Effect of postmortem changes and freezing on the viscoelastic properties of vocal fold tissues

It is common practice in laryngeal research laboratories to store excised larynges and vocal fold tissue specimens frozen, presumably to preserve viability and structural integrity of the specimens for physiologic and biomechanical experiments. However, little is known about the biomechanical effects of postmortem changes and frozen storage in vocal fold tissues. This study attempted to quantify the effects of postmortem changes and freezing on the viscoelastic shear properties of the canine vocal fold mucosa. Sixteen larynges were excised from adult dogs immediately postmortem in a viable state. Using a torsional rheometer, the complex shear modulus (G*) of the mucosal tissue from one vocal fold of each larynx was measured as a function of frequency (0.01-15 Hz) by linear small-amplitude oscillation experiments. Measurement was repeated for ten mucosal specimens after 24 h of postmortem storage in saline solution at room temperature. Eleven of the 16 larynges were frozen and stored at -20 degrees C, six of them at a slow rate of cooling (storage in a regular freezer) and five others by quick freezing (chilling by liquid nitrogen prior to frozen storage). The larynges were thawed slowly overnight after one month and the viscoelastic shear properties of the contralateral vocal fold mucosa were measured. Results showed that the elastic shear modulus (G') and dynamic viscosity (eta') of the vocal fold mucosa did not seem to change significantly after 24 h of storage in saline at room temperature, nor after one month of frozen storage following quick freezing, whereas both G' and eta' decreased significantly for tissues that were slowly frozen. These findings supported the feasibility of using quick freezing to preserve laryngeal tissues for in vitro biomechanical testing, for excised larynx experiments, and for tissue engineering applications.