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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]

Background: Treating large-volume bone defects (LVBD) remains a challenge for orthopedics and maxillofacial surgeons globally. Objective: The present study was to aimed investigate the role of Beta-naphthoflavone (BNF) a synthetic flavonoid on differentiation of osteoblast of human adipose derived stem cells. Results: BNF at 1 μM showed a significantly increased ALP activity and rate of cell proliferation compared to control on the 3rd, 7th and 14th day. Also, it was observed that, BNF at 1 μM resulted in significantly increased expression of BSP. On the 14th day of treatment, BNF at all the three treatments resulted in increased levels of OCN. Also, extracellular matrix mineralization levels were recorded to be highest for BNF 1 μM. The levels of Runx2 were higher in 1 μM treated group on the 3rd day compared to other, whereas in contrast the levels were lower on the 7th and 14th day compared to control. BNF at all the three treatments caused a significant decrease in levels of pSTAT3, C/EBP-α and PPAR-γ levels. Conclusion: BNF treatment resulted in increased differentiation of osteoblast of hASCs by targeting STAT3 pathway. [ABSTRACT FROM AUTHOR]

Proteasome inhibitors (PIs) can preferentially restore bone in bone-defective lesions of patients with multiple myeloma (MM) who respond favorably to these drugs. Most prior in vitro studies on PIs used continuous exposure to low PI concentrations, although pharmacokinetic analysis in patients has shown that serum concentrations of PIs change in a pulsatile manner. In the present study, we explored the effects of pulsatile treatment with PIs on bone metabolism to simulate in vivo PI pharmacokinetics. Pulsatile treatment with bortezomib, carfilzomib, or ixazomib induced MM cell death but only marginally affected the viability of osteoclasts (OCs) with F-actin ring formation. Pulsatile PI treatment suppressed osteoclastogenesis in OC precursors and bone resorption by mature OCs. OCs robustly enhanced osteoblastogenesis in cocultures with OCs and MC3T3-E1 pre-osteoblastic cells, indicating OC-mediated coupling to osteoblastogenesis. Importantly, pulsatile PI treatment did not impair robust OC-mediated osteoblastogenesis. These results suggest that PIs might sufficiently reduce MM cell-derived osteoblastogenesis inhibitors to permit OC-driven bone formation coupling while suppressing OC differentiation and activity in good responders to PIs. OC-mediated coupling to osteoblastogenesis appears to be a predominant mechanism for preferential occurrence of bone regeneration at sites of osteoclastic bone destruction in good responders. [ABSTRACT FROM AUTHOR]

Background: Hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, which includes bone-forming and bone-resorbing cells, i.e., osteoblasts (OBs) and osteoclasts (OCs). OBs originate from mesenchymal progenitors, while OCs are derived from HSCs. Self-renewal, proliferation and differentiation of HSCs are under the control of regulatory signals generated by OBs and OCs within the BM niche. Consequently, OBs and OCs control both bone physiology and hematopoiesis. Since the human developmental and bone marrow failure genetic syndrome fanconi anemia (FA) presents with skeletal abnormalities, osteoporosis and HSC impairment, we wanted to test the hypothesis that the main pathological abnormalities of FA could be related to a defect in OC physiology and/or in bone homeostasis. Results: We revealed here that the intrinsic differentiation of OCs from a Fanca−/− mouse is impaired in vitro due to overactivation of the p53–p21 axis and defects in NF-kB signaling. The OC differentiation abnormalities observed in vitro were rescued by treating Fanca−/− cells with the p53 inhibitor pifithrin-α, by treatment with the proinflammatory cytokine TNFα or by coculturing them with Fanca-proficient or Fanca-deficient osteoblastic cells. Conclusions: Overall, our results highlight an unappreciated role of Fanca in OC differentiation that is potentially circumvented in vivo by the presence of OBs and TNFα in the BM niche. [ABSTRACT FROM AUTHOR]

BACKGROUND: In recent years, there have been many studies on the prevention and treatment of femoral head necrosis with traditional Chinese medicine. A variety of single Chinese medicines, Chinese medicine monomers and Chinese medicine compounds can regulate bone metabolism, lipid metabolism, and oxidative stress by targeting signaling molecules. The prevention and treatment of femoral head necrosis by translation-related signaling pathways has become a research hotspot. OBJECTIVE: To expound the global research progress in hormone-induced femoral head necrosis treated with Chinese medicine, in order to provide some ideas for the treatment of hormone-induced femoral head necrosis. METHODS: CNKI, WanFang, VIP, SinoMed, PubMed, Embase, and Web of Science databases were searched with the search terms “glucocorticoids, avascular necrosis of the femoral head, ANFH, pathogenesis, signal path, Chinese medicine compound, oxidative stress” in Chinese and English. Literature was retrieved on Chinese medicine compounds and active ingredients for intervention of the signaling pathway related to hormone-induced femoral head necrosis. A total of 71 documents were included according to the inclusion and exclusion criteria. RESULTS AND CONCLUSION: Chinese medicine may treat hormone-induced femoral head necrosis through multiple signaling pathways. Total saponins of Panax notoginseng, total flavonoids of Rhizoma Drynariae, and resveratrol glucoside promote osteoblast proliferation and differentiation and inhibit osteoclast formation by regulating the level of β-catenin protein and the formation of β-catenin-TCF/LEF complex via the Wnt/β-catenin signaling pathway. Recipes for invigorating blood and reinforcing marrow, warming yang and tonifying kidney, and relieving bone bi-syndrome maintain osteoblast-osteoclast dynamic balance and bone homeostasis by regulating the OPG/RANK/RANKL pathway. Puerarin, salidroside, allicin, and icariin influence the proliferation and apoptosis of osseous cells and angiogenesis of microvascular endothelial cells in the femoral head via the PI3K/Akt signaling pathway. Ginsenosides can down-regulate the expressions of superoxide dismutase and catalase by interfering with the Keapl-Nrf2-ARE signaling pathway, thereby attenuating oxidative stress damage. Gastrodin, lutein, and astragaloside IV can promote the proliferation and differentiation of osteoblasts by intervening Nrf2/HO-1 signaling, thereby improving the level of oxidative substances. Naringin and polygonin balance the osteogenic/adipogenic differentiation of bone marrow mesenchymal stem cells via the Nrf2/ARE signaling pathway. Traditional Chinese medicine can regulate oxidative stress response of osteocytes, which is another important way to treat hormone-induced necrosis of the femoral head. [ABSTRACT FROM AUTHOR]

BACKGROUND: Piezo1 is a mechanosensitive channel that acts as an important target protein for mechanical stress stimulation. Piezo1 converts external mechanical forces into biological signals, regulating bone formation and bone resorption. Piezo1 plays an important role in anti-osteoporosis. OBJECTIVE: To summarize the mechanism of mechanical stress stimulation in anti-osteoporosis and research progress in Piezo1 molecular biology as well as the use of Piezo1 in the treatment of osteoporosis. METHODS: Relevant literature was retrieved in CNKI, WanFang, VIP, PubMed, and Web of Science databases using computers. The search terms were “osteoporosis, Piezo1, mechanical, osteoblast, osteoclast, chondrocyte, bone” in Chinese and English. Finally, 65 documents were included for review according to the inclusion and exclusion criteria. RESULTS AND CONCLUSION: The structure of Piezo1 has been continuously analyzed in the breakthrough of cryo-electron microscopy. Based on a three-bladed propeller 3D structure, Piezo1 exhibits different structural changes under silent and stressful states. Piezo1-mediated mechanical stress stimulation can affect bone formation by regulating the functions of osteoblasts, chondrocytes, endothelial cells and intestinal cells, and simultaneously affect bone resorption by regulating the function of osteoclasts, thereby playing an important role in anti-osteoporosis. Piezo1-mediated mechanical stress mainly affects bone formation by regulating the function of osteoblasts through the Wnt/β-catenin signaling pathway. And it has a two-way regulatory effect on osteoclasts due to different type, strength, and duration of mechanical force, which needs to be further quantified. For the use of Piezo1 in the treatment of osteoporosis, emphasis should be placed on clinical and animal model research for osteoporosis, osteoclast-related mechanisms, and new composite materials. [ABSTRACT FROM AUTHOR]

A gelatin hydrolysate with a hydrolysis degree of 13.7% was generated using the skin gelatin of chum salmon (Oncorhynchus keta) and papain-catalyzed enzymatic hydrolysis. The results of analysis demonstrated that four amino acids, namely Ala, Gly, Pro, and 4-Hyp, were the most abundant in the obtained gelatin hydrolysate with measured molar percentages ranging from 7.2% to 35.4%; more importantly, the four amino acids accounted for 2/3 of the total measured amino acids. However, two amino acids, Cys and Tyr, were not detected in the generated gelatin hydrolysate. The experimental results indicated that the gelatin hydrolysate at a dose of 50 µg/mL could combat etoposide-induced apoptosis in human fetal osteoblasts (hFOB 1.19 cells), causing a decrease in the total apoptotic cells from 31.6% to 13.6% (via apoptotic prevention) or 13.3% to 11.8% (via apoptotic reversal). Meanwhile, the osteoblasts exposed to the gelatin hydrolysate showed expression changes for 157 genes (expression folds > 1.5-fold), among which JNKK, JNK1, and JNK3 were from the JNK family with a 1.5–2.7-fold downregulated expression. Furthermore, the protein expressions of JNKK, JNK1, JNK3, and Bax in the treated osteoblasts showed a 1.25–1.41 fold down-regulation, whereas JNK2 expression was not detected in the osteoblasts. It is thus suggested that gelatin hydrolysate is rich in the four amino acids and has an in vitro antiapoptotic effect on etoposide-stimulated osteoblasts via mitochondrial-mediated JNKK/JNK(1,3)/Bax downregulation. [ABSTRACT FROM AUTHOR]

• Lack of TRIC-B in osteoblasts strongly impairs skeleton growth and structure, leading to bone fractures. • In absence of TRIC-B, the impaired Ca2+ flux from endoplasmic reticulum to cytosol is associated to delayed osteoblast differentiation and decreased collagen synthesis that cause reduced collagen incorporation in the extracellular matrix and poor mineralization. • An impaired SMAD signaling in mutant mice and in OI patient osteoblasts explains the osteoblast malfunction. • The reduced SMAD phosphorylation and nuclear translocation are mainly caused by alteration in Ca2+ calmodulin kinase II (CaMKII)-mediated signaling. Ca2+ is a second messenger that regulates a variety of cellular responses in bone, including osteoblast differentiation. Mutations in trimeric intracellular cation channel B (TRIC-B), an endoplasmic reticulum channel specific for K +, a counter ion for Ca2+flux, affect bone and cause a recessive form of osteogenesis imperfecta (OI) with a still puzzling mechanism. Using a conditional Tmem38b knock out mouse, we demonstrated that lack of TRIC-B in osteoblasts strongly impairs skeleton growth and structure, leading to bone fractures. At the cellular level, delayed osteoblast differentiation and decreased collagen synthesis were found consequent to the Ca2+ imbalance and associated with reduced collagen incorporation in the extracellular matrix and poor mineralization. The impaired SMAD signaling detected in mutant mice, and validated in OI patient osteoblasts, explained the osteoblast malfunction. The reduced SMAD phosphorylation and nuclear translocation were mainly caused by alteration in Ca2+ calmodulin kinase II (CaMKII)-mediated signaling and to a less extend by a lower TGF-β reservoir. SMAD signaling, osteoblast differentiation and matrix mineralization were only partially rescued by TGF-β treatment, strengthening the impact of CaMKII-SMAD axes on osteoblast function. Our data established the TRIC-B role in osteoblasts and deepened the contribution of the CaMKII-SMAD signaling in bone. [ABSTRACT FROM AUTHOR]

Osteoporosis has become a serious public health problem especially in postmenopausal women. This work aims to assess both the function and mechanism of SDH5 in osteoporosis. The animal model of osteoporosis in Sprague-Dawley rats was established by utilizing ovariectomy (OVX). The trabecular bone morphometry had been determined by micro-CT, and tibia injury of rats was detected through HE and alcian blue staining. Meanwhile, the levels of oxidative stress factors, including malondialdehyde, catalase, glutathione peroxidase (GSH-Px), and superoxide (SOD), were detected by ELISA. The proliferation and apoptosis of osteoblasts isolated from OVX-induced rats were found out by CCK-8 and flow cytometry, respectively. The expression of SDH5, Osterix, Type I collagen (CoL1A1), osteocalcin (OC), SOD1, SOD2, p-MyD88/MyD88, and p-NF-κB p65/NF-κB p65 was assessed by Western blot. The effect and mechanism of SDH5 knockdown on osteoporosis were verified by lipopolysaccharide treatment. In the osteoporosis rat model, the expression of SDH5 had an up-regulated tend. A higher bone mineral density value was found in the SDH5 knockdown group. SDH5 inhibition ameliorated bone loss, mitigated bone histopathological injury, alleviated oxidative stress, and elevated osteogenic marker protein expression in vivo and in vitro. SDH5 down-regulation also promoted the proliferation and restrained apoptosis of osteoblasts extracted from OVX-induced rats. Furthermore, we found that the underlying mechanism was associated with the inhibition of the MyD88/NF-κB pathway. Down-regulation of SDH5 mitigates the damage of osteoporosis both in vivo and in vitro via inhibiting the MyD88/NF-κB signaling activation. [ABSTRACT FROM AUTHOR]

Proper bone remodeling depends not only on a team of bone-resorbing osteoclasts and bone-forming osteoblasts. It also depends on the site-specific delivery of a large amount of osteoblast lineage cells to the bone remodeling site. How this delivery occurs is poorly known. Here, we gained insight into this mechanism by analyzing the distribution of markers of osteoblastogenesis on bone surfaces and in their bone marrow neighborhood in human cancellous bone. We found a CD271-positive/PDGFβ-R-positive cell layer surrounding the bone marrow that provides osteoblastogenic potential along all bone surfaces, whether quiescent or remodeling. This bone marrow envelope cell layer takes the appearance of a canopy above remodeling sites, where it then also shows an upregulation of the proliferation marker Ki67, smooth muscle actin (SMA), tenascin C, fibronectin, and MMP13. This indicates that the canopy is a region of the bone marrow envelope where early markers of osteoblastogenesis are activated concurrently with initiation of bone remodeling. Importantly, the high proliferation index in the canopy is not associated with increasing cell densities at the canopy level, but it is at the bone surface level, thereby supporting delivery of cells from the canopy to the bone surface. This delivery route explains why lack of canopies was previously found to coincide with lack of bone formation, and fits current knowledge on the canopies as a target for regulators of bone remodeling. We conclude that the coordination of bone marrow envelope activities and bone surface activities allows integrating osteoblastogenesis and bone remodeling into the same functional unit, and propose that the bone marrow envelope is critical for preserving bone health. [ABSTRACT FROM AUTHOR]

Background. The pathogenesis of Legg--Calve--Perthes disease (LCPD), a juvenile form of avascular necrosis of the femoral head (ANFH), is not fully understood. Objectives. The purpose of this work was to study the regulatory effect of R-spondin 1 (Rspo1) on osteoblastic apoptosis and evaluate the pre-clinical efficacy of recombinant human protein Rspo1 (rhRspo1) in treatment of LCPD. Materials and methods. This is an experimental study. In vivo rabbit ANFH model was established. Human osteoblast cell line hFOB1.19 (hFOB) was used to overexpress and silence Rspo1 in vitro. Additionally, hFOB cells were induced with glucocorticoid (GC) and methylprednisolone (MP), and treated with rhRspo1. The expres- sions of Rspo1, β-catenin, Dkk-1, Bcl-2, and caspase-3, and the apoptosis rate of hFOB cells were examined. Results. The expressions of Rspo1 and β-catenin were lower in ANFH rabbits. The expression of Rspo1 was decreased in GC-induced hFOB cells. Compared to the control group, after 1 μM MP induction for 72 h, the expressions of β-catenin and Bcl-2 were higher, while Dkk-1, caspase-3 and cleaved caspase-3 expressions were lower in Rspo1 overexpression and rhRspo1-treated groups. The apoptosis rate of GC-induced hFOB cells was decreased in Rspo1 overexpression and rhRspo1-treated groups compared to the control group. Conclusions. R-spondin 1 inhibited GC-induced osteoblast apoptosis via Wnt/β-catenin pathway, which might be associated with the development of ANFH. Moreover, rhRspo1 had a potential pre-clinical therapeutic effect on LCPD. [ABSTRACT FROM AUTHOR]

Prostate cancer is one of the most common cancers among men. Although many patients respond favorably to first-line treatments, castration—and chemotherapy—resistance arises after a few years, leading to metastasis. Thus, new approaches are being investigated using natural supplements to reinforce current therapies. Ocoxin is a plant-based mixture with antitumor properties that have been proved in several cancers. Here, we evaluated the cytotoxic capacity of this compound itself and combined with Docetaxel, Enzalutamide and Olaparib as an adjuvant agent. We observed that Ocoxin reduced tumor cell viability; slowed down cell cycles; altered the expression of genes involved in DNA replication, cell cycles and the p53 signaling pathway; and reduced migratory capacity after stimulation with cancer-associated fibroblasts (CAFs) and osteoblasts in vitro and reduced tumor volume in vivo. The combination of the nutritional supplement with chemotherapy showed a higher cytotoxic effect than chemotherapy alone and reverted chemoresistance conferred by CAFs and osteoblasts. Moreover, the adjuvant therapy also improved the outcome in vivo compared to the treatment with solo chemotherapy, where mice developed smaller tumors and less angiogenesis. Therefore, Ocoxin arises as a good candidate for further studies in combination with current treatments for prostate-cancer patients. [ABSTRACT FROM AUTHOR]

Bone cells are known to express multiple P2 receptor subtypes, and the functional effects of receptor activation have been described for many of these. One exception is the P2X4 receptor, which despite strong expression in osteoblasts and osteoclasts, has no defined functional activity. This study used the selective P2X4 receptor antagonists, 5-BDBD and PSB-12062, to investigate the role of this receptor in bone. Both antagonists (≥ 0.1 μM) dose-dependently decreased bone formation by 60–100%. This was accompanied by a ≤ 70% decrease in alkaline phosphatase activity, a ≤ 40% reduction in cell number, and a ≤ 80% increase in the number of adipocytes present in the culture. The analysis of gene expression showed that levels of osteoblast marker genes (e.g. Alpl, Bglap) were decreased in 5-BDBD treated cells. Conversely, expression of the adipogenic transcription factor PPARG was increased 10-fold. In osteoclasts, high doses of both antagonists were associated with a reduction in osteoclast formation and resorptive activity by ≤ 95% and ≤ 90%, respectively. Taken together, these data suggest that the P2X4 receptor plays a role in modulating bone cell function. In particular, it appears to influence osteoblast differentiation favouring the osteogenic lineage over the adipogenic lineage. [ABSTRACT FROM AUTHOR]

Titanium materials are essential treatment modalities in the medical field and serve as a tissue engineering scaffold and coating material for medical devices. Thus, there is a significant demand to improve the bioactivity of titanium for therapeutic and experimental purposes. We showed that ultraviolet light (UV)-pre-treatment changed the protein-adsorption ability and subsequent osteoconductivity of titanium. Fibronectin (FN) adsorption on UV-treated titanium was 20% and 30% greater after 1-min and 1-h incubation, respectively, than that of control titanium. After 3-h incubation, FN adsorption on UV-treated titanium remained 30% higher than that on the control. Osteoblasts were cultured on titanium disks after 1-h FN adsorption with or without UV-pre-treatment and on titanium disks without FN adsorption. The number of attached osteoblasts during the early stage of culture was 80% greater on UV-treated and FN-adsorbed (UV/FN) titanium than on FN-adsorbed (FN) titanium; osteoblasts attachment on UV/FN titanium was 2.6- and 2.1-fold greater than that on control- and UV-treated titanium, respectively. The alkaline phosphatase activity of osteoblasts on UV/FN titanium was increased 1.8-, 1.8-, and 2.4-fold compared with that on FN-adsorbed, UV-treated, and control titanium, respectively. The UV/FN implants exhibited 25% and 150% greater in vivo biomechanical strength of bone integration than the FN- and control implants, respectively. Bone morphogenetic protein-2 (BMP-2) adsorption on UV-treated titanium was 4.5-fold greater than that on control titanium after 1-min incubation, resulting in a 4-fold increase in osteoblast attachment. Thus, UV-pre-treatment of titanium accelerated its protein adsorptivity and osteoconductivity, providing a novel strategy for enhancing its bioactivity.

Effects of UV-photofunctionalization on bone-to-titanium integration under challenging systemic conditions remain unclear. We examined the behavior and response of osteoblasts from sham-operated and ovariectomized (OVX) rats on titanium surfaces with or without UV light pre-treatment and the strength of bone-implant integration. Osteoblasts from OVX rats showed significantly lower alkaline phosphatase, osteogenic gene expression, and mineralization activities than those from sham rats. Bone density variables in the spine were consistently lower in OVX rats. UV-treated titanium was superhydrophilic and the contact angle of ddH2O was ≤5°. Titanium without UV treatment was hydrophobic with a contact angle of ≥80°. Initial attachment to titanium, proliferation, alkaline phosphatase activity, and gene expression were significantly increased on UV-treated titanium compared to that on control titanium in osteoblasts from sham and OVX rats. Osteoblastic functions compromised by OVX were elevated to levels equivalent to or higher than those of sham-operated osteoblasts following culture on UV-treated titanium. The strength of in vivo bone-implant integration for UV-treated titanium was 80% higher than that of control titanium in OVX rats and even higher than that of control implants in sham-operated rats. Thus, UV-photofunctionalization effectively enhanced bone-implant integration in OVX rats to overcome post-menopausal osteoporosis-like conditions.

Titanium (Ti) is an osteoconductive material that is routinely used as a bulk implant to fix and restore bones and teeth. This study explored the effective use of Ti as a bone engineering scaffold. Challenges to overcome were: (1) difficult liquid/cell infiltration into Ti microfiber scaffolds due to the hydrophobic nature of Ti; and (2) difficult cell attachment on thin and curved Ti microfibers. A recent discovery of UV-photofunctionalization of Ti prompted us to examine its effect on Ti microfiber scaffolds. Scaffolds in disk form were made by weaving grade 4 pure Ti microfibers (125 µm diameter) and half of them were acid-etched to roughen the surface. Some of the scaffolds with original or acid-etched surfaces were further treated by UV light before cell culture. Ti microfiber scaffolds, regardless of the surface type, were hydrophobic and did not allow glycerol/water liquid to infiltrate, whereas, after UV treatment, the scaffolds became hydrophilic and immediately absorbed the liquid. Osteogenic cells from two different origins, derived from the femoral and mandibular bone marrow of rats, were cultured on the scaffolds. The number of cells attached to scaffolds during the early stage of culture within 24 h was 3-10 times greater when the scaffolds were treated with UV. The development of cytoplasmic projections and cytoskeletal, as well as the expression of focal adhesion protein, were exclusively observed on UV-treated scaffolds. Osteoblastic functional phenotypes, such as alkaline phosphatase activity and calcium mineralization, were 2-15 times greater on UV-treated scaffolds, with more pronounced enhancement on acid-etched scaffolds compared to that on the original scaffolds. These effects of UV treatment were associated with a significant reduction in atomic carbon on the Ti microfiber surfaces. In conclusion, UV treatment of Ti microfiber scaffolds tunes their physicochemical properties and effectively enhances the attachment and function of osteoblasts, proposing a new strategy for bone engineering.

Finely tuned cartilage mineralization, endochondral ossification, and normal bone formation are necessary for normal bone growth. Hypertrophic chondrocytes in the epiphyseal cartilage secrete matrix vesicles, which are small extracellular vesicles initiating mineralization, into the intercolumnar septa but not the transverse partitions of the cartilage columns. Bone-specific blood vessels invade the unmineralized transverse septum, exposing the mineralized cartilage cores. Many osteoblast precursors migrate to the cartilage cores, where they synthesize abundant bone matrices, and mineralize them in a process of matrix vesicle-mediated bone mineralization. Matrix vesicle-mediated mineralization concentrates calcium (Ca) and inorganic phosphates (Pi), which are converted into hydroxyapatite crystals. These crystals grow radially and are eventually get out of the vesicles to form spherical mineralized nodules, leading to collagen mineralization. The influx of Ca and Pi into the matrix vesicle is regulated by several enzymes and transporters such as TNAP, ENPP1, PiT1, PHOSPHO1, annexins, and others. Such matrix vesicle-mediated mineralization is regulated by osteoblastic activities, synchronizing the synthesis of organic bone material. However, osteocytes reportedly regulate peripheral mineralization, e.g., osteocytic osteolysis. The interplay between cartilage mineralization and vascular invasion during endochondral ossification, as well as that of osteoblasts and osteocytes for normal mineralization, appears to be crucial for normal bone growth.

Abstract Background Calcific aortic valve disease (CAVD) is the second leading cause of adult heart diseases. The purpose of this study is to investigate whether miR-101-3p plays a role in the human aortic valve interstitial cells (HAVICs) calcification and the underlying mechanisms. Methods Small RNA deep sequencing and qPCR analysis were used to determine changes in microRNA expression in calcified human aortic valves. Results The data showed that miR-101-3p levels were increased in the calcified human aortic valves. Using cultured primary HAVICs, we demonstrated that the miR-101-3p mimic promoted calcification and upregulated the osteogenesis pathway, while anti-miR-101-3p inhibited osteogenic differentiation and prevented calcification in HAVICs treated with the osteogenic conditioned medium. Mechanistically, miR-101-3p directly targeted cadherin-11 (CDH11) and Sry-related high-mobility-group box 9 (SOX9), key factors in the regulation of chondrogenesis and osteogenesis. Both CDH11 and SOX9 expressions were downregulated in the calcified human HAVICs. Inhibition of miR-101-3p restored expression of CDH11, SOX9 and ASPN and prevented osteogenesis in HAVICs under the calcific condition. Conclusion miR-101-3p plays an important role in HAVIC calcification through regulation of CDH11/SOX9 expression. The finding is important as it reveals that miR-1013p may be a potential therapeutic target for calcific aortic valve disease.

Abstract Background While transcription factor (TF) regulation is known to play an important role in osteoblast development, differentiation, and bone metabolism, the molecular features of TFs in human osteoblasts at the single-cell resolution level have not yet been characterized. Here, we identified modules (regulons) of co-regulated genes by applying single-cell regulatory network inference and clustering to the single-cell RNA sequencing profiles of human osteoblasts. We also performed cell-specific network (CSN) analysis, reconstructed regulon activity-based osteoblast development trajectories, and validated the functions of important regulons both in vivo and in vitro. Results We identified four cell clusters: preosteoblast-S1, preosteoblast-S2, intermediate osteoblasts, and mature osteoblasts. CSN analysis results and regulon activity-based osteoblast development trajectories revealed cell development and functional state changes of osteoblasts. CREM and FOSL2 regulons were mainly active in preosteoblast-S1, FOXC2 regulons were mainly active in intermediate osteoblast, and RUNX2 and CREB3L1 regulons were most active in mature osteoblasts. Conclusions This is the first study to describe the unique features of human osteoblasts in vivo based on cellular regulon active landscapes. Functional state changes of CREM, FOSL2, FOXC2, RUNX2, and CREB3L1 regulons regarding immunity, cell proliferation, and differentiation identified the important cell stages or subtypes that may be predominantly affected by bone metabolism disorders. These findings may lead to a deeper understanding of the mechanisms underlying bone metabolism and associated diseases.

Abstract Background Bone is the most common site of metastasis of prostate cancer (PCa). PCa invasion leads to a disruption of osteogenic-osteolytic balance and causes abnormal bone formation. The interaction between PCa and bone stromal cells, especially osteoblasts (OB), is considered essential for the disease progression. However, drugs that effectively block the cancer-bone interaction and regulate the osteogenic-osteolytic balance remain undiscovered. Methods A reporter gene system was constructed to screen compounds that could inhibit PCa-induced OB activation from 631 compounds. Then, the pharmacological effects of a candidate drug, Procoxacin (Pro), on OBs, osteoclasts (OCs) and cancer-bone interaction were studied in cellular models. Intratibial inoculation, micro-CT and histological analysis were used to explore the effect of Pro on osteogenic and osteolytic metastatic lesions. Bioinformatic analysis and experiments including qPCR, western blotting and ELISA assay were used to identify the effector molecules of Pro in the cancer-bone microenvironment. Virtual screening, molecular docking, surface plasmon resonance assay and RNA knockdown were utilized to identify the drug target of Pro. Experiments including co-IP, western blotting and immunofluorescence were performed to reveal the role of Pro binding to its target. Intracardiac inoculation metastasis model and survival analysis were used to investigate the therapeutic effect of Pro on metastatic cancer. Results Luciferase reporter gene consisted of Runx2 binding sequence, OSE2, and Alp promotor could sensitively reflect the intensity of PCa-OB interaction. Pro best matched the screening criteria among 631 compounds in drug screening. Further study demonstrated that Pro effectively inhibited the PCa-induced osteoblastic changes without killing OBs or PCa cells and directly killed OCs or suppressed osteoclastic functions at very low concentrations. Mechanism study revealed that Pro broke the feedback loop of TGF-β/C-Raf/MAPK pathway by sandwiching into 14–3-3ζ/C-Raf complex and prevented its disassociation. Pro treatment alleviated both osteogenic and osteolytic lesions in PCa-involved bones and reduced the number of metastases of PCa in vivo. Conclusions In summary, our study provides a drug screening strategy based on the cancer-host microenvironment and demonstrates that Pro effectively inhibits both osteoblastic and osteoclastic lesions in PCa-involved bones, which makes it a promising therapeutic agent for PCa bone metastasis. Graphical Abstract

This study aimed to explore the physiological activity of phosphorylated soybean polypeptide chelated calcium (PSPCC) and improve its calcium chelation capacity. To be specific，the preparation process of PSPCC was optimized by single factor test and response surface methodology. And the effect of phosphorylated soybean peptide chelated calcium on osteoblast activity was evaluated by in vitro experiments. The results showed that, the optimum process of PSPCC was as follows: Mass ratio of sodium tripolyphosphate to soybean polypeptide 1:2, phosphorylation reaction temperature 52 ℃, phosphorylation reaction pH7.0, phosphorylation reaction time 9.7 h, mass ratio of phosphorylated soybean polypeptide to calcium chloride 2:1, chelation reaction pH8.0, chelation reaction temperature 50 °C, and chelation reaction time 1.5 h. The maximum calcium chelation amount of PSPCC was 107.25±0.10 mg/g under the above optimal conditions. The results of MTT assay showed that the relative proliferation rate of osteoblasts in the PSPCC group (group D) was 1.6 times that of the soybean polypeptide group (group A) (the third day). In particular, the ALP staining experiment showed that the positive rate of staining in group D was 33.6 times that of the blank group. In addition, the ALP activity of each sample was detected by the ALP kit on the 7th day, and the results showed that the ALP activity of the group D was 6.2 times that of the blank group. And it was found that the number of calcium nodules in the group D was 94 times higher than that of the blank group through the alizarin red staining experiment. It was reasonable that the preparation process of PSPCC was optimized by response surface methodology, which was preliminarily proved that PSPCC had a significant effect on promoting proliferation and differentiation of osteoblasts (P

Background and Aim: Evaluation of the alkaline phosphatase expression is known as one of the diagnostic markers to confirm the stem cells differentiation into osteoblasts. Several methods, including immunocytochemistry, are used to measure alkaline phosphatase in different studies. Due to limitations of using this method, we decided to optimize the calorimetric method as an alternative, low cost and efficient method for quantitative assessment of alkaline phosphatase. Materials and Methods: Mesenchymal stem cells derived from human adipose tissue were used. Cells were differentiated into osteoblasts using an induction medium. Alkaline phosphatase levels were assessed on the days 0 and 21 of differentiation using both immunocytochemistry and calorimetric methods. Results: Immunocytochemistry findings demonstrated a significant increase (approximately 5-fold) in alkaline phosphatase expression level on the 21st day of differentiation compared to that on the day 0 (p

The imbalance of bone homeostasis is the root cause of osteoporosis. However current therapeutic approaches mainly focus on either anabolic or catabolic pathways, which often fail to turn the imbalanced bone metabolism around. Herein we reported that a SIRT-1 agonist mediated molecular therapeutic strategy to reverse the imbalance in bone homeostasis by simultaneously regulating osteogenesis and osteoclastogenesis via locally sustained release of SRT2104 from mineral coated acellular matrix microparticles. Immobilization of SRT2104 on mineral coating (MAM/SRT) harnessing their electrostatic interactions resulted in sustained release of SIRT-1 agonist for over 30 days. MAM/SRT not only enhanced osteogenic differentiation and mineralization, but also attenuated the formation and function of excessive osteoclasts via integrating multiple vital upstream signals (β-catenin, FoxOs, Runx2, NFATc1, etc.) in vitro. Osteoporosis animal model also validated that it accelerated osteoporotic bone healing and improved osseointegration of the surrounding bone. Overall, our work proposes a promising strategy to treat osteoporotic bone defects by reversing the imbalance in bone homeostasis using designated small molecule drug delivery systems.

In tissue engineering, a porous material is applied as the extracellular matrix or scaffold for cell growth. Then growth factors are added to this scaffold whose cytotoxicity must be assessed. In this study, the cytotoxicity effects of polyhydroxybutyrate/chitosan/bioglass nanocomposite scaffolds on human osteoblast-like cells (SAOS-2 cells) are explored.Materials&Methods:This study was experimental and was done in Isfahan Dental School. The polyhydroxybutyrate/chitosan/bioglass nanocomposite scaffold was fabricated using the electrospinning method. Scanning Electron Microscopy (SEM) analysis was used to evaluate the morphological characteristics of the nanofibers and their diameter distribution. Additionally, bioglass nanoparticles in the fibers were identified via SEM. The MTT assay was performed to assess cell viability after 3, 5, and 7 days.The collected data were analyzed using SPSS 20.0 through statistical tests including Kruskal-Wallis test (p value≤ 0.05).Results:According to SEM images, the scaffold fibers were fully porous and no beads were observed. Furthermore, the polyhydroxybutyrate/chitosan/bioglass scaffold showed greater cell viability and proliferation compared to the groups lacking bioglass.Conclusion:The polyhydroxybutyrate/chitosan/bioglass scaffold has no cytotoxic effect on osteoblast–like cells.

Studies from both humans and animal models indicated that maternal chronic poor-quality diet, especially a high fat diet (HFD), is significantly associated with reduced bone density and childhood fractures in offspring. When previously studied in a rat model, our data suggested that maternal HFD changes epigenetic marks such as DNA methylation and histone modifications to control osteoblast metabolism. In mouse embryonic and postnatal offspring bone samples, a ChIP-sequencing (ChIP-Seq)-based genome-wide method was used to locate the repressive histone mark H3K27me3 (mediated via the polycomb histone methyltransferase, Ezh2) and expressive histone mark H3K27ac (p300/CBP mediated) throughout the genome. Using isolated mouse embryonic cells from foetal calvaria (osteoblast-like cells), H3K27me3 ChIP-Seq showed that 147 gene bodies and 26 gene promoters in HFD embryotic samples had a greater than twofold increase in H3K27me peaks compared to controls. Among the HFD samples, Pthlh and Col2a1 that are important genes playing roles during chondro- and osteogenesis had significantly enriched levels of H3K27me3. Their decreased mRNA expression was confirmed by real-time PCR and standard ChIP analysis, indicating a strong association with Ezh2 mediated H3K27me3 epigenetic changes. Using embryonic calvaria osteoblastic cells and offspring bone samples, H3K27ac ChIP-Seq analysis showed that osteoblast inhibitor genes Tnfaip3 and Twist1 had significantly enriched peaks of H3K27ac in HFD samples compared to controls. Their increased gene expression and association with H3K27ac were also confirmed by real-time PCR and standard ChIP analysis. These findings indicate that chronic maternal HFD changes histone trimethylation and acetylation epigenetic marks to regulate expression of genes controlling osteoblastogenesis.

Osteosarcoma (OS) is a bone cancer exhibiting a 20% survival rate for metastatic patients, which motivates the development of new therapeutic options. Among the various new treatment approaches, modulation of autophagy is the subject of rising interest. In addition to its pro-survival role in established tumors, autophagy recently emerged as an active player in the crosstalk between tumor and stromal cells. In OS, although the knockdown of key autophagy genes in human cell lines demonstrates a protumoral role of autophagy, the analysis of patient tumors indicates that lack of LC3-positive punctae at resection following neoadjuvant chemotherapy is a poor prognostic marker, suggesting that loss of autophagy is not detrimental for the tumor. In the present work, we analyzed the consequences of autophagy inactivation in OS cells both on tumor development and on bone microenvironment in an orthotopic syngeneic model. We found that inactivation of the autophagy-essential gene Atg5 in OS cells decreases their tumorigenic properties in vitro. However, these effects were no longer observed in vivo, likely due to microenvironment modifications such as overexpression of the major OS-promoting factor TGF-β or increased infiltration of Foxp3-positive and CD31-positive cells in Atg5 KO tumors. In addition, autophagy-deficient tumor cells stimulate the in vitro formation of osteoclast, the cells in charge of bone resorption which can release bone matrix-embedded growth factors thereby stimulating tumor growth. Taken together, these results suggest that Atg5 inactivation in OS cells is associated with microenvironment modifications known as poor prognosis-associated factors in OS, and could thus balance the negative cell-autonomous effects of autophagy suppression. Abbreviations: ACTB -β-actin; Atg -autophagy-related; Baf-A1 - Bafilomycin-A1; CSC -cancer stem cells; Col1A -type 1a collagen; d -day; HBSS -Hank’s balanced salt solution; LC3 -microtubule-associated protein 1 light chain 3 protein; SQSTM1/p62 -sequestosome; OB -osteoblast; OC -osteoclast; OS -osteosarcoma; TEM -transmission electron microscopy; TGF-β -transforming growth factor β; TRAP -acid phosphatase 5, tartrate-resistant.

Introduction: To probe the impacts and biological roles of miR-4739/ITGA10 on the proliferation, differentiation and apoptosis of osteoblasts. Methods: Bioinformatics analysis was conducted to screen the key genes in osteoporosis. The upstream miRNAs of ITGA10 were predicted by TargetScan. KEGG pathway enrichment analysis was performed by DAVID database. The osteoblast proliferation and apoptosis were measured using CCK-8 and flow cytometry. The differentiation markers were measured by qRT-PCR and western blotting. The luciferase reporter assay was conducted to verify the binding of miR-4739 to ITGA10. Results: ITGA10 was down-regulated in patients with osteoporosis and identified as the key gene in osteoporosis by the bioinformatics analysis. Then the prediction provided by TargetScan indicated that miR-4739 was the potential upstream miRNA for ITGA10. And the following luciferase reporter assay showed that miR-4739 could bind to ITGA10 3′UTR. Furthermore, the miR-4739 inhibitor promoted osteoblasts proliferation, differentiation, and inhibited cell apoptosis by increasing the expression of ITGA10 and subsequently activating the PI3K/AKT signaling pathway. Conclusions: Overall, we proved that the higher expression of miR-4739 participated in the progression of osteoporosis by targeting ITGA10 and modulating PI3K/AKT signaling pathway, and perhaps miR-4739/ITGA10 axis could be potential diagnostic markers and therapeutic target for osteoporosis.

Abstract Background Tiger bone, which had been one of the most famous traditional Chinese medicine for 2000 years, was originate from the skeleton of Panthera tigris L., and had the actions of anti-inflammatory, analgesic, immune-regulatory and promoting healing of bone fracture, and was used for the treatment of osteoporosis and rheumatoid arthritis. Jin-Tian-Ge (JTG), the artificial tiger bone powder, were prepared from skeletons of several farmed animals to substitute the natural tiger bone, and has been used for the treatment of osteoporosis in clinical practice. However, the characteristic and mechanism of action of JTG for the therapy of osteoporosis need to be further evidenced by using modern pharmacological methods. The aim of this work is to investigate the bone-protective effects of JTG, and explore the possible underlying mechanism. Methods Ovariectomy (OVX) rats were orally administrated JTG or estradiol valerate (EV) for 12 weeks. We investigated the pharmacodynamic effects of JTG on anti-bone loss in OVX rats, and also investigated the role of JTG in promoting osteogenesis and inhibiting osteoclast differentiation. Results JTG increased the bone mineral density (BMD), improved the bone microarchitecture and biomechanical properties in ovariectomized rast, whereas reversed the bone high turnover in OVX rats as evidenced by serum biochemical markers in OVX rats. JTG increased osteogenic differentiation of BMSCs in vitro, and up-regulated the expression of the key proteins of BMP and Wnt/β-catenin pathways. JTG also inhibited the osteoclastogenesis of BMM as evidenced by the alteration of the TRAP activity, F-actin construction and the expression of nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), c-Fos, Cathepsin K (Ctsk) and matrix metallopeptidase 9 (MMP9) of OCs induced with RANKL and LPS, reduced the expression and phosphorylation of NF-κB in OCs. Conclusions JTG prevented bone loss in OVX rats and increased osteogenic differentiation of BMSCs through regulation of the BMP and Wnt/β-catenin pathway, inhibited osteoclastogenesis by suppressing the NF-κB pathway, suggesting that JTG had the potentials for prevention and treatment of osteoporosis by modulating formation and differentiation of osteoblast and osteoclast.

Beta-carotene (BC) is a nutrient in vegetables and a precursor of vitamin A. BC has been reported to have anticarcinogenic, antiaging, and antioxidation properties and prevents heart diseases. Recently, BC has gained significant attention due to stimulating effect on osteoblast differentiation. Poly(lactic acid)/hydroxyapatite (PLA/HAp) core-shell nanoparticles have been reported earlier with a load capacity of 250% for water-insoluble substances, using a surfactant-free emulsification method. In this work, PLA/HAp core-shell nanoparticles loaded with BC were prepared, and osteoblast differentiation behavior was evaluated. BC was successfully loaded into PLA/HAp core-shell nanoparticles with diameters of approximately 30 nm. BC/loaded PLA/HAp core-shell nanoparticles stimulated osteoblast differentiation by upregulating collagen type I, osteopontin, and osteocalcin expression. In addition, the gene expression levels of these osteoblasts were significantly larger than those stimulated by PLA/HAp core-shell nanoparticles without BC and cultured in a differential medium (with ascorbic acid and β-glycerophosphate). PLA/HAp core-shell nanoparticles showed satisfactory cytocompatibility because they were attached to the osteoblasts. Consequently, BC was effectively delivered to osteoblasts by nanoparticles. These results suggested that BC-loaded PLA/HAp core-shell nanoparticles could enhance bone formation.

Osteoporosis occurs due to a dysregulation in bone remodeling, a process requiring both bone-forming osteoblasts and bone-resorbing osteoclasts. Current leading osteoporosis therapies suppress osteoclast-mediated bone resorption but show limited therapeutic effects because osteoblast-mediated bone formation decreases concurrently. We developed a gene therapy strategy for osteoporosis that simultaneously promotes bone formation and suppresses bone resorption by targeting two microRNAs (miRNAs)—miR-214-3p and miR-34a-5p. We modulated the expression of these miRNAs using systemically delivered recombinant adeno-associated viral (rAAV) vectors targeting the bone. rAAV-mediated overexpression of miR-214-3p or inhibition of miR-34a-5p in the skeleton resulted in bone loss in adult mice, resembling osteoporotic bones. Conversely, rAAV-mediated inhibition of miR-214-3p or overexpression of miR-34a-5p reversed bone loss in mouse models for postmenopausal and senile osteoporosis by increasing osteoblast-mediated bone formation and decreasing osteoclast-mediated bone resorption. Notably, these mice did not show any apparent pathological phenotypes in non-skeletal tissues. Mechanistically, inhibiting miR-214-3p upregulated activating transcription factor 4 in osteoblasts and phatase and tensin homolog in osteoclasts, while overexpressing miR-34a-5p downregulated Notch1 in osteoblasts and TGF-β-induced factor homeobox 2 in osteoclasts. In summary, bone-targeting rAAV-mediated regulation of miR-214-3p or miR-34a-5p is a promising new approach to treat osteoporosis, while limiting adverse effects in non-skeletal tissues.

Abstract Background Osteoporosis (OP) is a common metabolic bone disease mainly involving bone remodeling and blood vessels. The current study aimed to explore the role of zinc finger E-box binding homeobox 1 (ZEB1) in OP. Methods First, gene expression microarrays for OP were downloaded from the Gene Expression Omnibus database and analyzed to screen for potential targets. Subsequently, a rat OP model was constructed using ovariectomy (OVX), and osteoblastic and osteoclastic differentiation and alterations in osteoporotic symptoms were observed upon intraperitoneal injection of oe-ZEB1 lentiviral vectors. DNA polymerase delta interacting protein 2 (POLDIP2) was predicted to be a downstream target of ZEB1, which was validated by ChIP-qPCR and dual-luciferase experiments. RAW264.7 cells were subjected to lentiviral vector infection of oe-ZEB1 and/or sh-POLDIP2, followed by RANKL treatment to induce osteoclast differentiation. Results ZEB1 was poorly expressed in blood samples of postmenopausal patients with OP and in bone tissues of OVX-treated rats. Overexpression of ZEB1 or POLDIP2 in OVX rats promoted osteoblastogenesis and inhibited osteoclast differentiation. In RANKL-treated RAW264.7 cells, the transcription factor ZEB1 enhanced the expression of POLDIP2, and silencing of POLDIP2 attenuated the inhibitory effect of oe-ZEB1 on the differentiation of macrophages RAW264.7 to osteoclasts. Conclusions ZEB1 promotes osteoblastogenesis and represses osteoclast differentiation, ultimately reducing the occurrence of postmenopausal OP by elevating the expression of POLDIP2.

Abstract Background Tophi are lesions commonly present at sites of bone erosion in gout-affected joints. The tophus comprises a core of monosodium urate (MSU) crystals surrounded by soft tissue that contains macrophages and other immune cells. Previous studies found that MSU crystals directly reduce osteoblast viability and function. The aim of the current study was to determine the indirect, macrophage-mediated effects of MSU crystals on osteoblasts. Methods Conditioned medium from the RAW264.7 mouse macrophage cell line cultured with MSU crystals was added to the MC3T3-E1 mouse osteoblastic cell line. Conditioned medium from the THP-1 human monocytic cell line cultured with MSU crystals was added to primary human osteoblasts (HOBs). Matrix mineralization was assessed by von Kossa staining. Gene expression was determined by real-time PCR, and concentrations of secreted factors were determined by enzyme-linked immunosorbent assay. Results In MC3T3-E1 cells cultured for 13 days in an osteogenic medium, the expression of the osteoblast marker genes Col1a1, Runx2, Sp7, Bglap, Ibsp, and Dmp1 was inhibited by a conditioned medium from MSU crystal-stimulated RAW264.7 macrophages. Mineral staining of MC3T3-E1 cultures on day 21 confirmed the inhibition of osteoblast differentiation. In HOB cultures, the effect of 20 h incubation with a conditioned medium from MSU crystal-stimulated THP-1 monocytes on osteoblast gene expression was less consistent. Expression of the genes encoding cyclooxygenase-2 and IL-6 and secretion of the proinflammatory mediators PGE2 and IL-6 were induced in MC3T3-E1 and HOBs incubated with conditioned medium from MSU crystal-stimulated macrophages/monocytes. However, inhibition of cyclooxygenase-2 activity and PGE2 secretion from HOBs indicated that this pathway does not play a major role in mediating the indirect effects of MSU crystals in HOBs. Conclusions Factors secreted from macrophages stimulated by MSU crystals attenuate osteoblast differentiation and induce the expression and secretion of proinflammatory mediators from osteoblasts. We suggest that bone erosion in joints affected by gout results from a combination of direct and indirect effects of MSU crystals.

Abstract Osteoclasts, the only cells that can resorb bone, play a central role in bone homeostasis as well as bone damage under pathological conditions such as osteoporosis, arthritis, periodontitis, and bone metastasis. Recent studies using single-cell technologies have uncovered the regulatory mechanisms underlying osteoclastogenesis at unprecedented resolution and shed light on the possibility that there is heterogeneity in the origin, function, and fate of osteoclast-lineage cells. Here, we discuss the current advances and emerging concepts in osteoclast biology.

Abstract A contiguous carbonate‐rich hydroxyapatite microcoating in a microfluidic device represents a substrate that has chemical and structural similarity to bone mineral. The present work describes a low‐temperature method to deposit a carbonate‐rich hydroxyapatite microcoating on a glass slide and its incorporation within the microchannels of a microfluidic device. A glass slide is covered/masked with polypropylene‐based tape and CaCO3 nanoparticles are deposited on exposed areas by convective self assembly. The precursor CaCO3 is converted to carbonate‐rich hydroxyapatite by dissolution‐recrystallization in phosphate‐buffered saline. The microcoating is aligned/incorporated within a microchannel when the underlying glass is bonded to a polydimethylsiloxane structure with the device layout. X‐ray diffraction, laser Raman microspectroscopy, and X‐ray photoelectron spectroscopy indicate that the microcoating was comprised of carbonate‐rich hydroxyapatite. Scanning electron microscopy and 3D laser confocal microscopy showed that was comprised of nanocrystalline rod‐like clusters that collectively exhibit a thickness of ∼20 µm. Monocultures/cocultures of osteoblast‐lineage (MC3T3‐E1, MG63) and preosteoclast‐lineage (RAW 264.7) cells were performed. Osteoblast‐lineage cells adhered to the microcoating and deposited an extracellular matrix of collagen fibrils and mineral accretions. Mineralization was detected in/near the inlet wells. The microcoating is analogous to bone mineral and could be applied to various layouts and mineral systems.

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. [ABSTRACT FROM AUTHOR]

BACKGROUND: Osteoporosis is a systemic metabolic disease and its incidence is increasing year by year, seriously harming the health of middle-aged and elderly. Melatonin is an endogenous hormone secreted by the body regulating circadian rhythms. In recent years, it has been found that melatonin can regulate the balance between bone resorption and bone formation, exerting a vital role in maintaining the stability of bone metabolism. OBJECTIVE: To summarize the research progress of melatonin in the treatment of osteoporosis and provide a novel foundation for the treatment of osteoporosis. METHODS: “Osteoporosis, melatonin, ovariectomized, pinealectomy, osteoblast, osteoclast, stem cells” were used as the key words. PubMed and Web of Science databases were searched for relevant articles addressing the treatment of osteoporosis with melatonin. Totally 696 articles were initially obtained and 62 articles were finally included for review RESULTS AND CONCLUSION: The gene expression of melatonin varies in different age groups. Melatonin can maintain the self-renewal and differentiation abilities of mesenchymal stem cells as well as inhibit cell senescence through promoting the expression of Sirt1, PRC1/2 and cell cycle-related proteins. Melatonin can regulate the differentiation of mesenchymal stem cells, stimulate the differentiation of stem cells into osteoblasts, and hinder the differentiation of mesenchymal stem cells into adipocytes and osteoclasts. Melatonin can effectively alleviate the cytotoxic damage triggered by oxidative stress and inflammatory response, inhibit the differentiation of osteoclasts, maintain the stability of mitochondria, and enhance the differentiation and mineralization of osteoblasts. Melatonin can block bone metabolism disorders resulted from RANKL and inducible nitric oxide synthase, lower the bone loss, increase the expression of alkaline phosphatase and osteocalcin, and exert a corresponding biological effect on the balance between osteoclasts and osteoblasts. Melatonin has generated a good therapeutic impact on osteoporosis whereas its target is too complex, involving anti-aging, stem cell differentiation, anti-inflammatory and antioxidant, which has not been completely explained and requires further research. [ABSTRACT FROM AUTHOR]

Introduction: Bisphosphonates are widely used for the treatment of osteoporosis, which could cause osteonecrosis of the jaw (also known as bisphosphonate-related osteonecrosis of the jaw [BRONJ]). Currently, there is no effective treatment for BRONJ. Here, we investigated the role of human recombinant semaphorin 4D (Sema4D) in BRONJ in vitro. Methods: MG-63 and RAW264.7 cells were used to determine the effects of Sema4D on BRONJ. Osteoclast and osteoblast were differentiated by treatment with 50 ng/mL RANKL for 7 days. In vitro BRONJ model was induced by treatment with ZOL (2.5 μm). The development of osteoclasts and osteoblasts was evaluated using ALP activity and ARS staining. qRT-PCR was used to measure the genes relative expression involved in the development of osteoclasts and osteoblasts. In addition, ZOL decreased TRAP-positive area; TRAP protein and mRNA expression were determined using Western blot and qTR-PCR. Results: ZOL treatment remarkedly suppressed Sema4D expression in RAW264.7 cells. Moreover, ZOL reduced TRAP-positive area and TRAP protein and mRNA expression. In parallel, genes involved in osteoclast formation were reduced by ZOL treatment. In contrast, osteoclast apoptosis was increased by ZOL treatment. Recombinant human Sema4D significantly abolished these effects of ZOL. In addition, ALP activity was reduced by recombinant human Sema4D. Discussions: Genes involved in osteoblast formation were decreased by recombinant human Sema4D in a dose-dependent manner. We demonstrated that ZOL treatment inhibited Sema4D expression in RAW264.7 cells. Conclusion: Recombinant human Sema4D treatment can effectively alleviate ZOL-induced inhibition of osteoclast formation and apoptosis and promote osteoblast formation. [ABSTRACT FROM AUTHOR]

Efficient coordination between Mg2+ and vitamin D maintains adequate Ca2+ levels during lactation. This study explored the possible interaction between Mg2+ (0.3, 0.8, and 3 mM) and 1,25-dihydroxyvitamin D3 (1,25D; 0.05 and 5 nM) during osteogenesis using bovine mesenchymal stem cells. After 21 days, differentiated osteocytes were subjected to OsteoImage analysis, alkaline phosphatase (ALP) activity measurements, and immunocytochemistry of NT5E, ENG (endoglin), SP7 (osterix), SPP1 (osteopontin), and the BGLAP gene product osteocalcin. The mRNA expression of NT5E, THY1, ENG, SP7, BGLAP, CYP24A1, VDR, SLC41A1, SLC41A2, SLC41A3, TRPM6, TRPM7, and NIPA1 was also assessed. Reducing the Mg2+ concentration in the medium increased the accumulation of mineral hydroxyapatite and ALP activity. There was no change in the immunocytochemical localization of stem cell markers. Expression of CYP24A1 was higher in all groups receiving 5 nM 1,25D. There were tendencies for higher mRNA abundance of THY1, BGLAP, and NIPA1 in cells receiving 0.3 mM Mg2+ and 5 nM 1,25D. In conclusion, low levels of Mg2+ greatly enhanced the deposition of bone hydroxyapatite matrix. The effect of Mg2+ was not modulated by 1,25D, although the expression of certain genes (including BGLAP) tended to be increased by the combination of low Mg2+ and high 1,25D concentrations. [ABSTRACT FROM AUTHOR]

MOTS-c, a mitochondrial-derived peptide (MDP), is an essential regulatory mediator of cell protection and energy metabolism and is involved in the development of specific diseases. Recent studies have revealed that MOTS-c promotes osteoblast proliferation, differentiation, and mineralization. Furthermore, it inhibits osteoclast production and mediates the regulation of bone metabolism and bone remodeling. Exercise effectively upregulates the expression of MOTS-c, but the specific mechanism of MOTS-c regulation in bone by exercise remains unclear. Therefore, this article reviewed the distribution and function of MOTS-c in the tissue, discussed the latest research developments in the regulation of osteoblasts and osteoclasts, and proposed potential molecular mechanisms for the effect of exercise on the regulation of bone metabolism. This review provides a theoretical reference for establishing methods to prevent and treat skeletal metabolic diseases. [ABSTRACT FROM AUTHOR]

Rheumatoid arthritis (RA) is a chronic inflammatory disease that leads to systemic and articular bone loss by activating bone resorption and suppressing bone formation. Despite current therapeutic agents, inflammation-induced bone loss in RA continues to be a significant clinical problem due to joint deformity and lack of articular and systemic bone repair. Here, we identify the suppressor of bone formation, Schnurri-3 (SHN3), as a potential target to prevent bone loss in RA. SHN3 expression in osteoblast-lineage cells is induced by proinflammatory cytokines. Germline deletion or conditional deletion of Shn3 in osteoblasts limits articular bone erosion and systemic bone loss in mouse models of RA. Similarly, silencing of SHN3 expression in these RA models using systemic delivery of a bone-targeting recombinant adenoassociated virus protects against inflammation-induced bone loss. In osteoblasts, TNF activates SHN3 via ERK MAPK-mediated phosphorylation and, in turn, phosphorylated SHN3 inhibits WNT/β-catenin signaling and up-regulates RANKL expression. Accordingly, knock-in of a mutation in Shn3 that fails to bind ERK MAPK promotes bone formation in mice overexpressing human TNF due to augmented WNT/β-catenin signaling. Remarkably, Shn3-deficient osteoblasts are not only resistant to TNF-induced suppression of osteogenesis, but also down-regulate osteoclast development. Collectively, these findings demonstrate SHN3 inhibition as a promising approach to limit bone loss and promote bone repair in RA. [ABSTRACT FROM AUTHOR]

Piper sarmentosum is a well-known traditional herbal plant in various diseases treatments. Multiple scientific studies have also reported various biological activities exhibited by the plant's extract, such as antimicrobial, anticarcinogenic and antihyperglycemic activities, and, in addition, a bone protective effect in ovariectomized rats has been reported. However, no known Piper sarmentosum extract is involved in osteoblast differentiation using stem cells. Our study aims to identify the potential of P. sarmentosum ethanolic extract to induce osteoblast differentiation of human peripheral blood stem cells. Prior to the assay, the proliferation ability of the cells was observed for 14 days and the presence of hematopoietic stem cells in the culture was determined by the expression of SLAMF1 and CD34 genes. During the differentiation assay, the cells were treated with P. sarmentosum ethanolic extract for 14 days. Osteoblast differentiation was examined using an (alkaline phosphatase) ALP assay, by monitoring the expression of osteogenic gene markers and by von Kossa staining. The untreated cells served as the negative control, while cells treated with 50 µg/mL ascorbic acid and 10 mM β-glycerophosphate acted as the positive control. Finally, the determination of the compound profile was performed using a gas chromatography-mass spectrometry (GC-MS) analysis. The isolated cells were able to proliferate for 14 days during the proliferation assay. The expression of hematopoietic stem cell markers was also upregulated during the 14 days assay. Following the differentiation induction, the ALP activity exhibited a significant increase (p < 0.05) from day 3 of the differentiation assay. A molecular analysis also showed that the osteogenic markers ALP, RUNX2, OPN and OCN were upregulated compared to the positive control. The presence of mineralized cells with a brownish-stained morphology was observed, indicating the mineralization process increased in a time-dependent manner regardless of the concentration used. There were 54 compounds observed in the GC-MS analysis, including β-asarones, carvacrol and phytol, which have been shown to possess osteoinductive capacities. Our results demonstrate that the ethanolic extract of P. sarmentosum can induce osteoblast differentiation of peripheral blood stem cells. The extract contains potent compounds which can potentially induce the differentiation of bone cells, i.e., osteoblasts. [ABSTRACT FROM AUTHOR]

The pathogenesis of medication-related osteonecrosis of the jaw (MRONJ) is multifactorial and there is a substantial consensus on the role of antiresorptive drugs (ARDs), including bisphosphonates (BPs) and denosumab (Dmab), as one of the main determinants. The time exposure, cumulative dose and administration intensity of these drugs are critical parameters to be considered in the treatment of patients, as cancer patients show the highest incidence of MRONJ. BPs and Dmab have distinct mechanisms of action on bone, but they also exert different effects on immune subsets which interact with bone cells, thus contributing to the onset of MRONJ. Here, we summarized the main effects of ARDs on the different immune cell subsets, which consequently affect bone cells, particularly osteoclasts and osteoblasts. Data from animal models and MRONJ patients showed a deep interference of ARDs in modulating immune cells, even though a large part of the literature concerns the effects of BPs and there is a lack of data on Dmab, demonstrating the need to further studies. [ABSTRACT FROM AUTHOR]