Steven M. Tommasini

Relationship Between Bone Morphology and Bone Quality in Male Tibias: Implications for Stress Fracture Risk†

Biomechanical properties were assessed from the tibias of 17 adult males 17‐46 years of age. Tissue‐level mechanical properties varied with bone size. Narrower tibias were comprised of tissue that was more brittle and more prone to accumulating damage compared with tissue from wider tibias.

Genetic randomization reveals functional relationships among morphologic and tissue-quality traits that contribute to bone strength and fragility

We examined femora from adult AXB/BXA recombinant inbred (RI) mouse strains to identify skeletal traits that are functionally related and to determine how functional interactions among these traits contribute to genetic variability in whole-bone stiffness, strength, and toughness. Randomization of A/J and C57BL/6J genomic regions resulted in each adult male and female RI strain building mechanically functional femora by assembling unique sets of morphologic and tissue-quality traits. A correlation analysis was conducted using the mean trait values for each RI strain. A third of the 66 correlations examined were significant, indicating that many bone traits covaried or were functionally related. Path analysis revealed important functional interactions among bone slenderness, cortical thickness, and tissue mineral density. The path coefficients describing these functional relations were similar for both sexes. The causal relationship among these three traits suggested that cellular processes during growth simultaneously regulate bone slenderness, cortical thickness, and tissue mineral density so that the combination of traits is sufficiently stiff and strong to satisfy daily loading demands. A disadvantage of these functional interactions was that increases in tissue mineral density also deleteriously affected tissue ductility. Consequently, slender bones with high mineral density may be stiff and strong but they are also brittle. Thus, genetically randomized mouse strains revealed a basic biological paradigm that allows for flexibility in building bones that are functional for daily activities but that creates preferred sets of traits under extreme loading conditions. Genetic or environmental perturbations that alter these functional interactions during growth would be expected to lead to loss of function and suboptimal adult bone quality.

Genetic Variation in Structure‐Function Relationships for the Inbred Mouse Lumbar Vertebral Body

Structure‐function relationships were determined for L5 vertebral bodies from three inbred mouse strains. Genetic variability in whole bone mechanical properties could be explained by a combination of the traits specifying the amount, distribution, and quality of the cortical and trabecular bone tissue.

Biological co-adaptation of morphological and composition traits contributes to mechanical functionality and skeletal fragility.

A path analysis was conducted to determine whether functional interactions exist among morphological, compositional, and microstructural traits for young adult human tibias. Data provided evidence that bone traits are co‐adapted during ontogeny so that the sets of traits together satisfy physiological loading demands. However, certain sets of traits are expected to perform poorly under extreme load conditions.

Targeting of androgen receptor in bone reveals a lack of androgen anabolic action and inhibition of osteogenesis A model for compartment-specific androgen action in the skeleton

Androgens are anabolic hormones that affect many tissues, including bone. However, an anabolic effect of androgen treatment on bone in eugonadal subjects has not been observed and clinical trials have been disappointing. The androgen receptor (AR) mediates biological responses to androgens. In bone tissue, both AR and the estrogen receptor (ER) are expressed. Since androgens can be converted into estrogen, the specific role of the AR in maintenance of skeletal homoeostasis remains controversial. The goal of this study was to use skeletally targeted overexpression of AR in differentiated osteoblasts as a means of elucidating the specific role(s) for AR transactivation in the mature bone compartment. Transgenic mice overexpressing AR under the control of the 2.3-kb alpha1(I)-collagen promoter fragment showed no difference in body composition, testosterone, or 17ss-estradiol levels. However, transgenic males have reduced serum osteocalcin, CTx and TRAPC5b levels, and a bone phenotype was observed. In cortical bone, high-resolution micro-computed tomography revealed no difference in periosteal perimeter but a significant reduction in cortical bone area due to an enlarged marrow cavity. Endocortical bone formation rate was also significantly inhibited. Biomechanical analyses showed decreased whole bone strength and quality, with significant reductions in all parameters tested. Trabecular morphology was altered, with increased bone volume comprised of more trabeculae that were closer together but not thicker. Expression of genes involved in bone formation and bone resorption was significantly reduced. The consequences of androgen action are compartment-specific; anabolic effects are exhibited exclusively at periosteal surfaces, but in mature osteoblasts androgens inhibited osteogenesis with detrimental effects on matrix quality, bone fragility and whole bone strength. Thus, the present data demonstrate that enhanced androgen signaling targeted to bone results in low bone turnover and inhibition of bone formation by differentiated osteoblasts. These results indicate that direct androgen action in mature osteoblasts is not anabolic, and raise concerns regarding anabolic steroid abuse in the developing skeleton or high-dose treatment in eugonadal adults.

Changes in intracortical microporosities induced by pharmaceutical treatment of osteoporosis as detected by high resolution micro-CT

Bones microporosities play important biologic and mechanical roles. Here, we quantified 3D changes in cortical osteocyte-lacunae and other small porosities induced by estrogen withdrawal and two different osteoporosis treatments. Unlike 2D measurements, these data collected via synchrotron radiation-based μCT describe the size and 3D spatial distribution of a large number of porous structures. Six-month old female Sprague-Dawley rats were separated into four groups of age-matched controls, untreated OVX, OVX treated with PTH, and OVX treated with Alendronate (ALN). Intracortical microporosity of the medial quadrant of the femoral diaphysis was quantified at endosteal, intracortical, and periosteal regions of the samples, allowing the quantification of osteocyte lacunae that were formed primarily before versus after the start of treatment. Across the overall thickness of the medial cortex, lacunar volume fraction (Lc.V/TV) was significantly lower in ALN treated rats compared to PTH. In the endosteal region, average osteocyte lacunar volume () of untreated OVX rats was significantly lower than in age-matched controls, indicating a decrease in osteocyte lacunar size in bone formed on the endosteal surface after estrogen withdrawal. The effect of treatment (OVX, ALN, PTH) on the number of lacunae per tissue volume (Lc.N/TV) was dependent on the specific location within the cortex (endosteal, intracortical, periosteal). In both the endosteal and intracortical regions, Lc.N/TV was significantly lower in ALN than in untreated OVX, suggesting a site-specific effect in osteocyte lacuna density with ALN treatment. There also were a significantly greater number of small pores (5-100 μm(3) in volume) in the endosteal region for PTH compared to ALN. The mechanical impact of this altered microporosity structure is unknown, but might serve to enhance, rather than deteriorate bone strength with PTH treatment, as smaller osteocyte lacunae may be better able to absorb shear forces than larger lacunae. Together, these data demonstrate that current treatments of osteoporosis can alter the number, size, and distribution of microporosities in cortical rat lamellar bone.

Phenotypic integration among trabecular and cortical bone traits establishes mechanical functionality of inbred mouse vertebrae.

Conventional approaches to identifying quantitative trait loci (QTLs) regulating bone mass and fragility are limited because they examine cortical and trabecular traits independently. Prior work examining long bones from young adult mice and humans indicated that skeletal traits are functionally related and that compensatory interactions among morphological and compositional traits are critical for establishing mechanical function. However, it is not known whether trait covariation (i.e., phenotypic integration) also is important for establishing mechanical function in more complex, corticocancellous structures. Covariation among trabecular, cortical, and compositional bone traits was examined in the context of mechanical functionality for L4 vertebral bodies across a panel of 16‐wk‐old female AXB/BXA recombinant inbred (RI) mouse strains. The unique pattern of randomization of the A/J and C57BL/6J (B6) genome among the RI panel provides a powerful tool that can be used to measure the tendency for different traits to covary and to study the biology of complex traits. We tested the hypothesis that genetic variants affecting vertebral size and mass are buffered by changes in the relative amounts of cortical and trabecular bone and overall mineralization. Despite inheriting random sets of A/J and B6 genomes, the RI strains inherited nonrandom sets of cortical and trabecular bone traits. Path analysis, which is a multivariate analysis that shows how multiple traits covary simultaneously when confounding variables like body size are taken into consideration, showed that RI strains that tended to have smaller vertebrae relative to body size achieved mechanical functionality by increasing mineralization and the relative amounts of cortical and trabecular bone. The interdependence among corticocancellous traits in the vertebral body indicated that variation in trabecular bone traits among inbred mouse strains, which is often thought to arise from genetic factors, is also determined in part by the adaptive response to variation in traits describing the cortical shell. The covariation among corticocancellous traits has important implications for genetic analyses and for interpreting the response of bone to genetic and environmental perturbations.

Percolation theory relates corticocancellous architecture to mechanical function in vertebrae of inbred mouse strains

Complex corticocancellous skeletal sites such as the vertebra or proximal femur are connected networks of bone capable of transferring mechanical loads. Characterizing these structures as networks may allow us to quantify the load transferring behavior of the emergent system as a function of the connected cortical and trabecular components. By defining the relationship between certain physical bone traits and mechanical load transfer pathways, a clearer picture of the genetic determinants of skeletal fragility can be developed. We tested the hypothesis that the measures provided by network percolation theory will reveal that different combinations of cortical, trabecular, and compositional traits lead to significantly different load transfer pathways within the vertebral bodies among inbred mouse strains. Gross morphologic, micro-architectural, and compositional traits of L5 vertebrae from 15 week old A/J (A), C57BL6/J (B6), and C3H/HeJ (C3H) inbred mice (n=10/strain) were determined using micro-computed tomography. Measures included total cross-sectional area, bone volume fraction, trabecular number, thickness, spacing, cortical area, and tissue mineral density. Two-dimensional coronal sections were converted to network graphs with the cortical shell considered as one highly connected node. Percolation parameters including correlation length (average number of connected nodes between superior and inferior surfaces), chemical length (minimum number of connected nodes between surfaces), and backbone mass (strut number) were measured. Analysis of the topology of the connected bone networks showed that A and B6 mice transfer load through trabecular pathways in the middle of the vertebral body in addition to the cortical shell. C3H mice transfer load primarily through the highly mineralized cortical shell. Thus, the measures provided by percolation theory provide a quantitative approach to study how different combinations of cortical and trabecular traits lead to mechanically functional structures. The data further emphasize the interdependent nature of these physical bone traits suggesting similar genetic variants may affect both trabecular and cortical bone. Therefore, developing a network approach to study corticocancellous architecture during growth should further our understanding of the biological basis of skeletal fragility and, thus, provide novel engineering approaches to studying the genetic basis of fracture risk.

Deletion of Rac in Mature Osteoclasts Causes Osteopetrosis, an Age‐Dependent Change in Osteoclast Number and a Reduced Number of Osteoblasts In Vivo

Rac1 and Rac2 are thought to have important roles in osteoclasts. Therefore, mice with deletion of both Rac1 and Rac2 in mature osteoclasts (DKO) were generated by crossing Rac1flox/flox mice with mice expressing Cre in the cathepsin K locus and then mating these animals with Rac2‐/‐ mice. DKO mice had markedly impaired tooth eruption. Bone mineral density (BMD) was increased 21% to 33% in 4‐ to 6‐week‐old DKO mice at all sites when measured by dual‐energy X‐ray absorptiometry (DXA) and serum cross‐linked C‐telopeptide (CTx) was reduced by 52%. The amount of metaphyseal trabecular bone was markedly increased in DKO mice, but the cortices were very thin. Spinal trabecular bone mass was increased. Histomorphometry revealed significant reductions in both osteoclast and osteoblast number and function in 4‐ to 6‐week‐old DKO animals. In 14‐ to 16‐week‐old animals, osteoclast number was increased, although bone density was further increased. DKO osteoclasts had severely impaired actin ring formation, an impaired ability to generate acid, and reduced resorptive activity in vitro. In addition, their life span ex vivo was reduced. DKO osteoblasts expressed normal differentiation markers except for the expression of osterix, which was reduced. The DKO osteoblasts mineralized normally in vitro, indicating that the in vivo defect in osteoblast function was not cell autonomous. Confocal imaging demonstrated focal disruption of the osteocytic dendritic network in DKO cortical bone. Despite these changes, DKO animals had a normal response to treatment with once‐daily parathyroid hormone (PTH). We conclude that Rac1 and Rac2 have critical roles in skeletal metabolism.

Novel anatomic adaptation of cortical bone to meet increased mineral demands of reproduction

The goal of this study was to investigate the effects of reproductive adaptations to mineral homeostasis on the skeleton in a mouse model of compromised mineral homeostasis compared to adaptations in control, unaffected mice. During pregnancy, maternal adaptations to high mineral demand include more than doubling intestinal calcium absorption by increasing calcitriol production. However, calcitriol biosynthesis is impaired in HYP mice, a murine model of X-linked hypophosphatemia (XLH). In addition, there is a paucity of mineralized trabecular bone, a primary target of bone resorption during pregnancy and lactation. Because the highest density of mineral is in mature cortical bone, we hypothesized that mineral demand is met by utilizing intracortical mineral reserves. Indeed, analysis of HYP mice revealed dramatic increases in intracortical porosity characterized by elevated serum PTH and type-I collagen matrix-degrading enzyme MMP-13. We discovered an increase in carbonate ion substitution in the bone mineral matrix during pregnancy and lactation of HYP mice, suggesting an alternative mechanism of bone remodeling that maintains maternal bone mass during periods of high mineral demand. This phenomenon is not restricted to XLH, as increased carbonate in the mineral matrix also occurred in wild-type mice during lactation. Taken together, these data suggest that increased intracortical perilacunar mineral turnover also contributes to maintaining phosphate levels during periods of high mineral demand. Understanding the mechanisms of skeletal contribution to mineral homeostasis is important to improving the treatment and prevention of fracture risk and bone fragility for female patients with XLH, but also provides important insight into the role and unique adaptations of the maternal skeleton to the demands of fetal development and the needs of postnatal nutrition.