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Extracellular vesicles derived from human dental mesenchymal stem cells stimulated with low-intensity pulsed ultrasound alleviate inflammation-induced bone loss in a mouse model of periodontitis

ABSTRACT: Extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) have emerged as a new mode of intercellular crosstalk and are responsible for many of the therapeutic effects of MSCs. To promote the application of MSC-EVs, recent studies have focused on the manipulation of MSCs to improve the production of EVs and EV-mediated activities. The current paper details an optimization method using non-invasive low-intensity pulsed ultrasound (LIPUS) as the stimulation for improving oral MSC-EV production and effectiveness. Stem cells from apical papilla (SCAP), a type of oral mesenchymal stem cell, displayed intensity-dependent pro-osteogenic and anti-inflammatory responses to LIPUS without significant cytotoxicity or apoptosis. The stimuli increased the secretion of EVs by promoting the expression of neutral sphingomyelinases in SCAP. In addition, EVs from LIPUS-induced SCAP exhibited stronger efficacy in promoting the osteogenic differentiation and anti-inflammation of periodontal ligament cells in vitro and alleviating oral inflammatory bone loss in vivo. In addition, LIPUS stimulation affected the physical characteristics and miRNA cargo of SCAP-EVs. Further investigations indicated that miR-935 is an important mediator of the pro-osteogenic and anti-inflammatory capabilities of LIPUS-induced SCAP-EVs. Taken together, these findings demonstrate that LIPUS is a simple and effective physical method to optimize SCAP-EV production and efficacy.

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PROVIDER: S-EPMC10311020 | biostudies-literature | 2022 Jul

REPOSITORIES: biostudies-literature

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Project description:BackgroundBone marrow-derived mesenchymal stem cells (BMSCs)-derived extracellular vesicles (EVs) have shown potent anti-inflammatory function in various pathological conditions, such as osteoarthritis and neurodegenerative diseases. Since the number of EVs naturally secreted by cells is finite and they usually bear specific repertoires of bioactive molecules to perform manifold cell-cell communication, but not one particular therapeutic function as expected, their practical application is still limited. Strategies are needed to increase the production of EVs and enhance their therapeutic function. Recent studies have suggested that low-intensity pulsed ultrasound (LIPUS) is a promising non-invasive method to increase the secretion of EVs and promote their anti-inflammatory effects. However, the effect of LIPUS stimulation of BMSCs on EVs derived from the cells remains unclear. The objective of this study was to investigate whether LIPUS stimulation on BMSCs could increase the secretion of EVs and enhance their anti-inflammatory effects.MethodsBMSCs were exposed to LIPUS (300 mW/cm2) for 15 min and EVs were isolated by ultracentrifugation. Anti-inflammatory effects of EVs were investigated on RAW264.7 cells in vitro and in the allogeneic skin transplantation model. Small RNA-seq was utilized to identify components difference in EVs with/without LIPUS irradiation.ResultsIn this study, we found that LIPUS stimulation could lead to a 3.66-fold increase in the EVs release from BMSCs. Moreover, both in vitro and in vivo experimental results suggested that EVs secreted from LIPUS-treated BMSCs (LIPUS-EVs) possessed stronger anti-inflammatory function than EVs secreted from BMSCs without LIPUS stimulation (C-EVs). RNA-seq analysis revealed that miR-328-5p and miR-487b-3p were significantly up-regulated in LIPUS-EVs compare with C-EVs. The suppression of MAPK signaling pathway by these two up-regulated miRNAs could be the potential mechanism of strengthened anti-inflammatory effects of LIPUS-EVs.ConclusionLIPUS stimulation on BMSCs could significantly increase the secretion of EVs. Moreover, EVs generated from LIPUS-treated BMSCs possessed much stronger anti-inflammatory function than C-EVs. Therefore, LIPUS could be a promising non-invasive strategy to promote the production of EVs from BMSCs and augment their anti-inflammatory effects.

Project description:Periodontitis is a widespread oral disease that results in the loss of alveolar bone. Low-intensity pulsed ultrasound (LIPUS), which is a new therapeutic option, promotes alveolar bone regeneration in periodontal bone injury models. This study investigated the protective effect of LIPUS on oxidative stress in periodontitis and the mechanism underlying this process. Methods: An experimental periodontitis model was induced by administering a ligature. Immunohistochemistry was performed to detect the expression levels of oxidative stress, osteogenic, and osteoclastogenic markers in vivo. Cell viability and osteogenic differentiation were analyzed using the Cell Counting Kit-8, alkaline phosphatase, and Alizarin Red staining assays. A reactive oxygen species assay kit, lipid peroxidation MDA assay kit, and western blotting were used to determine oxidative stress status in vitro. To verify the role of nuclear factor erythroid 2-related factor 2 (Nrf2), an oxidative regulator, during LIPUS treatment, the siRNA technique and Nrf2-/- mice were used. The PI3K/Akt inhibitor LY294002 was utilized to identify the effects of the PI3K-Akt/Nrf2 signaling pathway. Results: Alveolar bone resorption, which was experimentally induced by periodontitis in vivo, was alleviated by LIPUS via activation of Nrf2. Oxidative stress, induced via H2O2 treatment in vitro, inhibited cell viability and suppressed osteogenic differentiation. These effects were also alleviated by LIPUS treatment via Nrf2 activation. Nrf2 silencing blocked the antioxidant effect of LIPUS by diminishing heme oxygenase-1 expression. Nrf2-/- mice were susceptible to ligature-induced periodontitis, and the protective effect of LIPUS on alveolar bone dysfunction was weaker in these mice. Activation of Nrf2 by LIPUS was accompanied by activation of the PI3K/Akt pathway. The oxidative defense function of LIPUS was inhibited by exposure to LY294002 in vitro. Conclusions: These results demonstrated that LIPUS regulates alveolar bone homeostasis in periodontitis by attenuating oxidative stress via the regulation of PI3K-Akt/Nrf2 signaling. Thus, Nrf2 plays a pivotal role in the protective effect exerted by LIPUS against ligature-induced experimental periodontitis.

Project description:BackgroundAs a leading cause of vision decline and severe blindness in adults, diabetic retinopathy (DR) is characterized by the aggravation of retinal oxidative stress and apoptosis in the early stage. Emerging studies reveal that mesenchymal stem cells-derived small extracellular vesicles (MSC-sEV) treatment represents a promising cell-free approach to alleviate ocular disorders. However, the repairing effects of MSC-sEV in DR remain largely unclear. This study aimed at exploring the role and the underlying mechanism of MSC-sEV in hyperglycemia-induced retinal degeneration.MethodsIn vivo, we used streptozotocin (STZ) to establish diabetic rat model, followed by the intravitreal injection of MSC-sEV to determine the curative effect. The cell viability and antioxidant capacity of retinal pigment epithelium (RPE) cells stimulated with high-glucose (HG) medium after MSC-sEV treatment were analyzed in vitro. By detecting the response of cell signaling pathways in MSC-sEV-treated RPE cells, we explored the functional mechanism of MSC-sEV. Mass spectrometry was performed to reveal the bioactive protein which mediated the role of MSC-sEV.ResultsThe intravitreal injection of MSC-sEV elicited antioxidant effects and counteracted retinal apoptosis in STZ-induced DR rat model. MSC-sEV treatment also reduced the oxidative level and enhanced the proliferation ability of RPE cells cultured in HG conditions in vitro. Further studies showed that the increased level of phosphatase and tensin homolog (PTEN) inhibited AKT phosphorylation and nuclear factor erythroid 2-related factor 2 (NRF2) expression in RPE cells stimulated with HG medium, which could be reversed by MSC-sEV intervention. Through mass spectrometry, we illustrated that MSC-sEV-delivered neuronal precursor cell-expressed developmentally downregulated 4 (NEDD4) could cause PTEN ubiquitination and degradation, activate AKT signaling and upregulate NRF2 level to prevent DR progress. Moreover, NEDD4 knockdown impaired MSC-sEV-mediated retinal therapeutic effects.ConclusionsOur findings indicated that MSC-sEV ameliorated DR through NEDD4-induced regulation on PTEN/AKT/NRF2 signaling pathway, thus revealing the efficiency and mechanism of MSC-sEV-based retinal protection and providing new insights into the treatment of DR.

Project description:Ultrasound is considered a safe and non-invasive tool in regenerative medicine and has been used in the clinic for more than twenty years for applications in bone healing after the approval of the Exogen device, also known as low-intensity pulsed ultrasound (LIPUS). Beyond its effects on bone health, LIPUS has also been investigated for wound healing of soft tissues, with positive results for various cell processes including cell proliferation, migration and angiogenesis. As LIPUS has the potential to treat chronic skin wounds, we sought to evaluate the effects produced by a conventional therapeutic ultrasound device at low intensities (also considered LIPUS) on the migration capacity of mouse and human skin mesenchymal precursors (s-MPs). Cells were stimulated for 3 days (20 minutes per day) using a traditional ultrasound device with the following parameters: 100 mW/cm2 with 20% duty cycle and frequency of 3 MHz. At the parameters used, ultrasound failed to affect s-MP proliferation, with no evident changes in morphology or cell groupings, and no changes at the cytoskeletal level. Further, the migration and invasion ability of s-MPs were unaffected by the ultrasound protocol, and no major changes were detected in the gene/protein expression of ROCK1, integrin β1, laminin β1, type I collagen and transforming growth factor β1. Finally, RNA-seq analysis revealed that only 10 genes were differentially expressed after ultrasound stimulation. Among them, 5 encode for small nuclear RNAs and 2 encode for proteins belonging to the nuclear pore complex. Considering the results overall, while the viability of s-MPs was not affected by ultrasound stimulation and no changes were detected in proliferation/migration, RNA-seq analysis would suggest that s-MPs do respond to ultrasound. The use of 100 mW/cm2 intensity or conventional therapeutic ultrasound devices might not be optimal for the stimulation the properties of cell populations. Future studies should investigate the potential application of ultrasound using variations of the tested parameters.

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Extracellular vesicles derived from human dental mesenchymal stem cells stimulated with low-intensity pulsed ultrasound alleviate inflammation-induced bone loss in a mouse model of periodontitis

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