Osteocyte Activity Research Articles

The uremic toxin indoxyl sulfate decreases osteocyte RANKL/OPG and increases wnt inhibitor RNA expression that is reversed by PTH

Abstract Renal osteodystrophy (ROD) leads to increased fractures, potentially due to underlying low bone turnover in chronic kidney disease (CKD). We hypothesized that indoxyl sulfate (IS), a circulating toxin elevated in CKD and ligand for the aryl hydrocarbon receptor (AhR), may target the osteocytes leading to bone cell uncoupling in ROD. The IDG-SW3 osteocytes were cultured for 14 days (early) and 35 days (mature osteocytes) and incubated with 500 μM of indoxyl sulfate after dose finding studies to confirm AhR activation. Long-term incubation of IS for 14 days led to decreased expression of Tnfsf11/Tnfrsf11b ratio (RANKL/OPG), which would increase osteoclast activity, and increased expression of Wnt inhibitors Sost and Dkk1, which would decrease bone formation in addition to decreased mineralization and alkaline phosphatase (ALP) activity. When osteocytes were incubated with IS and the AhR translocation inhibitor CH223191, mineralization and ALP activity were restored. However, the Tnfsf11/Tnfrsf11b ratio and Sost, Dkk1 expression were not altered compared to IS alone, suggesting more complex signaling. In both early and mature osteocytes, co-culture with PTH and IS reversed the IS-induced upregulation of Sost and Dkk1, and IS enhanced the PTH-induced increase of the Tnfsf11/Tnfrsf11b ratio. Co-culture of IS with PTH additively enhanced the AhR activity assessed by Cyp1a1 and Cyp1b1 expression. In summary, IS in the absence of PTH, increased osteocyte mRNA Wnt inhibitor expression in both early and mature osteocytes and decreased mRNA expression ofTnfsf11/Tnfrsf11b ratio and mineralization in early osteocytes. These changes would lead to decreased resorption and formation resulting in low bone remodeling. These data suggest IS may be important in the underlying low turnover bone disease observed in CKD when PTH is not elevated. In addition, when elevated PTH, IS interacts to further increase Tnfsf11/Tnfrsf11b ratio for osteoclast activity in both early and mature osteocytes which would worsen bone resorption.

Distribution of Immunomodulation, Protection and Regeneration Factors in Cleft-Affected Bone and Cartilage.

Craniofacial clefts can form a significant defect within bone and cartilage, which can negatively affect tissue homeostasis and the remodeling process. Multiple proteins can affect supportive tissue growth, while also regulating local immune response and tissue protection. Some of these factors, like galectin-10 (Gal-10), nuclear factor kappa-light-chain-enhancer of activated B cells protein 65 (NF-κB p65), heat shock protein 60 (HSP60) and 70 (HSP70) and cathelicidin (LL-37), have not been well studied in cleft-affected supportive tissue, while more known tissue regeneration regulators like type I collagen (Col-I) and bone morphogenetic proteins 2 and 4 (BMP-2/4) have not been assessed jointly with immunomodulation and protective proteins. Information about the presence and interaction of these proteins in cleft-affected supportive tissue could be helpful in developing biomaterials and improving cleft treatment. Two control groups and two cleft patient groups for bone tissue and cartilage, respectively, were organized with five patients in each group. Immunohistochemistry with the semiquantitative counting method was implemented to determine Gal-10-, NF-κB p65-, HSP60-, HSP70-, LL-37-, Col-I- and BMP-2/4-positive cells within the tissue. Factor-positive cells were identified in each study group. Multiple statistically significant correlations were identified. A significant increase in HSP70-positive chondrocytes in cleft patients could indicate that HSP70 might be reacting to stressors caused by the local tissue defect. A significant increase in Col-I-positive osteocytes in cleft patients might indicate increased bone remodeling and osteocyte activity due to the presence of a cleft. Correlations between factors indicate notable differences in molecular interactions within each group.

Vitality of autologous retromolar bone grafts for alveolar ridge augmentation after a 3-months healing period: A prospective histomorphometrical analysis.

The incorporation of retromolar bone grafts used for alveolar ridge augmentation is not well understood. This prospective observational study aims to supply histomorphometrical data from bone graft biopsies taken at the time of retrieval and after a 3-month healing period using patient-matched biopsies. In 17 patients, trephine biopsies of the graft were acquired at the time of graft retrieval and after a 3-month healing period. The biopsies were compared histomorphometrically regarding the number of osteocytes, appearance of osteocyte lacunae, quantity, surface area, and activity of the Haversian canals. All grafts appeared clinically stable after screw removal and 17 implants were placed. Histomorphometric analysis revealed no significant difference in the number of osteocytes (p = .413), osteocyte lacunae (p = .611), the ratio of filled/empty osteocyte lacunae (p = .467) and active Haversian canals (p = .495) between the biopsies retrieved after a 3-months healing period with those at the time of grafting. The only significant difference was noted in the mean surface area of the Haversian canals (p = .002). Specifically, the grafts post 3-month healing showed a significantly larger mean area (0.069 mm2) compared to the time of grafting (0.029 mm2). This study demonstrates, compared to other data, a high rate of vital structures in retromolar bone block grafts after 3 months of healing, exhibiting the same histological features in comparison to the biopsies from the native alveolar ridge. Standard histomorphometrical parameters, e.g., the amount of filled or empty osteocyte lacunae for the description of the vitality of the graft need to be reappraised.

Mechanically strained osteocyte-derived exosomes contained miR-3110-5p and miR-3058-3p and promoted osteoblastic differentiation

BackgroundOsteocytes are critical mechanosensory cells in bone, and mechanically stimulated osteocytes produce exosomes that can induce osteogenesis. MicroRNAs (miRNAs) are important constituents of exosomes, and some miRNAs in osteocytes regulate osteogenic differentiation; previous studies have indicated that some differentially expressed miRNAs in mechanically strained osteocytes likely influence osteoblastic differentiation. Therefore, screening and selection of miRNAs that regulate osteogenic differentiation in exosomes of mechanically stimulated osteocytes are important.ResultsA mechanical tensile strain of 2500 με at 0.5 Hz 1 h per day for 3 days, elevated prostaglandin E2 (PGE2) and insulin-like growth factor-1 (IGF-1) levels and nitric oxide synthase (NOS) activity of MLO-Y4 osteocytes, and promoted osteogenic differentiation of MC3T3-E1 osteoblasts. Fourteen miRNAs differentially expressed only in MLO-Y4 osteocytes which were stimulated with mechanical tensile strain, were screened, and the miRNAs related to osteogenesis were identified. Four differentially expressed miRNAs (miR-1930-3p, miR-3110-5p, miR-3090-3p, and miR-3058-3p) were found only in mechanically strained osteocytes, and the four miRNAs, eight targeted mRNAs which were differentially expressed only in mechanically strained osteoblasts, were also identified. In addition, the mechanically strained osteocyte-derived exosomes promoted the osteoblastic differentiation of MC3T3-E1 cells in vitro, the exosomes were internalized by osteoblasts, and the up-regulated miR-3110-5p and miR-3058-3p in mechanically strained osteocytes, were both increased in the exosomes, which was verified via reverse transcription quantitative polymerase chain reaction (RT-qPCR).ConclusionsIn osteocytes, a mechanical tensile strain of 2500 με at 0.5 Hz induced the fourteen differentially expressed miRNAs which probably were in exosomes of osteocytes and involved in osteogenesis. The mechanically strained osteocyte-derived exosomes which contained increased miR-3110-5p and miR-3058-3p (two of the 14 miRNAs), promoted osteoblastic differentiation.

Tumor necrosis alpha (tnf-α) levels in the human gingival sulcus: rates and factors affecting its levels in healthy subjects

Background and objective: ​​ Gingival crevicular fluid (GCF) provides an exceptional window for investigation of the periodontal condition in which levels of inflammatory mediators, which, as a consequence of the increased local destruction of connective tissue structural elements, are ideal markers of disease activity can be appreciated in the GCF. This study aimed to investigate the levels of tumor necrosis factor-alpha (TNF-α) in the human gingival sulcus of healthy subjects and the influence of host factors such as age, sex, and tooth type used on pro-inflammatory biomarkers. Methods: Eighty-seven patients, 54 (62.1%) female and 33 (37.9%) males (aged 12–34 years; mean 19.58±4.4 years), participated in this study. Each subject underwent a session on professional oral hygiene and received oral hygiene instructions. Gingival crevicular fluid (GCF) sampling was conducted (baseline). GCF was collected from the central incisor, the lateral incisor, the Canine, the First premolar and the Second premolar in this study. The concentration of the pro-inflammatory cytokine (TNF-α) present in the GCF was evaluated by Enzyme-Linked Immunosorbent Assay (ELISA), following manufacturers' recommendations. Differences in mean TNF-α concentrations (pg/ml) of the teeth selected for sample collection for each individual with variables that included age, sex, and tooth type for GCF samples, were determined. Results: In total, the mean ± SD of central incisor TNF-α was 31.88 ± 4.99 pg/ml, and ranged from 20.22 to 40.47 pg/ml; the variance in all individual values ​​was significantly distributed on the normal curve with t-test of 59 and p < 0.001. For males the TNF-α concentration (mean ± SD) of central incisor was 39.25 ± 7.26 pg/ml Vs. 40.98 ± 9.24 pg/ml for females. For the lateral incisor, Canine, First premolar and Second premolar: total, males and females; the mean ± SD of TNF-α level were roughly similar to that of the central incisor. A lower level of TNF-α was in <16 years (37.54 ± 9.5 pg/ml). These findings differ from those of the 16-25 years age group and 26-34 age group in which a significantly higher value was recorded (p=0.035). Conclusion: This study provides the upper limit of normal values ​​for TNF-α levels for people aged 12-34 years in the GCF. These upper limits of normal values ​​will guide dentists in Yemen when considering the diagnosis of periodontal disease, as well as its role during orthodontic tooth movement as they play an important role in the activities of Osteocytes, and will provide useful baseline data for future studies of interventions against periodontal disease; and the movement of teeth by orthodontic appliances, in Yemen. Also, this data can also be applied to neighboring Yemeni cities and neighboring countries.

Influence of Muscle Mass and Strength on Bone Mineralisation with Consideration of Sclerostin Concentration.

Osteoporosis is a disease characterised by a reduction in bone strength due to increased porosity and impaired mineralisation. In our study, we investigated whether muscle strength and mass exert a significant effect on bone mineral density in young adult women. We also tested whether sclerostin can be used as an indicator in the assessment of bone mineralisation. The study included 111 patients. All patients had their bone mineral density determined in the L1-L4 section of the lumbar spine and in the whole skeleton. The parameters of fat mass (FM), lean body mass (LBM) and visceral fat mass (VF) were also determined. Metabolic activity of osteocytes was assessed by measuring the serum sclerostin concentration. There was a statistically significant association of both hands' muscle strength with all parameters expressing bone mineralisation. A statistically significant relationship was also obtained between BMD L1-L4 and the body mass components (FM, LBM). Sclerostin levels in the study did not differ between groups with normal and reduced bone mineral density. Muscle strength assessment may be a potential exponent of reduced bone mineral density, also used clinically in young adult women. The utility of sclerostin in the clinical assessment of bone mineralisation has not been demonstrated.

Pulsed electromagnetic fields as a promising therapy for glucocorticoid-induced osteoporosis.

Glucocorticoid-induced osteoporosis (GIOP) is considered the third type of osteoporosis and is accompanied by high morbidity and mortality. Long-term usage of glucocorticoids (GCs) causes worsened bone quality and low bone mass via their effects on bone cells. Currently, there are various clinical pharmacological treatments to regulate bone mass and skeletal health. Pulsed electromagnetic fields (PEMFs) are applied to treat patients suffering from delayed fracture healing and non-unions. PEMFs may be considered a potential and side-effect-free therapy for GIOP. PEMFs inhibit osteoclastogenesis, stimulate osteoblastogenesis, and affect the activity of bone marrow mesenchymal stem cells (BMSCs), osteocytes and blood vessels, ultimately leading to the retention of bone mass and strength. However, the underlying signaling pathways via which PEMFs influence GIOP remain unclear. This review attempts to summarize the underlying cellular mechanisms of GIOP. Furthermore, recent advances showing that PEMFs affect bone cells are discussed. Finally, we discuss the possibility of using PEMFs as therapy for GIOP.

Effects of Irradiation by Carbon Dioxide Laser Equipped With a Water Spray Function on Bone Formation in Rat Tibiae.

Irradiation of tissue with carbon dioxide (CO2) laser shows a characteristic thermal effect that causes vaporization of tissue in the target region. However, the thermal effect in places other than the target region induces tissue damage. Two methods are used: high reactive-level laser therapy (HLLT), aimed at surgical treatment, and low reactive-level laser therapy (LLLT), aimed at cell and tissue activation. In both, vaporization of tissue is induced by thermal damage. A water spray function may ameliorate thermal damage from CO2 laser irradiation. In this study, we irradiated CO2 laser on rat tibiae with or without a water spray function and examined the effects of this technique on bone metabolism. Bone defects were created in rat tibiae by dental bur in a Bur group and by laser in laser irradiation groups with (Spray group) and without (Air group) water spray function. At 1 week postoperatively, histological analyses of tibiae were performed using hematoxylin and eosin staining, immunohistochemical staining (IHC) with anti-sclerostin antibody, and 3-dimensional (3D) observation using micro-computed tomography. Histological findings and 3D observation confirmed induction of new bone formation following laser irradiation in both the Air and Spray groups. No bone formation was seen in the Bur group. IHC revealed that the activity of osteocytes in the region of irradiated cortical bone was markedly impaired in the Air group, but osteocyte impairment was ameliorated in the Spray group and absent in the Bur group. The water spray function appears effective in reducing thermal damage to tissues irradiated by CO2 laser. CO2 lasers with water spray function may be useful in bone regeneration therapy.

The effects of collagen cross-link deficiency on osseointegration process of pure titanium implants.

This study aimed to investigate the effect of collagen cross-link deficiency on collagen fiber formation around an implant and its effect on the osseointegration process. Wistar rats were fed 0.1% beta-aminopropionitrile (BAPN) dissolved in water to induce collagen cross-link deficiency. Custom-made mini-implants with machined surfaces were placed proximal to the tibia. At 1, 2, and 4 weeks postoperatively, the bone area around the implant, bone-implant contact ratio, osteoclast/osteocyte activity, and osseointegration strength were evaluated using histological and immunohistochemical analyses and biomechanical tests. Long-term disturbance of collagen cross-link formation in the BAPN group resulted in faster collagen fiber maturation than that in controls, with a defective collagen structure, low bone formation quantity, and low bone-implant contact values. Deficiency of collagen cross-links resulted in increased bone resorption and decreased osteocyte activity. Collagen cross-linking is important for the formation of the collagen matrix, and their deficiency may impair bone activity around implants, affecting the osseointegration process.

Study on mass transfer in the bone lacunar-canalicular system under different gravity fields.

The bone lacunar-canalicular system (LCS) is an important microstructural basis for signaling and material transport in bone tissue, guaranteeing normal physiological processes in tissues. Spaceflight astronauts and elderly osteoporosis are related to its function, so it is necessary to reveal the mass transfer laws in bone microstructure under different gravity fields to provide insight for effective clinical treatment. Using the natural LCS structure of bovine tibial cortical bone as the object, the mass transfer experiments on cortical bone were conducted by using sodium fluorescein tracer through different frequency pulsating pressure provided by dynamic perfusion loading device and different high G environments provided by high-speed centrifuge to analyze the mass transfer laws under different gravity fields and different pulsating pressures. The fluorescence intensity of lacunae within the osteon was lower the farther away from the Haversian canal. As the gravity field magnitude increased, the fluorescence intensity within each lacuna enhanced, and the more distant the lacunae from the Haversian canal, the greater the fluorescence intensity enhancement. High-frequency pulsating pressure simulated high-intensity exercise in humans can improve mass transfer efficiency in the LCS. High-intensity exercise may greatly increase solute molecules, nutrients, and signaling molecules in osteocytes and improve the activity of osteocytes. Hypergravity can enhance the transport of solute molecules, nutrients, and signaling molecules in the LCS, especially promoting mass transfer to deep layer lacunae. Conversely, mass transfer to deep layer lacunae may be inhibited under microgravity, causing bone loss and ultimately leading to osteoporosis.

Osteoclast and Sclerostin Expression in Osteocytes in the Femoral Head with Risedronate Therapy in Patients with Hip Fractures: A Retrospective Comparative Study.

Background and Objectives: The majority of research on the effects of osteoporosis drugs has measured the bone mineral density (BMD) of the spine and femur through dual-energy X-ray absorptiometry (DEXA) and compared and analyzed the effects of the drugs through changes in the BMD values. This study aims to compare osteoclast and sclerostin expression in osteocytes after risedronate therapy by obtaining femoral heads from patients with hip fractures. Materials and Methods: We obtained the femoral heads of 10 female patients (age: ≥65 years) who received risedronate therapy for at least 1 year through hip arthroplasty during 2019-2021 (risedronate group). Meanwhile, 10 patients who had never received osteoporosis treatment were selected as controls using propensity scores with age, body mass index, and bone density as covariates (control group). While the osteoclast count was evaluated using tartrate-resistant acid phosphatase (TRAP) staining, the sclerostin expression in osteocytes was assessed using immunohistochemistry. Moreover, Western blotting and polymerase chain reaction (PCR) were performed for receptor activation of nuclear factor kappa-Β ligand (RANKL), RANK, osteoprotegerin (OPG), sclerostin, and bone morphogenetic protein-2 (BMP2). Results: TRAP staining revealed significantly more TRAP-positive cells in the control group (131.75 ± 27.16/mm2) than in the risedronate group (28.00 ± 8.12/mm2). Moreover, sclerostin-positive osteocytes were expressed more in the control group (364.12 ± 28.12/mm2) than in the risedronate group (106.93 ± 12.85/mm2). Western blotting revealed that the expressions of RANKL, RANK, sclerostin, and BMP2 were higher in the control group than in the risedronate group (p < 0.05). Furthermore, RANK, sclerostin, and OPG protein levels were higher in the control group than in the risedronate group. Conclusions: In this study, the risedronate group demonstrated lower osteoclast activity and sclerostin expression in osteocytes in the femoral head than the control group.

Sirt3 mediates the benefits of exercise on bone in aged mice.

Exercise in later life is important for bone health and delays the progression of osteoporotic bone loss. Osteocytes are the major bone cells responsible for transforming mechanical stimuli into cellular signals through their highly specialized lacunocanalicular networks (LCN). Osteocyte activity and LCN degenerate with aging, thus might impair the effectiveness of exercise on bone health; however, the underlying mechanism and clinical implications remain elusive. Herein, we showed that deletion of Sirt3 in osteocytes could impair the formation of osteocyte dendritic processes and inhibit bone gain in response to exercise in vivo. Mechanistic studies revealed that Sirt3 regulates E11/gp38 through the protein kinase A (PKA)/cAMP response element-binding protein (CREB) signaling pathway. Additionally, the Sirt3 activator honokiol enhanced the sensitivity of osteocytes to fluid shear stress in vitro, and intraperitoneal injection of honokiol reduced bone loss in aged mice in a dose-dependent manner. Collectively, Sirt3 in osteocytes regulates bone mass and mechanical responses through the regulation of E11/gp38. Therefore, targeting Sirt3 could be a novel therapeutic strategy to prevent age-related bone loss and augment the benefits of exercise on the senescent skeleton.

Association between Molecular Mechanisms and Tooth Eruption in Children with Obesity.

Different works have reported earlier permanent teething in obese/overweight children compared to control ones. In contrast, others have reported a delayed permanent teething in undernutrition/underweight children compared to control one. It has been reported that becoming overweight or suffering from obesity can increase gingival pro-inflammatory drive and can affect orthodontic treatment (among other complications). In this sense, little is known about the molecular mechanisms affecting dental eruption timing. Leptin and adiponectin are adipocytokines signaling molecules released in overweight and underweight conditions, respectively. These adipocytokines can modulate osteocyte, odontoblast, and cementoblast activity, even regulating dental lamina initiation. The present review focuses on the molecular approach wherein leptin and adiponectin act as modulators of Runt-related transcription factor 2 (Runx 2) gene regulating dental eruption timing.

Osteocytes Serve as a Reservoir for Intracellular Persisting Staphylococcus aureus Due to the Lack of Defense Mechanisms.

Chronic staphylococcal osteomyelitis can persist for long time periods causing bone destruction. The ability of Staphylococcus aureus to develop chronic infections is linked to its capacity to invade and replicate within osteoblasts and osteocytes and to switch to a dormant phenotype called small colony variants. Recently, osteocytes were described as a main reservoir for this pathogen in bone tissue. However, the mechanisms involved in the persistence of S. aureus within these cells are still unknown. Here, we investigated the interaction between S. aureus and osteoblasts or osteocytes during infection. While osteoblasts are able to induce a strong antimicrobial response and eliminate intracellular S. aureus, osteocytes trigger signals to recruit immune cells and enhance inflammation but fail an efficient antimicrobial activity to clear the bacterial infection. Moreover, we found that extracellular signals from osteocytes enhance intracellular bacterial clearance by osteoblasts. Even though both cell types express Toll-like receptor (TLR) 2, the main TLR responsible for S. aureus detection, only osteoblasts were able to increase TLR2 expression after infection. Additionally, proteomic analysis indicates that reduced intracellular bacterial killing activity in osteocytes is related to low antimicrobial peptide expression. Nevertheless, high levels of lipid mediators and cytokines were secreted by osteocytes, suggesting that they can contribute to inflammation. Taken together, our results demonstrate that osteocytes contribute to severe inflammation observed in osteomyelitis and represent the main niche for S. aureus persistence due to their poor capacity for intracellular antimicrobial response.

Autophagy and bone diseases.

Autophagy is a ubiquitous cellular process, allowing the removal and recycling of damaged proteins and organelles. At the basal level, this process plays a role in quality control, thus participating in cellular homeostasis. Autophagy can also be induced by various stresses, such as nutrient deprivation or hypoxia, to allow the cell to survive until conditions improve. In recent years, the role of this process has been widely studied in many pathologies such as neurodegenerative diseases or cancers. In bone tissue, various studies have shown that autophagy is involved in the survival, differentiation and activity of osteoblasts, osteocytes and osteoclasts. The evolution of this knowledge has led to the identification of new molecular pathophysiological mechanisms in bone pathologies. This review reports the current state of knowledge on the role of autophagy in 4 bone diseases: osteoporosis, which seems to be associated with a decrease in autophagy, osteopetrosis and Paget's disease where the course of the autophagic process is disturbed, and finally osteosarcoma where autophagy seems to play a protumoral role. A better understanding of the involvement of autophagy in these pathologies should eventually lead to the identification of new potential therapeutic targets.

Symmetry breaking and effects of nutrient walkway in time-dependent bone remodeling incorporating poroelasticity

Bone is an extraordinary biological material that continuously adapts its hierarchical microstructure to respond to static and dynamic loads for offering optimal mechanical features, in terms of stiffness and toughness, across different scales, from the sub-microscopic constituents within osteons—where the cyclic activity of osteoblasts, osteoclasts, and osteocytes redesigns shape and percentage of mineral crystals and collagen fibers—up to the macroscopic level, with growth and remodeling processes that modify the architecture of both compact and porous bone districts. Despite the intrinsic complexity of the bone mechanobiology, involving coupling phenomena of micro-damage, nutrients supply driven by fluid flowing throughout hierarchical networks, and cells turnover, successful models and numerical algorithms have been presented in the literature to predict, at the macroscale, how bone remodels under mechanical stimuli, a fundamental issue in many medical applications such as optimization of femur prostheses and diagnosis of the risk fracture. Within this framework, one of the most classical strategies employed in the studies is the so-called Stanford’s law, which allows uploading the effect of the time-dependent load-induced stress stimulus into a biomechanical model to guess the bone structure evolution. In the present work, we generalize this approach by introducing the bone poroelasticity, thus incorporating in the model the role of the fluid content that, by driving nutrients and contributing to the removal of wastes of bone tissue cells, synergistically interacts with the classical stress fields to change homeostasis states, local saturation conditions, and reorients the bone density rate, in this way affecting growth and remodeling. Through two paradigmatic example applications, i.e. a cylindrical slice with internal prescribed displacements idealizing a tract of femoral diaphysis pushed out by the pressure exerted by a femur prosthesis and a bone element in a form of a bent beam, it is highlighted that the present model is capable to catch more realistically both the transition between spongy and cortical regions and the expected non-symmetrical evolution of bone tissue density in the medium–long term, unpredictable with the standard approach. A real study case of a femur is also considered at the end in order to show the effectiveness of the proposed remodeling algorithm.

Impact of degradable magnesium implants on osteocytes in single and triple cultures

In vitro triple cultures of human primary osteoblasts, osteocytes and osteoclasts can potentially help to analyze the effect of drugs and degradation products of biomaterials as a model for native bone tissue. In the present study, degradation products of Magnesium (Mg), which has been successfully applied in the biomedical field, were studied with respect to their impact on bone cell morphology and differentiation both in osteocyte single cultures and in the triple culture model. Fluorescence microscopic and gene expression analysis, analysis of osteoclast- and osteoblast-specific enzyme activities as well as osteocalcin protein expression were performed separately for the three cell types after cultivation in triple culture in the presence of extracts, containing 5 and 10 mM Mg2+. All three cell species were viable in the presence of the extracts and did not show morphological changes compared to the Mg-free control. Osteoblasts and osteoclasts did not show significant changes in gene expression of ALPL, BSPII, osteocalcin, TRAP, CTSK and CA2. Likewise on protein level, no significant changes in ALP-, TRAP-, CTSK- and CAII activities were detected. Osteocytes showed a significant downregulation of MEPE, which codes for a protein playing an important role in regulation of phosphate homeostasis by osteocytes. This study is the first to analyze the effects of Mg degradation products on primary osteocytes in vitro, both in single and triple culture. Even if promoting effects on the three examined bone cell species were not found in the applied triple culture setup, it was shown, that Mg degradation products do not interfere with the activity of osteoblasts, osteoclasts and osteocytes in vitro.

Osteocytic Connexin Hemichannels Modulate Oxidative Bone Microenvironment and Breast Cancer Growth.

Simple SummaryEstrogen deficiency with increased oxidative stress is associated with various pathological conditions, including cancers. Oxidative stress (OS) is elevated in the bone microenvironment with estrogen deficiency, but the impact of OS on tumor growth in bone remains largely unknown. In our research, we found that elevated oxidative stress inhibits tumor cell growth and the lack of Cx43 hemichannel function in bone osteocytes could mediate this effect. Impairment of Cx43 hemichannels either under oxidative stress or in Cx43-hemichannel-impaired transgenic mice under estrogen deficiency led to the inhibition of breast cancer growth. Treatment with an antioxidant reverses the effect, showing an increase in tumor growth in mice. This study unveiled a new mechanism regarding the role of osteocytic Cx43 hemichannels in modulating an oxidative bone microenvironment and the response of breast cancer cells to local oxidative stress.Osteocytes, the most abundant bone cell types embedded in the mineral matrix, express connexin 43 (Cx43) hemichannels that play important roles in bone remodeling and osteocyte survival. Estrogen deficiency decreases osteocytic Cx43 hemichannel activity and causes a loss in osteocytes’ resistance to oxidative stress (OS). In this study, we showed that OS reduced the growth of both human (MDA-MB-231) and murine (Py8119) breast cancer cells. However, co-culturing these cells with osteocytes reduced the inhibitory effect of OS on breast cancer cells, and this effect was ablated by the inhibition of Cx43 hemichannels. Py8119 cells were intratibially implanted in the bone marrow of ovariectomized (OVX) mice to determine the role of osteocytic Cx43 hemichannels in breast cancer bone metastasis in response to OS. Two transgenic mice overexpressing dominant-negative Cx43 mutants, R76W and Δ130-136, were adopted for this study; the former inhibits gap junctions while the latter inhibits gap junctions and hemichannels. Under normal conditions, Δ130-136 mice had significantly more tumor growth in bone than that in WT and R76W mice. OVX increased tumor growth in R76W but had no significant effect on WT mice. In contrast, OVX reduced tumor growth in Δ130-136 mice. To confirm the role of OS, WT and Δ130-136 mice were administered the antioxidant N-acetyl cysteine (NAC). NAC increased tumor burden and growth in Δ130-136 mice but not in WT mice. Together, the data suggest that osteocytes and Cx43 hemichannels play pivotal roles in modulating the oxidative microenvironment and breast cancer growth in the bone.

Effect of High Static Magnetic Fields on Biological Activities and Iron Metabolism in MLO-Y4 Osteocyte-like Cells.

There are numerous studies that investigate the effects of static magnetic fields (SMFs) on osteoblasts and osteoclasts. However, although osteocytes are the most abundant cell type in bone tissue, there are few studies on the biological effects of osteocytes under magnetic fields. Iron is a necessary microelement that is involved in numerous life activities in cells. Studies have shown that high static magnetic fields (HiSMF) can regulate cellular iron metabolism. To illustrate the effect of HiSMF on activities of osteocytes, and whether iron is involved in this process, HiSMF of 16 tesla (T) was used, and the changes in cellular morphology, cytoskeleton, function-related protein expression, secretion of various cytokines, and iron metabolism in osteocytes under HiSMF were studied. In addition, the biological effects of HiSMF combined with iron preparation and iron chelator on osteocytes were also investigated. The results showed that HiSMF promoted cellular viability, decreased apoptosis, increased the fractal dimension of the cytoskeleton, altered the secretion of cytokines, and increased iron levels in osteocytes. Moreover, it was found that the biological effects of osteocytes under HiSMF are attenuated or enhanced by treatment with a certain concentration of iron. These data suggest that HiSMF-regulated cellular iron metabolism may be involved in altering the biological effects of osteocytes under HiSMF exposure.

Peripheral Blood Mononuclear Cells (PBMCs) to Dissect the Underlying Mechanisms of Bone Disease in Chronic Kidney Disease and Rare Renal Diseases

To describe the methods that can be used to obtain functional and mature osteoclasts from peripheral blood mononuclear cells (PBMCs) and report the data obtained with this model in two peculiar diseases, namely pediatric chronic kidney disease-associated mineral and bone disorders (CKD-MBD) and nephropathic cystinosis. To discuss future research possibilities in the field. Bone tissue undergoes continuous remodeling throughout life to maintain bone architecture; it involves two processes: bone formation and bone resorption with the coordinated activity of osteoblasts, osteoclasts, and osteocytes. Animal models fail to fully explain human bone pathophysiology during chronic kidney disease, mainly due to interspecies differences. The development of in vitro models has permitted to mimic human bone-related diseases as an alternative to in vivo models. Since 1997, osteoclasts have been generated in cell cultures, notably when culturing PBMCs with specific growth factors and cytokines (i.e., M-CSF and RANK-L), without the need for osteoblasts or stromal cells. These models may improve the global understanding of bone pathophysiology. They can be been used not only to evaluate the direct effects of cytokines, hormones, cells, or drugs on bone remodeling during CKD-MBD, but also in peculiar genetic renal diseases inducing specific bone impairment.