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We previously reported that PRDM16 mediated the improvement in body composition in testosterone (T)-treated hypogonadal men by shifting adipogenesis to myogenesis. Previous preclinical studies suggest that Prdm16 regulates Runx2, an important osteoblastic transcription factor, expression and activity. However, the changes in PRDM16, and other genes/proteins involved in osteoblastogenesis with T therapy in hypogonadal men are unexplored. We investigated the role of PRDM16 in RUNX2 activation by measuring changes in gene expression in peripheral blood monocytes (PBMCs) and proteins in the serum of hypogonadal men after T therapy for 6 months. Likewise, we evaluated changes in the WNT10b—β-CATENIN signaling pathway by gene expression and protein analyses. We found significant increases in PRDM16 and RUNX2 expression in PBMCs together with significant increases in serum proteins at 6 months when compared to baseline. There were also increases in gene and protein expressions of WNT10b, and β-CATENIN at 6 months. Furthermore, we found a significant positive correlation between % changes in PRDM16 and WNT10b. Our results suggest that T therapy activates PRDM16, leading to enhanced signaling in the canonical WNT10b—β-CATENIN-RUNX2 pathway, the pathway involved in osteoblastogenesis. The above findings may account for the improvement in bone density and quality in hypogonadal men treated with T. [ABSTRACT FROM AUTHOR]

Infectious disease, neoplasms, congenital abnormalities, tooth extraction and physical injuries are factors that can cause loss of alveolar bone structure. The balance of various cytokines that play a role in the osteoblastogenesis process greatly influences the success of treatment for bone healing. The objective of this literature review is to understand the network of various cytokines that influence the process of osteoblastogenesis. A search of the MEDLINE/PubMed database produced an assessment of the pertinent literature for this literature review. Numerous Osteoblastogenesis is regulated by signaling pathways, which include Bone Morphogenetic Proteins (BMPs), TGF-β, interleukin and IFN-γ. The multifunctional growth factors known as BMPs are members of the TGF-β superfamily and it has been well reported that BMP-2 is important factor for osteblastogenesis. Osteoblast differentiation is induced by BMP-2, which binds to type II serine/threonine kinase receptors and opens type I receptors. These actions result in the production of R-Smads complexes including Smad1, 5 and 8, which are then imported into the nucleus. Together with Smad4, Smads complexes activate RUNX2 which resulting in increased osteoblastogenic marker expression. In this study, we reviewed the relationship between BMP-2 and osterix in osteoblastogenesis process. [ABSTRACT FROM AUTHOR]

Abstract Peri-prosthesis osteolysis (PPO) represents the most severe complication of total joint arthroplasty (TJA) surgery and imposes the primary cause of prosthesis failure and subsequent revision surgery. Antiresorptive therapies are usually prescribed to treat PPO, especially for elderly people. Nevertheless, the efficacy of anti-osteoporotic medications remains constrained. Recent therapeutic strategies to promote periprosthetic osseointegration by restoring osteoblast function are considered more effective approaches. However, the precise mechanism underlying the inhibition of osteogenesis triggered by wear particles remains enigmatic. Herein, we demonstrate that wear particles inhibit osteoblast function by inducing ferroptosis to sabotage extracellular mineralization and arouse periprosthetic osteolysis. The suppression of ferroptosis could significantly rescue osteogenesis thus alleviating PPO. Furthermore, Glutathione Peroxidase 4 (GPX4) has been identified as a key target in regulating osteoblastic ferroptosis. By utilizing virtual screening techniques, we have successfully conducted a comprehensive screening of a natural compound known as Urolithin A (UA), which exhibits remarkable inhibition of osteoblastic ferroptosis while simultaneously promoting the process of osteogenesis through its precise targeting mechanism on GPX4. Meanwhile, UA improves the osteolytic conditions significantly in vivo even when the adjunction of titanium (Ti) nanoparticles. This strategy has great potential in treating peri-prosthesis osteolysis and potentially broadens the scope of clinical therapy. Graphical Abstract

Bone metastasis is the primary cause of death among patients with advanced prostate cancer (PCa). PCa tends to spread to bones and acquire the bone‐like phenotype, causing osteoblastic bone metastasis. Unfortunately, there is no effective treatment for this condition. However, melatonin, which regulates our circadian rhythm, has been found to have anti‐tumor properties. It has yet to be established whether it is effective in treating osteoblastic PCa metastasis. Our findings show that melatonin inhibits the production of endothelin‐1 (ET‐1) in osteoblastic PCa cells, suppressing osteoblast differentiation. Clinical results indicate that bone metastatic PCa patients have higher levels of ET‐1 compared to nonmetastatic PCa patients. Furthermore, melatonin‐induced miR‐let‐7f‐5p inhibits ET‐1‐promoted osteoblast differentiation in osteoblastic PCa. Melatonin also suppresses the property of osteomimicry in osteoblastic PCa cells. Importantly, in the intratibia injection PCa metastasis model, melatonin decreased osteoblastic PCa tumor growth, inhibiting ET‐1 production and osteoblast differentiation in vivo. Taken together, melatonin inhibits osteoblastic PCa‐regulated osteoblastogenesis by reducing ET‐1 production through upregulation of miR‐let‐7f‐5p, while suppressing the property of osteomimicry in osteoblastic PCa. Melatonin therapy could be a promising approach to treating bone metastasis in osteoblastic PCa. [ABSTRACT FROM AUTHOR]

Background: Electromagnetic field therapy is gaining attention for its potential in treating bone disorders, with Extracorporeal Magnetotransduction Therapy (EMTT) emerging as an innovative approach. EMTT offers a higher oscillation frequency and magnetic field strength compared to traditional Pulsed Electromagnetic Field (PEMF) therapy, showing promise in enhancing fracture healing and non-union recovery. However, the mechanisms underlying these effects remain unclear. Results: This study demonstrates that EMTT significantly enhances osteoblast bone formation at multiple levels, from gene expression to extracellular matrix mineralization. Key osteoblastogenesis regulators, including SP7 and RUNX2, and bone-related genes such as COL1A1, ALPL, and BGLAP, were upregulated, with expression levels surpassing those of the control group by over sevenfold (p < 0.001). Enhanced collagen synthesis and mineralization were confirmed by von Kossa and Alizarin Red staining, indicating increased calcium and phosphate deposition. Additionally, calcium imaging revealed heightened calcium influx, suggesting a cellular mechanism for EMTT's osteogenic effects. Importantly, EMTT did not compromise cell viability, as confirmed by live/dead staining and WST-1 assays. Conclusion: This study is the first to show that EMTT can enhance all phases of osteoblastogenesis and improve the production of critical mineralization components, offering potential clinical applications in accelerating fracture healing, treating osteonecrosis, and enhancing implant osseointegration. [ABSTRACT FROM AUTHOR]

Background There is currently no pathogenetically based treatment for aseptic necrosis of the femoral head. One of the most promising areas of possible targeted therapy is the use of genetically engineered drugs, including monoclonal blockers of proinflammatory cytokines, aimed at inhibiting inflammation and indirectly reducing the activity of osteodestruction. The aim of the work is to evaluate the effectiveness of the use of the IL-6 monoclonal blocker in the course of aseptic necrosis of the femoral head in an experiment. Purpose Evaluate the preliminary results of the use of the IL-6 monoclonal blocker in the course of aseptic necrosis of the femoral head in an experiment. Materials and methods Surgical induction of aseptic necrosis of the femoral head was performed in 18 male Wistar rats. The animals were divided into two groups of 9 individuals each. The first group did not receive any treatment, the second received therapy with a monoclonal IL-6 receptor blocker, starting from the second week of the experiment, one injection once every two weeks. All animals were removed from the experiment at 4, 6 and 8 weeks after the induction of aseptic necrosis, 3 rats from each group at a time. Total RNA was isolated from the femoral head on the aseptic necrosis side and the conditionally healthy side as a control. The expression of genes of regulatory proteins of osteogenesis was studied by PCR. To study the features of osteodestructive processes, histological examination of femoral head preparations in all animals was conducted. Results Histological preparations of femoral heads of the second group animals were characterized by less pronounced osteodestructive, chondrodestructive processes compared to the animals that did not receive therapy. The mRNA profile of the rats of the second group displayed an increase in the expression of genes encoding proteins involved in osteoreparation at all stages of the experiment. At the same time, the activity of genes encoding proteins of proinflammatory cytokines, regulatory molecules of osteoclastogenesis was reduced relative to the first group. Discussion The data obtained indicate an important role of inflammation in the regulation of osteodestruction. Inhibition of the biological action of IL-6 contributed to inhibition of the expression of osteoclastogenesis genes, increased activity of bone metabolism genes, and caused a decrease in the intensity of osteodestruction and activation of osteoreparation. Conclusion Preliminary results of the use of a monoclonal blocker of the proinflammatory cytokine IL-6 indicate the inhibition of osteodestructive and strengthening of osteoreparative processes due to the correction of the expression of bone metabolism genes during the progression of aseptic necrosis of the femoral head in rats in an experimental model.

Osteoblastogenesis is regulated by several signaling pathways like hedgehog signaling. Of three types of mammalian Hedgehog genes, the Indian Hedgehog (Ihh) plays an important role in the formation of the skeleton. Mesenchymal stem cells (MSCs) isolated from adipose tissue have been considered a good source of osteoblast differentiation. Evidence also suggests that miRNAs play an important role in regulating key stages of osteoblast differentiation. In this study, two miRNAs targeting the Ihh were predicted by using bioinformatics analysis. ASCs were successfully derived, purified, and characterized from human adipose tissue. ASCs were chemically induced into osteoblast cells. Then, differentiation was confirmed by alkaline phosphatase (ALP) activity and Alizarin red staining. The relative expression of Ihh and related miRNAs was evaluated after 0, 7, 14, and 21 from the differentiation duration. The results of bioinformatics data showed that has-miR-195-5p and has-miR-15b-5p target the Ihh gene. The expression of Ihh significantly increased in a time-dependent manner in the differentiation process. In contrast, miR-195-5p and miR-15b-5p were significantly downregulated dependent on time duration (P < 0.01). Overall, the data indicate the antithetical regulation of Ihh versus has-miR-195-5p and has-miR-15b-5p during the differentiation process. These results support the hypothesis that these mi-RNAs could target the Ihh in the pathway of osteoblast differentiation derived from human ASCs. [ABSTRACT FROM AUTHOR]

RNA-binding proteins (RBPs), which regulate gene expression through post-transcriptional modifications of RNAs, play a role in diverse biological processes that include bone cell development and bone tissue formation. RBP dysregulation may result in aberrant bone homeostasis and contribute to various bone diseases. The function of RBPs in bone physiology and pathophysiology and the underlying molecular mechanisms have been extensively studied in recent years. This article provides a review of such studies, highlighting the potential of RBPs as pivotal targets for therapeutic intervention. [ABSTRACT FROM AUTHOR]

Royal jelly (RJ) is recognized as beneficial to mammalian health. Multilineage differentiation potential is an important property of mesenchymal stem cells (MSCs). C2C12 cells have an innate ability to differentiate into myogenic cells. Like MSCs, C2C12 cells can also differentiate into osteoblast- and adipocyte-lineage cells. We recently reported that RJ enhances the myogenic differentiation of C2C12 cells. However, the effect of RJ on osteoblast or adipocyte differentiation is still unknown. Here in this study, we have examined the effect of RJ on the osteoblast and adipocyte differentiation of C2C12 cells. Protease-treated RJ was used to reduce the adverse effects caused by RJ supplementation. To induce osteoblast or adipocyte differentiation, cells were treated with bone morphogenetic proteins (BMP) or peroxisome proliferator-activated receptor γ (PPARγ) agonist, respectively. RNA-seq was used to analyze the effect of RJ on gene expression. We found that RJ stimulates osteoblast and adipocyte differentiation. RJ regulated 279 genes. RJ treatment upregulated glutathione-related genes. Glutathione, the most abundant antioxidative factor in cells, has been shown to promote osteoblast differentiation in MSC and MSC-like cells. Therefore, RJ may promote osteogenesis, at least in part, through the antioxidant effects of glutathione. RJ enhances the differentiation ability of C2C12 cells into multiple lineages, including myoblasts, osteoblasts, and adipocytes. [ABSTRACT FROM AUTHOR]

Bone remodeling begins with bone resorption and culminates with the formation of new bone. Several variables influence bone remodeling, including oxidative stress, which results from an imbalance of oxidants, reactive oxygen species (ROS), and antioxidants. Herbal supplements may enhance interactions between antioxidants, growth factors, osteoclasts, and osteoblasts. The aim of this study was to investigate bioactive components in Stingless Bee's (Trigona biroi) propolis interact with antioxidants and biomarkers that enhance growth factor and osteoblastogenesis. The simulation of ligand interaction which attempts to identify a correlation between binding energy or binding affinity and ligand interaction patterns in the target domain. Bioinformatic approach employed is molecular docking screening, which screens compounds with the highest negative binding affinity using a grid that spans the full target area. PyRx v1.0.0 software with an academic license was used to simulate the binding of Stingless Bee's Propolis components to Heat Shock Protein (HSP)-10, HSP-70, Fibroblast Growth Factor-2 (FGF-2), Vascular Endothelial Growth Factor (VEGF), runt-related transcription factor-2 (RUNX2), alkaline phosphatase (ALP), Osteocalcin, and collagen type 1a1 (Coll1a1). Molecular docking simulations show that compounds from stingless bee's (T. biroi) propolis have the most negative binding affinity, such as 27-hydroxymangiferolic acid, which can trigger HSP-70 activity (-9.7 kcal/mol), 27-hydroxyisomangiferolic acid on FGF-2 (-8.3), VEGF (-7.3 kcal/mol), RUNX-2 (-7.7 kcal/mol), ALP (-7.9 kcal/mol), Osteocalcin (-7.3 kcal/mol), Coll1a1 (-5.7 kcal/mol), and (+)-Pinobaksin. 27-hydroxymangiferolic acid, 27-hydroxyisomangiferolic acid, and (+)-Pinobanksin in tingless bee (T. biroi) propolis are thought to activate targets and cause responses involving antioxidant activity, growth factors, and osteoblastogenesis. [ABSTRACT FROM AUTHOR]

Low bone mass is a pervasive global health concern, with implications for osteoporosis, frailty, disability, and mortality. Lifestyle factors, including sedentary habits, metabolic dysfunction, and an aging population, contribute to the escalating prevalence of osteopenia and osteoporosis. The application of mechanical load to bone through physical activity and exercise prevents bone loss, while sufficient mechanical load stimulates new bone mass acquisition. Osteocytes, cells embedded within the bone, receive mechanical signals and translate these mechanical cues into biological signals, termed mechanotransduction. Mechano-transduction signals regulate other bone resident cells, such as osteoblasts and osteoclasts, to orchestrate changes in bone mass. This review explores the mechanisms through which osteocytemediated response to mechanical loading regulates osteoblast differentiation and bone formation. An overview of bone cell biology and the impact of mechanical load will be provided, with emphasis on the mechanical cues, mechano-transduction pathways, and factors that direct progenitor cells toward the osteoblast lineage. While there are a wide range of clinically available treatments for osteoporosis, the majority act through manipulation of the osteoclast and may have significant disadvantages. Despite the central role of osteoblasts to the deposition of new bone, few therapies directly target osteoblasts for the preservation of bone mass. Improved understanding of the mechanisms leading to osteoblastogenesis may reveal novel targets for translational investigation. [ABSTRACT FROM AUTHOR]

AbstractContext Fructus Ligustri Lucidi (FLL), a commonly used herb of traditional Chinese medicine (TCM), is the fruit of Ligustrum lucidum Ait. (Oleaceae). The ethanol extract of FLL is a potential candidate for preventing and treating postmenopausal osteoporosis (PMOP) by nourishing the liver and kidneys.Objective This study determines whether an ethanol extract of FLL has anti-osteoporotic effects in ovariectomized (OVX) mice and explores the underlying mechanism.Materials and methods The OVX model of eight-week-old C57BL/6J female mice was taken, and ovariectomy was used as PMOP. Mice were divided into five groups: sham-operated group (n = 10), OVX group (n = 10), OVX + E2 group (n = 10; 0.039 mg/kg), OVX + FLL group (n = 10; 2 g/kg) and OVX + FLL group (n = 10; 4 g/kg). Mice were treated by gavage with FLL or CMCNa once daily for 8 weeks. We harvested uteri, femur, and tibias from mice; bone mineral density (BMD) and bone microstructure were obtained by X-ray absorptiometry and micro-CT. Furthermore, the effect of FLL on the balance of osteoblast and adipocyte differentiation was investigated using bone marrow mesenchymal stem cells (BMMSCs).Results The results indicated that FLL did not affect OVX-induced estradiol reduction. Compared with OVX mice, FLL significantly increased BMD (63.54 vs. 61.96), Conn. D (86.46 vs. 57.00), and left tibial strength (13.91 vs. 11.27), decreased Tb. Sp (0.38 vs. 0.44) and body fat content (4.19% vs. 11.24%). FLL decreased osteoclast activity and enhanced RUNX2 expression; inhibited perilipin peroxisome proliferator-activated receptor gamma (PPARγ) expression and adipocyte differentiation from BMMSCs.Conclusions FLL prevented additional bone loss and improved bone microstructure in OVX mice by modulating bone and fat balance, suggesting that FLL might be a therapeutic agent for PMOP.

Background: Electromagnetic field therapy is gaining attention for its potential in treating bone disorders, with Extracorporeal Magnetotransduction Therapy (EMTT) emerging as an innovative approach. EMTT offers a higher oscillation frequency and magnetic field strength compared to traditional Pulsed Electromagnetic Field (PEMF) therapy, showing promise in enhancing fracture healing and non-union recovery. However, the mechanisms underlying these effects remain unclear. Results: This study demonstrates that EMTT significantly enhances osteoblast bone formation at multiple levels, from gene expression to extracellular matrix mineralization. Key osteoblastogenesis regulators, including SP7 and RUNX2, and bone-related genes such as COL1A1, ALPL, and BGLAP, were upregulated, with expression levels surpassing those of the control group by over sevenfold (p < 0.001). Enhanced collagen synthesis and mineralization were confirmed by von Kossa and Alizarin Red staining, indicating increased calcium and phosphate deposition. Additionally, calcium imaging revealed heightened calcium influx, suggesting a cellular mechanism for EMTT’s osteogenic effects. Importantly, EMTT did not compromise cell viability, as confirmed by live/dead staining and WST-1 assays. Conclusion: This study is the first to show that EMTT can enhance all phases of osteoblastogenesis and improve the production of critical mineralization components, offering potential clinical applications in accelerating fracture healing, treating osteonecrosis, and enhancing implant osseointegration.

Low bone mass is a pervasive global health concern, with implications for osteoporosis, frailty, disability, and mortality. Lifestyle factors, including sedentary habits, metabolic dysfunction, and an aging population, contribute to the escalating prevalence of osteopenia and osteoporosis. The application of mechanical load to bone through physical activity and exercise prevents bone loss, while sufficient mechanical load stimulates new bone mass acquisition. Osteocytes, cells embedded within the bone, receive mechanical signals and translate these mechanical cues into biological signals, termed mechano-transduction. Mechano-transduction signals regulate other bone resident cells, such as osteoblasts and osteoclasts, to orchestrate changes in bone mass. This review explores the mechanisms through which osteocyte-mediated response to mechanical loading regulates osteoblast differentiation and bone formation. An overview of bone cell biology and the impact of mechanical load will be provided, with emphasis on the mechanical cues, mechano-transduction pathways, and factors that direct progenitor cells toward the osteoblast lineage. While there are a wide range of clinically available treatments for osteoporosis, the majority act through manipulation of the osteoclast and may have significant disadvantages. Despite the central role of osteoblasts to the deposition of new bone, few therapies directly target osteoblasts for the preservation of bone mass. Improved understanding of the mechanisms leading to osteoblastogenesis may reveal novel targets for translational investigation.

Fructus Ligustri Lucidi (FLL), a commonly used herb of traditional Chinese medicine (TCM), is the fruit of Ligustrum lucidum Ait. (Oleaceae). The ethanol extract of FLL is a potential candidate for preventing and treating postmenopausal osteoporosis (PMOP) by nourishing the liver and kidneys. This study determines whether an ethanol extract of FLL has anti-osteoporotic effects in ovariectomized (OVX) mice and explores the underlying mechanism. The OVX model of eight-week-old C57BL/6J female mice was taken, and ovariectomy was used as PMOP. Mice were divided into five groups: sham-operated group (n = 10), OVX group (n = 10), OVX + E2 group (n = 10; 0.039 mg/kg), OVX + FLL group (n = 10; 2 g/kg) and OVX + FLL group (n = 10; 4 g/kg). Mice were treated by gavage with FLL or CMCNa once daily for 8 weeks. We harvested uteri, femur, and tibias from mice; bone mineral density (BMD) and bone microstructure were obtained by X-ray absorptiometry and micro-CT. Furthermore, the effect of FLL on the balance of osteoblast and adipocyte differentiation was investigated using bone marrow mesenchymal stem cells (BMMSCs). The results indicated that FLL did not affect OVX-induced estradiol reduction. Compared with OVX mice, FLL significantly increased BMD (63.54 vs. 61.96), Conn. D (86.46 vs. 57.00), and left tibial strength (13.91 vs. 11.27), decreased Tb. Sp (0.38 vs. 0.44) and body fat content (4.19% vs. 11.24%). FLL decreased osteoclast activity and enhanced RUNX2 expression; inhibited perilipin peroxisome proliferator-activated receptor gamma (PPARγ) expression and adipocyte differentiation from BMMSCs. FLL prevented additional bone loss and improved bone microstructure in OVX mice by modulating bone and fat balance, suggesting that FLL might be a therapeutic agent for PMOP. [ABSTRACT FROM AUTHOR]

Abstract Background Knowledge about regulating transcription factors (TFs) for osteoblastogenesis from mesenchymal stem cells (MSCs) is limited. Therefore, we investigated the relationship between genomic regions subject to DNA-methylation changes during osteoblastogenesis and the TFs known to directly interact with these regulatory regions. Results The genome-wide DNA-methylation signature of MSCs differentiated to osteoblasts and adipocytes was determined using the Illumina HumanMethylation450 BeadChip array. During adipogenesis no CpGs passed our test for significant methylation changes. Oppositely, during osteoblastogenesis we identified 2462 differently significantly methylated CpGs (adj. p

Periodontitis is a progressive disease that ultimately leads to bone and tooth loss. A major consequence of periodontal disease is the inability to regain lost bone in the periodontium. The importance was demonstrated of glucose-regulated protein-78 (GRP78) in the osteogenic differentiation of periodontal ligament stem cells and their potential use for regeneration of the periodontium. Previous studies have shown the relationship between GRP78 and dentine matrix protein-1 (DMP1). The importance of this receptor-ligand complex in supporting the process of osteogenesis and angiogenesis was confirmed in this study. To show the function of GRP78 in mineralised tissues, transgenic periodontal ligament stem cells (PDLSCs) were generated in which GRP78 was either overexpressed or silenced. Gene expression analysis of the cells cultured under osteogenic conditions showed an increase in key osteogenic genes with the overexpression of GRP78. RNA-Seq analysis was also performed to understand the transcriptome profile associated with genotype changes. Using the database for annotation, visualisation, and integration discovery (DAVID) for the functional enrichment analysis of differentially expressed genes, the upregulation of genes promoting osteogenesis and angiogenesis with GRP78 overexpression was demonstrated. Alizarin red staining and scanning electron microscopy analysis revealed matrix mineralisation with increased calcium deposition in GRP78 overexpressing cells. The in vivo osteogenic and angiogenic function of GRP78 was shown using a subcutaneous implantation rodent model. The results suggested that GRP78 in PDLSCs can regulate the expression of both osteogenesis and angiogenesis. Therefore, GRP78 could be considered as a therapeutic target for repair of diseased periodontium.

Bi-allelic variants in ASCC1 cause the ultrarare bone fragility disorder “spinal muscular atrophy with congenital bone fractures-2” (SMABF2). However, the mechanism by which ASCC1 dysfunction leads to this musculoskeletal condition and the nature of the associated bone defect are poorly understood. By exome sequencing, we identified a novel homozygous deletion in ASCC1 in a female infant. She was born with severe muscular hypotonia, inability to breathe and swallow, and virtual absence of spontaneous movements; showed progressive brain atrophy, gracile long bones, very slender ribs, and a femur fracture; and died from respiratory failure aged 3 months. A transiliac bone sample taken postmortem revealed a distinct microstructural bone phenotype with low trabecular bone volume, low bone remodeling, disordered collagen organization, and an abnormally high bone marrow adiposity. Proteomics, RNA sequencing, and qPCR in patient-derived skin fibroblasts confirmed that ASCC1 was hardly expressed on protein and RNA levels compared with healthy controls. Furthermore, we demonstrate that mutated ASCC1 is associated with a downregulation of RUNX2, the master regulator of osteoblastogenesis, and SERPINF1, which is involved in osteoblast and adipocyte differentiation. It also exerts an inhibitory effect on TGF-b/SMAD signaling, which is important for bone development. Additionally, knockdown of ASCC1 in human mesenchymal stromal cells (hMSCs) suppressed their differentiation capacity into osteoblasts while increasing their differentiation into adipocytes. This resulted in reduced mineralization and elevated formation of lipid droplets. These findings shed light onto the pathophysiologic mechanisms underlying SMABF2 and assign a new biological role to ASCC1 acting as an important pro-osteoblastogenic and antiadipogenic regulator. [ABSTRACT FROM AUTHOR]

The balance between bone-resorbing osteoclasts and bone-forming osteoblasts is essential for the bone remodeling process. This study aimed to investigate the effect of Ishophloroglucin A (IPA) isolated from Ishige okamurae on the function of osteoclasts and osteoblasts in vitro. First, we demonstrated the effect of IPA on osteoclastogenesis in receptor activator of nuclear factor κB ligand (RANKL)-induced RAW 264.7 cells. IPA inhibited the tartrate-resistant acid phosphatase (TRAP) activity and osteoclast differentiation in RANKL-induced RAW 264.7 cells. Moreover, it inhibited the RANKL-induced osteoclast-related factors, such as TRAP, matrix metalloproteinase-9 (MMP-9), and calcitonin receptor (CTR), and transcription factors, such as nuclear factor of activated T cells 1 (NFATc1) and c-Fos. IPA significantly suppressed RANKL-activated extracellular signal-regulated kinase (ERK), and NF-κB in RAW 264.7 cells. Our data indicated that the ERK and NF-κB pathways were associated with the osteoclastogenesis inhibitory activity of IPA. Next, we demonstrated the effect of IPA on osteoblastogenesis in MG-63 cells. IPA significantly promoted alkaline phosphatase (ALP) activity in MG-63 cells, along with the osteoblast differentiation-related markers bone morphogenetic protein 2 (BMP2), type 1 collage (COL1), p-Smad1/5/8, and Runx2, by activating the MAPK signaling pathways. Taken together, the study indicated that IPA could be effective in treating bone diseases, such as osteoporosis. [ABSTRACT FROM AUTHOR]

Bone remodelling is mediated by orchestrated communication between osteoclasts and osteoblasts which, in part, is regulated by coupling and anti‐coupling factors. Amongst formally known anti‐coupling factors, Semaphorin 4D (Sema4D), produced by osteoclasts, plays a key role in downmodulating osteoblastogenesis. Sema4D is produced in both membrane‐bound and soluble forms; however, the mechanism responsible for producing sSema4D from osteoclasts is unknown. Sema4D, TACE and MT1‐MMP are all expressed on the surface of RANKL‐primed osteoclast precursors. However, only Sema4D and TACE were colocalized, not Sema4D and MT1‐MMP. When TACE and MT1‐MMP were either chemically inhibited or suppressed by siRNA, TACE was found to be more engaged in shedding Sema4D. Anti‐TACE‐mAb inhibited sSema4D release from osteoclast precursors by ~90%. Supernatant collected from osteoclast precursors (OC‐sup) suppressed osteoblastogenesis from MC3T3‐E1 cells, as measured by alkaline phosphatase activity, but OC‐sup harvested from the osteoclast precursors treated with anti‐TACE‐mAb restored osteoblastogenesis activity in a manner that compensates for diminished sSema4D. Finally, systemic administration of anti‐TACE‐mAb downregulated the generation of sSema4D in the mouse model of critical‐sized bone defect, whereas local injection of recombinant sSema4D to anti‐TACE‐mAb‐treated defect upregulated local osteoblastogenesis. Therefore, a novel pathway is proposed whereby TACE‐mediated shedding of Sema4D expressed on the osteoclast precursors generates functionally active sSema4D to suppress osteoblastogenesis. [ABSTRACT FROM AUTHOR]

Bi-allelic variants in ASCC1 cause the ultrarare bone fragility disorder “spinal muscular atrophy with congenital bone fractures-2” (SMABF2). However, the mechanism by which ASCC1 dysfunction leads to this musculoskeletal condition and the nature of the associated bone defect are poorly understood. By exome sequencing, we identified a novel homozygous deletion in ASCC1 in a female infant. She was born with severe muscular hypotonia, inability to breathe and swallow, and virtual absence of spontaneous movements; showed progressive brain atrophy, gracile long bones, very slender ribs, and a femur fracture; and died from respiratory failure aged 3 months. A transiliac bone sample taken postmortem revealed a distinct microstructural bone phenotype with low trabecular bone volume, low bone remodeling, disordered collagen organization, and an abnormally high bone marrow adiposity. Proteomics, RNA sequencing, and qPCR in patient-derived skin fibroblasts confirmed that ASCC1 was hardly expressed on protein and RNA levels compared with healthy controls. Furthermore, we demonstrate that mutated ASCC1 is associated with a downregulation of RUNX2, the master regulator of osteoblastogenesis, and SERPINF1, which is involved in osteoblast and adipocyte differentiation. It also exerts an inhibitory effect on TGF-β/SMAD signaling, which is important for bone development. Additionally, knockdown of ASCC1 in human mesenchymal stromal cells (hMSCs) suppressed their differentiation capacity into osteoblasts while increasing their differentiation into adipocytes. This resulted in reduced mineralization and elevated formation of lipid droplets. These findings shed light onto the pathophysiologic mechanisms underlying SMABF2 and assign a new biological role to ASCC1 acting as an important pro-osteoblastogenic and anti-adipogenic regulator.

Stem cells' self-renewal and multi-lineage differentiation are regulated by a complex network consisting of signaling factors, chromatin regulators, transcription factors, and non-coding RNAs (ncRNAs). Diverse role of ncRNAs in stem cell development and maintenance of bone homeostasis have been discovered recently. The ncRNAs, such as long non-coding RNAs, micro RNAs, circular RNAs, small interfering RNA, Piwi-interacting RNAs, etc., are not translated into proteins but act as essential epigenetic regulators in stem cells' self-renewal and differentiation. Different signaling pathways are monitored efficiently by the differential expression of ncRNAs, which function as regulatory elements in determining the fate of stem cells. In addition, several species of ncRNAs could serve as potential molecular biomarkers in early diagnosis of bone diseases, including osteoporosis, osteoarthritis, and bone cancers, ultimately leading to the development of new therapeutic strategies. This review aims to explore the specific roles of ncRNAs and their effective molecular mechanisms in the growth and development of stem cells, and in the regulation of osteoblast and osteoclast activities. Furthermore, we focus on and explore the association of altered ncRNA expression with stem cells and bone turnover. [ABSTRACT FROM AUTHOR]

Bone is a dynamic tissue composed of cells, an extracellular matrix, and mineralized portion. Osteoblasts are responsible for proper bone formation and remodeling, and function. These processes are endergonic and require cellular energy in the form of adenosine triphosphate (ATP), which is derived from various sources such as glucose, fatty acids, and amino acids. However, other lipids such as cholesterol have also been found to play a critical role in bone homeostasis and can also contribute to the overall bioenergetic capacity of osteoblasts. In addition, several epidemiological studies have found a link between elevated cholesterol, cardiovascular disease, an enhanced risk of osteoporosis, and increased bone metastasis in cancer patients. This review focuses on how cholesterol, its derivatives, and cholesterol-lowering medications (statins) regulate osteoblast function and bone formation. It also highlights the molecular mechanisms underlying the cholesterol–osteoblast crosstalk. [ABSTRACT FROM AUTHOR]

Background: Knowledge about regulating transcription factors (TFs) for osteoblastogenesis from mesenchymal stem cells (MSCs) is limited. Therefore, we investigated the relationship between genomic regions subject to DNA-methylation changes during osteoblastogenesis and the TFs known to directly interact with these regulatory regions. Results: The genome-wide DNA-methylation signature of MSCs differentiated to osteoblasts and adipocytes was determined using the Illumina HumanMethylation450 BeadChip array. During adipogenesis no CpGs passed our test for significant methylation changes. Oppositely, during osteoblastogenesis we identified 2462 differently significantly methylated CpGs (adj. p < 0.05). These resided outside of CpGs islands and were significantly enriched in enhancer regions. We confirmed the correlation between DNA-methylation and gene expression. Accordingly, we developed a bioinformatic tool to analyse differentially methylated regions and the TFs interacting with them. By overlaying our osteoblastogenesis differentially methylated regions with ENCODE TF ChIP-seq data we obtained a set of candidate TFs associated to DNA-methylation changes. Among them, ZEB1 TF was highly related with DNA-methylation. Using RNA interference, we confirmed that ZEB1, and ZEB2, played a key role in adipogenesis and osteoblastogenesis processes. For clinical relevance, ZEB1 mRNA expression in human bone samples was evaluated. This expression positively correlated with weight, body mass index, and PPARγ expression. Conclusions: In this work we describe an osteoblastogenesis-associated DNA-methylation profile and, using these data, validate a novel computational tool to identify key TFs associated to age-related disease processes. By means of this tool we identified and confirmed ZEB TFs as mediators involved in the MSCs differentiation to osteoblasts and adipocytes, and obesity-related bone adiposity. [ABSTRACT FROM AUTHOR]

Abstract Background Aucubin (AU), an iridoid glucoside isolated from many traditional herbal medicines, has anti-osteoporosis and anti-apoptosis bioactivities. However, the effect of AU on the treatment of bone-fracture remains unknown. In the present study, the aims were to investigate the roles and mechanisms of AU not only on osteoblastogenesis of human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) and anti-oxidative stress injury in vitro, but also on bone-fracture regeneration by a rat tibial fracture model in vivo. Methods CCK-8 assay was used to assess the effect of AU on the viability and proliferation of hBM-MSCs. The expression of specific genes and proteins on osteogenesis, apoptosis and signaling pathways was measured by qRT-PCR, western blotting and immunofluorescence analysis. ALP staining and quantitative analysis were performed to evaluate ALP activity. ARS and quantitative analysis were performed to evaluate calcium deposition. DCFH-DA staining was used to assess the level of reactive oxygen species (ROS). A rat tibial fracture model was established to validate the therapeutic effect of AU in vivo. Micro-CT with quantitative analysis and histological evaluation were used to assess the therapeutic effect of AU locally injection at the fracture site. Results Our results revealed that AU did not affect the viability and proliferation of hBM-MSCs. Compared with control group, western blotting, PCR, ALP activity and calcium deposition proved that AU-treated groups promoted osteogenesis of hBM-MSCs. The ratio of phospho-Smad1/5/9 to total Smad also significantly increased after treatment of AU. AU-induced expression of BMP2 signaling target genes BMP2 and p-Smad1/5/9 as well as of osteogenic markers COL1A1 and RUNX2 was downregulated after treating with noggin and LDN193189. Furthermore, AU promoted the translocation of Nrf2 from cytoplasm to nucleus and the expression level of HO1 and NQO1 after oxidative damage. In a rat tibial fracture model, local injection of AU promoted bone regeneration. Conclusions Our study demonstrates the dual effects of AU in not only promoting bone-fracture healing by regulating osteogenesis of hBM-MSCs partly via canonical BMP2/Smads signaling pathway but also suppressing oxidative stress damage partly via Nrf2/HO1 signaling pathway.

The nuclear receptor subfamily 4 group A member 1 (NR4A1) gene plays a crucial role in both osteoporosis and adipogenesis. The present study investigated the mechanisms by which NR4A1 influences osteoblastogenesis and adipogenesis in human bone marrow-derived mesenchymal stem cells (BMD-MSCs). NR4A1 was overexpressed or knocked down in mouse MC3T3-E1 osteoblast cells and 3T3-L1 adipocyte cells, as well as in PCS-500-012, a BMD-MSC line. The alkaline phosphatase (ALP) assay and Alizarin Red S staining were performed using MC3T3-E1 and BMD-MSCs to assess ALP activity and mineralization, while Oil Red O staining was used to assess the lipid content in 3T3-L1 cells and BMD-MSCs. Total RNA was isolated from control, NR4A1-overexpressing and NR4A1 small interfering RNA (siRNA; siNR4A1)-treated BMD-MSCs. RNA sequencing (RNA-seq) was performed to identify differentially expressed genes, followed by ingenuity pathway analysis (IPA) to determine the role of NR4A1 in osteoblastogenesis and adipogenesis. NR4A1 or Nr4a1 knockdown tended to increase ALP activity and significantly increased calcification in BMD-MSCs (P<0.005) and MC3T3-E1 cells (P<0.005), respectively. By contrast, NR4A1 or Nr4a1 overexpression significantly decreased ALP activity and calcification. NR4A1 or Nr4a1 knockdown and overexpression significantly decreased and increased adipogenesis, respectively, in BMD-MSCs (P<0.005 and <0.05, respectively) and 3T3-L1 cells (P<0.005 in both). Treatments of BMD-MSCs with an NR4A1 antagonist, 1,1-bis(3′-indolyl)-1-(p-hydroxyphenyl) methane and siNR4A1 showed similar results. RNA-seq and IPA in control, NR4A1 knockdown and NR4A1 overexpressing cells indicated that Notch signaling mediated the effects of NR4A1 in osteoblastogenesis and adipogenesis. Expression of mastermind-like transcriptional coactivator 3 was reduced in the Notch signaling pathway in cells treated with siNR4A1. In conclusion, NR4A1 suppressed osteoblastogenesis and promotes adipogenesis in human BMD-MSCs. The present study also suggested that NR4A1 plays a role in the progression of osteoporosis and adipogenesis by modulating the Notch signaling cascade. [ABSTRACT FROM AUTHOR]

Implants made of Ti and its alloys are widely utilized in orthopaedic surgeries. However, insufficient osseointegration of the implants often causes complications such as aseptic loosening. Our previous research discovered that disordered titanium dioxide nanorods (TNrs) had satisfactory antibacterial properties and biocompatibility, but TNrs harmed angiogenic differentiation, which might retarded the osseointegration process of the implants. Magnetic nanomaterials have a certain potential in promoting osseointegration, electromagnetic fields within a specific frequency and intensity range can facilitate angiogenic and osteogenic differentiation. Therefore, this study used Fe3O4 to endow magnetism to TNrs and explored the regulation effects of Ti, TNrs, and Fe3O4-TNrs under 1 ​mT 15 ​Hz sinusoidal electromagnetic field (SEMF) on osteoblastogenesis, osseointegration, angiogenesis, and its mechanism.We discovered that after the addition of SEMF treatment to VR-EPCs cultured on Fe3O4-TNrs, the calcineurin/NFAT signaling pathway was activated, which then reversed the inhibitory effect of Fe3O4-TNrs on angiogenesis. Besides, Fe3O4-TNrs with SEMF enhanced osteogenic differentiation and osseointegration. Therefore, the implant modification mode of Fe3O4-TNrs with the addition of SEMF could more comprehensively promote osseointegration and provided a new idea for the modification of implants.

In cases of severe bone loss, natural healing does not occur because the body's natural regenerative capacity is exceeded. Therefore, there is a need for alternative materials that can support the healing process with good osteointegrative properties. Osteointegration is the relationship between a synthetic biomaterial implanted in the body and the biological response of the host tissue. Osteoblasts play a role in the process of bone remodeling and cooperate with osteoclasts to balance bone formation and resorption and remove mature bone tissue to maintain bone homeostasis. RUNX2 is a member of a small domain of transcription factors that are important for differentiation and proliferation during the transition of mesenchymal stem cells to osteoprogenitor cells during osteoblast differentiation. The demineralized dentin material sponge derived from bovine dentin contains 70% hydroxyapatite, 20% organic matrix, and 10% water, contains organic and inorganic components similar to dentin and human bone, which enhances the expression of RUNX2. It can be used as a bone substitute material. In the process of osteoblast formation. The aim of this study was to determine the potential ability of demineralized dentin sponges to promote RUNX2 expression in osteoblastogenesis. [ABSTRACT FROM AUTHOR]

The macrolide erythromycin (ERM) inhibits excessive neutrophil accumulation and bone resorption in inflammatory tissues. We previously reported that the expression of developmental endothelial locus-1 (DEL-1), an endogenous anti-inflammatory factor induced by ERM, is involved in ERM action. Furthermore, DEL-1 is involved in the induction of bone regeneration. Therefore, in this study, we investigated whether ERM exerts an osteoblastogenic effect by upregulating DEL-1 under inflammatory conditions. We performed in vitro cell-based mechanistic analyses and used a model of Porphyromonas gingivalis lipopolysaccharide (LPS)-induced periodontitis to evaluate how ERM restores osteoblast activity. In vitro, P. gingivalis LPS stimulation suppressed osteoblast differentiation and bone formation. However, ERM treatment combined with P. gingivalis LPS stimulation upregulated osteoblast differentiation-related factors and Del1, indicating that osteoblast differentiation was restored. Alveolar bone resorption and gene expression were evaluated in a periodontitis model, and the results confirmed that ERM treatment increased DEL-1 expression and suppressed bone loss by increasing the expression of osteoblast-associated factors. In conclusion, ERM restores bone metabolism homeostasis in inflammatory environments possibly via the induction of DEL-1. [ABSTRACT FROM AUTHOR]

The extraction of bitumen from surface mining in the Athabasca Oil Sands Region (AOSR) produces large quantities of oil sands process‐affected water (OSPW) that needs to be stored in settling basins near extraction sites. Chemical constituents of OSPW are known to impair bone health in some organisms, which can lead to increased fracture risk and lower reproductive fitness. Naphthenic acid fraction components (NAFCs) are thought to be among the most toxic class of compounds in OSPW; however, the effect of NAFCs on osteoblast development is largely unknown. In this study, we demonstrate that NAFCs from OSPW inhibit osteoblast differentiation and deposition of extracellular matrix, which is required for bone formation. Extracellular matrix deposition was inhibited in osteoblasts exposed to 12.5–125 mg/L of NAFC for 21 days. We also show that components within NAFCs inhibit the expression of gene markers of osteoblast differentiation and function, namely, alkaline phosphatase (Alp), osteocalcin, and collagen type 1 alpha 1 (Col1a1). These effects were partially mediated by the induction of glucocorticoid receptor (GR) activity; NAFC induces the expression of the GR activity marker genes Sgk1 (12.5 mg/L) and p85a (125 mg/L) and inhibits GR protein (125 mg/L) and Opg RNA (12.5 mg/L) expression. This study provides evidence that NAFC concentrations of 12.5 mg/L and above can directly act on osteoblasts to inhibit bone formation and suggests that NAFCs contain components that can act as GR agonists, which may have further endocrine disrupting effects on exposed wildlife. Surface mining in the Athabasca Oil Sands Region (AOSR) produces large quantities of oil sands process‐affected water (OSPW), constituents of which are known to impair bone health in some organisms. Naphthenic acid fraction components (NAFCs) are thought to be responsible for most of the toxic effect of OSPW, but their effects on bone are unknown. In this study, we demonstrate that NAFCs extracted from the AOSR inhibit osteoblast differentiation and may act via the glucocorticoid receptor. [ABSTRACT FROM AUTHOR]

Background: Interferon-gamma (IFN-γ) is an immune-derived cytokines in the innate and adaptive immune responses, and functions as a major pro-inflammatory cytokine. IFNγ has previously been reported involving in the regulation of bone metabolism. However, contradictory results about the roles of IFN-γ in bone formation or bone resorption have been reported. It is possible that the functions of IFN-γ in bone formation is dose-dependent or time-dependent. In this study we examined the effect of IFN-γ on different stages of osteoblastogenesis and bone formation. Materials and methods: Cell proliferation, gene expression and protein levels of the critical effectors involving in different stages of differentiation were compared between differentiating preosteoblast MC3T3-E1 treated with or without IFN-γ at different stages. Cell proliferation were determined by MTT assay. Expression levels of osteoblast differentiation markers was performed by quantitative PCR assay. Also, western blot was conducted to investigate the protein levels in those effectors. Conclusion: IFN-γ regulates osteoblast and bone formation in a stage-dependent manner. IFN-γ did not alter and the expression of critical osteogenic transcription factors, such as Runx2 and Cbfb, suggesting that the differentiation was not disrupted by IFN-γ. The cell number and the levels of matrix proteins, including COL1A and BSP, at both early and late stage of osteoblastogenesis were downregulated by IFN-γ, indicating its negative regulating roles in early stages. In contrast, the mineralization protein ALP and OCN was upregulated at late stages. The results suggested that IFN-γ might act as a negative regulator in osteoblast differentiation and bone formation at early stages but switch into positive regulator at late stage. Our data revealed the complex features of the effects of IFN-γ on osteoblast differentiation. The detailed mechanisms of how IFN-γ influence on the bone formation and balance of bone remodeling will be further studied. [ABSTRACT FROM AUTHOR]

Mesenchymal stem cells (MSCs) osteogenic commitment before injection enhances bone regeneration therapy results. Piezoelectric stimulation may be an effective cue to promote MSCs pre-differentiation, and poly(vinylidene) fluoride (PVDF) cell culture supports, when combined with CoFe2O4 (CFO), offer a wireless in vitro stimulation strategy. Under an external magnetic field, CFO shift and magnetostriction deform the polymer matrix varying the polymer surface charge due to the piezoelectric effect. To test the effect of piezoelectric stimulation on MSCs, our approach is based on a gelatin hydrogel with embedded MSCs and PVDF-CFO electroactive microspheres. Microspheres were produced by electrospray technique, favouring CFO incorporation, crystallisation in β-phase (85%) and a crystallinity degree of around 55%. The absence of cytotoxicity of the 3D construct was confirmed 24 h after cell encapsulation. Cells were viable, evenly distributed in the hydrogel matrix and surrounded by microspheres, allowing local stimulation. Hydrogels were stimulated using a magnetic bioreactor, and no significant changes were observed in MSCs proliferation in the short or long term. Nevertheless, piezoelectric stimulation upregulated RUNX2 expression after 7 days, indicating the activation of the osteogenic differentiation pathway. These results open the door for optimising a stimulation protocol allowing the application of the magnetically activated 3D electroactive cell culture support for MSCs pre-differentiation before transplantation. [ABSTRACT FROM AUTHOR]

Melatonin, the primary hormone involved in circadian entrainment, plays a significant role in bone physiology. This study aimed to assess the role of MEK1/2 and MEK5 in melatonin‐mediated actions in mouse and human mesenchymal stem cells (MSCs) and on bone using small‐molecule inhibitors and CRISPR/Cas9 knockout approaches. Consistent with in vitro studies performed in mMSCs and hMSCs, nightly (25 mg/kg, i.p., 45 days) injections with PD184352 (MEK1/2 inhibitor) or Bix02189 (MEK5 inhibitor) or SC‐1‐151 (MEK1/2/5 inhibitor) demonstrated that MEK1/2 and MEK5 were the primary drivers underlying melatonin's actions on bone density, microarchitecture (i.e., trabecular number, separation, and connectivity density), and bone mechanical properties (i.e., ultimate stress) through increases in osteogenic (RUNX2, BMP‐2, FRA‐1, OPG) expression and decreases in PPARγ. Furthermore, CRISPR/Cas9 knockout of MEK1 or MEK5 in mMSCs seeded on PLGA scaffolds and placed into critical‐size calvarial defects in Balb(c) mice (male and female) revealed that treatment with melatonin (15 mg/L; p.o., nightly, 90 days) mediates sex‐specific actions of MEK1 and MEK5 in new bone formation. This study is the first to demonstrate a role for MEK1/2 and MEK5 in modulating melatonin‐mediated actions on bone formation in vivo and in a sex‐specific manner. [ABSTRACT FROM AUTHOR]

Endoplasmic reticulum (ER) is a multifunctional membrane-enclosed organelle. One of the major ER functions is cotranslational transport and processing of secretory, lysosomal, and transmembrane proteins. Impaired protein processing caused by disturbances in the ER homeostasis results in the ER stress. Restoration of normal ER functioning requires activation of an adaptive mechanism involving cell response to misfolded proteins, the so-called unfolded protein response (UPR). Besides controlling protein folding, UPR plays a key role in other physiological processes, in particular, differentiation of cells of connective, muscle, epithelial, and neural tissues. Cell differentiation is induced by the physiological levels of ER stress, while excessive ER stress suppresses differentiation and can result in cell death. So far, it remains unknown whether UPR activation induces cell differentiation or if UPR is initiated by the upregulated synthesis of secretory proteins during cell differentiation. Cell differentiation is an important stage in the development of multicellular organisms and is tightly controlled. Suppression or excessive activation of this process can lead to the development of various pathologies in an organism. In particular, impairments in the differentiation of connective tissue cells can result in the development of fibrosis, obesity, and osteoporosis. Recently, special attention has been paid to fibrosis as one of the major complications of COVID-19. Therefore, studying the role of UPR in the activation of cell differentiation is of both theoretical and practical interest, as it might result in the identification of molecular targets for selective regulation of cell differentiation stages and as well as the potential to modulate the mechanisms involved in the development of various pathological states. [ABSTRACT FROM AUTHOR]

The balance between bone-resorbing osteoclasts and bone-forming osteoblasts is essential for the bone remodeling process. This study aimed to investigate the effect of Ishophloroglucin A (IPA) isolated from Ishige okamurae on the function of osteoclasts and osteoblasts in vitro. First, we demonstrated the effect of IPA on osteoclastogenesis in receptor activator of nuclear factor κB ligand (RANKL)-induced RAW 264.7 cells. IPA inhibited the tartrate-resistant acid phosphatase (TRAP) activity and osteoclast differentiation in RANKL-induced RAW 264.7 cells. Moreover, it inhibited the RANKL-induced osteoclast-related factors, such as TRAP, matrix metalloproteinase-9 (MMP-9), and calcitonin receptor (CTR), and transcription factors, such as nuclear factor of activated T cells 1 (NFATc1) and c-Fos. IPA significantly suppressed RANKL-activated extracellular signal-regulated kinase (ERK), and NF-κB in RAW 264.7 cells. Our data indicated that the ERK and NF-κB pathways were associated with the osteoclastogenesis inhibitory activity of IPA. Next, we demonstrated the effect of IPA on osteoblastogenesis in MG-63 cells. IPA significantly promoted alkaline phosphatase (ALP) activity in MG-63 cells, along with the osteoblast differentiation-related markers bone morphogenetic protein 2 (BMP2), type 1 collage (COL1), p-Smad1/5/8, and Runx2, by activating the MAPK signaling pathways. Taken together, the study indicated that IPA could be effective in treating bone diseases, such as osteoporosis.

Background: Aucubin (AU), an iridoid glucoside isolated from many traditional herbal medicines, has anti-osteoporosis and anti-apoptosis bioactivities. However, the effect of AU on the treatment of bone-fracture remains unknown. In the present study, the aims were to investigate the roles and mechanisms of AU not only on osteoblastogenesis of human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) and anti-oxidative stress injury in vitro, but also on bone-fracture regeneration by a rat tibial fracture model in vivo. Methods: CCK-8 assay was used to assess the effect of AU on the viability and proliferation of hBM-MSCs. The expression of specific genes and proteins on osteogenesis, apoptosis and signaling pathways was measured by qRT-PCR, western blotting and immunofluorescence analysis. ALP staining and quantitative analysis were performed to evaluate ALP activity. ARS and quantitative analysis were performed to evaluate calcium deposition. DCFH-DA staining was used to assess the level of reactive oxygen species (ROS). A rat tibial fracture model was established to validate the therapeutic effect of AU in vivo. Micro-CT with quantitative analysis and histological evaluation were used to assess the therapeutic effect of AU locally injection at the fracture site. Results: Our results revealed that AU did not affect the viability and proliferation of hBM-MSCs. Compared with control group, western blotting, PCR, ALP activity and calcium deposition proved that AU-treated groups promoted osteogenesis of hBM-MSCs. The ratio of phospho-Smad1/5/9 to total Smad also significantly increased after treatment of AU. AU-induced expression of BMP2 signaling target genes BMP2 and p-Smad1/5/9 as well as of osteogenic markers COL1A1 and RUNX2 was downregulated after treating with noggin and LDN193189. Furthermore, AU promoted the translocation of Nrf2 from cytoplasm to nucleus and the expression level of HO1 and NQO1 after oxidative damage. In a rat tibial fracture model, local injection of AU promoted bone regeneration. Conclusions: Our study demonstrates the dual effects of AU in not only promoting bone-fracture healing by regulating osteogenesis of hBM-MSCs partly via canonical BMP2/Smads signaling pathway but also suppressing oxidative stress damage partly via Nrf2/HO1 signaling pathway. [ABSTRACT FROM AUTHOR]

Dickkopf-1 (Dkk-1) is a key regulator of bone remodeling in spondyloarthropathies. Nevertheless, data regarding its expression in cells of pathophysiologic relevance, such as mesenchymal stem cells (MSCs), are lacking. Herein, we aimed to address DKK1 gene expression and Wnt pathway activation in MSCs from patients with ankylosing spondylitis (AS) and explore the effect of IL-17 on MSCs with respect to DKK-1 expression and Wnt pathway activation. Primary MSCs were isolated from the bone marrow of the femoral head of two patients with AS and two healthy controls undergoing orthopedic surgery. MSCs were cultured for 7 days in expansion medium and for 21 days in osteogenic medium in the presence or absence of IL-17A. Gene expression of DKK-1 and osteoblastic markers was determined by RT-PCR. Alkaline phosphatase activity, alizarin red and Van Kossa staining were used to assess osteoblastic function and mineralization capacity. DKK-1 was significantly downregulated in MSCs and osteoblasts from patients with AS compared to controls. Moreover, MSCs and osteoblasts from AS patients displayed increased Wnt pathway activation and enhanced osteoblastic activity, as indicated by increased expression of osteoblast marker genes and alkaline phosphatase activity. IL-17 downregulated DKK-1 expression and increased osteoblastic activity and mineralization capacity. DKK-1 is underexpressed in MSCs from AS patients compared to controls, whereas IL-17 has an inhibitory effect on DKK-1 expression and stimulates osteoblastic function. These data may have pathogenetic and clinical implications in AS. [ABSTRACT FROM AUTHOR]

Abstract Background Inflammatory microenvironment is significant to the differentiation and function of mesenchymal stem cells (MSCs). It evidentially influences the osteoblastogenesis of MSCs. IL-34, a newly discovered cytokine, playing a key role in metabolism. However, the research on its functional role in the osteogenesis of MSCs was rarely reported. Here, we described the regulatory effects of low-dose IL-34 on both osteoblastogenesis and osteoclastogenesis. Methods We performed the osteogenic effects of hBMSCs by exogenous and overexpressed IL-34 in vitro, so were the osteoclastogenesis effects of mBMMs by extracellular IL-34. CCK-8 was used to assess the effect of IL-34 on the viability of hBMSCs and mBMMs. ALP, ARS, and TRAP staining was used to evaluate ALP activity, mineral deposition, and osteoclastogenesis, respectively. qRT-PCR and Western blotting analysis were performed to detect the expression of target genes and proteins. ELISA was used to evaluate the concentrations of IL-34. In vivo, a rat tibial osteotomy model and an OVX model were established. Radiographic analysis and histological evaluation were performed to confirm the therapeutic effects of IL-34 in fracture healing and osteoporosis. Statistical differences were evaluated by two-tailed Student’s t test, one-way ANOVA with Bonferroni’s post hoc test, and two-way ANOVA with Bonferroni multiple comparisons post hoc test in the comparison of 2 groups, more than 2 groups, and different time points of treated groups, respectively. Results Promoted osteoblastogenesis of hBMSCs was observed after treated by exogenous or overexpressed IL-34 in vitro, confirmed by increased mineral deposits and ALP activity. Furthermore, exogenous or overexpressed IL-34 enhanced the expression of p-AKT and p-ERK. The specific AKT and ERK signaling pathway inhibitors suppressed the enhancement of osteoblastogenesis induced by IL-34. In a rat tibial osteotomy model, imaging and histological analyses testified the local injection of exogenous IL-34 improved bone healing. However, the additional IL-34 has no influence on both osteoclastogenesis of mBMMs in vitro and osteoporosis of OVX model of rat in vivo. Conclusions Collectively, our study demonstrate that low-dose IL-34 regulates osteogenesis of hBMSCs partly via the PIK/AKT and ERK signaling pathway and enhances fracture healing, with neither promoting nor preventing osteoclastogenesis in vitro and osteoporosis in vivo.

Seventy million patients worldwide are suffering from epilepsy. The long-term use of antiepileptic drugs causes the alteration of the bone tissue and its metabolism, thus increasing the risk of fractures. Clinical and pre-clinical studies have highlighted conflicting data on the influence of the relatively new antiepileptic drug pregabalin (Lyrica®). The objective of the present study was therefore to investigate its cytotoxicity in primary human osteoblasts (hOB). HOB and human mesenchymal stem cells (hMSC) were isolated from patients. The human osteosarcoma cells MG63 were included as established cell line. Cells were incubated with pregabalin at concentrations ranging from 0 to 40 μg/mL. Time-dependent cell proliferation was measured by automatic cell counting, and metabolism was determined by XTT assay and osseous differentiation by alkaline phosphatase (ALP) activity. Histological examinations of calcium deposit were performed with ALP, Alizarin Red, and von Kossa staining. A concentration-dependent increase in the proliferation of hOB and hMSC was observed after treatment with pregabalin. All cells showed a significant increase in cell metabolism. The osteogenic differentiation, confirmed by the increase of calcium deposit, was promoted by the administration of pregabalin. This effect was already significant at the therapeutic plasma concentration of pregabalin (10 μg/mL). In contrast to the other antiepileptic drugs, pregabalin showed no osteocatabolic effects. Conflicting in-vivo data must therefore be attributed to systemic effects of pregabalin. [ABSTRACT FROM AUTHOR]

Introduction. In osteoporosis field, it had been clinically well established a given relationship between bone formation and lipid accumulation. Although numerous molecules had been well documented for adipogenesis and osteoblastogenesis (adipo-osteoblastogenesis), the reciprocal transcriptional regulation still remains to be explored. Material and methods. Here, we tried to identify the common candidate genes of adipocyte/osteoblastocyte differentiation at 3, 5, and 7 days using human mesenchymal stem cells (hMSCs) via RNA-Seq technique. By using RNA interference (RNAi), we further confirmed the function of candidate genes during adipo-osteoblastogenesis through Oil Red/Alizarin Red/alkaline phosphatase (ALPL) staining and qRT-PCR (quantitative real-time PCR). Results. The identified 275 significantly differentially expressed genes (DEGs), especially with the down-regulated genes most prevalent and PI3K-AKT signaling pathway mostly enriched, were simultaneously shared by both differentiation events. Using lentiviral system, we further confirmed that ANKRD1 (ankyrin repeat domain 1) promoted adipogenesis and inhibited osteoblastogenesis via RNA interference (RNAi), and IGF1 (insulin like growth factor 1) simultaneously facilitated adipo-osteoblastogenesis on the base of gene expression of biomarkers and cellular phenotype property. Conclusion. This study would provide the potential molecular switches to control the adipocyte/osteoblastocyte balance or hMSCs fate choices and clues to screen the study and therapy targets of metabolic bone disease osteoporosis. [ABSTRACT FROM AUTHOR]

Bone morphogenic protein (BMP-) 2 plays an important role in the regeneration of bone defects by promoting osteogenic differentiation. However, several animal studies have reported adverse side effects of BMP-2, including osteoclast activation, induction of peroxisome proliferator- activated receptor gamma (PPARG)expression, and inflammation. High BMP-2 concentrations are thought to be responsible for these side effects. For this reason, primary pre-osteoblasts were exposed to lower BMP-2 concentrations (1 and 2 µg/mL). Long-term exposure (up to 28 days) was performed to investigate whether this stimulation protocol may promote osteogenic differentiation without causing the side effects mentioned above. The results showed that BMP-2 treatment for 14 or 28 days resulted in increased osteogenesis, through an increase in runt-related transcription factor 2, osterix, alkaline phosphatase, and integrin-binding sialoprotein expression. However, an increase in tumor necrosis factor alpha and receptor activator of nuclear factor kappa-Β ligand protein levels was observed after BMP-2 exposure, indicating also an increased potential for osteoclast activation by osteoblasts. Additionally, morphological changes like intracellular, filled vacuoles could be detected. Enhanced PPARG and perilipin 1 mRNA transcripts and lipid droplets indicated an induced adipogenic differentiation. Overall, the data demonstrate that long-term BMP-2 exposure promotes not only osteogenic differentiation but also adipogenesis and regulates mediators involved in osteoclast activation in vitro. [ABSTRACT FROM AUTHOR]

Chromatin remodeling, specifically the tissue-specific regulation in mineralized tissues, is an understudied avenue of gene regulation. Here we show that Baf45a and Baf45d, two Baf45 homologs belong to ATPase-dependent SWI/SNF chromatin remodeling complex, preferentially expressed in osteoblasts and odontoblasts compared to Baf45b and Baf45c. Recently, biochemical studies revealed that BAF45A associates with Polybromo-associated BAF (PBAF) complex. However, the BAF45D subunit belongs to the polymorphic canonical BRG1-associated factor (cBAF) complex. Protein profiles of osteoblast and odontoblast differentiation uncovered a significant increase of BAF45A and PBAF subunits during early osteoblast and odontoblast maturation. Chromatin immunoprecipitation sequencing (ChIP-seq) during the bone marrow stromal cells (BMSCs) differentiation showed higher histone H3K9 and H3K27 acetylation modifications in the promoter of Baf45a and Baf45d and increased binding of bone and tooth specific transcription factor RUNX2. Overexpression of Baf45a in osteoblasts activates genes essential for the progression of osteoblast maturation and mineralization. Furthermore, shRNA-mediated knockdown of Baf45a in odontoblasts leads to markedly altered genes responsible for the proliferation, apoptosis, DNA repair, and modest decrease in dentinogenic marker gene expression. Assay for Transposase-Accessible Chromatin sequencing (ATAC-seq) assay in Baf45a knockout osteoblasts revealed a noticeable reduction in chromatin accessibility of osteoblast and odontoblast specific genes, along with transcription factor Atf4 and Klf4. Craniofacial mesenchyme-specific loss of Baf45a modestly reduced the mineralization of the tooth and mandibular bone. These findings indicated that BAF45A-dependent mineralized tissue-specific chromatin remodeling through PBAF-RUNX2 crosstalk results in transcriptional activation is critical for early differentiation and matrix maturation of mineralized tissues.

Chromatin remodeling, specifically the tissue-specific regulation in mineralized tissues, is an understudied avenue of gene regulation. Here we show that Baf45a and Baf45d , two Baf45 homologs belong to ATPase-dependent SWI/SNF chromatin remodeling complex, preferentially expressed in osteoblasts and odontoblasts compared to Baf45b and Baf45c. Recently, biochemical studies revealed that BAF45A associates with Polybromo-associated BAF (PBAF) complex. However, the BAF45D subunit belongs to the polymorphic canonical BRG1-associated factor (cBAF) complex. Protein profiles of osteoblast and odontoblast differentiation uncovered a significant increase of BAF45A and PBAF subunits during early osteoblast and odontoblast maturation. Chromatin immunoprecipitation sequencing (ChIP-seq) during the bone marrow stromal cells (BMSCs) differentiation showed higher histone H3K9 and H3K27 acetylation modifications in the promoter of Baf45a and Baf45d and increased binding of bone and tooth specific transcription factor RUNX2. Overexpression of Baf45a in osteoblasts activates genes essential for the progression of osteoblast maturation and mineralization. Furthermore, shRNA -mediated knockdown of Baf45a in odontoblasts leads to markedly altered genes responsible for the proliferation, apoptosis, DNA repair, and modest decrease in dentinogenic marker gene expression. Assay for Transposase-Accessible Chromatin sequencing (ATAC-seq) assay in Baf45a knockout osteoblasts revealed a noticeable reduction in chromatin accessibility of osteoblast and odontoblast specific genes, along with transcription factor Atf4 and Klf4. Craniofacial mesenchyme-specific loss of Baf45a modestly reduced the mineralization of the tooth and mandibular bone. These findings indicated that BAF45A-dependent mineralized tissue-specific chromatin remodeling through PBAF-RUNX2 crosstalk results in transcriptional activation is critical for early differentiation and matrix maturation of mineralized tissues. [ABSTRACT FROM AUTHOR]

Peroxisome proliferator-activated receptor-γ coactivator-1 (PGC-1) is an inducible co-regulator of nuclear receptors and is involved in a wide variety of biological responses. As the master regulators of mitochondrial biogenesis and function, PGC-1α and PGC-1β have been reported to play key roles in bone metabolism. They can be rapidly induced under conditions of increased metabolic activities, such as osteoblastogenesis and osteoclastogenesis, to fulfill greater energy demand or facilitate other biochemical reactions. PGC-1α and PGC-1β have both overlapping and distinct functions with each other among their target organs. In bone homeostasis, PGC-1α and PGC-1β promote the expression of genes required for mitochondrial biogenesis via coactivator interactions with key transcription factors, respectively regulating osteoblastogenesis and osteoclastogenesis. Here, we review the current understanding of how PGC-1α and PGC-1β affect osteoblastogenesis and osteoclastogenesis, how these two PGC-1 coactivators are regulated in bone homeostasis, and how we can translate these findings into therapeutic potential for bone metabolic diseases. [ABSTRACT FROM AUTHOR]

Introduction. With cellular lipid storage varying, the balance between lipid intake and lipid degradation was a must to keep healthy and determined the level of lipid droplets. Although lipid droplets accumulation had been well demonstrated in adipocytes, gene expression profiling and gene function during adipogenesis and osteoblastogenesis remain unknown. Material and methods. Here, this work profiled gene transcriptional landscapes of lipid droplets formation during adipogenesis from human mesenchymal stem cells (hMSCs) using RNA-Seq technique. By using RNA interference (RNAi) we investigated the function of candidate genes during adipogenesis and osteoblastogenesis using Oil Red/Alizarin Red/alkaline phosphatase (ALPL) staining and qRT-PCR (quantitative real-time PCR). Results. Eleven differentially up-regulated genes associated with lipid droplets formation were identified at 3, 5, 7, 14, 21, and 28 days during adipogenesis. Unexpectedly, APOB per se inhibiting adipogenesis weakened osteoblastogenesis and METTL7A facilitating adipogenesis negligibly inhibited osteoblastogenesis according to the phenotypic characterization of adipocytes and osteoblasts and transcriptional condition of biomarkers through lentivirus transfection assays. Conclusions. The establishment of the gene transcriptional profiling of lipid droplets formation would provide the molecular switches of hMSCs cell fate determination and the study targets for fat metabolic diseases. [ABSTRACT FROM AUTHOR]