Eldecalcitol protected osteocytes against ferroptosis of D-gal-induced senescent MLO-Y4 cells and ovariectomized mice.

BACKGROUND: Active vitamin D analog eldecalcitol is clinically applied in treatment of postmenopausal osteoporosis. This study aims to determine the role of eldecalcitol in the protection of osteocytes from senescence and the associated ferroptosis. METHODS: The MLO-Y4 osteocytes were exposed to D-gal inducing senescence. The ovariectomized (OVX) mice treated with D-gal using as an aging inducer were intraperitoneally injected with eldecalcitol. The multiplexed confocal imaging, fluorescence in situ hybridization and transmission electron microscopy were applied in assessing osteocytic properties. Immunochemical staining and immunoblotting were carried out to detect abundance and expression of molecules. RESULTS: The ablation of vitamin D receptor led to a reduction in amounts of osteocytes, a loss of dendrites, an increase in mRNA expression of SASP factors and in protein expression of senescent factors, as well as changes in mRNA expression of ferroptosis-related genes (PTGS2 & RGS4). Eldecalcitol reversed senescent phenotypes of MLO-Y4 cells shown by improving cell morphology and density, decreasing ß-gal-positive cell accumulation, and down-regulating protein expression (P16, P21 & P53). Eldecalcitol reduced intracellular ROS and MDA productions, elevated JC-1 aggregates, and up-regulated expression of Nrf2 and GPX4. Eldecalcitol exhibited osteopreserve effects in D-gal-induced aging OVX mice. The confocal imaging displayed its improvement on osteocytic network organization. Eldecalcitol decreased the numbers of senescent osteocytes at tibial diaphysis by SADS assay and attenuated mRNA expression of SASP factors as well as down-regulated protein expression of senescence-related factors and restored levels of ferroptotic biomarkers in osteocytes-enriched bone fraction. It reduced 4-HNE staining area, stimulated Nrf2-positive staining, and promoted nuclear translocation of Nrf2 in osteocytes of mice as well as inhibited and promoted protein expression of 4-HNE and Nrf2, respectively, in osteocytes-enriched bone fraction. CONCLUSIONS: The present study revealed the ameliorative effects of eldecalcitol on senescence and the associated ferroptosis of osteocytes, contributing to its preservation against osteoporosis of D-gal-induced senescent ovariectomized mice.

The Osteocyte with SB216763-Activated Canonical Wnt Signaling Constructs a Multifunctional 4D Intelligent Osteogenic Module.

The enhancement of bioactivity in materials has become an important focus within the field of bone tissue engineering. Four-dimensional intelligent osteogenic module, an innovative fusion of 3D printing with the time axis, shows immense potential in augmenting the bioactivity of these materials, thereby facilitating autologous bone regeneration efficiently. This study focuses on novel bone repair materials, particularly bioactive scaffolds with a developmental osteogenic microenvironment prepared through 3D bioprinting technology. This research mainly creates a developmental osteogenic microenvironment named "DOME". This is primed by the application of a small amount of the small molecule drug SB216763, which activates canonical Wnt signaling in osteocytes, promoting osteogenesis and mineralization nodule formation in bone marrow stromal cells and inhibiting the formation of adipocytes. Moreover, DOME enhances endothelial cell migration and angiogenesis, which is integral to bone repair. More importantly, the DOME-PCI3D system, a 4D intelligent osteogenic module constructed through 3D bioprinting, stably supports cell growth (91.2% survival rate after 7 days) and significantly increases the expression of osteogenic transcription factors in bone marrow stromal cells and induces osteogenic differentiation and mineralization for 28 days. This study presents a novel approach for bone repair, employing 3D bioprinting to create a multifunctional 4D intelligent osteogenic module. This innovative method not only resolves challenges related to shape-matching and biological activity but also demonstrates the vast potential for applications in bone repair.

Mechanical Stretch-Induced ATP Release from Osteocytes Promotes Osteogenesis of Bone Marrow Mesenchymal Stem Cells.

BACKGROUND: Mandibular distraction osteogenesis (MDO) is a highly effective method for bone regeneration, commonly employed in treating craniofacial defects and deformities. Osteocytes sense mechanical forces in the pericellular space, relay external stimuli to biochemical changes, and send signals to other effector cells, including bone marrow mesenchymal stem cells (BM-MSCs), to regulate bone resorption and formation. Piezo1 potentially affects the secretion signal molecules of bone cells under mechanical stretch. The primary aim of this study was to enhance our comprehension of the molecular biology underlying this therapeutic approach and to identify specific signaling molecules that facilitate bone formation in response to stretch forces. METHODS: Mechanical stretching was applied to negative controls and Piezo1 knockdown osteocyte-like MLO-Y4 cells. Alkaline phosphatase and Alizarin Red S staining were used to survey the osteogenic potential of BM-MSCs. The production and secretion content of adenosine triphosphate (ATP) was measured using ATP content determination analysis. Pathway-related and osteo-specific genes and proteins were evaluated using real-time polymerase chain reaction (RT-PCR), Western blots, and immunofluorescence. Mitochondrial organization was examined with a transmission electron microscope. RESULTS: The conditioned medium of stretch-exposed MLO-Y4s significantly upregulated osteogenesis-related indicators of BM-MSCs (p < 0.001). The upregulation of BM-MSC osteogenesis was associated with ATP release from osteocytes. Mechanically induced calcium transfer and transcriptional coactivator with PDZ-binding motif (TAZ) nuclear translocation mediated by Piezo1 could promote mitochondrial fission and ATP release. Osteocytes detected stretch forces through Piezo1, triggering calcium influx, TAZ nuclear translocation, and ATP production. CONCLUSIONS: The stretch stimulation of Piezo1 induces calcium influx, which in turn promotes calcium-related TAZ nuclear translocation, changes in mitochondrial dynamics, and the release of ATP in osteocytes. This signaling cascade leads to an up-regulation in the osteogenic capacity of BM-MSCs. Mitochondrial energy metabolism of mechanosensitive protein Piezo1-dependent and ATP release may provide a new effective intervention method for mechanically related bone remodeling.

Osteocyte mitochondria regulate angiogenesis of transcortical vessels.

Transcortical vessels (TCVs) provide effective communication between bone marrow vascular system and external circulation. Although osteocytes are in close contact with them, it is not clear whether osteocytes regulate the homeostasis of TCVs. Here, we show that osteocytes maintain the normal network of TCVs by transferring mitochondria to the endothelial cells of TCV. Partial ablation of osteocytes causes TCV regression. Inhibition of mitochondrial transfer by conditional knockout of Rhot1 in osteocytes also leads to regression of the TCV network. By contrast, acquisition of osteocyte mitochondria by endothelial cells efficiently restores endothelial dysfunction. Administration of osteocyte mitochondria resultes in acceleration of the angiogenesis and healing of the cortical bone defect. Our results provide new insights into osteocyte-TCV interactions and inspire the potential application of mitochondrial therapy for bone-related diseases.

Estimation of the in-plane ultimate stress of lamellar tissue as a function of bone mineral density and osteocyte lacunae porosity.

BACKGROUND AND OBJECTIVE: Detailed finite element models based on medical images (µ-CT) are commonly used to analyze the mechanical behavior of bone at microscale. In order to simulate the tissue failure onset, isotropic failure criteria of lamellar tissue are often used, despite its non-isotropic and heterogeneous nature. The main goal of the present work is to estimate the in-plane ultimate stress of lamellar bone, considering the influence of mineral content and the porosity due to the osteocyte lacunae concentration. METHODS: To this aim, a representative volume cell of lamellar tissue is modeled numerically, including: (1) non-isotropic elastic properties of tissue as a function of the bone mineral density and (2) explicit modeling of the osteocyte lacunae, considering the range of porosity content, size and orientation of ellipsoid-shaped lacunae. Firstly, the element size for the finite element models have been defined from a preliminary convergence analysis. Bounds on the ultimate stress of non-porous lamellar tissue are estimated for two values of bone mineral density, considering the results of tensile and compressive tests of wet osteons from the literature. Subsequently, the ultimate stress of lamellar tissue considering several values of micro-porosity is addressed. RESULTS: Results obtained in this work show that the strength of lamellar bone decreases exponentially with the increase of lacunae porosity concentration. Ultimate stress of non-porous tissue (p=0%) increases with high mineral content, reaching a value of S¯transc=355.40±39.80 MPa for compression in the transversal direction of the fiber bundles, being BMD=1.246g/cm3. The mean value for the longitudinal to transverse strength ratio evaluated for porosity p=0%,1% and 5% and a mineral content BMD=1.2g/cm3, is 2.47:1 for tension and 1.55:1 for compression. These values are in agreement with literature. CONCLUSIONS: Osteocyte lacunae act as stress concentrators, acting as potential stimulus for the bone regeneration process. A novel micromechanical model for the in-plane ultimate stress of lamellar tissue as a function of mineral content and lacunae concentration is presented. Additional considerations about the intralamellar shear stress evolution are also given.

Mechanically stimulated osteocytes maintain tumor dormancy in bone metastasis of non-small cell lung cancer by releasing small extracellular vesicles.

Although preclinical and clinical studies have shown that exercise can inhibit bone metastasis progression, the mechanism remains poorly understood. Here, we found that non-small cell lung cancer (NSCLC) cells adjacent to bone tissue had a much lower proliferative capacity than the surrounding tumor cells in patients and mice. Subsequently, it was demonstrated that osteocytes, sensing mechanical stimulation generated by exercise, inhibit NSCLC cell proliferation and sustain the dormancy thereof by releasing small extracellular vesicles with tumor suppressor micro-RNAs, such as miR-99b-3p. Furthermore, we evaluated the effects of mechanical loading and treadmill exercise on the bone metastasis progression of NSCLC in mice. As expected, mechanical loading of the tibia inhibited the bone metastasis progression of NSCLC. Notably, bone metastasis progression of NSCLC was inhibited by moderate exercise, and combinations with zoledronic acid had additive effects. Moreover, exercise preconditioning effectively suppressed bone metastasis progression. This study significantly advances the understanding of the mechanism underlying exercise-afforded protection against bone metastasis progression.

Pronounced cortical porosity and sex-specific patterns of increased bone and osteocyte lacunar mineralization characterize the human distal fibula with aging.

The high occurrence of distal fibula fractures among older women suggests a potential link to impaired bone health. Here we used a multiscale imaging approach to investigate the microarchitecture, mineralization, and biomechanics of the human distal fibula in relation to age and sex. Micro-computed tomography was performed to analyze the local volumetric bone mineral density and various microarchitectural parameters of the trabecular and the cortical compartment. Bone mineral density distribution and osteocyte lacunar parameters were quantified using quantitative backscattered electron imaging in periosteal, endocortical, and trabecular regions. Additionally, cortical hardness and Young's modulus were assessed by nanoindentation. While cortical porosity strongly increased with age independent of sex, trabecular microarchitecture remained stable. Notably, nearly half of the specimens showed non-bony hypermineralized tissue located at the periosteum, similar to that previously detected in the femoral neck, with no consistent association with advanced age. Independent of this finding, cortical and trabecular mineralization, i.e., mean calcium content, as well as endocortical tissue hardness increased with age in males but not females. Importantly, we also observed mineralized osteocyte lacunae that increased with age specifically in females. In conclusion, our results indicate that skeletal aging of the distal fibula is signified not only by pronounced cortical porosity but also by an increase in mineralized osteocyte lacunae in females. These findings may provide an explanation for the increased occurrence of ankle fractures in older women.

Numerical calculation of streaming potential around osteocytes under human gait loading.

Streaming potential is a type of stress-generated potential in bone that affects the electrical environment of osteocytes and may play a role in bone remodeling. Because the electrical environment around osteocytes has been difficult to measure experimentally until now, a numerical solid-liquid-streaming potential coupling method was proposed to analyze the streaming potential generated by bone deformation in the lacunae and canaliculus network (LCN) of the bone. Using this method, the cellular shear stress caused by liquid flow on the osteocyte surface was first calculated, and the results were consistent with those reported in the literature. Subsequently, the streaming potentials in the LCN caused by bone matrix deformation under an external gait load were calculated numerically. The results showed that the streaming potential increased slowly in the lacuna and relatively rapidly in the canaliculus and that the streaming potential increased with a decrease in the radius or an increase in the length of the canaliculus. The results also showed that relatively large gaps between the lacunae and osteocytes could induce higher streaming potentials under the same loading.

Influence of intramedullary pressure on Lacuno-Canalicular fluid flow: A systematic review.

While most of current models investigating bone remodelling are based on matrix deformation, intramedullary pressure also plays a role. Bone remodelling is orchestrated by the Lacuno-Canalicular Network (LCN) fluid-flow. The aim of this review was hence to assess the influence of intramedullary pressure on the fluid circulation within the LCN. Three databases (Science Direct, Web of Science, and PubMed) were used. The first phase of the search returned 731 articles, of which 9 respected the inclusion/exclusion criteria and were included. These studies confirm the association between intramedullary pressure and fluid dynamics in the LCN. Among the included studies, 7 experimental studies using animal models and 2 numerical models were found. The studies were then ranked according to the nature of the applied loading, either axial compression or direct cyclic intramedullary pressure. The current review revealed that there is an influence of intramedullary pressure on LCN fluid dynamics and that this influence depends on the magnitude and the frequency of the applied pressure. Two studies confirmed that the influence was effective even without bone matrix deformation. While intramedullary pressure is closely associated with LCN fluid, there is a severe lack of studies on this topic. STATEMENT OF SIGNIFICANCE: Since the 1990's, numerical models developed to investigate fluid flow in bone submicrometric porous network are based on the flow induced by matrix deformation. Bone fluid flow is known to be involved in cells stimulation and hence directly influences bone remodeling. Different studies have shown that intramedullary pressure is also associated with bone mechanosensitive adaptation. This pressure is developed in bone due to blood circulation and is increased during loading or muscle stimulation. The current article reviews the studies investigating the influence of this pressure on bone porous fluid flow. They show that fluid flow is involved by this pressure even without bone matrix deformation. The current review article highlights the severe lack of studies about this mechanism.

Controlled mechanical loading affects the osteocyte transcriptome in porcine trabecular bone in situ.

INTRODUCTION: Osteocytes modulate bone adaptation in response to mechanical stimuli imparted by the deforming bone tissue in which they are encased by communicating with osteoclasts and osteoblasts as well as other osteocytes in the lacuna-canalicular network through secreted cytokines and chemokines. Understanding the transcriptional response of osteocytes to mechanical stimulation in situ could identify new targets to inhibit bone loss or enhance bone formation in the presence of diseases like osteoporosis or metastatic cancer. We compared the mechanically regulated transcriptional response of osteocytes in trabecular bone following one or three days of controlled mechanical loading. METHODS: Porcine trabecular bone explants were cultured in a bioreactor for 48 h and subsequently loaded twice a day for one day or 3 days. RNA was isolated and sequenced, and the Tuxedo suite was used to identify differentially expressed genes and pathway analysis was conducted using Ingenuity Pathway Analysis (IPA). RESULTS: There were about 4000 differentially expressed genes following in situ culture relative to fresh bone. One hundred six genes were differentially expressed between the loaded and non-loaded groups following one day of loading compared to 913 genes after 3 d of loading. Only 45 of these were coincident between the two time points, indicating an evolving transcriptome. Clustering and principal component analysis indicated differences between the loaded and non-loaded groups after 3 d of loading. DISCUSSION: With sustained loading, there was a nine-fold increase in the number of differentially expressed genes, suggesting that osteocytes respond to loading through sequential activation of downstream genes in the same pathways. The differentially expressed genes were related to osteoarthritis, osteocyte, and chondrocyte signaling pathways. We noted that NFkB and TNF signaling are affected by early loading and this may drive downstream effects on the mechanobiological response. Moreover, these genes may regulate catabolic effects of mechanical disuse through their actions on pre-osteoclasts in the bone marrow niche.

TNF-α promotes osteocyte necroptosis by upregulating TLR4 in postmenopausal osteoporosis.

Postmenopausal osteoporosis (PMOP) is a common kind of osteoporosis that is associated with excessive osteocyte death and bone loss. Previous studies have shown that TNF-α-induced osteocyte necroptosis might exert a stronger effect on PMOP than apoptosis, and TLR4 can also induce cell necroptosis, as confirmed by recent studies. However, little is known about the relationship between TNF-α-induced osteocyte necroptosis and TLR4. In the present study, we showed that TNF-α increased the expression of TLR4, which promoted osteocyte necroptosis in PMOP. In patients with PMOP, TLR4 was highly expressed at skeletal sites where exists osteocyte necroptosis, and high TLR4 expression is correlated with enhanced TNF-α expression. Osteocytes exhibited robust TLR4 expression upon exposure to necroptotic osteocytes in vivo and in vitro. Western blotting and immunofluorescence analyses demonstrated that TNF-α upregulated TLR4 expression in vitro, which might further promote osteocyte necroptosis. Furthermore, inhibition of TLR4 by TAK-242 in vitro effectively blocked osteocyte necroptosis induced by TNF-α. Collectively, these results suggest a novel TLR4-mediated process of osteocyte necroptosis, which might increase osteocyte death and bone loss in the process of PMOP.

Aging impairs the osteocytic regulation of collagen integrity and bone quality.

Poor bone quality is a major factor in skeletal fragility in elderly individuals. The molecular mechanisms that establish and maintain bone quality, independent of bone mass, are unknown but are thought to be primarily determined by osteocytes. We hypothesize that the age-related decline in bone quality results from the suppression of osteocyte perilacunar/canalicular remodeling (PLR), which maintains bone material properties. We examined bones from young and aged mice with osteocyte-intrinsic repression of TGFß signaling (TßRIIocy-/-) that suppresses PLR. The control aged bone displayed decreased TGFß signaling and PLR, but aging did not worsen the existing PLR suppression in male TßRIIocy-/- bone. This relationship impacted the behavior of collagen material at the nanoscale and tissue scale in macromechanical tests. The effects of age on bone mass, density, and mineral material behavior were independent of osteocytic TGFß. We determined that the decline in bone quality with age arises from the loss of osteocyte function and the loss of TGFß-dependent maintenance of collagen integrity.

PTH 1-34 reduced apoptosis of MLO-Y4 osteocyte-like cells by activating autophagy and inhibiting ER stress under RPM conditions.

Osteocytes, as mechanosensitive cells residing within bone tissue, hold a pivotal role in averting the occurrence and progression of osteoporosis. The apoptosis of osteocytes induced by unloading is one of the contributing factors to osteoporosis, although the underlying molecular mechanisms have not been fully elucidated. PTH 1-34 is known to promote bone formation and inhibit bone loss by targeting osteoblasts and osteocytes. However, it is not known whether PTH 1-34 can inhibit osteocyte apoptosis under unloading conditions and the molecular mechanisms involved. In this study, we employed a Random Positioning Machine (RPM) to emulate unloading conditions and cultured MLO-Y4 osteocyte-like cells, in order to unravel the mechanisms through which PTH 1-34 constrains osteocyte apoptosis amidst unloading circumstances. Our findings revealed that PTH 1-34 activated autophagy while suppressing endoplasmic reticulum stress by curtailing the generation of reactive oxygen species (ROS) in MLO-Y4 osteocyte-like cells during unloading conditions. By shedding light on the osteoporosis triggered by skeletal unloading, this study contributes vital insights that may pave the way for the development of pharmacological interventions.

Iguratimod suppresses sclerostin and receptor activator of NF-κB ligand production via the extracellular signal-regulated kinase/early growth response protein 1/tumor necrosis factor alpha pathway in osteocytes and ameliorates disuse osteoporosis in mice.

Disuse osteoporosis is a prevalent complication among patients afflicted with rheumatoid arthritis (RA). Although reports have shown that the antirheumatic drug iguratimod (IGU) ameliorates osteoporosis in RA patients, details regarding its effects on osteocytes remain unclear. The current study examined the effects of IGU on osteocytes using a mouse model of disuse-induced osteoporosis, the pathology of which crucially involves osteocytes. A reduction in distal femur bone mass was achieved after 3 weeks of hindlimb unloading in mice, which was subsequently reversed by intraperitoneal IGU treatment (30 mg/kg; five times per week). Histology revealed that hindlimb-unloaded (HLU) mice had significantly increased osteoclast number and sclerostin-positive osteocyte rates, which were suppressed by IGU treatment. Moreover, HLU mice exhibited a significant decrease in osteocalcin-positive cells, which was attenuated by IGU treatment. In vitro, IGU suppressed the gene expression of receptor activator of NF-κB ligand (RANKL) and sclerostin in MLO-Y4 and Saos-2 cells, which inhibited osteoclast differentiation of mouse bone marrow cells in cocultures. Although IGU did not affect the nuclear translocation or transcriptional activity of NF-κB, RNA sequencing revealed that IGU downregulated the expression of early growth response protein 1 (EGR1) in osteocytes. HLU mice showed significantly increased EGR1- and tumor necrosis factor alpha (TNFα)-positive osteocyte rates, which were decreased by IGU treatment. EGR1 overexpression enhanced the gene expression of TNFα, RANKL, and sclerostin in osteocytes, which was suppressed by IGU. Contrarily, small interfering RNA-mediated suppression of EGR1 downregulated RANKL and sclerostin gene expression. These findings indicate that IGU inhibits the expression of EGR1, which may downregulate TNFα and consequently RANKL and sclerostin in osteocytes. These mechanisms suggest that IGU could potentially be used as a treatment option for disuse osteoporosis by targeting osteocytes.

Osteocyte-derived sclerostin impairs cognitive function during ageing and Alzheimer's disease progression.

Ageing increases susceptibility to neurodegenerative disorders, such as Alzheimer's disease (AD). Serum levels of sclerostin, an osteocyte-derived Wnt-ß-catenin signalling antagonist, increase with age and inhibit osteoblastogenesis. As Wnt-ß-catenin signalling acts as a protective mechanism for memory, we hypothesize that osteocyte-derived sclerostin can impact cognitive function under pathological conditions. Here we show that osteocyte-derived sclerostin can cross the blood-brain barrier of old mice, where it can dysregulate Wnt-ß-catenin signalling. Gain-of-function and loss-of-function experiments show that abnormally elevated osteocyte-derived sclerostin impairs synaptic plasticity and memory in old mice of both sexes. Mechanistically, sclerostin increases amyloid ß (Aß) production through ß-catenin-ß-secretase 1 (BACE1) signalling, indicating a functional role for sclerostin in AD. Accordingly, high sclerostin levels in patients with AD of both sexes are associated with severe cognitive impairment, which is in line with the acceleration of Αß production in an AD mouse model with bone-specific overexpression of sclerostin. Thus, we demonstrate osteocyte-derived sclerostin-mediated bone-brain crosstalk, which could serve as a target for developing therapeutic interventions against AD.

Dexamethasone Induced Osteocyte Apoptosis in Steroid-Induced Femoral Head Osteonecrosis through ROS-Mediated Oxidative Stress.

OBJECTIVE: Glucocorticoid (GC) overuse is strongly associated with steroid-induced osteonecrosis of the femoral head (SINFH). However, the underlying mechanism of SINFH remains unclear. This study aims to investigate the effect of dexamethasone (Dex)-induced oxidative stress on osteocyte apoptosis and the underlying mechanisms. METHODS: Ten patients with SINFH and 10 patients with developmental dysplasia of the hips (DDH) were enrolled in our study. Sixty rats were randomly assigned to the Control, Dex, Dex + N-Acetyl-L-cysteine (NAC), Dex + Dibenziodolium chloride (DPI), NAC, and DPI groups. Magnetic resonance imaging (MRI) was used to examine edema in the femoral head of rats. Histopathological staining was performed to assess osteonecrosis. Immunofluorescence staining with TUNEL and 8-OHdG was conducted to evaluate osteocyte apoptosis and oxidative damage. Immunohistochemical staining was carried out to detect the expression of NOX1, NOX2, and NOX4. Viability and apoptosis of MLO-Y4 cells were measured using the CCK-8 assay and TUNEL staining. 8-OHdG staining was conducted to detect oxidative stress. 2',7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) staining was performed to measure reactive oxygen species (ROS). The expression of NOX1, NOX2, and NOX4 in MLO-Y4 cells was analyzed by Western blotting. Multiple comparisons were performed using one-way analysis of variance (ANOVA). RESULTS: In patients and the rat model, hematoxylin-eosin (HE) staining revealed a significantly higher rate of empty lacunae in the SINFH group than in the DDH group. Immunofluorescence staining indicated a significant increase in TUNEL-positive cells and 8-OHdG-positive cells in the SINFH group compared to the DDH group. Immunohistochemical staining demonstrated a significant increase in the expression of NOX1, NOX2, and NOX4 proteins in SINFH patients compared to DDH patients. Moreover, immunohistochemical staining showed a significant increase in the proportion of NOX2-positive cells compared to the Control group in the femoral head of rats. In vitro, Dex significantly inhibited the viability of osteocyte cells and induced apoptosis. After Dex treatment, the intracellular ROS level increased. However, Dex treatment did not alter the expression of NOX proteins in vitro. Additionally, NAC and DPI inhibited the generation of intracellular ROS and partially alleviated osteocyte apoptosis in vivo and in vitro. CONCLUSION: This study demonstrates that GC promotes apoptosis of osteocyte cells through ROS-induced oxidative stress. Furthermore, we found that the increased expression of NOXs induced by GC serves as an important source of ROS generation.

How osteons form: A quantitative hypothesis-testing analysis of cortical pore filling and wall asymmetry.

Osteon morphology provides valuable information about the interplay between different processes involved in bone remodelling. The correct quantitative interpretation of these morphological features is challenging due to the complexity of interactions between osteoblast behaviour, and the evolving geometry of cortical pores during pore closing. We present a combined experimental and mathematical modelling study to provide insights into bone formation mechanisms during cortical bone remodelling based on histological cross-sections of quiescent human osteons and hypothesis-testing analyses. We introduce wall thickness asymmetry as a measure of the local asymmetry of bone formation within an osteon and examine the frequency distribution of wall thickness asymmetry in cortical osteons from human iliac crest bone samples from women 16-78 years old. Our measurements show that most osteons possess some degree of asymmetry, and that the average degree of osteon asymmetry in cortical bone evolves with age. We then propose a comprehensive mathematical model of cortical pore filling that includes osteoblast secretory activity, osteoblast elimination, osteoblast embedment as osteocytes, and osteoblast crowding and redistribution along the bone surface. The mathematical model is first calibrated to symmetric osteon data, and then used to test three mechanisms of asymmetric wall formation against osteon data: (i) delays in the onset of infilling around the cement line; (ii) heterogeneous osteoblastogenesis around the bone perimeter; and (iii) heterogeneous osteoblast secretory rate around the bone perimeter. Our results suggest that wall thickness asymmetry due to off-centred Haversian pores within osteons, and that nonuniform lamellar thicknesses within osteons are important morphological features that can indicate the prevalence of specific asymmetry-generating mechanisms. This has significant implications for the study of disruptions of bone formation as it could indicate what biological bone formation processes may become disrupted with age or disease.

Interpenetrating network hydrogels for studying the role of matrix viscoelasticity in 3D osteocyte morphogenesis.

During bone formation, osteoblasts are embedded in a collagen-rich osteoid tissue and differentiate into an extensive 3D osteocyte network throughout the mineralizing matrix. However, how these cells dynamically remodel the matrix and undergo 3D morphogenesis remains poorly understood. Although previous reports investigated the impact of matrix stiffness in osteocyte morphogenesis, the role of matrix viscoelasticity is often overlooked. Here, we report a viscoelastic alginate-collagen interpenetrating network (IPN) hydrogel for 3D culture of murine osteocyte-like IDG-SW3 cells. The IPN hydrogels consist of an ionically crosslinked alginate network to tune stress relaxation as well as a permissive collagen network to promote cell adhesion and matrix remodeling. Two IPN hydrogels were developed with comparable stiffnesses (4.4-4.7 kPa) but varying stress relaxation times (t1/2, 1.5 s and 14.4 s). IDG-SW3 cells were pre-differentiated in 2D under osteogenic conditions for 14 days to drive osteoblast-to-osteocyte transition. Cellular mechanosensitivity to fluid shear stress (2 Pa) was confirmed by live-cell calcium imaging. After embedding in the IPN hydrogels, cells remained highly viable following 7 days of 3D culture. After 24 h, osteocytes in the fast-relaxing hydrogels showed the largest cell area and long dendritic processes. However, a significantly larger increase of some osteogenic markers (ALP, Dmp1, hydroxyapatite) as well as intercellular connections via gap junctions were observed in slow-relaxing hydrogels on day 14. Our results imply that fast-relaxing IPN hydrogels promote early cell spreading, whereas slow relaxation favors osteogenic differentiation. These findings may advance the development of 3D in vivo-like osteocyte models to better understand bone mechanobiology.

Red light-emitting diode therapy minimizes the functional deleterious effects of the antiretroviral ritonavir on osteoblasts in vitro.

PURPOSE: Long-term human immunodeficiency virus (HIV)-infected patients are considered at higher risk for osteoporosis. Among the various causes that lead these patients to lower bone health, there is the use of antiretroviral drugs (ARVs), especially protease inhibitors (PI), such as ritonavir (RTV). In this context, emerge the potential benefits of LED therapy, whose effects on bone cells are currently being extensively studied, showing a modulation in cell differentiation. However, it remains unclear if photobiostimulation might interfere with RTV effects on osteoblast differentiation. METHODS: In the present study, we investigated the effects of red LED (625 nm) irradiation (15 mW/cm2, 0.2 J/cm2, and 8 mW/cm2, 0.12 J/cm2) on osteoblast cell line MC3T3-E1 treated with RTV (2.5, 5, and 10 µg/mL). RESULTS: Our results indicated that red LED irradiation was able to reverse, or at least minimize, the deleterious effects of RTV on the osteoblasts. Neither the ARV treatments 5 and 10 µg/mL (104.4% and 95.01%) nor the LED protocols (100.3% and 105.7%) statistically altered cell viability, assessed by the MTT assay. Also, the alkaline phosphatase activity and mineralization showed a decrease in osteoblast activity followed by ARV exposure (39.3-73%), which was attenuated by LED in more than 70% with statistical significance (p < 0.05). CONCLUSION: In conclusion, photobiostimulation with red LED at 625 nm was associated with improved beneficial biological effects as a potential inducer of osteogenic activity on RTV-affected cells. This is the first study that investigated the benefits of red LED irradiation over ARV-treated in vitro osteoblasts.

Osteocyte mitochondria inhibit tumor development via STING-dependent antitumor immunity.

Bone is one of the most common sites of tumor metastases. During the last step of bone metastasis, cancer cells colonize and disrupt the bone matrix, which is maintained mainly by osteocytes, the most abundant cells in the bone microenvironment. However, the role of osteocytes in bone metastasis is still unclear. Here, we demonstrated that osteocytes transfer mitochondria to metastatic cancer cells and trigger the cGAS/STING-mediated antitumor response. Blocking the transfer of mitochondria by specifically knocking out mitochondrial Rho GTPase 1 (Rhot1) or mitochondrial mitofusin 2 (Mfn2) in osteocytes impaired tumor immunogenicity and consequently resulted in the progression of metastatic cancer toward the bone matrix. These findings reveal the protective role of osteocytes against cancer metastasis by transferring mitochondria to cancer cells and potentially offer a valuable therapeutic strategy for preventing bone metastasis.