This study demonstrates that tissues retain significantly more water after acetone dehydration than previously assumed, challenging the long-held belief that acetone dehydration sufficient to reduce tissue water content to below 1% is necessary. Our initial aim was to set a stage for finding ways to better optimize the efficiency of acetone dehydration for plastination. Our evaporation experiments suggested that nearly all acetone evaporated from what we presumed was fully dehydrated tissue after 80 minutes on the benchtop at room temperature. In 7-day acetone stabilization experiments, we observed that tissue samples appeared to become fully dehydrated in only 1-2 days when using an overwhelming acetone-to-tissue ratio (5000:1). However, dehydration experiments revealed that despite the apparent stabilization, tissues surprisingly still retained between 10-20% water.
Our findings after 7-day stabilization differ from previous literature (Henry RW, 2007;von Hagens G,Tiedemann K and Kriz W, 1987). These differences could be due to the homogeneity of the tissue samples used in our experiments. Unlike whole-body or limb plastination, where different tissue types may dehydrate at different rates, our samples consisted of more uniform tissue types, such as myocardium, which may have dehydrated more quickly. Furthermore, the high acetone-to-tissue ratio (5000:1) likely accelerated dehydration compared to the 10:1 ratio used in other studies, contributing to the faster dehydration times observed in our study. Despite the expected faster dehydration times, the significant water retention observed in stabilized 99% acetone was unexpected. This retention may be linked to the extracellular matrix (ECM) of the tissue, which plays a key role in maintaining tissue structure and volume. The hydrophilic components of the ECM, such as proteoglycans and glycosaminoglycans, are known to attract and hold water, forming a hydrogel that can trap water molecules during the dehydration process (Frantz C et al., 2010). Different tissues and organs have varying ECM compositions, which could explain the differences in water retention between organ types and species. Additionally, the ECM composition can vary within different compartments of the same tissue, further complicating acetone dehydration efficiency (Hu M et al., 2022).
Several studies support the notion that proteins, particularly myofibrillar proteins like myosin and actin, may also contribute to water retention in tissue. These proteins, which form fibrous filaments in muscle and organ tissues, create small gaps (~20 nm) that sequester water molecules, making it difficult for complete water diffusion and dehydration (Miyanishi T et al., 2002). Research on lysozymes has also shown that proteins can promote local water sequestering via hydrogen-bond reorganization, creating hydration shells around the protein (Shi R, 2022). Such protein-water interactions may play a role in the water retention observed in our study. Even lyophilized (freeze-dried) proteins like lysozymes have been shown to retain water, suggesting that water binding to proteins persists even under extreme dehydration conditions (Phan-Xuan T et al., 2020). Further support for the biochemical role of water retention comes from thermodynamic studies on protein hydration networks. Water is essential for maintaining protein structure, and removing water could destabilize the protein. Molecular dynamic simulations have shown that ordered water molecules, which bridge proteins and ligands, are critical for binding stability (Rudling A et al., 2018).While our study did not focus on protein-ligand interactions, the presence of ordered water within proteins could explain why tissue retains more water than previously realized after the acetone dehydration. The thermodynamic cost of completely removing water from proteins may be too high, causing the tissue to retain water to preserve protein stability (Bellissent-Funel MC et al., 2016). To ensure that formalin fixation did not influence water retention, we performed additional experiments on fresh cow and horse heart tissues. The results of our study confirmed that water retention levels remained above 1% after reaching acetone bath stabilization at >99%, supporting our original findings and ruling out formalin as a confounding factor. These results suggest either that some levels of such excess water are removed during the freeze- or room temperature-substitution steps, or that the remaining water is sequestered in ways that do not inhibit subsequent monomer polymerizations.
Limitations should be noted in this preliminary study. If it is not possible to fully dehydrate tissue due to proteins retaining molecules of water even after freeze-drying, then our freeze dryer-based determination of tissue true dry mass may also not be fully precise. The sample size was relatively small, and scaling these findings to larger specimens, such as large body parts or whole bodies, may require different acetone-to-tissue ratios. Although we attempted to ensure sample homogeneity, variations in tissue composition, such as excess latex in veins or arteries, could have impacted results. Despite these limitations, our findings offer new insights into the acetone dehydration process and tissue equilibrium. The results suggest that water content remains above 1% in tissues post-dehydration, which challenges the traditional understanding of plastination. The finding of 10-20% water content in tissues removed from stabilized 99% acetone was surprising, and may suggest opportunity for successful plastination of specimens after less intense acetone dehydration. One of the present authors (SH) was the originator of the freeze dryer plastination (Holladay SD, 1988b) and has felt the freeze dryer likely continued some water sublimating activity during forced impregnation. In like manner, very similar traditional forced impregnation under vacuum and cold temperatures may also remove limited amounts of water molecules from specimens. Should or how this may vary under newer room temperature impregnation procedures remains unknown, and could influence amount of remaining water that would be tolerated by the different impregnation techniques. Optimizing the dehydration process based on this new understanding could improve the efficiency of plastination, as dehydration is a major portion of the overall process, thereby reducing the time, resources, and expenses required. This would be particularly beneficial in educational settings, where faster plastination could enable the production of more anatomical models, reducing reliance on dissection cadavers that are becoming increasingly difficult to obtain.
