In-situ mechanical tests in the microscope
The combination of mechanical characterization such as tensile testing and membrane inflation with microscopy generates what we call in-situ mechanical testing. This technique allows acquiring data on both the macroscopically acting loads (forces and displacements) at the boundary of a tissue sample and the corresponding changes of microstructure within the tissue. For example, deformation mechanisms in soft biological tissues often depend on the density and configuration of collagen fibers. The obtained information is therefore key in order to unravel the micrometer scale mechanisms that are responsible for the tissue scale mechanical characteristics. We further combine this information with microscopical anatomical information, and use this data to develop and validate physically based constitutive equations and computational models of the microstructure.
We introduced the in-situ testing technique in our lab about one decade ago to study the deformation and fracture behavior of fetal membranes [1-3], and to this end designed custom devices for uniaxial testing and membrane inflation in liquid environments within the sample chamber of a Fluoview 1000 MPE Olympus multi-photon microscope at the Center for Microscopy at the University of Zurich.
By now we have optimized the preparation and staining protocols, and developed a series of different devices to realize desired loading conditions and applied this technique to different materials and tissues, including the liver capsule [4,5], skeletal muscle [6] and skin [7]. We are constantly elaborating these methods and, together with collaborators, we have transferred the in-situ testing concept to other microscopy techniques and imaging methods such as ultrasound [8, 9] scanning electron microscopy [10] and magnetic resonance imaging [11] to test materials or under conditions not suitable for fluorescence imaging.
A special type of in-situ testing is used to analyze the behavior of cells on deformable substrates. For example, we have developed protocols and custom computational tools to determine the traction forces exerted by cells on the substrate through their focal adhesions [12-14], and we have analyzed the reorganization of endothelial cells layers upon mechanical loading [15,16].
[1] Mauri A., Perrini M., Mateos J.M., Maake, C., Ochsenbein-Kölble, N., Ehrbar, M., Mazza, E. Second harmonic generation microscopy of fetal membranes under deformation: Normal and altered morphology. (2013) Placenta 34, 1020-1026. DOI: 10.1016/j.placenta.2013.09.002
[2] Mauri, A., Ehret, A. E., Perrini, M., Maake, C., Ochsenbein-Kölble, N., Ehrbar, M., Oyen M.L., Mazza, E. (2015) Deformation mechanisms of human amnion: quantitative studies based on second harmonic generation microscopy. J. Biomech. 48, 1606-1613. DOI: 10.1016/j.jbiomech.2015.01.045
[3] Mauri A., Perrini M., Ehret A.E., De Focatiis D.S.A., Mazza E. (2015) Time-dependent mechanical behavior of human amnion: macroscopic and microscopic characterization. Acta Biomater. 11, 314-323. DOI: 10.1016/j.actbio.2014.09.012
[4] Bircher K., Ehret A.E., Mazza E. (2017) Microstructure based prediction of the deformation behavior of soft collagenous membranes. Soft Matter 13, 5107-5116. DOI: 10.1039/c7sm00101k
[5] Ehret A.E., Bircher K., Stracuzzi A., Marina V., Zündel M., Mazza E. (2017) Inverse poroelasticity as a fundamental mechanism in biomechanics and mechanobiology. Nat. Commun. 8, 1002. DOI: 10.1038/s41467-017-00801-3
[6] Kuravi R. Investigating the role of meso-scale structure on the mechanical response of skeletal muscle tissues. ETH Diss. Nr. 27212 (2021).
[7] Pensalfini M. Multiscale Investigations of Skin Biomechanics and Mechanobiology. ETH Diss. Nr. 25837 (2019).
[8] Weickenmeier J., Wu, R. Lecomte-Grosbras P., Witz, J.F., Brieu M., Winklhofer S., Andreisek G., Mazza E. (2014) Experimental Characterization and Simulation of Layer Interaction in Facial Soft Tissues. In: Bello F., Cotin S. (eds) Biomedical Simulation. ISBMS 2014. Lect. Notes Comp. Sci. 8789. DOI: 10.1007/978-3-319-12057-7_27
[9] Weickenmeier J., Investigation of the mechanical behavior of facial soft tissues. ETH Diss Nr. 22522 (2015).
[10] Domaschke S., Morel A., Kaufmann R., Hofmann J., Rossi R.M., Mazza E., Fortunato G., Ehret A.E. (2020) Predicting the macroscopic response of electrospun membranes based on microstructure and single fibre properties. J. Mech. Behav. Biomed. Mater. 104, 103634. DOI: 10.1016/j.jmbbm.2020.103634
[11] Sachs D., Wahlsten, A., Kozerke S., Restivo G., Mazza E. (2021) A biphasic multilayer computational model of human skin. Bioemch. Model. Mechanobiol. 20, 969-982. DOI: 10.1007/s10237-021-01424-w
[12] Bergert, M., Lendenmann, T., Zündel, M., Ehret, A. E., Panozzo, D., Richner, P., Kim D.K., Kress S.J.P., Norris D.J., Sorkine-Hornung O, Mazza E, Poulikakos D Ferrari, A. (2016) Confocal reference free traction force microscopy. Nat. Commun. 7, 12814. DOI: 10.1038/ncomms12814
[13] Zündel, M., Ehret, A. E., & Mazza, E. (2017) Factors influencing the determination of cell traction forces. PLoS One 12, e0172927. DOI: 10.1371/journal.pone.0172927
[14] Reyes Lua, A.M. Factors influencing the analysis of cell-substrate interaction. ETH Diss. Nr. 26609 (2020).
[15] Bernardi L., Giampietro C., Marina V., Genta M., Mazza E., Ferrari A. (2018) Adaptive reorientation of endothelial collectives in response to strain. Integ Biol, 10, 527-538. DOI: 10.1039/c8ib00092a
[16] Bachmann B.J., Bernardi L., Loosli C., Marschewski J., Perrini M., Ehrbar M., Ermanni P., Poulikakos D., Ferrari A., Mazza, E. (2016) A novel bioreactor system for the assessment of endothelialization on deformable surfaces. Sci. Rep. 6, 38861. DOI: 10.1038/srep38861