INTRODUCTION

Silicone plastination replaces tissue fluid with a curable polymer. Besides the Biodur™ products, other silicone polymers and chemicals, all well-known in the silicone industry (Henry et al., 2002a), have been developed for use in the plastination process (Henry et al., 2002b). Forced impregnation of silicone polymer into biological specimens is the common thread for all of the generic plastination products . Impregnation uses the same intermediary solvent (acetone) along with a decrease in pressure to extract the acetone (von Hagens, 1979a; 1979b; 1986; von Hagens et al., 1987; Henry, 2004). This resultant tissue void allows the impregnation-mix to be drawn into the specimen. Each generic product produces a durable, high quality plastinated specimen.

Chemicals used in the various "alternate" silicone­ plastination processes need to be similar to the Biodur™ SlO plastination technique :

• Acetone
• Methylene chloride
• Silicone polymer
• Cross-linker, enables side to side linkage and formation of a 3-D meshwork to the elongated silicone molecules
• Chain extender, promotes the silicone molecules in formation of longer-chain silicone molecules
• Catalyst, prepares the silicone molecules for elongation and cross-linking

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

MATERIALS AND METHODS

The basic steps of plastination are utilized for each plastination process:

Specimen preparation dehydration and defatting

Specimen preparation, dehydration and defatting are the same for all plastination methods . Please refer to: "The S10/15 cold-temperature plastination technique" for that information . As well, refer to the "Dow room­ temperature technique" for additional information.

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 or methylene chloride) in a specimen with a curable polymer. For this to happen, the products must meet the same conditions as for the Biodur™ S10 plastination process .

Impregnation equipment: Similar to Biodur™ S10 requirements [vacuum pump (oil preferred) and chamber with see through port and specimen basket, vacuum gauge, manometer , needle valves]. However, no deep freezer is required or used for impregnation. Deep freezers are necessary and strongly recommended for dehydration.

Preparing the impregnation-mixture: NCSX or NCSXI polymer is mixed with NCSVI (Cross-linker) at 100:8 to prepare the impregnation-mixture and stirred thoroughly. The main difference in this room temperature methodology is handling of the resultant reaction-mixture. This mixture is stable (does not become viscous) when stored and/or used at room temperature for an indefinite period of time as is the case with other generic room-temperature polymers.

Adjusting  the  vacuum: Speed of lowering the  pressure in the vacuum chamber is fast when  compared to the Biodur™ S10 Cold-temperature  Technique. The NCSX and NCSXI polymers have a much lower viscosity than Biodur™ S10 or Dow^TM   PR 10. The impregnation­ mixture is not reactive,  provided it is not exposed to catalyst. There-fore the  polymer-mix remains fluid at impregnation (room)  temperatures As well, since there is no increase of viscosity which results from placement in a cold   environment. When monitoring bubble formation, a  rapid boil is recommended. Remember the plastination   principal , if in doubt, decrease pressure slower, to prevent incomplete impregnation which can result in shrinkage.

Impregnation  regimen

Day 1: The dehydrated and degreased specimens are removed from the solvent (acetone or methylene chloride). Excess solvent  is drained and the dehydrated, solvent-filled specimens are placed in the room-temperature    polymer    impregnation-mixture. Submerge the specimens immediately to prevent solvent evaporation from their surface and hence, drying. The port (glass) on ·the vacuum chamber is closed and the specimens allowed to accommodate/ equilibrate in the polymer-mix overnight.

Day 2: The vacuum pump is warmed briefly. Close the needle valves and lower pressure (apply vacuum) to seal the chamber. Lower the pressure until bubble formation becomes rapid (decrease to around 30- 25cm/13 .5-13in Hg pressure) . When a rapid boil is achieved, maintain this pressure by opening the vacuum adjustment valves slowly until pressure is stabilized. The vapor pressure of acetone at  room temperature (+25°C) is 22cm/10in Hg and MeCl is 43cm/17in Hg. Continue to monitor the pressure and decrease the pressure as needed to maintain a rapid boil by closing the needle valves incrementally . Likely adjustment will be every one half hour for the first three or four hours.

Day 3: Continue to monitor bubble production (solvent extraction) and keep a rapid boil by decreasing pressure (closing the valves) as needed.

Day 4a: Small specimens - Impregnation will be complete as noted by cessation or reduction in number of l-2cm size bubbles forming and/or reaching < lcm Hg of pressure. Note: When pressure goes below 4 or 5 mm Hg, larger bubbles will rise and burst if the chamber is tilted or shaken. These are not likely the solvent (acetone) but likely water vapor or the cross­ linker, NCSVI vaporizing . Vacuum should be turned off when this occurs. Tum off pump and bring specimens to atmospheric pressure . Allow the impregnated specimens to sit in the polymer-mix over night at atmospheric pressure.

Day 5: Proceed to Step 4 - "Curing".

Day 4b: Large specimens or a large quantity of specimens . Continue to monitor solvent extraction by watching bubble formation and by reading and adjusting pressure to maintain a rapid boil (extraction of the solvent).

Day 5 or plus X: Continue to monitor solvent extraction (watch bubble formation) and maintain a rapid boil. If impregnation is completed , as noted by cessation or reduction of 1-2 cm size bubble formation and/or reaching 5mm Hg of pressure, tum off pump and bring specimens to atmospheric pressure (see note in Day 4a). Proceed to Step 4 - "Curing".

Rule: If 1-2 cm bubbles are actively rising to the top of the polymer and bursting, impregnation is not finished! Impregnation will be complete when both needle valves are closed, pressure has reached <5mm Hg and/or l- 2cm diameter bubble production is greatly diminished. Note, once the 1-2cm bubbles have subsided, larger 4-5 cm bubbles will explode to the top if you shake the vacuum chamber. These are likely not acetone bubbles but either water vapor or cross-linker (NCSVI) vaponzing. Nearly complete evacuation of acetone/solvent is necessary to avoid incomplete impregnation of the specimen with the polymer-mix which may lead to shrinkage.

Specimen removal and drainage of surface polymer impregnation-mix: Follow the S10 protocol.  Since the impregnation-mixture is stable, drain specimens into the room-temperature plastination  chamber.

Curing (hardening or cross-linking)

Equipment for curing:

• Absorbent paper to wipe excess polymer-mix from the
• Closed environment for NCSV application via
• Gloved finger, paint brush or mist bottle to apply NCSIII (cross-linker) to the Do not reintroduce gloved finger or brush into stock reservoir of catalyst if specimen has been touched. The stock solution will be contaminated with cross-linker and/or polymer and at first become viscous and eventually solidify .
• Foil (plastic wrap) to seal the specimen in an air­ tight environment and keep the Catalyst (NCSIII) next to the impregnated specimen.
• NCSV - chain extender and NCSIII - catalyst

Curing of the impregnation-mixture within the specimen is a three-step process:
Drain:  Drain  the  excess  impregnation  polymer-mix from the specimens.

Chain   elongation:  Chain  elongation  of  the  siliconepolymer molecules by end to end alignment of the molecules. Chain elongation occurs as the NCSV vapor is applied to the surface of the specimen. NCSV is vaporized in an enclosed chamber by using an aquarium pump or ventilator for a few minutes once a day for one to three days. This process is similar to the cross-linking application in the Biodur™ S10 Cold- or Ambient­ temperature  technique.

Catalyzing and cross-linking: Catalyzing and cross­ linking of the silicone polymer molecules. This reaction occurs when the catalyst (NCSIII) is applied to the surface of the impregnated specimen. It enables the NCSX or XI molecules in the impregnation-mix to react with the NCSVI which is in the impregnated specimen. Cross-linkage changes the polymer from a liquid to a solid. Mist or wipe NCSIII onto the surface of the specimen, wrap specimen with foil and leave over night. Wrapping in foil (plastic wrap) is necessary to facilitate  cross-linking.

RESULTS

The NCSX or XI impregnated and cured specimens are dry and durable. Thin specimens exhibit some flexibility. The room-temperature plastinates are not models but real specimens (Figs. 1, 2, 3, 4). They make excellent teaching and public relation aids.

Figure I. Lateral view of feline left thoracic limb - North Carolina Room-temperature silicone technique	Figure 3. Auricular surface of canine heart - North Carolina Room-temperature silicone technique .
Figure 2. Bowfin fish - North Carolina Room-temperature silicone technique.	Figure 4. Dorsal view of prosected equine brain stem - North Carolina Room-temperature silicone technique

DISCUSSION

As with all plastination processes, the room­ temperature plastination produces real specimens and not models. The specimens are dry and odorless. Only time will reveal if the NCSX develops a less than transparent surface which conceals intricate surface cellular detail as seen with the Dow™/Corcoran chemicals (Henry et al., 2004; Smodlaka et al., 2005a; 2005b). Room-temperature technique specimens are used as teaching aids (Latorre et al., 2001). They have been used to compile a library of specimens for normal, exotic (Fig. 2) and pathological anatomy (Henry, 2005) and is useful in research (Raoof, 2001).