Microengineered Osteoinductive and Vasculogenic Scaffold

描述(申请人提供)：重建大型骨缺损的主要挑战是成骨细胞和血管生成细胞的成熟需要互补的微环境。该项目的目的是开发一种具有空间组织微通道的3D多层细胞负载复合结构，以模拟骨髓中的成骨细胞-血管生态位，并解决组织结构和细胞微环境的问题。在骨组织中，骨髓基质允许的柔软的血管诱导微环境支持血管生成，而与骨组织结合的骨诱导内膜支持矿化和骨形成。假设如下：a)具有长降解时间的骨诱导性高模水凝胶(SPELA Gel)为骨髓基质细胞(MSCs)的矿化提供了微环境；b)短降解的高顺应性水凝胶(GelMA Gel)为内皮祖细胞(EPC)的血管化提供了允许的微环境；c)在SPELA凝胶中形成的软GelMA凝胶的微通道为复合材料中的血管形成和矿化提供了允许和指导的“成骨-血管”利基；以及d)微图案化过程可以重复，以产生3D多层结构。我们提出了以下目的来设计和评估用于骨段再生的细胞结构。在目标1.1中，我们将合成含有短丙交酯链段的SPELA水凝胶，作为一种具有强大压缩弹性的可降解基质，以支持MSCs的包裹和矿化。在Aim 1.2中，我们将在SPELA水凝胶中添加骨诱导基因重组人骨形态发生蛋白-2蛋白，并将其移植到自组装纳米颗粒中，以防止蛋白质迁移并将其骨诱导活性限制在SPELA基质中。在目标1.3中，我们将合成一种基于明胶的GelMA水凝胶，作为一种可允许的基质，以支持骨髓来源的EPC和MSCs的血管生成分化和成熟以及血管形成。在目标2.1中，我们将在MSC种子SPELA水凝胶中构建EPC/MSC种子GelMA凝胶的微通道，以形成在矿化SPELA水凝胶中具有空间有序微血管的“凝胶-凝胶”组织层。在目标2.2中，我们将设计大孔组织层，将这些层集成到具有空间组织的微通道的3D多层构建物中，并确定构建物中央部分嵌入的细胞的活性。在Aim 2.3中，我们将评估3D多层细胞负载结构在体外矿化和血管形成方面的作用。在目标3中，将在大鼠股骨节段性缺损处进行体内评估，以观察骨形成和愈合的程度。这是一种临床可行的方法，因为骨髓间充质干细胞和内皮祖细胞可以从患者的骨髓中分离出来，并在植入大型骨缺损处之前植入结构中。

DESCRIPTION (provided by applicant): The major challenge in reconstruction of large bone defects is that the maturation of osteogenic and vasculogenic cells require complementary microenvironments. The aim of this project is to develop a 3D multilayer cell-laden composite construct with spatially organized microchannels to mimic the "osteoblastic- vascular" niche in the bone marrow, and to address the issues of tissue architecture and cell microenvironment. In bone tissue, the soft permissive vasculoinductive microenvironment of the marrow stroma supports vasculogenesis while the osteoinductive endosteal layer bound to the osseous tissue supports mineralization and bone formation. The hypotheses are: a) an osteoinductive high modulus hydrogel with and long-degradation time (SPELA gel) provides a microenvironment for mineralization of marrow stromal cells (MSCs); b) a high compliance hydrogel with short degradation (GelMA gel) provides a permissive microenvironment for vascularization of endothelial progenitor cells (EPCs); c) microchannels of the soft GelMA gel patterned in the SPELA gel provide a permissive and instructive "osteoblastic-vascular" niche for concurrent vascularization and mineralization in the composite matrix; and d) the micropatterning process can be repeated to produce a 3D multilayer construct. We propose the following aims to engineer and evaluate the cellular constructs for regeneration of bone segments. In Aim 1.1, we will synthesize the SPELA hydrogel with short lactide segments as a degradable matrix with robust compressive modulus to support encapsulation and mineralization of MSCs. In Aim 1.2, we will supplement the SPELA hydrogel with osteoinductive rhBMP-2 protein grafted to self-assembled nanoparticles to prevent migration of the protein and confine its osteoinductivity to the SPELA matrix. In Aim 1.3, we will synthesize a gelatin-based GelMA hydrogel as a permissive matrix to support vasculogenic differentiation and maturation of bone marrow derived EPCs and MSCs and vessel formation. In Aim 2.1, we will fabricate microchannels of EPC/MSC- seeded GelMA gel in MSC-seeded SPELA hydrogel to form a "gel-in-gel" tissue layer with spatially organized microvessels in the mineralizing SPELA hydrogel. In Aim 2.2, we will engineer macroporous tissue layers, integrate the layers into 3D multilayer constructs with spatially organized microchannels, and determine viability of the embedded cells in the central part of the construct. In Aim 2.3, we will evaluate the 3D multilayer cell-laden constructs with respect to mineralization and vascularization in vitro. In Aim 3, the patterned cell-laden 3D constructs will be evaluated in vivo in rat segmental femur defect for the extent of bone formation and healing. This is a clinically viable approach as MSCs and EPCs can be isolated from the bone marrow of the patient and embedded in the construct prior to implantation in a large bone defect.