Gingiva-derived Mesenchymal Stem Cells Research Articles

Immunomodulatory differences between mesenchymal stem cells from different oral tissues

Mesenchymal stem cells (MSCs) have recently been identified as having potentially therapeutic immunomodulatory properties. MSCs isolated from different oral tissues have similar morphology and immunophenotypes, however, direct comparisons of their gene expression and immunomodulatory properties have not been conducted. We isolated alveolar bone-derived MSCs (aBMSCs), dental pulp stem cells (DPSCs) and gingiva-derived MSCs (GMSCs) from the same patients and compared their immunophenotypes and transcriptomes. Additionally, we compared their production of soluble immunomodulatory cytokines as well as their immunoregulatory properties in coculture with THP-1 human monocytic cells. RNA sequencing revealed distinct gene expression in DPSCs while aBMSCs and GMSCs had less differentially expressed genes. DPSCs also had significantly less secretion of osteopontin compared to aBMSCs and GMSCs. Finally, DPSCs did not exhibit an immunosuppresive effect on THP-1 cells to the same degree as aBMSCs and GMSCs. These findings demonstrate that MSCs from different oral tissues have distinct transcriptomes and immunoregulatory properties.

Influence of Topography and Composition of Commercial Titanium Dental Implants on Cell Adhesion of Human Gingiva-Derived Mesenchymal Stem Cells: An In Vitro Study

Influence of Topography and Composition of Commercial Titanium Dental Implants on Cell Adhesion of Human Gingiva-Derived Mesenchymal Stem Cells: An In Vitro Study

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

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. 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/50μL) 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 8weeks. 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 8weeks (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. 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.

Plant exosomes fused with engineered mesenchymal stem cell-derived nanovesicles for synergistic therapy of autoimmune skin disorders.

Existing therapeutics for autoimmune diseases remain problematic due to low efficacy, severe side effects, and difficulties to reach target tissues. Herein, we design multifunctional fusion nanovesicles that can target lesions for the treatment of autoimmune skin diseases. The grapefruit-derived exosome-like nanovesicles (GEVs) with anti-inflammatory and antioxidant effects are first encapsulated with CX5461, an immunosuppressant with anti-proliferative properties to form GEV@CX5461. In order to enhance therapeutic efficiency and safety, GEV@CX5461 are then fused with CCR6+ nanovesicles derived from membranes of engineered gingiva-derived mesenchymal stem cells (GMSCs). The resulting FV@CX5461 not only maintain the bioactivity of GEVs, CX5461, and GMSC membranes but also home to inflamed tissues rich in chemokine CCL20 through the chemotaxis function of CCR6 on FVs. Moreover, FV@CX5461 reduce the secretion of inflammatory factors, calm down Th17 cell activation, and induce Treg cell infiltration. Finally, impressive therapeutic efficiency in both psoriasis and atopic dermatitis disease models is demonstrated using FV@CX5461 to reshape the unbalanced immune microenvironment. A nanotherapeutic drug delivery strategy is developed using fusion nanovesicles derived from plant and animal cells with high clinical potential.

Single-cell analysis of immune cells on gingiva-derived mesenchymal stem cells in experimental autoimmune uveitis

Gingiva-derived mesenchymal stem cells (GMSCs) have shown astonishing efficacy in the treatment of various autoimmune diseases. However, the mechanisms underlying these immunosuppressive properties remain poorly understood. Here, we generated a lymph node single-cell transcriptomic atlas of GMSC-treated experimental autoimmune uveitis mice. GMSC exerted profound rescue effects on Tcells, B cells, dendritic cells, and monocytes. GMSCs rescued the proportion of T helper 17 (Th17) cells and increased the proportion of regulatory Tcells. In addition to globally altered transcriptional factors (Fosb and Jund), we observed cell type-dependent gene regulation (e.g., Il17a and Rac1 in Th17 cells), highlighting the GMSCs' cell type-dependent immunomodulatory capacity. GMSCs strongly influenced the phenotypes of Th17 cells, suppressing the formation of the highly inflammatory CCR6-CCR2+ phenotype and enhancing the production of interleukin (IL) -10 in the CCR6+CCR2+ phenotype. Integration of the glucocorticoid-treated transcriptome suggests a more specific immunosuppressive effect of GMSCs on lymphocytes.

The Emerging Biological Functions of Exosomes from Dental Tissue-Derived Mesenchymal Stem Cells.

Exosomes are one kind of small-cell extracellular membranous vesicles that can regulate intercellular communication and give rise to mediating the biological behaviors of cells, involving in tissue formation, repair, the modulation of inflammation, and nerve regeneration. The abundant kinds of cells can secret exosomes, among them, mesenchymal stem cells (MSCs) are very perfect cells for mass production of exosomes. Dental tissue-derived mesenchymal stem cells (DT-MSCs), including dental pulp stem cells, stem cells from exfoliated deciduous teeth, stem cells from apical papilla, stem cells from human periodontal ligament (PDLSCs), gingiva-derived mesenchymal stem cells, dental follicle stem cells, tooth germ stem cells, and alveolar bone-derived mesenchymal stem cells, are now known as a potent tool in the area of cell regeneration and therapy, more importantly, DT-MSCs can also release numerous types of exosomes, participating in the biological functions of cells. Hence, we briefly depict the characteristics of exosomes, give a detailed description of the biological functions and clinical application in some respects of exosomes from DT-MSCs through systematically reviewing the latest evidence, and provide a rationale for their use as tools for potential application in tissue engineering.

MiR-1260b inhibits periodontal bone loss by targeting ATF6β mediated regulation of ER stress.

The expression profiles of exosomal microRNAs (miRNAs) are regulated by the microenvironment, and appropriate priming with mesenchymal stem cells (MSCs) is one of the strategies to enhance the paracrine potency of MSCs. Our previous work demonstrated that exosomes from tumor necrosis factor (TNF)-α-primed human gingiva-derived MSCs (GMSCs) could be a therapeutic tool against periodontitis, and that TNFα-inducible exosomal miR-1260b is essential for the inhibition of alveolar bone loss. However, the precise molecular mechanism underlying miR-1260b-mediated inhibition of osteoclastogenesis is not yet fully understood. Here, we found that the activating transcription factor (ATF)-6β, a novel miR-1260b-targeting gene, is critical for the regulation of osteoclastogenesis under endoplasmic reticulum (ER) stress. An experimental periodontal mouse model demonstrated that induction of ER stress was accompanied by enhanced ATF6β expression, and local administration of miR-1260b and ATF6β siRNA using polyethylenimine nanoparticles (PEI-NPs) significantly suppressed the periodontal bone resorption. In periodontal ligament (PDL) cells, the ER stress inducer, tunicamycin, enhanced the expression of the receptor activator of NF-κB ligand (RANKL), while miR-1260b-mediated downregulation of ATF6β caused RANKL inhibition. Furthermore, the secretome from miR-1260b/ATF6β-axis-activated PDL cells inhibited osteoclastogenesis in human CD14+ peripheral blood-derived monocytes. These results indicate that the miR-1260b/ATF6β axis mediates the regulation of ER stress, which may be used as a novel therapeutic strategy to treat periodontal disease.

Insulin-producing cell clusters derived from human gingival mesenchymal stem cells as a model for diabetes research.

The human gingiva-derived mesenchymal stem cells (hGMSCs) possess a great potential to develop the cell-based therapy for diabetes due to its unscarred healing capacity and reparative potential. In this current study, we isolated, cultured and characterised the GMSCs and explored their potential to differentiate into Insulin Producing Cell Clusters (IPCCs). The cells derived from gingival tissues exhibited fibroblast-like morphology. The flow cytometric analysis revealed positive expression of CD73(97.43%), CD90(95.05%), and CD105(93.17%) and negative expression of CD34(0.05%), CD45(0.09%), and HLA-DR (0.025) surface markers. We then converted this adherent fibroblast-like GMSCs into floating IPCCs using a sequential three-step protocol containing a different combination of differentiating agents. Initially, the presence of insulin in IPCCs was confirmed by dithizone staining. Glucose-stimulated insulin secretion (GSIS) assay confirmed that IPCCs secrete insulin in response to glucose. Generated IPCCs express pancreatic markers such as insulin, pdx1, glucagon, GLUT4 and GLUT2 as evidenced by RT-PCR analysis. Our results unequivocally showed that IPCCs can be generated from gingiva which is a potential source of postnatal MSCs. Our results offer the IPCCs generated from hGMSCs a platform for screening anti-diabetic drugs and a new autologous source of tissue for islet transplantation for the treatment of diabetes. Our results unequivocally demonstrate for the first time that hGMSCs can be used as an attractive non-invasive tissue source for generating IPCCs, which can be employed in diabetes research for screening antidiabetic agents and also for transplantation in type 1 diabetic patients as autologous source without the need of immunosuppression.

Supramolecular Hydrogel-Wrapped Gingival Mesenchymal Stem Cells in Cutaneous Radiation Injury.

Radiation-induced skin wound/dermatitis is one of the common side effects of radiotherapy or interventional radiobiology. Gingiva-derived mesenchymal stem cells (GMSCs) were indicated to have therapeutic potentials in skin diseases. However, stem cells are prone to spread and difficult to stay in the skin for a long time, limiting their curative effects and application. This study investigated the therapeutic efficacy of Nap-GDFDFpDY (pY-Gel) self-assembled peptide hydrogel-encapsulated GMSCs to treat 137Cs γ-radiation-induced skin wounds in mice. The effects were evaluated by skin damage score, hind limb extension measurement and histological and immunohistochemical analysis. In vivo studies showed that pY-Gel self-assembled peptide hydrogel-encapsulated GMSCs could effectively improve wound healing in irradiated skin tissues. In addition, it was found that GMSCs conditioned medium (CM) could promote the proliferation, migration and DNA damage repair ability of skin cells after irradiation in human keratinocyte cell line HaCaT and normal human dermal fibroblasts (HFF). Mechanistically, GMSCs-CM can promote the expression of epidermal growth factor receptor (EGFR), signal transducers and activators of transcription 3 (STAT3) and matrix metalloproteinases (MMPs), suggesting that activation of the EGFR/STAT3 signaling pathway may be involved in the repair of skin cells after exposure to radiations. In conclusion, pY-Gel self-assembled peptide hydrogel-encapsulated GMSCs have a beneficial therapeutic effect on radiation-induced cutaneous injury and may serve as a basis of novel cells therapeutic approach.

Gingiva-Derived Mesenchymal Stem Cells Attenuate Imiquimod- (IMQ-) Induced Murine Psoriasis-Like Skin Inflammation.

Human gingiva-derived mesenchymal stem cells (GMSCs) are isolated from the gingival propria with promising regenerative, immunomodulatory, and anti-inflammatory properties. Recently, several studies, including ours, have found that GMSCs have the therapeutic potentials of nerve regeneration and skin disorders in various types such as the cell itself, cell-free conditioned medium, or extracellular vesicles (EVs). However, the mechanobiological behavior of GMSCs is closely related to the culture conditions. Therefore, the purpose of this study was to evaluate the function of human GMSCs on imiquimod- (IMQ-) induced murine psoriasis-like skin inflammation in two-dimensional (2D) and three-dimensional (3D) culture conditions. Here, we isolated and characterized GMSCs in 2D and 3D culture conditions and found that GMSCs in 2D and 3D infusion can significantly ameliorate the IMQ-induced murine psoriasis-like skin inflammation, reduce the levels of Th1- and Th17-related cytokines IFN-γ, TNF-α, IL-6, IL-17A, IL-17F, IL-21, and IL-22, and upregulate the percentage of spleen CD25+CD3+ T cells while downregulate the percentage of spleen IL-17+CD3+ T cells. In summary, our novel findings reveal that GMSCs in 2D and 3D infusion may possess therapeutic effects in the treatment of psoriasis.

Implantation of a nerve protector embedded with human GMSC-derived Schwann-like cells accelerates regeneration of crush-injured rat sciatic nerves

BackgroundPeripheral nerve injuries (PNIs) remain one of the great clinical challenges because of their considerable long-term disability potential. Postnatal neural crest-derived multipotent stem cells, including gingiva-derived mesenchymal stem cells (GMSCs), represent a promising source of seed cells for tissue engineering and regenerative therapy of various disorders, including PNIs. Here, we generated GMSC-repopulated nerve protectors and evaluated their therapeutic effects in a crush injury model of rat sciatic nerves.MethodsGMSCs were mixed in methacrylated collagen and cultured for 48 h, allowing the conversion of GMSCs into Schwann-like cells (GiSCs). The phenotype of GiSCs was verified by fluorescence studies on the expression of Schwann cell markers. GMSCs encapsulated in the methacrylated 3D-collagen hydrogel were co-cultured with THP-1-derived macrophages, and the secretion of anti-inflammatory cytokine IL-10 or inflammatory cytokines TNF-α and IL-1β in the supernatant was determined by ELISA. In addition, GMSCs mixed in the methacrylated collagen were filled into a nerve protector made from the decellularized small intestine submucosal extracellular matrix (SIS-ECM) and cultured for 24 h, allowing the generation of functionalized nerve protectors repopulated with GiSCs. We implanted the nerve protector to wrap the injury site of rat sciatic nerves and performed functional and histological assessments 4 weeks post-surgery.ResultsGMSCs encapsulated in the methacrylated 3D-collagen hydrogel were directly converted into Schwann-like cells (GiSCs) characterized by the expression of S-100β, p75NTR, BDNF, and GDNF. In vitro, co-culture of GMSCs encapsulated in the 3D-collagen hydrogel with macrophages remarkably increased the secretion of IL-10, an anti-inflammatory cytokine characteristic of pro-regenerative (M2) macrophages, but robustly reduced LPS-stimulated secretion of TNF-1α and IL-1β, two cytokines characteristic of pro-inflammatory (M1) macrophages. In addition, our results indicate that implantation of functionalized nerve protectors repopulated with GiSCs significantly accelerated functional recovery and axonal regeneration of crush-injured rat sciatic nerves accompanied by increased infiltration of pro-regenerative (M2) macrophages while a decreased infiltration of pro-inflammatory (M1) macrophages.ConclusionsCollectively, these findings suggest that Schwann-like cells converted from GMSCs represent a promising source of supportive cells for regenerative therapy of PNI through their dual functions, neurotrophic effects, and immunomodulation of pro-inflammatory (M1)/pro-regenerative (M2) macrophages.

Comparison of Cellular and Differentiation Characteristics of Mesenchymal Stem Cells Derived from Human Gingiva and Periodontal Ligament.

ABSTRACTObjectives:Dental tissues possess multipotent stem cells with varying biological properties. The present study was aimed to establish a primary culture of human gingiva-derived mesenchymal stem cells (GMSCs) and periodontal ligament-derived stem cells (PDLSCs) from periodontally healthy subjects and compare their biological characteristics.Materials and Methods:Gingival and periodontal ligament (PDL) tissues were collected from extracted premolar teeth of five healthy subjects and primary cultures were established. Basic biological characteristics, such as cell morphology, viability, proliferation capacity, and colony-forming units, and in vitro osteogenic and adipogenic differentiation potential were performed at passage 3 of GMSCs and PDLSCs. This was followed by immuno-phenotyping and flow cytometric analysis for identification of positive mesenchymal stem cell (MSC) markers, such as CD73, CD90, and CD105, and negative markers CD45 and CD34.Statistical Analysis Used:One-way analysis of variance (ANOVA).Results:Primary cultures of GMSCs and PDLSCs were successfully established. Cells exhibited a fibroblast-like morphology with a homogeneous population at passage 3. Cells derived from both tissues were highly viable (>95%), proliferative, and capable of forming colonies. Both cells did not exhibit any noticeable differences in cellular properties. Immunofluorescence and flow cytometric analyses showed positivity for MSC markers, CD73, CD90, and CD105, and negativity for CD34 and CD45. Furthermore, GMSCs and PDLSCs were capable of differentiating in vitro into osteocytes as evidenced by Alizarin red-S staining, and adipocytes as demonstrated by oil red O staining.Conclusions:The results of the present study indicate that both GMSCs and PDLSCs have similar cellular characteristics and mesenchymal differentiation potential. Therefore, they may serve as an equally potent source of stem cells for use in cell-based periodontal therapies.

Effects of noni on cellular viability and osteogenic differentiation of gingiva‑derived stem cells demonstrated by RNA sequencing and quantitative PCR

Noni fruit (Morinda citrifolia) has been widely used in traditional medicine across tropical and subtropical regions, and is now being paid more attention in Western medicine. The present study aimed to investigate the effects of noni extract on the change in the cellular morphology, maintenance of cellular viability and enhancement of osteogenic differentiation of stem cells. Stem cells obtained from gingiva were cultured where noni extracts existed at concentrations ranging from 10-200 ng/ml. Evaluations of cell morphology and cellular viability were performed. Alkaline phosphatase activity assays were performed to assess the osteogenic differentiation. Alizarin Red S staining was performed to evaluate the calcium deposits in the culture, with the addition of noni extract. Global gene expression was analyzed via next-generation mRNA sequencing. Gene ontology and pathway analyses were performed to determine the associated mechanisms. Validation procedures were performed via quantitative (q)PCR analysis. The addition of noni at concentrations ranging from 10-200 ng/ml did not produce significant morphological changes. There were significantly higher values of cellular viability, with the highest value at 100 ng/ml compared with the control (P<0.05). Furthermore, significantly higher values of alkaline phosphatase activity was noted in the 10 and 100 ng/ml groups compared with the 0 ng/ml group on day 7 (P<0.05). Alizarin Red S staining revealed calcium deposits in each group. In addition, the highest value for Alizarin Red S staining was observed at 100 ng/ml compared with the unloaded control (P<0.05). qPCR analysis demonstrated that the mRNA expression levels of RUNX2, BSP, OCN and COL1A1 increased following treatment with noni. Taken together, the results of the present study suggest that noni extract has enhancing effects on gingiva-derived mesenchymal stem cells, by enhancing cellular viability and osteogenic differentiation.

Dephosphorylation of Caveolin-1 Controls C-X-C Motif Chemokine Ligand 10 Secretion in Mesenchymal Stem Cells to Regulate the Process of Wound Healing.

Mesenchymal stem cells (MSCs) secrete cytokines in a paracrine or autocrine manner to regulate immune response and tissue regeneration. Our previous research revealed that MSCs use the complex of Fas/Fas-associated phosphatase-1 (Fap-1)/caveolin-1 (Cav-1) mediated exocytotic process to regulate cytokine and small extracellular vesicles (EVs) secretion, which contributes to accelerated wound healing. However, the detailed underlying mechanism of cytokine secretion controlled by Cav-1 remains to be explored. We show that Gingiva-derived MSCs (GMSCs) could secrete more C-X-C motif chemokine ligand 10 (CXCL10) but showed lower phospho-Cav-1 (p-Cav-1) expression than skin-derived MSCs (SMSCs). Moreover, dephosphorylation of Cav-1 by a Src kinase inhibitor PP2 significantly enhances CXCL10 secretion, while activating phosphorylation of Cav-1 by H2O2 restraints CXCL10 secretion in GMSCs. We also found that Fas and Fap-1 contribute to the dephosphorylation of Cav-1 to elevate CXCL10 secretion. Tumor necrosis factor-α serves as an activator to up-regulate Fas, Fap-1, and down-regulate p-Cav-1 expression to promote CXCL10 release. Furthermore, local applying p-Cav-1 inhibitor PP2 could accelerate wound healing, reduce the expression of α-smooth muscle actin and increase cleaved-caspase 3 expression. These results indicated that dephosphorylation of Cav-1 could inhibit fibrosis during wound healing. The present study establishes a previously unknown role of p-Cav-1 in controlling cytokine release of MSC and may present a potential therapeutic approach for promoting scarless wound healing.

Harnessing 3D collagen hydrogel-directed conversion of human GMSCs into SCP-like cells to generate functionalized nerve conduits

Achieving a satisfactory functional recovery after severe peripheral nerve injuries (PNI) remains one of the major clinical challenges despite advances in microsurgical techniques. Nerve autografting is currently the gold standard for the treatment of PNI, but there exist several major limitations. Accumulating evidence has shown that various types of nerve guidance conduits (NGCs) combined with post-natal stem cells as the supportive cells may represent a promising alternative to nerve autografts. In this study, gingiva-derived mesenchymal stem cells (GMSCs) under 3D-culture in soft collagen hydrogel showed significantly increased expression of a panel of genes related to development/differentiation of neural crest stem-like cells (NCSC) and/or Schwann cell precursor-like (SCP) cells and associated with NOTCH3 signaling pathway activation as compared to their 2D-cultured counterparts. The upregulation of NCSC-related genes induced by 3D-collagen hydrogel was abrogated by the presence of a specific NOTCH inhibitor. Further study showed that GMSCs encapsulated in 3D-collagen hydrogel were capable of transmigrating into multilayered extracellular matrix (ECM) wall of natural NGCs and integrating well with the aligned matrix structure, thus leading to biofabrication of functionalized NGCs. In vivo, implantation of functionalized NGCs laden with GMSC-derived NCSC/SCP-like cells (designated as GiSCs), significantly improved the functional recovery and axonal regeneration in the segmental facial nerve defect model in rats. Together, our study has identified an approach for rapid biofabrication of functionalized NGCs through harnessing 3D collagen hydrogel-directed conversion of GMSCs into GiSCs.

Microstructure and mechanical behaviors of tibia for collagen-induced arthritic mice treated with gingiva-derived mesenchymal stem cells

Rheumatoid arthritis (RA) is a systemic polyarticular arthritis that primarily affects the small joints but also causes bone erosion in large joints. None of the currently existing treatment approaches is curable. In this study, the effects of human gingiva-derived mesenchymal stem cells (GMSCs) on collagen-induced arthritis (CIA) mice are examined by experimentally assessing the microstructure and mechanical behaviors of tibia. Bone morphology and mineral density of mouse tibiae were assessed using micro-X-ray computed tomography (micro-CT). Compression testing was performed on mouse tibia to access its stiffness. The deformation and strain localized inside proximal tibia were mapped using mechanical testing coupled with micro-CT and digital volume correlation of micro-CT images. The results show that CIA disease caused bone erosion in epiphyseal cortical bone, which manifested into the adjacent epiphyseal trabecular bone, and also affected the metaphyseal cortical bone. CIA disease also weakened the load-bearing function of proximal tibia. GMSC treatment interfered with the progress of CIA, attenuated the bone erosion in epiphyseal and metaphyseal trabecular bone and resulted in improved load-bearing function of proximal tibia. GMSCs provide a promising potential treatment of autoimmune arthritis.

Role of ascorbic acid in the regulation of epigenetic processes induced by Porphyromonas gingivalis in endothelial-committed oral stem cells

Periodontitis is a common inflammatory disease that affects the teeth-supporting tissue and causes bone and tooth loss. Moreover, in a worldwide population, periodontal disease is often associated with cardiovascular diseases. Emerging studies have reported that one of the major pathogens related to periodontitis is Porphyromonas gingivalis (P. gingivalis), which triggers the inflammatory intracellular cascade. Here, we hypothesized a possible protective effect of ascorbic acid (AA) in the restoration of the physiological molecular pathway after exposure to lipopolysaccharide derived from P. gingivalis (LPS-G). In particular, human gingiva-derived mesenchymal stem cells (hGMSCs) and endothelial-differentiated hGMSCs (e-hGMSCs) exposed to LPS-G showed upregulation of p300 and downregulation of DNA methyltransferase 1 (DNMT1), proteins associated with DNA methylation and histone acetylation. The co-treatment of AA and LPS-G showed a physiological expression of p300 and DNMT1 in hGMSCs and e-hGMSCs. Moreover, the inflammatory process triggered by LPS-G was demonstrated by evaluation of reactive oxygen species (ROS) and their intracellular localization. AA exposure re-established the physiological ROS levels. Despite the limitations of in vitro study, these findings collectively expand our knowledge regarding the molecular pathways involved in periodontal disease, and suggest the involvement of epigenetic modifications in the development of periodontitis.

Effects of Connective Tissue Growth Factor on the Cell Viability, Proliferation, Osteogenic Capacity and mRNA Expression of Stem Cell Spheroids

Background: Connective tissue growth factor (CTGF) is a cellular communication network factor family protein involved in many cellular functions. The purpose of this study was to determine the effects of CTGF on the proliferation, osteogenic capacity, and mRNA expression of spheroids composed of gingiva-derived mesenchymal stem cells (GMSCs). Methods: CTGF was applied at final concentrations of 0, 25, 50, 100, and 200 ng/mL. Qualitative cell viability was determined using Live/Dead kit assay. Metabolic viability was determined with a colorimetric assay kit. Osteogenic activity was analyzed with alkaline phosphatase activity and Alizarin Red S staining. Quantitative polymerase chain reaction (qPCR) was used to assess the expression levels of RUNX2, BSP, OCN, and COL1A1. Results: In general, there was no significant difference in cell viability between the groups on Days 1, 4, and 7. Addition of CTGF produced an increase in Alizarin Red S staining. qPCR results demonstrated that the mRNA expression levels of RUNX2, BSP, OCN, and COL1A1 were significantly increased with the addition of CTGF. Conclusions: Based on these findings, we conclude that CTGF can be applied for increased osteogenic differentiation of stem cell spheroids.

WITHDRAWN: Human gingiva-derived mesenchymal stem cells promote osteogenic differentiation through their immunosuppressive function

Human gingiva-derived mesenchymal stem cells (GMSCs) have emerged as a new MSC population exhibiting robust immune regulatory functions, multipotent differentiation potential, and regenerative ability. However, the effects of GMSCs on T cells remain unexplored. Herein, we aimed to evaluate whether GMSCs promote osteogenic differentiation by regulating immune cells. The GMSC phenotype was confirmed using the colony-forming assay, immunophenotyping, Oil red O staining, and Alizarin red staining. mRNA expression levels of proinflammatory molecules (interleukin-1β [IL-1β] and tumor necrosis factor-α [TNF-α]) and anti-inflammatory factors (IL-10) were measured by quantitative reverse-transcription PCR (qRT-PCR). Then, MC3T3-E1 cells were treated with the collected co-culture supernatant, followed by alkaline phosphatase (ALP) and immunofluorescence staining to evaluate osteogenic differentiation of MC3T3-E1 cells. qRT-PCR and western blotting were employed to analyze the expression levels of osteogenic differentiation proteins, including collagen type I (COL-1), ALP, osteopontin (OPN), and runt-related transcription factor 2 (RUNX2). GMSCs were successfully isolated and identified. We observed that GMSCs suppressed the activated T-cell function by downregulating IL-1β and TNF-α and upregulating IL-10. Simultaneously, the expression levels of osteogenesis-related genes (COL-1, ALP, OPN, and RUNX2) were markedly lower in the co-culture supernatant and Jurkat T cell supernatant groups than those in the normal culture medium group; however, expression levels were significantly increased in the co-culture supernatant group when compared with the Jurkat T cell supernatant group. Our findings indicate that GMSCs could promote the osteogenic differentiation of MC3T3-E1 cells by inhibiting the biological activity of activated T cells.

The Gingiva from the Tissue Surrounding the Bone to the Tissue Regenerating the Bone: A Systematic Review of the Osteogenic Capacity of Gingival Mesenchymal Stem Cells in Preclinical Studies.

The current review aims to systematically assess the osteogenic capacity of gingiva-derived mesenchymal stem cells (GMSCs) in preclinical studies. A comprehensive electronic search of PubMed, Embase, Web of Science, and Scopus databases, as well as a manual search of relevant references, was performed in June 2020 without date or language restrictions. Eligibility criteria were the following: studies that compared mesenchymal stem cells (MSCs) derived from the gingiva with other MSC sources (in vitro or in vivo) or cell-free scaffold (in vivo) and studies that reported at least one of the following outcomes: osteogenic potential and new bone formation for in vitro and in vivo, respectively. Moreover, the assessment of included studies was conducted using appropriate guidelines. From 646 initial retrieved studies, 35 full-text articles were subjected to further screening and 26 studies were selected (20 in vitro studies and 6 in vivo studies). GMSCs showed great proliferation capacity and expressed recognized mesenchymal stem cell markers, particularly CD90. In vitro, MSC sources including GMSCs were capable of undergoing osteogenic differentiation with less ability in GMSCs, while most in vivo studies confirmed the capacity of GMSCs to regenerate bony defects. Concerning the assessment of methodological quality, in vitro studies met the relevant guideline except in five areas: the sample size calculation, randomization, allocation concealment, implementation, and blinding, and in vivo publications had probably low risk of bias in most domains except in three areas: allocation concealment, attrition, and blinding items.