ALTERING THE IMMUNE LANDSCAPE TO AUGMENT BONE REGENERATION

项目摘要 该项目的首要假设是，骨再生可以通过利用 疫苗接种状态，并通过控制释放一种 抗原的为了验证这一假设，本项目的一般方法是免疫小鼠， 然后进行体内骨组织工程研究，以测试来自骨组织的抗原递送的影响。 支架对骨形成和免疫反应的影响。这些老鼠将专门接种疫苗 由于美国人口的高年度疫苗覆盖率， 血凝素（HAG）肽将从植入的支架递送。测试将在 小鼠使用两种实验模型，颅骨缺损和股骨缺损。在这两种模型中， 缺陷将用单独释放HAG肽或与HAG肽组合的水凝胶支架治疗。 治疗或亚治疗剂量的重组人骨形态发生蛋白-2（BMP-2）。 所有处理将在未接种疫苗的对照组中使用雄性和雌性小鼠进行重复， 测试性别效应。有两个具体目标。目标1的重点是评价骨形成 和缺损再生，这将在治疗后3周和6周进行评估， 显微计算机断层扫描分析和组织学。结果将以缺陷为基准 用从胶原海绵递送的治疗剂量的BMP-2治疗，其将用作 临床对照治疗。目的2集中于评估HAG肽递送对细胞增殖的影响。 免疫反应将在3周时评价再生缺损中的免疫细胞浸润 治疗6周后行免疫组化染色。此外，将评价转录组学变化 治疗后1周通过单细胞RNA测序。如果成功，这个项目将导致新的 具有高转化潜力的再生免疫疗法。

PROJECT SUMMARY The overarching hypothesis of this project is that bone regeneration can be enhanced by exploiting vaccination status and recruiting adaptive immunity to the injury site through controlled release of an antigen. To test this hypothesis, the general approach in this project will be to immunize mice and then perform in vivo bone tissue engineering studies to test the impact of antigen delivery from the scaffold on bone formation and the immune response. The mice will specifically be vaccinated against influenza due to high annual vaccine coverage in the U.S. population, and influenza-derived hemagglutinin (HAG) peptide will be delivered from implanted scaffolds. Testing will be performed in mice using two experimental models, calvarial defects and femoral defects. In both models, the bone defects will be treated with hydrogel scaffolds releasing HAG peptide alone or in combination with therapeutic or sub-therapeutic doses of recombinant human bone morphogenetic protein-2 (BMP-2). All treatments will be duplicated in an unvaccinated control group using both male and female mice to test for gender effects. There are two Specific Aims. Aim 1 is focused on evaluating bone formation and defect regeneration, which will be evaluated at 3 weeks and 6 weeks after treatment by microcomputed tomography analysis and histology. The results will be benchmarked against defect treatment with a therapeutic dose of BMP-2 delivered from a collagen sponge, which will serve as a clinical control treatment. Aim 2 is focused on evaluating the effects of HAG peptide delivery on the immune response. Immune cell infiltration in the regenerating defects will be evaluated at 3 weeks and 6 weeks after treatment by immunostaining. In addition, transcriptomic changes will be evaluated 1 week after treatment by single-cell RNA sequencing. If successful, this project will lead to novel regenerative immunotherapies with high translational potential.