Jennifer M. MacLeay

Variations in nanomechanical properties and tissue composition within trabeculae from an ovine model of osteoporosis and treatment.

Osteoporosis and treatment may affect both composition and nanomechanical properties and their spatial distributions within the individual trabeculae of cancellous bone at length scales that cannot be captured by bulk measurements. This study utilized 25 mature adult ewes divided into 5 treatment groups. Four treatment groups were given a dietary model for human high-turnover osteoporosis, and two of these were treated with antiresorptive drugs, either zoledronate (ZOL) or raloxifene (RAL), to examine their effects on bulk tissue properties and nanoscale tissue composition and mechanical properties within trabeculae. Treatment effects were most pronounced at the nanoscale, where RAL increased indentation modulus and hardness throughout trabeculae by 10% relative to the osteoporosis model. In comparison, ZOL increased these properties exclusively at the surfaces of trabeculae (indentation modulus +12%, hardness +16%). Nanomechanical alterations correlated with changes in tissue mineralization, carbonate substitution, crystallinity, and aligned collagen. Despite only minimal changes in bulk tissue tBMD, the nanomechanical improvements within trabeculae with both treatments greatly improved the predicted theoretical bending stiffness of individual trabeculae when idealized as cylindrical struts. Hence, small tissue-level alterations in critical locations for resisting trabecular failure could account for some of the discrepancy between the large reductions in fracture risk and the only modest changes in BMD with antiresorptive treatments.

Effect of dietary-induced metabolic acidosis and ovariectomy on bone mineral density and markers of bone turnover.

Dietary-induced metabolic acidosis (DIMA) has been implicated as a significant confounder in the development of osteoporosis. Twenty-four mature ewes were randomly assigned to four groups of six sheep. Group 1 consumed a control diet (ND); group 2 consumed a normal diet (ND) and had ovariectomy (OVX), group 3 consumed a diet that induced metabolic acidosis (MA), without OVX, and group 4 consumed a diet that induced MA, with OVX. The study was conducted over 180 days and the sheep were maintained on the assigned diet throughout. Sheep were weighed and bone mineral density (BMD) was measured, using dual-energy X-ray absorptiometry (DEXA), on days 0 and 180. Serum bone alkaline phosphatase (BAP), urine deoxypyridinoline (DPD), and fractional excretions (FE) of Ca and P were determined on days 0, 90, and 180. Arterial blood pH was determined on day 180. Analysis consisted of a two-way analysis of variance for repeated measures with significance set at P ≤ 0.05. Body weights, serum BAP, and urine DPD were not influenced by either diet or OVX status. DIMA did significantly increase urinary FE of Ca and P and significantly decreased lumbar BMD and arterial pH. Arterial pH remained within physiologic normal limits. DIMA was a more potent cause of calcium wasting than OVX over the time frame of this study. Sheep appear to be sensitive to DIMA and will therefore be a useful animal model to study the influence of diet on the development of osteoporosis. The specific mechanisms through which DIMA exerts its influence are still unknown and are the subject of ongoing studies.

Dietary-Induced Metabolic Acidosis Decreases Bone Mineral Density in Mature Ovariectomized Ewes

Dietary-induced metabolic acidosis (DIMA) may be a significant confounder in the development of osteoporosis. Diets that are acidifying are typically rich in proteins and grains and relatively poor in fruits and vegetables. Previous studies have not examined whether an interaction between estrogen depletion and DIMA have a compounded affect on bone mineral density loss. Sheep have been used successfully in previous studies to examine the interaction of bone turnover and ovariectomy. Therefore, the goal of this pilot study was to determine if bone mineral density (BMD) loss could be induced using DIMA in skeletally mature ovariectomized (OVX) ewes.

The effect of osteoporosis treatments on fatigue properties of cortical bone tissue

Bisphosphonates are commonly prescribed for treatment of osteoporosis. Long-term use of bisphosphonates has been correlated to atypical femoral fractures (AFFs). AFFs arise from fatigue damage to bone tissue that cannot be repaired due to pharmacologic treatments. Despite fatigue being the primary damage mechanism of AFFs, the effects of osteoporosis treatments on fatigue properties of cortical bone are unknown. To examine if fatigue-life differences occur in bone tissue after different pharmacologic treatments for osteoporosis, we tested bone tissue from the femurs of sheep given a metabolic acidosis diet to induce osteoporosis, followed by treatment with a selective estrogen reception modulator (raloxifene), a bisphosphonate (alendronate or zoledronate), or parathyroid hormone (teriparatide, PTH). Beams of cortical bone tissue were created and tested in four-point bending fatigue to failure. Tissue treated with alendronate had reduced fatigue life and less modulus loss at failure compared with other treatments, while tissue treated with PTH had a prolonged fatigue life. No loss of fatigue life occurred with zoledronate treatment despite its greater binding affinity and potency compared with alendronate. Tissue mineralization measured by microCT did not explain the differences seen in fatigue behavior. Increased fatigue life with PTH suggests that current treatment methods for AFF could have beneficial effects for restoring fatigue life. These results indicate that fatigue life differs with each type of osteoporosis treatment.

Evaluation of Direct In Vivo Gene Transfer in an Equine Metacarpal IV Ostectomy Model Using an Adenoviral Vector Encoding the Bone Morphogenetic Protein-2 and Protein-7 Gene

OBJECTIVEnTo evaluate gene transfer in an equine metacarpal IV (MCIV) ostectomy model using adenoviral vectors encoding the human bone morphogenetic protein-2 and protein-7 gene (Ad-BMP-2/-7).nnnSTUDY DESIGNnnnnEXPERIMENTAL ANIMALSnHealthy adult horses (n = 15).nnnMETHODSnA plate stabilized, critical size 1.5 cm ostectomy was created in left and right MCIV. The ostectomy site was injected with either Ad-green fluorescent protein (Ad-GFP) or Ad-hBMP-2/-7 at completion of surgery; the same treatment was assigned to both the left and right forelimb of each horse (n = 5 horses/group). Bone healing was evaluated radiographically every 2 weeks for 16 weeks. Horses in a pilot study (n = 5) were used as untreated controls for radiographic evaluation to 8 weeks. After euthanasia at 16 weeks bone healing was evaluated using dual energy X-ray absorptiometry (DEXA) and histomorphometry. Data were analyzed using an ANOVA or Kruskal-Wallis test. Level of significance was P < .05.nnnRESULTSnAt 4 and 6 weeks, the Ad-GFP group had a significantly lower percentage defect ossification compared with the untreated control group. There was no significant difference between untreated and Ad-hBMP-2/-7 groups at any time point and no significant difference in bone healing radiographically, histologically, or using DEXA between any groups at 16 weeks.nnnCONCLUSIONSnAd-hBMP-2/-7 did not improve bone healing in horses at 16 weeks.

Fourier Transform Infrared Imaging Analysis of Cancellous Bone in Alendronate- and Raloxifene-Treated Osteopenic Sheep

Fourier transform infrared imaging spectroscopy (FTIRI)-assessed bone composition parameters (mineral content, collagen maturity, crystal size and perfection, and carbonate content) describe bone quality and correlate to bone fracture risk. The challenge with studying bone quality in patients treated with antiresorptive drugs such as bisphosphonates (e.g., alendronate) and selective estrogen receptor modulators (SERMs) (e.g. raloxifene) is being able to test bone mechanical performance and material properties pre- and posttreatment. The purpose of this study was to evaluate the FTIRI changes in a large animal model of osteoporosis (female sheep with dietary induced metabolic acidosis; MA). Previous studies have investigated the relationship between bone material properties and bone strength in humans and smaller animals and have shown that changes in compositional properties influence fracture risk. Here we characterize the MA model at 6 and 12 months, demonstrate the loss of bone and changes in compositional properties, and show that 6 months of treatment with both antiresorptives ameliorate the bone loss as assessed by bone mineral density and FTIRI. This preliminary data suggest that the MA sheep model allows investigation of whether drug treatments preserve bone properties that exist at the time of treatment or if they induce further beneficial changes.

Femoral Cortical Bone Mineral Density and Biomechanical Properties in Sheep Consuming an Acidifying Diet

Dietary acidity is a likely contributor to the development of osteoporosis. Dietary acidosis in an ovine model has effects on trabecular bone that have been previously shown to mimic human osteoporosis. Effects on cortical bone using this model have not been investigated. The objective of this study was to examine the effects of dietary acidosis on cortical bone mineral density and material properties. Skeletally mature ovariectomized (OVX) sheep consumed either a normal diet (ND) or a metabolic acidosis diet (MA) for 6 or 12 months. Whole femoral and cortical bone beam BMD was determined using dual energy x-ray absorptiometry (DEXA). Beams were then subjected to three point flexure monotonically to failure to determine strength and modulus and then ashed to determine percent mineralization. Femoral BMD in adult OVX ND 6 mo sheep was significantly greater than those in the non-OVX ND group. The BMD in the MA groups was lower than the control non-OVX ND group. Cortical beams had significantly decreased modulus in all MA and OVX groups when compared with the non-OVX ND group and a tendency towards decreased strength in all groups with significance only in the OVX ND 6 mo sheep. Percent mineralization increased in MA and OVX groups when compared to the non-OVX ND group and was significantly increased in the OVX ND 6 mo and OVX MA 12 mo groups. A significant correlation was seen between BMD of the beam and breaking strength and modulus. Dietary acidity impacts cortical bone and results in reduced material properties that may contribute to failure.