INTRODUCTION

The plastination process is a widely adopted technique for creating durable anatomical models, involving key steps such as dissection, dehydration, polymerization, and final curing of polymeric chemicals (e.g., silicone; polyester; epoxy) impregnated into biological tissue. The dehydration step is critical as it removes water and lipids from the tissue, the latter of these being unable to undergo direct polymer exchange (von Hagens et al., 1987). Adequate dehydration is also essential to inhibit tissue decomposition, as water is a fundamental requirement for microbial growth (Sierra et al., 2017).

When plastination was first developed, it was believed that tissue was "completely dehydrated" following the dehydration sequence (von Hagens et al., 1987). Others soon suggested that the water content in tissue after acetone-based dehydration can acceptably stabilize at around 1%, evidenced by acetone concentrations in the final dehydration bath of >99% ( Bickley et al., 1987; Ravi and Bhat, 2011). This low water content is critical because residual water can compromise silicone or other monomer polymerization following monomer exchange for acetone under vacuum. Previous studies used >99% acetone/tissue equilibrium as an indirect measure of water content in dehydrated tissues. However, this assumption has not been empirically tested, prompting interest in verifying tissue water content following standard dehydration procedures. Freeze drying was considered a potential alternative to acetone dehydration (Holladay, 1988).

Longstanding protocols recommend that the volume of acetone used in dehydration baths be at least ten times the volume of the tissue (Bickley et al., 1987; Ravi and Bhat, 2011). Reports suggest that dehydration in such conditions will generally take 3-5 weeks, often requiring multiple acetone changes (von Hagens et al., 1987). Other studies using similar ratios also estimated dehydration times ranging from 3 weeks to over 4 weeks (Henry, 2007), with some variations in success (Satte et al., 2017). However, the timelines reported in the literature vary widely, from as short as 2 days to as long as 8 weeks (Alpar et al., 2005; Barnett et al., 2005; Miller et al., 2017) or even 3 months when plastinating larger specimens, such as limbs of horses (Yu et al., 2015). Some researchers have bypassed time-based protocols altogether, focusing solely on acetone concentration (Smodlaka et al., 2005).

Despite widespread use of plastination, the dehydration step remains inconsistently employed as plastinators seek to maximize use of acetone.  The purpose of this study was to test the hypothesis that tissue water content reaches an equilibrium between the tissue and the acetone bath solution. A 5000:1 acetone-to-tissue ratio was chosen to allow rapid and confident progression to maximal tissue dehydration. The objective was to quickly achieve a stable acetone bath concentration of the plastination target >99%, implying tissue water content of <1%. The 5000:1 bath ensured dehydration with little to no dilution of the acetone solution. To establish these parameters, we first measured the times required for acetone stabilization and then for evaporation from dehydrated tissue samples. Diverse tissue types, including myocardium, liver, and kidney from equine, canine, and bovine specimens, were subjected to acetone dehydration using the high acetone-to-tissue volume ratio. After dehydration and acetone evaporation, the samples were weighed to determine water loss, then freeze-dried to assess the final dry weight that might be achieved (0% water), providing accurate measurements of residual water content after acetone dehydration. Resulting data offer a more precise understanding of dehydration and may have utility for developing more standardized plastination protocols.

MATERIALS AND METHODS

Tissue sample

Tissue water content after acetone dehydration was evaluated using myocardium, kidney, and liver tissue from bovine, equine, and canine specimens. Tissues were collected from formalin-fixed dissection cadavers in the University of Georgia’s veterinary college anatomy lab. Similar-sized samples from hearts, livers, and kidneys were excised from organs removed from the cadavers. Myocardial samples were collected from the left ventricular wall of all species due to the thickness and homogeneity of this segment as compared to other heart chamber tissues. Additionally, the samples from livers were collected from the left lateral lobe in all species to maintain homogeneity. Finally, the kidney samples were collected from the medulla and cortex from all species avoiding larger renal vessels. These regions were chosen to respectively contain similar structure and components. The intention was that any undesirable and potentially confounding aspects of the organs such as high fat, dried blood, or latex were avoided when the samples were being obtained.

Dehydration Determination Methodology

The weights of the samples used for the following experiments ranged between 2.5 g and 3.0 g, and were used to calculate the ratio of 5000:1 for acetone to tissue. All samples were approximately 1 cm³, their weights were recorded and then the specimens were positioned sequentially on a surgical suture line (n=15/line). This process was repeated several times for each experiment. The acetone concentration was never less than 99% concentration (-25°C). Samples were dehydrated in the acetone to determine the evaporation time interval (RT, 20°C to 22°C) as well as water and lipid weight loss. The minimum number of days required to fully dehydrate the tissues in acetone was determined and compared to corresponding tissues achieving >99% dehydration. This procedure is represented in Figure 1.

Figure 1. The experimental design for the controlled dehydration experiment. The process begins with dissection and the creation of samples fitting a weight range of 2.5-3.0 g (approximately 10 mm3). The initial weight of the samples was recorded. The samples were sutured into groups for convenience of handling. Grouped samples were dehydrated in acetone. Once dehydrated to <1% water content, the samples were allowed to evaporate their acetone content and the weight of the samples was recorded. Finally, the samples were freeze-dried to obtain absolute dry weight and determine water content after the acetone dehydration.

Rate of Acetone Evaporation in Tissue

Due to the volatility of acetone, benchtop evaporation was an effective method to distinguish water content from acetone in a tissue sample. The acetone content from the tissue dissipated prior to meaningful changes in residual water, giving the sample’s evaporated weight consisting of dry matter and water content. Water evaporation may have also occurred during the acetone evaporation but was assumed to be minimal. Subsequent experiments where acetone-dehydrated specimens are placed directly into the freeze dryer without desktop evaporation would make this determination. Such specimens would need to be small as in those presently studied, for solvent safety considerations.

Samples were excised, weighed, and later placed on a non-dissolvable string via suture, with there being 15 samples per suture. The suture-containing samples were then placed in acetone at all times >98% and stabilized to >99% concentration for 10 days to ensure stabilization. Samples were then taken out and placed on benchtop at room temperature which were weighed in 10-minute intervals for 120 minutes. ANOVA tests were used to determine the point where there was no longer a significant difference in water loss.

Determination of Time Required for Complete Acetone Dehydration

This experiment took place to find the minimum number of days the tissues needed to be in acetone to be fully stabilized. To begin, 24 samples of tissue were cut and weighed from an organ initially with 21 of them being placed on a suture in the same fashion as the previous experiment. Those samples were then placed in acetone. A HarvestRight Freeze-Dryer was used to determine the baseline dry weight of tissues to distinguish the liquid content remaining. Three randomly selected samples not placed in acetone were freeze-dried to determine the average water content in each sample. In 24-hour intervals from this point, 3 samples were removed from acetone and allowed to evaporate for the allotted time from the evaporation determination experiment. Samples were then weighed and placed in the freeze-dryer to obtain their dry weight. The evaporation weight lost eventually stayed the same and at this point, we concluded the samples had been fully dehydrated in acetone. These values were calculated by dividing the post-evaporation weight by the original weight, which showed the percentage remaining. Once this value remained constant, this indicated the tissue was as fully dehydrated of water as was going to occur. The post freeze-dried weight was divided by the original weight and then 1 was subtracted from that value. Averaging all of these values revealed the average water percentage in all of the tissue types.

Controlled Dehydration

From the ‘Seven-Day Stabilization’ experiment, it was discovered that the evaporation weights never reached the freeze-dried weights. From this we hypothesized that fully freeze-substitution dehydrated samples in >99% concentration acetone may actually contain >1% water.

For this experiment, 3 organs were used with 10 samples being cut and weighed from each, giving a total of 30 samples. 15 randomly selected samples, collected as 5 from each organ, were immediately freeze-dried as the control while the remaining 15 samples were placed on a suture and dehydrated in acetone. The samples were then removed from acetone and evaporated for the needed time established by the ‘Evaporation Determination’ experiments and weighed afterward. The samples were then freeze-dried as before and weighed to obtain the dry-weight percentage in every individual section. The post-evaporation weights were divided by the original weight of each individual piece to reveal the percent weight remaining, similar to the 7-day stabilization calculation. The post freeze-dried weights were also divided by the weights of the original samples which revealed the percentage of dry weight in every sample. After this, the first calculation was subtracted from the latter and divided by the average percent of water in that particular organ as a decimal. This revealed the percentage of water remaining in each section.

Statistical Analysis

All statistical analyses were run on PRISM software through multiple comparison ANOVA tests to determine any significant differences between the means of sample groups. Results are expressed as mean ± standard deviations. All outliers are denoted in results, with significance based on a value of p<0.05.

RESULTS

All experiments listed were performed sequentially in order to achieve an accurate remaining water percentage in

Figure 2. Data collected from the “Rate of acetone evaporation from tissue” experiment show the change in weight of dehydrated tissue at room temperature for 120 minutes in 10-minute intervals. As shown, there is a significant decrease in weight until the 80-minute mark, indicated by asterisks, where weight loss became no longer significant out to the 120-minute mark, p<0.05 * indicates statistically significant difference from value at 120 minutes.

each tissue. Evaporation determination displayed an approximate time of 60-80 minutes at which acetone was no longer evaporating (Fig. 2), leaving only dry weight and water remaining and again suggesting minimal weight loss during the same time from the much slower water evaporation. The ‘Seven-Day Stabilization’ experiment, the results of which are shown in Figure 3, was performed on all tissue types and revealed the minimum days necessary to fully dehydrate them.

Evaporation Determination Results

Seven-Day Stabilization Results

In this experiment, we collected data using myocardium, liver, and kidney tissue samples from three different species. Using multiple comparisons ANOVA test we assessed at which day the samples were fully dehydrated as their post-evaporation weight percent would not be significantly different from day 7. This day was determined to be day 2 for all kidney tissues and from horse heart; day 1 for all liver tissue and for cow and dog heart (Fig. 3). The dotted line indicates the absolute dry weight of the samples found by freeze-drying the samples leaving behind only dry matter.

Figure 3a. Horse heart data show significant weight retention between day 1 (42.56%) and day 7 (25.07%), but no significant difference from day 2 (24.26%) to day 7. Cow heart data show no significant weight reduction between day 2 (34.18%) and day 7 (28.40%). Dog heart data show no significant weight reduction between day 2 (32.50%) and day 7 (28.20%). 3b. Horse liver data show no significant weight reduction between day 1 (43.94%) and day 7 (42.25%). Cow liver data show no significant difference between day 1 (42.03%) and day 7 (29.20%). Dog liver data show no significant difference between day 1 (45.36%) and day 7 (31.83%). 3c. Horse kidney data show a significant difference from day 1 (29.42%) and day 7 (27.58%), but no significant reduction between day 2 (25.95%) and day 7. Cow kidney data show a significant difference between day 1 (34.52%) and day 7 (20.20%), but no difference from day 2 (19.99%) to day 7. Dog kidney data show a significant difference between day 1 (37.32%) and day 7 (25.73%), but no difference from day 2 (25.43%) to day 7. Significance: p<0.05, * indicates statistically significant difference from value at day 7

Controlled Dehydration Results

In this experiment, myocardium, liver, and kidney samples from three different species were acetone dehydrated and evaporated in preparation for freeze drying. These samples were then freeze-dried, leaving the tissue with 0% water content (Fig. 4). After comparing the differences between tissues post-acetone dehydration and freeze-drying we calculated the % of water remaining after acetone only. ANOVA was used to assess the percent water remaining in the samples of three different hearts per species. This procedure was repeated with fresh non-formalin-fixated hearts of 3 cows and 1 horse to ensure that formalin fixation was not a factor in these results. The key finding from these experiments was that the percent water content post-acetone dehydration was greater, sometimes considerably so, than 1%.

Figure 4a. Horse heart data show an average post-evaporation weight retention of 29.02% and average 11.47% water remaining, with heart three significantly lower at 7.96%. Cow heart data show 28.15% weight retention and an average 12.33% water remaining. Dog heart data show 22.81% weight retention and 6.93% water remaining. 4b. Horse kidney data show 23.79% weight retention and an average 13.03% water remaining, with no significant differences. Cow kidney data show 21.90% weight retention and an average 7.40% water remaining, with one kidney at 9.19%. Dog kidney data show 18.13% weight retention and an average 8.96% water remaining, with no significant differences. 4c. Horse liver data show 31.14% weight retention and an average 13.68% water remaining, with livers 1 and 3 at 10.03% and 17.19%. Cow liver data show 26.73% weight retention and an average 8.96% water remaining, with no significant differences. Dog liver data show 32.26% weight retention and an average 12.60% water remaining, with liver 1 higher at 15.72%. 4d. The fresh non-formalin fixed horse heart data show an average 8.87% water retention and on average 27.36% weight retention. The fresh non-formalin fixed cow heart data show an average 15.97% water retention and had on average 32.22% weight retention. H: Horse, C: Cow, D: Dog; Significance: p<0.05, * indicates statistically significant difference from other two values of the same group. Bracket indicates statistically significant difference between the two marked values

DISCUSSION

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 (von Hagens et al., 1987, Henry, 2007). 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 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 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 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, 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 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 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 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, 1988) and felt the freeze dryer likely contributed 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.

In conclusion, this study extends our understanding of water retention after acetone dehydration of specimens intended for plastination, and may suggest ways for more optimizing of what have become long-time standardized protocols. Future experiments are planned, to compare plastination outcomes of different specimens under cold or room temperatures, or in a freeze dryer with a water condensing chamber. If results are encouraging, we will then determine the ability to make greater re-use of acetone baths to achieve modestly lower stabilized acetone levels, which could lead to reduced solvent use under some conditions of plastination.