Parallel In Vitro and In Vivo Evaluation of Bone Tissue Engineering Constructs

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0101239GuldbergTissue engineering strategies have recently emerged as an alternative approach to bone grafting to augment the regeneration of bone in vivo. The basic elements required for successful regeneration of bone include an extracellular matrix scaffold, cells, and bioactive genes or proteins. Whether these elements are provided by the host or must be included within a tissue-engineered construct depends critically on the local biochemical, mechanical, and vascular environments at the defect site. Mesenchymal stem cell (MSC)-based approaches to bone regeneration have advanced rapidly in recent years in parallel with an increased understanding of musculoskeletal cell biology. Cellular augmentation is especially important for difficult clinical cases involving older patients, smokers, patients receiving chemotherapy or radiation, and patients with severely damaged wound beds where the endogenous cellular supply may be diminished. Well-characterized in vitro and in vivo test bed systems with quantitative outcome measures are required to evaluate the efficacy of these and other bone tissue-engineering technologies.The objective of this project is to quantifiably compare in vivo and in vitro bone formation within 3D mesenchymal stem cell constructs subjected to identical cyclic mechanical loading conditions. For all experiments, tissue-engineered constructs will be created by seeding demineralized trabecular bone allografts with MSCs purified from canine marrow aspirates. In vivo experiments will be conducted using a canine hydraulic bone chamber (HBC) implant model that has been used previously to test bone tissue engineering constructs. Cylindrical MSC constructs measuring 6.35 millimeters in diameter and length will be implanted within bilateral chambers located in the distal femoral metaphyses of canines. The HBC model has the ability to apply a controlled cyclic mechanical stimulus to constructs implanted within the chamber. Separate experiments will evaluate the effects of time, seeding density, and mechanical loading on MSC differentiation and mineralized matrix synthesis in vivo. The amount and organization of mineralized matrix formation will be quantified and compared using microtomography (microCT) imaging and 3D stereology.Parallel in vitro experiments will be conducted using a novel 3D tissue culture system with the ability to simultaneously perfuse cylindrical cell-seeded constructs in the transverse direction and apply a cyclic axial mechanical stimulus. As in the in vivo experiments, cylindrical MSC constructs measuring 6.35 millimeters in diameter and length will be tested. The effects of time, seeding density, and mechanical loading on in vitro bone formation will be quantified using microCT and 3D stereology. The hypothesis that the 3D tissue culture system will accurately predict the relative effect of experimental variables such as time, seeding density, and mechanical loading on mineralized bone formation in vivo will be tested. The proposed experiments provide a basis for better understanding the interaction between physical factors in vivo and the efficacy of cell-seeded constructs designed to enhance bone regeneration. Identifying aspects of the in vivo bone formation response that may be predicted by a 3D, load-bearing in vitro system may lead to improved in vitro screening protocols, potentially reducing the number or size of animal studies required to benchmark and optimize bone tissue engineering technologies. A validated 3D system would facilitate, for example, efficient evaluation of a wide range of design parameters that may influence overall construct efficacy such as the scaffold architecture, material, and mechanical properties as well as cell type and seeding density.