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Abstract Both dental pulp stem cells (DPSC) from permanent teeth and stem cells from human exfoliated deciduous teeth (SHED) have attracted tremendous interest recently by playing a major role in tissue engineering and regenerative medicine. This research focuses on studying stem cells isolated from he pulp of permanent teeth (DPSC) and exfoliated deciduous eeth (SHED) populations with regard to their proliferation and capability to differentiate into osteogenic lineage. Materials and Methods To determine the existence of such a cells in the dental pulp; we applied methodology that had been previously developed for the isolation and characterization of BMSCs and dental pulp stem cells ( Gronthos et al., 2000, Miura et al., 2003 ). Collected teeth were allocated into two main groups: I) DPSC Group:This group included human dental pulp tissues obtained from impacted third molars from patients aged 16–26 years II) SHED group: This group included human dental pulp tissues obtained from clinically healthy, but luxated deciduous incisors from children aged 6-8years. Cell Isolation and Culture: Extirpated pulp tissue was digested in a solution of 3 mg/mlcollagenase type I and 4 mg/ml dispase for 30–60 min at 37°Cand then stem cells were collected.Following, the isolated cellswere the cells were resuspended at density of 10.000 cells/cm2in complete culture media. The culture media consisted ofalpha modified Eagle‟s medium (aMEM) with L-glutamine,(Gibco, Invitrogen Life Technologies,USA)supplemented with 10 % fetal bovine serum, (Gibco,Invitrogen Life Technologies,USA), antibiotics;penicillin/streptomycin (Penicillin G100 unit/ml andSterptomycin 100 µg/ml) and finally antimycotic agent(Fungizone, 0.25 µg/ml) in sterile 25 cm2 polysterine filter capcell culture plates and incubated at 37°C in a humidifiedatmosphere of 5% CO2.The medium was changed once everyfour days. Passaging was performed when primary cell culture of adherent cells reached 70% confluence and was named passagezero (P0) later passages were named accordingly. The primary cell culture was thus propagated and expanded in repeated cell cultures. Cells were sub cultured every other week and culture medium was replaced every 3 days. Assesing Proliferation Capability: To assess the proliferation capacity of the isolated stem cells from both tissues; colony formation efficiency testing and MTT assay were done. The colony formation efficiency of both tissues was observed from the first day of culture to give cells from permanent and deciduous pulp tissues adequate time to demonstrate their difference in growth patterns since some tissues form colonies earlier than the other. Evaluation of Osteogenic Differentiation of Cultured Cells: In order to determine the differentiation ability of DPSC and SHED from passage three , Cells from the third passage were cultured in osteogenic medium consisting of 50 mg/ml Lascorbic acid, 10m M dexamethasone and 10 mM ßglycerophosphate. The cellswere maintained in differentiation cultures for 3 weeks. Also, the effect of BMP2 with the culture medium was also investigated as a potent osteogenic differentiation inducer. Osteogenic differentiation wasevaluated by Alizarin Red Stain and RT-PCR. To confirm osteogenesis, the cells were also examined by RT-PCR for the expression of several osteoblast-related genes including Bone Sialoprotien II and Osteocalcin (OC). |