Programa del congreso

1. Introduction

Extracellular matrix (ECM) mechanics play a critical role in directing cellular behavior. Considerable efforts focus on developing in vitro platforms that replicate the mechanical properties of biological tissues in health and disease (1). Natural hydrogels are promising as they resemble native environments but require mechanical tuning. This is particularly relevant for the myocardium, which sustains continuous loading. Yet their mechanical properties remain poorly characterized across scales, from the microscale sensed by cells to the macroscale of whole tissue, and especially under strain.

2. Objective

To characterize the multiscale mechanical properties of collagen hydrogels using atomic force microscopy (AFM) and rheometry with different crosslinking degrees and stretches applied.

3. Methodology

To measure the micromechanical stiffness with AFM, a device to stretch samples, compatible with simultaneous AFM measurements, was first designed and built (Figs. 1A,B). Collagen I was isolated from rat tails and diluted at 10 mg/mL. To produce photocrosslinkable hydrogels, 10 µL of Ru/SPS photocrosslinker (Advanced Biomatrix, #5248) was added to the collagen I mixture and then pipetted on top of the stretching device preparing gels of 50 µL volume and left it at 37 ºC for 30 min for thermal crosslinking. Afterwards, gels were illuminated with blue light (455 nm) for 0, 5 or 30 minutes. Then, the stretching device was placed on the AFM stage and the hydrogels were measured at strains up to 30% to extract the Young’s modulus. In parallel, myocardial ECM derived hydrogels from swine hearts were produced by following already described protocol (2). The produced hydrogels were further crosslinked with genipin (5 mM) and further tested with AFM at 0% stretch. Macromechanical properties of uncrosslinked hydrogels were assessed by rheometry using a HAAKE RheoStress 1 rheometer (ThermoFisher, MA) with 35 mm serrated parallel plates. 1 mL of hydrogel solution was loaded onto the rheometer Peltier plate (4 °C) with a plate gap of 200 µm. The storage modulus (G'), loss modulus (G''), and viscosity (η) were recorded at a constant frequency of 0.1 Hz and strain of 0.5%. The plate temperature was maintained at 4 °C for 15 min, then increased to 37 °C (to allow for thermal crosslinking of the hydrogels) and held constant until the end of the experiment.

4. Results and Discussion

The AFM-compatible stretching device (Figs. 1A,B), fabricated using 3D-printed polylactic acid, allowed successful micromechanical measurement of hydrogel Young’s modulus under stretch. Collagen I hydrogels exhibited a baseline stiffness of ~100 Pa (uncrosslinked, control), ~900 Pa (crosslinked, 5 min), and ~1200 Pa (crosslinked, 30 min) (Fig. 1C). The Young’s modulus displayed significant nonlinearity under strain, increasing ~3 to 5 fold at 30% strain (Fig. 1C), independently of the crosslinking degree. Uncrosslinked collagen hydrogels were softer than myocardial ECM hydrogels, but crosslinking increased collagen I stiffness beyond that of myocardial ECM (Fig. 1D). Finally, the rheometry results showed that thermally crosslinked myocardial ECM hydrogels are also stiffer than the collagen I hydrogels following the same tendency as the micromechanical data.

5. Conclusion

This study presents the first report of multiscale mechanical comparison between collagen I hydrogels and myocardial ECM hydrogels with different crosslinking degrees and stretches applied. The findings offer insights into developing novel in vitro platforms with tunable mechanical properties.

Figure 1. A) Schematics of the stretching device compatible with AFM. B) Photograph of the stretching device with collagen I hydrogel. C) Micromechanical properties of collagen I hydrogels under strain with different crosslinking degrees. **, * statistical significance compared to 0% stretch. $ statistical significance compared to different crosslinking degrees. D) Micromechanical properties of the collagen I and myocardial ECM hydrogels. E) Rheometry of the uncrosslinked collagen I and myocardial ECM hydrogels.

6. References

1. Jorba I, Gussenhoven S, van der Pol A, et al. Steering cell orientation through light-based spatiotemporal modulation of the mechanical environment. Biofabrication. 2024;16(3):10.1088/1758-5090/ad3aa6. Published 2024 Apr 16. doi:10.1088/1758-5090/ad3aa6.
2. Sanz-Fraile H, Herranz-Diez C, Ulldemolins A, et al. Characterization of Bioinks Prepared via Gelifying Extracellular Matrix from Decellularized Porcine Myocardia. Gels. 2023;9(9):745. Published 2023 Sep 13. doi:10.3390/gels9090745.