Cartilage graft engineering by co-culturing primary human articular chondrocytes with human bone marrow stromal cells

Co-culture of mesenchymal stromal cells (MSCs) with articular chondrocytes (ACs) has been reported to improve the efficiency of utilization of a small number of ACs for the engineering of implantable cartilaginous tissues. However, the use of cells of animal origin and the generation of small-scale micromass tissues limit the clinical relevance of previous studies. Here we investigated the in vitro and in vivo chondrogenic capacities of scaffold-based constructs generated by combining primary human ACs with human bone marrow MSCs (BM-MSCs). The two cell types were cultured in collagen sponges (2 x 6 mm disks) at the BM-MSCs: ACs ratios: 100: 0, 95: 5, 75: 25 and 0: 100 for 3 weeks. Scaffolds freshly seeded or further precultured in vitro for 2 weeks were also implanted subcutaneously in nude mice and harvested after 8 or 6 weeks, respectively. Static co-culture of ACs (25%) with BM-MSCs (75%) in scaffolds resulted in up to 1.4-fold higher glycosaminoglycan (GAG) content than what would be expected based on the relative percentages of the different cell types. In vivo GAG induction was drastically enhanced by the in vitro preculture and maximal at the ratio 95: 5 (3.8-fold higher). Immunostaining analyses revealed enhanced accumulation of type II collagen and reduced accumulation of type X collagen with increasing ACs percentage. Constructs generated in the perfusion bioreactor system were homogeneously cellularized. In summary, human cartilage grafts were successfully generated, culturing BM-MSCs with a relatively low fraction of non-expanded ACs in porous scaffolds. The proposed co-culture strategy is directly relevant towards a single-stage surgical procedure for cartilage repair. Copyright (C) 2012 John Wiley & Sons, Ltd.