INTRODUCTION

Plastination is a relatively new process (1982) that is now widely used to preserve perishable biological specimens with high water content. In this technique, tissue water and lipid are replaced with curable polymers. The completely impregnated specimen is cured by a gaseous vapor.

Dr. Gunther von Hagens of the University of Heidelberg, Germany, developed the suitable polymers and four variations of plastination techniques based on the same fundamental process providing for difference of specimens, (von Hagens, 1979a, b, 1985/1986, 1987). Silicone impregnated specimens (S-10) are resilient and flexible and are mainly used in teaching purposes. Whole organs, limbs, student prosections and even whole bodies may be plastinated with the silicone impregnation method. Specimens produced with polymerized emulsions (P.E.M.) are as opaque as the silicone specimens but are rigid and to some extent breakable. The use of this technique is in production of thick body slices exhibiting a sharp contrast be- tween fat tissue, which shows up white, and all other more intensively stained parenchyma. Transparent body or organ slices are produced with epoxy resins. This process is known as E-12. For research purposes, these 2.5 mm. to 4.5 mm. thin slices which allow macroscopic study of the topography of all body structures with the naked eye in an uncollapsed and non-dislo­cated state. In addition, the specimens are useful in advanced training programs in sectional topography (resident training in CT and NMR). As with specimens produced with polymerized emulsions, the epoxy imbedded specimens are cured with heat.  Opaque brain slices (P-35 process) of 4 mm. thickness, are impregnated with polyester resin, and allow a unique discrimina­tion between fiber and nuclear areas as a direct result of contrast properties within the resin activated by curing the specimen by U.V.A. light sources.

The general procedure of Standard silicone technique  S-10 (von Hagens, 1985/1986) has 4 steps.

1. Fixation by using formalin in concentrations be­ tween 5 to 20%. It takes about one week in this step. The lower the percentage of formalin the better the color retention within the tissue.

2. Dehydration by freeze-substitution in acetone (-25YC) until water content is below 1%. It takes from 1-5 weeks.

3. Impregnation with Biodur* Sl0/S3 in vacuum at -25YC for IO days to 5 weeks.

4. Remove from silicone polymer bath, drain, wipe off and harden by gas vapor curing It takes about 3 weeks. The total time required is about 12 weeks.

In Thailand, because of the limited availability of some equipment, especially the large deep freezer (-25°C), we try to modify the standard silicone technique to allow all steps to be carried out at room temperature (average room temperature is about +25 to +30°C).

MATERIALS AND METHODS

We use surgical specimens that are sent to the depart­ment of pathology for pathological diagnosis.   The specimens are  fixed  in   10%  buffered  formalin  at  room  temperature.  Polymer (S-10), hardener (S-3) and gas cure (S-6) are required . They are only available from Biodur (Heidelberg, F.R.G.)  Ethanol and acetone are also required.

FIXATION

The pathology specimens have been collected and fixed in  10% buffered  formalin  at room temperature for at least 1 week.  In this step, care is taken that the specimens maintain their natural shape, or the shape they will exhibit when finished.

DEHYDRATION  AND DEGREASING

We use stepwise dehydration in increasing graded etha­nol (50%, 60%, 70%, 80%, 90% and 100%, one week for each concentration), and then 2 baths of acetone for complete dehy­dration and degreasing. The specimens remain in each acetone bath for about 2 weeks at room temperature or until water con­ tent is below 1%.

FORCED IMPREGNATION

This is the most important step in plastination, in which the intermediate solvent (acetone) is replaced by curable poly­mers by means of a vacuum during forced impregnation.  The curable polymer is composed of a silicone base material (S-10) and hardener (S-3).  We use 10 Kg. of S-10 mixed with 0.1 L. of S-3.   Many kinds of tissue with varying thickness from 0.5-10 cm can be done at the same time. It takes about 2 weeks at room temperature.

GAS CURING

The specimen s are removed from the mixture of cur­ able polymers and then wiped of excess polymers. The speci­mens remain in pre-cure at room temperature for about 1 week to allow the excess polymer to drain off. After that, the speci­mens are cured by a silicate containing gas, evaporating from a fluid (S-6) for about 2 weeks. This step ends when the speci­mens become solid and dry to the touch.

The total time required is approximately  16 weeks.

RESULTS

This technique provides satisfactory results with mini­ mum equipment, because all steps can be done at room tempera­ture. This technique can be used in all laboratories without a large deep freezer (-25°C).

The resulting plastinated specimens are dry to touch, odorless, durable, lifelike and non-toxic. They do not deterio­ rate with time, are maintainance free and resistant to damage caused by handling. They maintain their original shape and are close in color and consistency (figure 1 and 2). They can be stored at room temperature indefinitely. They are useful in the teaching of pathology, especially when used in small groups and are suitable for exhibition purposes as museum specimens.

Figure1- Plastinated specimen of stomach showing stress ulcer.

Figure 2- Plastinated specimen of liver showing cholangiocarcinoma mass.

DISCUSSION

The specimens preserved by plastination  techniques  are  superior to those preserved in formalin. In this method we modify the standard silicone technique so that all steps can be done at  room temperature.  In the step of fixation we use surgical specimens that are sent to the department of pathology for pathologi­cal diagnosis. As the specimens can be used in the plastination process only after complete diagnosis, fixation in 10% buffered formalin at room temperature for at least 1 week is necessary. This makes the tissue firm, and reduces susceptibility to shrink­ age in the subsequent plastination process. However, little change of color can be observed. This problem can be solved by using the Kayserling-Fixation method or staining with BIODUR STAIN C (von Hagens 1985/1986).

Dehydration in graded ethanol causes considerable shrinkage and is time consuming (Schwab and von Hagens, 1981; von Hagens, 1987) This problem is reduced by using the speci­mens fixed in 10% buffered formalin for a long time and not over dehydrated. The specimens which have high fat content, such as breast tissue, still shows shrinkage.

Following dehydration and degreasing, the cellular spaces within the specimens originally filled with water and lip­ ids are now occupied by a volatile intermediary solvent (acetone). In forced impregnation the intermediary solvent is replaced by curable polymers by means of a vacuum. The intermediary solvent has a high vapor pressure and a low boiling point (ac­etone: +56°C), while the polymer solution has a low vapor pres­sure and a high boiling point. Therefore, when a vacuum is applied, only the intermediary solvent is continuously extracted gradually out of the specimens and through the surrounding poly­mer solution in the form of gaseous bubbles. (von Hagens, 1987). It takes about 2 weeks to complete forced impregnation. Vari­ous types of tissue with varying thicknesses (0.5-10 cm) can be done at the same time. The residual polymer in the force im­pregnation bath can be reused. In this experiment the step of forced impregnation is carried out at room temperature so the viscosity of the polymer increases. This problem is solved by mixing the curable polymers with acetone before reuse for a new batch of specimens.

Following forced impregnation, the specimens are cured by a silicate containing gas, evaporated from a fluid S-6. The hardener S-3 contained in the impregnation bath initiates the curing of the silicone molecules by end to end polymerization. Due to crosslinking during the final gas curing, the silicone rub­ber within the specimen will become solid and dry (von Hagens, 1987). This step ends when the specimens become dry to the touch. The degree of flexibility of the finished specimen is de­ pendent on the ratio of S-3 hardener to the number of days of the S-6 gas curing.

This technique provides both a satisfactory result with minimum equipment and can be done in all laboratories without a large deep freezer (-25YC).