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The official publication of the International Society for Plastination
The Journal of Plastination
I SSN 2 311 -77 61
Volume 30 (1); July 2018
Influence of the
Temperature on the
Viscosity of Different Types
of Silicone – p4
A Comparison of Different
De-plastination
Methodologies for
Preparing Histological
Sections – p10
Biomechanical Analysis of
The Skin and Jejunum of
Dog Cadavers Subjected
To A New Anatomical
Preservation Technique For
Surgical Teaching – p16
Bleaching of Specimens
Before Dehydration in
Plastination: A Small-scale
Pilot Study Using Human
Intestine – p24
General Issues of Safety in
Plastination – p27
IN THIS ISSUE:
The Journal of Plastination
ISSN 2311-7761 ISSN 2311-777X online The official publication of the International Society for Plastination
Editorial Board:
Rafael Latorre Murcia, Spain
Scott Lozanoff Honolulu, HI USA
Ameed Raoof. Ann Arbor, MI USA
Mircea-Constantin Sora Vienna, Austria
Hong Jin Sui Dalian, China
Carlos Baptista Toledo, OH USA
Philip J. Adds Editor-in-Chief Institute of Medical and Biomedical Education (Anatomy) St. George’s, University of London London, UK
Robert W. Henry Associate Editor Department of Comparative Medicine College of Veterinary Medicine Knoxville, Tennessee, USA
Selcuk Tunali Assistant Editor Department of Anatomy Hacettepe University Faculty of Medicine Ankara, Turkey
Executive Committee: Rafael Latorre, President Dmitry Starchik, Vice-President Selcuk Tunali, Secretary Carlos Baptista, Treasurer
Instructions for Authors
Manuscripts and figures intended for publication in The Journal of Plastination should be sent via e-mail attachment to: [email protected]. Manuscript preparation guidelines are on the last two pages of this issue.
On the Cover: Right atrium of a plastinated human heart showing pectinate muscles and portion of the crista terminalis
visualized by trans illumination. Specimen from the collection of the Liberato Didio and Peter Goldblatt Interactive
Museum of Anatomy and Pathology, University of Toledo. Photography courtesy of Dr. Telma Masuko.
The Journal of Plastination 30(1):1 (2018)
Journal of Plastination Volume 30 (1); July 2018
Contents
Letter from the President, Rafael Latorre 2
Letter from the Editor, Philip J. Adds 3
Influence of the Temperature on the Viscosity of Different Types of Silicone; Athelson S. Bittencourt, Yuri F. Monteiro, Laissa da S. Juvenato, et al
4
A Comparison of Different De-plastination Methodologies for Preparing Histological Sections of Material Plastinated with Biodur® S10 / S3; M. L. Ramos, et al
10
Biomechanical Analysis of The Skin And Jejunum Of Dog Cadavers Subjected To A New Anatomical Preservation Technique For Surgical Teaching; T.A. Rocha, C. Santos, A. Fechis, F. Oliveira, et al
16
Bleaching of specimens before dehydration in plastination: a small-scale pilot study using human intestine; Jie-Ru Chen, Hong-Jin Sui
24
General Issues of Safety in Plastination, V. K. Schill 27
Instructions for Authors 37
The Journal of Plastination 30(1):2 (2018)
LETTER FROM THE
PRESIDENT
Dear Friends and Plastinators:
It is with great pleasure that I present to you Volume 30, Issue 1, of the Journal of
Plastination. I would like to thank the reviewers for taking their time to review the
manuscripts.
In this issue, we present remarkable papers. The first paper from Dr. Bittencourt
is about the viscosity of different silicones for plastination, and the importance of
considering their physicochemical characteristics and dynamic viscosity, before
choosing the ideal silicone for our particular needs. The second study is about
deplastination for histology studies, from Dr. Moema Lopes Ramos. It presents a
very interesting result: that it is possible to produce histological sections directly
from plastinated specimens, without previous deplastination, at least in the three
tissues tested. The third paper, presented by Dr. Rocha, describes a new protocol
for tissue preservation, focused on surgical training applications. The fourth paper
of this issue is a very concise work presented by Dr. Sui, about the specific effects
of bleaching before plastination on the final appearance of the specimens. The last
paper, about General Issues of Safety in Plastination, by Mr. Volker Schill, is a very
important paper for all of us. We all are worry if we do not have a proper vacuum
pump or a good impregnation chamber for instance, however, we are not always
alert about how to work in a healthy ambience in our plastination lab. This paper
helps us to be aware of the potential hazards of the chemicals that we are working
with every single day.
I would like to welcome all new members of the International Society for
Plastination (ISP) and to invite all of you to participate in the Journal of
Plastination. Please, share with us your results, and your expertise in plastination
and other anatomical techniques.
With best regards from Murcia, Spain
Rafael Latorre
President
Rafael Latorre, DVM, PhD
The Journal of Plastination 30(1):3 (2018)
LETTER FROM THE EDITOR
Dear Colleagues,
Thank you to those who have submitted manuscripts to the Journal. We are pleased to
publish five new papers in this issue, on topics ranging from safety in the laboratory, to
technical reports on the plastination process. The world-wide reach of this technology is
evidenced by the fact that the papers in this issue come from Brazil, China and Germany,
with three coming from teams working in Brazil.
My main priority for next year is to get the Journal indexed. We have made changes to
the journal in the light of feedback we received from the National Library of Medicine,
which should help in making a strong case for getting the journal indexed on Medline in
the near future.
In the meantime, I have submitted an application to Scopus and Embase. “Over 8,500
journals are currently indexed in Embase, and each year several hundred additional
journals of potential interest are screened by an editorial committee entrusted with the
review and quality assessment of biomedical publications for Embase.”1
Scopus is the largest abstract and citation database of peer-reviewed literature:
scientific journals, books and conference proceedings, covering over 36,000 titles.2
The review process, however, is not fast, and it can take many months from application
to actual inclusion. The acknowledgement email from Scopus advised us to allow a
minimum period of 6-12 months for the review process to be completed. Yesterday I
received the following message in response to my request for an update: “The title
Journal of Plastination is in the final phase of the evaluation process and we are now
waiting for the final decision of our Content Selection & Advisory Board (CSAB). Once the
outcome of the evaluation by the CSAB is available, you will be contacted by us again.
We appreciate your patience in this matter.”
I hope to be able to report more positive news in the next issue.
Best wishes,
Philip J Adds Editor-in-Chief References
1. https://www.elsevier.com/solutions/embase-biomedical-research/journal-title-
suggestion?sb=1508153513999
2. https://www.elsevier.com/en-gb/solutions/scopus
Philip J. Adds, MSc, FIBMS, SSFHEA
The Journal of Plastination 30 (1): 4-9 (2018)
ORIGINAL RESEARCH
Influence of the Temperature on the Viscosity of Different Types of Silicone
YURI F. MONTEIRO1*,
LAISSA DA S. JUVENATO1,
ANA PAULA S. V.
BITTENCOURT2,3,
BRUNO M.M. SIQUEIRA2,
FLÁVIO C. MONTEIRO2,
CARLOS A C BAPTISTA4,
ATHELSON S.
BITTENCOURT1,2*
Department of Morfology,
Federal University of
Espirito Santo, Brazil
ABSTRACT:
The objective of this work was to test the influence of temperature on the viscosity of three
silicones of different molecular weights (Biodur® S10, Polisil® P10 and P1) commonly
used in the plastination technique. For the study, the RheolabQC model rotational
rheometer was used to measure the dynamic viscosities of the chosen polymers at the
following temperatures: -5, 0, 5, 10, 15, 20, 25, 30 and 35 °C. From the 9 measurements
of viscosities obtained from each sample, a viscosity vs. temperature graph was
constructed. The equation of the dynamic viscosity curve of each polymer was analyzed.
Polisil® P1 silicone had a much lower viscosity compared to other silicones (about 80
mPa.s at 25 °C and 550 mPa.s at -25 °C). Polisil® P10 silicone presented the highest
viscosity of the polymers analyzed (approximately 1180 mPa.s at 25 °C and 3730 mPa.s
at -25 °C). The Biodur®'s S10 silicone showed an intermediate viscosity (about 410
mPa.s at 25 ° C and 1500 mPa.s at -25 °C). We conclude that Polisil® P1 silicone
presented the best physico-chemical characteristics of the tested silicones for
plastination, because it has high fluidity and low viscosity. It is noteworthy that the
viscosity of Polisil® P1 in cold impregnation temperature (-15 °C) is still lower than the
viscosity of the Biodur® S10 (control) at room temperature (20-25 °C). We also conclude
that the knowledge of the intrinsic and extrinsic physicochemical characteristics of the
silicone and its dynamic viscosity is helpful in choosing the ideal silicone for use in the
cold or room temperature plastination techniques.
KEY WORDS: Viscosity, PDMS, temperature, silicone, plastination
* Correspondence to: Athelson S Bittencourt, Federal University of Espirito Santo Health Sciences Center, Marechal Campos Avenue, 1468 Maruipe, Vitoria- ES, Brazil Zip code: 29.043-900, Fax: +55 27 33357358, [email protected]
Introduction
The term silicone, or polysiloxane, was created in 1901 to
describe mixed polymers of organic and inorganic
materials, whose crude formula is [R2SiO]n, where R are
organic groups such as methyl, ethyl and phenyl. These
polymers are inert, odorless, insipid and resistant to
decomposition by heat, water or oxidizing agents, besides
being good electrical insulators. They exhibit good
resistance to high or low temperatures (-45 to +145 °C)
and have viscosities between 10 and 100,000 millipascal
second (mPa.s) (Milles et al., 1975).
A polymer is a macromolecule formed by repetitive
structural units, joined together by covalent bonds. In
silicone, the repeating unit is siloxane (subgroup of silica
compounds containing Si-O bonds with organic radicals
attached to the molecule) (Carraher, 2003).
Silicones, technically, are polymers that can be obtained
basically in three steps: synthesis of chlorosilanes,
hydrolysis of chlorosilanes to silanols and polymerization
of silanols. The first step occurs in a fluidized bed of metal
silicon powder treated with a flow of chloromethane,
generally at temperatures of 250 to 350 °C and pressures
of 1 to 5 atm. A mixture of different chlorosilanes is
obtained mainly containing the dimethyldichlorosilane
(Me2SiCl2), which represents the most important
monomer for the subsequent steps. In the second step,
polydimethylsiloxanes are obtained by the hydrolysis of
dimethyldichlorosilane, in the presence of excess water
(Hardman, 1989).
The products of this reaction are readily condensed, thus
leading to a mixture of linear and cyclic silicones. The
linear and cyclic oligomers obtained by hydrolysis of
dimethyldichlorosilane have still very short chains, for
Influence of the Temperature on the Viscosity - 5
most applications. In the third step, they must be
condensed (in the case of linear ones) or polymerized (in
the case of cyclic ones), to obtain macromolecules of
satisfactory lengths (Hardman, 1989). This last step is
decisive for the determination of the viscosity of the final
product, since the viscosity of the silicones is directly
related to their degree of polymerization (n). Depending
on the size of the polymer chain, the silicone can be
produced in three forms: liquid, gel and cohesive
(Hardman, 1989).
Viscosity is a characteristic of liquids that is related to their
ability to flow. The greater the viscosity of a liquid (or a
solution), the greater the difficulty of the liquid to flow and
more "viscous" the liquid is. One of the main external
factors influencing the viscosity of a silicone is the
temperature (Oliveira, Barros and Rossi, 2009). The
viscosity is directly proportional to the internal friction of
the silicone, and friction originates from the pulling force
of the silicone molecules themselves. As temperature
increase, this pulling force decrease, causing viscosity
reduction. This reduction of viscosity occurs due to the
increase of the intermolecular distances of the silicone by
the higher kinetic energy (caused by the heating),
reducing the attraction forces and, consequently, the
friction of molecules, allowing a faster flow (lower
viscosity) (Granjeiro et al, 2007).
For the rheological study of liquids, two factors are of
great importance and should always be observed: shear
stress and shear rate. Shear stress is a type of stress
generated by forces applied in opposite orientation, but in
similar directions in the analyzed material. On the other
hand, shear rate or deformation rate is defined by the
variation of the shear deformation in relation to the time
(Shiroma, 2012).The concepts of shear stress (applied
force) and shear rate (velocity gradient) are used to
describe the deformation and flow of a fluid. Fluids in
which the shear stress is directly proportional to the rate
of deformation are called Newtonian fluids, and the
viscosity is a constant for these fluids. However, if the
shear stress is not directly proportional to the shear rate,
the fluid is termed non-Newtonian; therefore, the viscosity
varies according to the shear stress applied to the fluid
(Schramm, 2006).
The main silicone used in the plastination process is
polydimethylsiloxane (PDMS), which is a linear polymer
whose radicals are methyl groups (Chaynes and
Mingotaud, 2004). A literature search on the subject of
viscosity of silicones for plastination reviewed a lack of
research in this area. This study aimed to show the
characteristics of three silicones of different molecular
weights (Biodur S10, Polisil P10 and P1), and the
influence of the temperature in their viscosity. Knowledge
obtained in this study will allow us to better understand
the variables influencing the impregnation process.
Materials and Methods:
The RheolabQC rotational rheometer, manufactured by
the Austrian multinational Company Anton Paar, was
used to test the influence of temperature on the viscosities
of the silicones. The equipment was used with a coaxial
cylinder measurement system to measure the dynamic
viscosities of the polymers Biodur® S10, Polisil® P10 and
P1 in the following temperatures: -5, 0, 5, 10, 15, 20, 25,
30, 35 °C. As recommended by the manufacturer
protocol, the samples of silicones were transferred one by
one to a specific vessel for use in the rheometer with a
cylindrical rod rotating within the sample, thus generating
a shear in the fluid. The vessel was attached to the
measuring head of the rheometer. In addition to the
dynamic viscosity and temperature, the apparatus also
measured the shear rate and the shear stress. The
apparatus has a cooling and heating system coupled to
the samples, with the minimum and maximum
temperature reached varying from -5 to 80 °C.
The dynamic viscosities of the three silicone samples
were measured in 9 different temperatures, and at each
pre-programmed temperature, the device made 100
measurements of the actual temperature and its
respective viscosity, with the shear rate varying from 100
to 600 seconds-1 (s-1). The shear rate and stress values
were also measured. The data were plotted in the
software Start Rheoplus® by the equipment itself and
later exported to Microsoft Excel®.
The measurements obtained by the rheometer, i.e. the
temperature averages and respective viscosities ±
standard deviation, were calculated and plotted. An
equation of the dynamic viscosity curve of the different
types of silicone was generated.
Results
Nine viscosity measurements were obtained from
each sample. The temperature averages and their
respective viscosities are showed in Table 1. A viscosity
vs. temperature graph was constructed, and the dynamic
viscosity curve equation of each polymer analyzed
(Graph 1).
6 - Bittencourt, Monteiro, Junvenato
Table 1. Average values of the temperatures (°C) and respective dynamic viscosities (Pa.s) of the
Biodur® S10, Polisil® P10 and P1 silicones measured by the RheolabQC model rotational rheometer.
The graphic of dispersion (XY) for each polymer from
table 1 was ploted and its curve equations were
calculated. The most suitable trend line that represents
the viscosity points of the silicones is the exponential type
(Giap, 2010; Romano et al., 2017). The use of 9
temperature points to generate the construction of a
dispersion graph gave greater reliability to the results.
Only three points in a scatter plot (XY) are sufficient to
make a trend line, although the higher the number of
measurements the more reliable the graph equation
would be (Skoog, 2014). The determination of the curve
equation of each silicone was done by Excel® software,
through point-to-point regression. Graphs 1, 2 and 3 show
the viscosity vs. temperature curves for each silicone. All
graphs present decreasing of viscosity with the increasing
of temperature.
Graph 1. Graph 1. Viscosity dispersion vs temperature of
polymer S10 ± standard deviation, including the curve
equation (y), the coefficient of determination value (R2).
Graph 2. Viscosity dispersion vs temperature of polymer
P10 ± standard deviation, including the curve equation (y),
the value of the determination coefficient (R2).
Graph 3. Viscosity dispersion vs temperature of polymer P1
± standard deviation, including the curve equation (y), the
coefficient of determination (R2) value.
SILICONE BIODUR® S10 SILICONE POLISIL® P10 SILICONE POLISIL® P1
ºC Pa.s ºC Pa.s ºC Pa.s
-5,07 0,918 -5,08 2,423 -5,04 0,260
-0,08 0,791 0,27 2,110 0,09 0,213
5,91 0,677 5,27 1,846 5,89 0,174
10,33 0,593 10,20 1,630 10,35 0,147
15,11 0,515 15,46 1,451 15,22 0,118
19,93 0,458 20,22 1,301 19,80 0,098
25,37 0,404 25,48 1,168 25,40 0,080
30,29 0,361 30,47 1,055 30,42 0,068
35,40 0,323 35,27 0,958 35,09 0,059
Influence of the Temperature on the Viscosity - 7
Equations of the curves found for the silicones S10, P10
and P1 are y = 0,785e-0,02x, y = 2,102e-0,02x and y =
0,213e-0,03x, respectively. The degree of accuracy
between the viscosity values calculated by the equation
and the actual values can be demonstrated by the
coefficient of determination (R2). R2 is a statistical real
data points. An R2 of 1 indicates that the regression line
perfectly fits the data.The closer R2 is to the 1, closer to
real are the values calculated from the equations of each
curve. The R2 values of the silicones graphs are: for P10
(0.997), S10 (0.996) and P1 (0.998). The values of R2
showed a degree of accuracy of the equation higher than
99.5%, a very high degree of association (Cosentino et al,
2013). The standard deviation was calculated for each
point of the graph, however some deviations were too
small to appear in the plot, denoting a high homogeneity
and precision of the measurements obtained.
The lack of linearity of the polymeric viscosity became
more evident with the decrease in the viscosity of the
silicone. The increase in the viscosity of the P10, S10 and
P1 silicones from the maximum temperature (35 °C) to the
minimum (-5 °C) measured on the rheometer was 153%,
184% and 340%, respectively.
Mathematical calculations were used to determine
viscosities in temperatures outside the range of the
equipment, but important for the plastination process.
Plotting a value to the curve equation, an approximate
value of viscosities of the silicone samples at any desired
temperature is obtained. Table 2 shows the viscosity
values of the three tested silicones at different
temperatures, calculated from the curve equation for each
polymer. Graph 4 shows the comparison of the viscosity
curves of the silicones Biodur® S10, Polisil® P10 and P1
calculated from the curve equations of the silicones.
Plotting the curves of the three types of silicones on the
same graph make it possible to observe the viscosity and
behavior of each silicone by changing the temperature
(graph 4); therefore, the viscosity values were calculated
from the curve equations of tested silicones.
Table 2. Comparative viscosity values (mPa.s) calculated
from the viscosity curve vs. temperature equation of the
silicates S10, P10 and P1 at different temperatures of
importance in plastination.
Temperature Biodur®
S10
Polisil®
P10
Polisil®
P1
-25° C 1505 3736 552
-20° C 1321 3330 457
-18° C 1254 3181 424
-15° C 1160 2969 378
-10° C 1019 2646 312
- 5° C 894 2359 258
0° C 785 2102 213
5° C 689 1874 176
10° C 605 1670 146
15° C 532 1489 121
20° C 467 1327 100
25° C 410 1183 83
30° C 360 1054 68
Graph 4: Comparison of the viscosity and temperature
curves of the S10, P10 and P1 silicones. The viscosity
values were calculated from each curve equation of the
tested silicones.
Discussion
Knowledge of the influence of temperature on the
viscosity of the silicones used in plastination is of great
importance. Through a detailed study of this topic, several
questions can be raised, such as: 1) what is the ideal
temperature of the silicone in low temperature (LT)
impregnation to decrease the retraction of the tissues and
also to avoid hardening over time? 2) what is the ideal
viscosity of the silicone for plastination at low and room
temperatures? 3) what is the rheological behavior of
silicones with different viscosities? 4) how does the
viscosity of different silicones behave over time when in
the reaction mixture for cold impregnation? and 5) does
the temperature increase present a linear relationship
with the viscosity increase?
8 - Bittencourt, Monteiro, Junvenato
The graphs (Graph 1, 2 & 3) for each silicone show the
behavior of each polymer against the temperature
gradient. An exponential increase in the viscosity of the
silicones was observed with each temperature decrease.
The shear rate and shear stress measurements made by
the apparatus were used to show that although the
polymers are considered non-Newtonian fluids, the flow
curves (shear rate vs. shear stress) of the tested silicones
showed that they are closer to the Newtonian fluids’
features (Orrah et al., 1988; Schneider et al., 2009). As
the flow curve (rate vs shear stress) becomes more linear,
the closer the sample will be to the behavior of Newtonian
fluids (Schramm, 2006).
The rheological characteristics of the three silicones used
in plastination allow us to define their dynamic viscosity
as the average of hundreds of measurements made at a
given temperature. Viscosities remained constant or had
a minimum change, regardless of the shear stress applied
to the sample at a given temperature.
The different viscosities found in the tested silicones are
determined by the degree of polymerization. The larger
the silicone chain (P10> S10> P1), the more
intermolecular bonds are made with adjacent molecules,
and thus the less fluidity of the chain. The P10 has the
largest chain (molecular weight), and consequently the
highest viscosity. The different viscosities found within the
same silicone sample were mainly found at different
temperatures. Molecular cohesion is the dominant cause
of viscosity, and, as the temperature of the silicone
increases, these cohesive forces decrease, resulting in a
decrease in viscosity (Granjeiro et al., 2007).
The curves of viscosity vs temperature of all analyzed
silicones (Graphs 1, 2 & 3) has an exponential trend,
showing that there is no linearity in the increase of the
viscosity as a function of temperature, and that these
values of viscosity grow with increasing rates. Therefore,
the lower the temperature, the steeper the viscosity curve
of the silicones becomes.
Silicone P1 has a higher proportional increase in viscosity
with decreasing temperature, making its curve steeper in
proportion to the others (Graph 3). However, P10 silicone
has the highest viscosity and the lowest increase
proportional to the decrease in temperature, that is, its
curve is proportionally less steep when compared to the
others (Graph 2).
Silicones of lower molecular weight are more sensitive to
the temperature gradient, when compared to those of
higher molecular weight. As expected, all graphs showed
a strong negative correlation, that is, the increase on the
temperature variable implies in a decrease on the
viscosity variable.
At 35 °C (maximum measured temperature), silicones
P10 and Biodur® S10 are respectively 16.2 and 5.5 X
more viscous than P1. At -5 °C, P10 and Biodur® S10 are
respectively 9.3 and 3.5 X more viscous than P1. The
lower the temperature, the lower the difference between
the viscosities of the silicones.
The viscosity data found are in accordance with the
values provided by the manufacturers (Polisil® P10 =
1000-1500 mPas, Biodur® S10 = 450-600 mPas and P1
= maximum of 100 mPas, all at 25 °C).
The P1 Silicone appeared to be a good alternative to the
reference silicone in plastination (Biodur® S10), since it
has a viscosity at least 4 X lower at room temperature and
3 X in cold temperature, when compared to S10. The
lower viscosity of P1 allows the silicone to flow more
quickly and easily into the biological tissues during forced
impregnation, therefore reducing shrinkage. The viscosity
of P1 at cold temperature is less than the viscosity of
Biodur® S10 at room temperature. Thus, plastination with
the silicone P1 at low temperatures is likely to produce
equal or less shrinkage than the Biodur® S10 at room
temperature. The advantages attributed to room
temperature plastination are described in the literature
(Starchik and Henry, 2015b). It is known that the higher
the viscosity of a silicone, whether by the size of the
polymer chain or the reduction in silicone temperature, the
greater the retraction of biological tissues in the forced
impregnation step of plastination (Starchik and Henry,
2015b). The use of low molecular weight silicones may be
preferable when a specimen with less shrinkage is
sought. Specimens more prone to shrinkage, such as the
brain and nervous system, will benefit from impregnation
using silicones of shorter chains (more fluid).
From the results presented by this research, we conclude
that the Polisil® P1 silicone presents the best physico-
chemical characteristics of the silicones tested for the
application in plastination, because it has high fluidity and
low viscosity. It is noteworthy that the viscosity of P1 at
cold impregnation temperature (-15 °C) is still lower than
the viscosity of Biodur® S10 at room temperature
(20 - 25 °C).
Influence of the Temperature on the Viscosity - 9
Financial support
This research was supported by CNPq (458328/2013-8),
undergraduate scholarship provided by UFES and
graduate scholarship provided by CAPES.
Acknowledgements
Special thanks to the scientific collaboration with
Processing and Characterization Laboratory/LabPetro -
UFES
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The Journal of Plastination 30(1):10 - 15 (2018)
ORIGINAL RESEARCH
A Comparison of Different De-plastination Methodologies for Preparing Histological Sections of Material Plastinated with Biodur® S10 / S3
M. L. RAMOS1,2
T. A. R. DE PAULA2,
M. F. ZERLOTINI2,
V. H. D. SILVA2,
L. B. CARAZO2,
M. F. DE PAULA2;
F. F. R. SILVA2
M. L. SANTANA2;
L. C. SILVA2;
L. B. C. FERREIRA2
1 Institute of Biological
and Health Sciences,
Federal University of
Viçosa, campus Rio
Paranaíba, Brazil.
2 Department of
Veterinary Medicine,
Federal University of
Viçosa, Brazil.
ABSTRACT:
Objectives - The objective of this study was the evaluation of different protocols to obtain
histological slides from silicone plastinated specimens.
Materials and Methods - Samples of pig aorta, heart, and kidney were used. Four
treatments for light microscopy (LM) were compared. Treatment 1 (control): fixation in
10% formalin for 48 h at room temperature; tissue samples were then processed for LM
histology. Treatment 2: plastinated fragments were directly embedded in paraffin wax.
Treatment 3: plastinated tissue samples were de-plastinated by immersion in 99% ethyl
alcohol for 24 hours, then in methylbenzene for 48 hours; samples were then processed
for LM histology. Treatment 4: plastinated samples were de-plastinated in 1,4-
dimethylbenzene for 36 hours, and then processed for LM histology.
Results - The renal capsule was preserved intact in all treatments. The renal cortex
showed some damage, and the epithelium of the renal tubule had some shrinkage in
treatments 3 and 4. Changes in the structure of the myocardium were visible in treatments
2, 3 and 4. It was not possible to visualize the vasa vasorum in the tunica adventitia of
the aorta of treatments 2, 3 and 4. All treatments showed elastic lamellae relatively well
organized following Verhoeff staining.
Conclusions - We found that de-plastination with 1,4-dimethylbenzene produced a
material similar in quality to de-plastination with methylbenzene, and plastinated tissue
without de-plastination produced histological material similar to de-plastinated
specimens..
KEY WORDS: de-plastination; plastination; histological architecture.
*Correspondence to: Moema Lopes Ramos, Institute of Biological and Health Sciences, Federal University of Viçosa, campus Rio Paranaíba, Brazil, Rodovia MG-230 – Km 7, Rio Paranaíba – MG, CEP: 38810-000. Caixa Postal 22. Tel +55 34 3855 9368; E-mail: [email protected]
Introduction
Formaldehyde is the main fixative solution employed
worldwide as a preserving solution in anatomy. It acts on
biological tissues, preventing their degradation (Hambeli
et al., 2010). However, the use of formaldehyde for the
preservation of bodies and parts for gross anatomy study
has been discouraged. In 2004, the International Agency
for Research on Cancer (IARC) of the World Health
Organization (WHO), classified formaldehyde as
carcinogenic (group 1), tumorigenic, and teratogenic to
humans (INCA, 2005).
Thus, it has become a matter of great importance to find
an alternative to formaldehyde for specimen preservation
in anatomy teaching. Plastination is an option to prevent
the exposure of students and anatomy staff to
formaldehyde (von Hagens, 1987; Latorre et al., 2007).
Plastination is based on the replacement of body fluids
and fats by a curable polymer. According to Ravi and Bhat
(2011), one of the most interesting, important, and
potentially useful qualities of silicone plastinated tissue is
that its microscopic structure remains intact. This implies
that the specimen can be preserved, almost indefinitely,
in a form that is easily stored, while still retaining the full
potential for histological examination. To access the
histological structure of plastinated specimens, authors
have described de-plastination with sodium methoxide
(Walker et al., 1988), and methylbenzene, methylene or
dichloroacetone (Ripani et al., 1996). However, the most
De-plastination Methodologies for Preparing Histological Sections - 11
effective substances for de-plastination, sodium
methoxide and methylbenzene, are very toxic. The aim of
this work, then, was to evaluate different protocols to
obtain histological sections from silicone plastinated
specimens.
Materials and Methods
For this study, samples of aorta, heart, and kidney of pigs
were used. Four different treatments were used to
investigate protocols for light microscopy (LM). Treatment
1: the fragments were fixed in 10% formalin for 48 hours
at room temperature. For treatments 2, 3 and 4, samples
from specimens plastinated with Biodur® S10/S3 were
used. Treatment 2: plastinated fragments were directly
embedded in paraffin wax without previous de-
plastination. Treatment 3: plastinated fragments were de-
plastinated by immersion in 99% ethyl alcohol for 24
hours, and then in methylbenzene for 48 hours.
Treatment 4: plastinated samples were de-plastinated in
1,4-dimethylbenzene for 36 hours.
The samples were processed for routine histological light
microscopy (LM) in a tissue processor (Leica TP 1020),
including dehydration with ethyl alcohol in increasing
concentrations from 70 to 100%; clearing in xylene;
paraffin wax impregnation at 58° C, and embedding.
Serial 5 μm sections were cut with a microtome, mounted
on glass slides, and stained with hematoxylin-eosin (H &
E) or Verhoeff’s stain.
The plastinated fragments used in this study came from
complete pig heart, aorta and kidney, plastinated using
the standardized Biodur® S10/S3 silicone method, with
forced impregnation at -25oC. The heart and aorta were
plastinated at the Veterinary Anatomy Laboratory,
Department of Anatomy and Comparative Pathological
Anatomy, Faculty of Veterinary Medicine, University of
Murcia, Spain. The kidney was plastinated at the Anatomy
Laboratory, Department of Morphology, Federal
University of Espírito Santo, Brazil.
The comparative and descriptive analysis of the
histological sections was performed under a conventional
light microscope using a Motic® BA 410 microscope,
observing any changes in the morphological structure of
the treatments compared to the control (treatment 1). The
histological images, (magnification 40X), were captured
as digital images using the program Motic Images Plus
2.0 ML, in the laboratory of Reproduction of Small
Animals and Wild Animals (REPAAS), at the morphology
sector of the Veterinary Department, Federal University of
Viçosa, Brazil.
Results
It was found that it is possible to make histological
sections of plastinated material, although some difficulties
were encountered, due to the rigidity of the plastinated
material, depending on the treatment employed.
Results are presented through the histology of each organ
in turn. Sections obtained from treatments 2, 3 and 4 were
compared with the control, treatment 1. In treatment 1,
constituent elements of the renal, cardiac and aortic
tissues preserved their morphological characteristics and
histoarchitecture.
Table 1. Treatments and main histological characteristics of the kidneys
Treatments Renal corpuscles
Areas of
distortion
Tubular
epithelium
morphology
Tubular
lumen
Macula densa
Control Preserved Absent Normal Normal Evident
Biodur Preserved Present Normal Normal Evident
De-plastinated
methylbenzene
Preserved Present Irregular Reduced Less evident
De-plastinated 1,4-
dimethylbenzene
Preserved Present Irregular Reduced Less evident
12 – Lopes Ramos et al.
A
B
C
D
Figure 1: Photomicrographs of the cortical region of pig
kidneys showing preserved renal corpuscles in all
treatments and macula densa (MD). A, Control; B,
Plastinated; C, De-plastinated with methylbenzene; D, De-
plastinated with 1,4-dimethylbenzene. H&E staining. 400X.
A
B
C
D
Figure 2: Figure 2. Photomicrographs of the cortical region
of pig kidney showing tubular cells presenting irregularities
in B and D that sometimes have loose epithelium in tubular
lumen. A, Control; B, Plastinated; C, De-plastinated with
methylbenzene; D, De-plastinated with 1,4-
dimethylbenzene. H&E staining. 400X. Arrows indicate
areas of distortion.
Table 1 lists the main changes observed in the kidney
histoarchitecture in all four treatments. In treatment 1,
histological staining of renal tissue with H & E showed
normal kidney structure, with renal corpuscles with a knot
of capillaries (the glomerulus), surrounded by Bowman’s
capsule (Fig 1). The tubules were observed with oval
luminal morphology, and the epithelial cells showed
eosinophilic cytoplasm, and central rounded nuclei (Fig.
2). The macula densa (MD) was observed in close
proximity to the vascular pole of the renal corpuscle.
Treatments 2 and 3 revealed renal corpuscles with
preserved Bowman’s space, with some distortion areas
presenting silicone. Epithelial cells from the parietal layer
of Bowman’s capsule were evident (Fig. 1). In treatment
2 the MD was observed in close proximity to the vascular
pole, with tubular architecture preserved. In treatment 3,
some renal tubules revealed reduced lumen, and irregular
morphology of the tubular epithelium. In treatments 3 and
4, histological changes, such as some cells of the renal
tubules with morphological alterations, reduction of the
tubular lumen, or capsular contour with distortion areas
were also seen (Fig. 2).
The three experimental treatments showed preservation
of the renal capsule, low affinity for hematoxylin staining,
and small fragmented areas of tissue, just beneath the
renal capsule.
Table 2. Treatments and main histological characteristics
of the heart
Treatments Organization of
muscle bundles
Areas of
distortion
Intercalated
discs
Control Preserved Absent Not observed
Biodur Lost Present Not observed
De-plastinated
methylbenzene
Lost Present Not observed
De-plastinated 1,4-
dimethylbenzene
Preserved Present Not observed
Table 2 lists the main changes observed in the
histoarchitecture of the heart in all treatments. Histological
examination of the heart sections showed clear
differences between treatment 1, the control, and the
experimental treatments. Analysis of the myocardium in
treatment 1 showed cells with a single, centrally placed
nucleus, well stained by hematoxylin, surrounded by
MD MD
MD
De-plastination Methodologies for Preparing Histological Sections - 13
myofibrils, with blood vessels in the connective tissue. In
treatments 2 and 4, the central nuclei were evident in
myocardial cells. Muscle bundles showed preserved
architecture. Treatments 2 and 3 revealed partial loss of
organization of the muscle bundles, and low affinity for H
& E staining. In treatments 2, 3 and 4, the blood vessels
were not evident in the interstitium. In all of the
treatments, transverse striations and intercalated discs
were seen (Fig. 3). In treatments 2, 3 and 4, areas of
distortion were seen in the slides. Slides from treatments
1 and 4 had similar staining with H & E, with both showing
greater affinity for eosin.
A - Control
B - Plastinated
C - De-plastinated with
methylbenzene
D - De-plastinated with
1,4-dimethylbenzene
Figure 3. Photomicrographs of left ventricular myocardium
of pig, showing low affinity for H&E staining with
plastinated fragments in B, and fragments after de-
plastination with methylbenzene in C. A and D show
standard staining with H&E. 200X.
Aorta
Table 3 lists the main changes observed in the
histoarchitecture of the aorta in all treatments. The
histological structure of transverse sections in treatment
1 demonstrates the three layers that constitute the wall of
the aorta. The tunica intima showed endothelium well
stained by H & E (Fig. 4). The internal elastic membrane,
however, was less obvious. The tunica media showed
several layers of elastic membranes and smooth muscle
cells. The elastic material was well evidenced by
Verhoeff's stain (Fig. 5). The tunica adventitia appeared
thinner than the tunica media, and the presence of vasa
vasorum in connective tissue was observed at random
intervals.
Table 3. Treatments and main histological characteristics of the aorta
Treatment Nucleus
endothelial
cells
Layers of
elastic
membranes
Vasa
vasorum
Areas of
distortion
Control Evident Defined Present Absent
Biodur Little evidence Defined Not seen Present
De-plastinated methylbenzene
Little evidence Defined Not seen Present
De-plastinated
1,4-dimethylbenzene
Little evidence Defined Not seen Present
A - Control
B B - Plastinated
C - De-plastinated with methylbenzene
D - – De-plastinated with 1,4 – dimethylbenzene
Figure 4: Photomicrographs of tunica adventitia of the pig aorta showing vasa vasorum (vv) in A.
H&E staining. 400X.
14 – Lopes Ramos et al.
A - Control
B – Plastinated
C – De-plastinated with methylbenzene
D – De-plastinated with 1,4 – dimethylbenzene
Figure 5: Photomicrographs of tunica intima of pig aorta with Verhoeff staining. 400X.
In Treatments 2, 3 and 4, the tunica intima was observed,
however, the endothelial nuclei were not distinct; in the
tunica adventitia it was not possible to visualize the vasa
vasorum (Fig. 5). In Treatments 2 and 4, areas of
distortion in the endothelium were observed. In Treatment
4, spaces between the elastic membranes in the tunica
media were observed. In all treatments, the elastic
laminae were preserved in the tunica media, with few
spaces between them. This was observed in both the H &
E and Verhoeff stains.
Discussion
In this study, we analyzed by light microscopy how the
histoarchitecture of different organs (kidney, heart and
aorta) was preserved after plastination. As in previous
published studies, we used a control (Treatment 1) to
compare and validate the different methods used to
process the plastinated tissue samples (Treatments 2, 3
and 4) (Dellmann and Brown, 1982; Young and Heath,
2000, Gartner and Hiatt, 2003, Junqueira and Carneiro,
2008, Ross and Pawlina, 2008).
Manjunatha et al. (2014) compared histological sections
of pig organs (liver, spleen and kidneys) embedded in
paraffin wax, with histological sections of uncured
silicone-plastinated tissues. Both were stained with H &
E. Using light microscopy, they found that the tissue
structure was maintained, without shrinkage, in the
plastinated specimens. López-Albors et al. (2004)
plastinated tissue fragments with and without the curing
process, and used de-plastination with sodium
methoxide, according to the protocol of Walker et al.
(1988). They found that the curing process influenced
tissue preservation. However, the results we report here
from cured samples, showed no disruption of the tissue
architecture.
Walker et al. (1988), describe de-plastination with sodium
methoxide, which achieved good quality results. Others
authors report the use of methylbenzene, methylene, and
bichloride acetone for the same process (Ripani et al.,
1988). However, our results in plastinated kidney samples
without de-plastination (Treatment 2) showed a well-
preserved histological structure of tubules and renal
corpuscles. Moreover, the cells of the macula densa had
a similar appearance to those from the control treatment
(Treatment 1). Surprisingly, the results from de-
plastinated kidney samples had renal tubules with
reduced lumen and thin macular densa cells (Treatment
3), and the capsular space was increased in some renal
corpuscles, probably due to shrinkage of the glomerular
capillaries (Treatment 4). These findings for Treatments 3
and 4 agree with the results of Ripani et al. (1996), who
reported lesions in the renal tubule epithelium and
Bowman's capsule, after de-plastination with
methylbenzene. In this study, Treatments 2, 3 and 4
showed some areas of distortion in the heart specimens,
probably due to the rigidity of the material (we used
fragments of the left ventricle), and also due to the
hardening effect of curing, which may have caused
difficulty in obtaining regular histological sections,
resulting in differences in the thickness of the sections,
and areas of distortion, in all three treatments.
Microscopic examination of the heart in Treatment 3
showed areas that were not well preserved, with loss of
organization of muscle bundles. This problem probably
occurred due to the rigidity of the cured material. It was
not possible visualize blood capillaries and transverse
striations in Treatments 2, 3, and 4. In addition, the slides
had several areas of distortion, and the sections were not
uniform. It is likely that this occurred due to the resistance
of the tissue during sectioning. Our findings corroborate
the results presented by Patil et al. (2016), in which clear
striations in Biodur-infiltrated cardiac tissue could not be
seen after de-plastination with methylbenzene.
In our findings, the tunica intima and endothelium of the
aorta were mostly preserved in Treatments 2, 3 and 4.
De-plastination Methodologies for Preparing Histological Sections - 15
The nuclei showed poor hematoxylin affinity. The tunica
media, in Treatments 2, 3 and 4, was composed of
smooth muscle cells interspersed with the large amount
of elastic fibers. The outer elastic limiting membrane could
not be identified and, in Treatment 4, gaps between the
elastic membranes were observed. This could have been
due to resistance to paraffin infiltration. In the tunica
adventitia of all three treatments, the non-visualization of
the vasa vasorum may have been due to the loss of the
surrounding connective tissue during the plastination
process. The areas of refractivity in Treatment 2 and 4
probably occurred due the presence of silicone in the
tissue.
According to Ravi and Bhat (2011), to achieve good
results with standard staining, slides of de-plastinated
samples need a slightly extended immersion time,
compared to unplastinated samples. In our results, we
observed a similar coloration in slides of both the de-
plastinated and plastinated treatments, when stained with
H & E. Both showed less evident hematoxylin staining
compared to the control. This may be due to the
plastination process changing the electronegativity of the
nucleus, and the basophilic pattern. However, for the
Verhoeff staining, the same pattern for the elastic
membranes was observed in all treatments, indicating
that the plastination process did not alter the uptake of the
stain.
In conclusion, our experiments showed that histological
slides can be made directly from Biodur® silicone
plastinated specimens, and embedded directly in paraffin.
This protocol presented satisfactory results, similar to
those found in specimens de-plastinated with 1,4-
dimethylbenzene and methylbenzene, which resulted in
incomplete removal of the Biodur® silicone from the
samples.
References
Dellmann H, Brown EM. 1982: Histologia Veterinária. Rio
de Janeiro: Guanabara Koogan, 397 p.
Gartner LP, Hiatt, JL. 2003: Tratado de Histologia em
Cores. 2a ed. Rio de Janeiro: Guanabara Koogan, 458 p.
Hambeli AT, Lombardi M, Prochownik M. 2010: Técnicas
de conservación de piezas cadavéricas. Tercera Epoca:
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Latorre RM, García-Sanz MP, Moreno M, Hernández F,
Gil F, López O, Ayala MD, Ramírez G, Vázquez JM,
Arencibia A, Henry RW. 2007: How useful is plastination
in learning anatomy? J Vet Med Educ 34: 172-176.
López-Albors O, Gil F, Orenes M, Ayala MªD, Abellán H,
Henry R, Latorre R. 2004: Curing influences the tissue
preservation of silicone plastinated organs. 12th Int Conf
Plast, Murcia, Spain, 2004. Abstract in J Int Soc
Plastination 19: 49-50.
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Suguna R, Ramkrishna V. 2014: Comparison of
histological architecture of paraffin embedded and
indigenously plastinated tissues. Indian J Vet Anat 26:
132-133.
Patil SK, Jamuna, KV, Badami, S, Ramkrishna, V. 2016:
Comparison of histology of cardiac Muscle using different
infiltrating media. Indian J Nat Sci 6: 10558-10563.
Ravi SB, Bhat VM. 2011: Plastination: a novel, innovative
teaching adjunct in oral pathology. J Oral Maxillofac
Pathol 15: 133-137.
Ripani M, Boccia L, Cervone P Macciucca DV. 1996: Light
microscopy of plastinated tissue. Can plastinated organs
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Ross MH, Pawlina W. 2008: Histologia: Texto e Atlas em
correlação com biologia celular e molecular. 5a ed. Rio
de Janeiro: Guanabara Koogan. 908 p.
von Hagens G, Tiedemann K, Kriz W. 1987: The current
potential of plastination. Anat Embryol (Berl) 175: 411-
421.
Walker AN, Jackson RL, Powell, S. 1988: Technical
communication: routine microscopy of deplastinated
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atlas em cores. 4a ed. Rio de Janeiro: Guanabara
Koogan. 415 p.
The Journal of Plastination 30(1):16 - 23 (2018)
ANATOMICAL
TECHNIQUE
Biomechanical Analysis of The Skin And Jejunum Of Dog
Cadavers Subjected To A New Anatomical Preservation
Technique For Surgical Teaching
Rocha TASS1
Yanagihara GR2
Shimano AC2
Rolim GS3
Santos CCC1
Fechis ADS1
Oliveira FS1
1Department of Animal
Morphology & Physiology, São Paulo
State University (Unesp), School of Agricultural and
Veterinarian Sciences, Jaboticabal, São Paulo,
Brazil
2Department of Biomechanics, Medicine
and Rehabilitation of Locomotor System,
College of Medicine, University of São Paulo
(Usp), Ribeirão Preto, Brazil
3Department of Exact Sciences, São Paulo State
University (Unesp), School of Agricultural and
Veterinarian Sciences, Jaboticabal, São Paulo,
Brazil
ABSTRACT:
Formaldehyde is a fixative and preservative widely used in anatomy laboratories, but it is
harmful to health, and poses an environmental risk. Ethyl alcohol (EtOH) has also been
used for effective fixation of bird muscles, and sodium chloride has been successfully
tested for the preservation of anatomical parts for more than five years. The objective of
this present study was to evaluate a new anatomical technique for teaching surgical
techniques using dog cadavers fixed in EtOH, and preserved in a 30% aqueous solution
of sodium chloride (ASSC). In addition, we aimed to determine the ideal time to stop the
fixation, so that the skin and jejunum present biomechanical characteristics as close as
possible to the control group of fresh animals. Five groups were used: a control group
(fresh animals without fixation or conservation), and the other 4 groups which differed in
the time of fixation in EtOH (30, 60, 90 and 120 days). Except for the controls, all groups
were conserved in 30% ASSC for 120 days. Statistical analysis of variance (ANOVA)
revealed no difference between treatments and times (P > 0.05) relative to the skin, and
showed at least one time significantly different from the others (P < 0.01) in relation to the
jejunum. The non-linear modeling test showed differences in the group fixed in EtOH for
30 days, suggesting that this was the best time period for fixing dog cadavers for use in
surgical training.
KEY WORDS: preservation, biomechanics, jejunum, dog Correspondence to: Fabrício S Oliveira, Department of Animal Morphology and Physiology, São Paulo State University (UNESP), Via de Acesso Paulo Donato Castelane, Jaboticabal, SP, 14884-900, Brazil. Telephone: 551632097333, e-mail: [email protected]
Introduction
Good conservation of anatomical specimens prevents
deterioration, and also prevents the proliferation of
pathogens that can spread diseases to laboratory
personnel (Corrêa, 2003). Formaldehyde is widely used
as an anatomical fixative and preservative, because it is
inexpensive and rapidly penetrates the tissues
(Rodrigues, 2010). However, it is hazardous to health,
and can contaminate the environment through improper
handling and through disposal of carcasses and effluent
(WHO, 1991). In 2006, the International Agency for
Research on Cancer (IARC) classified formaldehyde as
carcinogenic and teratogenic (IARC, 2006). Glycerin is a
preservative with dehydrating and antiseptic properties,
preventing fungi and bacterial growth (Alvarenga, 1992).
It does not have harmful fumes such as those released by
formaldehyde (Cury et al., 2013), but it is up to ten times
more expensive than formaldehyde (Krug et al., 2011).
AN
AT
OM
ICA
L TECH
NIQ
UE
Biomechanical Analysis of The Skin And Jejunum Of Dog Cadavers- 17
There are a number of other examples of fixative solutions
that are capable of preserving cadavers for surgical
training, among them Thiel solution (composed of boric
acid, ethylene glycol, potassium nitrate,
(chloromethyl)phenol, sodium sulfate, and formalin)
(Groscurth et al., 2001), Klotz's solution (sodium chloride,
sodium bicarbonate, chloral hydrate, formalin and water)
and Jores’ solution (distilled water, formaldehyde, sodium
sulfate, potassium sulfate, sodium chloride, sodium
bicarbonate, glycerin, and sodium or potassium acetate)
(Rodrigues, 2010), all of which contain formalin. Modified
Larssen solution contains 100 ml of 10% formaldehyde,
400 ml of glycerol, 200 g of chloral hydrate, 200 g of
sodium sulfate, 200 g of sodium bicarbonate, 180 g of
sodium chloride, and 2 liters of distilled water (Silva et al.
al., 2004). The original Larssen solution did not contain
liquid glycerin (Menezes, 2012). Laskowski solution
contains 800 ml of glycerine, 200 ml of ethanol, 50 g of
phenolic acid and 50 g of boric acid, and requires the
preservation of cadavers at 0° C until they are used in
surgery classes. Between classes, they must remain
frozen, and have to be thawed in water for 24 hours prior
to each class. With this solution, the tissues become
excessively dark (Silva et al., 2007). Of these, Larssen
solution is considered the best solution for maintaining the
original consistency, color and characteristics of the
biological material, and can also remove blood clots
(Sampaio, 1989).
Ethyl alcohol (96%) has been found to be efficient for
fixing and preserving the pectoral muscles of hens,
although they became almost five times more rigid during
the first six months, and three times more rigid after one
year of immersion in the preservative agent (Nunes et al.,
2011). The use of a 30% aqueous solution of sodium
chloride (ASSC) in the preservation of anatomical
specimens previously fixed by formaldehyde was
successfully evaluated for 5 years, with no visual
contamination, presence of putrefaction odors, or
alteration of tissue color and softness (Oliveira, 2014).
There is a need to find a preservation technique that
preserves the body realistically for a long time, serving not
only anatomy classes, but also surgical and clinical
studies, testing of new radiographic equipment, minimally
invasive surgery, and encouraging more scientific
research to develop protocols that will assist anatomists
in the preparation of preserved cadavers, in order to meet
these demands for high quality parts (Balta et al., 2015).
The objective of this study was to determine the viability
of a new anatomical technique using EtOH for fixation,
and 30% ASSC for conservation; and to establish the best
time to stop fixation in EtOH for the optimal tissue
resistance in comparison to fresh cadavers without
fixatives/preservatives.
Materials and Methods:
The animals
Forty (40) frozen, adult, dog cadavers, 14 male and 26
female were used. The dogs all died due to causes that
did not involve evident morphological alterations, such as
large tumors, extensive lacerating traumas or bone
fractures. All were from the Zoonoses Control Center of
Ribeirão Preto, São Paulo, Brazil, after approval from the
Municipal Law Department (process 02.2014/ 000027-1).
The ages of the animals were determined from their death
certificates.
The selected cadavers had a mean body weight of
7.6±2.7 kg, mean age 5.6±4.1 years, and body score of 4
to 5, which is considered ideal on a scale of 1 to 9
(Laflamme, 1997). They were thawed in a horizontal
refrigerator at 8° C, weighed, and then randomly
distributed into groups (Table 1).
Table 1. Division of dog cadaver groups in relation to fixation time in ethyl alcohol (ETOH) and storage in a 30% sodium chloride solution (ASSC 30%). Control group consisting of cadavers of dogs not subjected to fixation or conservation (fresh).
Group Fixation
(ETOH)
Conservation
(ASSC 30%)
Control (fresh cadavers) - -
1 30 days 120 days
2 60 days 120 days
3 90 days 120 days
4 120 days 120 days
For fixation, 96% EtOH containing 5% glycerin was
infused with a 60 ml plastic syringe, via the common
carotid artery, at a rate of 120 ml/kg. Each group
consisted of eight animals that were fixed for different
times (except for the control group).
After the fixative was injected, running water was used to
flush out surplus liquid accumulated in the cavities via two
incisions, one in the thorax (between the fourth and fifth
right intercostal space), and a median abdominal incision.
18 – Rocha, et al
The cadavers were then placed in plastic tanks (1 tank
per group) with a threaded cap (total capacity 310 liters –
Fig. 1), containing 180 litres of EtOH. The abdomen and
thorax were washed with water for 5 consecutive days.
The plastic boxes were kept in an area and with abundant
ventilation, away from sources of ignition, to avoid any risk
of accident. Storage in 30% ASSC for 120 days followed
the fixation period. Sodium chloride solution was placed
in plastic tubs with a capacity of 310 liters, and the same
volume (180 liters) was used for each fixation group.
Figure 1. Plastic box of 310 liters filled with 180 litres of EtOH (A). Dogs cadavers kept during ethylic fixation (B).
Collection of Material
A 1 x 7cm stainless steel template was made for the
collection of the skin and jejunum samples (chosen due to
the higher tissue sample availability and frequent use in
teaching of surgical techniques) during both the fixation in
EtOH and 30% ASSC conservation solution. A total of
1,152 samples were taken.
The cadaver was initially placed in right lateral decubitus
(left antimere, facing upwards). Using a scalpel (blade
number 23) and the template, three sequential, equally-
sized samples were collected transverse to the dog's skin
tension line (Kirpensteijn and Haar, 2013), on the lateral
side of the thorax, parallel to, and 5 cm from, the median
plane (Figure 2). Shaving had been performed throughout
the thorax. Samples from the control group were also
collected in the left antimere and immediately submitted
to biomechanical testing. During the fixation phase in
EtOH, samples were collected from the 4 groups in the
left antimere and, during the conservation phase in 30%
ASSC, from the right antimere.
For the collection of the jejunum samples, the animals
were positioned in the right lateral decubitus, exteriorizing
the jejunum by manual traction. After identification of the
duodenojejunal flexure, the steel template was positioned
to delimit the specimen, which was then sectioned
longitudinally with Metzenbaum scissors. Subsequently,
section of the mesenteric edge was performed, exposing
the lumen, under which the incision template was
positioned with a scalpel blade (Figure 3).
Figure 2. Skin samples collected transversally to the dog's skin tension line, on the lateral side of the thorax and using a steel mold, parallel to and 5 cm from the median plane after trichotomy.
Figure 3. Jejunum sample collected with the cadaver positioned in the right lateral decubitus, after manual traction and using a steel mold.
Tissue Strength Analysis
To evaluate tissue resistance, a Universal Testing
Machine (Instron®- EMIC® - DL2000) was used, with a
500 N load cell and electromechanical drive support, with
a speed of 100 mm/min. Traction claws were also used
by manual compression, in the Laboratory of Surgical
Anatomy of the Department of Morphology and Animal
Physiology of FCAV - UNESP, Jaboticabal Campus, SP,
Brazil (Figure 4). Tensile testing was conducted up to the
point of rupture of the skin and jejunum samples,
obtaining the values of the maximum force applied, in N.
Analysis of The Skin and Jejunum of Dog Cadavers - 19
Figure 4. Universal testing machine (A) for biomechanical analysis. A dog´s skin sample during a traction test (B).
Statistical Analysis
The data for the maximum force for the rupture of skin and
jejunal tissues at different times and treatments were
analyzed by analysis of variance (ANOVA) with
significance set at 0.05.
For a more detailed analysis of the maximum jejunum
breaking point as a function of time, nonlinear models
were fitted in exponential form by the ordinary least
squares methodology, according to the following
equations:
𝑌 = 𝑌𝑜 + 𝐴1 × 𝑒−(𝑋−𝑋𝑜)/𝑡1
𝑀𝐴𝑃𝐸 =∑ (|
𝑌𝑒𝑠𝑡𝑖 − 𝑌𝑜𝑏𝑠𝑖𝑌𝑜𝑏𝑠𝑖
| × 100)𝑁𝑖=1
𝑁,
where Y is the maximum force (N), Yo is the mean in the
stabilization of the maximum force (N) at the final times of
the treatment, A1 is the weighted amplitude of the values
of Y, Xo is the time of greatest decay rate of the maximum
force (time), t1 is the mean decay rate (N time-1), and X
is the measured time. MAPE is the mean percentage
error, Yesti is the estimated value of maximum force at
time i, Yobsi is the observed value of maximum force at
time i and N is the number of data.
An ANOVA test was also performed to verify the
differences between the adjusted parameters in the
different treatments.
Results
The preservation technique employed proved to be
efficient for the fixation and conservation of the animals
throughout the experiment. During the storage period in
the plastic boxes in ASSC 30%, there was a gradual
release of fat from cadavers in groups 3 and 4 (fixed 90
and 120 days in EtOH respectively). Because they
remained longer in the alcohol fixative, they were more
greasy and viscous at the end of storage in 30% ASSC,
making it difficult to manipulate them during sample
collection. During fixation in EtOH, stiffening of the skin
and jejunum was observed when handling the samples,
and later, there was an increase of the tissue malleability
during the period of conservation in 30% ASSC. At the
end of the period of alcohol fixation, the alcohol
concentration ranged from 80 to 83%, which is an
excellent amount of alcohol in the solution, and an
appropriate degree of fixation of the cadaver for
placement in the 30% ASSC for 120 days.
Table 2 - Absolute mean (± SD) of the maximum strength of rupture (N), of skin samples of groups 1, 2, 3 and 4, submitted to different fixation times in ethyl alcohol and preservation in an aqueous solution of sodium chloride.
Time: 0 (pre-fixation analysis); 1 (30, 60, 90 and 120 days of fixation in ethyl alcohol, for groups 1, 2, 3 and 4, respectively); 2 (30 days 30% ASSC); 3 (60 days 30% ASSC); 4 (90 days 30% ASSC) and 5 (120 days 30% ASSC).
From the general data of the maximum rupture forces of
the skin and jejunum samples submitted to the
biomechanical tensile test, the absolute mean and
Time G1 G2 G3 G4
0 131.3 ±
75.6
131.3 ±
75.6
131.3 ±
75.6
131.3 ±
75.6
1 152.6 ±
71.9
131.1 ±
53.6
177.5 ±
86.6
110.8 ±
56.7
2 139.3 ±
55.0
119.1 ±
52.2
148.7 ±
83.3
118.4 ±
65.9
3 130.3 ±
54.1
106.6 ±
51.0
148.7 ±
71.9
121.5 ±
78.8
4 148.7 ±
63.1
145.6 ±
69.6
116.4 ±
60.9
110.3 ±
64.0
5 115.1 ±
53.5
112.3 ±
78.3
112.3 ±
60.3
132.4 ±
65.5
20 – Rocha, et al
respective standard deviation of groups 1, 2, 3 and 4 were
obtained at different times of fixation in EtOH, and
conservation in 30% ASSC (Tables 2 and 3). The
maximum force required to rupture the skin and jejunum
samples (at all times) ranged from 106.7 N to 177.5 N
(mean 142.1 N) and from 12.9 N to 27.6 N (mean 20.2 N),
respectively.
Table 3 - Absolute mean (± SD) of the maximum force of rupture (N) of jejunum samples of groups 1, 2, 3, and 4 submitted to different fixation times in ethyl alcohol and preservation in an aqueous solution of sodium chloride at 30% (30% ASSC).
Time: 0 (analysis prior to fixation); 1 (30. 60. 90 and 120 days of fixation in ethyl alcohol. for groups 1. 2. 3 and 4. respectively); 2 (30 days 30% ASSC); 3 (60 days 30% ASSC); 4 (90 days 30% ASSC) and 5 (120 days 30% ASSC).
Table 4 - Analysis of variance (ANOVA) between treatments and time for skin samples from dog cadavers fixed at different times in ethyl alcohol and preserved in a 30% aqueous solution of sodium chloride for 120 days.
There was no difference between treatments (P > 0.05).
Source of variation
SS DF MS F P F critical
Times 5,676.82 5 1,135.36 0.24 0.94 2.27
Treatments 9,460.00 3 3,153.33 0.68 0.57 2.66
Interactions 3,8855.60 15 2,590.37 0.56 0.90 1.73
Total 836,112.50 191
*SS: sum of squares; DF: degrees of freedom; MS: mean square; F: ratio between the model and its error; P: significance level; F critical.
For the skin samples, ANOVA showed that there were no
differences between treatments and times (Table 4),
because the P value was always higher than 0.05. The
ANOVA test also indicated that there were no interactions
between the treatments and times. For the jejunum
samples, ANOVA showed that at least one time was
significantly different from the others (p <0.01) and that
there were no differences between treatments (G1 to G4)
(Table 5).
Table 5 - Analysis of variance (ANOVA) between treatments and times for jejunum samples from dog cadavers fixed for different times in ethyl alcohol and preserved in a 30% aqueous solution of sodium chloride for 120 days. There was at least one different time in the treatments (P < 0.01).
Source of variation SS DF MS F P F critical
Times 2,392.40 5 478.48 5.45
Treatments 273.064 3 91.021 1.037
Interactions 559.24 15 37.28 0.42
Total 17,969.80 191
SS: sum of squares; DF: degrees of freedom; MS: mean square; F: ratio between the model and its error; P: significance level; F critical.
As a different time was identified in the statistical analysis
of the jejunum, nonlinear modelling was performed,
aiming to determine the time. In the modelling, the
exponential decay model (equation 1) explained the
variation of the maximum force for rupture of jejunum
samples as a function of the different times in the
treatments (Figure 5), since general MAPE was 9.6%. All
values of Yo between the models were statistically similar,
indicating that the mean maximum strength at the final
times of the treatments were equal, thus confirming the
efficiency of the 30% sodium chloride solution as a
preservative. All other adjusted parameters showed at
least one difference between the treatments, indicating
that the response of the treatments varied in relation to
the mean amplitude (A1), the time of the highest decay
rate (Xo), and the decay rate (T1), to the maximum force
values.
In the jejunum samples, a gradual decay of the values
was observed for the maximum rupture force until the final
fixation time in ethyl alcohol and a later stabilization of the
maximum force for up to 120 days, thus confirming the
preservative effect of the 30% ASSC for this type of
tissue. Thus, in relation to jejunum, G1 presented A1 and
Xo differently from the other groups, as this is the time of
significance (Table 6).
Time G1 G2 G3 G4
0 27.6 ±
17.5
27.6
±17.5
27.6 ±
17.5
27.6 ±
17.5 1 14.0 ± 5.1 14.8 ± 6.8 18.5 ± 9.7 18.3 ± 6.1
2 15.4 ± 8.9 19.0 ±
11.9
20.3 ±
11.4
18.0 ±
13.0 3 18.9 ± 9.3 20.3 ±
11.7
19.6 ±
10.6
21.1 ±
17.6 4 20.5 ± 9.3 21.7 ±
12.4
20.1 ± 10 18.4 ±
16.0 5 12.9 ±
16.0
18.3 ±
11.6
19.7 ± 8.9 24.3 ±
15.5
Analysis of The Skin and Jejunum of Dog Cadavers - 21
Figure 5. Nonlinear models of exponential decay relating to the maximum force as a function of different treatment times and different fixation times in ethyl alcohol and preservation for 120 days in a solution of 30% sodium chloride. Note the stabilization of the values during storage in saline solution.
G1 (30 days in alcohol), G2 (60 days in alcohol),
G3 (90 days in alcohol), and G4 (120 days in alcohol).
M1: final moment of fixation; M2: after 30 days preserved
in saline solution; M3: after 60 days preserved in saline
solution; M4: after 90 days preserved in saline solution;
M5: after 120 days preserved in saline solution.
Table 6 - Adjusted values of the parameters of the exponential non-linear decay model. The general model indicates the fit for all treatments together. The letters in superscript indicate the ANOVA test verifies at least one difference of the parameters between treatments of different fixation times in ethyl alcohol and preservation for 120 days in a 30% aqueous solution of sodium chloride. G1 (30 days in alcohol), G2 (60 days in alcohol), G3 (90 days in alcohol) and G4 (120 days in alcohol).
MAPE: mean percentage error; Yo: mean in the
stabilization of the maximum force in the final times of the
treatment (N); A1: amplitude of the values of Y; Xo: time of
greatest decay rate of maximum force (N); T1: average
decay rate (N time-1).
Discussion
Ethyl alcohol was effective as a fixative of dog cadavers,
providing good conservation, and avoiding deterioration
of the material, per Rodrigues (2010). The method
described here is similar to the study involving alcohols
used to fix human cadavers for 6 months to 1 year, which
left the tissue quality like that of fresh tissue (Goyri-O’Neill
et al., 2013).
The 30% ASSC solution was extremely effective in the
preservation of the fixed tissues, as described by Oliveira
(2014). There was no apparent contamination similar to
that described in canine pericardium preserved for a
minimum period of 90 days in hyper-saturated sodium
chloride solution (Brun et al., 2002), and that described for
the canine phrenic center preserved in glycerin (Brun et
al., 2004). The 30% ASSC may be successful because
of the difficulty of survival for microorganisms in a medium
that requires an enormous osmoregulation capacity,
similarly to the oceans (Munro et al., 1989), and the Dead
Sea (Nissenbaum, 1975). Several concentrations of
fixatives and preservatives have been evaluated for
preservation of anatomical specimens, but the use of a
sodium chloride solution below 20% has failed to preserve
parts for use in tissue dissection (Friker et al., 2007).
Statistical modeling analysis evidenced the stability of the
data in this study, using 30% ASSC as a tissue
preservative for skin and jejunal samples for up to 120
days.
There was no generation of contaminated effluent,
commonly observed when toxic preservatives are used
(WHO, 1991), nor any health-damaging fumes, such as
those released by formaldehyde (Cury et al., 2013). In
addition, the appropriate waste management of
formaldehyde is costly in both financial and environmental
terms, requiring the search for low-cost and non-risky
alternatives (Janczyk et al., 2011).
Cadavers prepared for anatomical dissection were
described as having better quality when a mid-line
abdominal incision was performed (to enable the
preservative to enter the abdominal cavity, to improve
perfusion of the abdominal tissues) as was done in this
study, instead of not opening the abdominal cavity
(Janczyk et al., 2011).
12
14
16
18
20
22
24
26
28
0 2 4 6
Forc
e fo
r th
e ru
ptu
re o
f th
e je
jun
um
sam
ple
s (N
)
Moments
G1
G2
G3
G4
G1est
G2est
G3est
G4est
Model MAPE
Parameters
Yo A1 Xo T1
Geral 9.6 % 18.748a 1.916a.b 0.122a.b 0.079a.b
G1 16.7 % 16.393a 1.984a 0.129a 0.074b
G2 10.3 % 18.847a 1.703b 0.098b 0.060a
G3 2.5 % 19.681a 1.824b 0.111b 0.075b
G4 10.5 % 20.078 a 1.804b 0.111b 0.077b
22 – Rocha, et al
Tissues become stiffer with formaldehyde, which, in
biomechanical shear tests, caused a considerable
stiffening in chicken breasts fixed for up to one year (4.4
to 5 times higher) (Guastalli et al., 2012). When EtOH was
used as fixative, tissue stiffness increased, making them
almost five times more lilely to rupture during the first six
months, and three times more likely after one year of
immersion (Nunes et al., 2011). However, in this study,
the maximum tensile strength in the skin traction test
(mean of 142.1 N) and jejunum (mean of 20.2 N) did not
show a great variation in relation to the maximum strength
of skin (131.3 ± 75.6 N) and jejunum (27.6 ± 17.5 N) of
the unfixed cadaver control group.
Conventional procedures for fixing cadavers using
formaldehyde are of limited use for surgical practise, due
to the profound alteration in the staining, resistance, and
fragility of organs and tissues. Artificial anatomical models
are an alternative, and can be used mutiple times
(Groscurth et al., 2001). However, with the method
described here, each cadaver, prepared without the use
of formaldehyde, can be used for training in surgical
techniques for an entire semester, with life-like softness
and tissue malleability.
ANOVA showed that there were no differences between
treatments and times (P > 0.05 in all cases). Thus, among
the evaluated time periods (30, 60, 90 or 120 days), there
is no specific time that is better than others. Therefore, we
suggest the shortest time (30 days - G1) for the
preparation of the dog cadavers, because, due to the
rapidity of preparation and the lower occupational risk that
it confers, it is the best for cutaneous surgery.
Acknowledgement: FAPESP, process 2015/08259-9.
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cães e gatos. Jaboticabal: Fundação de Estudos e
Pesquisas em Agronomia da Universidade Estadual
Paulista. p 33-39.
Balta JY, Cronin M, Cryan JF, O´Mahony SM. 2015:
Human preservation techniques in anatomy: a 21st
century medical education perspective. Clin Anat 28:
725-734.
Brun MV, Pippi NL, Dreimeier D, Contesini EA, Beck
CAC, Cunha O, Pinto Filho SL, Roehsig C, Stedile R.
2002: Solução hipersaturada de sal como conservante
de pericárdio canino utilizado na reparação do músculo
reto abdominal de ratos Wistar. Ciên Rur 32: 1019-
1025.
Brun MV, Pippi NL, Driemeier D, Contesini EA, Beck
CAC, Cunha O, Pinto Filho SL, Roehsig C, Stedile R,
Silva TF. 2004: Solução hipersaturada de sal ou de
glicerina a 98% como conservantes de centros frênicos
caninos utilizados na reparação de defeitos musculares
em ratos Wistar. Ciên Rur 34: 147-153.
Corrêa WR. 2003: Isolation and identification of
filamentous fungi found in anatomical pieces preserved
in 10% formalin solution. Dissertation (Biological
Sciences). Institute of Research and Development,
University of the Valley of Paraíba. 59p.
Cury FS, Censoni JB, Ambrósio CE. 2013: Técnicas
anatômicas no ensino da prática de anatomia animal.
Pesq Vet Bras 5: 688-696.
Friker J, Zeiler E, McDaniel BJ. 2007: From formalin to
salt. Development and introduction on a salt-based
preserving solution for macroscopic anatomic
specimens. Tierärztl. Praxis 35: 243–248.
Guastalli BHL, Nunes TC, Gamón THM, Carmo LG, Del
Quiqui EM, Oliveira FS. 2012: Análise da textura de
músculos submetidos à fixação em formaldeído e
conservação em benzoato de sódio 0,5% e ácido
acético 0,5%. Acta SciVet 40: 1041.
Goyri-O’Neill J, Pais D, Freire De Andrade F, Ribeiro P,
Belo A, O’Neill A, Ramos S, Neves Marques C. 2013:
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Groscurth P, Eggli P, Kapfhammer J, Rager GJ,
Hornung P, Fasel JDH. 2001: Gross anatomy in the
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development. Anat Rec 265: 254–256.
IARC (International Agency for Research on Cancer).
2006: Formaldehyde, 2-butoxyethanol and 1-
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Janczyk P, Weignera P, Luebke-Beckerb A,
Kaessmeyera S, Plendla J. 2011: Nitrite pickling salt as
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Semmelmann C, Roman LI, Barreta MH. 2011:
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The Journal of Plastination 30(1):24 - 26 (2018)
TECHNICAL REPORT
Bleaching of specimens before dehydration in
plastination: a small-scale pilot study using human
intestine
Jie-Ru Chen1
Hong-Jin Sui1,2
1Department of
Anatomy, Dalian Medical University, No.9 West Section, Lushun South
Road, Dalian 116044, China
2Dalian Hoffen Bio-technique Co. Ltd., No.36,
Guangyuan Street, Lushunkou Economic
Department Zone, Dalian 116052, China
ABSTRACT:
Objective: The aim of this study was to explore the factors that influence bleaching of
specimens prior to plastination.
Materials and Methods: Four sections of formalin-fixed human intestines were divided
into two groups, to compare the effects of hydrogen peroxide concentration (5% and 10%)
and temperature (20 °C and 30 °C) on the effectiveness of bleaching.
Results: In the first group, a high concentration of bleach appeared to make a better
appearance. In the second group, a higher temperature gave a better appearance.
Conclusion: A high concentration of bleach and temperature can both lead to a better
appearance of the specimen.
KEY WORDS: Bleaching, intestine, plastination Correspondence to: *[email protected]
Introduction
Plastination is a technology that can preserve biological
tissues in a life-like state for long-term preservation
without unpleasant odors (Weber et al., 2007); as a result,
it has now become more and more popular both for
medical/veterinary teaching, and public exhibitions.
Plastination technology is a technique purely for
preservation, so in order to create a good specimen, it is
vital to start with a high quality dissection. This is because
plastination cannot make the specimen better if the quality
of the dissection is not good enough in the first place, and
a good specimen requires a clear structure and a natural
color (Smodlaka et al., 2005). Color is a very important
factor in a good specimen, because it can make the
surface of the specimen more natural, and improve the
appearance of the specimen. It can also give a better
experience for the person who is using the plastinated
specimen. Bleaching is a key technology, and is very
important in enhancing the color, as it can make the color
brighter (Sui and Henry, 2007). Hydrogen peroxide
solution is usually used as the bleaching agent; the
specimen is bleached for 2-5 days at a concentration of
10%, at a temperature of 24 °C in the hydrogen peroxide
bath. In this study, we investigated the effects of hydrogen
peroxide concentration and temperature on samples of
human intestines.
Materials and Methods
Four sections of formalin-fixed human intestines were
used in this experiment. The samples were divided into
two groups (Fig. 1); one group (Group 1) was used to
study the effect of concentration of hydrogen peroxide
on the color of the specimen, while the other group
(Group 2) was used to investigate the effect of
temperature.
In Group 1, investigating the effect of hydrogen peroxide
concentration, two lengths of intestine were placed into
separate baths of hydrogen peroxide at 24° C for 24
hours. The baths contained 5% and 10% hydrogen
peroxide, respectively.
In Group 2, the effect of temperature on the bleaching process was investigated. Two lengths of intestine were
TECH
NIC
AL R
EPO
RT
Bleaching of Specimens Before Dehydration - 25
placed into separate baths of 5% hydrogen peroxide at
20° C and 30° C, respectively, for 24 hours.
Figure 1. The four intestinal canals were dissected, and
divided into two groups prior to bleaching.
Results
In Group 1 (Fig. 2), we found that the intestine segment
which was bleached in 10% hydrogen peroxide had a
better appearance, and brighter color, than the other
specimen, which was bleached in 5% concentration. In
Group 2 (Fig. 3), the intestine segment which was placed
in 5% hydrogen peroxide at 30° C had a better
appearance than the specimen that was bleached at
20°C.
Figure 2. Group 1. The two intestine specimens after bleaching at 24° C for 24 hours in 5% hydrogen peroxide (left) and 10% hydrogen peroxide (right).
Figure 3. Group 2. The two intestine specimens after bleaching for 24 hours in 5% hydrogen peroxide at 20 °C (left), and at 30 °C (right).
Discussion
The color of an anatomical specimen is very important to
the people who use it, whether it is used in medical
teaching or in popular exhibitions. A good color in a
plastinate means that, technically, a better result has
been achieved. In plastination, bleaching is an important
step before dehydration, in which the color of the
specimen can be changed to enhance the appearance of
the specimen. From our own experience, we use
hydrogen peroxide bleaching solution of sufficient depth
to completely cover the specimen (Gao, et al., 2006). The
specimen is bleached for 2-5 days in a 10% hydrogen
peroxide bath, at a temperature of 24 °C. During the
bleaching process, the specimen should be inspected
regularly, until the specimen has turned white or pink.
There are three factors that can affect the bleaching
process: the concentration of the bleach, the temperature,
and, in our experience, sunlight can also be a factor.
Findings from the small-scale study reported here,
confirmed that when the concentration of bleach and the
ambient temperature are high (20%, and 30 °C,
respectively) the bleaching is fast and effective. We have
also found, through our own practical experience, that
with exposure to sunlight, the speed of bleaching is faster.
However, for those who are new to bleaching, it is advised
to use low concentration and low temperature, and a
longer period in the bleach solution. This is because the
contrast of the tissue may be very low if the bleaching is
too fast, and is not stopped in time.
Conclusion
Increasing the concentration or the temperature of the
hydrogen peroxide during the bleaching process can
enhance the appearance of anatomical specimens.
However, this is not advised for inexperienced
plastinators.
References
Gao H, Liu J, Yu S, Sui HJ. 2006: A new polyester
technique for sheet plastination. J Int Soc Plastination
21:7-10
Smodlaka H, Latorre R, Reed RB, Gil F, Ramirez R,
Vaquez-Auton JM, Lopez-Albors O, Ayala MD, Orenes M,
Cuellar R, Henry RW. 2005: Surface detail comparison of
26 – Sui, Chen
specimens impregnated using six current plastination
regimens. J Int Soc Plastination 20:20-30
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biological tissue: Hoffen P45 technique. J Int Soc
Plastination 22:78-81
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J Int Soc Plastination 22:50-58
The Journal of Plastination 30 (1): 27-36 (2018)
TECHNICAL REPORT
General Issues of Safety in Plastination
Schill VK
BIODUR® Products
GmbH, Im Bosseldorn 17,
69126 Heidelberg,
Germany
ABSTRACT:
When people intend to start plastination at their institute, they are sometimes unaware of
the scope of equipment, auxiliaries and chemicals they need. They may be even less
aware of the potential hazards which arise from plastination. Certain chemicals may pose
acute or chronic health hazards. Acetone, which is mostly used for dehydration and
defatting, is a flammable liquid and therefore brings about fire and explosion hazards.
In this paper, information about the characteristics of some commonly used chemicals in
plastination is provided. Suitable personal protective equipment must be used to allow for
safe working when handling these substances. For chemicals posing an inhalation
hazard, technical room ventilation or workplace ventilation is required to keep the
concentration of hazardous vapours below their respective workplace concentration
limits. If ventilation is not sufficient, respiratory protection must be worn.
Avoiding the risk of fire and explosion caused by handling of acetone or other flammable
liquids is achieved by a combination of measures: Proper laboratory furnishings
(ventilation system, electric installations, etc.) are of importance as well as the design of
the equipment used for plastination. Depending on the result of the local risk assessment,
some appliances like solvent pumps or fans should be designed to be explosion-proof.
Organisational protective measures support the technical measures in order to enhance
occupational safety. Here, proper instruction of staff is of particular importance..
KEY WORDS: plastination equipment; inhalation hazard; explosion hazard; explosion protection; occupational safety Correspondence to: V. Schill, telephone: +49 6221 331165; Fax: +49 6221 331112; Email: [email protected]
Introduction
The technique of plastination, invented by Gunther von
Hagens in 1977, offers the opportunity for scientists to
produce durable preparations in their own labs (von
Hagens et al., 1987). While plastinated specimens as final
products are non-hazardous, the handling of solvents and
some other specific chemicals during the production
process requires (1) awareness of their hazards and (2)
some technical, organisational, and personal protective
measures to achieve work safety. In the following article,
the main potential hazards related to plastination work, as
well as recommended measures to effectively avoid
explosion and health hazards are discussed.
Important note: work safety is a wide subject where
international, national, and regional regulations must be
observed. This paper naturally can’t cover all regulations,
thus, the following information should be considered as a
subjective selection, based on several years’ experience
in plastination work and on a number of visits to
laboratories in different countries, without any claim to be
exhaustive.
Hazard types
In plastination, we may be confronted with hazards of the
following types:
-Health hazards (acute toxicity, sensitisation,
carcinogenicity, etc.)
-Physical hazards (explosion hazard, mechanical
hazards, etc.)
-Biohazard (when handling fresh, unfixed specimens)
TECH
NIC
AL R
EPO
RT
28 - Schill
-Environmental hazard (in case chemicals are accidently
released)
Biohazard and environmental hazard are related to
plastination only in a wider context. Biohazard may occur
when handling fresh tissue, i.e. before fixation,
embalming, or transfer into the dehydration bath.
Environmental hazards mainly occur where chemicals
hazardous to the environment are accidently released,
and enter the soil or water system. Health and explosion
hazards are more closely associated with plastination
work, and will therefore be discussed in more detail below.
Gathering information
Information about chemicals presenting health hazards or
explosion hazards can be found
• on the product container’s label.
• in the Safety Data Sheet (SDS).
o In Europe, all SDS have a standardised
structure of 16 sections according to the
regulation no. 1907/2006 (“REACh”; The
European Parliament and the Council of the
European Union, 2006), and regulation (EU) no.
2015/830 (The European Commission, 2015),
respectively. Information about classification
and labelling of the product is found in section 2;
physical properties are given in section 9.
• in national or international chemicals inventories, e.g.
the “Classification and Labelling Inventory of the
European Chemicals Agency” which can be found at
https://echa.europa.eu/information-on-chemicals/cl-
inventory-database .
While information about specialised chemicals is usually
(and sometimes exclusively) found in the safety data
sheets provided by the suppliers, chemical inventories are
a good source to review properties of basic substances
like solvents.
Minimum information on a chemical product’s hazards
includes one or more hazard pictograms, hazard
statements (in short “H-statements”, e.g. H225 “Highly
flammable liquid and vapour”), and a signal word
(“Warning” or “Danger”). Precautionary statements (“P-
statements”) indicate measures that should be taken in
order to prevent exposure to the chemical (e.g. P284 “In
case of inadequate ventilation wear respiratory
protection”) or to respond to an exposure (e.g. P312 “Call
a POISON CENTER or doctor if you feel unwell”).
Health hazards
When discussing health hazards, we commonly
distinguish three different routes of exposure: skin/eye
contact, ingestion, and inhalation. Of these, inhalation is
of major importance when working in plastination.
How can we find out if a chemical poses an inhalation
hazard? The first approach is having a look at the product
label on the container and/or at the SDS. While the hazard
pictograms alone are often not very clear in their meaning,
the H-statements are more informative. For example, the
H-statements indicating acute toxicity through inhalation
are very clear. They are, in decreasing severity: H330
“Fatal if inhaled,” H331 “Toxic if inhaled,” H332 “Harmful
if inhaled,” and H333 “May be harmful if inhaled”. (Note:
H333 is included in the United Nations’ Globally
Harmonized System of Classification and Labelling of
Chemicals [“GHS”; United Nations, 2017], Annex 3, but
has not been adopted e.g. by the European CLP
regulation.)
Besides the H-statements related to acute toxicity, there
are numerous others that are, or can be, related to
inhalation hazard. Among these are:
-H334 “May cause allergy or asthma symptoms or
breathing difficulties if inhaled,” indicating the risk of
respiratory sensitisation.
-H335 “May cause respiratory irritation,” indicating a
specific target organ toxicity after single exposure.
-H372 “Causes damage to [state organ] [state route
of exposure],” indicating a specific target organ
toxicity after repeated exposure. The route of
exposure, e.g. “through exposure by inhalation,” may
be given only if other routes of exposure can be
reliably excluded.
Commonly used solvents for dehydration like acetone or
isopropanol either have H335 (“May cause respiratory
irritation”) or H336 (“May cause drowsiness or dizziness”).
BIODUR® gas cure S 6 has H332 (“Harmful if inhaled”).
H372 applies to all styrene-containing polyester resins
that are particularly used for plastination of brain slices,
with the wording “Causes damage to hearing organs
through prolonged or repeated exposure”.
Another approach (besides looking at the H-statements)
that can also be of help, is to get an idea about the
importance of avoiding inhalation: in the majority of the
SDS, very often in section 9, you can find information
General Issues of Safety in Plastination - 29
about the vapour pressure of the chemical. This value is
usually given for a temperature of 20° C, therefore you
can easily compare different chemicals. Acetone, for
instance, has a vapour pressure of nearly 250 hPa while
ethanol’s is 58 hPa (Table 1). These values indicate that
inhalation hazard has to be taken into consideration. On
the other hand, unsurprisingly, for a product like the
hardener BIODUR® S3 with a vapour pressure of far less
than 1 hPa, inhalation hazard is not of relevance under
normal working conditions.
While it is comparably easy to protect one’s skin and eyes
by wearing suitable safety goggles, protective gloves, arm
sleeves, etc., we have to consider several aspects when
we strive to avoid inhalation of hazardous vapours.
Depending on the chemicals we work with and the scale
of the plastination unit, we have to decide which technical
and organisational measures are to be taken, alongside
the use of personal protective equipment. As the
atmosphere holding vapours hazardous to health usually
is identical to the one that poses an explosion hazard,
technical and organisational protective measures will be
discussed below, under the heading “Explosion Hazard”.
Personal Protective Equipment (PPE)
Hand/skin protection: protective gloves should be
mandatory whenever one works in the plastination lab.
Disposable nitrile or latex gloves provide only minor
protection against solvents like acetone, though they are
most commonly used in laboratories. They are most
suited for protecting the hands in situations where there is
a risk of minor splashing. Whenever intensive contact with
solvent is expected and reliable protection against
permeation is required, one should choose a high-quality
glove material like butyl rubber (IFA, 2017). When
handling larger amounts of chemicals, disposable sleeve
protectors and aprons made of polyethylene are a
comparatively inexpensive and effective solution (Fig. 1).
Respiratory protection: if technical room ventilation is not
available, or is not powerful enough to keep the
concentration of hazardous vapours below the locally
prescribed limits, protective masks should be worn (Fig.
1). Manufacturers of respirators offer full face masks as
well as half-masks. Combination filters, which absorb
different kinds of vapours, render the purchase of several
filter cartridges superfluous.
Figure 1. Examples of personal protective equipment (PPE) to avoid exposure to chemicals: protective gloves made of butyl rubber (left), disposable apron (middle), full face piece respirator (right).
Physical Hazards
Physical factors leading to a potential hazard can arise,
for example when a safety glass plate as part of a
plastination kettle bursts, leading to mechanical impact
caused by small fragments (Fig. 2). Therefore, it is
important to inspect such glass plates from time to time
for damage, especially the edges. Damaged glass plates
should be replaced with new ones.
Working in cold temperatures carries the risk of cryogenic
burns caused by low-temperature impact (Fig. 2).
Wearing insulated gloves or chemical protective gloves
with cotton gloves underneath avoids this risk.
Figure 2. Physical hazards: when damaged, a safety glass
plate shatters into numerous small pieces (left). Working
in the cold with unprotected hands bears the risk of
cryogenic burns (right).
Explosion Hazard
The most severe hazard in plastination is explosion,
caused by the presence of vapour of a flammable liquid in
the ambient air at a concentration within its lower and
upper explosion limits (Fig. 3). If the temperature is above,
or near the flash point (see appendix) of this liquid, any
source of ignition able to set free enough energy (inside
or in proximity to the air-vapour-mixture) will ignite this
hazardous atmosphere, thus causing an explosion. The
occurrence of such situations during work procedures
30 - Schill
must be identified and considered in a risk assessment.
Areas where an explosion hazard may occur should be
defined. They are often named “hazard zones”.
Fig. 3: Potential explosion hazard occurs when the concentration of vapour in the surrounding air ranges between the lower explosion limit (LEL) and the upper explosion limit (UEL).
Measures to improve work safety consist of steps to
preferentially avoid the formation of a hazardous air-
vapour-mixture, e.g. by effective and reliable removal of
the vapour through ventilation. This approach is referred
to as “primary explosion protection”. In cases where
primary explosion protection is not consistently possible,
precautions should be taken so that the hazardous
atmosphere will not ignite (“secondary explosion
protection”). Secondary explosion protection consists of
identifying and eliminating all potential sources of ignition
(see below) from the hazard zone.
When working in plastination, we cannot completely
exclude the formation of an air-vapour mixture, especially
during the dehydration and defatting steps, where
acetone or other solvents are used. Some recurring steps
involve working at open containers. If we look at the
silicone standard method we find that handling of
flammable liquids happens during the following activities:
-Storing flammable liquids (mostly acetone)
-Transporting them within your department
-Filling /decanting
-Immersing specimens into or removing them from
the dehydration bath
-Taking acetone measurements with a density
areometer (“acetonometer”)
-Disposing of acetone
Thus, flammable liquids play a role in numerous steps
while working. All of them pose some risk of evaporation.
The likelihood of this evaporation, the subsequent
formation of a hazardous atmosphere, and the spatial
expansion of this atmosphere, must be considered when
determining the explosion hazard in the plastination lab.
Classification of flammable liquids into categories
The hazardous potential of different flammable liquids
varies markedly. In the UN “Globally Harmonized System
of Classification and Labelling of Chemicals” (GHS;
United Nations, 2017), flammable liquids are classified
into category 1, 2, or 3, according to their respective flash
points and boiling points (Table 2).
Category 1 represents the most severe hazard. It is linked
to the H-statement H224 “Extremely flammable liquid and
vapour”. If we look at some solvents commonly used in
dehydration we find that they all fall into category 2 with
H225 “Highly flammable liquid and vapour” (Table 1).
Table 1: Some characteristics of solvents used for
dehydration (source: “GESTIS” database of the
German Statutory Accident Insurance, 2017)
Characteristic Ethanol Acetone 2-Propanol
Boiling point 78 °C 56 °C 82 °C
Flash point 12 °C < -20 °C 12 °C
Vapour pressure
at 20 °C
58 hPa 246 hPa 42.6 hPa
Density at 20 °C 0.79 g/cm³ 0.79
g/cm³
0.78 g/cm³
Viscosity
(dynamic) at 20
°C
1.2 mPa*s 0.32
mPa*s
2.4 mPa*s
Upper explosion
limit (UEL)
27.7 Vol-% 14.3 Vol-% 13.4 Vol-%
Lower explosion
limit (LEL)
3.1 Vol-% 2.5 Vol-% 2.0 Vol-%
Partition
coefficient
n-octanol/water
(log POW)
-0.3 -0.24 0.05
Miscibility with
water
miscible miscible miscible
Some countries like the US have adopted a fourth
category of flammable liquids in addition to the GHS: if the
flash point of a liquid is higher than 60 °C but does not
exceed 93 °C it is classified into category 4 with H227
“Combustible liquid”.
General Issues of Safety in Plastination - 31
Table 2: Criteria for the classification of flammable liquids as defined by the Globally Harmonized System of Classification and Labelling of Chemicals” (GHS).
Determination of explosion hazard zones
In many countries it is mandatory to designate special
rooms or areas where people handle flammable liquids as
“explosion hazard zones”. An explosion hazard zone is an
area where an explosive air-vapour mixture may form with
a certain probability and frequency of occurrence. Within
Europe, directive 1999/92/EC gives the definition of
hazard zones 0, 1, and 2 (Table 3) (The European
Parliament and the Council of the European Union, 1999).
Table 3: Explosion hazard zones 0, 1, and 2 caused by
gas, vapour or mist, as defined by the European directive
1999/92/EC.
Hazard caused by
Explosion hazard zone 0
Explosion hazard zone 1
Explosion hazard zone 2
Gases, vapours or mists
A place in which an explosive atmosphere consisting of a mixture with air of flammable substances in the form of gas, vapour or mist is present continuously or for long periods or frequently.
A place in which an explosive atmosphere consisting of a mixture with air of flammable substances in the form of gas, vapour or mist is likely to occur in normal operation occasionally.
A place in which an explosive atmosphere consisting of a mixture with air of flammable substances in the form of gas, vapour or mist is not likely to occur in normal operation but, if it does occur, will persist for a short period only.
Note: explosion hazards not caused by the presence of
gas, vapour or mist, but by combustible dust in the air,
would lead to a classification into hazard zone 20, 21, or
22. This type of hazard is outside the scope of this article.
Figure 4 gives an example of explosion hazard zone
determination. Zone 0 is usually restricted to the interior
of conveyor pumps, barrels, and other kinds of
receptacles. However, there is no general information on
the spatial extent of zones 1 and 2. They must be defined
by the person who is responsible on-site.
Fig. 4: Example: determination of explosion hazard zones when pumping solvent from a drum into a smaller receptacle. Example drawing given by the German statutory accident insurer VBG (2010)
In the United States and Canada, safety officers in charge
of explosion hazard assessment follow either the zone
classification system or their traditional definition of
hazard class and division, given by the US National
Electrical Code (NEC 500) and the Canadian Electrical
Code (CEC J18), respectively (Stahl AG, 2016; Siemens
AG, 2010). In brief, NEC 500 and CEC J18 define Class I
locations as places in which flammable gases or vapours
are or may be present in the air in quantities sufficient to
produce explosive or ignitable mixtures. In class I, division
1 areas, ignitable concentrations of flammable gases,
vapours or liquids may exist under normal operating
conditions and during repair or maintenance works. In
division 2 areas, these ignitable concentrations should not
exist under normal operating conditions but only in case
of a malfunction, e.g. a container leakage.
Note: Class II refers to explosion hazard caused by
combustible dust in the air. Class III refers to explosion
hazard caused by easily ignitable fibres.
Potential sources of ignition
If reliable avoidance of a hazardous atmosphere is not
always possible, it will be necessary to consider potential
ignition sources. In theory, there are many different types,
however, not all of them are likely to occur in a plastination
laboratory. For instance, the European standard EN
Category Criteria
1 Flash point < 23 °C and initial boiling point ≤ 35 °C
2 Flash point < 23 °C and initial boiling point > 35 °C
3 Flash point ≥ 23 °C and ≤ 60 °C
32 - Schill
1127-1 lists 13 different types (German Institute for
Standardisation, 2011). Among these, flames and hot
gases, static electricity, hot surfaces, and electrical
equipment are probably of major importance in a typical
plastination laboratory (nevertheless, all types of ignition
sources and the likelihood of their occurrence have to be
considered!)
Types of ignition sources (examples)
Hot surfaces Electromagnetic waves
Flames and hot gases Optical radiation
Mechanically produced sparks Ultrasound
Electrical equipment Chemical reactions
Transient currents Lightning strikes
Static electricity
Flames and hot gases are present for example when
using a Bunsen burner.
Static electricity can occur during friction between certain
materials, for example when wearing clothes made of
synthetic fibre.
Hot surfaces are found in heating cabinets, magnetic
stirrers, and similar laboratory apparatus.
Electrical appliances, permanent installations or mobile
devices, have to be removed from the explosion hazard
zone unless they are manufactured with explosion-proof
design (Fig. 5).
Fig. 5: Electrical devices as potential ignition sources (examples): Magnetic stirrer with a heating plate, Computer.
Technical Protective Measures
Technical protective measures should always have
priority over other measures. For instance, installation of
a workplace ventilation system takes precedence over
having the staff wear respirators. The order of priority is
stated in the “T-O-P” principle, according to which,
technical measures have priority over organisational
measures which, in turn, have priority over personal
protective measures.
Effective room ventilation is of major importance when
running a plastination laboratory. At this point, people are
often uncertain about the appropriate air-change rate. The
information booklet “Working Safely in Laboratories –
Basic Principles and Guidelines” (German Statutory
Accident Insurance, 2008) can serve as a reference.
Therein, a value of 25 m3 per m2 of floor area per hour is
given. In other words: the air changes/hour should be
approximately 10 (assuming that the ceiling height of the
room is approx. 2.5 m). A ventilation system offering two,
or several, power levels is recommended, enabling the
operator to choose the extraction capacity according to
the current need.
If individual ventilation for the laboratory is to be newly
installed, one should consider that the vapours of
acetone, ethanol, and all other commonly used solvents
are heavier than air and therefore will gather near the
floor. For this reason, it is advantageous to provide for air
extraction openings close to the floor (Fig. 6).
Fig. 6: Room ventilation with a ventilation opening beside
a freezer chest. As solvent vapours are heavier than air, it
is advantageous to extract them close to ground level.
In some situations, e.g. when working with polyester resin
or when preparing epoxy for injecting vessels of organs,
workplace ventilation with a flexible extraction arm (Fig. 7)
or working under a fume hood is advisable.
A potential equalisation bar should be present in every
plastination laboratory, in order to provide an earth for
General Issues of Safety in Plastination - 33
pieces of equipment (Fig. 8). In this way, electrostatic
charging can be prevented, and the risk of spark
generation will be reduced.
Fig. 7: Extraction arm with explosion-proof fan on top, offering workplace ventilation.
Fig. 8: Wall-mounted potential equalisation bar with several connection options.
Choosing proper explosion-proof equipment
Primary explosion protection, i.e. avoiding the formation
of an explosive air-vapour-mixture, is always desirable
and has priority. However, as mentioned before, in some
steps of the plastination procedure it is necessary to
handle flammable liquids in open containers, and
therefore we need to apply secondary explosion
protection, i.e. avoiding ignition of the explosive
atmosphere. This means that all potential sources of
ignition are to be removed from the hazard zone. If
removal is not possible, as might be the case for some
fixed electrical installations, one should consider their
disconnection from the mains, thus achieving permanent
inactivation.
For electrical devices that are intended to be operated
inside an explosive atmosphere, those with explosion-
proof design should be chosen. The suitability of an
appliance for use in hazard zone 0, 1, or 2 is shown on
the type plate (Table 4) (The European Parliament and
the Council of the European Union, 2014):
Table 4: Marking of electrical devices according to directive 2014/34/EU (examples).
Equipment group and category for use in
II 1 G zone 0 or zone 1 or zone 2
II 2 G zone 1 or zone 2
II 3 G zone 2
The Roman numeral, I or II, indicates the equipment
group. For plastination, as well as for general laboratory
purposes, only group II comes into consideration. The
Arabic numeral, 1, 2, or 3, indicates the equipment
category, which determines the hazard zone where it is
approved for use.
The capital “G” indicates that the equipment is approved
for use in hazardous atmosphere caused by the presence
of gas, vapour or mist. See Figure 9 for an example type
plate. A capital “D”, on the other hand, would indicate the
fitness for use in a hazardous atmosphere cause by the
presence of dust.
Fig. 9: Example: type plate of an explosion proof fan approved for use in explosion hazard zone 1 or 2.
Organisational Protective Measures
Technical protective measures usually are accompanied
by organisational measures to enhance safety, especially
when working in a large plastination laboratory.
34 - Schill
Organizational measures mainly consist of meaningful
labelling, instructions, or restrictions, such as:
- Access restrictions – only authorised personnel
are allowed to enter the lab.
- Written and oral working instructions – instruct
the staff before they start working in the
plastination lab, and then recurrently, e.g. on a
yearly basis. Mandatory in many countries.
- -Working alone is not allowed (night time,
weekends) – two or more persons are more alert
than one, thus avoiding for instance, handling
errors. Also enables quick first aid.
- -Regular inspection of equipment for safety –
often prescribed at least once-yearly. To be done
by a qualified person, e.g. by an electrician.
- -No smoking! Relevant instructions and easily-
visible prohibition signs making it clear, even to
visitors, that smoking is not allowed in the area
(Fig. 10).
Fig. 10: Prohibition signs at the entrance to a solvent storage. The yellow-black warning sign indicates the potential explosion hazard.
Summary
The objective of this article was to review the major types
of hazards which may occur when working in plastination,
and to create awareness of the precautions that should
be taken before starting a plastination laboratory.
Information about the specific hazards of chemicals used
in plastination can be obtained from the product labels,
from safety data sheets, or from chemical substance
databases. Some protective measures for preventing
health hazards, such as wearing safety gloves and
protective glasses, are standard and are identical to those
required in a normal chemical laboratory. Working
underneath an air extraction arm or under a fume hood
avoids health hazards caused by inhalation, e.g. when
handling solvent-containing epoxy for vessel injection or
polyester resin.
The main characteristic of a typical plastination laboratory
is the fact that people store, transport, and handle unusual
amounts of solvent, mostly acetone. This requires more
specific measures in order to prevent (1) health hazards
caused by acetone vapour inhalation, and (2) explosion
hazards caused by the presence of a vapour-air-mixture.
Effective room or workplace ventilation is essential to
extract the vapour from the workspace. Despite the
ventilation system, however, whenever an explosive
atmosphere may form, it is essential to ensure that no
sources of ignition are present. Potential sources of
ignition include spark-producing electrical installations
such as light switches, or portable electrical appliances,
for instance stirrers with heating plates, or laptop
computers. Electrostatic charging of apparatus may also
lead to sparking. This charging can be reliably avoided by
earthing the equipment via a potential equalisation bar. In
some work steps when you need an electrical appliance
inside the explosion hazard zone, you have to use
explosion-proof equipment. The suitability of a pump, fan,
etc. for operation inside the hazard zone is given on the
equipment type plate. All technical protective measures
should be accompanied by organisational measures,
whereby comprehensive and repeated instructions to staff
are of particular importance.
When carrying out a risk assessment for a plastination
laboratory one always has to consider the overall
situation, including the lab dimensions, the types of
chemicals used, their quantities and their physical
properties, the capacity of the ventilation system, the
number of people who work in the lab (and their level of
knowledge), the items of equipment, etc. When estimating
the hazard caused by flammable liquids, their respective
flash points are the most important characteristic.
As the conditions differ from department to department,
and from one lab to the next, there is often no general
“right” or “wrong” when people ask, for example, about the
suitability of existing premises, or of a certain appliance
like a freezer, for plastination. It is necessary to know the
overall situation on site, or the specifics of a plastination
project before a decision can be made.
General Issues of Safety in Plastination - 35
It is quite possible to run a plastination lab safely, as
proven by a number of labs all over the world where
people have worked for decades without any accidents.
Everyone who is interested in plastination and is able to
provide a suitable surrounding to set up the equipment
should feel encouraged to start their own lab. In any case,
whether you are a novice or routinely plastinating, it is
good to be aware of the hazard situation during all steps
of the procedure.
Appendix
A brief explanation of the characteristics of gases or
vapours related to explosion protection follows. Values
thereof can sometimes be found in safety data sheets, or
in some online databases or reference books.
Flash point - the lowest temperature at which a liquid
generates flammable vapours above its surface which
can be ignited in air by a flame. Examples: acetone:
approx. -20° C; ethanol: 12° C; isopropanol: 12° C.
Lower explosion limit (LEL) – a substance-specific
concentration of vapour in air. Below the LEL, the quantity
of flammable gas in the air is not sufficient to propagate a
flame in the surroundings of the ignition source.
Practically, the atmosphere will not burn or explode.
Upper explosion limit (UEL) – a substance-specific
concentration of vapour in the air. Above the UEL, the
concentration of flammable gas or vapour in the air is so
high that there is not enough oxygen left to have the
reaction of combustion or explosion propagated.
Explosion group – a classification system for gases and
vapours, derived from a combination of two substance-
specific physical characteristics, “minimum ignition
current ratio” and “maximum experimental safe gap”.
Describes the “readiness” of a gas or vapour to ignite and
the ability of the explosion to pass a narrow gap of given
dimensions. Important for the design of certain explosion-
proof equipment.
Groups are, in increasing severity: IIA -> IIB -> IIC.
Examples: acetone: IIA; ethanol: IIB; isopropanol: IIA
(Table 5).
Note: The traditional North American system of
classification divides explosive gases, vapours or mists
into gas groups A, B, C or D, with A posing the most
severe hazard. Group A and B correlate with the
international group IIC, group C correlates with IIB, and
group D correlates with IIA.
(Auto-)Ignition temperature – a substance-specific
temperature at which an explosive air-vapour-atmosphere
will ignite, even in the absence of any flame or spark.
Examples: acetone: approx. 530 °C, ethanol: approx. 400
°C; isopropanol: 425 °C.
Temperature class – according to their ignition
temperatures, gases and vapours are classified into six
temperature classes, T1 -> -> T6, with T6 constituting the
lowest temperature range and therefore posing the most
severe hazard (Table 5).
Table 5: Explosion groups (IIA -> IIC) and temperature classes (T1 -> T6) of some gases and vapours. Example: The surface temperature of an electrical appliance labelled “T4” must not exceed 135 °C. Temperature subclasses may apply, especially in North America (e.g. T4A).
T1 Ti > 450 °C
T2 450 > Ti > 300 °C
T3 300 > Ti > 200 °C
T4 200 > Ti > 135 °C
T5 135 > Ti > 100 °C
T6 100 > Ti > 85 °C
IIA
Acetone Iso- propanol
Gasoline
IIB Ethanol Ethylene
BIODUR® S 6 Ethyl ether
IIC Hydrogen Acetylene Carbon disulphide
Ti = ignition temperature.
36 - Schill
References
German Institute for Standardisation (2011): DIN EN
1127-1 Explosive atmospheres – Explosion prevention
and protection – Part 1: Basic concepts and methodology;
German version. Beuth, Berlin
German Statutory Accident Insurance (2008): Working
Safely in Laboratories – Basic Principles and Guidelines.
German version. Jedermann-Verlag, Heidelberg.
German Statutory Accident Insurance (2017): “GESTIS”
Hazardous Substance Information System
http://www.dguv.de/ifa/gestis/gestis-
stoffdatenbank/index.jsp (in German). Viscosity data
taken from various safety data sheets from solvent
suppliers.
IFA - Institute For Occupational Safety And Health Of The
German Social Accident Insurance (as of 09/29/2017):
“Practical solutions for occupational health and safety at
company level”
http://www.dguv.de/ifa/praxishilfen/index.jsp, German
version.
Stahl AG (2016): Grundlagen Explosionsschutz
(“explosion protection basics,” brochure in German)
https://r-
stahl.com/fileadmin/user_upload/mitarbeiter/PDF/ex-
grundlagen-explosionsschutz-rstahl-b-de.pdf (as of
10/13/2017)
Siemens AG (2010): Explosion Protection – Answers for
Industry (brochure in German)
https://www.automation.siemens.com/salesmaterial-
as/brochure/de/brochure_explosion_protection_de.pdf
(as of 10/13/2017)
The European Commission (2015): Commission
Regulation (EU) 2015/830 of 28 May 2015 amending
Regulation (EC) No 1907/2006 of the European
Parliament and of the Council on the Registration,
Evaluation, Authorisation and Restriction of Chemicals
(REACH).
The European Parliament and the Council of the
European Union (2014): “Directive 2014/34/EU of the
European Parliament and of the Council on the
harmonisation of the laws of the Member States relating
to equipment and protective systems intended for use in
potentially explosive atmospheres (recast),” Official
Journal of the European Union, L 96, 309 – 356.
The European Parliament and the Council of the
European Union (2006): Regulation (EC) No 1907/2006
of 18 December 2006 concerning the Registration,
Evaluation, Authorisation and Restriction of Chemicals
(REACH), establishing a European Chemicals Agency,
amending Directive 1999/45/EC and repealing Council
Regulation (EEC) No 793/93 and Commission Regulation
(EC) No 1488/94 as well as Council Directive 76/769/EEC
and Commission Directives 91/155/EEC, 93/67/EEC,
93/105/EC and 2000/21/EC.
The European Parliament and the Council of the
European Union (1999): “Directive 1999/92/EC of the
European Parliament and of the Council on minimum
requirements for improving the safety and health
protection of workers potentially at risk from explosive
atmospheres.”
United Nations (2017) “Globally Harmonized System of
Classification and Labelling of Chemicals (GHS)” Seventh
revised edition
https://www.unece.org/fileadmin/DAM/trans/danger/publi
/ghs/ghs_rev07/English/ST_SG_AC10_30_Rev7e.pdf
(as of 10/13/2017)
VBG - Verwaltungs-Berufsgenossenschaft (2010):
Broschüre Explosionsschutz – Praxishilfe (brochure in
German). VBG, Hamburg.
Von Hagens G, Tiedemann K, Kriz W (1987): The current
potential of plastination. Anat Embryol 175:411-421.
The Journal of Plastination 30 (1):37 (2018)
Journal of Plastination Instructions for Authors
(Revised July 2017)
JOURNAL OF PLASTINATION is owned and controlled by the International Society for Plastination (ISP).
Goals - The Journal of Plastination (ISSN 1090-2171) aims to provide a medium for the publication of scientific papers dealing with all aspects of plastination and preservation of biological specimens.
Submission Guidelines All manuscripts must be submitted to the Editorial Office via the e-mail: [email protected]. If you experience any problems or need further information, please contact Philip J. Adds, [email protected].
Authors must have an e-mail address at which they may be reached.
Necessary Files for Submission Include:
• Cover letter
• Manuscript (including references and figure legends)
• Table(s) (when appropriate)
• Figure(s) (when appropriate)
• Copyright Release Form (after acceptance)
Note: The above items should be prepared as separate files. Each file must contain a file extension (.doc, tif, jpg, eps).
• File formats appropriate for text and table submissions: Microsoft Word
• File formats appropriate for figure submissions: TIFF, JPEG (JPG) and EPS
Categories of submissions: Articles published in Journal of Plastination are grouped into general article types (listed below). Final designation of a manuscript’s article type is determined by the EDITOR.
• Original Research – Plastination
• Original Research – preservation
• Education
• Case reports
• Technical brief notes
• Review - by invitation only
• Legacy – institutions and people
• Correspondence
• Editorial
Acceptance of a submission implies the transfer of copyright from the authors to the publisher. It is the author's responsibility to obtain permission to reproduce illustrations, tables and figures from other publications.
Copyright Transfer Form may be downloaded from http://www.journal.plastination.org/downloads/copyright.pdf. After the form is completed and signed by all the authors, it should be submitted to the Editorial Office ([email protected]) as a pdf or jpeg file via an e-mail attachment. Manuscript preparation
Cover Letter The cover letter should include a statement of authorship, notification of conflicts of interest, ethical adherence, and any financial disclosures. Cover letters may be addressed to the Editor-in-Chief, Journal of Plastination.
Manuscript The manuscript should consist of subdivisions in the following sequence:
Title Page Abstract with keywords Text Introduction Materials and methods Results Discussion References Figure Legends
Title Page The first page of the manuscript should include:
• Title of paper
• Each author’s name
• Institution from which paper emanated, with city, state, and postal code. Each affiliation should be listed as a separate entity, with a superscript number that links it to the individual author.
For example: S. D. HOLLADAY1*, B. L. BLAYLOCK2 and B. J. SMITH1 1Department of Biomedical Sciences and Pathobiology, Virginia Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0442, USA. 2College of Pharmacy and Health Sciences, University of Louisiana at Monroe, Monroe, LA 71209, USA.
• Corresponding Author’s name, address, telephone and telefax numbers, and e-mail address.
For example: *Correspondence to: Dr Shane D. Holladay, Department of Biomedical Sciences and Pathobiology, Virginia Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0442, USA. Tel.: +001 404 739 6403; Fax: +001 404 739 6492; E-mail: [email protected]
The Journal of Plastination 30 (1):38 (2018)
It is the corresponding author’s responsibility to notify the Editorial Office of changes of address. Only the corresponding author should communicate with the Editorial office for matters regarding each manuscript. Abstract & Key Words The abstract should be no longer than 250 words. It should contain a description of the objectives, materials and methods, results, and conclusions. The abstract should include a section on technique/technical development if the paper is significantly technical in nature. The abstract must be written in complete sentences and be intelligible without reference to the rest of the paper. No references should be used in the abstract. On the same page, list, in alphabetical order, five Key Words that reflect the content of the manuscript. Consult the Medical Subject Headings for appropriate key words. Key words should be set in lower case (except for essential capitals), separated by a semicolon and bolded. Text The body of the text should be written using American English spelling. Where quantities are specified, S.I. units should be used. Equivalent Imperial or U.S. units, if desired, should follow in parentheses e.g. 1 Kg (2.2 pounds). References
• References to published works, abstracts and books must include all that are relevant and necessary to the manuscript.
• Citations in the text should be in parentheses and listed chronologically; e.g. (Bickley et al., 1981; von Hagens, 1985; Henry and Haynes, 1989) except when the authors name is part of a sentence; e.g. "…von Hagens (1985) reported that…" When references are made to more than one paper by the same author published in the same year, designate each citation as 1999 a, b, c, etc.
• Literature cited may only include the publications, which are cited in the text. References are to be listed alphabetically using abbreviated journal names according to Index Medicus. Page numbers of the citation must be included.
• Examples of the reference style are as follows:
• For a journal article: Bickley HC, von Hagens G, Townsend FM. 1981: An improved method for preserving of teaching specimens. Arch Pathol Lab Med 105:674-676.
• For a book section: Henry R, Haynes C. 1989: The urinary system. In: Henry R, editor. An atlas and guide to the dissection of the pony, 4th ed. Edina, MN: Alpha Editions, p 8-17.
Von Hagens G. 1985: Heidelberg plastination folder: Collection of technical leaflets for plastination. Heidelberg: Anatomiches Institut 1, Universität Heidelberg, p 16-33.
• For other publications:
• Internet references: Author last name, initial(s). Year: Title of article. URL: Internet address [accessed month, year].
Figure legends
• Legends for all figures should be brief, specific and not be a substitute listing for the result section, and appear on a separate page at the end of the manuscript, following the list of references.
• Legends must be numbered consecutively as they first appear in the text. All symbols or abbreviations appearing in any figure must be defined in the legend.
Tables
• All tables must be cited in the text and have titles. Table titles should be complete but brief. Information other than that defining the data should be presented as footnotes.
• Create tables using the table creating and editing feature of Microsoft Word. Do not use Excel or comparable spreadsheet programs.
• Each table should be simple and uncomplicated, with NO vertical and as few horizontal lines as possible.
• Each table is to appear on a separate page and must include the table title and appropriate column heads.
• Save each table in a separate word document file and upload individually, like figures.
• Do not embed tables within the body of the manuscript. Figures
• All figures must be cited in the text and must have legends.
• Each figure should be attached as a separate file and labeled with the appropriate number.
• Figures should be created, saved and submitted as either a TIFF, JPEG (JPG) or an EPS file.
• Line drawings must have a resolution of at least 1200 dpi, and electronic photographs, scanned images, radiographs, CT and MRI scans must have a resolution of at least 300 dpi.
• The size of each figure should be at least 8.25 cm / 3.25 inches (one-column width) or 16 cm / 6 inches (two-column width).
• Magnification must be recorded and have a “scale bar” in the photo. Since reproduction of illustrations is costly, authors should limit the number of figures to those which adequately present the findings, and add to the understanding of the manuscript.
• Figures that are submitted in color must be published in color.
The Journal of Plastination 30 (1):39 (2018)
Statement of Publication and Research Ethics: This statement is based mainly on the Code of Conduct and Best-Practice Guidelines for Journal Editors (Committee on Publication Ethics, 2011). Responsibilities of the Editor and Editorial Board:
• Publication decisions
The editor (in consultation with the Editorial Board where appropriate) is responsible for deciding which of the manuscripts submitted to the Journal of Plastination will be accepted for publication, and into which category of submission they should be placed. The decision will be based solely on the paper's importance, originality and clarity, and the study's validity and its relevance to the scope of the journal. The Editor and Editorial Board will also consider, where appropriate, current legal requirements regarding libel, copyright infringement, and plagiarism.
• Confidentiality
The Editor undertakes not to disclose details about any submitted manuscripts to anyone other than the corresponding author, reviewers (and potential reviewers), and the publisher, as appropriate.
• Disclosure and conflicts of interest Unpublished materials disclosed in a submitted paper will not be used by the editor or the members of the editorial board for their own research purposes without the author's explicit written consent.
• Responsibilities of the Reviewers Contribution to editorial decisions The peer-reviewing process assists the Editor and the Editorial board in making editorial decisions and will also, where appropriate, inform the author of improvements that will, in the opinion of the reviewer, enhance the paper.
• Promptness Any selected referee who feels unqualified to review the research reported in a manuscript or knows that its prompt review will be impossible should notify the editor and withdraw from the review process.
• Confidentiality
Manuscripts sent for review must be treated by them as confidential documents. They must not be disclosed to or discussed with others unless specifically authorized by the Editor.
• Standards of objectivity Reviews must be conducted objectively, without personal criticisms of the author(s). Referees should express their opinions clearly, and justify their comments with examples and supporting arguments.
• References and reference citations Reviewers should check that published works cited in the manuscript have also been listed accurately in the References section, and that all references listed have also been correctly cited in the text. Reviewers may also wish to indicate other relevant papers in the literature of which the author(s) may not have been aware. Reviewers will notify the Editor of any substantial similarity or overlap between the manuscript under review and other published papers of which they are aware.
• Disclosure and conflict of interest Privileged information or ideas obtained through peer review must be kept confidential and not used for personal advantage. Reviewers should not consider a manuscript in which they have a conflict of interest resulting from competitive, collaborative, or other relationships, or connections with any of the authors, companies, or institutions associated with the manuscript. Any such conflict should be declared to the Editor before agreeing to undertake the review. Duties of the Authors
• Reporting standards Authors of original research reports should present an accurate account of the work performed as well as an objective discussion of its significance. Underlying data should be represented accurately in the paper. A paper should contain sufficient detail and references to permit others to replicate the work. Fraudulent or knowingly inaccurate statements constitute unethical behavior and are unacceptable.
• Data access and retention Authors may be asked to supply the raw data for their study, and should be prepared to make the data publicly available where appropriate and practicable.
• Plagiarism, originality, and acknowledgement of sources
The Journal of Plastination 30 (1):40 (2018)
Authors will submit only entirely original works. The work and/or words of others, where they have been used or quoted, will be appropriately acknowledged and cited.
• Multiple, redundant or concurrent publication In general, papers that describe essentially the same research should not be published in more than one journal. Submitting the same paper to more than one journal is considered to be unethical and is unacceptable. Manuscripts that have been published as copyrighted material elsewhere cannot be submitted. Manuscripts that are undergoing the review process should not be resubmitted elsewhere. By submitting a manuscript, the author(s) retain the rights to the published material, although in case of publication, copyright of the published paper passes to the Journal of Plastination.
• Authorship of the paper Authorship should be limited to those who have made a significant contribution to the conception, design, execution, or interpretation of the reported study and its subsequent write-up for publication. All those, and only those, who have made significant contributions should be listed as co-authors. The corresponding author must ensure that all contributing co-authors are included in the author list. The corresponding author will also verify that all co-authors have approved the final version of the paper and have agreed to its submission for publication.
• Disclosure and conflicts of interest The corresponding author should include a statement disclosing any financial or other substantive conflicts of interest that may be construed to influence the results or interpretation of the manuscript. All sources of financial support for the project should be disclosed. Where there are no conflicts of interest, a statement to that effect should be included.
• Fundamental errors in published works When an author subsequently discovers a significant error or inaccuracy in their own published work, it is the author's obligation promptly to notify the Editor of the Journal and to cooperate with the Editor to retract or correct the paper by issuing an erratum.
• Research involving human or animal subjects In research involving human subjects, The Journal of Plastination requires that all such studies adhere to the principles of the Declaration of Helsinki. Each manuscript must include details of the a) number of subjects, b) age and sex of the participants, c) inclusion and exclusion criteria, and f) a statement that ethical approval was obtained for the study, and that informed consent was given by the participants. For cadaveric studies, appropriate consent must be in place prior to utilizing the cadavers or specimens. Studies involving experimental animals must conducted in a humane manner and in accordance with relevant guidelines for the care and utilization of laboratory animals. Animal care should be in line with the NIH Guidelines for the Care and Use of Laboratory Animals (NIH, 2015). The manuscript must include a statement that ethical approval of the protocol was obtained. The Journal of Plastination will reject manuscripts if the Editor and/or Editorial Board are not satisfied with the standards of ethical use of animals or data from humans in research. References Committee on Publication Ethics (COPE). (2011, March 7). Code of Conduct and Best-Practice Guidelines for Journal Editors. Retrieved from: https://publicationethics.org/files/Code_of_conduct_for_journal_editors_Mar11.pdf (accessed 5th September 2017) NIH Office of Laboratory Animal Welfare - Public Health Service Policy on Humane Care and Use of Laboratory Animals (NIH, 2015). Retrieved from: https://grants.nih.gov/grants/olaw/references/phspol.htm
