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ENgineering DYnamic ViscoElasticity to study cell response

Periodic Reporting for period 1 - ENDYVE (ENgineering DYnamic ViscoElasticity to study cell response)

Reporting period: 2016-11-01 to 2018-06-30

The biomechanical properties of the extracellular matrix (ECM) play critical roles in directing pathophysiological cell behaviour via mechano-transduction. Although biological tissues generally exhibit a viscoelastic behaviour that changes over time during development, ageing and disease (here named dynamic viscoelasticity), the majority of mechanobiology studies have focused only on static (i.e. time-invariant) mechanical properties, typically by characterizing tissue elasticity and investigating cell behaviour as a function of substrate stiffness.
With the ultimate goal of contributing to a better understanding of pathophysiological cell mechano-transduction mechanisms, the ENDYVE project was aimed at engineering the dynamic viscoelasticity typical of tissue pathophysiological processes in-vivo to start exploring its role in the modulation of stem cell behaviour. This can have a number of societal and clinical implications, such as developing new strategies to control stem cell behaviour for obtaining mature differentiated cells for drug screening in-vitro, or limiting, if not preventing, tissue fibrosis and tumour progression in-vivo. Focusing on cardiac development and pathophysiology, the project goals were achieved through a series of multi-disciplinary activities divided into 4 work packages (WPs), outlined in the next section.
Complementary project-related studies were also carried out to:
-Develop of a new analytical method for estimating lumped parameter constants of linear viscoelastic models from strain rate tests;
-Compare frequency and strain-rate domain mechanical characterization;
-Analyse intimal, medial and adventitial stiffness in human aneurysmal abdominal aortas.
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.

-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.

-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.

-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.

-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.

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.

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
The achievements of the ENDYVE project have a significant impact on the scientific community, going beyond the state of the art. In particular, this project i) introduced the new topic of engineering dynamic viscoelasticity, ii) provided new insights into the micro-mechanical viscoelastic properties sensed by cells in developing, ageing and infarcted myocardium, iii) proposed a simple-yet-effective modular approach to design smart cell culture substrates with temporally tuneable viscoelastic properties (that can be adopted also for engineering the dynamic viscoelasticity of other biological tissues than the myocardium), and iv) showed that hPSC-CM contraction dynamics are not dependent on and do not respond to substrate viscoelasticity within the myocardial foetal-to-infarcted pathophysiologically-relevant range, nor slightly above the latter. The results obtained contribute to opening a new branch of mechanobiology research towards a better understating of cell-ECM interactions and the underlying biophysics of mechano-transduction.
Beyond the impact on the Researcher’s career, in the long term the findings and outcomes of the project can positively impact on the European biomedical, pharmaceutical and cell therapy research and business sectors, with numerous potential societal and clinical implications, possibly leading to a better control of pathophysiological cell behaviour and the establishment of new therapeutic strategies to improve human lives.