Osteoarthritis (OA), or degenerative joint disease, is the most prevalent of all musculoskeletal diseases and is characterised by the progressive destruction of articular cartilage. As mature articular cartilage has very limited regenerative potential, treatment options for OA are limited and ultimately the disease leads to total joint arthroplasty. Mesenchymal stem cells (MSCs) and gene therapy offer new possibilities for the treatment of OA by contributing to the regeneration or maintenance of connective tissues. The aim of this study will be to evaluate various strategies to promote repair within fibrillated OA cartilage, such as MSCs transfected, using non-viral delivery approaches, with microRNA (miR)-455 known to be highly expressed within normal cartilage, using a human OA cartilage explant culture model to advance tissue repair.
Human MSCs, obtained commercially from healthy donors, will be labelled fluorescently with Cell Tracker Red and DAPI, transfected with miR-455 using the non-viral gene delivery glycosaminoglycan enhanced transduction (GET) cell penetrating peptide from collaborator, Prof James Dixon in the University of Nottingham, and added to explants at a cell density of 4×106cells/ml at 37ºC with agitation. Unattached cells will be removed and explants will be either processed immediately or treated with chondrogenic media containing TGF-β3 (CCM) or ICM for up to 14 days. Cartilage disks incubated without cells will be used as controls. Formalin-fixed, paraffin-embedded sections will be stained with haematoxylin and eosin (H&E), Toluidine blue, immunohistochemistry for Coll II and and Coll X, or processed for fluorescence microscopy. Known targets of miR-455, such as hypoxia-inducible factor-2α (HIF-2α), will also be investigated. Bound cells will be quantified using FIJI software.
In conclusion, this system of explant culture, cell attachment and gene delivery will allow evaluation of the ability of targeted cells to repair the cartilage surface.