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

[101239 . guldberg]组织工程策略最近成为骨移植的一种替代方法，以增加体内骨的再生。成功再生骨的基本要素包括细胞外基质支架、细胞和生物活性基因或蛋白质。这些元素是由宿主提供的，还是必须包含在组织工程结构中，主要取决于缺陷部位的局部生化、机械和血管环境。近年来，基于间充质干细胞（MSC）的骨再生方法发展迅速，同时对肌肉骨骼细胞生物学的了解也在增加。对于老年患者、吸烟者、接受化疗或放疗的患者以及内源性细胞供应可能减少的伤口床严重受损的患者，细胞增强尤其重要。为了评估这些和其他骨组织工程技术的有效性，需要具有定量结果测量的体外和体内测试平台系统。该项目的目的是在相同的循环机械载荷条件下，定量比较三维间充质干细胞构建体体内和体外骨形成。在所有的实验中，组织工程结构将通过用从犬骨髓中纯化的间充质干细胞播种脱矿化骨小梁同种异体移植物来创建。体内实验将使用犬水力骨腔（HBC）植入模型进行，该模型先前已用于测试骨组织工程构建。直径和长度为6.35毫米的圆柱形MSC构建体将被植入位于犬股远端股骨干的双侧腔内。HBC模型能够对植入腔内的结构施加可控的循环机械刺激。单独的实验将评估时间、播种密度和机械负荷对骨髓间充质干细胞分化和矿化基质合成的影响。矿化基质形成的数量和组织将通过显微断层扫描（microCT）成像和三维立体成像进行量化和比较。平行体外实验将使用一种新型3D组织培养系统进行，该系统能够同时向横向灌注圆柱形细胞种子构建物，并施加循环轴向机械刺激。与体内实验一样，将测试直径和长度为6.35毫米的圆柱形MSC构建体。时间、播种密度和机械载荷对体外骨形成的影响将使用微ct和3D立体学进行量化。3D组织培养系统将准确预测实验变量（如时间、播种密度和机械载荷）对体内矿化骨形成的相对影响的假设将进行测试。本实验为更好地理解体内物理因素与细胞种子构建体增强骨再生功效之间的相互作用提供了基础。确定体内骨形成反应的各个方面，可以通过3D，承重体外系统预测，可能导致改进体外筛选方案，潜在地减少基准和优化骨组织工程技术所需的动物研究的数量或规模。例如，一个经过验证的3D系统将有助于对可能影响整体结构效率的各种设计参数（如支架结构、材料、机械性能以及细胞类型和播种密度）进行有效评估。

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.