Immobilization and long-term recovery results in large changes in bone structure and strength but no corresponding alterations of osteocyte lacunar properties

The ability of osteocytes to demineralize the perilacunar matrix, osteocytic osteolysis, and thereby participate directly in bone metabolism, is an aspect of osteocyte biology that has received increasing attention during the last couple of years. The aim of the present work was to investigate whether osteocyte lacunar properties change during immobilization and subsequent recovery. A rat cortical bone model with negligible Haversian remodeling effects was used, with temporary immobilization of one hindlimb induced by botulinum toxin. Several complementary techniques covering multiple length scales enabled correlation of osteocyte lacunar properties to changes observed on the organ and tissue level of femoral bone. Bone structural parameters measured by μCT and mechanical properties were compared to sub-micrometer resolution SR μCT data mapping an unprecedented number (1.85 million) of osteocyte lacunae.Immobilization induced a significant reduction in aBMD, bone volume, tissue volume, and load to fracture, as well as the muscle mass of rectus femoris. During the subsequent recovery period, the bone structural and mechanical properties were only partly regained in spite of a long-term (28 weeks) study period. No significant changes in osteocyte lacunar volume, density, oblateness, stretch, or orientation were detected upon immobilization or subsequent recovery. In conclusion, the bone architecture and not osteocyte lacunar properties or bone material characteristics dominate the immobilization response as well as the subsequent recovery.     

Quantifying load‐induced solute transport and solute‐matrix interaction within the osteocyte lacunar‐canalicular system

Osteocytes, the most abundant cells in bone, are essential in maintaining tissue homeostasis and orchestrating bone's mechanical adaptation. Osteocytes depend upon load‐induced convection within the lacunar‐canalicular system (LCS) to maintain viability and to sense their mechanical environment. Using the fluorescence recovery after photobleaching (FRAP) imaging approach, we previously quantified the convection of a small tracer (sodium fluorescein, 376 Da) in the murine tibial LCS under intermittent cyclic loading. In the present study, we first expanded the investigation of solute transport using a larger tracer (parvalbumin, 12.3 kDa), which is comparable in size to some signaling proteins secreted by osteocytes. Murine tibiae were subjected to sequential FRAP tests under rest‐inserted cyclic loading while the loading magnitude (0, 2.8, or 4.8 N) and frequency (0.5, 1, or 2 Hz) were varied. The characteristic transport rate k and the transport enhancement relative to diffusion (k/k₀) were measured under each loading condition, from which the peak solute velocity in the LCS was derived using our LCS transport model. Both the transport enhancement and solute velocity increased with loading magnitude and decreased with loading frequency. Furthermore, the solute‐matrix interaction, quantified in terms of the reflection coefficient through the osteocytic pericellular matrix (PCM), was measured and theoretically modeled. The reflection coefficient of parvalbumin (σ = 0.084) was derived from the differential fluid and solute velocities within loaded bone. Using a newly developed PCM sieving model, the PCM's fiber configurations accounting for the measured interactions were obtained for the first time. The present study provided not only new data on the micro‐fluidic environment experienced by osteocytes in situ but also a powerful quantitative tool for future study of the PCM, the critical interface that controls both outside‐in and inside‐out signaling in osteocytes during normal bone adaptation and in pathological conditions. © 2013 American Society for Bone and Mineral Research.     

Long-Term Immobilization in Elderly Females Causes a Specific Pattern of Cortical Bone and Osteocyte Deterioration Different From Postmenopausal Osteoporosis

Immobilization as a result of long-term bed rest can lead to gradual bone loss. Because of their distribution throughout the bone matrix and remarkable interconnectivity, osteocytes represent the major mechanosensors in bone and translate mechanical into biochemical signals controlling bone remodeling. To test whether immobilization affects the characteristics of the osteocyte network in human cortical bone, femoral diaphyseal bone specimens were analyzed in immobilized female individuals and compared with age-matched postmenopausal individuals with primary osteoporosis. Premenopausal and postmenopausal healthy individuals served as control groups. Cortical porosity, osteocyte number and lacunar area, the frequency of hypermineralized lacunae, as well as cortical bone calcium content (CaMean) were assessed using bone histomorphometry and quantitative backscattered electron imaging (qBEI). Bone matrix properties were further analyzed by Fourier transform infrared spectroscopy (FTIR). In the immobilization group, cortical porosity was significantly higher, and qBEI revealed a trend toward higher matrix mineralization compared with osteoporotic individuals. Osteocyte density and canalicular density showed a declining rate from premenopausal toward healthy postmenopausal and osteoporotic individuals with peculiar reductions in the immobilization group, whereas the number of hypermineralized lacunae accumulated inversely. In conclusion, reduced osteocyte density and impaired connectivity during immobilization are associated with a specific bone loss pattern, reflecting a phenotype clearly distinguishable from postmenopausal osteoporosis. Immobilization periods may lead to a loss of survival signals for osteocytes, provoking bone loss that is even higher than in osteoporosis states, whereas osteocytic osteolysis remains absent. © 2020 The Authors. Journal of Bone and Mineral Research published by American Society for Bone and Mineral Research.     

Reduced Bone Mass and Increased Osteocyte Tartrate-Resistant Acid Phosphatase (TRAP) Activity,But Not Low Mineralized Matrix Around Osteocyte Lacunae,Are Restored After Recovery From Exogenous Hyperthyroidism in Male Mice

Hyperthyroidism causes secondary osteoporosis through favoring bone resorption over bone formation, leading to bone loss with elevated bone fragility. Osteocytes that reside within lacunae inside the mineralized bone matrix orchestrate the process of bone remodeling and can themselves actively resorb bone upon certain stimuli. Nevertheless, the interaction between thyroid hormones and osteocytes and the impact of hyperthyroidism on osteocyte cell function are still unknown. In a preliminary study, we analyzed bones from male C57BL/6 mice with drug-induced hyperthyroidism, which led to mild osteocytic osteolysis with 1.14-fold larger osteocyte lacunae and by 108.33% higher tartrate-resistant acid phosphatase (TRAP) activity in osteocytes of hyperthyroid mice compared to euthyroid mice. To test whether hyperthyroidism-induced bone changes are reversible, we rendered male mice hyperthyroid by adding levothyroxine into their drinking water for 4 weeks, followed by a weaning period of 4 weeks with access to normal drinking water. Hyperthyroid mice displayed cortical and trabecular bone loss due to high bone turnover, which recovered with weaning. Although canalicular number and osteocyte lacunar area were similar in euthyroid, hyperthyroid and weaned mice, the number of terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling (TUNEL)-positive osteocytes was 100% lower in the weaning group compared to euthyroid mice and the osteocytic TRAP activity was eightfold higher in hyperthyroid animals. The latter, along with a 3.75% lower average mineralization around the osteocyte lacunae in trabecular bone, suggests osteocytic osteolysis activity that, however, did not result in significantly enlarged osteocyte lacunae. In conclusion, we show a recovery of bone microarchitecture and turnover after reversal of hyperthyroidism to a euthyroid state. In contrast, osteocytic osteolysis was initiated in hyperthyroidism, but its effects were not reversed after 4 weeks of weaning. Due to the vast number of osteocytes in bone, we speculate that even minor individual cell functions might contribute to altered bone quality and mineral homeostasis in the setting of hyperthyroidism-induced bone disease. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).     

Real‐time measurement of solute transport within the lacunar‐canalicular system of mechanically loaded bone: Direct evidence for load‐induced fluid flow

Since proposed by Piekarski and Munro in 1977, load‐induced fluid flow through the bone lacunar‐canalicular system (LCS) has been accepted as critical for bone metabolism, mechanotransduction, and adaptation. However, direct unequivocal observation and quantification of load‐induced fluid and solute convection through the LCS have been lacking due to technical difficulties. Using a novel experimental approach based on fluorescence recovery after photobleaching (FRAP) and synchronized mechanical loading and imaging, we successfully quantified the diffusive and convective transport of a small fluorescent tracer (sodium fluorescein, 376 Da) in the bone LCS of adult male C57BL/6J mice. We demonstrated that cyclic end‐compression of the mouse tibia with a moderate loading magnitude (–3 N peak load or 400 µε surface strain at 0.5 Hz) and a 4‐second rest/imaging window inserted between adjacent load cycles significantly enhanced (+31%) the transport of sodium fluorescein through the LCS compared with diffusion alone. Using an anatomically based three‐compartment transport model, the peak canalicular fluid velocity in the loaded bone was predicted (60 µm/s), and the resulting peak shear stress at the osteocyte process membrane was estimated (～5 Pa). This study convincingly demonstrated the presence of load‐induced convection in mechanically loaded bone. The combined experimental and mathematical approach presented herein represents an important advance in quantifying the microfluidic environment experienced by osteocytes in situ and provides a foundation for further studying the mechanisms by which mechanical stimulation modulates osteocytic cellular responses, which will inform basic bone biology, clinical understanding of osteoporosis and bone loss, and the rational engineering of their treatments. © 2011 American Society for Bone and Mineral Research.     

Alterations in the osteocyte lacunar-canalicular microenvironment due to estrogen deficiency

While reduced estrogen levels have been shown to increase bone turnover and induce bone loss, there has been little analysis of the effects of diminished estrogen levels on the lacunar-canalicular porosity that houses the osteocytes. Alterations in the osteocyte lacunar-canalicular microenvironment may affect the osteocyte's ability to sense and translate mechanical signals, possibly contributing to bone degradation during osteoporosis. To investigate whether reduced estrogen levels affect the osteocyte microenvironment, this study used high-resolution microscopy techniques to assess the lacunar-canalicular microstructure in the rat ovariectomy (OVX) model of postmenopausal osteoporosis. Confocal microscopy analyses indicated that OVX rats had a larger effective lacunar-canalicular porosity surrounding osteocytes in both cortical and cancellous bone from the proximal tibial metaphysis, with little change in cortical bone from the diaphysis or cancellous bone from the epiphysis. The increase in the effective lacunar-canalicular porosity in the tibial metaphysis was not due to changes in osteocyte lacunar density, lacunar size, or the number of canaliculi per lacuna. Instead, the effective canalicular size measured using a small molecular weight tracer was larger in OVX rats compared to controls. Further analysis using scanning and transmission electron microscopy demonstrated that the larger effective canalicular size in the estrogen-deficient state was due to nanostructural matrix-mineral level differences like loose collagen surrounding osteocyte canaliculi. These matrix-mineral differences were also found in osteocyte lacunae in OVX, but the small surface changes did not significantly increase the effective lacunar size. The alterations in the lacunar-canalicular surface mineral or matrix environment appear to make OVX bone tissue more permeable to small molecules, potentially altering interstitial fluid flow around osteocytes during mechanical loading.     

Osteocytes remove and replace perilacunar mineral during reproductive cycles

Lactation is associated with an increased demand for calcium and is accompanied by a remarkable cycle of bone loss and recovery that helps to supply calcium and phosphorus for milk production. Bone loss is the result of increased bone resorption that is due, in part, to increased levels of PTHrP and decreased levels of estrogen. However, the regulation of bone turnover during this time is not fully understood. In the 1960s and 1970s many observations were made to suggest that osteocytes could resorb bone and increase the size of their lacunae. This concept became known as osteocytic osteolysis and studies suggested that it occurred in response to parathyroid hormone and/or an increased systemic demand for calcium. However, this concept fell out of favor in the late 1970s when it was established that osteoclasts were the principal bone-resorbing cells. Given that lactation is associated with increased PTHrP levels and negative calcium balance, we recently examined whether osteocytes contribute to bone loss during this time. Our findings suggest that osteocytes can remodel their perilacunar and pericanalicular matrix and that they participate in the liberation of skeletal calcium stores during reproductive cycles. These findings raise new questions about the role of osteocytes in coordinating bone and mineral metabolism during lactation as well as the recovery of bone mass after weaning. It is also interesting to consider whether osteocyte lacunar and canalicular remodeling contribute more broadly to the maintenance of skeletal and mineral homeostasis.This article is part of a Special Issue entitled "The Osteocyte".     

Demonstration of osteocytic perilacunar/canalicular remodeling in mice during lactation

Osteoclasts are thought to be solely responsible for the removal of bone matrix. However, we show here that osteocytes can also remove bone matrix by reversibly remodeling their perilacunar/canalicular matrix during the reproductive cycle. In contrast, no osteocytic remodeling was observed with experimental unloading despite similar degrees of bone loss. Gene array analysis of osteocytes from lactating animals revealed an elevation of genes known to be utilized by osteoclasts to remove bone, including tartrate‐resistant acid phosphatase (TRAP) and cathepsin K, that returned to virgin levels upon weaning. Infusion of parathyroid hormone–related peptide (PTHrP), known to be elevated during lactation, induced TRAP activity and cathepsin K expression in osteocytes concurrent with osteocytic remodeling. Conversely, animals lacking the parathyroid hormone type 1 receptor (PTHR1) in osteocytes failed to express TRAP or cathepsin K or to remodel their osteocyte perilacunar matrix during lactation. These studies show that osteocytes remove mineralized matrix through molecular mechanisms similar to those utilized by osteoclasts. © 2012 American Society for Bone and Mineral Research.     

A histological assessment on the distribution of the osteocytic lacunar canalicular system using silver staining

Giving the complexity that characterizes the mechanisms of bone remodeling and the number of events that have to be in absolute harmony for it to occur flawlessly, the postulation that temporospatial distribution of osteocytes and their lacunar canalicular system might influence and be influenced by bone remodeling can be regarded, at least, as feasible. In this study, using Schoen's silver staining, we have examined the distribution of the osteocytic lacunar canalicular system (OLCS) in bones of developing mice. Trabecular bones of 3-day-old, 2-week-old, and 3-week-old mice displayed osteocytic cytoplasmic processes without any perceptible alignment. Also, many plump osteocytes were embedded in the mineralized bone matrix in a disorderly manner. At 4 weeks of age, however, mice bones showed some osteocytic processes that reached the bone surface on a right angle, while other osteocytes displayed the same features seen on 3-week specimens. Samples at 8 weeks of age featured osteocytes with their usual spindle shape, organized so as to parallel the longitudinal axis of trabecular bone. They also extended their cytoplasmic processes perpendicularly to the bone surface. However, several osteocytes immersed in older bone, i.e., a residual mix of cartilage and bone matrices, still showed a random pattern of distribution of their cytoplasmic processes. Up to 12 weeks of age, the majority of the osteocytes became flattened and were shown to be aligned with their long axis paralleling the bone surface. This tendency for such a gradual arrangement was also observed in cortical bones. We have further demonstrated that 8-week-old osteoprotegerin-deficient mice, which demonstrated histological evidence of higher than average bone turnover, revealed a disorganized OLCS. Given the data gathered in this work, the OLCS appears to assume an organized, probably function-related spatial distribution as normal bone remodeling goes on.     

Decline in osteocyte lacunar density in human cortical bone is associated with accumulation of microcracks with age

Despite osteocytes' ideal position to sense the local environment and thereby influence bone remodeling, the function of osteocytes in bone remains controversial. In this study, histomorphometric examination of male and female femoral middiaphyseal cortical bone was conducted to determine if bone's remodeling response, indicated by tissue porosity and accumulation of damage, is associated with osteocyte lacunar density (number of osteocyte lacunae per bone area). The results support the sensory role of the osteocyte network as the decline in osteocyte lacunar density in human cortical bone is associated with the accumulation of microcracks and increase in porosity with age. Porosity and microcrack density increased exponentially with a decline in osteocyte lacunar density indicating that a certain minimum number of osteocytes is essential for an "operational" network. No gender-related differences were found in the relationship of osteocyte lacunar density to age, porosity, or microcrack density. The coefficient of variation of osteocyte lacunar density increased linearly with age, indicating that aging bone tissue is characterized by increased heterogeneity in the spatial organization of osteocytes. Osteocyte lacunar density, porosity, and microcrack density exhibited the same exponential probability density distribution in the donor population, indicating their regulation by similar biological phenomena.     

Intravital bone imaging by two-photon excitation microscopy to identify osteocytic osteolysis in vivo

Bone is a highly dynamic organ in which several cell types function cooperatively. Among these, osteocytes have recently emerged as an important regulator of bone homeostasis, although their mechanism of regulation is unclear. Here, intravital bone imaging by two-photon excitation microscopy allowed us to directly visualize ‘osteocytic osteolysis’, or resorption of bone in the lacuno-canalicular system. Osteocyte lacunae and the canalicular network in the cortex of murine tibiae were imaged by in vivo calcein staining, and local acidification in these structures was monitored using a topically applied pH sensor. We also demonstrated that sciatic neurectomy causes significant acidification around osteocytic lacunae and enlargement of lacuno-canalicular areas. These results provide strong evidence for osteocytic osteolysis, and demonstrate that two-photon intravital microscopy is useful for analysis of this phenomenon.     

Structural changes of the periosteocytic area inVipera aspis (L.) (Ophidia,viperidae) bone tissue in various physiological conditions

Summary Microradiographs of ribs and vertebrae of the snakeVipera aspis, over an annual cycle, show a significant enlargement of the osteocytic lacunae in the winter months and, for the breeding females, during the period of embryo development. This enlargement is due to resorption of bone substance (periosteocytic osteolysis). The objection that such morphological findings could as well be explained by the formation of new, larger osteocytes derived from recent osteoblasts does not apply to the present animal model. No internal bone remodelling occurs during the annual seasonal cycle and therefore no new osteoblasts would have differentiated to osteocytes in the interior of the bone. In the vertebrae, an additional process is indicated as an area of decreased mineral density, termed demineralization halo, around the periosteocytic lacunae. An electron miscroscopy study suggests that this process of demineralization is not the first stage of periosteocytic resorption, but an additional process of demineralization. Thus, both osteolysis and demineralization halos in the perilacunar osteocytic region of the bone tissue represent reversible biological processes mediated by the osteocytes.     

Do osteocytes contribute to bone mineral homeostasis? Osteocytic osteolysis revisited

Osteocytes are cells buried in the bone matrix. They largely contribute to the regulation of bone remodeling in response to mechanical and microenvironmental changes. Much has been recognized in recent years regarding the role of osteocytes in bone homeostasis, nevertheless their ability to directly contribute to mineral equilibrium has been neglected. In the light of the renewed interest in their biology, we revisited the literature and discuss experimental evidence favoring the hypothesis that osteocytes are able to remove and replace the bone matrix according to the systemic needs of the body. We also reviewed reports against this theory, thus providing current views of what is known so far on the ability of osteocytes to mobilize bone mineral. This re-examination of osteocytic osteolysis might stimulate new interest and open new perspectives in osteocyte biology and in the cellular mechanisms that control bone homeostasis.     

Parallel mechanisms suppress cochlear bone remodeling to protect hearing

Bone remodeling, a combination of bone resorption and formation, requires precise regulation of cellular and molecular signaling to maintain proper bone quality. Whereas osteoblasts deposit and osteoclasts resorb bone matrix, osteocytes both dynamically resorb and replace perilacunar bone matrix. Osteocytes secrete proteases like matrix metalloproteinase-13 (MMP13) to maintain the material quality of bone matrix through perilacunar remodeling (PLR). Deregulated bone remodeling impairs bone quality and can compromise hearing since the auditory transduction mechanism is within bone. Understanding the mechanisms regulating cochlear bone provides unique ways to assess bone quality independent of other aspects that contribute to bone mechanical behavior. Cochlear bone is singular in its regulation of remodeling by expressing high levels of osteoprotegerin. Since cochlear bone expresses a key PLR enzyme, MMP13, we examined whether cochlear bone relies on, or is protected from, osteocyte-mediated PLR to maintain hearing and bone quality using a mouse model lacking MMP13 (MMP13^−/−). We investigated the canalicular network, collagen organization, lacunar volume via micro-computed tomography, and dynamic histomorphometry. Despite finding defects in these hallmarks of PLR in MMP13^−/− long bones, cochlear bone revealed no differences in these markers, nor hearing loss as measured by auditory brainstem response (ABR) or distortion product oto-acoustic emissions (DPOAEs), between wild type and MMP13^−/− mice. Dynamic histomorphometry revealed abundant PLR by tibial osteocytes, but near absence in cochlear bone. Cochlear suppression of PLR corresponds to repression of several key PLR genes in the cochlea relative to long bones. These data suggest that cochlear bone uniquely maintains bone quality and hearing independent of MMP13-mediated osteocytic PLR. Furthermore, the cochlea employs parallel mechanisms to inhibit remodeling by osteoclasts and osteoblasts, and by osteocytes, to protect hearing. Understanding the cellular and molecular mechanisms that confer site-specific control of bone remodeling has the potential to elucidate new pathways that are deregulated in skeletal disease.     

Effects of Vitamin K2 on Cortical and Cancellous Bone Mass, Cortical Osteocyte and Lacunar System, and Porosity in Sciatic Neurectomized Rats

The purpose of the present study was to examine the effects of vitamin K₂ on cortical and cancellous bone mass, cortical osteocyte and lacunar system, and porosity in sciatic neurectomized rats. Thirty-four female Sprague-Dawley retired breeder rats were randomized into three groups: age-matched control, sciatic neurectomy (NX), and NX + vitamin K₂ administration (menatetrenone, 30 mg/kg/day p.o., three times a week). At the end of the 8-week experiment, bone histomorphometric analysis was performed on cortical and cancellous bone of the tibial diaphysis and proximal metaphysis, respectively, and osteocyte lacunar system and porosity were evaluated on cortical bone of the tibial diaphysis. NX decreased cortical and cancellous bone mass compared with age-matched controls as a result of increased endocortical and trabecular bone erosion and decreased trabecular mineral apposition rate (MAR). Vitamin K₂ ameliorated the NX-induced increase in bone erosion, prevented the NX-induced decrease in MAR, and increased bone formation rate (BFR/bone surface) in cancellous bone, resulting in an attenuation of NX-induced cancellous bone loss. However, vitamin K₂ did not significantly influence cortical bone mass. NX also decreased osteocyte density and lacunar occupancy and increased porosity in cortical bone compared with age-matched controls. Vitamin K₂ ameliorated the NX-induced decrease in lacunar occupancy by viable osteocytes and the NX-induced increase in porosity. The present study showed the efficacy of vitamin K₂ for cancellous bone mass and cortical lacunar occupancy by viable osteocytes and porosity in sciatic NX rats.     

Anatomic variations of the lacunar–canalicular system influence solute transport in bone

Solute transport in the lacunar–canalicular system (LCS) is essential for bone metabolism and mechanotransduction. Using the technique of fluorescence recovery after photobleaching (FRAP) we have been quantifying solute transport in the LCS of murine long bone as a function of loading parameters and molecular size. However, the influence of LCS anatomy, which varies among animal species, bone type and location, age and health condition, is not well understood. In this study, we developed a mathematical model to simulate solute convection in the LCS during a FRAP experiment under a physiological cyclic flow. We found that the transport rate (the reciprocal time constant for refilling the photobleached lacuna) increased linearly with canalicular number and decreased with canalicular length for both diffusion and convection. As a result, the transport enhancement of convection over diffusion was much less sensitive to the variations associated with chick, mouse, rabbit, bovine, dog, horse, and human LCS anatomy, when compared with the rates of diffusion or convection alone. Canalicular density did not affect transport enhancement, while solute size and the lacunar density had more complicated, non-linear effects. This parametric study suggests that solute transport could be altered by varying LCS parameters, and that the anatomical details of the LCS need systemic examination to further understand the etiology of aged and osteoporotic bones.     

Osteocytic osteolysis observed in rats to which parathyroid hormone was continuously administered

In order to prove osteocytic osteolysis in vivo, human parathyroid hormone (hPTH (1–34), 749ng/h), or only solvent of the same volume, was continuously administered to 8-month-old rats by an infusion pump for 4 weeks, and then structural changes in osteocytes in the cortical bones of the tibiae were analyzed morphometrically, histologically, and histochemically. Based on contact microradiography (CMR) observations, the osteocyte lacunae in the PTH group tended to be enlarged, compared with those of the control, while the average lacuna area was 137.0µm² in the PTH group versus 93.9µm² in the control, suggesting evidence of osteocytic osteolysis. Acid phosphatase enzyme histochemical localization was observed in some osteocytes in the PTH group; therefore, lysosome systems may participate in the osteolytic mechanisms. On histological samples stained with hematoxylin-eosin or toluidine blue, the lacunae of the controls were surrounded by narrow areas of matrices both positive for hematoxylin and metachromatic for toluidine blue, while belt-like areas positive for hematoxylin were observed around the PTH-group lacunae. These findings suggested that, after osteocytic osteolysis, regenerated bone matrices may be added to the walls of osteocytes that possess enlarged lacunae.     

Defective cancellous bone structure and abnormal response to PTH in cortical bone of mice lacking Cx43 cytoplasmic C-terminus domain

Connexin 43 (Cx43) forms gap junction channels and hemichannels that allow the communication among osteocytes, osteoblasts, and osteoclasts. Cx43 carboxy-terminal (CT) domain regulates channel opening and intracellular signaling by acting as a scaffold for structural and signaling proteins. To determine the role of Cx43 CT domain in bone, mice in which one allele of full length Cx43 was replaced by a mutant lacking the CT domain (Cx43^ΔCT/fl) were studied. Cx43^ΔCT/fl mice exhibit lower cancellous bone volume but higher cortical thickness than Cx43^fl/fl controls, indicating that the CT domain is involved in normal cancellous bone gain but opposes cortical bone acquisition. Further, Cx43^ΔCT is able to exert the functions of full length osteocytic Cx43 on cortical bone geometry and mechanical properties, demonstrating that domains other than the CT are responsible for Cx43 function in cortical bone. In addition, parathyroid hormone (PTH) failed to increase endocortical bone formation or energy to failure, a mechanical property that indicates resistance to fracture, in cortical bone in Cx43^ΔCT mice with or without osteocytic full length Cx43. On the other hand, bone mass and bone formation markers were increased by the hormone in all mouse models, regardless of whether full length or Cx43^ΔCT were or not expressed. We conclude that Cx43 CT domain is involved in proper bone acquisition; and that Cx43 expression in osteocytes is dispensable for some but not all PTH anabolic actions.     

Perlecan‐Containing Pericellular Matrix Regulates Solute Transport and Mechanosensing Within the Osteocyte Lacunar‐Canalicular System

The pericellular matrix (PCM), a thin coating surrounding nearly all mammalian cells, plays a critical role in many cell‐surface phenomena. In osteocytes, the PCM is believed to control both “outside‐in” (mechanosensing) and “inside‐out” (signaling molecule transport) processes. However, the osteocytic PCM is challenging to study in situ because it is thin (～100 nm) and enclosed in mineralized matrix. To this end, we recently developed a novel tracer velocimetry approach that combined fluorescence recovery after photobleaching (FRAP) imaging with hydrodynamic modeling to quantify the osteocytic PCM in young murine bone. In this study, we applied the technique to older mice expressing or deficient for perlecan/HSPG2, a large heparan‐sulfate proteoglycan normally secreted in osteocytic PCM. The objectives were (1) to characterize transport within an altered PCM; (2) to test the sensitivity of our approach in detecting the PCM alterations; and (3) to dissect the roles of the PCM in osteocyte mechanosensing. We found that: (1) solute transport increases in the perlecan‐deficient (hypomorphic [Hypo]) mice compared with control mice; (2) PCM fiber density decreases with aging and perlecan deficiency; (3) osteocytes in the Hypo bones are predicted to experience higher shear stress (+34%), but decreased fluid drag force (?35%) under 3‐N peak tibial loading; and (4) when subjected to tibial loading in a preliminary in vivo experiment, the Hypo mice did not respond to the anabolic stimuli as the CTL mice did. These findings support the hypothesis that the PCM fibers act as osteocyte's sensing antennae, regulating load‐induced cellular stimulations and thus bone's sensitivity and in vivo bone adaptation. If this hypothesis is further confirmed, osteocytic PCM could be new targets to develop osteoporosis treatments by modulating bone's intrinsic sensitivity to mechanical loading and be used to design patient‐specific exercise regimens to promote bone formation. © 2014 American Society for Bone and Mineral Research.     

Glucocorticoid-treated mice have localized changes in trabecular bone material properties and osteocyte lacunar size that are not observed in placebo-treated or estrogen-deficient mice.

This study compares changes in bone microstructure in 6-month-old male GC-treated and female ovariectomized mice to their respective controls. In addition to a reduction in trabecular bone volume, GC treatment reduced bone mineral and elastic modulus of bone adjacent to osteocytes that was not observed in control mice nor estrogen-deficient mice. These microstructural changes in combination with the macrostructural changes could amplify the bone fragility in this metabolic bone disease. INTRODUCTION: Patients with glucocorticoid (GC)-induced secondary osteoporosis tend to fracture at higher bone mineral densities than patients with postmenopausal osteoporosis. This suggests that GCs may alter bone material properties in addition to BMD and bone macrostructure. MATERIALS AND METHODS: Changes in trabecular bone structure, elastic modulus, and mineral to matrix ratio of the fifth lumbar vertebrae was assessed in prednisolone-treated mice and placebo-treated controls for comparison with estrogen-deficient mice and sham-operated controls. Compression testing of the third lumbar vertebrae was performed to assess whole bone strength. RESULTS: Significant reductions in trabecular bone volume and whole bone strength occurred in both prednisolone-treated and estrogen-deficient mice compared with controls after 21 days (p < 0.05). The average elastic modulus over the entire surface of each trabecula was similar in all the experimental groups. However, localized changes within the trabeculae in areas surrounding the osteocyte lacunae were observed only in the prednisolone-treated mice. The size of the osteocyte lacunae was increased, reduced elastic modulus around the lacunae was observed, and a "halo" of hypomineralized bone surrounding the lacunae was observed. This was associated with reduced (nearly 40%) mineral to matrix ratio determined by Raman microspectroscopy. These localized changes in elastic modulus and bone mineral to matrix ratio were not observed in the other three experimental groups. CONCLUSIONS: Based on these results, it seems that GCs may have direct effects on osteocytes, resulting in a modification of their microenvironment. These changes, including an enlargement of their lacunar space and the generation of a surrounding sphere of hypomineralized bone, seem to produce highly localized changes in bone material properties that may influence fracture risk.