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BACKGROUND: The osteogenic differentiation ability of exosomes derived from osteogenic mesenchymal stem cells is well established. However, their impact on the osteogenic differentiation of human periodontal ligament stem cells in an inflammatory microenvironment remains unclear. OBJECTIVE: To examine the impact of exosomes derived from osteogenesis-induced human periodontal ligament stem cells on the osteogenic differentiation of human periodontal ligament stem cells within an inflammatory microenvironment. METHODS: Human periodontal ligament stem cells were isolated and cultured. After 3 days of osteogenic induction, exosomes were extracted. Human periodontal ligament stem cells were divided into four groups. Control group was treated with osteogenesis-induced medium. The exosome group was treated with osteogenesis-induced medium containing 5 μg/mL exosomes. Inflammatory model and inflammatory model + exosome groups were treated with 1 μg/ mL lipopolysaccharide for 24 hours to construct a cellular inflammatory microenvironment. The inflammatory model group was treated with osteogenesis induced medium after lipopolysaccharide intervention. The inflammatory model + exosome group was treated with osteogenesis-induced medium containing 5 μg/mL exosome. The osteogenic differentiation ability of human periodontal ligament stem cells was assessed using alkaline phosphatase staining and alizarin red staining. The expressions of Runt-related transcription factor 2, osteopontin, osteoblast-specific transcription factor Osterix (OSX) and wnt pathway-related protein β-catenin were detected by real-time fluorescence quantitative PCR and western blot assay. RESULTS AND CONCLUSION: Compared with the control group, the relative area stained by alkaline phosphatase, the relative area stained by mineralized nodules and the expression levels of Runx2, osteopontin, and OSX were significantly decreased in the inflammatory model group (P < 0.05). Compared with the inflammatory model group, the expression of Runx2, osteopontin, and OSX in the inflammatory model + exosome group was significantly increased in the relative area of alkaline phosphatase staining, the relative area of mineralized nodules staining (P < 0.05). Compared with the control group, the expression of wnt pathway-related protein β-catenin was significantly increased in the inflammatory model group (P < 0.05). Compared with the inflammatory model group, the expression of β-catenin in the inflammatory model + exosome group was significantly decreased (P < 0.05). These findings indicate that exosomes derived from human periodontal ligament stem cells induced by bone formation can enhance the osteogenic differentiation of human periodontal ligament stem cells within an inflammatory microenvironment, and the mechanism may be related to wnt/β-catenin signaling pathway. [ABSTRACT FROM AUTHOR]

Polymethylmethacrylate (PMMA)-based acrylic bone cement is commonly used to fix bone and metallic implants in orthopedic procedures. The polymerization initiator tri-n-butylborane (TBB) has been reported to significantly reduce the cytotoxicity of PMMA-based bone cement compared to benzoyl peroxide (BPO). However, it is unknown whether this benefit is temporary or long-lasting, which is important to establish given that bone cement is expected to remain in situ permanently. Here, we compared the biocompatibility of PMMA-TBB and PMMA-BPO bone cements over several days. Rat femur-derived osteoblasts were seeded onto two commercially-available PMMA-BPO bone cements and experimental PMMA-TBB polymerized for one day, three days, or seven days. Significantly more cells attached to PMMA-TBB bone cement during the initial stages of culture than on both PMMA-BPO cements, regardless of the age of the materials. Proliferative activity and differentiation markers including alkaline phosphatase production, calcium deposition, and osteogenic gene expression were consistently and considerably higher in cells grown on PMMA-TBB than on PMMA-BPO, regardless of cement age. Although osteoblastic phenotypes were more favorable on older specimens for all three cement types, biocompatibility increased between three-day-old and seven-day-old PMMA-BPO specimens, and between one-day-old and three-day-old PMMA-TBB specimens. PMMA-BPO materials produced more free radicals than PMMA-TBB regardless of the age of the material. These data suggest that PMMA-TBB maintains superior biocompatibility over PMMA-BPO bone cements over prolonged periods of at least seven days post-polymerization. This superior biocompatibility can be ascribed to both low baseline cytotoxicity and a further rapid reduction in cytotoxicity, representing a new biological advantage of PMMA-TBB as a novel bone cement material.

Piper sarmentosum or ‘kaduk’ is a well-known herb plant in Malaysia. Its extracts were found to exhibit a bone-protective effect against osteoporotic rats. Our study aim is to morphologically observe the effect of P. sarmentosum ethyl acetate extract on the differentiation of human peripheral blood stem cells (PBSCs) into osteoblasts. P. sarmentosum extracts (1-900 µg/mL) prepared using 1% dimethyl sulfoxide (DMSO) were used in the cytotoxicity assay. Then, the differentiation assay was performed using concentrations of 1-50 µg/mL. The untreated cells acted as the negative control, while the cells cultured in 50 µg/mL ascorbic acid and 10 mM β-glycerophosphate was a positive control. The cytotoxicity effect and proliferation capacity of the cells were analyzed using Trypan Blue exclusion method, while the differentiation of PBSCs was observed using von Kossa staining and ALP gene expression analysis. The result showed a decrease in cells’ viability in a dose-dependent manner during cytotoxicity assay. After 14 days of the differentiation, a constant rate of proliferation could be observed in the treated cells and positive control, while the untreated cells showed an increase in proliferation. The mineralization of extract-treated cells showed significant differences (p

Abstract Background Osteoporosis is a common systemic bone disease that leads to bone fragility and increases the risk of fracture. However, the pathogenesis of osteoporosis is considered to be highly complex. The exosomes can regulate the communication between cells. The specific mechanism of information transmission between osteoblasts and endothelial cells is worthy of further study. Methods Exosomes were isolated and verified from senescent osteoblasts. The source and properties of exosomes were determined by TEM, particle size analysis and western blot. We established the co-culture model of endothelial cells and senescent osteoblasts. We used qRT-PCR to identify differentially expressed miRNAs. The functional changes of vascular endothelial cells were verified by cell transfection. β-Galactosidase cell senescence assay, Hoechst cell apoptosis assay, Ki67 cell proliferation assay and Transwell migration assay were used to verify cell senescence, apoptosis, proliferation, and migration. The potential target gene of miRNA was detected by bio-informatics pathway and double luciferase report. Results We discovered that senescent osteoblasts could promote the senescence and apoptosis of vascular endothelial cells and inhibit their proliferation and migration. miR-214-3p was upregulated in senescent osteoblast-derived exosomes. miR-214-3p could effectively promote senescence and apoptosis of endothelial cells and inhibit proliferation and migration ability. L1CAM is a miR-214-3p direct target gene determined by bio-informatics and double luciferase report. Conclusions In conclusion, senescent osteoblast-derived exosomes can accelerate endothelial cell senescence through miR-214-3p/L1CAM pathway. Our experiments reveal the role of exosomes in the skeletal microenvironment.

Background: Periodontitis is an inflammatory disease of the teeth-supporting tissues caused by microorganisms. Robusta coffee bean extract has antibacterial properties due to its caffeine, flavonoids, trigonelline, and chlorogenic acid contents. The robusta coffee bean extract also regulates alveolar bone healing through bone remodelling. Aim: The study aimed to investigate robusta coffee bean extract to inhibit bacterial growth and accelerate bone repair in vitro and in vivo. Methods: This study used the paper disc diffusion method with the research group of robusta coffee bean extract with concentrations of 50%, 25%, 12.5%, 6.25%, and negative control, as much as 20 and dripped onto the disc paper then placed on the surface of the agar media that had been inoculated with bacteria. The diameter of the inhibition zone was measured. Twenty periodontitis rat models were given 0.05 ml of the robusta coffee bean extract on the molars and put in a periodontal pocket for seven days. Rats were decapitated, and alveolar bone tissues were stained with HE and IHC staining. The number of osteoclasts, osteoblasts and BMP-2 was counted using a microscope. Statistical test with Kruskal Wallis followed by Mann Whitney showed a p-value of

Abstract Background Despite extensive research, there is still a need for safe and effective agents to promote spinal fusion. Interleukin (IL)-1β is an important factor which influences the bone repair and remodelling. The purpose of our study was to determine the effect of IL-1β on sclerostin in osteocytes and to explore whether inhibiting the secretion of sclerostin from osteocytes can promote spinal fusion at early stages. Methods Small-interfering RNA was used to suppress the secretion of sclerostin in Ocy454 cells. MC3T3-E1 cells were cocultured with Ocy454 cells. Osteogenic differentiation and mineralisation of MC3T3-E1 cells were evaluated in vitro. SOST knock-out rat generated using the CRISPR-Cas9 system and rat spinal fusion model was used in vivo. The degree of spinal fusion was assessed by manual palpation, radiographic analysis and histological analysis at 2 and 4 weeks. Results We found that IL-1β level had a positive association with sclerostin level in vivo. IL-1β promoted the expression and secretion of sclerostin in Ocy454 cells in vitro. Inhibition of IL-1β-induced secretion of sclerostin from Ocy454 cells could promote the osteogenic differentiation and mineralisation of cocultured MC3T3-E1 cells in vitro. The extent of spinal graft fusion was greater in SOST-knockout rats than in wild-type rats at 2 and 4 weeks. Conclusions The results demonstrate that IL-1β contributes to a rise in the level of sclerostin at early stages of bone healing. Suppressing sclerostin may be an important therapeutic target capable of promoting spinal fusion at early stages.

Increasing the osteogenic differentiation ability and decreasing the adipogenic differentiation ability of bone marrow mesenchymal stem cells (BMSCs) is a potential strategy for the treatment of osteoporosis (OP). Naturally derived oligosaccharides have shown significant anti-osteoporotic effects. Nystose (NST), an oligosaccharide, was isolated from the roots of Morinda officinalis How. (MO). The aim of the present study was to investigate the effects of NST on bone loss in ovariectomized mice, and explore the underlying mechanism of NST in promoting differentiation of BMSCs to osteoblasts. Administration of NST (40, 80 and 160 mg/kg) and the positive control of estradiol valerate (0.2 mg/kg) for 8 weeks significantly prevented bone loss induced by ovariectomy (OVX), increased the bone mass density (BMD), improved the bone microarchitecture and reduced urine calcium and deoxypyridinoline (DPD) in ovariectomized mice, while inhibited the increase of body weight without significantly affecting the uterus weight. Furthermore, we found that NST increased osteogenic differentiation, inhibited adipogenic differentiation of BMSCs in vitro, and upregulated the expression of the key proteins of BMP and Wnt/β-catenin pathways. In addition, Noggin and Dickkopf-related protein-1 (DKK-1) reversed the effect of NST on osteogenic differentiation and expression of the key proteins in BMP and Wnt/β-catenin pathway. The luciferase activities and the molecular docking analysis further supported the mechanism of NST. In conclusion, these results indicating that NST can be clinically used as a potential alternative medicine for the prevention and treatment of postmenopausal osteoporosis.

Calcium and its putative receptor (CaSR) control skeletal development by pacing chondrocyte differentiation and mediating osteoblast (OB) function during endochondral bone formation-an essential process recapitulated during fracture repair. Here, we delineated the role of the CaSR in mediating transition of callus chondrocytes into the OB lineage and subsequent bone formation at fracture sites and explored targeting CaSRs pharmacologically to enhance fracture repair. In chondrocytes cultured from soft calluses at a closed, unfixed fracture site, extracellular [Ca2+ ] and the allosteric CaSR agonist (NPS-R568) promoted terminal differentiation of resident cells and the attainment of an osteoblastic phenotype. Knockout (KO) of the Casr gene in chondrocytes lengthened the chondrogenic phase of fracture repair by increasing cell proliferation in soft calluses but retarded subsequent osteogenic activity in hard calluses. Tracing growth plate (GP) and callus chondrocytes that express Rosa26-tdTomato showed reduced chondrocyte transition into OBs (by >80%) in the spongiosa of the metaphysis and in hard calluses. In addition, KO of the Casr gene specifically in mature OBs suppressed osteogenic activity and mineralizing function in bony calluses. Importantly, in experiments using PTH (1-34) to enhance fracture healing, co-injection of NPS-R568 not only normalized the hypercalcemic side effects of intermittent PTH (1-34) treatment in mice but also produced synergistic osteoanabolic effects in calluses. These data indicate a functional role of CaSR in mediating chondrogenesis and osteogenesis in the fracture callus and the potential of CaSR agonism to facilitate fracture repair. © 2019 American Society for Bone and Mineral Research.

In this study of the alterations of Glypicans 1 to 6 (GPCs) and Notum in plasma, bone marrow mesenchymal stromal cells (BM-MSCs) and osteoblasts in Osteoarthritis (OA), the levels of GPCs and Notum in the plasma of 25 patients and 24 healthy subjects were measured. In addition, BM-MSCs from eight OA patients and eight healthy donors were cultured over a period of 21 days using both a culture medium and an osteogenic medium. Protein and gene expression levels of GPCs and Notum were determined using ELISA and qPCR at 0, 7, 14 and 21 days. GPC5 and Notum levels decreased in the plasma of OA patients, while the BM-MSCs of OA patients showed downexpression of GPC6 and upregulation of Notum. A decrease in GPC5 and Notum proteins and an increase in GPC3 were found. During osteogenic differentiation, elevated GPCs 2, 4, 5, 6 and Notum mRNA levels and decreased GPC3 were observed in patients with OA. Furthermore, the protein levels of GPC2, GPC5 and Notum decreased, while the levels of GPC3 increased. Glypicans and Notum were altered in BM-MSCs and during osteogenic differentiation from patients with OA. The alterations found point to GPC5 and Notum as new candidate biomarkers of OA pathology.

Leukocyte and Platelet-Rich Fibrin (L-PRF) is part of the second generation of platelet-concentrates. L-PRF derived from nonsmokers has been used in surgical procedures, with its beneficial effects in wound healing being proven to stimulate biological activities such as cell proliferation, angiogenesis, and differentiation. Cigarette smoking exerts detrimental effects on tissue healing and is associated with post-surgical complications; however, evidence about the biological effects of L-PRF derived from smokers is limited. This study evaluated the impact of L-PRF secretome (LPRFS) derived from smokers and nonsmokers on angiogenesis and osteoblast differentiation. LPRFS was obtained by submerging L-PRF membranes derived from smokers or nonsmokers in culture media and was used to treat endothelial cells (HUVEC) or SaOs-2 cells. Angiogenesis was evaluated by tubule formation assay, while osteoblast differentiation was observed by alkaline phosphatase and osterix protein levels, as well as in vitro mineralization. LPRFS treatments increased angiogenesis, alkaline phosphatase, and osterix levels. Treatment with 50% of LPRFS derived from smokers and nonsmokers in the presence of osteogenic factors stimulates in vitro mineralization significantly. Nevertheless, differences between LPRFS derived from smokers and nonsmokers were not found. Both LPRFS stimulated angiogenesis and osteoblast differentiation in vitro; however, clinical studies are required to determine the beneficial effect of LPRFS in smokers.

The rapid aging of the population worldwide presents a significant social and economic challenge, particularly due to osteoporotic fractures, primarily resulting from an imbalance between osteoclast-mediated bone resorption and osteoblast-mediated bone formation. While conventional therapies offer benefits, they also present limitations and a range of adverse effects. This study explores the protective impact of Neorhodomela munita ethanol extract (EN) on osteoporosis by modulating critical pathways in osteoclastogenesis and apoptosis. Raw264.7 cells and Saos-2 cells were used for in vitro osteoclast and osteoblast models, respectively. By utilizing various in vitro methods to detect osteoclast differentiation/activation and osteoblast death, it was demonstrated that the EN’s potential to inhibit RANKL induced osteoclast formation and activation by targeting the MAPKs-NFATc1/c-Fos pathway and reducing H2O2-induced cell death through the downregulation of apoptotic signals. This study highlights the potential benefits of EN for osteoporosis and suggests that EN is a promising natural alternative to traditional treatments.

Antioxidant vanillin (4-hydroxy-3-methoxybenzaldehyde) is used as a flavoring in foods, beverages, and pharmaceuticals. Vanillin possesses various biological effects, such as antioxidant, anti-inflammatory, antibacterial, and anticancer properties. This study aimed to investigate the biological activities of vanillin purified from Adenophora triphylla var. japonica Hara on bone-forming processes. Vanillin treatment induced mineralization as a marker for mature osteoblasts, after stimulating alkaline phosphatase (ALP) staining and activity. The bone-forming processes of vanillin are mainly mediated by the upregulation of the bone morphogenetic protein 2 (BMP2), phospho-Smad1/5/8, and runt-related transcription factor 2 (RUNX2) pathway during the differentiation of osteogenic cells. Moreover, vanillin promoted osteoblast-mediated bone-forming phenotypes by inducing migration and F-actin polymerization. Furthermore, we validated that vanillin-mediated bone-forming processes were attenuated by noggin and DKK1. Finally, we demonstrated that vanillin-mediated antioxidant effects prevent the death of osteoblasts during bone-forming processes. Overall, vanillin has bone-forming properties through the BMP2-mediated biological mechanism, indicating it as a bone-protective compound for bone health and bone diseases such as periodontitis and osteoporosis.

Orally administered “tortoiseshell and deer antler gelatin” is a common traditional medicine for patients with osteoporosis or osteoarthritis. From the pepsin-digested gelatin, we previously isolated and identified the osteoblast-stimulating pentapeptide, TSKYR. Its trypsin digestion products include the dipeptide YR, enhancing calcium ion uptake, and tripeptide TSK, resulting in remarkable 30- and 50-fold increases in mineralized nodule area and density in human osteoblast cells. These peptides were chemically synthesized in this study. The composition of deer antler preparations comprises not only proteins and peptides but also a significant quantity of metal ion salts. By analyzing osteoblast growth in the presence of peptide YR and various metal ions, we observed a synergistic effect of calcium and strontium on the effects of YR. Those peptides could also stimulate the growth of C2C12 skeletal muscle cells and human chondrocytes, increasing collagen and glycosaminoglycan content in a three-dimensional environment. The maintenance of bone homeostasis relies on a balance between osteoclasts and osteoblasts. Deer antler peptides were observed to inhibit osteoclast differentiation, as evidenced by ROS generation, tartrate-resistant acid phosphatase (TRACP) activity assays, and gene expression in RAW264.7 cells. In summary, our findings provide a deep understanding of the efficacy of this folk medicine.

Fibrodysplasia ossificans progressiva (FOP) is a rare congenital disorder characterized by abnormal bone formation due to ACVR1 gene mutations. The identification of the molecular mechanisms underlying the ectopic bone formation and expansion in FOP is critical for the effective treatment or prevention of HO. Here we find that Hh signaling activation is required for the aberrant ectopic bone formation in FOP. We show that the expression of Indian hedgehog (Ihh), a Hh ligand, as well as downstream Hh signaling, was increased in ectopic bone lesions in Acvr1R206H; ScxCre mice. Pharmacological treatment with an Ihh-neutralizing monoclonal antibody dramatically reduced chondrogenesis and ectopic bone formation. Moreover, we find that the activation of Yap in the FOP mouse model and the genetic deletion of Yap halted ectopic bone formation and decreased Ihh expression. Our mechanistic studies showed that Yap and Smad1 directly bind to the Ihh promoter and coordinate to induce chondrogenesis by promoting Ihh expression. Therefore, the Yap activation in FOP lesions promoted ectopic bone formation and expansion in both cell-autonomous and non-cell-autonomous manners. These results uncovered the crucial role of the Yap-Ihh axis in FOP pathogenesis, suggesting the inhibition of Ihh or Yap as a potential therapeutic strategy to prevent and reduce HO.

Diabetes mellitus has been shown to delay osseointegration of titanium dental implants. This study tested the hypothesis that serum derived from diabetes negatively affects osteoblast adhesion to polystyrene and titanium surfaces, partly through the presence of advanced glycation end products (AGEs). Materials and Methods: Twenty-four Sprague-Dawley rats were divided into three groups: normoglycemic control, streptozotocin-induced diabetic group, and diabetic group treated with the AGE inhibitor aminoguanidine. Polystyrene or titanium disks were preincubated in serum derived from each group. Human osteoblasts transfected with green fluorescent protein (GFP) were cultured, and the number of adherent osteoblasts was quantified. High-pressure liquid chromatography (HPLC) was used to fractionate eluates, which were further characterized by western blot with AGE antibody and adhesion assays. In parallel, sera derived from healthy patients, patients with controlled diabetes, and patients with uncontrolled diabetes were utilized for osteoblast adhesion assay and western blot.Results: Diabetic serum significantly reduced the number of adherent osteoblast and osteoblast aggregates on titanium disks, whereas aminoguanidine-treated serum rescued the effect of diabetes on the number of adherent osteoblast aggregates. Fractionated diabetic serum revealed distinct AGE bands at ~100 kDa and 44 kDa, whereas healthy serum did not express any. In human serum samples, both controlled and uncontrolled diabetes led to a significant reduction in the number of adherent osteoblasts on polystyrene and titanium surfaces compared with normoglycemic serum. This correlated with presence of AGEs in western blot in diabetic but not in healthy serum. Conclusion: Osteoblast adhesion on the titanium surface was greatly reduced by the exposure of serum derived from diabetic rats or humans. Recovery of osteoblast aggregates by aminoguanidine treatment suggests that AGEs played a role in this negative effect. The correlating presence of AGEs from the fractionated sera of diabetic rats or humans and impaired osteoblast adhesion on the titanium surface further supports this role. [ABSTRACT FROM AUTHOR]

Purpose: Autogenous bone grafts are considered a "gold standard." The success of autografts mainly depends on their ability to promote an osteogenic response. The aim of this study was to collect autogenous bone during implant osteotomy preparation using two different drilling protocols and to evaluate and compare the proliferation and differentiation ability of the collected bone particles. Materials and Methods: Autogenous bone particles were harvested from 20 patients during implant osteotomy preparation using two different drilling protocols: (1) standard drilling protocol with saline irrigation (according to the manufacturer's recommendation) and (2) low-speed drilling protocol without saline irrigation (speed < 200 rpm). Collected bone samples were cultured in growth medium, and after 2 to 3 weeks, cells that grew out from bone grafts were cultured in the normal medium as well as in osteogenic medium for days 0, 4, 7, and 20. Scanning electron microscopy, alizarin red/toluidine blue staining, DNA, ALP, and calcium content measurements were performed. Repeated measures analysis of variance (ANOVA) with Bonferroni's test was employed to analyze the data of this study. Results: The total DNA content was significantly higher for the low-speed drilling samples compared with the standard drilling on day 4 (P < .05), day 7 (P < .01), and day 20 (P < .001) in the normal medium and on day 7 (P < .01) and day 20 (P < .01) in the osteogenic medium. Besides, calcium measurements and mineralized matrix formation observed with alizarin red/toluidine blue staining were significantly higher for the low-speed drilling group compared with the standard drilling group. Conclusion: Osteogenic efficacy (differentiation and proliferation) of autogenous bone particles collected using low-speed drilling was superior compared with standard drilling samples. [ABSTRACT FROM AUTHOR]

This article explores the effects of hemin supplementation on bone precursor cells. The study investigates how hemin affects the proliferation, differentiation, and migration of MC3T3-E1 pre-osteoblasts. The results show that low concentrations of hemin increase cell proliferation and migration, while higher concentrations adversely affect cell attachment and survival. Additionally, the study finds that hemin does not significantly impact osteoblastic differentiation after 7 days, but delays mineralization after 14 days. Further research is needed to understand the molecular mechanisms involved in hemin modulation of bone metabolism. [Extracted from the article]

ABSTRACT Bone has the fascinating ability to self‐regenerate. However, under certain conditions, such as type 2 diabetes mellitus (T2DM), this ability is impaired. T2DM is a chronic metabolic disease known by the presence of elevated blood glucose levels that is associated with reduced bone regeneration capability, high fracture risk, and eventual non‐union risk after a fracture. Several mechanical and biological factors relevant to bone regeneration have been shown to be affected in a diabetic environment. However, whether impaired bone regeneration in T2DM can be explained due to mechanical or biological alterations remains unknown. To elucidate the relevance of either one, the aim of this study was to investigate the relative contribution of T2DM‐related alterations on either cellular activity or mechanical stimuli driving bone regeneration. A previously validated in silico computer modeling approach that was capable of explaining bone regeneration in uneventful conditions of healing was further developed to investigate bone regeneration in T2DM. Aspects analyzed included the presence of mesenchymal stromal cells (MSCs), cellular migration, proliferation, differentiation, apoptosis, and cellular mechanosensitivity. To further verify the computer model findings against in vivo data, an experimental setup was replicated, in which regeneration was compared in healthy and diabetic after a rat femur bone osteotomy stabilized with plate fixation. We found that mechanical alterations had little effect on the reduced bone regeneration in T2DM and that alterations in MSC proliferation, MSC migration, and osteoblast differentiation had the highest effect. In silico predictions of regenerated bone in T2DM matched qualitatively and quantitatively those from ex vivo μCT at 12 weeks post‐surgery when reduced cellular activities reported in previous in vitro and in vivo studies were included in the model. The presented findings here could have clinical implications in the treatment of bone fractures in patients with T2DM. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Type H vessels are specialized blood vessels found in the bone marrow that are closely associated with osteogenic activity. They are characterized by high expression of endomucin and CD31. Type H vessels form in the cancellous bone area during long bone development to provide adequate nutritional support for cells near the growth plate. They also influence the proliferation and differentiation of osteoprogenitors and osteoclasts in a paracrine manner, thereby creating a suitable microenvironment to facilitate new bone formation. Because of the close relationship between type H vessels and osteogenic activity, it has been found that type H vessels play a role in the physiological and pathological processes of bone diseases such as fracture healing, osteoporosis, osteoarthritis, osteonecrosis, and tumor bone metastasis. Moreover, experimental treatments targeting type H vessels can improve the outcomes of these diseases. Here, we reviewed the molecular mechanisms related to type H vessels and their associated osteogenic activities, which are helpful in further understanding the role of type H vessels in bone metabolism and will provide a theoretical basis and ideas for comprehending bone diseases from the vascular perspective.

Abstract Background Adolescent idiopathic scoliosis (AIS) is a scoliotic deformity of unknown etiology that occurs during adolescent development. Abnormal bone metabolism is closely related to AIS, but the cause is uncertain. Recent studies have shown that heat shock protein 27 (HSP27) and its phosphorylation (pHSP27) play important roles in bone metabolism. However, whether HSP27 and pHSP27 are involved in abnormal bone metabolism in AIS is unclear. Methods Osteoblasts (OBs) and bone marrow stem cells (BMSCs) were extracted from the facet joints and bone marrow of AIS patients and controls who underwent posterior spinal fusion surgery. The expression levels of HSP27 and pHSP27, as well as the expression levels of bone formation markers in OBs from AIS patients and controls, were examined by quantitative real‐time PCR (qRT–PCR) and Western blotting. The mineralization ability of OBs from AIS patients and controls was analyzed by alizarin red staining after osteogenic differentiation. Heat shock and thiolutin were used to increase the levels of pHSP27 in OBs, and the levels of bone formation markers were also investigated. In addition, the levels of pHSP27 and the bone formation ability of BMSCs from AIS patients and controls were investigated after heat shock treatment. Results Lower pHSP27 levels and impaired osteogenic differentiation abilities were observed in the OBs of AIS patients than in those of controls. Thiolutin increased HSP27 phosphorylation and increased the mRNA levels of SPP1 and ALPL in OBs from AIS patients. Heat shock treatment increased SPP1 and HSP27 mRNA expression, pHSP27 levels, OCN expression, and mineralization ability of both OBs and BMSCs from AIS patients. Conclusion Heat shock treatment and thiolutin can increase the levels of pHSP27 and further promote the bone formation of OBs and BMSCs from AIS patients. Therefore, decreased pHSP27 levels may be associated with abnormal bone metabolism in AIS patients.

The endoplasmic reticulum (ER) is the main site for protein synthesis, folding, and secretion, and accumulation of the unfolded/misfolded proteins in the ER may induce ER stress. ER stress is an important participant in various intracellular signaling pathways. Prolonged- or high-intensity ER stress may induce cell apoptosis. Osteoporosis, characterized by imbalanced bone remodeling, is a global disease caused by many factors, such as ER stress. ER stress stimulates osteoblast apoptosis, increases bone loss, and promotes osteoporosis development. Many factors, such as the drug’s adverse effects, metabolic disorders, calcium ion imbalance, bad habits, and aging, have been reported to activate ER stress, resulting in the pathological development of osteoporosis. Increasing evidence shows that ER stress regulates osteogenic differentiation, osteoblast activity, and osteoclast formation and function. Various therapeutic agents have been developed to counteract ER stress and thereby suppress osteoporosis development. Thus, inhibition of ER stress has become a potential target for the therapeutic management of osteoporosis. However, the in-depth understanding of ER stress in the pathogenesis of osteoporosis still needs more effort.

Blood vessel formation is an important initial step for bone formation during development as well as during remodelling and repair in the adult skeleton. This results in a heavily vascularized tissue where endothelial cells and skeletal cells are constantly in crosstalk to facilitate homeostasis, a process that is mediated by numerous environmental signals, including mechanical loading. Breakdown in this communication can lead to disease and/or poor fracture repair. Therefore, this study aimed to determine the role of mature bone cells in regulating angiogenesis, how this is influenced by a dynamic mechanical environment, and understand the mechanism by which this could occur. Herein, we demonstrate that both osteoblasts and osteocytes coordinate endothelial cell proliferation, migration, and blood vessel formation via a mechanically dependent paracrine mechanism. Moreover, we identified that this process is mediated via the secretion of extracellular vesicles (EVs), as isolated EVs from mechanically stimulated bone cells elicited the same response as seen with the full secretome, while the EV-depleted secretome did not elicit any effect. Despite mechanically activated bone cell-derived EVs (MA-EVs) driving a similar response to VEGF treatment, MA-EVs contain minimal quantities of this angiogenic factor. Lastly, a miRNA screen identified mechanoresponsive miRNAs packaged within MA-EVs which are linked with angiogenesis. Taken together, this study has highlighted an important mechanism in osteogenic-angiogenic coupling in bone and has identified the mechanically activated bone cell-derived EVs as a therapeutic to promote angiogenesis and potentially bone repair. [ABSTRACT FROM AUTHOR]

The aim of this study was to develop a solvent‐free polylactic acid (PLA) open‐cell porous scaffold for bone tissue engineering using compression molding and a new chemical foaming compound, that we named CFCO. The latter is composed of a blend of PLA and azodicarbonamide (ADA) as a chemical foaming agent, with the addition of chitosan‐grafted PLA (CS‐g‐PLA) copolymer at various concentrations. The novelty of this approach is that during the foaming, that is, during the decomposition of the ADA foaming agent, the CS‐g‐PLA copolymer is projected toward the surface of the pores and strongly adhere there, which was confirmed by scanning electron microscopy and confocal microscopy. The results showed that at 6.90 wt% of CS‐g‐PLA copolymer, the immobilization of the latter on the surface of the pores led to a 52% increase in cell proliferation compared to the pure PLA control sample and a 26% increase compared to scaffolds that had 10 wt% of CS‐g‐PLA copolymer dispersed throughout the entire PLA matrix. [ABSTRACT FROM AUTHOR]

Introduction: Neuropilin 2 (NRP2) mediates the effects of class 3 semaphorins and vascular endothelial growth factor and is implicated in axonal guidance and angiogenesis. Moreover, NRP2 expression is suggested to be involved in the regulation of bone homeostasis. Indeed, osteoblasts and osteoclasts express NRP2 and male and female global Nrp2 knockout mice have a reduced bone mass accompanied by reduced osteoblast and increased osteoclast counts. Methods: We first examined the in vitro effect of the calciotropic hormone 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on Nrp2 transcription in osteoblasts. We next generated mice with a conditional deletion of Nrp2 in the osteoblast cell lineage under control of the paired related homeobox 1 promoter and mice with a conditional Nrp2 knockdown in osteoclasts under control of the Lysozyme promoter. Mice were examined under basal conditions or after treatment with either the bone anabolic vitamin D3 analog WY 1048 or with 1,25(OH)2D3. Results and discussion: We show that Nrp2 expression is induced by 1,25(OH)2D3 in osteoblasts and is associated with enrichment of the vitamin D receptor in an intronic region of the Nrp2 gene. In male mice, conditional deletion of Nrp2 in osteoblast precursors and mature osteoblasts recapitulated the bone phenotype of global Nrp2 knockout mice, with a reduced cortical cross-sectional tissue area and lower trabecular bone content. However, female mice with reduced osteoblastic Nrp2 expression display a reduced cross-sectional tissue area but have a normal trabecular bone mass. Treatment with the vitamin D3 analog WY 1048 (0.4 µg/kg/d, 14 days, ip) resulted in a similar increase in bone mass in both genotypes and genders. Deleting Nrp2 from the osteoclast lineage did not result in a bone phenotype, even though in vitro osteoclastogenesis of hematopoietic cells derived from mutant mice was significantly increased. Moreover, treatment with a high dose of 1,25(OH)2D3 (0.5 µg/kg/d, 6 days, ip), to induce osteoclast-mediated bone resorption, resulted in a similar reduction in trabecular and cortical bone mass. In conclusion, osteoblastic Nrp2 expression is suggested to regulate bone homeostasis in a sex-specific manner. [ABSTRACT FROM AUTHOR]

Apolipoprotein E (ApoE), a ligand for low-density lipoprotein receptors, is strongly induced during osteogenesis and has a physiologic role in regulating osteoblast function, but the mechanisms of its action are still unclear. The study aims to elucidate the influence and molecular mechanisms of ApoE on bone formation. An ovariectomy-induced osteoporotic model were conducted in ApoE knockout (ApoE-/-) mice to study the effect of ApoE on the bone system. Bone quality were assessed through bone mineral density and histomorphometric analysis. To investigate the underlying role and mechanisms of ApoE during osteogenesis, primary osteoblasts from the calvariums of newborn ApoE-/- or wild-type (WT) mice were cultured in the osteoblastic differentiation medium in vitro for further research. Our animal experiment data showed that ApoE-/- mice exhibited bone loss, exacerbated by estrogen deprivation after ovariectomy. ApoE deficiency attenuated osteoblast activity and inhibited osteoblast osteogenesis, accompanied by decreased osterix expression. ApoE deficiency did not affect primary osteoblast viability and collagen-1 expression. Moreover, osteoprotegerin expression in ApoE-/- osteoblasts was reduced compared to WT controls. Our study demonstrated that ApoE gene deficiency contributed to bone loss and attenuated osteogenesis by down-regulating osterix expression. [ABSTRACT FROM AUTHOR]

Background: Osteoid osteoma is a painful benign osteoblastic lesion occurring mainly in the long bones. On the one hand, some studies have declared that post-treatment CT cannot determine the success or failure of radiofrequency ablation, on the other hand, some studies have stated that imaging follow-up can be helpful or even mandatory in some cases. The present study aims to evaluate Osteoid Osteoma's imaging features before or after radiofrequency ablation with or without curettage and during the followup period. Methods: In this retrospective and cross-sectional study, we reviewed twenty seven Osteoid Osteoma patients with radiofrequency ablation who were assessed via drilling with or without curettage from March 2015 to December 2019 at Nemazi Hospital, Shiraz University of Medical Sciences. Results: Radiofrequency ablation with drilling was used in 22(81.5%) patients, and for the rest 5(18.5%), radiofrequency ablation with curettage and drilling was performed as a treatment procedure. The overall clinical success rate was 92.6%(25/27), with a low complication rate (7.4%). The mean diameter of nidus in pre/pos treatment was 3.46±2.02 and 2.22±1.75 mm, respectively. Femur 12(44.4%) was the most common bone in the patients. Significant differences between pre/post treatment follow up examinations in nidus size (P=0.03), nidus diameter (P=0.02), bone and calcification size (P=0.005) were detected. Additionally, it depicts that the mean values of tumor size and cortical thickening decreased after treatment. Conclusion: It is noteworthy that the present study had some limitations, including the small sample size and the relatively short follow-up period. There is no significant difference between radiofrequency ablation after drilling and curettage in treating Osteoid Osteoma. It is concluded that although there was a significant difference in pre/post-treatment imaging, there is no need for continuous imaging follow-up in treated patients without clinical complications such as pain to mitigate radiation dose risks and healthcare expenses. [ABSTRACT FROM AUTHOR]

Osteoporosis is the most common metabolic bone disease. Although the development of osteoporosis is mostly asymptomatic, its ultimate consequence, namely fragile fracture, brings a huge medical and economic challenge to patients and the society. It is of great significance to prevent and treat it. Reproductive hormones play an important role in the growth and maintenance of mammalian bones. It is found that the reproductive hormone kisspeptin stimulates osteoblast differentiation and inhibits osteoclasts. Therefore, it may have clinical therapeutic potential. This paper reviews the important research findings and progress in this field, with a view to providing some reference for subsequent research. [ABSTRACT FROM AUTHOR]

Senile osteoporosis is a common metabolic bone disease associated with age, and the prevalence rate increases year by year. The research on the pathological mechanism of osteoporosis has attracted much clinical attention. Current studies have confirmed that bone loss caused by bone formation and bone resorption imbalance is the main pathological mechanism of osteoporosis, in which the process of bone formation is closely related to osteoblast proliferation, extracellular matrix maturation and bone matrix mineralization disorders, and may involve the correlation between osteoblasts and different related signaling pathways. This paper reviews the mechanism of osteoblasts and their mediated signaling pathways in senile osteoporosis, with a view to providing new ideas for the study and treatment of senile osteoporosis's pathological mechanism. [ABSTRACT FROM AUTHOR]

Skeletal progenitor: collagen interactions are critical for bone development and regeneration. Both collagen-binding integrins and discoidin domain receptors (DDR1 and DDR2) function as collagen receptors in bone. Each receptor is activated by a distinct collagen sequence; GFOGER for integrins and GVMGFO for DDRs. Specific triple helical peptides containing each of these binding domains were evaluated for ability to stimulate DDR2 and integrin signaling and osteoblast differentiation. GVMGFO peptide stimulated DDR2 Y740 phosphorylation and osteoblast differentiation as measured by induction of osteoblast marker mRNAs and mineralization without affecting integrin activity. In contrast, GFOGER peptide stimulated focal adhesion kinase (FAK) Y397 phosphorylation, an early measure of integrin activation, and to a lesser extent osteoblast differentiation without affecting DDR2-P. Significantly, the combination of both peptides cooperatively enhanced both DDR2 and FAK signaling and osteoblast differentiation, a response that was blocked in Ddr2 -deficient cells. These studies suggest that the development of scaffolds containing DDR and integrin-activating peptides may provide a new route for promoting bone regeneration. A method for stimulating osteoblast differentiation of skeletal progenitor cells is described that uses culture surfaces coated with a collagen-derived triple-helical peptide to selectively activate discoidin domain receptors. When this peptide is combined with an integrin-activating peptide, synergistic stimulation of differentiation is seen. This approach of combining collagen-derived peptides to stimulate the two main collagen receptors in bone (DDR2 and collagen-binding integrins) provides a route for developing a new class of tissue engineering scaffolds for bone regeneration. [Display omitted] [ABSTRACT FROM AUTHOR]

miR‐196b‐5p plays a role in various malignancies. We have recently reported its function in regulating adipogenesis. However, it remains to be clarified whether and how miR‐196b‐5p affects bone cells and bone homeostasis. In this study, in vitro functional experiments showed an inhibitory effect of miR‐196b‐5p on osteoblast differentiation. Mechanistic explorations revealed that miR‐196b‐5p directly targeted semaphorin 3a (Sema3a) and inhibited Wnt/β‐catenin signaling. SEMA3A attenuated the impaired osteogenesis induced by miR‐196b‐5p. Osteoblast‐specific miR‐196b transgenic mice showed significant reduction of bone mass. Trabecular osteoblasts were reduced and bone formation was suppressed, whereas osteoclasts, marrow adipocytes, and serum levels of bone resorption markers were increased in the transgenic mice. The osteoblastic progenitor cells from the transgenic mice had decreased SEMA3A levels and exhibited retarded osteogenic differentiation, whereas those marrow osteoclastic progenitors exhibited enhanced osteoclastogenic differentiation. miR‐196b‐5p and SEMA3A oppositely regulated the expression of receptor activator of nuclear factor‐κB ligand and osteoprotegerin. The calvarial osteoblastic cells expressing the transgene promoted osteoclastogenesis, whereas the osteoblasts overexpressing Sema3a inhibited it. Finally, in vivo transfection of miR‐196b‐5p inhibitor to the marrow reduced ovariectomy‐induced bone loss in mice. Our study has identified that miR‐196b‐5p plays a key role in osteoblast and osteoclast differentiation and regulates bone homeostasis. Inhibition of miR‐196b‐5p may be beneficial for amelioration of osteoporosis. © 2023 American Society for Bone and Mineral Research (ASBMR). [ABSTRACT FROM AUTHOR]

Background and Objectives: The incidence of diabetic osteoporosis, an important complication of diabetes mellitus, is increasing gradually. This study investigated the combined effect of the Zuogui pill (ZGP) and eldecalcitol (ED-71), a novel vitamin D analog, on type 2 diabetic osteoporosis (T2DOP) and explored their action mechanism. Materials and Methods: Blood glucose levels were routinely monitored in db/db mice while inducing T2DOP. We used hematoxylin and eosin staining, Masson staining, micro-computed tomography, and serum biochemical analysis to evaluate changes in the bone mass and blood calcium and phosphate levels of mice. Immunohistochemical staining was performed to assess the osteoblast and osteoclast statuses. The MC3T3-E1 cell line was cultured in vitro under a high glucose concentration and induced to undergo osteogenic differentiation. Quantitative real-time polymerase chain reaction, Western blot, immunofluorescence, ALP, and alizarin red staining were carried out to detect osteogenic differentiation and PI3K–AKT signaling pathway activity. Results: ZGP and ED-71 led to a dramatic decrease in blood glucose levels and an increase in bone mass in the db/db mice. The effect was strongest when both were used together. ZGP combined with ED-71 promoted osteoblast activity and inhibited osteoclast activity in the trabecular bone region. The in vitro results revealed that ZGP and ED-71 synergistically promoted osteogenic differentiation and activated the PI3K–AKT signaling pathway. The PI3K inhibitor LY294002 or AKT inhibitor ARQ092 altered the synergistic action of both on osteogenic differentiation. Conclusions: The combined use of ZGP and ED-71 reduced blood glucose levels in diabetic mice and promoted osteogenic differentiation through the PI3K–AKT signaling pathway, resulting in improved bone mass. Our study suggests that the abovementioned combination constitutes an effective treatment for T2DOP. [ABSTRACT FROM AUTHOR]

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A series of copper(II) complexes with the formula [Cu2+Hy(x)Car%] varying the molecular weight (MW) of Hyaluronic acid (Hy, x = 200 or 700 kDa) conjugated with carnosine (Car) present at different loading were synthesized and characterized via different spectroscopic techniques. The metal complexes behaved as Cu, Zn-superoxide dismutase (SOD1) mimics and showed some of the most efficient reaction rate values produced using a synthetic and water-soluble copper(II)-based SOD mimic reported to date. The increase in the percentage of Car moieties parallels the enhancement of the I50 value determined via the indirect method of Fridovich. The presence of the non-functionalized Hy OH groups favors the scavenger activity of the copper(II) complexes with HyCar, recalling similar behavior previously found for the copper(II) complexes with Car conjugated using β-cyclodextrin or trehalose. In keeping with the new abilities of SOD1 to activate protective agents against oxidative stress in rheumatoid arthritis and osteoarthritis diseases, Cu2+ interaction with HyCar promotes the nuclear translocation of erythroid 2-related factor that regulates the expressions of target genes, including Heme-Oxigenase-1, thus stimulating an antioxidant response in osteoblasts subjected to an inflammatory/oxidative insult. [ABSTRACT FROM AUTHOR]

Background: Osteoporosis remains a significant clinical challenge worldwide. Recent studies have shown that exosomes stimulate bone regeneration. Thus, it is worthwhile to explore whether exosomes could be a useful therapeutic strategy for osteoporosis. The purpose of this study was to investigate the effects of exosomes derived from human umbilical cord mesenchymal stem cells (hucMSCs) on osteoblast proliferation and differentiation. Methods: Exosomes were isolated from hucMSCs. Bioinformatics analysis was performed to identify the differentially expressed lncRNAs in myeloma-derived mesenchymal stem cells. Plasmids encoding LINC00520 or short hairpin RNA of LINC00520 were transfected into hucMSCs and then exosomes were isolated. After human osteoblasts hFOB1.19 were exposed to the obtained exosomes, cell survival, cell cycle, apoptosis and calcium deposits of hFOB1.19 cell were detected by MTT, 7-aminoactinomycin D, Annexin V-FITC/propidium iodide and Alizarin red staining, respectively. Results: In hFOB1.19 cells, 10 × 109/mL hucMSC-derived exosomes inhibited cell proliferation, arrested cell cycle, and promoted apoptosis, while hucMSCs or 1 × 109/mL exosomes promoted cell proliferation, accelerated cell cycle, and promoted calcium deposits and the expression of OCN, RUNX2, collagen I and ALP. In hFOB1.19 cells, exosomes from hucMSCs with LINC00520 knockdown reduced the survival and calcium deposits, arrested the cell cycle, and enhanced the apoptosis, while exosomes from hucMSCs overexpressing LINC00520 enhance the proliferation and calcium deposits and accelerated the cell cycle. Conclusions: LINC00520 functions as a modulator of calcium deposits, and exosomes derived from hucMSCs overexpressing LINC00520 might be a novel therapeutic approach for osteoporosis. [ABSTRACT FROM AUTHOR]

BACKGROUND: Regenerative repair of large bone defects remains one of the great challenges for clinical orthopedic surgeons. Polyphosphates not only play an important role in osteoblast development and growth factor release, but also regulate osteogenic activity by affecting the structure of local blood clots. OBJECTIVE: To summarize the relationship between polyphosphates and bone tissue regeneration and to understand the mechanism of polyphosphates in osteogenesis. METHODS: CNKI, Wanfang, VIP, CBM and PubMed databases were searched for articles regarding research progress of polyphosphates in bone tissue regeneration. After preliminarily screened by reading abstracts, a total of 80 articles were finally included for review. RESULTS AND CONCLUSION: The mechanisms of action of polyphosphates in bone tissue regeneration can be classified into the following categories: (1) Polyphosphates enhance fibroblast growth factor-2-mediated cellular signaling pathways by inducing the expression of the related transcription factors Runx2, matrix metalloproteinase-1, osteopontin, osteocalcin and osteoprotegerin related genes, enhance the affinity between fibroblast growth factor-2 and the receptor, activate fibroblast growth factor-mediated cellular signaling pathways, and promote osteoblast differentiation of mesenchymal stem cells. (2) During osteoblast metabolism: polyphosphate catabolism provides a source of energy and regulation of calcitonin, osteogenesis-associated transcription factors, bone sialic acid protein, and tissue non-specific alkaline phosphatase gene expression, promoting cell mineralization. (3) Due to the presence of competitive inhibition: polyphosphate inhibits the enzymatic activity of tartrate resistant acid phosphatase in osteoclasts, contributing to the decrease in bone resorption activity of osteoclasts. (4) Polyphosphate activates the nuclear factor κB signaling pathway and upregulates the expression of vascular cell adhesion molecule-1, intercellular adhesion molecule-1, and selectin-E, enhancing monocyte adhesion to endothelial cells. (5) Polyphosphate also binds to histone 4 and high mobility group B1 with high affinity and enhances the expression of vascular permeability signaling factors, cell surface adhesion molecules, inflammatory cell migration, and apoptosis. (6) Polyphosphate regulates the inflammatory response by modulating the PI3K/AKT and PLC/PKC/Ca2+ signaling pathways and bradykinin system in endothelial cells. (7) By promoting the formation of coarse fibers of a blood clot, polyphosphates increase the pore size of the fibrous clot, promote the infiltration of relevant cells and factors, and promote local vascularization, and the repair and regeneration of bone tissue. [ABSTRACT FROM AUTHOR]

BACKGROUND: Sanhua Jiegu San is a common drug for clinical fracture and tendon injury, which have good effect. However, there is a lack of relevant experimental evidence, and its mechanism of action remains unclear. OBJECTIVE: To observe the effect of Sanhua Jiegu San on Wnt/β-catenin signaling pathway in osteoblasts. METHODS: Firstly, the optimal concentration of Sanhua Jiegu San for osteoblast intervention was screened by pre-experiment. Then, the proliferation of osteoblasts was tested by cell counting kit-8 method. The maturity of osteoblasts was detected by alkaline phosphatase assay. The mRNA expressions of alkaline phosphatase, Runt-related transcription factor 2, osteocalcin, osteopontin, collagenase I, Axin2, Dkk4, Lef-1, and Tcf-1 were detected by PCR. The protein expressions of β-Catenin and P-β-Catenin were detected by western blot method. RESULTS AND CONCLUSION: After 5 days of osteoblast culture, the number of osteoblasts reached the maximum at the concentration of 10 mg/L. After 21 days of osteoblast culture, the proliferation of cells in different concentration groups of Sanhua Jiegu San increased, which was obvious in the 10 mg/L group, but tended to decrease at 20 mg/L. After 7 days of osteoblast culture, alkaline phosphatase expression was increased in the low, medium and high concentration groups, especially in the high concentration group (20 mg/L). After 7 days of osteoblast culture, mRNA expressions of alkaline phosphatase, Runt-related transcription factor 2, osteocalcin, osteopontin, collagenase I were increased in the low, medium and high concentration groups, especially in the high concentration group (20 mg/L). After 21 days of osteoblast culture, the mRNA expressions of Axin2 and Dkk4 were decreased in the low, medium and high concentration groups, while the mRNA expressions of Lef-1 and Tcf-1 were increased, especially in the high concentration group (20 mg/L). After 21 days of osteoblast culture, the expression of P-β-Catenin protein was decreased in the low, medium and high concentration groups, while the expression of β-Catenin protein was increased, especially in the high concentration group (20 mg/L). To conclude, Sanhua Jiegu San can regulate the Wnt/β-Catenin signaling pathway in osteoblasts, promote the mRNA expression of alkaline phosphatase, Runt-related transcription factor 2, osteocalcin, osteopontin, collagenase I, Lef1, and Tcf-1 and the protein expression of β-Catenin, and decrease the mRNA expression of Axin2 and Dkk4 and the protein expression of P-β-Catenin, thereby promoting osteoblast proliferation. This may be the mechanism by which Sanhua Jiegu San promotes fracture healing. [ABSTRACT FROM AUTHOR]

BACKGROUND: Estrogen deficiency can inhibit osteoclast proliferation and differentiation. Epimedium is a phytoestrogen that acts as a traditional kidney tonic herb, and its active ingredient, icariin, can produce some effects of estrogen. OBJECTIVE: To investigate the possible signaling pathways involved in the non-nuclear effects of icariin in promoting osteoblast proliferation and differentiation, as well as the possible mechanisms of phosphorylated protein cascade regulation in the signaling pathway by phosphorylated proteomics techniques. METHODS: Primary osteoblasts were obtained from newborn Sprague-Dawley rats, cultured, and identified. The effect of icariin on osteoblast activity was measured by cell counting kit-8 method. The primary osteoblasts were passed to the third generation. After 24 hours, passaged cells were then divided into six groups: bovine serum albumin intervention group, PBS intervention group, icariin intervention group, icariin-bovine serum albumin coupling intervention group, ICI182780+icariin-bovine serum albumin coupling intervention group, and PD98059+icariin-bovine serum albumin coupling intervention group. The cells of each group were collected at 0, 5, 15 and 25 minutes and used for protein extraction, enzymatic digestion, peptide labeling and phosphorylated peptide enrichment. Phosphoproteomic analysis was performed using liquid chromatography-mass spectrometry technology RESULTS AND CONCLUSION: 10 μg/L Icariin significantly promoted the proliferation and differentiation of osteoblasts. Icariin-bovine serum albumin coupling was able to induce phosphorylation of ERK in osteoblasts without membrane permeation, confirming the non-nuclear effect. Compared with the icariinbovine serum albumin coupling intervention group, there were 971 differentially expressed proteins (499 up-regulated and 472 down-regulated) in the icariin intervention group, 974 (509 up-regulated and 465 down-regulated) in the bovine serum albumin intervention group, 836 (377 up-regulated and 459 downregulated) in the PBS intervention group, 231 (126 up-regulated and 105 down-regulated) in the ICI182780+icariin-bovine serum albumin coupling intervention group, and 150 (65 up-regulated and 85 down-regulated ) in the PD98059+icariin-bovine serum albumin coupling intervention group. Among them, 65 and 62 significantly expressed proteins were down-regulated (Fc value < 0.8) in the ICI182780+icariin-bovine serum albumin coupling intervention group and PD98059+icariin-bovine serum albumin coupling intervention group, respectively, most of which were involved in important biological processes, such as mRNA processing and stability regulation, RNA splicing, protein phosphorylation, and mitosis, as well as in signaling pathways analyzed by KEGG library, such as MAPK, protein ubiquitination, mTOR, and PI3K/Akt pathways. To conclude, icariin promotes osteoblast proliferation and differentiation through non-nuclear effects in relation to MAPK, protein ubiquitination, mTOR, and PI3K/Akt signaling pathways. [ABSTRACT FROM AUTHOR]

Glucocorticoid-induced bone loss is a toxic effect of long-term therapy with glucocorticoids resulting in a significant increase in the risk of fracture. Here, we find that glucocorticoids reciprocally convert osteoblast-lineage cells into endothelial-like cells. This is confirmed by lineage tracing showing the induction of endothelial markers in osteoblast-lineage cells following glucocorticoid treatment. Functional studies show that osteoblast-lineage cells isolated from glucocorticoid-treated mice lose their capacity for bone formation but simultaneously improve vascular repair. We find that the glucocorticoid receptor directly targets Foxc2 and Osterix, and the modulations of Foxc2 and Osterix drive the transition of osteoblast-lineage cells to endothelial-like cells. Together, the results suggest that glucocorticoids suppress osteogenic capacity and cause bone loss at least in part through previously unrecognized osteoblast–endothelial transitions. [ABSTRACT FROM AUTHOR]

The evaluated herbs plants (a-Mangostin, usnic acid, Piper betle L, and Daemonorops draco) have potency in the alveolar bone repair of mandibular on the periodontitis modeling. Its herbs were evaluated with the flavonoid and phenolic, and ionic values identified by a multi-tester meter. Mol-inspiration software was used to calculate the miLogP value of -mangostin and usnic acid. The evaluation of alveolar bone repair is based on H and E staining profiles of osteoblasts and osteoclasts. The a-Mangostin and Piper beetle L had higher total flavonoid and phenolic concentrations than usnic acid and Daemonorops draco. Based on the miLogP value, usnic acid has better absorption than a-mangostin. The ionic amounts of dissolved Oxygen, conductivity, and total dissolved solids have correlated with osteoblast and osteoclast expression in the bone repair process. The herbs extract can increase the stimulation to produce osteoclast and stability in 7 and 14 days. The ionic values, flavonoids, and phenolic totals of herbs affect the mandibular alveolar bone repair, which is marked to increase the new bone formation (osteoblast) and bone resorption (osteoclast). [ABSTRACT FROM AUTHOR]

Bone is a highly dynamic tissue, and the constant actions of bone-forming and bone-resorbing cells are responsible for attaining peak bone mass, maintaining bone mass in the adults, and the subsequent bone loss with aging and menopause, as well as skeletal complications of diseases and drug side-effects. It is now accepted that the generation and activity of bone-forming osteoblasts and bone-resorbing osteoclasts is modulated by osteocytes, osteoblast-derived cells embedded in the bone matrix. The interaction among bone cells occurs through direct contact and via secreted molecules. In addition to the regulation of bone cell function, molecules released by these cells are also able to reach the circulation and have effects in other tissues and organs in healthy individuals. Moreover, bone cell products have also been associated with the establishment or progression of diseases, including cancer and muscle weakness. In this review, we will discuss the role of bone as an endocrine organ, and the effect of selected, osteoblast-, osteocyte-, and osteoclast-secreted molecules on other tissues. [ABSTRACT FROM AUTHOR]

The bone marrow microenvironment contains a diverse array of cell types under extensive regulatory control and provides for a novel and complex mechanism for bone regulation. Megakaryocytes (MKs) are one such cell type that potentially acts as a master regulator of the bone marrow microenvironment due to its effects on hematopoiesis, osteoblastogenesis, and osteoclastogenesis. While several of these processes are induced/inhibited through MK secreted factors, others are primarily regulated by direct cell–cell contact. Notably, the regulatory effects that MKs exert on these different cell populations has been found to change with aging and disease states. Overall, MKs are a critical component of the bone marrow that should be considered when examining regulation of the skeletal microenvironment. An increased understanding of the role of MKs in these physiological processes may provide insight into novel therapies that can be used to target specific pathways important in hematopoietic and skeletal disorders. [ABSTRACT FROM AUTHOR]

Bisphenol A (BPA) analogs, like BPA, could have adverse effects on human health including bone health. The aim was to determine the effect of BPF, BPS and BPAF on the growth and differentiation of cultured human osteoblasts. Osteoblasts primary culture from bone chips harvested during routine dental work and treated with BPF, BPS, or BPAF for 24 h at doses of 10–5, 10–6, and 10–7 M. Next, cell proliferation was studied, apoptosis induction, and alkaline phosphatase (ALP) activity. In addition, mineralization was evaluated at 7, 14, and 21 days of cell culture in an osteogenic medium supplemented with BP analog at the studied doses. BPS treatment inhibited proliferation in a dose-dependent manner at all three doses by inducing apoptosis; BPF exerted a significant inhibitory effect on cell proliferation at the highest dose alone by an increase of apoptosis; while BPAF had no effect on proliferation or cell viability. Cell differentiation was adversely affected by treatment with BPA analogs in a dose-dependent, observing a reduction in calcium nodule formation at 21 days. According to the results obtained, these BPA analogs could potentially pose a threat to bone health, depending on their concentration in the organism. [ABSTRACT FROM AUTHOR]

Bone is a highly vascularized organ that not only plays multiple roles in supporting the body and organs but also endows the microstructure, enabling distinct cell lineages to reciprocally interact. Recent studies have uncovered relevant roles of the bone vasculature in bone patterning, morphogenesis, homeostasis, and pathological bone destruction, including osteoporosis and tumor metastasis. This review provides an overview of current topics in the interactive molecular events between endothelial cells and bone cells during bone ontogeny and discusses the future direction of this research area to find novel ways to treat bone diseases. [ABSTRACT FROM AUTHOR]

Transforming growth factor beta (TGF-β) is a key factor mediating the intercellular crosstalk between the hematopoietic stem cells and their microenvironment. Here, we investigated the skeletal phenotype of transgenic mice expressing constitutively active TGF-β receptor type I under the control of Mx1-Cre (Mx1;TβRICA mice). μCT analysis showed decreased cortical thickness, and cancellous bone volume in both femurs and mandibles. Histomorphometric analysis confirmed a decrease in cancellous bone volume due to increased osteoclast number and decreased osteoblast number. Primary osteoblasts showed decreased ALP and mineralization. Constitutive TβRI activation increased osteoclast differentiation. qPCR analysis showed that Tnfsf11/Tnfrsf11b ratio, Ctsk, Sufu, and Csf1 were increased whereas Runx2, Ptch1, and Ptch2 were decreased in Mx1;TβRICA femurs. Interestingly, Gli1, Wnt3a, Sp7, Alpl, Ptch1, Ptch2, and Shh mRNA expression were reduced whereas Tnfsf11/Tnfrsf11b ratio was increased in Mx1;TβRICA mandibles. Similarly, osteoclast-related genes were increased in Mx1;TβRICA osteoclasts whereas osteoblast-related genes were reduced in Mx1;TβRICA osteoblasts. Western blot analysis indicated that SMAD2 and SMAD3 phosphorylation was increased in Mx1;TβRICA osteoblasts, and SMAD3 phosphorylation was increased in Mx1;TβRICA osteoclasts. CTSK was increased while RUNX2 and PTCH1 was decreased in Mx1;TβRICA mice. Microindentation analysis indicated decreased hardness in Mx1;TβRICA mice. Our study indicated that Mx1;TβRICA mice were osteopenic by increasing osteoclast number and decreasing osteoblast number, possibly by suppressing Hedgehog signaling pathways. [ABSTRACT FROM AUTHOR]

Osteoblast is the key cell of bone repair and reconstruction, and its abnormal proliferation, differentiation and mineralization are the main causes of bone metabolic diseases such as osteoporosis. The regulatory role of mitochondria in the osteogenic function of osteoblasts can not be ignored. In recent years, it has been found that the ability of mitochondria to regulate osteoblasts is closely related to mitochondrial oxidative phosphorylation, mitochondrial biogenesis, mitochondrial dynamics, mitochondrial autophagy mediating the maintenance of mitochondrial quantity and function, providing energy for cells, and signal transmission. This paper reviews the research progress of the mechanism of mitochondria regulating osteoblast function in recent years. [ABSTRACT FROM AUTHOR]

Due to the general application of in vitro test, cell culture is generally selected to evaluate the cytocompatibility of devices and materials. The choice of test condition should depend on the probable site and clinical application. The oxygen content of human body could be estimated around 5%∼12%, and the oxygen level of healing bone fracture range from 0.8%∼3.8%%. However, materials for bone implant are traditionally evaluated under laboratory normoxia condition (21% O 2) in vitro. The aim was to study the effect of oxygen level on osteoblast upon high stiffness titanium with different roughness. After sandblasted and acid-etched (SLA) process, we create titanium surfaces with four different roughness. The differentiation and proliferation of MC3T3-E1 osteoblast cultured on SLA-treated specimens were evaluated in designed chamber with oxygen level of 1%, 5%, 10%, 21%. By scanning electron microscopy, all samples had sub-micro pit inside the micro-holes upon SLA-treated Ti disk surface. The decrease of oxygen level from 21% to 5% promoted osteoblast growth of SLA-treated specimens, but 1% O 2 delayed cell proliferation. The surface roughness of specimens influenced osteoblast cell differentiation. The differentiation and proliferation ability of the cells upon SLA-treated specimens is proportional to oxygen level. Our results demonstrated that 5% O 2 will easily discriminate osteoblasts responses on different SLA-treated specimens. These results suggest that hypoxia (5% O 2) environment is better model for biological evaluation of bone-related materials. [ABSTRACT FROM AUTHOR]