Bioengineered corneal endothelial graft using photodegradable device to induce graft-host integration

项目总结 尽管生物工程化的人角膜内皮细胞(HCEC)单层移植物已经显示出 在动物模型中，hCEC单层不能与宿主角膜结合，原因是 HCEC载体生物材料在前房中的不可调谐降解，这提供了机械 对单层的支撑。因此，移植的hCEC单层能否与宿主角膜结合。 在hCEC载体生物材料完全降解并此后保持功能之后， 未知。为了验证生物工程hCEC单层，显然需要开发一种生物材料 具有可调的体内降解率来评价hCECs的植入，因此具有很强的机械性能 生物材料膜/hCEC单层结构在移植过程中不会断裂。上一次 该团队的工作已经证实，细胞外地形信号可以显著调节hCEC 回应。该团队的初步工作已经开发出可光降解的水凝胶(PdGel)，可以 在移植后，利用组织穿透光，在几小时到几周内以可调的方式降解。 因此，本提案的目的是开发纳米形貌pdGel-hCEC单层接枝， 通过调节pdGel的体内降解率来评估单层与宿主角膜的结合，以及 体内验证hCEC-单层功能。据推测，纳米图案的pdGel将使 HCEC作为融合的单层生长，通过诱导hCEC单层的生长，改善hCEC的功能和稳定性 类天然细胞外基质(ECM)的沉积和载体的可调光降解将 提高hCEC单层的植入率。这个项目的基本原理是对 HCEC单层与角膜的植入和此后的功能将验证 生物工程hCEC移植物可能治疗一位供者的多个角膜患者。朝向 总体目标，在第一个目标中，hCEC在pdGel上的单层生长，光降解动力学， 降解产物的生物相容性和单层的植入将在体外进行评估 和体外实验。在第二个目标中，使用了高通量的地形平台，效果独特的pdGel 将根据hCEC单层功能来评估表面形貌，以确定最佳的移植物设计。在 第三个目标是，将在体内利用光暴露来调节光降解速率，以评估其对 HCEC植入。这项拟议的研究预计将具有重要意义，因为它将验证 利用一种新型的可光降解生物材料的生物工程hCEC-单层接枝技术，并将发展 新型生物材料和纳米地形平台，将在眼组织以外具有重要应用 工程学。本文的创新之处在于：(1)采用双光子光刻技术 开发创新的、高通量的纳米地形平台，以及(2)利用光解 菁染料在开发用于hCEC植入的创新光降解水凝胶方面的作用。

PROJECT SUMMARY Although the bioengineered human corneal endothelial cell (hCEC) monolayer graft have shown vision recovery in animal models, the hCEC monolayer do not integrate with the host cornea due to suboptimal and non-tunable degradation of the hCEC-carrier biomaterial in the anterior chamber, which provide mechanical support to the monolayer. Thus, whether the transplanted hCEC monolayer will integrate with the host cornea following the complete degradation of the hCEC-carrier biomaterial and remain functional thereafter is unknown. To validate the bioengineered hCEC monolayers, there is a clear need to develop a biomaterial that has tunable-degradation rate in-vivo to evaluate the engraftment of the hCECs, and is mechanically strong so that the biomaterial-film/hCEC-monolayer construct does not break during the transplantation. The previous work of the team has established that the extracellular-topography cues can significantly modulate hCEC responses. The preliminary work of the team has developed photodegradable hydrogel (pdGel), which can be degraded in a tunable manner after transplantation, within hours to weeks, using tissue-penetrative light. Accordingly, the objective of this proposal is to develop a nano-topography pdGel-hCEC monolayer graft, evaluate monolayer integration with host cornea by tuning the in-vivo degradation rate of the pdGel, and validate the hCEC-monolayer function in-vivo. It is hypothesized that the nano-patterned pdGel will enable the growth of hCECs as a confluent monolayer, improve the hCEC monolayer function and stability by inducing the deposition of native-like extracellular matrix (ECM), and the tunable photodegradation of the carrier will improve the engraftment of the hCEC monolayer. The rationale for this project is the evaluation of the engraftment of hCEC monolayer with the cornea and the function thereafter will validate the use of bioengineered hCEC grafts for potential treatment of multiple corneal patients with one donor. Towards the overall objective, in the first aim, the hCEC monolayer growth on the pdGel, photodegradation kinetics, the biocompatibility of the degradation products, and the engraftment of the monolayer will be evaluated in-vitro and ex–vivo. In the second aim, using a high-throughput topography platform, the effect of 253 unique pdGel topographies will be evaluated on the hCEC monolayer functions to identify the optimum graft design. In the third aim, the photodegradation rate will be tuned in-vivo using light exposure to evaluate its effect on the hCEC engraftment. The proposed research is expected to be significant because it will validate the bioengineered hCEC-monolayer graft technology using a new photodegradable biomaterial, and it will develop new biomaterial and nano-topography platform that will have significant applications beyond ocular tissue engineering. The proposed research is innovative because it, (1) uses two-photon lithography approach to develop an innovative, high throughput nano-topography platform, and (2) leverages the photo-decomposition liability of the cyanine dye to develop an innovative photodegradable hydrogel for hCEC engraftment.