| Dental pulp stem cells(DPSCs)are adult stem cells with self-renewal capacity and multi-lineage differentiation potential which means they may differentiate into certain cell types under conditions.Therefore,they have the potential to become seeding cells in tissue engineering for dental pulp restoration and regeneration and becoming pulp-dentin complex.Autologous concentrated growth factors(CGF)fibrin is considered to be the third generation of platelet concentrate which is made thorough continuous differential-speed centrifugalizing autologous venous blood.CGF contains a variety of growth factor and it has been proved to have improving effects to proliferation and mineralization of mesenchymal stem cells(MSCs).Objective: To isolate,culture,and identify human dental pulp stem cells(HDPSCs)by using the enzymes digesting method;For providing experimental basis for clinical practice of HDPSCs and autologous CGF fibrin,using CCK-8 method and Alkaline Phosphatase Assay Kit to test different effects to proliferation and mineralization of HDPSCs caused by different concentration of autologous CGF membrane.Methods:1 Isolation,cultivation,and identification of HDPSCsPulp tissue were extracted from third molars of healthy volunteers who were 18-25 years old,with good oral hygiene and indication,and the volunteers demanded of extraction of impacted teeth.All the volunteers had their impacted teeth extracted in Oral and Maxillofacial Surgery Department of The Second Hospital of Hebei Medical University.The extracted impacted teeth were completed,without caries,cracks,and periapical tissue diseases.The HDPSCs were isolated and cultured by using the enzymes digesting method and then the cells were cultured in high glucose DMEM contained 15% fetal bovine serum in vitro.The HDPSCs were digested when cells overgrowed the 80% of bottom of the bottle.The first generation followed the ratio of 1:1,then 1:3 for the after generations.The surface markers of HDPSCs were identified by using Flow Cytometry(FCM)and HDPSCs' growth curve was detected by using CCK-8 method.By inducing culturing osteogenesis and adipogenesis of cells,the multi-directional differentiation capacity of HDPSCs were tested by alizarin red and Oil Red O staining.2 Preparation of CGF membrane and groupsVenous blood was obtained from the patients and was centrifuged in 9 ml sterile Vacuette tubes for 13 minutes.After centrifuging,the venous blood was divided into three layers.The upper layer was discarded while the junction of the middle layer and the bottom layer was cut and was pressed into CGF membrane.The CGF membrane was cut into 3 mm square tissue and then preserved in sterile normal saline.Different concentrations of CGF were added to different experiment groups respectively,the group that was added with one piece of CGF was named 1 CGF group,the group that was added with two pieces of CGF was named 2 CGF group and the group that was added with three pieces of CGF was named 3 CGF group.In addition,the other one group that was not added with CGF was control group.3 The effects of CGF membrane on the proliferation activity of HDPSCsCells of 4th generation of HDPSCs with good biologic characters were incubated in 6-well plate by 1×104/porocyte.After 24 hours culture medium was discarded.Three concentration gradients of CGF membranes with DMEM solution,10% FBS were added in treatment groups,and DMEM solution,10% FBS,without CGF membrane was added in control group.The culture medium in the above 4 groups were discarded successively after 1 day,3 days,5 days,and 7 days,and incubated the cells from 6-well plates to 96-well plates.The absorbance values of the above groups were tested at 450 nm after 2-hour incubation with CCK-8 solution in incubator under 37?.4 The effect of CGF membrane on the mineralization activity of HDPSCsCells of 4th generation of HDPSCs with good biologic characters were incubated in 6-well plate by 2×105/porocyte.After 24 hours culture medium was discarded.Three concentration gradients of CGF membranes with mineralization introducing solution were added in treatment groups,and mineralization introducing solution,without CGF membrane was added in control group.The culture medium in the above 4 groups were discarded after 14 days.The activity of alkaline phosphatase(ALP)was calculated by following the instructions on Alkaline Phosphatase Assay Kit.5 Statistical analysesAll data were statistically analyzed utilizing SPSS 13.0 software by one-way analysis of variance,Dunnett's T3 and S-N-K for multiple comparisons.All data were expressed as mean±se.A probability value of P < 0.05 was considered statistically significant.Results:1 After primary culturing HDPSCs by using the enzymes digesting method,cellular morphology was observed under microscope as spindle,soma fullness,abundant cytoplasm.The round or elliptic nucleolus are in the center of cells.The HDPSCs' growth curve was “S” style.In test of flow cytometry,cell surface antigenicity CD44 and CD29 were positive,CD34 and CD45 were negative.After 4 weeks inducing culturing osteogenesis and adipogenesis of cells,calcium compounds were observed in cells with alizarin red,and red lipid droplet was observed in cells with Oil Red O staining.2 Compared to control group,all experimental groups with different concentrations had positive effect to HDPSCs proliferation,and with the increased of concentration of CGF,the promoting effect increased.3 Compared to control group,all experimental groups with different concentrations had positive effect to HDPSCs osteogenic differentiation,and with the increased of concentration of CGF,the promoting effect increased.Conclusions:1 The HDPSCs,cultured by using the enzymes digesting method,have met the standards of HDPSCs biological characters which include cellular morphology,cell surface antigenicity,and the differentiation capacity.2 CGF in a certain range of concentration has positive effect to proliferation and differentiation of HDPSCs.This reveals new opportunities for clinical practices and research of CGF and HDPSCs tissue engineering. |
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