Background: Stem cell-based therapies have shown great promise in regenerative medicine and continue to generate wide interest in future clinical applications. However, the issue of storage and preservation of stem cells, for future clinical applications, still requires extensive investigation. Objective: The purpose of this study was to evaluate the effect of cryopreservation on the regenerative capacity of bone marrow stem cells in periodontal defects in dogs. Materi als and Methods: Bone marrow mesenchymal stem cells (BMSCs) were obtained from 5 beagle dogs. After cryopreservation for 1 month, cell viability, surface adherence ability, alkaline phosphatase activity and mineralized nodule formation were assessed. Twenty-six periodontal fenestration defects (5 × 5 mm) were created at a location 5 mm apical to the cemento-enamel junction in experimental teeth. Cryopreserved BMSCs were transplanted into the defects using a collagen scaffold carrier. Freshly isolated BMSCs and collagen scaffold alone were used as controls. All animals were sacrificed 8 weeks after surgery, and specimens were evaluated by histomorphometry. Results: Cryopreservation had no discernible negative effect on BMSC growth and differentiation in vitro. Both freshly isolated and cryopreserved BMSC transplantations induced significantly better periodontal regeneration with newly formed cementum, alveolar bone and periodontal ligament compared with the application of collagen scaffold alone. Conclusion: Cryopreserved BMSCs showed no altered regenerative capacity compared with freshly isolated BMSCs in the application of periodontal regeneration.
To eliminate invading pathogens and keep homeostasis, host employs multiple approaches such as the non-inflammation associated-apoptosis, inflammation associated-necroptosis and pyroptosis, etc. Necroptosis is known as a highly pro-inflammatory form of cell death due to the release of massive damage-associated molecular patterns (DAMPs). For the first time, we reported that Porphyromonas gingivalis induced cellular necroptosis through receptor-interacting protein 1 (RIP1)/RIP3/mixed lineage kinase domain-like (MLKL) signaling pathway in monocytes. Necroptosis in THP-1 cells was induced by MLKL phosphorylation in vitro. P. gingivalis treated-THP-1 cells exhibited lower cell death rate with pretreatment of inhibitors RIP1 and MLKL, accompanied with attenuated TNF-α and IL-6 expressions. Moreover, the necroptosis risk was also reduced via gene silencing by RIP3 or MLKL in the P. gingivalis treated-THP-1 cell lines. We further explored P. gingivalis-induced necroptosis in animal models in vivo. Firstly, C57BL/6 mice were injected with P. gingivalis in the subcutaneous chamber model. Animals pretreated with MLKL inhibitor exhibited significantly enhanced P. gingivalis clearance; in addition, levels of TNF-α and IL-6 were notably decreased by 60% via MLKL inhibition. Secondly, P. gingivalis-induced periodontitis was utilized to investigate necroptosis related-periodontopathogensis. Positive staining of phosphorylated MLKL in mice periodontitis biopsies was detected to a higher degree, while larger amount of alveolar bone loss was observed in MLKL (-) group comparing to those in the MLKL (+) group. These findings may suggest that P. gingivalis play essential roles in necroptosis process during periodontitis, and our research may shed light on the further work on the related periodontopathogenesis investigation.
Loss of periodontal ligament fibroblasts (PDLFs) is one critical issue for regenerating lost periodontal tissues. A wide variety of regulated cell death pathways, such as apoptosis, pyroptosis, and necroptosis have been proposed in the periodontitis development. The aim of the present study was to explore whether long-term periodontitis-level butyrate may trigger ferroptosis, a newly characterized iron-dependent regulated cell death in PDLFs. Here, we showed that long-term treatment of butyrate, an important short-chain fatty acid in the periodontal pocket, induces the cargo receptor nuclear receptor coactivator 4 (NCOA4)-mediated ferritinophagy and ferroptosis in PDLFs. Butyrate-induced iron accumulation, reactive oxygen species (ROS) generation, glutathione depletion and lipid peroxidation in PDLFs, and the butyrate-induced ferroptosis can be blocked by the lipid peroxide scavenger ferrostatin-1. The NCOA4-mediated ferritinophagy is dependent on p38/hypoxia inducible factor-1α (HIF-1α) pathway activation as well as Bromodomain-containing protein (BRD) 4 and cyclin-dependent kinase 9 (CDK9) coordination. These lines of evidence provide a new mechanistic insight into the mechanism of loss of PDLFs during periodontitis development, showing that periodontitis-level butyrate disrupted iron homeostasis by activation of NCOA4-mediated ferritinophagy, leading to ferroptosis in PDLFs.
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