THE ANATOMICAL RECORD 204295-299 (1982) The Technique of Heart Plastination K. TIEDEMANN AND G.v. HAGENS Anatomical Institute I, Uniuersity of Heidelberg, Im Neuenheimr Feld 307, 6900 Heidelberg, Federal Republic of Germany ABSTRACT Plastinated hearts are natural specimens, preserved in a firm or flexible state, which thus can be grasped in the hand. They are fixed in a dilated state. Their tissue water and lipids are replaced by polymerized synthetic resins or elastomers. The final specimens show doubly injected coronary vessels, opened atria and ventricles, thus exhibiting all finer external and internal details. The procedure described consists of the removal of the heart; intermediate storage facilities; dilatation with water under hydrostatic pressure; color injection of the coronary vessels; fixation; dehydration in acetone under hydrostatic pressure; forced impregnation in a vacuum chamber; and hardening with aftercare. Technical variations and pitfalls are discussed, as well as the required equipment. In the teaching of anatomy, pathology, and surgery, the heart is one of the few organs which cannot be grasped through a lecture. As is the case of the brain, its complex external and internal shape is understood only when an open cut specimen is actually grasped in the hand. Artificial models do not reveal finer structural details. As they are uniformly manufactured, peculiarities of different species, congential malformations, and pathological alterations cannot be demonstrated. Dry specimens preserved with conventional methods, such as freeze drying (Hower, 1979) or paraffinization (Schmeidel, 1925; Hochstetter, 1927), are brittle and the intransparent paraffin masks finer details. Cleared and corroded heart specimens, plastic-embedded after a vascular injection (Wolfe, 1956) aim only a t the perfect demonstration of the heart's vascular system. Through the new process of plastination (v.Hagens, 1979a), real specimens are impregnated with thermosetting resins or elastomers of desired optical qualities (v.Hagens, 1979b) which replace the original tissue water and part of the lipids. A mechanically resistant flexible or firm specimen of approximately natural size which is odorless, dry, and durable is obtained. Plastinated hearts, which have been fixed in their dilated state to improve their instructive value, not only show doubly injected coronary vessels, but are fenestrated and thereby reveal all the internal structures (Fig. la, b). 0003-276x182/2043-0295$0200 fi) 1982 ALAN R. LISS, INC PROCEDURE The sequence of the main steps is as follows: 1)Removal from the body 2) Dilatation under hydrostatic pressure 3) Color injection of the coronary vessels 4)Fixation under hydrostatic pressure 5) Dehydration under hydrostatic pressure 6) Impregnation in a vacuum chamber 7) Hardening and aftercare Removal All public vessels must be cut far away from the heart, as they must be tightly tied off in order to accomplish dilatation (step 2). The large lung vessels must be loosened away several centimeters from the lung tissue. Removed hearts can be stored in a frozen state or processed after rigor mortis. Fresh hearts can be stored in tap water for 5-10 days in a refrigerator (+ 5°C). Dilatation In order to accomplish dilatation, a short polyethylene tube ($3 8-15 mm) is inserted into one of the lung veins and a second tube into the anterior vena cava as shown in Figure 2. The tubes, which hold better when they have a ridge made with a soldering iron, are connected t o a water tap. The water removes blood clots and fibrin and shows which of the apReceived January 13, 1982; accepted July 22, 1982. 296 K. TIEDEMANN AND G.v. HAGENS Fig. 1.a. Plastinated dog heart, left side view. Both ventricles are cut open, the right one exhibiting parts of the tricuspid valve. From the doubly injected coronary vessels, the red injected arteries are indistinct due to the black and white reproduction. b. Right side view of the same heart, the atrium fenestrated by a flap. proximately 25 vessels must be tied off. Cork stoppers are used to retain the rounded shape of larger vessels (Fig. 2). Dilatation is performed with tap water under hydrostatic pressure with the tubes connected to a water tank located 40-80 cm above the organ. The pressure at the origin of the aorta results in a complete rinsing of the coronary vessels, and the continued pressure dilatation, sustained for several hours in a dog heart and up to 2 days in a cattle heart, leads to a permanent relaxation of the heart muscle. jection of the red resin with a disposable syringe forces the resin as far as the arterial capillaries and is ended by tying off when the myocardium and the adipose tissue have taken a reddish color, which gives the whole specimen an almost natural tone. For the retrograde color injection of the heart veins with the blue epoxy resin we use the coronary sinus via the posterior vena cava. In small species, such as the dog, the tying off of the coronary sinus with a thread is difficult because of its tenuous wall. We therefore use polyethylene tubing, with a thicker piece of tubing attached (Fig. 3B),which, passed through the vena cava, can just be pressed into the coronary sinus. This tubing remains in place after the injection until the resin is gelated. In cattle, we tie off the coronary sinus around a thick cone of a needle (Fig. 3A); a finger can be inserted into this large vessel t o feel the tip of the curved tweezers which force the thread around the vessel. The great cardiac vein is easily filled;the resin can be forced further into the smaller branches by rubbing the bulging Color Znjection For the injection of the coronary vessels with a colored epoxy resin, red and blue BIODUR E 201with the hardener E 2 is used. The coronary arteries are injected from within by the use of a needle with a cone at its tip (Fig. 3A). This needle, introduced into the brachiocephalic artery, is placed into the orifice of the coronary artery. The stem of this vessel, hidden under the respective auricle, is lifted up with bent forceps, and a thread for its later tying off is layed around it. The groove in the cone of the needle permits a temporary fixation with this thread during the injection. The in- 'BIODUR Jahnstr. 8, D-6900 HeidelbergGermany TECHNIQUE OF HEART PLASTINATION 2 297 3 Fig. 2. Posterior view of a heart from above. One lung vein and the anterior cava vein carry a polyethylene tube. The other vessels (except the posterior cava vein) are tied off; the aorta and pulmonary arteries are closed with cork stoppers. Fig. 3. Instrumentsfor the color injection of the coronary vessels. A) Needles with a cone at their tip. B) Connected polyethylene tubing for the m u l a t i o n of the coronary sinus in smaller species. veins with the finger tips. The middle cardiac vein is frequently filled through anastomoses around the tip of the heart. Air bubbles found in the veins mainly travel the same way. The middle cardiac vein is tied off with a surgical needle and thread before the resin, which follows the air bubbles, empties into the right atrium. Alternatively, this vein-and in human hearts also the small cardiac vein-can be cannulated a t its opening in the orifice of the coronary sinus. After the injection, a short powerful water rinse via the two polyethylene tubes removes the small amounts of colored epoxy resin which may have entered the atria and ventricles from the injection sites or from veins opening directly into the atria. tubes and hydrostatic pressure. Fixation occurs after the color injection, when the resin is still flexible enough to allow expansion of the heart. The heart, whose tissue maintains some resiliency, becomes fixed in its dilated state for several hours and becomes temporarily dull in color as it loses its tissue transparency. Depending on the thickness of its ventricular walls, the heart remains immersed for 1 or several days in the formalin, where it can also be stored before further processing. Fixation Fixation with 20% formalin solution (20 parts 3538 wt.% formaldehyde solution in water and 80 parts tap water) proceeds in the same way as the dilatation with the insertion of the two Dehydration A water rinse before dehydration is useful to double-check whether or not all vessels are tied off. After emptying the heart, technical acetone in a grade between 96 and 100% is applied in the same manner as in the dilatation step. The continual hydrostatic pressure counteracts the shrinkage of the heart; only hollow organs can be dehydrated by this rapid method. A heart of a human being or dog requires 5 kg 298 K. TIEDEMANN AND G.v. HAGENS of acetone; a cattle heart requires up to 80 kg over a period of 8 hours. When acetone in an amount five times greater than the volume of the heart has seeped through its walls, the heart becomes rigid. In this time frame, the final shape of the heart and position of its large vessels still may be corrected. After the pressure-dehydration, the hardened heart is kept in pure acetone with several changes until a concentration of a t least 99% is maintained. During this time, all threads, cork stoppers, and the two tubes are removed. The atria are cut open in a semicircular line so that a flap can be turned up and fixed by a ligature. The fenestration of the right ventricle must not damage the transverse muscle of the heart. Similarly, the ovoid opening in the left ventricle should not destroy the anterior papillary muscle but must offer a view into the origin of the aorta below the septa1valve. Remnants of fibrin and colored resin are now removed from the interior of the organ. Impregnation Impregnation can be accomplished with three different curable resins depending on the desired mechanical properties and transparency of the final specimen. A solid but breakable specimen, best suited for display due to the clearness of its injected vessels, is impregnated with the inexpensive BIODUR PEM 27, a specially formulated epoxy-siliconecopolymer,plus the hardener E 6 and accelerator E 60. Flexible specimens better suited for the handling by students are obtained by impregnation with a more expensive silicone, whose opacity renders the vascular injection less clear. Two silicones have been approved. BIODUR S 20 is less opaque, but may be damaged by tearing, so that its use is more practical for a teacher rather than a student. The silicone BIODUR S 10 obscures finer details of the specimen, but is more easily processed, and its high tear resistance makes it ideal for student use. The heart remains in either solution in a vacuum chamber for 1-3 days. The basic principle of the forced impregnation process is that the acetone, which is continuously pumped out of the heart and through the surrounding polymer bath in its gaseous state, draws this polymer into the specimen. The vacuum is turned off when bubbles of the evaporating acetone cease to appear at a pressure of 5 mm Hg. Hardening and Aftercare After the hearts have been taken out of the polymer bath, excess resin is poured out and thoroughly wiped off from the BIODUR PEM 27 or S 20, while S 10 specimens need only to be drained. To maintain the cava veins in their correct position, they must be plugged before hardening, e.g., with silicone stoppers. The hardening is achieved for PEM 27 at 50°C, for BIODUR S 20 a t ambient temperature, and for BIODUR S 10 in a final curing bath, followed by drying at 50°C in order to remove excess curing agent. Polypropylene foil must be used to prevent sticking of the specimens to their underlying surface. DISCUSSION During the removal of the organ, many hearts are injured by piercing, or their vessels are cut too short so that they cannot be tied off. Failure to tie off only one of the intercostal or bronchial arteries still permits dilatation, but later causes costly leakage of acetone. Pressure dilatation of the heart with water, already described by Luciani (1905) as cited by Monckeberg in Abderhalden's manual (1924), leads to a relaxation of the heart muscle exceeding the physiological diastolic state. The most prominent dilatations are those of the pulmonary artery and the atria. This initial overexpansionbecomes markedly reduced during fixation and dehydration. Specimens showing insufficient dilatation of the left ventricle are more frequently seen than those with an overexpanded right heart. The pressure-dilatation of the heart ends when the original elastic consistency of the organ has distinctly changed into a flabby one. The color injection can also be performed after the fixation of the heart (and even on old perfusion-fixed specimens taken from the dissection lab). However, this results in a less complete filling of smaller vessels. Compensation for the rigidity of such fixed vessel walls can be partly achieved by the use of hardener E 3 instead of E 2, which gives the epoxy resin BIODUR E 20 a lower viscosity and a much longer pot life. This mixture is not used in fresh hearts because the elastic wall tension of the injected arteries propels it further into the capillaries after the completion of the injection and the larger branches almost empty themselves through this undesired process. During fixation, a green discoloration of the coronary sulcus is sometimes observed, as the hemolytic blood accumulates below the epicardium. This can be avoided by a prolonged water dilatation or by a rinse of the coronary arteries (via the aorta) with physiological saline immediately after removal of the heart. TECHNIQUE OF HEART PLASTINATION Dehydration can also be performed by freeze substitution with acetone a t - 30°C (Schwab and v.Hagens, 1981), a method recommended for specimens that cannot be dilated, such as old open cut formalin specimens. This alternative method requires less acetone than the pressure dehydration and avoids the shrinkage of the subepicardial fat. On the other hand, it is more time consuming and renders the subepicardial vessels less distinct. As the acetone consumption is an essential cost factor in the large-scale production of plastinated heart specimens, a distillation unit is desirable. In anatomical institutes, this can be achieved by the conversion of a bone degreasing unit. The impregnationprocess is explained in more detail in technical leaflets for the polymers employed. As all resins used for the impregnation can be reused after the removal of the impregnated specimens, a stock of fully dehydrated specimens for a second or third run is of economic importance. A common pitfall in heart plastination is focal discoloration of the organ due to threads or cork stoppers which have not been color-proofed or due to drying of the organ surface. The latter easily happens during the handling in acetone, and leads to an incomplete impregnation (v.Hagens 1979a; Bickley 1980) with typical white discolorations of the hardened specimen. An insufficient color injection occurs when 1) both large branches of the left coronary artery have not been completely filled, 2) fine atrial branches have been injured during the encircling of the respective coronary artery, or 31 vascular variations such as additional coronary arteries have not been considered. The ramification pattern of the coronary vessels is species-dependent;we found it most beautiful (for display specimens) in the dog and unattractive in the pig. The technical equipment necessary for heart plastination is often found in morphological institutes: a deep freezer, a vacuum chamber (best with cooling devices for -2O"C), an explosion-proof vacuum pump, and a hot cabinet. A bell jar and a water-jet vacuum pump can be used as minimal equipment for the beginner. The materials for the plastination of one dog heart cost between $30 and $40; for a cattle 299 heart, $100-$150. The economy of scale reduces these costs by a third when solvents and polymers are reused. Plastination is covered by several patents (v.Hagens, 1977-1981). Its use in public institutions like universities and schools, however, is virtually unrestricted because the use of BIODUR-polymers includes its use on a noncommercial basis. These polymers were developed especially for plastination over the last 5 years with major support by BAYER AG, Leverkusen, BAKELITE GmbH, Iserlohn-Lethmate and BASF, Ludwigshafen. Our most annoying experience with heart plastination is that plastinated specimens are more quickly stolen than produced. ACKNOWLEDGMENTS We thank the chairman of the Anatomical Institute I, Dr. W. Kriz for his kind support of this project. We highly appreciate the efforts of our chief technician, Mrs. Hiltraud Hosser, for doing the major part of the practical work involved. LITERATURE CITED Bickley, H.C. (1980) Preservation of gross tissue specimen by plastination. Bull. Pathol. Educ., 6:5-7. v.Hagens, G. (1979a) Impregnation of soft biological specimens with thermosetting resins and elastomers. Anat. Rec., 194.247-256. v.Hagens, G. (1979b) Emulsifying resins for plastination. Der Praparator, 25:43-50. v.Hagens, G.Patents: D.B. Pat. 27 10 147 (1977),Brit. Pat. 1 558 802 (19781, Belg. Pat. 863.949 (19781, R.S.A. Pat. 78/1330 (1978),Austr. Pat. 360 272 (1980),U S . Pat. 4,205, 059(1980),U.S.Pat. 4,244,992 (1981),U S . Pat. 4,278,701 (1981). Hochstetter, F. (1927) Die Paraffindurchtrankung zur Erhaltung von Tieren und Pflanzen in ihrem naturlichen Aussehen. Umsch., 31:650-652. Hower, R.O. (1979) Freeze-Drying Biological Specimens: A Laboratory Manual. Smithsonian Institution Press, Washington. Monckeberg,J.G. (1924) Die Methoden zur morphologischen Untersuchung erkrankter Herzen. In: Handbuch der Biologischen Arbeitamethoden.E. Abderhalden, ed.Urban und Schwanenberg, Berlin, Vol. VIII, pp. 635-650. Schmeidel, G. (1925) Wie Paraffinpraparatehergestellt werden. Erg.-H. Anat. Anz., 60:282-283. Schwab, K., G. v.Hagens (1981) Freeze substitution of macroscopic specimens for plastination. Acta Anat., 111:139-140. Wolfe, K. (1956) Plastic-embedded heartpcleared and corroded specimens. Arch. Pathol., 61~153-158.