Severe cancellous bone loss occurs after spinal cord injury (SCI), which increases fracture risk. Bodyweight-supported treadmill training (TM) and passive Cycle training are activity-based rehabilitation therapies that improve neuromuscular plasticity after SCI. However, the skeletal adaptations to these therapies remain unknown. PURPOSE: Determine whether TM or Cycle training alter the rate of cancellous bone loss in a rodent severe contusion SCI model. METHODS: 16-wk old male Sprague-Dawley rats received: 1) SHAM surgery (T9 laminectomy) (n=9), 2) T9 laminectomy plus severe contusion SCI (n=8), 3) SCI+TM (n=14), or 4) SCI+Cycle (n=7). TM and Cycle were initiated 1-wk post-SCI and consisted of two 20 min bouts/day for 3 wks. For TM, 40% bodyweight support was provided and the paralyzed hindlimbs were manually positioned into plantar stepping (3.5 m/min, increasing 0.1 m/min/day). For Cycle, the paralyzed hindlimbs were secured to pedals on a motor-driven bike and moved passively through a cycling motion that mimicked normal gait patterning (12 rotations/min). Distal femur cancellous bone was quantified before surgery (baseline), and at 2- and 4-wk post-surgery via in vivo microCT. Outcomes are reported as percent change from baseline. RESULTS: Across all groups, cancellous bone volume (cBV/TV) was reduced 52-75% at 2-wk and 54-84% at 4-wk, compared with baseline (p<0.01). cBV/TV loss was 22% greater in SCI at 2-wk and 29% greater at 4-wk vs SHAM (p<0.01), characterized by 28% lower trabecular number (Tb.N) and 90% higher trabecular separation (Tb.Sp) (p<0.01) and a higher trabecular pattern factor (Tb.Pf) (p<0.05) that indicates a less connected trabecular network. At 2-wk, neither TM nor Cycle prevented SCI-induced bone deficits. However, at 4-wk SCI+Cycle displayed 25-30% higher cBV/TV, 23-24% higher trabecular thickness (Tb.Th), 17-22% higher Tb.N, and lower Tb.Pf vs SCI and SCI+TM (p<0.01). Ultimately, no differences in cancellous bone outcomes were present between SCI+Cycle and SHAM at 4-wk, except for 16% higher Tb.Th in SCI+Cycle (p<0.01). CONCLUSION: Our data indicate Cycle better attenuated cancellous bone loss in rodents after severe SCI. The higher cBV/TV and Tb.Th in SCI+Cycle at 4-wk also suggests that this modality stimulated bone formation; although, further investigation is needed.

Loading and testosterone may influence musculoskeletal recovery after spinal cord injury (SCI). Our objectives were to determine (a) the acute effects of bodyweight-supported treadmill training (TM) on hindlimb cancellous bone microstructure and muscle mass in adult rats after severe contusion SCI and (b) whether longer-term TM with adjuvant testosterone enanthate (TE) delivers musculoskeletal benefit. In Study 1, TM (40 min/day, 5 days/week, beginning 1 week postsurgery) did not prevent SCI-induced hindlimb cancellous bone loss after 3 weeks. In Study 2, TM did not attenuate SCI-induced plantar flexor muscles atrophy nor improve locomotor recovery after 4 weeks. In our main study, SCI produced extensive distal femur and proximal tibia cancellous bone deficits, a deleterious slow-to-fast fiber-type transition in soleus, lower muscle fiber cross-sectional area (fCSA), impaired muscle force production, and levator ani/bulbocavernosus (LABC) muscle atrophy after 8 weeks. TE alone (7.0 mg/week) suppressed bone resorption, attenuated cancellous bone loss, constrained the soleus fiber-type transition, and prevented LABC atrophy. In comparison, TE+TM concomitantly suppressed bone resorption and stimulated bone formation after SCI, produced near-complete cancellous bone preservation, prevented the soleus fiber-type transition, attenuated soleus fCSA atrophy, maintained soleus force production, and increased LABC mass. 75% of SCI+TE+TM animals recovered voluntary over-ground hindlimb stepping, while no SCI and only 20% of SCI+TE animals regained stepping ability. Positive associations between testosterone and locomotor function suggest that TE influenced locomotor recovery. In conclusion, short-term TM alone did not improve bone, muscle, or locomotor recovery in adult rats after severe SCI, while longer-term TE+TM provided more comprehensive musculoskeletal benefit than TE alone.

Muscle atrophy is a major sequela occurring after spinal cord injury (SCI) that results from disuse. Additionally, other secondary complications of SCI (e.g., alterations in muscle blood flow) may contribute to muscle loss. PURPOSE: To determine the time course of muscle blood flow changes in relation to muscle atrophy in a rodent severe contusion SCI model. METHODS: Sixty-three 4-months-old (skeletally-mature) male Sprague-Dawley rats received SHAM surgery (T9 laminectomy) or severe (250 kdyne) contusion SCI using a computer-guided impactor. At 1-, 2-, and 4-weeks (wk) post-surgery, an intravenous catheter was implanted into the tail vein of SHAM and SCI animals. Colored microspheres (15μm diameter) were then infused into the circulation, allowing for the measurement of regional blood flow (ml/min/g tissue mass). Subsequently, the animals were euthanized and the mass of the dissected right and left soleus, gastrocnemius, and plantaris were taken. Concentrations of the colored microspheres within each muscle were determined via spectrophotometry, following chemical digestion of the muscle. Muscle blood flow calculations were then averaged across the contralateral hindlimbs for the aforementioned muscles. SCI vs SHAM comparisons were made at each time point using independent samples t-tests and Pearson’s correlation coefficients. RESULTS: SCI animals exhibited 23-41% lower soleus mass, 17-27% lower gastrocnemius mass, and 16-29% lower plantaris mass vs SHAM, at all time points (p<0.001). Soleus and gastrocnemius blood flow (corrected for tissue mass, ml/min/g) was 51% lower (p<0.001) and 25% lower (p<0.05) after SCI, respectfully, at 1-wk only. Additionally, a positive relationship between soleus mass and blood flow (corrected for mass) was identified at 1-wk (r= 0.687, p<0.01). No significant alterations in plantaris blood flow were identified at any time point. CONCLUSION: Hindlimb muscle atrophy and reduced muscle blood flow occurred within 1-wk of severe contusion SCI. Thereafter, muscle blood renormalized in comparison with SHAM animals. Further research is needed to determine whether the reductions in muscle blood flow occurring after SCI contribute to muscle loss and/or whether prevention of blood flow deficits preserves muscle mass.

Bone fractures are associated with considerable morbidity and increased mortality. A major limitation to healing is lack of bone blood flow, which is impaired by physical disruption of intraskeletal and/or periosteal vasculature by the fracture. Thus, pharmacological interventions are needed to improve osseous blood flow, thereby accelerating bone fracture closure. Relaxin is secreted by the ovary and circulates in rodents and humans during pregnancy. Because relaxin might benefit bone fracture healing by stimulating angiogenesis, vasculogenesis (and potentially osteogenesis) through mobilization and activation of bone marrow progenitor cells, and by increasing blood flow via vasodilation, we investigated whether relaxin administration would accelerate closure of a calvarial defect in mice. Whether administered systemically by osmotic pump or locally by collagen scaffolds for ~2 week period after lesioning, relaxin did not accelerate bone healing. Despite implementing relaxin doses that reached plasma concentrations spanning the physiological to supraphysiological range, testing the closure of two different sizes of calvarial lesions, allowing for different intervals of time from instigation of cranial lesion to euthanasia, and investigating mice of different ages, we did not observe a significant benefit of relaxin in bone lesion healing. Nor did we observe stimulation of blood vessel formation in the bone lesion by the hormone. An incidental finding was that relaxin appeared to enhance trabecular bone growth in an uninjured control bone (femur). Although the results of this study were not supportive of a therapeutic benefit for relaxin on calvarial defect closure, future investigation is needed employing different animal species and experimental models of bone fracture.

Bone loss following spinal cord injury (SCI) is a major contributor to bone fracture risk, particularly at the proximal tibia. PURPOSE: To determine longitudinal changes in trabecular bone integrity at the proximal tibia in a rodent contusion SCI model. METHODS: 16-week old male Sprague-Dawley rats (n = 28) were randomized to receive no surgery (Non-Surgical Controls), T9 laminectomy (SHAM), or T9 laminectomy plus severe (250 kdyne) contusion SCI using a computer-guided impactor and were euthanized 2- or 4-weeks (w) post-surgery. Hindlimb locomotion was assessed weekly using the BBB locomotor scale and trabecular bone integrity at the proximal tibia was assessed weekly using in vivo microCT. Comparisons were made using one-way ANOVAs and post-hoc analyses were done when appropriate. RESULTS: SCI animals exhibited significant losses in hindlimb locomotor function [BBB score < 6 (0-21 scale); p<0.01 vs SHAM at all time points]. Percent cancellous bone volume (cBV/TV) was 32% lower at 2-w and 42% lower at 4-w in SCI vs SHAM animals (p<0.01). This bone loss was exemplified by progressively lower trabecular thickness (Tb.Th) and trabecular number (Tb.N) at 2- and 4-w (p<0.01) and higher trabecular separation (Tb.Sp) (p<0.01 at 4-w only). No differences in trabecular pattern factor (Tb.Pf), an inverse indicator of trabecular network connectedness, were present at 1-w. However, Tb.Pf was higher at 2- and 4-w in SCI vs SHAM (p<0.01). Structure model index (SMI) was higher at 2- and 4-w in SCI vs SHAM (p<0.01), indicating transition from rod- to plate-shaped trabecular spicules. Similarly, in SCI animals, cBV/TV was 48-75% lower, Tb.Th was 15-27% lower, Tb.N was 36-62% lower across the 4-w period in comparison with Non-Surgical Controls (p<0.01 for all), while Tb.Sp was progressively higher in SCI animals (p<0.05 for 2- and 4-w). Higher Tb.Pf and SMI were also found in SCI vs Non-Surgical Controls at all timepoints (p<0.05). Only cBV/TV was lower in SHAM vs Non-Surgical Controls across the 4-w period (p<0.01). CONCLUSION: Our findings indicate that trabecular bone integrity at the proximal tibia was significantly impaired in rats following a severe contusion SCI due to both bone loss and diminished bone quality. As such, our rodent model may be useful to examine effectiveness of strategies intended to prevent SCI-induced bone loss.

AbstractObjectivesTo determine the extent that zoledronate (ZOL) dose and duration is associated with bisphosphonate‐related osteonecrosis of the jaw (BRONJ) prevalence in rice rats with generalized periodontitis (PD), characterize structural and tissue‐level features of BRONJ‐like lesions in this model, and examine the specific anti‐resorptive role of ZOL in BRONJ.Materials and MethodsRice rats (n = 228) consumed high sucrose‐casein diet to enhance generalized PD. Groups of rats received 0, 8, 20, 50 or 125 µg/kg IV ZOL/4 weeks encompassing osteoporosis and oncology ZOL doses. Rats from each dose group (n = 9–16) were necropsied after 12, 18, 24 and 30 weeks of treatment. BRONJ‐like lesion prevalence and tissue‐level features were assessed grossly, histopathologically and by MicroCT. ZOL bone turnover effects were assessed by femoral peripheral quantitative computed tomography, serum bone turnover marker ELISAs and osteoclast immunolabelling.ResultsPrevalence of BRONJ‐like lesions was significantly associated with (a) ZOL treatment duration, but plateaued at the lowest oncologic dose, and (b) there was a similar dose‐related plateau in the systemic anti‐resorptive effect of ZOL. ZOL and BRONJ‐like lesions also altered the structural and tissue‐level features of the jaw.ConclusionThe relationship between BRONJ‐like lesion prevalence and ZOL dose and duration varies depending on the co‐ or pre‐existing oral risk factor. At clinically relevant doses of ZOL, BRONJ‐like lesions are associated with anti‐resorptive activity.

Acquired brain injuries (ABI) have been known to lead to a host of neurological impairments including visual imbalances that can greatly impact everyday life. Along with headaches, dizziness, and photophobia individuals with ABI report difficulty with postural alignment, balance, and gait. It has been suggested that common symptoms associated with ABIs are due in part to poor integration of the sensory motor system. Recent research has shown that yoked prisms can alter spatial perception and if used correctly draw the visual midline to its corrected position. PURPOSE: To examine the effects of yoked prism lenses on individuals with acquired brain injuries to determine if there is a change in gait. METHODS: 13 individuals with ABI (mean age: 43.1±18.1 years) walked 2-4 times at their self-selected speed across a 14’ GAITRite walkway under two conditions with current vision correcting prescription: without additional yoked prisms (WOP) and with a yoked prism lens (WP). Spatiotemporal gait parameters consisting of gait speed, stride length, stride width, and single support percent were extracted and averaged across the trials within each condition. Paired samples t-test was used to compare the gait parameters across both the conditions. RESULTS: Wearing yoked prism lens resulted in increased gait speed (WOP: 92.3±25.9 cm/s; WP: 99.1±28.9 cm/s; p=0.007) and stride length (WOP: 102.1±17.7 cm; WP: 108.3±20.0 cm; p=0.035). Stride width (WOP: 10.8±3.8 cm; WP: 10.8±3.0 cm; p=0.855) and single support percent (WOP: 37.0±2.8; WP: 37.3±2.9; p=0.335) did not show significant changes. CONCLUSION: When participants with reported ABIs wore yoked prism lenses they may be experiencing better integration of their sensory motor systems including a corrective shift in visual midline which allows for an increase in gait speed and stride length, indicating an overall improvement in gait. Our findings suggest that yoked prism lenses have acute benefits to improve gait in individuals with ABIs. Long-term benefits need to be evaluated.

To elucidate mechanisms of bone loss after spinal cord injury (SCI), we evaluated the time-course of cancellous and cortical bone microarchitectural deterioration via microcomputed tomography, measured histomorphometric and circulating bone turnover indices, and characterized the development of whole bone mechanical deficits in a clinically relevant experimental SCI model. 16-weeks-old male Sprague–Dawley rats received T 9 laminectomy (SHAM, n = 50) or moderate–severe contusion SCI ( n = 52). Outcomes were assessed at 2-weeks, 1-month, 2-months, and 3-months post-surgery. SCI produced immediate sublesional paralysis and persistent hindlimb locomotor impairment. Higher circulating tartrate-resistant acid phosphatase 5b (bone resorption marker) and lower osteoblast bone surface and histomorphometric cancellous bone formation indices were present in SCI animals at 2-weeks post-surgery, suggesting uncoupled cancellous bone turnover. Distal femoral and proximal tibial cancellous bone volume, trabecular thickness, and trabecular number were markedly lower after SCI, with the residual cancellous network exhibiting less trabecular connectivity. Periosteal bone formation indices were lower at 2-weeks and 1-month post-SCI, preceding femoral cortical bone loss and the development of bone mechanical deficits at the distal femur and femoral diaphysis. SCI animals also exhibited lower serum testosterone than SHAM, until 2-months post-surgery, and lower serum leptin throughout. Our moderate–severe contusion SCI model displayed rapid cancellous bone deterioration and more gradual cortical bone loss and development of whole bone mechanical deficits, which likely resulted from a temporal uncoupling of bone turnover, similar to the sequalae observed in the motor-complete SCI population. Low testosterone and/or leptin may contribute to the molecular mechanisms underlying bone deterioration after SCI.

Sclerostin is a circulating osteocyte-derived glycoprotein that negatively regulates Wnt-signaling after binding the LRP5/LRP6 co-receptors. Pharmacologic sclerostin inhibition produces bone anabolic effects after spinal cord injury (SCI), however, the effects of sclerostin-antibody (Scl-Ab) on muscle morphology remain unknown. In comparison, androgen administration produces bone antiresorptive effects after SCI and some, but not all, studies have reported that testosterone treatment ameliorates skeletal muscle atrophy in this context. Our purposes were to determine whether Scl-Ab prevents hindlimb muscle loss after SCI and compare the effects of Scl-Ab to testosterone enanthate (TE), an agent with known myotrophic effects. Male Sprague-Dawley rats aged 5 months received: (A) SHAM surgery (T8 laminectomy), (B) moderate-severe contusion SCI, (C) SCI+TE (7.0 mg/wk, im), or (D) SCI+Scl-Ab (25 mg/kg, twice weekly, sc). Twenty-one days post-injury, SCI animals exhibited a 31% lower soleus mass in comparison to SHAM, accompanied by >50% lower soleus muscle fiber cross-sectional area (fCSA) (p<0.01 for all fiber types). Scl-Ab did not prevent soleus atrophy, consistent with the relatively low circulating sclerostin concentrations and with the 91-99% lower LRP5/LRP6 gene expressions in soleus versus tibia (p<0.001), a tissue with known anabolic responsiveness to Scl-Ab. In comparison, TE partially prevented soleus atrophy and increased levator ani/bulbocavernosus (LABC) mass by 30-40% (p<0.001 vs all groups). The differing myotrophic responsiveness coincided with a 3-fold higher androgen receptor gene expression in LABC versus soleus (p<0.01). This study provides the first direct evidence that Scl-Ab does not prevent soleus muscle atrophy in rodents after SCI and suggests that variable myotrophic responses in rodent muscles after androgen administration are influenced by androgen receptor expression.

Sublesional bone loss occurs rapidly following spinal cord injury (SCI) and contributes to a 20-100 fold greater bone fracture risk. PURPOSE: To determine the time course of bone strength deficits at different femoral test sites in a rodent contusion SCI model. METHODS: Sixty 16-week old male Sprague-Dawley rats received SHAM surgery or T9 laminectomy plus severe (250 kilodyne) contusion SCI using a computer-guided impactor and were euthanized 1-, 2-, or 3-months (m) post-surgery. Hindlimb locomotion was assessed weekly using the BBB locomotor scale and bone strength was assessed ex vivo at the distal femur, femoral midshaft, and femoral neck. SCI vs SHAM comparisons were made at each time point using independent t-tests. RESULTS: SCI animals exhibited persistent hindlimb locomotor deficits [BBB score < 6 (0-21 scale), p < 0.01 vs SHAMs at all time points], characterized by an inability to support the hindlimbs in stance or to perform hindlimb weight supported stepping. Bone strength deficits were observed at all testing sites after SCI in a somewhat variable pattern. At the distal femur, maximal breaking load (N) was 19% lower at 1-m (p < 0.05), 10% lower at 2-m (p < 0.05), and 16% lower at 3-m (p < 0.01) in SCI vs SHAM animals using a cantilever bending test. In addition, displacement at max load was 29% lower at 1-m (p < 0.05) and 22% lower at 3-m (p < 0.05). At the femoral neck, maximal breaking load was 22% lower at 1-m in SCI vs SHAM (p < 0.05), but was not different at 2-m or 3-m post-surgery. At the femoral midshaft, maximal breaking load was not different at 1-m post-surgery, but was 11% lower at 2-m (p < 0.05), and 23% lower at 3-m (p < 0.05) in SCI vs SHAMs, using a 3-point breaking test. No other differences in displacement or stiffness were observed among groups. CONCLUSION: In our SCI model, femoral skeletal integrity is compromised 1-m post-injury, with strength deficits dependent upon the skeletal site and the tests that were utilized. The distal femur cantilever test yielded less variability and typifies a common site of fracture in humans after SCI, suggesting this test is clinically-relevant. Interventions focused on preventing bone loss after SCI should initiate therapy soon after the injury occurs to ensure maintenance of skeletal integrity. Supported by VA RR&D SPiRE 1I21RX001373-01 to JFY.

We have reported that testosterone-enanthate (TE) prevents the musculoskeletal decline occurring acutely after spinal cord injury (SCI), but results in a near doubling of prostate mass. Our purpose was to test the hypothesis that administration of TE plus finasteride (FIN; type II 5α-reductase inhibitor) would prevent the chronic musculoskeletal deficits in our rodent severe contusion SCI model, without inducing prostate enlargement. Forty-three 16-week-old male Sprague-Dawley rats received: 1) SHAM surgery (T9 laminectomy); 2) severe (250 kdyne) contusion SCI; 3) SCI+TE (7.0 mg/week, intramuscular); or 4) SCI+TE+FIN (5 mg/kg/day, subcutaneous). At 8 weeks post-surgery, SCI animals exhibited reduced serum testosterone and levator ani/bulbocavernosus (LABC) muscle mass, effects that were prevented by TE. Cancellous and cortical (periosteal) bone turnover (assessed by histomorphometry) were elevated post-SCI, resulting in reduced distal femur cancellous and cortical bone mass (assessed by microcomputed tomography). TE treatment normalized cancellous and cortical bone turnover and maintained cancellous bone mass at the level of SHAM animals, but produced prostate enlargement. FIN coadministration did not inhibit the TE-induced musculoskeletal effects, but prevented prostate growth. Neither drug regimen prevented SCI-induced cortical bone loss, although no differences in whole bone strength were present among groups. Our findings indicate that TE+FIN prevented the chronic cancellous bone deficits and LABC muscle loss in SCI animals without inducing prostate enlargement, which provides a rationale for the inclusion of TE+FIN in multimodal therapeutic interventions intended to alleviate the musculoskeletal decline post-SCI.

Dietary-induced obesity (DIO) resulting from high-fat (HF) or high-sugar diets produces a host of deleterious metabolic consequences including adverse bone development. We compared the effects of feeding standard rodent chow (Control), a 30% moderately HF (starch-based/sugar-free) diet, or a combined 30%/40% HF/high-fructose (HF/F) diet for 12weeks on cancellous/cortical bone development in male Sprague–Dawley rats aged 8weeks. Both HF feeding regimens reduced the lean/fat mass ratio, elevated circulating leptin, and reduced serum total antioxidant capacity (tAOC) when compared with Controls. Distal femur cancellous bone mineral density (BMD) was 23–34% lower in both HF groups (p<0.001) and was characterized by lower cancellous bone volume (BV/TV, p<0.01), lower trabecular number (Tb.N, p<0.001), and increased trabecular separation versus Controls (p<0.001). Cancellous BMD, BV/TV, and Tb.N were negatively associated with leptin and positively associated with tAOC at the distal femur. Similar cancellous bone deficits were observed at the proximal tibia, along with increased bone marrow adipocyte density (p<0.05), which was negatively associated with BV/TV and Tb.N. HF/F animals also exhibited lower osteoblast surface and reduced circulating osteocalcin (p<0.05). Cortical thickness (p<0.01) and tissue mineral density (p<0.05) were higher in both HF-fed groups versus Controls, while whole bone biomechanical characteristics were not different among groups. These results demonstrate that “westernized” HF diets worsen cancellous, but not cortical, bone parameters in skeletally-immature male rats and that fructose incorporation into HF diets does not exacerbate bone loss. In addition, they suggest that leptin and/or oxidative stress may influence DIO-induced alterations in adolescent bone development.

ABSTRACT Spinal cord injury (SCI) results in rapid and extensive sublesional bone loss. Sclerostin, an osteocyte-derived glycoprotein that negatively regulates intraskeletal Wnt signaling, is elevated after SCI and may represent a mechanism underlying this excessive bone loss. However, it remains unknown whether pharmacologic sclerostin inhibition ameliorates bone loss subsequent to SCI. Our primary purposes were to determine whether a sclerostin antibody (Scl-Ab) prevents hindlimb cancellous bone loss in a rodent SCI model and to compare the effects of a Scl-Ab to that of testosterone-enanthate (TE), an agent that we have previously shown prevents SCI-induced bone loss. Fifty-five (n = 11–19/group) skeletally mature male Sprague-Dawley rats were randomized to receive: (A) SHAM surgery (T8 laminectomy), (B) moderate-severe (250 kilodyne) SCI, (C) 250 kilodyne SCI + TE (7.0 mg/wk, im), or (D) 250 kilodyne SCI + Scl-Ab (25 mg/kg, twice weekly, sc) for 3 weeks. Twenty-one days post-injury, SCI animals exhibited reduced hindlimb cancellous bone volume at the proximal tibia (via μCT and histomorphometry) and distal femur (via μCT), characterized by reduced trabecular number and thickness. SCI also reduced trabecular connectivity and platelike trabecular structures, indicating diminished structural integrity of the remaining cancellous network, and produced deficits in cortical bone (femoral diaphysis) strength. Scl-Ab and TE both prevented SCI-induced cancellous bone loss, albeit via differing mechanisms. Specifically, Scl-Ab increased osteoblast surface and bone formation, indicating direct bone anabolic effects, whereas TE reduced osteoclast surface with minimal effect on bone formation, indicating antiresorptive effects. The deleterious microarchitectural alterations in the trabecular network were also prevented in SCI + Scl-Ab and SCI + TE animals, whereas only Scl-Ab completely prevented the reduction in cortical bone strength. Our findings provide the first evidence indicating that sclerostin inhibition represents a viable treatment to prevent SCI-induced cancellous and cortical bone deficits and provides preliminary rationale for future clinical trials focused on evaluating whether Scl-Ab prevents osteoporosis in the SCI population. © 2014 American Society for Bone and Mineral Research.