Targeting mitochondrial dysfunction using methylene blue or mitoquinone to improve skeletal aging.

Methylene blue (MB) is a well-established antioxidant that has been shown to improve mitochondrial function in both in vitro and in vivo settings. Mitoquinone (MitoQ) is a selective antioxidant that specifically targets mitochondria and effectively reduces the accumulation of reactive oxygen species. To investigate the effect of long-term administration of MB on skeletal morphology, we administered MB to aged (18 months old) female C57BL/J6 mice, as well as to adult male and female mice with a genetically diverse background (UM-HET3). Additionally, we used MitoQ as an alternative approach to target mitochondrial oxidative stress during aging in adult female and male UM-HET3 mice. Although we observed some beneficial effects of MB and MitoQ in vitro, the administration of these compounds in vivo did not alter the progression of age-induced bone loss. Specifically, treating 18-month-old female mice with MB for 6 or 12 months did not have an effect on age-related bone loss. Similarly, long-term treatment with MB from 7 to 22 months or with MitoQ from 4 to 22 months of age did not affect the morphology of cortical bone at the mid-diaphysis of the femur, trabecular bone at the distal-metaphysis of the femur, or trabecular bone at the lumbar vertebra-5 in UM-HET3 mice. Based on our findings, it appears that long-term treatment with MB or MitoQ alone, as a means to reduce skeletal oxidative stress, is insufficient to inhibit age-associated bone loss. This supports the notion that interventions solely with antioxidants may not provide adequate protection against skeletal aging.

A Fluorescent Intravital Imaging Approach to Study Load-Induced Calcium Signaling Dynamics in Mouse Osteocytes.

Bone tissue is exquisitely sensitive to differences in mechanical load magnitude. Osteocytes, dendritic cells that form a syncytium throughout the bone, are responsible for the mechanosensory function of bone tissue. Studies employing histology, mathematical modeling, cell culture, and ex vivo bone organ cultures have greatly advanced the understanding of osteocyte mechanobiology. However, the fundamental question of how osteocytes respond to and encode mechanical information at the molecular level in vivo is not well understood. Intracellular calcium concentration fluctuations in osteocytes offer a useful target for learning more about acute bone mechanotransduction mechanisms. Here, we report a method for studying osteocyte mechanobiology in vivo, combining a mouse strain with a fluorescently genetically encoded calcium indicator expressed in osteocytes with an in vivo loading and imaging system to directly detect osteocyte calcium levels during loading. This is achieved with a three-point bending device that can deliver well-defined mechanical loads to the third metatarsal of living mice while simultaneously monitoring fluorescently indicated calcium responses of osteocytes using two-photon microscopy. This technique allows for direct in vivo observation of osteocyte calcium signaling events in response to whole bone loading and is useful in the endeavor to reveal mechanisms in osteocyte mechanobiology.

Lifelong Excess in GH Elicits Sexually Dimorphic Effects on Skeletal Morphology and Bone Mechanical Properties.

Excess in growth hormone (GH) levels, seen in patients with acromegaly, is associated with increases in fractures. This happens despite wider bones and independent of bone mineral density. We used the bovine GH (bGH) transgenic mice, which show constitutive excess in GH and insulin-like growth factor 1 (IGF-1) in serum and tissues, to study how lifelong increases in GH and IGF-1 affect skeletal integrity. Additionally, we crossed the acid labile subunit (ALS) null (ALSKO) to the bGH mice to reduce serum IGF-1 levels. Our findings indicate sexually dimorphic effects of GH on cortical and trabecular bone. Male bGH mice showed enlarged cortical diameters, but with marrow cavity expansion and thin cortices as well as increased vascular porosity that were associated with reductions in diaphyseal strength and stiffness. In contrast, female bGH mice presented with significantly smaller-diameter diaphysis, with greater cortical bone thickness and with a slightly reduced tissue elastic modulus (by microindentation), ultimately resulting in overall stronger, stiffer bones. We found increases in C-terminal telopeptide of type 1 collagen and procollagen type 1 N propeptide in serum, independent of circulating IGF-1 levels, indicating increased bone remodeling with excess GH. Sexual dimorphism in response to excess GH was also observed in the trabecular bone compartment, particularly at the femur distal metaphysis. Female bGH mice preserved their trabecular architecture during aging, whereas trabecular bone volume in male bGH mice significantly reduced and was associated with thinning of the trabeculae. We conclude that pathological excess in GH results in sexually dimorphic changes in bone architecture and gains in bone mass that affect whole-bone mechanical properties, as well as sex-specific differences in bone material properties. © 2022 American Society for Bone and Mineral Research (ASBMR).

Induction of somatopause in adult mice compromises bone morphology and exacerbates bone loss during aging.

Somatopause refers to the gradual declines in growth hormone (GH) and insulin-like growth factor-1 throughout aging. To define how induced somatopause affects skeletal integrity, we used an inducible GH receptor knockout (iGHRKO) mouse model. Somatopause, induced globally at 6 months of age, resulted in significantly more slender bones in both male and female iGHRKO mice. In males, induced somatopause was associated with progressive expansion of the marrow cavity leading to significant thinning of the cortices, which compromised bone strength. We report progressive declines in osteocyte lacunar number, and increases in lacunar volume, in iGHRKO males, and reductions in lacunar number accompanied by ~20% loss of overall canalicular connectivity in iGHRKO females by 30 months of age. Induced somatopause did not affect mineral/matrix ratio assessed by Raman microspectroscopy. We found significant increases in bone marrow adiposity and high levels of sclerostin, a negative regulator of bone formation in iGHRKO mice. Surprisingly, however, despite compromised bone morphology, osteocyte senescence was reduced in the iGHRKO mice. In this study, we avoided the confounded effects of constitutive deficiency in the GH/IGF-1 axis on the skeleton during growth, and specifically dissected its effects on the aging skeleton. We show here, for the first time, that induced somatopause compromises bone morphology and the bone marrow environment.

Estrogen depletion on In vivo osteocyte calcium signaling responses to mechanical loading.

Microstructural adaptation of bone in response to mechanical stimuli is diminished with estrogen deprivation. Here we tested in vivo whether ovariectomy (OVX) alters the acute response of osteocytes, the principal mechanosensory cells of bone, to mechanical loading in mice. We also used super resolution microscopy (Structured Illumination microscopy or SIM) in conjunction with immunohistochemistry to assess changes in the number and organization of "osteocyte mechanosomes" - complexes of Panx1 channels, P2X7 receptors and CaV3 voltage-gated Ca2+ channels clustered around αvß3 integrin foci on osteocyte processes. Third metatarsals bones of mice expressing an osteocyte-targeted genetically encoded Ca2+ indicator (DMP1-GCaMP3) were cyclically loaded in vivo to strains from 250 to 3000 µÎµ and osteocyte intracellular Ca2+ signaling responses were assessed in mid-diaphyses using multiphoton microscopy. The number of Ca2+ signaling osteocytes in control mice increase monotonically with applied strain magnitude for the physiological range of strains. The relationship between the number of Ca2+ signaling osteocytes and loading was unchanged at 2 days post-OVX. However, it was altered markedly at 28 days post-OVX. At loads up to 1000 µÎµ, there was a dramatic reduction in number of responding (i.e. Ca2+ signaling) osteocytes; however, at higher strains the numbers of Ca2+ signaling osteocytes were similar to control mice. OVX significantly altered the abundance, make-up and organization of osteocyte mechanosome complexes on dendritic processes. Numbers of αvß3 foci also staining with either Panx 1, P2X7R or CaV3 declined by nearly half after OVX, pointing to a loss of osteocyte mechanosomes on the dendritic processes with estrogen depletion. At the same time, the areas of the remaining foci that stained for αvß3 and channel proteins increased significantly, a redistribution of mechanosome components suggesting a potential compensatory response. These results demonstrate that the deleterious effects of estrogen depletion on skeletal mechanical adaptation appear at the level of mechanosensation; osteocytes lose the ability to sense small (physiological) mechanical stimuli. This decline may result at least partly from changes in the structure and organization of osteocyte mechanosomes, which contribute to the distinctive sensitivity of osteocytes (particularly their dendritic processes) to mechanical stimulation.

Skeletal Response to Insulin in the Naturally Occurring Type 1 Diabetes Mellitus Mouse Model.

Patients with type 1 diabetes mellitus (T1DM) exhibit reduced BMD and significant increases in fracture risk. Changes in BMD are attributed to blunted osteoblast activity and inhibited bone remodeling, but these cannot fully explain the impaired bone integrity in T1DM. The goal of this study was to determine the cellular mechanisms that contribute to impaired bone morphology and composition in T1DM. Nonobese diabetic (NOD) mice were used, along with µCT, histomorphometry, histology, Raman spectroscopy, and RNAseq analyses of several skeletal sites in response to naturally occurring hyperglycemia and insulin treatment. The bone volume in the axial skeleton was found to be severely reduced in diabetic NOD mice and was not completely resolved with insulin treatment. Decreased bone volume in diabetic mice was associated with increased sclerostin expression in osteocytes and attenuation of bone formation indices without changes in bone resorption. In the face of blunted bone remodeling, decreases in the mineral:matrix ratio were found in cortical bones of diabetic mice by Raman microspectroscopy, suggesting that T1DM did not affect the bone mineralization process per se, but rather resulted in microenvironmental alterations that favored mineral loss. Bone transcriptome analysis indicated metabolic shifts in response to T1DM. Dysregulation of genes involved in fatty acid oxidation, transport, and synthesis was found in diabetic NOD mice. Specifically, pyruvate dehydrogenase kinase isoenzyme 4 and glucose transporter 1 levels were increased, whereas phosphorylated-AKT levels were significantly reduced in diabetic NOD mice. In conclusion, in addition to the blunted bone formation, osteoblasts and osteocytes undergo metabolic shifts in response to T1DM that may alter the microenvironment and contribute to mineral loss from the bone matrix. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

A Bisphosphonate With a Low Hydroxyapatite Binding Affinity Prevents Bone Loss in Mice After Ovariectomy and Reverses Rapidly With Treatment Cessation.

Bisphosphonates (BPs) are a mainstay of osteoporosis treatment; however, concerns about bone health based on oversuppression of remodeling remain. Long-term bone remodeling suppression adversely affects bone material properties with microdamage accumulation and reduced fracture toughness in animals and increases in matrix mineralization and atypical femur fractures in patients. Although a "drug holiday" from BPs to restore remodeling and improve bone quality seems reasonable, clinical BPs have long functional half-lives because of their high hydroxyapatite (HAP) binding affinities. This places a practical limit on the reversibility and effectiveness of a drug holiday. BPs with low HAP affinity and strong osteoclast inhibition potentially offer an alternative approach; their antiresorptive effect should reverse rapidly when dosing is discontinued. This study tested this concept using NE-58025, a BP with low HAP affinity and moderate osteoclast inhibition potential. Young adult female C57Bl/6 mice were ovariectomized (OVX) and treated with NE-58025, risedronate, or PBS vehicle for 3 months to test effectiveness in preventing long-term bone loss. Bone microarchitecture, histomorphometry, and whole-bone mechanical properties were assessed. To test reversibility, OVX mice were similarly treated for 3 months, treatment was stopped, and bone was assessed up to 3 months post-treatment. NE-58025 and RIS inhibited long-term OVX-induced bone loss, but NE-58025 antiresorptive effects were more pronounced. Withdrawing NE-58025 treatment led to the rapid onset of trabecular resorption with a 200% increase in osteoclast surface and bone loss within 1 month. Cessation of risedronate treatment did not lead to increases in resorption indices or bone loss. These results show that NE-58025 prevents OVX-induced bone loss, and its effects reverse quickly following cessation treatment in vivo. Low-HAP affinity BPs may have use as reversible, antiresorptive agents with a rapid on/off profile, which may be useful for maintaining bone health with long-term BP treatment. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Apoptotic Osteocytes Induce RANKL Production in Bystanders via Purinergic Signaling and Activation of Pannexin Channels.

Localized apoptosis of osteocytes, the tissue-resident cells within bone, occurs with fatigue microdamage and activates bone resorption. Osteoclasts appear to target and remove dying osteocytes, resorbing damaged bone matrix as well. Osteocyte apoptosis similarly activates bone resorption with estrogen loss and in disuse. Apoptotic osteocytes trigger viable neighbor (ie, bystander) osteocytes to produce RANKL, the cytokine required for osteoclast activation. Signals from apoptotic osteocytes that trigger this bystander RANKL expression remain obscure. Studying signaling among osteocytes has been hampered by lack of in vitro systems that model the limited communication among osteocytes in vivo (ie, via gap junctions on cell processes and/or paracrine signals through thin pericellular fluid spaces around osteocytes). Here, we used a novel multiscale fluidic device (the Macro-micro-nano, or Mµn) that reproduces these key anatomical features. Osteocytes in discrete compartments of the device communicate only via these limited pathways, which allows assessment of their roles in triggering osteocytes RANKL expression. Apoptosis of MLOY-4 osteocytes in the Mµn device caused increased osteocyte RANKL expression in the neighboring compartment, consistent with in vivo findings. This RANKL upregulation in bystander osteocytes was prevented by blocking Pannexin 1 channels as well as its ATP receptor. ATP alone caused comparable RANKL upregulation in bystander osteocytes. Finally, blocking Connexin 43 gap junctions did not abolish osteocyte RANKL upregulation, but did alter the distribution of RANKL expressing bystander osteocytes. These findings point to extracellular ATP, released from apoptotic osteocytes via Panx1 channels, as a major signal for triggering bystander osteocyte RANKL expression and activating bone remodeling. © 2020 American Society for Bone and Mineral Research.

CITED2 mediates the cross-talk between mechanical loading and IL-4 to promote chondroprotection.

Osteoarthritis (OA) pathogenesis is mediated largely through the actions of proteolytic enzymes such as matrix metalloproteinase (MMP) 13. The transcriptional regulator CITED2, which suppresses the expression of MMP13 in chondrocytes, is induced by interleukin (IL)-4 in T cells and macrophages, and by moderate mechanical loading in chondrocytes. We tested the hypothesis that CITED2 mediates cross-talk between IL-4 signaling and mechanical loading-induced pathways that result in chondroprotection, at least in part, by downregulating MMP13. IL-4 induced CITED2 gene expression in human chondrocytes in a dose- and time-dependent manner through JAK/STAT signaling. Mechanical loading combined with IL-4 resulted in additive effects on inducing CITED2 expression and downregulating of MMP13 in human chondrocytes in vitro. In vivo, IL-4 gene knockout (KO) mice exhibited reduced basal levels of CITED2 expression in chondrocytes. While moderate treadmill running induced CITED2 expression and reduced MMP13 expression in wild-type mice, these effects were blunted (for CITED2) or abolished (for MMP13) in chondrocytes of IL-4 gene KO mice. Moreover, intra-articular injections of mouse recombinant IL-4 combined with regular cage activity mitigated post-traumatic OA to a greater degree compared to immobilized mice treated with IL-4 alone. These data suggest that using moderate loading to enhance IL-4 may be a potential therapeutic strategy for chondroprotection in OA.

Correction to: Green tea polyphenol treatment is chondroprotective, anti-inflammatory and palliative in a mouse posttraumatic osteoarthritis model.

Following publication of the original article [1], the authors reported an error in Figs. 2C and 5C.

Mitochondrial Function Is Compromised in Cortical Bone Osteocytes of Long-Lived Growth Hormone Receptor Null Mice.

Despite increased longevity and resistance to multiple stressors, growth hormone receptor null (GHRKO) mice exhibit severe skeletal impairment. The role of GHR in maintaining osteocyte mitochondrial function is unknown. We found that GHR ablation was detrimental to osteocyte mitochondrial function. In vivo multiphoton microscopy revealed significant reductions of >10% in mitochondrial membrane potential (MMP) in GHRKO osteocytes and reduced mitochondrial volumetric density. Reductions in MMP were accompanied by reductions in glucose transporter-1 levels, steady state ATP, NADH redox index, oxygen consumption rate, and mitochondrial reserve capacity in GHRKO osteocytes. Glycolytic capacity did not differ between control and GHRKO males' osteocytes. However, osteocytes from aged female GHRKO mice exhibited reductions in glycolytic parameters, indicating impairments in glucose metabolism, which may be sex dependent. GHRKO osteocytes exhibited increased levels of cytoplasmic reactive oxygen species (ROS) (both basal and in response to high glucose), insulin-like growth factor-1 (IGF-1), and insulin. Mitochondrial ROS levels were increased and correlated with reduced glutathione in GHRKO osteocytes. Overall, the compromised osteocyte mitochondrial function and responses to metabolic insults strongly correlated with skeletal impairments, suggesting that despite increased life span of the GHRKO mice, skeletal health span is decreased. © 2018 American Society for Bone and Mineral Research.

Role of pannexin 1 channels in load-induced skeletal response.

The pannexin 1 (Panx1) channel is a mechanosensitive channel that interacts with P2X7 receptors (P2X7R) to form a functional complex that has been shown in vitro to play an essential role in osteocyte mechanosignaling. While the participation of P2X7R in skeletal responses to mechanical loading has been demonstrated, the role of Panx1 and its interplay with P2X7R still remain to be determined. In this study, we use a global Panx1-/- mouse model and in vivo mechanical loading to demonstrate that Panx1 channels play an essential role in load-induced skeletal responses. We found that absence of Panx1 not only disrupts the P2X7R-Panx1 signaling complex, but also alters load-induced regulation of P2X7R expression. Moreover, lack of Panx1 completely abolished load-induced periosteal bone formation. Load-induced regulation of ß-catenin and sclerostin expression was dysregulated in Panx1-/- , compared to wild-type, bone. This finding suggests that Panx1 deficiency disrupts Wnt/ß-catenin signaling by lowering ß-catenin while favoring inhibition of bone formation by increasing load-induced sclerostin expression. This study demonstrates the existence of a Panx1-dependent mechanosensitive mechanism that not only modulates ATP signaling but also coordinates Wnt/ß-catenin signaling that is essential for proper skeletal response to mechanical loading.

Reduced Serum IGF-1 Associated With Hepatic Osteodystrophy Is a Main Determinant of Low Cortical but Not Trabecular Bone Mass.

Hepatic osteodystrophy is multifactorial in its pathogenesis. Numerous studies have shown that impairments of the hepatic growth hormone/insulin-like growth factor-1 axis (GH/IGF-1) are common in patients with non-alcoholic fatty liver disease, chronic viral hepatitis, liver cirrhosis, and chronic cholestatic liver disease. Moreover, these conditions are also associated with low bone mineral density (BMD) and greater fracture risk, particularly in cortical bone sites. Hence, we addressed whether disruptions in the GH/IGF-1 axis were causally related to the low bone mass in states of chronic liver disease using a mouse model of liver-specific GH-receptor (GHR) gene deletion (Li-GHRKO). These mice exhibit chronic hepatic steatosis, local inflammation, and reduced BMD. We then employed a crossing strategy to restore liver production of IGF-1 via hepatic IGF-1 transgene (HIT). The resultant Li-GHRKO-HIT mouse model allowed us to dissect the roles of liver-derived IGF-1 in the pathogenesis of osteodystrophy during liver disease. We found that hepatic IGF-1 restored cortical bone acquisition, microarchitecture, and mechanical properties during growth in Li-GHRKO-HIT mice, which was maintained during aging. However, trabecular bone volume was not restored in the Li-GHRKO-HIT mice. We found increased bone resorption indices in vivo as well as increased basal reactive oxygen species and increased mitochondrial stress in osteoblast cultures from Li-GHRKO and the Li-GHRKO-HIT compared with control mice. Changes in systemic markers such as inflammatory cytokines, osteoprotegerin, osteopontin, parathyroid hormone, osteocalcin, or carboxy-terminal collagen cross-links could not fully account for the diminished trabecular bone in the Li-GHRKO-HIT mice. Thus, the reduced serum IGF-1 associated with hepatic osteodystrophy is a main determinant of low cortical but not trabecular bone mass. © 2017 American Society for Bone and Mineral Research.

Potential role for a specialized ß3 integrin-based structure on osteocyte processes in bone mechanosensation.

Osteocyte processes are an order of magnitude more sensitive to mechanical loading than their cell bodies. The mechanisms underlying this remarkable mechanosensitivity are not clear, but may be related to the infrequent αV ß3 integrin sites where the osteocyte cell processes attach to canalicular walls. These sites develop dramatically elevated strains during load-induced fluid flow in the lacunar-canalicular system and were recently shown to be primary sites for osteocyte-like MLO-Y4 cell mechanotransduction. These αV ß3 integrin sites lack typical integrin transduction mechanisms. Rather, stimulation at these sites alters Ca2+ signaling, ATP release and membrane potential. In the current studies, we tested the hypothesis that in authentic osteocytes in situ, key membrane proteins implicated in osteocyte mechanotransduction are preferentially localized at or near to ß3 integrin-foci. We analyzed these spatial relationships in mouse bone osteocytes using immunohistochemistry combined with Structured Illumination Super Resolution Microscopy, a method that permits structural resolution at near electron microscopy levels in tissue sections. We discovered that the purinergic channel pannexin1, the ATP-gated purinergic receptor P2 × 7R and the low voltage transiently opened T-type calcium channel CaV3.2-1 all reside in close proximity to ß3 integrin attachment foci on osteocyte processes, suggesting a specialized mechanotransduction complex at these sites. We further confirmed this observation on isolated osteocytes in culture using STochasitc Optical Resonance Microscopy. These findings identify a possible structural basis for the unique mechanosensation and transduction capabilities of the osteocyte process. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:642-652, 2018.

Osteocyte calcium signals encode strain magnitude and loading frequency in vivo.

Osteocytes are considered to be the major mechanosensory cells of bone, but how osteocytes in vivo process, perceive, and respond to mechanical loading remains poorly understood. Intracellular calcium (Ca2+) signaling resulting from mechanical stimulation has been widely studied in osteocytes in vitro and in bone explants, but has yet to be examined in vivo. This is achieved herein by using a three-point bending device which is capable of delivering well-defined mechanical loads to metatarsal bones of living mice while simultaneously monitoring the intracellular Ca2+ responses of individual osteocytes by using a genetically encoded fluorescent Ca2+ indicator. Osteocyte responses are imaged by using multiphoton fluorescence microscopy. We investigated the in vivo responses of osteocytes to strains ranging from 250 to 3,000 [Formula: see text] and frequencies from 0.5 to 2 Hz, which are characteristic of physiological conditions reported for bone. At all loading frequencies examined, the number of responding osteocytes increased strongly with applied strain magnitude. However, Ca2+ intensity within responding osteocytes did not change significantly with physiological loading magnitudes. Our studies offer a glimpse into how these critical bone cells respond to mechanical load in vivo, as well as provide a technique to determine how the cells encode magnitude and frequency of loading.

Ablation of Hepatic Production of the Acid-Labile Subunit in Bovine-GH Transgenic Mice: Effects on Organ and Skeletal Growth.

Growth hormone (GH) and insulinlike growth factor 1 (IGF-1) are anabolic hormones that facilitate somatic and skeletal growth and regulate metabolism via endocrine and autocrine/paracrine mechanisms. We hypothesized that excess tissue production of GH would protect skeletal growth and integrity in states of reduction in serum IGF-1 levels. To test our hypothesis, we used bovine GH (bGH) transgenic mice as a model of GH hypersecretion and ablated the liver-derived acid-labile subunit, which stabilizes IGF-1 complexes with IGF-binding protein-3 and -5 in circulation. We used a genetic approach to create bGH/als gene knockout (ALSKO) mice and small interfering RNA (siRNA) gene-silencing approach to reduce als or igf-1 gene expression. We found that in both models, decreased IGF-1 levels in serum were associated with decreased body and skeletal size of the bGH mice. Excess GH produced more robust bones but compromised mechanical properties in male mice. Excess GH production in tissues did not protect from trabecular bone loss in response to reductions in serum IGF-1 (in bGH/ALSKO or bGH mice treated with siRNAs). Reduced serum IGF-1 levels in the bGH mice did not alleviate the hyperinsulinemia and did not resolve liver or kidney pathologies that resulted from GH hypersecretion. We concluded that reduced serum IGF-1 levels decrease somatic and skeletal growth even in states of excess GH.

Microdamage induced by in vivo Reference Point Indentation in mice is repaired by osteocyte-apoptosis mediated remodeling.

Reference Point Indentation (RPI) is a technology that is designed to measure mechanical properties that relate to bone toughness, or its ability to resist crack growth, in vivo. Independent of the mechanical parameters generated by RPI, its ability to initiate and propagate microcracks in bone is itself an interesting issue. Microcracks have a crucial biological relevance in bone, are central to its ability to maintain homeostasis. In healthy tissues, a process of targeted remodeling routinely repairs microcracks in a process mediated by osteocyte apoptosis. However, in diseases such as osteoporosis this process becomes deficient and microcracks can accumulate. Small animal models such are crucial for the study of such diseases, but it is technically challenging to create microcracks in these animals without causing outright failure. Therefore we sought to use RPI as a focal microdamage placement tool, to introduce microcracks to mouse long bones and investigate whether the same pathway mediates their repair as that described in other microdamage systems. We first used SEM to confirm that microdamage is formed RPI in mouse bone. Then, since RPI is carried out transdermally, we sought to confirm that no periosteal response occurred at the indented region. We then used a pan-caspase inhibitor (QVD) to determine whether osteocyte apoptosis plays the same pivotal role in microdamage repair in this model, as has been demonstrated in others. In conclusion, we validated that the microdamage-apoptosis-remodeling pathway is maintained with this method of microdamage induction in mice. We show that RPI can be used as a reliable and reproducible microdamage placement tool in living mouse long bones without inducing a periosteal response. We also used a caspase inhibitor, to block osteocyte apoptosis and thus abrogate the remodeling response to microdamage. This demonstrates that the well described microdamage repair system, involving targeted remodeling mediated by osteocyte apoptosis, is conserved in this novel mouse model using an in vivo RPI loading system.

Lactation-Induced Changes in the Volume of Osteocyte Lacunar-Canalicular Space Alter Mechanical Properties in Cortical Bone Tissue.

Osteocytes can remove and remodel small amounts of their surrounding bone matrix through osteocytic osteolysis, which results in increased volume occupied by lacunar and canalicular space (LCS). It is well established that cortical bone stiffness and strength are strongly and inversely correlated with vascular porosity, but whether changes in LCS volume caused by osteocytic osteolysis are large enough to affect bone mechanical properties is not known. In the current studies we tested the hypotheses that (1) lactation and postlactation recovery in mice alter the elastic modulus of bone tissue, and (2) such local changes in mechanical properties are related predominantly to alterations in lacunar and canalicular volume rather than bone matrix composition. Mechanical testing was performed using microindentation to measure modulus in regions containing solely osteocytes and no vascular porosity. Lactation caused a significant (∼13%) reduction in bone tissue-level elastic modulus (p < 0.001). After 1 week postweaning (recovery), bone modulus levels returned to control levels and did not change further after 4 weeks of recovery. LCS porosity tracked inversely with changes in cortical bone modulus. Lacunar and canalicular void space increased 7% and 15% with lactation, respectively (p < 0.05), then returned to control levels at 1 week after weaning. Neither bone mineralization (assessed by high-resolution backscattered scanning electron microscopy) nor mineral/matrix ratio or crystallinity (assessed by Raman microspectroscopy) changed with lactation. Thus, changes in bone mechanical properties induced by lactation and recovery appear to depend predominantly on changes in osteocyte LCS dimensions. Moreover, this study demonstrates that tissue-level cortical bone mechanical properties are rapidly and reversibly modulated by osteocytes in response to physiological challenge. These data point to a hitherto unappreciated role for osteocytes in modulating and maintaining local bone mechanical properties. © 2016 American Society for Bone and Mineral Research.

Regional differences in oxidative metabolism and mitochondrial activity among cortical bone osteocytes.

Metabolic oxidative stress has been implicated as a cause of osteocyte apoptosis, an essential step in triggering bone remodeling. However, little is known about the oxidative behavior of osteocytes in vivo. We assessed the redox status and distribution of total and active mitochondria in osteocytes of mouse metatarsal cortical bone in situ. Multiphoton microscopy (MPM) was used to measure fluorescence of reduced pyridine nucleotides (NADH) under normoxic conditions and acutely following extreme (postmortem) hypoxic stress. Under non-hypoxic conditions, osteocytes exhibited no detectable fluorescence, indicating rapid NADH re-oxidation. With hypoxia, NADH levels peaked and returned to near baseline levels over 3h. Cells near the periosteal surface reached maximum NADH levels twice as rapidly as osteocytes near the mid-cortex, due to the time required to initiate NADH accumulation; once started, NADH accumulation followed a similar exponential relationship at all sites. Osteocytes near periosteal and endosteal bone surfaces also had higher mitochondrial content than those in mid-cortex based on immunohistochemical staining for mitochondrial ATPase-5A (Complex V ATPase). The content of active mitochondria, assessed in situ using the potentiometric dye TMRM, was also high in osteocytes near periosteum, but low in osteocytes near endocortical surfaces, similar to levels in mid-cortex. These results demonstrate that cortical osteocytes maintain normal oxidative status utilizing mainly aerobic (mitochondrial) pathways but respond to hypoxic stress differently depending on their location in the cortex, a difference linked to mitochondrial content. An apparently high proportion of poorly functional mitochondria in osteocytes near endocortical surfaces, where increased apoptosis mainly occurs in response to bone remodeling stimuli, further suggest that regional differences in oxidative function may in part determine osteocyte susceptibility to undergo apoptosis in response to stimuli that trigger bone remodeling.