Bladder smooth muscle cell differentiation and induction of mesenchymal stem cell differentiation in a three dimensional hydrogel matrix

Abstract

Purpose To allow cell attachment and proliferation an adequate temporary scaffold must be provided, while synthesis of extra-cellular matrix proteins and tissue re-organisation take place. Bladder tissue engineering suffers from the formation of scar tissue partly due to the phenotypic switch of smooth muscle cells from a quiescent contractile to a synthetic proliferative phenotype. Material and methods Human bladder smooth muscle and mesenchymal stem cells were successfully cultured three-dimensionally in enzymatically degradable polyethylene-glycol hydrogels, modified with integrin binding peptides (e.g. RGD peptide sequence). Cell viability was analyzed using the the live/dead staining method (Rodamin reaction). Results On zymography (FACS) the smooth muscle cells showed an up-regulation of matrix metalloproteinase 2 (MMP-2) speaking in favour of differentiation into the quiescent, contractile type of smooth muscle cells. 3-dimensional growth of mesenchymal stem cells in the hydrogel induced up-regulation of smooth muscle alpha-actin and myosin and a down-regulation of CD 90, a marker for un-differentiated mesenchymal stem cells. All these findings are in favour of stem cell differentiation into smooth muscle cells. Furthermore an up-regulation of alpha1 beta1 integrin subunits was noted in favour of differentiation into quiescent contractile smooth muscle cells. Conclusions We have demonstrated the feasibility of a model for three-dimensional growth of human bladder smooth muscle cells, differentiating into the quiescent contractile type and of mesenchymal stem cells differentiating into functional smooth muscle cells. This model can be used to study reconstructive processes and can also contribute to the understanding of underlying basic mechanisms of human smooth muscle cell differentiation.