Modulation of MicroRNAs to Engineer a Layered Osteochondral Tissue Construct

项目摘要/摘要 该项目的目标是开发一种模仿天然骨软骨组织的骨软骨结构。基座 在先导数据上，可以预期microRNA调节可以用于指导软骨前体细胞的分化 细胞转化为天然的骨软骨组织。这里描述的研究将定义生物途径 在CPC软骨形成中受特定microRNAs过度表达的影响。他们还将发展一种文化 同时将组织结构暴露在两种不同介质中的系统。关节软骨缺损 治疗仅限于临时的、需要二次缺陷产生(自体移植)的解决方案，或 供应有限(同种异体移植)。即使接受治疗，这些缺陷也可能导致骨关节炎--骨关节炎的一个主要原因 美国和世界上的残疾问题。从大多数关节软骨中可以分离出软骨祖细胞。 组织，包括骨关节炎关节组织。CPC是一种多能细胞，可以分化为关节样细胞 或肥大的软骨，后者作为骨形成的模板。MicroRNAs(MiRs)为21- 25个核苷酸的表观遗传调节剂，已被用于指导成骨CPC的分化。以特定的目标 1，一种能够从CPC产生骨软骨组织的miR介导的分化系统将是 发展起来的。先前的研究表明，慢病毒介导的miR-138的过度表达可以用来维持 当上调miR-181a/b时，处于关节样软骨状态的CPC促进CPC分化为 肥大的软骨和骨骼。CPC已经被种植到脱钙的人骨支架上，以创造出 3D骨组织。这些支架还被用于开发3D软骨组织，使用的是骨髓来源的 基质细胞。一种双培养系统，促进基于CPC的软骨和骨在人工骨上的部位特异性生长 将对脱钙人骨支架进行测试。由此产生的结构将使用组织学进行表征 (von Kossa，Safranin-O)、µCT(骨矿化)和力学测试(软骨聚集体模数、 水力渗透性、剪切强度)。每个miR的生物学作用将在CPC软骨形成中进行评估 使用RNA-Seq和相关的生物信息学分析(GO、KEGG、GSEA)进行颗粒培养。在具体目标2中， 骨软骨结构将在体内和体外进行评估。从组织学和机械学的角度来看， 植入免疫缺陷小鼠背侧8周后进行体内检测。 同时，来自特定目标1的双向培养系统将用于保存人的骨软骨组织。 活着。将在这些组织外植体中产生缺陷，骨软骨结构将被植入 缺陷。组织学和机械测试将被用来评估修复的缺陷。这个项目的重点是 利用分子生物学技术生产生物组织工程构建物。实习生将获得 在分子和细胞生物学、组织工程、生物力学测试、生物信息学和 处理动物。位于圣路易斯的华盛顿大学肌肉骨骼研究中心提供专业知识， 设备，以及开发和表征这些骨软骨组织结构所需的合作。

PROJECT SUMMARY/ABSTRACT The goal of this project is to develop an osteochondral construct that mimics native osteochondral tissue. Based on pilot data, it is expected that microRNA modulation can be used to guide differentiation of cartilage progenitor cells into native-like osteochondral tissue. The studies described herein will define the biological pathways impacted by overexpressing specific microRNAs in CPC chondrogenesis. They will also develop a culture system that simultaneously exposes tissue constructs to two separate media. Articular cartilage defect treatments are restricted to solutions that are temporary, require secondary defect creation (autograft), or have limited supply (allograft). Even with treatment, these defects can lead to osteoarthritis – a leading cause of disability in the US and world. Cartilage progenitor cells (CPCs) can be isolated from most articular cartilage tissues including osteoarthritic joint tissues. CPCs are multi-potent cells that can differentiate into articular-like or hypertrophic cartilage, the latter of which serves as a template for bone formation. MicroRNAs (miRs) are 21- 25 nucleotide epigenetic regulators that have been used to guide osteogenic CPC differentiation. In Specific Aim 1, a miR-mediated differentiation system capable of producing osteochondral tissue from CPCs will be developed. Prior studies suggest that lentivirus-mediated overexpression of miR-138 can be used to maintain CPCs in an articular-like cartilage state while upregulating miR-181a/b promotes CPC differentiation into hypertrophic cartilage and bone. CPCs have been seeded onto demineralized human bone scaffolds to create 3D bone tissue. These scaffolds have also been used to develop 3D cartilage tissue using bone marrow-derived stromal cells. A bi-culture system that facilitates site-specific growth of CPC-based cartilage and bone on a demineralized human bone scaffold will be tested. The resulting construct will be characterized using histology (von Kossa, Safranin-O), µCT (bone mineralization), and mechanical testing (cartilage aggregate modulus, hydraulic permeability, shear strength). The biological role of each miR will be assessed in CPC chondrogenic pellet cultures using RNA-Seq and associated bioinformatic analyses (GO, KEGG, GSEA). In Specific Aim 2, osteochondral constructs will be evaluated in vivo and ex vivo. Constructs will be histologically and mechanically evaluated after being implanted into the dorsal flanks of immunodeficient mice for 8 weeks for in vivo testing. Meanwhile, the bi-culture system from Specific Aim 1 will be used to preserve human osteochondral tissue ex vivo. Defects will be created in these tissue explants and the osteochondral constructs will be implanted in the defects. Histology and mechanical testing will be used to evaluate repair of the defect. This project focuses on using molecular biology techniques to produce biological tissue engineering constructs. The trainee will gain experience in molecular and cellular biology, tissue engineering, biomechanical testing, bioinformatics, and animal handling. The Washington University in St. Louis Musculoskeletal Research Center provides expertise, equipment, and collaborations necessary to develop and characterize these osteochondral tissue constructs.