INTRODUCTION

Plastination is the process of creating nonperishable and easily handled biological specimens by replacing tissue fluid with a curable polymer, such as silicone, polyester, or epoxy (de Jong & Henry, 2007). The standard silicone technique is the method most frequently used in our and many other plastination laboratories. The silicone-plastination process consists of 4 major steps: specimen preparation/fixation, dehydration, forced impregnation, and curing (von Hagens et al., 1987; de Jong K & Henry, 2007). Cold dehydration (-20 to -25 °C) replaces water within the tissue with cold acetone (von Hagens et al., 1987; de Jong & Henry, 2007).

Formalin is the most common and recommended fixative agent for use with plastination (Oostrom, 1987; Riepertinger, 1988; Ulmer, 1994; Sargon & Tatar, 2014). Formalin-fixation of tissues is strongly recommended to prevent putrefaction and decrease the likelihood of the model becoming biohazardous or odorous (Bickley et al., 1987;Oostrom, 1987; Ravi & Bhat, 2011; Sargon & Tatar, 2014). Formalin-fixed tissue samples are then washed in tap water for 2-4 days to remove formalin (de Jong & Henry, 2007). Alternatively, plastination of fresh tissues may produce more flexible specimens that retain more of their natural color (Sargon & Tatar, 2014).

During acetone dehydration, tissues are submerged in acetone to facilitate the replacement of water in the tissue with acetone (de Jong & Henry, 2007). The replacement of water with acetone allows acetone to serve as a volatile intermediary solvent that can be replaced with silicone during forced vacuum impregnation (de Jong & Henry, 2007). The standard for acetone dehydration is freeze-substitution, which is carried out by submerging the samples in acetone at approximately -25 °C (von Hagens, 1986). The acetone dehydration step can also be carried out at room temperature with samples submerged in an acetone bath at 15-20 °C (Zheng et al., 1996; Zheng et al., 1998). Because acetone is volatile and flammable at room temperature, we utilize the freeze-substitution method to better ensure safety (Recovery OoRCa, 2024).

For freeze-substitution acetone dehydration, the samples are typically submerged in multiple acetone baths (Bickley et al., 1987; Fahlman, 1997; Brown et al., 2002; Dezse et al., 2020). Many factors affect dehydration dynamics, such as the ratio of acetone to tissue of the acetone baths used. Some laboratories recommend using three to four acetone baths of 99 to 100%  (Tiedemann & Ivic-Matijas, 1988; Ripani et al., 1994; Baeres & Møller, 2001; Sagoo & Adds, 2013) while others suggest using several graded acetone baths to fully dehydrate the tissue (Zheng et al., 1996; Tiedemann & Ivic-Matijas, 1988; Zheng et al., 1998).

Many laboratories mention the use of a 10:1 acetone-to-tissue ratio (Bickley et al., 1987; Brown  et al., 2002). Tiedemann and Ivic-Matijas (1988) worked to create a procedure that was time and solvent efficient, and their studies suggest that a ratio of 5:1 is sufficient. However, the latter authors also used three changes of acetone while dehydrating a specimen. Another laboratory worked to create a time and solvent efficient procedure while maintaining the specimen's structure (Ripani, 1994). This laboratory recommended a 5:1 ratio and passed each specimen through three or four acetone baths on average for 11-14 days (Ripani et al., 1994). Many laboratories employ methods where smaller ratios of acetone-to-tissue are used, but acetone baths must be replaced multiple times. This process can be time-consuming and labor intensive while not actually minimizing acetone use. Plastination practitioners agree that acetone waste should be minimized, as it is expensive and becomes hazardous when disposed of as waste or recycled (Breysse & Reh, 2022; Recovery OoRCa, 2024).

The present study was initiated with the goal of better defining acetone-to-tissue ratios necessary to effectively dehydrate specimens, with particular interest in situations where rapid preparation time may be a priority. Effects of various acetone-to-tissue ratios were explored by measuring dehydration in samples of the equine left ventricular wall. These samples were selected as the thickest regions of the equine heart, which we felt would dehydrate in a manner representative of other small specimens. We hypothesized that an as-of-now unknown point would be reached where increasing the acetone-to-tissue ratio becomes no longer beneficial to dehydration time or completeness. It is hoped that the results of the research will bring new information for planning acetone dehydration.

MATERIALS AND METHODS

Sample Preparation

Equine hearts were collected from formalin-fixed cadavers previously used for teaching veterinary anatomy at the UGA College of Veterinary Medicine. The hearts were removed from the thoracic cavity and cleaned of associated parietal pericardium, connective tissue, and fat. The organs were submerged in a wetting agent mixture of water, ethyl alcohol, ethylene glycol, phenol, and fabric softener for 2-4 days. Samples, approximately 10 mm³, weighing between 2.5 g and 2.9 g, were cut from the left ventricular wall (Fig. 2). All samples for all studies were produced in the same fashion.

Twenty samples were collected for each tested ratio, and two polyethylene sutures were used to gather and support 10 samples each. Placing the samples on sutures (Fig. 1) allowed for organization and to maintain the proper order of the samples throughout the dehydration. One assembly served as the treatment group (X:1), and the second as a within-treatment control group (X:1/4:1) used to ensure dehydration completeness.

Figure 1. 10 mm3 samples of equine left ventricular wall were strung on polyethylene sutures

Acetone Evaporation Test

After freeze-substitution dehydration, the samples contained the tissue specimen, acetone, and minimal remaining water. Typically, such dehydrated tissues would proceed to forced impregnation, but for our study, they underwent a lab bench acetone evaporation. This allowed for a comparison between the initial weight and evaporated weight and ensuring of dehydration completeness. The acetone evaporation testing was conducted to determine how long the samples needed to sit on the laboratory bench for evaporation stabilization (completeness) to occur. This is a time when significant acetone evaporation has finished, and further weight lost is due to the slower evaporation of water that may remain. Overall, the acetone evaporation goal was to determine how long tested samples should undergo evaporation to accurately test for remaining water content after dehydration in each ratio.

Nine samples were collected from an equine heart in accordance with the methods described above. The samples were then labeled, and their initial weights were recorded. A polyethylene suture was put through the samples from one to nine, and a loop was placed after the ninth piece as an indicator. The suture assembly was placed in a large bucket with 19 liters (five gallons) of 100% acetone for a week to ensure water had been replaced with acetone. After removal, the weight of each sample was recorded every 10 minutes for 150 minutes using a laboratory scale. The evaporation data were then analyzed using a Multiple Comparisons Test, and the time in which significant acetone evaporation stopped was found by comparing the weight at each time point to the weight at the final time point. The evaporation efficiency time point was then used for the following studies, as these required a similar level of acetone evaporation.

Determining Minimum and Maximum Acetone to Tissue Ratios

Thirty equine left ventricle heart samples were collected, as described above. The initial weight of each sample was recorded, and the samples were labeled. Three polyethylene suture string assemblies containing ten samples each were prepared. The total volume of samples was determined by volume displacement by individually submerging the samples in a graduated cylinder filled with acetone and taking the difference between the final (with samples) and initial (without samples) volume measurements. Suture assembly one was assigned to the 200:1 acetone-to-tissue treatment (volume of acetone was determined by multiplying the assembly volume by 200). It was hypothesized that a 200:1 ratio contained a large excess of acetone and would fully dehydrate the samples. To ensure that 200:1 was sufficient to fully dehydrate the samples, the aliquot of 100% acetone and tissue was placed in a large acetone-resistant container, covered, and stored in a deep freezer at -25 °C for five days. Suture assembly two was assigned to the 4:1 acetone-to-tissue treatment group (volume of acetone was determined by multiplying the assembly volume by 4). A 4:1 acetone to tissue ratio was chosen as the minimum because this is the lowest ratio of acetone-to-tissue in which the tissue could be fully submerged in acetone. The aliquot of 100% acetone and tissue was placed in a graduated cylinder and stored in a freezer at -25 °C for five days. Suture assemblies 1 and 2 remained submerged in their respective ratios for the five days. After the 5^th day, samples were removed from acetone and acetone concentrations were taken using an acetometer. Acetone concentrations were measured at room temperature (21 ± 2 °C) due to the temperature dependency of acetone’s density (Zheng et al., 1998).  Samples were placed on the lab bench for an 80-minute evaporation as indicated by the acetone evaporation testing. Final weights were taken following the acetone evaporation. It was hypothesized that a 4-day dehydration would be sufficient to fully dehydrate the samples of potentially remaining water. To ensure this was sufficient time for dehydration, a stabilization control was included. If the % weight remaining calculated for the experimental and control groups was not significantly different then dehydration was completed by the 4^th day. Suture assembly 3 served as the control to ensure that water and acetone reached an equilibrium by the 4^th day and stabilization had occurred.

The 3^rd suture assembly was placed in a large, graduated cylinder with 100% acetone in a 200:1 acetone-to-tissue ratio and stored in a deep freezer at -25 °C for four days. On the 5^th day, acetone concentration was taken using an acetonometer, and acetone was removed from the graduated cylinder until only a 4:1 ratio remained. After the 5^th day, samples were removed, and final acetone measurements were taken using an acetonometer. Samples were placed on the laboratory bench for an 80-minute evaporation. Following the evaporation, final weight measurements were recorded. Initial and final weight measurements were used to calculate the percent weight remaining (final weight/initial weight x 100%). Percent weight remaining across treatment groups was compared using a Multiple Comparisons Test.

Finding the Optimal Acetone to Tissue Ratio

We began with ratios near the extremes between our minimum and maximum ratios and narrowed down to the smallest ratio that could dehydrate samples as efficiently as a 200:1 ratio. Twenty samples were harvested, and two suture assemblies were prepared as outlined in the sample preparation guidelines seen above. The volume of each assembly was determined by volume displacement using a graduated cylinder and a wetting agent mixture of water, ethyl alcohol, ethylene glycol, phenol, and fabric softener. Suture assembly 1 and 2 were placed in large, graduated cylinders and submerged in 100% acetone at a 50:1 acetone to tissue ratio (acetone volume calculated by multiplying assembly volume by 50) and placed in a deep freezer for 5 and 4 days at -25 °C, respectively. After the 4^th day, suture assembly 2 was removed from its graduated cylinder, and acetone concentrations were taken using an acetonometer. Acetone was removed from the graduated cylinder until a 4:1 ratio remained, and the suture assemblies were returned to the graduated cylinder. After the 5^th day, both suture assemblies were removed from their graduated cylinders, and final acetone concentrations were taken using an acetometer. The suture assemblies were placed on the lab bench for an 80-minute evaporation. Following evaporation, final weights were taken, and the percent weight remaining was calculated (final weight/initial weight x 100%). The percent weight remaining for the 50:1 and 50:1/4:1 groups were compared to one another and to the 200:1 control group using the Multiple Comparisons Test. These steps were repeated for 60:1, 65:1, and 75:1 ratios.

Recycling 65:1 for Multiple Acetone Dehydrations

Ten samples were placed in 100% acetone for 4 days at our optimal ratio (65:1). After 4 days, the acetone concentration was measured. New 10 mm³ samples with weights between 2.5 g and 2.9 g from the left ventricle of the equine hearts were prepared and placed in the same acetone at the same ratio. These steps were repeated until the dehydration acetone reached below 98% purity.

Statistical Analysis

A Multiple Comparisons Test compares multiple samples to determine significant differences in the means to one or multiple other sample means. This test was used for the statistical analysis of the Acetone Evaporation Efficiency Point Test and Ratio Studies. For these studies, a confidence interval of 95% was used to determine significance. To determine the evaporation efficiency range, the weight of nine samples of the ventricular wall was recorded every ten minutes for 150 minutes. Using these data, a Multiple Comparisons Test was run, comparing the weight at each time point to the weight at t=150 and generating a p-value for each time increment. Multiple Comparison Tests were also run for the acetone dehydration ratios of 4:1, 200:1, 50:1, 75:1, 60:1, and 65:1. For each ratio, the initial weight remaining of the variable ratio was compared to the minimum effective ratio of 4:1, and the maximum effective ratio of 200:1. Another Multiple Comparisons Test was run on all of the tested ratios compared to one another. Lastly, a bar graph was made to represent acetone concentration loss after 3 uses of the optimal acetone in acetone dehydration.

RESULTS

Figure 2 shows the results of the heart acetone evaporation experiment. The graph shows the relationship between the weight (g) of 9 heart wall samples and evaporation time (min).

Figure 2. The Multiple Comparisons Test shows weight stabilization after 60 minutes when the p-value was no longer significantly different than the final time point of 150 minutes. The weights at t=0, 10, 20, 30, and 40 (p<0.0001) and at time point 50 (p=0.0024) were all significantly different from the final weight at t=150. On the other hand, the weights at t=60 (p=0.1372), t=70 (p=0.7201), t=80 (0.9843), t=90 (0.9996), and t=100, 110, 120, 130, and 140 (p>0.9999) were all not significantly different from the weight at t=150.

Sample weights were recorded in 10-minute increments from t=0 to t=150 min. A Multiple Comparisons Test was used to compare the mean weight of the samples at each time point with the mean weight of the samples at the final time point (t=150 min).  Starting at t=60 min the mean weight was not significantly different from the mean weight at t=150 min (p-value=0.1372).

The data from the minimum, maximum and intermediate ratio experiments are shown in Figure 4. All statistical comparisons in these figures were made using Multiple Comparisons Tests with a 95% confidence interval. The percent weight remaining values for the minimum and maximum ratios are represented in Figure 3. The percent weight remaining values of the 200:1 group and 200:1/4:1 stabilization control were not statistically different. However, a statistical difference between the 200:1 maximum group and the 4:1 minimum group was present.

A summary of the results obtained from the minimum, maximum, and intermediate ratio dehydration trials are shown in Figure 4. The percent weight remaining for each tested ratio was compared to that of the 200:1 control via the Multiple Comparisons Test. The percent weight remaining values calculated for 4:1, 50:1, and 60:1 were significantly different from that of the 200:1 ratio, indicating these ratios were too low to fully dehydrate the samples. The percent weight remaining values calculated from the 65:1 and 75:1 ratios were not significantly different than that of the 200:1 control, indicating these ratios were sufficient to fully dehydrate the samples.

Figure 3. The percent of initial weight remaining in the 4:1 ratio was significantly higher than 200:1 (p<0.0001), and the percent of initial weight remaining in the 200:1 /4:1 group was not significantly different than the 200:1 group (p=7.28).

Figure 4. The percent of initial weight remaining in 4:1, 50:1, and 60:1 ratios were all significantly higher than the percent of initial weight remaining in the 200:1 ratio (p<0.05). The percent of initial weight remaining in the 65:1 and 75:1 ratios were not significantly different from the percent of initial weight remaining in the 200:1 ratio.

A comparison of the intermediate ratios and their respective stabilization control group is shown in Figure 5. The percent weight remaining values of the 50:1 group and 50:1/4:1, stabilization control group, were not statistically different. This is true for the 60:1, 65:1, and 75:1 group as well, indicating an equilibrium was reached within the intermediate ratio before it was compared to our maximum ratio of 200:1.

Figure 6 showed the result of the study performed to determine if acetone used in a 65:1 acetone-to-tissue ratio can be reused. The study involved three, 4-day dehydration cycles and maintained the proper acetone-to-tissue ratio throughout. The acetone concentrations were taken following each dehydration cycle. After 1 dehydration the acetone concentration was 99.1%, after 2 it reached 97.7%, and following the 3^rd it was measured at 96.8%. This indicated the 65:1 ratio cannot be reused multiple times as the acetone concentration fell below 99% during the second dehydration.

Figure 5. The percent of initial weight remaining in the stabilization control groups (50:1 / 4:1, 60:1 / 4:1, 65:1 / 4:1, 75:1 / 4:1) were not significantly different (p>0.05) from their respective experimental groups (50:1, 60:1, 65:1, 75:1). This suggests that dehydration was complete before each ratio was examined.

Figure 6. Acetone in a 65:1 ratio dropped to 99.1% after one dehydration cycle of samples, 97.7% after two dehydrations, and 96.8% after the third dehydration

DISCUSSION

Commonly accepted methods of acetone dehydration include low ratios of acetone-to-tissue, such as 10:1 to 5:1, and multiple baths of acetone (Bickley et al., 1987; Tiedemann & Ivic-Matijas, 1988; Ripani et al., 1994; Brown etal., 2002). Our concern was that these procedures produce excessive acetone waste. The goal of this study was to determine the optimal acetone-to-tissue ratio for the cold dehydration step of silicone-plastination utilizing a one bath method. We hypothesized that increasing the acetone-to-tissue ratio would improve the efficiency of acetone dehydration until a point at which increasing the ratio no longer had an effect. The motivation to find an optimal acetone-to-tissue ratio for a dehydration method that does not include using multiple acetone baths was driven by the desire to reduce the amount of acetone waste produced.

Acetone evaporation testing was conducted to determine how long samples should undergo benchtop evaporation when testing each ratio's effectiveness. This defines a time at which significant acetone evaporation has finished, and further weight lost is due to the evaporation of water. The results from the Multiple Comparisons Test for acetone evaporation showed weight stabilization within the range of 60 to 150 minutes for the ventricular wall samples. Therefore, we used 80 minutes as our acetone evaporation time in all studies to ensure acetone evaporation had occurred.

Next, the dehydration effectiveness of our minimum and maximum acetone to tissue ratios were compared. Results of our maximum (200:1) and minimum (4:1) ratio comparison showed that 4:1 retained significantly more of its initial weight when compared to both the 200:1 / 4:1 and 200:1 group. This showed that the 4:1 ratio was not as efficient for dehydrating the tissues, as a larger portion of the initial water weight remained in the tissue after the five-day dehydration period.

After comparing our maximum and minimum ratios, the dehydration efficiencies of various intermediate ratios were compared to that of the maximum ratio. This is because the optimal method will include the smallest ratio that dehydrates samples as effectively as the 200:1 ratio. In all ratios, the stabilization control (X:1/4:1) and experimental ratios were not significantly different in terms of percent weight remaining, suggesting that dehydration was complete before each ratio was examined. The results of all ratios 60:1 and below showed that the percent of initial weight remaining was significantly higher than 200:1. This suggests that these ratios retained a significantly higher percentage of water in comparison to that of the 200:1 ratio. Therefore, these ratios are not sufficient for full acetone dehydration. Alternatively, the results for all ratios 65 and above showed that the percent of initial weight remaining was not significantly different from 200:1, suggesting these ratios were large enough to effectively dehydrate the sample. Therefore, our optimal ratio was 65:1, as it was the smallest ratio that fully dehydrated the samples. The results also validate utilizing the percent of initial weight remaining for 200:1 as a target result to test ratio effectiveness. This is because our results show that beyond 65:1, increasing the ratio had no significant effect on the dehydration dynamics. Additionally, studies show that the water content in the tissue should drop below 1% for optimum results during dehydration (Ravi & Bhat, 2011). Therefore, we took acetone measurements of each acetone bath and ensured that the acetone concentration did not drop below 99% after complete dehydration at our optimal ratio and beyond.

However, there are limitations to this method of acetone dehydration. This ratio would require a potentially prohibitive amount of acetone for dehydrating larger specimens and thus may not be a practical approach for dehydrating these samples. Additionally, the acetone from the 65:1 ratio cannot be reused for multiple single bath dehydrations because the acetone concentration dropped below 99% in the process of the second dehydration. Furthermore, this study only employed formalin-fixed specimens, so specimens prepared using alternative fixatives or methods could have different dehydration dynamics. Lastly, this study did not explore tissue types beyond the equine left ventricular wall. Other tissue types may have different dehydration dynamics due to variations, such as tissue density. It is plausible that less dense tissues may be more permeable to acetone which could increase the rate at which dehydration occurs. Future studies should use a variety of different tissue types to discover if there are differences in their optimal acetone to tissue ratios for dehydration so that the results may be applied broadly. Therefore, these results are intended as a starting point and other optimal ratios may need defining for plastination of other tissue types.

In conclusion, this study identified an optimal acetone-to-tissue ratio for the freeze-substitution dehydration of formalin-fixed tissues. By focusing on a single acetone bath method, the results also provide new information for more rapid plastination of small specimens. These findings offer new information in plastination by presenting a time-efficient and resource-conscious method for small specimen dehydration. Future research should explore the optimal ratios for larger specimens and diverse tissue types, to further advance plastination techniques while prioritizing sustainability.