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

Guldberg组织工程策略最近已经作为骨移植的替代方法出现，以增加体内骨再生。骨成功再生所需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。这些元件是否由宿主提供或必须包含在组织工程构建体中，关键取决于缺损部位的局部生物化学、机械和血管环境。间充质干细胞（MSC）为基础的方法，骨再生近年来取得了迅速进展，同时增加了对肌肉骨骼细胞生物学的理解。细胞扩增对于涉及老年患者、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严重受损伤口床的患者的困难临床病例尤其重要。良好的特点，在体外和体内试验床系统与定量结果的措施，需要评估这些和其他骨组织工程technologies. This项目的目的是定量比较在体内和体外骨形成内的三维间充质干细胞结构进行相同的循环机械负荷条件下的有效性。 对于所有实验，将通过用从犬骨髓抽吸物纯化的MSC接种脱矿小梁骨同种异体移植物来创建组织工程化构建体。将使用先前用于测试骨组织工程结构的犬液压骨腔（HBC）植入物模型进行体内实验。直径和长度为6.35 mm的圆柱形MSC结构将植入位于犬股骨远端干骺端的双侧腔室内。HBC模型能够对植入腔室内的结构施加受控的循环机械刺激。单独的实验将评估时间、接种密度和机械负荷对体内MSC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被量化，并使用显微断层扫描（microCT）成像和3D体视学进行比较。平行的体外实验将使用一种新型的3D组织培养系统进行，该系统能够同时灌注圆柱形细胞接种的结构在横向方向和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 mm的圆柱形MSC结构。将使用microCT和3D体视学定量时间、接种密度和机械负荷对体外骨形成的影响。将测试3D组织培养系统将准确预测实验变量（如时间、接种密度和机械载荷）对体内矿化骨形成的相对影响的假设。所提出的实验提供了一个基础，更好地了解体内的物理因素之间的相互作用和细胞接种的结构，旨在提高骨再生的功效。识别可以通过3D、承载体外系统预测的体内骨形成反应的方面可以导致改进的体外筛选方案，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。一个经过验证的3D系统将有助于，例如，有效评估广泛的设计参数，这些参数可能影响整体构建体的功效，如支架结构、材料和机械性能以及细胞类型和接种密度。

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.