Elliott Goff

Clinical studies indicate that microvascular disease (MVD) affects bone microstructure and decreases bone strength in type 2 diabetes mellitus (T2D). Osteocytes are housed in small voids within the bone matrix and lacunae and act as sensors of mechanical forces in bone. These cells regulate osteoclastic bone resorption and osteoblastic bone formation as well as osteocytic perilacunar remodeling. We hypothesized that MVD changes morphometric osteocyte lacunar parameters in individuals with T2D. We collected iliac crest bone biopsies from 35 individuals (10 female, 25 male) with T2D with MVD (15%) or without MVD (21%) with a median age of 67 years (interquartile range [IQR] 62–72 years). The participants were included based on c‐peptide levels >700 pmol L⁻¹, absence of anti‐GAD65 antibodies, and glycated hemoglobin (HbA1c) levels between 40 and 82 mmol mol⁻¹ or 5.8% and 9.7%, respectively. We assessed osteocyte lacunar morphometric parameters in trabecular and cortical bone regions using micro‐computed tomography (micro‐CT) at a nominal resolution of 1.2 μm voxel size. The cortical osteocyte lacunar volume (Lc.V) was 7.7% larger (p = 0.05) and more spherical (Lc.Sr, p < 0.01) in the T2D + MVD group. Using linear regression, we found that lacunar density (Lc.N/BV) in trabecular but not cortical bone was associated with HbA1c (p < 0.05, R² = 0.067) independently of MVD. Furthermore, Lc.V was larger and Lc.Sr higher in the center than in the periphery of the trabecular and cortical bone regions (p < 0.05). In conclusion, these data imply that MVD may impair skeletal integrity, possibly contributing to increased skeletal fragility in T2D complicated by MVD. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Bone’s adaptation ability is governed by the network of embedded osteocytes that inhabit individual crevasses called lacunae. The morphology of these lacunae and their resident osteocytes are known to change with age and diseases such as postmenopausal osteoporosis. However, it is unclear whether alterations in lacunar morphology are present in younger populations with osteoporosis. To investigate this, we implemented a previously validated methodology to image and quantify the three-dimensional morphometries of lacunae on a large scale (26.2 million cells) with ultra-high-resolution micro-computed tomography (microCT) in transiliac bone biopsies from three groups of premenopausal women: control n=39; idiopathic osteoporosis (IOP) n=45; idiopathic low BMD (ILBMD) n=19. Important lacunar morphometric parameters were measured in both trabecular and cortical bone: lacunar density (Lc.N/BV), lacunar porosity (Lc.TV/BV), lacunar number (Lc.N), lacunar volume (Lc.V), lacunar surface area (Lc.S), lacunar alignment (Lc.θ), lacunar stretch (Lc.St), lacunar oblateness (Lc.Ob), lacunar equancy (Lc.Eq), and lacunar sphericity (Lc.Sr). These were then compared against each other and also with previously measured tissue morphometries including: bone volume density (BV/TV), trabecular separation (Tb.Sp), trabecular number (Tb.N), and trabecular thickness (Tb.Th), structure model index (SMI), cortical porosity (Ct.Po) and cortical pore spacing (Ct.Sp). We detected no differences in lacunar morphology between the IOP, ILBMD and healthy premenopausal women. In contrast, we did find significant differences between lacunar morphologies in cortical and trabecular regions within all three groups, which was consistent with our previous findings on a subgroup of the healthy group. Furthermore, we discovered strong correlations between Lc.Sr from both trabecular and cortical regions with the measured BV/TV. The findings and comprehensive lacunar dataset we present here will be a crucial foundation for future investigations of the relationship between osteocyte lacunar morphology and disease.

Ultra-high-resolution imaging of the osteocyte lacuno-canalicular network (LCN) three-dimensionally (3D) in a high-throughput fashion has greatly improved the morphological knowledge about the constituent structures – positioning them as potential biomarkers. Technologies such as serial focused ion beam/scanning electron microscopy (FIB/SEM) and confocal scanning laser microscopy (CLSM) can image in extremely high resolution, yet only capture a small number of lacunae. Synchrotron radiation computed tomography (SR-CT) can image with both high resolution and high throughput but has a limited availability. Desktop micro-computed tomography (micro-CT) provides an attractive balance: high-throughput imaging on the micron level without the restrictions of SR-CT availability. Over the past decade, desktop micro-CT has been used to image osteocyte lacunae in a variety of animals, yet few studies have employed it to image human lacunae using clinical biopsies. In this study, accuracy, reproducibility, and sensitivity of large-scale quantification of human osteocyte lacunar morphometries were assessed by ultra-high-resolution desktop micro-computed tomography. For this purpose, thirty-one transiliac human bone biopsies containing trabecular and cortical regions were imaged using ultra-high-resolution desktop micro-CT at a nominal isotropic voxel resolution of 1.2μm. The resulting 3D images were segmented, component labeled, and the following morphometric parameters of 7.71 million lacunae were measured: Lacunar number (Lc.N), density (Lc.N/BV), porosity (Lc.TV/BV), volume (Lc.V), surface area (Lc.S), surface area to volume ratio (Lc.S/Lc.V), stretch (Lc.St), oblateness (Lc.Ob), sphericity (Lc.Sr), equancy (Lc.Eq), and angle (Lc.θ). Accuracy was quantified by comparing automated lacunar identification to manual identification. Mean true positive rate (TPR), false positive rate (FPR), and false negative rate (FNR) were 89.0%, 3.4%, and 11.0%, respectively. Regarding the reproducibility of lacunar morphometry from repeated measurements, precision errors were low (0.2 – 3.0%) and intraclass correlation coefficients were high (0.960 – 0.999). Significant differences between cortical and trabecular regions (p<0.001) existed for Lc.N/BV, Lc.TV/BV, local lacunar surface area (), and local lacunar volume (), all of which demonstrate the sensitivity of the method and are possible biomarker candidates. This study provides the foundation required for future large-scale morphometric studies using ultra-high-resolution desktop micro-CT and high-throughput analysis of millions of osteocyte lacunae in human bone samples. Furthermore, the validation of this technology for imaging of human lacunar properties establishes the quality and reliability required for the accurate, precise, and sensitive assessment of osteocyte morphometry in clinical bone biopsies.