The Anabolic - Anti-catabolic Paradigm of Augmenting Skeletal Regeneration: A Bone Quality Perspective

Abstract: Successful fracture repair is a complex series of coordinated processes leading to regeneration of new bone to bridge the fracture site, remodelling of the newly formed bone and restoration of skeletal function. Disruption of these processes occurs in approximately 10% of all fractures and leads to delayed or failed healing requiring surgical interventions, increased complication rates, prolonged rehabilitation and high morbidity. In instances of fracture non-unions, the healing process could be augmented through the use of potent anabolic agents such as bone morphogenetic proteins (BMPs) or anti-catabolic agents such as the bisphosphonate Zoledronate (ZA). Use of BMP alone or synergistically combined with ZA is known to substantially increase bone formation and enhance fracture healing. Such interventions necessitate an understanding of both the mechanisms that contribute to impaired or delayed healing and the influence of the administered treatments in countering these processes. This thesis presents a multi-scale characterization of the synergistic efficacy of BMP (specifically BMP-7) and ZA using a femoral open-fracture rodent model of recalcitrant repair. The aim is to characterize the quality of the bone regenerated in response to these specific interventions that stimulate bone formation and inhibit bone resorption. Bone quality is assessed using conventional radiography, ex vivo and in vivo micro-computed tomography (μCT), three-point bending mechanical testing, histology, Fourier-transform infrared (FTIR) spectroscopy, synchrotron-based small-/wide-angle X-ray scattering (SAXS/WAXS) and positron emission tomography (PET). In the first study of this thesis, the synergistic efficacy of allograft in combination with BMP-7 and ZA is investigated. Evidence is presented for an enhanced synergistic efficacy with callus volumes and peak force measures of mechanical strength doubling relative to the gold standard of autograft treatment. Subsequently, an extensive analysis of osteoporotic fracture repair is conducted in an ovariectomized (OVX) rat model of osteoporosis with a particular emphasis on high resolution compositional and nanostructural characterization. The hypothesis of osteoporotic fractures constituting a greater challenge for skeletal repair is refuted. Radiography, μCT, mechanical testing, FTIR and SAXS/WAXS measures of bone quality were found to be equivalent in untreated healthy and osteoporotic bone environments. However, the BMP-7 induced osteogenic response appears to differ with calluses doubling in volume and exhibiting significantly greater heterogeneity in nanostructural crystal dimensions in oestrogen-deficient rats compared to corresponding healthy control rats. Moreover, a comprehensive study of fracture repair at the nanoscale is presented. Callus remodelling in BMP-7 and ZA treated fractures are mapped using in vivo μCT to follow the progression of healing at 6 week intervals over 24 weeks. Remodelling was found to be prolonged as a consequence of either treatment. Finally, the potential of positron-emitting 18F-fluoride isotope as a predictive indicator of skeletal regeneration is assessed. Specifically, the hypothesis that 18F-fluoride tracer activity at an earlier time point is predictive of CT measured bone formation at a later time point is investigated using an in vivo PET/CT instrument. The statistical analysis demonstrates that 18F-fluoride activity is strongly correlated to bone formation and should be further investigated as a potential diagnostic measure to predict bone regeneration.Cumulatively, these studies support the rationale for an anabolic – anti-catabolic paradigm to augment skeletal regeneration. Conventional techniques of assessing the characteristics of bone that confers resistance to fracture are complemented with innovative, state-of-the-art techniques that span multiple length scales. The synergistic efficacy of BMP-7 in combination with ZA is underscored but caution is advised in relation to the osteogenic response induced by BMP-7 in oestrogen-deficient bone environments and the prolongation of remodelling with either treatment. Finally, the potential of 18F-fluoride isotope is highlighted as a possible prognostic tool to monitor complex high-risk fractures and intervene early to reduce healing time with recalcitrant fractures.