The Establishment And Evolution Of 3D Acellular Matrix Model For Dermal Papilla Cells

Background:Dermal papilla,considered as a highly specific cluster of fibroblasts at the bottom of hair follicles,secrets a variety of growth factors to regulate the morphogenesis and growth cycle of hair follicles.Low-passaged DPCs(dermal papilla cells)cultured in vitro still remain the inductive ability of hair follicle regeneration.But the DPCs tend to loss its inductive ability during serial culture.Therefore,with the in vitro cultivation the maintaining inductive function of DPCs is the key to reconstruct tissue-engineered hair follicles.We believe that a large number of biologically-functional DPCs can be obtained by establishing a three-dimensional culture model of newborn mouse acellular dermal matrix(ADM)to provide sufficient sources of DPCs for reconstruction of tissue-engineered hair follicles.Objective1.To establish a 3D newborn mouse ADM model for DPCs2.To evaluation of biological and functional characteristics of DPCs cultured on ADM.3.To reconstruct hair follicle in vivo by utilizing DPCs cultured on ADM.Methods1.The establishment of a 3D newborn mouse ADM model for DPCsThe neonatal mouse dermis was decellularized by a method that combined physical method with chemical method.The adult mouse vibrissal DPCs were isolated by a method that combined anatomical method with collagenase digestion method.The morphology of DPCs cultured in two-dimensional an three-dimensional conditions were observed with a microscope.2.The evaluation of biological and functional characteristics of DPCs cultured on ADM.The evaluation of biological characteristics:the gene expression level of specific markers which are ALP and Versican in DPCs was detect by qRT-PCR.the protein expression level of ALP and Versican in DPCs was detect by immunofluorescence and Western blotting.3.The reconstruction of hair follicle in vivo by utilizing DPCs cultured on ADM.Biological function test:DPCs were mixed with neonatal mouse epidermal cells in the following groups for in vivo transplantation.2 weeks later,the grafts were observed and stained by H&E staining.Result1.The establishment a 3D newborn mouse ADM model for DPCsThe ADM decellularized by a method that combined physical method with chemical method was stained with DAPI and H&E staining.The results showed that there was no nuclear shown in ADM in both DAPI and H&E staining.There were fibers arranged in a grid pattern shown in ADM by electron microscope observation.2.The evaluation of biological and functional characteristics of DPCs cultured on ADM.The result of qRT-PCR,immunofluorescence staining and western blotting showed that the expression of ALP and Versican were down-regulated in high-passaged DPC cultured in two-dimensional culture conditions,while the ones of ALP and Versican were restored in high-passaged DPC cultured on ADM.3.The reconstruction hair follicle in vivo by utilizing DPCs cultured on ADM.The co-transplantation of 2D-P8-DPCs with fresh epidermal cells was failed to reconstruct hair follicles,while the one of 3D-P8-DPCs with fresh epidermal cells was succeed to reconstruct hair follicles.Conclusion1.ADM was obtained by decellularization by a method that combined physicalmethod with chemical method for culturing DPC.2.The expression of ALP and Versican in high-passaged DPC were restored by culturing on ADM.3.The high-passaged DPC cultured by ADM regained the inductive ability for hair follicle reconstruction in vivo.