A Cell-free Approach to the Engineering of Corneal Stroma

角膜基质工程的无细胞方法

• 批准号: 10222698
• 负责人: Nima Saeidi
• 金额: $ 41.62万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-30 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10222698
• 关键词: Address; Allografting; Biological; Biomimetics; Biophysics; Blindness; Caliber; Cell Culture System; Cells; Characteristics; Cicatrix; Clinical; Clinical Engineering; Collagen; Collagen Fibril; Collagen Type I; Complex; Cornea; Corneal Stroma; Crowding; Development; Endothelium; Engineering; Epithelial; Exhibits; Extracellular Matrix; Eye; Foundations; Generations; Goals; Grain; Human; In Vitro; Keratoplasty; Liquid substance; Maps; Mechanics; Modeling; Molecular; Morphology; Nature; Optics; Oryctolagus cuniculus; Outcome; Patients; Production; Property; Proteoglycan; Research; Stromal Cells; Structure; System; Techniques; Testing; Tissue Engineering; Tissues; Transplantation; Weight-Bearing state; Work; analog; base; condensed matter physics; corneal scar; crystallinity; decorin; flexibility; gain of function; immunoreaction; improved; in vivo; lumican; mechanical properties; mimetics; novel; predictive modeling; self assembly; self organization; sight restoration; simulation; stem cell fate; stem cells; success; theories; wound; wound healing

ABSTRACT. Over 25 million people worldwide suffer from corneal blindness in one or both eyes. Corneal transplantation is the only option available to restore vision. However, there is a worldwide donor shortage, and fewer than 1% of patients with corneal blindness receive a transplant each year. Despite the great promise of corneal tissue engineering research to fill this gap, efforts to develop a clinically viable corneal substitute are hindered primarily due to the inability of the existing in vitro culture systems to reproduce the intricate extracellular matrix structure (ECM) of the stroma. Approximately 95% of the stroma is composed of type I collagen-based ECM in which regularly packed collagen fibrils have a uniform diameter and are arranged as orthogonal lamellae, providing the cornea with its unique mechanical and optical properties. The predominantly acellular nature of the stroma has motivated us to pursue a cell-free approach to the engineering of the stroma. We have recently demonstrated that the planar confinement of crowded collagen molecules induces self-organization of the collagen into highly ordered structures. Our objective here is to engineer an acellular stromal analog. Our central hypothesis is that a fully functional corneal stroma can be developed by harnessing the inherent physicochemical properties of collagen molecules. Our efforts to pursue the goals of this proposal will be pursued in three specific aims: In Aim 1, the effects of confining and crowding conditions on the long and short-range organization of collagen fibrils will be determined. Next, the impact of lumican and decorin proteoglycans on collagen ultrastructure (i.e. diameter and spacing) will be elucidated. In Aim 2, we will use a combination of theoretical and numerical modeling to provide an understanding of how crowding and confining conditions, as well as interactions with proteoglycans, impact collagen organization, morphology, and self-assembly. The predictive nature of the model would also enable identifying additional experimental parameters to further optimize the stromal-mimetic collagenous structures. In Aim 3, the long-term in vivo function of the acellular stromal analogs will be delineated by transplanting them into rabbits with deep corneal scars. In addition, in a set of exploratory studies we will investigate whether the native-like physical characteristics of the constructs (e.g., fibrillary organization, diameter and mechanical properties), would improve the differentiation of corneal stromal stem cells into native corneal stromal keratocytes. If successful, the work described here is expected to result in the development of first generation acellular corneal stromal analogs which can restore corneal function upon transplantation. The acellular stroma could be used directly for lamellar transplant in vivo or they could be integrated with the epithelial and endothelial layers to produce a fully functional cornea. Furthermore, the versatile collagen organizing technique that we propose to develop could be used for the production of biomimetic substrates to be integrated into the existing in vitro platforms to mechanistically investigate how various properties of ECM (e.g., organization and diameter) impacts cellular fate and function.

抽象的。全世界有超过2500万人在一只或两只眼睛中都遭受角膜失明。角膜 移植是恢复视力的唯一选择。但是，世界范围的捐助者短缺， 每年不到1％的角膜失明患者接受移植。尽管有很大的希望 角膜组织工程研究以填补这一空白，开发临床可行的角膜替代品的努力是 主要是由于现有的体外培养系统无法再现了复杂的细胞外 基质的基质结构（ECM）。大约95％的基质由I型胶原蛋白组成 ECM定期包装的胶原蛋白原纤维的直径均匀，并作为正交薄片排列， 为角膜提供其独特的机械和光学特性。主要的细胞性质 Stroma激励了我们采取无细胞的方法来进行基质的工程。我们最近有 证明了拥挤的胶原蛋白分子的平面限制会引起自组织 胶原蛋白成高度有序的结构。我们的目的是设计一个细胞基质类似物。我们的中心 假设是可以通过利用固有的物理化学来开发功能齐全的角膜基质 胶原分子的性能。我们追求该提案目标的努力将在三个特定的 目的：在AIM 1中，限制和拥挤条件对长期和短期组织的影响 将确定胶原原纤维。接下来，Lumican和Decorin蛋白聚糖对胶原蛋白的影响 将阐明超微结构（即直径和间距）。在AIM 2中，我们将结合理论和 数值建模，以理解拥挤和限制条件以及 与蛋白聚糖，影响胶原蛋白组织，形态和自组装的相互作用。预测性 该模型的性质还将使识别其他实验参数以进一步优化 基质模拟胶原结构。在AIM 3中，细胞基质类似物的长期体内功能 将通过将它们移植到带有深层疤痕的兔子中来描绘它们。另外，在一系列探索性中 研究我们将研究构建体的天然样物理特征（例如，纤维纤维 组织，直径和机械性能）将改善角膜基质茎的分化 细胞进入天然角膜基质角膜细胞。如果成功，此处描述的工作将导致 开发第一代细胞角膜基质类似物，可以恢复角膜功能 移植。细胞基质可直接用于体内层状移植，或者可以是 与上皮和内皮层集成以产生功能齐全的角膜。此外， 我们建议开发的多功能胶原蛋白组织技术可用于生产 仿生底物将集成到现有的体外平台中，以机械学研究如何 ECM（例如，组织和直径）的各种特性会影响细胞命运和功能。