Modulation of MicroRNAs to Engineer a Layered Osteochondral Tissue Construct

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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.