Osteogenic/Oblastogenic Differentiation Ability Of Dental Follicle Stem Cells Derived From Type ? Dentine Dysplasia In Vitro

ObjectiveDentin dysplasia type I(DD-I)is a type of autosomal dominant hereditary disease with poor root development and reduced mineralization of dentin.The main characteristics are that the root is short or absent,the medullary cavity is completely or incompletely closed,and some of the root tips have carious shadow or not.At present,the research mainly focuses on the ultrastructure of teeth,and abnormal dentins have been reported.In 2010,our group collected a DD-I family whose oral and pathological examination revealed that the alveolar bone is insufficient and the cementum is thin than the nomal teeth.Therefore,this study is proposed to isolate and cultured the dental follicle stem cells(DFCs)of the proband of DD-I family in vitro,and observe their biological characteristics and osteogenesis/cementum differentiation ability.So as to provide new clues for DD-I root development pathological mechanism in cell biology.Materials and MethodsPart I: Study on the In Vitro Isolation,Culture and Proliferation Ability of DD-I DFCs1.Removal of the third molars with incompletely developed root tips from DD-I patient and healthy people,then isolate and culture the DFCs individually by using the method“reformed enzymatic digestion + tissue explant”.Label the groups as DD-I group and healthy control group,and the morphology of each group are observed via an inverted microscope.2.Identification of the source of DFCs with the methods of immunofluorescence by detecting the anti-VIM and anti-CK;3.Detection of the colony-forming efficiency and proliferation ability of each group using limiting dilution assay(LDA)and MTT assay.Part II: Exploration of Osteogenic Differentiation Ability of DD-I DFCs in vitro1.Observation of the formation of extracorporeal mineralized nodules with the method of alizarin red staining after the induction of mineralization in both groups for 14 d,21d,and 28d;2.Detection of the alkaline phosphatase activity of each group using ALP staining(improved Gomori calcium cobalt method)after the induction of mineralization in both groups for 7d,14 d,and 21d;3.Examination of the expression level of mineral-associated proteins,like ALP,OCN,BSP,RUNX-2 in both m RNA and protein level.Results1.The morphology of DFCs which was observed under the inverted microscope showed typical fibroid cell with a long spindle and a longer pole.Though the morphology of the two groups were similar,the generation time differed.Generally,cells in DD-I group required 3-4 days for generation,while 5-6 days were required in the other group.2.Dental follicle stem cells Vim(+)and CK(-)were detected by immunofluorescence which means cells derive from mesenchymal stem cells(MSCs);3.Clonal and population analyses showed that the colony-forming efficiency in DD-I group was approximately 12.5%,whereas in the healthy control group was 10.5%;4.MTT assay showed that the growth curves of DFCs in both groups were "S-like".However,the rate of cell proliferation in DD-I group was significantly higher(P<0.05)than that in healthy control group.5.Alizarin red staining after 14-days of mineralization induction indicated that both groups showed orange calcification nodules and black calcium salts,when compare with the calcium nodules in healthy control group,smaller number,less area and less calcium salt content were detected in DD-I group.6.ALP staining results showed that the alkaline phosphatase positive cells in both groups elevated with the increase of mineralized induction time,however,the number of alkaline phosphatase positive cells in DD-I group was lower than that of the control group at each time point.7.Real time PCR results showed that the m RNA expression level of ALP,OCN,BSP and RUNX-2 in both two groups rose gradually with the increasing of osteogenic induction time.Besides,the expression of each gene in DD-I group was lower than the control group at every time point,and the difference was statistically significant(P<0.05).8.Western blot results showed that the expression of ALP,OCN,BSP and RUNX-2 began to express from the 7d,and the expression level of osteogenic protein increased gradually of the two groups.The expression of each gene group in DD-I was lower compared with the control group at every time point,and the difference was statistically significant(P<0.05).Conclusion1.DFCs of DD-I were successfully isolated and cultured with the method of “reformed enzymatic digestion + tissue explant”.The cells showed basically normal morphology and strong independent survival and proliferation ability.2.The osteogenesis/cementum differentiation ability of DFCs from the DD-I patient was reduced.The reduction above would show negative effect on the insufficient formation of cementum and alveolar bone in the process of root development,and further resulted in insufficient root development.