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

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

Crosstalk within a brain-breast-bone axis regulates mineral and skeletal metabolism during lactation.

To support the increased calcium demands for milk production during lactation, a dramatic and reversible physiological response occurs to alter bone and mineral metabolism. This coordinated process involves a brain-breast-bone axis that integrates hormonal signals that allow for adequate calcium delivery to milk yet also protects the maternal skeletal from excessive bone loss or decreases in bone quality or function. Here, we review the current knowledge on the crosstalk between the hypothalamus, mammary gland, and skeleton during lactation. We discuss the rare entity of pregnancy and lactation associated osteoporosis and consider how the physiology of bone turnover in lactation may impact the pathophysiology of postmenopausal osteoporosis. Further understanding of the regulators of bone loss during lactation, particularly in humans, may provide insights into new therapies for osteoporosis and other diseases of excess bone loss.

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).

Histochemical assessment on osteocytic osteolysis in lactating mice fed with a calcium-insufficient diet.

OBJECTIVES: This study aimed to examine if feeding lactating mice a calcium-insufficient diet while simultaneously administering alendronate (ALN) could potentially induce osteocytic osteolysis. METHODS: Lactating mice were fed calcium (Ca)-insufficient diets with or without ALN administration, and then their femurs were examined for TRAP and ALP, and observed by Kossa staining and transmission electron microscopy (TEM). Mice that had been fed a Ca-insufficient diet were then fed a 44Ca-containing diet, and their tibial sections were examined by isotope microscopy. RESULTS: Mice fed a Ca-insufficient diet had a reduced number of TRAP-positive osteoclasts after ALN administration. ALN-treated, lactating mice fed a Ca-insufficient diet had enlarged lacunae in their cortical bones, and TEM imaging demonstrated expanded regions between osteocytes and lacunar walls. In ALN-treated lactating mice fed a Ca-insufficient diet, huge areas of demineralized bone matrix occurred, centered around blood vessels in the cortical bone. Isotope microscopy showed 44Ca in the vicinity of the osteocytic lacunae, and in the broad, previously demineralized region around the blood vessels in the cortical bone of lactating mice fed a 44Ca-sufficient diet. CONCLUSIONS: Bone demineralization likely takes place in the periphery of the osteocytic lacunae and in the broad regions around the blood vessels of lactating mice when they are exposed to severely reduced serum Ca through a Ca-insufficient diet coupled with ALN administration.

Osteocytes and Cancer.

PURPOSE OF REVIEW: While the function of osteocytes under physiologic conditions is well defined, their role and involvement in cancer disease remains relatively unexplored, especially in a context of non-bone metastatic cancer. This review will focus on describing the more advanced knowledge regarding the interactions between osteocytes and cancer. RECENT FINDINGS: We will discuss the involvement of osteocytes in the onset and progression of osteosarcoma, with the common bone cancers, as well as the interaction that is established between osteocytes and multiple myeloma. Mechanisms responsible for cancer dissemination to bone, as frequently occur with advanced breast and prostate cancers, will be reviewed. While a role for osteocytes in the stimulation and proliferation of cancer cells has been reported, protective effects of osteocytes against bone colonization have been described as well, thus increasing ambiguity regarding the role of osteocytes in cancer progression and dissemination. Lastly, supporting the idea that skeletal defects can occur also in the absence of direct cancer dissemination or osteolytic lesions directly adjacent to the bone, our recent findings will be presented showing that in the absence of bone metastases, the bone microenvironment and, particularly, osteocytes, can manifest a clear and dramatic response to the distant, non-metastatic tumor. Our observations support new studies to clarify whether treatments designed to preserve the osteocytes can be combined with traditional anticancer therapies, even when bone is not directly affected by tumor growth.

Non-bone metastatic cancers promote osteocyte-induced bone destruction.

The effects of bone metastatic cancer on the skeleton are well described, whereas less is known regarding the effects of non-metastatic bone cancer on bone. Here we investigated the effects of three non-bone metastatic cancer cachexia models, namely Colon-26 adenocarcinoma (C26), ES-2 ovarian cancer (ES-2), and Lewis lung carcinoma (LLC). Even though C26, ES-2 and LLC tumor growth resulted in comparable weight and muscle loss, the ES-2 and LLC hosts exhibited severe bone loss, whereas only modest bone loss was observed in the C26 bearers, correlating with increased TRAP+ osteoclasts in the femurs of ES-2 and LLC but not C26 hosts. Surprisingly, all three showed increased osteocyte lacunar area indicating osteocytic osteolysis and displayed dramatically increased osteocyte death, as well as empty lacunae. To test whether tumor-secreted factors were responsible for the observed effect, IDG-SW3 osteocyte cells were co-cultured with cancer cells in permeable trans-wells. Apoptosis was observed in the osteocyte cells exposed to all three cancer cell lines suggesting that all tumors were cytotoxic for osteocytes. In addition, the expression of the osteoclastic markers, Acp5, CtsK, Atp6v0d2 and Mmp13, was elevated in IDG-SW3 osteocytes exposed to tumor factors, supporting the in vivo observations of increased lacunar size due to osteocytic osteolysis. For the first time, we describe osteocytic bone destruction and extensive osteocyte cell death in non-bone metastatic cancer. These bone alterations, in conjunction with muscle wasting, may create a musculoskeletal system that is incapable of full recovery upon eradication of tumor. Co-treatment with bone preserving therapies should be considered.

Potential Role of Perilacunar Remodeling in the Progression of Osteoporosis and Implications on Age-Related Decline in Fracture Resistance of Bone.

PURPOSE OF REVIEW: We took an interdisciplinary view to examine the potential contribution of perilacunar/canalicular remodeling to declines in bone fracture resistance related to age or progression of osteoporosis. RECENT FINDINGS: Perilacunar remodeling is most prominent as a result of lactation; recent advances further elucidate the molecular players involved and their effect on bone material properties. Of these, vitamin D and calcitonin could be active during aging or osteoporosis. Menopause-related hormonal changes or osteoporosis therapies affect bone material properties and mechanical behavior. However, investigations of lacunar size or osteocyte TRAP activity with age or osteoporosis do not provide clear evidence for or against perilacunar remodeling. While the occurrence and potential role of perilacunar remodeling in aging and osteoporosis progression are largely under-investigated, widespread changes in bone matrix composition in OVX models and following osteoporosis therapies imply osteocytic maintenance of bone matrix. Perilacunar remodeling-induced changes in bone porosity, bone matrix composition, and bone adaptation could have significant implications for bone fracture resistance.

No Role of Osteocytic Osteolysis in the Development and Recovery of the Bone Phenotype Induced by Severe Secondary Hyperparathyroidism in Vitamin D Receptor Deficient Mice.

Osteocytic osteolysis/perilacunar remodeling is thought to contribute to the maintenance of mineral homeostasis. Here, we utilized a reversible, adult-onset model of secondary hyperparathyroidism to study femoral bone mineralization density distribution (BMDD) and osteocyte lacunae sections (OLS) based on quantitative backscattered electron imaging. Male mice with a non-functioning vitamin D receptor (VDRΔ/Δ) or wild-type mice were exposed to a rescue diet (RD) (baseline) and subsequently to a low calcium challenge diet (CD). Thereafter, VDRΔ/Δ mice received either the CD, a normal diet (ND), or the RD. At baseline, BMDD and OLS characteristics were similar in VDRΔ/Δ and wild-type mice. The CD induced large cortical pores, osteomalacia, and a reduced epiphyseal average degree of mineralization in the VDRΔ/Δ mice relative to the baseline (-9.5%, p < 0.05 after two months and -10.3%, p < 0.01 after five months of the CD). Switching VDRΔ/Δ mice on the CD back to the RD fully restored BMDD to baseline values. However, OLS remained unchanged in all groups of mice, independent of diet. We conclude that adult VDRΔ/Δ animals on an RD lack any skeletal abnormalities, suggesting that VDR signaling is dispensable for normal bone mineralization as long as mineral homeostasis is normal. Our findings also indicate that VDRΔ/Δ mice attempt to correct a calcium challenge by enhanced osteoclastic resorption rather than by osteocytic osteolysis.

Osteocytic Osteolysis in PTH-treated Wild-type and Rankl-/- Mice Examined by Transmission Electron Microscopy, Atomic Force Microscopy, and Isotope Microscopy.

To demonstrate the ultrastructure of osteocytic osteolysis and clarify whether osteocytic osteolysis occurs independently of osteoclastic activities, we examined osteocytes and their lacunae in the femora and tibiae of 11-week-old male wild-type and Rankl-/- mice after injection of human parathyroid hormone (PTH) [1-34] (80 µg/kg/dose). Serum calcium concentration rose temporarily 1 hr after PTH administration in wild-type and Rankl-/- mice, when renal arteries and veins were ligated. After 6 hr, enlargement of osteocytic lacunae was evident in the cortical bones of wild-type and Rankl-/- mice, but not so in their metaphyses. Von Kossa staining and transmission electron microscopy showed broadly demineralized bone matrix peripheral to enlarged osteocytic lacunae, which contained fragmented collagen fibrils and islets of mineralized matrices. Nano-indentation by atomic force microscopy revealed the reduced elastic modulus of the PTH-treated osteocytic perilacunar matrix, despite the microscopic verification of mineralized matrix in that region. In addition, 44Ca deposition was detected by isotope microscopy and calcein labeling in the eroded osteocytic lacunae of wild-type and Rankl-/- mice. Taken together, our findings suggest that osteocytes can erode the bone matrix around them and deposit minerals on their lacunar walls independently of osteoclastic activity, at least in the murine cortical bone. (J Histochem Cytochem 68: -XXX, 2020).

Large osteocyte lacunae in iliac crest infantile bone are not associated with impaired mineral distribution or signs of osteocytic osteolysis.

The enlargement of osteocyte lacunae via osteocytic osteolysis was previously detected in situations of increased calcium demand (e.g., lactation, vitamin D deficiency). However, it is unclear whether similar processes occur also in the growing infantile skeleton and how this is linked to the mineral distribution within the bone matrix. Human iliac crest biopsies of 30 subjects (0-6 months, n = 14; 2-8 years, n = 6 and 18-25 years, n = 10) were acquired. Bone microarchitecture was assessed by micro-CT, while cellular bone histomorphometry was performed on undecalcified histological sections. Quantitative backscattered electron imaging (qBEI) was conducted to determine the bone mineral density distribution (BMDD) as well as osteocyte lacunar size and density. We additionally evaluated cathepsin K positive osteocytes using immunohistochemistry. Infantile bone was characterized by various signs of ongoing bone development such as higher bone (re)modeling, lower cortical and trabecular thickness compared to young adults. Importantly, a significantly higher osteocyte lacunar density and increased lacunar area were detected. Large osteocyte lacunae were associated with a more heterogeneous bone mineral density distribution of the trabecular bone matrix due to the presence of hypermineralized cartilage remnants, whereas the mean mineralization (i.e., CaMean) was not different in infantile bone. Absence of cathepsin K expression in osteocyte lacunae indicated nonexistent osteocytic osteolysis. Taken together, we demonstrated that the overall mineralization distribution in infantile bone is not altered compared to young adults besides high trabecular mineralization heterogeneity. Our study also provides important reference values for bone microstructure, BMDD and osteocyte characteristics in infants, children and young adults. Infantile bone displays large osteocyte lacunae indicating a developmental phenomenon rather than osteocytic osteolysis. Larger osteocytes may have superior mechanosensory abilities to enable bone adaption during growth.

Evidence for Gender-Specific Bone Loss Mechanisms in Periprosthetic Osteolysis.

Osteolysis adjacent to total hip replacement (THR) prostheses is a major cause of their eventual failure. Periprosthetic osteolysis is associated with the production of bioactive particles, produced by the wear of articulating prosthesis surfaces. Wear particles invade the periprosthetic tissue, inducing inflammation and bone resorption. Previous studies have shown that osteocytes, the most numerous cell type in mineralised bone, can respond to wear particles of multiple orthopaedic material types. Osteocytes play important roles in bone resorption, regulating bone resorption by osteoclasts and directly through osteocytic osteolysis, also known as perilacunar remodelling. In this study, we perform a histological analysis of bone biopsies obtained from cohorts of male and female patients undergoing either primary THR surgery or revision THR surgery for aseptic loosening. The osteocyte lacunae area (Ot.Lac.Ar) and percentage lacunar area/bone area (%Ot.Lac.Ar/B.Ar) were significantly larger overall in revision THR bone than bone from similar sites in primary THR. Analysis by patient gender showed that increased Ot.Lac.Ar, indicative of increased perilacunar remodelling, was restricted to female revision samples. No significant differences in osteoclast parameters were detectable between the cohorts. These findings suggest previously unrecognised gender-specific mechanisms of bone loss in orthopaedic wear particle-induced osteolysis in humans.

No Signature of Osteocytic Osteolysis in Cortical Bone from Lactating NMRI Mice.

The roles of osteocytes in bone homeostasis have garnered increasing attention since it has been realized that osteocytes communicate with other organs. It has long been debated whether and/or to which degree osteocytes can break down the bone matrix surrounding them in a process called osteocytic osteolysis. Osteocytic osteolysis has been indicated to be induced by a number of skeletal challenges including lactation in CD1 and C57BL/6 mice, whereas immobilization-induced osteocytic osteolysis is still a matter of controversy. Motivated by the wish to understand this process better, we studied osteocyte lacunae in lactating NMRI mice, which is a widely used outbred mouse strain. Surprisingly, no trace of osteocytic osteolysis could be detected in tibial or femoral cortical bone either by 3D investigation by synchrotron nanotomography, by studies of lacunar cross-sectional areas using scanning electron microscopy, or by light microscopy. These results lead us to conclude that osteocytic osteolysis does not occur in NMRI mice as a response to lactation, in turn suggesting that osteocytic osteolysis may not play a generic role in mobilizing calcium during lactation.

The contribution of the pericanalicular matrix to mineral content in human osteonal bone.

The osteocyte lacunar-canalicular network (LCN) penetrates bone and houses the osteocytes and their processes. Despite its rather low volume fraction, the LCN represents an outstanding large surface that is possibly used by the osteocytes to interact with the surrounding mineralized bone matrix thereby contributing to mineral homeostasis. The aim of this study was to quantitatively describe such contributions by spatially correlating the local density of the LCN with the mineral content at the same location in micrometer-sized volume elements in human osteons. For this purpose, 65 osteons from the femur midshaft from healthy adults (n = 4) and children (n = 2) were structurally characterized with two different techniques. The 3D structure of the LCN in the osteons was imaged with confocal laser scanning microscopy after staining the bone samples with rhodamine. Subsequent image analysis provided the canalicular length density, i.e. the total length of the canaliculi per unit volume (µm/µm3). Quantitative information on the mineral content (wt%Ca) from the identical regions was obtained using quantitative backscattered electron imaging. As the LCN-porosity lowers the mineral content, a negative correlation between Ca content and network density was expected. Calculations predict a reduction of around -0.97 fmol Ca per µm of network. However, the experiment revealed for 62 out of 65 osteons a positive correlation resulting in an average additional Ca loading of +1.15 fmol per µm of canalicular network, i.e. an accumulation of mineral has occurred at dense network regions. We hypothesize that this accumulation happens in the close vicinity of canaliculi forming mineral reservoirs that can be utilized by osteocytes. Significant differences found between individuals indicate that the extent of mineral loading of the reservoir zone reflects an important parameter for mineral homeostasis.

Recombinant sclerostin antagonizes effects of ex vivo mechanical loading in trabecular bone and increases osteocyte lacunar size.

Sclerostin has emerged as an important regulator of bone mass. We have shown that sclerostin can act by targeting late osteoblasts/osteocytes to inhibit bone mineralization and to upregulate osteocyte expression of catabolic factors, resulting in osteocytic osteolysis. Here we sought to examine the effect of exogenous sclerostin on osteocytes in trabecular bone mechanically loaded ex vivo. Bovine trabecular bone cores, with bone marrow removed, were inserted into individual chambers and subjected to daily episodes of dynamic loading. Cores were perfused with either osteogenic media alone or media containing human recombinant sclerostin (rhSCL) (50 ng/ml). Loaded control bone increased in apparent stiffness over time compared with unloaded bone, and this was abrogated in the presence of rhSCL. Loaded bone showed an increase in calcein uptake as a surrogate of mineral accretion, compared with unloaded bone, in which this was substantially inhibited by rhSCL treatment. Sclerostin treatment induced a significant increase in the ionized calcium concentration in the perfusate and the release of ß-CTX at several time points, an increased mean osteocyte lacunar size, indicative of osteocytic osteolysis, and the expression of catabolism-related genes. Human primary osteocyte-like cultures treated with rhSCL also released ß-CTX from their matrix. These results suggest that osteocytes contribute directly to bone mineral accretion, and to the mechanical properties of bone. Moreover, it appears that sclerostin, acting on osteocytes, can negate this effect by modulating the dimensions of the lacunocanalicular porosity and the composition of the periosteocyte matrix.

The Skeleton Is a Storehouse of Mineral That Is Plundered During Lactation and (Fully?) Replenished Afterwards.

During lactation, mammals resorb mineral from the maternal skeleton to provide calcium to milk. Rodents lose 25% to 35% of skeletal ash weight, ash calcium content, and bone mineral content as measured by dual-energy X-ray absorptiometry (DXA), and have compromised material properties of bone as assessed by crushing vertebrae and 3-point bend tests of femora or tibias. The strength, stiffness, and toughness of vertebrae, femora, and tibias are reduced by as much as 60%. The effects of lactation are not uniform throughout the skeleton, but instead resorption is much more marked in the trabecular-rich spine than in the appendicular skeleton or whole body. Women who breastfeed exclusively lose an average of 210 mg calcium in milk each day, whereas nursing of twins or triplets can double and triple the output of calcium. Clinical data are also consistent with skeletal calcium being released during lactation to provide much of the calcium needed for milk production. Lumbar spine bone mineral density (BMD), as assessed by DXA, declines by a mean of 5% to 10% among numerous studies during 3 to 6 months of exclusive lactation, whereas largely cortical sites (hip, forearm, whole body) show half that loss or no significant changes. Micro-CT of rodents and high-resolution peripheral quantitative computed tomography (HR-pQCT) imaging of women confirm that lactation causes microarchitectural deterioration of bone. These skeletal losses occur through two pathways: upregulated osteoclast-mediated bone resorption and osteocytic osteolysis, in which osteocytes remove mineral from their lacunae and pericanalicular spaces. After weaning, the skeleton is fully restored to its prior mineral content and strength in both animal models and humans, despite persistent microarchitectural changes observed in high-resolution imaging. Osteoblasts upregulate to lay down new osteoid, while osteocytes remineralize their surroundings. The factors that stimulate this post-weaning skeletal recovery remain unclear. In most studies, a history of lactation does not increase the risk, but may protect against, low BMD and fragility fractures. © 2017 American Society for Bone and Mineral Research.

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.

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 (28weeks) 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.

Evidence that osteocyte perilacunar remodelling contributes to polyethylene wear particle induced osteolysis.

Periprosthetic osteolysis (PO) leading to aseptic loosening, is the most common cause of failure of total hip replacement (THR) in the mid- to long-term. Polyethylene (PE) particulates from the wear of prosthesis liners are bioactive and are implicated in the initiation and or progression of osteolysis. Evidence exists that cells of the osteoblast/osteocyte lineage are affected by PE particles and contribute to the catabolic response by promoting osteoclastic bone resorption. In this study, we hypothesised that osteocytes contribute directly to PO by removing bone from their perilacunar matrix. Osteocyte responses to ultra-high molecular weight PE (UHMWPE) particles were examined in vitro in human primary osteocyte-like cultures, in vivo in the mouse calvarial osteolysis model, and in the acetabulum of patients undergoing revision total hip replacement (THR) surgery for PO. Osteocytes exposed to UHMWPE particles showed upregulated expression of catabolic markers, MMP-13, carbonic anhydrase 2 (CA2), cathepsin K (CTSK) and tartrate resistant acid phosphatase (TRAP), with no effect on cell viability, as assessed by Caspase 3 activity. Consistent with this catabolic activity causing perilacunar bone loss, histological analysis of calvarial sections from mice exposed to UHMWPE revealed a significant (p<0.001) increase in osteocyte lacunar area (Lac.Ar) compared to sham-operated animals. Furthermore, acetabular biopsies from patients with PO also showed significantly (p<0.001) increased osteocyte lacunar size in trabecular bone adjacent to PE particles, compared with osteocyte lacunar size in bone from primary THR patients. Together, these findings suggest a previously unrecognised action of UHMWPE wear particles on osteocytes, which directly results in a loss of osteocyte perilacunar bone. This action may exacerbate the indirect pro-osteoclastic action of UHMWPE-affected osteocytes, previously shown to contribute to aseptic loosening of orthopaedic implants. STATEMENT OF SIGNIFICANCE: This study addresses the clinical problem of periprosthetic osteolysis, bone loss in response to polyethylene wear particles derived from materials used in orthopaedic implants. Periprosthetic osteolysis has been thought to be due largely to wear particles stimulating the activity of bone resorbing osteoclasts. However, in this study we demonstrate for the first time that polyethylene particles stimulate another type of bone loss, mediated by the direct activity of bone mineral embedded osteocytes, termed osteocytic osteolysis or osteocyte perilacunar remodelling. This study provides new mechanistic insight into wear-particle mediated bone loss and represents a new paradigm for the way in which bone cells, namely osteocytes, the key controlling cell type in bone, react to biomaterials.

Osteocyte-directed bone demineralization along canaliculi.

The mammalian skeleton stores calcium and phosphate ions in bone matrix. Osteocytes in osteocyte lacunae extend numerous dendrites into canaliculi less than a micron in diameter and which are distributed throughout bone matrix. Although osteoclasts are the primary bone-resorbing cells, osteocytes also reportedly dissolve hydroxyapatite at peri-lacunar bone matrix. However, robust three-dimensional evidence for peri-canalicular bone mineral dissolution has been lacking. Here we applied a previously reported Talbot-defocus multiscan tomography method for synchrotron X-ray microscopy and analyzed the degree of bone mineralization in mouse cortical bone around the lacuno-canalicular network, which is connected both to blood vessels and the peri- and endosteum. We detected cylindrical low mineral density regions spreading around canaliculi derived from a subset of osteocytes. Transmission electron microscopy revealed both intact and demineralized bone matrix around the canaliculus. Peri-canalicular low mineral density regions were also observed in osteopetrotic mice lacking osteoclasts, indicating that osteoclasts are dispensable for peri-canalicular demineralization. These data suggest demineralization can occur from within bone through the canalicular system, and that peri-canalicular demineralization occurs not uniformly but directed by individual osteocytes. Blockade of peri-canalicular demineralization may be a therapeutic strategy to increase bone mass and quality.

Neural crest-mediated bone resorption is a determinant of species-specific jaw length.

Precise control of jaw length during development is crucial for proper form and function. Previously we have shown that in birds, neural crest mesenchyme (NCM) confers species-specific size and shape to the beak by regulating molecular and histological programs for the induction and deposition of cartilage and bone. Here we reveal that a hitherto unrecognized but similarly essential mechanism for establishing jaw length is the ability of NCM to mediate bone resorption. Osteoclasts are considered the predominant cells that resorb bone, although osteocytes have also been shown to participate in this process. In adults, bone resorption is tightly coupled to bone deposition as a means to maintain skeletal homeostasis. Yet, the role and regulation of bone resorption during growth of the embryonic skeleton have remained relatively unexplored. We compare jaw development in short-beaked quail versus long-billed duck and find that quail have substantially higher levels of enzymes expressed by bone-resorbing cells including tartrate-resistant acid phosphatase (TRAP), Matrix metalloproteinase 13 (Mmp13), and Mmp9. Then, we transplant NCM destined to form the jaw skeleton from quail to duck and generate chimeras in which osteocytes arise from quail donor NCM and osteoclasts come exclusively from the duck host. Chimeras develop quail-like jaw skeletons coincident with dramatically elevated expression of TRAP, Mmp13, and Mmp9. To test for a link between bone resorption and jaw length, we block resorption using a bisphosphonate, osteoprotegerin protein, or an MMP13 inhibitor, and this significantly lengthens the jaw. Conversely, activating resorption with RANKL protein shortens the jaw. Finally, we find that higher resorption in quail presages their relatively lower adult jaw bone mineral density (BMD) and that BMD is also NCM-mediated. Thus, our experiments suggest that NCM not only controls bone resorption by its own derivatives but also modulates the activity of mesoderm-derived osteoclasts, and in so doing enlists bone resorption as a key patterning mechanism underlying the functional morphology and evolution of the jaw.