Local Delivery of Neurogenic Factors via Polymeric Microparticles for Enhanced Endochondral Bone Repair in the Mandible

项目摘要/摘要 大约有几种。每年有1500万次骨折，下颌骨支撑着绝大多数的颅面 骨折。目前，临床方法，如颌面部固定，是非常有侵入性的，而且 愈合受损的情况仍然普遍存在。因此，这笔赠款的目的是解决临床上对 下颌骨骨折的平移和临床相关入路。实现这一目标需要交叉- 学科方法，利用生物材料和药物输送方面的专业知识，同时了解 驱动功能性骨修复的机制。下颌骨主要通过软骨内愈合。 骨化，在此过程中软骨中间形成，然后被骨取代。近年来，许多 包括我们在内的研究小组已经发表了重要的证据，表明软骨细胞转分化为 骨发育和骨折愈合过程中的成骨细胞。软骨细胞的作用机制 到目前为止，转分化还没有得到彻底的研究。然而，我的初步数据，以及 先前发表的工作表明，β-连环蛋白信号是软骨细胞来源的关键介质 成骨细胞的形成。已观察到β-连环蛋白由神经生长因子/TrkA信号激活在不同类型的细胞和 有趣的是，我们的初步数据显示，骨折骨痂中NGF和TrkA的表达增加。最后， 我们的初步数据显示，在软骨期，NGF在骨折上的应用加速 骨修复。在此期间，我致力于了解NGF在软骨细胞转分化中的作用，以及 开发一种局部和持续释放“无痛”NGF NGFR100W的治疗性给药系统。这个 该项目的中心假设是通过PEGDMA微粒持续释放NGFR100W将 通过激活肥大软骨细胞中的β-连环蛋白信号来加速软骨内骨折的愈合。 在第一个目标中，我将以我们的初步数据为基础，通过工程学方法增强NGF治疗小鼠的骨修复 NGFR100W-洗脱PEGDMA微粒以加速愈合。NGFR100W负载的PEGDMA微粒将 经皮注射到骨折痂上，然后评估组织成分，生物力学 通过组织学、显微CT成像、三点弯曲试验和体视学检查，评估骨折的强度和愈合率。 在第二个目标中，我将确定NGF刺激成骨的机制。我会用一个体外的 应用神经生长因子培养的骨折愈伤组织软骨检测其下游标志物 RT-qPCR法研究成骨、血管生成和候选通路，包括β-连环蛋白、Sox2和Hedgehog 和西方的污点。在体内，我将有条件地通过交叉TrkAfl/fl和TrkA从软骨细胞中删除TrkA Aggrecan-Creer转基因小鼠测试骨折过程中软骨细胞转分化是否需要NGF 使用目标1中描述的相同的功能结果衡量标准进行治疗。

Project Summary/Abstract There are approx. 15 million bone fractures annually and the mandible sustains the vast majority of craniofacial bone fractures. Currently clinical approaches, such as maxillofacial fixation, are exceedingly invasive and the prevalence of impaired healing remains. Therefore, the objective of this grant is to address the clinical need for a translational and clinically relevant approach to mandibular fractures. Accomplishing this goal requires cross- disciplinary methods that harness expertise in biomaterials and drug delivery coupled with an understanding of the mechanisms that drive functional bone repair. The mandible primarily heals through endochondral ossification, in which a cartilage intermediate forms and is later replaced by bone. In recent years, many groups, including ours, have published significant evidence to show that chondrocytes transdifferentiate into osteoblasts during bone development and fracture healing. The mechanisms underlying chondrocyte transdifferentiation have thus far not been thoroughly explored. However, my preliminary data, along with previously published work, indicate that β-catenin signaling is a critical mediator of chondrocyte-derived osteoblastogenesis. Activation of β-catenin by NGF/TrkA signaling has been observed in various cell types and interestingly; our preliminary data show an increase in NGF and TrkA expression in fracture calluses. Finally, our preliminary data show that NGF administration onto fractures during the cartilaginous phase accelerates bone repair. During this fellowship I aim to understand the role of NGF in chondrocyte transdifferentiation, and develop a therapeutic delivery system for local and sustained release of a “painless” NGF, NGFR100W. The central hypothesis for this project is that sustained release of NGFR100W via PEGDMA microparticles will accelerate endochondral fracture healing by activating β-catenin signaling in hypertrophic chondrocytes. In the first Aim I will build on our preliminary data of enhanced bone repair in NGF-treated mice by engineering NGFR100W-eluting PEGDMA microparticles to accelerate healing. NGFR100W-loaded PEGDMA microparticles will be injected percutaneously onto fracture calli followed by assessment of tissue composition, biomechanical strength, and rate of healing by using histology, microCT imaging, three-point bending tests, and stereology. In the second Aim, I will determine the mechanism by which NGF stimulates osteogenesis. I will use an ex vivo system of fracture callus-derived cartilage cultured with NGF to measure downstream markers of osteogenesis, angiogenesis, and candidate pathways including β-Catenin, Sox2, and hedgehog by RT-qPCR and western blot. In vivo I will conditionally delete TrkA from chondrocytes by crossing the TrkAfl/fl and aggrecan-CreER transgenic mice to test if NGF is required for chondrocyte transdifferentiation during fracture healing using the same functional outcome measures described in Aim 1.