Gingiva-derived Stem Cells Research Articles

Impact of Polydeoxyribonucleotides on the Morphology, Viability, and Osteogenic Differentiation of Gingiva-Derived Stem Cell Spheroids

Background and Objectives: Polydeoxyribonucleotides (PDRN), composed of DNA fragments derived from salmon DNA, is widely recognized for its regenerative properties. It has been extensively used in medical applications, such as dermatology and wound healing, due to its ability to enhance cellular metabolic activity, stimulate angiogenesis, and promote tissue regeneration. In the field of dentistry, PDRN has shown potential in promoting periodontal healing and bone regeneration. This study aims to investigate the effects of PDRN on the morphology, survival, and osteogenic differentiation of gingiva-derived stem cell spheroids, with a focus on its potential applications in tissue engineering and regenerative dentistry. Materials and Methods: Gingiva-derived mesenchymal stem cells were cultured and formed into spheroids using microwells. The cells were treated with varying concentrations of PDRN (0, 25, 50, 75, and 100 μg/mL) and cultivated in osteogenic media. Cell morphology was observed over seven days using an inverted microscope, and viability was assessed with Live/Dead Kit assays and Cell Counting Kit-8. Osteogenic differentiation was evaluated by measuring alkaline phosphatase activity and calcium deposition. The expression levels of osteogenic markers RUNX2 and COL1A1 were quantified using real-time polymerase chain reaction. RNA sequencing was performed to assess the gene expression profiles related to osteogenesis. Results: The results demonstrated that PDRN treatment had no significant effect on spheroid diameter or cellular viability during the observation period. However, a PDRN concentration of 75 μg/mL significantly enhanced calcium deposition by Day 14, suggesting increased mineralization. RUNX2 and COL1A1 mRNA expression levels varied with PDRN concentration, with the highest RUNX2 expression observed at 25 μg/mL and the highest COL1A1 expression at 75 μg/mL. RNA sequencing further confirmed the upregulation of genes involved in osteogenic differentiation, with enhanced expression of RUNX2 and COL1A1 in PDRN-treated gingiva-derived stem cell spheroids. Conclusions: In summary, PDRN did not significantly affect the viability or morphology of gingiva-derived stem cell spheroids but influenced their osteogenic differentiation and mineralization in a concentration-dependent manner. These findings suggest that PDRN may play a role in promoting osteogenic processes in tissue engineering and regenerative dentistry applications, with specific effects observed at different concentrations.

Impact of 17β-Estradiol on the Shape, Survival, Osteogenic Transformation, and mRNA Expression of Gingiva-Derived Stem Cell Spheroids.

Background and Objectives: Mesenchymal stem cells hold promise for tissue regeneration, given their robust growth and versatile differentiation capabilities. An analysis of bone marrow-sourced mesenchymal stem cell proliferation showed that 17β-estradiol could enhance their growth. This study aims to investigate the influence of 17β-estradiol on the shape, survival, osteogenic differentiation, and mineralization of human mesenchymal stem cells. Materials and Methods: Spheroids made from human gingiva-derived stem cells were cultivated with varying concentrations of 17β-estradiol: 0, 0.01, 0.1, 1, and 10 nM. Morphology was assessed on days 1, 3, and 5. The live/dead kit assay was employed on day 3 for qualitative cell viability, while cell counting kit-8 was used for quantitative viability assessments on days 1, 3, and 5. To evaluate the osteogenic differentiation of the spheroids, a real-time polymerase chain reaction assessed the expressions of RUNX2 and COL1A1 on day 7. Results: The stem cells formed cohesive spheroids, and the inclusion of 17β-estradiol did not noticeably alter their shape. The spheroid diameter remained consistent across concentrations of 0, 0.01, 0.1, 1, and 10 nM of 17β-estradiol. However, cellular viability was boosted with the addition of 1 and 10 nM of 17β-estradiol. The highest expression levels for RUNX2 and COL1A1 were observed with the introduction of 17β-estradiol at 0.1 nM. Conclusions: In conclusion, from the results obtained, it can be inferred that 17β-estradiol can be utilized for differentiating stem cell spheroids. Furthermore, the localized and controlled use, potentially through localized delivery systems or biomaterials, can be an area of active research. While 17β-estradiol holds promise for enhancing stem cell applications, any clinical use requires a thorough understanding of its mechanisms, careful control of its dosage and delivery, and extensive testing to ensure safety and efficacy.

Vitamin E Enhances Cell Viability and the Osteogenic Differentiation of Cell Spheroids Made of Gingiva-Derived Stem Cells.

Background and Objectives: Vitamin E is reported to expedite new bone formation in animal models, and this has led to a decrease in the time needed for treatment. In this study, human gingiva-derived stem cell-derived spheroids were examined to determine the effects of vitamin E on cell survival, osteogenic differentiation, and mineralization. Materials and Methods: Human gingiva-derived stem cells were used to create spheroids, which were then cultivated with vitamin E at doses of 0, 0.1, 1, 10, and 100 ng/mL. The morphological examination and the qualitative and quantitative vitality of the cells were assessed. Alizarin Red S staining and alkaline phosphatase activity assays were performed on days 7 and 14 to evaluate the osteogenic differentiation. The expression levels of RUNX2 and COL1A1 were assessed using a real-time polymerase chain reaction. Results: The addition of vitamin E did not appear to alter the spheroid's shape at the measured quantities without altering the diameter. During the culture time, the majority of the cells in the spheroids fluoresced green. Regardless of concentration, there were substantial increases in cell viability in the vitamin E-loaded groups on day 7 (p < 0.05). On day 14, the Alizarin Red S staining was statistically higher in the 1 ng/mL group compared to the unloaded control (p < 0.05). The addition of vitamin E to the culture enhanced the mRNA expression levels of RUNX2, OCN, and COL1A1 based on the real-time polymerase chain reaction data. Conclusions: We draw the conclusion that vitamin E may be used to promote the osteogenic differentiation of stem cell spheroids in light of these data.

Effects of Enamel Matrix Derivative on Cell Spheroids Made of Stem Cells Obtained from the Gingiva on Osteogenic Differentiation.

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 Effects of Transforming Growth Factor-β1 on the Differentiation of Cell Organoids Composed of Gingiva-Derived Stem Cells.

This study was aimed at evaluating the effects of transforming growth factor-β on the differentiation and mRNA expression of organoids made out of human mesenchymal stem cells. Cell organoids composed of gingiva-derived stem cells were cultured in the presence of transforming growth factor-β1 at concentrations ranging from 0, 1, 10, to 20 ng/ml. Evaluations of the cell morphology of the organoids were performed on days 7, 9, 11, and 14. Quantitative cellular viability was completed on day 14. Alkaline phosphatase activity assays were performed to evaluate the differentiation of stem cells on day 14. Real-time polymerase chain reactions were used to determine the expression levels of TGF-β1, RUNX2, OCN, SOX9, and COL1A1 mRNA on day 14. The stem cells produced well-formed organoids on day 7, and the addition of transforming growth factor-β1 did not result in relevant changes in their shape. The organoids grew in size and became more intact with longer incubation times. On day 14, the diameters were 222.2 ± 9.6, 186.1 ± 4.8, 197.2 ± 9.6, and 211.1 ± 19.2 m for transforming growth factor-β1 at final concentrations of 0, 1, 10, and 20 ng/ml, respectively. Quantitative cell viability results from day 14 exhibited no significant difference between the groups (P > 0.05). There was significantly higher alkaline phosphatase activity with the addition of transforming growth factor-β1 with the highest value for the 1 ng/ml group (P < 0.05). Real-time polymerase chain reaction results demonstrated that the mRNA expression levels of RUNX2, OCN, and SOX were higher in 1 ng/ml but did not reach statistical significance. Treatment with 1 ng/ml of transforming growth factor-β1 significantly increased COL1A1 mRNA expression at day 14. The application of transforming growth factor-β1 increased differentiation, which was confirmed by alkaline phosphatase activity and mRNA expression while maintaining cell viability.

NELL-1 Increased the Osteogenic Differentiation and mRNA Expression of Spheroids Composed of Stem Cells.

Background and objectives: NELL-1 is a competent growth factor and it reported to target cells committed to the osteochondral lineage. The secreted, osteoinductive glycoproteins are reported to rheostatically control skeletal ossification. This study was performed to determine the effects of NELL-1 on spheroid morphology and cell viability and the promotion of osteogenic differentiation of stem cell spheroids. Materials and Methods: Cultures of stem cell spheroids of gingiva-derived stem cells were grown in the presence of NELL-1 at concentrations of 1, 10, 100, and 500 ng/mL. Evaluations of cell morphology were performed using a microscope, and cell viability was assessed using a two-color assay and Cell Counting Kit-8. Evaluation of the activity of alkaline phosphatase and calcium deposition assays involved anthraquinone dye assay to determine the level of osteogenic differentiation of cell spheroids treated with NELL-1. Real-time quantitative polymerase chain reaction (qPCR) was used to evaluate the expressions of RUNX2, BSP, OCN, COL1A1, and β-actin mRNAs. Results: The applied stem cells produced well-formed spheroids, and the addition of NELL-1 at tested concentrations did not show any apparent changes in spheroid shape. There were no significant changes in diameter with addition of NELL-1 at 0, 1, 10, 100, and 500 ng/mL concentrations. The quantitative cell viability results derived on Days 1, 3, and 7 did not show significant disparities among groups (p > 0.05). There was statistically higher alkaline phosphatase activity in the 10 ng/mL group compared with the unloaded control on Day 7 (p < 0.05). A significant increase in anthraquinone dye staining was observed with the addition of NELL-1, and the highest value was noted at 10 ng/mL (p < 0.05). qPCR results demonstrated that the mRNA expression levels of RUNX2 and BSP were significantly increased when NELL-1 was added to the culture. Conclusions: Based on these findings, we conclude that NELL-1 can be applied for increased osteogenic differentiation of stem cell spheroids.

Morphological stability, cellular viability and stem cell marker expression of three-dimensional cultures of stem cells from bone marrow and periodontium.

The aim of the present study was to evaluate the morphology, cellular viability and stem cell marker expression of three-dimensional cultures of bone marrow and gingiva-derived stem cells in different ratios. Stem cell spheroids were made with bone marrow and gingiva-derived stem cells using ratios of 6:0 (Group 1), 4:2 (Group 2), 3:3 (Group 3), 2:4 (Group 4) and 0:6 (Group 5), respectively. The viability of cell spheroids was analyzed using a Live/Dead kit assay and a Cell Counting Kit-8 assay. Total RNA extraction and reverse transcription-quantitative PCR were performed to detect the mRNA expression levels of Nanog and β-actin in each group. Stem cell spheroids were well formed in silicone elastomer-based concave microwells with different ratios of bone marrow and gingiva-derived stem cells. The shape of the spheroids and their viability were maintained throughout the entirety of the experimental procedure. Statistically significant increases in spheroid diameters were noted in Groups 4 and 5 on day 1 when compared with Group 1 on day 1. There was a significant increase in the cell viability values seen in Group 3 on day 1 when compared with Group 1 on day 1. Highest levels of Nanog expression was seen in Group 3 on day 10, but the increase was not significant when compared with Group 1 on day 1. Co-culturing with higher ratios of gingiva-derived stem cells produced stem cell spheroids with larger diameters and increased cellular viability. This co-culture technique may be used in stem cell therapy with allogenic stem cell transplantation.

The Effects of Bone Morphogenetic Protein-4 on Cellular Viability, Osteogenic Potential, and Global Gene Expression on Gingiva-Derived Stem Cell Spheroids

Bone morphogenetic protein-4 (BMP-4) is engaged in the migration ability of mesenchymal stem cells and the transition of mesenchymal stem cells into osteogenic and adipocytic lines. The aim of this study was to evaluate the effects of BMP-4 on the cellular viability, osteogenic differentiation, and genome-wide mRNA levels using three-dimensional cell spheroids composed of stem cells. Stem cell spheroids were formed using concave microwells in the presence of BMP-4 with final concentrations of 0, 2, 6, and 10 ng/mL. Cellular viability was measured qualitatively using a microscope and quantitatively using an assay kit based on water-soluble tetrazolium salt. Osteogenic differentiation was assessed by measuring the level of alkaline phosphatase activity. Global gene expression was assessed using next-generation mRNA sequencing and performing gene ontology and pathway analyses. Spheroids were well-maintained with the addition of BMP-4 up to Day 7. No significant differences were observed in cell viability between each group. There were significantly higher alkaline phosphatase values in the 2 ng/mL BMP-4 groups when compared with the control (p &lt; 0.05). A total of 25,737 mRNAs were differentially expressed. Expression of β-catenin (CTNNB1) was increased with higher dosages of BMP-4. The expression of runt-related transcription factor 2 (RUNX2) was increased up to 6 ng/mL. The phosphoinositide-3-kinase–protein kinase B/Akt signaling pathway was associated with the target genes. This study demonstrates that the application of BMP-4 enhanced alkaline phosphatase activity and the expression of CTNNB1 and RUNX2 without affecting cellular viability.

The Effects of Morinda citrifolia (Noni) on the Cellular Viability and Osteogenesis of Stem Cell Spheroids.

Background and objectives: Morinda citrifolia (Noni) has been widely used in herbal remedies to treat and prevent various kinds of diseases. We conducted this study to evaluate the effects of Noni extract on the maintenance of morphology, the improvement of cellular viability, and the enhancement of osteogenesis of stem cell spheroids. Materials and Methods: We cultured stem cell spheroids made with gingiva-derived stem cells in the presence of Noni extract at concentrations of 10, 100 and 200 ng/mL. We performed analysis of the cell morphology and changes in the cellular viability. We conducted alkaline phosphatase activity assays using a kit, and mineralization assays using an anthraquinone dye to evaluate the osteogenesis of stem cell spheroids with the addition of Noni extract. Results: The applied cells formed spheroids well, and the addition of Noni at 10, 100 and 200 ng/mL concentrations did not produce significant morphological changes. The quantitative values for cellular viability on Day 3 showed that the absorbance values at 450 nm were 0.314 ± 0.013, 0.318 ± 0.008, 0.304 ± 0.000 and 0.300 ± 0.011 for Noni at 0, 10, 100 and 200 ng/mL concentrations, respectively. The results of alkaline phosphatase activity with absorbance values at 405 nm were 0.189 ± 0.019, 0.174 ± 0.023, 0.192 ± 0.014 and 0.210 ± 0.062 for Noni at 0, 10, 100 and 200 ng/mL concentrations, respectively, on Day 4. There were significantly higher values of Alizarin Red S staining for Noni in the 10, 100 and 200 ng/mL groups, with the highest value at 100 ng/mL when compared with the unloaded control on Day 14. Conclusions: Based on these findings, we concluded that Noni extract might be applied for the enhanced osteogenic differentiation of stem cell spheroids.

Cytotoxicity and Mineralization Potential of Four Calcium Silicate-Based Cements on Human Gingiva-Derived Stem Cells

The aim of this study was to evaluate the cytotoxicity and mineralization potential of four calcium silicate-based cements on human gingiva-derived stem cells (GDSCs). The materials evaluated in the present study were ProRoot MTA (Dentsply Tulsa Dental Specialties), Biodentine (Septodont), Endocem Zr (Maruchi), and RetroMTA (BioMTA). Experimental disks of 6 mm in diameter and 3 mm in height were produced and placed in a 100% humidified atmosphere for 48 h to set. We evaluated the cytotoxic effects of the cements using methyl-thiazoldiphenyl-tetrazolium (MTT) and live/dead staining assays. We used a scratch wound healing assay to evaluate cell migratory ability. Mineralization potential was determined with an Alizarin red S (ARS) staining assay. In the MTT assay, no significant differences were found among the ProRoot MTA, Biodentine, and control groups during the test period (p &gt; 0.05). The Endocem Zr and RetroMTA groups showed relatively lower cell viability than the control group at day 7 (p &lt; 0.05). In the wound healing assay, no significant differences were found among the ProRoot MTA, Biodentine, Endocem Zr, and control groups during the test period (p &gt; 0.05). The RetroMTA group had slower cell migration compared to the control group at days 3 and 4 (p &lt; 0.05). In the ARS assay, the ProRoot MTA, Biodentine, and RetroMTA groups exhibited a significant increase in the formation of mineralized nodules compared to the Endocem Zr and control groups on day 21 (p &lt; 0.05). In conclusion, the four calcium silicate-based cements evaluated in the present study exhibited good biological properties on GDSCs. ProRoot MTA, Biodentine, and RetroMTA showed higher mineralization potential than the Endocem Zr and control groups.

Time-Course Transcriptome Analysis of Gingiva-Derived Mesenchymal Stem Cells Reveals That Fusobacterium nucleatum Triggers Oncogene Expression in the Process of Cell Differentiation.

Fusobacterium nucleatum has pathogenic effects on oral squamous cell carcinoma and colon cancer, while the effects of continuously altered gene expression in normal human cells, as induced by persistent exposure to F. nucleatum, remain unclear. In this study, a microarray Significant Profiles (maSigPro) analysis was used to obtain the transcriptome profile of gingiva-derived mesenchymal stem cells (GMSCs) stimulated by F. nucleatum for 3, 7, 14, and 21 day, and the results revealed 790 (nine clusters) differentially expressed genes (DEGs), which were significantly enriched in cell adherens junctions and cancer-related pathways. On the basis of a short time-series expression miner (STEM) analysis, all the expressed genes in the GMSCs were grouped into 50 clusters according to dynamic gene expression patterns, and the expression levels of three gene clusters in the F. nucleatum-treated GMSCs were significantly different than the predicted values. Among the 790 DEGs, 50 tumor-associated genes (TAGs; such as L3MBTL4, CD163, CCCND2, CADM1, BCL7A, and IGF1) and five core dynamic DEGs (PLCG2, CHI3L2, L3MBTL4, SH2D2A, and NLRP3) were identified during F. nucleatum stimulation. Results from a GeneMANIA database analysis showed that PLCG2, CHI3L2, SH2D2A, and NLRP3 and 20 other proteins formed a complex network of which 12 genes were enriched in cancer-related pathways. Based on the five core dynamic DEGs, the related microRNAs (miRNAs) and transcription factors (TFs) were obtained from public resources, and an integrated network composed of the related TFs, miRNAs, and mRNAs was constructed. The results indicated that these genes were regulated by several miRNAs, such as miR-372-3p, miR-603, and miR-495-3p, and several TFs, including CREB3, GATA2, and SOX4. Our study suggests that long-term stimulation by F. nucleatum may trigger the expression of cancer-related genes in normal gingiva-derived stem cells.

Lovastatin increases the proliferation and osteoblastic differentiation of human gingiva-derived stem cells in three-dimensional cultures.

Lovastatin is a cholesterol-lowering agent that also has effects of cell proliferation and apoptosis. The present study was performed to evaluate the effects of lovastatin on the proliferation and osteogenic differentiation of three-dimensional cell spheroids formed from human gingiva-derived stem cells (GDSCs) using concave microwells. GDSCs were plated on polydimethylsiloxane-based concave micromolds and grown in the presence of lovastatin at concentrations of 0, 2 and 6 µM. The morphology of the cells was viewed under an inverted microscope, and cell viability was determined with Cell Counting kit-8 on days 2, 7 and 14. Alkaline phosphatase activity assays were performed to evaluate the osteogenic differentiation on days 2 and 8. Alizarin red-S staining was also used to assess the mineralization of the stem cell spheroids at day 14. The results confirmed that GDSCs formed spheroids in concave microwells. No significant changes were noted with longer incubation time, and no significant differences in cell viability were noted between the three lovastatin groups at each time point. Higher osteogenic differentiation was observed in the 2 µM group when compared with the control. Mineralized extracellular deposits were visible after Alizarin red-S staining, and higher mineralization was noted in the 2 and 6 µM lovastatin groups when compared with the 0 µM control. The relative mineralization values of the 0, 2 and 6 µM groups on day 14 were 39.0±9.6, 69.3±6.0 and 60.9±7.5, respectively. This study demonstrated that the application of lovastatin enhanced the osteogenic differentiation of cell spheroids formed from GDSCs. This suggests that combinations of lovastatin and stem cell spheroids may have the potential for use in tissue engineering.

Bone morphogenetic protein‑7 upregulates genes associated with osteoblast differentiation, including collagen I, Sp7 and IBSP in gingiva‑derived stem cells

The present study was performed to evaluate the effects of short-term application of bone morphogenetic protein-7 (BMP-7) on human gingiva-derived mesenchymal stem cells with next-generation sequencing. Human gingiva-derived stem cells were treated with a final concentration of 100 ng/ml BMP-7 and the same concentration of a vehicle control. mRNA sequencing and data analysis were performed along using gene ontology and pathway analysis. RT-qPCR of mRNA of collagen I, Sp7, IBSP and western blot analysis of collagen I, osterix and bone sialoprotein was also performed. A total of 25,737 mRNAs were identified to be differentially expressed. Regarding osteoblast differentiation, 14 mRNAs were upregulated and 10 were downregulated when the results of the BMP-7 at 3 h were compared with the control at 3 h. The expression of collagen I was increased following the application of BMP-7 at 3 h, and this increase was also observed following western blot analysis. The effects of BMP-7 on stem cells were evaluated with mRNA sequencing, and the expression was validated with RT-qPCR and western blot analysis. The short-term application of BMP-7 produced an increased expression of collagen I, which was associated with target genes selected for osteoblast differentiation. This study may provide novel insights into the role of BMP-7 using mRNA sequencing.

Dental Pulp Stem Cell-Derived Factors Alleviate Subarachnoid Hemorrhage-Induced Neuroinflammation and Ischemic Neurological Deficits.

Aneurysmal subarachnoid hemorrhage (aSAH), characterized by the extravasation of blood into the subarachnoid space caused by an intracranial aneurysm rupture, may lead to neurocognitive impairments and permanent disability and usually carries poor outcome. Dental or gingiva-derived stem cells have been shown to contribute to immune modulation and neuroregeneration, but the underlying mechanisms are unclear. In the present study, we sought to investigate whether dental pulp stem cells (DPSCs) secrete certain factor(s) that can ameliorate the neural damage and other manifestations in a rat aSAH model. Twenty-four hours after the induction of aSAH, microthrombosis, cortical vasoconstriction, and the decrease in microcirculation and tissue oxygen pressure were detected. Intrathecal administration of DPSC-derived conditioned media (DPSC-CM) ameliorated aSAH-induced vasoconstriction, neuroinflammation, and improved the oxygenation in the injured brain. Rotarod test revealed that the aSAH-induced cognitive and motor impairments were significantly improved by this DPSC-CM administration. Cytokine array indicated the major constituent of DPSC-CM was predominantly insulin growth factor-1 (IGF-1). Immunohistochemistry staining of injured brain tissue revealed the robust increase in Iba1-positive cells that were also ameliorated by DPSC-CM administration. Antibody-mediated neutralization of IGF-1 moderately deteriorated the rescuing effect of DPSC-CM on microcirculation, Iba1-positive cells in the injured brain area, and the cognitive/motor impairments. Taken together, the DPSC-derived secretory factors showed prominent therapeutic potential for aSAH. This therapeutic efficacy may include improvement of microcirculation, alleviation of neuroinflammation, and microglial activation; partially through IGF-1-dependent mechanisms.

Dimethyl Sulfoxide Leads to Decreased Osteogenic Differentiation of Stem Cells Derived from Gingiva via Runx2 and Collagen I Expression.

Objectives Dimethyl sulfoxide (DMSO) plays various functions, including cellular functions such as cellular growth. The aim of this study was to evaluate the effects of DMSO on the proliferation and osteogenic differentiation of human gingiva-derived stem cells. Materials and Methods Stem cells derived from gingiva were cultured in the presence of DMSO at concentrations ranging from 0.01 to 10%. Statistical Analysis We performed a one-way analysis of variance (ANOVA) with post hoc test to determine the differences between the groups using a commercially available program and the level of significance was 0.05. Results The cells in the control group showed normal fibroblast morphology. The cells treated with 0.01%, 0.01%, 0.1%, and 1% DMSO were morphologically similar to those from the control group on each day. Statistically significant decreases in cell counting kit-8 (CCK-8) values were seen in the 3% and 10% DMSO groups ( p < 0.05). A statistically significant decrease in alkaline phosphatase activity was seen in the 3% DMSO group. ( p < 0.05). The application of DMSO produced a decrease in alizarin red S staining. The expression of Runx2 and collagen I by immunofluorescence decreased as the dose of lovastatin increased. Conclusion The effects of DMSO on the viability of osteogenic differentiation among stem cells derived from human gingiva were evaluated. Applying DMSO produced decreased cell viability and decreased osteogenic differentiation in this experimental setting. This should be considered when designing and interpreting the data, and a DMSO-free method may be considered for bone regeneration applications.

Cellular viability and osteogenic differentiation potential of human gingiva-derived stem cells in 2D culture following treatment with anionic, cationic, and neutral liposomes containing doxorubicin.

The effects of doxorubicin, particularly doxorubicin liposome, on stem cells have remained to be fully elucidated. The aim of the present study was to evaluate the effects of anionic, cationic and neutral liposomes loaded with doxorubicin on the viability and osteogenic differentiation potential of human gingiva-derived stem cells in two-dimensional culture. Doxorubicin-loaded liposomes were prepared using the traditional thin-lipid-film hydration method. Stem cells were seeded on a culture plate and maintained in osteogenic media. The morphology of the stem cells was observed under an inverted microscope. The number of viable cells was determined using a Cell-Counting Kit-8 assay. The alkaline phosphatase activity was assessed and Alizarin Red S staining was performed to evaluate osteogenic differentiation. A higher concentration of doxorubicin caused noticeable changes in the morphology of the stem cells. Decreases in cellular viability were observed after applying doxorubicin. The application of doxorubicin, particularly at higher concentrations, produced a noticeable decrease in alkaline phosphatase activity and Alizarin Red S staining. The present study indicated that application of doxorubicin with or without liposomes reduced the cellular viability and osteogenic differentiation. Among the different treatments, the doxorubicin-loaded cationic liposomes induced the strongest reduction in the cellular viability and osteogenic differentiation in the stem cell culture.

Osteogenic potential of cell spheroids composed of varying ratios of gingiva-derived and bone marrow stem cells using concave microwells.

The aim of the current study was to evaluate cell viability and osteogenic differentiation potential in cell spheroids composed of varying ratios of gingiva-derived and bone marrow stem cells cultured in concave microwells. Cell spheroids were established from bone marrow and gingiva-derived stem cells in ratios of 6:0 (Group 1), 2:1 (Group 2), 3:3 (Group 3), 1:2 (Group 4), and 0:6 (Group 5). On days 3 and 5, the viability of the cell spheroids was qualitatively analyzed using a calcein acetoxymethyl ester working solution and an ethidium homodimer-1 live/dead assay. On days 1, 3, 5 and 7, a quantitative cell viability analysis was performed using a Cell Counting Kit-8. Alkaline phosphatase activity assays were performed using a commercially available kit on day 7 to assess osteogenic differentiation. In addition, reverse transcription-quantitative polymerase chain reaction and western blot analysis were performed to evaluate runt-related transcription factor 2 (Runx2) and osteocalcin expression. The ratio of gingiva-derived to bone marrow stem cells did not affect the stem cell spheroid morphology. No significant changes in cell viability were noted among the different groups following incubation for 7 days. A consistent alkaline phosphatase activity was measured in co-cultured gingiva-derived and bone marrow stem cell spheroids of varying compositions. Runx2 and osteocalcin expression was increased when co-cultured compared with pure gingiva-derived or bone marrow stem cells. In conclusion, stem cell spheroids established by co-culturing maintained morphology, viability and a high osteogenic differentiation potential during the experimental period of 7 days. These spheroids containing human gingiva-derived and bone marrow stem cells may enhance the osteogenic differentiation potential. The use of multicell spheroids may be a simple and effective strategy for improving stem cell therapy.

Dexamethasone Leads to Upregulation of BMP6 and ACHE Suppression of SMAD3 and ESR1 Genes in Human Mesenchymal Stem Cells

The purpose of this study is to evaluate the effects of dexamethasone on mesenchymal stem cells from human gingiva using next-generation sequencing. The verification and suggestion of possible mechanisms was performed. Human gingiva-derived stem cells were treated with a final concentration of 10-7M dexamethasone at 2 and 24 hours. Extraction of RNA, sequencing of mRNA, gene ontology and pathway analysis were performed. Quantification by real-time polymerase chain reaction was conducted for validation. A fold change of two was applied for this study, and a log2 normalized read count of 5 or greater was applied to minimize false counts. Expression of SMAD3 and ESR1 was decreased in dexamethasone at 24 hours. Increased expression of BMP6 and ACHE was noted in dexamethasone at 24 hours. TGF-β signaling was involved in the target genes chosen for osteoblast differentiation. It was clear that the application of dexamethasone produced reduced expression of SMAD3 and ESR1 and enhanced expression of ACHE and BMP6 of human gingiva-derived mesenchymal stem cells. An RNA sequencing strategy can provide new insights into the mechanism of dexamethasone in stem cells originating from dental areas.

Evaluation of the Effects of Biodegradable Microspheres Loaded with Quercetin on Adipogenic and Chondrogenic Differentiation of Cellular Spheroids

This study was performed to evaluate the effects of quercetin application and the biodegradable poly(lactic-co-glycolic acid)-based microspheres loaded with quercetin on the adipogenic and chondrogenic differentiation of three-dimensional cells composed of gingiva-derived stem cells and osteoblast-like cells. Three-dimensional cell spheroids were fabricated using silicon elastomer-based concave microwells and cultured in adipogenic and chondrogenic media. Quercetin at 1 μg/mL (Q1) and microspheres loaded with quercetin at 1 μg/mL (M1) were used. Quantitative cellular viability was evaluated using Cell Counting Kit-8 assay. Adipogenesis was determined after oil red O staining and chondrogensis was evaluated by measuring relative intensity of Alcian blue staining. Spheroids were well maintained irrespective of quercetin application in silicon elastomer-based concave microwells. The relative Cell Counting Kit-8 assay values for unloaded, Q1, and M1 groups in adipogenic media on Day 1 were 100.0±7.6%, 153.6±11.3%, and 104.7±6.3%, respectively. Relative values of adipogenesis were 114.6%, 116.2%, and 113.4% for unloaded, Q1, and M1 groups on Day 7, respectively, and relative values of adipogenesis were 131.7%, 137.5%, and 133.6% for unloaded, Q1, and M1 groups on Day 14, respectively. The relative Cell Counting Kit-8 assay values for unloaded, Q1, and M1 groups in chondrogenic media on Day 1 were 100.0±3.2%, 162.1±13.0%, and 96.6±3.7%, respectively. Relative values of chondrogensis were 103.6%, 111.0%, and 103.7% for unloaded, Q1, and M1 groups on Day 7, respectively, and the relative values of chondrogensis were 157.4%, 165.3%, and 160.9% for unloaded, Q1, and M1 groups on Day 14, respectively. Application of quercetin produced quercetin at 1 μg/mL and promoted the viability of cell spheroids cultured in adipogenic and chondrogenic media. However, application of quercetin did not reach statistically significant enhancement of adipogenisis or chondrogenesis at the experimental condition.

Enhanced Osteogenic Differentiation Potential of Stem-Cell Spheroids Created From a Coculture of Stem Cells and Endothelial Cells.

This study was performed to fabricate stem-cell spheroids formed with human gingiva-derived stem cells and endothelial cells and to evaluate their viability and osteogenic differentiation potential. Gingiva-derived stem cells were isolated, and stem cells and endothelial cells with a total of 6 × 10 cells were seeded into concave micromolds with different ratios of 6:0 (group 1), 4:2 (group 2), 3:3 (group 3), and 2:4 (group 4). Gingiva-derived stem cells and/or endothelia cells formed spheroids in concave microwells. There was a decreasing trend in the diameter of spheroids with increasing amounts of endothelial cells, but there were no statistically significant differences between the groups. The secretion of vascular endothelial growth factor from the spheroids was noted. The results of the alkaline phosphatase activity assays showed significantly higher values for groups 2, 3, and 4 when compared with the value of group 1. Conclusively, stem-cell spheroids formed with human gingiva-derived stem cells and endothelial cells using concave microwells enhanced osteogenic differentiation potential, and multicell spheroid-based cell delivery could be a simple and effective strategy for improving stem-cell therapy.