Microfluidic Formation of Ultrathin, Handleable Collagen Sheets Exhibiting Toe-heel Tensile Behavior

The extracellular matrix (ECM) of cardiovascular tissues displays a non-linear, strain-dependent elastic modulus, attributed to the hierarchical organization of collagen. At low loads, these tissues exhibit compliance, permitting contraction or dilation, while at high loads, they stiffen considerably, increasing their mechanical strength by at least tenfold. Although collagen gels are widely used in 3D cell culture, tissue engineering, and biofabrication, current engineering techniques fail to replicate this hierarchical organization at the microscale. As a result, they lack both the non-linear tensile behavior and the physiologically relevant strength of native tissues. To address this limitation, we present templated collagen sheets that are 1.8 microns thin and 10 mm wide that demonstrate non-linear tensile behavior. Collagen sheets are obtained from an acidic collagen solution via a microfluidic flow focusing process, incorporating and subsequently removing emulsified oil droplets (mean diameters 2.1 microns and 5.0 microns, volume concentration 2.25%). Templated collagen sheets exhibit a two-fold increase in fibril alignment dispersion compared with non-templated ones. When assessed along their length, the Young’s modulus of templated sheets increases 62-fold at 90% failure strain, closely matching the properties of native load-bearing tissues. We anticipate that these ultrathin templated collagen sheets will have broad applications as a substrate material for the bottom-up fabrication of load-bearing biomaterials and tissue structures for in vitro applications and implantation.