Speaker
Description
Introduction
Bone defects above a critical size exhaust the self-healing capacity of bone and in these circumstances intervention is needed to promote regeneration. In recent decades, an increasing number of tissue engineered bone grafts have been developed. However, expensive and laborious screenings in vivo are necessary to assess their safety and efficacy. Rodents are the first choice for initial in vivo screens but their size limits the dimensions and number of bone grafts that can be tested in orthotopic locations. Here, we report the development of a refined subcutaneous semi-orthotopic bone defect model coupled with a semi-automated longitudinal in vivo micro-CT registration method1.
Methodology
The model is based on four bovine bone implants, which are subcutaneously implanted on the back of immunodeficient mice, where bone healing potential of grafts can be evaluated in an artificially created 4mm wide defect in vivo. Crucially, these defects are “critical size” and unable to heal within the timeframe of the study without intervention. Various grafts were prepared to modulate bone regeneration inside the defect; cortical bone chips, tissue engineered constructs consisting of MSC pellets, collagen scaffolds or a combination, and cartilage grafts. Animals were scanned by micro-CT after implantation and then every two weeks until sacrifice at week 8. For analysis of architectural changes in bone structure, a semi-automatic algorithm for longitudinal micro-CT imaging was developed. Micro-CT scans were segmented into binary datasets and afterwards a co-registration method was performed to assess bone morphometric parameters of the implanted defect over time, followed by histological assessment.
Results
Firstly it was assessed if a graft composed of cortical bone chips, the current gold-standard, would increase bone regeneration in the defect of our model. After 8 weeks, empty defects filled their mineralised bone volume, analysed by micro-CT, by 4±3%, while the defects implanted with bone chips showed a net bone volume increase of 26±8%. Histological analysis confirmed that bone formation was stimulated by bone chips. In this study we demonstrated that bone regeneration can be assessed and that osteogenic performance of grafts composed of solely biomaterial, cells or a combination can be studied effectively. Additionally, with the use of image registration a method to analyse the testing pocket only was developed, which was combined with bone morphometric analysis to monitor defect healing longitudinally in the same animal, thus limiting the number of animals needed .
Conclusion(s)
Our novel semi-orthotopic in vivo model suggests that it is possible to overcome some of the current limitations that rodent bone defect models pose, in particular regarding number and size, since in previous mouse critical-size defect models defects with a size of 3mm3 were reported, while in our model each of the four grafts contains a 50mm3 defect. With the semi-automated micro-CT method we have developed a quantitative technique for assessing the testing pocket of our bone defect model. Our data supports that the semi-orthotopic model combined with the novel micro-CT registration method provides an excellent approach for assessment of new biomaterials or tissue engineered constructs for large bone defect repair.
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