Helen R. Buie

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.

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 (µCT) 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 µCT measurements, resulting in r2 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 μCT 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 ∼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.

Signs of irreversible architectural changes occur early in the development of experimental osteoporosis as assessed by in vivo micro-CT

SummaryUsing in vivo micro-computed tomography, we assessed bone loss in the rat during the first twelve weeks after ovariectomy when structural changes were most rapid. Significant changes to the trabecular architecture were observed, including irreversible changes reflected by reduction in connectivity after only two weeks. This highlights that topological changes to the structure occur early in this experimental model of osteoporosis.IntroductionThe purpose of this study was to establish a longitudinal time course of bone loss in the ovariectomized (OVX) rat model during the initial twelve-week period where structural changes are most rapid, and to identify when irreversible changes occur using in vivo micro-computed tomography (micro-CT).MethodsThe proximal tibiae of OVX (N = 10) and sham (N = 10) operated mature female Wistar rats were micro-CT scanned every two weeks from week 0 to week 12, excluding week 10. Changes in architecture were quantified using direct three-dimensional techniques and serum osteocalcin and CTX-I was measured at weeks 0, 6 and 12. Biomechanical properties were determined from femoral three-point bending and L-4 vertebral compression at the end of the protocol. ANOVA and paired t-tests were used to analyze the longitudinal and endpoint data, respectively.ResultsAll of the measured architectural parameters changed significantly over the study in the OVX group, including irreversible changes reflected by connectivity density after two weeks. Osteocalcin concentration was elevated in the OVX group. Moderate changes in the mechanical properties of the femora midshaft and vertebrae were observed.ConclusionsChanges to the bone architecture and mechanics within twelve weeks after OVX highlight the importance of early diagnosis and treatment of osteoporosis.

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.

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

BackgroundHigh 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.Methods12 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).ResultsBone 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% 95varied 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.ConclusionIn 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.

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.

Embryonic stem cell therapy improves bone quality in a model of impaired fracture healing in the mouse; tracked temporally using in vivo micro-CT

In the current study, we used an estrogen-deficient mouse model of osteoporosis to test the efficacy of a cell-generated bone tissue construct for bone augmentation of an impaired healing fracture. A reduction in new bone formation at the defect site was observed in ovariectomized fractures compared to the control group using repeated measures in vivo micro-computed tomography (μCT) imaging over 4 weeks. A significant increase in the bone mineral density (BMD), trabecular bone volume ratio, and trabecular number, thickness and connectivity were associated with fracture repair in the control group, whereas the fractured bones of the ovariectomized mice exhibited a loss in all of these parameters (p<0.001). In a separate group, ovariectomized fractures were treated with murine embryonic stem (ES) cell-derived osteoblasts loaded in a three-dimensional collagen I gel and recovery of the bone at the defect site was observed. A significant increase in the trabecular bone volume ratio (p<0.001) and trabecular number (p<0.01) was observed by 4 weeks in the fractures treated with cell-loaded collagen matrix compared to those treated with collagen I alone. The stem cell-derived osteoblasts were identified at the fracture site at 4 weeks post-implantation through in situ hybridization histochemistry. Although this cell tracking method was effective, the formation of an ectopic cellular nodule adjacent to the knee joints of two mice suggested that alternative in vivo cell tracking methods should be employed in order to definitively assess migration of the implanted cells. To our knowledge, this study is the first of its kind to examine the efficacy of stem cell therapy for fracture repair in an osteoporosis-related fracture model in vivo. The findings presented provide novel insight into the use of stem cell therapies for bone injuries.

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 (β=0.974, p<0.0001) to be a significant predictor of the defect volume (R(2)=0.949) and Spmax (β=0.712, p<0.0001) and SpSD (β=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.

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.