Osteocalcin affects bone mineral and mechanical properties in female mice.

Osteocalcin is one of the most abundant noncollagenous proteins in bone. Phenotypes of osteocalcin knock-out mice (OC-/-) may vary on different backgrounds and with sex. Previous studies using adult female (OC-/-) mice on a mixed genetic background (129/B6) showed osteocalcin inhibited bone formation leading to weaker bone in wild-type (OC+/+). Yet on a pure (B6) genetic background male mice revealed osteocalcin improved fracture resistance and OC-/- bones were more prone to fracture. Osteocalcin is decreased with age and in some diseases (diabetes) where bone weakness is observed. The effect of osteocalcin in adult female bone from mice on a pure B6 background is unknown. We investigated differences in bone mineral properties and bone strength in female adult (6 months) (OC+/+) and (OC-/-) mice on a pure C57BL/6J background using Fourier Transform Infrared Imaging (FTIRI), micro-computed tomography (uCT), biomechanical measurements, histomorphometry and serum turnover markers (P1NP, CTX). Similar to female age matched mice on the (129/C57) background we found B6 OC-/- mice had a higher bone formation rate, no change in bone resorption, more immature mineral, decreased crystallinity and increased trabecular bone as compared to OC+/+. In contrast, the OC-/- mice on a pure B6 background had a lower bone mineral density, lower mineral to matrix ratio resulting in reduced stiffness and weaker bone strength. Our results demonstrate some properties of the OC-/- phenotype are dependent on genetic background. This may suggest that reduced osteocalcin may contribute to fracture and weaker bone in some groups of elderly and adults with diseases where osteocalcin is low.

Low-level cyclic tibial compression attenuates early osteoarthritis progression after joint injury in mice.

OBJECTIVE: Mechanical loading and joint health have a unique relationship in osteoarthritis (OA) onset and progression. Although high load levels adversely affect cartilage health, exercise that involves low to moderate load levels can alleviate OA symptoms. We sought to isolate the beneficial effects of mechanical loading using controlled in vivo cyclic tibial compression. We hypothesized that low-level cyclic compression would attenuate post-traumatic OA symptoms induced by destabilization of the medial meniscus (DMM). METHODS: 10-week-old C57Bl/6J male mice underwent DMM surgery (n = 51). After a 5-day post-operative recovery period, we applied daily cyclic tibial compression to the operated limbs at low (1.0N or 2.0N) or moderate (4.5N) magnitudes for 2 or 6 weeks. At the completion of loading, we compared cartilage and peri-articular bone features of mice that underwent DMM and loading to mice that only underwent DMM. RESULTS: Compared to DMM alone, low-level cyclic compression for 6 weeks attenuated DMM-induced cartilage degradation (OARSI score, P = 0.008, 95% confidence interval (CI): 0.093 to 0.949). Low-level loading attenuated DMM-induced osteophyte formation after 2 weeks (osteophyte size, P = 0.033, 95% CI: 3.27-114.45 µm), and moderate loading attenuated subchondral bone sclerosis after 6 weeks (tissue mineral density (TMD), P = 0.011, 95% CI: 6.32-70.60 mg HA/ccm) compared to limbs that only underwent DMM. Finally, loading had subtle beneficial effects on cartilage cellularity and aggrecanase activity after DMM. CONCLUSION: Low-level cyclic compression is beneficial to joint health after an injury. Therefore, the progression of early OA may be attenuated by applying well controlled, low-level loading shortly following joint trauma.

The effects of metabolic syndrome, obesity, and the gut microbiome on load-induced osteoarthritis.

OBJECTIVE: Metabolic syndrome is characterized by obesity, hyperglycemia, hypertension, insulin resistance, and dyslipidemia. Metabolic syndrome is associated with osteoarthritis (OA), but it is unclear if the association is attributable to increased mechanical loading on joints caused by obesity or other aspects of metabolic syndrome. Here we examined the effects of altered metabolism, obesity, and the gut microbiome on load-induced OA. DESIGN: Cartilage damage was induced through cyclic compressive loading in four groups of adult male mice: Toll-like receptor-5 deficient (TLR5KO) mice that develop metabolic syndrome due to alterations in the gut microbiome, TLR5KO mice submitted to chronic antibiotics to prevent metabolic syndrome (TLR5KOΔMicrobiota), C57BL/6J mice fed a high fat diet to cause obesity (HFD), and untreated C57BL/6J mice (WT). Loading was applied for 2 weeks (n = 10-11/group) or 6 weeks (n = 10-11/group). RESULTS: After 2 weeks of loading, cartilage damage (OARSI score) was not different among groups. After 6 weeks of loading, HFD mice had increased load-induced cartilage damage, while TLR5KO mice had cartilage damage comparable to WT mice. TLR5KOΔMicrobiota mice had less cartilage damage than other groups. HFD mice had elevated serum inflammatory markers. Each group had a distinct gut microbiome composition. CONCLUSIONS: Severe obesity increased load-induced cartilage damage, while milder changes in adiposity/metabolic syndrome seen in TLR5KO mice did not. Furthermore, the effects of systemic inflammation/obesity on cartilage damage depend on the duration of mechanical loading. Lastly, reduced cartilage damage in the TLR5KOΔMicrobiota mice suggests that the gut microbiome may influence cartilage pathology.

Role of subchondral bone properties and changes in development of load-induced osteoarthritis in mice.

OBJECTIVE: Animal models recapitulating post-traumatic osteoarthritis (OA) suggest that subchondral bone (SCB) properties and remodeling may play major roles in disease initiation and progression. Thus, we investigated the role of SCB properties and its effects on load-induced OA progression by applying a tibial loading model on two distinct mouse strains treated with alendronate (ALN). DESIGN: Cyclic compression was applied to the left tibia of 26-week-old male C57Bl/6 (B6, low bone mass) and FVB (high bone mass) mice. Mice were treated with ALN (26 µg/kg/day) or vehicle (VEH) for loading durations of 1, 2, or 6 weeks. Changes in articular cartilage and subchondral and epiphyseal cancellous bone were analyzed using histology and microcomputed tomography. RESULTS: FVB mice exhibited thicker cartilage, a thicker SCB plate, and higher epiphyseal cancellous bone mass and tissue mineral density than B6 mice. Loading induced cartilage pathology, osteophyte formation, and SCB changes; however, lower initial SCB mass and stiffness in B6 mice did not attenuate load-induced OA severity compared to FVB mice. By contrast, FVB mice exhibited less cartilage damage, and slower-growing and less mature osteophytes. In B6 mice, inhibiting bone remodeling via ALN treatment exacerbated cartilage pathology after 6 weeks of loading, while in FVB mice, inhibiting bone remodeling protected limbs from load-induced cartilage loss. CONCLUSIONS: Intrinsically lower SCB properties were not associated with attenuated load-induced cartilage loss. However, inhibiting bone remodeling produced differential patterns of OA pathology in animals with low compared to high SCB properties, indicating that these factors do influence load-induced OA progression.

Non-invasive mouse models of post-traumatic osteoarthritis.

Animal models of osteoarthritis (OA) are essential tools for investigating the development of the disease on a more rapid timeline than human OA. Mice are particularly useful due to the plethora of genetically modified or inbred mouse strains available. The majority of available mouse models of OA use a joint injury or other acute insult to initiate joint degeneration, representing post-traumatic osteoarthritis (PTOA). However, no consensus exists on which injury methods are most translatable to human OA. Currently, surgical injury methods are most commonly used for studies of OA in mice; however, these methods may have confounding effects due to the surgical/invasive injury procedure itself, rather than the targeted joint injury. Non-invasive injury methods avoid this complication by mechanically inducing a joint injury externally, without breaking the skin or disrupting the joint. In this regard, non-invasive injury models may be crucial for investigating early adaptive processes initiated at the time of injury, and may be more representative of human OA in which injury is induced mechanically. A small number of non-invasive mouse models of PTOA have been described within the last few years, including intra-articular fracture of tibial subchondral bone, cyclic tibial compression loading of articular cartilage, and anterior cruciate ligament (ACL) rupture via tibial compression overload. This review describes the methods used to induce joint injury in each of these non-invasive models, and presents the findings of studies utilizing these models. Altogether, these non-invasive mouse models represent a unique and important spectrum of animal models for studying different aspects of PTOA.

Mechanical load increases in bone formation via a sclerostin-independent pathway.

Sclerostin, encoded by the Sost gene, is an important negative regulator of bone formation that has been proposed to have a key role in regulating the response to mechanical loading. To investigate the effect of long-term Sclerostin deficiency on mechanotransduction in bone, we performed experiments on unloaded or loaded tibiae of 10 week old female Sost-/- and wild type mice. Unloading was induced via 0.5U botulinum toxin (BTX) injections into the right quadriceps and calf muscles, causing muscle paralysis and limb disuse. On a separate group of mice, increased loading was performed on the left tibiae through unilateral cyclic axial compression of equivalent strains (+1200 µe) at 1200 cycles/day, 5 days/week. Another cohort of mice receiving equivalent loads (-9.0 N) also were assessed. Contralateral tibiae served as normal load controls. Loaded/unloaded and normal load tibiae were assessed at day 14 for bone volume (BV) and formation changes. Loss of BV was seen in the unloaded tibiae of wild type mice, but BV was not different between normal load and unloaded Sost-/- tibiae. An increase in BV was seen in the loaded tibiae of wild type and Sost-/- mice over their normal load controls. The increased BV was associated with significantly increased mid-shaft periosteal mineralizing surface/bone surface (MS/BS), mineral apposition rate (MAR), and bone formation rate/bone surface (BFR/BS), and endosteal MAR and BFR/BS. Notably, loading induced a greater increase in periosteal MAR and BFR/BS in Sost-/- mice than in wild type controls. Thus, long-term Sclerostin deficiency inhibits the bone loss normally induced with decreased mechanical load, but it can augment the increase in bone formation with increased load.

A rare case of a bisphosphonate-induced peri-prosthetic femoral fracture.

An 81-year-old woman presented with a fracture in the left femur. She had well-fixed bilateral hip replacements and had received long-term bisphosphonate treatment. Prolonged bisphosphonate use has been recently linked with atypical subtrochanteric and diaphyseal femoral fractures. While the current definition of an atypical fracture of the femur excludes peri-prosthetic fractures, this case suggests that they do occur and should be considered in patients with severe osteopenia. Union of the fracture followed cessation of bisphosphonates and treatment with teriparatide. Thus, this case calls into question whether prophylactic intramedullary nailing is sufficient alone to treat early or completed atypical femoral fractures.

MicroCT morphometry analysis of mouse cancellous bone: intra- and inter-system reproducibility.

The agreement between measurements and the relative performance reproducibility among different microcomputed tomography (microCT) systems, especially at voxel sizes close to the limit of the instruments, is not known. To compare this reproducibility 3D morphometric analyses of mouse cancellous bone from distal femoral epiphyses were performed using three different ex vivo microCT systems: GE eXplore Locus SP, Scanco µCT35 and Skyscan 1172. Scans were completed in triplicate at 12 µm and 8 µm voxel sizes and morphometry measurements, from which relative values and dependence on voxel size were examined. Global and individual visually assessed thresholds were compared. Variability from repeated scans at 12 µm voxel size was also examined. Bone volume fraction and trabecular separation values were similar, while values for relative bone surface, trabecular thickness and number varied significantly across the three systems. The greatest differences were measured in trabecular thickness (up to 236%) and number (up to 218%). The relative dependence of measurements on voxel size was highly variable for the trabecular number (from 0% to 20% relative difference between measurements from 12 µm and 8 µm voxel size scans, depending on the system). The intra-system reproducibility of all trabecular measurements was also highly variable across the systems and improved for BV/TV in all the systems when a smaller voxel size was used. It improved using a smaller voxel size in all the other parameters examined for the Scanco system, but not consistently so for the GE or the Skyscan system. Our results indicate trabecular morphometry measurements should not be directly compared across microCT systems. In addition, the conditions, including voxel size, for trabecular morphometry studies in mouse bone should be chosen based on the specific microCT system and the measurements of main interest.

The effect of lead on bone mineral properties from female adult C57/BL6 mice.

Lead toxicity is a significant problem in the U.S. with elevated blood lead levels being highest among very young children and older adults >50 years old. Bone is the major reservoir of body lead, accounting for 75% in children and 90% in adults. Very little is known about the effect of lead on bone mineral properties in adults. We investigated the effect of lead on the femora from adult, 6 month old female C57/BL6 mice who were administered lead in the drinking water (250 ppm, blood lead 33 µg/dL) for 4 months. Bone mineral properties were examined using Fourier Transform Infrared Microscopy (FTIRM), quantitative microcomputed tomography (microCT) and whole bone mechanical testing. Lead significantly decreased the bone mineral density in the cortical and proximal cancellous bone and increased the marrow area in the cortical bone with microCT. Whole bone three-point bending showed a trend of decreased maximum and failure moments in the lead treated bones compared to controls. Lead significantly decreased the mineral/matrix ratio, collagen maturity and crystallinity in the trabecular bone as measured by FTIRM. In the cortical bone lead significantly decreased collagen maturity and bone crystal size by FTIRM. In contrast to cell culture studies, lead significantly increased serum osteocalcin levels. Lead also significantly increased the bone formation and resorption markers suggesting increased bone turnover. These data show that lead increases bone turnover resulting in weaker cortical bone in adult female mice and suggest that lead may exacerbate bone loss and osteoporosis in the elderly.

Association of low-energy femoral fractures with prolonged bisphosphonate use: a case control study.

SUMMARY: Recent evidence has linked long-term bisphosphonate use with insufficiency fractures of the femur in postmenopausal women. In this case-control study, we have identified a significant association between a unique fracture of the femoral shaft, a transverse fracture in an area of thickened cortices, and long-term bisphosphonate use. Further studies are warranted. INTRODUCTION: Although clinical trials confirm the anti-fracture efficacy of bisphosphonates over 3-5 years, the long-term effects of bisphosphonate use on bone metabolism are unknown. Femoral insufficiency fractures in patients on prolonged treatment have been reported. METHODS: We performed a retrospective case-control study of postmenopausal women who presented with low-energy femoral fractures from 2000 to 2007. Forty-one subtrochanteric and femoral shaft fracture cases were identified and matched by age, race, and body mass index to one intertrochanteric and femoral neck fracture each. RESULTS: Bisphosphonate use was observed in 15 of the 41 subtrochanteric/shaft cases, compared to nine of the 82 intertrochanteric/femoral neck controls (Mantel-Haenszel odds ratio (OR), 4.44 [95% confidence interval (CI) 1.77-11.35]; P = 0.002). A common X-ray pattern was identified in ten of the 15 subtrochanteric/shaft cases on a bisphosphonate. This X-ray pattern was highly associated with bisphosphonate use (OR, 15.33 [95% CI 3.06-76.90]; P < 0.001). Duration of bisphosphonate use was longer in subtrochanteric/shaft cases compared to both hip fracture controls groups (P = 0.001). CONCLUSIONS: We found a significantly greater proportion of patients with subtrochanteric/shaft fractures to be on long-term bisphosphonates than intertrochanteric/femoral neck fractures. Bisphosphonate use was highly associated with a unique X-ray pattern. Further studies are warranted.

Full-field transmission x-ray microscopy for bio-imaging.

A full-field hard-x-ray microscope at SSRL has successfully imaged samples of biological and environmental origin at 40 nm resolution. Phase contrast imaging of trabeculae from a female mouse tibia, loaded in vivo to study the effects of weight-bearing on bone structure, revealed a complex network of osteocytes and canaliculi. Imaging of cordgrass roots exposed to mercury revealed nanoparticles with strong absorption contrast. 3D tomography of yeast cells grown in selenium rich media showed internal structure.

Loading induces site-specific increases in mineral content assessed by microcomputed tomography of the mouse tibia.

Adaptation to mechanical loading has been studied extensively in cortical, but not cancellous bone. However, corticocancellous sites are more relevant to osteoporosis and related fracture risk of the hip and spine. We tested the hypotheses that adaptation in a long bone would be greater at cancellous than cortical sites and would depend on the term of daily in vivo cyclic axial loading. We applied compressive loads to the adolescent, 10-week old, male C57BL/6 mouse tibia to examine the skeletal response immediately prior to attainment of peak bone mass. Adaptation was quantified at the completion of either 2-week (n = 8) or 6-week (n = 12) loading terms by directly comparing volumetric bone mineral content between loaded and contralateral limbs by microcomputed tomography. The increase in mineral content was site specific with a greater response found in the corticocancellous proximal metaphysis (14%) than the cortical mid-shaft (2%) after 6 weeks of loading. Furthermore, bone volume fraction and average trabecular thickness of cancellous bone in the proximal tibia increased after 6 weeks by 15% and 12% respectively. Diaphyseal response was only evident proximal to the mid-shaft as indicated by an 8% increase in maximum principal moment of inertia. Both loading terms produced similar results for mineral content, volume fraction, and moments of inertia. Our finding that non-invasive loading increases the bone volume and fraction at a corticocancellous site by as much as 15% motivates exploring the use of mechanical loading to attain greater peak bone mass and inhibit osteoporosis.

In vitro biomechanical comparison of limited contat dynamic compression plate and locking compression plate.

The locking compression plate (LCP) supports biological osteosynthesis by functioning as an internal fixator, rather than as a full or limited contact bone plate which must be adequately contoured and affixed directly to the bone for stable internal fixation of the fracture. In order to help justify the use of the LCP in our veterinary patients, in vitro biomechanical testing was performed comparing the LCP to the conventional limited contact dynamic compression plate (LC-DCP) in canine femurs. We hypothesized that the LCP construct would be at least as stiff under bending and torsional loads as the LC-DCP. The LCP and LC-DCP were applied over a 20-mm osteotomy gap to contralateroal bones within each pair of 14 femora. Non-destructive four-point bending and torsion, and cyclical testing in torsion were performed. The constructs were then loaded to failure in torsion. In medial-lateral and lateral-medial structural bending, significant differences were not found between the LCP and LC-DCP, however, at the gap, the LCP construct was stiffer than the LC-DCP in lateral-medial bending. Significant differences in behaviour over time were not noted between the plate designs during cyclical testing. When loading the constructs to failure in internal rotation, the LC-DCP failed at a significantly lower twist angle (P = .0024) than the LCP. Based on the similar performance with loading, the locking compression plate is a good alternative implant for unstable diaphyseal femoral fracture repair in dogs.

Effects of disrupted beta1-integrin function on the skeletal response to short-term hindlimb unloading in mice.

The study was designed to determine whether beta1-integrin plays a role in mediating the acute skeletal response to mechanical unloading. Transgenic (TG) mice were generated to express a dominant negative form of beta1-integrin under the control of the osteocalcin promoter, which targets expression of the transgene to mature osteoblasts. At 63 days of age, wild-type (WT) and TG mice were subjected to hindlimb unloading by tail suspension for 1 wk. Pair-fed, normally loaded WT and TG mice served as age-matched controls. Bone samples from each mouse were processed for quantitative bone histomorphometry and biomechanical testing. The skeletal phenotype of TG mice was characterized by lower cancellous bone mass in the distal femoral metaphysis (-52%) and lumbar vertebral body (-20%), reduced curvature of the proximal tibia (-20%), and decreased bone strength (-20%) and stiffness (-23%) of the femoral diaphysis with relatively normal indexes of cancellous bone turnover. Hindlimb unloading for only 1 wk induced a 10% decline in tibial curvature and a 30% loss of cancellous bone in the distal femur due to a combination of increased bone resorption and decreased bone formation in both WT and TG mice. However, the strength and stiffness of the femoral diaphysis were unaffected by short-term hindlimb unloading in both genotypes. The observed increase in osteoclast surface was greater in unloaded TG mice (92%) than in unloaded WT mice (52%). Cancellous bone formation rate was decreased in unloaded WT (-29%) and TG (-15%) mice, but, in contrast to osteoclast surface, the genotype by loading interaction was not statistically significant. The results indicate that altered integrin function in mature osteoblasts may enhance the osteoclastic response to mechanical unloading but that it does not have a major effect on the development of cancellous osteopenia in mice during the early stages of hindlimb unloading.

Skeletal phenotype of growing transgenic mice that express a function-perturbing form of beta1 integrin in osteoblasts.

Skeletal modeling entails the deposition of large amounts of extracellular matrix (ECM) to form structures tailored to withstand increasing mechanical loads during rapid growth. Specific ECM molecules bind to integrin receptors on the cell surface, thereby triggering a cascade of signaling events that affect critical cell functions. To evaluate the role of integrins during skeletal growth, transgenic mice were engineered to express a function-perturbing fragment of beta1 integrin consisting of the transmembrane domain and cytoplasmic tail under the control of the osteocalcin promoter (TG mice). Thus, transgene expression was targeted to mature cells of the osteoblast lineage, and herein we show that cultured cells resembling osteocytes from 90-day-old TG mice display impaired adhesion to collagen I, a ligand for beta1 integrin. To determine the influence of beta1 integrin on bones that are responsible for providing structural support during periods of rapid growth, we examined the phenotype of the appendicular skeleton in TG mice compared to wild type (WT) mice. According to radiographs, bones from mice of both genotypes between 14 and 90 days of age appeared similar in gross structure and density, although proximal tibiae from 35-90 days old TG mice were less curved than those of WT mice (72-92% TG/WT). Although there were only mild and transient differences in absolute bone mass and strength, once normalized to body mass, the tibial dry mass (79.1% TG/WT females), ash mass (78.5% TG/WT females), and femoral strength in torsion (71.6% TG/WT females) were reduced in TG mice compared to WT mice at 90 days of age. Similar effects of genotype on bone mass and curvature were observed in 1-year-old retired breeders, indicating that these phenotypic differences between TG and WT mice were stable well into adulthood. Effects of genotype on histomorphometric indices of cancellous bone turnover were minimal and evident only transiently during growth, but when present they demonstrated differences in osteoblast rather than osteoclast parameters. Together, these results suggest that integrin signals generated during growth enhance the acquisition of a skeletal mass, structure, and strength to withstand the mechanical loads generated by weight-bearing.