Osteogenic potency of dental stem cell-composite scaffolds in an animal cleft palate model.

Objective: To evaluate the osteogenic potency of stem cells isolated from human exfoliated deciduous teeth (SHED) in polycaprolactone with gelatin surface modification (PCL-GE) and poly (lactic-co-glycolic acid)-bioactive glass composite (PLGA-bioactive glass (BG)) scaffolds after implantation in a rat cleft model. Methods: Cleft palate-like lesions were induced in Sprague-Dawley rats by extracting the right maxillary first molars and drilling the intact alveolar bone. Rats were then divided into five groups: Control, PCL-GE, PCL-GE-SHED, PLGA-BG, and PLGA-BG-SHED, and received corresponding composite scaffolds with/without SHED at the extraction site. Tissue samples were collected at 2, 3, and 6 months post-implantation (4 rats per group). Gross and histological analyses were conducted to assess osteoid or bone formation. Immunohistochemistry for osteocalcin and human mitochondria was performed to evaluate bone components and human stem cell viability in the tissue. Results: Bone tissue formation was observed in the PCL-GE and PLGA-BG groups compared to the control, where no bone formation occurred. PLGA-BG scaffolds demonstrated greater bone regeneration potential than PCL-GE over 2-6 months. Additionally, scaffolds with SHED accelerated bone formation compared to scaffolds alone. Osteocalcin expression was detected in all rats, and positive immunoreactivity for human mitochondria was observed in the regenerated bone tissue with PCL-GE-SHED and PLGA-BG-SHED. Conclusion: PCL-GE and PLGA-BG composite scaffolds effectively repaired and regenerated bone tissue in rat cleft palate defects. Moreover, scaffolds supplemented with SHED exhibited enhanced osteogenic potency. Clinical significance: PCL-GE and PLGA-BG scaffolds, augmented with SHED, emerge as promising biomaterial candidates for addressing cleft repair and advancing bone tissue engineering endeavors.

Nanocrystal hydroxyapatite carrying traditional Chinese medicine for osteogenic differentiation.

Developing biomaterials with high osteogenic properties is crucial for achieving rapid bone repair and regeneration. This study focuses on the application of nanocrystal hydroxyapatite (nHAp) as a drug carrier to load Fu Yuan Huo Xue Decoction (FYHXD), a traditional Chinese medicine derived from Angelica sinensis, aiming to achieve improved efficacy in treating bone diseases such as osteoporosis. Through a facile physical adsorption approach, the FTIR result emerges new characteristic absorption peaks in the range of 1200-950 cm-1, proving the successful absorption of FYHXD onto the nHAp with a loading efficiency of 39.76 %. The modified nHAp exhibits a similar shape to the bone-derived hydroxyapatite nanocrystals, and their diameter increases slightly after modification. The drug release assay implies the rapid release of FYHXD in the first 10 h, followed by a continuously slow release within 70 h. The developed nHAp effectively enhances the adhesion, spreading, and proliferation of MC3T3-E1 cells in vitro, and significantly promotes their osteogenic differentiation, as indicated by increased alkaline phosphatase activity. Overall, the biocomposites hold great promise as active ingredients for integration into bone-associated biomaterials, offering the potential to stimulate spontaneous osteogenesis without requiring exogenous osteogenic factors.

Enhanced osteogenic differentiation in 3D hydrogel scaffold via macrophage mitochondrial transfer.

To assess the efficacy of a novel 3D biomimetic hydrogel scaffold with immunomodulatory properties in promoting fracture healing. Immunomodulatory scaffolds were used in cell experiments, osteotomy mice treatment, and single-cell transcriptomic sequencing. In vitro, fluorescence tracing examined macrophage mitochondrial transfer and osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). Scaffold efficacy was assessed through alkaline phosphatase (ALP), Alizarin Red S (ARS) staining, and in vivo experiments. The scaffold demonstrated excellent biocompatibility and antioxidant-immune regulation. Single-cell sequencing revealed a shift in macrophage distribution towards the M2 phenotype. In vitro experiments showed that macrophage mitochondria promoted BMSCs' osteogenic differentiation. In vivo experiments confirmed accelerated fracture healing. The GAD/Ag-pIO scaffold enhances osteogenic differentiation and fracture healing through immunomodulation and promotion of macrophage mitochondrial transfer.

CB1 Promotes Osteogenic Differentiation Potential of Periodontal Ligament Stem Cells by Enhancing Mitochondrial Transfer of Bone Marrow Mesenchymal Stem Cells.

OBJECTIVE: To reveal the role and mechanism of cannabinoid receptor 1 (CB1) and mitochondria in promoting osteogenic differentiation of periodontal ligament stem cells (PDLSCs) in the inflammatory microenvironment. METHODS: Bidirectional mitochondrial transfer was performed in bone mesenchymal stem cells (BMSCs) and PDLSCs. Laser confocal microscopy and quantitative flow cytometry were used to observe the mitochondrial transfer and quantitative mitochondrial transfer efficiency. Realtime reverse transcription polymerase chain reaction (RT-PCR) was employed to detect gene expression. Alkaline phosphatase (ALP) activity, alizarin red staining (ARS) and quantitative calcium ion analysis were used to evaluate the degree of osteogenic differentiation of PDLSCs. RESULTS: Bidirectional mitochondrial transfer was observed between BMSCs and PDLSCs. The indirect co-culture system could simulate intercellular mitochondrial transfer. Compared with the conditioned medium (CM) for BMSCs, that for HA-CB1 BMSCs could significantly enhance the mineralisation ability of PDLSCs. The mineralisation ability of PDLSCs could not be enhanced after removing the mitochondria in CM for HA-CB1 BMSCs. The expression level of HO-1, PGC-1α, NRF-1, ND1 and HK2 was significantly increased in HA-CB1 BMSCs. CONCLUSION: CM for HA-CB1 BMSCs could significantly enhance the damaged osteogenic differentiation ability of PDLSCs in the inflammatory microenvironment, and the mitochondria of CM played an important role. CB1 was related to the activation of the HO-1/PGC-1α/NRF-1 mitochondrial biogenesis pathway, and significantly increased the mitochondrial content in BMSCs.

Modulation of osteoblastogenesis by NRF2: NRF2 activation suppresses osteogenic differentiation and enhances mineralization in human bone marrow-derived mesenchymal stromal cells.

Mesenchymal stromal stem cells (MSCs) or skeletal stem cells (SSCs) play a major role in tissue repair due to their robust ability to differentiate into osteoblasts, chondrocytes, and adipocytes. Complex cell signaling cascades tightly regulate this differentiation. In osteogenic differentiation, Runt-related transcription factor 2 (RUNX2) and ALP activity are essential. Furthermore, during the latter stages of osteogenic differentiation, mineral formation mediated by the osteoblast occurs with the secretion of a collagenous extracellular matrix and calcium deposition. Activation of nuclear factor erythroid 2-related factor 2 (NRF2), an important transcription factor against oxidative stress, inhibits osteogenic differentiation and mineralization via modulation of RUNX2 function; however, the exact role of NRF2 in osteoblastogenesis remains unclear. Here, we demonstrate that NRF2 activation in human bone marrow-derived stromal cells (HBMSCs) suppressed osteogenic differentiation. NRF2 activation increased the expression of STRO-1 and KITLG (stem cell markers), indicating NRF2 protects HBMSCs stemness against osteogenic differentiation. In contrast, NRF2 activation enhanced mineralization, which is typically linked to osteogenic differentiation. We determined that these divergent results were due in part to the modulation of cellular calcium flux genes by NRF2 activation. The current findings demonstrate a dual role for NRF2 as a HBMSC maintenance factor as well as a central factor in mineralization, with implications therein for elucidation of bone formation and cellular Ca2+ kinetics, dystrophic calcification and, potentially, application in the modulation of bone formation.

Osteogenic and angiogenic potential of molybdenum-containing mesoporous bioactive glass nanoparticles: An ionic approach to bone tissue engineering.

Biomaterials intended for application in bone tissue engineering (BTE) ideally stimulate osteogenesis and angiogenesis simultaneously, as both mechanisms are of critical importance for successful bone regeneration. Mesoporous bioactive glass nanoparticles (MBGNs) can be tailored towards specific biological needs, for example by addition of ions like Molybdenum (Mo). While Mo has been shown to enhance osteogenic differentiation of human bone marrow-derived mesenchymal stromal cells (BMSCs) as well as their ability to form and mature a primitive osseous extracellular matrix (ECM), there are contradictory findings regarding its impact on angiogenesis. In this study, the effects of Mo-MBGNs (mol%: 70 SiO2, 25 CaO, 5 MoO3) on viability, proliferation, osteogenic differentiation, ECM formation and angiogenic response of BMSCs were compared to undoped MBGNs (in mol%: 70 SiO2, 30 CaO) and a control group of BMSCs. Furthermore, a human umbilical vein endothelial cells tube formation assay and a chorioallantoic membrane-assay using fertilized chicken eggs were used to analyze angiogenic properties. Mo-MBGNs were cytocompatible and promoted the proliferation of BMSCs. Furthermore, Mo-MBGNs showed promising osteogenic properties as they enhanced osteogenic differentiation, ECM formation and maturation as well as the gene expression and protein production of relevant osteogenic factors in BMSCs. However, despite the promising outcome on osteogenic properties, the addition of Mo to MBGNs resulted in anti-angiogenic effects. Due to the high relevance of vascularization in-vivo, the anti-angiogenic properties of Mo-MBGNs might hamper their osteogenic properties and therefore might restrict their performance in BTE applications. These limitations can be overcome by the addition of ions with distinct pro-angiogenic properties to the Mo-MBGNs-composition. Due to their promising osteogenic properties, Mo-MBGNs constitute a suitable basis for further research in the field of ionic (growth factor free) BTE.

Borate bioactive glass enhances 3D bioprinting precision and biocompatibility on a sodium alginate platform via Ca2+ controlled self-solidification.

Sodium alginate (SA) has gained widespread acclaim as a carrier medium for three-dimensional (3D) bioprinting of cells and a diverse array of bioactive substances, attributed to its remarkable biocompatibility and affordability. The conventional approach for fabricating alginate-based tissue engineering constructs entails a post-treatment phase employing a calcium ion solution. However, this method proves ineffectual in addressing the predicament of low precision during the 3D printing procedure and is unable to prevent issues such as non-uniform alginate gelation and substantial distortions. In this study, we introduced borate bioactive glass (BBG) into the SA matrix, capitalizing on the calcium ions released from the degradation of BBG to incite the cross-linking reaction within SA, resulting in the formation of BBG-SA hydrogels. Building upon this fundamental concept, it unveiled that BBG-SA hydrogels greatly enhance the precision of SA in extrusion-based 3D printing and significantly reduce volumetric contraction shrinkage post-printing, while also displaying certain adhesive properties and electrical conductivity. Furthermore, in vitro cellular experiments have unequivocally established the excellent biocompatibility of BBG-SA hydrogel and its capacity to actively stimulate osteogenic differentiation. Consequently, BBG-SA hydrogel emerges as a promising platform for 3D bioprinting, laying the foundation for the development of flexible, biocompatible electronic devices.

NOR1 promotes the osteoblastic differentiation of human periodontal ligament stem cells via TGF-ß signaling pathway.

Alveolar bone loss is a main manifestation of periodontitis. Human periodontal ligament stem cells (PDLSCs) are considered as optimal seed cells for alveolar bone regeneration due to its mesenchymal stem cell like properties. Osteogenic potential is the premise for PDLSCs to repair alveolar bone loss. However, the mechanism regulating osteogenic differentiation of PDLSCs remain elusive. In this study, we identified Neuron-derived orphan receptor 1 (NOR1), was particularly expressed in PDL tissue in vivo and gradually increased during osteogenic differentiation of PDLSCs in vitro. Knockdown of NOR1 in hPDLSCs inhibited their osteogenic potential while NOR1 overexpression reversed this effect. In order to elucidate the downstream regulatory network of NOR1, RNA-sequencing was used. We found that downregulated genes were mainly enriched in TGF-ß, Hippo, Wnt signaling pathway. Further, by western blot analysis, we verified that the expression level of phosphorylated-SMAD2/3 and phosphorylated-SMAD4 were all decreased after NOR1 knockdown. Additionally, ChIP-qPCR and dual luciferase reporter assay indicated that NOR1 could bind to the promoter of TGFBR1 and regulate its activity. Moreover, overexpression of TGFBR1 in PDLSCs could rescue the damaged osteogenic potential after NOR1 knockdown. Taken together, our results demonstrated that NOR1 could activate TGF-ß/SMAD signaling pathway and positively regulates the commitment of osteoblast lineages of PDLSCs by targeting TGFBR1 directly.

Zinc phosphate glass microspheres promoted mineralization and expression of BMP2 in MC3T3-E1 cells.

Degradable phosphate glasses have shown favorable properties for tissue engineering. By changing the composition of the glasses, the degradation rate, and ion release are controllable. Zinc oxide can function as a glass network modifier and has been shown to play a positive role in bone formation. Also, phosphate glasses can easily be processed into microspheres, which can be used as microcarriers. This study aims to develop zinc phosphate glasses microspheres and explore the optimized size and composition for applications in bone tissue engineering. Zinc-titanium-calcium-sodium phosphate glasses with 0, 1, 3, 5, or 10 mol % zinc oxide were prepared and processed into microspheres. The smaller microspheres ranged in size from 50 to 106 µm, while the larger ones ranged from 106 to 150 µm. The characteristics of glasses were examined. The osteoblastic cell line MC3T3-E1 was cultured on the surface of microspheres and the cell viability was examined. To evaluate osteogenic differentiation, Alizarin Red S staining, quantitative reverse transcription polymerase chain reaction, and western blot analysis were performed after 14 days. Different sizes of zinc phosphate glass microspheres were successfully made. The glass microspheres with <10 mol % zinc oxide were able to support the adhesion and proliferation of MC3T3-E1 cell lines. The relative gene expression of BMP2 was significantly upregulated in the smaller glass microspheres containing 3 mol % zinc oxide (26-fold, p < .001) and both sizes of microspheres containing 5 mol % zinc oxide (smaller: 27-fold, p < .001; larger: 35-fold, p < .001). Additionally, cluster formation was observed in glass microspheres after 14 days, and the mineralization of MC3T3-E1 cell lines was promoted. Based on these findings, the glass microspheres containing 3-5 mol % of zinc oxide can promote osteogenic differentiation for MC3T3-E1 cells.

Exosomal lncRNA HCP5 derived from human bone marrow mesenchymal stem cells improves chronic periodontitis by miR-24-3p/HO1/P38/ELK1 pathway.

Objective: The present study aimed to explore the function of human bone marrow mesenchymal stem cells (hBMMSCs)-derived exosomal long noncoding RNA histocompatibility leukocyte antigen complex P5 (HCP5) in the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) to improve chronic periodontitis (CP). Methods: Exosomes were extracted from hBMMSCs. Alizarin red S staining was used to detect mineralised nodules. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to measure HCP5 and miR-24-3p expression. The mRNA and protein levels of alkaline phosphatase (ALP), osteocalcin, osterix, runt-related transcription factor 2, bone morphogenetic protein 2, osteopontin, fibronectin, collagen 1, heme oxygenase 1 (HO1), P38, and ETS transcription factor ELK1 (ELK1) were detected using RT-qPCR and Western blot. Enzyme-linked immunosorbent assay (ELISA) kits were used to determine the HO1 and carbon monoxide concentrations. Heme, biliverdin, and Fe2+ levels were determined using detection kits. Micro-computed tomography, hematoxylin and eosin staining, ALP staining, tartrate-resistant acid phosphatase staining, ELISA, and RT-qPCR were conducted to evaluate the effect of HCP5 on CP mice. Dual luciferase, RNA immunoprecipitation, and RNA pulldown experiments were performed to identify the interactions among HCP5, miR-24-3p, and HO1. Results: The osteogenic ability of hPDLSCs significantly increased when co-cultured with hBMMSCs or hBMMSCs exosomes. Overexpression of HCP5 and HO1 in hBMMSCs exosomes promoted the osteogenic differentiation of hPDLSCs, and knockdown of HCP5 repressed the osteogenic differentiation of hPDLSCs. HCP5 knockdown enhanced the inflammatory response and repressed osteogenesis in CP mice. MiR-24-3p overexpression diminished the stimulatory effect of HCP5 on the osteogenic ability of hPDLSCs. Mechanistically, HCP5 acted as a sponge for miR-24-3p and regulated HO1 expression, and HO1 activated the P38/ELK1 pathway. Conclusion: HBMMSCs-derived exosomal HCP5 promotes the osteogenic differentiation of hPDLSCs and alleviates CP by regulating the miR-24-3p/HO1/P38/ELK1 signalling pathway.

Bioinformatics analysis and validation of RNA methylation-related genes in osteogenic and adipogenic differentiation of rat bone marrow mesenchymal stem cells.

BACKGROUND: The regulatory mechanisms of RNA methylation during the processes of osteogenic and adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) have yet to be fully understood. The objective of our study was to analyze and validate the contribution of RNA methylation regulators to the mechanisms underlying the osteogenic and adipogenic differentiation of rat BMSCs. METHODS: We downloaded the GSE186026 from the Gene Expression Omnibus (GEO). Differentially expressed genes (DEGs) were screened using the DESeq2 package in R software (version 3.6.3). A total of 50 RNA methylation genes obtained from literature review and summary were intersected with the previous DEGs to obtain RNA methylation genes, which have different expressions (RM-DEGs). Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were utilized to reveal the functional enrichment. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to validate RM-DEGs. Protein-protein interaction network (PPI) analysis and visual analysis were performed using STRING and Cytoscape. RM-DEGs regulatory network was constructed to analyze the top 10 hub genes. The relationship between RM-DEGs, some enriched GO and pathways was also been analyzed. The miRNAs and RM-DEGs regulatory networks were established by using miRWalk and TargetScan. RESULTS: As part of our research, we detected varying levels of expression for m6A regulators Mettl3 and Rbm15, as well as m7G regulators Mettl1 and Wdr4, in relation to osteogenic differentiation, along with m6A regulator Fmr1 in adipogenic differentiation. The protein-protein interaction (PPI) networks were constructed for 49 differentially expressed genes (DEGs) related to RNA methylation during the process of osteogenic differentiation, and 13 DEGs for adipogenic differentiation. Moreover, top10 hub genes were calculated. In osteogenic differentiation, Mettl3 regulated the Wnt pathway and Hippo pathway by regulating Smad3, Rbm15 regulated the Notch pathway by Notch1, Mettl1 regulated the PI3K-Akt pathway by Gnb4. In adipogenic differentiation, Fmr1 regulated the PI3K-Akt pathway by Egfr. M6A methylation sites of Smad3, Notch1 and Gnb4 were predicted, and the results showed that all three genes were possibly methylated by m6A, and more than 9 sites per gene were possibly methylated. Finally, we constructed the regulatory networks of Mettl3, Rbm15, Mettl1, and Wdr4 and 109 miRNAs in osteogenic differentiation, Fmr1 and 118 miRNAs in adipogenic differentiation. CONCLUSIONS: Mettl3(m6A), Rbm15(m6A), Wdr4 and Mettl1(m7G) were differentially expressed in osteogenic differentiation, while Fmr1(m6A) was differentially expressed in adipogenic differentiation. These findings offered potential candidates for further research on the involvement of RNA methylation in the osteogenic and adipogenic differentiation of BMSCs.

Qianggu concentrate: unlocking bone protection power via antioxidative SIRT1/NRF2/HO-1 pathways in type 2 diabetic osteoporosis.

Background: Qianggu Concentrate (QGHJ), a traditional Chinese medicine, is extensively used to treat Type 2 Diabetic Osteoporosis (T2DOP). Despite its widespread use, research on its therapeutic mechanisms within T2DOP is notably scarce. Objective: To explore QGHJ's osteoprotection in T2DOP rats and BMSCs, focusing on the antioxidant activation of SIRT1/NRF2/HO-1 and NRF2 nuclear migration. Methods: QGHJ constituent analysis was performed using UPLC-HRMS. Safety, bone-health efficacy, and glucose metabolic effects in T2DOP rats were evaluated via general condition assessments, biomarker profiling, micro-CT, biomechanics, staining methods, and ELISA, supplemented by RT-qPCR and Western blot. BMSCs' responses to QGHJ under oxidative stress, including viability, apoptosis, and osteogenic differentiation, were determined using CCK-8, flow cytometry, ALP/ARS staining, and molecular techniques. The modulation of the SIRT1/NRF2/HO-1 pathway by QGHJ was explored through oxidative stress biomarkers, immunofluorescence, and Western blot assays. Results: UPLC-HRMS identified flavonoids, monoterpenes, and isoflavones as QGHJ's key compounds. In vivo, QGHJ proved safe and effective for T2DOP rats, enhancing bone mineral density, microenvironment, and biomechanical properties without impairing vital organs. It modulated bone markers PINP, TRACP 5b, RUNX2 and PPARγ, favoring bone anabolism and reduced catabolism, thus optimizing bone integrity. QGHJ also regulated glycemia and mitigated insulin resistance. In vitro, it preserved BMSCs' viability amidst oxidative stress, curbed apoptosis, and fostered osteogenesis with regulated RUNX2/PPARγ expression. Mechanistic insights revealed QGHJ activated the SIRT1/NRF2/HO-1 pathway, augmented NRF2 nuclear translocation, and enhanced the antioxidative response, promoting bone health under stress. Conclusion: In T2DOP rat and BMSCs oxidative stress models, QGHJ's bone protection is anchored in its antioxidative mechanisms via the SIRT1/NRF2/HO-1 pathway activation and NRF2 nuclear translocation.

Nocardamine mitigates cellular dysfunction induced by oxidative stress in periodontal ligament stem cells.

BACKGROUND: The role of periodontal ligament stem cells (PDLSCs) in repairing periodontal destruction is crucial, but their functions can be impaired by excessive oxidative stress (OS). Nocardamine (NOCA), a cyclic siderophore, has been shown to possess anti-cancer and anti-bacterial properties. This study aimed to investigate the protective mechanisms of NOCA against OS-induced cellular dysfunction in PDLSCs. METHODS: The cytotoxicity of NOCA on PDLSCs was assessed using a CCK-8 assay. PDLSCs were then treated with hydrogen peroxide (H2O2) to induce OS. ROS levels, cell viability, and antioxidant factor expression were analyzed using relevant kits after treatment. Small molecule inhibitors U0126 and XAV-939 were employed to block ERK signaling and Wnt pathways respectively. Osteogenic differentiation was assessed using alkaline phosphatase (ALP) activity staining and Alizarin Red S (ARS) staining of mineralized nodules. Expression levels of osteogenic gene markers and ERK pathway were determined via real-time quantitative polymerase chain reaction (RT-qPCR) or western blot (WB) analysis. ß-catenin nuclear localization was examined by western blotting and confocal microscopy. RESULTS: NOCA exhibited no significant cytotoxicity at concentrations below 20 µM and effectively inhibited H2O2-induced OS in PDLSCs. NOCA also restored ALP activity, mineralized nodule formation, and the expression of osteogenic markers in H2O2-stimulated PDLSCs. Mechanistically, NOCA increased p-ERK level and promoted ß-catenin translocation into the nucleus; however, blocking ERK pathway disrupted the osteogenic protection provided by NOCA and impaired its ability to induce ß-catenin nuclear translocation under OS conditions in PDLSCs. CONCLUSIONS: NOCA protected PDLSCs against H2O2-induced OS and effectively restored impaired osteogenic differentiation in PDLSCs by modulating the ERK/Wnt signaling pathway.

Long non-coding TRPM2-AS regulates fracture healing by targeting miR-545-3p/Bmp2.

OBJECTIVE: Delayed fracture healing increases the suffering of patients. An in-depth investigation of the pathogenesis of delayed fracture healing may offer new direction for the prevention and treatment. METHODS: The study included 63 normal healing tibial fractures and 58 delayed healing tibial fractures patients. Long non-coding RNA (lncRNA)TRPM2-AS, microRNA-545-3p (miR-545-3p), bone morphogenetic protein 2 (Bmp2) mRNA and osteogenic differentiation markers, including runt-related transcription factor 2 (Runx2), osteocalcin (Ocn), and alkaline phosphatase (Alp) mRNA expression were determined by Real-time quantitative reverse transcription-polymerase chain reaction in serum and MC3T3-E1 cells. The prediction potential of TRPM2-AS in delayed healing fracture patients was verified by receiver operating characteristic curves. The binding relationship of TRPM2-AS/miR-545-3p/Bmp2 was evaluated by dual luciferase reporter gene assay. Cell proliferation and apoptosis were detected by CCK-8 and flow cytometry. RESULTS: TRPM2-AS was remarkably down-regulated in patients with delayed fracture healing and could better predict the fracture healing status. TRPM2-AS downregulation inhibited osteogenic markers mRNA expression, restrained proliferation, and promoted apoptosis of MC3T3-E1 cells (p < 0.05). In delayed fracture healing, miR-545-3p was dramatically up-regulated and was negatively regulated by TRPM2-AS. Reducing miR-545-3p eliminate the negative effect of TRPM2-AS down-regulation on osteoblast proliferation and differentiation (p < 0.05). miR-545-3p targets Bmp2, which plays a positive role in osteoblast differentiation (p < 0.05). CONCLUSION: This study found that TRPM2-AS has the potential to be a diagnostic marker for delayed fracture healing and revealed that the TRPM2-AS/miR-545-3p/Bmp2 axis affects fracture healing by regulating osteoblast.

F-actin microfilaments affect the LIPUS-promoted osteogenic differentiation of BMSCs through TRPM7.

The differentiation of bone marrow mesenchymal stem cells (BMSCs) toward osteogenesis can be induced by low-intensity pulsed ultrasound (LIPUS). However, the molecular mechanisms responsible for LIPUS stimulation are unclear. The possible molecular mechanisms by which LIPUS promotes osteogenic differentiation of BMSCs were investigated in this study. The quantification of alkaline phosphatase (ALP) activity, Alizarin Red S staining, ALP staining, and the establishment of a calvarial defect model were used to evaluate osteogenic effects. Immunofluorescence was performed to observe the expression of microfilaments and transient receptor potential melastatin 7 (TRPM7). The levels of F-actin/G-actin and osteogenesis-related proteins under LIPUS alone or LIPUS combined with cytoskeleton interfering drugs (Cytochalasin D [CytoD] or Jasplakinolide [JA]) were assayed by western blot. Quantitative real-time reverse transcription polymerase chain reaction was utilized to measure the expression of Trpm7 mRNA. Moreover, adenoviral Trpm7 knockdown was verified using western blot. The results demonstrated that LIPUS promoted bone formation in vivo. Under osteogenic induction in vitro, the osteogenesis of BMSCs induced by LIPUS was accompanied by the depolymerization and rearrangement of microfilaments and increased levels of TRPM7. By perturbing intracellular actin dynamics, CytoD enhanced the pro-osteogenicity of LIPUS and increased TRPM7 level, while JA inhibited the pro-osteogenicity of LIPUS and reduced TRPM7 level. Additionally, the knockdown of Trpm7 suppressed the osteogenic promotion of BMSCs induced by LIPUS. The transient depolymerization and rearrangement of the cytoskeleton microfilaments mediated by LIPUS can affect TRPM7 expression and subsequently promote the osteogenesis of BMSCs. This study provides further direction for exploring the molecular mechanism of LIPUS, as a mechanical stress, in facilitating the osteogenic differentiation of BMSCs.

Electrospun polycaprolactone-chitosan nanofibers on a zinc mesh as biodegradable guided bone-regeneration membranes with enhanced mechanical, antibacterial, and osteogenic properties for alveolar bone-repair applications.

Guided bone-regeneration membrane (GBRM) is commonly used in bone-repair surgery because it blocks fibroblast proliferation and provides spatial support in bone-defect spaces. However, the need for removal surgery and the lack of antibacterial properties of conventional GBRM limit its therapeutic applicability for alveolar bone defects. Here we developed a GBRM for alveolar bone-repair and -regeneration applications through double-sided electrospinning of polycaprolactone and chitosan layers on a Zn mesh surface (denoted DSZM). The DSZM showed a UTS of ∼25.6 MPa, elongation of ∼16.1%, strength-elongation product of ∼0.413 GPa%, and ultrahigh spatial maintenance ability, and the UTS was over 6 times higher than that of commercial Bio-Gide membrane. The DSZM exhibited a corrosion rate of ∼17 µm/y and a Zn ion concentration of ∼0.23 µg/ml after 1 month of immersion in Hanks' solution. The DSZM showed direct and indirect cytocompatibility with exceptional osteogenic differentiation and calcium deposition toward MC3T3-E1 cells. Further, the DSZM showed strongly sustained antibacterial activity against S. aureus and osteogenesis in a rat critical-sized maxillary defect model. Overall, the DSZM fits the requirements for alveolar bone-repair and -regeneration applications as a biodegradable GBRM material due to its spatial support, suitable degradability, cytocompatibility, and antibacterial and osteogenic capabilities. STATEMENT OF SIGNIFICANCE: This work reports the mechanical properties, antibacterial ability and osteogenic properties of electrospun PCL-CS nanofiber on Zn mesh as biodegradable guided bone-regeneration membrane for alveolar bone-repair applications. Our findings demonstrate that the DSZM prepared by double-sided electrospinning of PCL-CS layers on Zn mesh showed a UTS of ∼25.6 MPa, elongation of ∼16.1%, strength-elongation product of ∼0.413 GPa%, and ultrahigh spatial maintenance ability, and the UTS was over 6 times greater than that of commercial Bio-Gide® membrane. The DSZM showed direct and indirect cytocompatibility with exceptional osteogenic differentiation and calcium deposition toward MC3T3-E1 cells. Further, the DSZM showed strongly sustained antibacterial activity against S. aureus and osteogenesis in a rat critical-sized maxillary defect model.

Osteoclast-derived exosomes influence osteoblast differentiation in osteoporosis progression via the lncRNA AW011738/ miR-24-2-5p/ TREM1 axis.

AIMS: To investigate the molecular mechanism of osteoclast-derived exosomes in osteoporosis. MAIN METHODS: RANKL induced osteoclast model was screened for significantly differentially expressed lncRNAs and mRNAs by whole RNA sequencing. Exosomes were characterized using electron microscopy, western blotting and nanosight. Overexpression or knockdown of AW011738 was performed to explore its function. The degree of osteoporosis in an osteoporosis model was assessed by mirco-CT. The osteoclast model, osteoblast differentiation ability and the molecular mechanism of lncRNA AW011738/miR-24-2-5p/TREM1 axis in osteoporosis were assessed by dual luciferase reporter gene assay, Western blotting (WB), immunofluorescence and ALP staining. Bioinformatics was used to predict interactions of key osteoporosis-related genes with miRNAs, transcription factors, and potential drugs after upregulation of AW011738. To predict the protein-protein interaction (PPI) network associated with key genes, GO and KEGG analyses were performed on the key genes. The ssGSVA was used to predict changes in the immune microenvironment. KEY FINDINGS: Osteoclast-derived exosomes containing lncRNA AW011738 decreased the osteogenesis-related markers and accelerated bone loss in OVX mice. Osteoclast (si-AW011738)-derived exosomes showed a significant increase in biomarkers of osteoblast differentiation in vitro compared to the si-NC group. As analyzed by mirco-CT, tail vein injected si-AW011738 OVX mice were less osteoporotic than the control group. AW011738 inhibited osteoblast differentiation by regulating TREM1 expression through microRNA. Meanwhile, overexpression of miR-24-2-5p inhibited TREM1 expression to promote osteoblast differentiation. SIGNIFICANCE: Osteoclast-derived exosomes containing lncRNA AW011738 inhibit osteogenesis in MC3T3-E1 cells through the lncRNA AW011738/miR-24-2-5p/TREM1 axis and exacerbate osteoporosis in OVX mice.

Construction of a Titanium-Magnesium Composite Internal Fixation System for Repairing Bone Defects.

The repair and regeneration of maxillofacial bone defects are major clinical challenges. Titanium (Ti)-magnesium (Mg) composites are a new generation of revolutionary internal fixation materials encompassing the mechanical strength and bioactive advantages of Ti and Mg alloys, respectively. This study was aimed to construct a Ti-Mg composite internal plate/screw fixation system to fix and repair bone defects. Further, the effects of different internal fixation systems on bone repair were analyzed through radiological and histological analyses. Notably, Ti6Al4V with rolled Mg foil was used as the experimental group, and a bone defect model of transverse complete amputation of the ulna in rabbits similar to the clinical condition was established. The internal fixation system with the highest osteogenic efficiency was selected based on in vivo results, and the direct and indirect bone repair abilities of the selected materials were evaluated in vitro. Notably, the thin Mg foil-Ti6Al4V internal fixation system exhibited the best fixation effect in the bone defect model and promoted the formation of new bone and early healing of bone defect areas. In vitro, the thin Mg foil-Ti6Al4V composite enhanced the activity of MC3T3-E1 cells; promoted the proliferation, adhesion, extension, and osteogenic differentiation of MC3T3-E1 cells; and regulated new bone formation. Further, it also promoted the polarization of RAW264.7 cells to M2 macrophages, induced the osteogenic immune microenvironment, and indirectly regulated the bone repair process. Therefore, a internal fixation system holds a promising potential for the internal fixation of maxillofacial bone defects. Our findings provide a theoretical and scientific basis for the design and clinical application of Ti-Mg internal fixation systems.

Diagnostic significance of LncRNA MIAT in periodontitis and the molecular mechanisms influencing periodontal ligament fibroblasts via the miR-204-5p/DKK1 axis.

OBJECTIVE: This study investigated the clinical importance of long noncoding RNA myocardial infarction-associated transcript (MIAT) in periodontitis and its impact on the functional regulation of human periodontal ligament fibroblasts (hPDLFs). METHODS: Ninety-eight periodontitis patients and 74 healthy controls were enrolled. In vitro cellular models were created using Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) to stimulate hPDLFs. Real-time quantitative polymerase chain reaction was used to measure mRNA levels of MIAT and osteogenic factors. Inflammation factor concentration was assessed using an enzyme-linked immunosorbent assay. Cell viability and apoptosis were examined by cell counting kit -8 and flow cytometry assay. The targeting relationship was verified by the dual-luciferase reporter and RNA Immunoprecipitation assay. RESULTS: Highly expressed MIAT and Dicckopf-1 (DDK1), and lowly expressed miR-204-5p were found in the gingival crevicular fluid of periodontitis patients and Pg-LPS induced hPDLFs. MIAT has a sensitivity of 76.53 % and a specificity of 86.49 % for identifying patients with periodontitis among healthy individuals. MIAT acts as a sponge for miR-204-5p and upregulates DDK1 mRNA expression. Silencing of MIAT diminished the promotion of apoptosis and inflammation in hPDLFs by Pg-LPS and enhanced osteogenic differentiation. However, a miR-204-5p inhibitor significantly reversed the effect of silenced MIAT. CONCLUSIONS: MIAT may act as a promising biomarker for periodontitis. It modulates apoptosis, inflammation, and osteogenic differentiation of PDLFs by focusing on the miR-204-5p/DKK1 axis, indicating its potential as a new therapeutic target for treating periodontitis.

Baicalin Plays an Anti-Osteosarcoma Role in Vitro and Promotes Osteogenic Differentiation by Inhibiting NF-κB Signaling.

BACKGROUND: Osteosarcoma (OS) is commonly recognized as a malignant cancer originating from bone-forming mesenchymal stem cells, comprising approximately 20% of sarcomas. Baicalin, a bioactive flavonoid glycoside isolated from Scutellaria baicalensis, has been demonstrated to possess potent anti-inflammatory and neuroprotective properties. OBJECTIVE: To explore the potential mechanisms through which baicalin exerts anti-osteosarcoma effects and facilitates osteogenesis in vitro. METHODS: Cell Counting Kit-8 (CCK-8), scratch assay, and transwell assay were employed to assess the effects of baicalin at varying concentrations (20, 40, and 80 µM) on U2OS cell proliferation, invasion, and migration, respectively. Western blot and qRT-PCR analyses were conducted to evaluate the influence of baicalin on the osteogenic potential of OS cells by examining osteoblast markers such as osteocalcin (OCN), osteopontin (OPN), and runt-related transcription factor 2 (RUNX2), as well as the osteoclast marker-receptor activator of nuclear factor kappa B ligand (RANKL). Additionally, the impact of baicalin on epithelial-mesenchymal transition (EMT) markers (N-cadherin, E-cadherin, Vimentin) and proteins related to the Nuclear factor κB (NF-κB) signaling pathway (p-p65, p-IκBα, p65, IκBα) in OS cells was evaluated via western blot analysis. The activity and mineralization capacity of Alkaline Phosphatase (ALP) in baicalin-treated cells were examined through ALP staining and Alizarin Red S (ARS) staining. RESULTS: Baicalin exhibited significant suppression of OS cell U2OS invasion (p < 0.01), migration (p < 0.01), and proliferation (p < 0.05) at various concentrations. Additionally, baicalin treatment notably increased the E-cadherin protein level, while decreasing the expression levels of Vimentin and N-cadherin proteins (p < 0.01), thus promoting EMT. Following baicalin treatment, there was a marked elevation in the protein and mRNA expression levels of RUNX2, OPN, and OCN, while the expression level of RANKL protein was reduced (p < 0.05), indicating enhanced osteogenic differentiation. The groups treated with baicalin exhibited higher ALP activity and mineralization ability (p < 0.01). Moreover, baicalin treatment significantly reduced the expression levels of p-IκBα and p-p65 proteins, as well as the ratios of p-IκBα/IκBα and p-p65/p65 (p < 0.01). These effects of baicalin were concentration-dependent, with higher concentrations yielding stronger effects. CONCLUSION: In vitro, baicalin demonstrates anti-OS effects and facilitates osteogenic differentiation, potentially by inhibiting NF-κB pathway activity.