الفهرس 
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Abstract
Bone marrow stem cells are the principal source of adult mesenchymal stem cells, possessing the ability for differentiating into several cell phenotypes (Fitzsimmons et al., 2018). BM-MSCs isolated from the iliac crest, known as the primary source of BM-MSCs, can differentiate into myogenic, chondrogenic, osteogenic, adipogenic, and non-mesenchymal neurogenic lineages (Mosaddad et al., 2022).
Due to the fact that BM-MSCs present some dermits, investigations have been going on to search a more suitable cell source for bone tissue engineering (Liao & Chen, 2014).
GMSCs lately have gained extensive attention, as they were found to be easily extracted from human gingival tissue, which is usually removed as biological waste in the clinic. It was found to exist in quantities that are suitable for transplantation needs as well as demonstrate rapid proliferation in vitro (Adel et al., 2022).
GMSCs can be easily isolated from the gums, with minimal pain and discomfort. (Venkatesh et al., 2017). GMSCs are characterized by clonogenicity, self-renewability, multipotent differentiation capacity, as well as display stem cell-like and immune-regulating properties (Al Bahrawy et al., 2020).
The self-scaffolding PRF gel is described as a 3-dimensional (3D) fibrin biomaterial, composed of a high concentration of growth factors, which include TGF-b, BMP-2, insulin-like growth factor, and platelet-derived growth factor. in addition to that, it possesses a honeycomb-like microstructure which typically entraps platelets, resulting in the steady release of growth factors, for a sustained time period. By in vivo PRF implantation, the scaffold’s interconnecting porosity not only offers contact guidance for recruited cells, but also sustains a favorable microenvironment, which promotes cell growth and proliferation. (Wong et al., 2020b).
PRF is seen as a reservoir of bioactive materials that promote wound healing and bone regeneration. Although the cellular mechanisms involved remain unclear, in vitro research provides possible explanations. The overall findings show that PRF induces cell proliferation, migration, adhesion, and differentiation as well as possessing anti-inflammatory features, which offer a therapeutic potential in wound healing and bone regeneration (Strauss et al., 2020).
MATERIAL AND METHODS
1. Tissue Collection
Rats were given Ketamine and Xylazine by a dose of 50-75 mg/kg and 10 mg/kg i/p respectively. They were then euthanized by cervical dislocation. The required tissue samples from the bone marrow of the femur and tibia, as well as the gingival oral mucosa were aseptically collected, after which all remaining bodies of the deceased rats were incinerated at the of the National Research Center incinerator. The collected tissue samples were then allocated into two groups
- group (BM-MSCs): where 10ml bone marrow sample was aspirated from the rat femur and tibia.
- group (GMSCs): where part of the oral mucosal gingiva was excised from the oral cavity of the rat.
After the collection of samples, the rats were given ketoprofen (5 mg/kg S.C) to relieve the post-operative pain. Incineration of the deceased rat were done at the incineration of the National Research Center.
2. Stem cell isolation and culture
Collected samples were kept in DMEM to which 10% FBS and antibiotics were added to eliminate any contamination.
The tissues were enzymatically digested using a preparation of collagenase enzyme in a solution containing phosphate buffered saline to generate single cell suspensions.
After inactivation of enzymes, the cells obtained were washed and isolated, guided by their ability to adhere to plastic plates. The isolated cells were identified by flow cytometric analysis.
The isolated cells were expanded. Three passages of the cells were done to distribute proliferating cells on additional culture plates.
3. Preparation of PRF
To prepare PRF, fresh blood was collected from the tail vein of a rat was collected into glass-coated tubes without anticoagulants, after the approval of the Ethics Committee of the National Research Centre, Cairo, Egypt.
Samples were immediately centrifuged at 1300 rpm (400g)/8 minutes spin time. The PRF clot was concentrated between the red blood cell corpuscles at the bottom of the centrifuge tubes, and acellular plasma called PPP at the top of the tubes, which were discarded. After PPP removal, PRF clots were mechanically separated from red blood cells, using a sterile syringe and scissors and gently compressed using gauze to drain the remaining serum, in order to obtain a stable fibrin membrane. All cell culture wells were treated with one whole PRF clot consisting of identical amounts of fibrin, platelets, and white blood cells, and each PRF clot is freshly prepared from blood.
4. Stem cell characterization
The isolated stem cells were tested for CD29, CD90 and CD105, by flow cytometry.
5. Assessment of Cell Proliferation
Isolated cells were divided into four groups, where each group, (group BM-MSCs) and (group GMSCs) were divided into 2 subgroups, each seeded into T25 flasks. One subgroup, control media subgroup, was cultured in plain culture media containing, DMEM + 10% FBS for 8 hours, and the other subgroup, PRF membrane subgroup, was cultured in plain culture media + PRF.
After cell attachment, each well was washed twice with phosphate-buffered saline solution (PBS) and the cells were counted via MTT assay.
6. Induction of Osteogenic Differentiation
Primary cultures of cells were allowed to proliferate until reaching confluence (70-80% adherent cells). Isolated stem cells from the two groups, were seeded into 6-well plates. After cell attachment the medium was changed to an osteogenic differentiation medium. According to the media applied for osteogenic treatment, each of the two groups was further subdivided into:
a- Control media subgroup: which contained plain culture media DMEM + 10% FBS (to serve as a control).
b- PRF membrane subgroup: which contained PRF membranes added to DMEM supplied with 10% FBS.
c- Osteogenic medium subgroup: which contained osteogenic media (D-MEM, 10 mmol/L β-glycerophosphate, 0.2 mmol/L ascorbate-2-phosphated and 100 nmol/L dexamethasone) supplied with 10% FBS.
7. Evaluation of Osteogenic Differentiation
For evaluation of mineralized nodules all sub groups were:
a- assessed by PCR (polymerase chain reaction), for the identification of genes specific for osteoblasts.
b- fixed with methanol and stained with 10% alizarin red solution. Mineralized nodules were identified as red spots on the culture dish. Alizarin red S staining was performed after two weeks.
8. Statistical analysis:
Statistical assessment was carried out using significant tests to compare between different groups, for the two osteogenic genes Runx2 and OSN, where the assessment was done on the 7th and 14th day.
RESULTS
1- Flow Cytometry Results
By running a flow cytometric analysis, (group BM-MSCs) showed a high expression of the three markers, where it showed a 97.7% expression of CD29, 94.7% expression of CD90, and 96.4% expression of CD105.
However, (group GMSCs) displayed a lower expression of the tested markers, where it showed a 93.7% expression of CD29, a 72.5% expression of CD90, and a 68.4 % expression of CD105.
2- MTT Assay Results
Culturing of the stem cells in PRF resulted in an increase in proliferation when compared to the plain media, throughout the first and second week. However, when comparing the bone marrow stem cells to the gingival stem cells, the gingival stem cells were found to show a higher proliferative rate throughout the two weeks.
3- RT-qPCR Results
a- OSN gene: Both the osteogenic media and PRF resulted in an increased expression of OSN, throughout the first and second week, when compared to the plain media.
In case of the BM-MSCS, the PRF membrane caused a comparable effect compared to osteogenic media in the first week, however, during the second week the effect of the osteogenic media became stronger, resulting in a higher OSN expression compared to the PRF membrane.
On the other hand, in case of the GMSCs, the effect of culturing in PRF resulted in a more pronounced effect, where the stem cells showed a higher OSN expression, when compared to osteogenic media, especially during the 1st week.
b- Runx2 gene: Both the osteogenic media and PRF resulted in an increased expression of Runx2, throughout the first and second week, when compared to the plain media.
The osteogenic media caused a more pronounced effect, on the BM-MSCS, compared to the GMSCs, where there was a higher demonstration of Runx2 expression, during both weeks.
During the first week, the PRF also caused a more pronounced effect on the BM-MSCs, compared to the GMSCs, However, during the 2nd week no significant difference in Runx2 expression between the two stem cells was noted, which reflects that the PRF’s effect on GMSCs during the 2nd week was strong enough to be comparable that of the osteogenic media.
4- Alizarin Red staining:
In case of the BM-MSCs, the osteogenic media showed a stronger expression in the second week compared to the first week. In addition to that, the osteogenic media’s effect seemed to be more pronounced than that of the PRF membrane.
In case of the GMSCs, the osteogenic media showed a stronger expression in the second week compared to the first week, as well. However, in case of PRF membrane culture, a strong expression of the alizarin red stain was observed, where the effect was as strong as that demonstrated by the osteogenic media especially in the 2nd week.