Osteocyte-derived exosomes regulate the DLX2/wnt pathway to alleviate osteoarthritis by mediating cartilage repair.

BACKGROUND: Chondrocyte viability, apoptosis, and migration are closely related to cartilage injury in osteoarthritis (OA) joints. Exosomes are identified as potential therapeutic agents for OA. OBJECTIVE: This study aimed to investigate the role of exosomes derived from osteocytes in OA, particularly focusing on their effects on cartilage repair and molecular mechanisms. METHODS: An injury cell model was established by treating chondrocytes with IL-1ß. Cartilage repair was evaluated using cell counting kit-8, flow cytometry, scratch test, and Western Blot. Molecular mechanisms were analyzed using quantitative real-time PCR, bioinformatic analysis, and Western Blot. An OA mouse model was established to explore the role of exosomal DLX2 in vivo. RESULTS: Osteocyte-released exosomes promoted cell viability and migration, and inhibited apoptosis and extracellular matrix (ECM) deposition. Moreover, exosomes upregulated DLX2 expression, and knockdown of DLX2 activated the Wnt pathway. Additionally, exosomes attenuated OA in mice by transmitting DLX2. CONCLUSION: Osteocyte-derived exosomal DLX2 alleviated IL-1ß-induced cartilage repair and inactivated the Wnt pathway, thereby alleviating OA progression. The findings suggested that osteocyte-derived exosomes may hold promise as a treatment for OA.

BIGH3 mediates apoptosis and gap junction failure in osteocytes during renal cell carcinoma bone metastasis progression.

Renal cell carcinoma (RCC) bone metastatis progression is driven by crosstalk between tumor cells and the bone microenvironment, which includes osteoblasts, osteoclasts, and osteocytes. RCC bone metastases (RCCBM) are predominantly osteolytic and resistant to antiresorptive therapy. The molecular mechanisms underlying pathologic osteolysis and disruption of bone homeostasis remain incompletely understood. We previously reported that BIGH3/TGFBI (transforming growth factor-beta-induced protein ig-h3, shortened to BIGH3 henceforth) secreted by colonizing RCC cells drives osteolysis by inhibiting osteoblast differentiation, impairing healing of osteolytic lesions, which is reversible with osteoanabolic agents. Here, we report that BIGH3 induces osteocyte apoptosis in both human RCCBM tissue specimens and in a preclinical mouse model. We also demonstrate that BIGH3 reduces Cx43 expression, blocking gap junction (GJ) function and osteocyte network communication. BIGH3-mediated GJ inhibition is blocked by the lysosomal inhibitor hydroxychloroquine (HCQ), but not osteoanabolic agents. Our results broaden the understanding of pathologic osteolysis in RCCBM and indicate that targeting the BIGH3 mechanism could be a combinational strategy for the treatment of RCCBM-induced bone disease that overcomes the limited efficacy of antiresorptives that target osteoclasts.

Spatial control of perilacunar canalicular remodeling during lactation.

Osteocytes locally remodel their surrounding tissue through perilacunar canalicular remodeling (PLR). During lactation, osteocytes remove minerals to satisfy the metabolic demand, resulting in increased lacunar volume, quantifiable with synchrotron X-ray radiation micro-tomography (SRµCT). Although the effects of lactation on PLR are well-studied, it remains unclear whether PLR occurs uniformly throughout the bone and what mechanisms prevent PLR from undermining bone quality. We used SRµCT imaging to conduct an in-depth spatial analysis of the impact of lactation and osteocyte-intrinsic MMP13 deletion on PLR in murine bone. We found larger lacunae undergoing PLR are located near canals in the mid-cortex or endosteum. We show lactation-induced hypomineralization occurs 14 µm away from lacunar edges, past a hypermineralized barrier. Our findings reveal that osteocyte-intrinsic MMP13 is crucial for lactation-induced PLR near lacunae in the mid-cortex but not for whole-bone resorption. This research highlights the spatial control of PLR on mineral distribution during lactation.

Prkd1 regulates the formation and repair of plasma membrane disruptions (PMD) in osteocytes.

We and others have seen that osteocytes sense high-impact osteogenic mechanical loading via transient plasma membrane disruptions (PMDs) which initiate downstream mechanotransduction. However, a PMD must be repaired for the cell to survive this wounding event. Previous work suggested that the protein Prkd1 (also known as PKCµ) may be a critical component of this PMD repair process, but the specific role of Prkd1 in osteocyte mechanobiology had not yet been tested. We treated MLO-Y4 osteocytes with Prkd1 inhibitors (Go6976, kbNB 142-70, staurosporine) and generated an osteocyte-targeted (Dmp1-Cre) Prkd1 conditional knockout (CKO) mouse. PMD repair rate was measured via laser wounding and FM1-43 dye uptake, PMD formation and post-wounding survival were assessed via fluid flow shear stress (50 dyn/cm2), and in vitro osteocyte mechanotransduction was assessed via measurement of calcium signaling. To test the role of osteocyte Prkd1 in vivo, Prkd1 CKO and their wildtype (WT) littermates were subjected to 2 weeks of unilateral axial tibial loading and loading-induced changes in cortical bone mineral density, geometry, and formation were measured. Prkd1 inhibition or genetic deletion slowed osteocyte PMD repair rate and impaired post-wounding cell survival. These effects could largely be rescued by treating osteocytes with the FDA-approved synthetic copolymer Poloxamer 188 (P188), which was previously shown to facilitate membrane resealing and improve efficiency in the repair rate of PMD in skeletal muscle myocytes. In vivo, while both WT and Prkd1 CKO mice demonstrated anabolic responses to tibial loading, the magnitude of loading-induced increases in tibial BMD, cortical thickness, and periosteal mineralizing surface were blunted in Prkd1 CKO as compared to WT mice. Prkd1 CKO mice also tended to show a smaller relative difference in the number of osteocyte PMD in loaded limbs and showed greater lacunar vacancy, suggestive of impaired post-wounding osteocyte survival. While P188 treatment rescued loading-induced increases in BMD in the Prkd1 CKO mice, it surprisingly further suppressed loading-induced increases in cortical bone thickness and cortical bone formation. Taken together, these data suggest that Prkd1 may play a pivotal role in the regulation and repair of the PMD response in osteocytes and support the idea that PMD repair processes can be pharmacologically targeted to modulate downstream responses, but suggest limited utility of PMD repair-promoting P188 in improving bone anabolic responses to loading.

Staphylococcus aureus persistence in osteocytes: weathering the storm of antibiotics and autophagy/xenophagy.

Staphylococcus aureus is a major causative pathogen of osteomyelitis. Intracellular infections of resident bone cells including osteocytes can persist despite gold-standard clinical intervention. The mechanisms by which intracellular S. aureus evades antibiotic therapy are unknown. In this study, we utilised an in vitro S. aureus infection model of human osteocytes to investigate whether antibiotic-mediated dysregulation of autophagy contributes to this phenomenon. Infected or non-infected osteocyte-like cells were exposed to combinations of rifampicin, vancomycin, and modulators of autophagy. Intracellular bacterial growth characteristics were assessed using colony-forming unit (CFU) analysis, viable bacterial DNA abundance, and the rate of escape into antibiotic-free medium, together with measures of autophagic flux. Rifampicin, alone or in combination with vancomycin, caused a rapid decrease in the culturability of intracellular bacteria, concomitant with stable or increased absolute bacterial DNA levels. Both antibiotics significantly inhibited autophagic flux. However, modulation of autophagic flux did not affect viable bacterial DNA levels. In summary, autophagy was shown to be a factor in the host-pathogen relationship in this model, as its modulation affected the growth state of intracellular S. aureus with respect to both their culturability and propensity to escape the intracellular niche. While rifampicin and vancomycin treatments moderately suppressed autophagic flux acutely, this did not explain the paradoxical response of antibiotic treatment in decreasing S. aureus culturability whilst failing to clear bacterial DNA and hence intracellular bacterial load. Thus, off-target effects of rifampicin and vancomycin on autophagic flux in osteocyte-like cells could not explain the persistent S. aureus infection in these cells.

Local E-rhBMP-2/ß-TCP Application Rescues Osteocyte Dendritic Integrity and Reduces Microstructural Damage in Alveolar Bone Post-Extraction in MRONJ-like Mouse Model.

The pathology of medication-related osteonecrosis of the jaw (MRONJ), often associated with antiresorptive therapy, is still not fully understood. Osteocyte networks are known to play a critical role in maintaining bone homeostasis and repair, but the exact condition of these networks in MRONJ is unknown. On the other hand, the local application of E-coli-derived Recombinant Human Bone Morphogenetic Protein 2/ß-Tricalcium phosphate (E-rhBMP-2/ß-TCP) has been shown to promote bone regeneration and mitigate osteonecrosis in MRONJ-like mouse models, indicating its potential therapeutic application for the treatment of MRONJ. However, the detailed effect of BMP-2 treatment on restoring bone integrity, including its osteocyte network, in an MRONJ condition remains unclear. Therefore, in the present study, by applying a scanning electron microscope (SEM) analysis and a 3D osteocyte network reconstruction workflow on the alveolar bone surrounding the tooth extraction socket of an MRONJ-like mouse model, we examined the effectiveness of BMP-2/ß-TCP therapy on the alleviation of MRONJ-related bone necrosis with a particular focus on the osteocyte network and alveolar bone microstructure (microcrack accumulation). The 3D osteocyte dendritic analysis showed a significant decrease in osteocyte dendritic parameters along with a delay in bone remodeling in the MRONJ group compared to the healthy counterpart. The SEM analysis also revealed a notable increase in the number of microcracks in the alveolar bone surface in the MRONJ group compared to the healthy group. In contrast, all of those parameters were restored in the E-rhBMP-2/ß-TCP-treated group to levels that were almost similar to those in the healthy group. In summary, our study reveals that MRONJ induces osteocyte network degradation and microcrack accumulation, while application of E-rhBMP-2/ß-TCP can restore a compromised osteocyte network and abrogate microcrack accumulation in MRONJ.

Aging decreases osteocyte peri-lacunar-canalicular system turnover in female C57BL/6JN mice.

Osteocytes engage in bone resorption and mineralization surrounding their expansive lacunar-canalicular system (LCS) through peri-LCS turnover. However, fundamental questions persist about where, when, and how often osteocytes engage in peri-LCS turnover and how these processes change with aging. Furthermore, whether peri-LCS turnover is associated with natural variation in cortical tissue strain remains unexplored. To address these questions, we utilized confocal scanning microscopy, immunohistochemistry, and scanning electron microscopy to characterize osteocyte peri-LCS turnover in the cortical (mid-diaphysis) and cancellous (metaphysis) regions of femurs from young adult (5 mo) and early-old-age (22 mo) female C57BL/6JN mice. LCS bone mineralization was measured by the presence of perilacunar fluorochrome labels. LCS bone resorption was measured by immunohistochemical marker of bone resorption. The dynamics of peri-LCS turnover were estimated from serial fluorochrome labeling, where each mouse was administered two labels between 2 and 16 days before euthanasia. Osteocyte participation in mineralizing their surroundings is highly abundant in both cortical and cancellous bone of young adult mice but significantly decreases with aging. LCS bone resorption also decreases with aging. Aging has a greater impact on peri-LCS turnover dynamics in cancellous bone than in cortical bone. Lacunae with recent peri-LCS turnover are larger in both age groups. While peri-LCS turnover is associated with variation in tissue strain between cortical quadrants and intracortical location for 22 mo mice, these associations were not seen for 5 mo mice. The impact of aging on decreasing peri-LCS turnover may have significant implications for bone quality and mechanosensation.

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.

ATP6AP2, a regulator of LRP6/ß-catenin protein trafficking, promotes Wnt/ß-catenin signaling and bone formation in a cell type dependent manner.

Wnt/ß-catenin signaling is critical for various cellular processes in multiple cell types, including osteoblast (OB) differentiation and function. Exactly how Wnt/ß-catenin signaling is regulated in OBs remain elusive. ATP6AP2, an accessory subunit of V-ATPase, plays important roles in multiple cell types/organs and multiple signaling pathways. However, little is known whether and how ATP6AP2 in OBs regulates Wnt/ß-catenin signaling and bone formation. Here we provide evidence for ATP6AP2 in the OB-lineage cells to promote OB-mediated bone formation and bone homeostasis selectively in the trabecular bone regions. Conditionally knocking out (CKO) ATP6AP2 in the OB-lineage cells (Atp6ap2Ocn-Cre) reduced trabecular, but not cortical, bone formation and bone mass. Proteomic and cellular biochemical studies revealed that LRP6 and N-cadherin were reduced in ATP6AP2-KO BMSCs and OBs, but not osteocytes. Additional in vitro and in vivo studies revealed impaired ß-catenin signaling in ATP6AP2-KO BMSCs and OBs, but not osteocytes, under both basal and Wnt stimulated conditions, although LRP5 was decreased in ATP6AP2-KO osteocytes, but not BMSCs. Further cell biological studies uncovered that osteoblastic ATP6AP2 is not required for Wnt3a suppression of ß-catenin phosphorylation, but necessary for LRP6/ß-catenin and N-cadherin/ß-catenin protein complex distribution at the cell membrane, thus preventing their degradation. Expression of active ß-catenin diminished the OB differentiation deficit in ATP6AP2-KO BMSCs. Taken together, these results support the view for ATP6AP2 as a critical regulator of both LRP6 and N-cadherin protein trafficking and stability, and thus regulating ß-catenin levels, demonstrating an un-recognized function of osteoblastic ATP6AP2 in promoting Wnt/LRP6/ß-catenin signaling and trabecular bone formation.

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.

Subchondral Bone Osteocyte Lacunae Morphology in End-Stage Osteoarthritis of the Human Tibial Plateau.

Subchondral bone remodeling, mediated by osteocytes within the lacuno-canalicular network, plays a crucial role in osteoarthritis (OA) progression. Following cell death, lacunae preserve integrity, offering insights into bone remodeling mechanisms. Limited and controversial data on osteocyte lacuna morphology in OA result from small sample sizes and two-dimensional (2D) techniques that have been used thus far. This study aimed to quantify three-dimensional (3D) osteocyte lacunar characteristics at well-defined tibial plateau locations, known to be differently affected by OA. Specifically, 11 tibial plateaus were obtained from end-stage knee-OA patients with varus deformity. Each plateau provided one sample from the less affected lateral compartment and two samples from the medial compartment, at minimum and maximum bone volume fraction (BV/TV) locations. High-resolution desktop micro-computed tomography (micro-CT) at 0.7 µm voxel resolution imaged the 33 samples. Lacuna number density (Lc.N/BV) and lacuna volume density (Lc.TV/BV) were significantly lower (p < 0.02) in samples from the medial side with maximum BV/TV compared to lateral side samples. In the medial compartment at maximum local BV/TV, mean lacuna volume (Lc.V), total lacuna volume (Lc.TV), and Lc.TV/BV were significantly (p < 0.001) lower than in the region with minimum BV/TV. Lc.N/BV was also significantly lower (p < 0.02) at the maximum local BV/TV location compared to the region with minimum BV/TV. Our findings suggest that subchondral bone lacunae adapt to the changing loads in end-stage OA.

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

BACKGROUND: Osteocytes 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. RESULTS: A mechanical tensile strain of 2500 µÎµ 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). CONCLUSIONS: In osteocytes, a mechanical tensile strain of 2500 µÎµ 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.

Runx2 and Polycystins in Bone Mechanotransduction: Challenges for Therapeutic Opportunities.

Bone mechanotransduction is a critical process during skeletal development in embryogenesis and organogenesis. At the same time, the type and level of mechanical loading regulates bone remodeling throughout the adult life. The aberrant mechanosensing of bone cells has been implicated in the development and progression of bone loss disorders, but also in the bone-specific aspect of other clinical entities, such as the tumorigenesis of solid organs. Novel treatment options have come into sight that exploit the mechanosensitivity of osteoblasts, osteocytes, and chondrocytes to achieve efficient bone regeneration. In this regard, runt-related transcription factor 2 (Runx2) has emerged as a chief skeletal-specific molecule of differentiation, which is prominent to induction by mechanical stimuli. Polycystins represent a family of mechanosensitive proteins that interact with Runx2 in mechano-induced signaling cascades and foster the regulation of alternative effectors of mechanotransuction. In the present narrative review, we employed a PubMed search to extract the literature concerning Runx2, polycystins, and their association from 2000 to March 2024. The keywords stated below were used for the article search. We discuss recent advances regarding the implication of Runx2 and polycystins in bone remodeling and regeneration and elaborate on the targeting strategies that may potentially be applied for the treatment of patients with bone loss diseases.

Spatial variations in the osteocyte lacuno-canalicular network density and analysis of the connectomic parameters.

Osteocyte lacuno-canalicular network (LCN) is comprised of micrometre-sized pores and submicrometric wide channels in bone. Accumulating evidence suggests multiple functions of this network in material transportation, mechanobiological signalling, mineral homeostasis and bone remodelling. Combining rhodamine staining and confocal laser scanning microscopy, the longitudinal cross-sections of six mouse tibiae were imaged, and the connectome of the network was quantified with a focus on the spatial heterogeneities of network density, connectivity and length of canaliculi. In-vivo loading and double calcein labelling on these tibiae allowed differentiating the newly formed bone from the pre-existing regions. The canalicular density of the murine cortical bone varied between 0.174 and 0.243 µm/µm3, and therefore is three times larger than the corresponding value for human femoral midshaft osteons. The spatial heterogeneity of the network was found distinctly more pronounced across the cortex than along the cortex. We found that in regions with a dense network, the LCN conserves its largely tree-like character, but increases the density by including shorter canaliculi. The current study on healthy mice should serve as a motivating starting point to study the connectome of genetically modified mice, including models of bone diseases and of reduced mechanoresponse.

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.

In vivo study of porous NiTi cryotweezers for bone tissue cryotherapy.

This study examined the effects of liquid nitrogen vapor on osteogenesis in the rabbit femur. Cryotweezers made of porous nickel titanium alloy (nitinol or NiTi) obtained by self-propagating high temperature synthesis were used in this experiment. The porous structure of the cryotweezers allows them to hold up to 10 g of liquid nitrogen after being immersed for 2 min, which completely evaporates after 160 s. To study the effects of liquid nitrogen evaporation on osteogenesis, a rabbit femur was perforated. The formed holes were subjected to cryotherapy with varying exposure times. It was found that a 3 s exposure time stimulates osteogenesis, which was manifested in a greater number of osteoblasts in the regenerate compared to the control sample without liquid nitrogen. It was observed that increasing the exposure to 6, 9 or 12 s had a destructive effect, to varying degrees. The most severe damage was exerted by a 12 s exposure, which resulted in the formation of osteonecrosis areas. In the samples exposed to 6 and 9 s of cryotherapy, destruction of the cytoplasm of osteocytes and osteoclasts was observed.

Perception and response of skeleton to mechanical stress.

Mechanical stress stands as a fundamental factor in the intricate processes governing the growth, development, morphological shaping, and maintenance of skeletal mass. The profound influence of stress in shaping the skeletal framework prompts the assertion that stress essentially births the skeleton. Despite this acknowledgment, the mechanisms by which the skeleton perceives and responds to mechanical stress remain enigmatic. In this comprehensive review, our scrutiny focuses on the structural composition and characteristics of sclerotin, leading us to posit that it serves as the primary structure within the skeleton responsible for bearing and perceiving mechanical stress. Furthermore, we propose that osteocytes within the sclerotin emerge as the principal mechanical-sensitive cells, finely attuned to perceive mechanical stress. And a detailed analysis was conducted on the possible transmission pathways of mechanical stress from the extracellular matrix to the nucleus.

Deletion of FNDC5/irisin modifies murine osteocyte function in a sex-specific manner.

Irisin, released from exercised muscle, has been shown to have beneficial effects on numerous tissues but its effects on bone are unclear. We found significant sex and genotype differences in bone from wildtype (WT) mice compared to mice lacking Fndc5 (knockout [KO]), with and without calcium deficiency. Despite their bone being indistinguishable from WT females, KO female mice were partially protected from osteocytic osteolysis and osteoclastic bone resorption when allowed to lactate or when placed on a low-calcium diet. Male KO mice have more but weaker bone compared to WT males, and when challenged with a low-calcium diet lost more bone than WT males. To begin to understand responsible molecular mechanisms, osteocyte transcriptomics was performed. Osteocytes from WT females had greater expression of genes associated with osteocytic osteolysis and osteoclastic bone resorption compared to WT males which had greater expression of genes associated with steroid and fatty acid metabolism. Few differences were observed between female KO and WT osteocytes, but with a low-calcium diet, the KO females had lower expression of genes responsible for osteocytic osteolysis and osteoclastic resorption than the WT females. Male KO osteocytes had lower expression of genes associated with steroid and fatty acid metabolism, but higher expression of genes associated with bone resorption compared to male WT. In conclusion, irisin plays a critical role in the development of the male but not the female skeleton and protects male but not female bone from calcium deficiency. We propose irisin ensures the survival of offspring by targeting the osteocyte to provide calcium in lactating females, a novel function for this myokine.

Toll-like receptor-2 induced inflammation causes local bone formation and activates canonical Wnt signaling.

It is well established that inflammatory processes in the vicinity of bone often induce osteoclast formation and bone resorption. Effects of inflammatory processes on bone formation are less studied. Therefore, we investigated the effect of locally induced inflammation on bone formation. Toll-like receptor (TLR) 2 agonists LPS from Porphyromonas gingivalis and PAM2 were injected once subcutaneously above mouse calvarial bones. After five days, both agonists induced bone formation mainly at endocranial surfaces. The injection resulted in progressively increased calvarial thickness during 21 days. Excessive new bone formation was mainly observed separated from bone resorption cavities. Anti-RANKL did not affect the increase of bone formation. Inflammation caused increased bone formation rate due to increased mineralizing surfaces as assessed by dynamic histomorphometry. In areas close to new bone formation, an abundance of proliferating cells was observed as well as cells robustly stained for Runx2 and alkaline phosphatase. PAM2 increased the mRNA expression of Lrp5, Lrp6 and Wnt7b, and decreased the expression of Sost and Dkk1. In situ hybridization demonstrated decreased Sost mRNA expression in osteocytes present in old bone. An abundance of cells expressed Wnt7b in Runx2-positive osteoblasts and ß-catenin in areas with new bone formation. These data demonstrate that inflammation, not only induces osteoclastogenesis, but also locally activates canonical WNT signaling and stimulates new bone formation independent on bone resorption.

Combining anabolic loading and raloxifene improves bone quantity and some quality measures in a mouse model of osteogenesis imperfecta.

Osteogenesis imperfecta (OI) increases fracture risk due to changes in bone quantity and quality caused by mutations in collagen and its processing proteins. Current therapeutics improve bone quantity, but do not treat the underlying quality deficiencies. Male and female G610C+/- mice, a murine model of OI, were treated with a combination of raloxifene and in vivo axial tibial compressive loading starting at 10 weeks of age and continuing for 6 weeks to improve bone quantity and quality. Bone geometry and mechanical properties were measured to determine whole bone and tissue-level material properties. A colocalized Raman/nanoindentation system was used to measure chemical composition and nanomechanical properties in newly formed bone compared to old bone to determine if bone formed during the treatment regimen differed in quality compared to bone formed prior to treatment. Lastly, lacunar geometry and osteocyte apoptosis were assessed. OI mice were able to build bone in response to the loading, but this response was less robust than in control mice. Raloxifene improved some bone material properties in female but not male OI mice. Raloxifene did not alter nanomechanical properties, but loading did. Lacunar geometry was largely unchanged with raloxifene and loading. However, osteocyte apoptosis was increased with loading in raloxifene treated female mice. Overall, combination treatment with raloxifene and loading resulted in positive but subtle changes to bone quality.