Sarah L. Manske

Human trabecular bone microarchitecture can be assessed independently of density with second generation HR-pQCT

The second generation HR-pQCT scanner (XtremeCTII, Scanco Medical) can assess human bone microarchitecture of peripheral limbs with a 61 μm nominal isotropic voxel size. This is a marked improvement from the first generation HR-pQCT that had a nominal isotropic voxel size of 82 μm, which is at the limit to accurately determine the thickness of individual human trabeculae. We sought to determine the accuracy of a direct morphometric approach to measure trabecular bone microarchitecture with three-dimensional morphological techniques using second generation HR-pQCT, and to compare this with the approach currently applied by the first generation HR-pQCT scanner based on derived indices using ex vivo scans of human cadaveric radii. We also compared images acquired and resampled to mimic the first generation HR-pQCT with those obtained directly from the first generation HR-pQCT. We evaluated 20 human cadaveric radii and a micro-CT performance phantom using the first (XtremeCT, Scanco Medical) and second generation HR-pQCT scanner (XtremeCTII) and compared a patient evaluation (XCTII, 61 μm) with a high resolution ex vivo protocol (HR, 30μm). We generated 82 μm scans of the same specimens to mimic a first-generation HR-pQCT evaluation (XCTIM, 82 μm) and compared these with a first-generation patient evaluation (XCTI, 82 μm). A standard structural extraction approach was applied to both XCTII and HR evaluations for assessment of bone volume fraction (BV/TV), and a distance transform was used to assess trabecular number (Tb.N), trabecular thickness (Tb.Th) and trabecular separation (Tb.Sp). For XCTI and XCTIM evaluations we followed the manufacturers standard procedure and assessed bone mineral density (BMD), Tb.N with a distance transform, and then derived bone volume ratio (BV/TV(d)), trabecular thickness (Tb.Th(d)) and separation (Tb.Sp(d)). The spatial resolution (10% MTF) was 142.2 μm for XCTI, 108.9 μm for XCTIM, 95.2μm for XCTII, and 55.9 μm for HR. XCTI and XCTIM provided strongly associated measurements of BMD and microarchitectural outcomes (R(2)>0.97), however there were systematic differences in all outcomes. The Tb.N was highly associated with HR by both XCTII (R(2)=0.93, mean error=-0.12 mm(-1)) and XCTIM (R(2)=0.98, mean error=0.25 mm(-1)). Also, both XCTII (R(2)=0.99, mean error=0.20mm) and XCTIM (R(2)=0.99, mean error=-0.18 mm) had Tb.Sp that were strongly related to HR. For Tb.Th, the XCTII was more closely related to HR (R(2)=0.94, mean error=0.04 mm) than the relatively weak XCTIM (R(2)=0.16, mean error=- 0.076 mm). We found that trabecular microarchitecture assessment following the XCTII direct morphometric approach accurately represented the HR data. In particular, the measure of Tb.Th was markedly improved for XCTII compared with the derived approach of XCTIM. These data support the application of analysis techniques in HR-pQCT that are analogous to those traditionally used for micro-CT to assess trabecular microarchitecture. The decreased dependence of structural outcomes on density provides a new, important opportunity to monitor human in vivo bone microarchitecture.

Muscle and bone follow similar temporal patterns of recovery from muscle-induced disuse due to botulinum toxin injection

If muscle force is a primary source for triggering bone adaptation, with disuse and reloading, bone changes should follow muscle changes. We examined the timing and magnitude of changes in muscle cross-sectional area (MCSA) and bone architecture in response to muscle inactivity following botulinum toxin (BTX) injection. We hypothesized that MCSA would return to baseline levels sooner than bone properties following BTX injection. Female BALB mice (15 weeks old) were injected with 20 muL of BTX (1 U/100 g body mass, n=18) or saline (SAL, n=18) into the posterior calf musculature of one limb. The contralateral limb (CON) served as an internal control. MCSA and bone properties were assessed at baseline, 2, 4, 8, 12, and 16 weeks post-injection using in vivo micro-CT at the tibia proximal metaphysis (bone only) and diaphysis. Muscles were dissected and weighed after sacrifice. Significant GroupxLegxTime interactions indicated that the maximal decrease in MCSA (56%), proximal metaphyseal BV/TV (38%) and proximal diaphyseal Ct.Ar (7%) occurred 4 weeks after injection. There was no delay prior to bone recovery as both muscle and bone properties began to recover after this time, but MCSA and BV/TV remained 15% and 20% lower, respectively, in the BTX-injected leg than the BTX-CON leg 16 weeks post-injection. Gastrocnemius mass (primarily fast-twitch) was 14% lower in the BTX-injected leg than the SAL-injected leg, while soleus mass (primarily slow-twitch) was 15% greater in the BTX group than the SAL group. Our finding that muscle size and bone began to recover at similar times after BTX injection was unexpected. This suggested that partial weight-bearing and/or return of slow-twitch muscle activity in the BTX leg may have been sufficient to stimulate bone recovery. Alternatively, muscle function may have recovered sooner than MCSA. Our results indicated that muscle cross-sectional area, while important, may not be the primary factor associated with bone loss and recovery when muscle atrophy is induced through BTX injection. To understand the nature of the interaction between muscle and bone, future work should focus on the functional recovery of individual muscles in relation to bone.

High-frequency, low-magnitude vibration does not prevent bone loss resulting from muscle disuse in mice following botulinum toxin injection.

High-frequency, low-magnitude vibration enhances bone formation ostensibly by mimicking normal postural muscle activity. We tested this hypothesis by examining whether daily exposure to low-magnitude vibration (VIB) would maintain bone in a muscle disuse model with botulinum toxin type A (BTX). Female 16–18 wk old BALB/c mice (N = 36) were assigned to BTX-VIB, BTX-SHAM, VIB, or SHAM. BTX mice were injected with BTX (20 µL; 1 U/100 g body mass) into the left hindlimb posterior musculature. All mice were anaesthetized for 20 min/d, 5 d/wk, for 3 wk, and the left leg mounted to a holder. Through the holder, VIB mice received 45 Hz, ±0.6 g sinusoidal acceleration without weight bearing. SHAM mice received no vibration. At baseline and 3 wk, muscle cross-sectional area (MCSA) and tibial bone properties (epiphysis, metaphysis and diaphysis) were assessed by in vivo micro-CT. Bone volume fraction in the metaphysis decreased 12±9% and 7±6% in BTX-VIB and BTX-SHAM, but increased in the VIB and SHAM. There were no differences in dynamic histomorphometry outcomes between BTX-VIB and BTX nor between VIB and SHAM. Thus, vibration did not prevent bone loss induced by a rapid decline in muscle activity nor produce an anabolic effect in normal mice. The daily loading duration was shorter than would be expected from postural muscle activity, and may have been insufficient to prevent bone loss. Based on the approach used in this study, vibration does not prevent bone loss in the absence of muscle activity induced by BTX.

Quantitative in vivo assessment of bone microarchitecture in the human knee using HR-pQCT

OBJECTIVE High-resolution peripheral quantitative computed tomography (HR-pQCT) is a novel imaging modality capable of visualizing bone microarchitecture in vivo at human peripheral sites such as the distal radius and distal tibia. This research has extended the technology to provide a non-invasive assessment of bone microarchitecture at the human knee by establishing new hardware, imaging protocols and data analysis. DESIGN A custom leg holder was developed to stabilize a human knee centrally within a second generation HR-pQCT field of view. Five participants with anterior cruciate ligament reconstructions had their knee joint imaged in a continuous scan of 6cm axially. The nominal isotropic voxel size was 60.7μm. Bone mineral density and microarchitecture were assessed within the weight-bearing regions of medial and lateral compartments of the knee at three depths from the weight-bearing articular bone surface, including both the cortical and trabecular bone regions. RESULTS Scan duration was approximately 18min per knee and produced 5GB of projection data and 10GB of reconstructed image data (2304×2304 image matrix, 1008 slices). Motion during the scan was minimized by the leg holder and was similar in magnitude as a scan of the distal tibia. Bone mineral density and microarchitectural parameters were assessed for 16 volumes of interest in the tibiofemoral joint. CONCLUSIONS This is a new non-invasive in vivo assessment tool for bone microarchitecture in the human knee that provides an opportunity to gain insight into normal, injured and surgically reconstructed human knee bone architecture in cross-sectional or longitudinal studies.

Vertical ground reaction forces diminish in mice after botulinum toxin injection.

We examined changes in weight-bearing ability in mice after injection with botulinum toxin type A (BTX) to determine whether BTX can be used to isolate the effects of muscle on bone. As ambulation patterns were previously shown to improve within two weeks post-injection, we hypothesized that BTX injection to the posterior hindlimb would not significantly affect the mouses ability to bear weight in the affected limb one week post-injection. Female BALB/c mice (N=13, 16-17 week old) were injected with either 20 μL of BTX (1U/100 g) or saline (SAL) in the left posterior hindlimb. Vertical ground reaction forces (GRF), hindlimb muscle cross-sectional area (MCSA), and tibial bone micro-architecture were assessed for 42 d following injection. Peak and average vertical GRF were 11±1% and 23±3% lower, respectively, in the BTX-injected hindlimb within 4d post-injection and remained lower than the SAL-injected hindlimb 14-21 d post-injection (15±4% and 10±2%, respectively). Time between forelimb and hindlimb peaks was 30-40% greater in the BTX-injected hindlimb than SAL-injected hindlimb 4-14 d post-injection. Peak vertical GRF recovered earlier following BTX injection than MCSA or bone volume fraction. These results indicate that weight-bearing ability recovered despite persistent muscle atrophy, and that weight-bearing alone was insufficient to maintain bone in the absence of muscle activity. We suggest that the absence of high-frequency signals typically associated with fast-twitch muscle activity may be contributing to the ongoing degradation of bone after BTX injection.

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.

Skim milk powder enhances trabecular bone architecture compared with casein or whey in diet-induced obese rats

OBJECTIVE We previously showed that skim milk powder (SMP) prevents weight gain more so than casein or whey alone. Dairy foods and changes in body mass can affect bone architecture; therefore, our objective was to examine the effect of dairy proteins on bone structure in the tibia of dietary-induced obese rats. METHODS Twelve-week-old diet-induced obese Sprague-Dawley rats were randomized to one of six diets that varied in protein source (casein, whey, or SMP), Ca level (0.67% or 2.4%), and energy density (high-fat/high-sucrose [HFHS], or normal energy density [NE]). After 8 wk, body composition was assessed via dual energy x-ray absorptiometry and trabecular and cortical bone parameters of the tibia were assessed using micro-computed tomography and mixed model analysis. RESULTS Rats fed SMP with 2.4% calcium had significantly lower body mass and fat mass than all other groups. The ratio of bone volume to total volume (BV/TV) was significantly higher when the HFHS diet was supplemented with SMP and 2.4% calcium compared with whey (+66.7%) or casein (+32.6%). The HFHS diet group had 49.3% greater BV/TV compared with the NE groups. Increasing the amount of calcium resulted in a significant increase in BV/TV (188.9%) in the HFHS diet groups but not in the NE groups. CONCLUSION The intake of skim milk powder supplemented with calcium enhances trabecular bone architecture in obese rats consuming HFHS diet to a greater extent than with either casein or whey protein alone. Bioactive ingredients in complete dairy may contribute to these effects.

Lower Bone Density, Impaired Microarchitecture, and Strength Predict Future Fragility Fracture in Postmenopausal Women: 5-Year Follow-up of the Calgary CaMos Cohort: BASELINE SKELETAL PARAMETERS PREDICT FUTURE FRAGILITY FRACTURES

The aim of this prospective study was to use high‐resolution peripheral quantitative computed tomography (HR‐pQCT) to determine if baseline skeletal parameters can predict incident fragility fracture in women and, secondly, to establish if women that fracture lose bone at a faster rate than those who do not fracture. Women older than 60 years who experienced a fragility fracture during the 5‐year follow‐up period (incident fracture group, n = 22) were compared with those who did not experience a fragility fracture during the study (n = 127). After image registration between baseline and follow‐up measures, standard and cortical morphological analyses were conducted. Odds ratios were calculated for baseline values and annualized percent change of HR‐pQCT and finite element variables. At the radius, baseline HR‐pQCT results show women who fractured had lower total bone mineral density (Tt.BMD; 19%), trabecular bone mineral density (Tb.BMD; 25%), and trabecular number (Tb.N; 14%), with higher trabecular separation (Tb.Sp; 19%) than women who did not fracture. At the tibia, women with incident fracture had lower Tt.BMD (15%), Tb.BMD (12%), cortical thickness (Ct.Th; 14%), cortical area (Ct.Ar; 12%), and failure load (10%) with higher total area (Tt.Ar; 7%) and trabecular area (Tb.Ar; 10%) than women who did not fracture. Odds ratios (ORs) at the radius revealed every SD decrease of Tt.BMD (OR = 2.1), Tb.BMD (OR = 2.0), and Tb.N (OR = 1.7) was associated with a significantly increased likelihood of fragility fracture. At the tibia, every SD decrease in Tt.BMD (OR = 2.1), Tb.BMD (OR = 1.7), Ct.Th (OR = 2.2), Ct.Ar (OR = 1.9), and failure load (OR = 1.7) were associated with a significantly increased likelihood of fragility fracture. Irrespective of scanning modality, the annualized percent rate of bone loss was not different between fracture groups. The results suggest baseline bone density, microarchitecture, and strength rather than change in these variables are associated with incident fragility fractures in women older than 60 years. Furthermore, irrespective of fragility fracture status, women experienced changes in skeletal health at a similar rate.

Bone health: part 2, physical activity.

Mechanical loading is a crucial factor for maintaining skeletal health. Physical activities, exercise, and sports provide a wealth and variety of mechanical loads to bones, through muscle forces, ground reaction forces, and other contact or impact forces. Weightbearing activities can be effective exercises to enhance bone health—particularly, those that involve jumping and impact loads (with greater strain magnitudes, rates, and frequencies). Physical activity appears to be acutely beneficial for enhancing bone health in the early pubertal period and in older age, such as in postmenopausal women. In preparing this article, PubMed, Web of Science, and relevant edited books (English language) were reviewed from 1961 to present.

Bilateral Asymmetry of Radius and Tibia Bone Macroarchitecture and Microarchitecture: A High-Resolution Peripheral Quantitative Computed Tomography Study

Studies assessing bone health often select the dominant or nondominant limb to scan, but not both, for efficiency reasons. New scanning technology allows 3-dimensional (3D) visualization of the microarchitecture in bone, but it is not well understood whether there are differences between the dominant and nondominant limbs. Using 3D high-resolution peripheral quantitative computed tomography (HR-pQCT), the aim of this study is to investigate the effect of limb dominance on bone macroarchitecture and microarchitecture. Healthy male and female participants (N=100; 59 female, 41 male), mean age 30.7±12.1 years, were scanned at both radii and tibiae using HR-pQCT. Hand and foot dominance were determined by the participants self-report. Most participants were right hand dominant (94.0%) and right foot dominant (91.0%). In the pooled cohort, the dominant radius had significantly greater cortical area (2.11%; p=0.002) and failure load (3.00%; p=0.001). At the tibia, the dominant foot had significantly lower bone mineral density (-0.77%; p=0.042), cortical area (-1.05%; p=0.031), and thickness (-1.51%; p=0.017). For females, there were no differences at the radius, but at the tibia, the dominant side had greater cross-sectional area (1.03%; p=0.044). Our data suggest that dominance has a small yet significant effect on macroarchitecture at both the ultradistal radius and tibia but not microarchitecture. This work emphasizes that it is important to be consistent in the selection of either dominant or nondominant limbs for HR-pQCT cohort studies; however, in the case where the opposite limb needs to be scanned, there would be small differences in macroarchitecture and no significant differences in microarchitecture anticipated.