Engineering Immunomodulatory Scaffolds for Dental Pulp Regeneration

项目摘要 该K 01奖项描述了一项为期3年的培训计划，以支持弗兰卡博士向科学 独立弗兰卡博士是一名牙医科学家，她的长期目标是领导一个研究项目，以（i） 开发用于牙齿和颅面再生的免疫调节生物材料;（ii）创建组织 了解和治疗口腔疾病的芯片。K 01的科学目标是开发一种 用于牙髓再生免疫调节支架。尽管不可否认的进步， 牙髓的再生策略，坏死的牙齿提出了具有挑战性的再生条件。 即使经过仔细消毒，先前感染的根管微环境仍有残留 脂多糖（LPS），激活免疫细胞进入与组织不相容的促炎状态 再生最近，免疫反应的早期事件被证明对组织具有高度影响， 工程战略的成功。特别是，巨噬细胞对生物材料的反应已被确定为 支架重塑的基本介质和免疫介导的再生的有前途的目标 战略布局这些细胞可以通过将其表型从促炎性改变为促炎性来响应外部刺激。 在称为M1/M2极化的过程中，具有许多中间状态的前再生。这两种状态 具有高度的可塑性，协同作用，并且可以通过支架的特性进行调节。当一个脚手架 用于牙髓再生的材料与LPS污染的牙本质接触，可以预期M1 激活将持续存在而不提供组织形成所必需的促再生环境。 小抗体或纳米抗体已被用于中和革兰氏阴性感染中的LPS，并靶向 病原体在难以接近的组织微环境中。不幸的是，这种治疗工具还没有被 用于牙齿再生。我们的首要假设是免疫调节支架 中和LPS并增加支架-组织界面中的M2/M1比率将提供可持续的 促再生微环境更有利于牙髓组织的形成。因此，我们的目标是 设计一种新型的载有纳米抗体的可光交联水凝胶， 中和牙本质LPS。该支架将能够可控地引导M1巨噬细胞活化， 最大限度地减少它们在根管内的存在，使得进入的单核细胞可以进入M2细胞， 渗透水凝胶，并分泌抗炎细胞因子环境以促进牙髓组织形成。 本研究的主要目的是：（1）研究支架刚度对M1/M2巨噬细胞增殖的影响 极化，（目的2）中和牙本质LPS并减少M1极化，以及（目的3）评估 纳米抗体负载支架的体内免疫调节能力。成功实现这些目标将 生成关于支架特性和巨噬细胞功能之间相互作用的数据，为 R 01提交和其他新型免疫调节材料的开发。

PROJECT SUMMARY This K01 award describes a 3-year training program to support Dr. Franca's transition to scientific independence. Dr. Franca is a dentist-scientist, and her long-term goal is to lead a research program to (i) develop immunomodulatory biomaterials for dental and craniofacial regeneration and (ii) to create tissue chips to understand and treat oral diseases. The scientific goal of this K01 is to develop an immunomodulatory scaffold for dental pulp regeneration. Despite undeniable progress to develop regenerative strategies for the dental pulp, necrotic teeth present challenging conditions for regeneration. Even after careful disinfection, a previously infected root canal's microenvironment has remnant lipopolysaccharide (LPS) that activates immune cells into a pro-inflammatory state incompatible with tissue regeneration. Recently, early events of the immune response were shown to highly influence tissue engineering strategies' success. Especially, macrophage responses to biomaterials have been identified as essential mediators of scaffold remodeling and promising targets for immune-mediated regenerative strategies. These cells can respond to external stimuli by changing their phenotype from pro-inflammatory to pro-regenerative, with many intermediate states, in a process known as M1/M2 polarization. These two states are highly plastic, function synergistically, and can be modulated by the scaffold's properties. When a scaffold for dental pulp regeneration is placed in contact with LPS-contaminated dentin, it can be expected that M1 activation will persist without providing the pro-regenerative environment necessary for tissue formation. Small antibodies or nanobodies have been used to neutralize LPS in Gram-negative infections and target pathogens in scarcely accessible tissue microenvironments. Unfortunately, this therapeutic tool has not been used for dental regeneration purposes. Our overarching hypothesis is that an immunomodulatory scaffold that neutralizes LPS and increases the M2/M1 ratio in the scaffold-tissue interface will provide a sustainable pro-regenerative microenvironment more favorable to dental pulp tissue formation. Hence, our goal is to engineer a novel nanobody-laden photocrosslinkable hydrogel with fine-tuned stiffness and the ability to neutralize dentin LPS. This scaffold will enable controllable guidance of M1 macrophages activation, minimizing their presence within the root canal so that incoming monocytes can polarize into M2 cells, infiltrate the hydrogel, and secret an anti-inflammatory cytokine milieu to foster dental pulp tissue formation. We propose the following aims: (Aim 1) to determine the effect of scaffold's stiffness on M1/M2 macrophage polarization, (Aim 2) to neutralize dentin LPS and reduce M1 polarization, and (Aim 3) to evaluate the nanobody-laden scaffold's immunomodulatory capacity in vivo. Successful completion of these aims will generate data on the interplay between scaffold's properties and macrophage function, forming the basis for an R01 submission and the development of additional novel immunomodulatory materials.