Cell membrane vesicles derived from hBMSCs and hUVECs enhance bone regeneration.

Bone tissue renewal can be enhanced through co-transplantation of bone mesenchymal stem cells (BMSCs) and vascular endothelial cells (ECs). However, there are apparent limitations in stem cell-based therapy which hinder its clinic translation. Hence, we investigated the potential of alternative stem cell substitutes for facilitating bone regeneration. In this study, we successfully prepared cell membrane vesicles (CMVs) from BMSCs and ECs. The results showed that BMSC-derived cell membrane vesicles (BMSC-CMVs) possessed membrane receptors involved in juxtacrine signaling and growth factors derived from their parental cells. EC-derived cell membrane vesicles (EC-CMVs) also contained BMP2 and VEGF derived from their parental cells. BMSC-CMVs enhanced tube formation and migration ability of hUVECs, while EC-CMVs promoted the osteogenic differentiation of hBMSCs in vitro. Using a rat skull defect model, we found that co-transplantation of BMSC-CMVs and EC-CMVs could stimulate angiogenesis and bone formation in vivo. Therefore, our research might provide an innovative and feasible approach for cell-free therapy in bone tissue regeneration.

Sorafenib inhibits ossification of the posterior longitudinal ligament by blocking LOXL2-mediated vascularization.

Ossification of the Posterior Longitudinal Ligament (OPLL) is a degenerative hyperostosis disease characterized by the transformation of the soft and elastic vertebral ligament into bone, resulting in limited spinal mobility and nerve compression. Employing both bulk and single-cell RNA sequencing, we elucidate the molecular characteristics, cellular components, and their evolution during the OPLL process at a single-cell resolution, and validate these findings in clinical samples. This study also uncovers the capability of ligament stem cells to exhibit endothelial cell-like phenotypes in vitro and in vivo. Notably, our study identifies LOXL2 as a key regulator in this process. Through gain-and loss-of-function studies, we elucidate the role of LOXL2 in the endothelial-like differentiation of ligament cells. It acts via the HIF1A pathway, promoting the secretion of downstream VEGFA and PDGF-BB. This function is not related to the enzymatic activity of LOXL2. Furthermore, we identify sorafenib, a broad-spectrum tyrosine kinase inhibitor, as an effective suppressor of LOXL2-mediated vascular morphogenesis. By disrupting the coupling between vascularization and osteogenesis, sorafenib demonstrates significant inhibition of OPLL progression in both BMP-induced and enpp1 deficiency-induced animal models while having no discernible effect on normal bone mass. These findings underscore the potential of sorafenib as a therapeutic intervention for OPLL.

Peripheral nerve-derived Sema3A promotes osteogenic differentiation of mesenchymal stem cells through the Wnt/ß-catenin/Nrp1 positive feedback loop.

Sensory nerves play a crucial role in maintaining bone homeostasis by releasing Semaphorin 3A (Sema3A). However, the specific mechanism of Sema3A in regulation of bone marrow mesenchymal stem cells (BMMSCs) during bone remodelling remains unclear. The tibial denervation model was used and the denervated tibia exhibited significantly lower mass as compared to sham operated bones. In vitro, BMMSCs cocultured with dorsal root ganglion cells (DRGs) or stimulated by Sema3A could promote osteogenic differentiation through the Wnt/ß-catenin/Nrp1 positive feedback loop, and the enhancement of osteogenic activity could be inhibited by SM345431 (Sema3A-specific inhibitor). In addition, Sema3A-stimulated BMMSCs or intravenous injection of Sema3A could promote new bone formation in vivo. To sum up, the coregulation of bone remodelling is due to the ageing of BMMSCs and increased osteoclast activity. Furthermore, the sensory neurotransmitter Sema3A promotes osteogenic differentiation of BMMSCs via Wnt/ß-catenin/Nrp1 positive feedback loop, thus promoting osteogenesis in vivo and in vitro.

Hem1 is essential for ruffled border formation in osteoclasts and efficient bone resorption.

Bone resorption is highly dependent on the dynamic rearrangement of the osteoclast actin cytoskeleton to allow formation of actin rings and a functional ruffled border. Hem1 is a hematopoietic-specific subunit of the WAVE-complex which regulates actin polymerization and is crucial for lamellipodia formation in hematopoietic cell types. However, its role in osteoclast differentiation and function is still unknown. Here, we show that although the absence of Hem1 promotes osteoclastogenesis, the ability of Hem1-/- osteoclasts to degrade bone was severely impaired. Global as well as osteoclast-specific deletion of Hem1 in vivo revealed increased femoral trabecular bone mass despite elevated numbers of osteoclasts in vivo. We found that the resorption defect derived from the morphological distortion of the actin-rich sealing zone and ruffled border deformation in Hem1-deficient osteoclasts leading to impaired vesicle transport and increased intracellular acidification. Collectively, our data identify Hem1 as a yet unknown key player in bone remodeling by regulating ruffled border formation and consequently the resorptive capacity of osteoclasts.

[The relationship between the expression of PGE2 and COX-2 and the osteogenic activity and oxygen level of alveolar bone].

PURPOSE: To study the relationship between the expression of prostaglandin E2 (PGE2) and cyclooxygenase-2 (COX-2) and the osteogenic activity and oxygen level of alveolar bone. METHODS: The alveolar bones of 56 patients with chronic periodontitis who received dental treatment from March 2021 to March 2023 were collected as the experimental (periodontitis) group, and the healthy alveolar bones of 53 patients who received dental treatment during the same period were selected as the control group. The osteoblasts were cultured by tissue block culture, and modified Kaplow's alkaline phosphatase (ALP) staining was used to identify the cells. COX-2, PGE2 and osteoclastogenesis inhibitory factor (OPG) receptor activator of nuclear factor-κb ligand (RANKL) and other indicators were determined by ELISA. PGE2, COX-2, OPG, internal oxygen level, ALP, RANKL and their correlation were compared between the two groups. Statistical analysis was performed with SPSS 27.0 software package. RESULTS: PGE2, COX-2 and RANKL in periodontitis group were significantly higher than those in the control group, but OPG, internal oxygen level and ALP were significantly lower than those in the control group (P＜0.05). PGE2 and COX2 were highly positively correlated with OPG, internal oxygen level and ALP, but were highly positively correlated with RANKL(P＜0.05). CONCLUSIONS: The expression of PGE2 and COX-2 is highly negatively correlated with ALP and oxygen levels. Clinical treatment may consider increasing oxygen levels, increasing oxygen partial pressure, and regulating ALP levels by drugs, so as to change the inflammatory condition of periodontitis or other dental diseases.

Multiscale architecture design of 3D printed biodegradable Zn-based porous scaffolds for immunomodulatory osteogenesis.

Reconciling the dilemma between rapid degradation and overdose toxicity is challenging in biodegradable materials when shifting from bulk to porous materials. Here, we achieve significant bone ingrowth into Zn-based porous scaffolds with 90% porosity via osteoinmunomodulation. At microscale, an alloy incorporating 0.8 wt% Li is employed to create a eutectoid lamellar structure featuring the LiZn4 and Zn phases. This microstructure optimally balances high strength with immunomodulation effects. At mesoscale, surface pattern with nanoscale roughness facilitates filopodia formation and macrophage spreading. At macroscale, the isotropic minimal surface G unit exhibits a proper degradation rate with more uniform feature compared to the anisotropic BCC unit. In vivo, the G scaffold demonstrates a heightened efficiency in promoting macrophage polarization toward an anti-inflammatory phenotype, subsequently leading to significantly elevated osteogenic markers, increased collagen deposition, and enhanced new bone formation. In vitro, transcriptomic analysis reveals the activation of JAK/STAT pathways in macrophages via up regulating the expression of Il-4, Il-10, subsequently promoting osteogenesis.

Microdroplets Encapsulated with NFATc1-siRNA and Exosomes-Derived from MSCs Onto 3D Porous PLA Scaffold for Regulating Osteoclastogenesis and Promoting Osteogenesis.

Introduction: Osteoporotic-related fractures remains a significant public health concern, thus imposing substantial burdens on our society. Excessive activation of osteoclastic activity is one of the main contributing factors for osteoporosis-related fractures. While polylactic acid (PLA) is frequently employed as a biodegradable scaffold in tissue engineering, it lacks sufficient biological activity. Microdroplets (MDs) have been explored as an ultrasound-responsive drug delivery method, and mesenchymal stem cell (MSC)-derived exosomes have shown therapeutic effects in diverse preclinical investigations. Thus, this study aimed to develop a novel bioactive hybrid PLA scaffold by integrating MDs-NFATc1-silencing siRNA to target osteoclast formation and MSCs-exosomes (MSC-Exo) to influence osteogenic differentiation (MDs-NFATc1/PLA-Exo). Methods: Human bone marrow-derived mesenchymal stromal cells (hBMSCs) were used for exosome isolation. Transmission electron microscopy (TEM) and confocal laser scanning microscopy were used for exosome and MDs morphological characterization, respectively. The MDs-NFATc1/PLA-Exo scaffold was fabricated through poly(dopamine) and fibrin gel coating. Biocompatibility was assessed using RAW 264.7 macrophages and hBMSCs. Osteoclast formations were examined via TRAP staining. Osteogenic differentiation of hBMSCs and cytokine expression modulation were also investigated. Results: MSC-Exo exhibited a cup-shaped structure and effective internalization into cells, while MDs displayed a spherical morphology with a well-defined core-shell structure. Following ultrasound stimulation, the internalization study demonstrated efficient delivery of bioactive MDs into recipient cells. Biocompatibility studies indicated no cytotoxicity of MDs-NFATc1/PLA-Exo scaffolds in RAW 264.7 macrophages and hBMSCs. Both MDs-NFATc1/PLA and MDs-NFATc1/PLA-Exo treatments significantly reduced osteoclast differentiation and formation. In addition, our results further indicated MDs-NFATc1/PLA-Exo scaffold significantly enhanced osteogenic differentiation of hBMSCs and modulated cytokine expression. Discussion: These findings suggest that the bioactive MDs-NFATc1/PLA-Exo scaffold holds promise as an innovative structure for bone tissue regeneration. By specifically targeting osteoclast formation and promoting osteogenic differentiation, this hybrid scaffold may address key challenges in osteoporosis-related fractures.

The dinosaurs that weren't: osteohistology supports giant ichthyosaur affinity of enigmatic large bone segments from the European Rhaetian.

Very large unidentified elongate and rounded fossil bone segments of uncertain origin recovered from different Rhaetian (Late Triassic) fossil localities across Europe have been puzzling the paleontological community since the second half of the 19th century. Different hypotheses have been proposed regarding the nature of these fossils: (1) giant amphibian bones, (2) dinosaurian or other archosaurian long bone shafts, and (3) giant ichthyosaurian jaw bone segments. We call the latter proposal the 'Giant Ichthyosaur Hypothesis' and test it using bone histology. In presumable ichthyosaur specimens from SW England (Lilstock), France (Autun), and indeterminate cortical fragments from Germany (Bonenburg), we found a combination of shared histological features in the periosteal cortex: an unusual woven-parallel complex of strictly longitudinal primary osteons set in a novel woven-fibered matrix type with intrinsic coarse collagen fibers (IFM), and a distinctive pattern of Haversian substitution in which secondary osteons often form within primary ones. The splenial and surangular of the holotype of the giant ichthyosaur Shastasaurus sikanniensis from Canada were sampled for comparison. The results of the sampling indicate a common osteohistology with the European specimens. A broad histological comparison is provided to reject alternative taxonomic affinities aside from ichthyosaurs of the very large bone segment. Most importantly, we highlight the occurrence of shared peculiar osteogenic processes in Late Triassic giant ichthyosaurs, reflecting special ossification strategies enabling fast growth and achievement of giant size and/or related to biomechanical properties akin to ossified tendons.

Atorvastatin reduces calcification in valve interstitial cells via the NF-κB signalling pathway by promoting Atg5-mediated autophagy.

Aortic valve calcification (AVC) is a common cardiovascular disease and a risk factor for sudden death. However, the potential mechanisms and effective therapeutic drugs need to be explored. Atorvastatin is a statin that can effectively prevent cardiovascular events by lowering cholesterol levels. However, whether atorvastatin can inhibit AVC by reducing low-density lipoprotein (LDL) and its possible mechanism of action require further exploration. In the current study, we constructed an in vitro AVC model by inducing calcification of the valve interstitial cells. We found that atorvastatin significantly inhibited osteogenic differentiation, reduced the deposition of calcium nodules in valve interstitial cells, and enhanced autophagy in calcified valve interstitial cells, manifested by increased expression levels of the autophagy proteins Atg5 and LC3B-II/I and the formation of smooth autophagic flow. Atorvastatin inhibited the NF-κB signalling pathway and the expression of inflammatory factors mediated by NF-κB in calcified valve interstitial cells. The activation of the NF-κB signalling pathway led to the reversal of atorvastatin's effect on enhancing autophagy and alleviating valve interstitial cell calcification. In conclusion, atorvastatin inhibited the NF-κB signalling pathway by upregulating autophagy, thereby alleviating valve interstitial cell calcification, which was conducive to improving AVC.

[A sericin hydrogel scaffold for sustained dexamethasone release modulates macrophage polarization to promote mandibular bone defect repair in rats].

OBJECTIVE: To evaluate the efficacy of a modified sericin hydrogel scaffold loaded with dexamethasone (SMH-CD/DEX) scaffold for promoting bone defect healing by stimulating anti-inflammatory macrophage polarization. METHODS: The light-curable SMH-CD/DEX scaffold was prepared using dexamethasone-loaded NH2-ß-cyclodextrin (NH2-ß-CD) and sericin hydrogel and characterized by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), biocompatibility assessment and drug release test. THP-1 macrophages incubated with the scaffold were examined for protein expressions of iNOS and Arg-1, mRNA expressions of IL-6, Il-10, Arg-1 and iNOS, and surface markers CD86 and CD206 using Western blotting, RT-qPCR, and flow cytometry. In a co-culture system of human periodontal ligament stem cells (HPDLSCs) and THP-1 macrophages, the osteogenic ability of the stem cells incubated with the scaffold was evaluated by detecting protein expressions of COL1A1 and Runx2 and expressions of ALP, Runx2, OCN and BMP2 mRNA, ALP staining, and alizarin red staining. In a rat model of mandibular bone defect, the osteogenic effect of the scaffold was assessed by observing bone regeneration using micro-CT and histopathological staining. RESULTS: In THP-1 macrophages, incubation with SMH-CD/DEX scaffold significantly enhanced protein expressions of Arg-1 and mRNA expressions of IL-10 and Arg-1 and lowered iNOS protein expression and IL-6 and iNOS mRNA expressions. In the co-culture system, SMH-CD/DEX effectively increased the protein expressions of COL1A1 and Runx2 and mRNA expressions of ALP and BMP2 in HPDLSCs and promoted their osteogenic differentiation. In the rat models, implantation of SMH-CD/DEX scaffold significantly promoted bone repair and bone regeneration in the bone defect. CONCLUSION: The SMH-CD/DEX scaffold capable of sustained dexamethasone release promotes osteogenic differentiation of stem cells and bone defect repair in rats by regulating M2 polarization.

Comprehensive assessment of goat adipose tissue-derived mesenchymal stem cells cultured in different media.

Mesenchymal stem cells (MSCs) have demonstrated potential in treating livestock diseases that are unresponsive to conventional therapies. MSCs derived from goats, a valuable model for studying orthopaedic disorders in humans, offer insights into bone formation and regeneration. Adipose tissue-derived MSCs (ADSCs) are easily accessible and have a high capacity for expansion. Although the choice of culture media significantly influences the biological properties of MSCs, the optimal media for goat ADSCs (gADSCs) remains unclear. This study aimed to assess the effects of four commonly used culture media on gADSCs' culture characteristics, stem cell-specific immunophenotype, and differentiation. Results showed that MEM, DMEM/F12, and DMEM-LG were superior in maintaining cell morphology and culture parameters of gADSCs, such as cell adherence, metabolic activity, colony-forming potential, and population doubling. Conversely, DMEM-HG exhibited poor performance across all evaluated parameters. The gADSCs cultured in DMEM/F12 showed enhanced early proliferation and lower apoptosis. The cell surface marker distribution exhibited superior characteristics in gADSCs cultured in MEM and DMEM/F12. In contrast, the distribution was inferior in gADSCs cultured in DMEM-LG. DMEM/F12 and DMEM-LG culture media demonstrated a significantly higher potential for chondrogenic differentiation and DMEM-LG for osteogenic differentiation. In conclusion, DMEM/F12 is a suitable culture medium for propagating gADSCs as it effectively maintains cell morphology, growth parameters, proliferation and lower apoptosis while exhibiting desirable expression patterns of MSC-specific markers. These findings contribute to optimising culture conditions for gADSCs, enhancing their potential applications in disease treatment and regenerative medicine.

Magnetic Graphene Oxide Nanocomposites Boosts Craniomaxillofacial Bone Regeneration by Modulating circAars/miR-128-3p/SMAD5 Signaling Axis.

Background: The ability of nanomaterials to induce osteogenic differentiation is limited, which seriously imped the repair of craniomaxillofacial bone defect. Magnetic graphene oxide (MGO) nanocomposites with the excellent physicochemical properties have great potential in bone tissue engineering. In this study, we aim to explore the craniomaxillofacial bone defect repairment effect of MGO nanocomposites and its underlying mechanism. Methods: The biocompatibility of MGO nanocomposites was verified by CCK8, live/dead staining and cytoskeleton staining. The function of MGO nanocomposites induced osteogenic differentiation of BMSCs was investigated by ALP activity detection, mineralized nodules staining, detection of osteogenic genes and proteins, and immune-histochemical staining. BMSCs with or without MGO osteogenic differentiation induction were collected and subjected to high-throughput circular ribonucleic acids (circRNAs) sequencing, and then crucial circRNA circAars was screened and identified. Bioinformatics analysis, Dual-luciferase reporter assay, RNA binding protein immunoprecipitation (RIP), fluorescence in situ hybridization (FISH) and osteogenic-related examinations were used to further explore the ability of circAars to participate in MGO nanocomposites regulation of osteogenic differentiation of BMSCs and its potential mechanism. Furthermore, critical-sized calvarial defects were constructed and were performed to verify the osteogenic differentiation induction effects and its potential mechanism induced by MGO nanocomposites. Results: We verify the good biocompatibility and osteogenic differentiation improvement effects of BMSCs mediated by MGO nanocomposites. Furthermore, a new circRNA-circAars, we find and identify, is obviously upregulated in BMSCs mediated by MGO nanocomposites. Silencing circAars could significantly decrease the osteogenic ability of MGO nanocomposites. The underlying mechanism involved circAars sponging miR-128-3p to regulate the expression of SMAD5, which played an important role in the repair craniomaxillofacial bone defects mediated by MGO nanocomposites. Conclusion: We found that MGO nanocomposites regulated osteogenic differentiation of BMSCs via the circAars/miR-128-3p/SMAD5 pathway, which provided a feasible and effective strategy for the treatment of craniomaxillofacial bone defects.

KAT2A-mediated succinylation modification of notch1 promotes the proliferation and differentiation of dental pulp stem cells by activating notch pathway.

BACKGROUND: Dental pulp stem cells (DPSCs) are a kind of undifferentiated dental mesenchymal stem cells with strong self-renewal ability and multi-differentiation potential. This study aimed to investigate the regulatory functions of succinylation modification in DPSCs. METHODS: DPSCs were isolated from the dental pulp collected from healthy subjects, and then stem cell surface markers were identified using flow cytometry. The osteogenic differentiation ability of DPSCs was verified by alkaline phosphatase (ALP) and alizarin red staining methods, while adipogenic differentiation was detected by oil red O staining. Meanwhile, the mRNA of two desuccinylases (SIRT5 and SIRT7) and three succinylases (KAT2A, KAT3B, and CPT1A) in DPSCs before and after mineralization induction were detected using quantitative real-time PCR. The cell cycle was measured by flow cytometry, and the expression of bone-specific genes, including COL1a1 and Runx2 were evaluated by western blotting and were combined for the proliferation and differentiation of DPSCs. Co-immunoprecipitation (co-IP) and immunofluorescence were combined to verify the binding relationship between proteins. RESULTS: The specific markers of mesenchymal stem cells were highly expressed in DPSCs, while the osteogenic differentiation ability of isolated DPSCs was confirmed via ALP and alizarin red staining. Similarly, the oil red O staining also verified the adipogenic differentiation ability of DPSCs. The levels of KAT2A were found to be significantly upregulated in mineralization induction, which significantly decreased the ratio of G0/G1 phase and increased S phase cells; converse results regarding cell cycle distribution were obtained when KAT2A was inhibited. Moreover, overexpression of KAT2A promoted the differentiation of DPSCs, while its inhibition exerted the opposite effect. The elevated KAT2A was found to activate the Notch1 signaling pathway, which succinylated Notch1 at the K2177 site to increase their corresponding protein levels in DPSCs. The co-IP results showed that KAT2A and Notch1 were endogenously bound to each other, while inhibition of Notch1 reversed the effects of KAT2A overexpression on the DPSCs proliferation and differentiation. CONCLUSION: KAT2A interacted directly with Notch1, succinylating the Notch1 at the K2177 site to increase their corresponding protein levels in DPSCs. Similarly, KAT2A-mediated succinylation modification of Notch1 promotes the DPSCs proliferation and differentiation, suggesting that targeting KAT2A and Notch1 may contribute to tooth regeneration.

Effects of surface nanomorphology on the senescence of periodontal ligament stem cells.

OBJECTIVES: The effect of TiO2 nanotube morphology on the differentiation potency of senescent periodontal ligament stem cells was investigated. METHODS: Two types of titanium sheets with TiO2 nanotube morphology (20V-NT and 70V-NT) were prepared via anodic oxidation at 20 and 70 V separately, and their surface morphology was observed. Young periodontal ligament stem cells were cultivated in an osteogenic induction medium, and the most effective surface morphology in promoting osteogenic differentiation was selected. RO3306 and Nutlin-3a were used to induce the aging of young periodontal ligament stem cells, and senescent periodontal ligament stem cells were obtained. The osteogenic differentiation of senescent periodontal ligament stem cells was induced, and the effect of surface morphology on osteogenic differentiation was observed. RESULTS: Nanotube morphology was achieved on the surfaces of titanium sheets through anodic oxidation, and the diameters of the nanotubes increased with voltage. A significant difference in the effect of nanotube morphology was found among nanotubes with different diameters in the young periodontal ligament stem cells. The surface nanotube morphology of 20V-NT had a more significant effect that promoted osteogenic differentiation. Compared with a smooth titanium sheet, the surface nanotube morphology of 20V-NT increased the number of alkaline phosphatase-positive senescent periodontal ligament stem cells and promoted calcium deposition and the expression of osteogenic marker genes Runt-related transcription factor 2, osteopontin, and osteocalcin. CONCLUSIONS: A special nanotube morphology enhances the differentiation ability of senescent periodontal ligament stem cells, provides an effective method for periodontal regeneration, and further improves the performance of implants.

Synthetic biology-based bacterial extracellular vesicles displaying BMP-2 and CXCR4 to ameliorate osteoporosis.

Osteoporosis (OP) is a systematic bone disease characterized by low bone mass and fragile bone microarchitecture. Conventional treatment for OP has limited efficacy and long-term toxicity. Synthetic biology makes bacterial extracellular vesicle (BEVs)-based therapeutic strategies a promising alternative for the treatment of OP. Here, we constructed a recombinant probiotics Escherichia coli Nissle 1917-pET28a-ClyA-BMP-2-CXCR4 (ECN-pClyA-BMP-2-CXCR4), in which BMP-2 and CXCR4 were overexpressed in fusion with BEVs surface protein ClyA. Subsequently, we isolated engineered BEVs-BMP-2-CXCR4 (BEVs-BC) for OP therapy. The engineered BEVs-BC exhibited great bone targeting in vivo. In addition, BEVs-BC had good biocompatibility and remarkable ability to promote osteogenic differentiation of BMSCs. Finally, the synthetic biology-based BEVs-BC significantly prevented the OP in an ovariectomized (OVX) mouse model. In conclusion, we constructed BEVs-BC with both bone-targeting and bone-forming in one-step using synthetic biology, which provides an effective strategy for OP and has great potential for industrialization.

Current perspectives on the multiple roles of osteoclasts: Mechanisms of osteoclast-osteoblast communication and potential clinical implications.

Bone remodeling is a complex process involving the coordinated actions of osteoblasts and osteoclasts to maintain bone homeostasis. While the influence of osteoblasts on osteoclast differentiation is well established, the reciprocal regulation of osteoblasts by osteoclasts has long remained enigmatic. In the past few years, a fascinating new role for osteoclasts has been unveiled in promoting bone formation and facilitating osteoblast migration to the remodeling sites through a number of different mechanisms, including the release of factors from the bone matrix following bone resorption and direct cell-cell interactions. Additionally, considerable evidence has shown that osteoclasts can secrete coupling factors known as clastokines, emphasizing the crucial role of these cells in maintaining bone homeostasis. Due to their osteoprotective function, clastokines hold great promise as potential therapeutic targets for bone diseases. However, despite long-standing work to uncover new clastokines and their effect in vivo, more substantial efforts are still required to decipher the mechanisms and pathways behind their activity in order to translate them into therapies. This comprehensive review provides insights into our evolving understanding of the osteoclast function, highlights the significance of clastokines in bone remodeling, and explores their potential as treatments for bone diseases suggesting future directions for the field.

Oxidized phospholipid POVPC contributes to vascular calcification by triggering ferroptosis of vascular smooth muscle cells.

Vascular calcification is an actively regulated biological process resembling bone formation, and osteogenic differentiation of vascular smooth muscle cells (VSMCs) plays a crucial role in this process. 1-Palmitoyl-2-(5'-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC), an oxidized phospholipid, is found in atherosclerotic plaques and has been shown to induce oxidative stress. However, the effects of POVPC on osteogenic differentiation and calcification of VSMCs have yet to be studied. In the present study, we investigated the role of POVPC in vascular calcification using in vitro and ex vivo models. POVPC increased mineralization of VSMCs and arterial rings, as shown by alizarin red staining. In addition, POVPC treatment increased expression of osteogenic markers Runx2 and BMP2, indicating that POVPC promotes osteogenic transition of VSMCs. Moreover, POVPC increased oxidative stress and impaired mitochondria function of VSMCs, as shown by increased ROS levels, impairment of mitochondrial membrane potential, and decreased ATP levels. Notably, ferroptosis triggered by POVPC was confirmed by increased levels of intracellular ROS, lipid ROS, and MDA, which were decreased by ferrostatin-1, a ferroptosis inhibitor. Furthermore, ferrostatin-1 attenuated POVPC-induced calcification of VSMCs. Taken together, our study for the first time demonstrates that POVPC promotes vascular calcification via activation of VSMC ferroptosis. Reducing the levels of POVPC or inhibiting ferroptosis might provide a novel strategy to treat vascular calcification.

[Biological characteristics and osteogenic differentiation of magnesium-doped nanoporous titanium coating].

PURPOSE: Bioactive magnesium ions were successfully incorporated into the nanoporous titanium base coating by micro-arc oxidation(MAO), and its physical properties and osteogenic effects were explored. METHODS: Non-magnesium-containing and magnesium-containing titanium porous titanium coatings(MAO, MAO-mg) were prepared by changing the composition of MAO electrolyte and controlling the doping of magnesium in porous titanium coatings. The samples were characterized by scanning electron microscope (SEM), roughness, contact angle and energy dispersive X-ray spectrometer (EDS). Mg2+ release ability of magnesium-doped nanoporous titanium coatings was determined by inductively coupled plasma/optical emission spectrometer(ICP-OES). The structure of the cytoskeleton was determined by live/dead double staining, CCK-8 detection of material proliferation-toxicity, and staining of ß-actin using FITC-phalloidin. The effects of the coating on osteogenic differentiation in vitro were determined by alizarin red (ARS), alkaline phosphatase (ALP) staining and real-time polymerase chain reaction (qRT-PCR). SPSS 25.0 software package was used for statistical analysis. RESULTS: The MAO electrolyte with magnesium ions did not change the surface characteristics of the porous titanium coating. Each group prepared by MAO had similar microporous structure(P＞0.05). There was no significant difference in surface roughness and contact angle between MAO treatment group (MAO, MAO-mg)(P＞0.05), but significantly higher than that of Ti group (P＜0.05). With the passage of cell culture time, MAO-mg group promoted cell proliferation (P＜0.05). MAO-mg group was significantly higher than other groups in ALP and ARS staining. The expression of Runx2 mRNA (P＜0.05), ALP(P＜0.05) and osteocalcin OCN(P＜0.05) in MAO-mg group was significantly higher than that in Ti and MAO groups. CONCLUSIONS: MAO successfully prepared magnesium-containing nanoporous titanium coating, and showed a significant role in promoting osteogenic differentiation.

[Genistein loaded by PRF improved bone healing in obese mice].

PURPOSE: To clarify the effect of genistein(GEN) on osteogenic differentiation and explore the effect of GEN loaded by platelet-rich fibrin (PRF) on the repair process of bone defects in obese mice. METHODS: In in vitro experiments, the effect of GEN(0, 0.1, 1, 10, 50 µmol/L) on the proliferation of mouse embryonic osteoblast precursor cells (MC3T3-E1) was determined by CCK 8. Alkaline phosphatase(ALP) staining and quantitative detection of ALP activity were performed to determine the changes of ALP activity in cells; RNA and protein expression levels of ALP, osteopontin (OPN) and osteocalcin (OCN) were detected by quantitative real-time PCR(qRT-PCR) and Western blot. Alizarin red staining was used to define the effect of GEN on mineralization of MC3T3-E1. To verify the feasibility of the PRF drug loading, the ultrastructure of PRF was subsequently observed under SEM. In in vivo experiments, obese C57 mouse models were established by high-fat diet feeding. On this basis, skull defect models with a diameter of 2.8 mm were established, and the prepared GEN/PRF complexes were placed into the bone defect area. The effects of GEN on skull defect repair in obese mice were evaluated by Micro-CT scanning and hematoxylin-eosin(H-E) staining. Statistical analysis was performed with GraphPad Prism 5.0 software package. RESULTS: CCK 8 results showed that 0.1, 1 µmol/L GEN promoted cell proliferation within 7 days(P＜0.05); 10 µmol/L GEN had no significant effect on the process of cell proliferation. From the second day, 50 µmol/L GEN significantly inhibited cell growth and showed cytotoxicity(P＜0.05). These two concentrations had similar effects in promoting cellular osteogenic differentiation. SEM results showed that PRF presented a 3-dimensional network structure, providing space for loading drug molecules. In in vivo experiments, the body weight of mice in the high-fat diet (HFD) group was 27.7% greater than that in the normal diet group(P＜0.05) and had abnormal glucose tolerance (P＜0.05). Micro-CT showed that compared with the normal diet group, the number of bone trabeculae in the femur of obese mice was decreased(P＜0.05), the distance between bone trabeculae was widened(P＜0.05), and the bone density was decreased (P＜0.05). In addition, GEN (0.1, 1.0 µmol/L) loaded by PRF increased bone volume fraction in the skull of obese mice (P＜0.05). H-E results showed that GEN/PRF promoted the healing of the bone defects. CONCLUSIONS: GEN promotes osteogenic differentiation of MC3T3-E1, and it can effectively accelerate the healing of cranial bone defects after loading with PRF in obese mice.

Melt Electrowriting Combined with Fused Deposition Modeling Printing for the Fabrication of Three-Dimensional Biomimetic Scaffolds for Osteotendinous Junction Regeneration.

Purpose: This study aims to explore a novel scaffold for osteotendinous junction regeneration and to preliminarily verify its osteogenic and tenogenic abilities in vitro. Methods: A polycaprolactone (PCL) scaffold with aligned and orthogonal fibers was created using melt electrowriting (MEW) and fused deposition modeling (FDM). The scaffold was coated with Type I collagen, and hydroxyapatite was carefully added to separate the regions intended for bone and tendon regeneration, before being rolled into a cylindrical shape. Human adipose-derived stem cells (hADSCs) were seeded to evaluate viability and differentiation. Scaffold characterization was performed with Scanning Electron Microscope (SEM). Osteogenesis was assessed by alkaline phosphatase (ALP) and Alizarin red staining, while immunostaining and transcription-quantitative polymerase chain reaction (RT-qPCR) evaluated osteogenic and tendogenic markers. Results: Scaffolds were developed in four variations: aligned (A), collagen-coated aligned (A+C), orthogonal (O), and mineral-coated orthogonal (O+M). SEM analysis confirmed surface morphology and energy-dispersive X-ray spectroscopy (EDS) verified mineral coating on O+M types. Hydrophilicity and mechanical properties were optimized in modified scaffolds, with A+C showing increased tensile strength and O+M improved in compression. hADSCs demonstrated good viability and morphology across scaffolds, withO+M scaffolds showing higher cell proliferation and osteogenic potential, and A and A+C scaffolds supporting tenogenic differentiation. Conclusion: This study confirms the potential of a novel PCL scaffold with distinct regions for osteogenic and tenogenic differentiation, supporting the regeneration of osteotendinous junctions in vitro.