Mohammad W. Aref

In vivo reference point indentation reveals positive effects of raloxifene on mechanical properties following 6 months of treatment in skeletally mature beagle dogs

Raloxifene treatment has been shown previously to positively affect bone mechanical properties following 1 year of treatment in skeletally mature dogs. Reference point indentation (RPI) can be used for in vivo assessment of mechanical properties and has been shown to produce values that are highly correlated with properties derived from traditional mechanical testing. The goal of this study was to use RPI to determine if raloxifene-induced alterations in mechanical properties occurred after 6 months of treatment. Twelve skeletally mature female beagle dogs were treated for 6 months with oral doses of saline vehicle (VEH, 1 ml/kg/day) or a clinically relevant dose of raloxifene (RAL, 0.5 mg/kg/day). At 6 months, all animals underwent in vivo RPI (10N force, 10 cycles) of the anterior tibial midshaft. RPI data were analyzed using a custom MATLAB program, designed to provide cycle-by-cycle data from the RPI test and validated against the manufacturer-provided software. Indentation distance increase (IDI), a parameter that is inversely related to bone toughness, was significantly lower in RAL-treated animals compared to VEH (-16.5%), suggesting increased bone toughness. Energy absorption within the first cycle was significantly lower with RAL compared to VEH (-21%). These data build on previous work that has documented positive effects of raloxifene on material properties by showing that these changes exist after 6 months.

Development of an in vivo rabbit ulnar loading model.

Ulnar and tibial cyclic compression in rats and mice have become the preferred animal models for investigating the effects of mechanical loading on bone modeling/remodeling. Unlike rodents, rabbits provide a larger bone volume and normally exhibit intracortical Haversian remodeling, which may be advantageous for investigating mechanobiology and pharmaceutical interventions in cortical bone. Therefore, the objective of this study was to develop and validate an in vivo rabbit ulnar loading model. Ulnar tissue strains during loading of intact forelimbs were characterized and calibrated to applied loads using strain gauge measurements and specimen-specific finite element models. Periosteal bone formation in response to varying strain levels was measured by dynamic histomorphometry at the location of maximum strain in the ulnar diaphysis. Ulnae loaded at 3000 microstrain did not exhibit periosteal bone formation greater than the contralateral controls. Ulnae loaded at 3500, 4000, and 4500 microstrain exhibited a dose-dependent increase in periosteal mineralizing surface (MS/BS) compared with contralateral controls during the second week of loading. Ulnae loaded at 4500 microstrain exhibited the most robust response with significantly increased MS/BS at multiple time points extending at least 2weeks after loading was ceased. Ulnae loaded at 5250 microstrain exhibited significant woven bone formation. Rabbits required greater strain levels to produce lamellar and woven bone on periosteal surfaces compared with rats and mice, perhaps due to lower basal levels of MS/BS. In summary, bone adaptation during rabbit ulnar loading was tightly controlled and may provide a translatable model for human bone biology in preclinical investigations of metabolic bone disease and pharmacological treatments.

Cx43 Overexpression in Osteocytes Prevents Osteocyte Apoptosis and Preserves Cortical Bone Quality in Aging Mice: OSTEOCYTIC Cx43 AND APOPTOTIS IN AGED MICE

Young, skeletally mature mice lacking Cx43 in osteocytes exhibit increased osteocyte apoptosis and decreased bone strength, resembling the phenotype of old mice. Further, the expression of Cx43 in bone decreases with age, suggesting a contribution of reduced Cx43 levels to the age‐related changes in the skeleton. We report herein that Cx43 overexpression in osteocytes achieved by using the DMP1‐8kb promoter (Cx43OT mice) attenuates the skeletal cortical but not trabecular bone phenotype of aged, 14‐month‐old mice. The percentage of Cx43‐expressing osteocytes was higher in Cx43OT mice, whereas the percentage of Cx43‐positive osteoblasts remained similar to wild‐type (WT) littermate control mice. The percentage of apoptotic osteocytes and osteoblasts was increased in aged WT mice compared with skeletally mature, 6‐month‐old WT mice, and the percentage of apoptotic osteocytes, but not osteoblasts, was decreased in age‐matched Cx43OT mice. Aged WT mice exhibited decreased bone formation and increased bone resorption as quantified by histomorphometric analysis and circulating markers compared with skeletally mature mice. Further, aged WT mice exhibited the expected decrease in bone biomechanical structural and material properties compared with young mice. Cx43 overexpression prevented the increase in osteoclasts and decrease in bone formation on the endocortical surfaces and the changes in circulating markers in the aged mice. Moreover, the ability of bone to resist damage was preserved in aged Cx43OT mice both at the structural and material level. All together, these findings suggest that increased Cx43 expression in osteocytes ameliorates age‐induced cortical bone changes by preserving osteocyte viability and maintaining bone formation, leading to improved bone strength.

Raloxifene Improves Bone Mechanical Properties in Mice Previously Treated with Zoledronate.

Bisphosphonates represent the gold-standard pharmaceutical agent for reducing fracture risk. Long-term treatment with bisphosphonates can result in tissue brittleness which in rare clinical cases manifests as atypical femoral fracture. Although this has led to an increasing call for bisphosphonate cessation, few studies have investigated therapeutic options for follow-up treatment. The goal of this study was to test the hypothesis that treatment with raloxifene, a drug that has cell-independent effects on bone mechanical material properties, could reverse the compromised mechanical properties that occur following zoledronate treatment. Skeletally mature male C57Bl/6J mice were treated with vehicle (VEH), zoledronate (ZOL), or ZOL followed by raloxifene (RAL; 2 different doses). At the conclusion of 8 weeks of treatment, femora were collected and assessed with microCT and mechanical testing. Trabecular BV/TV was significantly higher in all treated animals compared to VEH with both RAL groups having significantly higher BV/TV compared to ZOL (+21%). All three drug-treated groups had significantly more cortical bone area, higher cortical thickness, and greater moment of inertia at the femoral mid-diaphysis compared to VEH with no difference among the three treated groups. All three drug-treated groups had significantly higher ultimate load compared to VEH-treated animals (+14 to 18%). Both doses of RAL resulted in significantly higher displacement values compared to ZOL-treated animals (+25 to +50%). In conclusion, the current work shows beneficial effects of raloxifene in animals previously treated with zoledronate. The higher mechanical properties of raloxifene-treated animals, combined with similar cortical bone geometry compared to animals treated with zoledronate, suggest that the raloxifene treatment is enhancing mechanical material properties of the tissue.

Assessment of regional bone tissue perfusion in rats using fluorescent microspheres

Disturbances in bone blood flow have been shown to have deleterious effects on bone properties yet there remain many unanswered questions about skeletal perfusion in health and disease, partially due to the complexity of measurement methodologies. The goal of this study was use fluorescent microspheres in rats to assess regional bone perfusion by adapting mouse-specific fluorescent microsphere protocol. Ten fifteen-week old Sprague Dawley rats were injected with fluorescent microspheres either via cardiac injection (n = 5) or via tail vein injection (n = 5). Femora and tibiae were harvested and processed to determine tissue fluorescence density (TFD) which is proportional to the number of spheres trapped in the tissue capillaries. Right and left total femoral TFD (2.77 ± 0.38 and 2.70 ± 0.24, respectively) and right and left tibial TFD (1.11 ± 0.26 and 1.08 ± 0.34, respectively) displayed bilateral symmetry in flow when assessed in cardiac injected animals. Partitioning of the bone perfusion into three segments along the length of the bone showed the distal femur and proximal tibia received the greatest amount of perfusion within their respective bones. Tail vein injection resulted in unacceptably low TFD levels in the tibia from 4 of the 5 animals. In conclusion this report demonstrates the viability of cardiac injection of fluorescent microspheres to assess bone tissue perfusion in rats.

Reversal of loss of bone mass in old mice treated with mefloquine.

Aging is accompanied by imbalanced bone remodeling, elevated osteocyte apoptosis, and decreased bone mass and mechanical properties; and improved pharmacologic approaches to counteract bone deterioration with aging are needed. We examined herein the effect of mefloquine, a drug used to treat malaria and systemic lupus erythematosus and shown to ameliorate bone loss in glucocorticoid-treated patients, on bone mass and mechanical properties in young and old mice. Young 3.5-month-old and old 21-month-old female C57BL/6 mice received daily injections of 5 mg/kg/day mefloquine for 14 days. Aging resulted in the expected changes in bone volume and mechanical properties. In old mice mefloquine administration reversed the lower vertebral cancellous bone volume and bone formation; and had modest effects on cortical bone volume, thickness, and moment of inertia. Mefloquine administration did not change the levels of the circulating bone formation markers P1NP or alkaline phosphatase, whereas levels of the resorption marker CTX showed trends towards increase with mefloquine treatment. In addition, and as expected, aging bones exhibited an accumulation of active caspase3-expressing osteocytes and higher expression of apoptosis-related genes compared to young mice, which were not altered by mefloquine administration at either age. In young animals, mefloquine induced higher periosteal bone formation, but lower endocortical bone formation. Further, osteoclast numbers were higher on the endocortical bone surface and circulating CTX levels were increased, in mefloquine- compared to vehicle-treated young mice. Consistent with this, addition of mefloquine to bone marrow cells isolated from young mice led to increased osteoclastic gene expression and a tendency towards increased osteoclast numbers in vitro. Taken together our findings identify the age and bone-site specific skeletal effects of mefloquine. Further, our results highlight a beneficial effect of mefloquine administration on vertebral cancellous bone mass in old animals, raising the possibility of using this pharmacologic inhibitor to preserve skeletal health with aging.

Skeletal vascular perfusion is altered in chronic kidney disease

Patients with chronic kidney disease (CKD) are at an alarming risk of cardiovascular disease and fracture-associated mortality. CKD has been shown to have negative effects on vascular reactivity and organ perfusion. Although alterations in bone blood flow are linked to dysregulation of bone remodeling and mass in multiple conditions, changes to skeletal perfusion in the setting of CKD have not been explored. The goal of this study was to establish the effect of CKD on skeletal perfusion in a rat model of CKD. In two experiments with endpoints at 30 and 35 weeks of age, respectively, normal (NL) and Cy/+ (CKD) animals (n = 6/group) underwent in vivo intra-cardiac fluorescent microsphere injection to assess bone tissue perfusion. These two separate time points aimed to describe skeletal perfusion at 30 and 35 weeks based on previous studies demonstrating significant progression of hyperparthyroid bone disease during this timeframe. CKD animals had blood urea nitrogen (BUN) levels significantly higher than NL at both 30 and 35 weeks. At 30 weeks, perfusion was significantly higher in the femoral cortex (+259%, p < 0.05) but not in the tibial cortex (+140%, p = 0.11) of CKD animals relative to NL littermates. Isolated tibial marrow perfusion at 30 weeks showed a trend toward being higher (+183%, p = 0.08) in CKD. At 35 weeks, perfusion was significantly higher in both the femoral cortex (+173%, p < 0.05) and the tibial cortex (+241%, p < 0.05) in CKD animals when compared to their normal littermates. Isolated tibial marrow perfusion (−57%, p <0.05) and vertebral body perfusion (−71%, p <0.05) were lower in CKD animals. The current study demonstrates two novel findings regarding bone perfusion in an animal model of high turnover CKD. First, cortical bone perfusion in CKD animals is higher than in normal animals. Second, alterations in bone marrow perfision differed among the stages of CKD and were distinct from perfusion to the cortical bone. Determining whether these changes in bone perfusion are drivers, propagators, or consequences of skeletal deterioration in CKD will necessitate further work.

Effects of combination treatment with alendronate and raloxifene on skeletal properties in a beagle dog model.

A growing number of studies have investigated combination treatment as an approach to treat bone disease. The goal of this study was to investigate the combination of alendronate and raloxifene with a particular focus on mechanical properties. To achieve this goal we utilized a large animal model, the beagle dog, used previously by our laboratory to study both alendronate and raloxifene monotherapies. Forty-eight skeletally mature female beagles (1–2 years old) received daily oral treatment: saline vehicle (VEH), alendronate (ALN), raloxifene (RAL) or both ALN and RAL. After 6 and 12 months of treatment, all animals underwent assessment of bone material properties using in vivo reference point indentation (RPI) and skeletal hydration using ultra-short echo magnetic resonance imaging (UTE-MRI). End point measures include imaging, histomorphometry, and mechanical properties. Bone formation rate was significantly lower in iliac crest trabecular bone of animals treated with ALN (-71%) and ALN+RAL (-81%) compared to VEH. In vivo assessment of properties by RPI yielded minimal differences between groups while UTE-MRI showed a RAL and RAL+ALN treatment regimens resulted in significantly higher bound water compared to VEH (+23 and +18%, respectively). There was no significant difference among groups for DXA- or CT-based measures lumbar vertebra, or femoral diaphysis. Ribs of RAL-treated animals were smaller and less dense compared to VEH and although mechanical properties were lower the material-level properties were equivalent to normal. In conclusion, we present a suite of data in a beagle dog model treated for one year with clinically-relevant doses of alendronate and raloxifene monotherapies or combination treatment with both agents. Despite the expected effects on bone remodeling, our study did not find the expected benefit of ALN to BMD or structural mechanical properties, and thus the viability of the combination therapy remains unclear.

Effects of daily restraint with and without injections on skeletal properties in C57BL/6NHsd mice

imaging, and bone mechanical testing (Supplementary Methods). Our data (Figs. 1–3; Supplementary Tables 1–5) clearly showed no effect of saline injections or animal handling on skeletal morphology or mechanical properties following an 8-week study period. Several prior studies have documented the effects of handling and/or injection on animal stress levels1,2. These studies and others have utilized a variety of outcome measures including assessing body/organ weight4-7, animal activity/behavior6,8, and biochemical assays9. We found no significant effect among the three experimental groups in body or organ weight (Fig. 1a,b; Supplementary Table 1). Blood levels of leukocytes were also similar across the three groups and were within normal limits10. The ratio of neutrophils to lymphocytes has been used as an index of animal stress in multiple species11, and this study demonstrated no significant differences between the three groups (Fig. 1c; Supplementary Table 2). Finally, qualitative evaluation of several in addition to a placebo group (administration of vehicle), though changes of bone are relatively slow and likely not influenced by the stressors associated with restraint and injection. The goal of the current study was to examine the effects of saline injections in mice, specifically on skeletal properties, and to determine if a reduction of animal use can be affected through the elimination of a placebo group that does not provide meaningful comparison data. Our working hypothesis was that daily handling and injection would not significantly alter bone mass or mechanical properties compared to non-intervention controls. We tested 60 female C57BL/6NHsd mice that were randomly assigned to one of three experimental groups: animals that were only handled during weekly cage changes (CON, n=20); animals that were restrained but not injected 5 days per week (SHAM, n=20); and animals that were restrained and given an intraperitoneal (IP) injection (0.15 cc 0.9% saline solution) 5 days per week (INJ, n=20).The experiment lasted 8 weeks and included Animal model experiments have an essential place in the infrastructure of biomedical science. The literature is replete with papers studying various physiological and organ systems in which animals are manipulated via administration of novel compounds. These studies typically involve groups of animals that are injected with placebo compounds. As there are studies that demonstrate that restraint and injection can affect behavior and corticosteroid levels in rodents1,2, the basis of such placebo injections is to control for any potential effects caused by handling and injecting the experimental animals. But these stressors may not adversely affect all studies equally. While placebo injections make sense for studies that are focused on outcomes which may be directly or indirectly affected by stress hormones, the value of placebo injections is less clear for other studies. If placebo groups are not necessary for some studies, this would reduce both the number of animals used in research and the need to handle/inject a significant number of animals. Bone is a dynamic organ that undergoes continual renewal throughout life3. The breakdown of this process leads to conditions such as osteoporosis, where bones lose mass and mechanical properties, ultimately leading to fracture. The mouse has become a highly utilized research model in skeletal biology due to the ease of genetic manipulation to answer mechanistic questions. Due to the relatively slow changes that occur in bone, most interventional studies involve treatment durations that last weeks or months. Studies of bone physiology often include a control group (no manipulation) Effects of daily restraint with and without injections on skeletal properties in C57BL/6NHsd mice