Multiphoton Excited Fabrication of Collagen Matrixes Cross-Linked by a Modified Benzophenone Dimer:  Bioactivity and Enzymatic Degradation

Abstract

Multiphoton excited (MPE) photochemistry is used to fabricate model tissue engineering scaffolds directly from types I, II, and IV collagen. A modified benzophenone dimer (BPD) provides the photoactivation and becomes incorporated into the resulting collagen matrixes. Unlike xanthene photochemistries, the benzophenone dimer can be used in acidic environments, where most forms of collagen have the greatest solubility. The minimum feature sizes are investigated by using two- and three-photon excitation, where the latter provides for superior "resolution" and suggests that collagen structures can be fabricated on the size scales of focal contacts. The resulting structures display excellent retention of bioactivity as evidenced by highly specific cell adhesion as well as immunofluorescence labeling. Structural and chemical aspects of the collagen matrixes are probed through measuring the enzymatic degradation through specific and nonspecific proteases, as the resulting relative rates are consistent with the activity of these enzymes. The degradation rates can also be controlled through varying the cross-link density in the matrixes, which is achieved through tuning the exposure dose during the fabrication process. The degradation rates are also found to be consistent with swelling/shrinking measurements and thus the average mesh size of the matrixes. In all cases the enzymatic degradations are well-fit single exponentials, suggesting that the matrixes can be fabricated with a priori knowledge of their structural properties. These results coupled with the resulting bioactivity suggest that the multiphoton fabrication process may be a powerful tool for the creation of cell-sized tissue engineering scaffolds.

Spatial and Temporal Analysis of Extracellular Matrix Proteins in the Developing Murine Heart: A Blueprint for Regeneration

“…While the example here used a synthetic polymer, we have previously used this method to crosslink many ECM proteins, with excellent biocompatibility. [56][57][58] The images and quantitative analyses generated within this work will better inform efforts in cardiac tissue engineering and regenerative medicine.…”

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

“…The use of effective photoinitiators could improve the fabrication process and also expand the range of applicable proteins. As an example, a benzophenone derivative (BPD) was used to cross-link small amounts of various types of collagen 52 , while the water-soluble compound sodium 4-[2-(4-morpholino)benzoyl-2-dimethylamino]butylben zenesulfonate (MBS) was utilized to cross-link enhanced green fluorescent protein (EGFP) 37 . A three-component system comprising a protein, rhodamine B and flavin adenine dinucleotide (FAD) significantly improved both fabrication speed and feature sizes 66 .…”

“…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.…”

Two-photon polymerization of hydrogels – versatile solutions to fabricate well-defined 3D structures

“…Consequently, they reported a newly synthesized benzophenone dimer that could act as a photoinitiator for both BSA and collagen cross-linking. 73,74 However, cytotoxicity was not tested for the fabricated structures. In Campagnola's group, other proteins, such as brinogen, bronectin and concanavalin A, were also processed using Rose Bengal as the photoinitiator.…”