The development of a small animal model for assessing the 3D implications of loading on bone microarchitecture

It is well established that bone is capable of adapting to changes in its environment; however, little is known regarding how environmental stimuli, specifically loading, are associated with the internal 3D microarchitecture of cortical bone. The aim of this thesis was to develop a small animal model that can be used to experimentally test hypotheses regarding bone adaptation. High resolution micro-CT was validated and employed as a novel method for the visualization and quantification of rat cortical bone microarchitecture in 3D. The use of this imaging method allowed for the measurement of primary vascular canal orientation in 3D, which had never been achieved before. Using this measure along with an immobilization model for unloading allowed me to test how loading is associated with the orientation of these vascular canals. Normally ambulating rat bones (from 10 female rats) had a canal structure that was 9.9° more longitudinal than their immobilized counterparts. This finding that loading has an effect on primary canal orientation brought to light the need to induce remodeling and therefore, secondary vascular canals, in the rat to increase its novelty as a model for looking at bone adaptation. Remodeling was induced by increasing the calcium demands of female rats, either through a calcium restricted diet (n=2) or pregnancy and lactation coupled with a calcium restricted diet (n=2). Mean cortical thickness for the calcium restricted rats and the pregnant and lactating rats that were on a calcium restricted diet were 622 µm and 419 µm, respectively. The mean BMU count for calcium restricted rats seemed to be higher than that of the pregnant and lactating rats; however, the calcium restricted rats seemed to have a lower BMU density. Once this full-scale study is executed the rat will provide a more representative model for studying human bone adaptation

The Effect of Genetic Variation on Mouse Bone Strength

Osteoporosis is a heritable bone disease which is characterized by decreased bone mass and a deterioration in bone microarchitecture. This leads to a decrease in bone strength and therefore the risk of fracturing is greater in people who have been diagnosed as being osteoporotic. The mechanism by which osteoporosis is inherited is still unknown, as are the underlying microarchitectural causes of changes in bone strength. Without knowing more about these two mechanisms we cannot begin to develop more effective treatment plans or even a cure for this disease. The aim of this dissertation was to determine whether bone strength can be influenced by vascular canal microarchitecture and whether bone strength is a heritable trait, which is determined by specific quantitative trait loci. Using the CC founder strains and their F1 crosses in a diallel analysis we found that non-additive variance accounted for the same amount or more of the heritability than additive variance. Using the same CC mice we also found that BMD, canal connectivity, canal diameter, canal orientation, cortical area, cortical thickness, and percent porosity were all heritable determinants of bone strength. The bone strength measures used in this thesis were found to be highly correlated (0.702) and there was no statistical difference between methods (p=1.000). Employing the DO population we found a significant QTL for Imax on Chromosome 1 that is 1.43 cM wide and contained 19 candidate genes, of which Cacna1e looks the most promising. The QTL analysis for MaxF and BMD also found suggestive and near suggestive QTLs on Chromosome 1, which would indicate that Chromosome 1 is important in the genetic determination of bone strength

Stem Cell–Derived Endochondral Cartilage Stimulates Bone Healing by Tissue Transformation

Although bone has great capacity for repair, there are a number of clinical situations (fracture non-unions, spinal fusions, revision arthroplasty, segmental defects) in which auto- or allografts attempt to augment bone regeneration by promoting osteogenesis. Critical failures associated with current grafting therapies include osteonecrosis and limited integration between graft and host tissue. We speculated that the underlying problem with current bone grafting techniques is that they promote bone regeneration through direct osteogenesis. Here we hypothesized that using cartilage to promote endochondral bone regeneration would leverage normal developmental and repair sequences to produce a well-vascularized regenerate that integrates with the host tissue. In this study, we use a translational murine model of a segmental tibia defect to test the clinical utility of bone regeneration from a cartilage graft. We further test the mechanism by which cartilage promotes bone regeneration using in vivo lineage tracing and in vitro culture experiments. Our data show that cartilage grafts support regeneration of a vascularized and integrated bone tissue in vivo, and subsequently propose a translational tissue engineering platform using chondrogenesis of mesenchymal stem cells (MSCs). Interestingly, lineage tracing experiments show the regenerate was graft derived, suggesting transformation of the chondrocytes into bone. In vitro culture data show that cartilage explants mineralize with the addition of bone morphogenetic protein (BMP) or by exposure to human vascular endothelial cell (HUVEC)-conditioned medium, indicating that endothelial cells directly promote ossification. This study provides preclinical data for endochondral bone repair that has potential to significantly improve patient outcomes in a variety of musculoskeletal diseases and injuries. Further, in contrast to the dogmatic view that hypertrophic chondrocytes undergo apoptosis before bone formation, our data suggest cartilage can transform into bone by activating the pluripotent transcription factor Oct4A. Together these data represent a paradigm shift describing the mechanism of endochondral bone repair and open the door for novel regenerative strategies based on improved biology