The Influence Of Surface Modification By Chitosan And Raloxifene On The Cellular Morphology, Proliferation, Differentiation And Migration

Tissue engineering, applying the theory of molecular biology, cytobiology and engineering, is a science of study to develop biologic substitutes for reparing tissue defects and improving organ function. It contents three problems at least:①The selection of biomaterials;②the application of cellular regulators;③the obtaining of seed cells. Many studies have shown that the materials surface interacts with the cellular regulators, such as extracellular matrix (ECM) protein and growth factors govern the cellular behaviors and intracellular signal transduction has been key in the development polymers for use within biomedical applications. Despite this improved understanding, the surface characteristics and their effect on governing host responses still remains to be fully elucidated. Surface modification of biomaterials is an effective treatment technique and widely applicated in tissue engineering field. In this study chitosan was explored on poly-L-lactic acid (PLLA) films surface. Compared to pristine PLLA substrate, chitosan grafted PLLA surface can promote the proliferation rate of L929 murine fibroblasts, however we found that cells cultured on this film could hardly spread and tend to be round shape. It was well documented that ECM protein adsorbed on the biomaterials surface bind to integrin receptors in the cell membrane and influence cytoskeletal organization, cell spreading and cell proliferation. Chitosan is a hydrophilic biomaterial that is not conducive to ECM protein adsorption, therefore unlike pristine PLLA and glass substrate, chitosan grafted PLLA films limited L929 cells spreading. Previous studies suggested that prevention of cell spreading or disruption of cytoskeletal organization will inhibit cell proliferation and block cell cycle progression correlated with a failure to increase cyclin D1 and down-regulate the cell cycle inhibitors p27Kip1 expression. To investigate the stimulatory effect on fibroblast proliferation by chitosan, we proposed that because growth factors or components in serum which contribute to cell proliferation are unstable and easily inactive by the condition of heat, proteolysis and acid, chitosan molecular may protect their activity and potentiate their effect. Furthermore, with the interaction with chitosan molecular, growth factors in serum was more effective to bind to specific receptors on cell membrane and stimulate downstream extracellular signal-regulated kinase (ERK1/2) phosphorylation and cyclin D1 expression. In summary, our results demonstrated that chitosan can accelerate L929 cell proliferation and drive cell cycle progression via the effect of growth factor signal and independent of cell round morphology and disorganized cytoskeleton.Seed cell is the first step and essential element of construction and application of tissue engineering. Because adipose-derived stem cells (ADSCs) can be easily obtained in large quantities and minimal discomfort, there has been increased interest in ADSCs for tissue engineering. In our work, ADSCs were obtained from SD rats according to the technique reported and identified their proliferation and differentiation potential. The high proliferation rate and multilineage differentiation capacity of ADSCs were comfired in vitro. Although the osteogenic potential of ADSCs has been characterized, the underlying molecular mechanism responsible for their osteogenic lineage commitment remains to be revealed.. Nitric oxide (NO) is a free radical signaling molecule that has been indicated as a secondary messenger in many biological pathways. It’s generated by conversion of the amino acid, L-arginine, catalysed by nitric oxide synthase. Recently the effect of NO on the metabolism and activity of osteoblast are being investigated. It has been shown NO functions as a crucial factor for regulating the bone formation. However, there’s little report the effect of NO in the ADSCs osteogenic differentiation. Raloxifene is a selective estrogen receptor modulator (SERM). In present study, we demonstrated that treatment of rat ADSCs with raloxifene (0.1-1μmol/L) led to a dose-dependent increase of NO production. The release of NO by raloxifene resulted in increased osteoblastic differentiation of ADSCs, the induction of both mRNA and protein levels expression of osteoblast-specific markers such as Cbfal/Runx2, alkaline phosphatase (ALP), bone morphogenetic protein (BMP2) and typeⅠcollagen (Col-Ⅰ) as well as cell mineralization, calcium deposition into extracellular matrix. The increment of NO production was also correlated with the upregulation of osteoblastic markers in ADSCs cultures. These effects by raloxifene were diminished by concurrent treatment involving the endothelial nitric oxide synthase (eNOS) inhibitor L-nitro-L-arginine methyl ester (L-NAME, 1mM). To investigate the mechanism, because of its estrogen-agonistic effects on bone, raloxifene can bind to the estrogen receptors (ERs) and activate the phosphatidylinositol-3-kinase (PI3K)/Akt and extracellular signal-regulated protein kinase (ERK) cascades. These processes were involved in phosphorylation of endothelial nitric oxide synthase (eNOS), enhancing release of nitric oxide (NO) and its downstream effector soluble guanylyl cyclase (sGC) and cGMP content. The accumulation of cGMP may subsequently regulate osteoblastic markers expression, cell mineralization and calcium deposition. In conclusion, our findings suggested that raloxifene can induce the osteoblastic differentiation of ADSCs via activation of the NO/cGMP pathway.