Protein-cell assemblies as tissue-mimics

描述(申请人提供)：蛋白质-细胞组件作为组织模拟物建议将活细胞和重组结构蛋白直接组装成组织模拟物，用于随后的组织再生、治疗性筛选和其他生物医学应用。近年来，静电纺丝技术已经发展成为一种强有力的纤维形成工具，用于制备具有优异力学性能和增强细胞-支架相互作用的纳米/微米级结构的蛋白质纤维支架。然而，电纺蛋白质支架的过度细小的毛孔阻碍了细胞高效地迁移到支架中并再生3D组织。最近，通过一种复杂的、基于喷气的3D打印技术，细胞和蛋白质的直接组装得以实现。与电纺支架相比，这种方法制备的支架的力学性能要差得多。在这项提议中，我们计划将细胞打印的概念融入到电纺技术中，以制造类似于天然组织的结构和机械特征的蛋白质-细胞组件。组织模拟蛋白-细胞组合(TMPCA)的发展可能是构建功能组织的重要一步。此外，TMPCA可能会为研究人员提供一种新型的体外细胞培养系统，用于研究生化途径、疾病机制和药物代谢，有助于包括癌症和动脉粥样硬化在内的许多疾病的治疗开发。具体目的1.同轴电纺重组真丝-弹性蛋白样蛋白(SELP)和交联剂或凝固剂，使其成为坚固的纤维支架，无需后电纺处理。优化电纺工艺参数，提高支架的力学性能。同样，将合成不同多肽序列和交联点的SELP，并定义这些结构决定因素调节纤维形成和支架性能的能力。具体目的2.同时电纺SELP和电喷雾成纤维细胞，将3T3成纤维细胞(作为模型系统)同时整合到SEP纳米纤维支架中。为了提高细胞的活力，将寻求用海藻酸盐包裹细胞和快速蒸发交联剂/凝固剂。将制造一种机械坚固的模拟高细胞密度的组织。具体目标3.在长达一周的制造后期间，检查SELP-成纤维细胞组件的结构完整性、细胞活性和支架重塑。特别是，将在静态和动态加载条件下获取机械性能。同样，细胞的存活、生长和增殖将在制造后的一周内进行研究。在这个过程中，将使用数学本构模型来帮助进行力学表征。与公共卫生相关：蛋白质-细胞组合作为组织模拟原生组织是由活细胞和结构蛋白以有组织的方式组成的。我们建议将重组结构蛋白和活细胞组装成一个健壮的、有组织的结构作为组织模拟物。这种组织模拟物可以用来培养人造组织，并提供一个模拟的组织环境来研究疾病机制和药物代谢，有助于开发各种疾病的新疗法。

DESCRIPTION (provided by applicant): Protein-Cell Assemblies as Tissue Mimics It is proposed is to directly assemble living cells and recombinant structural proteins into tissue mimics for subsequent tissue regeneration, therapeutic screening, and other biomedical applications. Electrospinning techniques have recently evolved into a powerful fiber formation tool for fabricating protein fibrous scaffolds of nano/micro-scale structures with excellent mechanical properties and enhanced cell-scaffold interaction. However, the overly small pores of an electrospun protein scaffold inhibit cells from efficiently migrating into the scaffold and regenerating a 3D tissue. A direct assembling of cells and proteins was recently achieved through a sophisticated, jet-based, 3D printing technology. Compared to an electrospun scaffold, the mechanical properties of a scaffold prepared by this method are far inferior. In this proposal, we plan to incorporate the cell printing concept into an electrospinning technique for the fabrication of protein-cell assemblies, which can resemble the structural and mechanical characteristics of native tissues. The development of tissue-mimetic protein-cell assemblies (TMPCA) may be an important step in engineering a functional tissue. In addition, TMPCA may provide researchers with a novel, in vitro cell- culture system for the study of biochemical pathways, disease mechanisms, and drug metabolisms, aiding in therapy development for many diseases including cancer and atherosclerosis. Specific Aim 1. Coaxially electrospin recombinant silk-elastin-like proteins (SELPs) and crosslinking agents or coagulants into a robust fibrous scaffold without post-electrospinning treatments. The electrospinning parameters will be optimized to enhance the scaffold mechanical properties. Likewise, SELPs of varying polypeptide sequences and crosslinking sites will be synthesized, and the ability of these structural determinants to modulate the fiber formation and scaffold properties will be defined. Specific Aim 2. Simultaneously integrate 3T3 fibroblasts (as a model system) into a SELP nanofibrous scaffold by concurrently electrospinning SELPs and electrospraying fibroblasts. Cell encapsulation by alginate and fast evaporation of crosslinking agents/coagulants will be pursued, in order to enhance cell viability. A mechanically robust tissue mimic with high cell densities will be fabricated. Specific Aim 3. Examine the structural integrity, cell activities, and scaffold remodeling of the SELP- fibroblast assemblies during a post-fabrication period of up to one week. In particular, the mechanical properties will be accessed under both static and dynamic loading conditions. Likewise, cell viability, growth, and proliferation will be investigated during one week of post-fabrication follow-up. In this process, mathematical constitutive models will be used to aid in mechanical characterization. PUBLIC HEALTH RELEVANCE: Protein-Cell Assemblies as Tissue Mimics Native tissues are comprised of living cells and structural proteins in an organized way. We propose to assembly recombinant structural proteins and living cells into a robust, organized structure as a tissue mimic. Such a tissue mimic can be used to grow artificial tissues, and provide a mimetic tissue- environment to study disease mechanisms and drug metabolisms, aiding in the development of new therapy for various diseases.