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BACKGROUND OF INVENTION
This invention relates generally to the preservation of large sections of
biological tissues, and more particularly to a method which converts such
a section, e.g. cross section of a rabbit, into a plastinated sheet. In a
method in accordance with the invention, the tissue water of the large
section is replaced by a polymer, thereby 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 often required for teaching and
scientific purposes in the fields of anatomy, pathology, forensic
medicine, biology and the like.
Large sections have heretofore been preserved by the following methods:
(1) Gelatine--and Paper method: With gelatine preservation, 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 Whimster, W. F., in Human Pathology 1, 1, 1970).
(2) Embedment: The sections are embedded in blocks of plastic. To this end,
a bottom, a specimen and a top layer must be poured (A Simple Method for
Embedding Anatomical Specimens, by Grimsrud, O. K., and Dugstad, G.,
NEURORADIOLOGY 10 143-145, 1975; Preparation of Plastic Mounted Brain
Specimens, by Deonar, V., NEURORADIOLOGY 4, 197-201, 1972).
(3) Polymer impregnation: A method of impregnating large sections with
uncured polymers with subsequent curing between separating foils is
described in "Impregnation of Soft Biological Specimens with Thermosetting
Resins and Elastomers," by v. Hagens, G., THE ANATOMICAL RECORD 194,
247,256, 1979.
These known methods as well as the preserved products attained thereby
suffer from serious disadvantages, as will now be explained:
(1) Gelatine--and Paper method: The preserved large section is not
resistant to scratches (gelatine surface) or to mechanical stress (paper
as supporting medium). Because of the acrylic sheet functioning as a
supporting medium, the preserved large section is considerably thicker
than the tissue section.
(2) Embedment: Due to the bottom and the 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 pour three layers of resin
at different times and the surface of the cured block has to be ground and
polished, this method is quite time-consuming and costly.
(3) Polymer impregnation: Because of the firmness of the polymer material
and the minimal thickness of the resulting sections, this method appears
to be quite advanced. However, this technique for preserving a large
section lacks uniform thickness and surfaces which are even or polished.
Moreover, it is very difficult to remove air bubbles in the vicinity of
the tissue section.
It is also 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) in a cell defined by glass plates. The glass plates
are separated from each other along their outer edges by an elastomeric
material in order to prevent leakage of the uncured polymer. These plates
are subjected to a constant pressure which causes the plates to move
toward each other in the course of polymer shrinkage during
polymerization. The resulting transparent sheets of organic glass exhibit
a uniform thickness and surfaces which are even and polished. These sheet
properties are also desirable in the context of a plastinated sheet
preserving a large section of biological tissue.
SUMMARY OF INVENTION
Accordingly, the main object of this invention is to provide a method for
converting a large section of biological tissue into a plastinated sheet
having smooth surfaces and a uniform thickness.
A significant aspect of the invention is that it combines the known
technology of preserving large sections with the otherwise unrelated art
of casting sheets of organic glass to produce a unique plastinated sheet.
We shall now summarize the major advantages to be gained by
sheet-plastination.
The method makes it possible, without difficulty, to preserve a very large
section (e.g., from a cow), because during the curing step the large
section is pressed between plates acting as a supporting medium. Since the
closed cell with the impregnated tissue section therein can be tilted,
this causes air bubbles in the vicinity of the section to rise and
disappear. The method, if performed by a skilled person, may be carried
out quickly. Moreover, the method is inexpensive as regards consumption of
polymer and biological material.
As regards the product: The surface of the plastinated sheet is smooth,
thereby obviating the need for time-consuming polishing operations. A
plastinated sheet can be made as thin as 0.2 mm. Such very thin
plastinated sheets afford optimum light transmittance. The plastinated
sheet is of uniform thickness. The structure of the plastinated sheet can
be viewed and directly marked. As a consequence, explanatory indicia can
be inscribed in the immediate vicinity of the structures of interest. Thus
the invention affords a highly valuable aid for teaching and demonstration
purposes in morphology.
OUTLINE OF FIGURE
For a better understanding of the invention as well as other objects and
further features thereof, reference is made to the following detailed
description to be read in conjunction with the accompanying drawing, whose
single FIGURE illustrates a cell adapted to carry out a method in
accordance with the invention to produce a plastinated sheet.
DESCRIPTION OF INVENTION
The terminology used herein 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 thickness and size not being
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 with a thickness between 1.mu. and 50.mu. 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 a minimal
amount of additional polymer is situated around the impregnated section,
its size is about equal to that of the large section in its fresh state.
"Plastinated sheet" is intended to mean a large section impregnated with a
cured polymer, which is surrounded by additional cured polymer almost
exclusively around its outer periphery. The surface of the plastinated
sheet is smooth, its thickness uniform and about equal to that of the
large section before treatment.
"Uncured polymer" is intended to mean a fluid precursor composition capable
of being polymerized into a solid synthetic resin or firm elastomer. The
uncured polymer is taken from the class of thermosets and elastomers. It
experiences a certain amount of shrinkage during polymerization, ranging
from less than 1% to more than 20% by volume. More specifically, compounds
taken from 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, the invention encompasses
polymers which are uncured in a fluid state and which are capable of being
cured into a solid synthetic resin or firm elastomer.
"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 of the
type described in the foregoing paragraphs. Optically clear polymers are
preferable for this purpose.
"Specimen" is intended to mean whole organs, bodies or parts thereof,
including extremities and virtually all kinds of biological material whose
conversion into plastinated sheets is desirable.
"Last intermedium" is intended to mean an organic solvent such as xylene,
acetone, methylene chloride or propylene oxide which is miscible with the
uncured polymer used for impregnation and which serves as an immersion
bath after the dehydration procedure and before impregnation with the
uncured polymer.
From the standpoint of processing, the different procedures entailed in a
method in accordance with the invention shall be classified for
convenience into three distinct stages--namely: (I) pretreatment of large
section, (II) impregnation with polymers, and (III) casting the
plastinated sheets. These stages shall now be set forth in greater detail.
Stage I--Pretreatment of large 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.degree. C. Specimens consisting uniformly of soft, putrifiable tissue
(e.g., a lung section) are preferably cut by means of a rotary slicing
machine in their frozen and/or fixed state.
Fixation: Either the specimen or the large sections obtained therefrom are
fixed with known fixatives like formaldehyde or glutaraldehyde.
Preservation of color and/or staining: The natural color can be preserved
by known techniques such as by the addition of sodium nitrite and ascorbic
acid to the fixing solution. For staining, known staining techniques for
organic material may be used for this purpose. 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 must be exchanged with an intermedium that is compatible or at
best fully miscible with the uncured polymer that is used. 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 proven to be very suitable. At the conclusion
of pretreatment, the large section is imbued with an organic solvent,
named "last intermedium" which is substantially free of water, which has
preferably a high vapor pressure such as acetone or methylene chloride,
and is miscible with the uncured polymer that is used.
Stage II--Impregnation with uncured polymer
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 where 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 section's
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 vapor pressure like acetone or methylene chloride. The large section,
imbued with this kind of last intermedium, is then immersed in a vat
containing the uncured polymer which 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 removed in
its gaseous state by a vacuum pump and gradually replaced by the still
uncured polymer that is used. The infiltration of the polymer into the
tissue is facilitated by the presence of the last intermedium because of
its properties as a solvent.
This technique of impregnating biological specimen with curable polymers is
known and described in greater detail in my U.S. Pat. No. 4,205,059, whose
entire disclosure is incorporated herein by reference. Once the large
section is impregnated, it is kept in the uncured polymer for processing
in stage III or put between separating foils and cured until the polymer
is gelated or until it is fully cured. At the end of the impregnation
procedure, the large section is uniformly impregnated with an uncured,
partially or fully cured polymer.
Stage III--Casting plastinated sheets
This stage is the crux of my invention. The critical procedure of this
stage is illustrated in the drawing, where it will be seen that two plates
(a), preferably in the form of clean, toughened glass plates of about the
same size, are employed. If necessary, these plates are treated with a
release agent. A selected uncured polymer which is de-aired is poured as a
thin layer (c) in the center of one of the glass plates. Formation of air
bubbles should be avoided.
The large section which has been prepared by the procedures in stages I and
II, impregnated with cured, partially cured (e.g., jelled) or uncured
polymer, is now placed onto the poured layer of uncured polymer. The
entrapment of air bubbles must be avoided. Thereafter, a flexible gasket
(d) of elastomeric material, preferably made out of especially graded
polyvinylchloride and with a thickness about equal to that of the
impregnated section, is now placed on the glass plate.
For an inexperienced operator, it may be helpful to hold the gasket in its
position with the aid of a very thin polyamid-thread which is held in turn
with pressure-sensitive tape on the reverse side of the glass plate.
Thereafter, the impregnated large section is covered with an additional
thin layer of uncured polymer.
The first glass plate (a) with the impregnated large section (b), the
flexible gasket (d) and the poured polymer (c) is now covered by the
second glass plate (a). During this procedure, one must be careful to
avoid the entrapment of air bubbles above the impregnated large section.
As the next step in this procedure, the two glass plates (a) are pressed
together by specially designed clamps (e) which hold the impregnated large
section and the flexible gasket in position, thereby defining a flat cell
in which the opposing cut surfaces of the impregnated large section (b)
abut the inner surfaces of the two glass plates (a). When pressing plates
(a) together, polymer (c) is squeezed out into the region surrounding the
section.
Now, a specially designed funnel (f) is inserted into the upper slot of
this flat cell. The flat cell is brought to an upright position, as shown
in the drawing, and additional polymer (g) is poured into the cell to
merge therein with the polymer (c) previously introduced to fill the cell
completely. Air bubbles (h) are allowed to rise. To permit manipulation of
the cell in order to direct the air bubbles away from the section therein,
both ends of the flexible gasket are fused together with a soldering tool
and squeezed fully between the glass plates to close the cell and thereby
prevent leakage of the polymer therefrom during manipulation.
Polymerization of the uncured polymer can now be effected. This may be
done, depending upon the type of uncured polymer used, by means of
elevated temperature, ultraviolet radiation or other known methods.
During polymerization, the two plates move towards each other, this
movement being facilitated by the elasticity of the materials involved;
i.e., the flexible gasket, the glass plates and the impregnated large
section. After curing, the clamps are removed and the glass plates of the
flat cell are taken apart. The plastinated sheet so produced is now cut
into a convenient shape and, if desired, inscribed for educational or
other purposes.
The casting of plastinated sheets directly on glass plates with polymers
which do not have a distinct gelating phase, e.g., methacrylates, causes
the plastinated sheet to have glass-like surfaces. Casting of plastinated
sheets directly on glass plates with polymers which exhibit a distinct
gelating phase, combined with substantial shrinkage during final curing in
hampered by the formation of boundary lines on the surface of the cured
plastinated sheet. Boundary lines will especially appear if there is rapid
curing. These lines usually arise in the direct vicinity and around the
impregnated large section and in the surrounding cast polymer region.
In general, there are two ways to prevent the formation of boundary lines.
If casting is done directly on glass plates, it is advisable to remove the
plastinated sheet from the plates when still in its partially cured state
in order to hang it up or wrap it up 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 the latter case, a much thicker
foil (e.g. 350.mu.) is most useful. A preferred material for a separating
foil is PETP (polyethylene terephthalate). The term foil as used herein is
intended to cover thin plastic film sheets such as those made of Mylar.
The use of separating foils as coverage for the glass plates gives rise to
certain adverse effects: While the use of separating foils is of advantage
to prevent the formation of air bubbles because the foil can be bent if
the impregnated section is covered therewith, the surface of the
plastinated sheet is not as smooth when coming from foils rather than
directly from the glass plates.
In casting plastinated sheets, bubble formation must be avoided. There are
several ways of preventing bubbles. For example, if a section impregnated
with cured 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 on the surface of the cured large section with the uncured polymer
and thereby prevent the formation of air bubbles which otherwise may
expand and appear when the temperature of the polymer rises during curing.
The glass plates can be wrapped with very thin foils (thickness, e.g.
12.mu.) in order to prevent adhesion of polymers to glass and covered with
an additional separating foil. It is also possible to cover the glass
plates with a foil having adhesive properties on its outer face. Once the
polymer of the plastinated sheet is cured, it adheres permanently to these
foils. This modification of the method is especially useful should
delicate silicone-rubber be used for casting plastinated sheets.
In those instances where a large section is to be formed--e.g., whole
sections of a cow--it may be advisable in order to stabilize the section
during processing, to cut or saw the large section to be impregnated in a
slice which is quite thick; e.g., 8 mm. After impregnation and curing of
this large section, it can be ground with emery paper to a thickness of,
e.g., 2.5 mm. Thereafter, casting is carried out with this ground-down
impregnated large section.
While the above description includes many specific details, these should
not be construed as limitations on the scope of the invention, but rather
as preferred embodiments thereof. Many other modifications in the
procedure are possible. For example, the impregnation of large sections
with uncured polymer may be facilitated by vibration. If a plastinated
sheet is of reduced quality, as a result, for example, of bubble formation
taking place during curing, it can be excised, possibly reground and again
casted. It is also understood that several steps described herein can be
omitted or performed in another sequence. For example, the staining of the
large section, if desired, can be carried out at various stages or it may
be entirely omitted.
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