Increased proportion of hypermineralized osteocyte lacunae in osteoporotic and osteoarthritic human trabecular bone: implications for bone remodeling

Hypermineralized osteocyte lacunae (micropetrosis) have received little research attention. While they are a known aspect of the aging human skeleton, no data are available for pathological bone. In this study, intertrochanteric trabecular bone cores were obtained from patients at surgery for osteoporotic (OP) femoral neck fracture (10F, 4M, 65-94 years), for hip osteoarthritis (OA; 7F, 8M, 62-87 years), and femora at autopsy (CTL; 5F, 11M, 60-84 years). Vertebral trabecular bone cores were also obtained from the vertebra of autopsy cases (CVB; 3F, 6M, 53-83 years). Specimens were resin-embedded, polished, and carbon coated for quantitative backscattered electron imaging (qBEI), energy dispersive X-ray (EDX) spectrometry, and imaging analysis. Bone mineralization (Wt %Ca) was not different between OP, OA, and CTL; but was greater in femoral CTL than in CVB. The percent of hypermineralized osteocyte lacunae relative to the total number (HL/TL) was greater in OP and OA than in CTL. However, relative to bone mineral area, OP was characterised by increased hypermineralized osteocyte lacunar number density (Hd.Lc.Dn), whereas OA was characterised by decreased osteocyte lacunar number density (Lc.Dn) and total osteocyte lacunar number density (Tt.Lc.Dn). Lc.Dn was higher in CVB than in femoral CTL. The calcium-phosphorus ratio (R(Ca/P)) was not different between hypermineralized osteocyte lacunae and bone matrix in each group. In addition, this study focused on the phenomenon of osteocyte lacunae hypermineralization using qBEI. Seven morphological types of osteocyte lacunae hypermineralization were described according to the presence of one or several hypermineralized spherites, associated or not with a hypermineralized lacunar ring. This study has described, for the first time, the morphology of hypermineralized osteocyte lacunae in OP and OA human bone. Further studies are suggested to investigate the functional influence of hypermineralized osteocyte lacunae on bone remodeling and bone biomechanical properties.     

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.     

Differences in Osteocyte Density and Bone Histomorphometry Between Men and Women and Between Healthy and Osteoporotic Subjects

Bone defects related to osteoporosis develop with increasing age and differ between males and females. It is currently thought that the bone remodeling process is supervised by osteocytes in a strain-dependent manner. We have shown an altered response of osteocytes from osteoporotic patients to mechanical loading, and osteocyte density is reduced in osteoporotic patients, which might relate to imperfect bone remodeling, leading to lack of bone mass and strength. Hence, information on osteocyte density will contribute to a better understanding of bone biology in males and females and to the assessment of osteoporosis. Osteocyte density as well as conventional histomorphometric parameters of trabecular bone were determined in cancellous iliac crest bone of healthy postmenopausal women and men and of osteoporotic women and men. Osteocyte density was higher in healthy females than in healthy males and lower in osteoporotic females than in healthy females. Bone mass was reduced in osteoporotic patients, both male and female. In females, trabecular number was reduced, whereas in males, trabecular thickness was reduced and eroded surface was increased. There were no correlations between the parameter groups bone architecture, bone formation, bone resorption, and osteocyte density. These results are consistent with impaired osteoblast function in osteoporotic patients and with a different mechanism of bone loss between men and women, in which osteocyte density might play a role. The reduced osteocyte numbers in female osteoporotic patients might relate to imperfect bone remodeling leading to lack of bone mass and strength. M. G. Mullender and S. D. Tan contributed equally to this work.     

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).     

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.     

Effect of Raloxifene Treatment on Osteocyte Apoptosis in Postmenopausal Women

Increased osteocyte apoptosis, as the result of estrogen deficiency, could play a role in the decrease of bone mass and bone strength seen in postmenopausal osteoporosis. We investigated whether treatment with raloxifene of postmenopausal women with osteoporosis affects osteocyte apoptosis. Transiliac bone biopsies were obtained from 26 osteoporotic women at baseline and after 2 years of treatment with placebo or raloxifene. Immunohistochemical detection of cleaved caspase-3 was performed on sections from nondecalcified bone biopsies to visualize apoptosis. In the trabecular bone total osteocytes, positively stained osteocytes and empty lacunae were counted and percent positive cells and percent empty lacunae determined. Statistical evaluation was performed by Wilcoxon’s paired t-test and Spearman’s rank correlations. There was no significant difference in percentage positive osteocytes between baseline and follow-up biopsies in both the placebo and the raloxifene groups. The percentage empty lacunae increased significantly in the placebo group (11.20 ± 1.43 vs. 9.00 ± 2.25, P = 0.014) but not in the raloxifene group. At baseline in both groups combined, there was a negative correlation between indices of bone remodeling and the percentage positive osteocytes (bone formation rate/bone volume r = −0.67, P = 0.001). We found no direct evidence for an effect of raloxifene treatment on osteocyte apoptosis, but small effects of raloxifene treatment cannot be excluded. The percent of apoptotic osteocytes was dependent on the level of bone remodeling in an individual.     

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.     

Osteocyte Lacunar Occupancy in the Femoral Neck Cortex: An Association with Cortical Remodeling in Hip Fracture Cases and Controls.

In adult humans, osteocytes die and disappear from their lacunae in the cortex of bones which remodel slowly, such as the proximal femur, and osteocyte death is particularly prevalent in the elderly. We have investigated the statistical determinants of osteocyte density in microscopic fields (0.71 mm2) within thin, complete femoral neck cross-sections cut from biopsies embedded in methyl methacrylate and stained with solochrome cyanine R. Lacunae were counted under phase contrast and osteocytes within lacunae were counted in the same fields under epifluorescence. The percentage of lacunae containing an osteocyte varied between 12.4% and 99.2%, according to subject and quadrantic region of the cortex examined. The microscopic determinants of field-specific osteocyte density included the porosity measured in the field itself and the regional measurement of the proportion of cortical canals bearing osteoid. There was significant variation between subjects and, within subjects, between cortical regions. Also the inferior region showed a significantly higher density of lacunae than the superior region (+8.2%; P = 0.013). However, cases of fracture were not significantly different from controls with respect to osteocyte lacunar occupancy after adjusting for osteoid-bearing canals and porosity. It is concluded that in subjects in their 7th-9th decades of age, osteocyte lacunar occupancy is statistically associated with bone turnover, implying that high turnover (locally young bone age) might favor lacunar occupancy (ln% osteoid; P = 0.021). Alternative explanations of the association are that porosity reflects a better nutritional supply via the vasculature or that porosity of the cortex is associated with osteocyte density through an effect of osteocytes on bone remodeling.     

Differences in osteocyte and lacunar density between Black and White American women

We examined the differences in osteocyte and lacunar density between Black and White women, using previously obtained iliac bone biopsies from 34 healthy Black women, aged 21-70 years, and 94 White women, aged 20-73 years. For each subject, the density of osteocytes (Ot.N/B.Ar), empty lacunae (EL.N/B.Ar), and total lacunae (Tt.L.N/B.Ar) and the proportion of osteocyte-occupied lacunae (Ot.N/Tt.L.N) were separately measured in whole trabeculae, superficial bone (<25 microm from the bone surface), and deep bone (>45 microm from the bone surface). Compared with White women, Black women had higher values for osteocytes, empty lacunae, and total lacunae and lower values for percent occupied lacunae in superficial bone and whole trabeculae (P < 0.01 to <0.001). In deep bone there were more osteocytes and total lacunae in Black women, but the other measurements did not differ significantly between the two groups. As in White women, there were fewer osteocytes and total lacunae and more empty lacunae in deep than in superficial bone. The regressions of osteocyte and total lacunar density on age were not significant in Black women, but postmenopausal Black women had fewer osteocytes than premenopausal Black women, and percent occupied lacunae declined significantly with age in whole trabeculae and deep bone, which could only have resulted from osteocyte death. In contrast to White women, there was no inverse relationship between bone formation rate and osteocyte density in superficial bone and the observed bone formation rate was lower than predicted by osteocyte density. We conclude the following: (1) Cancellous bone is made with more osteocytes in Black than in White women, most likely because of diminished apoptosis of osteoblasts; this could contribute to increased bone strength in Black women. (2) In Black women, as in White women, there are fewer osteocytes and total lacunae and more empty lacunae in deep than in superficial bone. (3) There was moderate age-related loss of osteocytes in deep bone in Black women, indicating that osteocyte density depends more on the age of the bone than on the age of the subject. (4) The higher osteocyte density in Black women was not responsible for their lower bone formation rate.     

Autocrine and Paracrine Regulation of the Murine Skeleton by Osteocyte‐Derived Parathyroid Hormone‐Related Protein

Parathyroid hormone–related protein (PTHrP) and parathyroid hormone (PTH) have N‐terminal domains that bind a common receptor, PTHR1. N‐terminal PTH (teriparatide) and now a modified N‐terminal PTHrP (abaloparatide) are US Food and Drug Administration (FDA)‐approved therapies for osteoporosis. In physiology, PTHrP does not normally circulate at significant levels, but acts locally, and osteocytes, cells residing within the bone matrix, express both PTHrP and the PTHR1. Because PTHR1 in osteocytes is required for normal bone resorption, we determined how osteocyte‐derived PTHrP influences the skeleton. We observed that adult mice with low PTHrP in osteocytes (targeted with the Dmp1(10kb)‐Cre) have low trabecular bone volume and osteoblast numbers, but osteoclast numbers were unaffected. In addition, bone size was normal, but cortical bone strength was impaired. Osteocyte‐derived PTHrP therefore stimulates bone formation and bone matrix strength, but is not required for normal osteoclastogenesis. PTHrP knockdown and overexpression studies in cultured osteocytes indicate that osteocyte‐secreted PTHrP regulates their expression of genes involved in matrix mineralization. We determined that osteocytes secrete full‐length PTHrP with no evidence for secretion of lower molecular weight forms containing the N‐terminus. We conclude that osteocyte‐derived full‐length PTHrP acts through both PTHR1 receptor‐mediated and receptor‐independent actions in a paracrine/autocrine manner to stimulate bone formation and to modify adult cortical bone strength. © 2017 American Society for Bone and Mineral Research.     

Alendronate increases degree and uniformity of mineralization in cancellous bone and decreases the porosity in cortical bone of osteoporotic women

The strength of bone is correlated with bone mass but is also influenced significantly by other factors such as structural properties of the matrix (e.g., collagen mutations) and the mineral. Changes at all levels of this organization could contribute to fracture risk. We investigated the effects of alendronate (Aln) treatment on the density of mineralization and the ultrastructure of the mineral/collagen composite, size and habitus of mineral particles in iliac cancellous bone, as well as on the porosity of iliac cortical bone from postmenopausal osteoporotic women. Twenty-four transiliac bone biopsies from Phase III Aln (10 mg/day) trials (placebo and Aln after 2 and 3 years of treatment, n = 6 per group) were studied. The mineral structure was investigated by quantitative backscattered electron imaging (qBEI) and by scanning small-angle X-ray scattering (scanning-SAXS). qBEI histograms reflect the bone mineralization density distribution (BMDD), whereas SAXS patterns characterize the size and arrangement of the mineral particles in bone. We found that: (i) the relative calcium content of osteoporotic bone was significantly lower than that of data-base controls; (ii) mineralization was significantly higher and more uniform after Aln treatment; (iii) size and habitus of the mineral particles was not different between placebo and Aln-treated groups; and (iv) the porosity of cortical bone was reduced significantly by Aln treatment. We conclude that Aln treatment increases the degree and uniformity of bone matrix mineralization without affecting the size and habitus of the mineral crystals. It also decreases the porosity of the corticalis. Together these effects may contribute to the observed reduction in fractures.     

Childhood Cortical Porosity Is Related to Microstructural Properties of the Bone‐Muscle Junction

Childhood cortical porosity is attributable to giant asymmetrical drifting osteonal canals that arise predominantly along the primary‐secondary bone interface (PSBI). Bone from the external iliac crest cortex of 92 subjects aged 0 to 25 years was examined histomorphometrically for differences in microstructural properties between primary and secondary bone that might account for features of drifting osteonal canals. Primary compared with secondary bone showed greater numbers of osteocyte lacunae, thinner collagen lamellae, and a scaffold of elastic perforating fibers (PFs). The greater number of osteocyte lacunae compounded by known perilacunar strain amplification and the presence of elastic PFs are expected to be associated with greater bone tissue strain in primary than in secondary bone and thus with strain gradients at the PSBI. Strain gradients may lead local osteocytes to originate resorption canals and to promote transverse drift of the resorption front into lower‐strain secondary bone, thus creating giant asymmetrical drifting osteonal canals that remodel primary to secondary bone. PFs extended from muscle fibers through periosteum and primary bone to the PSBI, where they were resorbed by origination of drifting canals. Growth modeling by periosteal osteoblasts proceeds in the gaps between PFs. Through the direct connection between muscle and the PSBI via PFs, muscle forces may influence not only modeling by raising strain but also remodeling of primary to secondary bone by increasing strain gradients at the PSBI. With reduction in primary bone width after the mid‐teens, numbers of drifting canals and porosity declined. Differences in microstructural properties between primary and secondary bone are expected to generate strain gradients at the PSBI that contribute to site, transverse drift, asymmetry and large size of drifting canals, and, hence, to cortical porosity. Cortical porosity in children is a physiological feature of bone growth in width. Advisability of therapeutic intervention remains to be defined. © 2014 American Society for Bone and Mineral Research.     

Osteocyte morphology in human tibiae of different bone pathologies with different bone mineral density — Is there a role for mechanosensing?

Matrix strains due to external loading are different in bones of different pathologies with different bone mineral density (BMD), and are likely sensed by the osteocytes, the putative bone mechanosensors. The mechanosensitivity of osteocytes appears to be strongly influenced by their morphology. In this study, we explored the possibility that osteocyte morphology might play a role in various bone pathologies with different BMD.Confocal laser scanning microscopy and nano-CT were used to quantitatively determine 3D morphology and alignment of osteocytes and osteocyte lacunae in human proximal tibial bone with relatively low (osteopenic), medium (osteoarthritic), and high (osteopetrotic) BMD.Osteopenic osteocytes were relatively large and round (lengths 8.9:15.6:13.4 μm), osteopetrotic osteocytes were small and discoid shaped (lengths 5.5:11.1:10.8 μm), and osteoarthritic osteocytes were large and elongated (lengths 8.4:17.3:12.2 μm). Osteopenic osteocyte lacunae showed 3.5 fold larger volume and 2.2 fold larger surface area than osteoarthritic lacunae, whereas osteopetrotic lacunae were 1.9 fold larger and showed 1.5 fold larger surface area than osteoarthritic lacunae. Osteopetrotic osteocyte lacunae had lower alignment than osteopenic and osteoarthritic lacunae as indicated by their lower degree of anisotropy.The differences in 3D morphology of osteocytes and their lacunae in long bones of different pathologies with different BMD might reflect an adaptation to matrix strain due to different external loading conditions. Moreover, since direct mechanosensing of matrix strain likely occurs by the cell bodies, the differences in osteocyte morphology and their lacunae might indicate differences in osteocyte mechanosensitivity. The exact relationship between osteocyte morphology and bone architecture, however, is complex and deserves further study.     

Matrix metalloproteinase-13 is required for osteocytic perilacunar remodeling and maintains bone fracture resistance

Like bone mass, bone quality is specified in development, actively maintained postnatally, and disrupted by disease. The roles of osteoblasts, osteoclasts, and osteocytes in the regulation of bone mass are increasingly well defined. However, the cellular and molecular mechanisms by which bone quality is regulated remain unclear. Proteins that remodel bone extracellular matrix, such as the collagen-degrading matrix metalloproteinase (MMP)-13, are likely candidates to regulate bone quality. Using MMP-13–deficient mice, we examined the role of MMP-13 in the remodeling and maintenance of bone matrix and subsequent fracture resistance. Throughout the diaphysis of MMP-13–deficient tibiae, we observed elevated nonenzymatic cross-linking and concentric regions of hypermineralization, collagen disorganization, and canalicular malformation. These defects localize to the same mid-cortical bone regions where osteocyte lacunae and canaliculi exhibit MMP-13 and tartrate-resistant acid phosphatase (TRAP) expression, as well as the osteocyte marker sclerostin. Despite otherwise normal measures of osteoclast and osteoblast function, dynamic histomorphometry revealed that remodeling of osteocyte lacunae is impaired in MMP-13^−/− bone. Analysis of MMP-13^−/− mice and their wild-type littermates in normal and lactating conditions showed that MMP-13 is not only required for lactation-induced osteocyte perilacunar remodeling, but also for the maintenance of bone quality. The loss of MMP-13, and the resulting defects in perilacunar remodeling and matrix organization, compromise MMP-13^−/− bone fracture toughness and postyield behavior. Taken together, these findings demonstrate that osteocyte perilacunar remodeling of mid-cortical bone matrix requires MMP-13 and is essential for the maintenance of bone quality. © 2012 American Society for Bone and Mineral Research.     

The amazing osteocyte

The last decade has provided a virtual explosion of data on the molecular biology and function of osteocytes. Far from being the “passive placeholder in bone,” this cell has been found to have numerous functions, such as acting as an orchestrator of bone remodeling through regulation of both osteoclast and osteoblast activity and also functioning as an endocrine cell. The osteocyte is a source of soluble factors not only to target cells on the bone surface but also to target distant organs, such as kidney, muscle, and other tissues. This cell plays a role in both phosphate metabolism and calcium availability and can remodel its perilacunar matrix. Osteocytes compose 90% to 95% of all bone cells in adult bone and are the longest lived bone cell, up to decades within their mineralized environment. As we age, these cells die, leaving behind empty lacunae that frequently micropetrose. In aged bone such as osteonecrotic bone, empty lacunae are associated with reduced remodeling. Inflammatory factors such as tumor necrosis factor and glucocorticoids used to treat inflammatory disease induce osteocyte cell death, but by different mechanisms with potentially different outcomes. Therefore, healthy, viable osteocytes are necessary for proper functionality of bone and other organs. © 2011 American Society for Bone and Mineral Research.     

Conductive Hearing Loss in the Hyp Mouse Model of X-Linked Hypophosphatemia Is Accompanied by Hypomineralization of the Auditory Ossicles

X-linked hypophosphatemia (XLH) is a hereditary musculoskeletal disorder caused by loss-of-function mutations in the PHEX gene. In XLH, increased circulating fibroblast growth factor 23 (FGF23) levels cause renal phosphate wasting and low concentrations of 1,25-dihydroxyvitamin D, leading to an early clinical manifestation of rickets. Importantly, hearing loss is commonly observed in XLH patients. We present here data from two XLH patients with marked conductive hearing loss. To decipher the underlying pathophysiology of hearing loss in XLH, we utilized the Hyp mouse model of XLH and measured auditory brain stem responses (ABRs) and distortion product otoacoustic emissions (DPOAEs) to functionally assess hearing. As evidenced by the increased ABR/DPOAE threshold shifts in the mid-frequency range, these measurements indicated a predominantly conductive hearing loss in Hyp mice compared to wild-type (WT) mice. Therefore, we carried out an in-depth histomorphometric and scanning electron microscopic analysis of the auditory ossicles. Quantitative backscattered electron imaging (qBEI) indicated a severe hypomineralization of the ossicles in Hyp mice, evidenced by lower calcium content (CaMean) and higher void volume (ie, porosity) compared to WT mice. Histologically, voids correlated with unmineralized bone (ie, osteoid), and the osteoid volume per bone volume (OV/BV) was markedly higher in Hyp mice than WT mice. The density of osteocyte lacunae was lower in Hyp mice than in WT mice, whereas osteocyte lacunae were enlarged. Taken together, our findings highlight the importance of ossicular mineralization for hearing conduction and point toward the potential benefit of improving mineralization to prevent hearing loss in XLH. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).     

Glucocorticoid‐induced autophagy in osteocytes

Glucocorticoid (GC) therapy is the most frequent cause of secondary osteoporosis. In this study we have demonstrated that GC treatment induced the development of autophagy, preserving osteocyte viability. GC treatment resulted in an increase in autophagy markers and the accumulation of autophagosome vacuoles in vitro and in vivo promoted the onset of the osteocyte autophagy, as determined by expression of autophagy markers in an animal model of GC‐induced osteoporosis. An autophagy inhibitor reversed the protective effects of GCs. The effects of GCs on osteocytes were in contrast to tumor necrosis factor α (TNF‐α), which induced apoptosis but not autophagy. Together this study reveals a novel mechanism for the effect of GC on osteocytes, shedding new insight into mechanisms responsible for bone loss in patients receiving GC therapy. © 2010 American Society for Bone and Mineral Research.     

Compound Heterozygous Frameshift Mutations in MESD Cause a Lethal Syndrome Suggestive of Osteogenesis Imperfecta Type XX

Multiple genes are known to be associated with osteogenesis imperfecta (OI), a phenotypically and genetically heterogenous bone disorder, marked predominantly by low bone mineral density and increased risk of fractures. Recently, mutations affecting MESD, which encodes for a chaperone required for trafficking of the low-density lipoprotein receptors LRP5 and LRP6 in the endoplasmic reticulum, were described to cause autosomal-recessive OI XX in homozygous children. In the present study, whole-exome sequencing of three stillbirths in one family was performed to evaluate the presence of a hereditary disorder. To further characterize the skeletal phenotype, fetal autopsy, bone histology, and quantitative backscattered electron imaging (qBEI) were performed, and the results were compared with those from an age-matched control with regular skeletal phenotype. In each of the affected individuals, compound heterozygous mutations in MESD exon 2 and exon 3 were detected. Based on the skeletal phenotype, which was characterized by multiple intrauterine fractures and severe skeletal deformity, OI XX was diagnosed in these individuals. Histological evaluation of MESD specimens revealed an impaired osseous development with an altered osteocyte morphology and reduced canalicular connectivity. Moreover, analysis of bone mineral density distribution by qBEI indicated an impaired and more heterogeneous matrix mineralization in individuals with MESD mutations than in controls. In contrast to the previously reported phenotypes of individuals with OI XX, the more severe phenotype in the present study is likely explained by a mutation in exon 2, located within the chaperone domain of MESD, that leads to a complete loss of function, which indicates the relevance of MESD in early skeletal development. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR)..     

The microscopic morphology of fluoride-induced bone

To characterize further the bone changes in osteoporotic patients treated by a combined calcium, vitamin D, and sodium fluoride therapy regimen, full-thickness transilial undecalcified bone biopsy specimens from ten postmenopausal white women treated for idiopathic osteoporosis for 18-24 months were compared with those from ten age-, sex-, and race-matched untreated control subjects using standard light microscopy and histomorphometry. Statistically significant bone changes in the treated group consisted of cortical and trabecular new bone formation juxtaposed on underlying normal lamellar bone (p less than 0.001). The new bone showed increased osteocytic cellularity (p less than 0.001), irregular arrangement of osteocytes (p less than 0.001), enlarged osteocyte lacunae (p less than 0.001), and periosteocytic hematoxylinophilic staining intensity (p less than 0.001). Increases were also noted in trabecular bone volume (p less than .025), trabecular osteoid surface (p less than 0.001), and trabecular osteoid volume (p less than 0.001). Osteoid calculations were significantly less than those in the clinical and chemical osteomalacia observed in the authors' laboratory (p less than 0.01). Osteoclastic resorptive activity was increased (p less than .001), but no evidence of hyperparathyroidism was noted. These histologic and histomorphometric changes indicate accretion of new bone but with distinctly abnormal matrix characteristics. These are changes considered characteristic of the treatment and are pathologic markers of fluoride-induced abnormal bone formation.     

Quantitative regional associations between remodeling, modeling, and osteocyte apoptosis and density in rabbit tibial midshafts

Evidence suggests that osteocyte apoptosis is involved in the adaptive response of bone, although the specific role of osteocytes in the signaling mechanism is unknown. Here, we examined and correlated regional variability in indices of remodeling, modeling, osteocyte apoptosis, and osteocyte density in rabbit tibia midshafts. Histomorphometric analysis indicated that remodeling parameters (BMU activation frequency, osteon density, forming osteon density, and resorption cavity density) were lower in the cranial region compared to other quadrants. In addition, pericortical subregions displayed less remodeling relative to intracortical and endocortical ones. Modeling indices also demonstrated regional variability in that periosteal surfaces exhibited a greater extent of bone forming surface than endosteal ones across all anatomic quadrants. In contrast, endosteal surfaces demonstrated significantly greater surface mineral apposition rates compared to periosteal surfaces in caudal, medial, and lateral but not cranial quadrants. Using TUNEL analysis to detect osteocytes undergoing apoptosis, the density of apoptotic osteocytes was found to be lower in cranial quadrants relative to medial ones. In addition, the densities of osteocyte lacunae, empty lacunae, and total osteocytes were higher in lateral fields relative to caudal quadrants. There was a strong, statistically significant linear correlation between the remodeling indices and apoptotic osteocyte density, supporting the theory that osteocytes undergoing apoptosis produce signals that attract or direct bone remodeling. In contrast, the modeling parameters did not exhibit a correlation with apoptotic osteocytes, although there was a strong correlation between the modeling indices and the density of empty osteocyte lacunae, corroborating previous studies that have found that osteocytes inhibit bone formation. It was found that osteocyte density and osteocyte lacunar density did not significantly correlate with modeling or remodeling parameters, suggesting that cell viability should be examined in studies correlating bone turnover parameters with the functional role of osteocytes in bone adaptation.