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.

DMP-1-mediated Ghr gene recombination compromises skeletal development and impairs skeletal response to intermittent PTH.

Bone minerals are acquired during growth and are key determinants of adult skeletal health. During puberty, the serum levels of growth hormone (GH) and its downstream effector IGF-1 increase and play critical roles in bone acquisition. The goal of the current study was to determine how bone cells integrate signals from the GH/IGF-1 to enhance skeletal mineralization and strength during pubertal growth. Osteocytes, the most abundant bone cells, were shown to orchestrate bone modeling during growth. We used dentin matrix protein (Dmp)-1-mediated Ghr knockout (DMP-GHRKO) mice to address the role of the GH/IGF axis in osteocytes. We found that DMP-GHRKO did not affect linear growth but compromised overall bone accrual. DMP-GHRKO mice exhibited reduced serum inorganic phosphate and parathyroid hormone (PTH) levels and decreased bone formation indices and were associated with an impaired response to intermittent PTH treatment. Using an osteocyte-like cell line along with in vivo studies, we found that PTH sensitized the response of bone to GH by increasing Janus kinase-2 and IGF-1R protein levels. We concluded that endogenously secreted PTH and GHR signaling in bone are necessary to establish radial bone growth and optimize mineral acquisition during growth.

Matrix-dependent adhesion mediates network responses to physiological stimulation of the osteocyte cell process.

Osteocytes are bone cells that form cellular networks that sense mechanical loads distributed throughout the bone tissue. Interstitial fluid flow in the lacunar canalicular system produces focal strains at localized attachment sites around the osteocyte cell process. These regions of periodic attachment between the osteocyte cell membrane and its canalicular wall are sites where pN-level fluid-flow induced forces are generated in vivo. In this study, we show that focally applied forces of this magnitude using a newly developed Stokesian fluid stimulus probe initiate rapid and transient intercellular electrical signals in vitro. Our experiments demonstrate both direct gap junction coupling and extracellular purinergic P2 receptor signaling between MLO-Y4 cells in a connected bone cell network. Intercellular signaling was initiated by pN-level forces applied at integrin attachment sites along both appositional and distal unapposed cell processes, but not initiated at their cell bodies with equivalent forces. Electrical coupling was evident in 58% of all cell pairs tested with appositional connections; coupling strength increased with the increasing number of junctional connections. Apyrase, a nucleotide-degrading enzyme, suppressed and abolished force-induced effector responses, indicating a contribution from ATP released by the stimulated cell. This work extends the understanding of how osteocytes modulate their microenvironment in response to mechanical signals and highlights mechanisms of intercellular relay of mechanoresponsive signals in the bone network.

Mechanosensory responses of osteocytes to physiological forces occur along processes and not cell body and require αVß3 integrin.

Osteocytes in the lacunar-canalicular system of the bone are thought to be the cells that sense mechanical loading and transduce mechanical strain into biomechanical responses. The goal of this study was to evaluate the extent to which focal mechanical stimulation of osteocyte cell body and process led to activation of the cells, and determine whether integrin attachments play a role in osteocyte activation. We use a novel Stokesian fluid stimulus probe to hydrodynamically load osteocyte processes vs. cell bodies in murine long bone osteocyte Y4 (MLO-Y4) cells with physiological-level forces <10 pN without probe contact, and measured intracellular Ca(2+) responses. Our results indicate that osteocyte processes are extremely responsive to piconewton-level mechanical loading, whereas the osteocyte cell body and processes with no local attachment sites are not. Ca(2+) signals generated at stimulated sites spread within the processes with average velocity of 5.6 µm/s. Using the near-infrared fluorescence probe IntegriSense 750, we demonstrated that inhibition of αVß3 integrin attachment sites compromises the response to probe stimulation. Moreover, using apyrase, an extracellular ATP scavenger, we showed that Ca(2+) signaling from the osteocyte process to the cell body was greatly diminished, and thus dependent on ATP-mediated autocrine signaling. These findings are consistent with the hypothesis that osteocytes in situ are highly polarized cells, where mechanotransduction occurs at substrate attachment sites along the processes at force levels predicted to occur at integrin attachment sites in vivo. We also demonstrate the essential role of αVß3 integrin in osteocyte-polarized mechanosensing and mechanotransduction.

Primary cilia exist in a small fraction of cells in trabecular bone and marrow.

Primary cilia are potent mechanical and chemical sensory organelles in cells of bone lineage in tissue culture. Cell culture experiments suggest that primary cilia sense fluid flow and this stimulus is translated through biochemical signaling into an osteogenic response in bone cells. Moreover, in vivo, primary cilia knockout in bone cells attenuates bone formation in response to loading. However, understanding the role of the primary cilium in bone mechanotransduction requires knowledge of its incidence and location in vivo. We used immunohistochemistry to quantify the number of cells with primary cilia within the trabecular bone tissue and the enclosed marrow of ovine cervical vertebrae. Primary cilia were identified in osteocytes, bone lining cells, and in cells within the marrow, but were present in only a small fraction of cells. Approximately 4% of osteocytes and 4.6% of bone lining cells expressed primary cilia. Within the marrow space, only approximately 1% of cells presented primary cilia. The low incidence of primary cilia may indicate that cilia either function as mechanosensors in a selected number of cells, function in concert with other mechanosensing mechanisms, or that the role of primary cilia in mechanosensing is secondary to its role in chemosensing or cellular attachment.

Changes of elastic constants and anisotropy patterns in trabecular bone during disuse-induced bone loss assessed by poroelastic ultrasound.

Currently, the approach most widely used to examine bone loss is the measurement of bone mineral density (BMD) using dual X-ray absorptiometry (DXA). However, bone loss due to immobilization creates changes in bone microarchitecture, which in turn are related to changes in bone mechanical function and competence to resist fracture.Unfortunately, the relationship between microarchitecture and mechanical function within the framework of immobilization and antiresorptive therapy has not being fully investigated. The goal of the present study was to investigate the structure­function relationship in trabecular bone in the real-world situations of a rapidly evolving osteoporosis(disuse), both with and without antiresorptive treatment. We evaluated the structure­function relationship in trabecular bone after bone loss (disuse-induced osteoporosis)and bisphosphonate treatment (antiresorptive therapy using risedronate) in canine trabecular bone using lCT and ultrasound wave propagation. Microstructure values determined from lCT images were used into the anisotropic poroelastic model of wave propagation in order to compute the apparent elastic constants (EC) and elastic anisotropy pattern of bone. Immobilization resulted in a significant reduction in trabecular thickness (Tb.Th) and bone volume fraction (BV/TV), while risedronate treatment combined with immobilization exhibited a lesser reduction in Tb.Th and BV/TV, suggesting that risedronate treatment decelerates bone loss, but it was unable to fully stop it. Risedronate treatment also increased the tissue mineral density (TMD), which when combined with the decrease in Tb.Th and BV/TV may explain the lack of significant differences invBMD in both immobilization and risedronate treated groups. Interestingly, changes inapparent EC were much stronger in the superior­inferior (SI) direction than in the medial­lateral (ML) and anterior­posterior (AP) anatomical directions, producing changes in elastic anisotropy patterns. When data were pooled together, vBMD was able to explain 58% of ultrasound measurements variability, a poroelastic wave propagation analytical model (i.e., BMD modulated by fabric directionality) was able to predict 81%of experimental wave velocity variability, and also explained 91% of apparent EC and changes in elastic anisotropy patterns. Overall, measurements of vBMD were unable to distinguish changes in apparent EC due to immobilization or risedronate treatment.However, anisotropic poroelastic ultrasound (PEUS) wave propagation was able to distinguish functional changes in apparent EC and elastic anisotropy patterns due to immobilization and antiresorptive therapy, providing an enhanced discrimination of anisotropic bone loss and the structure­function relationship in immobilized and risedronate-treated bone, beyond vBMD.

Osteocytes: master orchestrators of bone.

Osteocytes comprise the overwhelming majority of cells in bone and are its only true "permanent" resident cell population. In recent years, conceptual and technological advances on many fronts have helped to clarify the role osteocytes play in skeletal metabolism and the mechanisms they use to perform them. The osteocyte is now recognized as a major orchestrator of skeletal activity, capable of sensing and integrating mechanical and chemical signals from their environment to regulate both bone formation and resorption. Recent studies have established that the mechanisms osteocytes use to sense stimuli and regulate effector cells (e.g., osteoblasts and osteoclasts) are directly coupled to the environment they inhabit-entombed within the mineralized matrix of bone and connected to each other in multicellular networks. Communication within these networks is both direct (via cell-cell contacts at gap junctions) and indirect (via paracrine signaling by secreted signals). Moreover, the movement of paracrine signals is dependent on the movement of both solutes and fluid through the space immediately surrounding the osteocytes (i.e., the lacunar-canalicular system). Finally, recent studies have also shown that the regulatory capabilities of osteocytes extend beyond bone to include a role in the endocrine control of systemic phosphate metabolism. This review will discuss how a highly productive combination of experimental and theoretical approaches has managed to unearth these unique features of osteocytes and bring to light novel insights into the regulatory mechanisms operating in bone.

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.

Physiological loading of joints prevents cartilage degradation through CITED2.

Both overuse and disuse of joints up-regulate matrix metalloproteinases (MMPs) in articular cartilage and cause tissue degradation; however, moderate (physiological) loading maintains cartilage integrity. Here, we test whether CBP/p300-interacting transactivator with ED-rich tail 2 (CITED2), a mechanosensitive transcriptional coregulator, mediates this chondroprotective effect of moderate mechanical loading. In vivo, hind-limb immobilization of Sprague-Dawley rats up-regulates MMP-1 and causes rapid, histologically detectable articular cartilage degradation. One hour of daily passive joint motion prevents these changes and up-regulates articular cartilage CITED2. In vitro, moderate (2.5 MPa, 1 Hz) intermittent hydrostatic pressure (IHP) treatment suppresses basal MMP-1 expression and up-regulates CITED2 in human chondrocytes, whereas high IHP (10 MPa) down-regulates CITED2 and increases MMP-1. Competitive binding and transcription assays demonstrate that CITED2 suppresses MMP-1 expression by competing with MMP transactivator, Ets-1 for its coactivator p300. Furthermore, CITED2 up-regulation in vitro requires the p38δ isoform, which is specifically phosphorylated by moderate IHP. Together, these studies identify a novel regulatory pathway involving CITED2 and p38δ, which may be critical for the maintenance of articular cartilage integrity under normal physical activity levels.

Osteocyte signaling in bone.

Osteocytes, the cells residing within the bone matrix and comprising 90% to 95% of the all bone cells, have long been considered quiescent bystander cells compared to the osteoblasts and osteoclasts whose activities cause bone gain and loss, and whose dysfunction lead to growth defects and osteoporosis. However, recent studies show that osteocytes play a crucial, central role in regulating the dynamic nature of bone in all its diverse functions. Osteocytes are now known to be the principal sensors for mechanical loading of bone. They produce the soluble factors that regulate the onset of both bone formation and resorption. Osteocytes regulate local mineral deposition and chemistry at the bone matrix level, and they also function as endocrine cells producing factors that target distant organs such as the kidney to regulate phosphate transport. Osteocytes appear to be the major local orchestrator of many of bone's functions.

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).

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.

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.

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.

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.

Serum complexes of insulin-like growth factor-1 modulate skeletal integrity and carbohydrate metabolism.

Serum insulin-like growth factor (IGF) -1 is secreted mainly by the liver and circulates bound to IGF-binding proteins (IGFBPs), either as binary complexes or ternary complexes with IGFBP-3 or IGFBP-5 and an acid-labile subunit (ALS). The purpose of this study was to genetically dissect the role of IGF-1 circulatory complexes in somatic growth, skeletal integrity, and metabolism. Phenotypic comparisons of controls and four mouse lines with genetic IGF-1 deficits-liver-specific IGF-1 deficiency (LID), ALS knockout (ALSKO), IGFBP-3 (BP3) knockout, and a triply deficient LID/ALSKO/BP3 line-produced several novel findings. 1) All deficient strains had decreased serum IGF-1 levels, but this neither predicted growth potential or skeletal integrity nor defined growth hormone secretion or metabolic abnormalities. 2) IGF-1 deficiency affected development of both cortical and trabecular bone differently, effects apparently dependent on the presence of different circulating IGF-1 complexes. 3) IGFBP-3 deficiency resulted in increased linear growth. In summary, each IGF-1 complex constituent appears to play a distinct role in determining skeletal phenotype, with different effects on cortical and trabecular bone compartments.

Sustained release of multiple growth factors from injectable polymeric system as a novel therapeutic approach towards angiogenesis.

PURPOSE: The aim was to investigate that a bio-degradable alginate and poly lactide-co-glycolide (PLG) system capable of delivering growth factors sequentially would be superior to single growth factor delivery in promoting neovascularization and improving perfusion. METHODS: Three groups of apoE null mice underwent unilateral hindlimb ischemia surgery and received ischemic limb intramuscular injections of alginate (Blank), alginate containing VEGF(165) (VEGF), or alginate containing VEGF(165) combined with PLG microspheres containing PDGF-BB (VEGF/PDGF). Vascularity in the ischemic hindlimb was assessed by morphologic and immunohistochemical end-points, while changes in blood flow were assessed by Laser Doppler Perfusion Index. Muscle VEGF and PDGF content was assessed at multiple time points. RESULTS: In the VEGF/PDGF group, local tissue VEGF and PDGF levels peaked at week 2 and 4, respectively, with detectable PDGF levels at week 6. At week 6, mean vessel mean diameter was significantly greater in the VEGF/PDGF group compared to the VEGF or Blank groups with evidence of well-formed smooth muscle-lined arterioles. CONCLUSIONS: Sequential delivery of VEGF and PDGF using an injectable, biodegradable platform resulted in stable and sustained improvements in perfusion. This sustained, control-released, injectable alginate polymer system is a promising approach for multiple growth factor delivery in clinical application.

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.