Abstract
As in humans, osteoarthritis (OA) causes considerable economic loss to the equine industry. New hopes for cartilage repair have emerged with the matrix-associated autologous chondrocyte implantation (MACI). Nevertheless, its limitation is due to the dedifferentiation occurring during the chondrocyte amplification phase, leading to the loss of its capacity to produce a hyaline extracellular matrix (ECM). To enhance the MACI therapy efficiency, we have developed a strategy for chondrocyte redifferentiation, and demonstrated its feasibility in the equine model. Thus, to mimic the cartilage microenvironment, the equine dedifferentiated chondrocytes were cultured in type I/III collagen sponges for 7 days under hypoxia in the presence of BMP-2. In addition, chondrocytes were transfected by siRNA targeting Col1a1 and Htra1 mRNAs, which are overexpressed during dedifferentiation and OA. To investigate the quality of the neo-synthesized ECM, specific and atypical cartilage markers were evaluated by RT-qPCR and Western blot. Our results show that the combination of 3D hypoxia cell culture, BMP-2 (Bone morphogenetic protein-2), and RNA interference, increases the chondrocytes functional indexes (Col2a1/Col1a1, Acan/Col1a1), leading to an effective chondrocyte redifferentiation. These data represent a proof of concept for this process of application, in vitro, in the equine model, and will lead to the improvement of the MACI efficiency for cartilage tissue engineering therapy in preclinical/clinical trials, both in equine and human medicine.
Highlights
Cartilage defects caused by traumas, focal lesions, or articular overstress, are the leading cause of osteoarthritis (OA) development
Data were normalized to P0 cells in these experiments, and very weak amounts of Col1a1 mRNA were detected in primary cells (P0) in some cases
We supposed that enzymatic digestion for cell isolation induces a slight stress to chondrocytes, which express Col1a1 mRNA de novo
Summary
Cartilage defects caused by traumas, focal lesions, or articular overstress, are the leading cause of osteoarthritis (OA) development. This incurable pathology generates pain, walking-related disability, and high morbidity. The poor intrinsic cartilage self-repair is not able to prevent the ineluctable tissue degradation. Chondrocytes, the major cartilage cell type, synthesize an abundant extracellular matrix (ECM), mainly composed of two specific phenotypic markers (type II collagen and aggrecan). During OA, the primary chondrocyte response to the inflammatory microenvironment is a change in its normal secretome, leading to the decreased expression of cartilage specific markers and matrix metalloproteinases synthesis. Chondrocytes dedifferentiate and express the fibrotic type I collagen, leading to fibrocartilage of poor quality
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