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    Effects of combination TGF-B1 transfection and platelet rich plasma (PRP) on three-dimension chondrogenic differentiation of rabbit dental pulp-derived mesenchymal stem cells
    (Taylor & Francis Inc, 2021) Salkin, Hasan; Gonen, Zeynep Burcin; Ozcan, Servet; Bahar, Dilek; Lekesizcan, Ayca; Taheri, Serpil; Kutuk, Nukhet
    Aim: The aim of this study was to evaluate the effects of standard culture medium and chondrogenic differentiation medium with PRP on chondrogenic differentiation of rabbit dental pulp-derived mesenchymal stem cells (rabbit DPSCs) that are transfected with transforming growth factor-beta 1 (TGF-B1) gene, based on the hypothesis of TGF- B1 and PRP can be effective on the chondrogenesis of stem cells. Materials and Methods: Rabbit DPSCs were characterized by using flow cytometry, immunofluorescent staining, quantitative Real Time Polymerase Chain Reaction (qRT-PCR) and differentiation tests. For the characterization, CD29, CD44 and CD45 mesenchymal cell markers were used. Rabbit DPSCs were transfected with TGF-B1 gene using electroporation technique in group 1; with PRP 10% in group 2; with chondrogenic medium in group 3; with both chondrogenic medium and PRP in group 4. DPSCs were cultured in medium with 10% inactive PRP in group 5, chondrogenic medium in group 6, chondrogenic medium with PRP 10% in group 7. SOX9, MMP13 and Aggrecan gene expression levels were evaluated in 3, 6, 12. and 24. days by qRT-PCR. Results: The expression levels of SOX9, MMP13 and Aggrecan were higher in group 2, 3 and group 7 in 3th day however in 24th day group 7 and group 2 were found higher. The expression levels changed by time-dependent. The extracellular matrix of the cells in experimental groups were positively stained with safranin O and toluidine blue. Conclusion: The combination in culture medium of TGF-B1 gene transfection and 10% PRP accelerates the chondrogenic differentiation of DPSCs.
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    Effects of TGF-?1 Overexpression on Biological Characteristics of Human Dental Pulp-derived Mesenchymal Stromal Cells
    (Korean Society for Stem Cell Research, 2019) Salkın, Hasan; Gönen, Zeynep Burçin; Ergen, Ergül; Bahar, Dilek; Çetin, Mustafa
    Objective: The aim of our study was to investigate the effect of Transforming growth factor beta-1 (TGF-?1) genetherapy on the surface markers, multilineage differentiation, viability, apoptosis, cell cycle, DNA damage and senescence of human Dental Pulp-derived Mesenchymal Stromal Cells (hDPSC). Methods: hDPSCs were isolated from human teeth, and were cultured with 20% Fetal Bovine Serum (FBS) in minimum essential media-alpha (?-MEM). TGF-?1 gene transfer into hDPSCs was performed by electroporation method after the plasmid was prepared. The transfection efficiency was achieved by using western blot and flow cytometry analyses and GFP transfection. Mesenchymal stem cell (MSC) markers, multilineage differentiation, cell proliferation, apoptosis, cell cycle, DNA damage and cellular senescence assays were performed by comparing the transfected and non-transfected cells. Statistical analyses were performed using GraphPad Prism. Results: Strong expression of TGF-?1 in pCMV-TGF-?1-transfected hDPSCs was detected in flow cytometry analysis. TGF-?1 transfection efficiency was measured as 95%. Western blot analysis showed that TGF-?1 protein levels increased at third and sixth days in pCMV-TGF-?1-transfected hDPSCs. The continuous TGF-?1 overexpression in hDPSCs did not influence the immunophenotype and surface marker expression of MSCs. Our results showed that TGF-?1 increased osteogenic and chondrogenic differentiation, but decreased adipogenic differentiation. Overexpression of TGF-?1 increased the proliferation rate and decreased total apoptosis in hDPSCs (p<0.05). The number of cells at “S” phase was higher with TGF-?1 transfection (p?0.05). Cellular senescence decreased in TGF-?1 transfected group (p?0.05). Conclusions: These results reflect that TGF-?1 has major impact on MSC differentiation. TGF-?1 transfection has positive effect on proliferation, cell cycle, and prevents cellular senescence and apoptosis.