WORK PERFORMED
WP1: software/hardware adaptation of a commercial nano-indenter (PIUMA, Optics11, NL) and development of a new control mode to characterize micro-mechanical viscoelastic properties of soft tissues and (bio)materials at typical cell length-scales, with no pre-stress and in physiological-like conditions via the nano-epsilon dot method (a strain-rate domain testing method, previously proposed by the Researcher);
WP2: characterization of pathophysiological myocardial dynamic viscoelasticity by performing nano-epsilon dot measurements on left ventricle samples harvested from healthy mice at different stages of development (embryonic day E15 and E18.5) and ageing (month 3, 4, 10, 11, 20), and from adult (3 months old) myocardial infarcted (MI) mice;
WP3: design, development and characterization of gelatin-based smart biomaterials with temporally tuneable mechanical properties that recapitulate pathophysiological myocardial dynamic viscoelasticity to be used as substrates for cell experiments in-vitro. A two-step crosslinking approach was investigated: first a chemical crosslinking to obtain hydrogels mimicking the desired initial myocardial viscoelastic properties (e.g. foetal, adult), then a second biocompatible enzymatic crosslinking to alter substrate viscoelasticity on-demand during cell culture towards e.g. infarcted one;
WP4: investigation of human pluripotent stem cell-derived cardiomyocyte (hPSC-CM) contraction dynamics on gelatin substrates mimicking pathophysiological myocardial viscoelasticity.
MAIN RESULTS
WP1:
-New PIUMA control mode enabled constant indentation (or loading) rate measurements, required for the nano-epsilon dot viscoelastic characterisation;
-Actual sample temperature (T) can be controlled in real-time via a new sample T sensor and a master-slave control loop. Typically, the physiological T (37 °C) was reached within a few minutes, with neglectable overshoot and good steady-state stability over time;
-New scripts to improve the user-independency and the automation of the nano-epsilon dot analyses for deriving material viscoelastic parameters.
WP2:
-Myocardial tissue not only stiffened during development, ageing (up to adult stage, i.e. month 3) and after infarction, but there was also a concomitant change in its viscoelastic behaviour towards a more elastic one. Surprisingly, tissue softening was observed with senescent ageing from month 4 to 20.
WP3:
-After the first step of crosslinking, hydrogels were stiffer and more elastic with increasing chemical crosslinker concentration;
-The second-step enzymatic crosslinking made all chemical-crosslinked samples stiffer and more elastic. Control samples (not exposed to enzyme) were fairly stable over time. Good candidates to mimic foetal-to-infarcted and adult-to-infarcted myocardial viscoelastic transitions were identified.
WP4:
-No significant differences in temporal contraction parameters (namely the time to reach the contraction peak from the baseline, TTP; the relaxation time from contraction peak to the baseline, RT; and the contraction duration, defined as TTP+RT) were observed with changes in substrates viscoelastic properties within the myocardial foetal-to-infarcted pathophysiologically-relevant range, nor slightly above that range.
EXPLOITATION
Results obtained in WP1 about the new PIUMA Nanoindenter control mode have been commercially exploited by Optics11, while those about the new sample T control are currently in the pipeline to be commercially exploited by the company. No commercial exploitation nor patentability was foreseen for the other results obtained.
DISSEMINATION
The Researcher disseminated the ENDYVE results uniformly during the project through a number of activities, including i) 2 co-authored scientific papers published in peer-reviewed journals (plus 1 currently under review and 2 in preparation), ii) participation to 7 international conferences/meetings delivering presentations (plus 1 scheduled in July 2018) and to regular meetings at both host institutions, iii) 1 invited lesson at the University Libre de Bruxelles (ULB), iv) students training and supervision activities and v) outreach activities to reach and engage also the general non-academic public outside the scientific community.
