Local application of gingiva-derived mesenchymal stem cells on experimental periodontitis in rats.

BACKGROUND: Stem cell-based approaches in regenerative periodontal therapy have been used in different experimental models. In this study, the effect of local application of gingival mesenchymal stem cells (GMSC) in fibroin/chitosan oligosaccharide lactate hydrogel (F/COS) on periodontal regeneration was evaluated using experimental periodontitis model in rats. METHODS: Mesenchymal stem cells were isolated from the gingiva of rats and characterized. Viability tests and confocal imaging of GMSC in hydrogels were performed. Healthy control without periodontitis (Health; H; n=10), control with periodontitis but no application (Periodontitis; P; n=10), only hydrogel application (F/COS; n=10), and GMSC+F/COS (n=10) four groups were formed for in vivo studies. Experimental periodontitis was created with silk sutures around the maxillary second molars. GMSC labeled with green fluorescent protein (GFP) (250,000 cells/50 µL) in F/COS were applied to the defect. Animals were sacrificed at 2nd and 8th weeks and maxillae of the animals were evaluated by micro-computed tomography (micro-CT) and histologically. The presence of GFP-labeled GMSC was confirmed at the end of 8 weeks. RESULTS: Micro-CT analysis showed statistically significant new bone formation in the F/COS+GMSC treated group compared with the P group at the end of 8 weeks (p < 0.05). New bone formation was also observed in the F/COS group, but the statistical analysis revealed that this difference was not significant when compared with the P group (p > 0.05). Long junctional epithelium formation was less in the F/COS+GMSC group compared with the P group. Periodontal ligament and connective tissue were well-organized in F/COS+GMSC group. CONCLUSION: The results showed that local GMSC application in hydrogel contributed to the formation of new periodontal ligament and alveolar bone in rats with experimental periodontitis. Since gingiva is easly accessible tissue, it is promising for autologous cell-based treatments in clinical applications.

Mesenchymal stem cell-derived conditioned medium and Methysergide give rise to crosstalk inhibition of 5-HT2A and 5-HT7 receptors in neuroblastoma cells.

OBJECTIVE: (s): We aimed to investigate the effects of mesenchymal stem cell secretome and methysergide combination on 5-hydroxytryptamine 2A, (5-HT2AR), 5-hydroxytryptamine 7 (5-HT7R), adenosine 2A (A2AR) receptors and CD73 on neuroblastoma cell line and how they affect biological characteristics. Methysergide was used as a serotonin antagonist on the neuroblastoma cells. MATERIALS AND METHODS: Human dental pulp-derived stem cells (hDPSCs) used to obtain conditioned medium (CM). Methysergide drug was prepared in CM and applied to neuroblastoma cells. Analysis of 5-HT7R, 5-HT2AR, A2AR and CD73 expressions was performed by western blot and immunofluorescence staining. Total apoptosis, mitochondrial membrane depolarization, Ki-67 proliferation test, viability analysis, DNA damage and cell cycle analysis were performed in accordance with the product procedure by using biological activity test kits. RESULTS: Our results showed that neuroblastoma cancer cells are normally on the Gs signaling axis via the serotonin 7 receptor and the adenosine 2A receptor. CM and Methysergide inhibited the 5-HT7 and A2A receptor levels in neuroblastoma cells. We found that CM and methysergide formed crosstalk inhibition between 5-HT2AR, 5-HT7R, A2AR and CD73. CM and Methysergide increased the total apoptosis in neuroblastoma cells and induced the mitochondrial membrane depolarization. CM and Methysergide induced the DNA damage and arrested in G0/G1 phase of cell cycle of the neuroblastoma cells. CONCLUSION: These findings suggest that the combination of CM and methysergite may exert a therapeutic effect on neuroblastoma cancer cells, and future in vivo studies may be important in area of neuroblastoma research to support the findings.

Effects of non-steroidal anti-inflammatory drug (ibuprofen) in low and high dose on stemness and biological characteristics of human dental pulp-derived mesenchymal stem cells.

PURPOSE: The effect of ibuprofen, an NSAID, on biological characteristics such as proliferation, viability, DNA damage and cell cycle in dental pulp derived stem cells (DPSCs) can be important for regenerative medicine. Our aim is to investigate how low and high doses of ibuprofen affect stem cell characteristics in DPSCs. MATERIALS AND METHODS: DPSCs were isolated from human teeth and characterized by flow cytometry and differentiation tests. Low dose (0.1 mmol/L) and high dose (3 mmol/L) ibuprofen were administered to DPSCs. Surface markers between groups were analyzed by immunofluorescence staining. Membrane depolarization, DNA damage, viability and cell cycle analysis were performed between groups using biological activity test kits. Cellular proliferation was measured by the MTT and cell count kit. Statistical analyzes were performed using GraphPad Prism software. RESULTS: High dose ibuprofen significantly increased CD44 and CD73 expression in DPSCs. High-dose ibuprofen significantly reduced mitochondrial membrane depolarization in DPSCs. It was determined that DNA damage in DPSCs decreased significantly with high dose ibuprofen. Parallel to this, cell viability increased significantly in the ibuprofen applied groups. High-dose ibuprofen was found to increase mitotic activity in DPSCs. Proliferation in DPSCs increased in parallel with the increase in mitosis stage because of high-dose ibuprofen administration compared to the control and low-dose ibuprofen groups. Our proliferation findings appeared to support cell cycle analyses. CONCLUSION: High dose ibuprofen improved the immunophenotypes and biological activities of DPSCs. The combination of ibuprofen in the use of DPSCs in regenerative medicine can make stem cell therapy more effective.

TGF-B1-over-expressed adipose stem cells-derived secretome exhibits CD44 suppressor and anti-cancer properties via antagonistic effects against SMAD4 in breast cancer cells.

OBJECTIVES: This study aimed to investigate the effect of TGF-B1-transfected adipose-derived mesenchymal stem cell (AD-MSC) conditional medium (TGF-B1-CM) on CD44 expression and biological activities in MCF-7 and MDA-MB-231 cells. METHODS: In the study, the experimental groups were created as a standard medium, AD-MSC-CM, TGF-B1-CM, and TGF-B1 recombinant protein. The medium and proteins specified in these groups were applied to MCF-7 and MDA-MB-231 cells separately at 24, 48 and 72 hours. Western blot and immunofluorescent staining were performed with antibodies suitable for CD44 and canonical smad signaling pathway analyses between groups. Cellular proliferation in MCF-7 and MDA-MB-231 cells was measured by MTT. Biological activity analyses such as apoptosis, cell cycle, proliferation, DNA damage, and membrane depolarization between groups were tested on the Muse Cell Analyzer using appropriate kits. Cellular migration between groups was determined by showing cells that migrated to the scar area with in vitro scar formation. Statistics were performed with GraphPad Prism 8.02 software. RESULTS: It was determined that TGF-B1-CM activates the smad signaling pathway in MCF-7 and MDA-MB-231 cells. TGF-B1-CM increased pSMAD2/3 expression and decreased SMAD4 expression in breast cancer cells. A decrease in CD44 expression was found at points of increase in pSMAD2/3 expression. Decreased expression of SMAD4 in breast cancer cells with TGF-B1-CM was associated with decreased expression of CD44. In MCF-7 and MDA-MB-231 cells, TGF-B1-CM was found to increase apoptosis, decrease proliferation, disrupt membrane depolarization, and arrest cells at G0/G1 stage. TGF-B1-CM suppressed MCF-7 and MDA-MB-231 migrations. CONCLUSION: SMAD4-targeted therapeutic strategies may be considered to suppress CD44 expression in breast cancer cells. Both the anti-tumorigenic factors released by AD-MSCs and the secretomes obtained as a result of supporting these factors with the overexpression of TGF-B1, severely suppressed breast cancer cells. With this study, it was planned to obtain a targeted biological product that suppresses breast cancer cells in vitro.

Effects of combination TGF-B1 transfection and platelet rich plasma (PRP) on three-dimension chondrogenic differentiation of rabbit dental pulp-derived mesenchymal stem cells.

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.

Comparison of the effect of postoperative care agents on human gingival fibroblasts: a preliminary study.

PURPOSE: The aim of this study is to compare effects of postoperative care agents; chlorhexidine, octenidine dihydrochloride and hyaluronic acid on human gingival fibroblasts' viability, proliferation, apoptosis and migration. MATERIALS AND METHODS: After cell culturing; chlorhexidine, octenidine dihydrochloride and hyaluronic acid solutions were applied on cells and nothing was applied for control group. The cells were monitored to investigate cytotoxicity; the percentage of apoptotic, living and dead cells at the time of 24, 48, and 72 hours (h). A scratch wound assay was performed to detect cell migration and cells were monitored at baseline, at 24 and 48h. RESULTS: At 24h, chlorhexidine showed statistically lower percentage of total apoptotic cells' than octenidine dihydrochloride (p=0.049), hyaluronic acid (p=0.049) and control (p=0.049). At 48h, hyaluronic acid showed statistically lower percentage than chlorhexidine (p=0.049), and control (p=0.049). All agents were found to have statistically and significantly more cytotoxic than control. However, there was no difference between experimental groups for proliferation rate. Octenidine dihydrochloride showed statistically negative effects on cell migration than chlorhexidine and hyaluronic acid at 24h. Chlorhexidine and hyaluronic acid maintained migration ability of cells than octenidine dihydrochloride at 48h. CONCLUSION: All agents have similar effects on cell behavior such as viability, apoptosis and cell proliferation. However, octenidine dihydrochloride showed statistically negative effects on migration ability than chlorhexidine and hyaluronic acid.

Effects of TGF-ß1 Overexpression on Biological Characteristics of Human Dental Pulp-derived Mesenchymal Stromal Cells.

OBJECTIVE: The aim of our study was to investigate the effect of Transforming growth factor beta-1 (TGF-ß1) gene therapy 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 (α-MEM). TGF-ß1 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-ß1 in pCMV-TGF-ß1-transfected hDPSCs was detected in flow cytometry analysis. TGF-ß1 transfection efficiency was measured as 95%. Western blot analysis showed that TGF-ß1 protein levels increased at third and sixth days in pCMV-TGF-ß1-transfected hDPSCs. The continuous TGF-ß1 overexpression in hDPSCs did not influence the immunophenotype and surface marker expression of MSCs. Our results showed that TGF-ß1 increased osteogenic and chondrogenic differentiation, but decreased adipogenic differentiation. Overexpression of TGF-ß1 increased the proliferation rate and decreased total apoptosis in hDPSCs (p<0.05). The number of cells at “S” phase was higher with TGF-ß1 transfection (p＜0.05). Cellular senescence decreased in TGF-ß1 transfected group (p＜0.05). CONCLUSIONS: These results reflect that TGF-ß1 has major impact on MSC differentiation. TGF-ß1 transfection has positive effect on proliferation, cell cycle, and prevents cellular senescence and apoptosis.

Comparative Analysis of Mesenchymal Stem Cells from Bone Marrow, Adipose Tissue, and Dental Pulp as Sources of Cell Therapy for Zone of Stasis Burns.

Introduction: The implantation of mesenchymal stem cells (MSCs) has been shown to exert benefits for the survival of the zone-of-stasis. However, the clinical experience indicates the importance of selecting the right source and type of stem cells. Therefore, we planned the current study to perform a quantitative comparison of MSCs isolated from three different sources to provide information useful in selection of the optimal source and to see whether critical mechanisms are conserved between different populations. Methods: The protective effects of MSCs derived from bone marrow, adipose tissue and dental pulp were compared in a rat model of thermal trauma. The stasis zones were evaluated 72 h after the burn using histochemistry, immunohistochemistry and biochemistry. Results: Gross evaluation of burn wounds revealed that the differences between the mean percentages of the calculated necrotic areas weren't statistically significant. Semi-quantitative grading of the histopathological findings revealed that there were no significant differences between damage scores. Immunohistochemical assessment of apoptotic and necrotic cell deaths revealed that the differences between the mean numbers of apoptotic and necrotic cells weren't statistically significant. Myeloperoxidase activity was found to be significantly lower in the adipose tissue group. Biochemical and immunohistochemical assessment of tissue malondialdehyde revealed that the differences between the groups weren't statistically significant. Finally, the number of neo-vessels in the dental pulp group was found to be significantly higher. Conclusion: Our findings suggest that bone marrow, adipose tissue and dental pulp may serve as a universal donor MSC source for the prevention of burn wound progression.