSX, regulating osteoblast differentiation, also showed a marked increase in expression on chitosan coated plates. (*p<0.05, **p < 0.01 and ***p< 0.001). 119

14 7.4 Discussion In this study, the potential of a natural polymer, chitosan for its beneficial effect on bone marrow derived mesenchymal stem cell s osteogenesis has been explored. Here, we have examined the optimum coating density of chitosan for hmscs culture. Human MSCs are plastic adherent and grow as a fibroblast-like adherent monolayer on tissue culture plates (Pittenger et al., 1999; Caplan, 1991). Interestingly, we have observed the formation of viable non-adherent cell aggregates of hmscs on chitosan coated plates. In our experiments, we observed that this aggregate formation took place at higher coating density of chitosan. The cells in the aggregates were found to be viable and were able to differentiate into osteogenic lineage. Similar results have been reported with melanocyte culture on chitosan coated plates (Lin et al., 2005). It was hypothesized that melanocyte aggregate on higher concentration of chitosan, may be due to the change in cell-chitosan and cell-cell interacting forces. It is known that hmscs adhere to a biomaterial via the integrin expressed on the cell surface by an indirect mechanism and mediated by the specific serum proteins adsorbed to the material surface (Clegg et al., 2005). The type and amount of proteins adsorbed depends on the chemical nature of the material (Hallab et al., 1995). One possible explanation for the difference in the cell adhesion properties of chitosan at different coating density may be due to the difference in the adsorbed protein layer on it. This altered protein layer at higher coating density did not allow the cells to attach to the plate but instead increased the cell-cell interactions resulting in the aggregation of the cells. We propose that, this property of chitosan can be utilised in cell culture applications like generation of neurosphere and islet like cell aggregates (ICAs), where non- adherent 120

15 mesenchymal stem cell populations are required. Enhanced ICA formation in vitro is obtained on non-adherent surfaces (Chandra et al., 2009). This is a first report on the evaluation of the osteogenic potential of chitosan, in a two dimensional culture system. Since we used tissue culture plates coated with chitosan, we were able to evaluate the osteogenic potential by various parameters like osteoblast differentiation associated gene expression levels, calcium quantification, ALP assay and histochemical staining for mineral deposits. ALP is an early and one of the most frequently used marker for osteoblast differentiation (Sila-Ansa et al., 2007; Allen, 2003). The ALP found in bone tissue is called tissue non-specific alkaline phosphatase (TNAP) which have three isozymes namely ALP/liver/bone/kidney abbreviated as ALPL. Though it is a widely used marker, their exact roles in osteogenesis are not yet very clear (Siffert, 1951; Golub and Boeze- Battaglia, 2007). Evidence suggests that it hydrolyses phosphate substrates releasing P i and is associated with the initiation of mineralization (Balcerzak et al., 2003). The NBT/BCIP staining showed more number of ALP positive cells in the periphery of the culture plate, which formed the proliferative and hypertrophic zone. In the ALP assay both the untreated and chitosan coated plates showed similar pattern of ALP activity giving a peak at day 14 of differentiation. The phase contrast microscopy as well as the histochemical staining also confirmed the initiation of mineralization in the second week of differentiation. Interestingly, the level of mrna expression for ALP on these plates did not give similar results. The pattern and level of the mrna expression was different for cells on both untreated and chitosan coated plates. Overall ALP gene expression in the chitosan coated plates was twofold higher compared to the cells differentiated on the untreated plates. However, similar results were not observed at the protein level. In their study on the effect of alcohol on the osteogenic differentiation of hmscs, Gong et al 121

16 found that although alcohol significantly reduced alkaline phosphatase activity, there was no effect on the alkaline phosphatase mrna level (Gong and Wezeman, 2004). Perhaps, an unknown control mechanism lies between ALP mrna expression and its protein level activity. The final phase of osteoblast differentiation is mineralization where mineral matrix containing mainly calcium phosphate in the form of hydroxyapatite is secreted and deposited by mature osteoblasts (Clarke, 2008). In our studies the matrix deposition was initiated from second week of osteoblast differentiation in both untreated and chitosan coated plates. We used Alizarin Red S and von Kossa, two widely used staining methods for the analysis of mineral matrix deposition. Alizarin Red S staining gave us an early evaluation of osteogenesis, which was confirmed by von Kossa staining at the later stage. Both the staining methods confirmed marked enhancement of mineralization by the cells undergoing osteoblast differentiation on chitosan coated plates. This data clearly establishes the well-known osteogenic potential of chitosan scaffolds in a two dimensional culture system. Chitosan coated plates showed a different yet definite pattern of mineralization as there were more discrete patches with relatively high intensity of staining. One of our experiments even showed the formation of bone nodule like structure by the cells only on chitosan coated plates. This could be due to the higher population of differentiated cells at a particular area depositing large amount of calcium in the secreted extra-cellular matrix. Previous data had shown cell migration/spreading on chitosan or its derivatives (Paer et al., 2009; Scanga et al., 2010). Fakhry et al (2004) reported that chitosan preferentially supported initial adhesion and spreading of osteoblasts over fibroblasts. Our data of osteoblast differentiation on chitosan coated plate demonstrated better mineralization and higher calcium content in the mineral deposits, which is essential for proper bone healing. 122

17 The master switch responsible for the initiation of osteoblast differentiation from MSCs is RUNX2, which is also known as osteoblast specific factor (Osf2) or core binding factor α (Cbfa1) (Komori, 2002; Lian et al., 2004). The subsequent stages of differentiation can be divided into three phases; early stage (the proliferative phase), midstage (the extracellular matrix synthesis and maturation phase) and late stage (the mineralization phase). Each stage is characterized by the expression of specific osteoblast differentiation genes. Higher expression of OPN IBSP and OSX genes were appeared at early stage of differentiation indicating proliferative phase. COL1A1 and ON appeared at mid-stage differentiation indicated a phase of synthesis and deposition of extra-cellular matrix. Up-regulation of OCN was seen at late stage of differentiation, which indicated the maturation phase or final phase of differentiation, where synthesis and deposition of minerals by matured osteoblast occurred. Collagen, which constitutes about 90% of the organic matrix, is known as the most important marker associated with the formation of the inorganic matrix of bone (Zhu et al., 2009). It is considered as one of the early and mid-stage marker of osteoblast differentiation associated with the binding of calcium. IBSP is associated with ECM matrix synthesis at the maturation phase and have the potential to act as a nucleation point for hydroxyapatite and there by initiating and promoting the mineral deposition (Ganss et al., 1999). Like IBSP, OPN (which is also known as Bone sialoprotein1, BSP1), is also considered as an early marker of bone matrix synthesis and is associated with initiation of mineralization (Roach, 1994). OCN and ON is reported to be present in the fully mineralized matrix and thus considered as the later markers of osteoblast differentiation. ON is a bone specific protein that binds selectively to collagen and hydroxyapatite and help in active mineralization (Termine et al., 1981). OCN is a final stage differentiation marker, which is exclusively secreted by mature osteoblasts towards 123

18 the end of mineralization (Kim, 1993; Malaval et al., 1999). OSX is the downstream mediator for the actions of RUNX2 and is considered as a more specific marker of osteoblasts (Nishio et al., 2006). Our attempt to understand the osteogenic property of chitosan at a molecular level revealed interesting findings. This is the first study showing the relative fold difference in mrna levels of hmscs undergoing osteoblast differentiation on a chitosan coated culture plate. Cells cultured on chitosan coated plate did not show any changes in expression level of RUNX2 gene, the gene responsible for the osteoblast lineage commitment. However, its downstream effecter OSX showed marked increase in expression especially in the initial phases of differentiation and later at the late stage of differentiation. OSX is considered as the key transcription factor required for the differentiation of pre-osteoblasts to mature osteoblasts (Zhang, 2010). Other genes such as COL1A1, ALPL, IBSP, OPN, ON and OCN, which are mostly associated with mineralization showed several fold increase in the level of expression on chitosan coated plates. More COL1A1 expression indicated more calcium binding and thus enhanced mineral matrix deposition on the chitosan coated plates. Both IBSP and OPN genes showed higher expression in the early stages of differentiation on chitosan coated plates. These plates also showed several fold difference in the expression of OCN in the late stage of differentiation as compared to the untreated plate. These findings suggest that chitosan enhances osteoblasts differentiation mainly by up-regulating most of the genes associated with calcium binding proteins, which resulted in more bone matrix deposition on chitosan coated plates. The gene expression profile also indicates that chitosan follows the normal pathway of osteoblast differentiation by human mesenchymal stem cells and embryonic stem cells (Kulterer et al., 2007; Karner et al., 2007). 124

19 To summarise, we report the formation of non-adherent, viable and functional cell aggregates by bone marrow derived hmscs on chitosan coated tissue culture plates at higher coating density. We also found that hmscs adhered to the surface of the plates with lower coating density of chitosan. In this study, we demonstrate for the first time that chitosan enhanced mineralization by up regulating the genes associated with mineralization and calcium binding proteins. This data will be useful in designing chitosan based culture systems for various cell culture and bone tissue engineering applications. Importantly, our data could be of clinical significance advocating the use of chitosan in treating non-union bone fractures. 125