Evidence for peri-lacunar remodeling and altered osteocyte lacuno-canalicular network in mouse models of myeloma-induced bone disease.

Myeloma bone disease (MBD) affects ~90% of multiple myeloma patients, but current treatment options are suboptimal. Therefore, to successfully develop new therapies or optimize current ones, we must improve our fundamental knowledge of how myeloma affects bone microstructure and function. Here, we have investigated the osteocyte lacuno-canalicular network (LCN) in MBD, as bone porosity affects bone quality and resilience. We used the syngeneic 5TGM1-C57BL-Kalwrij and the xenograft U266-NSG models at end stage and compared them to healthy controls (naïve). Micro-computed tomography (µCT) and histomorphometry indicated the 5TGM1 and U266 models developed mild and extensive MBD, respectively, with the U266 model producing large osteolytic lesions. High-resolution synchrotron micro-CT (SR-µCT) revealed significant osteocyte lacunae changes in U266 bones but not 5TGM1, with a reduction in lacunae number and sphericity, and an increase in lacunae volume compared with naïve. Canalicular length, visualized using histological Ploton silver staining, appeared significantly shorter in 5TGM1 and U266 bones compared with naïve. Canalicular area as a proportion of the bone was also decreased by 24.2% in the U266 model. We observed significant upregulation of genes implicated in peri-lacunar remodeling (PLR), but immunohistochemistry confirmed that the osteocyte-specific protein sclerostin, a known driver of PLR, was unchanged between MBD and naïve bones. In summary, we have demonstrated evidence of PLR and altered organization of the osteocyte LCN in MBD mouse models. The next step would be to further understand the drivers and implications of PLR in MBD, and whether treatments to manipulate PLR and the LCN may improve patient outcomes.

Altered extracellular matrix and mechanotransduction gene expression in rat bone tissue following long-term estrogen deficiency.

Osteoporosis is primarily associated with bone loss, but changes in bone tissue matrix composition and osteocyte mechanotransduction have also been identified. However, the molecular mechanisms underlying these changes and their relation to bone loss are not fully understood. The objectives of this study were to (1) conduct comprehensive temporal gene expression analyses on cortical bone tissue from ovariectomized rats, with a specific focus on genes known to govern matrix degradation, matrix production, and mechanotransduction, and (2) correlate these findings with bone mass, trabecular and cortical microarchitecture, and mineral and matrix composition. Microarray data revealed 35 differentially expressed genes in the cortical bone tissue of the ovariectomized cohort. We report that catabolic gene expression abates after the initial accelerated bone loss period, which occurs within the first 4 wk of estrogen deficiency. However, in long-term estrogen deficiency, we report increased expression of genes associated with extracellular matrix deposition (Spp1, COL1A1, COL1A2, OCN) and mechanotransduction (Cx43) compared with age-matched controls and short-term estrogen deficiency. These changes coincided with increased heterogeneity of mineral-to-matrix ratio and collagen maturity, to which extracellular matrix markers COL1A1 and COL1A2 were positively correlated. Interestingly, mineral heterogeneity and collagen maturity, exhibited a negative correlation with PHEX and IFT88, associated with mechanosensory cilia formation and Hedgehog (Hh) signaling. This study provides the first insight into the underlying mechanisms governing secondary mineralization and heterogeneity of matrix composition of bone tissue in long-term estrogen deficiency. We propose that altered mechanobiological responses in long-term estrogen deficiency may play a role in these changes.

Macrophage-to-osteocyte communication: Impact in a 3D in vitro implant-associated infection model.

Macrophages and osteocytes are important regulators of inflammation, osteogenesis and osteoclastogenesis. However, their interactions under adverse conditions, such as biomaterial-associated infection (BAI) are not fully understood. We aimed to elucidate how factors released from macrophages modulate osteocyte responses in an in vitro indirect 3D co-culture model. Human monocyte-derived macrophages were cultured on etched titanium disks and activated with either IL-4 cytokine (anti-inflammatory M2 phenotype) or Staphylococcus aureus secreted virulence factors to simulate BAI (pro-inflammatory M1 phenotype). Primary osteocytes in collagen gels were then stimulated with conditioned media (CM) from these macrophages. The osteocyte response was analyzed by gene expression, protein secretion, and immunostaining. M1 phenotype macrophages were confirmed by IL-1ß and TNF-α secretion, and M2 macrophages by ARG-1 and MRC-1.Osteocytes receiving M1 CM revealed bone inhibitory effects, denoted by reduced secretion of bone formation osteocalcin (BGLAP) and increased secretion of the bone inhibitory sclerostin (SOST). These osteocytes also downregulated the pro-mineralization gene PHEX and upregulated the anti-mineralization gene MEPE. Additionally, exhibited pro-osteoclastic potential by upregulating pro-osteoclastic gene RANKL expression. Nonetheless, M1-stimulated osteocytes expressed a higher level of the potent pro-osteogenic factor BMP-2 in parallel with the downregulation of the bone inhibitor genes DKK1 and SOST, suggesting a compensatory feedback mechanisms. Conversely, M2-stimulated osteocytes mainly upregulated anti-osteoclastic gene OPG expression, suggesting an anti-catabolic effect. Altogether, our findings demonstrate a strong communication between M1 macrophages and osteocytes under M1 (BAI)-simulated conditions, suggesting that the BAI adverse effects on osteoblastic and osteoclastic processes in vitro are partly mediated via this communication. STATEMENT OF SIGNIFICANCE: Biomaterial-associated infections are major challenges and the underlying mechanisms in the cellular interactions are missing, especially among the major cells from the inflammatory side (macrophages as the key cell in bacterial clearance) and the regenerative side (osteocyte as main regulator of bone). We evaluated the effect of macrophage polarization driven by the stimulation with bacterial virulence factors on the osteocyte function using an indirect co-culture model, hence mimicking the scenario of a biomaterial-associated infection. The results suggest that at least part of the adverse effects of biomaterial associated infection on osteoblastic and osteoclastic processes in vitro are mediated via macrophage-to-osteocyte communication.

[Effect and mechanism of Gusong Qianggu Decoction on reducing bone loss in ovariectomized mice by targeting endoplasmic reticulum stress-induced apoptosis of osteocytes].

This study aims to investigate the role and mechanism of Gusong Qianggu Decoction(GSQG) in attenuating bone loss in ovariectomized mice by targeting the endoplasmic reticulum stress(ERS)-induced apoptosis of osteocytes. After the modeling of osteoporosis in mice with bilateral ovary removal(OVX), 60 mice were randomized by the random number method into six groups: sham,model, low-, medium-, and high-dose GSQG(GSQG-L, GSQG-M, and GSQG-H, respectively), and estradiol(E_2), with 10 mice in each group. The mice in each group were administrated with corresponding drugs by gavage one month after surgery and the administration lasted for 3 months. Enzyme-linked immunosorbent assay(ELISA) was employed to determine the serum levels of osteocalcin(OCN), procollagen type Ⅰ N-terminal propeptide(PINP), carboxy-terminal cross-linked telopeptide of type Ⅰ collagen(CTX),and anti-tartarte acid phosphatase 5b(TRAcP-5b). Micro-CT was employed to observe the changes in bone microstructure of the distal femur. Hematoxylin-eosin(HE) staining was employed to observe the morphology of the bone tissue. RT-qPCR was conducted to determine the m RNA levels of tibial stem osteogenesis-associated genes [type Ⅰ collagen(Col-Ⅰ), alkaline phosphatase(ALP), Runtrelated transcription factor-2(Runx2), bone sialoprotein(BSP), and OCN] and bone-breaking related genes [tartrate-resistant acid phosphatase(TRAP), nuclear factor-activated T cell 1(NFATc1), and cathepsin K(CATK)]. TUNEL staining and immunohistochemistry were employed to detect the apoptosis of osteoblasts. Western blot was employed to measure the expression of ERS-related proteins glucose-regulated protein 78( Grp78), protein kinase RNA-like endoplasmic reticulum kinase( PERK), phosphorylated PERK(p-PERK),eukaryotic translation initiation factor 2 alpha(eIF2α), phosphorylated e IF2α(p-eIF2α), inositol-requiring enzyme 1 alpha(IRE1α), phosphorylated IRE1α(p-IRE1α), and activating transcription factor 6(ATF6) in the proximal tibial bone tissue. The results showed that GSQG significantly recovered the levels of OCN, PINP, TRAc P-5b, and CTX in the serum of ovariectomized mice, and Micro-CT showed that GSQG improved the bone microstructure of distal femur in a dose-dependent manner. Compared with the model group, GSQG widened and increased the bone trabeculae, restored the reticular structure with neat arrangement and enlarged interstitial gaps, and reduced the number of TUNEL-positive cells(P<0. 05, P<0. 01). Furthermore, GSQG down-regulated the expression levels of cysteine aspartate protease-3( caspase-3) and factor Bcl-2-associated X protein( Bax)(P< 0. 05,P<0. 01) and up-regulated the expression level of Bcl-2(P<0. 05, P<0. 01). The GSQG groups showed up-regulated m RNA levels of Col-Ⅰ, ALP, Runx2, BSP, and OCN(P< 0. 01) and down-regulated m RNA levels of TRAP, NFATc1, and CATK(P< 0. 05,P<0. 01). In addition, GSQG, especially GSQG-H, down-regulated the protein levels of Grp78, p-PERK, p-eIF2, p-IRE1α, and ATF6(P< 0. 05, P< 0. 01). In conclusion, GSQG can inhibit the apoptosis of osteocytes by inhibiting the Grp78/PERK/e IF2α/IRE1α/ATF6 signaling pathway in the proximal tibia tissue, thus reducing bone loss in ovariectomized mice.

A 3D bioreactor model to study osteocyte differentiation and mechanobiology under perfusion and compressive mechanical loading.

Osteocytes perceive and process mechanical stimuli in the lacuno-canalicular network in bone. As a result, they secrete signaling molecules that mediate bone formation and resorption. To date, few three-dimensional (3D) models exist to study the response of mature osteocytes to biophysical stimuli that mimic fluid shear stress and substrate strain in a mineralized, biomimetic bone-like environment. Here we established a biomimetic 3D bone model by utilizing a state-of-art perfusion bioreactor platform where immortomouse/Dmp1-GFP-derived osteoblastic IDG-SW3 cells were differentiated into mature osteocytes. We evaluated proliferation and differentiation properties of the cells on 3D microporous scaffolds of decellularized bone (dBone), poly(L-lactide-co-trimethylene carbonate) lactide (LTMC), and beta-tricalcium phosphate (ß-TCP) under physiological fluid flow conditions over 21 days. Osteocyte viability and proliferation were similar on the scaffolds with equal distribution of IDG-SW3 cells on dBone and LTMC scaffolds. After seven days, the differentiation marker alkaline phosphatase (Alpl), dentin matrix acidic phosphoprotein 1 (Dmp1), and sclerostin (Sost) were significantly upregulated in IDG-SW3 cells (p = 0.05) on LTMC scaffolds under fluid flow conditions at 1.7 ml/min, indicating rapid and efficient maturation into osteocytes. Osteocytes responded by inducing the mechanoresponsive genes FBJ osteosarcoma oncogene (Fos) and prostaglandin-endoperoxide synthase 2 (Ptgs2) under perfusion and dynamic compressive loading at 1 Hz with 5 % strain. Together, we successfully created a 3D biomimetic platform as a robust tool to evaluate osteocyte differentiation and mechanobiology in vitro while recapitulating in vivo mechanical cues such as fluid flow within the lacuno-canalicular network. STATEMENT OF SIGNIFICANCE: This study highlights the importance of creating a three-dimensional (3D) in vitro model to study osteocyte differentiation and mechanobiology, as cellular functions are limited in two-dimensional (2D) models lacking in vivo tissue organization. By using a perfusion bioreactor platform, physiological conditions of fluid flow and compressive loading were mimicked to which osteocytes are exposed in vivo. Microporous poly(L-lactide-co-trimethylene carbonate) lactide (LTMC) scaffolds in 3D are identified as a valuable tool to create a favorable environment for osteocyte differentiation and to enable mechanical stimulation of osteocytes by perfusion and compressive loading. The LTMC platform imitates the mechanical bone environment of osteocytes, allowing the analysis of the interaction with other cell types in bone under in vivo biophysical stimuli.

Standardized Testing for Thermal Evaluation of Bone Drilling: Towards Predictive Assessment of Thermal Trauma.

To ensure the prevention of thermal trauma and tissue necrosis during bone drilling in surgical procedures, it is crucial to maintain temperatures below the time- and temperature-dependent threshold of 50 °C for 30 s. However, the absence of a current standard for assessing temperatures attained during bone drilling poses a challenge when comparing findings across different studies. This article aims to address this issue by introducing a standardized testing method for acquiring thermal data during experimental bone drilling. The method requires the use of three controlled variables: infrared thermography, standard bone blocks, and a regulated drilling procedure involving a drill press with irrigation that simulates a surgeon. By utilizing this setup, we can obtain temperature data that can be effectively applied in the evaluation of other variables, such as surgical techniques or drill bit design, and translate the data into bone damage/clinical outcomes. Two surgical drill bits (2.0 mm-diameter twist drill bit and 3.3 mm-diameter multi-step drill bit) are compared using this experimental protocol. The results show the 2.0 mm bit reached significantly higher temperatures compared to the 3.3 mm bit when preparing an osteotomy (p < 0.05). The 2.0 mm drill bit reached temperatures over 100 °C while the 3.3 mm drill bit did not exceed 50 °C.

Fluid shear stress-mediated Piezo1 alleviates osteocyte apoptosis by activating the PI3K/Akt pathway.

Glucocorticoid-induced osteoporosis serves as a primary cause for secondary osteoporosis and fragility fractures, representing the most prevalent adverse reaction associated with prolonged glucocorticoid use. In this study, to elucidate the impact and underlying mechanisms of fluid shear stress (FSS)-mediated Piezo1 on dexamethasone (Dex)-induced apoptosis, we respectively applied Dex treatment for 6 h, FSS at 9 dyne/cm2 for 30 min, Yoda1 treatment for 2 h, and Piezo1 siRNA transfection to intervene in MLO-Y4 osteocytes. Western blot analysis was used to assess the expression of Cleaved Caspase-3, Bax, Bcl-2, and proteins associated with the PI3K/Akt pathway. Additionally, qRT-PCR was utilized to quantify the mRNA expression levels of these molecules. Hoechst 33258 staining and flow cytometry were utilized to evaluate the apoptosis levels. The results indicate that FSS at 9 dyne/cm2 for 30 min significantly upregulates Piezo1 in osteocytes. Following Dex-induced apoptosis, the phosphorylation levels of PI3K and Akt are markedly suppressed. FSS-mediated Piezo1 exerts a protective effect against Dex-induced apoptosis by activating the PI3K/Akt pathway. Additionally, downregulating the expression of Piezo1 in osteocytes using siRNA exacerbates Dex-induced apoptosis. To further demonstrate the role of the PI3K/Akt signaling pathway, after intervention with the PI3K pathway inhibitor, the activation of the PI3K/Akt pathway by FSS-mediated Piezo1 in osteocytes was significantly inhibited, reversing the anti-apoptotic effect. This study indicates that under FSS, Piezo1 in MLO-Y4 osteocytes is significantly upregulated, providing protection against Dex-induced apoptosis through the activation of the PI3K/Akt pathway.

PPARG in osteocytes controls cell bioenergetics and systemic energy metabolism independently of sclerostin levels in circulation.

OBJECTIVE: The skeleton is one of the largest organs in the body, wherein metabolism is integrated with systemic energy metabolism. However, the bioenergetic programming of osteocytes, the most abundant bone cells coordinating bone metabolism, is not well defined. Here, using a mouse model with partial penetration of an osteocyte-specific PPARG deletion, we demonstrate that PPARG controls osteocyte bioenergetics and their contribution to systemic energy metabolism independently of circulating sclerostin levels, which were previously correlated with metabolic status of extramedullary fat depots. METHODS: In vivo and in vitro models of osteocyte-specific PPARG deletion, i.e. Dmp1CrePparγflfl male and female mice (γOTKO) and MLO-Y4 osteocyte-like cells with either siRNA-silenced or CRISPR/Cas9-edited Pparγ. As applicable, the models were analyzed for levels of energy metabolism, glucose metabolism, and metabolic profile of extramedullary adipose tissue, as well as the osteocyte transcriptome, mitochondrial function, bioenergetics, insulin signaling, and oxidative stress. RESULTS: Circulating sclerostin levels of γOTKO male and female mice were not different from control mice. Male γOTKO mice exhibited a high energy phenotype characterized by increased respiration, heat production, locomotion and food intake. This high energy phenotype in males did not correlate with "beiging" of peripheral adipose depots. However, both sexes showed a trend for reduced fat mass and apparent insulin resistance without changes in glucose tolerance, which correlated with decreased osteocytic responsiveness to insulin measured by AKT activation. The transcriptome of osteocytes isolated from γOTKO males suggested profound changes in cellular metabolism, fuel transport, mitochondria dysfunction, insulin signaling and increased oxidative stress. In MLO-Y4 osteocytes, PPARG deficiency correlated with highly active mitochondria, increased ATP production, and accumulation of reactive oxygen species (ROS). CONCLUSIONS: PPARG in male osteocytes acts as a molecular break on mitochondrial function, and protection against oxidative stress and ROS accumulation. It also regulates osteocyte insulin signaling and fuel usage to produce energy. These data provide insight into the connection between osteocyte bioenergetics and their sex-specific contribution to the balance of systemic energy metabolism. These findings support the concept that the skeleton controls systemic energy expenditure via osteocyte metabolism.

Exogenous iron caused osteocyte apoptosis, increased RANKL production, and stimulated bone resorption through oxidative stress in a murine model.

Iron overload is a risk factor for osteoporosis due to its oxidative toxicity. Previous studies have demonstrated that an excessive amount of iron increases osteocyte apoptosis and receptor activator of nuclear factor κ-B ligand (RANKL) production, which stimulates osteoclast differentiation in vitro. However, the effects of exogenous iron supplementation-induced iron overload on osteocytes in vivo and its role in iron overload-induced bone loss are unknown. This work aimed to develop an iron overloaded murine model of C57BL/6 mice by intraperitoneal administration of iron dextran for two months. The iron levels in various organs, bone, and serum, as well as the microstructure and strength of bone, apoptosis of osteocytes, oxidative stress in bone tissue, and bone formation and resorption, were assessed. The results showed that 2 months of exogenous iron supplementation significantly increased iron levels in the liver, spleen, kidney, bone tissue, and serum. Iron overload negatively affected bone microstructure and strength. Osteocyte apoptosis and empty lacunae rate were elevated by exogenous iron. Iron overload upregulated RANKL expression but had no significant impact on osteoprotegerin (OPG) and sclerostin levels. Static and dynamic histologic analyses and serum biochemical assay showed that iron overload increased bone resorption without significantly affecting bone formation. Exogenous iron promoted oxidative stress in osteocytes in vivo and in vitro. Additional supplementation of iron chelator (deferoxamine) or N-acetyl-l-cysteine (NAC) partially alleviated bone loss, osteocyte apoptosis, osteoclast formation, and oxidative stress due to iron overload. These findings, in line with prior in vitro studies, suggest that exogenous iron supplementation induces osteoclastogenesis and osteoporosis by promoting osteocyte apoptosis and RANKL production via oxidative stress.

Part II: A new perspective for modeling the bone remodeling process: Biology, mechanics, and pathologies.

In this paper, we explore the effects of biological (pathological) and mechanical damage on bone tissue within a benchmark model. Using the Finite Element Methodology, we analyze and numerically test the model's components, capabilities, and performance under physiologically and pathologically relevant conditions. Our findings demonstrate the model's effectiveness in simulating bone remodeling processes and self-repair mechanisms for micro-damage induced by biological internal conditions and mechanical external ones within bone tissue. This article is the second part of a series, where the first part presented the mathematical model and the biological and physical significance of the terms used in a simplified benchmark model. It explored the bone remodeling model's application, implementation, and results under physiological conditions.

Cell type-specific network analysis in Diversity Outbred mice identifies genes potentially responsible for human bone mineral density GWAS associations.

Genome-wide association studies (GWASs) have identified many sources of genetic variation associated with bone mineral density (BMD), a clinical predictor of fracture risk and osteoporosis. Aside from the identification of causal genes, other difficult challenges to informing GWAS include characterizing the roles of predicted causal genes in disease and providing additional functional context, such as the cell type predictions or biological pathways in which causal genes operate. Leveraging single-cell transcriptomics (scRNA-seq) can assist in informing BMD GWAS by linking disease-associated variants to genes and providing a cell type context for which these causal genes drive disease. Here, we use large-scale scRNA-seq data from bone marrow-derived stromal cells cultured under osteogenic conditions (BMSC-OBs) from Diversity Outbred (DO) mice to generate cell type-specific networks and contextualize BMD GWAS-implicated genes. Using trajectories inferred from the scRNA-seq data, we identify networks enriched with genes that exhibit the most dynamic changes in expression across trajectories. We discover 21 network driver genes, which are likely to be causal for human BMD GWAS associations that colocalize with expression/splicing quantitative trait loci (eQTL/sQTL). These driver genes, including Fgfrl1 and Tpx2, along with their associated networks, are predicted to be novel regulators of BMD via their roles in the differentiation of mesenchymal lineage cells. In this work, we showcase the use of single-cell transcriptomics from mouse bone-relevant cells to inform human BMD GWAS and prioritize genetic targets with potential causal roles in the development of osteoporosis.

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

OBJECTIVES: 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. MATERIALS AND METHODS: 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. RESULTS: 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). CONCLUSION: 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.

The Expression of a Subset of Aging and Antiaging Markers Following the Chondrogenic and Osteogenic Differentiation of Mesenchymal Stem Cells of Placental Origin.

Mesenchymal stem cells (MSCs) of placental origin hold great promise in tissue engineering and regenerative medicine for diseases affecting cartilage and bone. However, their utility has been limited by their tendency to undergo premature senescence and phenotypic drift into adipocytes. This study aimed to explore the potential involvement of a specific subset of aging and antiaging genes by measuring their expression prior to and following in vitro-induced differentiation of placental MSCs into chondrocytes and osteoblasts as opposed to adipocytes. The targeted genes of interest included the various LMNA/C transcript variants (lamin A, lamin C, and lamin A∆10), sirtuin 7 (SIRT7), and SM22α, along with the classic aging markers plasminogen activator inhibitor 1 (PAI-1), p53, and p16INK4a. MSCs were isolated from the decidua basalis of human term placentas, expanded, and then analyzed for phenotypic properties by flow cytometry and evaluated for colony-forming efficiency. The cells were then induced to differentiate in vitro into chondrocytes, osteocytes, and adipocytes following established protocols. The mRNA expression of the targeted genes was measured by RT-qPCR in the undifferentiated cells and those fully differentiated into the three cellular lineages. Compared to undifferentiated cells, the differentiated chondrocytes demonstrated decreased expression of SIRT7, along with decreased PAI-1, lamin A, and SM22α expression, but the expression of p16INK4a and p53 increased, suggesting their tendency to undergo premature senescence. Interestingly, the cells maintained the expression of lamin C, which indicates that it is the primary lamin variant influencing the mechanoelastic properties of the differentiated cells. Notably, the expression of all targeted genes did not differ from the undifferentiated cells following osteogenic differentiation. On the other hand, the differentiation of the cells into adipocytes was associated with decreased expression of lamin A and PAI-1. The distinct patterns of expression of aging and antiaging genes following in vitro-induced differentiation of MSCs into chondrocytes, osteocytes, and adipocytes potentially reflect specific roles for these genes during and following differentiation in the fully functional cells. Understanding these roles and the network of signaling molecules involved can open opportunities to improve the handling and utility of MSCs as cellular precursors for the treatment of cartilage and bone diseases.

A role for sirtuin 1 in FGF23 activation following ß-glycerophosphate treatment.

Phosphate homeostasis is vital for many biological processes and disruptions in circulating levels can be detrimental. While the mechanisms behind FGF23 regulation have been regularly studied, the role of extracellular phosphate sensing and its impact on fibroblast growth factor 23 (FGF23) expression remains unclear. This study aimed to investigate the involvement of reactive oxygen species (ROS), silent information regulator 1 (SIRT1), and Hairy and Enhancer of Split-1 (HES1) in regulating FGF23 in FGF23 expressing MC3T3-E1 cells. MC3T3-E1 cells treated with ß-glycerophosphate (BGP) resulted in increased Fgf23 expression. Inhibition of ROS formation by inhibition of NADPH oxidase, which is essential for ROS production, did not affect this response to BGP, suggesting ROS is not involved in this process. Moreover, treatment with tert-butyl hydroperoxide (TBHP), a ROS-inducing agent, did not increase Fgf23 expression. This suggests that ROS machinery is not involved in FGF23 stimulation as previously suggested. Nonetheless, inhibition of SIRT1 using Ex527 eliminated the Fgf23 response to BGP, indicating its involvement in FGF23 regulation after BGP treatment. Indeed, activation of SIRT1 using SRT1720 increased Fgf23 expression. Moreover, transcription factor Hes1 was upregulated by BGP treatment, which was diminished when cells were treated with Ex527 implying it is also regulated through SIRT1. These findings suggest the existence of an upstream SIRT1-HES1 axis in the regulation of FGF23 by phosphate, though we were unable to find a role for ROS in this process. Further research should provide insights into phosphate homeostasis and potential therapeutic targets for phosphate-related disorders.

Osteoporosis treatments for intervertebral disc degeneration and back pain: a perspective.

Low back pain derived from intervertebral disc (IVD) degeneration is a debilitating spinal condition that, despite its prevalence, does not have any intermediary guidelines for pharmacological treatment between palliative care and invasive surgery. The development of treatments for the IVD is complicated by the variety of resident cell types needed to maintain the regionally distinct structural properties of the IVD that permit the safe, complex motions of the spine. Osteoporosis of the spine increases the risk of vertebral bone fracture that can increase the incidence of back pain. Fortunately, there are a variety of pharmacological treatments for osteoporosis that target osteoblasts, osteoclasts and/or osteocytes to build bone and prevent vertebral fracture. Of particular note, clinical and preclinical studies suggest that commonly prescribed osteoporosis drugs like bisphosphonates, intermittent parathyroid hormone, anti-sclerostin antibody, selective estrogen receptor modulators and anti-receptor activator of nuclear factor-kappa B ligand inhibitor denosumab may also relieve back pain. Here, we cite clinical and preclinical studies and include unpublished data to support the argument that a subset of these therapeutics for osteoporosis may alleviate low back pain by also targeting the IVD.

Swim training induces distinct osseous gene expression patterns in anosteocytic and osteocytic teleost fish.

The traditional understanding of bone mechanosensation implicates osteocytes, canaliculi, and the lacunocanalicular network in biomechanical adaptation. However, recent findings challenge this notion, as shown in advanced teleost fish where anosteocytic bone lacking osteocytes are nevertheless responsive to mechanical load. To investigate specific molecular mechanisms involved in bone mechanoadaptation in osteocytic and anosteocytic fish bone, we conducted a 5-min single swim-training experiment with zebrafish and ricefish, respectively. Through RNASeq analysis of fish spines, analyzed at various time points following swim training, we uncovered distinct gene expression patterns in osteocytic and anosteocytic fish bones. Notably, osteocytic fish bone exhibited an early response to mechanical load, contrasting to a delayed response observed in anosteocytic fish bones, both within 8 h following stimulation. We identified an increase in osteoblast differentiation in anosteocytic bone following training, while chordoblast activity was delayed. This temporal response suggests a time-dependent adaptation in anosteocytic bone, indicating the presence of intricate feedback networks within bone that lacks osteocytes.

Gut microbial alterations in arginine metabolism determine bone mechanical adaptation.

Although mechanical loading is essential for maintaining bone health and combating osteoporosis, its practical application is limited to a large extent by the high variability in bone mechanoresponsiveness. Here, we found that gut microbial depletion promoted a significant reduction in skeletal adaptation to mechanical loading. Among experimental mice, we observed differences between those with high and low responses to exercise with respect to the gut microbial composition, in which the differential abundance of Lachnospiraceae contributed to the differences in bone mechanoresponsiveness. Microbial production of L-citrulline and its conversion into L-arginine were identified as key regulators of bone mechanoadaptation, and administration of these metabolites enhanced bone mechanoresponsiveness in normal, aged, and ovariectomized mice. Mechanistically, L-arginine-mediated enhancement of bone mechanoadaptation was primarily attributable to the activation of a nitric-oxide-calcium positive feedback loop in osteocytes. This study identifies a promising anti-osteoporotic strategy for maximizing mechanical loading-induced skeletal benefits via the microbiota-metabolite axis.

Osteocytes/Osteoblasts Produce SAA3 to Regulate Hepatic Metabolism of Cholesterol.

Hypercholesterolaemia is a systemic metabolic disease, but the role of organs other than liver in cholesterol metabolism is unappreciated. The phenotypic characterization of the Tsc1Dmp1 mice reveal that genetic depletion of tuberous sclerosis complex 1 (TSC1) in osteocytes/osteoblasts (Dmp1-Cre) triggers progressive increase in serum cholesterol level. The resulting cholesterol metabolic dysregulation is shown to be associated with upregulation and elevation of serum amyloid A3 (SAA3), a lipid metabolism related factor, in the bone and serum respectively. SAA3, elicited from the bone, bound to toll-like receptor 4 (TLR4) on hepatocytes to phosphorylate c-Jun, and caused impeded conversion of cholesterol to bile acids via suppression on cholesterol 7 α-hydroxylase (Cyp7a1) expression. Ablation of Saa3 in Tsc1Dmp1 mice prevented the CYP7A1 reduction in liver and cholesterol elevation in serum. These results expand the understanding of bone function and hepatic regulation of cholesterol metabolism and uncover a potential therapeutic use of pharmacological modulation of SAA3 in hypercholesterolaemia.

Single-cell Transcriptome Analysis Identifies Senescent Osteocytes as Contributors to Bone Destruction in Breast Cancer Metastasis.

Breast cancer bone metastases increase fracture risk and are a major cause of morbidity and mortality among women. Upon colonization by tumor cells, the bone microenvironment undergoes profound reprogramming to support cancer progression that disrupts the balance between osteoclasts and osteoblasts, leading to bone lesions. Whether such reprogramming affects matrix-embedded osteocytes remains poorly understood. Here, we demonstrate that osteocytes in breast cancer bone metastasis develop premature senescence and a distinctive senescence-associated secretory phenotype (SASP) that favors bone destruction. Single-cell RNA sequencing identified osteocytes from mice with breast cancer bone metastasis enriched in senescence and SASP markers and pro-osteoclastogenic genes. Using multiplex in situ hybridization and AI-assisted analysis, we detected osteocytes with senescence-associated distension of satellites, telomere dysfunction, and p16Ink4a expression in mice and patients with breast cancer bone metastasis. In vitro and ex vivo organ cultures showed that breast cancer cells promote osteocyte senescence and enhance their osteoclastogenic potential. Clearance of senescent cells with senolytics suppressed bone resorption and preserved bone mass in mice with breast cancer bone metastasis. These results demonstrate that osteocytes undergo pathological reprogramming by breast cancer cells and identify osteocyte senescence as an initiating event triggering bone destruction in breast cancer metastases.

Multi-scale cortical bone traits vary in females and males from two mouse models of genetic diversity.

Understanding the genetic basis of cortical bone traits can allow for the discovery of novel genes or biological pathways regulating bone health. Mice are the most widely used mammalian model for skeletal biology and allow for the quantification of traits that cannot easily be evaluated in humans, such as osteocyte lacunar morphology. The goal of our study was to investigate the effect of genetic diversity on multi-scale cortical bone traits of 3 long bones in skeletally-mature mice. We measured bone morphology, mechanical properties, material properties, lacunar morphology, and mineral composition of mouse bones from 2 populations of genetic diversity. Additionally, we compared how intrabone relationships varied in the 2 populations. Our first population of genetic diversity included 72 females and 72 males from the 8 inbred founder strains used to create the Diversity Outbred (DO) population. These 8 strains together span almost 90% of the genetic diversity found in mice (Mus musculus). Our second population of genetic diversity included 25 genetically unique, outbred females and 25 males from the DO population. We show that multi-scale cortical bone traits vary significantly with genetic background; heritability values range from 21% to 99% indicating genetic control of bone traits across length scales. We show for the first time that lacunar shape and number are highly heritable. Comparing the 2 populations of genetic diversity, we show that each DO mouse does not resemble a single inbred founder, but instead the outbred mice display hybrid phenotypes with the elimination of extreme values. Additionally, intrabone relationships (eg, ultimate force vs. cortical area) were mainly conserved in our 2 populations. Overall, this work supports future use of these genetically diverse populations to discover novel genes contributing to cortical bone traits, especially at the lacunar length scale.