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In AFM experiments (Veeco Multimode AFM), the AV explants were adhered to a microscope slide and submerged in culture media. A cantilever (?0.12?N/m spring constant) with borosilicate spherical tip (5?��m diameter) was used to indent tissue samples. The cantilever was likely contacting cells (not ECM) with such as small tip; however, the ECM proteins that surround the cell impact the overall cell stiffness as well (Asparuhova et al., 2009). The tip was indented approximately 1?��m into the tissue at a rate of 1?��m/s. The cantilever deflection was measured and converted to force using the tip's associated Hook's spring constant (Vandiver et al., 2005). Data analysis was performed for each sample to determine tissue stiffness. First, the Young's modulus was calculated YES1 from the force�Cdistance curves using the Hertz Model (Darling et al., Icotinib purchase 2006?and?Hemmer et al., 2008). The modulus, which characterizes the stiffness of a linearly elastic material, should be lower for softer tissues. As with most biological tissues, nanoindentation of the AV explants showed a viscoelastic mechanical responses; however, the modulus values calculated from this simple Hertzian analysis is a useful first approximation of tissue nanoindentation (Darling et al., 2006) and can be used to broadly compare differences in tissue stiffness between AV cushion samples. Qualitative analysis of representative force�Cdistance curves was also performed to investigate changes in stiffness. For this analysis, a representative AFM curve was isolated from each of the data sets and the curves were then compared. The sharper the incline on the AFM curve is indicative of greater tissue stiffness (e.g., the higher the slope the stiffer the tissue). All data were expressed as Mean +/? SEM. Statistical see more comparisons were performed using ANOVA or independent t-test by SPSS 11.0 (SPSS Inc., Chicago, IL). P values