الفهرس 
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Abstract
Stem cell biology has become an important field in regenerative medicine and tissue engineering therapy since the discovery and characterization of mesenchymal stem cells (MSCs). MSCs can be isolated from many tissues, including bone marrow, adipose tissue, placenta and umbilical cord. All of these MSCs show fibroblast-like cell morphology, self-renewal capacities and multilineage differentiation potentiality.
MSCs have also been isolated from human dental tissues, including dental pulp stem cells, stem cells from human exfoliated deciduous teeth, stem cells from apical papilla, dental follicle cells and periodontal ligament stem cells. Those dental tissue-derived stem cells have potent capacities to differentiate into chondrogenic, osteogenic and odontogenic lineages and generate dental tissue structures.
Many people have an impacted third molar that does not cause occlusion and have that impacted tooth extracted to avoid inflammation or orthodontic therapy. Such extracted teeth usually contain dental follicle and are commonly discarded as medical waste. Dental follicle contains MSCs in the tooth germ at various stages of development in different species. Therefore, the DF is a candidate source for isolating MSCs.
Comparatively, a population of stem cells within gingival tissue, termed gingiva-derived mesenchymal stem cells constitutes more attractive alternatives to other dental-derived MSCs for the accessibility and availability and minimal discomfort.
This research was performed to assess and compare stem cells isolated from dental follicle and gingiva with regard to their characterization, proliferation and capability to differentiate into osteogenic and odontogenic lineages.
The gingival tissue sample was obtained from healthy patient undergoing gingivectomy for crown lengthening above 20 years, under local anesthesia after providing written informed consent. Human dental follicle sample was obtained from patient above 20 years undergoing surgical removal of impacted wisdom, under local anesthesia after providing written informed consent.
MSCs isolation procedures of both gingival and dental follicle tissues were done. The minced pieces were collected in sterile, labeled 15 ml Falcon tubes to which a digesting solution consisting of collagenase type II and dispase mixture was added. The digested solution used was 3 mg/ml collagenase type II and 4 mg/ml dispase (Sigma-Aldrich, USA) for 60 minutes at 37°C.
Propagation was performed when the primary cell culture of adherent cells reached 70% confluence and was named passage zero (P0). Later passages were named accordingly. Cells were sub-cultured every other week and the culture medium was replaced every 2 - 3 days over a 12 - 14 day period.
Cells were identified as being MSCs by their morphology (fibroblast like cells) and plastic adherence. Adherent cells were trypsinized and adjusted to 1×106 cells/ml. Then were incubated with 10μl of monoclonal antibodies: CD45, CD90 and CD105, (Beckman coulter, USA) at 4 ◦C in the dark. The results show that successful isolation of stem cells from dental follicle and gingiva based on their ability to adhere to plastic plates was achieved. Cells were successfully sub-cultured and expanded up to passage three. Cells positively expressed CD90 and CD105 and negatively expressed CD45.
Cells were allowed to proliferate and then the proliferation capacity was assessed using MTT assay to compare the proliferation of the two cell types. Although the highest mean was found for DFSCS, there was no significant difference between GMSCs and DFSCs in their proliferative capability.
Then the cells were induced for differentiation: odontogenic differentiation by placing the cells in odontogenic induction media for 21 days and osteogenic differentiation by placing the cells in osteogenic induction media for 30 days.
Odontogenic and osteogenic differentiation were evaluated by Alizarin red stain. By the 14th day, the aggregated cells were the first to be stained indicating beginning of mineralization in these cells. By the 21st day, the staining became more intense and multiple isolated mineralized extracellular nodules/sites appeared.
Our results were statistically evaluated using Independent sample-t test for two independent samples. Paired-wise sample t-test was used to compare between two related samples.
The RT-PCR results for Nanog gene expression indicated a significant difference between undifferentiated and differentiated MSCs in both gingival and dental follicle stem cells while no significant difference between undifferentiated GMSCs and DFSCs and differentiated osteoblasts and odontoblasts of both groups was found.
The expression of Dentine Sialophosphoprotein (DSPP) showed high significant difference between differentiated odontoblasts derived from DFSCs and GMSCs. The highest mean value was found for odontoblasts differentiated from DFSCs.
For Runx2 gene expression, it was expressed in both osteoblasts and odontoblasts in DFSCs. No statistical difference between the gene expression in osteoblasts from GMSCs and DFSCs was found but the highest mean was found for GMSCs.
The results also showed that the gene expression of osteopontin was highly expressed in the osteoblasts than the odontoblasts with significant difference while no statistical significant difference was found between osteoblasts differentiated from GMSCs and DFSCs with the highest mean was found for the DFSCS.
from the above results it can be concluded that discarded tissues as dental follicle and gingival tissue are promising sources of stem cells. DFSCs showed high proliferation capacity than GMSCs. Both DFSCS and GMSCS showed the ability to be induced into odontogenic and osteogenic lineages with the preference to DFSCs which showed the highest mean for odontogenic and osteogenic gene expression.