Modulation of MicroRNAs to Engineer a Layered Osteochondral Tissue Construct

项目总结/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构建体。基于 在试验数据上，预期microRNA调节可用于指导软骨祖细胞的分化， 细胞转化为天然的骨软骨组织。本文描述的研究将定义生物学途径 在CPC软骨形成中受到过表达特定microRNA的影响。他们也会发展出一种文化 该系统同时将组织结构暴露于两种单独的介质。关节软骨缺损 治疗仅限于暂时性的、需要二次缺损形成（自体移植物）或具有 有限供应（同种异体移植物）。即使经过治疗，这些缺陷也会导致骨关节炎-骨关节炎的主要原因之一。 残疾人在美国和世界。软骨祖细胞（CPC）可以从大多数关节软骨中分离出来 组织，包括骨关节炎关节组织。CPC是多能细胞，可以分化为关节样细胞， 或肥大软骨，后者用作骨形成的模板。microRNAs（miRs）是21- 25个核苷酸表观遗传调节因子，已用于指导成骨CPC分化。具体目标 1，miR-介导的分化系统能够从CPC产生骨软骨组织， 开发先前的研究表明，慢病毒介导的miR-138过表达可用于维持 处于关节样软骨状态的CPC同时上调miR-181 a/B促进CPC分化为 肥大软骨和骨骼。CPC已经被接种到脱矿物质的人骨支架上， 3D骨组织。这些支架也已被用于使用骨髓衍生的胶原蛋白来开发3D软骨组织。 基质细胞本发明提供了一种促进CPC基软骨和骨在骨基质上的位点特异性生长的双培养系统， 将测试脱矿物质的人骨支架。将使用组织学表征所得结构 (von Kossa，番红-O）、µ CT（骨矿化）和机械测试（软骨聚集体模量， 水力渗透性、剪切强度）。将在CPC软骨形成中评估每种miR的生物学作用。 使用RNA-Seq和相关生物信息学分析（GO、KEGG、GSEA）的沉淀培养物。在具体目标2中， 将在体内和离体评价骨软骨构建体。结构将在组织学和机械学上 在植入免疫缺陷小鼠的背侧8周后进行体内测试。 同时，从Specific Aim 1中获得的双培养系统将用于保存人骨软骨组织， vivo.将在这些组织外植体中产生缺陷，并将骨软骨构建体植入骨软骨中。 缺陷将使用组织学和机械测试来评价缺损的修复。该项目的重点是 利用分子生物学技术生产生物组织工程构建体。学员将获得 在分子和细胞生物学、组织工程、生物力学测试、生物信息学方面的经验， 动物处理。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.