Mechanism of Cyclic Tensile Stress in Osteogenic Differentiation of Human Periodontal Ligament Stem Cells
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PDLSCs (periodontal ligament stem cells), derived from dental tissues, are candidate cells for regeneration of dental tissues. MiRNAs could regulate osteogenic differentiation and the transformation into osteoblasts. This study was conducted to figure out how miR-184 regulates osteoblastic differentiation in PDLSCs.PDLSCs were isolated from premolars, and the osteoblastic differentiation was validated via Alizarin red staining and determination of ALP (alkaline phosphatase) activity. Expression of osteogenic specific genes were evaluated by western blot, and the expression pattern of miR-184 was determined by qRT-PCR. Target gene of miR-184 was then verified by dual luciferase reporter assay.Osteogenic-induced PDLSCs were successfully established with increased mineral deposition, ALP activity and protein expression of RUNX2 (runt-related transcription factor 2), osterix and BSP (bone sialoprotein). MiR-184 was reduced during osteoblastic differentiation of PDLSCs, and over-expression of miR-184 suppressed osteoblastic differentiation, as evidenced by reduction in mineral deposition, ALP activity and protein expression of RUNX2, osterix and BSP. MiR-184 could target NFI-C (nuclear factor I-C), and inhibit NFI-C expression in PDLSCs. NFI-C was enhanced during osteoblastic differentiation of PDLSCs, suggesting negative correlation with miR-184. Forced NFI-C expression promoted osteoblastic differentiation, and counteracted with the suppressive effects of miR-184 on osteoblastic differentiation.Downregulation of miR-184 facilitates osteoblastic differentiation in PDLSCs by modulating NFI-C, providing novel therapeutic strategy for regeneration of dental tissues.
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Diabetes mellitus (DM) is a chronic metabolic disease that gives rise to impaired bone remodeling. Increasing evidences have shown that miRNAs are associated with osteogenic differentiation of stem cells. However, the underlying mechanism that links DM-induced HG conditions and impaired osteogenic differentiation capacity of periodontal ligament stem cells (PDLSCs) still remains unclear. In this study, we found that diabetic mice with increased miR-31 level in periodontal ligaments exhibited greater bone loss. In vitro, the high expression of miR-31 is associated with the impaired osteogenic differentiation ability of PDLSCs in high glucose environment. Furthermore, miR-31 inhibitors increased mineralized bone matrix formation and raised Runx2, Osx and OCN expression at both mRNA and protein levels. However, PDLSCs pretreated with miR-31 mimics decreased bone matrix formation and reduced Runx2, Osx and OCN expression level in high glucose microenvironment. Moreover, Satb2 was identified as a target of miR-31 which directly binds to its 3'-untranslated region. To further elucidate the effect of Satb2 in miR-31-mediated osteogenic differentiation, PDLSCs were transfected with Satb2 siRNA and miR-31 inhibitors. The results showed that Satb2 siRNA inhibited osteogenic differentiation of PDLSCs in HG, whereas miR-31 inhibitors reversed the repression of osteogenic differentiation in Satb2 siRNA transfected PDLSCs. Taken together, these results demonstrate that miR-31 is involved in the high glucose-suppressed osteogenic differentiation of PDLSCs by targeting Satb2.
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Periodontal ligament stem cells (PDLSCs)-based regeneration therapy has received attention for its potential alternative applications in hard tissue and tooth. However, the environmental diversity of oral cavity that regulates PDLSCs differentiation has made it difficult to develop. Therefore, we investigated how high calcium concentrations in the oral environment influence osteogenic differentiation of human PDLSCs (hPDLSCs).hPDLSCs collected from human molars were isolated and cultured with CaCl2. First, multi lineage differentiation potentials to osteogenic, chondrogenic, and adipogenic cells were investigated. Then, the effects of CaCl2 on both alkaline phosphatase (ALP) activity and bone mineralization were analyzed and the expression of mRNA and protein for osteogenic marker was explored. Further, luciferase assay was performed to evaluate CaCl2 could regulate the transcriptional activity on osteogenic differentiation in hPDLSCs.CaCl2 treatment at normal to high concentrations showed similar suppression of ALP activity, while mineralized nodule formation was decreased by CaCl2 treatment dose-dependently without affecting proliferation or cytotoxicity in hPDLSCs. We also observed that CaCl2 treatment repressed the mRNA expression and protein abundance of osteogenic genes and transcriptional factors. Notably, repression of the Runx2 level was significant, and CaCl2 treatment inhibited Runx2-mediated transcriptional activity on the osteoblast-specific element (OSE) and ALP promoters.High concentrations of calcium negatively regulate osteogenic differentiation of hPDLSCs, by repressing osteogenic gene expressions and transcriptional activity. Therefore, these conditions may be applicable to determine the physiologically appropriate concentration of calcium.
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The periodontal ligament (PDL) is an essential fibrous tissue for tooth retention in the alveolar bone socket. PDL tissue further functions to cushion occlusal force, maintain alveolar bone height, allow orthodontic tooth movement, and connect tooth roots with bone. Severe periodontitis, deep caries, and trauma cause irreversible damage to this tissue, eventually leading to tooth loss through the destruction of tooth retention. Many patients suffer from these diseases worldwide, and its prevalence increases with age. To address this issue, regenerative medicine for damaged PDL tissue as well as the surrounding tissues has been extensively investigated regarding the potential and effectiveness of stem cells, scaffolds, and cytokines as well as their combined applications. In particular, PDL stem cells (PDLSCs) have been well studied. In this review, I discuss comprehensive studies on PDLSCs performed in vivo and contemporary reports focusing on the acquisition of large numbers of PDLSCs for therapeutic applications because of the very small number of PDLSCs available in vivo.
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Abstract Periodontal ligament stem cells (PDLSCs) are important mesenchymal stem cells contributing to regenerating lost periodontal tissues and repairing bone defects. Studies on the molecular mechanism affecting the osteogenic differentiation of PDLSCs are necessary. Scopolamine (SCO) is known as a regulator of neural cell damage. The focus of the current study is on unveiling the role of SCO‐mediated molecular mechanism in the osteogenic differentiation of PDLSCs. Through CCK‐8 assay and LDH detection, we confirmed that SCO enhanced the viability of PDLSCs. Moreover, we determined that SCO induced the PDLSCs osteogenic differentiation, according to data of ALP activity measurement and ARS staining. Mechanistically, we performed western blot and identified that SCO could promote the lactylation of runt‐related transcription factor 2 (RUNX2). We also found through rescue assays that knockdown of RUNX2 could reverse the effect of SCO treatment on the osteogenic differentiation of PDLSCs. Further mechanism investigation revealed that lactylation of RUNX2 at K176 site enhances the protein stability of RUNX2 through deubiquitination. Collectively, our present study unveils that SCO stabilizes RUNX2 to promote the osteogenic differentiation of PDLSCs through the lactylation modification of RUNX2.
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Deficiency of Macf1 inhibits bone formation by attenuating Bmp2/Smad/Runx2 signalling in osteoblasts. Macf1f/fOsx-Cre mice showed decreased bone mass, deteriorated bone microarchitecture and impaired bone strength. We demonstrated that Macf1 affected differentiation of primary osteoblasts through Bmp2/Smad/Runx2 signalling pathway.
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