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.

当角膜因严重的创伤或疾病而受损时，其通常光滑轮廓和光学 清晰度通常会受到损害，从而导致视力丧失。在这些情况下，护理标准涉及角膜 移植；但是，供体组织在世界大部分地区仍然有限，而对于许多人来说， 尽管已经有望在生物合成组织移植物材料中产生结果，但 概括人角膜的长期透明度，生物力学和再生能力 仍然是一个巨大的挑战。先前的研究表明，干细胞疗法可能是 供体组织的替代选择，并表明移植的角膜基质干细胞（CSSC）可以 预防角膜疤痕形成并恢复角膜透明度。但是，遵循简单的细胞活力 注射过程中的机械损伤在注射过程中保持较低。此外，CSSC表型 移植后及其再生潜力背后的机制仍然未知。因此，新的 交付CSSC并了解它们如何应对细胞外环境以促进的方法 需要角膜透明度。 在这项拟议的研究中，我将与可调的生物胸部交联的胶原蛋白凝胶开发 我假设的机械性能可以成功地将CSSC传递给受伤的角膜，稳定 伤口并促进快速重新上述化，同时保持角膜透明度。我们以前有 证明生物正交交联可以改善胶原蛋白的机械稳定性，同时避免 典型交联化学的潜在脱靶和细胞毒性作用。在这里，我将确定如何 凝胶基质中的CSSC培养CSSC会影响凝胶的机械性能，并随着时间的推移延长透明度 作为生物正交交联密度的函数。我还将表征交联密度如何影响 CSSC向角膜细胞的表型过渡，并确定3D培养条件如何影响 CSSCS转录组。然后，我将评估CSSC在损坏的重新上述中的旁分泌作用 角膜，包括生长因子表达的差异以及如何将工程凝胶整合到 天然组织。这将提高我们对CSSC如何促进角膜透明度的基本理解 重塑其细胞外基质以及周围的微环境如何影响他们 分化和再生潜力。最终，这项工作可以提供平台技术来启用 伤口在眼睛内外愈合。