Characterization of corneal stromal stem cells encapsulated within bioorthogonally crosslinked collagen gels for delivery to the ocular surface

当角膜因严重创伤或疾病而受损时，其正常光滑的轮廓和光学 清晰度通常会受到影响，导致失明。这些病例的护理标准涉及角膜。 移植；然而，捐献组织在世界大多数地区仍然有限，许多人无法获得 我需要它。尽管在生物合成组织移植材料的创造方面已经有了令人振奋的结果， 概述人类供体角膜的长期透明性、生物力学和再生能力 这仍然是一项艰巨的挑战。此前的研究表明，干细胞疗法可能是一种 替代供体组织的选择，研究表明，移植的角膜基质干细胞(CSSCs)可以 防止角膜疤痕形成，恢复角膜透明度。然而，细胞活力遵循简单的 由于注射过程中的机械损坏，注射保持在较低水平。此外，CSSC表型 移植后及其再生潜力背后的机制仍不清楚。因此，新的 传递CSSCs的方法以及了解它们如何响应细胞外环境以促进 角膜透明度是必要的。 在这项拟议的研究中，我将开发一种生物医用交联胶原蛋白凝胶，可调 我假设的机械性能可以成功地将CSSCs输送到受伤的角膜，稳定 在保持角膜透明度的同时，促进快速再上皮化。我们之前已经 证明了生物正交交联剂可以提高胶原蛋白的机械稳定性，同时避免 典型交联剂的潜在脱靶和细胞毒性效应。在这里，我将决定如何 在凝胶基质中培养CSSCs会影响凝胶的机械性能和随时间延长的透明度 作为生物正交交联剂密度的函数。我还将描述交联度如何影响 CSSCs向角质形成细胞的表型转变以及3D培养条件如何影响角质形成细胞 CSSCs转录组。然后我将评估CSSCs在损伤的再上皮化中的旁分泌作用。 角膜，包括生长因子表达的差异以及工程凝胶如何整合到 天然组织。这将促进我们对CSSCs如何通过以下方式促进角膜透明度的基本理解 重塑它们的细胞外基质以及周围的微环境如何影响它们 分化和再生潜力。最终，这项工作可以提供一种平台技术来实现 眼睛内外的伤口都在愈合。

When the cornea is damaged due to severe trauma or disease, its normally smooth contour and optical clarity are often compromised, resulting in loss of vision. The standard of care in these cases involves corneal transplantation; however, donor tissue remains limited in most parts of the world and unavailable to many who need it. Although there have been promising results in the creation of biosynthetic tissue graft materials, recapitulating the long-term transparency, biomechanics, and regenerative capacity of a human donor cornea remains a formidable challenge. Previous research has demonstrated that stem cell therapy could be an alternative option to donor tissue, and it was shown that transplanted corneal stromal stem cells (CSSCs) can prevent corneal scar formation and restore corneal transparency. However, cell viability following simple injection remains low due to mechanical damage during the injection procedure. In addition, CSSC phenotype post-transplantation and the mechanism behind their regenerative potential remain unknown. Therefore, new methods for delivering CSSCs and understanding how they respond to extracellular environment to facilitate corneal transparency are needed. In this proposed research, I will develop a bioothogonally crosslinked collagen gel with tunable mechanical properties that I hypothesize can successfully deliver CSSCs to the wounded cornea, stabilize the wound, and promote rapid re-epithelization while maintaining corneal transparency. We have previously demonstrated that bioorthogonal crosslinking can improve the mechanical stability of collagen while avoiding the potential off-target and cytotoxic effects of typical crosslinking chemistries. Here, I will determine how the culturing CSSCs in the gel matrix affects the gel’s mechanical properties and prolonged transparency over time as a function of bioorthogonal crosslinking density. I will also characterize how the crosslinking density affects the phenotypic transition of CSSCs to keratocytes and determine how the 3D culture conditions affect the CSSCs transcriptome. Then I will evaluate the paracrine role of the CSSCs in re-epithelization of the damaged cornea, including differences in the expression of growth factors and how the engineered gel is integrated into native tissue. This will advance our basic understanding of how CSSCs contribute to corneal transparency by remodeling their extracellular matrix and how their surrounding microenvironment influences their differentiation and regenerative potential. Ultimately, this work could provide a platform technology to enable wound healing inside and outside the eye.