Rib biomechanical properties exhibit diagnostic potential for accurate ageing in forensic investigations
Rib biomechanical properties exhibit diagnostic potential for accurate ageing in forensic investigations
Andrea Bonicelli 0 1
Bledar Xhemali 1
Elena F. Kranioti 0 1
Peter Zioupos 1
0 Edinburgh Unit for Forensic Anthropology, School of History Classics and Archaeology, University of Edinburgh , Edinburgh , United Kingdom , 2 Musculoskeletal & Medicolegal Research Group, Cranfield Forensic Institute, Defence Academy of the UK , Shrivenham , United Kingdom , 3 Forensic Institute, Department of Forensic Medicine, Tirana, Albania, 4 Department of Forensic Sciences, Faculty of Medicine, University of Crete , Heraklion, Crete , Greece
1 Editor: Gwendolen Reilly, University of Sheffield , UNITED KINGDOM
Age estimation remains one of the most challenging tasks in forensic practice when establishing a biological profile of unknown skeletonised remains. Morphological methods based on developmental markers of bones can provide accurate age estimates at a young age, but become highly unreliable for ages over 35 when all developmental markers disappear. This study explores the changes in the biomechanical properties of bone tissue and matrix, which continue to change with age even after skeletal maturity, and their potential value for age estimation. As a proof of concept we investigated the relationship of 28 variables at the macroscopic and microscopic level in rib autopsy samples from 24 individuals. Stepwise regression analysis produced a number of equations one of which with seven variables showed an R2 = 0.949; a mean residual error of 2.13 yrs ±0.4 (SD) and a maximum residual error value of 2.88 yrs. For forensic purposes, by using only bench top machines in tests which can be carried out within 36 hrs, a set of just 3 variables produced an equation with an R2 = 0.902 a mean residual error of 3.38 yrs ±2.6 (SD) and a maximum observed residual error 9.26yrs. This method outstrips all existing age-at-death methods based on ribs, thus providing a novel lab based accurate tool in the forensic investigation of human remains. The present application is optimised for fresh (uncompromised by taphonomic conditions) remains, but the potential of the principle and method is vast once the trends of the biomechanical variables are established for other environmental conditions and circumstances.
Competing interests: The authors have declared
that no competing interests exist.
Age-at-death estimation remains one of the most challenging tasks in forensic practice when
establishing a biological profile from unknown heavily fragmented or skeletonised human
remains. The methodological choice is subject to the general pattern of preservation of the
remains and the specific nature of the case[
During childhood, morphological methods based on developmental traits of bone can
provide extremely accurate results, but taphonomic changes increase the difficulty of the
procedure[1±3]. In adulthood, morphological methods, although easily applicable, are often
inaccurate for ages over 35 years old when all developmental markers disappear and thus
cannot be accepted by the legal system. These facts have led to the development of methods based
on the quantifiable degeneration of bone[1±8]. Furthermore, each method is highly population
and sex specific[
] and individual differences must be considered when interpreting the results
Garvin and Passalacqua[
] compared three of the most commonly used morphological age
estimation methods and studied the effect of the level of experience of different operators on
the application of the methods and the final age estimates. The results show that the
interobserver bias in the methodological application and consequent age estimation are not
predictable, which makes the application of such methods in a forensic situation problematic.
When the skeleton is preserved intact, a number of methods based on the pelvis, skull, rib
cage, and dentition can be applied and the final age estimate will be based on the combination
of these age estimates. In numerous occasions, though, the remains are only partially retrieved
and several important age estimation markers may be missing. This is one of the reasons that
numerous age estimation techniques were developed on ribs, a small skeletal element that can
be easily obtained and examined during a forensic examination. For instance, Işcan et al.[
developed a method based on the qualitative observation of metamorphic changes of the
sternal end of the fourth rib caused by a progressive age-related ossification of the cartilage tissue
connecting the ribs to the sternum. It has been proven to be generally very accurate (95% of
accurate estimation) and suitable for different populations[
]. However, this method, as well
as other morphological methods, has a high level of inter-observer error and a decreased
accuracy when aging older individuals[
The rib was also employed in histomorphometric studies based on bone remodelling[4±5].
The original method by Stout and Paine[
] on the sixth rib gave extremely accurate results
especially when combined with the clavicle. What is highlighted by the authors themselves is
the fact that, although the estimation has a 95% of accuracy, the method fails to give a reliable
age prediction for individuals over 40 years-old due to the overlapping of secondary osteons
]. Moreover, this method underestimates age significantly when applied to other populations
]. Cho et al. [
] have provided an updated histomorphometric method which employs
several additional parameters and seems to account for older ages and ethnicity. This method was
tested using a large sample (N = 213) from South Africa and reported 6±11% of the sample
falling out of the 95% confidence prediction range (+/-24.4 years) of the original study[
authors favour the use of the unknown-ethnicity formula and reject the hypothesis that
population specific-equations are needed, since equations based on their sample did not perform
any better than Cho's equations. This paper is one of the few validation studies with such a
large sample and contains criticisms as to the value of regression in the analysis of
Other laboratory-based methods include the aspartic acid racemization techniques that are
based on the heat-dependent gradual transformation of specific proteins during life[13±16].
Several studies confirm the high accuracy of such techniques, however, they have significant
limitations: high demand in time, equipment and expertise, poor results for particular
categories, such as mature females and are ineffective for post-mortem interval of more than 20 years
Radiocarbon dating methods use the variation of atmospheric 14C levels, which are
ultimately incorporated into living tissues to date the formation of proteins in the lens, brain
neurons and bone. The dramatic increase of the amount of atmospheric 14C from 1955 to 1963,
due to nuclear bomb testing allows for the accurate estimation of the time that tissues with
slow 14C turnover were formed, thus producing an accurate estimation of the date of birth.
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Alkass et al.[
] used dental enamel to combine aspartic acid racemization and radiocarbon
dating techniques on a known age sample. According to the results, radiocarbon analysis
showed an overall absolute error of 1.0 +/- 0.6 years while aspartic acid racemization showed
an overall absolute error of 5.4 +/- 4.2 years.
DNA methylation, one well-known epigenetic modification, has also been shown to
correlate with age[
]. More specifically, the global level of methylated genomic DNA decreases
with increased age. A recent study on Chinese Han female monozygotic twins identified
2,957 novel age-associated DNA methylation sites (P<0.01 and R2>0.5) in blood. Eleven CpG
sites were used to develop an age regression model which exhibited a mean absolute deviation
from real chronological age of 2.8 years and an average accuracy of 4.7 years[
]. It must be
stressed though that population, sex and environmental exposure (e.g. smoking, alcohol
consumption) influences DNA methylation[
In addition to the existing methodologic analytical methods mentioned above, Zioupos
] studied numerous physical characteristics of femoral bone at the macro- and
microscopic level and proposed a method that could approximate age with accuracy of +/-1 year.
These characteristics are based on changes in the biomechanical properties of bone and the
properties of the bone matrix, which change with age even after skeletal maturity, and include
traceable features such as the wet and dry apparent density, porosity, organic/mineral/water
fractions, collagen thermal degradation and the osteonal and matrix micro-hardness[22±23].
The authors offered several alternative procedures as well as various combinations of variables
in order to present an accurate method that was versatile enough to be carried out in less than
24 hours, or without the need for expensive equipment. The current paper presents a study
that follows the same methodological approach [
] for a larger sample of ribs. The underlying
hypothesis is that a methodology based on biomechanical properties and biomechanically
related structural characteristics may be more widely applicable and for more skeletal sites. An
alternative pertinent site for forensics is the rib and for that a different set of biomechanical
variables and parameters are needed that suit rib anatomy and physiology. Physical
characteristics of ribs are less influenced by mechanical stress compared to the femur throughout life
[24±25], but more influenced by hormonal and metabolic changes.
After the complete maturation of the skeletal system, the process of remodelling maintains
the structural integrity of the bone, which is constantly subjected to mechanical stress.
According to Martin[
], cyclic loading is the main cause for remodelling. When remodelling does
not succeed in maintaining the integrity of the bone then, for excessive loads or pathological
conditions, there is an accumulation of visible microdamage on the bone surface that results in
a deterioration of mechanical properties[27±28]. Ageing causes local hypermineralisation
patterns, not dissimilar to bio-mineralisation patterns seen in some extreme biological examples
], where the material becomes extremely brittle. In±vivo fatigue microcracks have been
seen to accumulate in such hypermineralised areas. The accumulation of microdamage is
related to cyclic loading, and previous studies have related this phenomenon to physiological
aging[22,30±32] and in ribs in particular[
]. Furthermore, collagen, the main component of
the organic matrix in bone, has been found to play a key part in maintaining the toughness
and the structural integrity of bone and its deterioration has been repeatedly demonstrated
with age[34±36]. Compositional and structural properties of the mineral matrix are also
affected, resulting too in a decrease of the overall mechanical integrity of the tissue[
ribs are convenient to access from the thoracic cage during autopsy, which would increase the
applicability of the method.
This project, therefore, focuses on the analysis of the rib bone matrix to propose a
laboratory-based method that optimises time and resources to produce an accurate age estimation
method that can be easily replicated without any forensic expertise. The small sample suggests
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the potential of this type of analysis in predicting age at death but does not guarantee that the
method would not be affected by other factors as further analysed in the discussion. Due to
the small sample size, this study should be treated as a proof of concept, and upon positive
results, a follow-up study with adequate sample size and reduced biases would be designed and
Material & methods
This study used autopsy material (N = 24) from two forensic departments in Albania and
Greece (Table 1 for details). The sample was divided into two sets of twelve 4th ribs each. The
Greek sample derived from the Dept of Forensic Sciences of the University of Crete and was
composed of 10 males (age 20±68; mean = 41.1/-17.1 yrs) and 2 females (age 22,40; mean =
31/-12.7 yrs). The Albanian specimens came from the Forensic Institute of the Ministry of
Justice in Tirana, Albania and consisted of 8 males (age 30±57; mean = 41.3 +/-10.7 yrs) and 4
females (age 29±58; mean = 45/-13.4 yrs).
Ethical approvals and permits
The study protocol was approved by the Ethics Committee of the School of History, Classics
and Archaeology of the University of Edinburgh (Ethics Assessment Level 2), the Ethics
Cause of death
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Committee of the University Hospital of Heraklion, Crete, Greece (Protocol Number 530) and
the General Prosecution Office of the Ministry of Justice (Protocol Number 1797/3 A. Xh.),
and the Institute of Forensic Medicine (Protocol Number 795) in Tirana, Albania. The study
used fragments of ribs from routine autopsies of individuals for whom their next of kin signed
an informed consent form or cases of unidentified remains for which rib sampling was
conducted for diagnostic purposes using standard histomorphometric techniques with permits
from the relevant judicial authorities. All methods were carried out in accordance with the
approved guidelines and the appropriate standards applying in the medicolegal context.
The samples were shipped in a polyesterene box with dry ice in order to preserve their native
]. Each was ~5 cm in length and was taken from the straightest portion of the
shaft. For the entire preparation and experimental examination the specimens were stored in
labelled airtight plastic bags at -20ÊC. The soft tissue on the bone was precisely removed using
a disposable surgical knife and making sure to completely eliminate the periosteum without
compromising the integrity of the. A thin transverse session (~5 mm) from each specimen was
obtained using a Struers1 Accutom wafering saw equipped with diamond impregnated blade
(300 μm) and was cooled down using deionised water. The same machine was employed to
divide the rest of the bone into two halves. When curvature or thickness did not allow for the
procedure to be completed as stated, a Dremel1 3000 drill equipped with an abrasive cutting
disk was used under continuous irrigation with deionised water in order to produce the thin
The transverse sections were stained in plastic vials on a spinning mixer with a solution of
Basic Fuchsin and Ethanol 70% for 14 days, with the solution replaced after seven days. The
rest of the tissue was immersed in two baths of Ethanol 100% and 50%, respectively for three
hours and then was left spinning overnight in deionised water. The stained sections were dried
completely at room temperature (~12 hrs) and then embedded in epoxy resin (Metprep
KleerSet Type SSS) to make the histological surface visible. After 24 hours the resin blocks were
metallographically polished using an automatic Struers RotoPol-15 with 203 mm silicon
carbide abrasive disks grinding paper of decreasing grit size (400, 800, 1200, 2500) on a MasterTex
cloth with Alumina 3B 6OZ.
Nanoindentation was performed using a CSM-NHT (system v.3.75, CSM, 2034 Peseux,
Switzerland) instrument, at 10 mN maximum hold load (20 mN/min loading/unloading speed)
with 30s long load/hold/unload stages. For each specimen, six indentations were performed on
each of three different secondary osteons and in three surrounding interstitial bone areas
nearby (see Fig 1). Each osteon was chosen from different sectors of the bone (pleural surface,
cutaneous surface and one of the two edges chosen according with the regularity of the
surface). Universal Hardness (H in MPa) was calculated from load and contact area, Elastic
Modulus (EIT in GPa) was obtained (assuming a Poisson's ratio value of ν = 0.3) in the
unloading phase as per the Oliver and Pharr method[
]. Indentation creep (CIT in %) was
calculated by the proportional increase in depth occurring while the load is held at its maximum
level (for 30s) and its measurement reflects the viscoelasticity of the tissue. The elastic portion
(nIT in %) of the indentation work was obtained by examining the percentage ratio of the
elastically recovered energy over the total energy (elastic + plastic) input during performing an
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Fig 1. Detail of cortical bone matrix showing micro- and nano-indentations in an interstitial bone area.
indentation sequence. During the test, attention was taken in avoiding factors that create
`experimental noise', such as levelling the sample to allow the indenter to penetrate at right
angles, minimising the external vibrations and discarding any asymmetric or problematic
]. An INDENTEC HWDM-7 instrument, equipped with a square-shaped pyramid
diamond tip of θ = 136Ê, was employed to produce Vickers microhardness (HV in Vickers)
values for osteonal and interstitial bone areas for each specimen. The maximum load in these
tests was set at 50 mN.
Optical porosity (%Po.Ar in %) was obtained from three pictures (respectively from cutaneous
surface, pleural surface and from one of the two edges) taken for each specimen with a
confocal transmitted light microscope 50× and the use of ImageJ RBS. The picture was cropped to
select areas completely occupied by bone, then converted into 16-bit and a threshold mask was
applied to highlight the voids in the tissue (Fig 2). If any of the osteonal canals was not stained
properly a correction was made manually. The volume fraction was calculated using the open
source software BoneJ and was then transformed into a percentage value. The three values for
each individual were averaged in order to obtain a unique measurement for porosity.
Osteocytic lacunae were included when automatically selected by the software.
A NIKON A1R with a 10× Plan Fluor/NA 0.3 objective was used to produce the numerical
density (Cr.Dn in nÊ/mm2) and surface density (Cr.S.Dn in mm/mm2) of in-vivo microcracks.
Three pictures were taken on the cutaneous surface, pleural surface and on one of the two
edges. For the numerical density, the area of the bone was calculated without removing the
porosity. Cracks were identified and counted for the three areas added up and the value
divided by the total area inspected. The same procedure was followed for surface density with
the exception that length of each crack was recorded in order to calculate the total length and
the value was divided by the total surface area of bone examined. In-vivo microcracks were
labelled using basic Fuchsin fluorescent sodium salt (in water) solutions. In order to accurately
distinguish between genuine cracks and artefacts, the following features were deemed
necessary to be present: (1) they must have sharp edges and must be stained all around their
length and width; (2) changing the depth focus the halo of Fuchsin and Fluorescein dyes must
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Fig 2. Pictures of a cortical bone section after conversion into 16-bit and the application of the
threshold mask with ImageJ.
penetrate the depth of the crack under visible light (predominantly for Fuchsin) and UV light
for Fluorescein; (3) genuine damage cracks ought to be larger than canaliculi but smaller than
the vascular canal[
]. Consequently, cracks stained and verified by both Fuchsin and
Fluorescein dyes (Fig 3) were counted and measured. Quantitative measurements were taken using
Fiji open source software.
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Rib fragments (~20 mg) were dried off completely at room temperature for 24 hours and then
tested using a helium pycnometer (Mycrometric Accupyc 1330). Their weight was recorded
using an electronic balance (Mettler Toledo1 College B154) in order to obtain the first density
measurement (Dnpyc in g/cm3).
Fig 3. Examples of verified in-vivo damage micro-cracks (white arrows) visualised in a fluorescence microscope.
8 / 20
Thermal stability of bone collagen
The remaining bone was dried out completely at room temperature and in order to powder it
was processed using a Retsch Mixer miller 2000 by cycling for 1 minute and at 60 Hz. In
between the two different cycles the powder was filtered using a 106 μm sieve in order to
obtain a fine and homogeneous sample. The powder was stored at -20ÊC and was left resting at
room temperature the night before the test. Differential scanning calorimetry (DSC1 Mettler
Toledo1, Indium calibrated) was used to assess thermal stability degeneration of bone
collagen. Twenty mL aluminium pans with flat bases were filled with ~10 mg of powder and the
weight was recorded using a microbalance (Sartorius Genius ME235), while an empty crucible
was used as a reference. The experiment was performed by steadily increasing the temperature
from 25ÊC to 550ÊC at a rate of 10ÊC/min. The output curve was normalised and analysed
with Stare V 10.00 software. This showed a first endothermic peak between 50ÊC and 120ÊC,
which relates to the well-known collagen thermal shrinkage phenomenon[
] and a second
exothermic peak between 200ÊC and 500ÊC that results from the combustion of the organic
matrix[40±41]. For both episodes, enthalpy between a fixed temperature range, 30ÊC to 140ÊC
and 200ÊC to 540ÊC, was calculated through integration and called respectively LΔH and
CΔH. Furthermore, for each episode onset (LOnset and COnset), peak (LPeak and CPeak)
and endset (LEndset and CEndset) was recorded. Finally, through a derivative the point of
maximum steepness was detected as symptomatic of the collagen stability threshold
(DerPeak1, DerPeak2) and for the combustion of the collagenous matrix (DerPeak3) [
Gravimetric analysis (TGA 50 Mettler Toledo1, Curie Point calibrated) was applied to a
sample of bone powder in the same conditions. A slightly bigger crucible (40 mL) was filled with
~20 mg of milled bone and the test was performed as previously described with a hold of the
temperature at 550ÊC for the 10 final minutes in order to obtain ash weight. Quantitative
investigation of the two main (percentage) weight losses with respect to final weight was
recorded (Ash%) and that was carried out in Stare V 10.00 software by using horizontal
tangents on the normalised curve. The first episode is believed to correspond to the complete
dehydration of the bone (W%) while the second represents the combustion of the organic
Statistical significance was set at P 0.05 for both correlation values between the 23 parameters
which we empirically measured and for the multifactorial regression analysis to predict
biological age of the sample. One-way ANOVA was used to check for sex or ethnicity differences and
once it was established that there were none the whole cohort of 24 donors was used for the
subsequent analysis. The analysis consisted of producing a number of equations, through
stepwise regressions, with consideration taken for the required degree of accuracy, time to
complete the round of tests and resource availability. The entire statistical analysis was performed
in Minitab v.17 and SPSS v.22.
A total of 28 physical parameters of interstitial bone (In) and osteons (On) were tested in ribs
from 24 donors. Table 2 shows a list of the parameters, abbreviations, units, descriptive
statistics and a list of experimental methods for data acquisition.
9 / 20
TLM = Transmitted Light Microscope, DSC = Differential Scanning Calorimeter, TGA = Thermo-Gravimetric Analysis, ImageJ = Image processing software.
Signi®cant (p<0.05) single correlations with age are shown with a * and in bold symbols.
Six parameters (%Po.Ar, OnH, Cr.Dn, Cr.S.Dn, Lpeak and DerPeak3) correlated singularly
and significantly with age. The third derivative peak (DerPeak3) decreased, indicating a clear
degeneration of the quality of the organic bone matrix and its amount in the bone tissue. The
nanohardness of the osteonal area (OnH) also decreased with age and showed a strong
correlation (p = 0.029) and this change is linked in literature to the effect of age on bone plasticity
]. Finally, optical porosity (%Po.Ar) drastically increased as a consequence of the
physiological loss in bone mass that occurs throughout life. Although the study did not aim to
investigate this relationship and did not perform any micromechanical test, no significant
correlations were seen between numerical or crack length density and the nanoindentation
parameters with age.
Significant positive correlation was observed between porosity and the percentage of
organic loss during TGA analysis (Or%). The relationship between the parameters of
differential scanning calorimetry (DSC) and those of the gravimetric analysis (TGA) denote a clear
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correlation between collagen integrity and amount of organic matrix present in the bone,
which is in agreement with other studies. Finally, the microhardness of the interstitial
bone areas (InHV) appeared to be affected by the amount of organic matrix of the tissue that is
present, which is expected.
Bone physicochemical characteristics not only have a direct influence on the mechanical
integrity of the bone tissue but also carry useful information in order to investigate chronological
age of human bone material. As shown in Table 2, only six parameters demonstrated
significant correlations with age when singularly considered. This could be due to the limited
variation of each parameter and its variance of the mean with regard to age for each individual
]. Multifactorial stepwise analysis exhibited the predictive potential of different
combinations of factors, as shown in Table 3. Different sets of variables were considered with
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Fig 4. Plot of real age vs. predicted age for E1-E6. (Regression line with the 95% prediction interval for the data).
12 / 20
forensic application and practice in mind and also bootstrapping methods were employed
based on 1000 bootstraps.
Unrestricted parameter selection
Firstly, equations were sought by considering all 28 available parameters. In forensic terms this
would be a situation in which time and resources were unlimited and the final goal was to
reach the maximum accuracy and reliability so as to meet the increasing standards required
for the admission of expert witness testimony. The best performing model by stepwise
regression was Equation 1 (E1, Table 2) with a R2 = 0.949 (Radj2 adjusted for the degrees of freedom
0.927) showing good general accuracy; the maximum residual error of 2.88 years was observed
for a 58-year-old individual (A8). The average residual error is 2.14±0.40 (SD). When
bootstrapping is applied, the error becomes 2.77±1.43 (SD). E1 was created without taking into
consideration time or resources availability; in fact, it required a two-week long preparation
and the use of DSC, nanoindentation and fluorescence microscopy. E2 is the next stepwise
regression and is based on just three variables (%Por.Ar, ItCIT and Cr.Dn) produced by using
the same instruments. The model shows Radj2 0.912. The average residual error after
bootstrapping is 2.75±1.47 (SD) and all coefficients present statistical significance.
Restricted parameter selection
In further analysis we applied stepwise regression by using two specific subsets (combinations)
of parameters in order to address practical problems that can occur in forensic context, such as
limitations in time, technical resources, or available bone material. Equation 3 was derived by
using the parameters produced by a DSC instrument, a Nanoindenter and the open source
software ImageJ. The result was an equation (R2 = 0.902, Radj2 = 0.875) which was produced
entirely by using bench top machines, and the entire experimental analysis can be conducted
in 36 hours. For this predictive equation the residual errors ranged between 0.30 and 9.26
years. Certain other combinations were: E4 with four variables (%Po.Ar, Der Peak3, LPeak
and CEndset) an R2 = 0.848 (Radj2 = 0.816); and E5 which involves the use of just one instrument
a nanoindenter equipped with a microscope. E5 uses optical porosity (%Po.Ar) and the
indentation creep (InCIT) values for the interstitial bone areas with R2 = 0.820 (Radj2 = 0.804).
Although it requires just one instrument the preparation time may not be rapid enough for
use in urgent legal cases. Finally, E6 was produced which requires the use of helium
pycnometer and optical porosity (%Po.Ar). E6 could potentially be produced within 24 hours from the
collection of samples. All six equations are shown in Fig 4.
The previous models consider the whole cohort of samples and provide in essence the
maximum possible prediction power of the approach we have implemented. In reality any
unknown sample will be other than the samples which produced the calibration relationship. To
simulate this, we applied a leave-one-out method where it turns we kept one sample out and
produced the analysis from the other 23 samples. This was done 24 times x 6 predictive
equations (6 sets of parameters). As expected cross-validation reduced the accuracy for E1 now
having a mean average error of 4.41±2.58(SD) with errors in the range [0.28±9.42]yrs and highest
error for a 39-year-old individual (Table 4).
Fig 5 shows the performance of E1 for the cross-validation study. The least performing E6,
in cross-validation, showed mean residual error of 6.08 +/- 4.54 (SD) and the `worst' single
result (a residual age error of 20 years) was noted for a 52-year-old individual (C13) who
suffered from hypertension.
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<RE>: mean absolute error of the estimation; SD: standard deviation
Bone matrix undergoes de-/re-generation throughout life. Some changes such as bone mass
loss and increase in porosity are easily quantifiable. Otherchanges that are of a more qualitative
nature are caused by physicochemical alterations that affect both the mineral and the organic
]. All of these modifications can be quantified to a greater or lesser degree and can be
correlated with the age of an individual. However, the rate of change decreases and is more
difficult to detect in individuals who have already reached skeletal maturity (over 35 years old)
]. It is believed that ribs are far less prone to remodelling from biomechanical stress
compared to the femur[24±25, 42±43]; hence, they are the target of many histomorphometric
studies for age-at-death estimation[
]. It is, however, well acknowledged that ribs are
metabolically active and subject to many hormonal changes which also affect remodelling rates
]. A few studies have focused on the material properties of the ribs in relation to age and
fracture risk[44±46]. To date though, there is not a lot of information about nano-mechanical
properties of the rib in relation to age. The current work attempts to explore the value of
nanomaterial properties of the rib in forensic age estimation.
According to our results, several parameters exhibited significant correlations (P<0.001)
with age thus making them potentially efficient age predictors. In addition, age estimates were
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Fig 5. Plot of real age vs. predicted age for E1 in the cross-validation. Line of 1:1 equality with the 95%
prediction interval for the data. Residual errors are shown on the x-axis for each donor (+ for overestimation;Ð
for underestimated values).
fairly accurate for all age ranges, which is a significant advantage as compared to other age
estimation methods based on ribs[4±5,8,11]. Lastly, preliminary observations suggest no influence
of sex and population, which would make it ideal for global applicability. It is clear that further
investigation should employ a large and well balanced sample in terms of age, sex and
population to have a better understanding of the effect of these demographic features on the
assessment and obtain a substantial understanding of the applicability in forensic setting.
In the present approach we employed a range of biomechanical and histomorphometric
techniques that were listed in the methodology recently proposed by Zioupos et al.[
addition, we also employed a microscopic examination of the bone matrix for the presence of
in-vivo fatigue microdamage, a well-known detectable feature for ribs [
]. It is commonly
accepted that the cyclic loading, which bone undergoes throughout life, results in the
formation of microcracks at the microscopic level on both cortical and trabecular bone. What has
been investigated here is the effect of cyclic loading on the mechanical quality of the cortical
bone of the 4th rib and its quantitative changes with age. Decreases in toughness, strength and
stiffness have been conclusively shown to correlate with the accumulation of micro-damage in
the bone matrix[
]. Other physicochemical properties (such as mineral content,
porosity and bone density) also relate to the accumulation of these cracks either as a cause or
as an effect.
This study proposed six different regression equations for age estimation in the forensic
context, according to the available time, specialised equipment and expertise. The best equation in
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Rib end morphology
Rib end morphology
Racemization of aspartic acid from rib cartilage
Ossi®cation of the ®rst rib through radiographs
R2: coef®cient of determination; <RE>: mean absolute residual error; SDE: standard deviation of absolute residual errors.
terms of accuracy was found to be E1, which was produced from the entire heterogeneous
cohort of parameters and presented a coefficient of determination R2 = 0.949; the maximum
observed residual (error) was 5.15 years for a 58-year-old individual. When time and resources
are limited, we recommend the use of E5, which can be completed in only 24 hours with the use
of a nanoindenter and the open source software ImageJ. E5 had a coefficient of determination
R2 = 0.830 and exhibited average residual error of 4.70±0.33 (SD), with a maximum of 12.57
years in one case. To put this into the forensic context and practice we have collated information
for other laboratory-based age estimation methods[6,7,13,48±53] and age estimation methods
based on the rib[4,5,8,11,54±57] from the literature including the present one. Comparing R2,
which is the proportion of the variance in age that is explained by the regression model in each
case and the mean residual error, E1 ranks third (see Table 5), making this, to our knowledge,
the most accurate laboratory-based age estimation methods for ribs.
Despite the potential of the present study in assessing age at death, it remains a preliminary
study and there are naturally limitations which need to be discussed. The sample comprised of
individuals from two countries with similar dietary habits and customs. our results showed no
effect of population specificity as the difference between the two population is negligible to
obtain final results. Nevertheless, the composition of the sample did not allow us to explore
potential sex-related differences or the effect of systematic pathologies affecting bone
metabolism. Follow up research will investigate a larger and more balanced sample size in terms of
sex, population and age ranges in order to reduce noise, taking into consideration dietary
habits and metabolic diseases affecting bone, such as osteoporosis, that may interfere with the
calibration of the method. Intra- and inter-observer error must be quantified in order to achieve
standardisation of the technique, especially in relation to histomorphometric analysis.
Furthermore, in order to understand better the relationship between microscopic and macroscopic
structures in relation to the mechanical behaviour of bone, dynamic mechanical analysis
16 / 20
(DMA) could also be tested. This can be performed by a bench top instrument and can be run
efficiently for specimens of small size and mass. DMA would also provide variables that relates
to the viscoelastic nature of bone[
] potentially adding to number of parameters used to
predict age from skeletal remains. Finally, the post-mortem interval and diagenesis may play an
important role in affecting bone quality (most likely through the organic matrix), and could
put temporal limitations on the application of the present method. Restriction in time and
resources availability did not allow for further investigation into this aspect, and remains a
central issue that needs to be addressed.
This study introduces a profoundly novel lab-based analytical method of `age at death'
estimation of skeletonised remains from the bone matrix properties of the human rib. For the
development of the procedure we analysed the trends with age of as many as 28 biomechanical and
material features of the human rib and created a mathematical model for age estimation that
outstrips all previous published methods, whether these were phenomenological or analytical
at the macroscopic or the microscopic level, or even chemical methods based on the analysis of
proteins in soft and hard tissue. More importantly this approach can be easily replicated
without need for the usual person centred high skilled forensic expertise. Its potential applicability
ranges from unidentified skeletonised bodies to multiple victims of mass disasters or mass
graves that are lacking identification and for which an accurate biological profile needs to be
established. This makes the method relevant to chemists, biologists and medical experts that
specialise in the field of forensics but it can also be relevant to any judicial personnel that deals
with reliability and evidence admittance in the court of law. The present application was
optimised for fresh (uncompromised by taphonomic conditions) remains, but the potential of the
method is vast once the trends of the biomechanical variables are established for other
environmental conditions and circumstances.
The authors would like to thank Professor Manolis Michalodimitrakis and the Ethical
Committee of the University Hospital in Heraklion, Crete, Greece for providing permission for the
Greek samples and Dr Despoina Nathena and Mr. Stratos Kougios for collecting the samples
and providing the demographic information. The authors are grateful to Mr. Kostaq Beluri,
Head of Control Department Investigation and Prosecution of the General Prosecutor,
Ministry of Justice, Tirana, Albania for granting permission to carry out the project. Special thanks
to Mara Karell for undertaking the linguistic review.
Conceptualization: AB EFK PZ.
Data curation: EFK PZ.
Formal analysis: EFK PZ.
Funding acquisition: PZ.
Investigation: AB BX EFK PZ.
Methodology: AB PZ.
Project administration: PZ.
17 / 20
Software: AB PZ.
Supervision: EFK PZ.
Writing ± review & editing: AB BX EFK PZ.
18 / 20
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