Fabrication of 2D protein microstructures and 3D polymer–protein hybrid microstructures by two-photon polymerization

Abstract

Two-photon polymerization (TPP) offers the possibility of creating artificial cell scaffolds composed of micro- and nanostructures with spatial resolutions of less than 1 µm. For use in tissue engineering, the identification of a TPP-processable polymer that provides biocompatibility, biofunctionality and appropriate mechanical properties is a difficult task. ECM proteins such as collagen or fibronectin, which could mimic native tissues best, often lack the mechanical stability. Hence, by generating polymer-protein hybrid structures, the beneficial properties of proteins can be combined with the advantageous characteristics of polymers, such as sufficient mechanical stability. This study describes three steps toward facilitated application of TPP for biomaterial generation. (1) The efficiency of a low-cost ps-laser source is compared to a fs-laser source by testing several materials. A novel photoinitiator for polymerization with a ps-laser source is synthesized and proved to enable increased fabrication throughput. (2) The fabrication of 3D-microstructures with both systems and the fabrication of polymer-protein hybrid structures are demonstrated. (3) The tissue engineering capabilities of TPP are demonstrated by creating cross-linked gelatin microstructures, which clearly forced porcine chondrocytes to adapt their cell morphology.

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

“…There are two established means of obtaining protein-polymer hybrid structures that combine the mechanical strength and chemical inertness of the polymer with the functionality of the protein. One approach is to conjugate the protein with acrylate, allowing the polymer chemistry to govern the cross-linking process 44 . This procedure has been successful employed with high molecular weight cellular matrix proteins such as collagen.…”

“…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.…”

“…Consequently, the resulting protein structures have been applied to fabricate functional microdevices, including pH-sensitive actuators 39,40 and optical microcomponents 41,42 . The integration of proteinaceous microstructures into commercial polymers 43 and protein-polymer hybrids 44 has also been found to increase mechanical strength.…”

“…As well, swollen structures are even less mechanically robust than those in standard proteinaceous structures, which restricts potential mechanical applications. The mechanical strength could be increased by using polymer-protein hybrid structures 43 or higher cross-linking densities 44 .…”

Development of Multi-Dimensional 3D Printed Vascular Networks for Self-Healing Materials

“…One example of an advantage this technology can provide to the self-healing community is the ability to create networks that are designed to ensure minimum resistance to flow (such as according to Murray's Law [25]), enabling higher degrees of healing efficiency to be achieved with a reduced time to infusion of more viscous healing agents. The use of extrusion and stereolithographic AM techniques to fabricate 3D vascular networks has previously been reported within tissue engineering studies [26][27][28][29][30][31][32][33][34]. However, the application of these technologies to create self-healing vasculature has not yet been extensively explored.…”