## 3 Results  
### Spatial Distribution and Characterization of the Neo-Synthesized Matrix  
We first evaluated the distribution of the neo-synthetized cartilage ECM produced by HACs in fibrin hydrogels. Following a 21 days culture period in the presence of BIT, static and perfused constructs were stained with fast green and counterstained with safranin-O to detect the presence of glycosaminoglycans (GAGs) of sulfated proteoglycans (Figure 2A). The presence of GAGs was detected in the outer periphery of the static gels, whereas sections of perfused gels showed a greater distribution of GAGs through the construct, as confirmed with the isolation of safranin-O positive area with ImageJ software(Figure 2B). Quantification of the safranin-O positive area in the histological sections (Figure 2C) showed a significant difference between static and perfused gels where GAG positive area covered 3.6 \(\pm\) 5.5% and 58 \(\pm\) 17% (median \(\pm\) SD) of the section respectively. High magnification of the periphery and core of the hydrogels confirmed GAG deposition only at the very periphery of the static gels, the intensity of the safranin-O staining rapidly decreasing towards the core where no GAG deposition was observed (Figure 2D). An intense and homogeneous safranin-O staining was found at the periphery of perfused gels whereas the core exhibited a lower staining intensity associated with strong ectopic staining. Thus, our observations clearly showed that perfusion resulted in superior accumulation and homogenous distribution of GAGs compared to static conditions. Similar observations were made for type II collagen, although mainly located around the cells with a limited diffusion in the extracellular space. Of note, intense ectopic accumulations of type II collagen were found in the core of the perfused gels. In contrast, immunostaining for type I collagen appeared similarly weak in static and perfused gels. Figure 2: Perfusion enhances cartilage matrix deposition by HACs in fibrin hydrogel. HACs seeded in fibrin hydrogel were cultured in a well plate (static) or a direct perfusion bioreactor in the presence of BIT for 3 weeks. (**A**): Static and perfusion cultures stained for safranin-O fast green to reveal the presence of glycosaminoglycans (GAGs) of sulfated proteoglycans. (**B**): Isolation of safranin-O positive area (orange to red) within the histological sample with the color threshold plugin of ImageJ software. (**C**): Safranin-O positive surface area normalized to the total surface area of the histological section. Each single data point in the dot plot is the mean value of several histological slices; bars represent the median of the data points. (**D**): Representative images of safranin-O fast green staining (GAGs) and collagen types I and II immunohistochemical staining of the engineered cartilage constructs. The level of significance of the statistical analysis (\(p\)-value) is indicated (scale bars: A = 1 mm, D = 100 μm) (\(n\) = 4). In parallel cultures, we analyzed by Western blotting the proteins newly synthesized by the chondrocytes encapsulated within hydrogels. Type II and type IX collagen were detected in static and perfusion gels but perfusion significantly stimulated their synthesis (\(p<0.05\)) (Figure 3A,B), with a relative fold change of 4 and 2.7, respectively. Type I collagen was produced to equivalent levels in the two culture conditions (\(p>0.1\), relative fold change of 1.3) (Figure 3A,B). Consequently, the type II/type I collagen ratio measured by densitometric analysis of the Western blots and taken as an index of chondrocyte differentiation was significantly higher in perfused gels than in static gels (\(p<0.05\)) (Figure 3C). Of note, type II collagen was present as mature forms but also abundantly present as unprocessed pro-forms in all conditions, indicating that cells were still actively producing type II collagen after 21 days of culture. In parallel, the presence of type I collagen was restricted as mature forms, but not as total unprocessed chains (Figure 3A).