Multiphoton fabrication is a powerful technique for three-dimensional (3D) printing of structures at the microscale. Many polymers and proteins have been successfully structured and patterned using this method. Type I collagen comprises a large part of the extracellular matrix for most tissue types and is a widely used cellular scaffold material for tissue engineering. Current methods for creating collagen tissue scaffolds do not allow control of local geometry on a cellular scale. This means the environment experienced by cells may be made up of the native material but unrelated to native cellular-scale structure. In this study, we present a novel method to allow multiphoton crosslinking of type I collagen with flavin mononucleotide photosensitizer. The method detailed allows full 3D printing of crosslinked structures made from unmodified type I collagen and uses only demonstrated biocompatible materials. Resolution of 1 μm for both standing lines and high-aspect ratio gaps between structures is demonstrated and complex 3D structures are fabricated. This study demonstrates a means for 3D printing with one of the most widely used tissue scaffold materials. High-resolution, 3D control of the fabrication of collagen scaffolds will facilitate higher fidelity recreation of the native extracellular environment for engineered tissues.
Photopolymerization methods such as multiphoton polymerization have been used successfully to create bioactive patterned scaffolds with micron-scale resolution capable of supporting cell growth and differentiation for engineered tissue. [1] They have also been shown effective for fabrication of a variety of MEMS devices. [2] Currently, multiphoton polymerization and similar technologies require a bulky and expensive optical system based on a femto- or picosecond pulsed laser and an XYZ arrangement of high-resolution translating stages. [3] Such systems are currently prohibitive in both cost and effort required to assemble, calibrate, and maintain. Consolidating optical components and motors into a smaller, less-complex device may facilitate the manufacture of customized tissue engineered constructs and MEMS devices on-site in more remote locations on an as-needed basis.
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