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Decellularization of cartilage enables the use of cartilage allografts or xenografts as natural scaffolds for repair and regeneration of injured cartilage. The preservation of the extracellular matrix ultrastructure of the graft makes this a promising tool for cartilage tissue engineering. We have optimized the decellularization protocol by enzymatically digesting proteoglycans while preserving the native collagen architecture. Here we describe our methods for cartilage decellularization and cell labeling for the tracking of infiltration for recellularization in detail.

The intricate architecture of the extracellular matrix (ECM) may play an important role in providing the mechanical strength necessary to cushion and lubricate the joints. Therefore, one approach to cartilage tissue engineering is to decellularize cartilage so as to preserve the complex collagen network while removing immunogenic chondrocytes and use the remaining structure as a scaffold. In this study, porcine articular cartilage plugs were subjected to freeze-thaw cycles and detergent cycles to decellularize the ECM. Three variations of this protocol were tested, with chondroitinase ABC (chABC) added at a different time point in each protocol. ChABC was added to degrade glycosaminoglycans (GAGs) that may block cell exit, infiltration, or both. Decellularized samples were seeded with synoviocytes then cultured for 72 h. Later chABC treatment generally resulted in more GAG removal without affecting the DNA or collagen content significantly. H&E staining showed some successful chondrocyte removal from lacunae across all groups, but cellular remains were still present in the ECM.

Articular cartilage has a limited capacity to heal itself and thus focal defects often result in the development of osteoarthritis. Current cartilage tissue engineering strategies seek to regenerate injured tissue by creating scaffolds that aim to mimic the unique structure and composition of native articular cartilage. Decellularization is a novel strategy that aims to preserve the bioactive factors and 3D biophysical environment of the native extracellular matrix while removing potentially immunogenic factors. The purpose of this study was to develop a procedure that can enable decellularization and recellularization of intact articular cartilage matrix. Full-thickness porcine articular cartilage plugs were decellularized with a series of freeze-thaw cycles and 0.1% (w/v) sodium dodecyl sulfate detergent cycles. Chondroitinase ABC (ChABC) was applied before the detergent cycles to digest glycosaminoglycans in order to enhance donor chondrocyte removal and seeded cell migration. Porcine synovium-derived mesenchymal stem cells were seeded onto the decellularized cartilage scaffolds and cultured for up to 28 days. The optimized decellularization protocol removed 94% of native DNA per sample wet weight, while collagen content and alignment were preserved. Glycosaminoglycan depletion prior to the detergent cycles increased removal of nuclear material. Seeded cells infiltrated up to 100 μm into the cartilage deep zone after 28 days in culture. ChABC treatment enhances decellularization of the relatively dense, impermeable articular cartilage by reducing glycosaminoglycan content. ChABC treatment did not appear to affect cell migration during recellularization under static, in vitro culture, highlighting the need for more dynamic seeding methods.