INTRODUCTION

Silicone plastination replaces tissue fluid with a curable polymer. Several other silicone polymers and additives, other than the Biodur^® products, have been developed to carry out the silicone plastination process (Henry et al., 2001). The basic ingredients of each process are well known products of the silicone industry (Henry et al., 2002). Principally, methods of placing the silicone into the biological specimen are similar: Using an intermediary solvent (acetone) along with a decrease in pressure to remove the acetone (von Hagens, 1986; Henry and Nel, 1993; Henry, 2004). Removal of the solvent produces a tissue void and the polymer-mix is drawn into the cells. Each of these variants of the cold temperature silicone plastination technique produces high quality durable plastinates.

Chemicals used in this "generic" silicone-plastination process are similar to the ones used in the Biodur^®  S10 plastination  process:

• Acetone and possibly methylene chloride
• Silicone polymer
• Catalyst to prepare the silicone molecules for elongation and cross-linkage
• Chain extender to cause the silicone molecules to form longer-chain silicone molecules
• Cross-linker to form a 3-D meshwork of the elongated silicone molecules by side to side linkage

The general steps of silicone plastination are described earlier in this volume. This manuscript will highlight the differences with respect to this generic process which occur with impregnation and curing of the plastinated specimens.

MATERIALS AND METHODS

The basic steps of plastination are utilized for each plastination technique : Specimen   preparation,   dehydration     and   defatting (degreasing)

Specimen preparation, dehydration and defatting are similar  for  all  plastination  methods.  However,  it  is necessary to prepare high quality dissected specimens to produce excellent plastinates . Please refer to:  "The SI0/15 Plastination Technique" for the basic, as well as, additional and more in depth information for specimen preparation and cold dehydration.

North  Carolina  products  for silicone plastination: NCSX (lowest viscosity): Silicone polymer NCSXI (low viscosity): Silicone polymer NCSIII: Catalyst NCSV: Chain extender NCSVI:  Cross-linker

Forced Impregnation

Replacing the volatile solvent (acetone/methylene chloride) in a biological specimen or tissue with a curable polymer. For this to occur, the chemicals must meet similar criteria as the chemicals used in the "Biodur® S10 Cold-temperature Technique".

Impregnation equipment: Similar to Biodur® S10 requirements - Vacuum chamber (kettle), Vacuum pump (oil preferred), Vacuum gauge, Vacuum tubing, Needle valves, Manometer, Specimen basket and Deep freezer.

Preparing     the     reaction-mixture:    NCSX    or   NCSXI polymer is thoroughly mixed with NCSIII  (Catalyst)  at 100:20-30    to    prepare     the    impregnation    reaction­ mixture .  The  reaction-mixture   is stored  lower  than  - 25°C  and  used   for   impregnation in a - l 5°C deep freezer. The  impregnation-mix  is  not  stable  at  room temperature  and will become  too viscous  over a 6-10 month    interval   if   stored   and/or    used    at    room temperature  continually.

Adjusting the vacuum: Pressure is generally lowered by closing the air intake valves which are located in line between the vacuum kettle and pump. Speed  of lowering the pressure in the vacuum  chamber  is slow and comparable to the Biodur^® S10 process.  The NCSX and XI polymers have a lower viscosity than Biodur^® S10 and hence, impregnation may be faster. The impregnation-mixture is  reactive  and  therefore needs to be kept at cold temperature. When monitoring bubble formation, a slow boil is recommended . As with any plastination procedure, if in doubt, reduce pressure slower, to prevent incomplete impregnation which will result in shrinkage.

Impregnation regimen: Similar to the Biodur^® S 10 Cold-temperature technique (Table 1).

Rule: If bubbles are actively rising to the top of the polymer and bursting, do not decrease pressure! It is better to decrease pressure too slow rather than too fast.

Once bubbles are active, solvent vapor pressure has been reached. More pressure decrease is not recommended  until  bubbling  activity  slows  or  nearly ceases. Bubbles should rise slowly but continually as simmering water, not as rapid boiling water. Acetone removal and hence polymer impregnation at cold temperature will likely take 3 to 5 weeks depending on volume of specimens and pump speed. **If bubbles cease to rise and the pressure is >3mm Hg, close the needle valve to decrease the pressure  until  active bubbles start to rise again. Usually it is necessary to lower pressure only 1 mm to resume active bubble production (vaporization of solvent); but it may take a few minutes before bubble production is observed.

Evacuation of acetone/solvent too quickly will result in incomplete impregnation of the polymer-mix into the specimen and result in shrinkage.

Impregnation at -15°C is complete  when  needle valves are closed and no more acetone bubbles appear at the surface of the reaction-mixture for several hours and/or near zero pressure has been maintained for a day. Specimen removal:  Similar to Biodur^® S 10 Cold­ technique . Drain excess surface polymer back into the impregnation kettle.

Curing and Hardening/Cross-linking

Equipment for curing:

• Similar to the Biodur^® S10 technique. However, the chain extender (NCSV) is utilized separately after impregnation, prior to cross-linking.
• Closed environment for NCSV application - The same type or same chamber as for cross-linking is used: Air-tight closed container, large enough to contain the
• Aquarium pump or small ventilator (fan, spark free): To volatilize the NCSV or
• Desiccant (CaS04): To remove moisture  from  the air around the specimen in the curing/chain extension  chamber.
• Compressed air connection, tubing and connectors: To dilate specimens and/or to evacuate polymer trapped in the organ.
•  Absorbent paper: To wipe excess polymer-mix from the specimen surface.

Curing, hardening and cross-linking the reaction-mix within the specimen to make the specimen dry is a two­ step process.

Chain extension of the silicone molecules  is  end  to end linkage of the silicone molecules. After the impregnated specimens have been drained  of  their excess polymer and wiped, the chain extender (NCSV) is applied via vaporization of the  NCSV in a closed environment for two or three days. During this time, longer silicone molecules  are forming.  After  exposure of NCSV to the specimens, the  specimens  should remain at room temperature for a few days to a few weeks to allow maximum chain extension (slow cure). During this time the specimens must be positioned anatomically correct and dilated to assure  correct position as chain extension progresses.

Cross-linking or connecting the silicone polymer molecules side to side, thus forming a firm  3-D meshwork of the silicone polymer, is caused by the NCSVI component. This reaction and process is similar to that of Biodur^® S 10 cold-technique. The drained and chain lengthened specimens are place in a closed environment with 10 - 20ml of NCSVI (cross-linker). The NCSVI is volatilized for 5 -10 minutes which commences the cross-linking of the silicone on the surface of and in the specimen.

RESULTS

The NCSX or XI cold-impregnated specimens have the same great qualities of the Biodur^® S10 cold­ technique specimens, including clarity of surface detail (Fig. 1). They are dry, durable, free of offensive odors and thin specimens exhibit some flexibility . North Carolina plastinated specimens are the  real  specimen and not a model (Figs. 1-3). NC cold-temperature specimens are used as teaching aids both in the class room and in clinics. When assembled in a group, they make  an  impressive  library  of  specimens  of  normal, exotic and pathological anatomy. Silicone preserved specimens are useful in research, especially to preserve findings.

Figure 1. Pathological specimen: Cat Kidney Lymphosarcoma metastasis. North Carolina Cold-temperature technique.

Figure 2. Camel liver, parietal surface. North Carolina Cold­ temperature technique.

Figure 3. Camel liver, visceral surface. North Carolina Cold­ temperature technique

Figure 4. Dog heart auricular surface. North Carolina Ambient-temperature technique.

DISCUSSION

NCSX or NCSXI cold (Figs. 1-3) or ambient­ temperature (Fig. 4) impregnated specimens have the great qualities of specimens produced by the Biodur® S10 ambient and cold-techniques, as well as superb clarity of surface detail (Smodlaka et al., 2005). They are dry, durable and free of offensive odors. As well, flexibility is inversely proportional to  specimen thickness. Both cold and  ambient-temperature specimens have been used as teaching aids both in the class room and the clinical setting (Latorre et al., 2001). Also, specimens have been used to compile a library of specimens for normal, exotic and pathological anatomy (Henry, 2005a; Sakamoto et al., 2006) and in research.

It should be understood that in cold, impregnation, using a deep pool of  polymer >40cm, makes it  difficult to get the pressure low enough for the solvent (acetone) to vaporize from the bottom specimens (Henry and Thompson, 1992; Henry, 2005b). The departing bubbles have to overcome the weight of the overlying polymer (von Horst, 2005). Therefore impregnation should be monitored closely.

The NCSX or XI polymer and NCSIII catalyst may also be used for impregnation at ambient-temperature . The protocol is similar to that of the "Biodur^® S10 ambient-temperature technique" (von Hagens, 1986). Impregnation can be completed in one week . The same precaution with the polymer impregnation-mixture must be adhered to. After impregnation , the mix must be  returned to at least a minimum of a -25°C freezer to minimize thickening of the impregnation reaction-mix .