Protein-cell assemblies as tissue-mimics

描述（由申请人提供）：蛋白质细胞组装作为组织模拟物。建议直接将活细胞和重组结构蛋白组装成组织模拟物，用于随后的组织再生，治疗筛选和其他生物医学应用。静电纺丝技术最近发展成为一种强大的纤维形成工具，用于制造具有优异力学性能和增强细胞-支架相互作用的纳米/微尺度结构的蛋白质纤维支架。然而，电纺丝蛋白支架的过小孔隙抑制了细胞有效地迁移到支架中并再生3D组织。最近，一种复杂的、基于喷射的3D打印技术实现了细胞和蛋白质的直接组装。与电纺丝支架相比，这种方法制备的支架的机械性能要差得多。在这个提议中，我们计划将细胞打印概念融入到一种静电纺丝技术中，用于制造蛋白质细胞组件，这种组件可以类似于天然组织的结构和机械特征。组织模拟蛋白-细胞组装（TMPCA）的发展可能是工程组织功能的重要一步。此外，TMPCA可以为研究人员提供一种新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，有助于癌症和动脉粥样硬化等许多疾病的治疗开发。具体目标同轴电纺丝重组丝弹性蛋白样蛋白（selp）和交联剂或凝血剂制成坚固的纤维支架，无需电纺丝后处理。优化静电纺丝工艺参数，提高支架的力学性能。同样，不同多肽序列和交联位点的selp将被合成，这些结构决定因素调节纤维形成和支架性能的能力将被定义。具体目标2。同时通过静电纺丝SELP和电喷涂成纤维细胞将3T3成纤维细胞（作为模型系统）整合到SELP纳米纤维支架中。海藻酸盐包封细胞和快速蒸发的交联剂/混凝剂将被追求，以提高细胞活力。将制造具有高细胞密度的机械健壮的组织模拟物。具体目标3。在制造后长达一周的时间内，检查SELP-成纤维细胞组装的结构完整性、细胞活性和支架重塑。特别是，在静态和动态加载条件下，将获得力学性能。同样地，细胞活力、生长和增殖将在制作后一周的随访中进行调查。在这个过程中，数学本构模型将用于帮助机械表征。公共卫生相关性：蛋白质细胞组装作为组织模拟物天然组织由活细胞和结构蛋白以有组织的方式组成。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。这种组织模拟物可用于人工组织的生长，并为研究疾病机制和药物代谢提供模拟组织环境，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.