Gingiva-derived Mesenchymal Stem Cells Research Articles

A Gingiva-Derived Mesenchymal Stem Cell-Laden Porcine Small Intestinal Submucosa Extracellular Matrix Construct Promotes Myomucosal Regeneration of the Tongue.

In the oral cavity, the tongue is the anatomic subsite most commonly involved by invasive squamous cell carcinoma. Current treatment protocols often require significant tissue resection to achieve adequate negative margins and optimal local tumor control. Reconstruction of the tongue while preserving and/or restoring its critical vocal, chewing, and swallowing functions remains one of the major challenges in head and neck oncologic surgery. We investigated the in vitro feasibility of fabricating a novel combinatorial construct using porcine small intestinal submucosa extracellular matrix (SIS-ECM) and human gingiva-derived mesenchymal stem cells (GMSCs) as a GMSC/SIS-ECM tissue graft for the tongue reconstruction. We developed a rat model of critical-sized myomucosal defect of the tongue that allowed the testing of therapeutic effects of an acellular SIS-ECM construct versus a GMSC/SIS-ECM construct on repair and regeneration of the tongue defect. We showed that the GMSC/SIS-ECM construct engrafted at the host recipient site, promoted soft tissue healing, and regenerated the muscular layer, compared to the SIS-ECM alone or nontreated defect controls. Furthermore, our results revealed that transplantation of the GMSC/SIS-ECM construct significantly increased the expression of several myogenic transcriptional factors and simultaneously suppressed the expression of type I collagen at the wounded area of the tongue. These compelling findings suggest that, unlike the tongue contracture and fibrosis of the nontreated defect group, transplantation of the combinatorial GMSC/SIS-ECM constructs accelerates wound healing and muscle regeneration and maintains the overall tongue shape, possibly by both enhancing the function of endogenous skeletal progenitor cells and suppressing fibrosis. Together, our findings indicate that GMSC/SIS-ECM potentially served as a myomucosal graft for tongue reconstruction postsurgery of head and neck cancer.

Transplantation of gingiva-derived mesenchymal stem cells ameliorates collagen-induced arthritis.

BackgroundRheumatoid arthritis (RA) is a chronic, progressive, and inflammatory autoimmune disease which primarily affects the small arthrodial joints. The aim of this study was to test whether transplantation of mesenchymal stem cells derived from gingival tissue (GMSCs) could ameliorate collagen-induced arthritis (CIA), and to explore the role of the FasL/Fas pathway in the underlying mechanism.MethodsDBA/1 mice with collagen II-induced arthritis were treated with GMSCs from the C57BL/6 J mouse, the B6Smn.C3-FasLgld/J mouse (FasL–/– GMSCs), and FasL overexpressed FasL–/– GMSCs (FasL TF GMSCs). Inflammation was evaluated by measuring clinical score, tumor necrosis factor (TNF)-α and anti-collagen II antibody levels, and histological analyses. The levels of CD4+ Th cell subsets in spleens and draining lymph nodes were assessed by flow cytometric analysis.ResultsSystemic infusion of GMSCs can significantly reduce the severity of experimental arthritis, and resume the balance of Th cell subsets. FasL–/– GMSCs failed to induce apoptosis of activated T cells in vitro and in vivo, and therefore show no therapeutic effects, whereas FasL TF GMSCs can rescue the immunosuppressant effects in the treatment of CIA.ConclusionsGMSC-based therapy induces T-cell apoptosis via the FasL/Fas pathway and results in immune tolerance and amelioration of the CIA inflammation.

Isolation and characterization of human gingiva-derived mesenchymal stem cells using limiting dilution method

Background/purposeGingiva-derived mesenchymal stem cells (GMSCs) are attractive alternative MSC sources because of their relative abundance of sources and ease of accessibility. However, the isolation method for harboring GMSCs remains under discussion. The aim of the study was to isolate and explore in vitro characterization of human GMSCs, and compare stem cell properties with bulk-cultured gingival fibroblasts (GFs). Materials and methodsGMSCs were isolated with limiting dilution method. Tissue-matched bulk-cultured GFs and GMSCs were evaluated in terms of their colony-forming abilities, population doubling capacities, cell surface epitopes, and multilineage differentiation potentials. ResultsGMSCs showed a significantly higher number of colony-forming units-fibroblast (P < 0.001) than bulk-cultured GFs, while the population doubling capacity of GMSCs reduced. Both types of cells were uniformly positive for MSC-associated makers CD44, CD73, CD90, CD105, and CD166, and were negative for hematopoietic markers CD14, CD34, and CD45. The only distinct marker was STRO-1, which was more highly expressed in GMSCs (13.4%) than in bulk-cultured GFs (0.02%). Upon induction, GMSCs displayed the capacity to undergo osteogenic, adipogenic, and chondrogenic differentiation. Real-time polymerase chain reaction showed related gene levels were significantly upregulated (P < 0.001). By contrast, bulk-cultured GFs lacked the capacity to undergo multilineage differentiation, and related gene levels showed no significant difference when compared with control groups. ConclusionThe data validate the effectiveness of limiting dilution method for GMSCs isolation. GMSCs, in contrast to bulk-cultured GFs, harbor stem cell characteristics and can act as alternative cell sources for tissue engineering.

Effects of risedronate on the morphology and viability of gingiva-derived mesenchymal stem cells.

Risedronate has been used for the prevention and treatment of postmenopausal and corticosteroid-induced osteoporosis. The present study was performed to evaluate the effects of risedronate on the morphology and viability of human stem cells derived from the gingiva. Stem cells derived from the gingiva were grown in the presence of risedronate at concentrations that ranged from 1 to 10 µM. The morphology of the cells was viewed under an inverted microscope, and cell proliferation was analyzed with a cell counting kit-8 (CCK-8) on days 2, 4 and 7. The untreated control group showed a spindle-shaped, fibroblast-like morphology. The shapes of the cells treated with 1 and 5 µM risedronate were similar to that of the control group on day 2. However, morphology of the 10 µM group markedly differed from that of the control group. The shapes of the cells in the 1, 5 and 10 µM groups were rounder, and pronounced alterations when compared with the untreated control group were noted in all groups on day 7. The cultures growing in the presence of risedronate showed decreased CCK-8 values on day 7. In conclusion, risedronate produced notable alterations in the morphology of the cells and reduced the viability of gingival mesenchymal stem cells.

GMSCs suppress osteoclastogenesis and bone erosion in collagen-induced arthritis through curbing NF-KB and RANKL signals (THER5P.900)

Abstract Osteoclasts are responsible for bone destruction in rheumatoid arthritis (RA) and gingiva-derived mesenchymal stem cells (GMSCs) can inhibit experimental collagen-induced-arthritis (CIA) model. This study aims to determine if GMSCs also suppress osteolastogenesis and bone erosion in CIA. Osteoclasts were induced from bone-marrow CD11b+ cells with RANKL and macrophage colony-stimulating factor (M-CSF), and assessed with tartrate-resistant acid phosphatase (TRAP) staining. Human osteoclasts were induced from human CD14+ cells. GMSCs were generated and added to cultures with different ratios with CD11b+ or CD14+ cells. NF-κB activation was determined by western blot. 2×106 GMSCs or fibroblast cells were adoptively transferred to DBA1/J mice on day 14 after immunization with CII/CFA. CIA onset and severity were monitored and bone erosion was examined by HE staining. We showed that GMSCs but not fibroblast cells completely suppressed osteoclastogenesis in vitro for human and mice cells. GMSCs injected after immunization and before of onset of CIA significantly suppressed disease development. Treatment with GMSCs dramatically decreased the levels of NF-κB p65/p50 in osteoclasts in vitro and P65/50 and RANKL expression by synovial tissues in vivo. These results suggest that the manipulation of GMSCs may have therapeutic effects on rheumatoid arthritis and other bone erosion related diseases.

Characteristics and Tissue Regeneration Properties of Gingiva-Derived Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) have 2 specific characteristics: self-renewal and multi- or pluripotency. Extensive studies have demonstrated the regenerative capability of MSCs both in vitro and in vivo. Gingiva-derived MSCs (GMSCs) represent a unique population of MSCs that can be easily isolated and obtained. GMSCs, which maintain a normal karyotype and telomerase activity in long-term cultures, display a stable phenotype and rapidly proliferate in vitro. In addition, GMSCs can be induced to differentiate into osteogenic, chondrogenic, and adipogenic lineages. Therefore, GMSCs are a promising alternative cell source for tissue regeneration in dentistry. In this article we review studies of the characterization, differentiation capacities, and regenerative role of GMSCs derived from the gingiva of humans and other species, focusing on the mechanisms of differentiation and tissue regeneration of human GMSCs. We anticipate that GMSC-based therapies will significantly contribute to regenerative medicine for the treatment of human dental diseases and improve human health.

The effects of tetracycline-loaded silk fibroin membrane on proliferation and osteogenic potential of mesenchymal stem cells

BackgroundThe main objective of this study was to investigate the effect of tetracycline-loaded silk fibroin membranes (TC-SFMs) on the proliferation and the osteogenic differentiation of human mesenchymal stem cells. Materials and methodsFour groups (0, 1, 5, and 10% concentration) of TC-SFMs were prepared for the experiments. We investigated cumulative tetracycline (TC) release profile for 7 d. Human gingiva-derived mesenchymal stem cells (GMSCs) were isolated from our previous study and seeded to the TC-SFMs. WST-8 assay (Cell Counting Kit-8; SigmaeAldrich Co, St. Louis, MO), staining of Phalloidin-FITC, and scanning electron microscope analyzed the cellular attachment and viability. Staining of Alizarin Red S (Sigma-Aldrich Co.) and osteogenic marker (osteocalcin) analyzed osteogenic differentiation. Additionally, quantitative polymerase chain reaction measured the expression of osteogenic lineage genes, including bone gamma-carboxyglutamic acid protein, bone sialoprotein, runt-related transcription factor 2, and collagen type I α1 according to TC concentration (0.05, 0.1, 0.25, and 0.5 mg/mL). ResultsThe release of TC from TC-SFMs plateaued and neared completion in 24 h. Significantly higher viability was noted achieved in 1% and 5% TC-SFMs. The morphology of GMSCs on TC-SFMs at 0% and 1% concentration showed spindle shapes, but cells in 10% TC-SFMs appeared spheroid. During Alizarin Red S staining at 21 d of osteogenic differentiation, calcium and osteocalcin formation were significantly lower in the 10% TC-SFM group than in the 0, 1, and 5 groups. Compared with the control group, bone gamma-carboxyglutamic acid protein showed significantly low expression rate at TC concentration ≥0.05 mg/mL. Bone sialoprotein was low at TC concentration ≥0.1 mg/mL. Likewise, runt-related transcription factor 2 and collagen type I α1 were low at TC concentration of 0.5 mg/mL. ConclusionsWithin the limits of this study, 1% and 5% TC-SFMs showed higher proliferation and osteogenic potential of GMSCs than 10% TC-SFM. Therefore, the use of 1% to 5% range of TC may be more suitable to silk fibroin membrane for stem cell tissue engineering.

Effects of enamel matrix derivative on the proliferation and osteogenic differentiation of human gingival mesenchymal stem cells.

IntroductionGingiva-derived mesenchymal stem cells (GMSCs) have recently been harvested and applied for rebuilding lost periodontal tissue. Enamel matrix derivative (EMD) has been used for periodontal regeneration and the formation of new cementum with inserting collagen fibers; however, alveolar bone formation is minimal. Recently, EMD has been shown to enhance the proliferation and mineralization of human bone marrow mesenchymal stem cells. Because the gingival flap is the major component to cover the surgical wound, the effects of EMD on the proliferation and mineralization of GMSCs were evaluated in the present study.MethodsAfter single cell suspension, the GMSCs were isolated from the connective tissues of human gingiva. The colony forming unit assay of the isolated GMSCs was measured. The expression of stem cell markers was examined by flow cytometry. The cellular telomerase activity was identified by polymerase chain reaction (PCR). The osteogenic, adipogenic and neural differentiations of the GMSCs were further examined. The cell proliferation was determined by MTS assay, while the expression of mRNA and protein for mineralization (including core binding factor alpha, cbfα-1; alkaline phosphatase, ALP; and osteocalcin, OC; ameloblastin, AMBN) were analyzed by real time-PCR, enzyme activity and confocal laser scanning microscopy.ResultsThe cell colonies could be easily identified and the colony forming rates and the telomerase activities increased after passaging. The GMSCs expressed high levels of surface markers for CD73, CD90, and CD105, but showed low expression of STRO-1. Osteogenic, adipogenic and neural differentiations were successfully induced. The proliferation of GMSCs was increased after EMD treatment. ALP mRNA was significantly augmented by treating with EMD for 3 hours, whereas AMBN mRNA was significantly increased at 6 hours after EMD treatment. The gene expression of OC was enhanced at the dose of 100 μg/ml EMD at day 3. Increased protein expression for cbfα-1 at day 3, for ALP at day 5 and 7, and for OC at week 4 after the EMD treatments were observed.ConclusionsHuman GMSCs could be successfully isolated and identified. EMD treatments not only induced the proliferation of GMSCs but also enhanced their osteogenic differentiation after induction.

Adoptive transfer of human gingiva-derived mesenchymal stem cells ameliorates collagen-induced arthritis via suppression of Th1 and Th17 cells and enhancement of regulatory T cell differentiation.

Current approaches offer no cures for rheumatoid arthritis (RA). Accumulating evidence has revealed that manipulation of bone marrow-derived mesenchymal stem cells (BM-MSCs) may have the potential to control or even prevent RA, but BM-MSC-based therapy faces many challenges, such as limited cell availability and reduced clinical feasibility. This study in mice with established collagen-induced arthritis (CIA) was undertaken to determine whether substitution of human gingiva-derived mesenchymal stem cells (G-MSCs) would significantly improve the therapeutic effects. CIA was induced in DBA/1J mice by immunization with type II collagen and Freund's complete adjuvant. G-MSCs were injected intravenously into the mice on day 14 after immunization. In some experiments, intraperitoneal injection of PC61 (anti-CD25 antibody) was used to deplete Treg cells in arthritic mice. Infusion of G-MSCs in DBA/1J mice with CIA significantly reduced the severity of arthritis, decreased the histopathology scores, and down-regulated the production of inflammatory cytokines (interferon-γ and interleukin-17A). Infusion of G-MSCs also resulted in increased levels of CD4+CD39+FoxP3+ cells in arthritic mice. These increases were noted early after infusion in the spleens and lymph nodes, and later after infusion in the synovial fluid. The FoxP3+ Treg cells that were increased in frequency mainly consisted of Helios-negative cells. When Treg cells were depleted, infusion of G-MSCs partially interfered with the progression of CIA. Pretreatment of G-MSCs with a CD39 or CD73 inhibitor significantly reversed the protective effect of G-MSCs on CIA. The role of G-MSCs in controlling the development and severity of CIA mostly depends on CD39/CD73 signals and partially depends on the induction of CD4+CD39+FoxP3+ Treg cells. G-MSCs provide a promising approach for the treatment of autoimmune diseases.

Three-Dimensional Spheroid Culture of Human Gingiva-Derived Mesenchymal Stem Cells Enhances Mitigation of Chemotherapy-Induced Oral Mucositis

Mesenchymal stem cells (MSCs) are capable of regenerative and immunomodulatory functions in cell-based therapies in a variety of human diseases and injuries; however, their therapeutic efficacy and potential side effects remain major obstacles in clinical applications. We report here a 3D spheroid culture approach to optimize stem cell properties and therapeutic effects of human gingiva-derived mesenchymal stem cells (GMSCs) in mitigation of experimental oral mucositis. Under growth condition of ultra-low attachment, GMSCs spontaneously aggregated into 3D spheroids and exhibited distinct early stem cell phenotype characterized by elevated expression Stro-1 and CXC chemokine receptor 4 (CXCR-4) as well as OCT-4 and Nanog, 2 important transcriptional factors relevant to stem cell properties, and decreased expression of MSC-associated markers, including CD29, CD90, and CD105. Functionally, spheroid GMSCs are capable of enhanced multipotency and augmented secretion of several chemokines and cytokines relevant to cell migration, survival, and angiogenesis. More importantly, spheroid GMSCs expressed increased levels of reactive oxygen species, hypoxia-inducible factor (HIF)-1 and -2α, and manganese superoxide dismutase, which correlated with improved resistance to oxidative stress-induced apoptosis. Using an in vivo murine model of chemotherapy-induced oral mucositis, we demonstrated that spheroid-derived GMSCs possessed better therapeutic efficacy than their adherent cells in reversing body weight loss and promoting the regeneration of disrupted epithelial lining of the mucositic tongues. These findings suggest that 3D spheroid culture allows early stemness preservation and potentially precondition GMSCs for enhanced mitigation of oral mucositis.

Gingiva-Derived Mesenchymal Stem Cell-Mediated Therapeutic Approach for Bone Tissue Regeneration

Up to now, the gingiva-derived mesenchymal stem cells (GMSCs) as a new postnatal stem cells have been isolated and characterized with multipotential differentiation capabilities in vitro. However, the in vivo efficacy of utilizing the GMSCs in bone regeneration remains obscure. First of all, we identified canonical MSCs in human gingival tissue, which possessed homogenous immunophenotype (CD34(-)CD45(-)CD29(+)CD105(+)CD90(+) STRO-1(+)) and had tri-lineage differentiation potential (osteoblasts, adipocytes, and chondrocytes). Next, we examined the efficacy of utilizing these stem cells in bone tissue regeneration; the enhanced green fluorescent protein-labeled GMSCs seeded on type I collagen gel were implanted into the mandibular defects as well as the critical-sized calvarial defects in Sprague Dawley rats. We first demonstrated that GMSCs could repair the mandibular wounds and calvarial defects at 2 months in rats postsurgical reconstruction. Histomorphological analysis and image of fluorescence microscope certified that new bone in the defect areas was derived from the transplanted GMSCs. Immunohistochemical analysis of green fluorescent protein, human collagen I, and osteopontin further confirmed our conclusion. The above results implied that mesenchymal stem cells derived from gingival tissue could be a novel source for stem cell-based therapy in bone reconstruction in clinical applications.

Stem cells

Recently, basic biology and clinical application of mesenchymal stem cells have become one of the most major subjects of scientific interest. Stem cell-based therapies for regeneration of various tissues allow an alternative therapeutic solution for a number of diseases. Many researches are concerned with the application of adipose tissue-derived stem cells, which share almost similar characteristics with bone marrow mesenchymal stem cells. Another new population of stem cells from human gingiva, namely, gingiva-derived mesenchymal stem cells is recently applied instead of bone marrow mesenchymal stem cells in the repair of injured organs.

Human gingiva-derived mesenchymal stem cells are superior to bone marrow-derived mesenchymal stem cells for cell therapy in regenerative medicine

Mesenchymal stem cells (MSCs) are capable of self-renewal and differentiation into multiple cell lineages. Presently, bone marrow is considered as a prime source of MSCs; however, there are some drawbacks and limitations in use of these MSCs for cell therapy. In this study, we demonstrate that human gingival tissue-derived MSCs have several advantages over bone marrow-derived MSCs. Gingival MSCs are easy to isolate, homogenous and proliferate faster than bone marrow MSCs without any growth factor. Importantly, gingival MSCs display stable morphology and do not loose MSC characteristic at higher passages. In addition, gingival MSCs maintain normal karyotype and telomerase activity in long-term cultures, and are not tumorigenic. Thus, we reveal that human gingiva is a better source of MSCs than bone marrow, and large number of functionally competent clinical grade MSCs can be generated in short duration for cell therapy in regenerative medicine and tissue engineering.

Mesenchymal Stem Cells Derived from Human Gingiva Are Capable of Immunomodulatory Functions and Ameliorate Inflammation-Related Tissue Destruction in Experimental Colitis

Aside from the well-established self-renewal and multipotent differentiation properties, mesenchymal stem cells exhibit both immunomodulatory and anti-inflammatory roles in several experimental autoimmune and inflammatory diseases. In this study, we isolated a new population of stem cells from human gingiva, a tissue source easily accessible from the oral cavity, namely, gingiva-derived mesenchymal stem cells (GMSCs), which exhibited clonogenicity, self-renewal, and multipotent differentiation capacities. Most importantly, GMSCs were capable of immunomodulatory functions, specifically suppressed peripheral blood lymphocyte proliferation, induced expression of a wide panel of immunosuppressive factors including IL-10, IDO, inducible NO synthase (iNOS), and cyclooxygenase 2 (COX-2) in response to the inflammatory cytokine, IFN-gamma. Cell-based therapy using systemic infusion of GMSCs in experimental colitis significantly ameliorated both clinical and histopathological severity of the colonic inflammation, restored the injured gastrointestinal mucosal tissues, reversed diarrhea and weight loss, and suppressed the overall disease activity in mice. The therapeutic effect of GMSCs was mediated, in part, by the suppression of inflammatory infiltrates and inflammatory cytokines/mediators and the increased infiltration of regulatory T cells and the expression of anti-inflammatory cytokine IL-10 at the colonic sites. Taken together, GMSCs can function as an immunomodulatory and anti-inflammatory component of the immune system in vivo and is a promising cell source for cell-based treatment in experimental inflammatory diseases.