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

封装在生物正交交联胶原凝胶内用于递送至眼表的角膜基质干细胞的表征

• 批准号: 10357733
• 负责人: Sarah Hull
• 金额: $ 3.98万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-30 至 2022-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10357733
• 关键词: Affect; Air; Area; Biocompatible Materials; Biomechanics; Blindness; Cadaver; Cell Survival; Cells; Chemistry; Cicatrix; Collagen; Copper; Cornea; Corneal Injury; Defect; Disease; Economics; Encapsulated; Engineering; Environment; Epithelial; Extracellular Matrix; Eye; Foundations; Future; Gel; Gene Expression; Goals; Growth Factor; HGF gene; Human; Hydrogels; Immunoassay; In Situ; In Vitro; Injections; Keratoplasty; Light; Liquid substance; Mechanics; Mediator of activation protein; Messenger RNA; Methods; Modeling; Operative Surgical Procedures; Optics; Outcome; Output; Phenotype; Procedures; Prosthesis; Proteins; Rattus; Reaction; Refractory; Regenerative capacity; Research; Research Proposals; Risk; Role; Side; Surface; System; Technology; Testing; Therapeutic; Thick; Thinness; Time; Tissue Donors; Tissue Grafts; Tissues; Transplantation; Trauma; Ulcer; Visual Pathways; Water; Work; base; biological systems; catalyst; cell behavior; chronic ulcer; corneal epithelial wound healing; corneal scar; crosslink; cytotoxic; density; epithelial wound; extracellular; improved; improved outcome; in vivo; keratinocyte growth factor; mechanical properties; novel; ocular surface; paracrine; post-transplant; prevent; regeneration potential; sight restoration; skin ulcer; standard of care; stem cell differentiation; stem cell therapy; stem cells; three dimensional cell culture; transcriptome; transcriptome sequencing; translational applications; wound; wound closure; wound healing

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.

当角膜由于严重的创伤或疾病而受损时，其正常光滑的轮廓和光学特性会发生变化。 清晰度经常受到损害，导致视力丧失。这些病例的标准治疗涉及角膜 然而，在世界大部分地区，供体组织仍然有限， 尽管在创造生物合成组织移植材料方面已经取得了可喜的成果， 概括了人类供体角膜的长期透明度、生物力学和再生能力 仍然是一项艰巨的挑战。以前的研究表明，干细胞治疗可能是一种治疗方法。 作为供体组织的替代选择，移植的角膜基质干细胞（CSCs）可以 防止角膜瘢痕形成，恢复角膜透明度。然而，细胞活力， 由于注射过程中的机械损坏，注射保持较低。此外，CSSC表型 移植后的再生能力及其再生潜力背后的机制仍然未知。因此，新 用于递送CSCs并了解它们如何响应细胞外环境以促进 需要角膜透明度。 在这项拟议的研究中，我将开发一种具有可调 我假设可以成功地将CSSC输送到受伤的角膜，稳定 伤口，并促进快速上皮再生，同时保持角膜透明度。我们先前已经 表明生物正交交联可以提高胶原的机械稳定性，同时避免 典型交联化学的潜在脱靶效应和细胞毒性效应。在这里，我将确定如何 在凝胶基质中培养CSSC影响凝胶的机械性质和随时间延长的透明度 作为生物正交交联密度的函数。我还将描述交联密度如何影响 CSCs向角膜基质细胞的表型转变，并确定3D培养条件如何影响 CSSC转录组。然后，我将评估CSSC在受损组织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CSCs如何通过以下方式促进角膜透明度的基本理解： 重塑他们的细胞外基质，以及他们周围的微环境如何影响他们的细胞外基质。 分化和再生潜力。最终，这项工作可以提供一个平台技术， 眼睛内外的伤口愈合。