Multiphoton crosslinking for biocompatible 3D printing of type I collagen

Abstract

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.

Fabrication of three-dimensional proteinaceous micro- and nano-structures by femtosecond laser cross-linking

“…Reports of cell culture scaffolds have focused on utilizing proteins to promote cell adhesion, with the specific protein dependent on the cell type, and including collagen type I, type II, type IV, fibronectin and BSA mixtures with such proteins 43,44,48,50,52,59,66,67 . Figures 7a and 7b show two recent examples of 3D cell culture scaffolds composed of at least two different materials that control cells spatially via adhesion.…”

Guided Homing of Cells in Multi‐Photon Microfabricated Bioscaffolds

“…[11] Of existing methods, multi-photon photolithography (MPP) holds the greatest promise for generating arbitrarily shaped 3D microchannels and patterns of cues down to the single-micron resolutions exhibited by the smallest capillaries (Figure 1a,i). MPP has been used to microfabricate a variety of biomaterials, including collagen, [12,13] bovine serum albumin (BSA), [14] poly(ethylene-glycol) diacrylate (PEGDA), [15,16] fibrin, [17] and poly(lactic acid). [18] MPP has also been used to pattern chemical cues inside bulk scaffolds (Figure 1a,ii).…”

Emerging Trends in Information‐Driven Engineering of Complex Biological Systems

“…[205] Through coordinated control, physical properties such as density and structure can be tuned to investigate cell behavior. [210] A critical outstanding question for all of these materials is whether they retain any of the biologically relevant protein configurations following the multiphoton crosslinking process. [157,207] Similarly, synthetic polymers and naturally derived proteins are being explored for their potential in 2PP.…”