Effects of Chitosan/Chondroitin Sulfate/Glutamic Acid Composite Materials on the Differentiation of Bone Marrow Mesenchymal Stem Cells

Suitable stem cell sources with good expansion and differentiation technique, as well as excellent biomaterials are important for tissue engineering applications. In this study, we aimed at the development of new biomaterials for the in vitro expansion and differentiation of bone marrow mesenchymal stem cell (BMSCs). Chondroitin sulfate and glutamic acid were used to modify chitosan in order to prepare composite biomaterials. Inverted microscopy and cellular DNA quantification were used to detect the influence of the biomaterials on the proliferation of BMSCs. Real-time PCR was also used to detect the gene expressions in BMSCs. The gene expressions of Oct-4 and Nanog in BMSCs were lower when they were exposed to a certain differentiation stimuli, thus revealing that cells morphology changed and specific differentiation started. We examine the gene expression of osteocalcin (OCN) and use Alizarin red S staining to detect calcium deposit after BMSCs exposed to osteogenic stimulus (OS), the gene expressions of peroxisome proliferator-activator receptor-γ2 (PPAR-γ2), lipoprotein lipase (LPL) and use of Oil red O staining to detect oil secretion when BMSC were exposed to adipogenic stimulus (AS), the gene expression of aggrecan and type II collagen and use of Toluidine blue and Safranin O staining to detect glycosaminoglycan (GAG) when BMSCs were exposed to chondrogenic stimulus (CS) respectively.
In summary, although the addition of chondroitin sulfate and glutamic acid to biomaterial did not improve the attachment and proliferation of BMSCs, the chitosan-based composite biomaterials developed in this study had superior effect than tissue culture polystyrene (TCPS) material in cell differentiation. The chitosan biomaterial was suitable for osteogeic differentiation, while chitosan/chondroitin sulfate/glutamic acid composite biomaterial was suitable for adipogenic and chondrogenic differentiation. Therefore, these biomaterials can be good candidates for bone- and cartilage-repairing tissue engineering applications.