15502-m-pleumeekers

RESULTS Decellularization of bovine ear cartilage Bovine cartilage samples were decellularized according to the protocol of Kheir, et al. , [101] that was further optimized to specifically decellularize b EC by the addition of a treatment with a low concentration elastase solution. Bovine articular cartilage ( b AC) samples were taken as controls, since AC decellularization has been performed by Kheir, et al . (Supplementary figure 2) After decellularization, b EC scaffolds and bAC control scaffolds retained their cartilage-like appearance, although samples seemed more translucent after the decellularization process. After decellularization, wet weight reduced by 26.1±4.9% in b EC and an 8.4±2.6% wet weight reduction was measured in bAC. The thickness of decellularized b EC scaffolds was significantly reduced ( p <0.001) by 23.5% (1.37 ± 0.32 mm) when compared to untreated b EC scaffolds (1.72 ± 0.40 mm), while no obvious reduction in sample diameter was observed. (Figure 1A). To assess decellularization efficiency, cell content was analyzed histologically (H&E stain) and biochemically. DNA content was significantly reduced ( p <0.001) and undetectable (<10 ng/sample) after decellularization, compared to untreated b EC. Similar results were obtained in the decellularized b AC control scaffolds; DNA was significantly reduced ( p <0.001) and undetectable after decellularization compared to untreated b AC. Histological analysis showed that the cell remnants were diminished after decellularization and those that were still present were clearly reduced in size and weakly stained for H&E. The ECM itself was weakly stained compared to the untreated scaffolds, although the overall structure of the ECM was virtually intact. (Figure 1B) SEM analysis showed no obvious changes in the extracellular matrix after decellularization compared to untreated cartilage. The decellularized b AC and b EC scaffolds retained their dense matrix consistent of fine, intact collagen fibers similar to that of untreated cartilage. In untreated b EC, the thick elastic fibers were deeply embedded and intertwined within a homogeneous collagen network and this 3D organization was retained after decellularization. (Figure 1C) Scaffold characterization To characterize the matrix properties of the decellularized b EC scaffolds, the sGAG, total collagen and elastin contents were measured biochemically in addition to histological evaluation. The sGAG content of decellularized b EC scaffolds significantly reduced to 3% ( p <0.001) compared to untreated bEC , which was confirmed by histological analysis when stained for Safranin-O. The total collagen content of untreated b EC did not reduce after decellularization, but significantly increased ( p =0.011). This phenomenon appears to be due to the normalization of the collagen content to the sample wet weight, since wet weight was reduced after decellularization while the collagen content most likely did not. As sGAG content was strongly reduced by the decellularization procedure, the relative contribution of collagens to the overall wet weight increased, resulting in the observed increase in collagen content. Furthermore, immunohistochemical analysis confirmed the retention of collagen type II after decellularization. As for the retention of elastin, no statistical difference was 137 DECELLULARIZED CARTILAGE: AN ECM-DERIVED SCAFFOLD 7