Measurement of layer-specific mechanical properties in multilayered biomaterials by micropipette aspiration

Many biomaterials and tissues are complex multilayered structures in which the individual layers have distinct mechanical properties that influence the mechanical behavior and define the local cellular microenvironment. Characterization of the mechanical properties of individual layers in intact tissues is technically challenging. Micropipette aspiration (MA) is a proven method for the analysis of local mechanical properties of soft single-layer biomaterials, but its applicability for multilayer structures has not been demonstrated. We sought to determine and validate MA experimental parameters that would permit measurement of the mechanical properties of only the top layer of an intact multilayer biomaterial or tissue. To do so, we performed parametric nonlinear finite-element (FE) analyses and validation experiments using a multilayer gelatin system. The parametric FE analyses demonstrated that measurement of the properties of only the top layer of a multilayer structure is sensitive to the ratio of the pipette inner diameter (D) to top layer thickness (t_top), and that accurate measurement of the top layer modulus requires D/t_top < 1. These predictions were confirmed experimentally by MA of the gelatin system. Using this approach and an inverse FE method, the mean effective modulus of the fibrosa layer of intact porcine aortic valve leaflets was determined to be greater than that of the ventricularis layer (P < 0.01), consistent with data obtained by tensile testing of dissected layers. This study provides practical guidelines for the use of MA to measure the mechanical properties of single layers in intact multilayer biomaterials and tissues.