Construction Of Rabbit Tibial Periosteal Cell Sheet In Vitro

The repair of maxillofacial bone defects is a challenge for clinicians due to the complexity of its structure.The development of tissue engineering technology provides a new treatment direction for the repair of maxillofacial bone defects.Cell sheet technology(CST)has recently been recognized as a scaffold free method,which preserve intact extracellular matrix(ECM).It has great application prospects in the repair of bone defects.Objective To isolate and culture rabbit tibia periosteal cells(PCs),to construct rabbit tibia PCs sheet and evaluate its fundamental biological property.Methods1.Rabbit tibia PCs were isolated and cultured in vitro,CCK-8 and clone formation test were performed to detect the capacity of cell proliferation and cloning.Alkaline phosphatase(ALP)staining and alizarin red staining were applied to detect osteogenic differentiation.Adipogenic differentiation was identified by oil red O staining.Alcian blue staining was used to detect chondrogenic differentiation.2.Rabbit tibia PCs were constructed by culture medium supplemented with Vitamin C.Hematoxylin-eosin staining(HE)and Masson staining were employed for morphological observation of the cell sheet.The live and dead cell assay was applied to detect the activity of cell sheet.3.ALP staining,ALP activity detection and real-time fluorescent quantitative polymerase chain reaction(Real-time PCR)were performed to detect osteogenic differentiation ability and ECM formation ability of the cell sheet.4.CCK-8,β-galactosidase staining,apoptosis flow cytometry and Real-time PCR were used to detect cell proliferation activity,senescence and apoptosis during the formation of cell sheet.Results1.Rabbit tibia PCs were successfully isolated and cultured in vitro,which has proliferation ability and clone formation ability;ALP staining of PCs was obvious and mineralized nodules were red by alizarin red staining after culturing in osteogenic differentiation medium for 7 days and 21 days.Oil red O positive lipid droplet could be observed after adipogenic induction for 21 days.After 21 days’ chondrogenic induction,alcian blue staining were positive.2.Construct the rabbit tibia PCs sheet by Vitamin C successfully,which was milky white and translucent film-like structure.HE and Masson staining showed that the cell sheet was composed of cells and abundant ECM,and there was a good intercellular connection;live and dead fluorescence staining showed that the cell sheet has good activity.3.ALP staining and ALP activity test showed that ALP content of the rabbit tibia PCs sheet were higher than the rabbit tibia PCs.The mRNA expression of OPN,ALP,Runx2,COL-Ⅰ,FN were up-regulated in rabbit tibia PCs sheet.4.CCK-8 results showed that the proliferation rate of the rabbit tibia PCs sheet was higher than rabbit tibia PCs;β-galactosidase staining showed that the number of positive cells stained by β-galactosidase in the the rabbit tibia PCs sheet was less;flow cytometry results showed that the cell sheet apoptotic rate was lower than the rabbit tibia PCs;the mRNA expression of p21,p53,Caspase 3,Bax was down-regulated in rabbit tibia PCs sheet,and the mRNA expression of Bcl2 was up-regulated in rabbit tibia PCs sheet.Conclusions1.Rabbit tibia PCs possess proliferation and colony forming ability and can differentiated into osteoblasts,adipocyte and chondrocyte under specific conditions in vitro.2.Using Vitamin C the rabbit tibia PCs sheet was constructed successfully.The cell sheet is composed of a large number of living cells and abundant extracellular matrix and it’s expected to be applied to maxillofacial bone regeneration.