## 4 Discussion
Our WB analyses showed that type II collagen, a typical marker of native cartilage [39], was produced both in static and in dynamic conditions. Undoubtedly, the combination of BIT and perfusion did enhance the deposition of type II collagen; it also enhanced that of type IX collagen, these being two main components of the collagen fibrils present in hyaline type of cartilage [40]. It is well-known that the amplification of HACs induces dedifferentiation, resulting in a progressive increase in type I collagen at the expense of type II collagen expression [41]. This \"side effect\" of HAC expansion is exacerbated with the FI cocktail despites its proliferative effect on HACs [27]. Thus, the chondrocytes were active in type I collagen production at the start of their culture in fibrine gel. However, no sign of precursor forms of type I collagen was detected, probably as a result of BIT treatment [24]. This stop in type I collagen production is particularly relevant since it indicates that the combination of fibrin and BIT favors the production of hyaline type cartilage as articular cartilage and not fibrocartilage (which has inferior mechanical properties). In a previous work, we showed that perfusion inhibited type I collagen production from HACs in collagen sponges [26]. Here, we observed such an effect only for one donor of four, type I collagen production being mainly equivalent between static and dynamic conditions. However, our differentiation index (type II collagen/type I collagen ratio) is in favor of a higher production of type II collagen and a differentiated phenotype in dynamic cultures. This is well supported by our histological and immunohistochemical analyses showing the abundant presence of GAGs, type II collagen, aggrecan core protein, and type VI collagen, whereas type I collagen was barely detected in dynamic cultures. The particular interest in type VI collagen in the current study arises from its unique structure and location. In articular cartilage, type VI collagen is almost exclusively located in the PCM. Its amino acid structure contains sequences that are active in binding to cellular receptors [42], and other sequences that are involved in the creation of multimeric fibrillar assemblies with collagen type II and aggrecan [43], which connect the chondrocytes to the macromolecular framework of the ECM. Such intimate interactions between the PCM components and the cell surface are likely involved in mechanotransduction to the cell surface; engineered constructs with non-native PCM structure could exhibit alteredmetabolic responses to physicochemical signals, affecting the ultimate success of such approaches. As a consequence, the presence of type VI collagen in our tissue-engineered constructs is an important indicator of advanced tissue maturation and demonstrates that cells emancipate themself from the biomaterial to create intimate contacts with the matrix they produce. The absence of type VI collagen in static cultures also indicates that perfusion was the main determinant of the type VI collagen secretion observed in our study. These results together demonstrate that the combination of perfusion and BIT substantially improved the reconstruction of hyaline cartilage-specific ECM and PCM in fibrin hydrogels. Perfusion can enhance the transport of molecules through large gels [15], but it also exerts mechanical forces such as fluid shear stress [44] and the expression of several ECM proteins, including type II and IX collagen and aggrecan, as well as transcription factors like Sox9, is sensitive to the mechanical forces in cartilage [45]. We also observed a statistically significant increase in the expression of type II collagen, type IX collagen, aggrecan, and Sox9 with direct perfusion (Figure S2). Thus, we can hypothesize that the fluid flow perfused here may have mechanically stimulated the chondrocytes seeded within fibrin gels.