ORIGINAL ARTICLE

Skeletal age estimation based on medial clavicle—a testof the method reliability

Petar Milenkovic & Ksenija Djukic & Danijela Djonic &

Petar Milovanovic & Marija Djuric

Received: 4 July 2012 /Accepted: 25 October 2012 /Published online: 18 January 2013# Springer-Verlag Berlin Heidelberg 2013

Abstract In order to establish a reliable age indicator in theperiod when all other epiphyseal age indicators have alreadybeen inactivated, medial clavicle as the bone with the lon-gest period of growth became the object of various inves-tigations. However, the lack of population-specific methodoften made it unreliable in some regions. The current studyinvolved a Balkan population and it was designed in order toexamine whether morphological, radiological, and histolog-ical analyses of medial clavicles could be applied withsuccess in age assessment of individuals beyond theirtwenties in anthropological and forensic practice. The me-dial clavicular specimens were collected from contemporarySerbian population, autopsied in the period from 1998 to2001, encompassing 67 individuals (42 males and 25females) with the age range from 20 to 90 years. Theconducted analyses of morphological features identifiedthe epiphyseal union timing, signs of lipping in the regionof the notch for the first rib as well as exostoses and boneovergrowths of the articular surface margin as age-dependent attributes. Trabecular bone volume fraction andminimum trabecular width were also highlighted as age-distinctive microscopic features. Sex difference was ascer-tainable in epiphyseal union timing, morphology of thenotch for the first rib, margin of the articular surface, andbasic morphology of articular surface as well as in two

microscopic characteristics: trabecular bone volume fractionand minimum trabecular width. The study managed to iden-tify several age- and sex-related features that could be ap-plied as additional guidance for age estimation in Serbianpopulation.

Keywords Medial clavicle . Morphology . Histology . Sexdependence . Age assessment

Introduction

Age estimation represents one of the main objects in the fieldof forensic practice and physical anthropology. Throughoutthe literature, scholars outlined different skeletal markers thatcould be considered age specific. The thoroughly conductedmacroscopic analyses of skeletons led to the development ofseveral discerning age-related patterns mostly emphasizingmorphological changes observed in cartilaginous joints inthe body mid-line such as pubic symphyses and sternal endof the ribs [1–3]. Nevertheless, the histology-based methodswere also conducted encompassing the specimens from thesame body region [4, 5]. Thus, the sternal clavicular endrepresented an interesting object of numerous investigations.Even more, clavicles used to be considered as the most reli-able specimen for age estimation in radiological studies [6].

Contrary to pubic symphysis, clavicles could not be put in acorrelation either with pregnancy or weight bearing; they areinvolved in locomotion just occasionally. The medial clavic-ular epiphysis is also the last to fuse among all long bones[7–9]. Therefore, with the longest period of growth the medialclavicle retains particular opportunity to become reliable ageindicator in the period when all other epiphyseal age indica-tors have already been inactivated [10]. In addition, its posi-tion in human body makes it accessible for examinations inliving individuals as well as in cadaveric material.

P. Milenkovic :K. Djukic :D. Djonic : P. Milovanovic :M. Djuric (*)School of Medicine, Institute of Anatomy,Laboratory for Anthropology, University of Belgrade,4/2 Dr Subotica,11000 Belgrade, Serbiae-mail: marijadjuric5@gmail.com

P. MilenkovicInstitute for Oncology and Radiology of Serbia,Pasterova 14,11000 Belgrade, Serbia

Int J Legal Med (2013) 127:667–676DOI 10.1007/s00414-012-0791-6

The recent requirement of post mortem age determinationwas accompanied by a growing demand for age estimationof living individuals in regard to legal relevance of genuineage in criminal, civil, and asylum proceedings. The recom-mended process of age assessment of young individualsinvolves physical and dental examinations with radiograph-ic analyses of dentition and left hand [11–15]. The sameauthors considered additional examination of the medialclavicle as the most appropriate. The criteria were basedon an official publication adopted in 2000 by the membersof the Study Group on Forensic Age Diagnostics of theGerman Association of Forensic Medicine—Arbeitsge-meinschaft für Forensische Altersdiagnostik, AGFAD [11].Thus, the most of this methodology was based on determi-nation of defining features for age legality boundary of21 years [12, 16, 17]. Furthermore, development of theexisting and new methods was induced [4, 18–20].

The influence of socioeconomic conditions on skeletalepiphyseal union phases was recently emphasized as moreimportant than interethnic variation [8, 21–23]. The years ofrecent war in the Balkans with subsequent process of foren-sic identification outlined the importance of developingreliable population-specific age estimation methodologyfor this region [24–26]. Moreover, criminal and asylumissues in Europe not so rarely enroll individuals from Bal-kan populations [12].

The broad range of macroscopic, histomorphometric, andradiological analyses were conducted in order to establishthe reliable age range for the phases of clavicular epiphysealfusion. However, those methods were successfully testedmainly in individuals under 35 years of age [12, 16, 17,27, 28]. It remains unclear whether various types of analysesof the medial clavicle can be beneficial for discriminatingbetween different age categories in adults. Hence, this studyin a Balkan population has been carried out with the aim toexamine whether morphological, radiological, and histolog-ical analyses of medial clavicles could be applied withsuccess in age assessment of individuals beyond theirtwenties in anthropological and forensic practice.

Materials and methods

The medial clavicular specimens were collected from acontemporary Serbian population; their socioeconomic sta-tus was considered in boundaries of upper-middle-incomecountry [29, 30]. All encompassed individuals were autop-sied in the period from 1998 to 2001 at the Institute ofForensic Medicine in Belgrade. The data about sex andage of the individuals were provided from the death certif-icates. The study sample encompassed 67 individuals (42males and 25 females). The age at death ranged from 20 to90 years and it was distributed in five age groups (Table 1).

As previous studies did not report statistically significantside differences in attaining complete union of medialclavicles, the present study disregarded specimen lateraliza-tion [10, 17, 31].

After the medial clavicles were obtained from the cadav-ers, the adherent soft tissues were carefully removed with ablunt scalpel, followed by 2-week storage in 3 % hydrogenperoxide solution for defatination.

The exclusion criteria encompassed the presence of anyvisible deformity, signs of pathological conditions, or frac-ture of the clavicle. Each specimen was subject to analysesof macroscopic morphological features of the articular sur-face and radiographic appearance.

During the assessment of macroscopic morphologicalfeatures (Fig. 1), the progress of epiphyseal union wasrecorded by applying a three-phase scoring system [25,32]. A “non-union” phase was assigned in cases withouttraces of fusion (Fig. 1a). The second one, marked as “par-tial union” phase, was recorded if the signs of active unionwere observed followed by an epiphyseal line in the processof obliteration (Fig. 1b), often creating a characteristic pic-ture of “epiphyseal flake” (Fig. 1c). The final phase (“com-plete union”) was recognized by obliterated line of fusion(Fig. 1d), although signs of an epiphyseal scar might still bevisible.

Further macroscopic analyses of the specimens involvedobservation of the following morphological features:

1. Basic morphology of the articular surface (convex,plane, concave) (Fig. 1d, i, j)

2. Relief of the articular surface (smooth, rugged) (Fig. 1e,f)

3. Porosity of the articular surface (not present, present)(Fig. 1e, g)

4. Shape of the articular surface (triangular, round, oval)(Fig. 1e, f, g)

5. Presence of ossific nodule (not present, present)(Fig. 1g)

6. Morphology of the margin (obtuse, sharp, with lipping,with exostoses and bone overgrowths) (Fig. 1d, j, l)

Table 1 Age and sex distribution of individuals within the sample

Age categories Whole sample Males Females

n % n % n %

25 and younger 12 17.9 10 23.8 2 8.0

26–35 15 22.4 11 26.2 4 16.0

36–45 11 16.4 5 11.9 6 24.0

46–55 13 19.4 7 16.7 6 24.0

56 and older 16 23.9 9 21.4 7 28.0

Total 67 100.0 42 100 25 100.0

668 Int J Legal Med (2013) 127:667–676

7. Morphology of the notch for the first rib (without out-standing margins, presence of outstanding margins, withlipping) (Fig. 1i, j, k).

The process of assessment of macroscopic morphologicalfeatures involved two experienced investigators who inde-pendently examined the study sample without knowingactual individual age.

After the adequate orientation and positioning, the radio-graphs (Fig. 2c, d) of each specimen were made by standardradiographic unit SELDIX 550 at 52 kV, 3.2 mAs, and 1-sexposure time, in frontal projection. The observationsencompassed the following features: thickness of the ante-rior and posterior cortex, translucency and architecture ofsternal metaphyses as well as enlargement of the medullarcanal [33].

After completing macroscopic morphological and radio-logical observations, histological analyses were performedon 11 male and 11 age-matched female specimens. Theselected male individuals were aged from 21 to 89 years,averagely 50.18 years (SD 20.92). Similarly, the female partof the sample which was subjected to histological analyseswas in age range between 23 and 89 years, averagely49.55 years (SD 20.16). The specimens were embedded in

epoxide resins (Mecaprex KM-U) and cut using a water-cooled diamond-saw microtome (Leica SP1600) to 70-μmcross-sections at the level positioned 5 mm from the loweredge of the articular surface. Subsequently, the images ofthree low-power fields of each prepared specimen wereacquired by a Leica camera using a Leica polarizing micro-scope. The fields were chosen randomly since field-to-fieldindividual histological variation in the clavicle’s sectionswas less expressed than in other long bones, as reportedpreviously [34–37]. All of the obtained images (Fig. 2a, b)were analyzed using KVI-POPOVAC V 2.2 software. Theimaging system was calibrated by measuring a 1-mm divi-sion scale. The following bone histomorphometric parame-ters were calculated: cortical width (Ct.Wi), cancellous bonearea/tissue area (Cn.B.Ar/T.Ar), and minimum trabecularwidth (Tr.Wi), in accordance with the standards of theAmerican Society for Bone and Mineral Research [38].The bone area and the tissue area were detected in the imageprocessing software. The cortical width and minimum tra-becular width were measured at ten randomly determinedsites, and average values for all acquired measurementswere calculated for each individual.

Statistical analyses were performed using SPSS for Win-dows, version 15. The Kolmogorov–Smirnov test was used

Fig. 1 Analyzed macroscopic morphological features: a “Non-union”phase. b “Partial union” phase. c “Partial union” phase with character-istic picture of “epiphyseal flake”. d “Complete union” phase withvisible convex articular surface and obtuse margins. e Smooth relief,triangular shape with no signs of porosity of the articular surface. fRugged relief and round shape of the articular surface. g Oval shape ofthe articular surface with present ossific nodule. h Round shape with

present porosity of the articular surface. i Plane articular surface with-out outstanding margins in the region of the notch for the first rib. jConcave articular surface with outstanding margins in the region of thenotch for the first rib and sharp margins of the articular surface. kLipping in the region of the notch for the first rib. l Margins of thearticular surface with exostoses and bone overgrowths

Int J Legal Med (2013) 127:667–676 669

to assess the normality of data distribution. Pearson’s chi-square test was used for comparing macroscopic featuresbetween different age groups, while Mann–Whitney U testwas applied in order to identify discrepancy of epiphysealunion phases between male and female populations.Cohen’s kappa test was used for estimation of interobserverreliability of observed macroscopic features, following theinstructions by Landis and Koch [39]. The relation betweenposterior cortex thickness and age was tested by Spearmancorrelation during analyses of radiological features. Linearregression analysis was used to determine the relation be-tween age and histomorphometric parameters as well aswith anterior cortex thickness and medullar canal enlarge-ment observed on obtained radiographs. Student’s t test wasused to estimate the differences of histomorphometric databetween the sexes. In all analyses, the significance level wasset at 0.05.

Results

Macroscopic morphology

The macroscopic morphological analysis of epiphysealunion (Table 2) determined “non-union” phase in only twosubjects aged 20 and 22 years. Conversely, the features of“partial union” were recorded in all age groups, but pre-dominantly in individuals of first two categories. The youn-gest individual with this phase was 21 years old, while it hadalso been detected in a 65-year-old individual. Morpholog-ical characteristics of “complete union” were noticed in themajority of individuals older than 36 years. The earliestdetection was at the age of 31. Statistical analysis confirmed

significant difference in stage of epiphyseal union betweendifferent age groups, either in the whole sample (p<0.001)or separately the males (p<0.001) and females (p00.001).Furthermore, females showed noticeable tendency towardshigher epiphyseal union stages than the males of the sameage (p00.001).

The significant difference in morphology of the notch forthe first rib (p00.002) and the margin of the articular surface(p00.001) were verified between age groups within thewhole sample. Namely, morphological analyses alloweddetection of lipping in the region of the notch for the firstrib (Table 3) in individuals above 44 years of age, while theabsence and presence of the signs of outstanding margins inthe same region were recorded at a much younger age (21and 22 years, respectively). The youngest individual withobserved exostoses and bone overgrowths of the articularsurface margin (Table 4) was 53 years old. On the contrary,

Fig. 2 Images of low-powerfields of histological cross-sections in young (a) and old(b) individuals. The fine-grained trabecular architectureobserved on radiographs ofyounger individuals (c), and thepresence of coarse trabecularpattern on radiographs of olderindividuals (d)

Table 2 Distribution of individuals by the age category and theprogress of epiphyseal union

Age categories Non-unionphase

Partial unionphase

Complete unionphase

n % n % n %

25 and younger 2 16.7 10 83.3 0 0

26–35a 0 0 12 85.7 2 14.3

36–45 0 0 3 27.3 8 72.7

46–55 0 0 1 7.7 12 92.3

56 and older 0 0 1 6.3 15 93.8

Total 2 3.0 27 40.9 37 56.1

a The damage of the medial clavicular end excluded one individualfrom this analysis

670 Int J Legal Med (2013) 127:667–676

Tab

le3

Distributionof

analyzed

morph

olog

ical

changesof

articular

surfaceby

theagecatego

ry(partI)

Age

catego

ries

Morph

olog

yof

theno

tchforthefirstrib

Bon

eov

ergrow

ths

With

outou

tstand

ingmargins

Presenceof

outstand

ingmargins

Lipping

MF

M&

FM

FM

&F

MF

M&

FM

FM

&F

n%

n%

n%

n%

n%

n%

n%

n%

n%

n%

n%

n%

25andyo

ungera,b

00

00

00

512

.82

8.0

777

.82

5.1

00

222

.20

00

00

0

26–35

00

00

00

820

.51

4.0

960

.03

7.7

312

.06

40.0

00

00

00

36–45

00

00

00

410

.33

12.0

763

.61

2.6

28.0

327

.30

01

4.0

19.1

46–55

00

00

00

717

.91

4.0

861

.50

04

16.0

430

.80

01

4.0

17.7

56andolder

00

00

00

25.1

00

212

.52

5.1

416

.06

37.5

512

.83

12.0

850

.0

Total

00

00

00

2666

.77

28.0

3351

.68

20.5

1352

.021

32.8

512

.85

20.0

1015

.6

Mmale,Ffemale

aBothindividu

alswith

non-un

ionepiphy

seal

stagewereexclud

edfrom

furthermorph

olog

ical

observations

bOne

individu

alwas

exclud

edfrom

analyses

ofmorph

olog

yof

theno

tchforthefirstribdu

eto

damaged

medialclavicular

end

Tab

le4

Distributionof

analyzed

morph

olog

ical

changesof

articular

surfaceby

theagecatego

ry(partII)

Age

catego

ries

Morph

olog

yof

themargin

Obtuse

Sharp

Lipping

Exo

stoses

MF

M&

FM

FM

&F

MF

M&

FM

FM

&F

n%

n%

n%

n%

n%

n%

n%

n%

n%

n%

n%

n%

25andyo

ungera

00

29.1

220

.08

20.0

00

880

.00

00

00

00

00

00

0

26–35

b1

2.5

29.1

321

.410

25.0

14.5

1178

.60

00

00

00

00

00

0

36–45

25.0

313

.65

45.5

37.5

313

.66

54.5

00

00

00

00

00

00

46–55

410

.05

22.7

969

.23

7.5

00

323

.10

00

00

00

01

4.5

17.7

56andolderc

820

.05

22.7

1392

.90

00

00

00

00

00

01

2.5

00

17.1

Total

1537

.517

77.3

3251

.624

60.0

418

.228

45.2

00

00

00

12.5

14.5

23.2

Mmale,Ffemale

aBothindividu

alswith

non-un

ionepiphy

seal

stagewereexclud

edfrom

furthermorph

olog

ical

observations

bOne

individu

alwas

exclud

edfrom

analyses

ofthemarginmorph

olog

ydu

eto

damaged

medialclavicular

end

cTwoindividu

alswereexclud

edfrom

analyses

ofthemarginmorph

olog

ydu

eto

damaged

medialclavicular

end

Int J Legal Med (2013) 127:667–676 671

the earliest signs of obtuse and sharp clavicular marginswere recorded at the age of 21. However, these agedifferences were significant only in males (p00.004and p00.001, respectively). In contrast, significant dif-ferences in basic morphology of the articular surface(Table 5) were evident between different age groupsonly in females (p00.010).

The analysis of other features (relief, porosity, and shapeof the articular surface, and presence of ossific nodule)showed no relationship (p>0.05) to age groups neitherwithin the whole sample nor males and females separately(Tables 5 and 6).

The applied Cohen’s kappa test estimated interobserv-er agreement in the range from fair to almost perfectconcerning observed macroscopic features. Thus, thefair agreement was measured only in examination ofpresence of ossific nodule (kappa00.367; p<0.001).The moderate level was calculated the most frequentlyand it was recorded in analyses of following features:basic morphology (kappa00.510; p<0.001) and relief ofthe articular surface (kappa00.445; p<0.001), morphol-ogy of the margin (kappa00.422; p<0.001), and mor-phology of the notch for the first rib (kappa00.559; p<0.001). The substantial agreement was present in obser-vations of shape of the articular surface (kappa00.700;p<0.001). The almost perfect agreement was estimatedin analyses of epiphyseal union progress (kappa00.851;p<0.001) and porosity of the articular surface (kappa00.877; p<0.001).

Radiology

The most prominent variety observed on the radiographsof the clavicles was general increase in translucency inboth sexes, after 45 years. The fine-grained trabeculararchitecture of the sternal metaphyses was predominant-ly observed in the younger individuals, while the pres-ence of the trabecular involution followed with coarsetrabecular pattern was detected after fourth decade(Fig. 2c, d). However, the pronounced irregular variabil-ity in the thickness of the anterior and posterior cortexwith mean values of 1.3 mm (SD 0.6) and 1.9 mm (SD0.7), respectively, prevented estimation of cortex alter-ation with age (p>0.05). Similarly, the correlation be-tween medullar canal enlargement and age in theanalyzed sample could not be distinguished (p>0.05)with the observed mean value of 11 mm (SD 3.6) ofmedullar canal diameter.

Histology

Cortical width (Table 7) did not show any correlationwith age either in males (R00.245; p00.468) or T

able

5Distributionof

analyzed

morph

olog

ical

changesof

articular

surfaceby

theagecatego

ry(partIII)

Age

catego

ries

Basic

morph

olog

yShape

Con

vex

Plane

Con

cave

Triangu

lar

Rou

ndOval

MF

M&

FM

FM

&F

MF

M&

FM

FM

&F

MF

M&

FM

FM

&F

n%

n%

n%

n%

n%

n%

n%

n%

n%

n%

n%

n%

n%

n%

n%

n%

n%

n%

25andyo

ungera

00

28.0

220

.04

10.0

00

440

.04

10.0

00

440

.02

5.0

00

220

.01

2.5

00

110

.05

12.5

28.7

770

.0

26–35

b0

00

00

02

5.0

416

.06

40.0

922

.50

09

60.0

615

.01

4.3

750

.02

5.0

28.7

428

.63

7.5

00

321

.4

36–45

00

00

00

00

520

.05

45.5

512

.51

4.0

654

.52

5.0

14.3

327

.31

2.5

28.7

327

.32

5.0

313

.05

45.5

46–55

00

14.0

17.7

410

.05

20.0

969

.23

7.5

00

323

.13

7.5

313

.06

46.2

12.5

00

17.7

37.5

313

.06

46.2

56andolderb

00

00

00

410

.05

20.0

956

.35

12.5

28.0

743

.83

7.5

14.3

426

.73

7.5

28.7

533

.33

7.5

313

.06

40.0

Total

00

312

.03

4.6

1435

.019

76.0

3350

.826

65.0

312

.029

44.6

1640

.06

26.1

2234

.98

20.0

626

.114

22.2

1640

.011

47.8

2742

.9

Mmale,Ffemale

aBothindividu

alswith

non-un

ionepiphy

seal

stagewereexclud

edfrom

furthermorph

olog

ical

observations

bOne

individu

alwas

exclud

edfrom

shapeanalysisdu

eto

damaged

medialclavicular

end

672 Int J Legal Med (2013) 127:667–676

females (R00.022; p00.949). Nevertheless, corticalwidth maintained higher values in men than in women(p00.004).

Trabecular bone volume fraction was 29 % on average inmales and 26 % in females (Table 8), however withoutstatistically significant intersex differences (p00.438). Ouranalysis revealed a significant correlation between bonevolume fraction and individual’s age in the male part ofthe sample (R00.701; SE00.06; p00.016), and not infemales (R00.292; p00.383). Thus, the relation betweenage and bone volume fraction in males was given in theequation:

Age ¼ 0:416� Cn :B:Ar= T :Ar

0:003

Minimum trabecular width differed (p00.053) be-tween the sexes (males 0150.7 μm, females 0134.5 μm) (Table 7). The conducted analyses couldnot determine correlation between minimum trabecularwidth and age in the female part of the sample (R00.168; p00.622). However, the correlation was ascer-tainable in the whole sample (R00.423; SE018.41; p00.050) due to significant age-related trabecular thinningin the male part of the sample (R00.717; SE014.62;p00.013). The obtained relations allowed implementa-tion of the following equations:

– for the whole sample: Age ¼ 163:446�Tr :Wi0:418

– for the male part of the sample: Age ¼ 185:773�Tr :Wi0:708

Discussion

Although the previously conducted morphological anal-yses (macroscopic and radiological) of the claviculararticular surface allowed differentiation of several age-related features, some inconsistencies remained mostlydue to differences in methodology. Thus, as the devel-oped multiphase scoring systems favored subjective er-ror, the three-phase staging system was proposed [8,25, 32]. In order to reduce all potential errors, therecommended three-phase system was applied in thecurrent study.T

able

6Distributionof

analyzed

morph

olog

ical

changesof

articular

surfaceby

theagecatego

ry(partIV

)

Age

catego

ries

Relief

Porosity

Ossific

nodu

le

Smoo

thRug

ged

Present

Not

present

Present

Not

present

MF

M&

FM

FM

&F

MF

M&

FM

FM

&F

MF

M&

FM

FM

&F

n%

n%

n%

n%

n%

n%

n%

n%

n%

n%

n%

n%

n%

n%

n%

n%

n%

n%

25andyo

ungera

717

.52

8.0

990

.01

2.5

00

110

.00

00

00

08

20.0

28.0

1010

00

00

00

08

20.0

28.0

1010

0

26–35

615

.04

16.0

1066

.75

12.5

00

533

.33

7.5

00

320

.08

20.0

416

.012

80.0

12.5

00

16.7

1025

.04

16.0

1493

.3

36–45

512

.55

20.0

1090

.90

01

4.0

19.1

12.5

00

19.1

410

.06

24.0

1090

.91

2.5

00

19.1

410

.06

24.0

1090

.9

46–55

512

.56

24.0

1184

.62

5.0

00

215

.42

5.0

14.0

323

.15

12.5

520

.010

76.9

00

00

00

717

.56

24.0

1310

0

56andolder

512

.56

24.0

1168

.84

10.0

14.0

531

.34

10.0

416

.08

50.0

512

.53

12.0

850

.02

5.0

14.0

318

.87

17.5

624

.013

81.3

Total

2870

.023

92.0

5178

.512

30.0

28.0

1421

.510

25.0

520

.015

23.1

3075

.020

80.0

5076

.94

10.0

14.0

57.7

3690

.024

96.0

6092

.3

Mmale,Ffemale

aBothindividu

alswith

non-un

ionepiphy

seal

stagewereexclud

edfrom

furthermorph

olog

ical

observations

Table 7 The average values of recorded cortical width and minimumtrabecular width

Min Max Mean SD

Female Cortical width (μm) 40.8 1,260.9 383.5 201.9

Min trabecular width (μm) 57.0 363.7 134.5 48.2

Male Cortical width (μm) 90.8 1,709.5 643.1 327.8

Min trabecular width (μm) 32.0 639.9 150.7 68.4

Int J Legal Med (2013) 127:667–676 673

Other interpretations emphasized method-specific dif-ferences in age ranges that could be ascribed to certainfeatures [7, 8, 10, 14–17]. Such impediments wereintroduced by disposition of particular procedure. In thismanner, radiological approach can affect age estimationdue to intrinsic optical and tissue superimpositions es-pecially in PA projections, but it is more prone to earlydetection of ossification. However, the subtle morpho-logical changes during the process of epiphyseal unionare more easily detected for a longer period of time byanatomical preparation. Overall, wherever it is applica-ble as the most reliable approach was proposed macro-scopic morphological examinations by Singh andChavali [10].

Additional problem in age estimation based on me-dial clavicle was introduced with population-specificvariety. Heretofore no definitive conclusions have beendrawn whether the age ranges established in a certainpopulation could be useful in age assessment of anoth-er. Nevertheless, the differences of attained completeunion in different populations were reported by Blackand Scheuer as well as Schaefer and Black [7, 24].Shirley not only emphasized the importance of ethnic-ity and socioeconomic influence for epiphyseal matura-tion but also alluded to reasonableness in applyingcontemporary age-related standards only to the modernpopulation [8].

Heretofore the only analyses which actually involvedsimilar population as in the current study encompassedBosnian population and were carried out by Schaeferand Black [24, 25]. Bosnia represents the nearby coun-try of similar economic and healthcare system as Serbia[29, 30]. Schaefer and Black noticed that the “non-union” stage was present until 23 years of age while“partial” and “complete” union were obtained from theage of 17 and 21, respectively [24, 25]. Those findingswere fairly in concordance with the results of Singh andChavali, and McKern and Stewart who also applieddirect skeletal inspection but in different populations[10, 31]. The results of the present study reported“non-union” phase until 22 years of age, whereas “par-tial” and “complete” union were present since 21 and31 years, respectively. Similar age ranges were recordedin a historical Portuguese sample from Black and Sche-uer’s study [7]. The observed discrepancy between our

studies and those of the other authors (Table 9) proba-bly arose since they analyzed predominantly youngmales while the current study enrolled a female popula-tion as well as older individuals. Furthermore, a pleth-ora of specified constraints considerably limitedcomparability of published studies. Thus, most otheranalyses were focused just on epiphyseal union timingand enrolled only population younger than 30 years [7,10, 15–17, 24, 25, 31].

The additional analyses conducted in the present studyallowed differentiation of a couple more age indicators. Theresults suggested that signs of lipping in the region of thenotch for the first rib (Fig. 1k) as well as exostoses and boneovergrowths of the articular surface margin (Fig. 1l) shouldbe considered as attributes of older individuals, as thesefeatures were noticed in individuals after 44 and 53 years,respectively.

Even though the current study identified females’tendency towards the higher epiphyseal union stages,the literature stayed ambiguous whether sexual matura-tion has any influence on the epiphyseal union progress.The differences among sexes in some phases of epiph-yseal union were reported by Schmeling et al. [16].However, the others could not affirm such impact [10,15, 17]. The current study also identified several moremorphological features with expressed sex dependence.For instance, the morphology of the notch for the firstrib, the margin of the articular surface as well as basicmorphology of articular surface differed significantlybetween male and female samples.

Although the remarkable correlations between radiologi-cal features of clavicles and age could be found in theliterature, the authors could not achieve the same [6, 33].The broad range of variety resulted in the inability to createappropriate patterns. Hence, the further appliance of thetested radiological methods could not be advisable in theprocess of age estimation.

The current study also involved histomorphometricanalyses as the same were previously applied in variousskeletal sites in order to assess age-related deteriorationof bone micro-architecture, particularly in femur andvertebrae [40, 41]. In our study, trabecular bone volumefraction and trabecular width linearly declined with agein males, demonstrating significant age-related deterio-ration of trabecular bone in the medial end of clavicle

Table 8 The average values ofrecorded trabecular bone areawith derived bone–tissue arearatio

Cancellous bone area (μm2) Cancellous bone area/tissue area

Min Max Mean SD Mean SD

Female 619,248.0 2,032,784.0 1,290,041.0 344,794.7 0.26 0.06

Male 724,992.0 2,245,664.0 1,395,039.0 427,289.2 0.29 0.08

674 Int J Legal Med (2013) 127:667–676

in men. Our results suggest that quantitative architectur-al parameters of trabecular bone may represent an age-distinctive feature in clavicles (Fig. 2a, b).

Conclusion

Age estimation methods developed from clavicles were pre-dominantly applied in population under 30 years of age.However, the application of the method beyond this boundarywas neglected in previous forensic and archaeological context.

Apart from the epiphyseal union timing, the current studymanaged to identify several other age-related features thatcould be applied as additional guidance for age estimation inanthropological and forensic analyses. However, the smallnumber of individuals encompassed in this study limited itscredibility, especially considering histology analyses, there-fore the obtained results should be used with caution.

Among investigated morphological features, signs of lip-ping in the region of the notch for the first rib as well asexostoses and bone overgrowths of the articular surface mar-gin should be considered as age-dependent attributes of me-dial clavicles. Our results also suggested that trabecular bonevolume fraction and minimum trabecular width are age-distinctive microscopic features of medial clavicles in men.

Analyses of sex differences in observed macroscopic fea-tures demonstrated that epiphyseal union timing, morphologyof the notch for the first rib, margin of the articular surface,and basic morphology of articular surface were sex-dependentattributes. Moreover, sex difference was ascertainable in twomicroscopic characteristics: trabecular bone volume fractionand minimum trabecular width. However, considerable over-lapping and inconsistent age and sex dependence of analyzedmorphological and histological features precluded the creationof accurate age-specific phases and sex-dependence criteria.

Acknowledgment This study is supported by the Ministry of Sci-ence of Republic of Serbia, Grant No. 45005.

Conflict of interest Authors declare that they have no conflicts ofinterest.

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