IN VITRO ANALYSIS AND DIFFERENTIAL CHARACTERISATION OF NUCLEUS PULPOSUS DERIVEDMESENCHYMAL STEM CELLS AND ITS ASSOCIATED STEMNESS AND GENE EXPRESSION CHANGES SUBJECTED TO INFLAMMATORY MARKERS IN HYPOXIC AND NORMOXIC CULTURE CONDITION

Abstract

Degenerative disc disease (DDD) is generally considered as a chronic, progressive and painful disease that is described by inflammation and breakdown of tissue within the intervertebral disc. In recent days, nucleus pulposus mesenchymal cells (NPMSCs) have been proved to be a successful in producing the intended result in therapy of degenerative disc disease (DDD). The aim of the present study was to evaluate the biological characteristics analysis of normal and degenerativenucleus pulposus derived mesenchymal stem cells and also the role of inflammatory cytokines on mouse derived NPMSCs and its related gene expression in normoxic and hypoxic culture setting. NP Tissues were collected from the mouse and expanded in cell culture in normoxic and hypoxic culture setting, NP derived mesenchymal stem cells phenotypic characterization, Cell cycle and apoptosis was confirmed with Flowcytometry. The multipotent differentiation potential of NPMSCs was analyzed with immuno histochemistry and its specific gene expression analysis was confirmed by gel electrophoresis and reverse transcriptase PCR. NPMSCs from non - degenerative and degenerative mice nucleus pulposus shows the similar phenotypic characteristics, But the expression of markers like stemness, differentiation, chemotactic migration and colony formation was high compared to the degenerative NPMSCs. Here proinflammatory markers suppress the expression of hypoxia resistant genes in both normoxic and hypoxic culture environments. Therefore the pro-inflammatory cytokines plays vital role in maintenance of biological behavior of NPMSCs. The findings of this study suggest that NPMSCs shows better proliferation in normoxic and hypoxic culture environment, though the NPMSCs showed the good proliferation in both types of culture condition in the presence of pro-inflammatory cytokines it’s proliferation, multipotent differentiation apoptosis highly increased.