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Circular RNA (circRNA) is a unique closed ring structure of noncoding RNA. Although many human diseases have been confirmed to be inextricably linked with circRNAs, whether circRNAs have a potential regulatory function in the osteogenic differentiation of bone marrow mesenchymal stem cells (BMMSCs) remains to be further elucidated. In a previous study by our team, all the differentially expressed circRNAs and messenger RNAs (mRNAs) involved in the osteogenic differentiation of BMMSCs were identified via high-throughput sequencing, and a competing endogenous RNA (ceRNA) regulatory network was constructed via bioinformatics analysis. The circRNA₁809/miR-370-3p/Kitlg axis may be involved in regulating the osteogenic differentiation of BMMSCs. In this study, gene knockdown/overexpression, small interfering RNA (siRNA) transfection and mimic/inhibitor treatment were used to evaluate the regulatory effects of circRNA₁809 on the miR-370-3p/Kitlg pathway and the osteogenic differentiation of rat bone marrow mesenchymal stem cells (rBMMSCs) in vitro. The results revealed that circRNA₁809 was upregulated and that miR-370-3p was downregulated during the osteogenic differentiation of rBMMSCs. The low expression of circRNA₁809 significantly downregulated the expression of Kitlg and decreased the protein expression of ALP and RUNX2. The expression of miR-370-3p was negatively correlated with the expression of Kitlg in rBMMSCs and the ability to undergo osteogenic differentiation. In addition, a dual-luciferase assay confirmed the binding of miR-370-3p and circRNA₁809, and si-circRNA₁809 + miR-370-3p inhibitor cotransfection reversed some of the downregulation of Kitlg induced by si-circRNA₁809, whereas si-circRNA₁809 + miR-370-3p mimic increased the downregulation of Kitlg. Therefore, circRNA₁809 may promote the expression of Kitlg by regulating miR-370-3p and subsequently promote the osteogenic differentiation of rBMMSCs.

Summary Bone marrow‐derived mesenchymal stem cells (BMMSCs) exhibit degenerative changes, including imbalanced differentiation and reduced proliferation during aging, that contribute to age‐related bone loss. We demonstrate here that autophagy is significantly reduced in aged BMMSCs compared with young BMMSCs. The autophagy inhibitor 3‐methyladenine (3‐MA) could turn young BMMSCs into a relatively aged state by reducing their osteogenic differentiation and proliferation capacity and enhancing their adipogenic differentiation capacity. Accordingly, the autophagy activator rapamycin could restore the biological properties of aged BMMSCs by increasing osteogenic differentiation and proliferation capacity and decreasing adipogenic differentiation capacity. Possible underlying mechanisms were explored, and the analysis revealed that autophagy could affect reactive oxygen species and p53 levels, thus regulating biological properties of BMMSCs. In an in vivo study, we found that activation of autophagy restored bone loss in aged mice. In conclusion, our results suggest that autophagy plays a pivotal role in the aging of BMMSCs, and activation of autophagy could partially reverse this aging and may represent a potential therapeutic avenue to clinically treat age‐related bone loss.

Previously, we have shown hyperhomocysteinemia (HHcy) to have a detrimental effect on bone remodeling, which is associated with osteoporosis. During transsulfuration, Hcy is metabolized into hydrogen sulfide (H 2 S), a gasotransmitter molecule known to regulate bone formation. Therefore, in the present study, we examined whether H 2 S ameliorates HHcy induced epigenetic and molecular alterations leading to osteoporotic bone loss. To test this mechanism, we employed cystathionine-beta-synthase heterozygote knockout mice, fed with a methionine rich diet (CBS +/− +Met), supplemented with H 2 S-donor NaHS for 8 weeks. Treatment with NaHS, normalizes plasma H 2 S, and completely prevents trabecular bone loss in CBS +/− mice. Our data showed that HHcy caused inhibition of HDAC3 activity and subsequent inflammation by imbalancing redox homeostasis. The mechanistic study revealed that inflammatory cytokines (IL-6, TNF-α) are transcriptionally activated by an acetylated lysine residue in histone (H3K27ac) of chromatin by binding to its promoter and subsequently regulating gene expression. A blockade of HDAC3 inhibition in CBS +/− mice by HDAC activator ITSA-1, led to the remodeling of histone landscapes in the genome and thereby attenuated histone acetylation-dependent inflammatory signaling. We also confirmed that RUNX2 was sulfhydrated by administration of NaHS. Collectively, restoration of H 2 S may provide a novel treatment for CBS-deficiency induced metabolic osteoporosis.

Microfluidic experimental models allow to study the mutual interrelation between tumor development and the microvasculature avoiding animal use and lacking interspecies differences. This study aimed to develop and characterize a 3D tissue culture model employing a two-compartment microfluidic chip-perfused platform to visualize and quantify human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and MCF-7 breast cancer cell-cell interactions in real time. MCF-7 cells were implanted in the tumor chamber and hBM-MSCs were injected into microvascular channels. hBM-MSCs culture media was perfused into microvascular compartments. The microfluidic device was microscopically examined weekly for four weeks. VE- and E-cadherin immunofluorescence validated hBM-MSCs differentiation into endothelial cells and MCF-7 cell tumor formation. hBM-MSCs differentiation was highly heterogeneous along the microvascular channels, due to different perfusion flow. hBM-MSCs lining microvascular channels acquired VE-cadherin positive endothelial phenotype and continuously covered microchannels as an endothelium like layer. MCF-7 cells were constantly grown as spheroidal aggregates and later formed a compact area of E-cadherin-positive tumor cells inside tumor compartment. Our study provides valuable knowledge on the properties of hBM-MSCs as vasculogenesis-supporting cells when co-cultured with MCF-7 cells on a 3D perfused biomimetic microfluidic device. This newly established model may serve as an experimental platform for testing anti-tumor/anti-angiogenic drugs.

Abstract N6-methyladenosine (m6A) methylation, a well-known modification with new epigenetic functions, has been reported to participate in the progression of osteoporosis (OP), providing novel insights into the pathogenesis of OP. However, as the key component of m6A methylation, Wilms tumor 1-associated protein (WTAP) has not been studied in OP. Here we explored the biological role and underlying mechanism of WTAP in OP and the differentiation of bone marrow mesenchymal stem cells (BMMSCs). We demonstrated that WTAP was expressed at low levels in bone specimens from patients with OP and OVX mice. Functionally, WTAP promoted osteogenic differentiation and inhibited adipogenic differentiation of BMMSCs in vitro and in vivo. In addition, microRNA-29b-3p (miR-29b-3p) was identified as a downstream target of WTAP. M6A modifications regulated by WTAP led to increased miR-29b-3p expression. WTAP interacted with the microprocessor protein DGCR8 and accelerated the maturation of pri-miR-29b-3p in an m6A-dependent manner. Target prediction and dual-luciferase reporter assays identified the direct binding sites of miR-29b-3p with histone deacetylase 4 (HDAC4). WTAP-mediated m6A modification promoted osteogenic differentiation and inhibited adipogenic differentiation of BMMSCs through the miR-29b-3p/HDAC4 axis. Furthermore, WTAP-mediated m6A methylation negatively regulates osteoclast differentiation. Collectively, our study first identified a critical role of WTAP-mediated m6A methylation in BMMSC differentiation and highlighted WTAP as a potential therapeutic target for OP treatment. Graphical Abstract Graphical Abstract

Circular RNA ( circRNA ) is a unique closed ring structure of noncoding RNA. Although many human diseases have been confirmed to be inextricably linked with circRNAs , whether circRNAs have a potential regulatory function in the osteogenic differentiation of bone marrow mesenchymal stem cells (BMMSCs) remains to be further elucidated. In a previous study by our team, all the differentially expressed circRNAs and messenger RNAs ( mRNAs ) involved in the osteogenic differentiation of BMMSCs were identified via high-throughput sequencing, and a competing endogenous RNA ( ceRNA ) regulatory network was constructed via bioinformatics analysis. The circRNA_1809/miR-370-3p/Kitlg axis may be involved in regulating the osteogenic differentiation of BMMSCs. In this study, gene knockdown/overexpression, small interfering RNA (siRNA) transfection and mimic/inhibitor treatment were used to evaluate the regulatory effects of circRNA_1809 on the miR-370-3p / Kitlg pathway and the osteogenic differentiation of rat bone marrow mesenchymal stem cells (rBMMSCs) in vitro. The results revealed that circRNA_1809 was upregulated and that miR-370-3p was downregulated during the osteogenic differentiation of rBMMSCs. The low expression of circRNA_1809 significantly downregulated the expression of Kitlg and decreased the protein expression of ALP and RUNX2. The expression of miR-370-3p was negatively correlated with the expression of Kitlg in rBMMSCs and the ability to undergo osteogenic differentiation. In addition, a dual-luciferase assay confirmed the binding of miR-370-3p and circRNA_1809 , and si-circRNA_1809  +  miR-370-3p inhibitor cotransfection reversed some of the downregulation of Kitlg induced by si-circRNA_1809 , whereas si-circRNA_1809  +  miR-370-3p mimic increased the downregulation of Kitlg . Therefore, circRNA_1809 may promote the expression of Kitlg by regulating miR-370-3p and subsequently promote the osteogenic differentiation of rBMMSCs.

Background iRoot BP Plus is a novel bioceramic endodontic material. Recently, it has been considered as an alternative to MTA which is the most popular scaffold cover during regenerative endodontic therapy. This study aimed to evaluate the effects of iRoot BP Plus on the osteo/odontogenic capacity of bone marrow mesenchymal stem cells (BMMSCs), including the underlying mechanisms. Methods BMMSCs were collected by a whole marrow method and treated with iRoot BP Plus-conditioned medium (BP-CM). The proliferation ability was evaluated by cell counting kit 8 and flow cytometry. Complete medium was used as a blank control, and 2 mg/ml MTA-conditioned medium was served as a positive control. Alkaline phosphatase (ALP) activity assay, ALP staining, western blot, real-time RT-PCR, Alizarin Red S staining, and immunofluorescence staining were performed to explore the osteo/odontogenic potential and the involvement of MAPK pathways. Besides, autophagy was investigated by western blot, immunofluorescence staining, and transmission electron microscopy. Results

Bone marrow mesenchymal stem cells (BMMSCs) have garnered attention as promising therapeutic modalities for spinal cord injury (SCI) due to their neuroregenerative, anti-apoptotic, and functional recovery-enhancing properties. The central role of microRNAs (miRNAs) in mediating the beneficial outcomes resulting from BMMSCs in SCI has been highlighted in recent studies, suggesting that targeted modulation of specific miRNAs holds potential for augmenting SCI recovery. Our previous investigation implicated miR-202-3p in the reparative processes of injured spinal cords, although the precise mechanistic underpinnings remain elusive. In vivo, BMMSCs were administered to SCI rats, while in vitro, miR-202-3p was transfected into PC-12 cells. Motor capabilities recovery was assessed via Basso-Beattie-Bresnahan (BBB) scores and footprinting tests; the evaluation of neuronal and spinal cord tissue repair was conducted using Nissl staining, TUNEL staining, hematoxylin and eosin (HE) staining, and immunofluorescence; and the impacts of miR-202-3p on cellular autophagy, neuronal apoptosis, and relevant pathways were evaluated using Western blotting, quantitative polymerase chain reaction (qPCR), and transmission electron microscopy (TEM). Functionally, BMMSCs utilized miR-202-3p to improve motor recovery in SCI rats. Histopathologically, they contributed to the repair of damaged spinal cords and the regeneration of nerve axons. At the molecular level, BMMSCs stimulated autophagy and suppressed neuronal apoptosis by regulating the AMPK, MAPK, and PI3K/AKT/mTOR pathway. Collectively, our findings demonstrate that BMMSCs coordinate miR-202-3p to inhibit mTOR activation via the AMPK, MAPK, and PI3K/AKT pathways, thereby promoting TFEB dephosphorylation, modulating autophagy and neuronal apoptosis, and ultimately fostering functional recovery post-SCI.

Poly(N-isopropylacrylamide) (PNIPAM) offers a promising platform for non-invasive and gentle cell detachment. However, conventional PNIPAM-based substrates often suffer from limitations including limited stability and reduced reusability, which hinder their widespread adoption in biomedical applications. In this study, PNIPAM copolymer films were formed on the surfaces of glass slides or silicon wafers using a two-step film-forming method involving coating and grafting. Subsequently, a comprehensive analysis of the films’ surface wettability, topography, and thickness was conducted using a variety of techniques, including contact angle analysis, atomic force microscopy (AFM), and ellipsometric measurements. Bone marrow mesenchymal stem cells (BMMSCs) were then seeded onto PNIPAM copolymer films prepared from different copolymer solution concentrations, ranging from 0.2 to 10 mg·mL−1, to select the optimal culture substrate that allowed for good cell growth at 37 °C and effective cell detachment through temperature reduction. Furthermore, the stability and reusability of the optimal copolymer films were assessed. Finally, AFM and X-ray photoelectron spectroscopy (XPS) were employed to examine the surface morphology and elemental composition of the copolymer films after two rounds of BMMSC adhesion and detachment. The findings revealed that the surface properties and overall characteristics of PNIPAM copolymer films varied significantly with the solution concentration. Based on the selection criteria, the copolymer films derived from 1 mg·mL−1 solution were identified as the optimal culture substrates for BMMSCs. After two rounds of cellular adhesion and detachment, some proteins remained on the film surfaces, acting as a foundation for subsequent cellular re-adhesion and growth, thereby implicitly corroborating the practicability and reusability of the copolymer films. This study not only introduces a stable and efficient platform for stem cell culture and harvesting but also represents a significant advance in the fabrication of smart materials tailored for biomedical applications.

Periodontal bone regeneration using bone marrow mesenchymal stem cell (BMMSC) transplantation is a promising method; however, the method for osteogenic differentiation of BMMSCs needs to be improved. In this research, we sought to identify the roles of let‐7a in the osteogenesis of BMMSCs and to provide a potential method for periodontal bone regeneration. Our previous study revealed that Fas/FasL is a target of let‐7a. In this study, we demonstrated that let‐7a overexpression significantly enhanced BMMSC‐CAs osteogenesis both in vitro and in vivo. Mechanistically, upregulation of Fas/FasL using the rfas/rfaslg plasmid obstructed the osteogenesis of BMMSCs by inhibiting autophagy. Furthermore, we confirmed that overexpression of let‐7a activated autophagy and alleviated the inhibited osteogenesis by the autophagy inhibitor 3‐MA and the rfas/rfaslg plasmid of BMMSCs. In general, our findings showed that let‐7a promoted the osteogenesis of BMMSCs through the Fas/FasL‐autophagy pathway, suggesting that the application of let‐7a in BMMSC‐CAs based periodontal bone regeneration could be a promising strategy.