The goal of this study is to measure the glass transition temperature of a biological tissue as a whole and to analyze the effect of trehalose and other sugars on the measurement of the glass transition temperature. The effect of trehalose on isolated proteins was studied by differential scanning calorimetry from the viewpoint of glass transition temperature (Mazzobre and Buera 1999; Attanasio et al. 2007; Hedoux et al. 2009). Those studies demonstrated that trehalose raised the melting temperature of lyophilized proteins but showed ambiguous results regarding the glass transition temperature (Mazzobre and Buera 1999; Attanasio et al. 2007; Hedoux et al. 2009). To the best of our knowledge, there has been no preceding report to measure the glass transition temperature of a biological tissue as a whole. We chose porcine lens as a model biological tissue to measure the glass transition temperature since the lens is an isolated small tissue in the eyeball and can be easily removed from the other surrounding tissues.
As a first step to understand the mechanism underlying the protection of a biological tissue by trehalose, we measured the melting temperature of the dried lens tissue under the presence or the absence of trehalose or cyclic tetrasaccharide in our previous study (Sun et al. 2011). That study was unique in that the melting temperature of a biological tissue as a whole, even in the dried condition, was measured by differential scanning calorimetry (Sun et al. 2011). In that study, the presence of trehalose, but not cyclic tetrasaccharide, led to an elevation of the melting temperature of the dried lens tissue (Sun et al. 2011). As an ideal condition of experiments, the melting temperature should be measured in the natural state of a biological tissue with water. However, the presence of water leads to the evaporation of water in the process of rising temperature, and hence, the evaporation hinders the measurement of the melting temperature in an accurate manner.
In the present study, we tried to measure the glass transition temperature of a biological tissue as a whole. The glass transition temperature is more difficult to measure than the melting temperature because of the subtlety of the glass transition. To overcome this limitation of the subtlety in measuring the glass transition temperature, the amount of the dried lens tissue, placed in a platform pan for the measurement, was increased about twice, compared with the amount used for the measurement of the melting temperature in the previous study (Sun et al. 2011). We could successfully measure the glass transition temperature of the dried lens tissue under this condition for the first time.
We chose maltose as a control ingredient for the pretreatment before drying of the lens tissue since maltose is another disaccharide with different bonding sites between two glucose molecules, compared with trehalose. We also chose cyclic tetrasaccharide as the pretreatment because we used this substance in the measurement of melting temperature of the dried lens tissue in the previous study (Sun et al. 2011). The pretreatment with trehalose did not lead to significant changes in the glass transition temperature, compared with saline pretreatment or pretreatment with maltose or cyclic tetrasaccharide. No significant change in the glass transition temperature of the dried lens tissue with trehalose pretreatment in the present study is in marked contrast with the elevation in the melting temperature by trehalose pretreatment in the previous study (Sun et al. 2011).
The concentration of trehalose and other sugars was set at 100 mM, based on the preceding studies to show the protective effect on cells in culture (Matsuo 2001) and also on the isolated lens tissues (Matsuo 2005). We also tested 10-times higher concentration of trehalose and other sugars, set at 1000 mM, as the pretreatment to expect better detection of the glass transition temperature. Trehalose at 1000 mM concentration was also used in thermal analysis of isolated proteins (Attanasio et al. 2007). The present study showed no difference between the different concentrations of the pretreatment with the sugars. As far as the results in this study were concerned, we could not prove the hypothesis that the stabilizing effect of trehalose on a biological tissue under the dried condition be underlain by changes in the glass transition temperature of the tissue.
The reason why the pretreatment with trehalose did not change the glass transition temperature of the dried lens tissue remains unknown at present. Based on our previous study to determine the melting temperature, the ratio of glass transition temperature over melting temperature (Tg/Tm) of the dried lens tissue falls in the range between 0.5 and 0.75 of the ratio of Tg/Tm of most polymers, as a rule of thumb in materials science. In our previous study, the melting temperature of the dried lens tissue was elevated by about 10°C with trehalose pretreatment (Sun et al. 2011). The elevation of melting temperature, combined with no change in glass transition temperature of the dried lens tissue with trehalose pretreatment in this study, indicates that the tissue would stay in the glassy state for a wider range of temperature under the presence of trehalose. These facts might explain the protective effect of trehalose on the tissues or cells in desiccation. A rise in melting temperature by trehalose for lyophilized proteins in a preceding study also supports this line of reasoning (Attanasio et al. 2007).
One possible limitation in this study would be that the residual water content in each dried tissue might show trivial difference among the samples even under the circumstances that the lens tissues were dried thoroughly in a desiccator until no weight change. The larger standard deviation for glass transition temperature of saline or cyclic tetrasaccharide-pretreated samples was noted in contrast with the smaller standard deviation for glass transition temperature of trehalose or maltose-pretreated samples. The large variation of the glass transition temperature, measured in the condition of saline or cyclic tetrasaccharide pretreatment, might be attributed to the difference in residual water content among the dried tissue samples. Preceding studies on thermal analysis of isolated proteins also mentioned that trehalose would keep moisture in lyophilized proteins and might influence the measurement of glass transition temperature (Mazzobre and Buera 1999; Hedoux et al. 2009).