Improvement Of Cultivating Technology Of Dental Follicle Cells From Rat Molars And Primary Research Of Their Characteristics

Dental follicle is one layer of loose connective tissue which surround the enamel organ and dental papilla .Dental follicle has potency of multi-directional differentiation.It can differentiate to periodontal ligament, cementum and proper alveolar bone throughing epithelium-mesenchyme action.So dental follicle cells is becoming one kind of seeding cells of periodontal tissue engineering because of its strong differentiation potency.Now the internal rearch for rat dental follicle cells is concentrate in the methods of cultivation in vitro and the study of their characteristics.My experiment is major in the improment of cultivating technology of dental follicle cells from rat molars,aiming at exploring a easy and fast method of cultivating rat dental follicle cells,and studying their characteristics including vimentin(Vim), keratin(CK),collagenI(COL I), collagen III(COL III), osteopontin(OPN) andosteocalcin(OCN).1 .Detachment,cultivation and passage of rat dental follicle cellsMy experiment use a method combinding two ways of "direct peel off method" and "alternative digestion between collagenase and trypsin":Peel off dental follicle directly from the surface of dental germ in the condition of sterilitas,and cut into tiny tissue about 1mm³,then use 0.2% collagenase to digest in warm water(37°C),1 hour later,add 0.25% trypsin,continue digesting for 5 minutes,then the tissue become cell suspension,centrifugate,add 3ml nutrient solution (DMEM containing 15% FBS)into sediment, cultivate in the condition of 5%CO₂, saturated humidity,37°C.12 hours later,major cells adherent.Cells coverage the floor of culture flask about 80% 6-7 days later,then it can passage,while it need 2 weeks using traditional method,so it become more efficient.The cell shape looks like nonhomogeneity,are major in polygonal and spindle-shaped cells,with the shape of fibroblast-like cells.In some cells,their cytoplasm are filled with abundant granules.2. Research of their characteristics Cells of passage 4 were fixed,and study their characteristics using immunohistochemistry.The expression of Vim was strong,its cytoplasm was stained buffy,while CK did not stain,which certify the cells are come from mesenchyme.More over,the expression of COL I ,COL III,OPN and OCN were positive.The results indicated that dental follicle cells not only exhibit fibroblastic characteristics, but also have some characteristics of osteoblasts or cementoblasts.Based on the above fmdings,we can conclude that my method can cultivate rat dental follicle cells in a easy and fast way,and study their characteristics from full aspects.Thus we can do some primary research for seeding cells of periodontal tissue engineering.