Fig. 3

Graphic display of decellularization process. Decellularization treatment consisted of freeze-thaw treatment, osmotic treatment and multi-cyclic detergent-enzymatic treatment. To identify necessary cumulative incubation time for cell removal while preserving ECM-components, detergent-enzymatic treatment was performed in cycles, each consisting of trypsin-, Triton X-100- and DNase-treatment. Detergent-enzymatic decellularization cycles were continued for 5 cycles. Analytics showed complete cell removal in histology and significant DNA reduction while preserving sGAG content after three detergent-enzymatic decellularization cycles. SGAG wash out was observed starting at four or more detergent-enzymatic decellularization cycles, therefore standard decellularization treatment (SDT) was defined as freeze-thaw-treatment, osmotic treatment and three consecutive detergent-enzymatic decellularization cycles (cycles 4 and 5 are crossed out in figure). All decellularized specimens were sterilized in peracetic-acid-ethanol-solution prior to recellularization