Extracellular Matrix Membrane Induces Cementoblastic/Osteogenic Properties of Human Periodontal Ligament Stem Cells.
ABSTRACT: Objective: Periodontitis affects nearly 90% of adults over the age of 70, resulting to periodontal tissue infection, destruction, and ultimately tooth loss. Guided tissue regeneration (GTR) is a method widely used to treat severe periodontal disease, and involves placement of an occlusive barrier to facilitate regeneration of the damaged area by periodontal ligament stem cells (PDLSCs). In this study, we evaluate natural extracellular matrix (ECM) as a scaffold material to provide a suitable microenvironment to support the proliferation, differentiation, and tissue-regenerating properties of PDLSCs. Design: The viability, proliferation, apoptosis, and migration of PDLSCs cultured on ECM membrane, that was isolated from porcine urinary bladders, were compared with those cultured on type I collagen membrane, a commonly used scaffold in GTR. To evaluate the effects of ECM vs. type I collagen on the tissue-regenerating properties of PDLSCs, the bio-attachment and cementoblastic/osteogenic differentiation of PDLSCs were evaluated. Results: Incubation of PDLSCs with ECM resulted in increased viability, proliferation, and reduced apoptosis, compared with type I collagen treated PDLSCs. Co-culture with ECM membrane also increased the migration and bio-attachment of PDLSCs. Incubation of PDLSCs with ECM membrane increased expression of the cementoblastic/osteogenic differentiation markers BSP, RUNX2, ALP, OPN, OCN, and periostin. Conclusion: ECM membrane enhances the proliferation and regenerative properties of PDLSCs, indicating that ECM membrane can serve as a suitable scaffold in the application of GTR to treat periodontal disease.
Project description:Periodontal ligament stem cells (PDLSCs) can repair alveolar bone defects in periodontitis in a microenvironment context-dependent manner. This study aimed to determine whether different extracellular matrices (ECMs) exert diverse effects on osteogenic differentiation of PDLSCs and accurately control alveolar bone defect repair. Methods: The characteristics of PDLSCs and bone marrow mesenchymal stem cells (BMSCs) with respect to surface markers and multi-differentiation ability were determined. Then, we prepared periodontal ligament cells (PDLCs)-derived and bone marrow cells (BMCs)-derived ECMs (P-ECM and B-ECM) and the related decellularized ECMs (dECMs). Transmission electron microscopy (TEM), scanning electron microscopy (SEM), atomic force microscopy (AFM), and protein mass spectrometry were used to distinguish the ECMs. The expression of Type IV collagen A2 (COL4A2) in the ECMs was inhibited by siRNA or activated by lentiviral transduction of relevant cells. The stemness, proliferation, and differentiation of PDLSCs were determined in vitro in different dECMs. For the in vivo analysis, different dECMs under the regulation of COL4A2 mixed with PDLSCs and Bio-Oss bone powder were subcutaneously implanted into immunocompromised mice or in defects in rat alveolar bone. The repair effects were identified by histological or immunohistochemical staining and micro-CT. Results: B-dECM exhibited more compact fibers than P-dECM, as revealed by TEM, SEM, and AFM. Protein mass spectrometry showed that COL4A2 was significantly increased in B-dECM compared with P-dECM. PDLSCs displayed stronger proliferation, stemness, and osteogenic differentiation ability when cultured on B-dECM than P-dECM. Interestingly, B-dECM enhanced the osteogenic differentiation of PDLSCs to a greater extent than P-dECM both in vitro and in vivo, whereas downregulation of COL4A2 in B-dECM showed the opposite results. Furthermore, the classical Wnt/?-catenin pathway was found to play an important role in the negative regulation of osteogenesis through COL4A2, confirmed by experiments with the Wnt inhibitor DKK-1 and the Wnt activator Wnt3a. Conclusion: These findings indicate that COL4A2 in the ECM promotes osteogenic differentiation of PDLSCs through negative regulation of the Wnt/?-catenin pathway, which can be used as a potential therapeutic strategy to repair bone defects.
Project description:Periodontal regeneration is an important part of regenerative medicine, with great clinical significance; however, the effects of nanotopography on the functions of periodontal ligament (PDL) stem cells (PDLSCs) and on PDLSC sheet based periodontal regeneration have never been explored. Titania nanotubes (NTs) layered on titanium (Ti) provide a good platform to study this. In the current study, the influence of NTs of different tube size on the functions of PDLSCs was observed. Afterward, an ectopic implantation model using a Ti/cell sheets/hydroxyapatite (HA) complex was applied to study the effect of the NTs on cell sheet based periodontal regeneration. The NTs were able to enhance the initial PDLSC adhesion and spread, as well as collagen secretion. With the Ti/cell sheets/HA complex model, it was demonstrated that the PDLSC sheets were capable of regenerating the PDL tissue, when combined with bone marrow mesenchymal stem cell (BMSC) sheets and HA, without the need for extra soluble chemical cues. Simultaneously, the NTs improved the periodontal regeneration result of the ectopically implanted Ti/cell sheets/HA complex, giving rise to functionally aligned collagen fiber bundles. Specifically, much denser collagen fibers, with abundant blood vessels as well as cementum-like tissue on the Ti surface, which well-resembled the structure of natural PDL, were observed in the NT5 and NT10 sample groups. Our study provides the first evidence that the nanotopographical cues obviously influence the functions of PDLSCs and improve the PDLSC sheet based periodontal regeneration size dependently, which provides new insight to the periodontal regeneration. The Ti/cell sheets/HA complex may constitute a good model to predict the effect of biomaterials on periodontal regeneration.
Project description:BACKGROUND:Periodontitis, which progressively destroys tooth-supporting structures, is one of the most widespread infectious diseases and the leading cause of tooth loss in adults. Evidence from preclinical trials and small-scale pilot clinical studies indicates that stem cells derived from periodontal ligament tissues are a promising therapy for the regeneration of lost/damaged periodontal tissue. This study assessed the safety and feasibility of using autologous periodontal ligament stem cells (PDLSCs) as an adjuvant to grafting materials in guided tissue regeneration (GTR) to treat periodontal intrabony defects. Our data provide primary clinical evidence for the efficacy of cell transplantation in regenerative dentistry. METHODS:We conducted a single-center, randomized trial that used autologous PDLSCs in combination with bovine-derived bone mineral materials to treat periodontal intrabony defects. Enrolled patients were randomly assigned to either the Cell group (treatment with GTR and PDLSC sheets in combination with Bio-oss(®)) or the Control group (treatment with GTR and Bio-oss(®) without stem cells). During a 12-month follow-up study, we evaluated the frequency and extent of adverse events. For the assessment of treatment efficacy, the primary outcome was based on the magnitude of alveolar bone regeneration following the surgical procedure. RESULTS:A total of 30 periodontitis patients aged 18 to 65 years (48 testing teeth with periodontal intrabony defects) who satisfied our inclusion and exclusion criteria were enrolled in the study and randomly assigned to the Cell group or the Control group. A total of 21 teeth were treated in the Control group and 20 teeth were treated in the Cell group. All patients received surgery and a clinical evaluation. No clinical safety problems that could be attributed to the investigational PDLSCs were identified. Each group showed a significant increase in the alveolar bone height (decrease in the bone-defect depth) over time (p < 0.001). However, no statistically significant differences were detected between the Cell group and the Control group (p > 0.05). CONCLUSIONS:This study demonstrates that using autologous PDLSCs to treat periodontal intrabony defects is safe and does not produce significant adverse effects. The efficacy of cell-based periodontal therapy requires further validation by multicenter, randomized controlled studies with an increased sample size. TRIAL REGISTRATION:NCT01357785 Date registered: 18 May 2011.
Project description:Periodontal disease is chronic inflammation that leads to the destruction of tooth-supporting periodontal tissues. We devised a novel method ("cell transfer technology") to transfer cells onto a scaffold surface and reported the potential of the technique for regenerative medicine. The aim of this study is to examine the efficacy of this technique in periodontal regeneration and the fate of transplanted cells. Human periodontal ligament stem cells (PDLSCs) were transferred to decellularized amniotic membrane and transplanted into periodontal defects in rats. Regeneration of tissues was examined by microcomputed tomography and histological observation. The fate of transplanted PDLSCs was traced using PKH26 and human Alu sequence detection by PCR. Imaging showed more bone in PDLSC-transplanted defects than those in control (amnion only). Histological examination confirmed the enhanced periodontal tissue formation in PDLSC defects. New formation of cementum, periodontal ligament, and bone were prominently observed in PDLSC defects. PKH26-labeled PDLSCs were found at limited areas in regenerated periodontal tissues. Human Alu sequence detection revealed that the level of Alu sequence was not increased, but rather decreased. This study describes a novel stem cell transplantation strategy for periodontal disease using the cell transfer technology and offers new insight for cell-based periodontal regeneration.
Project description:Cell sheet technology is a novel tissue engineering technology that has been rapidly developed in recent years. As a novel technology, cell sheet technology is expected to become one of the preferred methods for cell transplantation. The present study investigated the biological effects of rutin on the formation of periodontal ligament stem cell (PDLSC) sheets and their resultant osteogenic properties. The results of Cell Counting Kit?8 (CCK?8) assay demonstrated that a concentration of 1x10?6 mol/l rutin promoted the proliferation of PDLSCs more effectively compared with other designed concentrations. Rutin?modified cell sheets could be induced by complete medium supplemented with 20 µg/ml vitamin C (VC) and 1x10?6 mol/l rutin. Rutin?modified cell sheets appeared thicker and more compact compared with the VC?induced PDLSC sheets, demonstrating more layers of cells (3 or 4 layers), which secreted a richer extracellular matrix (ECM). Furthermore, the improved cell sheets exhibited varying degrees of increases in the mRNA and protein expression of collagen type I (COL1), alkaline phosphatase (ALP), runt?related transcription factor 2 (RUNX2) and osteopontin (OPN). Combined treatment with VC and rutin promoted the formation of PDLSC sheets and enhanced the osteogenic differentiation potential of the cell sheets. Therefore, rutin?modified cell sheets of PDLSCs are expected to play an important role in the treatment of periodontal tissue regeneration by stem cells.
Project description:Repair or regeneration of damaged nerves is still a challenging clinical task in reconstructive surgeries and regenerative medicine. Here, it is demonstrated that periodontal ligament stem cells (PDLSCs) and gingival mesenchymal stem cells (GMSCs) isolated from adult human periodontal and gingival tissues assume neuronal phenotype in vitro and in vivo via a subcutaneous transplantation model in nude mice. PDLSCs and GMSCs are encapsulated in a 3D scaffold based on alginate and hyaluronic acid hydrogels capable of sustained release of human nerve growth factor (NGF). The elasticity of the hydrogels affects the proliferation and differentiation of encapsulated MSCs within scaffolds. Moreover, it is observed that PDLSCs and GMSCs are stained positive for ?III-tubulin, while exhibiting high levels of gene expression related to neurogenic differentiation (?III-tubulin and glial fibrillary acidic protein) via quantitative polymerase chain reaction (qPCR). Western blot analysis shows the importance of elasticity of the matrix and the presence of NGF in the neurogenic differentiation of encapsulated MSCs. In vivo, immunofluorescence staining for neurogenic specific protein markers confirms islands of dense positively stained structures inside transplanted hydrogels. As far as it is known, this study is the first demonstration of the application of PDLSCs and GMSCs as promising cell therapy candidates for nerve regeneration.
Project description:During tooth development, the jawbone interacts with dental germ and provides the development microenvironment. Jawbone-derived mesenchymal stem cells (JBMSCs) maintain this microenvironment for root and periodontium development. However, the effect of the jawbone microenvironment on periodontium tissue regeneration is largely elusive. Our previous study showed that cell aggregates (CAs) of bone marrow mesenchymal stem cells promoted periodontium regeneration on the treated dentin scaffold. Here, we found that JBMSCs enhanced not only the osteogenic differentiation of periodontal ligament stem cells (PDLSCs) but also their adhesion to titanium (Ti) material surface. Importantly, the compound CAs of PDLSCs and JBMSCs regenerated periodontal ligament-like fibers and mineralized matrix on the Ti scaffold surface, both in nude mice ectopic and minipig orthotopic transplantations. Our data revealed that an effective regenerative microenvironment, reconstructed by JBMSCs, promoted periodontium regeneration by regulating PDLSCs function on the Ti material.
Project description:Objective:Per-implantitis is one of the implant treatment complications. Dentists have failed to restore damaged periodontium by using conventional therapies. Tissue engineering (stem cells, scaffold and growth factors) aims to reconstruct natural tissues. The paper aimed to isolate both periodontal ligament stem cells (PDLSCs) and bone marrow mesenchymal stem cells (BMMSCs) and use them in a co-culture method to create three-layered cell sheets for reconstructing natural periodontal ligament (PDL) tissue. Materials and methods:BMMSCs were isolated from rabbit tibia and femur, and PDLSC culture was established from the lower right incisor. The cells were co-cultured to induce BMMSC differentiation into PDL cells. Cell morphology, stem cells and PDL-specific markers (CD90, CD34, and periostin) were also detected using immunofluorescent assay. Co-cultured cell monolayers were detached using temperature-responsive tissue culture dishes and collagen graft to create the three-layer construct. The 3D-engineered tissue was examined histologically and by field emission scanning electron microscopy (FESEM). Results:BMMSCs co-cultured with PDLSCs successfully induced more PDL cells. The newly induced PDL cells exhibited periostin and CD90 expression. Fluorescence green intensity was measured for the co-cultured cells that were stained with periostin, the mean fluorescence green intensity (periostin expression) was significantly higher for the newly induced PDL cells after 1, 2, and 3 weeks when compared with control (BM-MSCs), at 21 days non-significant difference was measured when compared with control (PDLSCs).The results showed the successful formation of 3D multilayer PDL tissue. Histological cross-section showed cell sheets and the stable adhesion between them. FESEM examination was conducted for the cross-section, showing three-layered cell sheets with stable adhesion between cells. Conclusions:The results of this paper report that the three layered-cell sheets were successfully constructed by the novel use of collagen graft as a scaffold to be used in treatment of periodontitis and to envelop the dental implants to create biohybrid implant.
Project description:Objective:This research is aimed at investigating how high glucose affects the proliferation and apoptosis in periodontal ligament stem cells (PDLSCs) in the presence of TNF-?. Methods:PDLSCs obtained from periodontal healthy permanent teeth were treated under either high-glucose condition (30?mmol/L, G30 group) or normal glucose condition (5.6?mmol/L, G5.6 group) in the presence or absence of TNF-?. ?. ?. Results:CCK-8 assay showed that high glucose exacerbated TNF-?. ?. ?. ?. ?. ?. Conclusion:High glucose exacerbates TNF-?-induced proliferative inhibition in human periodontal ligament stem cells through the upregulation and activation of TNF receptor 1. Inhibition of intracellular ROS expression by vitamin C partially rescues PDLSCs in terms of cell proliferation.?.
Project description:Gene expression alterations in periodontal ligament stem cells (PDLSCs) during bisphosphonate (BP) usage and the transcriptomic mechanism underlying BP?related osteonecrosis of the jaw have not been fully elucidated. In the present study, human PDLSCs were isolated from adults with no history of periodontal disease, and subsequently incubated and treated with zoledronate on days 3 and 5. Subsequently, PDLSCs from all timepoints were screened using an Affymetrix Gene Expression Array. Limma differential expression analysis was performed on a normalized gene expression matrix, followed by cluster analysis, pathway and network analyses. Overall, 906 genes (352 upregulated and 554 downregulated) exhibited differential expression levels between days 0 and 5, and these were termed slow?response genes. These slow?response genes were enriched in cellular stress response signaling pathways (upregulated genes), as well as proliferation? and ossification?associated signaling pathways (downregulated genes). Furthermore, 168 (day 3 vs. 0) and 105 (day 5 vs. 3) genes were differentially expressed between adjacent timepoints. These genes were also enriched in stress response? and proliferation?associated signaling pathways, but not in ossification?associated signaling pathways. Poly(ADP?ribose) polymerase 1 (PARP1) and CYLD lysine 63 deubiquitinase (CYLD) had the most protein?protein interaction partners among the slow?response genes and were connected with both stress? (e.g. caspase?1) and ossification?associated genes [e.g. secreted phosphoprotein 1 and collagen type I ?1 chain (COL1A1)]. BP treatment induced stress response?like transcriptional alterations in PDLSCs, followed by inhibition of proliferation and ossification. These alterations may contribute to the onset of jaw osteonecrosis. PARP1 and CYLD may be two key genes involved in this pathological procedure.