Osteoporosis is a metabolic skeletal disorder, which primarily affects postmenopausal women and with 200 million female patients world-wide represents the second most significant health threat in women after breast cancer. The disease is characterised by severe bone loss and degradation of the microarchitecture of bone, resulting in reduced mechanical integrity of the bone and an increased risk of fracture. Current antiresorptive therapies for osteoporosis mainly target prevention of bone loss, yet these only reduce fracture risk by 50%. More recently, the focus for osteoporosis treatment strategies has shifted towards developing anabolic therapies, which aim to stimulate new bone formation and increase osteocyte activity. Neutralising antibodies to sclerostin (Scl-Ab) increase osteoblast bone formation activity by inhibiting sclerostin, a negative regulator of bone formation. Studies of Scl-Ab therapy have demonstrated positive effects on bone mass and bone mineral density (BMD) in ovariectomised animal models and recent research has indicated the benefit of extended dosing intervals in prolonging the positive bone forming effects of Scl-Ab therapy. However, the potential of Scl-Ab therapy in preventing alterations to bone mineral distribution during estrogen deficiency is not fully understood.
Extensive research has demonstrated that the mechanical properties of bone are highly dependent on bone mass, microarchitecture and tissue composition. In particular, it is well understood that trabecular bone is subject to dramatic bone loss during estrogen deficiency. Interestingly, recent research has revealed that osteoporosis is not simply a disease of bone loss and microarchitectural degradation, but that tissue composition is also altered. The organic collagen matrix of bone tissue serves as a scaffold for bone mineralisation and contributes to tensile strength, elasticity and toughness of bone. It has been reported that during osteoporosis the amount of collagen type I found in bone tissue is decreased while collagen synthesis is increased, which may suggest alterations in the quality of the organic matrix. However, the spatial and temporal nature of changes to bone matrix composition, relative to bone loss, and their implication on the mechanical properties of the bone are not yet fully understood.
The ovariectomised rat model provides an effective model of post-menopausal osteoporosis, which allows for the study of extensive periods of estrogen deficiency over relatively short periods of time. The first study of this thesis conducted longitudinal in vivo micro-CT analysis (15µm resolution), to assess changes in trabecular microarchitecture and bone mineral density distribution in the proximal tibia of an estrogen deficient female rat at specific time points (weekly in the first four weeks, week 8, 14 and 34 after ovariectomy). This study found that although trabecular bone volume and architecture are significantly deteriorated within the first four weeks of estrogen deficiency, there is no change in the distribution of trabecular mineral content during this initial period. The rate of bone loss in OVX animals dramatically reduced between weeks 4 and 14 and reached a homeostasis thereafter up to 34 weeks, which coincided with the initiation of increased bone mineral density, altered mineral distribution and trabecular thickening. On the basis of these results, it was proposed that altered loading conditions, arising in the depleted trabecular structure after bone loss, provoke a secondary mineralisation response and new bone formation at trabecular surfaces in long term estrogen deficiency, in order to reinforce the remaining trabecular structure.
The second study of this thesis involved a temporal and spatial examination of OVX-induced changes to trabecular microarchitecture, tissue matrix composition and tissue-level mechanical properties. Four distinct sub-regions of the proximal tibial metaphysis were analysed by high resolution ex-vivo micro-CT (3µm resolution), while individual trabeculae from three of the sub-regions were analysed by Raman spectroscopy and nanoindentation. The results of this study revealed that bone loss within the first four weeks of estrogen deficiency is ubiquitous across the proximal tibia but no change to tissue mineralisation or collagen maturity occur during this time. After 14 weeks of estrogen deficiency, region-specific increases in mineral-to-matrix heterogeneity and heterogeneity of collagen maturity were observed. Finally, nanoindentation revealed a region-specific decrease in tissue elastic modulus and hardness after 14 weeks of estrogen deficiency. Based on these findings, it was proposed that in the initial phase of bone loss expendable trabeculae are removed. Furthermore, the results suggest that when bone loss reaches a plateau, new bone formation and mineralisation occur at specific trabecular regions in response to altered loading conditions. This results in a highly heterogeneous matrix composition, across individual trabeculae and distinct trabecular regions, which may contribute to diminished mechanical integrity of the trabecular structure in long term estrogen deficiency.
The current FDA approved treatment regimen for Romosozumab, the human antibody to sclerostin, is two subcutaneous injections of 210 mg once per month. This frequency of treatment might not be favourable for patients compared to alternative osteoporotic injected drug therapies, such as Denosumab, which is administered once every 6 months, thus lower frequency treatment should be studied. However, the effe ct of low doses of Scl-Ab therapy, with extended dosing intervals has not been investigated. Furthermore, localised delivery of Scl-Ab with a biomaterial scaffold could be used to prevent the site-specific alteration of bone mineral distribution that has previously been reported, but this has never been investigated. Therefore, the final study of this thesis sought to assess the effectiveness of monthly systemic delivery of 2 mg/kg of Scl-Ab therapy or localised delivery of 0.5 mg of Scl-Ab via gelatin- nHA scaffold in preventing adverse changes in trabecular microarchitecture and tissue mineralisation in the proximal tibia of an estrogen deficient rat model. Longitudinal in vivo micro-CT was conducted on ovariectomised and Scl-Ab treated animals from monthly low-dose systemic and localised delivery animals at week 4, 8 and 14 of estrogen deficiency. The findings of this study indicate that, in OVX rats, neither monthly systemic delivery of 2 mg/kg of Scl-Ab nor localised delivery of 0.5 mg of Scl-Ab, were effective in preventing changes in trabecular architecture or mineralisation over 11 weeks compared to untreated age-matched OVX animals.
Together, these studies provide an advanced understanding of the time sequence and spatial variation of changes to trabecular microarchitecture, tissue composition and tissue-level mechanical properties in the estrogen deficient rat model. Using longitudinal in vivo microCT analysis, the sequence of changes in trabecular microarchitecture and tissue mineralisation were elucidated during long term estrogen deficiency. Additionally, spectroscopic methods and nanoindentation revealed the temporal effects of estrogen deficiency on the organic matrix and the implications of changes to bone composition on tissue-level mechanical properties. Collectively, the research presented in this thesis provides an important, but previously unrecognised, insight into the longitudinal changes in trabecular microarchitecture, tissue composition, tissue-level mechanical properties and response to Scl-Ab treatment which occur during estrogen deficiency. The information gained from this work may inform future mechanobiological treatments for osteoporosis.