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Since many soft tissues function in an isotonic in-vivo environment, it is expected that physiological osmolarity will be maintained when conducting experiments on these tissues ex-vivo. In this study, we aimed to examine how not adhering to such a practice may alter the mechanical response of the tricuspid valve (TV) anterior leaflet. Tissue specimens were immersed in deionized (DI) water prior to quantification of the stress–strain responses using an in-plane biaxial mechanical testing device. Following a two-hour immersion in DI water, the tissue thickness increased an average of 107.3% in the DI water group compared to only 6.8% in the control group, in which the tissue samples were submerged in an isotonic phosphate buffered saline solution for the same period of time. Tissue strains evaluated at 85 kPa revealed a significant reduction in the radial direction, from 34.8% to 20%, following immersion in DI water. However, no significant change was observed in the control group. Our study demonstrated the impact of a hypo-osmotic environment on the mechanical response of TV anterior leaflet. The imbalance in ions leads to water absorption in the valvular tissue that can alter its mechanical response. As such, in ex-vivo experiments for which the native mechanical response of the valves is important, using an isotonic buffer solution is essential.

Elastin is present in the extracellular matrix (ECM) of connective tissues, and its mechanical properties are well documented. In Marfan syndrome, however, the inability to properly code for the protein fibrillin-1 prematurely leads to the degradation and loss of elastin fiber integrity in the ECM. In this study, the role of elastin in the ECM of the anterior leaflet of the tricuspid valve was investigated by examining the biomechanical behavior of porcine leaflets before and after the application of the enzyme elastase. Five loading protocols were applied to the leaflet specimens in two groups (elastase-treated and control samples). The mechanical response following elastase application yielded a significantly stiffer material in both the radial and circumferential directions. At a physiological level of stress (85 kPa), the elastase group had an average strain of 26.21% and 6.32% in the radial and circumferential directions, respectively, at baseline prior to elastase application. Following elastase treatment, the average strain was 5.28% and 0.97% in the radial and circumferential directions, respectively. No statistically significant change was found in the control group following sham treatment with phosphate-buffered saline (PBS). Two-photon microscopy images confirmed that after the removal of elastin, the collagen fibers displayed a loss of undulation. With a significant reduction in radial compliance, the ability to withstand physiological loads may be compromised. As such, an extracellular matrix that is structurally deficient in elastin may hinder normal tricuspid valve function.