Background andObjectives: Human bone marrow-derived mesenchymal stem cells (BMSCs) are promising sources for cell-based regenerative therapy. The purpose of the present study was to elucidate the roles of age and sex on the cellular viability and osteogenic potential of BMSCs cultured in osteogenic media. Materials and Methods: Human BMSCs were isolated and expanded from 3 age groups—20s, 30s, and 50s—from both sexes. The total number of aspirates was ten, and each subgroup had five for 20s (two females and three males), three for 30s (one female and two male), and two for 50s (one female and one male). Analyses of the cell morphology, the cell viability, the expression of the stem cell marker SSEA-4, the secretion of human vascular endothelial growth factor (VEGF), the expression of Runx2 and collagen I, the metabolic activity, and the formation of mineralization nodules were performed. Results: No significant differences were found in the cell viability of human BMSCs cultured in osteogenic media among the different age groups. There were no significant differences in the expression of SSEA among the age groups or between males and females. There were no significant differences in the secretion of human VEGF between males and females. No significant differences in Runx2 or collagen I expression were noted by age or gender. Moreover, no significant differences were shown in osteogenesis by alizarin red staining. Conclusions: The human BMSCs showed no age-related decreases in cellular viability or osteogenic differentiation potential.

Background and Objectives: Cuminum cyminum L. has long been used in the treatment of various diseases in multiple geographical regions. This study was performed to determine the effects of C. cyminum methanolic extract (CCT) on the cellular viability, alkaline phosphatase activity and mineralization of human mesenchymal stem cells. Materials and Methods: Bone marrow-derived stem cells were cultured in the presence of CCT at concentrations of 0, 0.001, 0.01, 0.1 and 1 μg/mL. Evaluations of cell morphology were performed on days 1, 3, 7 and 14. Cellular viability was evaluated on days 1, 3, 5 and 7. On the 7th and 14th day, alkaline phosphatase activity measurements and Alizarin red S staining were conducted to assess the osteogenic differentiation of stem cells. A real-time polymerase chain reaction was used to determine the expression levels of RUNX2, BSP, OCN, COL2A1 and β-catenin mRNAs. Results: Stem cells in the control group showed fibroblast-like morphology and the addition of CCT at 0.001, 0.01, 0.1 and 1 μg/mL did not generate noticeable changes in morphology compared with the untreated control group. The application of CCT did not produce significant changes in cellular viability or alkaline phosphatase activity compared with controls. Alizarin Red S staining was significantly increased with the application of CCT. Treatment with CCT increased the expressions of RUNX2, BSP and OCN. Conclusions: These results indicate that CCT enhanced the osteogenic differentiation of stem cells derived from bone marrow by regulating the expressions of RUNX2, BSP and OCN. Thus, the use of CCT may be applied to achieve beneficial effects on the mineralization of stem cells.

Background and Objectives: A derivative of the enamel matrix was used to speed up periodontal regeneration, including the formation of new cementum, alveolar bone, and periodontal ligament. In this study, human gingiva-derived stem cell–derived cell spheroids were used to assess the effects of an enamel matrix derivative on cell viability, osteogenic differentiation, and mineralization. Materials and Methods: Human gingiva-derived stem cells were used to create spheroids, which were then coupled with unloaded control groups and an enamel matrix derivative at a final concentration of 2.7, 27, 270, and 2700 μg/mL. The morphological examination of the created stem cell spheroids took place on days 1, 3, 5, and 7. The Live/Dead Kit assay was used to determine the qualitative viability of cells on days 3 and 7. Using the Cell Counting Kit-8, the quantitative vitality of the cell spheroids was assessed on days 1, 3, and 5. On days 7 and 14, alkaline phosphatase activity assays and Alizarin Red S staining were carried out to examine the osteogenic differentiation of the cell spheroids. RUNX2 and COL1A1 expression levels on days 7 and 14 were determined using real-time polymerase chain reaction. Results: The added enamel matrix derivative at the tested concentrations did not significantly alter the morphology of the applied stem cells’ well-formed spheroids on day 1. On days 3 and 7, the majority of the spheroids’ cells fluoresced green while they were being cultivated. Alkaline phosphatase activity data revealed a substantial rise in the 2700 μg/mL group on day 7 when compared to the unloaded control (p < 0.05). On days 7 and 14, calcium deposits were distinctly seen in each group. In the 27 and 2700 μg/mL groups, the treatment with the enamel matrix derivative resulted in noticeably higher values for the Alizarin Red S staining (p < 0.05). qPCR results showed that adding an enamel matrix derivative to the culture of the 27 μg/mL group raised the level of RUNX2 mRNA expression. Conclusions: These results lead us to the conclusion that a derivative of the enamel matrix may be used to promote osteogenic differentiation in stem cell spheroids.

The growth of bone morphogenetic protein 7 (BMP-7) has been applied for tissue regeneration due to its osteoinductive properties. The aim of this research is to analyze the enhancing effects of BMP-7 on the osteogenic differentiation and mineralization of human bone marrow-derived stem cells cultured on the bovine bone particle. After the stem cells were loaded onto the bone graft material, their morphology was observed on day 7. Viability assays based on the application of fluorescent stains were used for qualitative analyses. Alkaline phosphatase activity assays and Alizarin red staining were used for the assessment of osteogenic differentiation on days 7 and 14. Next-generation mRNA sequencing was applied to evaluate global gene expression. Gene ontology and pathway analysis was used to propose the underlying mechanism. Fibroblast-like morphology was attained with the stem cells. The cells were shown to be firmly attached to the bone particle. Most of the stem cells produced an intense green fluorescence. The relative cellular viability assay values for BMP-7 groups at 0, 10, and 100 ng/mL on day 7 were 0.295 ± 0.003, 0.250 ± 0.002, and 0.240 ± 0.003, respectively (p < 0.05). Alkaline phosphatase activity was significantly higher in BMP-7 groups at concentration of 100 ng/mL compared to the control on days 7 and 14 (p < 0.05). The results of the mineralization assay showed significantly higher values for BMP-7 groups at 100 ng/mL concentration when compared with the control (p < 0.05). The expression of RUNX2 was increased with application of BMP-7 and mitogen-activated protein kinase pathway was associated with the target genes. Overall, this study shows that in vitro application of BMP-7 increases alkaline phosphorylase activity and mineralization of stem cells culture on deproteinized bovine bone mineral. The study suggests that combining stem cells with osteoinductive growth factors with scaffolds can have synergy effects on osteogenic differentiation.

Insulin-like growth factors (IGFs) plays various roles, including differentiation and mitogenesis, and IGFs are reported to regulate the bone growth and maintenance. This study was performed to analyze the enhancing effects of IGF-2 on osteogenic differentiation and the mineralization of stem cells cultured on deproteinized bovine bone mineral. Stem cell loaded bone graft material was cultured in the presence of the IGF-2 at final concentrations of 10 and 100 ng/mL and the morphology of the cells was observed on Days 1, 3, and 7. The commercially available, two-color assay based on plasma membrane integrity and esterase activity was also used for qualitative analyses on Days 1, 3, and 7. The level of alkaline phosphatase activity and anthraquinone dye assay were used to evaluate osteogenic differentiation on Days 7 and 14. Real-time polymerase chain reaction was applied in order to identify the mRNA expression of BGLAP, Runx2, and β-catenin. The stem cells were well-attached with fibroblast morphology and most of the stem cells produced a high intensity of green fluorescence, indicating that there were live cells on Day 1. The relative cellular viability assay values for IGF-2 groups at 0, 10, and 100 ng/mL on Day 1 were 0.419 ± 0.015, 0.427 ± 0.013, and 0.500 ± 0.030, respectively (p < 0.05). The absorbance values at 405 nm for alkaline phosphatase activity on Day 7 for IGF-2 at 0, 10, and 100 ng/mL were 2.112 ± 0.152, 1.897 ± 0.144, and 2.067 ± 0.128, respectively (p > 0.05). The mineralization assay results at Day 7 showed significantly higher values for IGF-2 groups at 10 and 100 ng/mL concentration when compared to the control (p < 0.05). The application of IGF-2 groups of 10 and 100 ng/mL produced a significant increase of BGLAP. Conclusively, this study indicates that the use of IGF-2 on stem cell loaded bone graft increased cellular viability, Alizarin red staining, and BGLAP expression of stem cells. This report suggests the combined approach of stem cells and IGF-2 with scaffold may have synergistic effects on osteogenesis.