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conditions in the absence of a scaffold. This indicates that decellularized b EC scaffolds are non- cytotoxic and suitable for cell seeding. To evaluate survival of human BMSCs in contact with decellularized b EC scaffolds, a LIVE/DEAD® assay was performed after 21 days of culture. Living BMSCs emitted a bright green fluorescence and showed a stretched morphology. Evaluation of z-stacks indicated that the seeded BMSCs were present on the surface of the scaffold. Unseeded, decellularized b EC scaffolds served as a control and no sign of living cells was observed in these controls. Histological sections showed that after 21 days, BMSCs were attached to the decellularized scaffolds, yet no migration into the scaffolds was observed. (Figure 3A) Gene-expression analysis of GAPDH, B2M and HPRT1 , confirmed the presence of BMSCs on decellularized b EC scaffolds after cell-seeding. In decellularized, non-seeded control scaffolds, the expression of either housekeeping gene was non-detectable (CT-values >40). The chondrogenic potential of BMSCs in pellet culture was confirmed by the expression of the chondrogenic-specific genes SOX9, COL2A1 and ACAN, while low expression presented after culturing in monolayer (negative control). Gene expression levels after seeding and culturing on decellularized b EC scaffolds, were similar to pellet culture. This shows that decellularized b EC scaffolds support the retention of the chondrogenic capacity of human BMSCs in vitro . (Figure 3B) Figure 3. Decellularized cartilage supports chondrogenesis. (A) H&E stain of decellularized ear cartilage scaffolds seeded with BMSCs shows attachment but limited migration (left) . Fluorescent imaging (right) confirms viability after 21 days of culture. Images are representative for all samples. Control image consists of an unseeded, decellularized b EC scaffold. (B) Relative expression of the chondrogenic markers SRY (sex determining region Y)-box 9 ( SOX9) , Collagen type II ( COL2A1) and aggrecan ( ACAN) are similar to that of BMSCs in pellet culture (positive control). Dotted line represents the relative expression after culturing in monolayer (negative control). Data shown as mean ± SD for 1 donor in 6-fold, relative to the best housekeeper index (BHKi) determined by the expression of Glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) , Beta-2-Microglobulin ( B2M ) and hypoxanthine phosphoribosyltransferase 1 ( HPRT1 ) . 140 CHAPTER 7