Cell-Free Assembly of Organized Collagen Arrays

描述（由申请人提供）：胶原蛋白是主要的承重分子，也是体内最丰富的蛋白质。胶原变性疾病包括骨关节炎（影响10%的60岁或以上的美国人），骨质疏松症（影响2800万美国人），动脉瘤和椎间盘疾病。美国每年进行50，000例角膜移植和200，000例初次ACL重建，证明需要合适的机械强度高的移植材料。胶原蛋白相关的遗传疾病同样具有破坏性;例子包括成骨不全（在美国影响27，000人），许多软骨发育不良（4，000名新生儿中有1名）和各种综合征（即Ehlers-Danlos，Alport和Bethlem肌病）。这些疾病的经济影响是巨大的;仅骨关节炎一项，每年就给美国经济造成600多亿美元的损失。尽管如此，很少有人知道的机制，在体内的胶原蛋白组装，限制了组织工程师在实验室中复制胶原承重组织的能力。相反，组织工程利用细胞接种的、可降解的合成或自组装的胶原支架，其需要再吸收和重塑才能发挥功能。支架的再吸收和重塑与体内疤痕重塑或伤口愈合非常相似，即使在理想条件下，也可能需要数年时间才能完成。因此，没有功能性承重组织工程等同物获得临床成功。构建机械强度强的胶原基质的方法应提高产生功能性结缔组织等同物的机会。胶原基质必须高度有序和高度浓缩以具有足够的强度。因此，有必要以类似于胚胎发育的方式在纳米级组织胶原蛋白，其中成纤维细胞对胶原蛋白的原纤维形成进行精密控制。尽管对这一过程的详细机制知之甚少，但有两种相互竞争的假设表明胶原蛋白是：a）由单个细胞通过细胞表面纤维定位器合成、组装和组织化的，或B）以单体形式分泌，然后通过细胞增殖效应组装成有组织的基质。在拟议的工作中，我们将观察有组织的矩阵组装使用延时活动态差分干涉对比成像的单个细胞在一个新的体外模型的角膜基质ECM的发展。然后，我们将通过模仿我们实验室目前正在开发的两种体外无细胞系统之一的发育机制，从头产生有组织的胶原基质。如果成功，拟议的研究将回答一个关于发育的关键基础科学问题，然后将这些结果转化为能够产生承重胶原组织等同物的过程。

DESCRIPTION (provided by applicant): Collagen is the principle load-bearing molecule and most abundant protein in the body. Collagen degenerative diseases include osteoarthritis (affecting 10% of Americans 60 years or older), osteoporosis (affecting 28 million Americans), arterial aneurysms and intervetebral disc disease. There are 50,000 corneal transplants and 200,000 primary ACL reconstructions performed in the US each year demonstrating the need for suitable, mechanically strong graft material. Collagen-related genetic disorders are equally devastating; examples include osteogenesis imperfecta (affecting 27,000 people in the US), many chondrodysplasias (1 in 4,000 births), and various syndromes (i.e. Ehlers-Danlos, Alport and Bethlem myopathy). The economic impact of these diseases is enormous; osteoarthritis alone costs the US economy more than $60 billion annually. In spite of this, little is known about the mechanisms that govern collagen assembly in vivo which has limited the ability of tissue engineers to reproduce collagenous load-bearing tissue in the laboratory. Instead, tissue engineering utilizes cell-seeded, degradable synthetic or self-assembled, collagenous scaffolds which require resorption and remodeling to be functional. Resorption and remodeling of scaffolds closely resembles in vivo scar remodeling or wound healing which can take years to complete, even under ideal conditions. Consequently, no functional load-bearing tissue-engineered equivalents have met with clinical success. A method to construct mechanically strong, collagenous matrix should improve the chances of producing functional connective tissue equivalents. Collagen matrices must be highly ordered and highly concentrated to possess adequate strength. It is thus necessary to organize collagen at the nanoscale in a manner similar to embryonic development where fibroblast cells exercise exquisite control over the fibrillogenesis of collagen. Though little is known about the detailed mechanics of this process there are two competing hypotheses which suggest that collagen is either: a) synthesized, assembled and organized by individual cells via cell-surface fibropositors or b) secreted in monomer form and then assembled into an organized matrix via cholesteric effects. In the proposed work, we will observe organized matrix assembly using time-lapse live dynamic differential interference contrast imaging of individual cells in a novel in vitro model of corneal stromal ECM development. We will then produce organized collagenous matrices de novo by mimicking the development mechanism in one of two in vitro cell-free systems currently under development in our laboratory. If successful, the proposed investigation will answer a critical basic science question regarding development and then translate those results into a process capable of producing load-bearing collagenous tissue equivalents.