###### Abstract  
Articular cartilage has limited potential for self-repair, and cell-based strategies combining scaffolds and chondrocytes are currently used to treat cartilage injuries. However, achieving a satisfying level of cell redifferentiation following expansion remains challenging. Hydrogels and perfusion bioreactors are known to exert beneficial cues on chondrocytes; however, the effect of a combined approach on the quality of cartilage matrix deposited by cells is not fully understood. Here, we combined soluble factors (BMP-2, Insulin, and Triiodothyonine, that is, BIT), fibrin hydrogel, direct perfusion and human articular chondrocytes (HACs) to engineer large cartilage tissues. Following cell expansion, cells were embedded in fibrin gels and cultivated under either static or perfusion conditions. The nature of the matrix synthesized was assessed by Western blotting and immunohistochemistry. The stability of cartilage grafts and integration with native tissue were also investigated by subcutaneous implantation of human osteochondral cylinders in nude mice. Perfusion preconditioning improved matrix quality and spatial distribution. Specifically, perfusion preconditioning resulted in a matrix rich in type II collagen but not in type I collagen, indicating the reconstruction of hyaline cartilage. Remarkably, the production of type VI collagen, the main component of the pericellular matrix, was also increased, indicating that chondrocytes were connecting to the hyaline matrix they produced.  
chordrocyte; cartilage tissue engineering; perfusion bioreactor; fibrin; type VI collagen +
Footnote †: journal: Medical  
excellent candidates for this due to their ability to encapsulate the cells, along with a high intrinsic water content (60-90%) [10], a feature found in native cartilage [11] which offers the tissue its \"shock absorber\" capabilities.  
Nutrient availability to cells throughout three-dimensional (3D) scaffolds occurs in vitro by diffusion, and therefore depends on the distance from the surface, diffusional constraints, and cellular utilization at the periphery [12]. These diffusional limitations become exacerbated when attempting to engineer larger cartilage tissues. Perfusion bioreactors have been shown to enhance cell access to oxygen and nutrients as well as the homogeneity of neo-synthesized ECM in 3D scaffolds [13; 14]; however, understanding of its impact on the quality of the ECM deposited by chondrocytes encapsulated in hydrogels remains limited [15; 16; 17; 18; 19].  
Following this view, this work aimed to evaluate whether direct perfusion preconditioning could enhance cartilage matrix deposition by human articular chondrocytes (HACs) in fibrin hydrogel, a clinically relevant material that has been shown to support and maintain chondrocyte redifferentiation [20; 21]. We used cocktails of specific factors to expand the cells on plastic (fibroblast growth factor (FGF)-2 and insulin, designated FI) and to allow their redifferentiation in fibrin hydrogel (bone morphogenetic protein (BMP)-2, insulin, and triiodothyronine T3, designated BIT). This sequential addition of factors has been proven efficient to amplify human chondrocytes isolated from diverse anatomical sites (including articular cartilage) and to induce their redifferentiation in collagen [22], agarose [23], self-assembling peptide [24; 25] gels and collagen sponges [26; 27]. Special attention was given to the quality and spatial distribution of the neosynthesized matrix through analysis of the pericellular matrix (PCM) components.