Effect of three-dimensional expansion and cell seeding density on the cartilage-forming capacity of human articular chondrocytes in type II collagen sponges

Chondrocytes for tissue engineering strategies are typically expanded in monolayer (2D), leading to cell dedifferentiation but allowing to generate large cell numbers for seeding into scaffolds. Direct chondrocyte culture in scaffolds, instead, may support better maintenance of the differentiated phenotype but reduce the extent of proliferation and thus the resulting cell density. This study investigates whether the quality of cartilaginous tissues generated in vitro by human articular chondrocytes (HAC) on type II collagen sponges is enhanced (1) by direct expansion on the scaffolds (3D), as compared with standard 2D, or (2) by increasing cell seeding density, which in turn requires extensive 2D expansion. Three-dimensional expansion of HAC on the scaffolds, as compared with 2D expansion for the same number of doublings, better maintained the chondrocytic phenotype of the expanded cells (13.7-fold higher levels of type II collagen mRNA) but did not enhance their accumulation of glycosaminoglycan (GAG) following chondrogenic culture. Instead, increasing the HAC seeding density in the scaffolds (from 25 × 10(3) to 66 × 10(3) cells/mm(3)) significantly improved chondrogenesis (up to 3.3-fold higher GAG accumulation and up to 9.3-fold higher type II collagen mRNA), even if seeded cells had to be expanded and dedifferentiated more extensively in 2D to reach the required cell numbers. This study indicates that, under the specific conditions tested, a high-seeding density of HAC in 3D scaffolds is more critical for the generation of cartilaginous constructs than the stage of cell differentiation reached following expansion.