Sex-Linked Skeletal Phenotype of Lysyl Oxidase Like-1 Mutant Mice.

Lysyl oxidases are required for collagen and elastin cross-linking and extracellular matrix maturation including in bone. The lysyl oxidase family consists of lysyl oxidase (LOX) and 4 isoforms (LOXL1-4). Here we investigate whether deletion of LOXL1, which has been linked primarily to elastin maturation, leads to skeletal abnormalities. Left femurs (n = 8), L5 vertebrae (n = 8), and tibiae (n = 8) were analyzed by micro-computed tomography in 13-week-old wild-type (WT) and LOXL1-/- male and female mice. Right femurs (n = 8) were subjected to immunohistochemistry for LOXL1, and histochemical/histology analyses of osteoclasts and growth plates. Sera from all mice were analyzed for bone turnover markers. Results indicate strong expression of LOXL1 in wild-type growth plates in femurs. Significant deterioration of trabecular bone structure in long bones and vertebrae from female was observed but not from male, mutant mice compared with WT. Decreases in BV/TV, Conn.D, trabecular thickness, and number in the femoral distal metaphysis were observed in female, but not in male, mutant mice. Trabecular spacing was increased significantly in femurs of female mutant mice. Findings were similar in trabeculae of L5 vertebrae from female mutant mice. The number of TRAP positive osteoclasts at the trabecular bone surface was increased in female mutant mice compared with WT females, consistent with increased serum RANKL and decreased OPG levels. Analysis of bone turnover markers confirmed increased bone resorption as indicated by significantly elevated CTX-1 in the serum of female LOXL1-/- mice compared to their wild-type counterparts, as well as decreased bone formation as measured by decreased serum levels of PINP. Picrosirius red staining revealed a loss of heterogeneity in collagen organization in female LOXL1-/- mice only, with little to no yellow and orange birefringence. Organization was also impaired in chondrocyte columns in both female and male LOXL1-/- mice, but to a greater extent in females. Data indicate that LOXL1-/- mutant mice develop appendicular and axial skeletal phenotypes characterized by decreased bone volume fraction and compromised trabecular microstructure, predominantly in females.

Presence of intervertebral discs alters observed stiffness and failure mechanisms in the vertebra.

Ex vivo mechanical testing is an essential tool for study of vertebral mechanics. However, the common method of testing vertebral bodies in the absence of adjacent intervertebral discs (IVDs) may limit the physiological relevance of the results. The goal of this study was to determine the influence of IVDs on vertebral mechanical properties and failure mechanisms. Rabbit thoracic vertebral bodies were tested with and without IVDs in a stepwise fashion that incorporated a micro-computed tomography scan at each loading step. The image sequences were analyzed using digital volume correlation to quantify deformations throughout the vertebral body. The observed deformation patterns differed substantially between the groups. Specimens tested with IVDs exhibited a slow increase in strain in the inferior and posterior regions, followed by a sudden increase in strain in the anterior cortex right at the yield point. In contrast, the highest strains in the isolated vertebral bodies were in the posterior regions throughout the test. Specimens tested with IVDs had lower stiffness (507.49±184.73N/mm vs. 845.61±296.09N/mm; p=0.044), higher ultimate displacement (2.00±0.68mm vs. 1.17±0.54mm; p=0.043), and higher maximum shear strains (e.g. top 25th percentile: 0.19±0.11 vs. 0.06±0.07mm/mm; p<0.0458), and tended to have lower ultimate force (690.28±160.25N vs. 873.81±131.48N; p=0.056). Similar work to failure (648.15±317.86N-mm vs. 603.49±437.95 N-mm; p=0.844) was observed between the two groups. These results indicate that testing vertebral bodies in the absence of IVDs can elicit artifactual failure mechanisms. These artifacts may be more prominent than the effects on vertebral strength and toughness.

Comparison of prosthetic feet prescribed to active individuals using ISO standards.

BACKGROUND: Little research has been done on the robustness of prosthetic feet prescribed to military personnel, and manufacturers are not required to test their products prior to sale. This is problematic because the prosthetic feet used by active individuals are subjected to loading conditions not seen in normal gait. OBJECTIVES: To evaluate whether commercially available heavy-duty prosthetic feet intended for use by military personnel meet ISO 10328 standards. STUDY DESIGN: Bench testing of heavy-duty prosthetic feet using ISO 10328 standards. METHODS: Prosthetic feet from three different manufacturers were tested according to ISO 10328 standards, using a testing frame fitted with axial load and displacement transducers. Pass/fail information was recorded as well as the stiffness and creep of each foot before and after cyclic testing. RESULTS: All feet passed the ISO 10328 standards at the highest loading level, and some significant differences were found within a given model of prosthesis when comparing stiffness and creep before and after cyclic testing. CONCLUSIONS: This study demonstrated that manufacturers of heavy-duty prosthetic feet adhere to the voluntary ISO 10328 standards. However, these standards may be insufficient because the tests simulate only idealized gait. Further development of the standards may be necessary to reproduce the circumstances that occur during extreme usage to ensure that prosthetic feet do not fail.

Micro-computed tomography assessment of fracture healing: relationships among callus structure, composition, and mechanical function.

Non-invasive characterization of fracture callus structure and composition may facilitate development of surrogate measures of the regain of mechanical function. As such, quantitative computed tomography- (CT-) based analyses of fracture calluses could enable more reliable clinical assessments of bone healing. Although previous studies have used CT to quantify and predict fracture healing, it is unclear which of the many CT-derived metrics of callus structure and composition are the most predictive of callus mechanical properties. The goal of this study was to identify the changes in fracture callus structure and composition that occur over time and that are most closely related to the regain of mechanical function. Micro-computed tomography (microCT) imaging and torsion testing were performed on murine fracture calluses (n=188) at multiple post-fracture timepoints and under different experimental conditions that alter fracture healing. Total callus volume (TV), mineralized callus volume (BV), callus mineralized volume fraction (BV/TV), bone mineral content (BMC), tissue mineral density (TMD), standard deviation of mineral density (sigma(TMD)), effective polar moment of inertia (J(eff)), torsional strength, and torsional rigidity were quantified. Multivariate statistical analyses, including multivariate analysis of variance, principal components analysis, and stepwise regression were used to identify differences in callus structure and composition among experimental groups and to determine which of the microCT outcome measures were the strongest predictors of mechanical properties. Although calluses varied greatly in the absolute and relative amounts of mineralized tissue (BV, BMC, and BV/TV), differences among timepoints were most strongly associated with changes in tissue mineral density. Torsional strength and rigidity were dependent on mineral density as well as the amount of mineralized tissue: TMD, BV, and sigma(TMD) explained 62% of the variation in torsional strength (p<0.001); and TMD, BMC, BV/TV, and sigma(TMD) explained 70% of the variation in torsional rigidity (p<0.001). These results indicate that fracture callus mechanical properties can be predicted by several microCT-derived measures of callus structure and composition. These findings form the basis for developing non-invasive assessments of fracture healing and for identifying biological and biomechanical mechanisms that lead to impaired or enhanced healing.

Mechanical stimulation alters tissue differentiation and molecular expression during bone healing.

Further understanding of how mechanical cues modulate skeletal tissue differentiation can identify potential means of enhancing repair following injury or disease. Prior studies examined the effects of mechanical loading on osteogenesis, chondrogenesis, and fibrogenesis in an effort to enhance bony union. However, exploring how mechanical stimuli can divert the bone healing process towards formation of other mesenchymal tissues, as an endpoint, may elucidate new avenues for repair and regeneration of tissues such as cartilage and fibrous tissue. This study investigated the use of mechanical stimulation to promote cartilage rather than bone formation within an osteotomy. Our overall goal was to define skeletal tissue distribution and molecular expression patterns induced by the stimulation. Retired breeder Sprague-Dawley rats (n = 85) underwent production of a mid-diaphyseal, transverse femoral osteotomy followed by external fixation. Beginning on postoperative day 10 and continuing for 1, 2, or 4 weeks, a cyclic bending motion (+35 degrees/-25 degrees at 1 Hz) was applied in the sagittal plane for 15 min/day for 5 consecutive days/week. Control animals experienced continuous rigid fixation. Histological and molecular analyses indicated that stimulation substantially altered normal bone healing. Stimulated specimens exhibited an increase in cartilage volume over time, while control specimens demonstrated bony bridging. Stimulation induced upregulation of cartilage-related genes (COL2A1 and COL10A1) and downregulation of bone morphogenetic proteins (BMPs) -4, -6 and -7. However, BMP-3 was upregulated with stimulation. These findings illustrate that mechanical cues can selectively modulate osteogenesis and chondrogenesis in vivo, and suggest a potential basis for treatment regimens for injured or diseased cartilaginous tissues.

Comparison of effects of the bisphosphonate alendronate versus the RANKL inhibitor denosumab on murine fracture healing.

The role of osteoclast-mediated resorption during fracture healing was assessed. The impact of two osteoclast inhibitors with different mechanisms of action, alendronate (ALN) and denosumab (DMAB), were examined during fracture healing. Male human RANKL knock-in mice that express a chimeric (human/murine) form of RANKL received unilateral transverse femur fractures. Mice were treated biweekly with ALN 0.1 mg/kg, DMAB 10 mg/kg, or PBS (control) 0.1 ml until death at 21 and 42 days after fracture. Treatment efficacy assessed by serum levels of TRACP 5b showed almost a complete elimination of TRACP 5b levels in the DMAB-treated animals but only approximately 25% reduction of serum levels in the ALN-treated mice. Mechanical testing showed that fractured femurs from both ALN and DMAB groups had significantly increased mechanical properties at day 42 compared with controls. muCT analysis showed that callus tissues from DMAB-treated mice had significantly greater percent bone volume and BMD than did both control and ALN-treated tissues at both 21 and 42 days, whereas ALN-treated bones only had greater percent bone volume and BMC than control at 42 days. Qualitative histological analysis showed that the 21-and 42-day ALN and DMAB groups had greater amounts of unresorbed cartilage or mineralized cartilage matrix compared with the controls, whereas unresorbed cartilage could still be seen in the DMAB groups at 42 days after fracture. Although ALN and DMAB delayed the removal of cartilage and the remodeling of the fracture callus, this did not diminish the mechanical integrity of the healing fractures in mice receiving these treatments. In contrast, strength and stiffness were enhanced in these treatment groups compared with control bones.

Combined effects of recombinant human BMP-7 (rhBMP-7) and parathyroid hormone (1-34) in metaphyseal bone healing.

Fracture healing involves multiple stages of repair and coordinated actions of multiple cell types. Consequently, it may be possible to enhance healing through treatment strategies that target more than one repair process or cell type. The goal of this study was to determine the combined effects of recombinant human bone morphogenetic protein 7 (rhBMP-7) and parathyroid hormone (PTH(1-34)) on metaphyseal bone healing. A wedge-shaped defect was created in the lateral aspect of the distal tibia in female New Zealand white rabbits (n=64) and was filled with tricalcium phosphate (TCP). Animals were assigned to four groups: 1) BMP-7 and PTH; 2) BMP-7; 3) PTH; and 4) control (TCP alone). In groups 1 and 2, 200 microg rhBMP-7 was incorporated into the TCP. Animals received daily subcutaneous injections of 10 microg/kg PTH(1-34) (groups 1 and 3) or saline (groups 2 and 4). Healing at 4 weeks was assessed using micro-computed tomography, histology, immunohistochemistry, and mechanical testing. Combined treatment with rhBMP-7 and PTH resulted in increased callus total volume (TV), mineralized volume (BV), average cross-sectional area, and bone mineral content (BMC) as compared to the control group (p<0.02). BV and BMC were also higher in the combined treatment group as compared to the BMP-7 group (p<0.02); however, tissue mineral density was highest in the BMP-7 group (p=0.002). New bone formation in the BMP-7 group was largely restricted to the defect site, while PTH promoted bone formation throughout the defect and surrounding regions. Combined treatment led to greater quantities of woven trabecular bone, increased trabecular thickness, decreased trabecular separation (p<0.04), and a trend towards increased numbers of osteoclasts (p=0.09). Combined treatment also resulted in increased torsional rigidity and compressive strength as compared to the control and BMP-7 groups (p<0.001). These results suggest that the improvements in mechanical function obtained with the combined treatment resulted from differing biological activities of rhBMP-7 and PTH. While the activities of rhBMP-7 appeared to be strictly anabolic, those of PTH appeared to work in the context of coupled remodeling. The combination of both agents led to greater bone volume as well as better microstructural organization and integration of this bone with the surrounding tissues.

Bone formation during distraction osteogenesis is dependent on both VEGFR1 and VEGFR2 signaling.

INTRODUCTION: Distraction osteogenesis (DO) is characterized by the induction of highly vascularized new bone formation through an intramembranous process largely devoid of the formation of cartilage. MATERIALS AND METHODS: To test the hypothesis that DO is strictly dependent on vascualrization, we inhibited vascular endothelial growth factor (VEGF) activity by antibody blockade of both receptors VEGFR1 (Flt-1) and VEGFR2 (Flk-1) or only VEGFR2 (Flk-1) in a previously developed murine tibia DO model. During normal DO, VEGFR1 (Flt-1), VEGFR2 (Flk-1), VEGFR3 (Flt4) and all four VEGF ligand (A, B, C, and D) mRNAs are induced. RESULTS: The expression of mRNA for the receptors generally paralleled those of the ligands during the period of active distraction. Bone formation, as assessed by muCT, showed a significant decrease with the double antibody treatment and a smaller decrease with single antibody treatment. Vessel volume, number, and connectivity showed progressive and significant inhibition in all of these of parameters between the single and double antibody blockade. Molecular analysis showed significant inhibition in skeletal cell development with the single and double antibody blockade of both VEGFR1 and 2. Interestingly, the single antibody treatment led to selective early development of chondrogenesis, whereas the double antibody treatment led to a failure of both osteogenesis and chondrogenesis. CONCLUSIONS: Both VEGFR1 and VEGFR2 are functionally essential in blood vessel and bone formation during DO and are needed to promote osteogenic over chondrogenic lineage progression.