INTRODUCTION

The plastination process for impregnation of biological tissues has remained nearly the same since its inception twenty-seven years ago by von Hagens (von Hagens, 1979a, 1979b, 1980, 1981, 1985; Bickley et al..1981, 1987; Tiedemann and von Hagens, 1982; Henry.1987, 1995, 1998; Oostrom, 1987b; von Hagens et al. 1987; Nicaise et al., 1990; Henry and Nel, 1993; Weiglein and Henry, 1996; von Hagens and Whalley, 2000). The various polymers, catalysts, chain extenders and cross-linkers used for plastination are all products currently used in the silicone polymer industry (Holladay et al., 2001; Henry et al., 2002b; Henry, 2004). There are at least six methodologies widely used today: SI0 (von Hagens', Biodur™), Corcoran (Dow), Chinese (Su-Yi), YisDocta™ (Italy), North Carolina A (NC A) and B (NC B). The major difference in these processes is how the components are combined and used during the process. Combination of polymer and catalyst (S 10, YisDocta™, NC A) yields an unstable impregnation reaction-mixture which may be semi­ stabilized and hence the rate of thickening (chain elongation) remarkably retarded if the reaction-mixture is stored at -20"C or less. Impregnation is recommended at - 15°C but may be carried out at slightly lower or much higher temperatures. However, the combination of polymer and cross-linker (Corcoran) yields a stable impregnation reaction-mixture even at room temperature and hence impregnation with such a product is routinely done at room temperature (Glover et al., 1998; Henry et al., 2001; Latorre et al., 2001; Raoof. 2001). Generally, China and NC B use only the polymer for impregnation (a stable impregnation bath at room temperature) and later a catalyst/cross-linker may be added. However, on hairy specimens it is desirable to use no additives. Any of these combinations have been recognized to produce useful, durable specimens (von Hagens 1979a, 1979b, 1980, 1981a, 1982, 1985; Bickley et al., 1981, 1987; Tiedemann and von Hagens, 1982; von Hagens et al., 1987; Weiglein, 1996; Zheng et al., 1996, 1998b, 2001; Glover et al., 1998; Henry, 1998: von Hagens and Whalley, 2000; Henry et al., 2002a, 2004; Latorre et al., 2002). However, it has been noted that surface clarity appears altered in specimens produced using cross-linker and polymer as the impregnation-mixture (Henry et al., 2001; Latorre et al., 2001; Henry, 2004). However, surface clarity is altered in specimens produced using cross-linker and polymer as the impregnation-mixture (Henry et al., 2001; Latorre et al., 2001; Henry, 2004). This problem continually appeared in routinely plastinated specimens (Fig. I). Therefore, it was decided to have attendees of the 12^th International Congress on Plastination evaluate the surface of representative specimens from the six plastination methodologies. The results of these evaluations are presented in this paper.

MATERIALS AND METHODS

Over a five-year period, various specimens from many species (human, domestic animals and exotic animals) were prepared in Knoxville, Tennessee and Murcia, Spain using classic techniques (prosection, dilation, vascular inJection, etc.) for rendering anatomical specimens suitable for plastination (Oostrom, 1987; Henry et al., 1997) and each method recorded. Fresh rather than embalmed tissue was used in preparation of most specimens. Fresh tissue was usually fixed in five to ten percent formaldehyde solution for a short time either prior to, du1ing or after prosection. A smaller number of embalmed specimens (primarily brains embalmed in I 0% formaldehyde solution) were collected and prosected. After fixation and prosection were completed, fixative was rinsed from the tissue via flowing tap water. Cool specimens were placed into either room temperature acetone (Brown et al., 2002) or cold acetone (-l5°C) (Schwab and von Hagens, 1981; von Hagens, 1985; Tiedemann and lvic-Matijas, 1988; Ripani et al., 1994; Henry et al., 1998) of at least 80% purity. Weekly changes into a higher percent acetone were performed until >99% purity was maintained. After dehydration in acetone. specimens from each type of processing were placed into plastination (vacuum) chambers containing one of the six silicone impregnation recipes for forced impregnation (von Hagens, 1985; Bickley et al., 1987; Henry and Nel, 1993).

Six recipes and techniques were carried out at various times over a period of five years in order to compare the quality of the finished plastinated specimens. as well as. provide new specimens for our collection. The six formularies were as follows:

1. The classic cold S 10 (BiodurTM, German, von Hagens) method and chemicals were used. Silicone polymer (SR 10) and catalyst and chain extender (SH 03) at a ratio of I 00: I were thoroughly mixed for the impregnation reaction-mixture (von Hagens. 1985: Bickley et al., 1987: Henry and Nel. 1993). Impregnation, at both -15"C and room temperature (25°C), was carried out. When the room temperature reaction-mixture was not in use. it was stored at -20°C to retard elongation of the silicone molecules and hence thickening of the reaction-mixture. After impregnation, excess polymer was drained from the specimens and the specimens remained at room temperature for several days to allow any remaining surface polymer to drain from them. Once drainage was deemed complete. the specimens were placed in a contained environment and gas cure (SH 06) was added to the environment and vaporized for 10 minutes a day with an aquarium pump (Weiglein and Henry. 1993). Specimens were manicured daily until all weeping had ceased and their surfaces were dry.
2. The VisDocta™ (Italy) method and chemicals were used: Silicone polymer (SH1) and catalyst (SH1/D) at a ratio of 100: 1 for the impregnation reaction-mixture. After thorough mixing, impregnation was carried out at -15°C and room temperature (25°C) (Shahar and Pace, 2003). When the room temperature reaction-mixture was not in use, it was stored at -20°C to retard elongation of the silicone molecules and hence slow thickening of the reaction-mixture. After impregnation, the excess polymer was drained from the specimens and the specimens remained at room temperature for several days to allow excess polymer to drain from them. Once drainage was deemed complete, the specimens were placed in a contained environment and gas curing agent (SH1/G) was added to the environment and vaporized for 10 minutes a day with an aquarium pump. Specimens were manicured daily until all weeping had ceased and their surface was dry.

3. The Corcoran (Dow™) method and chemicals were used. Silicone polymer (PR10) and cross-linker (CR 22) at a ratio of 100 to 8 were thoroughly mixed for the impregnation reaction-mixture. Impregnation at room temperature (25"C) was carried out (Glover et , 1998: Latorre et al., 2001; Raoof, 2001). When the room temperature reaction-mixture was not in use, it was stored at room temperature as this mixture is stable perhaps forever but for certain at least 8 years with no thickening of the reaction-mixture. After impregnation, the excess polymer was drained from the specimens and the specimens remained at room temperature for a few days to allow excess polymer to drain from them. Once drainage was deemed thorough, the specimens were misted with catalyst (CT 30), wrapped with foil/plastic wrap for twenty-four hours, and then inspected for extent of curing. Usually more curing time was needed. If so, the specimens were misted or wiped with more catalyst and rewrapped with foil until the next day. Their surface was checked to assert they were dry.
4. The Chinese (Su-Yi) method and chemicals were used: Silicone polymer (SuYi) alone was used for the impregnation bath (Zheng et , 1996, 1998a, 1998b, 2001). Impregnation was carried out at room temperature (25°C). When the room temperature impregnation bath was not in use, it was stored at room temperature as this bath is stable perhaps forever but for certain at least 3 years with no thickening of the impregnation bath. After impregnation, the excess polymer was drained from the specimens and the specimens remained at room temperature for several months to allow excess polymer to drain from them. At a later time some of these specimens were placed in a contained environment and the accompanying Chinese chemical (chemical composition unknown by authors but is likely a catalyst and/or cross-linker) was added to the environment and vaporized for 10 minutes a day with an aquarium pump or wiped onto the specimen. Specimens were manicured daily to determine if the damp, oily texture would decrease and yield a dry surface. The remaining specimens (Chinese) were not exposed to the catalyst/cross-linker but were allowed to remain a semi-damp.

5. The North Carolina A method and chemicals were used: Silicone polymer (Neat 285 or 295) and catalyst (S3) were combined at a ratio of 100:3. After thorough mixing, impregnation was carried out at -15°C or at room temperature (25°C). When the room temperature reaction-mixture was not in use, it was stored at -20°C to retard elongation of the silicone molecules and hence thickening of the reaction-mixture. After impregnation, the excess polymer was drained from the specimens and the specimens remained at room temperature for several days to allow excess polymer to drain from them. Once drainage seemed complete, the specimens were placed in a contained environment and 2.0 ml of chain extender (S7) was added to the environment daily for one week and vaporized for ten minutes a day with an aquarium pump. Specimens were manicured daily to alleviate weeping polymer. Then the specimens were exposed to gas cure (S6). Three milliliters of gas cure were added daily to the environment and vaporized for ten minutes a day with an aquarium pump. Specimens were manicured daily until all weeping had ceased and their surface was dry.
6. The North Carolina B method (Henry et al., 2004) and chemicals were used: Silicone polymer (Neat 285 or 295) by itself was used for the impregnation bath. Impregnation was carried out at room temperature (25"C). When the room temperature impregnation bath was not in use, it was stored at room temperature as this bath is stable perhaps forever but for certain at least five years with no thickening of the impregnation bath. After impregnation, the excess polymer was drained from the specimens and the specimens remained at room temperature for several months to allow excess polymer to drain from them. Once drainage appeared complete. some specimens (NC B₁) were placed in a contained environment and 2.0 ml of super-catalyst was added to the environment and vaporized for 10 minutes a day with an aquarium pump. Most specimens were manicured daily until all weeping had ceased and their surface was dry. The remaining specimens ( NC B₂ ). primarily hair covered, were not exposed to catalyst or cross-linker but were allowed to remain semi-damp.

As a group of specimens completed the plastination process, the cured and non-cured specimens were stored in appropriate cabinets and used routinely as needed for teaching, demonstration or display. The surfaces of the cured specimens, aged from four and one half years to ten days, were graded by attendees of the 12^th International Congress on Plastination at Murcia, Spain. For this evaluation, the specimens were assigned and tagged with random numbers and placed on tables for voluntary participation by attendees. The evaluators were asked to examine the surface of the specimens for quality and clarity of surface detail. They were asked to disregard obvious blemished areas due to handling, shipping or abuse of the specimens. They were to record their results as follows: B - For best surface detail, G - For good surface detail and O - For okay surface detail. The results were recorded for each specimen followed by the number of responses for each category of evaluation grade.

ln addition, the authors described the surfaces of the specimens which were graded in this study to provide the reader with an idea of what the evaluators were seeing and upon which they presumably based their evaluations. The authors did not participate in the evaluation at the 12^th International Congress on Plastination. The authors also described the surfaces of these specimens in their non-cured state prior to the evaluation that occurred in Spain.

RESULTS

Plastination procedures

Figure l. Plastinated camel fetuses. The fetus at top was plastinated using the classic S10 process. The fetus on bottom was plastinated using the Corcoran process

Occasionally a cured specimen would weep polymer and need to be wiped and exposed to additional curing agent to complete the curing process. Specimens with no catalyst or curing agent added needed to be wiped of weeping polymer. All specimens prior to curing exhibited great surface detail. Room temperature vs. cold dehydration of specimens and room temperature impregnation vs. cold impregnation of specimens yielded no discernable difference in surface quality. Between one week and one month after exposure to catalyst, a distortion of the surface of specimens impregnated with the polymer cross-linker reaction­ mixture was noted on all of this type specimen. The decrease in clarity of surface detail peaked by three months post impregnation and then remained at a constant level. This decrease in clarity was due to polymer invading the surface of the specimens produced via this plastination methodology. This resulted in a dull, gritty and/or blistered appearance to the surface of these specimens. The process in which specimens were impregnated with polymer only and not exposed to any catalyst/chain extender/cross-linker was especially user friendly for hair covered specimens. Because the polymer bath contained no additives, the polymer remained fluid and drained freely from the hair and prolonged or extra manicuring was not necessary.

Specimen evaluation

All classic S10 method, VisDocta TM method and North Carolina A method specimens exhibited excellent surface clarity at any time throughout the process.

The serosal surface of the bovine uterus (Figs. 2, 3) and North American opossum liver (Fig. 4) plastinated using the classic S 10 method are smooth and exhibit surface detail as is seen on fresh tissue. The margins of the liver are sharp and prominent (Fig. 4).

The VisDocta TM specimens have clear smooth margins with no excessive accumulation of polymer allowing clear visualization of all structures (Figs. 5a, 6. 7). The canine heart has a transparent epicardium such that the myocardium is visualized (Fig. 6). Brain exhibited sharp surface detail (Fig. 7).

The North Carolina processes also produced specimens with surface detail representative of fresh tissue. A feline stomach and liver display clear surface detail with sharp margins (Fig. 8). The surface detail of a porcine uterus (Fig. 9) and a canine heart (Fig. 10) is remarkable. Individual muscle fibers of the muscularis mucosa are visible through the serosa of tubular organs (Fig. 11 ).

Figure 2. Dorsal view of bovine uteri. The specimen on the left was plastinated using the Corcoran process. The specimen on the right was plastinated using the classic S10 process	Figure 3. Close up of serosal surface of bovine uteri from figure 2. The specimen on the left was plastinated using the Corcoran process. The specimen on the right was plastinated using the classic S10 process.	Figure 4. Surface of North American opossum liver plastinated using the classic S10 process.
Figure 5. Canine tibial plateau with menisci and ligaments plastinated using the VisDoctaTM process (A). Canine tibial plateau with menisci and ligaments plastinated using the Corcoran process (B).	Figure 6. Plastinated canine hearts. Specimen on left was plastinated using the Corcoran process. Specimen on right was plastinated using the YisDoctaTM process.	Figure 7. Mid-sagittal view of equine brain plastinated using the YisDoctaTM process.
Figure 8. Feline liver (top) and stomach plastinated using the North Carolina A process.	Figure 9. Serosal surface of porcine uterine horn plastinated using the North Carolina A process.	Figure 10. Canine heart plastinated with the North Carolina A process. Dark regions at arrows are blood remaining in tissue.

Figure 11. Serosal surface of tubular organs plastinated using the classic S10 process (A), VisDoctaTM process (B) and the Corcoran process (C).

All plastinated specimens produced with the Corcoran process exhibited altered clarity of surface detail with a slightly granular, translucent appearance after completion of polymerization (Figs. 2, 3, 5b, 6, 11). Raised. lumpy areas appeared on some specimens (Fig. 1). On transverse section, a heart is seen with subserosal accumulation of cured polymer (Fig. 12b). In many instances, surface detail is hidden by this subserosal accumulation of polymer (Figs. 6, 11 ). Nothing similar to this was found on any specimens produced using the other procedures of this study.

The Chinese process produced specimens on which surface detail appeared as it would on fresh tissue (Figs. 13. 14 ). The demarcation between the cortex and medulla on the cut surface of a feline kidney was excellent (Fig. 13). Occasionally, dry, white areas would appear on specimens produced with this method (Fig. 14). The surfaces of all specimens produced using the Chinese process remain slightly damp or oily feeling even three and one half years after impregnation.

The North Carolina B_I and B₂ processes produced specimens that exhibit remarkable surface detail (Figs. 15, 16, 17). One NC B₁ specimen had two (2 x 3mm) raised areas of cured polymer which had oozed and not been wiped prior to curing. This common curing artifact detracted from that portion of the surface. The surface of NC B₂ specimens, which received no curing. had great surface detail but remain slightly damp to touch.

Using the process in which hair covered specimens were impregnated with polymer only and not exposed to any catalyst/chain extender/cross-linker produced specimens with hair that looked and felt like it would on a living animal (Fig. 17). The drawback to this method is the specimens will remain damp for approximately one year.

Most of the finished specimens were used routinely as needed for teaching or demonstration prior to their use in this evaluation and hence some specimens exhibited small use/trauma artifacts.

Figure 12. Cross section of canine heart plastinated with the classic SIO process (A). Cross section of canine heart plastinated with the Corcoran process (B). Subserosal accumulation of polymer between arrows. Outer surface of myocardium at black arrows. Epicardium at white arrows.	Figure 13. Feline kidney plastinated using the Chinese process.	Figure 14. Canine duodenum and pancreas plastinated using the Chinese process. White area on left lobe of pancreas is a curing artifact.
Figure 15. Feline stomach plastinated using the North Carolina B1 process.	Figure 16. Rainbow trout (longitudinal and transverse section views) plastinated using the North Carolina B1 process	Figure 17. Hair on Jersey calf (A) and alpaca limb (B) which were plastinated using the North Carolina B2 process with no additives.

The results of the evaluation by the attendees of the 12^th International Congress on Plastination, Murcia, Spain are listed in table I.

DISCUSSION

All specimens produced during this project period were of good to superior quality and durable. Each of the three general types of impregnation has their own unique qualities and attributes. Since its invention, the classic SI 0 method is the most reliable method for production of high quality plastinated specimens. As well, the generic copies of the classic S IO method (VisDocta^TM and North Carolina A) provide equally as high quality plastinated specimens. Room temperature vs. cold dehydration of specimens and room temperature impregnation vs. cold impregnation of specimens yielded no discernable difference in surface quality or durability. Hence these specimens were not distinguished as a separate group for evaluation at the ISP congress.

The other two general types of plastination [impregnation reaction-mixture of polymer and cross­ linker (Corcoran) and polymer only in the impregnation bath (Chinese and North Carolina B)] also produced good to high quality specimens. However. the quality or specimens produced by these processes seems to be less predictable and yet all have their bright spots including a shortened impregnation time, a decreased need for freezer space for impregnation as well as the ease of polymer drainage and manicuring. The benefits in the ease of polymer drainage were most evident on hair covered specimens. The hair on specimens produced by other processes needed to be wiped excessively to remove the polymer and, even so, the hair of these cured specimens often contained some cured polymer giving the hair a matted, unnatural appearance. Currently. curing of the polymer is less predictable with these types of impregnation. If the specimens are left with a damp surface. they may not be as aesthetically pleasing to some. However, the benefit of life-like hair is great for hairy creatures.

The Chinese method recommends not to cure the impregnated specimen. Attempts to cure a few Chinese specimens by vaporizing the intended product as well as wiping it on the specimen mixed with polymer were not successful. The feline pancreas/duodenum was an example of this curing method and the surface detail was obscured in a few areas. Zheng and colleagues ( 1998a) recommend to mix old polymer with 1-5% hardener and wipe it onto the specimen su1face to produce a dry specimen. This methodology was not used in our studies as it yields specimens with a high sheen and coat of polymer on the surface.

The negative feature of the Corcoran method (polymer cross-linker impregnation reaction-mixture) is that there is poor clarity of surface detail of the impregnated specimens after curing. The appearance of the semi-translucent to opaque surface accumulation of polymer after polymerization to date has not been explained. This granular surface detracts from the beauty of the specimen and renders less prominent anatomical detail more difficult to visualize. Beyond this, nothing else has been observed that decreases the usefulness of these specimens.

The evaluation by the attendees of the 12^th International Congress on Plastination tends to support the findings of the authors. However, there is a high number of "G's" (mid-grade) among all types of specimens. This is a bit worrisome, but it may be that many felt more comfortable staying near the median rather than committing to either extreme. Also, it was not known that this evaluation was for part of a publication as we did not want to bias the evaluators. Additionally. some specimens had some "wear and tear" from usage which we hoped would not be included for or against the specimen during its evaluation. If we were to do a similar evaluation again, to narrow the choices to two or to widen to five choices may have moved the "G's" to one pole or the other. However. the high number of "G's" may support the fact that surface clarity may not be a major issue for the person new to plastination or even to an established plastinator.