Construction Of Osteoporotic Rat Bone Marrow Stromal Cell Sheet In Vitro

Periodontitis is characterized by destruction of dental support tissue which is a major cause of tooth loss in adults.Osteoporosis(OP)is characterized by reductions in bone mass and share several risk factors with periodontitis.OP-related bone changes in structure accelerate the progression of periodontitis.The transplantation efficiency of seed cells and scaffold related issues lead to the limited application of traditional tissue engineering technology.Cell sheet technology(CST)has recently been recognized as a scaffold free method,which harvest cell non-invasively and preserve intact extracellular matrix(ECM),cell surface adhesion molecules,cell-cell and cell-ECM interactions.Objective To construct osteoporotic rat bone marrow stromal cell(O-r BMSCs)sheet and evaluate it's fundamental biological property.Methods1.The OP model of SD rats were established by bilateral ovariectomy.The body weight,serum estradiol concentration and femoral histomorphology were used to identify the OP model.2.O-r BMSCs were isolated and cultured in vitro,and the ability of cell cloning was detected by crystal violet staining.Alkaline phosphatase staining and alizarin red staining were performed to detect osteogenic differentiation.Adipogenic differentiation was identified by oil red staining.3.O-r BMSC sheet were constructed by culture medium supplemented with vitamin C.Visual observation,HE staining,scaning electron microscopy and transmission electron microscopy were employed for morphological observation of the cell sheet.4.In order to analyze the effect of enzyme digestion on ECM,the expression of fibronectin(FN)in O-r BMSCs and O-r BMSC sheet were compared by Western Blot.5.We further analyzed the m RNA and protein expression of collagen type?(Col-?),FN,and periostin(PN)using Real-time PCR and immunohistochemistry to investigate the bioactivity of O-r BMSC sheet.Results1.OP models were successively established.The osteoporotic rat exhibited significant weight gain and serum estradiol concentration,as well as histological sparse femur.2.O-r BMSCs were successfully isolated and cultured with the manifestation that the single cell grew in clony.3.ALP staining was obvious after 7days osteogenic induction.On the 21 st day,alizarin red staining showed red mineralized nodules and vacuole-like droplets were dyed dark red after oil red O staining.4.O-r BMSC sheet was successfully constructed with the appearance of milky white and translucent film-like structure.HE staining showed O-r BMSC sheet was composed of multilayer cells and ECM,collagen fibers were stained blue in Masson staining.5.Abundant ECM were observed among cells under scanning electron microscope.Transmission electron microscopy showed cells,ECM,intercellular junctions and organelles.6.The protein level of FN in O-r BMSC sheet was significant higher than O-r BMSCs(P <0.05).7.Compared with day 5,the m RNA and protein expression of Col-?,FN,PN in O-r BMSC sheet were up-regulated at day 10(P <0.05).Conclusions1.O-r BMSCs possess colony forming ability and can differentiated into osteoblasts and adipocytes under specific conditions in vitro.2.The O-r BMSC sheet can be successfully constructed by vitamin C continuous induction.The O-r BMSC sheet is made up of a large number of vigorous cells and rich autocrine ECM,which is expected to be applied to tissue regeneration in osteoporosis.