Biomaterial regulation of cell spheroids to synergistically enhance bone healing

 描述(由申请人提供)：在美国每年发生的600多万个骨折中，高达20%会导致骨不连或延迟愈合，因此需要对骨再生进行干预。间充质干细胞(MSCs)具有良好的成骨潜能和强大的促血管生成营养因子分泌能力，是骨愈合细胞治疗的理想细胞来源。培养维度对细胞的各种功能有着深远的影响。与分离的MSCs相比，我们最近的数据显示，MSC球体分泌的血管生成因子水平是分离的MSCs的100倍，在保持成骨能力的同时更好地抵抗细胞凋亡。球体的形成是凝聚力和粘附力之间的竞争，通过在工程生物材料中的包埋来优化这种平衡提供了一个令人兴奋的机会来指导MSCs移植后的再生潜力。水凝胶的性质，如粘附性、硬度和降解性，会影响被包裹的细胞的功能和由此产生的组织形成。海藻酸盐是一种高度细胞相容性的天然聚合物，可以通过组成和交联来控制初始机械性能，以及通过将Arg-Gly-Asp(RGD)等肽序列共价偶联到结合细胞受体的聚合物骨架上来控制粘附性。因此，藻酸盐水凝胶是探索底物性质对球体功能影响的理想工具。我们的中央 假说是，利用具有工程化生物物理特性的藻酸盐水凝胶，可以增强MSC球体用于骨再生的治疗潜力。目的1.藻酸盐水凝胶中的黏附配体密度是否影响包裹的MSC球体的存活率、促血管生成和成骨能力？我们将合成不同密度的RGD藻酸盐水凝胶。粘附力和凝聚力的增加对球体功能的影响将被确定。目的2.水凝胶生物力学特性是否影响包裹的MSC球体的功能反应？使用具有不同生物物理特性的复合水凝胶，我们将检测基质硬度和降解对包裹的MSC球体存活、促血管生成和成骨能力的影响。目的3.将MSC球体移植到生物物理性能优化的RGD修饰的水凝胶中，能否加速临界大小的颅骨缺损区的骨形成？与分离的MSCs相比，我们将研究移植到RGD修饰的藻酸盐水凝胶中的MSC球体在促进原位缺损骨修复方面的能力。植入细胞的作用以及骨形成的质量将使用非侵入性成像方式进行评估。这项拟议的研究具有创新性，因为它利用细胞聚集和黏附的平衡来驱动细胞命运，使用可注射的、可生物降解的水凝胶来增强MSCs的修复潜力。这项研究将提供一种新的方法来驱动未愈合或缓慢愈合的骨折中的骨形成，这些策略在提高基于材料的治疗组织修复的有效性方面具有潜力。

 DESCRIPTION (provided by applicant): Of the greater than 6 million fractures occurring yearly in the US, up to 20% will result in nonunion or delayed union, thereby requiring intervention for bone regeneration. Mesenchymal stem/stromal cells (MSCs) are an attractive cell source for cell-based therapies of bone healing because of their osteogenic potential and robust secretion of proangiogenic trophic factors. Culture dimensionality has a profound impact on a myriad of cell functions. Compared to dissociated MSCs, our recent data demonstrate that MSC spheroids secrete 100- fold higher levels of angiogenic factors and better resist apoptosis while maintaining osteogenic potential. Spheroid formation is a competition between cohesion and adhesion, and optimizing this balance through the entrapment in engineered biomaterials provides an exciting opportunity to instruct the regenerative potential of MSCs after transplantation. Hydrogel properties such as adhesivity, stiffness, and degradation influence the function of entrapped cells and resulting tissue formation. Alginate is a highly cytocompatible natural polymer that is amenable to control of initial mechanical properties through composition and crosslinking, as well as adhesivity by covalently coupling peptide sequences such as Arg-Gly-Asp (RGD) to the polymer backbone that bind cellular receptors. Thus, alginate hydrogels represent an ideal tool to probe the role of substrate properties on spheroid function. Our central hypothesis is that the therapeutic potential of MSC spheroids for bone regeneration can be enhanced using alginate hydrogels with engineered biophysical properties. Aim 1. Does adhesion ligand density within alginate hydrogels affect the survival, proangiogenic, and osteogenic potential of entrapped MSC spheroids? We will synthesize alginate hydrogels with varying densities of RGD. The influence of increased adhesion versus cohesion on spheroid function will be determined. Aim 2. Do hydrogel biomechanical properties influence the functional response of entrapped MSC spheroids? Using composite hydrogels with distinct biophysical properties, we will examine the role of substrate stiffness and degradation on survival, proangiogenic and osteogenic potential of entrapped MSC spheroids. Aim 3. Can MSC spheroids transplanted in RGD-modified hydrogels with optimized biophysical properties accelerate bone formation in a critical-sized calvarial bone defect? We will characterize the capacity of MSC spheroids transplanted in RGD-modified alginate hydrogels to accelerate bone repair in an orthotopic defect compared to dissociated MSCs. The role of implanted cells, as well as quality of bone formation will be assessed using noninvasive imaging modalities. The proposed research is innovative because it exploits the balance of cellular aggregation versus adhesion to drive cell fate using an injectable, biodegradable hydrogel to potentiate the reparative potential of MSCs. This research will provide a new approach to drive bone formation in nonhealing or slow healing bone fractures, and the strategies have potential in enhancing the efficacy of materials-based therapies for tissue repair.