Biomaterial regulation of cell spheroids to synergistically enhance bone healing

 描述（由申请人提供）：在美国每年发生的超过600万例骨折中，高达20%将导致骨不连或延迟愈合，因此需要进行骨再生干预。间充质干/基质细胞（MSC）是一个有吸引力的细胞来源，为基于细胞的治疗骨愈合，因为他们的成骨潜力和强大的分泌促血管生成营养因子。文化维度对无数的细胞功能有着深远的影响。与分离的MSC相比，我们最近的数据表明MSC球状体分泌100倍高水平的血管生成因子，更好地抵抗细胞凋亡，同时保持成骨潜力。球体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，为指导移植后MSC的再生潜力提供了一个令人兴奋的机会。水凝胶的性质，如粘附性、刚度和降解影响截留细胞的功能和所产生的组织形成。藻酸盐是一种高度细胞相容性的天然聚合物，其可通过组成和交联来控制初始机械性能，以及通过将肽序列如Arg-Gly-Asp（RGD）共价偶联到结合细胞受体的聚合物主链来控制粘附性。因此，藻酸盐水凝胶代表了一种理想的工具，以探测球体功能的基板性能的作用。我们的中央 一种假设是，MSC球状体用于骨再生的治疗潜力可以使用具有工程化生物物理特性的藻酸盐水凝胶来增强。目标1。藻酸盐水凝胶内的粘附配体密度是否影响包埋MSC球体的存活、促血管生成和成骨潜力？我们将合成具有不同密度的RGD的海藻酸盐水凝胶。将确定增加的粘附力与内聚力对球体功能的影响。目标2.水凝胶的生物力学特性是否会影响包埋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，我们将研究基质硬度和降解对包埋MSC球体的存活、促血管生成和成骨潜力的作用。目标3：MSC球状体移植到生物物理性能优化的RGD改性水凝胶中能否加速临界尺寸颅骨缺损的骨形成？我们将表征移植到RGD修饰的藻酸盐水凝胶中的MSC球状体与解离的MSC相比加速原位缺损中的骨修复的能力。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。这项研究具有创新性，因为它利用细胞聚集与粘附的平衡，使用可注射的可生物降解的水凝胶来驱动细胞命运，以增强MSC的修复潜力。这项研究将提供一种新的方法来驱动不愈合或愈合缓慢的骨折中的骨形成，并且这些策略有可能提高基于材料的组织修复疗法的疗效。

 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.