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Abstract

Plastination suits very good for 3D reconstruction and morphometric measurements. Digital representation of anatomical features has provided a useful source of data for computer-based instructional development. The validation of 3D reconstructions and measurements needs a thoroughly knowledge about shrinkage. The aim of this paper is to determine the average slice shrinkage rate occurring during sheet plastination (E12 and P40). Fresh human brain slices for P40, pelvis slices for the standard E12 technique and a shoulder tissue block for the E12 thin slice technique were used for this study. In order to determine the shrinkage rate, the slice areas were measured before and after plastination for al samples. For the E12 thin slice technique, the shoulder tissue block was measured before and after plastination and a volumetric shrinkage rate was determined. The shrinkage rate was for P 40 technique 5.74%, for standard E12 technique 6.54% and for the E12 thin slices technique 6.23%. Therefore, plastinated slices showed a decreased shrinkage rate and fit best for research purposes. All shrinkage rates were under 10%, so we believe that every shrinkage below this value is appropriate and actually is fitted for a correct plastination procedure.
MATERIALE PLASTICE 52 No. 2 2015http://www.revmaterialeplastice.ro186
Slice Plastination and Shrinkage
MIRCEA-CONSTANTIN SORA
1
, MAXIMILLIAN BINDER
1
, PETRU MATUSZ
2
, HORIA PLES
3
*, IOAN SAS
4
1
Medical University of Vienna, Plastination Laboratory, Center for Anatomy and Cell Biology, Plastination and Topographic
Anatomy, Waehringerstr. 13/ 3, A-1090 Wien, Austria
2
"Victor Babes” University of Medicine and Pharmacy Timisoara, Department of Anatomy, 2 Eftimie Murgu Sq., 300041, Timisoara,
Romania
3
"Victor Babes” University of Medicine and Pharmacy Timisoara, Department of Neurosurgery, 2 Eftimie Murgu Sq., 300041,
Timisoara, Romania
4
"Victor Babes” University of Medicine and Pharmacy Timisoara, Department of Obstetrics and Gynecology, 2 Eftimie Murgu Sq.,
300041, Timisoara, Romania
Plastination suits very good for 3D reconstruction and morphometric measurements. Digital representation
of anatomical features has provided a useful source of data for computer-based instructional development.
The validation of 3D reconstructions and measurements needs a thoroughly knowledge about shrinkage.
The aim of this paper is to determine the average slice shrinkage rate occurring during sheet plastination
(E12 and P40). Fresh human brain slices for P40, pelvis slices for the standard E12 technique and a shoulder
tissue block for the E12 thin slice technique were used for this study. In order to determine the shrinkage rate,
the slice areas were measured before and after plastination for al samples. For the E12 thin slice technique,
the shoulder tissue block was measured before and after plastination and a volumetric shrinkage rate was
determined. The shrinkage rate was for P 40 technique 5.74%, for standard E12 technique 6.54% and for the
E12 thin slices technique 6.23%. Therefore, plastinated slices showed a decreased shrinkage rate and fit best
for research purposes. All shrinkage rates were under 10%, so we believe that every shrinkage below this
value is appropriate and actually is fitted for a correct plastination procedure.
Keywords: plastination, polymer E12, polymer P40, shrinkage
As we all know, the process of plastination has, since its
inception, taken giant strides and have been applied in a
variety of fields. It has been used in the field of teaching to
create examples for display purposes and, of course,
plastination has been used as a tool in research [1-7]. It
became quite clear that the process of plastination has
the potential to be used in a numerous and especially
various fields of interest. The most powerful plastination
tool to be applied in research is the sheet plastination. The
use of the sheet plastination method provides more
accurate and precise data than those obtained with the
use of dissection. Slice plastination performs no
modifications or deformations of anatomic tissue, thereby
making this technique a valuable tool in the evaluation of
topographic relations. For keen anatomists, one of the main
challenges is to obtain accurate 3D reconstruction of
specific complex anatomical regions [8-10]. Surgeons
require these to obtain both precise anatomical knowledge
and surgical orientation [11-14]. Surgeons and radiologists
can view these 2D images and usually create a mental
model of 3D structures in the study. However, many
anatomical structures have a complex morphology that
passes in and out of the cross sections [15, 16]. Plastination
has been proposed as a suitable method to provide
geometrical data for 3D reconstruction of hard and soft
tissue. Transparent serially-sectioned plastinated slices
reveal visual clarity of gross structures under
submacroscopic levels [16]. One of the most common
critics when dealing with plastination in research is
shrinkage. The main fields of using slices plastination in
research are morphometric measurements or 3D
reconstructions. In order to validate the obtained data, it is
needed to know the shrinkage rate of the specimens. As it
is known, shrinkage occurs during plastination, but the
question to be answered is: How high should be an
acceptable shrinkage? The purpose of this article was to
evaluate the shrinkage during sheet plastination of P40
brain slices, E12 body slices and E12 thin slice plastination.
Experimental part
P40 brain slices
A human brain was obtained post-mortem The brain
was maintained for one month in 5% formalin. Before being
serially sectioned, the brain was washed in running tap
water for one day. By using a meat slicer, the brain was
sliced into 16 sagittal slices with a thickness of 4 mm. The
obtained slices were placed into two grid baskets and into
distilled water at +5 °C overnight [17]. The slices were
placed then into an acetone bath at -25 °C. After two days
the grid baskets containing the slices were moved into
another acetone bath, also at -25°C. Dehydration was
completed after one week and the slices were scanned.
The slices were placed with the grid basket into the
immersion bath of P40 at -25 C for one day. Following this
procedure, the brain slices were taken from the immersion
bath (-25 °C) and placed in cold P40 at -25 °C in the vacuum
chamber. The vacuum chamber was then placed in a
freezer at -25°C. Impregnation was undertaken for 12 h
and completed under reduced pressure (2mm Hg) [18].
All slices were casted in flat chambers and the chambers
subject to UV irradiation for 3 h. The finished plastinated
slices were once again scanned in order to determine the
shrinkage rate between the fresh and plastinated slices.
Standard E12 body slices (3mm)
One female pelvis used for this study was removed from
a fresh unfixed cadaver and then frozen at –80 °C for one
week. In the next step slices with an average thickness of
* email: horia.ples@neuromed.ro; Tel.: 0744554540
MATERIALE PLASTICE 52 No. 2 2015 http://www.revmaterialeplastice.ro 187
3 mm were cut by using a band saw. Numbering markers
were placed on the cranial side. The slices were stored at
-25 °C overnight. The slices were plastinated according to
the standard E12 slice plastination method [1, 9]. Freeze
substitution is the standard dehydration procedure for
plastination, the shrinkage is minimized when cooled
acetone is used. The slices were submerged in cold (-25
°C) pure acetone for dehydration. Degreasing was
performed by using methylene chloride. Impregnation was
performed using the following epoxy Biodur (Heidelberg,
Germany) mixture: E12 (resin)/ E1 (hardener) [9] .The
original size of the frozen slices and their size after
dehydration were scanned with an Epson GT-10000+ Color
Image Scanner.
Thin slice E12 plastination (1mm)
One male shoulder was frozen at –80 °C for one week.
Afterwards, it was plastinated according to the standard
ultra-thin E12 slice plastination method [4, 19]. The tissue
block was submerged in cold (-25 °C) pure acetone for
dehydration. Degreasing was performed by using
methylene chloride. Impregnation was performed using
the following epoxy Biodur (Heidelberg, Germany) mixture:
E12 (resin)/ E6 (hardener)/ E600 (accelerator) [19]. When
impregnation was completed, the tissue block was
removed from the vacuum chamber. A mould of styrofoam
was constructed and lined with polyethylene foil. The tissue
block was inserted into the mould. The mould containing
the impregnated specimen and resin-mix was kept at 65
°C in oven, for four days to assure the hardening of the
resin-mix. The tissue/resin block was cooled at room
temperature and the mould was removed. A contact point
diamond blade saw, Exact 310 CP (Exact Apparatebau
GmbH, Norderstedt, Germany) was used for cutting the
block in 1 mm slices. Prior to plastination, the length, width
and depth of the frozen tissue block was measured after
placing markers into the tissue block. Following
plastination, the same parameters were measured at the
same spots as before plastination.
Results and discussions
P40 brain slices
In order to calculate shrinkage, the surface area of each
brain slice was measured before and after plastination.
Each slice was scanned into the computer. The finished
plastinated slices were once again scanned to determine
the shrinkage rate between the fresh and plastinated slices.
The surface area of each slice was calculated by using the
UTHSCSA Image Tool software for Windows, version 3.0
(University of Texas Health Science Centre at San Antonio,
San Antonio, TX). By comparing the area of the fresh and
of the plastinated slices a two dimensional shrinkage was
calculated. P40 slices had a shrinkage rate of 5.74%. (table
1).
Standard E12 body slices (3mm)
The surface area of each slice was measured before
and after plastination. Each slice was scanned into the
computer. The average shrinkage after standard E12
plastination was 6.54 %. This value is reasonable, with
regard to the plastination conditions. Once again, we
observed that some slices shrinked more than others. A
possible explanation could be the fact that these slices
contain more lipid tissue than others. In order to investigate
the shrinkage of different tissue types, we measured on
the slices the muscle areas and connective tissue area
before and after plastination. The connective tissue had a
shrinkage rate of 7.22% and the muscles had shrinkage of
6.82%.
Table 1
MEASUREMENTS OF BRAIN SLICES BEFORE AND AFTER P40
PLASTINATION (n = 10)
Fig.1. Mediosagital section of the brain: A-fresh cut brain slice; B-
P40 plastinated slice
Table 2.
MEASUREMENTS OF BODY SLICES BEFORE AND AFTER STANDARD
E12 PLASTINATION (n = 14)
Thin slice plastination (1mm)
To determine shrinkage resulting from the plastination
process, the tissue block from the body was first measured
immediately after removal and subsequently following
plastination. Prior to plastinaton, the length of the shoulder
block from the superior pole to the inferior pole was
measured and the depth and width were determined in
the middle of the anterior surface. Needles were used as
markers to define the measurements points. By comparing
the data of the fresh tissue block and the measurement
data of the plastinated block, the amount of shrinkage of
the specimen was calculated to be 6.23%.
In plastination studies, shrinkage is a main topic.
Independent of the technique used (S10, E12 or P40), the
process will generate shrinkage. Numerous investigators
studied the shrinkage rate of specimens during the S10
technique [20-25], but the shrinkage in E12 and P40
technique has been studied briefly [26-28]. The question
is: Why is shrinkage so relevant? Firstly, because a
considerable shrinkage will decrease the value of the
specimens and distort the initial shape. Secondly, because
shrinkage determines considerable deviations from the
original specimen size. This second reason is very important
when dealing with morphometric measurements and 3D
reconstructions. As we all know, slice plastination (E12,
P40) has the highest impact in applying plastination as a
research tool. Only by considering the shrinkage rate in our
calculations, the morphometric measurements and 3D
reconstructions could be valid.
Since the beginning of plastination, the E12 technique
was, and still is, the elected method for producing
MATERIALE PLASTICE 52 No. 2 2015http://www.revmaterialeplastice.ro188
transparent body slices. Transparent body or organ slices
are used for teaching and research purposes, because they
allow studying the topography of all body structures in a
non-collapsed and non-dislocated state. In addition, the
specimens are useful in advanced training programs in
sectional topography (resident training in CT and NMR).
Most of the research studies deal with the topography of
anatomical structures. However, if distances between
structures or calibers of vessels are to be measured on
plastinated slices, correct results can only be obtained
when the shrinkage rate is considered. Two factors
contribute to the amount of shrinkage: the shrinkage of
the epoxy polymer itself and the shrinkage of the tissue
slices during the plastination process. The observed
shrinkage of the E12 resin was found to be less than 0.2%.
These results are comparable to data we obtained from
the CIBA Company (Ciba Spezialtätenchemie GmBH,
Vienna, Austria). The shrinkage values determined in the
present study represent only the two-dimensional
shrinkage. The shrinkage rate of 6.54% represents actually
the shrinkage of the tissue slice during dehydration,
degreasing and impregnation. As the slice consists of
different tissue types we observe that the connective tissue
shrink most, having a shrinkage rate of 7.42% followed by
the muscular tissue with a shrinkage rate of 6.92%. Bony
tissue does not shrink and contribute to the shape
conservation of the slice.
In the last decade, plastinators started to use more
frequently P40 as an alternative for E12. There is mainly
one reason for this, i.e. the epoxy resin turns yellow in time
and the colour of the slices get dark, but polyester remains
clear, without changing the colour. A disadvantage of
polyester resin is the lower breaking index, so the
transparency of the connective tissue will be diminished.
We know that unsaturated polyester resins can usually
shrink 5 to 8% during the transition from the liquid to the
solid state [29-30], but this is a 3D shrinkage. Our
measurements determined a shrinkage value of 5.74%,
which is bidimensional and seems to be acceptable in this
context. P40 plastination is, in our opinion, the best method
to preserve brain slices. Furthermore, we consider that the
P40 technique is much better than the S10 standard
technique for plastination of brain slices, where shrinkage
can reach up to 10% [31].
Digital representation of anatomical features has
provided a useful source of data for computer-based
instructional development. In fact, the development of
multimedia tools for anatomical learning has received
much attention, spawning the new field of anatomical
informatics [32]. However, an initial cadaveric data source
is necessary to facilitate creation of multimedia resources.
Plastination serves as one human tissue preservation
technique that involves the replacement of water and lipids
with curable polymers that are subsequently hardened [33].
The E12 thin slice technique has proved particularly useful
for 3D reconstruction of anatomical structures and should
serve well as a data source for multimedia [16]. In order to
generate 3D structures, we need firstly to build up a
plastinated tissue block and then to cut it in thin (1mm)
slices. In this case, we can calculate only a 3D volumetric
shrinkage of our tissue block, but not shrinkage of different
tissue types. The calculated shrinkage (length/ width/
depth) in our study was 6.23% and there are not literature
data to compare to. We know that epoxy resin practically
do not shrink, so we could regard this shrinkage as the
shrinkage of the tissue block during plastination. When
dealing with 3D reconstruction, the main parameters are
length and width. These parameters will be always being
asked at the beginning of each reconstruction. The third
dimension, the thickness of the slice, is always the
thickness of the original slices cut from the fresh tissue
block, which is known from the beginning. In regard of
this, the main shrinkage that has to be known is the
bidimensional (length/ width) one.
But how high could be an acceptable shrinkage rate?
Theoretically, if we calculate it for every slices and tissue
type, we can accept every shrinkage rate that occurs. On
the other hand, for high shrinkage rates, the original
specimen size is lost. Combined with the tissue lost during
specimen sawing, all calculations or 3D reconstructions
could deviate and be distort. That is the reason why this
study tries to define an acceptable shrinkage rate for E12
and P40 plastination. As von Hagens [17] stated that up to
10% is unavoidable during plastination, we believe that
every shrinkage rate, for the E12 and P40 method, which
is lower than this value is very good and actually is a
feedback for a correct plastination procedure.
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Manuscript received: 22.09.2014
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