A comparison of methods for in vivo assessment of cortical porosity in the human appendicular skeleton.

The recent advent of high-resolution peripheral quantitative computed tomography (HR-pQCT) provides new opportunities to measure in vivo human bone microarchitecture. Increasingly, cortical porosity (CtPo) is of particular interest due to its relationship with bone quality and turnover. The two approaches that have emerged to measure CtPo from HR-pQCT are threshold-based and density-based methods, and the purpose of this work was to compare the performance of each against a gold-standard synchrotron radiation micro-computed tomography (SRµCT) measurement. Human cadaveric cortical bone specimens (N=23) were measured by SRµCT and HR-pQCT, and high correlations were found for both methods. The density-based approach had an r2=0.939 (95% confidence interval (CI) of +6.17% to +20.99%) and consistently overestimated porosity as measured by SRµCT, while the threshold-based approach had an r2=0.977 and consistently underestimated porosity (95% CI of -2.60% to -10.76%). The density-based approach is prone to beam hardening artifacts and susceptible to natural variations of tissue mineral density (TMD), but is less affected by motion artifacts that may occur in in vivo scans. The threshold-based method has the advantage that it provides structural information that complements the cortical porosity measure, such as number of pores and connectivity, and can accurately detect the larger pores which are the most relevant to bone biomechanical strength. With the first generation HR-pQCT systems the accuracy of detecting pores larger than 140 µm diameter is excellent (r2=0.983; 95% CI of -4.88% to +2.45%). The accuracy of the threshold-based method will improve as new HR-pQCT systems emerge and provide a robust quantitative approach to measure cortical porosity.

Bone quality in prehistoric, Cis-Baikal forager femora: a micro-CT analysis of cortical canal microstructure.

Bone quality, a contributor to bone strength, is determined by structural and mechanical properties, which may be analyzed by gross and/or microscopic methods. Variables that contribute to bone quality, such as porosity, can provide insight into the health and lifestyles of people in prehistory. This study tests the ability of microcomputed tomography (µCT) to capture and characterize cortical canal systems in archaeological bone. Seven variables and 71 femora are analyzed to explore bone dynamics in prehistoric foragers from Lake Baikal, Siberia. The results indicate that canal number and canal separation differ significantly (P < 0.05) between age-at-death categories, but only for the pooled and male samples. When merged into a new variable by means of principal components analysis, canal diameter and canal surface to canal volume are also able to discriminate amongst age-at-death categories, as well as between the sexes. However, the overall lack of significant differences between the sexes and amongst age-at-death categories indicates that Baikal forager bone quality (i.e., canal architecture) did not change drastically throughout the lifespan. Interestingly, principal component one identified an untested variable that contributes to canal microstructure variability, and a sexual division of labor may promote divergent trends in canal degree of anisotropy between the sexes. Overall, µCT provides an alternate method for exploring bone quality in archaeological remains, complementing existing methods such as thin-sectioning and gross morphological analyses.

A customized protocol to assess bone quality in the metacarpal head, metacarpal shaft and distal radius: a high resolution peripheral quantitative computed tomography precision study.

BACKGROUND: High Resolution-Peripheral Quantitative Computed Tomography (HR-pQCT) is an emerging technology for evaluation of bone quality in Rheumatoid Arthritis (RA). However, there are limitations with standard HR-pQCT imaging protocols for examination of regions of bone commonly affected in RA. We developed a customized protocol for evaluation of volumetric bone mineral density (vBMD) and microstructure at the metacarpal head (MH), metacarpal shaft (MS) and ultra-ultra-distal (UUD) radius; three sites commonly affected in RA. The purpose was to evaluate short-term measurement precision for bone density and microstructure at these sites. METHODS: 12 non-RA participants, individuals likely to have no pre-existing bone damage, consented to participate [8 females, aged 23 to 71 y [median (IQR): 44 (28) y]. The custom protocol includes more comfortable/stable positioning and adapted cortical segmentation and direct transformation analysis methods. Dominant arm MH, MS and UUD radius scans were completed on day one; repeated twice (with repositioning) three to seven days later. Short-term precision for repeated measures was explored using intraclass correlational coefficient (ICC), mean coefficient of variation (CV%), root mean square coefficient of variation (RMSCV%) and least significant change (LSC%95). RESULTS: Bone density and microstructure precision was excellent: ICCs varied from 0.88 (MH2 trabecular number) to .99 (MS3 polar moment of inertia); CV% varied from < 1 (MS2 vBMD) to 6 (MS3 marrow space diameter); RMSCV% varied from < 1 (MH2 full bone vBMD) to 7 (MS3 marrow space diameter); and LSC%95 varied from 2 (MS2 full bone vBMD to 21 (MS3 marrow space diameter). Cortical porosity measures were the exception; RMSCV% varying from 19 (MS3) to 42 (UUD). No scans were stopped for discomfort. 5% (5/104) were repeated due to motion during imaging. 8% (8/104) of final images had motion artifact graded > 3 on 5 point scale. CONCLUSION: In our facility, this custom protocol extends the potential for in vivo HR-pQCT imaging to assess, with high precision, regional differences in bone quality at three sites commonly affected in RA. Our methods are easy to adopt and we recommend other users of HR-pQCT consider this protocol for further evaluations of its precision and feasibility in their imaging facilities.

Micro-CT evaluation of bone defects: applications to osteolytic bone metastases, bone cysts, and fracture.

Bone defects can occur in various forms and present challenges to performing a standard micro-CT evaluation of bone quality because most measures are suited to homogeneous structures rather than ones with spatially focal abnormalities. Such defects are commonly associated with pain and fragility. Research involving bone defects requires quantitative approaches to be developed if micro-CT is to be employed. In this study, we demonstrate that measures of inter-microarchitectural bone spacing are sensitive to the presence of focal defects in the proximal tibia of two distinctly different mouse models: a burr-hole model for fracture healing research, and a model of osteolytic bone metastases. In these models, the cortical and trabecular bone compartments were both affected by the defect and were, therefore, evaluated as a single unit to avoid splitting the defects into multiple analysis regions. The burr-hole defect increased mean spacing (Sp) by 27.6%, spacing standard deviation (SpSD) by 113%, and maximum spacing (Spmax) by 72.8%. Regression modeling revealed SpSD (ß=0.974, p<0.0001) to be a significant predictor of the defect volume (R(2)=0.949) and Spmax (ß=0.712, p<0.0001) and SpSD (ß=0.271, p=0.022) to be significant predictors of the defect diameter (R(2)=0.954). In the mice with osteolytic bone metastases, spacing parameters followed similar patterns of change as reflected by other imaging technologies, specifically bioluminescence data which is indicative of tumor burden. These data highlight the sensitivity of spacing measurements to bone architectural abnormalities from 3D micro-CT data and provide a tool for quantitative evaluation of defects within a bone.

Reproducible metacarpal joint space width measurements using 3D analysis of images acquired with high-resolution peripheral quantitative computed tomography.

OBJECTIVE: Joint space narrowing is an important feature of progressive joint damage and functional impairment in rheumatoid arthritis (RA). Methods to provide a continuous measurement of joint space width have not been adopted in research or clinical settings. High-resolution peripheral quantitative computed tomography (HR-pQCT) (Scanco Medical AG, Brüttisellen, Switzerland) accurately and reproducibly images bone microstructure at a nominal isotropic voxel dimension of 82 µm. Given the ability of HR-pQCT to detect bone margins with high precision, we developed methodology to measure a three-dimensional (3D) metacarpophalangeal (MCP) joint space width and tested the reproducibility of the scan protocol with hand repositioning. MATERIALS AND METHODS: Consecutive HR-pQCT scans of the 2nd and 3rd MCP joints of ten subjects with early RA (70% female, mean age 45 years), with repositioning between scans, were obtained. The periosteal edges of the metacarpal head and proximal phalanx base were detected using the µCT Evaluation Program V6.0 (Scanco Medical AG). Using the method of 'fitting maximal spheres', the joint space width and distribution of joint space thickness was estimated. RESULTS: The mean minimum joint space width of the 2nd MCP was 1.82 mm (SD 0.20) and of the 3rd MCP 1.84 mm (SD 0.23). Reproducibility with repositioning was reliable, with overlapping filtered histograms and a root square mean coefficient of variance of 4.8%. CONCLUSIONS: We provide reproducible methodology for evaluating the joint space width of the MCP joints. When combined with the assessment of erosions and periarticular bone density, HR-pQCT may be the ideal technology to assess disease activity and progression in RA.

Embryonic stem cells incorporate into newly formed bone and do not form tumors in an immunocompetent mouse fracture model.

Embryonic stem (ES) cells are a uniquely self-renewing, pluripotent population of cells that must be differentiated before being useful for cell therapy. Since most studies utilize subcutaneous implantation to test the in vivo functionality of ES cell-derived cells, the objective of the current study was to develop an appropriate and clinically relevant in vivo implantation system in which the behavior and tumorigenicity of ES cell-derived cells could be effectively tested in a tissue-specific (orthotopic) site. Male ES cells were differentiated either into osteoblasts or chondrocytes using protocols that were previously developed and published by our laboratory. The differentiated cells were implanted into a burr-hole fracture created in the proximal tibiae of immunocompetent female mice, strain matched to the ES cell line. The ability of the differentiated ES cell-derived cells (bearing the Y chromosome) to incorporate into the newly formed bone was assessed by micro-computed tomography imaging and histochemistry. ES cells differentiated with either osteogenic or chondrogenic medium supplementation formed a soft tissue mass that disrupted the normal bone architecture by 4 weeks after implantation in some mice. In contrast, mice receiving osteoblastic cells that were differentiated in a three-dimensional type 1 collagen gel showed evidence of new bone formation at the defect site without evidence of tumor formation for up to 8 weeks after implantation. In this injury model, type 1 collagen is more effective than medium supplementation at driving more complete differentiation of ES cells, as evidenced by reducing their tumorigenicity. Overall, the current study emphasizes the importance of using an appropriate orthotopic implantation system to effectively test the behavior and tumorigenicity of the cells in vivo.

Quantitative ex-vivo micro-computed tomographic imaging of blood vessels and necrotic regions within tumors.

Techniques for visualizing and quantifying the microvasculature of tumors are essential not only for studying angiogenic processes but also for monitoring the effects of anti-angiogenic treatments. Given the relatively limited information that can be gleaned from conventional 2-D histological analyses, there has been considerable interest in methods that enable the 3-D assessment of the vasculature. To this end, we employed a polymerizing intravascular contrast medium (Microfil) and micro-computed tomography (micro-CT) in combination with a maximal spheres direct 3-D analysis method to visualize and quantify ex-vivo vessel structural features, and to define regions of hypoperfusion within tumors that would be indicative of necrosis. Employing these techniques we quantified the effects of a vascular disrupting agent on the tumor vasculature. The methods described herein for quantifying whole tumor vascularity represent a significant advance in the 3-D study of tumor angiogenesis and evaluation of novel therapeutics, and will also find potential application in other fields where quantification of blood vessel structure and necrosis are important outcome parameters.

Reproducibility of direct quantitative measures of cortical bone microarchitecture of the distal radius and tibia by HR-pQCT.

Quantitative cortical microarchitectural end points are important for understanding structure-function relations in the context of fracture risk and therapeutic efficacy. This technique study details new image-processing methods to automatically segment and directly quantify cortical density, geometry, and microarchitecture from HR-pQCT images of the distal radius and tibia. An automated segmentation technique was developed to identify the periosteal and endosteal margins of the distal radius and tibia and detect intracortical pore space morphologically consistent with Haversian canals. The reproducibility of direct quantitative cortical bone indices based on this method was assessed in a pooled data set of 56 subjects with two repeat acquisitions for each site. The in vivo precision error was characterized using root mean square coefficient of variation (RMSCV%) from which the least significant change (LSC) was calculated. Bland-Altman plots were used to characterize bias in the precision estimates. The reproducibility of cortical density and cross-sectional area measures was high (RMSCV <1% and <1.5%, respectively) with good agreement between young and elder medians. The LSC for cortical porosity (Ct.Po) was somewhat smaller in the radius (0.58%) compared with the distal tibia (0.84%) and significantly different between young and elder medians in the distal tibia (LSC: 0.75% vs. 0.92%, p<0.001). The LSC for pore diameter and distribution (Po.Dm and Po.Dm.SD) ranged between 15 and 23 microm. Bland-Altman analysis revealed moderate bias for integral measures of area and volume but not for density or microarchitecture. This study indicates that HR-pQCT measures of cortical bone density and architecture can be measured in vivo with high reproducibility and limited bias across a biologically relevant range of values. The results of this study provide informative data for the design of future clinical studies of bone quality.

Reduced bone mass accrual in swim-trained prepubertal mice.

INTRODUCTION: Clinical studies have found that nonimpact non-weight-bearing sports are associated with normal to low bone mineral densities. The objective of this study was to quantify structural bone adaptations of the mouse to swimming using in vivo micro-computed tomography. METHODS: Prepubertal female mice underwent a 16-wk training program, in which they swam for progressively increasing durations up to 55 min for 5 d·wk(-1). A sham group was subjected to the water, but they did not perform the swimming exercise. Skeletal sites that were assessed included the proximal humerus, lumbar spine, midshaft and distal femur, proximal tibia, and the skull. RESULTS: Normal bone mass accrual was suppressed in the swim group during the initial 4 wk of training, during rapid growth. Swim mice gained 31.8% less (P = 0.001) and 16.7% less (P = 0.003) trabecular bone volume at the proximal tibial and humeral metaphyses, respectively. In the 12 wk that followed, low trabecular bone volume persisted in the swimmers (P < 0.001). Trabecular properties were reduced primarily at the metaphysis but not the epiphysis, and cortical bone was reduced at both compartments. At the femoral midshaft, swim mice had less cortical porosity (SHAM = 32% ± 3% vs SWIM = 22% ± 3%, P = 0.043). Despite the bone microarchitectural adaptations, swimming did not induce detectable changes to macroarchitecture, including bone length and total volume. Tissue mineral density was unaffected by swimming, suggesting that material changes did not occur. CONCLUSIONS: In conclusion, the response of prepubertal mice to swimming was adaptation at the bone microarchitectural level and resulted in an altered topology that was sustained until the end of the training program. Low bone mass was systemic throughout the mouse, with the largest depressions in the hindlimbs and spine.

Postmenopausal women with osteopenia have higher cortical porosity and thinner cortices at the distal radius and tibia than women with normal aBMD: an in vivo HR-pQCT study.

Increases in cortical porosity (Ct.Po) and decreases in cortical thickness (Ct.Th) are associated with increased bone fragility. The purpose of this study was to validate an autosegmentation method for high-resolution peripheral quantitative computed tomography (HR-pQCT) scans to measure Ct.Po and Ct.Th and use it to compare Ct.Po and Ct.Th between pre- and postmenopausal women with normal, osteopenic, and osteoporotic areal bone mineral density (aBMD). The Ct.Po and Ct.Th measurements were validated using cadaver forearms (n = 10) and micro-computed tomography (microCT) as the gold standard. The analysis was applied to distal radius and tibia HR-pQCT scans from a subset of participants from the Calgary, Alberta, cohort of the Canadian Multicentre Osteoporosis Study (n = 280, 18 to 90 years). Analysis of covariance compared Ct.Po and Ct.Th outcomes between 63 normal premenopausal (dual-energy X-ray absorptiometry femoral neck T-score > -1), 87 normal postmenopausal, 121 osteopenic postmenopausal, and 9 osteoporotic postmenopausal women. Linear regression analysis and Bland-Altman plots were used to assess the agreement between the HR-pQCT and microCT measurements, resulting in r(2) values of 0.80 for Ct.Po and 0.98 for Ct.Th. At both sites, Ct.Po was higher in postmenopausal (all groups) than in premenopausal women (3.2% to 12.9%, p < .001). Ct.Th was not significantly different between normal premenopausal and postmenopausal women at either site; however, both osteopenic and osteoporotic women had thinner (-12.8% to -30.3%, p < .01), more porous (2.1% to 8.1%, p < .001) cortices than normal postmenopausal women. Our method offers promise as a valuable tool to measure Ct.Po and Ct.Th in vivo and investigate associations among cortical bone structure, age, and disease status.

Postpubertal architectural developmental patterns differ between the L3 vertebra and proximal tibia in three inbred strains of mice.

An understanding of normal microarchitectural bone development patterns of common murine models is needed. Longitudinal, structural, and mineralization trends were evaluated by in vivo microCT over 12 time points from 6-48 wk of age at the vertebra and tibia of C3H/HeN, C57BL/6, and BALB/C mice. Longitudinal growth occurred rapidly until 8-10 wk, slowed as the growth plate bridged, and fused at 8-10 mo. Structural augmentation occurred through formation of trabeculae at the growth plate and thickening of existing ones. In the vertebrae, BV/TV increased rapidly until 12 wk in all strains. Between 12 and 32 wk, the architecture was stable with BV/TV deviating <1.1%, 1.6%, and 3.4% for the C57BL/6, BALB/C, and C3H/HeN mice. In contrast, the tibial architecture changed continuously but more moderately for BV/TV and TbTh compared with the vertebra and with comparable or larger changes for TbN and TbSp. Age-related trabecular deterioration (decreased BV/TV and TbN; increased TbSp and structure model index) was evident at both sites at 32 wk. In all strains, the cortex continued to develop after trabecular values peaked. The temporal plateau of BMD was variable across mouse strains and site, whereas tissue mineral density was attained at approximately 6 mo for all sites and strains. Geometric changes at the tibial diaphysis occurred rapidly until 8-10 wk, providing the C57BL/6 mice and C3H/HeN mice with the highest torsional and compressive rigidity, respectively. In summary, key skeletal development milestones were identified, and architectural topology at the vertebra was found to be more stable than at the tibia.

Automatic segmentation of cortical and trabecular compartments based on a dual threshold technique for in vivo micro-CT bone analysis.

The use of high resolution peripheral quantitative computed tomography (HR-pQCT) and in vivo micro-CT for studies of bone disease and treatment has become increasingly common, and with these methods comes large quantities of data requiring analysis. A simple, robust, and fully-automated segmentation algorithm is presented that efficiently segments bone regions. The dual threshold technique refers to two required threshold inputs that are used to extract the periosteal and endosteal surfaces of the cortex. The proposed method was tested against the gold standard, semi-automated hand contouring, using 45 datasets: mouse, rat, human, and cadaver data from the tibia or radius with nominal isotropic resolutions of 10-82 microm. The performance of the proposed method to segment cortical and trabecular compartments was evaluated qualitatively from visualizations and quantitatively based on morphological measurements. Visual inspection confirmed successful segmentation of all datasets using the new method, with qualitatively better results when applied to the human and cadaver data compared to the gold standard. The dual threshold algorithm was able to extract thin and porous cortices, whereas some clipping and perforations occurred for the gold standard. Morphological parameters measured for segmentation by the proposed method versus the gold standard agreed (95% confidence) for Tb.Th, Tb.Sp, and Tb.N, but not Ct.Th and BV/TV for the human and cadaver datasets. Nonetheless, correlations ranged from 0.95 to 1.00 for all morphological parameters except the cadaver Ct.Th because systematic errors were present. Poor agreement for Ct.Th and BV/TV was due to qualitatively incorrect segmentation by the gold standard when the cortex was thin compared to trabeculae, or operator bias during hand contouring. Since Tb.Th, Tb.Sp, and Tb.N were insensitive to segmentation method, despite operator bias, they are robust parameters for inter-site comparisons. The dual threshold method offers a robust and fully-automated alternative to the gold standard that can efficiently segment bone regions with accurate and repeatable results. The algorithm can be easily implemented since it uses simple image analysis tools. Two input thresholds allow adjustment of the masked output, and are easily determined by trial and error. Using the same input thresholds for similar datasets assures maximal consistency while alleviating time consuming semi-automated contouring.