Tauroursodeoxycholic Acid Mitigates Oxidative Stress and Promotes Differentiation in High Salt-Stimulated Osteoblasts via NOX1 Mediated by PGC-1α.

BACKGROUND: High-salt diet (HSD) is a pivotal risk factor for osteoporosis (OP). Accumulating evidence has supported that tauroursodeoxycholic acid (TUDCA), a naturally produced hydrophilic bile acid, exerts positive effects on the treatment of OP. This study is committed to shedding light on the impacts of TUDCA on high salt-treated osteoblasts and probing into its underlying mechanisms of action. METHODS: Cell counting kit-8 (CCK-8) assay was used to determine the viability of osteoblasts. Alkaline phosphatase (ALP) staining and Alizarin red S (ARS) staining were used to measure osteoblast differentiation. Reverse transcription-quantitative PCR (RT-qPCR) and western blot were used to examine the expression of osteogenic markers. Western blot was also used to analyze the expression of superoxide dismutase-2 (SOD2), peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1α), and NADPH oxidase 1 (NOX1). The production of reactive oxygen species (ROS) was evaluated via dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay. Following PGC-1α knockdown in TUDCA-pretreated osteoblasts exposed to NaCl, the aforementioned functional experiments were implemented again. RESULTS: MC3T3-E1 cell viability was not significantly impacted by increasing concentrations of TUDCA. However, in NaCl-exposed MC3T3-E1 cells, the viability loss, oxidative stress, and decline of differentiation were all dose-dependently obstructed by TUDCA treatment. Moreover, NaCl exposure reduced PGC-1α expression and increased NOX1 expression, which was then reversed by TUDCA. PGC-1α deletion partially abolished the effects of TUDCA on PGC-1α and NOX1, differentiation, and oxidative stress in NaCl-treated osteoblasts. CONCLUSIONS: TUDCA might protect against high salt-induced OP via modulation of NOX1 mediated by PGC-1α.

The effects of keratin-coated titanium on osteoblast function and bone regeneration.

Wool derived keratin, due to its demonstrated ability to promote bone formation, has been suggested as a potential bioactive material for implant surfaces. The aim of this study was to assess the effects of keratin-coated titanium on osteoblast functionin vitroand bone healingin vivo. Keratin-coated titanium surfaces were fabricated via solvent casting and molecular grafting. The effect of these surfaces on the attachment, osteogenic gene, and osteogenic protein expression of MG-63 osteoblast-like cells were quantifiedin vitro. The effect of these keratin-modified surfaces on bone healing over three weeks using an intraosseous calvaria defect was assessed in rodents. Keratin coating did not affect MG-63 proliferation or viability, but enhanced osteopontin, osteocalcin and bone morphogenetic expressionin vitro. Histological analysis of recovered calvaria specimens showed osseous defects covered with keratin-coated titanium had a higher percentage of new bone area two weeks after implantation compared to that in defects covered with titanium alone. The keratin-coated surfaces were biocompatible and stimulated osteogenic expression in adherent MG-63 osteoblasts. Furthermore, a pilot preclinical study in rodents suggested keratin may stimulate earlier intraosseous calvaria bone healing.

The extracts of osteoblast developed from adipose-derived stem cell and its role in osteogenesis.

Cell-based therapy has become an achievable choice in regenerative medicines, particularly for musculoskeletal disorders. Adipose-derived stem cells (ASCs) are an outstanding resource because of their ability and functions. Nevertheless, the use of cells for treatment comes with difficulties in operation and safety. The immunological barrier is also a major limitation of cell therapy, which can lead to unexpected results. Cell-derived products, such as cell extracts, have gained a lot of attention to overcome these limitations. The goal of this study was to optimize the production of ASC-osteoblast extracts as well as their involvement in osteogenesis. The extracts were prepared using a freeze-thaw method with varying temperatures and durations. Overall, osteogenic-associated proteins and osteoinductive potential of the extracts prepared from the osteogenic-induced ASCs were assessed. Our results demonstrated that the freeze-thaw approach is practicable for cell extracts production, with minor differences in temperature and duration having no effect on protein concentration. The ASC-osteoblast extracts contain a significant level of essential specialized proteins that promote osteogenicity. Hence, the freeze-thaw method is applicable for extract preparation and ASC-osteoblast extracts may be beneficial as an optional facilitating biologics in bone anabolic treatment and bone regeneration.

Protecting Orthopaedic Implants from Infection: Antimicrobial Peptide Mel4 Is Non-Toxic to Bone Cells and Reduces Bacterial Colonisation When Bound to Plasma Ion-Implanted 3D-Printed PAEK Polymers.

Even with the best infection control protocols in place, the risk of a hospital-acquired infection of the surface of an implanted device remains significant. A bacterial biofilm can form and has the potential to escape the host immune system and develop resistance to conventional antibiotics, ultimately causing the implant to fail, seriously impacting patient well-being. Here, we demonstrate a 4 log reduction in the infection rate by the common pathogen S. aureus of 3D-printed polyaryl ether ketone (PAEK) polymeric surfaces by covalently binding the antimicrobial peptide Mel4 to the surface using plasma immersion ion implantation (PIII) treatment. The surfaces with added texture created by 3D-printed processes such as fused deposition-modelled polyether ether ketone (PEEK) and selective laser-sintered polyether ketone (PEK) can be equally well protected as conventionally manufactured materials. Unbound Mel4 in solution at relevant concentrations is non-cytotoxic to osteoblastic cell line Saos-2. Mel4 in combination with PIII aids Saos-2 cells to attach to the surface, increasing the adhesion by 88% compared to untreated materials without Mel4. A reduction in mineralisation on the Mel4-containing surfaces relative to surfaces without peptide was found, attributed to the acellular portion of mineral deposition.

Porous Nano-Fiber Structure of Modified Electrospun Chitosan GBR Membranes Improve Osteoblast Calcium Phosphate Deposition in Osteoblast-Fibroblast Co-Cultures.

Desirable characteristics of electrospun chitosan membranes (ESCM) for guided bone regeneration are their nanofiber structure that mimics the extracellular fiber matrix and porosity for the exchange of signals between bone and soft tissue compartments. However, ESCM are susceptible to swelling and loss of nanofiber and porous structure in physiological environments. A novel post-electrospinning method using di-tert-butyl dicarbonate (tBOC) prevents swelling and loss of nanofibrous structure better than sodium carbonate treatments. This study aimed to evaluate the hypothesis that retention of nanofiber morphology and high porosity of tBOC-modified ESCM (tBOC-ESCM) would support more bone mineralization in osteoblast-fibroblast co-cultures compared to Na2CO3 treated membranes (Na2CO3-ESCM) and solution-cast chitosan solid films (CM-film). The results showed that only the tBOC-ESCM retained the nanofibrous structure and had approximately 14 times more pore volume than Na2CO3-ESCM and thousands of times more pore volume than CM-films, respectively. In co-cultures, the tBOC-ESCM resulted in a significantly greater calcium-phosphate deposition by osteoblasts than either the Na2CO3-ESCM or CM-film (p < 0.05). This work supports the study hypothesis that tBOC-ESCM with nanofiber structure and high porosity promotes the exchange of signals between osteoblasts and fibroblasts, leading to improved mineralization in vitro and thus potentially improved bone healing and regeneration in guided bone regeneration applications.

Extracellular sodium regulates fibroblast growth factor 23 (FGF23) formation.

The bone-derived hormone fibroblast growth factor-23 (FGF23) has recently received much attention due to its association with chronic kidney disease and cardiovascular disease progression. Extracellular sodium concentration ([Na+]) plays a significant role in bone metabolism. Hyponatremia (lower serum [Na+]) has recently been shown to be independently associated with FGF23 levels in patients with chronic systolic heart failure. However, nothing is known about the direct impact of [Na+] on FGF23 production. Here, we show that an elevated [Na+] (+20 mM) suppressed FGF23 formation, whereas low [Na+] (-20 mM) increased FGF23 synthesis in the osteoblast-like cell lines UMR-106 and MC3T3-E1. Similar bidirectional changes in FGF23 abundance were observed when osmolality was altered by mannitol but not by urea, suggesting a role of tonicity in FGF23 formation. Moreover, these changes in FGF23 were inversely proportional to the expression of NFAT5 (nuclear factor of activated T cells-5), a transcription factor responsible for tonicity-mediated cellular adaptations. Furthermore, arginine vasopressin, which is often responsible for hyponatremia, did not affect FGF23 production. Next, we performed a comprehensive and unbiased RNA-seq analysis of UMR-106 cells exposed to low versus high [Na+], which revealed several novel genes involved in cellular adaptation to altered tonicity. Additional analysis of cells with Crisp-Cas9-mediated NFAT5 deletion indicated that NFAT5 controls numerous genes associated with FGF23 synthesis, thereby confirming its role in [Na+]-mediated FGF23 regulation. In line with these in vitro observations, we found that hyponatremia patients have higher FGF23 levels. Our results suggest that [Na+] is a critical regulator of FGF23 synthesis.

Resveratrol induces proliferation and differentiation of mouse pre-osteoblast MC3T3-E1 by promoting autophagy.

BACKGROUND: In mouse, it was discovered that resveratrol (Res) enhanced osteoporosis (OP) by boosting osteogenesis. Besides, Res can also have an impact on MC3T3-E1 cells, which are crucial for the control of osteogenesis and thus increase osteogenesis. Although some articles have discovered that Res enhanced autophagy to promote the value-added differentiation of MC3T3, it is unclear exactly how this affects the process of osteogenesis in mouse. Therefore, we will show that Res encourages MC3T3-E1 proliferation and differentiation in mouse pre-osteoblasts and further investigate the autophagy-related mechanism for this impact. METHODS: (1) MC3T3-E1 cells were separated into blank control group and various concentrations (0.01, 0.1, 1, 10, 100µmol/L) of group in order to determine the ideal Res concentration. In the Res group, Cell Counting Kit-8 (CCK-8) was used to measure the proliferation activity of pre-osteoblasts in mice in each group after resveratrol intervention. Alkaline Phosphatase (ALP) and alizarin red staining were used to gauge the degree of osteogenic differentiation, and RT-qPCR was used to measure the expression levels of Runx2 and OCN in the osteogenic differentiation ability of the cells. (2) In the experiment, four groups were set up: the control group, 3MA group, Res group, and Res + 3MA group. To examine cell mineralization, ALP and alizarin red staining were utilized. RT-qPCR and Western blot detection of cell autophagy activity levels and osteogenic differentiation capacity in each group following intervention. RESULTS: (1) Resveratrol might increase the number of mice pre-osteoblast, with the impact being most pronounced at 10µmol/L (P < 0.05). The nodules developed substantially more often than in the blank control group, and Runx2 and OCN expressions significantly increased (P < 0.05). (2) In contrast to the Res group, after 3MA purine blocked autophagy, the Res + 3MA group's alkaline phosphatase staining and the development of mineralized nodules were reduced. Runx2, OCN, LC3II / LC3I expression decreased, p62 expression increased (P < 0.05). CONCLUSION: The present study partially or indirectly demonstrated that Res may, through increased autophagy, induce osteogenic differentiation of MC3T3-E1 cells.

Jintiange proteins promote osteogenesis and inhibit apoptosis of osteoblasts by enhancing autophagy via PI3K/AKT and ER stress pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Tiger bone, which had long been used in traditional Chinese medicine, had the action of removing wind and alleviating pain, strengthening the sinews and bones, and often used to treat bone impediment, and atrophic debility of bones in TCM clinical practice. As a substitute of natural bone tiger, artificial tiger bone Jintiange (JTG), has been approved by the State Food and Drug Administration of China for relief the symptom of osteoporosis, such as lumbago and back pain, lassitude in loin and legs, flaccidity and weakness legs, and walk with difficulty based on TCM theory. JTG has similar chemical profile to natural tiger bone, and contains mineral substance, peptides and proteins, and has been shown to protect bone loss in ovariectomized mice and exert the regulatory effects on osteoblast and osteoclast activities. But how the peptides and proteins in JTG modulate bone formation remains unclear. AIM: To investigate the stimulating effects of JTG proteins on osteogenesis and explore the possible underlying mechanisms. MATERIALS AND METHODS: JTG proteins were prepared from JTG Capsules by extracting calcium, phosphorus and other inorganic elements using SEP-PaktC18 desalting column. MC3T3-E1 cells were treated with JTG proteins to evaluate their effects and explore the underlying mechanisms. Osteoblast proliferation was detected by CCK-8 method. ALP activity was detected using a relevant assay kit, and bone mineralized nodules were stained with alizarin red-Tris-HCl solution. Cell apoptosis was analyzed by flow cytometry. Autophagy was observed by MDC staining, and autophagosomes were observed by TEM. Nuclear translocations of LC3 and CHOP were detected by immunofluorescence and observed under a laser confocal microscope. The expression of key proteins related to osteogenesis, apoptosis, autophagy and PI3K/AKT and ER stress pathways was analyzed by Western Blot analysis. RESULTS: JTG proteins improved osteogenesis as evidenced by the alteration of proliferation, differentiation and mineralization of MC3T3-E1 osteoblasts, inhibited their apoptosis, and enhanced autophagosome formation and autophagy. They also regulated the expression of key proteins of PI3K/AKT and ER stress pathways. In addition, PI3K/AKT and ER stress pathway inhibitors could reverse the regulatory effects of JTG proteins on osteogenesis, apoptosis, autophagy and PI3K/AKT and ER stress pathways. CONCLUSION: JTG proteins increased the osteogenesis and inhibited osteoblast apoptosis by enhancing autophagy via PI3K/AKT and ER stress signaling pathways.

[Teriparatide regulates osteoblast differentiation in high-glucose microenvironment through the cAMP/PKA/CREB signaling pathway].

OBJECTIVE: To investigate the effect of teriparatide on the differentiation of MC3T3-E1 cells in high-glucose microenvironment and explore the possible mechanism. METHODS: MC3T3-E1 cells cultured in normal glucose or high-glucose (25 mmol/L) medium were treated with 10 nmol/L teriparatide with or without co-treatment with H-89 (a PKA inhibitor). CCK-8 assay was used to detect the changes in cell proliferation, and cAMP content in the cells was determined with ELISA. Alkaline phosphatase (ALP) activity and mineralized nodules in the cells were detected using ALP kit and Alizarin red staining, respectively. The changes in cell morphology were detected by cytoskeleton staining. Real-time PCR was used to detect the mRNA expressions of PKA, CREB, RUNX2 and Osx in the treated cells. RESULTS: The treatments did not result in significant changes in proliferation of MC3T3-E1 cells (P > 0.05). Compared with the cells in routine culture, the cells treated with teriparatide showed significantly increased cAMP levels (P < 0.05) with enhanced ALP activity and increased area of mineralized nodules (P < 0.05). Teriparatide treatment also resulted in more distinct visualization of the cytoskeleton in the cells and obviously up-regulated the mRNA expressions of PKA, CREB, RUNX2 and Osx (P < 0.05). The opposite changes were observed in cells cultured in high glucose. In cells exposed to high glucose, treatment with teriparatide significantly increased cAMP levels (P < 0.05), ALP activity and the area of mineralized nodules (P < 0.05) and enhanced the clarity of the cytoskeleton and mRNA expressions of PKA, CREB, RUNX2 and Osx; the effects of teriparatide was strongly antagonized by co-treatment with H-89 (P < 0.05). CONCLUSION: Teriparatide can promote osteoblast differentiation of MC3T3-E1 cells in high-glucose microenvironment possibly by activating the cAMP/PKA/CREB signaling pathway.

Zoledronic Acid Blocks Overactive Kir6.1/SUR2-Dependent KATP Channels in Skeletal Muscle and Osteoblasts in a Murine Model of Cantú Syndrome.

Cantú syndrome (CS) is caused by the gain of function mutations in the ABCC9 and KCNJ8 genes encoding, respectively, for the sulfonylureas receptor type 2 (SUR2) and the inwardly rectifier potassium channel 6.1 (Kir6.1) of the ATP-sensitive potassium (KATP) channels. CS is a multi-organ condition with a cardiovascular phenotype, neuromuscular symptoms, and skeletal malformations. Glibenclamide has been proposed for use in CS, but even in animals, the drug is incompletely effective against severe mutations, including the Kir6.1wt/V65M. Patch-clamp experiments showed that zoledronic acid (ZOL) fully reduced the whole-cell KATP currents in bone calvaria cells from wild type (WT/WT) and heterozygous Kir6.1wt/V65MCS mice, with IC50 for ZOL block < 1 nM in each case. ZOL fully reduced KATP current in excised patches in skeletal muscle fibers in WT/WT and CS mice, with IC50 of 100 nM in each case. Interestingly, KATP currents in the bone of heterozygous SUR2wt/A478V mice were less sensitive to ZOL inhibition, showing an IC50 of ~500 nM and a slope of ~0.3. In homozygous SUR2A478V/A478V cells, ZOL failed to fully inhibit the KATP currents, causing only ~35% inhibition at 100 µM, but was responsive to glibenclamide. ZOL reduced the KATP currents in Kir6.1wt/VMCS mice in both skeletal muscle and bone cells but was not effective in the SUR2[A478V] mice fibers. These data indicate a subunit specificity of ZOL action that is important for appropriate CS therapies.

Xianling Gubao attenuates high glucose-induced bone metabolism disorder in MG63 osteoblast-like cells.

Diabetes mellitus (DM) patients are prone to osteoporosis, and high glucose (HG) can affect bone metabolism. In the present study, we investigated the protective effects of traditional Chinese herbal formulation Xianling Gubao (XLGB) on HG-treated MG63 osteoblast-like cells. MG63 cells were incubated with control (mannitol), HG (20 mM glucose) or HG + XLGB (20 mM glucose+200 mg/L XLGB) mediums. Cell proliferation, apoptosis, migration and invasion were examined using CCK8, colony-formation, flow cytometry, Hoechst/PI staining, wound-healing and transwell assays, respectively. ELISA, RT-PCR and western blot analysis were used to detect the levels of osteogenesis differentiation-associated markers such as ALP, OCN, OPN, RUNX2, OPG, and OPGL in MG63 cells. The levels of the PI3K/Akt signaling pathway related proteins, cell cycle-related proteins, and mitochondrial apoptosis-related proteins were detected using western blot analysis. In HG-treated MG63 cells, XLGB significantly attenuated the suppression on the proliferation, migration and invasion of MG63 cells caused by HG. HG downregulated the activation of the PI3K/Akt signaling pathway and the expressions of cell cycle-related proteins, while XLGB reversed the inhibition of HG on MG63 cells. Moreover, XLGB significantly reduced the promotion on the apoptosis of MG63 cells induced by HG, the expressions of mitochondrial apoptosis-related proteins were suppressed by XLGB treatment. In addition, the expressions of osteogenesis differentiation-associated proteins were also rescued by XLGB in HG-treated MG63 cells. Our data suggest that XLGB rescues the MG63 osteoblasts against the effect of HG. The potential therapeutic mechanism of XLGB partially attributes to inhibiting the osteoblast apoptosis and promoting the bone formation of osteoblasts.

Apigenin Modulates AnxA6- and TNAP-Mediated Osteoblast Mineralization.

Mineralization-competent cells like osteoblasts and chondrocytes release matrix vesicles (MVs) which accumulate Ca2+ and Pi, creating an optimal environment for apatite formation. The mineralization process requires the involvement of proteins, such as annexins (Anx) and tissue-nonspecific alkaline phosphatase (TNAP), as well as low molecular-weight compounds. Apigenin, a flavonoid compound, has been reported to affect bone metabolism, but there are doubts about its mechanism of action under physiological and pathological conditions. In this report, apigenin potency to modulate annexin A6 (AnxA6)- and TNAP-mediated osteoblast mineralization was explored using three cell lines: human fetal osteoblastic hFOB 1.19, human osteosarcoma Saos-2, and human coronary artery smooth muscle cells HCASMC. We compared the mineralization competence, the morphology and composition of minerals, and the protein distribution in control and apigenin-treated cells and vesicles. The mineralization ability was monitored by AR-S/CPC analysis, and TNAP activity was determined by ELISA assay. Apigenin affected the mineral structure and modulated TNAP activity depending on the concentration. We also observed increased mineralization in Saos-2 cells. Based on TEM-EDX, we found that apigenin influenced the mineral composition. This flavonoid also disturbed the intracellular distribution of AnxA6 and TNAP, especially blocking AnxA6 aggregation and TNAP attachment to the membrane, as examined by FM analysis of cells and TEM-gold analysis of vesicles. In summary, apigenin modulates the mineralization process by regulating AnxA6 and TNAP, as well as through various effects on normal and cancer bone tissues or atherosclerotic soft tissue.

UPLC-ESI-Q-TOF-MSE-based metabolomics analysis of Acer mono sap and evaluation of osteogenic activity in mouse osteoblast cells.

Investigation of phytochemicals and bioactive molecules is tremendously vital for the applications of new plant resources in chemistry, food, and medicine. In this study, the chemical profiling of sap of Acer mono (SAM), a Korean syrup known for its anti-osteoporosis effect, was performed using UPLC-ESI-Q-TOF-MSE analysis. A total of 23 compounds were identified based on the mass and fragmentation characteristics and most of the compounds have significant biomedical applications. The in vitro antioxidant assessment of SAM indicated excellent activity by scavenging DPPH and ABTS-free radicals and were found to be 23.35 mg mL-1 and 29.33 mg mL-1, respectively, as IC50 concentrations. As well, the in vitro proliferation effect of the SAM was assessed against mouse MC3T3-E1 cells, and the results showed that the SAM enhanced the proliferation of the cells, and 12.5 mg mL-1 and 25 mg mL-1 of SAM were selected for osteogenic differentiation. The morphological analysis clearly evidenced the SAM enhanced the osteogenic activity in MC3T3-E1 cells by the increased deposition of extracellular calcium and nodule formation. Moreover, the qRT-PCR analysis confirmed the increased expression of osteoblast marker gene expression including ALP, osteocalcin, osteopontin, collagen1α1, Runx2, and osterix in SAM-treated MC3T3-E1 cells. Together, these results suggest that SAM possesses osteogenic effects and can be used for bone regeneration and bone loss-associated diseases such as osteoporosis.

Effects of Human Deciduous Dental Pulp-Derived Mesenchymal Stem Cell-Derived Conditioned Medium on the Metabolism of HUVECs, Osteoblasts, and BMSCs.

In this study, we assessed the effects of human deciduous dental pulp-derived mesenchymal stem cell-derived conditioned medium (SHED-CM) on the properties of various cell types. The effects of vascular endothelial growth factor (VEGF) in SHED-CM on the luminal architecture, proliferative ability, and angiogenic potential of human umbilical vein endothelial cells (HUVECs) were determined. We also investigated the effects of SHED-CM on the proliferation of human-bone-marrow mesenchymal stem cells (hBMSCs) and mouse calvarial osteoblastic cells (MC3T3-E1) as well as the expression of ALP, OCN, and RUNX2. The protein levels of ALP were examined using Western blot analysis. VEGF blockade in SHED-CM suppressed the proliferative ability and angiogenic potential of HUVECs, indicating that VEGF in SHED-CM contributes to angiogenesis. The culturing of hBMSCs and MC3T3-E1 cells with SHED-CM accelerated cell growth and enhanced mRNA expression of bone differentiation markers. The addition of SHED-CM enhanced ALP protein expression in hBMSCs and MT3T3-E1 cells compared with that of the 0% FBS group. Furthermore, SHED-CM promoted the metabolism of HUVECs, MC3T3-E1 cells, and hBMSCs. These findings indicate the potential benefits of SHED-CM in bone tissue regeneration.

In vitro osteoblast activity is decreased by residues of chemicals used in the cleaning and viral inactivation process of bone allografts.

Allograft bone tissue has a long history of use. There are two main ways of preserving allografts: by cold (freezing), or at room temperature after an additional cleaning treatment using chemicals. These chemicals are considered potentially harmful to humans. The aim of the study was (i) to assess the presence of chemical residues on processed bone allografts and (ii) to compare the in vitro biocompatibility of such allografts with that of frozen allografts. The presence of chemical residues on industrially chemically treated bone was assessed by high performance liquid chromatography (HPLC) after extraction. Biocompatibility analysis was performed on primary osteoblast cultures from Wistar rats grown on bone disks, either frozen (F-bone group) or treated with supercritical carbon dioxide with no added chemical (scCO2-bone group) or industrially treated with chemicals (CT-bone group). Cell viability (XTT) was measured after one week of culture. Osteoblastic differentiation was assessed after 1, 7 and 14 days of culture by measuring alkaline phosphatase (ALP) activity directly on the bone discs and indirectly on the cell mat in the vicinity of the bone discs. Residues of all the chemicals used were found in the CT-bone group. There was no significant difference in cell viability between the three bone groups. Direct and indirect ALP activities were significantly lower (-40% to -80%) in the CT-bone group after 7 and 14 days of culture (p < 0.05). Residues of chemical substances used in the cleaning of bone allografts cause an in vitro decrease in their biocompatibility. Tissue cleaning processes must be developed that limit or replace these chemicals to favor biocompatibility.

Dexamethasone promotes osteoblast apoptosis through the Chk2/p53 signaling pathway.

BACKGROUND: Glucocorticoids (GCs) are widely used to treat inflammatory or autoimmune diseases. However, several studies have reported that the use of GCs can lead to numerous complications, the most serious of which are osteoporosis and osteonecrosis of the femoral head (ONFH). Osteoblast apoptosis has been identified as an important event in the development of GC-induced osteoporosis and ONFH. However, the mechanisms underlying the regulation of these processes have not yet been explored. OBJECTIVES: To observe the effect of dexamethasone (Dex) on the apoptosis of osteoblasts and explore its mechanism, as well as provide a new therapeutic idea for GC­induced osteoporosis and ONFH. MATERIAL AND METHODS: Cell proliferation and apoptosis of MC3T3-E1 cells after Dex treatment were determined using the CellTiter-Glo® Luminescent Cell Viability Assay kit and Annexin V-FITC/PI Double Staining Apoptosis Detection Kit, respectively. The expression of caspase-3/cleaved caspase-3 and poly(ADP-ribose) polymerase (PARP)/cleaved PARP in MC3T3-E1 cells after Dex treatment was determined with western blotting. The expression of p53 and checkpoint kinase 2 (Chk2) in MC3T3-E1 cells after Dex treatment was analyzed using western blotting and polymerase chain reaction (PCR). The effects of p53 knockdown and Chk2 knockdown on Dex-induced apoptosis of MC3T3-E1 cells were also characterized. RESULTS: Dexamethasone remarkably inhibited cell growth and induced the apoptosis of MC3T3-E1 cells. We also observed that Dex induced osteoblast apoptosis by promoting p53 expression. The regulatory effect of Dex on p53 expression is mediated by the upregulation of Chk2, which interacted with p53 and inhibited p53 degradation. The knockdown of p53 alleviated Dex-induced MC3T3-E1 cell apoptosis by decreasing the expression of cleaved caspase-3 and cleaved PARP. CONCLUSIONS: We demonstrated that Dex increased Chk2 protein expression, which stabilized the protein expression of p53, and in turn promoted osteoblast apoptosis.

ED-71 inhibited osteoclastogenesis by enhancing EphrinB2-EphB4 signaling between osteoclasts and osteoblasts in osteoporosis.

BACKGROUND: Osteoporosis is a degenerative skeletal disease essentially caused by bone remodeling disorder. EphrinB2-EphB4 signaling play critical regulatory roles in bone remodeling via communication between osteoclasts and osteoblasts. Eldecalcitol (ED-71), a new vitamin D analog, is a high-potential drug for treating osteoporosis; however, its mechanism has yet to be determined. This study aims to investigate whether EphrinB2-EphB4 signal mediates the process of osteoporosis improved by ED-71. MATERIALS AND METHODS: An ovariectomized (OVX) rat model was constructed in vivo. ED-71 at 30 ng/kg was orally administered once daily for 8 weeks. Osteoclast activity and EphrinB2-EphB4 expression were evaluated by hematoxylin and eosin staining, tartrate-resistant acid phosphatase (TRAP) staining, and immunohistochemical staining. The mRNA levels of oxidation stress factors in the bone tissue were tested by reverse transcription polymerase chain reaction (RT-PCR). An H2O2-stimulated model in vitro was established to simulate the status of osteoporosis. Osteoclastogenesis and associated protein were detected by TRAP staining, F-actin ring formation assay, PCR, and Western blot analysis. EprhrinB2 and EphB4 levels were determined by immunofluorescence, PCR, and Western blot analysis. EprhrinB2 small-interfering RNA knocked down the EprhrinB2 in osteoclasts, and an EphB4 antibody blocked EphB4 in osteoblasts. RESULTS: ED-71 prevented bone loss and decreased the number of osteoclasts in vivo relative to the OVX group. In addition, the bone tissue of OVX rat displayed as an increased level of oxidation stress, which could be inhibited by ED-71. In vitro, in the simulation of osteoporosis with H2O2, ED-71 reversed the increase H2O2-induced oxidative stress. ED-71 then inhibited osteoclastogenesis and osteoclast function, accompanied by increased EphrinB2 expression in osteoclasts. Notably, EphrinB2 knockdown reversed the inhibitory effect of ED-71 on osteoclasts. ED-71 also enhanced EphB4 expression in osteoblasts in vivo and in vitro. Further research showed that ED-71 inhibited osteoclastogenesis in co-culture systems, which was weakened by blocking EphB4 in osteoblasts. CONCLUSIONS: ED-71 inhibited osteoclastogenesis by enhancing EphrinB2-EphB4 signaling between osteoclasts and osteoblasts, preventing osteoporosis. This theory explains the role of ED-71 in the treatment of osteoporosis.

Selenium nanoparticles stimulate osteoblast differentiation via BMP-2/MAPKs/ß-catenin pathway in diabetic osteoporosis.

Aim: To evaluate whether selenium nanoparticles (SeNPs) can stimulate bone formation and inhibit the bone loss involved in hyperglycemia-induced osteoporosis. Methods: Rat osteoblastic UMR-106 cells were used for in vitro studies and female Sprague-Dawley rats were used for type 2 diabetes-associated osteoporosis in vivo study. Results:In vitro studies show that SeNPs promote osteoblast differentiation via modulating alkaline phosphatase (ALP) activity, and promoting calcium nodule formation and collagen content. The authors also provide evidence regarding the involvement of the BMP-2/MAPKs/ß-catenin pathway in preventing diabetic osteoporosis. Further, in vivo and ex vivo studies suggested that SeNPs can preserve mechanical and microstructural properties of bone. Conclusion: To the best of our knowledge, this study provides the first evidence regarding the therapeutic benefits of SeNPs in preventing diabetes-associated bone fragility.

Osteoporosis is a common complication for people with diabetes. High glucose causes oxidative stress, and the antioxidant and anti-inflammatory properties of selenium nanoparticles (SeNPs) make them useful in the treatment of metabolic disorders associated with high glucose levels. The results of this paper report the protective effects of SeNPs in diabetic osteoporosis using rat osteoblastic UMR-106 cells and female Sprague­Dawley rats with type-2 diabetes-induced osteoporosis. SeNPs promote osteoblast differentiation and mineralization in osteoblasts, preserve bone microstructure and improve biomechanical stability, which suggests that SeNPs could be used therapeutically in the maintenance of diabetic osteoporosis.

Induced pluripotent stem cells from homozygous Runx2-deficient mice show poor response to vitamin D during osteoblastic differentiation.

Cleidocranial dysplasia (CCD) is a hereditary disorder associated with skeletal dysplasia and dental abnormalities. CCD arises from heterozygous loss of function mutations in the Runt-related transcription factor 2 (RUNX2) gene. Osteoporosis is often observed in CCD patients and conventional vitamin D supplementation is recommended. However, sufficient evidences have not been presented yet. This study investigated the role of RUNX2 in osteoblastic differentiation and sought to identify potential target genes for the treatment of osteoporosis associated with CCD, using induced pluripotent stem cell (iPSC) technology. We successfully established Runx2-/-, Runx2+/- and wild-type miPSCs from litter-matched mice and found poor Vdr expression in Runx2-/-cells. Significant down-regulation of osteoblastic differentiation in Runx2-/- miPSCs was observed. Gene expression array revealed unexpected results such as remarkable increase of Rankl expression and decrease of Vdr in Runx2-/- cells. Insufficient response to vitamin D in Runx2-/- cells was also observed. Our results suggest that RUNX2 functions as a regulator of Rankl and Vdr and thereby controls bone density. These findings also suggest that conventional vitamin D supplementation may not be as effective as previously expected, in the treatment of osteoporosis associated with CCD, and that inhibiting RANKL function might be worth considering as an alternative treatment strategy.

Dipeptidyl peptidase-4 is associated with myogenesis in patients with adolescent idiopathic scoliosis possibly via mediation of insulin sensitivity.

BACKGROUND: Abnormal metabolic features have been previously described in adolescent idiopathic scoliosis (AIS) patients. As an important regulator involved in energy metabolism, DPP-4 activity was reported to be remarkably decreased in osteoblasts of AIS patients. To date, there was still a lack of knowledge concerning the role of DPP-4 in the myogenesis of AIS. METHODS: Circulation DPP-4 level was assessed in the serum of 80 AIS girls and 50 healthy controls by ELISA. Myoblasts were purified from muscle specimens of AIS patients and LDH controls, and then treated with metabolic effectors including glucose and insulin. CCK-8 assay was used to assess the cell viability and myotube fusion index was calculated to evaluate myogenesis ability. Gene expressions of downstream signals of DPP-4 were evaluated by RT-qPCR and Western blot respectively. RESULTS: AIS girls had remarkably down-expressed DPP-4 in both serum level (0.76 fold) and tissue (0.68 fold) level. Treatment with metabolic effectors led to significantly increased DPP-4 expression in the control cells, while there was no increase of DPP-4 in AIS cells. CCK-8 assay showed that the proliferation rate of control cells was significantly increased after being treated. Remarkably higher fusion index was also observed in the treated control cells. By contrast, the fusion index and cell proliferation rate were comparable between the treated and the untreated AIS cells. CONCLUSIONS: Our study suggested a potential role of DPP-4 in abnormal metabolic condition of AIS patients. Compared with control cells, AIS myoblasts presented obviously impaired sensitivity to the treatment of glucose and insulin. Aberrant DPP-4 expression could lead to impaired insulin sensitivity in myoblasts and further influence the cell viability during myogenesis. The molecular mechanism connecting DPP-4 and insulin-related signaling in AIS is worthy of further investigation.