Osteogenesis-related Gene Expression Research Articles

ANXA2 promotes osteogenic differentiation and inhibits cellular senescence of periodontal ligament cells (PDLCs) in high glucose conditions

Background Periodontal ligament cells (PDLCs) are a major component of the periodontal ligament and have an important role in the regeneration of periodontal tissue and maintenance of homeostasis. High glucose can affect the activity and function of PDLCs in a variety of ways; therefore, it is particularly important to find ways to alleviate the effects of high glucose on PDLCs. Annexin A2 (ANXA2) is a calcium- and phospholipid-binding protein involved in a variety of cellular functions and processes, including cellular cytokinesis, cytophagy, migration, and proliferation. Aim The aim of this study was to exploring whether ANXA2 attenuates the deleterious effects of high glucose on PDLCs and promotes osteogenic differentiation capacity. Methods and results Osteogenic differentiation potential, cellular senescence, oxidative stress, and cellular autophagy were detected. Culturing PDLCs with medium containing different glucose concentrations (CTRL, 8 mM, 10 mM, 25 mM, and 40 mM) revealed that high glucose decreased the protein expression of ANXA2 (p < 0.0001). In addition, high glucose decreased the osteogenic differentiation potential of PDLCs as evidenced by decreased calcium deposition (p = 0.0003), lowered ALP activity (p = 0.0010), and a decline in the expression of osteogenesis-related genes (p = 0.0008). Moreover, β-Galactosidase staining and expression of p16, p21 and p53 genes showed that it increased cellular senescence in PDLCs (p < 0.0001). Meanwhile high glucose increased oxidative stress in PDLCs as shown by ROS (p < 0.0001). However, these damages caused by high glucose were inhibited after the addition of 1 µM recombinant ANXA2 (rANXA2), and we found that rANXA2 enhanced autophagy in PDLCs under high glucose conditions. Conclusions and discussion Therefore, our present study demonstrates that alterations in ANXA2 under high glucose conditions may be a factor in the decreased osteogenic differentiation potential of PDLCs. Meanwhile, ANXA2 is associated with autophagy, oxidative stress, and cellular senescence under high glucose conditions.

Si and Zn dual ions upregulate the osteogenic differentiation of mBMSCs: mRNA transcriptomic sequencing analysis

Both silicon (Si) and zinc (Zn) ions are essential elements to bone health and their mechanisms for promoting osteogenesis have aroused the extensive attention of researchers. Thereinto, the mechanism by which dual ions promote osteogenic differentiation remains to be elucidated. Herein, the effects of Si and Zn ions on the cytological behaviors of mBMSCs were firstly studied. Then, the molecular mechanism of Si-Zn dual ions regulating the osteogenic differentiation of mBMSCs was investigated via transcriptome sequencing technology. In the single-ion system, Si ion at the concentration of 1.5 mM (Si-1.5) had better comprehensive effects of cell proliferation, ALP activity and osteogenesis-related gene expression levels (ALP, Runx2, OCN, Col-I and BSP); Zn ion at the concentration of 50 μM (Zn-50) demonstrated better combining effects of cell proliferation, ALP activity and same osteogenic genes expression levels. In the dual-ion system, the Si (1.5 mM)-Zn (50 μM) group (Si1.5-Zn50) synthetically enhanced ALP activity and osteogenesis genes compared with single-ion groups. Analysis of the transcriptome sequencing results showed that Si ion had a certain effect on promoting the osteogenic differentiation of mBMSCs; Zn ion had a stronger effect of contributing to a better osteogenic differentiation of mBMSCs than that of Si ion; the Si-Zn dual ions had a synergistic enhancement on conducting to the osteogenic differentiation of mBMSCs compared to single ion (Si or Zn). This study offers a blueprint for exploring the regulation mechanism of osteogenic differentiation by dual ions.Graphical

Modified implant with dual functions of antioxidant and extracellular matrix reconstruction for regulating MSCs senescence

Bone repair in elderly patients is significantly weaker compared to normal bone repair, and one of the main reasons for this is the senescence of bone marrow mesenchymal stem cells (MSCs), which play a key role in the bone repair process. Therefore, we considered the corresponding surface modification of orthopedic implants to cope with the practical problem of bone defects in elderly patients. In this thesis, we prepared TiO2 nanotubes loaded with metformin on Ti substrates, followed by self-assembly layer by layer using chitosan-catechol and gelatin on the surface, to delay MSCs senescence by eliminating excessive reactive oxygen species (ROS) and reconstructing extracellular matrix (ECM), thereby achieving osteogenesis. The results showed a significant reduction in senescence-associated secretory phenotype (SASP) generation, an enhancement of PINK1/Parkin-mediated mitochondrial autophagy, and a decrease in intracellular and extracellular ROS levels in MSCs under the dual effects of antioxidants and ECM reconstruction, suggesting that the degree of senescence in MSCs was significantly reduced. Osteogenesis was also demonstrated by expression of osteogenesis-related genes and staining of animal sections. In conclusion, our material can indeed promote the proliferation and osteogenic differentiation of MSCs by delaying cellular senescence, which is expected to provide a novel approach for clinical solutions to tissue repair problems in elderly patients with bone defects.

Enhancing bone regeneration: Unleashing the potential of magnetic nanoparticles in a microtissue model.

Bone tissue engineering addresses the limitations of autologous resources and the risk of allograft disease transmission in bone diseases. In this regard, engineered three-dimensional (3D) models emerge as biomimetic alternatives to natural tissues, replicating intracellular communication. Moreover, the unique properties of super-paramagnetic iron oxide nanoparticles (SPIONs) were shown to promote bone regeneration via enhanced osteogenesis and angiogenesis in bone models. This study aimed to investigate the effects of SPION on both osteogenesis and angiogenesis and characterized a co-culture of Human umbilical vein endothelial cells (HUVEC) and MG-63 cells as a model of bone microtissue. HUVECs: MG-63s with a ratio of 4:1 demonstrated the best results among other cell ratios, and 50 μg/mL of SPION was the optimum concentration for maximum survival, cell migration and mineralization. In addition, the data from gene expression illustrated that the expression of osteogenesis-related genes, including osteopontin, osteocalcin, alkaline phosphatase, and collagen-I, as well as the expression of the angiogenesis-related marker, CD-31, and the tube formation, is significantly elevated when the 50 μg/mL concentration of SPION is applied to the microtissue samples. SPION application in a designed 3D bone microtissue model involving a co-culture of osteoblast and endothelial cells resulted in increased expression of specific markers related to angiogenesis and osteogenesis. This includes the design of a novel biomimetic model to boost blood compatibility and biocompatibility of primary materials while promoting osteogenic activity in microtissue bone models. Moreover, this can improve interaction with surrounding tissues and broaden the knowledge to promote superior-performance implants, preventing device failure.

Advanced biomimetic design strategies for porous structures promoting bone integration with additive-manufactured Ti6Al4V scaffolds

The natural bone structure exhibits a radial gradient with dense cortical bone externally and porous cancellous bone internally. However, previous studies proposing scaffold designs have predominantly focused on homogeneous porous structures. Moreover, no consensus exists on the optimal structure for gradient porous scaffolds. Our scaffold closely imitated the natural bone structure by incorporating a gradient of pillar diameters. Additionally, we introduced a inverse gradient structure and three uniform diameter pillar structures (400 μm, 600 μm, and 1000 μm) for comparative analysis. Mechanical testing revealed that the compressive strength and elastic modulus of the Ti6Al4V porous scaffolds gradually increased with an increase in pillar diameter. In vitro experiments demonstrated that both the biomimetic gradient and inverse gradient scaffolds promoted osteogenic differentiation, with higher ALP activity (alkaline phosphatase) and osteogenesis-related gene expression compared to the uniform pillar structure scaffolds. The in vivo experiments confirmed these results, highlighting the superior ability of the biomimetic gradient Ti6Al4V porous scaffold to induce new bone formation. Based on our findings, we suggest that the optimal porous structure should have fine-diameter pillars (approximately 400 μm) internally to enhance cell penetration, while coarse-diameter pillars (approximately 800 μm) should be used externally to increase the cell attachment area and enhance mechanical strength. Overall, our study contributes to the field of bone tissue engineering by providing a biomimetic scaffold design that closely imitates the structure of natural bone and promotes osteogenic activity.

Fibrin-konjac glucomannan-black phosphorus hydrogel scaffolds loaded with nasal ectodermal mesenchymal stem cells accelerated alveolar bone regeneration

BackgroundEffective treatments for the alveolar bone defect remain a major concern in dental therapy. The objectives of this study were to develop a fibrin and konjac glucomannan (KGM) composite hydrogel as scaffolds for the osteogenesis of nasal mucosa-derived ectodermal mesenchymal stem cells (EMSCs) for the regeneration of alveolar bone defect, and to investigate the osteogenesis-accelerating effects of black phosphorus nanoparticles (BPNs) embedded in the hydrogels.MethodsPrimary EMSCs were isolated from rat nasal mucosa and used for the alveolar bone recovery. Fibrin and KGM were prepared in different ratios for osteomimetic hydrogel scaffolds, and the optimal ratio was determined by mechanical properties and biocompatibility analysis. Then, the optimal hydrogels were integrated with BPNs to obtain BPNs/fibrin-KGM hydrogels, and the effects on osteogenic EMSCs in vitro were evaluated. To explore the osteogenesis-enhancing effects of hydrogels in vivo, the BPNs/fibrin-KGM scaffolds combined with EMSCs were implanted to a rat model of alveolar bone defect. Micro-computed tomography (CT), histological examination, real-time quantitative polymerase chain reaction (RT-qPCR) and western blot were conducted to evaluate the bone morphology and expression of osteogenesis-related genes of the bone regeneration.ResultsThe addition of KGM improved the mechanical properties and biodegradation characteristics of the fibrin hydrogels. In vitro, the BPNs-containing compound hydrogel was proved to be biocompatible and capable of enhancing the osteogenesis of EMSCs by upregulating the mineralization and the activity of alkaline phosphatase. In vivo, the micro-CT analysis and histological evaluation demonstrated that rats implanted EMSCs-BPNs/fibrin-KGM hydrogels exhibited the best bone reconstruction. And compared to the model group, the expression of osteogenesis genes including osteopontin (Opn, p < 0.0001), osteocalcin (Ocn, p < 0.0001), type collagen (Col , p < 0.0001), bone morphogenetic protein-2 (Bmp2, p < 0.0001), Smad1 (p = 0.0006), and runt-related transcription factor 2 (Runx2, p < 0.0001) were all significantly upregulated.ConclusionsEMSCs/BPNs-containing fibrin-KGM hydrogels accelerated the recovery of the alveolar bone defect in rats by effectively up-regulating the expression of osteogenesis-related genes, promoting the formation and mineralisation of bone matrix.

Black Phosphorus and E7-Functionalized Sulfonated Polyetheretherketone with Effective Osteogenicity and Antibacterial Activity

Given its excellent biological properties and the matching of its elastic modulus with that of human bone tissue, medical polyetheretherketone (PEEK) is considered a desirable candidate for bone-implant materials. However, its poor osseointegrative and antibacterial properties greatly limit its clinical application. To address these concerns, a functional PEEK implant is needed. Herein, a novel photo-responsive multifunctional PEEK-based implant material (sPEEK/BP/E7) with both effective osteogenesis and good disinfection properties was constructed via the self-assembly of black phosphorus (BP) nanosheets, mussel-inspired polydopamine (PDA), and bioactive short peptide E7 on sulfonated PEEK (sPEEK). The versatile micro-/nano-structured PEEK surface provides superior hydrophilicity, a favorable osteogenic microenvironment, and excellent photothermal effects under near-infrared (NIR) irradiation. The in vitro results showed that sPEEK/BP/E7 displays enhanced cytocompatibility and osteogenicity in terms of cell adhesion, proliferation, alkaline phosphatase (ALP) activity, matrix mineralization, and osteogenesis-related gene expression, superior to those of the sPEEK and sPEEK/BP samples. In addition to osteogenesis, the multifunctional coating exhibited strong antibacterial activity against both Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). Furthermore, it was confirmed in a rat femoral infection model that sPEEK/BP/E7 effectively resisted infection caused by S. aureus under NIR light irradiation and promoted osseointegration in vivo. Thus, this work presents a facile strategy to realize improvement of the “functional integration” of new polymer bone-implant materials and provide new ideas for their clinical application.

Copper nanoparticles incorporated visible light-curing chitosan-based hydrogel membrane for enhancement of bone repair

Alveolar bone defects caused by tumor, trauma and inflammation can lead to the loss of oral function and complicate denture restoration. Currently, guided bone regeneration (GBR) barrier membranes for repairing bone defect cannot effectively promote bone regeneration due to their unstable degradation rate and poor antibacterial properties. Furthermore, they require additional tailoring before implantation. Therefore, this study developed a visible light-curing hydrogel membrane (CF–Cu) comprising methacrylated carboxymethyl chitosan (CMCS-MA), silk fibroin (SF), and copper nanoparticles (Cu NPs) to address these shortcomings of commercial membranes. The CF-Cu hydrogel, characterized by scanning electron microscopy (SEM), a universal testing machine, and swelling and degradation tests, demonstrated a smooth porous network structure, suitable swelling ratio, biodegradability, and enhanced mechanical strength. Cytotoxicity and hemolysis tests in vitro demonstrated excellent cyto- and hemo-compatibility of the CF-Cu hydrogel extracts. Additionally, evaluation of antibacterial properties in vitro, including colony forming unit (CFU) counts, MTT assays, and live/dead fluorescence staining, showed that the CF-Cu hydrogel exhibited excellent antibacterial properties, inhibiting over 80% of S. aureus, S. mutans, and P. gingivalis with CF-1Cu hydrogel compared to the control group. Moreover, evaluation of osteogenic differentiation of rBMSCs in vitro suggested that the CF-1Cu hydrogel significantly improved alkaline phosphatase (ALP) activity and the mineralization of extracellular matrix, up-regulating the expressions of osteogenesis-related genes (Runx2, ALP, Col-1, OPN and BSP). In summary, these results indicated that CF-1Cu hydrogel exhibited excellent cytocompatibility, antibacterial and osteogenic properties in vitro. Therefore, the CF-1Cu hydrogel holds potential as a viable material for application in GBR procedures aimed at addressing bone defects.

Electrical Responsive Coating with a Multilayered TiO2-SnO2-RuO2 Heterostructure on Ti for Controlling Antibacterial Ability and Improving Osseointegration.

The bacterial infection and poor osseointegration of Ti implants could significantly compromise their applications in bone repair and replacement. Based on the carrier separation ability of the heterojunction and the redox reaction of pseudocapacitive metal oxides, we report an electrically responsive TiO2-SnO2-RuO2 coating with a multilayered heterostructure on a Ti implant. Owing to the band gap structure of the TiO2-SnO2-RuO2 coating, electron carriers are easily enriched at the coating surface, enabling a response to the endogenous electrical stimulation of the bone. With the formation of SnO2-RuO2 pseudocapacitance on the modified surface, the postcharging mode can significantly change the surface chemical state of the coating due to the redox reaction, enhancing the antibacterial ability and osteogenesis-related gene expression of the human bone marrow mesenchymal stem cells. Owing to the attraction for Ca2+, only the negatively postcharged SnO2@RuO2 can promote apatite deposition. The in vivo experiment reveals that the S-SnO2@RuO2-NP could effectively kill the bacteria colonized on the surface and promote osseointegration with the synostosis bonding interface. Thus, negatively charging the electrically responsive coating of TiO2-SnO2-RuO2 is a good strategy to endow modified Ti implants with excellent antibacterial ability and osseointegration.

Salvianolic Acid B Accelerates Osteoporotic Fracture Healing via LncRNA-MALAT1/miR-155-5p/HIF1A Axis

Background: Osteoporotic fracture is a pathological fracture secondary to osteoporosis, causing disabilities and a heavy burden to the patients. In the previous study, we found that salvianolic acid B (SalB) promoted the osteogenesis of Mesenchymal Stem Cells (MSCs). Objective: This study aimed to explore the role of SalB in osteoporotic fracture healing, as well as the potential molecular mechanism. Methods: Human bone marrow mesenchymal stem cells (hMSCs) were treated with SalB or PBS in vitro, and an osteoporotic fracture model in Sprague-Dawley (SD) rats was established successfully. SalB or PBS was locally injected at the fracture. Eight weeks later, microCT was used to compare the healing of osteoporotic fractures with or without SalB. The relative expressions of mRNAs were measured by qRT-PCR. Bioinformatics analysis, RT-PCR, and dual luciferase reporter assay were utilized to detect the interrelation of genes. Immunohistochemistry staining was used to test expressions of proteins. Results: In the present study, we found that SalB significantly increased the level of lncRNA-- MALAT1 in a dose-dependent manner. Additionally, silencing lncRNA-MALAT1 inhibited the expressions of osteogenesis-related marker genes and abolished the effect of SalB on osteogenesis. Also, we found that lncRNA-MALAT1 sponged miR-155-5p, and miR-155-5p directly targeted HIF-1α. Using the osteoporotic fracture healing model, our result demonstrated that local administration of SalB could promote both bone and type H vessel formation in the calluses. The mechanical test further showed that SalB could improve the mechanical properties of fractured femurs. Conclusion: Taken together, our study reported that SalB could promote osteogenesis and type H vessel formation to accelerate osteoporotic fracture healing through the lncRNA-- MALAT1/miR-155-5p/HIF-1α axis.

Emerging perfluoroalkyl substances retard skeletal growth by accelerating osteoblasts senescence via ferroptosis

Due to the persistent nature and significant negative impacts of perfluorooctanoic acid (PFOA) on human health and other organisms, the emergence of new PFOA alternatives, such as perfluoro (2-methyl-3-oxhexanoic) acid (GenX) and perfluoro-3,6,9-trioxyundecanoic acid (PFO3TDA), have drawn significant attention. However, the toxic effects of PFOA and its substitutes on bones remain limited. In this study, we administered different concentrations of PFOA, GenX, and PFO3TDA via gavage to 3-week-old male BALB/C mice for four weeks. X-ray and micro-CT scans revealed shortening of the femur and tibia and significant reduction in bone density. Additionally, PFOA, GenX, and PFO3TDA promoted osteoblast senescence and impaired osteogenic capabilities. This was characterized by a decrease in the expression of osteogenesis-related genes (OCN, ALP, Runx2, etc.) and an increase in the expression of aging and inflammation-related factors (p16INK4a, P21, MMP3, etc). Furthermore, RNA sequencing revealed activation of the ferroptosis pathway in PFOA-treated osteoblasts, characterized by notable lipid peroxidation and excessive iron accumulation. Finally, by inhibiting the ferroptosis pathway with ferrostatin-1 (Fer-1), we effectively alleviated the senescence of MC3T3-E1 cells treated with PFOA, GenX, and PFO3TDA, and improved their osteogenic capabilities. Therefore, our study provides a new therapeutic insight into the impact of PFOA and its substitutes on bone growth and development.

Cytocompatibility, cell-material interaction, and osteogenic differentiation of MC3T3-E1 pre-osteoblasts in contact with engineered Mg/PLA composites.

Bioabsorbable Mg wire-reinforced poly-lactic acid (PLA) matrix composites are potential candidate for load-bearing orthopedic implants offering tailorable mechanical and degradation properties by stacking sequence, volume fraction and surface modification of Mg wires. In this study, we investigated the cytocompatibility, cell-material interaction, and bone differentiation behavior of MC3T3-E1 pre-osteoblast cells for medical-grade PLA, Mg/PLA, and PEO-Mg/PLA (having PEO surface modification on Mg wires) composites. MTT and live/dead assay showed excellent biocompatibility of both composites while cell-material interaction analysis revealed that cells were able to adhere and proliferate on the surface of composites. Cells on the longitudinal surface of composites showed a high and uniform cell density while those on transversal surfaces initially avoided Mg regions but later migrated back after the formation of the passivation layer. Bone differentiation tests showed that cells in extracts of PLA and composites were able to initiate the differentiation process as osteogenesis-related gene expressions, alkaline phosphatase protein quantity, and calcium mineralization increased after 7 and 14 days of culture. Interestingly, the bone differentiation response of PEO-Mg/PLA composite was found to be similar to medical-grade PLA, proving its superiority over Mg/PLA composite.

Fabrication of a coral-like barium titanate nano-piezoelectric coating and its effect on promoting osteogenic differentiation of rat bone marrow mesenchymal stem cells in vitro

Objective: To investigate the biocompatibility of coral-like barium titanate nano-piezoelectric coatings and the influence of ultrasound-excited piezoelectric effect on the early osteogenic differentiation. Methods: The barium titanate nano-piezoelectric coating (the coating group) was prepared on the surface of titanium metal by anodic oxidation, hydrothermal reaction and high-temperature annealing, and polished titanium specimens were used as control group. The surface morphology, composition, and crystal phase and hydrophilicity of the two groups of titanium specimens were characterized using scanning electron microscopy, X-ray photoelectron spectroscopy, Raman spectroscopy and contact angle meter. The piezoelectric properties of the materials were characterized by piezoresponse force microscopy. Rat bone marrow mesenchymal stem cells (BMSC) were cultured and identified and seeded the surface of titanium specimens in two groups. The cells seeded on blank culture plates were used as blank group. After low intensity pulsed ultrasound intervention, cell proliferation and live/dead staining were detected to evaluate cytocompatibility of the coatings. Alkaline phosphatase (ALP) activity of each group was detected by ALP staining kit, and the expression of osteogenesis-related genes [integrin, bone morphogenetic protein 2 (BMP-2), Runt-related transcription factor 2 (RUNX2)] was detected by real-time fluorescent quantitative PCR (RT-qPCR) to evaluate the effect of the coating on promoting the early osteogenic differentiation of BMSC. Results: The surface of titanium specimens in the coating group showed a uniform coral-like morphology, and the diameter of the coral tentacles was 70-100 nm. The main component was tetragonal barium titanate. The surface hydrophilicity of the coating group (water contact angle 10.12°± 0.93°) was significantly better than that of the control group (water contact angle 78.32°±0.71°) (F= 10 165.91, P<0.001). The coating has a stable piezoelectric property with a piezoelectric constant of about 5 pC/N. Cell experiments showed that, with or without ultrasound, the cell proliferation activity of the coating group was significantly lower than that of the blank group and the control group on the third day (P<0.05). On the fifth day, with or without ultrasound, there was no significant difference in cell proliferation activity between the three groups (P>0.05). After 7 days of culture, the ALP activity of the coating group was significantly higher than that of the blank group and the control group (P<0.05). The results of RT-qPCR showed that the mRNA expression of integrin and BMP-2 in the coating group with ultrasound was significantly higher than that in the other groups with ultrasound, and was higher than that of the coating group without ultrasound (P<0.05). The expression of integrin mRNA in the control group with ultrasound was significantly higher than that in the control group without ultrasound (P<0.05). The expression of RUNX2 mRNA in the coating group with ultrasound was significantly higher than that in the coating group without ultrasound (P<0.05). Conclusions: The coral-like barium titanate nano-piezoelectric coating exhibits favorable biocompatibility and stable piezoelectric property, and facilitates the early osteogenic differentiation of BMSC under the excitation of low-intensity pulsed ultrasound.

Biocompatibility of Subperiosteal Dental Implants: Changes in the Expression of Osteogenesis-Related Genes in Osteoblasts Exposed to Differently Treated Titanium Surfaces.

The use of endosseous dental implants may become unfeasible in the presence of significant maxillary bone atrophy; thus, surgical techniques have been proposed to promote bone regeneration in such cases. However, such techniques are complex and may expose the patient to complications. Subperiosteal implants, being placed between the periosteum and the residual alveolar bone, are largely independent of bone thickness. Such devices had been abandoned due to the complexity of positioning and adaptation to the recipient bone site, but are nowadays witnessing an era of revival following the introduction of new acquisition procedures, new materials, and innovative manufacturing methods. We have analyzed the changes induced in gene and protein expression in C-12720 human osteoblasts by differently surface-modified TiO2 materials to verify their ability to promote bone formation. The TiO2 materials tested were (i) raw machined, (ii) electropolished with acid mixture, (iii) sand-blasted + acid-etched, (iv) AlTiColorTM surface, and (v) anodized. All five surfaces efficiently stimulated the expression of markers of osteoblastic differentiation, adhesion, and osteogenesis, such as RUNX2, osteocalcin, osterix, N-cadherin, β-catenin, and osteoprotegerin, while cell viability/proliferation was unaffected. Collectively, our observations document that presently available TiO2 materials are well suited for the manufacturing of modern subperiosteal implants.

Siwu decoction exerts a phytoestrogenic osteoprotective effect on postmenopausal osteoporosis via the estrogen receptor/phosphatidylinositol 3-kinase/serine/threonine protein kinase pathway

Ethnopharmacological relevanceSiwu decoction (SWD) is widely used in gynecological diseases, such as peripheral menopause syndrome, premature ovarian failure, and menstrual disorder. However, the mechanism of SWD on postmenopausal osteoporosis (PMOP) remains unclear. Aim of the studyTo discover the phytoestrogenic osteoprotective effect of SWD on PMOP. Materials and methodsThe potential mechanism of SWD on PMOP was filtered through network pharmacology research. The potential mechanism was verified in MC3T3-E1 cell lines in vitro. CCK8 assay was conducted to assess cell proliferation and the expressions of ER/PI3K/AKT pathway were analyzed using Western blot. Female F-344 rats were chosen to set up the PMOP model. The osteoprotective effect of SWD in vivo was evaluated using Hematoxylin-eosin staining, TRAP staining, Goldner staining and DXA. The potential mechanism was verified in vivo through Western blot and immunohistochemistry. RT-qPCR was conducted to unveil the expressions of osteogenesis genes. ResultsNetwork pharmacology research showed that ER/PI3K/AKT pathway may be the potential mechanism of SWD on PMOP. SWD promoted the proliferation of osteoblasts and regulated the protein expressions of ER/PI3K/AKT pathway in vitro. SWD improved the morphological structure, bone mineralization and bone mineral density of femurs and suppressed osteoclastogenesis in PMOP rat model via ER/PI3K/AKT pathway in vivo. In addition, SWD regulated the mRNA expressions of osteogenesis-related genes. ConclusionsSWD exerts a phytoestrogenic osteoprotective on PMOP by regulating ER/PI3K/AKT pathway, which marks it as a valuable medicine or supplement of PMOP.

A Composite Hydrogel Functionalized by Borosilicate Bioactive Glasses and VEGF for Critical-Size Bone Regeneration.

Critical-size bone defects pose a formidable challenge in clinical treatment, prompting extensive research efforts to address this problem. In this study, an inorganic-organic multifunctional composite hydrogel denoted as PLG-g-TA/VEGF/Sr-BGNPs is developed, engineered for the synergistic management of bone defects. The composite hydrogel demonstrated the capacity for mineralization, hydroxyapatite formation, and gradual release of essential functional ions and vascular endothelial growth factor (VEGF) and also maintained an alkaline microenvironment. The composite hydrogel promoted the proliferation and osteogenic differentiation of rat bone marrow mesenchymal stem cells (rBMSCs), as indicated by increased expression of osteogenesis-related genes and proteins in vitro. Moreover, the composite hydrogel significantly enhanced the tube-forming capability of human umbilical vein endothelial cells (HUVECs) and effectively inhibited the process of osteoblastic differentiation of nuclear factor kappa-B ligand (RANKL)-induced Raw264.7 cells and osteoclast bone resorption. After the implantation of the composite hydrogel into rat cranial bone defects, the expression of osteogenic and angiogenic biomarkers increased, substantiating its efficacy in promoting bone defect repair in vivo. The commendable attributes of the multifunctional composite hydrogel underscore its pivotal role in expediting hydrogel-associated bone growth and repairing critical bone defects, positioning it as a promising adjuvant therapy candidate for large-segment bone defects.

Unlocking the potential of iron-containing mesoporous bioactive glasses: Orchestrating osteogenic differentiation in bone marrow mesenchymal stem cells and osteoblasts

Iron (Fe) is a crucial element in the human body, playing a significant role in bone metabolism. The release of Fe ions at bone defect sites can promote bone regeneration. In this study, we synthesized Fe-containing mesoporous bioactive glasses (Fe-MBGs) in SiO2-CaO-Fe2O3 composition using a sol-gel method. Regardless of the amount of incorporated Fe2O3 (up to 5 mol%), the Fe-MBGs maintained a mesoporous structure, and the inclusion of Fe2O3 did not alter their amorphous characteristics. However, the presence of Fe2O3 led to a reduction in both pore volume and specific surface area of Fe-MBGs. Notably, Fe-MBGs demonstrated degradability in physiological fluids and could sustain release of Si, Fe, and Ca ions. Higher concentrations of incorporated Fe2O3 were found to reduce the degradation of Fe-MBGs. All Fe-MBGs exhibited favorable bioactivity, as evidenced by the rapid formation of hydroxyapatite when exposed to simulated body fluid. Fe-MBGs also demonstrated concentration-dependent effects on BMSCs and Saos-2 cells. Extracts of Fe-MBGs at 0.1 and 1 mg/mL exhibited non-cytotoxicity and promoted cell proliferation. Additionally, extracts of Fe-MBGs at 1 mg/mL significantly enhanced the alkaline phosphatase activity of BMSCs and Saos-2 cells, along with an upregulation of the expression of osteogenesis-related genes. These findings unlock the significant potential of Fe-MBGs as functional biomaterials for bone regeneration applications. The controlled release of Fe ions from these mesoporous bioactive glasses orchestrates osteogenic differentiation in bone marrow mesenchymal stem cells and osteoblasts.

Unveiling the role of hypoxia-inducible factor 2alpha in osteoporosis: Implications for bone health.

Osteoporosis (OP) has become a major public health problem worldwide. Most OP treatments are based on the inhibition of bone resorption, and it is necessary to identify additional treatments aimed at enhancing osteogenesis. In the bone marrow (BM) niche, bone mesenchymal stem cells (BMSCs) are exposed to a hypoxic environment. Recently, a few studies have demonstrated that hypoxia-inducible factor 2alpha (HIF-2α) is involved in BMSC osteogenic differentiation, but the molecular mechanism involved has not been determined. To investigate the effect of HIF-2α on the osteogenic and adipogenic differentiation of BMSCs and the hematopoietic function of hematopoietic stem cells (HSCs) in the BM niche on the progression of OP. Mice with BMSC-specific HIF-2α knockout (Prx1-Cre;Hif-2αfl/fl mice) were used for in vivo experiments. Bone quantification was performed on mice of two genotypes with three interventions: Bilateral ovariectomy, semilethal irradiation, and dexamethasone treatment. Moreover, the hematopoietic function of HSCs in the BM niche was compared between the two mouse genotypes. In vitro, the HIF-2α agonist roxadustat and the HIF-2α inhibitor PT2399 were used to investigate the function of HIF-2α in BMSC osteogenic and adipogenic differentiation. Finally, we investigated the effect of HIF-2α on BMSCs via treatment with the mechanistic target of rapamycin (mTOR) agonist MHY1485 and the mTOR inhibitor rapamycin. The quantitative index determined by microcomputed tomography indicated that the femoral bone density of Prx1-Cre;Hif-2αfl/fl mice was lower than that of Hif-2αfl/fl mice under the three intervention conditions. In vitro, Hif-2αfl/fl mouse BMSCs were cultured and treated with the HIF-2α agonist roxadustat, and after 7 d of BMSC adipogenic differentiation, the oil red O staining intensity and mRNA expression levels of adipogenesis-related genes in BMSCs treated with roxadustat were decreased; in addition, after 14 d of osteogenic differentiation, BMSCs treated with roxadustat exhibited increased expression of osteogenesis-related genes. The opposite effects were shown for mouse BMSCs treated with the HIF-2α inhibitor PT2399. The mTOR inhibitor rapamycin was used to confirm that HIF-2α regulated BMSC osteogenic and adipogenic differentiation by inhibiting the mTOR pathway. Consequently, there was no significant difference in the hematopoietic function of HSCs between Prx1-Cre;Hif-2αfl/fl and Hif-2αfl/fl mice. Our study showed that inhibition of HIF-2α decreases bone mass by inhibiting the osteogenic differentiation and increasing the adipogenic differentiation of BMSCs through inhibition of mTOR signaling in the BM niche.

Young osteocyte-derived extracellular vesicles facilitate osteogenesis by transferring tropomyosin-1

BackgroundBone marrow mesenchymal stem cells (BMSCs) can undergo inadequate osteogenesis or excessive adipogenesis as they age due to changes in the bone microenvironment, ultimately resulting in decreased bone density and elevated risk of fractures in senile osteoporosis. This study aims to investigate the effects of osteocyte senescence on the bone microenvironment and its influence on BMSCs during aging.ResultsPrimary osteocytes were isolated from 2-month-old and 16-month-old mice to obtain young osteocyte-derived extracellular vesicles (YO-EVs) and senescent osteocyte-derived EVs (SO-EVs), respectively. YO-EVs were found to significantly increase alkaline phosphatase activity, mineralization deposition, and the expression of osteogenesis-related genes in BMSCs, while SO-EVs promoted BMSC adipogenesis. Neither YO-EVs nor SO-EVs exerted an effect on the osteoclastogenesis of primary macrophages/monocytes. Our constructed transgenic mice, designed to trace osteocyte-derived EV distribution, revealed abundant osteocyte-derived EVs embedded in the bone matrix. Moreover, mature osteoclasts were found to release osteocyte-derived EVs from bone slices, playing a pivotal role in regulating the functions of the surrounding culture medium. Following intravenous injection into young and elderly mouse models, YO-EVs demonstrated a significant enhancement of bone mass and biomechanical strength compared to SO-EVs. Immunostaining of bone sections revealed that YO-EV treatment augmented the number of osteoblasts on the bone surface, while SO-EV treatment promoted adipocyte formation in the bone marrow. Proteomics analysis of YO-EVs and SO-EVs showed that tropomyosin-1 (TPM1) was enriched in YO-EVs, which increased the matrix stiffness of BMSCs, consequently promoting osteogenesis. Specifically, the siRNA-mediated depletion of Tpm1 eliminated pro-osteogenic activity of YO-EVs both in vitro and in vivo.ConclusionsOur findings suggested that YO-EVs played a crucial role in maintaining the balance between bone resorption and formation, and their pro-osteogenic activity declining with aging. Therefore, YO-EVs and the delivered TPM1 hold potential as therapeutic targets for senile osteoporosis.Graphical abstract

High-viscosity driven modulation of biomechanical properties of human mesenchymal stem cells promotes osteogenic lineage

Biomechanical cues could effectively govern cell gene expression to direct the differentiation of specific stem cell lineage. Recently, the medium viscosity has emerged as a significant mechanical stimulator that regulates the cellular mechanical properties and various physiological functions. However, whether the medium viscosity can regulate the mechanical properties of human mesenchymal stem cells (hMSCs) to effectively trigger osteogenic differentiation remains uncertain. The mechanism by which cells sense and respond to changes in medium viscosity, and regulate cell mechanical properties to promote osteogenic lineage, remains elusive. In this study, we demonstrated that hMSCs, cultured in a high-viscosity medium, exhibited larger cell spreading area and higher intracellular tension, correlated with elevated formation of actin stress fibers and focal adhesion maturation. Furthermore, these changes observed in hMSCs were associated with activation of TRPV4 (transient receptor potential vanilloid sub-type 4) channels on the cell membrane. This feedback loop among TRPV4 activation, cell spreading and intracellular tension results in calcium influx, which subsequently promotes the nuclear localization of NFATc1 (nuclear factor of activated T cells 1). Concomitantly, the elevated intracellular tension induced nuclear deformation and promoted the nuclear localization of YAP (YES‐associated protein). The concurrent activation of NFATc1 and YAP significantly enhanced alkaline phosphatase (ALP) for pre-osteogenic activity. Taken together, these findings provide a more comprehensive view of how viscosity-induced alterations in biomechanical properties of MSCs impact the expression of osteogenesis-related genes, and ultimately promote osteogenic lineage.