Microengineered Osteoinductive and Vasculogenic Scaffold

描述（由申请人提供）：骨缺损重建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。本项目旨在开发一种具有空间组织微通道的三维多层细胞负载复合结构，以模拟骨髓中的“成骨-血管”生态位，并解决组织结构和细胞微环境问题。在骨组织中，骨髓基质的柔软纵容血管诱导微环境支持血管发生，而骨诱导内层与骨组织结合支持矿化和骨形成。假设是：a)具有长降解时间的骨诱导高模量水凝胶（SPELA凝胶）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凝胶）为内皮祖细胞（EPCs）的血管化提供了一个允许的微环境；c) SPELA凝胶中的软GelMA凝胶的微通道为复合基质中的血管形成和矿化提供了一个允许和指导性的“成骨-血管”生态位；d)可以重复微图化过程以产生3D多层结构。我们提出以下目标来设计和评估用于骨段再生的细胞结构。在Aim 1.1中，我们将合成具有短丙交酯片段的SPELA水凝胶，作为具有强大压缩模量的可降解基质，以支持MSCs的包封和矿化。在Aim 1.2中，我们将在SPELA水凝胶中添加自组装纳米颗粒上的骨诱导性rhBMP-2蛋白，以防止蛋白质的迁移，并将其骨诱导性限制在SPELA基质上。在Aim 1.3中，我们将合成一种基于明胶的GelMA水凝胶作为允许基质，以支持骨髓来源的内皮祖细胞和间充质干细胞的血管分化和成熟以及血管形成。在Aim 2.1中，我们将在MSC种子的SPELA水凝胶中制造EPC/MSC种子的GelMA凝胶微通道，在矿化的SPELA水凝胶中形成具有空间组织微血管的“凝胶中”组织层。在Aim 2.2中，我们将设计大孔组织层，将这些层整合成具有空间组织的微通道的3D多层结构，并确定结构中心部分嵌入细胞的活力。在Aim 2.3中，我们将评估三维多层细胞负载结构在体外矿化和血管化方面的作用。在Aim 3中，将在大鼠股骨节段性缺损的体内评估带有图案细胞的3D构建体的骨形成和愈合程度。这是一种临床可行的方法，因为MSCs和EPCs可以从患者的骨髓中分离出来，并在植入大骨缺损之前嵌入结构中。

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.