الفهرس 
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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 the pulp of permanent teeth (DPSC) and exfoliated deciduous teeth (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/ml collagenase type I and 4 mg/ml dispase for 30–60 min at 37°C and then stem cells were collected.Following, the isolated cells were the cells were resuspended at density of 10.000 cells/cm2 in complete culture media. The culture media consisted of alpha modified Eagle’s medium (αMEM) 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 and Sterptomycin 100 µg/ml) and finally antimycotic agent (Fungizone, 0.25 µg/ml) in sterile 25 cm2 polysterine filter cap cell culture plates and incubated at 37°C in a humidified atmosphere of 5% CO2.The medium was changed once every four days.
Passaging was performed when primary cell culture of adherent cells reached 70% confluence and was named passage zero (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 L-ascorbic acid, 10m M dexamethasone and 10 mM β-glycerophosphate. The cells were 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 was evaluated 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).
Results
Successful isolation of stem cells from permanent and deciduous pulp tissue based on their ability to adhere to plastic plates was achieved. Cells were successfully sub-cultured and expanded up to passage three. Colonies were identified as early as 6 days from the beginning of culture in SHED in comparison to 10 days in DPSC. As for the MTT assay, our results demonstrated that SHED proliferated faster than DPSC. Both SHED and DPSC proliferated faster in the first week than they did in the second week.
Our results also showed that earlier nodule formation can be found at day 10 of Alizarin Red Stain in the SHED group supplemented with osteogenic media and at day12 in DPSC group supplemented with osteogenic media. Also the nodule formation was present in the two subgroups at days 22. Also, BMP2 was able to induce osteogenic differentiation of both SHED and DPSC with subsequent calcium deposition however a longer time was taken. RT-PCR results indicated that both DPSC and SHED cells can efficiently differentiate into bone tissue and express specific bone tissue genes; Bone Sialoprotein II and Osteocalcin.
Conclusion
It can be concluded that dental pulp contains cells that are clonogenic, highly proliferative, and capable of osteogenic differentiation. These properties effectively define them as stem cells. Moreover, SHED possesses a higher proliferative ability than DPSC and can differentiate readily into osteogenic lineage.