People living with HIV have low trabecular bone mineral density, high bone marrow adiposity, and poor trabecular bone microarchitecture at the proximal femur.

People living with HIV (PLWH) have increased risk of osteoporosis and fractures. We assessed the proximal femur of PLWH and age-matched seronegative controls using quantitative computed tomography and magnetic resonance imaging. Results suggest that the trabecular compartment is compromised at fracture-prone regions in the proximal femur of PLWH. INTRODUCTION: People living with HIV (PLWH) have increased risk of osteoporosis and fractures. However, studies assessing the main determinants of bone strength in the proximal femur exclude this vulnerable population. We assessed the proximal femur of 40 PLWH and 26 age-matched seronegative controls using quantitative computed tomography and magnetic resonance imaging. METHODS: We examined cortical volumetric bone mineral density (Ct.vBMD), trabecular vBMD (Tb.vBMD), cortical thickness (Ct.Th), bone marrow adiposity (BMA), and trabecular number, separation, and bone volume fraction. Parametric comparisons between the two groups were made for the femoral head, femoral neck, trochanter, and total hip using linear regression adjusting for several covariates, including metrics of body composition. In addition, we investigated the associations of BMA with Tb.vBMD and trabecular microarchitecture with Spearman's rank partial correlations. RESULTS: PLWH had lower Tb.vBMD and deteriorated trabecular microarchitecture in the femoral neck, trochanter and total hip, and elevated BMA in the femoral head, femoral neck, and total hip. Ct.vBMD and Ct.Th were not significantly different between the two groups. BMA was significantly associated with lower Tb.vBMD and deteriorated trabecular microarchitecture in both groups albeit at different femoral regions. CONCLUSIONS: Our findings suggest that the trabecular, and not the cortical, compartment is compromised in the proximal femur of PLWH. The observed impairments in fracture-prone regions in PLWH indicate lower femoral strength and suggest higher fracture risk. The inverse associations of BMA with trabecular bone density and microarchitecture quality agree with findings at other anatomic sites and in other populations, suggesting that excess BMA possibly due to a switch from the osteoblast to the adipocyte lineage may be implicated in the pathogenesis of bone fragility at the femur in PLWH.

Bone and cartilage demonstrate changes localized to bone marrow edema-like lesions within osteoarthritic knees.

OBJECTIVE: Our objective is to understand the biological and mechanical pathways linking cartilage, bone, and marrow changes in the progression of osteoarthritis (OA). The aim of the present study was to evaluate bone structure and composition within bone marrow edema-like lesion (BMEL) regions associated with knee OA. METHODS: Tibial plateau specimens (n = 18) were collected from 10 subjects with knee OA during total knee arthroplasty (TKA). Magnetic resonance (MR) imaging was used to identify BMEL and quantify metrics of cartilage composition. Micro-computed tomography (µCT) and high-resolution peripheral quantitative computed tomography (HR-pQCT) were used to quantify density and microstructure of the subchondral trabecular bone. Fourier transform infrared (FTIR) spectroscopy was used to quantify tissue composition. RESULTS: Trabecular bone within BMEL was higher in volume fraction, with more and thicker trabeculae that were more plate-like in structure compared to unaffected regions. BMEL trabecular tissue composition had decreased phosphate and carbonate content. Marrow infiltration by a fibrous collagen network and evidence of increased bone remodeling were present. Structural and compositional changes were specifically localized to regions underlying cartilage degradation. CONCLUSION: These results support the paradigm of focal interactions among bone, marrow, and cartilage in the progression of knee OA. Quantitative evaluation of tissue changes and interactions may aid in the understanding of disease pathophysiology and provide imaging markers for disease progression.

Mineral composition is altered by osteoblast expression of an engineered G(s)-coupled receptor.

Activation of the G(s) G protein-coupled receptor Rs1 in osteoblasts increases bone mineral density by 5- to 15-fold in mice and recapitulates histologic aspects of fibrous dysplasia of the bone. However, the effects of constitutive G(s) signaling on bone tissue quality are not known. The goal of this study was to determine bone tissue quality in mice resulting from osteoblast-specific constitutive G(s) activation, by the complementary techniques of FTIR spectroscopy and synchrotron radiation micro-computed tomography (SRµCT). Col1(2.3)-tTA/TetO-Rs1 double transgenic (DT) mice, which showed osteoblast-specific constitutive G(s) signaling activity by the Rs1 receptor, were created. Femora and calvariae of DT and wild-type (WT) mice (6 and 15 weeks old) were analyzed by FTIR spectroscopy. WT and DT femora (3 and 9 weeks old) were imaged by SRµCT. Mineral-to-matrix ratio was 25% lower (P = 0.010), carbonate-to-phosphate ratio was 20% higher (P = 0.025), crystallinity was 4% lower (P = 0.004), and cross-link ratio was 11% lower (P = 0.025) in 6-week DT bone. Differences persisted in 15-week animals. Quantitative SRµCT analysis revealed substantial differences in mean values and heterogeneity of tissue mineral density (TMD). TMD values were 1,156 ± 100 and 711 ± 251 mg/cm(3) (mean ± SD) in WT and DT femoral diaphyses, respectively, at 3 weeks. Similar differences were found in 9-week animals. These results demonstrate that continuous G(s) activation in murine osteoblasts leads to deposition of immature bone tissue with reduced mineralization. Our findings suggest that bone tissue quality may be an important contributor to increased fracture risk in fibrous dysplasia patients.

Three-dimensional surface texture visualization of bone tissue through epifluorescence-based serial block face imaging.

Serial block face imaging is a microscopy technique in which the top of a specimen is cut or ground away and a mosaic of images is collected of the newly revealed cross-section. Images collected from each slice are then digitally stacked to achieve 3D images. The development of fully automated image acquisition devices has made serial block face imaging more attractive by greatly reducing labour requirements. The technique is particularly attractive for studies of biological activity within cancellous bone as it has the capability of achieving direct, automated measures of biological and morphological traits and their associations with one another. When used with fluorescence microscopy, serial block face imaging has the potential to achieve 3D images of tissue as well as fluorescent markers of biological activity. Epifluorescence-based serial block face imaging presents a number of unique challenges for visualizing bone specimens due to noise generated by sub-surface signal and local variations in tissue autofluorescence. Here we present techniques for processing serial block face images of trabecular bone using a combination of non-uniform illumination correction, precise tiling of the mosaic in each cross-section, cross-section alignment for vertical stacking, removal of sub-surface signal and segmentation. The resulting techniques allow examination of bone surface texture that will enable 3D quantitative measures of biological processes in cancellous bone biopsies.

Automated simulation of areal bone mineral density assessment in the distal radius from high-resolution peripheral quantitative computed tomography.

SUMMARY: An automated image processing method is presented for simulating areal bone mineral density measures using high-resolution peripheral quantitative computed tomography (HR-pQCT) in the ultra-distal radius. The accuracy of the method is validated against clinical dual X-ray absorptiometry (DXA). This technique represents a useful reference to gauge the utility of novel 3D quantification methods applied to HR-pQCT in multi-center clinical studies and potentially negates the need for separate forearm DXA measurements. INTRODUCTION: Osteoporotic status is primarily assessed by measuring areal bone mineral density (aBMD) using 2D dual X-ray absorptiometry (DXA). However, this technique does not sufficiently explain bone strength and fracture risk. High-resolution peripheral quantitative computed tomography (HR-pQCT) has been introduced as a method to quantify 3D bone microstructure and biomechanics. In this study, an automated method is proposed to simulate aBMD measures from HR-pQCT distal radius images. METHODS: A total of 117 subject scans were retrospectively analyzed from two clinical bone quality studies. The distal radius was imaged by HR-pQCT and DXA on one of two devices (Hologic or Lunar). Areal BMD was calculated by simulation from HR-pQCT images (aBMD(sim)) and by standard DXA analysis (aBMD(dxa)). RESULTS: The reproducibility of the simulation technique was 1.1% (root mean-squared coefficient of variation). HR-pQCT-based aBMD(sim) correlated strongly to aBMD(dxa) (Hologic: R (2) = 0.82, Lunar: R (2) = 0.87), though aBMD(sim) underestimated aBMD(dxa) for both DXA devices (p < 0.0001). Finally, aBMD(sim) predicted aBMD at the proximal femur and lumbar spine with equal power compared to aBMD(dxa). CONCLUSION: The results demonstrate that aBMD can be simulated from HR-pQCT images of the distal radius. This approach has the potential to serve as a surrogate forearm aBMD measure for clinical HR-pQCT studies when axial bone mineral density values are not required.

Assessment of bone tissue mineralization by conventional x-ray microcomputed tomography: comparison with synchrotron radiation microcomputed tomography and ash measurements.

Assessment of bone tissue mineral density (TMD) may provide information critical to the understanding of mineralization processes and bone biomechanics. High-resolution three-dimensional assessment of TMD has recently been demonstrated using synchrotron radiation microcomputed tomography (SRmuCT); however, this imaging modality is relatively inaccessible due to the scarcity of SR facilities. Conventional desktop muCT systems are widely available and have been used extensively to assess bone microarchitecture. However, the polychromatic source and cone-shaped beam geometry complicate assessment of TMD by conventional muCT. The goal of this study was to evaluate muCT-based measurement of degree and distribution of tissue mineralization in a quantitative, spatially resolved manner. Specifically, muCT measures of bone mineral content (BMC) and TMD were compared to those obtained by SRmuCT and gravimetric methods. Cylinders of trabecular bone were machined from human femoral heads (n = 5), vertebrae (n = 5), and proximal tibiae (n = 4). Cylinders were imaged in saline on a polychromatic muCT system at an isotropic voxel size of 8 microm. Volumes were reconstructed using beam hardening correction algorithms based on hydroxyapatite (HA)-resin wedge phantoms of 200 and 1200 mg HA/cm3. SRmuCT imaging was performed at an isotropic voxel size of 7.50 microm at the National Synchrotron Light Source. Attenuation values were converted to HA concentration using a linear regression derived by imaging a calibration phantom. Architecture and mineralization parameters were calculated from the image data. Specimens were processed using gravimetric methods to determine ash mass and density, muCT-based BMC values were not affected by altering the beam hardening correction. Volume-averaged TMD values calculated by the two corrections were significantly different (p = 0.008) in high volume fraction specimens only, with the 1200 mg HA/cm3 correction resulting in a 4.7% higher TMD value. MuCT and SRmuCT provided significantly different measurements of both BMC and TMD (p < 0.05). In high volume fraction specimens, muCT with 1200 mg HA/cm3 correctionteg resulted in BMC and TMD values 16.7% and 15.0% lower, respectively, than SRmuCT values. In low volume fraction specimens, muCT with 1200 mg HA/cm3 correction resulted in BMC and TMD values 12.8% and 12.9% lower, respectively, than SRmuCT values. MuCT and SRmuCT values were well-correlated when volume fraction groups were considered individually (BMC R2 = 0.97-1.00; TMD R2 = 0.78-0.99). Ash mass and density were higher than the SRmuCT equivalents by 8.6% in high volume fraction specimens and 10.9% in low volume fraction specimens (p < 0.05). BMC values calculated by tomography were highly correlated with ash mass (ash versus muCT R2 = 0.96-1.00; ash versus SRmuCT R2 = 0.99-1.00). TMD values calculated by tomography were moderately correlated with ash density (ash versus muCT R2 = 0.64-0.72; ash versus SRmuCT R2 = 0.64). Spatially resolved comparisons highlighted substantial geometric nonuniformity in the muCT data, which were reduced (but not eliminated) using the 1200 mg HA/cm3 beam hardening correction, and did not exist in the SRmuCT data. This study represents the first quantitative comparison of muCT mineralization evaluation against SRnuCT and gravimetry. Our results indicate that muCT mineralization measures are underestimated but well-correlated with SRmuCT and gravimetric data, particularly when volume fraction groups are considered individually.

Validity of serial milling-based imaging system for microdamage quantification.

Understanding the three-dimensional distribution of microdamage within trabecular bone may help provide a better understanding of the mechanisms of bone failure. Toward that end, a novel serial milling-based fluorescent imaging system was developed for quantifying microscopic damage in three dimensions throughout cores of trabecular bone. The overall goal for this study was to compare two-dimensional (2D), surface-based measures of microdamage extracted from this new imaging system against those from more conventional histological section analyses. Human vertebral trabecular cores were isolated, stained en bloc with a series of chelating fluorochromes, monotonically loaded, and underwent microdamage quantification via the two methods. Bone area fraction measured by the new system was significantly correlated to that measured by histological point counting (p<0.001, R(2)=0.80). Additionally, the new system produced statistically equivalent (p=0.021) measures of damage fraction (mean+/-SD), Dx.AF=0.047+/-0.021, to that obtained from stereological point counting, Dx.AF=0.048+/-0.017, at a 10% difference level. These results demonstrate that this serial milling-based fluorescent imaging system provides a destructive yet practical alternative to more conventional histologic section analysis in addition to its ability to provide a better understanding of the three-dimensional nature of microdamage.

Automated high-resolution three-dimensional fluorescence imaging of large biological specimens.

We describe a novel automated technique for visualizing the three-dimensional distribution of fluorochrome-labelled components, in which image resolution is uncoupled from specimen size. This method is based on computer numerically controlled milling technology and combines an arrayed imaging technique with fluorescence capabilities. Fluorescent signals are segmented by emission spectra such that multiple fluorochromes present within a single specimen may be reconstructed and visualized individually or as a group. The automated nature of the system minimizes the workload and time involved in image capture and volume reconstruction. As an application, the system was used to image zones of fluorochrome-labelled microdamage within an 8-mm diameter cylinder of trabecular bone at a voxel size of 3 x 3 x 8 microm3. Our reconstruction of this specimen provides a visual map and quantitative measures of the volume of damage present throughout the cylinder, clearly demonstrating the interpretive power afforded by three-dimensional visualization. The three-dimensional nature of this highly automated and adaptable system has the potential to facilitate new diagnostic tools and techniques with application to a wide range of biological and medical research fields.