Osteocyte Lacunar Density Research Articles

Quantitative insights into osteocyte density developmental patterns in fossilized bone — A new method

This study presents a new methodology that combines quantitative image analysis, clustering, and statistical techniques to examine the 2D distribution of osteohistological features in an extinct stem-group bird. The driving force of our research was the need to map ontogenetic changes in the spatial density of osteocyte lacunae in the type specimen of Musivavis amabilis from the Lower Cretaceous of China. We particularly focused on developing tools to reveal quantitative aspects of the dynamics in the formation of avascular bone in this active flier. We achieve this goal by proposing an algorithm with the following methodological steps: 1) We obtain relevant coordinate details for pixel locations selected by thresholding the original image with shading as a criterion. 2) We estimate density using the Gaussian kernel estimator and refined it through observations and regression analysis. 3) After slicing the image, we apply k-means clustering to obtain one-dimensional representations of lacunar density. 4) We proceed by implementing weighted averaging employing the k-nearest neighbor approach. Having applied these steps, we are able to quantitatively disclose growth processes previously unnoticed and reveal dynamics in the formation of lacunar bone tissue in the enantiornithine birds capable of power flight.

The effect of caponization on tibia bone histomorphometric properties of crossbred roosters

The negative effect of caponization on the structural, geometric and mechanical parameters of femur and tibia has been shown in a few studies. Nevertheless, its influence on tibia bone microarchitecture is still largely unknown. Therefore, this study aimed to assess the effect of castration on the microstructural parameters of the trabecular and compact bone of tibia bone in crossbred chickens. The experiment involved 96 roosters derived from crossing Yellowleg Partridge hens (Z˙\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$${\\dot{\ ext{Z}}}$$\\end{document}-33) and Rhode Island Red cockerels (R-11) fattened until the 16th, 20th and 24th week of life. Animals were randomly divided into 2 groups of 48 each. Group I (control) consisted of intact roosters and group II (experimental) consisted of birds subjected to caponization at the 8th week of age. The castration surgery had no influence on some properties within compact bone such as osteon diameter On.Dm, osteon perimeter On.Pm, osteon area On.Ar, osteocyte lacunar number Ot.Lc.N, osteon bone area On.B.Ar, osteon wall thickness On.W.Th as well as thick-mature collagen content in all analyzed age groups of animals. Nevertheless, our results demonstrate that castration caused a decrease of Haversian canal area Hc.Ar, osteocyte lacunar area Ot.Lc.Ar and osteocyte lacunar porosity Ot.Lc.Po among the 16-week-old birds, decrease of Haversian canal perimeter Hc.Pm and increase of fraction of bone area On.B.Ar/On.Ar among 16- and 24-week-old individuals and also an increase of osteocyte lacunar density Ot.Lc.Dn in the osteons of the oldest roosters. Additionally, some microstructural parameters of trabecular bone show the negative effect of caponization. The youngest 16-week-old capons were characterized by thinnin the trabecular in the epiphysis part of tibia. Moreover, in the case of 24-week-old, there is an increase in the trabecular separation Tb.Sp with simultaneous decrease of trabecular number Tb.N compared to roosters, which may suggest the increase of the bone resorption among the oldest individuals. The increased bone turnover in the epiphysis part of the tibia bone also indicates changes in the collagen fibers distribution, where among 20-week-old animals there is a decrease in the content of immature thin collagen fibers with simultaneous increase in the content of mature thick collagen fibers. Furthermore, among the oldest 24-week-old individuals we can observe the increased thick-to-thin collagen ratio, which may be a sign of slowing down in bone formation.

Comparison of the 3D-microstructure of human alveolar and fibula bone in microvascular autologous bone transplantation: a synchrotron radiation μ-CT study.

Introduction: Autologous bone transplantation is successfully used in reconstructive surgery of large/critical-sized bone defects, whereby the microvascular free fibula flap is still regarded as the gold standard for the reconstruction of such defects in the head and neck region. Here, we report the morphological and lacunar properties of patient-paired bone samples from eight patients from the jaw (AB; recipient site) and the fibula (FB; donor site) on the micron length-scale using Synchrotron µ-CT. Insights into differences and similarities between these bone structures could offer a better understanding of the underlying mechanism for successful surgical outcomes and might clear the path for optimized, nature-inspired bone scaffold designs. Methods: Spatial vessel-pore arrangements, bone morphology, fluid-simulation derived permeability tensor, osteocyte lacunar density, and lacunar morphology are compared. Results: The orientation of the vessel system indicates a homogenous vessel orientation for AB and FB. The average mineral distance (50%) to the closest vessel boundary is higher in AB than in FB (the mean is 96μm for AB vs. 76μm for FB; p = 0.021). Average osteocyte lacunar density is found to be higher in AB than in FB (mean 22,874mm3 vs. 19,376mm3 for FB; p = 0.038), which might compensate for the high distance from the mineral to the nearest vessel. No significant differences in lacunar volume are found between paired AB and FB. Discussion: A comparable vessel network and similar distribution of vessel porosity between AB and FB may allow the FB graft to exhibit a high regeneration potential when connected to AB, and this might correlate with a high osteoinductive and osteoconductive potential of FB when connected to AB. Since widely used and potent synthetic bone grafts exist, new insight into the bone structure of well-established autologous bone grafts, such as the free fibula flap, could help to improve the performance of such materials and therefore the design of 3D scaffolds.

Microstructural and cellular characterisation of the subchondral trabecular bone in human knee and hip osteoarthritis using synchrotron tomography

It is unclear if different factors influence osteoarthritis (OA) progression and degenerative changes characterising OA disease in hip and knee. We investigated the difference between hip OA and knee OA at the subchondral bone (SCB) tissue and cellular level, relative to the degree of cartilage degeneration. Bone samples were collected from 11 patients (aged 70.4±10.7years) undergoing knee arthroplasty and 8 patients (aged 62.3±13.4years) undergoing hip arthroplasty surgery. Trabecular bone microstructure, osteocyte-lacunar network, and bone matrix vascularity were evaluated using synchrotron micro-CT imaging. Additionally, osteocyte density, viability, and connectivity were determined histologically. The associations between severe cartilage degeneration and increase of bone volume fraction (%) [-8.7, 95% CI (-14.1, -3.4)], trabecular number (#/mm) [-1.5, 95% CI (-0.8, -2.3)], osteocyte lacunar density (#/mm3) [4714.9; 95% CI (2079.1, 7350.6)] and decrease of trabecular separation (mm) [-0.07, 95% CI (0.02, 0.1)] were found in both knee and hip OA. When compared to knee OA, hip OA was characterised by larger (µm3) but less spheric osteocyte lacunae [47.3; 95% CI (11.2, 83.4), -0.04; 95% CI (-0.06, -0.02), respectively], lower vascular canal density (#/mm3) [-22.8; 95% CI (-35.4, -10.3)], lower osteocyte cell density (#/mm2) [-84.2; 95% CI (-102.5, -67.4)], and less senescent (#/mm2) but more apoptotic osteocytes (%) [-2.4; 95% CI (-3.6, -1.2), 24.9; 95% CI (17.7, 32.1)], respectively. SCB from hip OA and knee OA exhibits different characteristics at the tissue and cellular levels, suggesting different mechanisms of OA progression in different joints.

Bone Tissue Evaluation Indicates Abnormal Mineralization in Patients with Autoimmune Polyendocrine Syndrome Type I: Report on Three Cases

Autoimmune polyendocrine syndrome type-1 (APS1) is characterized by autoimmune manifestations affecting different organs from early childhood on. Immunological abnormalities, the resulting endocrinopathies, and their treatments may compromise bone health. For the first time in APS1, we analyzed transiliac bone biopsy samples by bone histomorphometry and quantitative backscattered electron imaging in three adult patients (female P1, 38 years; male P2, 47 years; male P3, 25 years). All had biallelic mutations in the autoimmune regulator gene and in addition to endocrinopathies, also significant bone fragility. Histomorphometry showed bone volume in the lower normal range for P1 (BV/TV, − 0.98 SD) and P3 (− 1.34 SD), mainly due to reduced trabecular thickness (TbTh, − 3.63 and − 2.87 SD). In P1, osteoid surface was low (OS/BS, − 0.96 SD); active osteoblasts and double labeling were seen only on cortical bone. P3 showed a largely increased bone turnover rate (BFR/BV, + 4.53 SD) and increased mineralization lag time (Mlt, + 3.40 SD). Increased osteoid surface (OS/BS, + 2.03 and + 4.71 SD for P2 and P3) together with a large proportion of lowly mineralized bone area (Trab CaLow, + 2.22 and + 9.81 SD for P2 and P3) and focal mineralization defects were consistent with abnormal mineralization. In all patients, the density and area of osteocyte lacunae in cortical and trabecular bone were similar to healthy adults. The bone tissue characteristics were variable and included decreased trabecular thickness, increased amount of osteoid, and abnormal mineralization which are likely to contribute to bone fragility in patients with APS1.

Alterations of bone material properties in growing Ifitm5/BRIL p.S42 knock-in mice, a new model for atypical type VI osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a heterogenous group of heritable connective tissue disorders characterized by high bone fragility due to low bone mass and impaired bone material properties. Atypical type VI OI is an extremely rare and severe form of bone dysplasia resulting from a loss-of-function mutation (p.S40L) in IFITM5/BRIL,the causative gene of OI type V and decreased osteoblast secretion of pigment epithelium-derived factor (PEDF), as in OI type VI. It is not yet known which alterations at the material level might lead to such a severe phenotype. We therefore characterized bone tissue at the micrometer level in a novel heterozygous Ifitm5/BRIL p.S42L knock-in murine model at 4 and 8 weeks of age. We evaluated in female mice, total body size, femoral and lumbar bone mineral density (BMD) by dual-energy X-ray absorptiometry. In the femoral bone we examined osteoid deposition by light microscopy, assessed bone histomorphometry and mineralization density distribution by quantitative backscattered electron imaging (qBEI). Osteocyte lacunae were examined by qBEI and the osteocyte lacuno-canalicular network by confocal laser scanning microscopy. Vasculature was examined indirectly by qBEI as 2D porosity in cortex, and as 3D porosity by micro-CT in third trochanter. Collagen orientation was examined by second harmonic generation microscopy. Two-way ANOVA was used to discriminate the effect of age and genotype. Ifitm5/BRIL p.S42L female mice are viable, do not differ in body size, fat and lean mass from wild type (WT) littermates but have lower whole-body, lumbar and femoral BMD and multiple fractures. The average and most frequent calcium concentration, CaMean and CaPeak, increased with age in metaphyseal and cortical bone in both genotypes and were always higher in Ifitm5/BRIL p.S42L than in WT, except CaMean in metaphysis at 4 weeks of age. The fraction of highly mineralized bone area, CaHigh, was also increased in Ifitm5/BRIL p.S42L metaphyseal bone at 8 weeks of age and at both ages in cortical bone. The fraction of lowly mineralized bone area, CaLow, decreased with age and was not higher in Ifitm5/BRIL p.S42L, consistent with lack of hyperosteoidosis on histological sections by visual exam. Osteocyte lacunae density was higher in Ifitm5/BRIL p.S42L than WT, whereas canalicular density was decreased. Indirect measurements of vascularity revealed a higher pore density at 4 weeks in cortical bone of Ifitm5/BRIL p.S42L than in WT and at both ages in the third trochanter. Importantly, the proportion of bone area with disordered collagen fibrils was highly increased in Ifitm5/BRIL p.S42L at both ages. Despite normal skeletal growth and the lack of a collagen gene mutation, the Ifitm5/BRIL p.S42L mouse shows major OI-related bone tissue alterations such as hypermineralization of the matrix and elevated osteocyte porosity. Together with the disordered lacuno-canalicular network and the disordered collagen fibril orientation, these abnormalities likely contribute to overall bone fragility.

Comparison of the 3D-Microstructure Between Alveolar and Iliac Bone for Enhanced Bioinspired Bone Graft Substitutes.

In oral- and maxillofacial bone augmentation surgery, non-vascularized grafts from the iliac crest demonstrate better clinical performance than alveolar bone grafts. The underlying mechanisms are not fully understood but are essential for the enhancement of bone regeneration scaffolds. Synchrotron Radiation µ-CT at a pixel size of 2.3 μm was used to characterize the gross morphology and the vascular and osteocyte lacuna porosity of patient-matched iliac crest/alveolar bone samples. The results suggest a difference in the spatial distribution of the vascular pore system. Fluid simulations reveal the permeability tensor to be more homogeneous in the iliac crest, indicating a more unidirectional fluid flow in alveolar bone. The average distance between bone mineral and the closest vessel pore boundary was found to be higher in alveolar bone. At the same time, osteocyte lacunae density is higher in alveolar bone, potentially compensating for the longer average distance between the bone mineral and vessel pores. The present study comprehensively quantified and compared the 3D microarchitecture of intraindividual human alveolar and iliac bone. The identified difference in pore network architecture may allow a bone graft from the iliac crest to exhibit higher regeneration potential due to an increased capacity to connect with the surrounding pore network of the residual bone. The results may contribute to understanding the difference in clinical performance when used as bone grafts and are essential for optimization of future scaffold materials.

Impaired bone quality in the superolateral femoral neck occurs independent of hip geometry and bone mineral density

Skeletal adaptation is substantially influenced by mechanical loads. Osteocytes and their lacuno-canalicular network have been identified as a key player in load sensation and bone quality regulation. In the femoral neck, one of the most common fracture sites, a complex loading pattern with lower habitual loading in the superolateral neck and higher compressive stresses in the inferomedial neck is present. Variations in the femoral neck-shaft angle (NSA), i.e., coxa vara or coxa valga, provide the opportunity to examine the influence of loading patterns on bone quality. We obtained femoral neck specimens of 28 osteoarthritic human subjects with coxa vara, coxa norma and coxa valga during total hip arthroplasty. Bone mineral density (BMD) was assessed preoperatively by dual energy X-ray absorptiometry (DXA). Cortical and trabecular microstructure and three-dimensional osteocyte lacunar characteristics were assessed in the superolateral and inferomedial neck using ex vivo high resolution micro-computed tomography. Additionally, BMD distribution and osteocyte lacunar characteristics were analyzed by quantitative backscattered electron imaging (qBEI). All groups presented thicker inferomedial than superolateral cortices. Furthermore, the superolateral site exhibited a lower osteocyte lacunar density along with lower lacunar sphericity than the inferomedial site, independent of NSA. Importantly, BMD and corresponding T-scores correlated with microstructural parameters at the inferomedial but not superolateral neck. In conclusion, we provide micromorphological evidence for fracture vulnerability of the superolateral neck, which is independent of NSA and BMD. The presented bone qualitative data provide an explanation why DXA may be insufficient to predict a substantial proportion of femoral neck fractures. Statement of significanceThe femoral neck, one of the most common fracture sites, is subject to a complex loading pattern. Site-specific differences (i.e., superolateral vs. inferomedial) in bone quality influence fracture risk, but it is unclear how this relates to hip geometry and bone mineral density (BMD) measurements in vivo. Here, we examine femoral neck specimens using a variety of high-resolution imaging techniques and demonstrate impaired bone quality in the superolateral compared to the inferomedial neck. Specifically, we found impaired cortical and trabecular microarchitecture, mineralization, and osteocyte properties, regardless of neck-shaft angle. Since BMD correlated with bone quality of the inferomedial but not the superolateral neck, our results illustrate why bone densitometry may not predict a substantial proportion of femoral neck fractures.

Palaeohistological inferences of resting metabolic rates inConcornisandIberomesornis(Enantiornithes, Ornithothoraces) from the Lower Cretaceous of Las Hoyas (Spain)

AbstractThe bone histology of non‐avian theropods such asTroodon, early pygostylians such asConfuciusornis, and neornithines, is characterized by the post‐hatching formation of fibrolamellar complex. In contrast, the cortex of enantiornithine birds, likeConcornisandIberomesornis, is made of poorly vascularized parallel‐fibred tissue. The cortex of metatarsals ofConcornis lacustrisMCCM‐LH21006 is composed of a thin layer of lamellar endosteal bone and a thick layer of almost avascular parallel‐fibred periosteal bone organized into an inner part containing large spherical osteocyte lacunae and a few secondary osteons, and an outer part containing flattened osteocyte lacunae and lines of arrested growth. The cortex of metatarsals ofIberomesornissp. MCCM‐LH09681 is similar to the inner layer described inConcornis. Endosteal bone, and the outer layer of periosteal bone containing flattened osteocytes and lines of arrested growth, are missing inIberomesornis, suggesting that this specimen is a sub‐adult. Here we test statistically a hypothesis suggesting that the absence of post‐hatching formation of fibrolamellar bone in Enantiornithes may be a side effect of their low resting metabolic rates (RMR). For this, we construct a palaeobiological inference model including osteocyte lacunar density (explaining a significant fraction of variation in RMR) and phylogenetic eigenvectors as predictor variables. RMR inferred forConcornisandIberomesornisare significantly higher than those measured in extant ectotherms, but they are not significantly different from the RMR measured in a sample of extant arboreal perching Neornithes of similar body size. We conclude that the ‘intermediate physiological condition’ hypothesis is rejected.

Free-fall landing and interval running have different effects on trabecular bone mass and microarchitecture, serum osteocalcin, biomechanical properties, SOST expression and on osteocyte-related characteristics.

The effects of treadmill interval training (IT) and free-fall exercise were evaluated on bone parameters including osteocyte related characteristics. Thirty-eight 4-month-old male Wistar rats were randomly divided into a control (C) group and exercise groups: IT, 10 free-fall impacts/day with a 10-s (FF10) or 20-s interval between drops (FF20), 5 days/week, for 9 weeks. We assessed bone mineral density (BMD); microarchitecture by µCT; mechanical strength by a 3-point bending test; density and occupancy of the osteocyte lacunae by toluidine blue staining; osteocalcin and NTx systemic levels by ELISA; and bone tissue Sost messenger RNA (mRNA) expression by RT-PCR. NTx levels were significantly lower in exercise groups as compared with the C group. In exercise groups the Sost mRNA expression was significantly lower than in C. Tb.N was significantly higher for IT and FF20 compared with the C group. Tb.Sp was significantly lower in FF10 compared with the C group. Both IT and FF20 were associated with higher tibial lacunar density as compared with FF10. compared with FF10, IT fat mass was lower, while tibial osteocyte lacunae occupancy and systemic osteocalcin level were higher. All exercise modes were efficient in reducing bone resorption. Both IT and free-fall impact with appropriate recovery periods, which may be beneficial for bone health and osteocyte-related characteristics. Novelty: Interval training is beneficial for bone mineral density. Exercises decreased both bone resorption and inhibition of bone formation (Sost mRNA). Longer interval recovery time favors osteocyte lacunae density.

Greater heterogeneity of the bone mineralisation density distribution and low bone matrix mineralisation characterise tibial subchondral bone marrow lesions in knee osteoarthritis patients.

Tibial subchondral bone marrow lesions (BMLs) identified by MRI have been recognised as potential disease predictors in knee osteoarthritis (KOA), and may associate with abnormal bone matrix mineralisation and reduced bone quality. However, these tissue-level changes of BMLs have not been extensively investigated. Thus, the aim of this study was to quantify the degree of subchondral bone matrix mineralisation (both plate and trabeculae) in relation to histomorphometric parameters of bone remodelling and osteocyte lacunae (OL) characteristics in the tibial plateau (TP) of KOA patients with and without BMLs (OA-BML and OA No-BML, respectively) in comparison to nonOA cadaveric controls (CTL). Osteochondral (cartilage-bone) tissue was sampled from the BML signal region within the medial compartment for each OA-BML TP, and from a corresponding medial region for OA No-BML and CTL TPs. The tissue samples were embedded in resin, and sections stained with Von-Kossa Haematoxylin and Eosin (H&E) for quantitation of static indices of bone remodelling. Resin blocks were then further polished, and carbon-coated for quantitative backscattered electron imaging (qBEI) to determine the bone mineralisation density distribution (BMDD), as well as OL characteristics. It was found that OA-BML contained higher osteoid volume per tissue volume (OV/TV; %) and per bone volume (OV/BV; %) in both subchondral plate and trabecular bone compared to OA No-BML and CTL. The BMDD of OA-BML in both subchondral plate and trabecular bone was shifted toward a lower degree of mineralisation. Typically, an increase in both the heterogeneity of mineralisation density (Ca Width; wt%Ca) and the percentage of lower calcium (Ca Low; % B.Ar) in trabecular bone with OA-BML versus CTL was observed. Further, unmineralised OL density (#/mm2) in subchondral plate was distinctly higher in OA-BML samples compared to CTL. The KOA patients with and without BMLs had significantly decreased density of mineralised OL (#/mm2) in trabecular bone compared to CTL. Taken together, these findings indicate that tibial BMLs in advanced KOA patients are characterised by significantly hypo-mineralised subchondral bone compared with CTL. These differences associated with evidence of increased bone remodelling in OA-BML, and may influence the mechanical properties of the subchondral bone, with implications for the overlying cartilage.

Increased Osteocyte Lacunae Density in the Hypermineralized Bone Matrix of Children with Osteogenesis Imperfecta Type I.

Osteocytes are terminally differentiated osteoblasts embedded within the bone matrix and key orchestrators of bone metabolism. However, they are generally not characterized by conventional bone histomorphometry because of their location and the limited resolution of light microscopy. OI is characterized by disturbed bone homeostasis, matrix abnormalities and elevated bone matrix mineralization density. To gain further insights into osteocyte characteristics and bone metabolism in OI, we evaluated 2D osteocyte lacunae sections (OLS) based on quantitative backscattered electron imaging in transiliac bone biopsy samples from children with OI type I (n = 19) and age-matched controls (n = 24). The OLS characteristics were related to previously obtained, re-visited histomorphometric parameters. Moreover, we present pediatric bone mineralization density distribution reference data in OI type I (n = 19) and controls (n = 50) obtained with a field emission scanning electron microscope. Compared to controls, OI has highly increased OLS density in cortical and trabecular bone (+50.66%, +61.73%; both p < 0.001), whereas OLS area is slightly decreased in trabecular bone (−10.28%; p = 0.015). Correlation analyses show a low to moderate, positive association of OLS density with surface-based bone formation parameters and negative association with indices of osteoblast function. In conclusion, hyperosteocytosis of the hypermineralized OI bone matrix associates with abnormal bone cell metabolism and might further impact the mechanical competence of the bone tissue.

Interstitial fluid velocity is decreased around cortical bone vascular pores and depends on osteocyte position in a rat model of disuse osteoporosis.

Muscle paralysis induced with botulinum toxin (Botox) injection increases vascular porosity and reduces osteocyte lacunar density in the tibial cortical bone of skeletally mature rats. These morphological changes potentially affect interstitial fluid flow in the lacunar-canalicular porosity, which is thought to play a role in osteocyte mechanotransduction. The aim of this study was to investigate the effects of disuse-induced morphological changes on interstitial fluid velocity around osteocytes in the bone cortex. Micro-CT images from a previous study that quantified the effects of Botox-induced muscle paralysis on bone microarchitecture in skeletally mature rats were used to create high-resolution, animal-specific finite element models that included the vascular pores and osteocyte lacunae within the tibial metaphysis of Botox-injected (BTX, n = 8) and saline-injected control (CTRL, n = 8) groups. To quantify fluid flow, lacunar and canalicular porosities were modeled as fluid-saturated poroelastic materials, and boundary conditions were applied to simulate physiological loading. This modeling approach allowed a detailed quantification of the fluid flow velocities around osteocytes in a relatively large volume of bone tissue. The analysis demonstrated that interstitial fluid velocity at the vascular pore surfaces was significantly lower in BTX compared to CTRL because of the decreased vascular canal separation. No significant differences in average fluid velocity were observed at the osteocyte lacunae and no correlation was found between the fluid velocity and the lacunar density, which was significantly lower in BTX. Instead, the lacunar fluid velocity was dependent on the osteocyte's specific position in the bone cortex and its proximity to a vascular pore.

Bone material properties and response to teriparatide in osteoporosis due to WNT1 and PLS3 mutations.

Patients with osteoporosis-associated WNT1 or PLS3 mutations have unique bone histomorphometric features and osteocyte-specific hormone expression patterns. To investigate the effects of WNT1 and PLS3 mutations on bone material properties. Transiliac bone biopsies were evaluated by quantitative backscattered electron imaging, immunohistochemistry, and bone histomorphometry. Ambulatory patients. Three pediatric and eight adult patients with WNT1 or PLS3 mutations. Bone mineralization density distribution and osteocyte protein expression was evaluated in 11 patients and repeated in six patients who underwent repeat biopsy after 24months of teriparatide treatment. Bone mineralization density distribution and protein expression. Children with WNT1 or PLS3 mutations had heterogeneous bone matrix mineralization, consistent with bone modeling during growth. Bone matrix mineralization was homogenous in adults and increased throughout the age spectrum. Teriparatide had very little effect on matrix mineralization or bone formation in patients with WNT1 or PLS3 mutations. However, teriparatide decreased trabecular osteocyte lacunae size and increased trabecular bone FGF23 expression. The contrast between preserved bone formation with heterogeneous mineralization in children and low bone turnover with homogenous bone mineral content in adults suggests that WNT1 and PLS3 have differential effects on bone modeling and remodeling. The lack of change in matrix mineralization in response to teriparatide, despite clear changes in osteocyte lacunae size and protein expression, suggests that altered WNT1 and PLS3 expression may interfere with coupling of osteocyte, osteoblast, and osteoclast function. Further studies are warranted to determine the mechanism of these changes.

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).

Osteocyte lacuno‐canalicular microstructure across the mid‐shaft femur in adult males from medieval England

AbstractArchaeological human bone histology can reveal well‐preserved osteocyte lacunae, which are indicators of bone remodelling activity. Analyses of these lacunae can be useful when reconstructing past human mechanical loading histories or metabolic fluctuations from bone microstructure. However, the relationship between osteocyte lacunae density and morphology, and bone anatomical variation within archaeological samples is largely unknown. We examined osteocyte lacuno‐canalicular network morphology in medieval human femora to test if osteocyte lacunae change with anatomical site location. Osteocyte lacunae density (Ot.Dn) data were analysed statistically in 10 middle‐aged (35–50 years old) males dated to the 11th–16th century AD (Canterbury, England). A subsequent case study (n = 2) was conducted using two well‐preserved samples from which canaliculi number per lacuna (Ci.N) and canaliculi‐rich lacunae density (Ci.Dn) were preliminarily examined descriptively. The data were collected from cortical bone regions encompassing intracortical to subperiosteal mid‐shaft femoral bone, comparing anterior, posterior, medial and lateral locations interindividually and intraindividually. Results show that Ot.Dn varied significantly between the four anatomical regions (p = 0.001), with the medial and lateral femoral regions showing the highest median Ot.Dn. The median of Ci.N was also the highest on the medial aspect, but Ci.Dn did not change greatly across the four apects of the bone. The combination of these results suggests that mid‐shaft femoral anatomical location, which undergoes morphological change with biomechanical load, affects the expression of bone microstructure at the osteocyte lacuna level. This knowledge will benefit future osteoarchaeological methods that infer past behaviour from the human femur.

Elevated Bone Hardness Under Denosumab Treatment, With Persisting Lower Osteocyte Viability During Discontinuation.

Denosumab is a potent osteoclast inhibitor targeted to prevent osteoporotic bone loss and thereby reduce fractures in the aging population. Recently, an elevated risk of rebound fractures following denosumab discontinuation was identified, unless patients were transitioned to an alternative antiresorptive medication. How denosumab affects the interaction of mechanosensitive osteocytes and bone quality remains unknown. We hypothesized that denosumab influences osteocyte function contributing to bone reorganization and increased fractures during discontinuation. Bone quality and osteocytes were assessed in archived iliac crest bone biopsies obtained from patients with high fracture occurrence from 2011 to 2016. Biopsies were obtained due to high fracture occurrence prior and during osteoporosis therapy from (i) patients with at least two semiannual subcutaneous injections of 60 mg denosumab, (ii) patients with rebound fractures during discontinuation, and (iii) patients of a treatment-naive group. In total, biopsies from 43 individuals were analyzed (mean age, 65.5 ± 12.1 years). Our results showed that during denosumab treatment, iliac cortical bone had a higher bone tissue hardness compared to treatment-naive bone (p = 0.0077) and a higher percentage of mineralized osteocyte lacunae (p = 0.0095). The density of empty osteocyte lacunae was higher with denosumab compared to treatment-naive (p = 0.014) and remained high in trabecular bone during discontinuation (p = 0.0071). We conclude that during denosumab treatment, increased bone hardness may contribute to improved fracture resistance. In biopsies from patients with high fracture occurrence, denosumab treatment reduced osteocyte viability, an effect that persisted during treatment discontinuation. High-resolution imaging of osteocyte viability indicates a role for osteocytes as a potential future mechanistic target to understand rebound bone loss and increased fractures with denosumab discontinuation.

Histological characteristics of advanced peri-implantitis bone defects in humans

BackgroundInflammatory osteolysis is the clinical hallmark of peri-implantitis. The morphology of the remaining peri-implant bone and the level of osseointegration, however, remain unknown. Our aim was to characterize advanced peri-implantitis bone defects in humans.MethodsFour patients (3 female and 1 male) were diagnosed with peri-implantitis. A total of 5 implants with machined surfaces and a mean loading time of 12 ± 6 years were removed due to advanced bone loss. The defect extension, the peri-implant bone density (bone area per tissue area in percentage), bone-to-implant contact (%), and the number of filled and empty osteocyte lacunae were calculated based on undecalcified histological specimens.ResultsThe defect extension was on average 4.2 mm (95% CI 0.8–3.4). Remaining peri-implant bone showed a high density of 85.5% (95% CI 79.1–91.3) and covered in total 74% (95% CI 70.5–77.5) of the implant surface. Filled and empty osteocyte lacunae density was on average 191 and 165/mm2 (95% CI 132–251; 103–225), respectively. Histology further revealed signs of ongoing bone formation and resorption.ConclusionThere are signs that suggest that once the original cortical bone is lost due to peri-implantitis, the remaining apical trabecular bone is reinforced and transformed into cortical bone that might take over the functional load.

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 (0-6months, n=14; 2-8years, n=6 and 18-25years, 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.

Morphology and osteohistology of a large‐bodied caenagnathid (Theropoda, Oviraptorosauria) from the Hell Creek Formation (Montana): implications for size‐based classifications and growth reconstruction in theropods

AbstractOviraptorosaurs, like many coelurosaurians, are frequently diagnosed using incomplete or fragmentary skeletal remains, with factors such as body size often used to assign material to a particular taxon or as a basis for describing new species. Here we describe a partial skeleton,ROM VP65884, from the Hell Creek Formation (Montana,USA), and through morphological and phylogenetic comparisons identify it as belonging to Caenagnathidae, and probably referable toAnzu wyliei. We perform multi‐element osteohistological sampling of the tibia, fibula, manual phalanx, rib and gastralium ofROM VP65884 to determine the ontogenetic status of the individual, and to perform a detailed assessment of the intra‐skeletal variation present in a series of histological proxies for growth and development. Skeletochronological analysis suggestsROM VP65884 was still actively growing at time of death. Lacunar area was variable, but consistent with values reported for other dinosaurs and vertebrates. Osteocyte lacunar density was found to be relatively similar to the few previous measurements known from coelurosaurian dinosaurs, and preserved no ontogenetic pattern. Osteocyte lacunar density (OLD) was much higher in sampled rib and gastralia elements when compared to limbs, and the high variability inOLDoverall lends further support against the hypothesis of this feature acting as a proxy for mass‐specific growth rate. Our results stress the importance of using osteohistological analyses to constrain variation and permit ontogenetic assessment, and suggest caution be taken when using size as a determinant for referral of disassociated elements to specific or new coelurosaur taxa.