A Cell-free Approach to the Engineering of Corneal Stroma

角膜基质工程的无细胞方法

基本信息

批准号：
10455311
负责人：
Nima Saeidi
金额：
$ 12万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-08-01 至 2023-07-31
项目状态：
已结题

项目摘要

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。全球有超过 2500 万人患有单眼或双眼角膜失明。角膜移植是恢复视力的唯一选择。然而，全球范围内的捐助者短缺，每年只有不到 1% 的角膜失明患者接受移植手术。尽管有很大的承诺角膜组织工程研究填补了这一空白，努力开发临床上可行的角膜替代品主要由于现有的体外培养系统无法复制复杂的细胞外细胞而受到阻碍基质的基质结构（ECM）。大约 95% 的基质由 I 型胶原蛋白组成 ECM，其中规则堆积的胶原原纤维具有均匀的直径并排列为正交片层，为角膜提供独特的机械和光学特性。主要的非细胞性质基质促使我们追求一种无细胞的基质工程方法。我们最近有证明拥挤的胶原蛋白分子的平面限制诱导了胶原蛋白的自组织将胶原蛋白转化为高度有序的结构。我们的目标是设计一种无细胞基质类似物。我们的中央假设是，可以通过利用固有的物理化学性质来开发功能齐全的角膜基质胶原蛋白分子的特性。我们将在三个具体方面努力实现本提案的目标目标：在目标 1 中，限制和拥挤条件对长程和短程组织的影响将测定胶原纤维。接下来，lumican和decorin蛋白多糖对胶原蛋白的影响将阐明超微结构（即直径和间距）。在目标 2 中，我们将结合理论和数值建模，以了解拥挤和限制条件以及与蛋白聚糖的相互作用，影响胶原组织、形态和自组装。预测性的该模型的性质还可以识别额外的实验参数，以进一步优化基质模拟胶原结构。在目标 3 中，无细胞基质类似物的长期体内功能将通过将它们移植到有深角膜疤痕的兔子体内来描绘。此外，在一系列探索性的研究中，我们将调查构建体（例如，原纤维）是否具有类似天然的物理特征组织、直径和机械性能），将提高角膜基质干的分化细胞转化为天然角膜基质角膜细胞。如果成功，这里描述的工作预计将导致开发第一代脱细胞角膜基质类似物，可恢复角膜功能移植。无细胞基质可直接用于体内层状移植，或者它们可以与上皮层和内皮层结合形成功能齐全的角膜。此外，我们建议开发的多功能胶原组织技术可用于生产仿生基质将集成到现有的体外平台中，以机械地研究如何 ECM 的各种特性（例如组织和直径）影响细胞的命运和功能。