GB2082041A - Method for preserving large sections of biological tissue with polymers - Google Patents

Abstract

Method of preserving large sections of biological tissue with curable polymers e.g. acrylic resins, allyl diglycol carbonate, unsaturated polyester resins, epoxy resins, polyurethanes or silicone rubber, by impregnating these large sections with the polymer and pressing the impregnated large sections (b) between flat plates (a), preferably toughened glass plates. These plates are additionally separated near their edges by an elastic material (d), thus providing a flat cell, wherein both cut surfaces of the impregnated large section are abutting against the inner surfaces of the two plates of the flat cell. Thereafter, the cell is filled up with uncured polymer (c) and (g). Air bubbles (h) are allowed to rise, subsequently the polymer is cured whereby the plates are moving toward each other due to the polymers shrinkage during curing. Afterwards the plates are removed. The resulting plastinated sheet is a permanently preserved large section of biological tissue, whose tissue water is completely replaced by a cured polymer. The surface is even, the thickness uniform and about that one of the large section before treatment. Many variations of the technique are possible. For example the glass-plates of the flat cells can be covered with separating foils or casting can be implemented with a large section which is already impregnated with cured polymer. <IMAGE>

Description

SPECIFICATION Method for preserving large sections of biological tissue with polymers This invention relates to the preservation of large sections of biological tissues, specifically to a method which turns such a section, e.g. cross section of a rabbit, into a plastinated sheet, which is a preserved tissue containing polymer sheet of smooth surface and uniform thickness.

In several steps, the tissue water of the large section is replaced by polymer, thus preserving the large section of biological tissue permanently.

Large sections of biological tissue, especially sections of whole organs and bodies of animal or human origin are requested for teaching and scientific purposes in the fields of Anatomy, Pathology, forensic medicine, Biology and the like.

Up to now, large sections are preserved by the following methods: (1) Gelatine- and Paper method; With gelatine impregnated sections are glued on plates of acrylic resin (Improved Plastic Embedding of Wet Biological Specimens, by Simmons, E.M. et al. in MEDICAL AND BIOLOGICAL ILLUSTRATIONS, 18, 260-262) or on paper (Rapid Paper Sections of Solid Organs, by WhimsterW.F. in Human Pathology 1, 1, 1970); (2) embedment; The sections are embedded in blocks of plastic. To achieve this, a bottom, a specimen and a top layer have to be poured (A Simple Method for Embedding Anatomical Specimens, by Grimsrud, O.K. and Dugstad, G., NEURORADIOL OGY 10, 143-145, 1975; Preparation of Plastic Mounted Brain Specimens, by Deonar, V., NEURORADlOLOGY4, 197-201, 1972).

(3) Polymer impregnation; I have described recently a method of impregnating large sections with uncured polymers with subsequent curing between separating foils (Impregnation of Soft Biological Specimens with Thermosetting Resins and Elastomers, by v. Hagens, G., THE ANATOMATICAL RECORD 194,247-256, 1979).

These methods as well as the preserved products achieved therewith show each remarkable disadvantages: (1) Gelatine- and Paper method: The preserved large section is not resistent against scratches (gelatine surface) or mechanical stress (paper as supporting medium). Due to the acrylic sheet as supporting medium, the preserved large section is considerably thicker than the tissue section.

(2) Embedment: Due to the bottom and top layer of cured resin, the thickness of the resulting block is considerably thicker than the large section itself.

Details of the specimen cannot be viewed directly with the aid of a magnifier. Because it is necessary to pourthree layers of resin at different times and the surface of the cured block has to be grinded and polished, this method is quite time consuming.

(3) Polymer impregnation: Because of the firmness of the polymer material and the minimal thickness of the resulting sections, this method seems quite advanced. However, this kind of preserved large section lacks uniform thickness and even or polished surfaces. Moreover it is very difficult to remove air bubbles in the vicinity of the tissue section.

It is known from the literature (POLYMER PROCESSES, Vol. X, by Schildknecht, C.E., Interscience Publishers Inc., New York, p.35, 1956) that acrylic sheets can be made by casting uncured polymer (catalyzed prepolymer or monomer) between glass plate cells. The glass plates are separated from each other in their outer circumference by an elastic material in order to avoid leakage of the uncured polymer. These plates are pressed constantly together, enabling them to move toward each other due to the polymers shrinkage during polymerization. The resulting transparent sheets of organic glass do have uniform thickness, even, polished surface and hardness. These are all properties which are most wanted for preserved large section of biological tissue.

Accordingly the object of my invention is to combine the known art of preserving large sections with the art of casting sheets of organic glass (smooth surface, uniform thickness).

In the following, the major advantages of the new process, named sheet-plastination (publication pending) as well as the major advantages of the resulting new product, named plastinated sheet will be named.

As regards the method: (1) Preservation of very large sections (e.g. from a cow) is not difficult, because during curing the large section is pressed between plates as supporting medium; (2) since the closed cell with the impregnated section enclosed in can be tilted, air bubbles in the vicinity of the section will rise and disappear; (3) The method, if performed by a skilled person, is fast to carry out and inexpensive as regards consumption of polymer and biological material. As regards the product: (1) the surface of the plastinated sheet is smooth, time consuming polishing is therefore not necessary; (2) Plastinated sheets can be as thin as 0.2 mm. Very thin plastinated sheets provide ultimate light transmittance; (3) the plastinated sheet is of uniform thickness; (4) the structures of the plastinated sheet can be viewed and marked directly.Explanations can be written down in the direct vicinity of structures of interest.

In summary, my invention provides a most valuable aid for teaching and demonstration purposes in morphology.

Further objects and advantages of my invention will become apparent from a consideration of the drawing and ensuing description thereof. The drawing demonstrates the main features of the invented method.

The terminology used in describing the several materials, substances and actions is to be construed in the light of the following definitions: "large section" is intended to mean one or several sections of biological tissue, especially of human or animal tissue, as obtainable with a rotary slicing machine or a saw. Large sections are usually obtained from whole organs, bodies or parts thereof. The thickness of large sections ranges between 0.2 mm and 5 mm, their maximal size is not limited. It is apparent that large sections in this sense are quite different from tissue sections as known from histology which are obtained with the aid of a microtome at a thickness between 1 CL and 50 CL and which are preserved permanently between glass plates.

"Impregnated section" or impregnated large section is intended to mean a large section whose tissue water is completely removed by an uncured, partially cured (i.e. gelated) or cured polymer. Because only minimal amount of additional polymer is situated around the impregnated section, its size is about that one of the large section in its fresh state.

The term "Plastinatedsheet" is intended to mean a large section impregnated with cured polymers, which is surrounded with additional cured polymer almost exclusively around its outer circumference.

The surface of the plastinated sheet is smooth, its thickness uniform and about that one of the large section before treatment.

"Uncuredpolymer" is intended to mean a fluid precursor composition, capable of being polymerized into a solid synthetic resin or firm elastomer.

The uncured polymer comes from the class of thermosets and elastomers. They experience a certain amount of shrinkage during polymerization, ranging from less than 1 % to more than 20% of volume.

More specifically, up to now compounds out of the following classes of polymers have been used successfully: Acrylic resins (e.g. methylmethacrylate), allyl compounds (e.g. copolymers of allyl diglycol carbonate), epoxy resins, polyester resins, polyurethanes and silicone rubber. However all kinds of polymer which are uncured in a fluid state and capable of being cured into a solid synthetic resin or firm elastomer can be implemented.

"Partially cured polymer" is intended to mean a fluid precursor composition as defined in the foregoing paragraph which is polymerized to its gelated state.

"Cured polymer" is intended to mean a cured thermoset or elastomer from the kind described in the foregoing paragraphs. Optically clear polymers are meant preferably.

The term "specimen" is intended to mean whole organs, bodies or parts thereof, including extremities and virtually all kinds of biological material of which it is worthwhile to obtain plastinated sheets.

"Lastintermedium" is intended to mean an organic solvent like xylene, acetone, methylene chloride or propylene oxide which is miscible with the uncured polymer used for impregnation and which serves as immersion bath after the dehydration procedure and before impregnation with the uncured polymer.

From the point of view of processing the different procedures of the new method shall be classified for convenience into three stages: (1) pretreatment of large sections, (2) impregnation with polymers, (3) casting plastinated sheets. These stages will now be setforth in greater detail.

Stage I-Pretreatment oflarge sections. Pretreatment of large sections includes cutting, fixation, preservation of color and/or staining and dehydration. Cutting of large sections: Specimens which contain solid substances like bone or horn are cut into large sections in their frozen state, preferably at a temperature below - 20"C. Specimens consisting uniformly of soft, putrifiabletissue (eg. a lung section) are cut preferably by means of a rotary slicing machine in theirfrozen anclorfixed state.Fixation: Eitherthe specimen or the large sections obtained thereof are fixed with known fixatives like formaldehyde orglutaraldehyde. Preservation of color andlor staining: The natural color can be preserved by known techniques like addition of sodium nitrite and ascorbic acid to the fixing solution. For staining nearly all kinds of known staining techniques for organic material can be implemented. It has been proven useful to stain with techniques known from paraffin-histology. Dehydration: Because almost all polymers which are useful for this invention are not miscible with water, the tissue water of the large sections has to be exchanged against an intermedium which is compatible or at best fully miscible with the uncured polymer implemented.

The kind of dehydration is of free choice. Dehydration by sequential immersion in e.g. five aqueous ethanol solutions ranging in concentration from 50 to 96% and final immersion in acetone as well known from paraffin-technology has been proven very suitable. As main point, at the end of the pretreatment the large section is imbued with an organic solvent, named "last intermedium" which is substantially free of water, which does have preferably a high vapour pressure like acetone or methylene chloride and which is miscible with the uncured polymer to be used.

Stage 11Impregnation with uncuredpolymer.

Preserved and dehydrated large sections which have been prepared by the procedures in Stage I are immersed into a solution of uncured polymer. In the rare case, that the uncured polymer used is miscible with water (e.g. hydroxaethylmethacrylate) the large section can be transferred from water directly into the water compatible polymer. Impregnation of the large section is achieved either by simple immersion in the uncured polymer and thus impregnated by diffusion or, especially if the large sections thickness exceeds 0.5 mm, by forced impregnation. To achieve this, as last intermedium an organic solvent has to be used which is characterized by a high vapour pressure like acetone or methylene chloride.The large section, imbued with this kind of last inter medium is then immersed in a vat, containing the uncured polymerwhich is ready for curing (e.g. by addition of a hardener) and placed into a vacuum chamber which is evacuated gradually. The last intermedium is now continuously pumped off by the vacuum pump in its gaseous state and gradually replaced by the still uncured polymer used. The infiltration of the polymer into the tissue is facilitated by the presence of the last intermedium because of its properties as solvent. This technique of impre gnating biological specimen with curable polymers is known and described more detailed in my U.S.

Patent 4,205,059. Once, the large section is impre- gnated, it is kept in the uncured polymer for proces sing in stage III or put between separating foils and cured until the polymer is gelated or until it is fully cured. As main point, at the end of the impregnation procedure the large section is uniformly impre gnated with an uncured, partially orfully cured polymer.

Stage Ill-Casting plastinated sheets This stage is the core of my invention. The critical procedure of this stage is shown in the drawing.

Therefore several references to the drawing are made in the ensuing description of this stage. The indicating letters of the drawing will be placed in brackets behind the items named.

Two plates, preferably clean toughened glass plates of about the same size are taken and, if necessary, treated with an release agent. A chosen, uncured polymer is taken, deaired and poured as a thin layer in the center of one of the glass plates.

A formation of air bubbles should be avoided. The large section which has been prepared by the procedures in stage I and II, impregnated with cured, partially cured (i.e. jelled) or uncured polymer is now placed on the poured layer of uncured polymer. The entrapment of air bubbles has to be avoided. Thereafter a flexible, elastic gasket, preferably made out of especially graded polyvinylchloride and with a thickness of about that one of the impregnated section, is placed on the glass plate, around the impregnated section as it is obvious from the drawing (flexible gasket indicated by letter "d"). For an unexperienced person, it may be helpful to fix the gasket in its position with the aid of very thin polyamidthread which is fixed in turn with pressure sensitive adhesive tape on the reverse side of the glass plate.

Thereafter the impregnated large section is coved with an additional thin layer of uncured polymer.

The first glass plate with the impregnated large section (b), the flexible gasket (d) and the poured polymer on it (c) is now covered by the second glass plate. Both glass plates are indicated with letter "a" in the drawing. During this procedure attention has to be paid, to avoid the entrapment of air bubbles above the impregnated large section. As next step in Wblis procedure the two glass plates (a) are pressed gether by especially designed clamps (e), holding she impregnated large section and the flexible gasket in its position, thus providing a flat cell, whereby both cut surfaces of the impregnated large section ) are albutring against the inner surfaces of the two glass plates (a).Now an especially designed funnel (1) is placed into the upper slot of this flat cell. The llaW cell is tilted upright as it is obvious from the drawing and additional polymer (g) is poured into the cell, merging therein with the previously, beneath and above the section poured polymer (c).

fir bubbles (h) are allowed to rise and if allowed to do so both ends of the flexible gaskets are melted together with a soldering tool and squeezed fully between the glass plates. This may be done with te aid of a wooden tongue blade. Polymerization of the uncured polymer can now be implemented. This may be done, dependent upon the kind of uncured ,polymer, by means of elevated temperature, light or other methods.

During polymerization the two plates are moving towards each other, facilitated by the elasticity of the materials envolved i.e. flexible gasket, glass plates and impregnated large section. After curing the clamps are removed and the glass plates of the flat cell are taken apart. The manufactured plastinated sheet is now cut into a convenient shape and, if wanted, inscribed for educational or other purposes.

Casting of plastinated sheets directly on glass plates with polymers which do not have a distinct gelating phase, e.g. methacrylates, does result in glass-like surfaces of the plastinated sheet Casting of plastinated sheets directly on glass plates with polymers which show a distinct gelating phase, combined with major shrinkage during final curing is hampered by the formation of boundary lines on the surface of the cured plastinated sheet. Boundary lines will appear especially, if rapid curing is implemented. These lines usually arise in the direct vicinity and around the impregnated large section and the surrounding, casted polymer.

There are two general ways to prevent the formation of boundary lines. If casting is done directly on glass plates, it is advised to deform the plastinated sheet in its cured state in order to hang it up or enwrap it in flannel for final curing. Secondly, the use of separating foils will prevent the formation of boundary lines. The separating foil may be either sealed upon the glass plate or just put on the glass plate. In this case a much thicker foil (e.g. 350 IL) is most useful. As material for a separating foil PETP (Polyethylene Terphthalate) has been proven most useful.

The implementation of separating foils as coverage of the glass plates results in two other adverse effects: on the one hand, the additional use of separating foils is of advantage in respect to the formation of air-bubbles, because the foil can be bent if the impregnated section is covered therewith.

On the other hand the surface of the plastinated sheet is not as smooth when coming from foils instead from the glass plate directly. Another challenge for casting plastinated sheets is the avoidance of bubble formation. There are several ways of preventing the bubbles. For example, if a section which is impregnated with polymer is used for casting a plastinated sheet, it is advisable to impregnate this cured large section with the setup of uncured polymer for casting the plastinated sheet. This procedure will fill up all pores at the surface of the cured large section with the uncured polymer and thus prevent the formation of air-bubbles, which otherwise may expand and appear at the time the temperature of the polymer rises during curing.

The glass plates can be enwrapped with very thin foils (thickness e.g. 12 IL) in order to prevent adhe simon of polymers to glass and covered with an addi tonal separating foil.

tt is also possible to cover the glass plates with a loil, having adhesive properties on its upper side.

Once the polymer of the plastinated sheet is cured, it cefheres permanently to these foils. This variation of the method is especially useful if delicate siliconerubber is used for casting plastinated sheets.

In case especially large sections shall be worked up, e.g. whole sections of a cow, it may be advisable for reason of stability of the section during the procedures, to cut or saw the large sections to be impregnated quite thick, e.g. 8 mm. After impregnation and curing of these large section, they can be grinded down with emery paper to a thickness of e.g.

2, 5 mm. Thereafter casting is done with these grinded, impregnated large sections again.

While the above description contains many specificities, these should not be construed as limitations on the scope of the invention, but rather as an exemplification of one preferred embodiment thereof. Many other variations are possible, for example the impregnation of large sections with uncured polymer can be facilitated by vibration. If a plastinated sheet is of lower quality, e.g. because bubble formation took place during curing, it can be cut out, grinded or not and casted again. It is also understood, that several steps described can be omitted or carried out in another sequence. For example the staining of the large section, if wanted, can be done at various stages or not at all.

Accordingly, the scope of the invention should be determined not by the embodiment illustrated, but by the appended claims and their legal equivalents in the light of the definitions made at the beginning of the description.

Claims (15)

CLAIMS 1. A method of preserving large sections of biological tissue or parts thereof which comprises: (a) if necessary, pretreatment, e.g. fixation and dehydration; and (b) impregnating said section with a fluid precursor composition such as acrylic resins, allyl diglycol carbonate, unsaturated polyester resins, epoxy resins, polyurethanes or silicone rubber, if occasion arises polymerizing said fluid precursor composition to its gelated or cured state, pressing said uncured, partially cured or fully cured section between plates, preferably glass plates, optionally covered with foils, whereby both cut surfaces of the impregnated large section are abutting against the inner surfaces of the two plates, which plates are additionally separated around said impregnated large section by an elastic material, thus providing a flat cell, filling said flat cell with a fluid precursor composition which may or may not be identical with that fluid precursor composition used for impregnating said large section, curing all curable content of said flat cell to its gelated or full cured state, thus allowing the plates to move toward each other due to the shrinkage caused by curing of any fluid precursor composition within said flat cell, thereafter removing the plates, in cases where the said polymerizable content of said flat cell is in its gelated state optionally implementing full curing of the resulting product separately, e.g. in a hanging position or enwrapped in flannel or the like, if occasion arises cutting and inscribing the resulting product, termed plastinated sheet, which is characterized by smooth surfaces and uniform thickness. 2. A method of preserving a biological specimen substantially as hereinbefore described other than in connection with the prior art methods 1,2 and 3 noted hereinbefore. Application No.8026418 New claims filed on 29/5/81 plus amendments to claimsfiled on 1 116/81. Superseded claims 1 and 2. CLAIMS

1. A method of preserving a large section of biological tissue our a part thereof which comprises: (a) if necessary, pretreating the section to render it suitable for impregnation; (b) impregnating the section with a fluid precur-: sor composition; (c) optionally polymerizing the fluid precursor composition to its gelated state or cured state; (d) compressing the impregnated section (which section is impregnated by uncured, partially cured or cured polymer) between plates, the inner surfaces of the plates optionally being covered with foil, whereby opposing cut surfaces of the section abut the inner surfaces of the plates of the foils, if present, the plates being separated by a compressible gasket around the section thus providing a flat cell; (e) filling the resulting flat cell with a fluid precursor composition;; (f) curing fluid precursor composition in the cell so that shrinkage occurs and the plates move towards each other to form a sheet in the cell of substantially uniform thickness; (g) removing the plates; (h) if necessary, implementing full curing of the resulting sheet.

2. A method as claimed in claim 1, wherein the fluid precursor composition is an allyl diglycol carbonate, an acrylic resin, an unsaturated polyester resin, an epoxy resin, a polyurethane or a silicone rubber.

3. A method, as claimed in claim 1 or claim 2, wherein the fluid precursor used for filling the flat cell in step (e) is identical with the fluid precursor composition used for impregnating the section in step (b).

4. A method, as claimed in any one of claims 1 to 3, wherein the fluid precursor composition used for filling the flat cell in step (e) is different from the fluid precursor composition used for impregnating the section in step (b).

5. A method, as claimed in any one of claims 1 to 4, wherein the pretreatment comprises fixation, dehydration or staining.

6. A method, as claimed in any one of claims 1 to 5, wherein the plates are of glass.

7. A method, as claimed in any one of claims 1 to 6, wherein the plates are covered with foils.

8. A method, as claimed in any one of claims 1 to 7, wherein step (h) is performed, implementation of full curing being achieved by hanging the sheet or wrapping the sheet in flannel.

9. A method, as claimed in any one of claims 1 to 8, wherein the sheet resulting from steps (g) or (h) is cut and inscribed.

10. An examinable plastinated sheet constituted by a cut section of biological tissue impregnated with a cured polymer, the periphery of the section being surrounded by a region of cured polymer to define a sheet of uniform thickness which is substantially equal to that of the section in its untreated state, said sheet having smooth surfaces.

11. A sheet as claimed in claim 10, wherein said cured polymer is transparent.

12. A sheet as claimed in claim 10. wherein the cured polymer is an acrylic resin, an epoxy resin or a polyester resin.

13. A sheet as claimed in any one of claims 10 to 12, wherein the cured polymer impregnating thetis- sue is of a composition that differs from that of the surrounding polymer.

14. A method of preserving large sections of biological tissue or a partthereof substantially as hereinbefore described other than in connection with the prior art methods 1,2 and 3.

15. An examinable plastinated sheet substantially as hereinbefore described other than in connection with the prior art methods 1,2 and 3.