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A review of bone preparation techniques for anatomical studies

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 Malaya Journal of Biosciences 2016, 3(2):76-80
ISSN 2348-6236 print /2348-3075 online

Malaya
Journal of REVIEW ARTICLE
Biosciences
www.malayabiosciences.com

Open Access Full Text Article

A Review of Bone Preparation Techniques for Anatomical

Studies
Ajayi A*, Edjomariegwe O, Iselaiye O T

Department of Anatomy, Faculty of Basic Medical Sciences, College of Health Sciences, Kogi State University,
Anyigba, PMB 1008, Anyigba, Nigeria.
*
For correspondence: abajayi2003@yahoo.co.in

Article Info: Received 24 Nov 2016; Revised: 18 Dec 2016; Accepted 19 Dec 2016.

ABSTRACT
Bones are essential part of anatomy teaching curriculum and are unsurpassed in the ability to provide three-
dimensional instruction in osteology as well as understanding the sites of soft tissue insertion and the course
of neurovascular structures in a region. A number of techniques have been employed over the years in the
preparation of bones for anatomical studies. These methods include the use of insects, chemicals and
enzyme. In this review, we discuss the evolution of these maceration techniques their application in the study
of human anatomy.

Keywords: Maceration, bones, anatomy, osteology

1. INTRODUCTION

Anatomy teaching has become a challenging task.
Anatomy evolved as a keystone of medical education
through ages. Bones are very essential part of
anatomy teaching curriculum [1]. Human bones are
unsurpassed in the ability to provide three-
dimensional instruction in osteology as well as
understanding the sites of soft tissue insertion and the
course of neurovascular structures in the region. The
important aspect of bone anatomy can be most
efficiently learned by utilizing a dried bone in
combination with textbooks and atlases as well as
laboratory dissection [2].
Bone preparation involves soft tissue removal,
bone bleaching, bone articulation and labelling. The
amount of time required for these processes vary -
depending on the size of the animal [3]. There are
different techniques or methods used in bone
preparation which include insect consumption, cold-
or warm-water maceration, which have been used as
standard maceration techniques and requires between
2 days and 8 weeks depending on the amount of
bacteria present, the size of material being macerated
and the temperature of the environment during the
maceration. Boiling and subsequent mechanical
cleaning of skeletal material is also used [4].
Solutions of organic and inorganic chemicals are also
used to remove soft tissue from bones. Inorganic
chemicals used are antiformin, ammonium
hydroxide, sodium hydroxide and other alkaline
solutions. Maceration with organic chemicals can be
performed with enzymes such as papain or pepsin or

A Review of Bone Preparation Techniques for Anatomical Studies

Copyright © 2016 MJB
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 Ajayi A et al., / Malaya Journal of Biosciences 2016, 3(2):76-80
with washing powders containing enzymes [5] as
well as burying in soil [1]. This article presents the
different methods of obtaining bones from cadavers
and animals using the various bone cleaning
techniques for anatomical studies.
1.1. Maceration
Animal skeletons are very useful in the teaching of
vertebrate anatomy and also for identification of
unknown specimens. The preparation of skeletons for
long-term curation and study entails plenty of effort
but does not involve difficult techniques or expensive
materials. Boyle describes the step-by- step process
and documents the preparation of a bobcat and a fox
acquired from a trapper in Texas [3]. The specimens
were boiled to loosen the soft tissues, which were
then removed manually. Additional simmering in
borax helped to break down cartilage and collagen,
followed by soaking in xyol to remove residual fats.
Upon completion, these specimens were labelled and
added to the collections at the University of
Indianapolis Archeology & Forensics Laboratory.
Bones were macerated in solutions of varying pH
to observe the effects of varying fresh water pH on
bone. Since little is known about the decomposition
of remains in aquatic environments of varying pH,
and even less is known about the specific effects of
these environments on bone. Bovine bones were
placed in solutions of pH 1, 4, 7, 10, and 14 and
observed over a period of 1 year. All solutions
eventually removed or dissolved the soft tissues from
the external surface of the bone. The pH 7 and pH 10
solutions had little effect on the bone, but the other
solutions affected the bone to varying degrees [6].
Extreme pH levels were the most destructive, while
more moderate pH levels had lesser but significant
and interesting effects. Empirical data on post-
mortem aquatic changes may be extremely useful in
forensic contexts for both improving time since death
estimates and also for providing better information to
underwater recovery experts thereby potentially
increasing the quantity and quality of remains
recovery.
2. USE OF INSECTS
It has been shown that dermestid beetles are useful as
a technique to clean bones, especially for the parts of
the skeleton which are difficult to dissect by hand. He
cleaned various parts of the skeleton of normal and
osteoporotic rats using Dermestes maculates beetles
or manually and compared them with respect to their
A Review of Bone Preparation Techniques for Anatomical Studies
dry weight, ash weight, and calcium content. Both
techniques gave similar results. This was also true
when whole body calcium measured by neutron
activation and total skeletal calcium from bones [7].
Three geographically dispersed Middle and Later
Stone Age cave sites in South Africa, and a Middle
Stone Age cave site in Ethiopia, share a similar
taphonomic signature that includes destruction of
bones associated with variable forms of star shaped
features, clusters of microscopically visible sub-
parallel striations, edge gnawing, pits, and etching of
the bone surface. Similar traces preserved on Plio-
Pleistocene fauna are interpreted by different authors
as the possible work of termites or ants. Considering
that ambiguity exists in the interpretation of these
traces and that there are no modern examples
available for comparison, an experiment with the
harvester termite: Trinervitermes trinervoides
(Sjöstedt) (Isoptera: Termitidae) in the Sterkfontein
Valley, South Africa to create a reference collection
of bones modified by southern African termites
showed that within six months all bones were
approximately half covered with a dark surface
residue, had an etched surface appearance and
recorded boreholes and destruction, particularly of
less dense elements and epiphyses. Star-shaped
marks, edge gnawing, and clusters of sub-parallel
striations on the periosteal surface were faint after six
months, but became clearly visible and more plentiful
after 12 months, a finding attributed to the termites
being more active in austral autumn and spring [8].
This demonstrates the pertinence of insect
modification studies to our understanding of regional
palaeoenvironment, palaeoecology and
palaeoclimate. This experiment has demonstrated that
T. trinervoides can destroy bone in all stages of
preservation, favouring fresh thin cortical and spongy
bone with meat and marrow. The presence of
appreciable quantities of calcium carbonate in termite
mounds suggests that termites may be drawn to
dolomitic cavesites and the calcium-rich bones they
contain to satisfy their mineral requirements. They
therefore have the potential to bias taxonomic and
element representation, minimum number of
individuals, and age profiles in a faunal assemblage,
and may account in part for the patchy preservation
of faunal remains, including hominins, in fossil
deposits, and/or the lack of bone artefacts at some
Middle Stone Age cave sites preserving long
sequences of occupation.
3. USE OF CHEMICALS
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 Ajayi A et al., / Malaya Journal of Biosciences 2016, 3(2):76-80
Human skulls are important in osteology because
they aid in understanding the sites of soft tissue
insertion and their intricate course of neurovascular
structures in the skull base. Recent geopolitical
developments in Asia have led to extreme difficulty
in obtaining human skull specimens. A method for
the preparation of dried human skulls from fresh and
frozen cadavers using commonly available chemicals
has been described [2]. The technique, requiring
about 8 weeks total time and basic equipment,
consists of maceration accelerated by several
enzymes followed by defatting, washing and
bleaching. The skulls produced are of excellent
quality and durability with no preparation artifacts.
An economical source of skulls has now been re-
established to facilitate learning of the intricate
relationships of the skull base.
In another study, the effect of 10 maceration
methods on gross bone structure and the preservation
of DNA in ribs of 12 pigs (Sus scrofa) was evaluated
[9]. This is because forensic anthropologists use a
number of maceration techniques to facilitate skeletal
analysis of personal identity and trauma, but they
may unwittingly eliminate valuable DNA evidence in
the process. In the study a scoring system was
applied to evaluate the ease of maceration and
resulting bone quality while DNA purity was
quantified by optical densitometry analysis, followed
by polymerase chain reaction (PCR) amplification of
three mitochondrial and three nuclear loci. The
results demonstrated that while mitochondrial DNA
could be amplified for all experiments, cleaning
treatments using bleach, hydrogen peroxide,
ethylenediaminetetraacetic acid/papain, room
temperature water and detergent/sodium carbonate
followed by degreasing had low DNA concentrations
and failed to generate nuclear PCR products. This
study shows that traditionally "conservative"
maceration techniques are not necessarily the best
methods to yield DNA from skeletal tissue.
Maceration techniques remove soft tissue by the
destruction of biomolecules, but the applied
techniques may also affect the morphology and the
molecular integrity of the hard tissue itself. The
impact of seven different techniques for soft tissue
removal on morphological and biomolecular
parameters of teeth and dental tissues was
systematically examined [5]. Six different chemical
maceration methods and soft tissue removal by the
feeding of Dermestes sp. were investigated. All
methods tested showed significant changes in dental
morphology and in the molecular integrity of DNA
A Review of Bone Preparation Techniques for Anatomical Studies
and the dental proteins, as revealed by aspartic acid
racemisation (AAR). In forensic casework this may
have severe impacts on the results of morphological
methods (e.g. age estimation based on root
translucency) and of biomolecular analyses (e.g. age
estimation based on AAR and DNA analysis).
Therefore, age estimation based on AAR should not
be applied to tissue treated in such a manner, and it is
recommended that teeth for analysis should be
extracted before soft tissue removal. DNA in the hard
tissue seems to be less susceptible to soft tissue
removal than proteins, and several of the tested
maceration techniques appear not to have a damaging
effect on DNA. Generally, the indication for soft
tissue removal demands a careful case management
to avoid methodological collisions.
Using 12 Adult African Giant Pouched rats of both
sexes, to deternime the effect of three different
methods of bone preparation on the bones of the
African Giant Rat (Cricetomys gambianus), chemical
preparation with sodium hydroxide was found to be
the best method in terms of time required to complete
the procedure, number of bones recovered, colour of
the bones and odour of the preparation. However, the
chemical method has the disadvantage of dissolving
and cracking the bones if the concentration used is
high and prompt attention is not given to the
preparation [10].
3.1. Detergent Maceration
Osteological assessment of human remains forms an
essential part of forensic work, especially during the
examination of extensively decomposed,
dismembered, or burnt bodies. Currently employed
methods for removal of adherent soft tissue reflect
practices often used by museum curators, notably
insect consumption, enzymatic maceration, or boiling
of the bones, with subsequent manual removal of
material. In a study to assess the effectiveness of
detergents for the purpose of soft-tissue removal from
animal-derived specimens, it was shown that such a
means is comparable to enzymatic maceration but
with fewer health and safety issues and greater
advantages regarding transportation and availability
of materials when an investigator is in a fieldwork
scenario [11].
The forensic pathologist increasingly relies on the
forensic anthropologist to be the consulting expert in
human identification. Likewise, if identification is
not possible from visual inspection of skeletal
remains, the forensic biologist may be called upon to
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 Ajayi A et al., / Malaya Journal of Biosciences 2016, 3(2):76-80
conduct DNA analysis. The possibility of
downstream DNA testing needs to be considered
when skeletal preparation techniques are employed to
deflesh human remains, as they have the potential to
strongly impact genetic analyses and subsequent
identification. Rennick et al., (2005) employed three
cleaning techniques, boiling bone in water, in bleach,
and in powdered detergent/sodium carbonate, to test
for their effect on nuclear and mtDNA recovery from
a variety of human and non-human bones. A
statistically significant reduction in DNA yields
occurred in non-human bones cleaned with bleach,
and DNA degradation was apparent
electrophoretically. The human bones also showed
much lower yields from bleach cleaning, while the
detergent/carbonate method allowed the largest
segments of DNA to be amplified, indicating it may
have a less degradative effect on bone DNA than
either of the other cleaning processes [12].
3.2. Enzyme Maceration Technique
In 1991, Bartels and Meyer carried out a study that
described a method which uses enzymatic additions
of detergents (household washing products) to
macerate small vertebrates or their heads,
respectively, in a rapid, most effective, careful, rather
odourless way and without any pollution of the
environment. When applied to fresh or deep-frozen
specimens and ethanol-fixed material, the maceration
solution produced excellent results, but the result
with formalin-fixed specimens were unsatisfying
[13].
Enzyme maceration has been shown to be
remarkably fast (1–3 h) compared to the traditional
warm-water procedure, which requires up to several
days. In addition, the enzyme maceration eliminates
the odor problem associated with the traditional
procedure. It is shown that stirring of the enzyme
maceration bath is the main factor which determines
the speed of the maceration. For mice, the time
required for enzyme maceration can vary from 1 to
8 h depending on the stirring speed [13]. This method
allows preparation of skeletal material in an
essentially odorless way within a matter of hours,
making the method useful in particular for forensic
science, private conservation workshops, and
educational purposes.
More recently, in an investigation into the
conservation of mitochondria DNA (mtDNA) from
skeletal material prepared by enzyme maceration, ten
individuals of Stone Marten (Martes foina) were
A Review of Bone Preparation Techniques for Anatomical Studies
enzyme-macerated using a mixture of protease and
lipase. After using a fast enzyme preparation method
the skeletal material was stored for two years in order
to see if degradation of DNA had taken place. As
warm-water maceration is the traditional maceration
technique used for example at The Natural History
Museum of Denmark, ten different individuals of
Stone Marten were warm-water macerated so a
comparison of the two different maceration
techniques’ amplifiable DNA outcome could be
made. Samples for DNA analysis were taken from
two different kinds of bone (pelvic and costa) from
each individual. The analyses showed that the
mtDNA was intact and all PCR products could be
indentified to the right species without
contamination, demonstrating that both the warm-
water maceration and the fast enzyme preparation
method had not compromised the DNA [4].
Maceration is time consuming especially with
larger animals and elicits strong odours. Insects are
useful as a technique to clean bones because they
perform an excellent debridement of smallest cavities
but several thousand are required to produce rapid
cleaning and if left with bones for a long period of
time can eat and destroy them. Soft tissue removal by
solutions of organic and inorganic chemicals was
found to be the most effective since it macerates bone
in a remarkably fast and odourless way. And it also
has a less degradative effect on bone DNA than the
other cleaning processes. But prompt attention should
be given to the preparation when using the chemical
method so that the concentration used is not high in
order to avoid dissolving and cracking the bones.
Conflict of Interest
The authors declare that they have no conflicts of
interest.
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A Review of Bone Preparation Techniques for Anatomical Studies

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