INTRODUCTION

Tissue slices plastinated with epoxy polymer are finding their way into medical research (Guhr et  al., 1987; Phillips et al., 2002) and education (Cook, 1997a; Cook 1997b). Being used in these disciplines, it is necessary to minimize artifacts introduced during  and after the plastination procedure to ensure accurate assessment of tissue samples. Attempts have been made to increase the transparency of specimens  plastinated with epoxy polymer (Mathura and Satyapal,  2000)  as well as to decrease the yellowing of cured epoxy following plastination (Latorre et al., 2002). Improved techniques for epoxy plastination  have  been  reported but do not include methods for reducing or eliminating the discoloration of cured epoxy (Fasel et al., 1988; Alston et al., 1997). Shrinkage of tissues plastinated via the E12 technique has been measured and documented (Sora et al., 2002) yet no information is currently available documenting the discoloration of cured epoxy polymer.

Acetone has classically been used as the dehydrating agent for epoxy plastination (von Hagens, 1989; Weber and Henry, 1993; Sora and Cook, 2007; Cook, 2007). Experiments have been performed to determine the optimum method for using  acetone as a  dehydrating agent for tissue (Holladay, 1988; Brown et al., 2002). However, investigations are lacking in the area of determining possible unwanted side effects  of  the acetone  dehydration  on  different  plastination procedures. This experiment was designed to determine if acetone is  involved  in  the  yellow  discoloration evident in cured epoxy polymer.

MATERIALS AND METHODS

E1 and E12 were mixed at 95 parts of polymer to 26 parts of hardener by weight as per von Hagens (1989). Glass separator (AE30) was intentionally omitted from the reaction-mixture to eliminate any involvement  it may have in the discoloration of epoxy polymer. Forty milliliters of this epoxy reaction-mixture  was  poured into each of fifteen 50 ml glass beakers. The beakers were randomly divided into five groups of three beakers each. The beakers in group 1 were used as experimental controls. One milliliter of acetone was added to each of the beakers in groups 2 and 3 and stirred into the epoxy reaction-mixture. Eight milliliters of acetone was added to each of the beakers in groups 4 and 5 and stirred into the epoxy reaction-mixture. The beakers in groups 1, 3 and 5 were left uncovered on a counter top for 24 hours.

The beakers in groups 2 and 4 were placed into a vacuum chamber. The pressure in the vacuum chamber was slowly decreased over six hours to the point of full vacuum. The beakers remained at the point of full vacuum for 24 hours. The epoxy reaction-mixture in the beakers of all groups was examined for curing after 24 hours.

The hardened epoxy reaction-mixture in  all  groups was stored in the dark and evaluated for signs of discoloration at 24 hours, six months and twelve months post  experimentation.

RESULTS

The epoxy contents of all beakers had cured after 24 hours. All reaction-mixtures appeared clear 24 hours following curing regardless of the exposure or non- exposure to acetone or vacuum. The control beakers containing epoxy reaction-mixture in the absence of acetone remained clear six months after curing (Figs. 1 and 2).

Figure 1. Epoxy reaction-mixture cured for 6 months. Reaction-mixture control (A), reaction-mixture with 1.0ml acetone and subjected to vacuum (B), reaction-mixture with 1.0ml acetone (C), reaction-mixture with 8.0ml acetone and subjected to vacuum (D), reaction-mixture with 8.0ml acetone (E).

At  six  months  post-experimentation,  all  beakers containing the reaction-mixture and 1.0ml of acetone, regardless of whether they were placed under vacuum or not, showed only a  very  slight  yellowing  that  was best observed when viewing the beaker from  the side (Fig. 1). The cured reaction-mixture exhibited a slightly darker yellow discoloration at its surface (Fig. 1). This greater discoloration extended 2.0mm into the cured polymer and was not visible when viewing the cured epoxy from above (Fig. 2).

The  beakers  containing  epoxy  reaction-mixture  and 8.0 ml of acetone that were exposed to vacuum showed a yellowing of the cured mixture that was greater than that evident in beakers treated with 1.0ml of  acetone (Figs. 1 and 2).

Figure 2. Epoxy reaction-mixture cured for 6 months (same samples as those in figure 1). Reaction-mixture control (A), reaction-mixture with 1.0ml acetone and subjected to vacuum (B), reaction-mixture with 1.0ml acetone (C), reaction-mixture with 8.0ml acetone and subjected to vacuum (D), reaction-mixture with 8.0ml acetone (E).

These beakers also exhibited yellowing at the surface at six months post-experimentation to a greater degree than did the beakers containing 1.0ml of acetone (Fig. 1). The yellowing was first evident at four months post-experimentation. This yellow discoloration extended 6.0 - 8.0mm into the cured polymer. The yellow discoloration of the cured  epoxy  reaction- mixture was  evident  in these  beakers when viewing them from above (Fig. 2).

The beakers containing epoxy reaction-mixture and 8.0mls of acetone and allowed to cure at 1.0 atmosphere exhibited a marked yellow discoloration throughout the extent of the cured epoxy at six months post- experimentation when compared to all other test mixtures. (Figs. 1 and  2).  This  discoloration  first became evident 3 months post-experimentation.

Between six months and one year post- experimentation, the glass beakers  began to crack and subsequently fall away from the cured reaction-mixture. At  one  year  post-experimentation,  control  beakers and all beakers containing 1.0ml of acetone showed a very  light  yellowing  throughout  the  cured

Figure 3. Epoxy reaction-mixture cured for 1 year (same samples as those in figure 1). Reaction-mixture control (A), reaction- mixture with 1.0ml acetone and subjected to vacuum (B), reaction-mixture with 1.0ml acetone (C), reaction-mixture with 8.0ml acetone and subjected to vacuum (D), reaction-mixture with 8.0ml acetone (E).

reaction- mixture (Figs. 3 and 4).

The yellowing was slightly less evident  in  the  control  beakers  when  viewed  from  the side  (Fig.  3).  The  beakers  containing  the  reaction- mixture with 8.0mls of acetone that were placed under vacuum   exhibited   a   deep   yellowing   of   the   cured reaction-mixture (Figs. 3 and 4). Beakers containing the reaction-mixture  with  8.0mls  of  acetone  that  were  not subjected to vacuum showed a marked discoloration of the cured reaction-mixture to the point it appeared orange (Figs. 3 and 4).

Figure 4. Epoxy reaction-mixture cured for 1 year (same samples as those in figure 1). Reaction-mixture control (A), reaction- mixture with 1.0ml acetone and subjected to vacuum (B), reaction-mixture with 1.0ml acetone (C), reaction-mixture with 8.0ml acetone and subjected to vacuum (D), reaction-mixture with 8.0ml acetone (E).

DISCUSSION

The epoxy  reaction-mixture  control  samples remained clear longer and yellowed to a lesser degree than all the others due to the lack of effects of acetone on the cured mixture. The fact that  it  did  discolor  to some degree suggests that acetone is not required to be present for epoxy reaction-mixture to discolor in some manner.

The reaction-mixtures to which 1.0ml  of  acetone was added cured no differently from one another, whether or not vacuum was applied. When these were compared to the control beakers, they did discolor to a greater degree which can only be attributed to the presence of the acetone. The fact that there was no discernable difference between those beakers to which vacuum   was   applied   and those which were not subjected to vacuum would suggest that  the  mere presence of 1.0ml of acetone was enough to discolor the surface of the cured reaction-mixture at six months and discolor the entire mixture at one year whether the acetone was forcibly removed or not. As  there was a difference  between  beakers  containing  8.0mls  of acetone when subjected to vacuum compared to those that cured at 1.0 atmosphere, we may conclude that the vacuum was able to remove some acetone from the reaction-mixture. In comparing the beakers to which 1.0ml of acetone was added to those with 8.0mls, it appears that vacuum is able to remove acetone to some degree yet  not  entirely.  It  cannot  be determined from these experiments if the vacuum was unable to remove all of the acetone prior to the acetone affecting the reaction-mixture or if vacuum simply cannot remove all acetone before the  reaction-mixture  cures  and  that which is left will then act to discolor the reaction- mixture. Additionally, is vacuum unable  to  extract acetone once acetone decreases to an undetermined concentration in the reaction-mixture.

The reaction-mixtures to which 8.0mls of acetone were added were markedly discolored by the presence of the acetone regardless of the effects of the vacuum. The results using 8.0mls of acetone clearly demonstrate that if acetone can be removed from the reaction- mixture before it cures, specimens will exhibit less discoloration after the passage of time.

Epoxy reaction-mixture used for impregnation is regularly used for casting the impregnated  tissue.  As soon as tissue ceases to release acetone bubbles, impregnation is considered complete, however, the reaction-mixture will be  saturated  with  acetone.  It would stand to reason that the sooner the impregnation reaction-mixture is used for casting following the completion of impregnation, the greater the probability for epoxy yellowing following curing. In actuality, the time that exists between completion  of  impregnation and curing of the reaction-mixture is most  likely  too short for much acetone to escape through the surface of an ever hardening mixture. This technique  will encapsulate the tissue slice with reaction-mixture containing acetone which will eventually lead to discoloration of the specimen. If cost is not an issue in specimen preparation, it would be best to use freshly mixed epoxy reaction-mixture which has never been in contact with acetone to plate the tissue slices. This should keep discoloration to a minimum. If a vacuum is applied to reaction-mixture which has been used for impregnation before it is used for casting, it should eliminate some acetone. This application  of  vacuum will also cause the release of bubbles from the reaction- mixture  which  are  not  related  to  acetone  and  which should not be confused for a highly acetone saturated reaction-mixture as this bubbling will not cease until the curing  reaction-mixture  becomes  so  viscous  that bubbles can no longer escape (Reed, 2003).

The cured epoxy reaction-mixtures shown in figures 3 and 4 are not contained within a glass beaker. Over time, the contracting reaction-mixture pulled on the glass and caused the glass beakers to crack and subsequently fall away as there was no glass separator included in the experiment.