Plastination: an overview of the techniques and its application in pathologic tissue preservation

INTRODUCTION

Plastination is a technique or process used to preserve bodies or body parts thus permitting the preservation of anatomical specimens in a physical state, approaching that of the living condition.

This technique was first developed by Dr. Gunther von Hagens in 1977.

The term “Plastination” is derived from the greek word “Plassein”- meaning “to shape, to form”. This technique which was first adopted in Europe and then North America has now been rapidly emerging to be of great use in anatomy halls and pathology laboratories and is now only entering the field of oral pathology.¹

Using this technique, allows preservation of anatomical and surgical specimens for a long time without surface morphological modifications. The resulting specimens are dry, odourless, durable, non-hazardous and maintenance free.

Principle of Plastination:

Plastination consists of forced impregnation of biological tissues with plastic resins. The principle involves removal of water and lipids from the tissues and their replacement by a plastic (curable polymer), thus yielding specimens which we can touch, do not smell or decay and retaining properties of the original sample.

Curable polymers are used such as epoxy, polyester or silicone and each of these resins produce variations in rigidity and opacity of the final product. (von Hagens 1979)

The class of polymers used determine the optical properties (transparency or opacity) and mechanical properties (flexibility or firmness) of the impregnated specimens.

Gross specimens, dissected specimens and crosssectional slices can be preserved permanently into specimens which are clean, dry and practical to use. Hence the irritating and harmful effects of older preservative liquids like formaldehyde are prevented by the plastination process.

The standard process of plastination involves four steps:

1. Fixation. 
2. Dehydration. 
3. Forced impregnation in vacuum. 
4. Curing and hardening.

(1) Fixation

Fixation of tissues is done by pumping formalin into the specimen preferably through arteries. Formalin kills all bacteria and chemically stops the decay of tissues. Using dissection tools, the skin, fatty and connective tissues are removed in order to prepare the individual anatomical structures. Smaller specimens are immersed directly in the fixative.

(2) Dehydration

Dehydration removes specimen fluid as well as some fat. In this step, tissue fluid is replaced with an organic solvent. Alcohol or acetone is used as a dehydrating agent for plastination. Acetone is preferred as it also serves as intermediary solvent during the next step. Cold acetone (-150c to 250c) used for dehydration minimizes specimen shrinkage.

If fat also has to be removed, the dehydrated specimen must be kept in acetone at room temperature for the same time. Best results are obtained if acetone used is 10 times the specimen weight. When the water content is less than 1%, dehydration is said to be complete.

(3) Forced impregnation in a vacuum

The central step in plastination. A reactive polymer, e.g.: silicone rubber replaces the acetone by forced impregnation. The specimen is immersed in a polymer solution and placed in a vacuum chamber. The vacuum removes the acetone from the specimen and helps the polymer to penetrate every cell.

(4) Curing and hardening After vacuum

impregnation, the specimen is positioned as required. Anatomical structures are properly aligned and fixed with the help of wires, needles, clamps and foam blocks.The specimen is hardened with gas, light or heat, depending upon the polymer used.

Silicone plastination of Pathology specimens^{2, 3, 4, 5, 6}

Used for whole specimen, thick body and organ slices. Is commonly used for plastination of head and neck pathological specimens. Silicone rubber polymer is most widely accepted for teaching purposes.

Chemicals used:

1. Acetone. 
2. Silicone polymer (* NCS X or XI ) 
3. Catalyst ( NCS III ) – to prepare molecules for elongation and cross linkage. 
4. Chain extender (vapour NCS V ) – promotes function of longer chains of silicone molecules. 
5. Cross linker (NCS VI ) – by side to side linkage forms a 3D meshwork of elongated silicone molecules.

The silicone polymer mixture was composed of NCS10 and the cross-linker, NCS6 (North Carolina products) at a ratio of 90:10. [*NCS- North Carolina products for silicone plastination]

Procedure

1. Specimen preparation (Dissection and fixation). 
2. Dehydration.
3. Defatting (Degreasing).
4. Forced impregnation. 
5. Curing and hardening. 
6. Finishing and storage.

Specimen preparation (Dissection and fixation)

Fresh tissues yield more flexible and pristine samples. Embalmed, formalin fixed specimens may be used. Specimen fixed with Kaiserling fluid enhances colour of the specimen.

Composition of Kaiserling fluid-

300 gm – potassium acetate

150gm – potassium nitrate.

200ml – formalin.

800ml- demineralised water.

Volume of Kaiserling fluid used should be 10-20 times the volume of specimen.5-20% formalin can also be used for fixation. (fig 1)

Fixation bath is maintained at 50 c to enhance colour retention. In order to maintain proper shape of specimen and also to ensure proper curing, specimens are fixed in their desired contour.

After fixation is complete the specimen is checked for any loosened and / or unwanted fragments. Form / shape of the specimen and surface detail are examined for any discrepancy.

The following criteria has to be noted:

• Specimens such as brain which autolyze rapidly or which get contaminated with pathogens should always be fixed.
• Fixatives containing alcohols, glycerine, glycols, and / or phenol should not be used as they cause brittleness or interfere with curing.
• Fixation imparts some rigidity, so the specimen should be fixed in the form that it will exhibit when finished. Eg: Small solid organs such as tongue, should first be rinsed in tap water over night, infiltrated with fixative (5-20% formalin) and then immersed in 5% formalin until fixed.

(2) Dehydration

Dehydration replaces tissue fluid/ water with an organic solvent. Cold acetone is the universal dehydrating solvent. Alcohol may be used and in such cases the specimen must be saturated with acetone or methylene chloride to act as intermediatery solvent.

Dehydration at -250c acetone causes the water in the specimen to freeze and stabilize the form, structure and size of specimen hence minimizing shrinkage. This technique is known as “freeze substitution” and is the most preferred method of dehydration for plastination. (Schwab and von Hagens 1981, Tiedemann and Ivic 1988, Brown et al 2002). (Fig 2)

Principle of dehydration

1. Pure acetone with a 1:10 specimen:acetone ratio is used. A fully dehydrated specimen is obtained after 3 weekly changes. 
2. Steering or agitating the acetone specimen mixture daily usually hastens dehydration with equilibrium resulting in 4 or 5 days. 

(3) Defatting (Degreasing)

The removal of excess fat/lipid from the specimen is defatting. The dehydrated specimen is removed from the deep freezer and placed at room temperature for several days to weeks. The receptacle with specimen is sealed with plastic wrap (foil) to decrease loss of solvent by evaporation. The receptacle is checked weekly for colour change of acetone from clear to yellow. The transparent acetone turns yellow as lipid accumulates in the solution. (Fig 3)

The specimen is transferred to a fresh 98%-100% acetone bath at room temperature to continue the process of defatting until the fat on the specimen begins to appear opaque from the usual white colour.

After defatting, the specimen is ready for impregnation.⁷

(4) Forced impregnation

It is the process of replacement of the volatile solvent in the biological specimen with a curable polymer. Forced impregnation creates a force (vacuum) to get the reaction mixture inside the specimen.

The solvent fixed specimens are submerged in the liquid silicone reaction mixture in a vacuum kettle (plastination chamber) which is in the -150c deep freezer. The vacuum pump is turned on and the solvent then evaporates and leaves the specimen by being pumped out through the vacuum pump exhaust.

Due to vaporization of acetone from the specimen, a tissue void or negative pressure is created inside the specimen and the reaction mixture is drawn into the specimen.

Small bubbles rise through the polymer mix and impregnation is said to be complete when bubbles stop to rise on the specimen surface. The specimen is now ready for curing. Rapid boiling results in incomplete impregnation.

(5) Curing and hardening

During curing, the impregnation-reaction mix within the specimen is cross linked and the specimen is made dry. This is a two-step process consisting of chain extension and cross linkage of polymer.

Chain extension

Chain extension of the silicone molecules is an end to end alignment forming longer chains via the chain extender (vapour NCS V). Chain elongation occurs as the NCS V vapour is applied to the surface of specimen.j

NCS V is vaporised in an enclosed chamber by using a pump or ventilator for few minutes once a day for 1-3 days. As vapour is used for curing it is known as “gas curing”.

Cross linking:

Cross linking is connecting the silicone polymer molecules side to side forming a firm 3D meshwork of the silicone polymer. Cross linking starts on the specimen surface and proceeds inwards to the depths of the specimen. Larger specimens may take longer time than the smaller specimens.

Cross linking starts when the catalyst (NCS III) is applied to the surface of the impregnated specimen. It enables the silicone polymer molecules (NCS X or NCS XI molecules) to react with the cross-linker molecules (NCS VI) which are already present in the impregnated specimen.

The catalyst is applied to the specimen surface via a mist bottle or brush and then wrapped in the foil. The foil is removed next day and the specimen is checked to see if it is cured (dry). If curing is incomplete, the catalyst is reapplied after wiping the specimen, wrapped in foil and checked the next day for complete curing. Depending on specimen size, the final deep curing may take a week or two. The specimen is ready to use once curing is completed.

(6) Finishing and storage

The specimen is trimmed with scalpel using dilute detergent as a lubricant. The specimen is then mounted for display purposes.

Plastination using epoxy and polyester resins are also effective in obtaining good specimens.

Epoxy and Polyester plastination:^7,8

Epoxy plastination(Sheet plastination):

1. Epoxy resins plastination is used for thin, transparent body and organ slices of 2-4mm thickness. Specimens produced are of excellent visual acuity.
2. Polymerisation is done by heat treatment at 250 c for few days.

3) Chemicals used:

a) Acetone.

b) Methylene chloride(defatting)

c) Epoxy resin.

d) Hardener , to link epoxy molecules.

4) Curing Method:

It can be done either in flat chamber or by stacking the specimen in foil at a temperature of >18o c for seven days, but 25o c is considered to be the best and maximum temperature for curing. After curing, specimens may be tempered by placing in a 50o c oven for five days to increase the durability of the specimen. After thorough curing and hardening, the specimen is ready for permanent use.

Polyester plastination:

1.  Polyester-copolymer plastination is exclusively used for brain slices (4-8mm thickness) to gain an excellent distinction of grey and white matter. Clear nervous tissue is also obtained by this technique.
2. Polymerisation is done by using UV light exposure for 45 min & is completed in the oven at 450 c for few days.

3) Chemicals used:

a) Acetone.

b) Polyester resin.

c) Catalyst.

4) Curing Method:

Once the specimen is properly impregnated, it is then cured and hardened suitably. UV light serves as a catalyst for polyester plastination. Exposure time is 45 min. During UV light exposure it is necessary to cool the chambers either by ventilator or by blowing compressed air. Cooling is important because the UV light commences an exothermic reaction that may harm the specimen. Following light curing, specimens are placed in an oven at 40o c for 4-5 days. Heat finishes the curing of the resin into the depths of the specimen. After curing is complete, the excess cured resin is trimmed off and the edges are smoothened which gives the final plastinated specimen for permanent use.

Advantages of Plastination

1. Aesthetically pleasing specimens and dry to touch.
2. Odourless and life like specimens.
3. Easy to handle.
4. Structural integrity of specimens remains sound.
5. Structures remain intact.
6. Non toxic.
7. Rare or unusual specimen can be made available for study when not available in clinical practise.
8. No maintenance required.
9. Specimens can be retained for years together with free potential for histological examination

Disadvantages of Plastination

1. Process is technique sensitive. 
2. Time consuming. 
3. More equipment required.
4. Polymers used may be costly. 
5. Specialised personnel required (technician or pathologist). 
6. Lots of posturing work required such as trimming, polishing, colouring and mounting. 
7. Wastage of rare specimens may occur if beginners do lots of trial and error.

Uses of Plastination^7,8,9

1. As teaching aids in Anatomy and Pathology.⁷ 
2. As patient education tools as an augmentation to MRI (Magnetic Resonance Imaging) and CT (Computer tomography). 
3. When adequate fixation protocol is followed plastinated specimens can be used for electron and light microscopic studies. (Giles Grondin et al) 
4. Plastinated specimens can be of great use in retrospective epidemiological studies of archived pathology specimens. 
5. In practise of arthroscopic techniques.(Tiedemann, 1988) 
6. Plastinated specimens have also become popular in zoological and botanical fields for the preservation of insects, fish and fungi. (Bukley et al,1981) (von Hagens et al 1985, 1987) 
7. Valuable in medico-legal cases. 
8. For preservation of autopsy or surgical tissue samples to be used as teaching aids.⁹ 
9. For prosthetic replacement of facial organs eg: nose and ear.

SUMMARY AND CONCLUSION

The preservation and demonstration of anatomical specimens that retain much of their natural features has been the goal of anatomists, pathologists and other teachers of medical education.

Plastinated specimens require little storage space and no maintenance.

Utilising long term fixed specimens for plastination results in good preservation of specimens for teaching and observation. A good histopathological laboratory can develop a simple plastination plant and by developing the technique of plastination can also maintain a student-oriented museum for preserving rare specimens for post graduate use. Thus, a library of specimens for normal, exotic and pathologic anatomy can be maintained.