Regenerative Immunotherapy using light triggered in vivo activation of adhesive peptides

腭裂修复术后的口鼻瘘仍是临床上经常遇到的一个具有挑战性的问题。 来自我们实验室的临床前证据和多份文献报告表明，免疫反应，包括单核细胞 招募，在ONF中调节失调，导致无法愈合的环境，留下疤痕和大的瘘管。这是一名少校 儿科人群中的一种问题，导致这些儿童说话和进食困难，需要重复手术 然后仍然会留下永久性的缺陷。先前的研究表明，非经典单核细胞是有偏见的 在切除的皮肤和口腔创面中，促血管生成、抗纤维化的巨噬细胞，它们在 损伤壁龛，促进微血管网扩张。这项建议旨在开发新型可降解聚乙烯 乙二醇)-马来酰亚胺(PEG)水凝胶，用可光激活的笼状RGD多肽官能化。初步数据 表明调节皮肤组织中免疫细胞黏附的时间可以促进再生。这些发现表明 光触发刺激响应型水凝胶生物材料是一种有效的启动促再生的新技术 免疫信号。由于光引发的生物材料反应在口腔中的广泛潜在应用 (例如光固化树脂)，这项提案将调查口腔中局部的、有时间调节的RGD表现 修复模式可以预防口腔伤口愈合过程中的并发症，类似于观察到的腭裂 修理。这些研究的主要假设是，口述过程中对光触发的RGD呈现的时间控制 空洞创面愈合会增加非经典单核细胞的黏附性，增强其促再生能力 与血管形成、组织重塑和再生相关的贡献。这一假设将在 具体目标如下：目标1：研究光触发的空间图案化和梯度呈现效应 RGD多肽在体内对促再生单核/巨噬细胞亚群的募集作用。这一目标将使用背部 重复、无创活体显微镜分析单核细胞募集的皮肤折叠窗腔室模型 光触发后的原位动力学。目的2：研究时间调节的聚乙二醇水凝胶呈现RGD的方式 促再生单核/巨噬细胞亚群募集和血管化对小鼠腭裂修复的影响 模特。这一目标将研究腭部伤口的免疫渗透和修复机制，并将决定如何 聚乙二醇水凝胶中黏附配体的时间调节作用影响伤口修复。这一目标还将包括 使用FTY720递送增加促再生Ly6clo单核细胞的新功能增强实验 积累。这些实验将确定光触发RGD的曝光量是否与增加 修复免疫细胞的组织积聚将促进腭部手术后缺损的愈合。这些创新之处 研究将确定刺激反应水凝胶材料如何单独使用或与免疫联合使用 口腔中的调节性治疗，以促进伤口愈合。这项提议的成功也将表明 临床上可用的药物，如FTY720，可以作为一种新的药物被重新用于局部靶向宿主的内源性修复细胞 再生免疫疗法的一种形式。

Oronasal fistulas (ONF) following cleft palate repair remain a challenging problem that is frequently encountered clinically. Preclinical evidence from our lab and multiple literature reports show that immune response, including monocyte recruitment, is dysregulated in ONF, leading to a non-healing environment with scarring and a large fistula. This is a major problem in the pediatric population which causes trouble talking and eating and requires repeat surgeries in these children who are then still left with permanent defects. Prior research shows that non-classical monocytes are biased progenitors of pro-angiogenic, anti-fibrotic macrophages within excisional skin and oral cavity wounds, and that they function within the injury niche to enhance microvascular network expansion. This proposal seeks to develop new degradable poly(ethylene glycol)-maleimide (PEG) hydrogels that are functionalized with photoactivatable caged RGD peptide. Preliminary data shows that regulating the timing of immune cell adhesion in cutaneous tissues enhances regeneration. These findings suggest that light triggering of stimulus-responsive hydrogel biomaterials is a potent new technology for initiating pro-regenerative immune signaling. Because of the widespread potential application of light-initiated biomaterial responses in the oral cavity (e.g. light-cured resin), this proposal will investigate whether local, time-regulated presentation of RGD in an oral cavity repair model can prevent complications during oral cavity wound healing, similar to those observed following cleft palate repair. The overarching hypothesis of these studies is that temporal control of light-triggered RGD presentation during oral cavity wound healing will increase the adhesion of non-classical monocytes and enhance their pro-regenerative contributions associated with vascularization, tissue remodeling and regeneration. This hypothesis will be addressed in the following specific aims: Aim 1: To investigate the effects of spatial patterning and gradient presentation of light-triggered RGD peptides in vivo on the recruitment of pro-regenerative monocyte / macrophage subsets. This aim will employ a dorsal skinfold window chamber model for repetitive, non-invasive intravital microscopy analysis of monocyte recruitment kinetics in situ after light-triggering. Aim 2: To investigate how time-regulated presentation of RGD from PEG hydrogels influences pro-regenerative monocyte / macrophage subset recruitment and vascularization in a murine cleft palate repair model. This aim will investigate immune infiltration and repair mechanisms in palatal wounds and will determine how the time-regulated presentation of adhesive ligands from PEG hydrogels influences wound repair. This aim will also include novel enhancement-of-function experiments using FTY720 delivery to increase pro-regenerative Ly6clo monocyte accumulation. These experiments will determine whether light-triggering the exposure of RGD combined with increased tissue accumulation of reparative immune cells will improve healing in post-surgical palatal defects. These innovative studies will establish how stimulus response hydrogel materials can be used alone or in combination with immune modulatory treatments in the oral cavity to enhance wound healing. Success of this proposal will also demonstrate how clinically available drugs such as FTY720 can be re-purposed to locally target endogenous repair cells in the host as a novel form of regenerative immunotherapy.