Micropatterned supramolecular hydrogels promote cell alignment for myocardium regeneration

S. F. Gomes^1,2, A. M. Brito^1,2, D. Caballero^1,2, D. S. Costa^1,2, V. Brancato^1,2, S. C. Kundu^1,2, A. M. Martins^1,2, I. Pashkuleva^1,2, R. L. Reis^1,2,3, R. A. Pires^1,2,3

¹ 3B's Research Group, I3Bs – Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Parque de Ciência e Tecnologia, Zona Industrial da Gandra, 4805-017 Barco, Guimarães, Portugal;

² ICVS/3B’s – PT Government Associate Laboratory, Braga/Guimarães, Portugal;

³ The Discoveries Centre for Regenerative and Precision Medicine, Headquarters at University of Minho, Avepark, 4805-017 Barco, Guimarães, Portugal

Cardiovascular diseases are the worldwide leading cause of death. They are responsible for over 3.9 million deaths/year in Europe (45% of all deaths) and 35.000 deaths/year in Portugal (30% of all deaths). No available methodologies stimulate the autonomous regeneration of the myocardium and restore its normal function. We propose to develop a micropatterned electroactive self-assembled nanofibrous patches as an integrated conductive and biomimetic interface, which will be able to guide the cell alignment through a micropatterned surface and to promote their concerted electrical coupling and simulating the heartbeats.

To mimic the biofunctionality of the myocardium extracellular matrix, we test the use of supramolecular hydrogel composed of a network of self-assembled nanofibers of fmoc-diphenylalanine (fmoc-FF). We select this amphiphilic short peptide due to its action as a morphological mimic of the proteins present in the ECM [1,2] and its similarity to FF, which is reported to present semi-conductive character [3,4] - an important characteristic to promote the electrical coupling of the cardiomyocytes [5]. We fabricate the supramolecular hydrogels using an already established protocol [1,6]. In brief, we dilute the solution of fmoc-FF (in DMSO) with ultrapure water with final fmoc-FF concentrations of 5 mM; 10 mM and 20 mM and final DMSO volume proportion of 2.5%, 5.0% and 10%, respectively. The nanofibrous structure of the developed hydrogel is confirmed by atomic force microscopy (AFM). Rheology testing is carried out to determine the elastic modulus of the different compositions, resulting in an increment of modulus with the augmentation of the fmoc-FF concentration: 5 mM (≈1.5 kPa); 10 mM (≈6.5 kPa); and 20 mM (≈22.5 kPa). In addition, the cytocompatibility of the hydrogels is confirmed by culturing L929 cells onto their surface; the results show that all the tested compositions are not toxic. A higher cell density is observed for the stiffer hydrogels. We also carried out the micropatterning of these hydrogels with grooves of 1x1x1 μm (height, width, pitch) using replica moulding technique. We demonstrate that these microstructures promote cell alignment, supporting its further evaluation targeting myocardium regeneration.

References:

1. Mahler, A., et al. Advanced Materials 2006, 18 (11), 1365-1370.

2. Jayawarna, V., et al. Advanced Materials 2006, 18 (5), 611-614.

3. Kholkin, A., et al. Acs Nano 2010, 4 (2), 610-614.

4. Tao, K., et al. Science 2017, 358 (6365).

5. Martins, A. M., et al. Biomacromolecules 2014, 15 (2), 635-643.

6. Raeburn, J., et al. Soft Matter 2012, 8 (4), 1168-1174.

Acknowledgements: