Regenerative Immunotherapy using light triggered in vivo activation of adhesive peptides

腭裂修复术后的口鼻瘘一直是临床上经常遇到的难题。 我们实验室的临床前证据和多篇文献报告表明，免疫反应，包括单核细胞 在ONF中，细胞募集失调，导致非愈合环境，伴有瘢痕和大瘘管。这是一个重大 儿科人群中的问题，导致说话和进食困难，并需要在这些儿童中重复手术 他们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祖细胞， 促血管生成、抗纤维化巨噬细胞在切除皮肤和口腔伤口内，并且它们在 损伤小生境增强微血管网扩张。本提案旨在开发新的可降解聚乙烯 乙二醇）-马来酰亚胺（PEG）水凝胶，其用可光活化的笼状RGD肽官能化。初步数据 表明调节皮肤组织中免疫细胞粘附的时间可以增强再生。这些发现表明 刺激响应水凝胶生物材料的光触发是启动促再生的有效新技术， 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， (e.g.光固化树脂），该提案将调查是否在口腔中的局部，时间调节的呈现 修复模型可以预防口腔伤口愈合期间的并发症，类似于腭裂术后观察到的并发症 修复.这些研究的主要假设是，在口腔中，光触发的RGD呈递的时间控制 空腔伤口愈合将增加非经典单核细胞的粘附，并增强其促再生能力。 与血管形成、组织重塑和再生相关的贡献。这一假设将在 以下具体目标：目标1：研究光触发的空间图案和梯度呈现的影响， RGD肽在体内对促再生单核细胞/巨噬细胞亚群的募集。这一目标将采用背 用于单核细胞募集的重复、非侵入性活体显微镜分析的皮褶窗室模型 光触发后的原位动力学。目的2：研究PEG水凝胶中RGD的时间调控呈递 影响小鼠腭裂修复中促再生单核细胞/巨噬细胞亚群的募集和血管形成 模型这一目标将调查免疫浸润和修复机制在腭伤口，并将确定如何 PEG水凝胶的粘附配体的时间调节呈递影响伤口修复。这一目标还将包括 使用FTY 720递送增加促再生Ly 6clo单核细胞的新的功能增强实验 积累这些实验将确定是否光触发曝光的RGD结合增加 修复性免疫细胞的组织积累将改善手术后腭缺损的愈合。这些创新 研究将确定刺激响应水凝胶材料如何可以单独使用或与免疫调节剂组合使用。 口腔中的调节治疗以促进伤口愈合。这项提案的成功也将表明， 临床上可获得的药物如FTY 720可以被重新用于局部靶向宿主中的内源性修复细胞，作为一种新的治疗方法。 一种再生免疫疗法

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.