A microphysiological system of tendon inflammation and fibrosis for drug screening and efficacy testing

急性和慢性肌腱损伤是最常见的肌肉骨骼健康问题之一。通常， 受伤的肌腱经历纤维化瘢痕， 使关节功能受损的粘连。在纤维化的肌腱瘢痕中，细胞-细胞和旁分泌信号 炎症细胞，如巨噬细胞和肌腱成纤维细胞之间的相互作用激活后者， 纤维增生性肌成纤维细胞，最终分化成衰老表型。我们以前的研究 使用下一代测序和基因集富集分析， 在具有TGF-β激活的mTOR信号传导的损伤的小鼠肌腱中的衰老。为了进一步阐明这一点， 病理学，该提案的目标是设计一个微流体人类肌腱芯片（hToC）系统，并使用 它可以更准确地模拟受伤肌腱中炎症和纤维化的生物学方面。在 UG 3阶段的这一建议，芯片将制造具有多室设计和微流体 通道以分别掺入成纤维细胞接种的胶原水凝胶和模拟血管血流。 超薄，高渗透性，光学透明的多孔硅膜（SiM）将分离 并在其间提供用于内皮屏障基底。之间的信令 成纤维细胞、水凝胶驻留的和循环的巨噬细胞以及内皮细胞将通过 纳米多孔SiM（~60 nm），而微孔SiM（~ 8 µm）将允许循环血液外渗。 巨噬细胞和TGF-β刺激下的水凝胶浸润。为了实现以病人为中心的芯片， 肌腱成纤维细胞将被用于创建肌腱水凝胶，并重新编程供体匹配的iPSC， 内皮细胞和巨噬细胞。另一个创新将是标签的整合- 将自由光子传感器引入微流体装置中以允许芯片上感测，这长期以来一直被认为是 这是对器官芯片设备的一个关键的、未满足的需求。UG 3研究将使用该芯片来验证 mTOR在疾病模型中的作用，并鉴定生物学相关的生物标志物。在UH 3阶段，我们将利用 芯片作为临床前试验平台，用于测试FDA批准的mTOR抑制剂的有效性和安全性， 疾病修饰药物，并作为药物筛选平台，以识别和优先考虑更安全，更有效的药物 用于临床试验的mTOR抑制剂和肌腱损伤中的衰老。为了成功地完成这一创新 项目中，我们聚集了一批在肌腱组织工程和外科方面有成就的专家， 免疫学、iPSC技术、GMP细胞制造、纳米和微米制造、传感器技术，以及 高通量筛选。拟议的研究将开发一种人类微生理系统， 临床试验和加速药物发现的急性和慢性肌腱损伤。

Acute and chronic tendon injuries are among the most common musculoskeletal health problems. Typically, an injured tendon experiences fibrotic scarring that leaves the tissue mechanically compromised and prone to debilitating adhesions that impair joint function. In a fibrotic tendon scar, the cell-cell and paracrine signaling between inflammatory cells, such as macrophages, and tendon fibroblasts activate the latter into fibroproliferative myofibroblasts, ultimately differentiating into a senescent phenotype. Our previous studies using next-generation sequencing and gene set enrichment analysis mechanistically linked fibrosis and senescence in injured mouse tendons with TGF-beta activated mTOR signaling. To further elucidate this pathology, the goal of this proposal is to engineer a microfluidic human tendon-on-chip (hToC) system and use it to more accurately model the biological aspects of the inflammation and fibrosis in injured tendons. In the UG3 phase of this proposal, the chip will be fabricated featuring a multicompartmental design and microfluidic channels to incorporate a fibroblast-seeded collagen hydrogel and simulate vascular blood flow, respectively. Ultrathin, highly permeable, and optically transparent porous silicon membranes (SiM) will separate the hydrogel from circulation and provide a substrate for an endothelial barrier in between. The signaling between the fibroblasts, hydrogel-resident- and circulating-macrophages, and endothelial cells will be enabled through nanoporous SiM (~60 nm), while a microporous SiM (~ 8 µm) will allow extravasation of circulating macrophages and infiltration of the hydrogel under TGF-beta stimulation. To allow for a patient-centric chip, tendon fibroblasts will be used to create the tendon hydrogel and to reprogram donor-matching iPSCs to derive the endothelial cells and macrophages, respectively. An additional innovation will be the integration of label- free photonic sensors into the microfluidic device to allow on-chip sensing, which has been long appreciated as a critical, unmet need for organ-on-chip devices. The UG3 studies will use the chip to validate the role of mTOR in the disease model and identify biologically relevant biomarkers. In the UH3 phase, we will utilize the chip as a pre-clinical trial platform for testing efficacy and safety of FDA-approved mTOR inhibitors as potential disease modifying drugs, and as a drug screening platform to identify and prioritize safer and more potent inhibitors of mTOR and senescence in tendon injury for clinical trials. To successfully complete this innovative project, we have assembled a team of accomplished experts in tendon tissue engineering and surgery, immunology, iPSC technology, GMP cell manufacturing, nano- and micro-fabrication, sensor technology, and high throughput screening. The proposed studies will develop a human microphysiological system to catalyze clinical trials and accelerate drug discovery for acute and chronic tendon injuries.