OPTIMIZING BONE MARROW AS A BONE GRAFT

这项建议的主要目的是通过确定可应用于骨髓来源结缔组织前体细胞(CTP)的快速采集和术中移植的一般原则和技术，推动骨组织工程领域的发展。最初资助期的工作已经确定了一些方法，通过这些方法，可以使用可植入的基质作为选择性细胞附着的表面，从人骨髓中快速浓缩和积极地选择CTP。我们还表明，以这种方式制备的复合细胞移植物显著提高了植骨程序的效果。这种快速、简单的CTPs浓缩和输送方法，通过细胞复合移植物的受控制备和输送，有望改善许多临床移植程序和其他组织工程应用。要实现这一承诺，需要更好地理解和控制细胞基质表面和细胞移植物内产生的代谢环境之间的相互作用。利用分子表面设计方法，获得了对调控CTP附着和选择以及存活、增殖和分化的表面性质的实验控制。根据与CTPs相互作用的证据，从文献中战略性地选择了一组小肽配体。这些多肽被呈现在化学稳定但可降解的底物聚己内酯(PCL)上，并筛选出对这些生物参数的特定剂量依赖效应。由于植入部位的低氧将对移植细胞的生物学性能产生深远的影响，而配体的作用有望调节细胞对低氧的反应，因此，我们评估了低氧对CTP存活和生物学性能的影响。CTPs和其他骨髓细胞亚群的选择性附着在3D多孔基质中进行评估，提供对配体呈现、结构和孔隙度的精确控制。然后，将对选定的细胞3D复合移植物进行体内评估，以通过控制CTP浓度、细胞密度、细胞组成和基质孔隙度来改进优化移植效果的有效策略。体内定量评估包括移植物部位的氧分压、代谢需求和血运重建。定量显微CT和组织学用于评估骨和组织的形成。使用这种独特的技术组合，我们将定义基本的生物学原理。涉及细胞-基质相互作用、基质表面化学、基质结构和局部缺氧的影响，可应用于在广泛的材料和组织工程应用中设计含有骨髓来源细胞的细胞植入物。

The Broad Aim of this proposal is to advance the field of Bone Tissue Engineering by defining general principles and techniques that can be applied to the rapid harvest and intra- operative transplantation of bone marrow derived connective tissue progenitors (CTPs). Work in the initial funding period has defined methods by which CTPs can be rapidly concentrated and positively selected from human bone marrow using an implantable matrix as a surface for selective cell attachment. We have also shown that composite cellular grafts prepared in this way significantly improve the efficacy of bone grafting procedures. This rapid and simple method for concentration and delivery of CTPs offers promise to improve many clinical grafting procedures and other tissue engineering applications through controlled preparation and delivery of cellular composite grafts. Realizing this promise requires a improved understanding and control over the interaction between cells matrix surfaces and the metabolic environment created within a cellular graft. Experimental control over surface properties modulating CTP attachment and selection, as well as survival, proliferation and differentiation is obtained using a molecular surface design approach. A set of small peptide ligands have been strategically selected from the literature based on evidence of interaction with CTPs. These peptides are be presented on a chemically stable but degradable substrate, polycaprolactone(PCL), and screened for specific dose dependent effects on these biologic parameters. Because hypoxia at the site of implantation will have profound effects on the biologic performance of transplanted cells, and the effects of ligands are expected to modulate cellular response to hypoxia, the effect of hypoxia on CTP survival and biologic performance is assessed. The selective attachment of CTPs and other subsets of marrow cells are evaluated in 3D porous matrices providing precise control over ligand presentation, structure and porosity. Selected cellular 3D composite grafts will then be evaluated in vivo to refine effective strategies for optimizing graft efficacy, through control of CTP concentration, cell density, cell composition, and matrix porosity. Quantitative in vivo assessment is made of oxygen tension, metabolic demand, revascularization in the graft site. Quantitative microCT and histology are used to assess bone and tissue formation. Using this unique combination of technologies, we will define fundamental biologic principles. related to cell-matrix interaction, matrix surface chemistry, matrix architecture and effects of local hypoxia that can be applies to the design of cellular implants containing marrow derived cells in a broad range of materials and tissue engineering applications.