Laminin fragments conjugated with perlecan's growth factor-binding domain differentiate human induced pluripotent stem cells into skin-derived precursor cells.

Deriving stem cells to regenerate full-thickness human skin is important for treating skin disorders without invasive surgical procedures. Our previous protocol to differentiate human induced pluripotent stem cells (iPSCs) into skin-derived precursor cells (SKPs) as a source of dermal stem cells employs mouse fibroblasts as feeder cells and is therefore unsuitable for clinical use. Herein, we report a feeder-free method for differentiating iPSCs into SKPs by customising culture substrates. We immunohistochemically screened for laminins expressed in dermal papillae (DP) and explored the conditions for inducing the differentiation of iPSCs into SKPs on recombinant laminin E8 (LM-E8) fragments with or without conjugation to domain I of perlecan (PDI), which binds to growth factors through heparan sulphate chains. Several LM-E8 fragments, including those of LM111, 121, 332, 421, 511, and 521, supported iPSC differentiation into SKPs without PDI conjugation. However, the SKP yield was significantly enhanced on PDI-conjugated LM-E8 fragments. SKPs induced on PDI-conjugated LM111-E8 fragments retained the gene expression patterns characteristic of SKPs, as well as the ability to differentiate into adipocytes, osteocytes, and Schwann cells. Thus, PDI-conjugated LM-E8 fragments are promising agents for inducing iPSC differentiation into SKPs in clinical settings.

A novel agonist with homobivalent single-domain antibodies that bind the FGF receptor 1 domain III functions as an FGF2 ligand.

Fibroblast growth factor (FGF) is a multifunctional protein that exhibits a wide range of biological effects. Most commonly, it acts as a mitogen, but it also has regulatory, morphological, and endocrine effects. The four receptor subtypes of FGF are activated by more than 20 different FGF ligands. FGF2, one of the FGF ligands, is an essential factor for cell culture in stem cells for regenerative medicine; however, recombinant FGF2 is extremely unstable. Here, we successfully generated homobivalent agonistic single-domain antibodies (variable domain of heavy chain of heavy chain antibodies referred to as VHHs) that bind to domain III and induce activation of the FGF receptor 1 and thus transduce intracellular signaling. This agonistic VHH has similar biological activity (EC50) as the natural FGF2 ligand. Furthermore, we determined that the agonistic VHH could support the proliferation of human-induced pluripotent stem cells (PSCs) and human mesenchymal stem cells, which are PSCs for regenerative medicine. In addition, the agonistic VHH could maintain the ability of mesenchymal stem cells to differentiate into adipocytes or osteocytes, indicating that it could maintain the properties of PSCs. These results suggest that the VHH agonist may function as an FGF2 mimetic in cell preparation of stem cells for regenerative medicine with better cost effectiveness.

Effect of Oxidative Stress-Induced Apoptosis on Active FGF23 Levels in MLO-Y4 Cells: The Protective Role of 17-ß-Estradiol.

The discovery that osteocytes secrete phosphaturic fibroblast growth factor 23 (FGF23) has defined bone as an endocrine organ. However, the autocrine and paracrine functions of FGF23 are still unknown. The present study focuses on the cellular and molecular mechanisms involved in the complex control of FGF23 production and local bone remodeling functions. FGF23 was assayed using ELISA kit in the presence or absence of 17ß-estradiol in starved MLO-Y4 osteocytes. In these cells, a relationship between oxidative stress-induced apoptosis and up-regulation of active FGF23 levels due to MAP Kinases activation with involvement of the transcriptional factor (NF-kB) has been demonstrated. The active FGF23 increase can be due to up-regulation of its expression and post-transcriptional modifications. 17ß-estradiol prevents the increase of FGF23 by inhibiting JNK and NF-kB activation, osteocyte apoptosis and by the down-regulation of osteoclastogenic factors, such as sclerostin. No alteration in the levels of dentin matrix protein 1, a FGF23 negative regulator, has been determined. The results of this study identify biological targets on which drugs and estrogen may act to control active FGF23 levels in oxidative stress-related bone and non-bone inflammatory diseases.

Osteonecrosis development by tooth extraction in zoledronate treated mice is inhibited by active vitamin D analogues, anti-inflammatory agents or antibiotics.

Invasive dental treatment such as tooth extraction following treatment with strong anti-bone resorptive agents, including bisphosphonates and denosumab, reportedly promotes osteonecrosis of the jaw (ONJ) at the extraction site, but strategies to prevent ONJ remain unclear. Here we show that in mice, administration of either active vitamin D analogues, antibiotics or anti-inflammatory agents can prevent ONJ development induced by tooth extraction during treatment with the bisphosphonate zoledronate. Specifically, tooth extraction during treatment with zoledronate induced osteonecrosis in mice, but administration of either 1,25(OH)2D3 or ED71, both active vitamin D analogues, significantly antagonized osteonecrosis development, even under continuous zoledronate treatment. 1,25(OH)2D3 or ED71 administration also significantly inhibited osteocyte apoptosis induced by tooth extraction and bisphosphonate treatment. Administration of either active vitamin D analogue significantly inhibited elevation of serum inflammatory cytokine levels in mice in response to injection of lipopolysaccharide, an infection mimetic. Furthermore, administration of either anti-inflammatory or antibiotic reagents significantly blocked ONJ development following tooth extraction and zoledronate treatment. These findings suggest that administration of active vitamin D, anti-inflammatory agents or antibiotics could prevent ONJ development induced by tooth extraction in patients treated with zoledronate.

Bisphenol A induces pyroptotic cell death via ROS/NLRP3/Caspase-1 pathway in osteocytes MLO-Y4.

Bisphenol A (BPA), a ubiquitous endocrine-disrupting chemical, is commonly used as a plasticizer to manufacture various food packaging materials. Evidence has demonstrated that BPA disturbed bone health. However, few studies focused on the effect of BPA on osteocytes, making up over 95% of all the bone cells. Here, we reported that BPA inhibited the cell viability of MLO-Y4 cells, and increased apoptosis in a dose-dependent manner. Furthermore, BPA up-regulated protein expressions of speck-like protein containing CARD (ASC), NLRP3, cleaved caspase-1 (Casp-1 p20) and cleaved gasdermin D (GSDMD-N), and increased the ratios of interleukin (IL)-1ß/pro-IL-1ß and IL-18/pro-IL-18 in MLO-Y4 cells. BPA enhanced levels of lactate dehydrogenase (LDH), IL-1ß and IL-18 in culture supernatants. This pyroptotic death and the NLPR3 inflammasome activation were reversed by the caspase-1 inhibitor VX765 or the NLRP3 inflammasome inhibitor MCC950. Furthermore, BPA stimulated the production of intracellular reactive oxygen species (ROS), mitochondrial ROS (mtROS), elevated malondialdehyde (MDA) level and decreased superoxide dismutase (SOD) activity, which led to oxidative damage in MLO-Y4 cells. The ROS scavenger N-acetylcysteine (NAC) or the mitochondrial antioxidant Mito-TEMPO inhibited the NLPR3 inflammasome activation and pyroptotic death induced by BPA. Collectively, our data suggest that BPA causes pyroptotic death of osteocytes via ROS/NLRP3/Caspase-1 pathway.

Soluble epoxide hydrolase inhibitor can protect the femoral head against tobacco smoke exposure-induced osteonecrosis in spontaneously hypertensive rats.

Exposure to tobacco smoke (TS) has been considered a risk factor for osteonecrosis of the femoral head (ONFH). Soluble epoxide hydrolase inhibitors (sEHIs) have been found to reduce inflammation and oxidative stress in a variety of pathologies. This study was designed to assess the effect of sEHI on the development of ONFH phenotypes induced by TS exposure in spontaneously hypertensive (SH) rats. SH and normotensive Wistar Kyoto (WKY) rats were exposed to filtered air (FA) or TS (80 mg/m3 particulate concentration) 6 h/day, 3 days/week for 8 weeks. During this period, sEHI was delivered through drinking water at a concentration of 6 mg/L. Histology, immunohistochemistry, and micro-CT morphometry were performed for phenotypic evaluation. As results, TS exposure induced significant increases in adipocyte area, bone specific surface (BS/BV), and trabecular separation (Tb.SP), as well as significant decreases in bone mineral density (BMD), percent trabecular area (Tb.Ar), HIF-1a expression, bone volume fraction (BV/TV), trabecular numbers (Tb.N), and trabecular thickness (Tb.Th) in both SH and WKY rats. However, the protective effects of sEHI were mainly observed in TS-exposed SH rats, specifically in the density of osteocytes, BMD, Tb.Ar, HIF-1a expression, BV/TV, BS/BV, Tb.N, and Tb.SP. Our study confirms that TS exposure can induce ONFH especially in SH rats, and suggests that sEHI therapy may protect against TS exposure-induced osteonecrotic changes in the femoral head.

Parathyroid hormone signaling in mature osteoblasts/osteocytes protects mice from age-related bone loss.

Aging is accompanied by osteopenia, characterized by reduced bone formation and increased bone resorption. Osteocytes, the terminally differentiated osteoblasts, are regulators of bone homeostasis, and parathyroid hormone (PTH) receptor (PPR) signaling in mature osteoblasts/osteocytes is essential for PTH-driven anabolic and catabolic skeletal responses. However, the role of PPR signaling in those cells during aging has not been investigated. The aim of this study was to analyze the role of PTH signaling in mature osteoblasts/osteocytes during aging. Mice lacking PPR in osteocyte (Dmp1-PPRKO) display an age-dependent osteopenia characterized by a significant decrease in osteoblast activity and increase in osteoclast number and activity. At the molecular level, the absence of PPR signaling in mature osteoblasts/osteocytes is associated with an increase in serum sclerostin and a significant increase in osteocytes expressing 4-hydroxy-2-nonenals, a marker of oxidative stress. In Dmp1-PPRKO mice there was an age-dependent increase in p16Ink4a/Cdkn2a expression, whereas it was unchanged in controls. In vitro studies demonstrated that PTH protects osteocytes from oxidative stress-induced cell death. In summary, we reported that PPR signaling in osteocytes is important for protecting the skeleton from age-induced bone loss by restraining osteoclast's activity and protecting osteocytes from oxidative stresses.

A simple method for decellularizing a cell-derived matrix for bone cell cultivation and differentiation.

The extracellular matrix regulates cell survival, proliferation, and differentiation. In vitro two-dimensional cell experiments are typically performed on a plastic plate or a substrate of a single extracellular matrix constituent such as collagen or calcium phosphate. As these approaches do not include extracellular matrix proteins or growth factors, they fail to mimic a complex cell microenvironment. The cell-derived matrix is an alternative platform for better representing the in vivo microenvironment in vitro. Standard decellularization of a cell-derived matrix is achieved by combining chemical and physical methods. In this study, we compared the decellularization efficacy of several methods: ammonium hydroxide, sodium dodecyl sulfate (SDS), or Triton X-100 with cold or heat treatment on a matrix of Saos-2 cells. We found that the protocols containing SDS were cytotoxic during recellularization. Heat treatment at 47 °C was not cytotoxic, removed cellular constituents, inactivated alkaline phosphatase activity, and maintained the levels of calcium deposition. Subsequently, we investigated the differentiation efficiency of a direct bone coculture system in the established decellularized Saos-2 matrix, an inorganic matrix of calcium phosphate, and a plastic plate as a control. We found that the decellularized Saos-2 cell matrix obtained by heat treatment at 47 °C enhanced osteoclast differentiation and matrix mineralization better than the inorganic matrix and the control. This simple and low-cost method allows us to create a Saos-2 decellularized matrix that can be used as an in vivo-like support for the growth and differentiation of bone cells.

Oxidative Stress Induced Osteocyte Apoptosis in Steroid-Induced Femoral Head Necrosis.

OBJECTIVE: To investigate the effect and mechanism of Glucocorticoids (GCs) induced oxidative stress and apoptosis on necrosis of the femoral head in patients and rats. METHODS: Eight patients with steroid-induced avascular necrosis of the femoral head (SINFH) and eight patients with developmental dysplasia of the hips (DDH) were enrolled in our study. In animal model, twenty male Sprague-Dawley rats were randomly divided into two groups (SINFH group and NS group). The SINFH model group received the methylprednisolone (MPS) injection, while control group was injected with normal saline (NS). MRI was used to confirm SINFH rat model was established successfully. Then, the rats were sacrificed 4 weeks later and femoral head samples were harvested. Histopathological staining was preformed to evaluate osteonecrosis. TUNEL staining was performed with 8-OHdG and DAPI immunofluorescence staining to evaluate oxidative injury and osteocyte apoptosis. Immunohistochemistry staining was used to detect Nox1, Nox2, and Nox4 protein expression. RESULTS: MRI showed signs of typical osteonecrosis of femoral head in SIHFH patients. Histopathological staining showed that the rate of empty lacunae in SINFH patients was significantly higher (56.88% ± 9.72% vs 19.92% ± 4.18%, T = -11.04, P < 0.001) than that in DDH patients. The immunofluorescence staining indicated that the TUNEL-positive cell and 8-OHdG-positve cell in SINFH patients were significantly higher (49.32% ± 12.95% vs 8.00% ± 2.11%, T = -7.04, P = 0.002, 54.6% ± 23.8% vs 9.75% ± 3.31%, T = -4.17, P = 0.003) compared to the DDH patients. The immunohistochemistry staining showed that the protein expression of NOX1, NOX2 and NOX4 in SINFH patients were significantly increased (64.50% ± 7.57% vs 37.58% ± 9.23%, T = -3.88, P = 0.018, 90.84% ± 2.93% vs 49.56% ± 16.47%, T = -5.46, P = 0.001, 85.46% ± 9.3% vs 40.69% ± 6.77%, T = -8.03, P = 0.001) compared to the DDH patients. In animal model, MRI showed signs of edema of femoral head in MPS group, which represents SINFH rat model was established successfully. Histological evaluation showed the rate of empty lacunae in MPS group was significantly higher (25.85% ± 4.68% vs 9.35% ± 1.99%, T = -7.96, P < 0.001) than that in NS group. The immunofluorescence staining indicated that the TUNEL-positive cell and 8-OHdG-positve cell (in MPS group were significantly increased (31.93% ± 1.01% vs 11.73% ± 1.16%, T = -32.26, P < 0.001, 47.59% ± 1.39% vs 22.07% ± 2.45%, T = -22.18, P < 0.001) compared to the NS group. The immunohistochemistry staining showed that the expression of NOX2 in MPS group was significantly increased (76.77% ± 8.34% vs 50.32% ± 10.84%, T = -4.74, P = 0.001) compare with NS group. CONCLUSION: Our findings indicated that GC-induced NOXs expression may be an important source of oxidative stress, which could lead to osteocyte apoptosis in the process of SINFH.

CSBD Healing in Rats after Application of Bovine Xenogeneic Biomaterial Enriched with Magnesium Alloy.

Xenogeneic biomaterials Cerbone® and OsteoBiol® are widely used in oral implantology. In dental practice, xenogeneic biomaterial is usually combined with autologous bone to provide bone volume stability needed for long-term dental implants. Magnesium alloy implants dissolve and form mineral corrosion layer that is directly in contact with bone tissue, allowing deposition of the newly formed bone. CSBD heals by intramembranous ossification and therefore is a convenient model for analyses of ostoconductive and osteoinductive properties of different type of biomaterials. Magnesium alloy-enriched biomaterials have not yet been applied in oral implantology. Therefore, the aim of the current study was to investigate biological properties of potentially new bovine xenogeneic biomaterial enriched with magnesium alloy in a 5 mm CSBD model. Osteoconductive properties of Cerabone®, Cerabone® + Al. bone, and OsteoBiol® were also analyzed. Dynamics of bone healing was followed up on the days 3, 7, 15, 21, and 30. Calvary bone samples were analyzed by micro-CT, and values of the bone morphometric parameters were assessed. Bone samples were further processed for histological and immunohistochemical analyses. Histological observation revealed CSBD closure at day 30 of the given xenogeneic biomaterial groups, with the exception of the control group. TNF-α showed high intensity of expression at the sites of MSC clusters that underwent ossification. Osx was expressed in pre-osteoblasts, which were differentiated into mature osteoblasts and osteocytes. Results of the micro-CT analyses showed linear increase in bone volume of all xenogeneic biomaterial groups and also in the control. The highest average values of bone volume were found for the Cerabone® + Mg group. In addition, less residual biomaterial was estimated in the Cerabone® + Mg group than in the Cerabone® group, indicating its better biodegradation during CSBD healing. Overall, the magnesium alloy xenogeneic biomaterial demonstrated key properties of osteoinduction and biodegradidibility during CSBD healing, which is the reason why it should be recommended for application in clinical practice of oral implantology.

Novel Aptamer-Based Small-Molecule Drug Screening Assay to Identify Potential Sclerostin Inhibitors against Osteoporosis.

A novel aptamer-based competitive drug screening platform for osteoporosis was devised in which fluorescence-labeled, sclerostin-specific aptamers compete with compounds from selected chemical libraries for the binding of immobilized recombinant human sclerostin to achieve high-throughput screening for potential small-molecule sclerostin inhibitors and to facilitate drug repurposing and drug discovery. Of the 96 selected inhibitors and FDA-approved drugs, six were shown to result in a significant decrease in the fluorescence intensity of the aptamer, suggesting a higher affinity toward sclerostin compared with that of the aptamer. The targets of these potential sclerostin inhibitors were correlated to lipid or bone metabolism, and several of the compounds have already been shown to be potential osteogenic activators, indicating that the aptamer-based competitive drug screening assay offered a potentially reliable strategy for the discovery of target-specific new drugs. The six potential sclerostin inhibitors suppressed the level of both intracellular and/or extracellular sclerostin in mouse osteocyte IDG-SW3 and increased alkaline phosphatase activity in IDG-SW3 cells, human bone marrow-derived mesenchymal stem cells and human fetal osteoblasts hFOB1.19. Potential small-molecule drug candidates obtained in this study are expected to provide new therapeutics for osteoporosis as well as insights into the structure-activity relationship of sclerostin inhibitors for rational drug design.

Prednisolone induces osteocytes apoptosis by promoting Notum expression and inhibiting PI3K/AKT/GSK3ß/ß-catenin pathway.

The apoptosis of mature osteocytes is the main factor causing damage to the microstructure of cortical bone in glucocorticoid-induced osteoporosis (GIOP). Our previous research found damaged areas and empty osteocytes lacunae in the tibial cortical bone of GIOP mice. However, the specific mechanism has not been clarified. Recently, a study showed that the quality of the cortical bone significantly increased by knocking out Notum, a gene encoding α/ß hydrolase. However, it is not clear whether Notum affects cortical bone remodeling by participating in glucocorticoids (GCs)-induced apoptosis of osteocytes. The present study aimed to explore the correlation between Notum, osteocytes apoptosis, and cortical bone quality in GIOP. Prednisolone acetate was intragastrically administered to mice for two weeks. Histochemical staining was applied to evaluate changes in GIOP and Notum expression. Osteocytes were stimulated with prednisolone, and cell viability was assessed via CCK8. Hoechst 33342/PI staining, flow cytometry, RT-PCR, and western blot were used to detect osteocytes apoptosis, siRNA transfection efficiency, and expressions of pathway related factors. The results showed that the number of empty osteocytes lacunae increased in GIOP mice. TUNEL-stained apoptotic osteocytes and Notum immuno-positive osteocytes were also observed. Furthermore, prednisolone was found to promote Notum expression and osteocytes apoptosis in vitro. Knocking down Notum via siRNA partially restored osteocytes apoptosis and phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/glycogen synthase kinase-3ß (GSK3ß)/ß-catenin pathway. These findings showed GCs-induced osteocytes apoptosis by promoting Notum expression and inhibiting PI3K/AKT/GSK3ß/ß-catenin pathway. Thus, Notum might be a potential therapeutic target for the treatment of GIOP.

Adiponectin regulates osteocytic MLO-Y4 cell apoptosis in a high-glucose environment through the AMPK/FoxO3a signaling pathway.

Clinical studies have shown that persistent hyperglycemia following oxidative stress is associated with the apoptosis of osteocytes in diabetics. Adiponectin (APN) can ameliorate oxidative stress, and its receptors have been identified in bone-forming cells. However, the relationship between APN and osteocyte apoptosis has not been fully elucidated. This study aimed to investigate whether APN could prevent osteocyte apoptosis and regulate reactive oxygen species (ROS) generation in a high-glucose environment. Hoechst staining and fluorescence microscopy were used to observe the apoptosis of osteocytic MLO-Y4 cells. Real-time quantitative polymerase chain reaction and Western blot analysis were used to detect the expression of Caspase 3, Caspase 8, and Bcl-2. ROS generation was investigated with an active oxygen kit and fluorescence microscopy. Furthermore, the expression of proteins in the AMPK/FoxO3A signaling pathway was also studied by Western blot analysis. In a high-glucose environment, APN promoted the proliferation of MLO-Y4 osteocytes and the expression of Bcl-2 but inhibited the expression of Caspase 3, Caspase 8, and ROS in a dose-dependent manner. APN promoted the activation of p-AMPK and p-AMPK/AMPK, which reached their highest levels at 10 min and returned to baseline at 30 min. The expression of p-FoxO3A/FoxO3A in both the cytoplasm and nucleus peaked at 15 min, and this expression was returned to baseline at 60 min. In summary, APN has an antiapoptotic effect and regulates ROS generation in MLO-Y4 osteocytes in a high-glucose environment. The AMPK/FoxO3A signaling pathway might be a key signaling pathway that participates in the effect of APN on regulating osteocyte apoptosis in diabetics.

Puerarin facilitates osteogenesis in steroid-induced necrosis of rabbit femoral head and osteogenesis of steroid-induced osteocytes via miR-34a upregulation.

The present study investigated the effect of puerarin on promoting the osteogenesis in steroid-induced necrosis of the femoral head (SONFH). New Zealand rabbits were administrated with horse serum and methylprednisolone (MPS) for establishing SONFH in vivo model, which was then treated with puerarin treatment. Histo-morphological changes in the femoral head were examined by hematoxylin-eosin staining. Osteoblasts were isolated from healthy rabbits and treated by individual or combined administration of dexamethasone and puerarin. Osteoblast viability was measured by CCK-8 assay. Mineralized nodule formation was evaluated by alizarin red assay. Expressions of RUNX family transcription factor 2 (RUNX2), Type-I collagen α 1 (COL1A1), ALP and miR-34a in the femoral head were determined by qRT-PCR and Western blot. Puerarin attenuated the effect of SONFH on promoting histopathological abnormalities and counteracted SONFH inhibition on the expressions of ALP, RUNX2, COL1A1 and miR-34a in the rabbits. Rabbit osteoblasts were successfully isolated, as they showed red mineralized nodules. Dexamethasone exposure decreased osteoblast viability, which was increased by puerarin treatment. Furthermore, puerarin treatment attenuated dexamethasone-induced inhibition on the viability, osteoblastic differentiation, and the expressions of ALP, RUNX2, COL1A1 and miR-34a in the osteoblasts. Puerarin facilitated osteogenesis of steroid-induced necrosis of rabbit femoral head and osteogenesis of steroid-induced osteocytes via miR-34a upregulation.

Evaluating Poly(Acrylamide-co-Acrylic Acid) Hydrogels Stress Relaxation to Direct the Osteogenic Differentiation of Mesenchymal Stem Cells.

The aim of this study is to investigate polyacrylamide-based hydrogels stress relaxation and the subsequent impact on the osteogenic differentiation of human mesenchymal stem cells (hMSCs). Different hydrogels are synthesized by varying the amount of cross-linker and the ratio between the monomers (acrylamide and acrylic acid), and characterized by compression tests. It has been found that hydrogels containing 18% of acrylic acid exhibit an average relaxation of 70%, while pure polyacrylamide gels show an average relaxation of 15%. Subsequently, hMSCs are cultured on two different hydrogels functionalized with a mimetic peptide of the bone morphogenetic protein-2 to enable cell adhesion and favor their osteogenic differentiation. Phalloidin staining shows that for a constant stiffness of 55 kPa, a hydrogel with a low relaxation (15%) leads to star-shaped cells, which is typical of osteocytes, while a hydrogel with a high relaxation (70%) presents cells with a polygonal shape characteristic of osteoblasts. Immunofluorescence labeling of E11, strongly expressed in early osteocytes, also shows a dramatically higher expression for cells cultured on the hydrogel with low relaxation (15%). These results clearly demonstrate that, by fine-tuning hydrogels stress relaxation, hMSCs differentiation can be directed toward osteoblasts, and even osteocytes, which is particularly rare in vitro.

Advanced oxidation protein products aggravate age­related bone loss by increasing sclerostin expression in osteocytes via ROS­dependent downregulation of Sirt1.

Advanced oxidation protein products (AOPPs) induce intracellular oxidative stress (OS) and are involved in numerous diseases. AOPPs accumulate with age, and our previous study revealed that AOPPs accelerated bone deterioration in aged rats. However, the underlying mechanism remains unknown. The present study demonstrated that AOPPs aggravated bone loss in aging male mice by increasing the resorptive activity and decreasing the formative activity of bone tissues. In addition, SOST mRNA (encoding sclerostin) and sclerostin protein levels were increased in the bone tissues of AOPP­treated mice, which was associated with enhanced OS status as well as decreased Sirtuin 1 (SIRT1) mRNA and protein expression levels. Incubation of MLO­Y4 cells with AOPPs induced the accumulation of reactive oxygen species (ROS) via the activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases. The accumulated ROS then upregulated sclerostin expression in MLO­Y4 cells by decreasing Sirt1 expression. In vivo, AOPP­challenged mice co­treated with apocynin (an inhibitor of NADPH oxidases), N­acetyl­L­cysteine (a ROS scavenger) or SRT3025 (a Sirt1 activator) displayed improved bone mass and microstructure. Moreover, sclerostin expression in the bone tissues of the co­treated groups was significantly lower compared with that in groups treated with AOPPs alone. Collectively, these data suggested that AOPPs aggravated age­related bone loss by increasing the expression of sclerostin in osteocytes via ROS­dependent downregulation of Sirt1. The present findings provide novel insights into the pathogenesis of senile osteoporosis.

Cumulative Effects of Strontium Ranelate and Impact Exercise on Bone Mass in Ovariectomized Rats.

OBJECTIVE: To explore the effect of physical exercise (EXE), strontium ranelate (SR), or their combination on bone status in ovariectomized (OVX) rats. DESIGN: Sixty female Wistar rats were randomized to one of five groups: sham (Sh), OVX (O), OVX+EXE (OE), OVX+SR (OSR), and OVX+EXE+SR (OESR). Animals in EXE groups were subjected to 10 drops per day (45 cm in height); rats in SR groups received 625 mg/kg/day of SR, 5 days/week for 8 weeks. Bone mineral density (BMD) and bone mineral content (BMC, dual-energy X-ray absorptiometry (DXA)), mechanical strength of the left femur (three-point bending test), and femur microarchitecture of (micro-computed tomography imaging, microCT) analyses were performed to characterize biomechanical and trabecular/cortical structure. Bone remodeling, osteocyte apoptosis, and lipid content were evaluated by ELISA and immunofluorescence tests. RESULTS: In OVX rats, whole-body BMD, trabecular parameters, and osteocalcin (OCN) levels decreased, while weight, lean/fat mass, osteocyte apoptosis, and lipid content all increased. EXE after ovariectomy improved BMD and BMC, trabecular parameters, cross-sectional area (CSA), moment of inertia, and OCN levels while decreasing osteocyte apoptosis and lipid content. SR treatment increased BMD and BMC, trabecular parameters, CSA, stiffness, OCN, and alkaline phosphatase (ALP) levels. Furthermore, fat mass, N-telopeptide (NTX) level, osteocyte apoptosis, and lipid content significantly decreased. The combination of both EXE and SR improved bone parameters compared with EXE or SR alone. CONCLUSION: EXE and SR had positive and synergistic effects on bone formation and resorption.

Resistance of bone marrow mesenchymal stem cells in a stressed environment - Comparison with osteocyte cells.

Introduction: Recently, the efficacy of mesenchymal stem cells (MSCs) mediated by their tissue repair and anti-inflammatory actions in the prevention and therapy of various disorders has been reported. In this research, our attention was focused specifically on the prevention and therapy of glucocorticoid-induced osteonecrosis. We investigated the stress resistance of MSC against glucocorticoid administration and hypoxic stress, which are factors known to induce osteocytic cell death. Materials and Methods: Mouse bone cells (MLO-Y4) and bone-marrow derived mouse MSCs were exposed to dexamethasone (Dex), hypoxia of 1% oxygen or both in vitro. Mitochondrial membrane potentials were estimated by mitochondria labeling with a cell-permeant probe (Mito tracker red); expression of these apoptosis-inducing molecules, oxidative stress marker (8-hydroxy-2'-deoxyguanosine), caspase-3, -9, and two apoptosis-inhibiting molecules, energy-producing ATP synthase (ATP5A) and X-linked inhibitor of apoptosis protein (XIAP), were analyzed by both immunofluorescence and western blot. Results: With exposure to either dexamethasone or hypoxia, MLO-Y4 showed reduced mitochondrial membrane potential, ATP5A and upregulation of 8-OHdG, cleaved caspases and XIAP. Those changes were significantly enhanced by treatment with dexamethasone plus hypoxia. In MSCs, however, mitochondrial membrane potentials were preserved, while no significant changes in the pro-apoptosis or anti-apoptosis molecules analyzed were found even with exposure to both dexamethasone and hypoxia. No such effects induced by treatment with dexamethasone, hypoxia, or both were demonstrated in MSCs at all. Discussion: In osteocyte cells subjected to the double stresses of glucocorticoid administration and a hypoxic environment osteocytic cell death was mediated via mitochondria. In contrast, MSC subjected to the same stressors showed preservation of mitochondrial function and reduced oxidative stress. Accordingly, even under conditions sufficiently stressful to cause the osteocytic cell death in vivo, it was thought that the function of MSC could be preserved, suggesting that in the case of osteonecrosis preventative and therapeutic strategies incorporating their intraosseous implantation may be promising.

Osteocytic Connexin43 Channels Regulate Bone-Muscle Crosstalk.

Bone-muscle crosstalk plays an important role in skeletal biomechanical function, the progression of numerous pathological conditions, and the modulation of local and distant cellular environments. Previous work has revealed that the deletion of connexin (Cx) 43 in osteoblasts, and consequently, osteocytes, indirectly compromises skeletal muscle formation and function. However, the respective roles of Cx43-formed gap junction channels (GJs) and hemichannels (HCs) in the bone-muscle crosstalk are poorly understood. To this end, we used two Cx43 osteocyte-specific transgenic mouse models expressing dominant negative mutants, Δ130-136 (GJs and HCs functions are inhibited), and R76W (only GJs function is blocked), to determine the effect of these two types of Cx43 channels on neighboring skeletal muscle. Blockage of osteocyte Cx43 GJs and HCs in Δ130-136 mice decreased fast-twitch muscle mass with reduced muscle protein synthesis and increased muscle protein degradation. Both R76W and Δ130-136 mice exhibited decreased muscle contractile force accompanied by a fast-to-slow fiber transition in typically fast-twitch muscles. In vitro results further showed that myotube formation of C2C12 myoblasts was inhibited after treatment with the primary osteocyte conditioned media (PO CM) from R76W and Δ130-136 mice. Additionally, prostaglandin E2 (PGE2) level was significantly reduced in both the circulation and PO CM of the transgenic mice. Interestingly, the injection of PGE2 to the transgenic mice rescued fast-twitch muscle mass and function; however, this had little effect on protein synthesis and degradation. These findings indicate a channel-specific response: inhibition of osteocytic Cx43 HCs decreases fast-twitch skeletal muscle mass alongside reduced protein synthesis and increased protein degradation. In contrast, blockage of Cx43 GJs results in decreased fast-twitch skeletal muscle contractile force and myogenesis, with PGE2 partially accounting for the measured differences.

A Review of Recent Developments in the Molecular Mechanisms of Bone Healing.

Between 5 and 10 percent of fractures do not heal, a condition known as nonunion. In clinical practice, stable fracture fixation associated with autologous iliac crest bone graft placement is the gold standard for treatment. However, some recalcitrant nonunions do not resolve satisfactorily with this technique. For these cases, biological alternatives are sought based on the molecular mechanisms of bone healing, whose most recent findings are reviewed in this article. The pro-osteogenic efficacy of morin (a pale yellow crystalline flavonoid pigment found in old fustic and osage orange trees) has recently been reported, and the combined use of bone morphogenetic protein-9 (BMP9) and leptin might improve fracture healing. Inhibition with methyl-piperidino-pyrazole of estrogen receptor alpha signaling delays bone regeneration. Smoking causes a chondrogenic disorder, aberrant activity of the skeleton's stem and progenitor cells, and an intense initial inflammatory response. Smoking cessation 4 weeks before surgery is therefore highly recommended. The delay in fracture consolidation in diabetic animals is related to BMP6 deficiency (35 kDa). The combination of bioceramics and expanded autologous human mesenchymal stem cells from bone marrow is a new and encouraging alternative for treating recalcitrant nonunions.