Multiphoton-based Fabrication of 3D ECM Scaffolds via Modulated Raster Scanning

Visar Ajeti,1 Ping-Jung Su,1 Jayne Squirrell,1 Brenda M. Ogle,1, 2 and Paul J. Campagnola1*

1Department of Biomedical Engineering, University of Wisconsin-Madison, 1550 Engineering

Dr., Madison WI, 53706, USA

2Department of Biomedical Engineering, University of Minnesota-Twin Cities, 312 Church Street

SE, Minneapolis, MN 55439, USA

Background. Extracellular matrices change dramatically with cardiac tissue development and maintenance. The oft-made conclusion is that these changes reflect passive accommodations to support differentiating cell types, including cardiomyocytes, smooth muscle cells and endothelial cells. We posit that, in addition or instead, changes of ECM actively direct cardiac differentiation. Approach. To test a portion of this hypothesis, that ECM distribution contributes to cardiomyocyte specification, we present here the development of a multiphoton-based fabrication technology to print 3D matrices based on tissue-specific ECM patterns and composed entirely of whole ECM proteins, with high spatial resolution. The method is analogous to multi-photon laser scanning microscopy (MPLSM) in that the excitation, and thus the photochemistry, is restricted to the focal volume. To this end, we implemented a new form of instrument control, termed modulated raster scanning, where rapid laser shuttering (40 MHz) is used to directly map the greyscale image data to the resulting protein concentration in the fabricated scaffold. Results. To assess the fidelity of the modulated raster scanning approach, individual optical sections of murine cardiac tissue immunostained for type IV collagen were compared to structures fabricated according to this template. Images of the immunostained tissue were overlain with images of fabricated structures and pixel by pixel co-localization tests using Fiji (ImageJ) yielded 95% or greater fidelity of both spatial localization and intensity. These results are substantially improved over more commonly used STL-based approaches that, in our hands, realize a fidelity of only 75% or greater. We used this approach to fabricate

3D scaffolds from a mixture of BSA and FN where the design was derived directly from a 3D confocal immunofluorescence (FN) image stack of the left ventricle of mouse at postnatal day 2. The image stack, (taken near and around a blood vessel), was comprised of 22 optical sections, taken 1 micron apart (Figure 1). Conclusion. 3D, ECM-based structures fabricated using this approach preserved most of the microarchitecture of the original images stack with the resulting relative protein concentration corresponding to relative intensities of the original pattern. In
addition, we have seeded induced pluripotent stem cells and cardiac progenitor cells (Irx4+

cells) into the scaffolds and show that cells survive within and are capable of remodeling structures fabricated in this way. Future Work. Future studies will discern whether and to which extent ECM distribution, especially as it relates to the distribution of ECM with development, directs cardiac differentiation. Further, this approach could be utilized to study cell-ECM interactions of other tissue and organ systems and will likely influence the way we culture cells outside the body and

Figure 1. Submicron 3D printing using MPE from myocardial tissue blueprint. Scale bar = 50 µm

our approaches to transplanting them back to the body.