# The new Coimbra method for recording entheseal changes and the effect of age-at-death

Charlotte Yvette Henderson, Valentina Mariotti, Frédéric Santos, Sébastien

Villotte, Cynthia Wilczak

► To cite this version:

Charlotte Yvette Henderson, Valentina Mariotti, Frédéric Santos, Sébastien Villotte, Cynthia Wilczak. The new Coimbra method for recording entheseal changes and the effect of age-at-death. Bulletins et Mémoires de la Société d'anthropologie de Paris, 2017, 29 (3-4), pp.140-149. 10.1007/s13219-017-0185-x. hal-02266521

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# The New Coimbra Method for Recording Entheseal Changes and the Effect of Age-at-Death

## La nouvelle méthode Coimbra : changement au niveau des enthèses et influence de l’âge au décès

C.Y. Henderson · V. Mariotti · F. Santos · S. Villotte · C.A. Wilczak

Abstract Entheseal changes have been widely used in anthropology to study activity patterns, but there is an increasing awareness that ageing is associated with these changes. The aim of this study was to test each feature of the new Coimbra method for its variability, side asymmetry and its relationship with age. In addition to this, an overall relationship with age was tested for a larger sample. Males 16 and over from the Coimbra skeletal collection of historically identified individuals were recorded using the new method (N = 260). To reduce the impact of occupation, side variability in asymmetry and age were only tested in the labourers (N = 51). All occupation groups were included to test the overall relationship with age using a random forest test. The results show that scores lack variability for many of the features and entheses. Where there is side asymmetry this is typically in favour of higher scores in the right side,

regression showed that ageing explains at most 44% of the variability. This alongside the side asymmetry may indicate that biomechanics has an explanatory role.

Keywords Asymmetry · Activity-patterns · Ageing · Degeneration

Résumé Les changements au niveau des enthèses ont été largement utilisés en anthropologie biologique pour discuter des patterns d'activités, malgré les études de plus en plus fréquentes associant ces changements principalement au vieillissement. L'objectif de cette étude est d'illustrer, pour chacune des modifications enregistrées avec la nouvelle méthode de Coimbra, la distribution générale des scores, l'asymétrie et leur relation à l'âge. Une étude plus globale sur l'effet du vieillissement a également été menée. L'analyse porte sur un échantillon de squelettes de sujets masculins décédés à 16 ans ou plus issus de la collection de squelettes identifiés de Coimbra (n = 260). Pour réduire l'influence de l'activité physique, seuls les sujets avec la profession de « trabalhador » (travailleur) ont été utilisés dans les tests sur l'asymétrie et l'âge (n = 51). Pour l'étude globale sur l'effet du vieillissement, toutes les professions ont été incluses dans une analyse utilisant les forêts aléatoires. Les résultats montrent que la variabilité des scores est faible pour la plupart des changements et des enthèses. Il existe une asymétrie assez claire avec des scores plus élevés du côté droit, sauf pour l'insertion du biceps brachii. La plupart des changements enregistrés présentent une corrélation positive avec l'âge au décès, sans toutefois être systématiques pour tous les changements ou toutes les enthèses considérées. Certains changements sont plus fréquents chez les sujets âgés (formation osseuse, érosion), alors que d'autres se retrouvent plus souvent chez les jeunes sujets (porosité fine et changement mineur de surface). Une régression logistique montre que le vieillissement explique au mieux 44 % de la variabilité perçue. Cela, ainsi que l'asymétrie directionnelle observée, pourrait indiquer que les phénomènes biomécaniques jouent un rôle dans l'apparition de ces changements.

Mots clés Asymétrie · Pattern d'activités · Vieillissement · Dégénération

C.Y. Henderson (*)
CIAS – Research Centre for Anthropology and Health,
Department of Life Sciences, University of Coimbra,
Calçada Martim de Freitas, Edifício de São Bento,
3000-456 Coimbra, Portugal e-
mail: c.y.henderson@uc.pt

V. Mariotti
Laboratorio di Bioarcheologia ed Osteologia Forense – Antropologia,
Dipartimento di Scienze Biologiche, Geologiche e
AmbientaliAlma Mater Studiorum Università di Bologna, 3 via
Selmi, I-40126 Bologna, Italia

ADÉS, UMR 7268 CNRS/Aix-Marseille université/EFS,
Aix-Marseille université, CS80011, boulevard Pierre-Dramard, F-
13344 Marseille cedex 15, France

F. Santos · S. Villotte
CNRS, UMR 5199 PACEA, bâtiment B8,
allée Geoffroy-Saint-Hilaire, université de Bordeaux,
CS, 50023, F-33615 Pessac cedex, France

C.A. Wilczak
Department of Anthropology, San Francisco State University,
San Francisco, CA United States

excepting the biceps brachii insertion. Most of the features scored show a relationship with ageing, but this is not uniform for all features or entheses. Some features are associated with an increase in age (bone formation and erosions), while others generally occur in younger individuals (fine porosity and textural change). Logistic
Introduction

The most common method for identifying specific types of
activities remains the observation and recording of enthesal
changes (ECs) [1–3]. These are changes from the normal
biological appearance of the attachment of tendons and
ligaments to the bone [4]. They are frequently interpreted as
indicators of repetitive loading of specific muscles, or
muscle groups, and are therefore considered of good
specificity to particular movements, which are then
interpreted as tasks [3,5–7].

The enthesis itself is a poorly understood region of the
muscle-bone complex, with limited current clinical research
in this area. The research that exists focuses on changes
associated with diseases, e.g. the seronegative
spondyloarthropathies, which include diseases such as
ankylosing spondylitis [8–11]. While the development of the
enthesis and its normal appearance are well described in the
literature [8,12], there has been little research on how the
enthesis relates to its neighbouring structures, for example
the interplay of tendon cross-sectional growth and enthesis
size, or, tendon injury and response within the enthesis. The
latter is of particular concern, as the inferences for using
entheses as indicators of repetitive stress are reliant on a
model based upon a direct interplay between tendon loading
and enthesis damage.

What is clear from anthropological research is that ECs
are associated with increasing age [13–22]. This has been
shown for most recording methods when tested on identified
skeletal collections (ibid.). It has also been used to support
the argument that the changes represent cumulative
repetitive stress, but this fails to take into account normal
degeneration caused by increasing age, which is a cause of
tendon tears [23]. Cumulative one-off trauma may also play
a role, often in association with underuse because regular
use promotes healthy tendon morphology [24]. The concept
of underuse injury is beginning to be discussed in sports
medicine where injuries often associated with high loads are
found in those not accustomed to activity [25].

The new Coimbra method was developed from the
original Coimbra method with the aim of creating a
standardised scoring system for ECs while taking into
account the normal biology and scoring a broad range of
features seen at fibrocartilaginous entheses [16,26–27].
These entheses consist of four tissues of differing
mechanical properties that mediate the transfer of stress
from the tendon to the bone [8]. The method separates
entheses into two zones based on the anatomy of the
enthesis: i.e. zone 2 the most fibrocartilaginous part and
zone 1, which is more fibrous running along the margin of
the enthesis at the most obtuse angle of tendon attachment.
Alongside this, it focuses on recording the types of changes

seen macroscopically, "called features" that are textural
change, bone formation, erosions, fine porosity,
macroporosity and cavitations. The scoring consists of three
scores (as well as a score for unobservable) for most features
except textural change that is only scored as present or
absent as it is hard to identify unless it covers a relatively
large surface area.

The original method was tested on a sample of 31
individuals with similar occupations curated as part of the
SIMON collection in Geneva [16]. It demonstrated first that
left and right sides typically showed similar changes and that
most of the changes were associated with ageing; although
the small sample size and limited variability meant that the
effects could not be fully interpreted. This paper repeats that
approach using the new Coimbra method [26] on a larger
sample of male skeletons, all with the same occupation. The
aims are to test the relationship between ECs and age for
each feature and determine bilateral asymmetry for ECs. To
test the overall relationship between EC features and age a
random forest approach was used, creating a prediction of
age that could be compared to the real age, enabling the age
effect to be determined.

Materials and methods

The Coimbra identified skeletal collection contains skeletal
remains and documentary evidence of age-at-death, sex,
occupation and cause of death for over 500 individuals who
died in the early 20th century [28]. Diverse occupations are
represented, but the predominant male occupation is
'trabalhador', meaning labourer. These individuals are likely
to have predominantly worked the land as farm labourers
undertaking seasonal work [29]. To fully capture age
effects, male individuals aged from 16 upwards were
included in this study. The minimum age of 16 was chosen
because by this age all entheses examined in this study are
fully developed and observable.

Several diseases, e.g. seronegative spondyloarthropathies
and diffuse idiopathic skeletal hyperostosis, are widely
described in the clinical literature as causing changes to the
entheses [8–11]. To exclude individuals whose ECs may be
pathological, all individuals with more than two vertebrae
ankylosed by new osseous tissue along the line of the
anterior longitudinal ligament were excluded from the
analysis [30]. Individuals with ligamentous ankylosis of the
sacroiliac joint with at least two vertebrae ankylosed were
also excluded (ibid.).

The fibrocartilaginous entheses included in this analysis
were the infra- and supraspinatus insertion, subscapularis
insertion, common extensor origin, common flexor origin
(all located on the humerus) and biceps brachii insertion (on
the radius). All ECs were recorded using the new Coimbra method [26], although the biceps brachii insertion was recorded using the footprint published in the first version of the method [16]. The infra- and supraspinatus insertions were scored as one insertion because the fibres are known to merge in some areas close to the enthesis [31]. These entheses were all recorded by one observer (CH) with inter- and intraobserver error for this new method previously reported [32].

There are a total of six features recorded in the new Coimbra method [26]. Two features, bone formation [BF(Z1)] and erosion [ER(Z1)], are scored in zone 1. In zone 2, these features (labelled Z2) plus textural change (TC), fine porosity (FPO), macroporosity (MPO) and cavitations are scored. All, apart from textural change, are scored from 0 (absence of change) to 2 (maximal expression), while textural change is only scored as absence (0) or presence (1). The scoring has been simplified and standardised to three scores since the original method's publication [16] and textural change, which was scored as a type of bone formation, is now scored separately.

The variability of scores for each enthesis and feature is described, as it has been found that some features are more common at some entheses than others. Asymmetry of scores by enthesis and feature was calculated by counting the number of individuals with equal scores on both sides; with right side scores higher and with left side scores higher [16]. Frequencies for these results were also reported to enable comparison.

Ordinal regression was used to test the effect of age for each enthesis and feature within a single occupation group, as was done in the 2013 paper [16] to reduce potential biomechanical effects. The skeletons (N = 51) meeting the above criteria had an age range from 16 to 66 (mean = 41.29, standard deviation = 13.94). The Shapiro-Wilk test of normality showed a normal distribution of age-at-death W = 0.96, P = 0.97. To study the effect of age, means with standard errors were plotted for each enthesis, alongside descriptive statistics. Where there was sufficient variability, ordinal regression was performed using a cumulative link model [33] in R version 3.2.2. A log-log link was used, as in the previous study, because lower scores were more probable than higher ones [16]. Ordinal regression was performed for each feature, enthesis and side. Nagelkerke's pseudo-R-square was calculated to determine the effect of age using an R script [34]. Ordinal regression was only performed where there was variability of scores based on the presence of at least five occurrences of changes (score 1 or 2).

Previous analyses using this recording method have only tested each enthesis and feature individually for the effect of

age. While it was demonstrated that each feature relates to age differently [16], no test has yet been performed using the variability from all entheses and all features simultaneously to study the impact of age. Our new approach is to create a prediction model of age, which can then be compared with true age to determine its effect. This could not be achieved with the labourers alone due to the relatively small sample size and its limited variability. For this study all males (N = 260) without signs of diseases associated with ECs (see above) were included. The age range was the same as for the ordinal regression study (mean = 44.8, standard deviation = 17.9). Random forests are a combination of regression trees and a bootstrap approach. Regression trees aim to partition the space into small homogeneous regions [35]. The trees are designed to build subsets with low within-variance and high between-variance, thereby partitioning the data according to an algorithm of rules. Thus, the algorithm will define some homogeneous regions of the feature space where the individuals have similar ages based on their overall EC expression. For random forests, several hundred regression trees are built, each of them using only a given portion of individuals and variables [36]. The overall prediction is obtained by taking the mean of the predictions of all trees. Random forests can handle ordinal predictors and have their own algorithm for missing value imputation. For this statistical analysis, the R package random forest was used [37]. The age of each individual was predicted using leave one out cross-validation. To determine the effect of age, the predicted age is plotted against the actual age such that if age is a major aetiology of these changes, the predicted age and the actual age should overlap, showing strong concordance.

## Results

The overall distribution of changes (Table 1) demonstrates the low variability of scores with very few of the highest scores present. Only bone formation in zone 1 [BF(Z1)] shows a consistent presence of score 2 with the majority of individuals having scores of zero. No doubt this is also the reason most of the scores are equal between left and right sides (Table 2). Where there is asymmetry, the right side tends to have higher scores. It is noteworthy that the left side scores are more commonly higher than the right for all biceps brachii features.
<table><caption>Table 1 Descriptive statistics showing the variability of entheses scores / Statistiques descriptives illustrant la variabilité des scores</caption><thead><tr><th>Enthesis</th><th>Side</th><th>Enthesis score</th><th>BF(Z1)</th><th>ER(Z1)</th><th>BF(Z2)</th><th>FPO</th><th>MPO</th><th>ER(Z2)</th><th>CA</th><th>TC</th></tr></thead><tbody><tr><td rowspan="6">Infra-an supraspinatus</td><td rowspan="3">Left</td><td>NA</td><td>17</td><td>17</td><td>8</td><td>8</td><td>8</td><td>8</td><td>8</td><td>8</td></tr><tr><td>0</td><td>31</td><td>33</td><td>33</td><td>19</td><td>37</td><td>26</td><td>42</td><td>42</td></tr><tr><td>1</td><td>3</td><td>1</td><td>9</td><td>21</td><td>6</td><td>16</td><td>1</td><td>1</td></tr><tr><td rowspan="3">Right</td><td>NA</td><td>16</td><td>16</td><td>9</td><td>9</td><td>10</td><td>10</td><td>9</td><td>9</td></tr><tr><td>0</td><td>31</td><td>32</td><td>30</td><td>19</td><td>38</td><td>20</td><td>41</td><td>41</td></tr><tr><td>1</td><td>3</td><td>3</td><td>10</td><td>22</td><td>2</td><td>17</td><td>1</td><td>1</td></tr><tr><td rowspan="6">Subscapularis</td><td rowspan="3">Left</td><td>NA</td><td>4</td><td>4</td><td>4</td><td>4</td><td>4</td><td>4</td><td>4</td><td>4</td></tr><tr><td>0</td><td>28</td><td>41</td><td>16</td><td>24</td><td>39</td><td>27</td><td>45</td><td>44</td></tr><tr><td>1</td><td>12</td><td>6</td><td>26</td><td>23</td><td>8</td><td>19</td><td>2</td><td>3</td></tr><tr><td rowspan="3">Right</td><td>NA</td><td>9</td><td>9</td><td>5</td><td>5</td><td>5</td><td>5</td><td>5</td><td>5</td></tr><tr><td>0</td><td>23</td><td>35</td><td>17</td><td>20</td><td>42</td><td>27</td><td>41</td><td>46</td></tr><tr><td>1</td><td>13</td><td>6</td><td>24</td><td>25</td><td>4</td><td>18</td><td>5</td><td>0</td></tr><tr><td rowspan="6">Common extensor</td><td rowspan="3">Left NA</td><td>15</td><td>15</td><td>12</td><td>12</td><td>12</td><td>12</td><td>12</td><td>12</td><td>12</td></tr><tr><td>12</td><td>12</td><td>12</td><td>12</td><td>12</td><td>12</td><td>12</td><td>12</td><td>12</td></tr><tr><td>12</td><td>12</td><td>12</td><td>12</td><td>12</td><td>12</td><td>12</td><td>12</td><td>12</td></tr><tr><td rowspan="3">Right</td><td>NA</td><td>17</td><td>17</td><td>9</td><td>9</td><td>9</td><td>9</td><td>9</td><td>9</td></tr><tr><td>0</td><td>23</td><td>34</td><td>29</td><td>37</td><td>41</td><td>34</td><td>42</td><td>42</td></tr><tr><td>1</td><td>6</td><td>0</td><td>11</td><td>2</td><td>1</td><td>8</td><td>0</td><td>0</td></tr><tr><td rowspan="6">Common flexor</td><td rowspan="3">Left NA</td><td>18</td><td>13</td><td>13</td><td>13</td><td>13</td><td>13</td><td>13</td><td>13</td><td>13</td></tr><tr><td>18</td><td>13</td><td>13</td><td>13</td><td>13</td><td>13</td><td>13</td><td>13</td><td>13</td></tr><tr><td>13</td><td>13</td><td>13</td><td>13</td><td>13</td><td>13</td><td>13</td><td>13</td><td>13</td></tr><tr><td rowspan="3">Right</td><td>NA</td><td>20</td><td>20</td><td>15</td><td>14</td><td>14</td><td>14</td><td>14</td><td>14</td></tr><tr><td>0</td><td>25</td><td>31</td><td>27</td><td>32</td><td>37</td><td>33</td><td>37</td><td>36</td></tr><tr><td>1</td><td>4</td><td>0</td><td>7</td><td>5</td><td>0</td><td>4</td><td>0</td><td>1</td></tr><tr><td rowspan="6">Biceps brachii</td><td rowspan="3">Left</td><td>NA</td><td>6</td><td>6</td><td>2</td><td>2</td><td>2</td><td>2</td><td>2</td><td>2</td></tr><tr><td>0</td><td>25</td><td>43</td><td>30</td><td>34</td><td>44</td><td>44</td><td>48</td><td>31</td></tr><tr><td>1</td><td>10</td><td>2</td><td>15</td><td>11</td><td>4</td><td>5</td><td>1</td><td>18</td></tr><tr><td rowspan="3">Right</td><td>NA</td><td>6</td><td>6</td><td>7</td><td>7</td><td>7</td><td>7</td><td>7</td><td>7</td></tr><tr><td>0</td><td>26</td><td>45</td><td>33</td><td>31</td><td>43</td><td>42</td><td>44</td><td>34</td></tr><tr><td>1</td><td>9</td><td>0</td><td>10</td><td>9</td><td>1</td><td>0</td><td>0</td><td>9</td></tr><tr><td></td><td></td><td>2</td><td>10</td><td>0</td><td>1</td><td>4</td><td>0</td><td>2</td><td>1</td></tr></tbody></table>

Z1 refers to zone 1, Z2 to zone 2; BF: bone formation; ER: erosions; FPO: fine porosity; MPO: macroporosity; CA: cavitations; TC: textural change. For definitions, see Henderson et al. [26] / Z1 correspond à zone 1, Z2 à zone 2 ; BF : formation osseuse ; ER : érosions ; FPO : porosité fine ; MPO : macroporosité ; CA : géodes ; TC : changement de texture. Pour les définitions, voir Henderson et al. [26]

The effect of age typically showed an increase in mean age with higher scores for features, but this was not the case for FPO or TC where higher scores often occurred in

younger individuals (Fig. 1). The limited variability of scores meant that ordinal regression could not be performed for
all entheses and features. The feature with the most variable scores, bone formation both BF(Z1) and BF(Z2), had the most consistent statistical significance (at 95%) with age,

but the pseudo-R-squared showed that the effect of age was typically minimal, at most explaining 44% of the variability

<table><caption>Table 2 Asymmetry scores for entheses by feature. Bold indicates a large number of higher scores on the left side / Score d'asymétrie pour chaque enthèse, par type de changement. Une dominance à gauche est indiquée en gras</caption><thead><tr><th rowspan="2">Enthesis</th><th rowspan="2">Asymmetry</th><th rowspan="2">BF(Z1)</th><th rowspan="2">ER(Z1)</th><th rowspan="2">BF(Z2)</th><th rowspan="2">FPO</th><th rowspan="2">MPO</th><th rowspan="2">ER(Z2)</th><th rowspan="2">CA</th><th rowspan="2">TC</th><th colspan="10">N % N % N % N % N % N % N % N % N %</th></tr><tr><th>28</th><th>28</th><th>39</th><th>39</th><th>38</th><th>38</th><th>39</th><th>39</th><th>39</th><th>39</th><th>39</th><th>39</th><th>39</th><th>39</th><th>39</th><th>39</th></tr></thead><tbody><tr><td rowspan="3">Infra- and supra-spinatus</td><td>N</td><td>28</td><td>28</td><td>39</td><td>39</td><td>38</td><td>38</td><td>39</td><td>39</td><td>39</td><td>39</td><td>39</td><td>39</td><td>39</td><td>39</td><td>39</td><td>39</td><td>39</td><td>39</td><td>39</td></tr><tr><td>Right higher</td><td>1</td><td>3.6</td><td>0</td><td>0.0</td><td>6</td><td>15.4</td><td>8</td><td>20.5</td><td>2</td><td>5.3</td><td>9</td><td>23.7</td><td>1</td><td>2.6</td><td>1</td><td>2.6</td><td>1</td><td>2.6</td><td>1</td><td>2.6</td></tr><tr><td>Equal</td><td>27</td><td>96.4</td><td>28</td><td>100.0</td><td>30</td><td>76.9</td><td>23</td><td>59.0</td><td>32</td><td>84.2</td><td>26</td><td>68.4</td><td>37</td><td>94.9</td><td>38</td><td>97.4</td><td>38</td><td>97.4</td><td>38</td><td>97.4</td></tr><tr><td rowspan="3">Subscapularis</td><td>Left higher</td><td>0</td><td>0.0</td><td>0</td><td>0.0</td><td>3</td><td>7.7</td><td>8</td><td>20.5</td><td>4</td><td>10.5</td><td>3</td><td>7.9</td><td>1</td><td>2.6</td><td>0</td><td>0.0</td><td>0</td><td>0.0</td><td>0</td><td>0.0</td></tr><tr><td>N</td><td>42</td><td>42</td><td>45</td><td>45</td><td>45</td><td>45</td><td>45</td><td>45</td><td>45</td><td>45</td><td>45</td><td>45</td><td>45</td><td>45</td><td>45</td><td>45</td><td>45</td><td>45</td><td>45</td></tr><tr><td>Right higher</td><td>8</td><td>19.0</td><td>5</td><td>11.9</td><td>4</td><td>8.9</td><td>13</td><td>28.9</td><td>4</td><td>8.9</td><td>6</td><td>13.3</td><td>4</td><td>8.9</td><td>0</td><td>0.0</td><td>0</td><td>0.0</td><td>0</td><td>0.0</td></tr><tr><td rowspan="3">Common extensor origin</td><td>Equal</td><td>30</td><td>71.4</td><td>34</td><td>81.0</td><td>35</td><td>77.8</td><td>23</td><td>51.1</td><td>33</td><td>73.3</td><td>31</td><td>68.9</td><td>40</td><td>88.9</td><td>42</td><td>93.3</td><td>42</td><td>93.3</td><td>42</td><td>93.3</td></tr><tr><td>Left higher</td><td>4</td><td>9.5</td><td>3</td><td>7.1</td><td>6</td><td>13.3</td><td>9</td><td>20.0</td><td>8</td><td>17.8</td><td>8</td><td>17.8</td><td>1</td><td>2.2</td><td>3</td><td>6.7</td><td>3</td><td>6.7</td><td>3</td><td>6.7</td></tr><tr><td>N</td><td>31</td><td>31</td><td>34</td><td>34</td><td>34</td><td>34</td><td>34</td><td>34</td><td>34</td><td>34</td><td>34</td><td>34</td><td>34</td><td>34</td><td>34</td><td>34</td><td>34</td><td>34</td><td>34</td><td>34</td></tr><tr><td rowspan="3">Common flexor origin</td><td>Right higher</td><td>7</td><td>22.6</td><td>0</td><td>0.0</td><td>8</td><td>23.5</td><td>4</td><td>11.8</td><td>1</td><td>2.9</td><td>4</td><td>11.8</td><td>0</td><td>0.0</td><td>0</td><td>0.0</td><td>0</td><td>0.0</td><td>0</td><td>0.0</td></tr><tr><td>Equal</td><td>24</td><td>77.4</td><td>31</td><td>100.0</td><td>25</td><td>73.5</td><td>28</td><td>82.4</td><td>33</td><td>97.1</td><td>26</td><td>76.5</td><td>34</td><td>100.0</td><td>34</td><td>100.0</td><td>34</td><td>100.0</td><td>34</td><td>100.0</td></tr><tr><td>Left higher</td><td>0</td><td>0.0</td><td>0</td><td>0.0</td><td>1</td><td>2.9</td><td>2</td><td>5.9</td><td>0</td><td>0.0</td><td>4</td><td>11.8</td><td>0</td><td>0.0</td><td>0</td><td>0.0</td><td>0</td><td>0.0</td><td>0</td><td>0.0</td></tr><tr><td rowspan="3">Biceps b.</td><td>N</td><td>25</td><td>25</td><td>31</td><td>31</td><td>32</td><td>32</td><td>32</td><td>32</td><td>32</td><td>32</td><td>32</td><td>32</td><td>32</td><td>32</td><td>32</td><td>32</td><td>32</td><td>32</td><td>32</td><td>32</td></tr><tr><td>Right higher</td><td>5</td><td>20.0</td><td>0</td><td>0.0</td><td>5</td><td>16.1</td><td>4</td><td>12.5</td><td>0</td><td>0.0</td><td>3</td><td>9.4</td><td>0</td><td>0.0</td><td>1</td><td>3.1</td><td>1</td><td>3.1</td><td>1</td><td>3.1</td></tr><tr><td>Equal</td><td>17</td><td>68.0</td><td>25</td><td>100.0</td><td>21</td><td>67.7</td><td>26</td><td>81.3</td><td>32</td><td>100.0</td><td>29</td><td>90.6</td><td>32</td><td>100.0</td><td>31</td><td>96.9</td><td>31</td><td>96.9</td><td>31</td><td>96.9</td></tr><tr><td rowspan="3">Biceps b.</td><td>Left higher</td><td>3</td><td>12.0</td><td>0</td><td>0.0</td><td>5</td><td>16.1</td><td>2</td><td>6.3</td><td>0</td><td>0.0</td><td>0</td><td>0.0</td><td>0</td><td>0.0</td><td>0</td><td>0.0</td><td>0</td><td>0.0</td><td>0</td><td>0.0</td></tr><tr><td>N</td><td>41</td><td>41</td><td>43</td><td>43</td><td>43</td><td>43</td><td>43</td><td>43</td><td>43</td><td>43</td><td>43</td><td>43</td><td>43</td><td>43</td><td>43</td><td>43</td><td>43</td><td>43</td><td>43</td><td>43</td></tr><tr><td>Right higher</td><td>7</td><td>17.1</td><td>0</td><td>0.0</td><td>4</td><td>9.3</td><td>7</td><td>16.3</td><td>0</td><td>0.0</td><td>2</td><td>4.7</td><td>0</td><td>0.0</td><td>1</td><td>2.3</td><td>1</td><td>2.3</td><td>1</td><td>2.3</td></tr><tr><td rowspan="3">Biceps b.</td><td>Equal</td><td>26</td><td>63.4</td><td>39</td><td>95.1</td><td>28</td><td>65.1</td><td>27</td><td>62.8</td><td>40</td><td>93.0</td><td>37</td><td>86.0</td><td>42</td><td>97.7</td><td>35</td><td>81.4</td><td>35</td><td>81.4</td><td>35</td><td>81.4</td></tr><tr><td>Left higher</td><td>8</td><td>19.5</td><td>2</td><td>4.9</td><td>11</td><td>25.6</td><td>9</td><td>20.9</td><td>3</td><td>7.0</td><td>4</td><td>9.3</td><td>1</td><td>2.3</td><td>7</td><td>16.3</td><td>7</td><td>16.3</td><td>7</td><td>16.3</td></tr></tbody></table>

Abbreviations: See Table 1 / Abréviations : voir tableau 1
<figure><img src="image_2.png" /><figcaption>Fig. 1 Mean age by score for each enthesis showing the variability in the relationship between age and score. Abbreviations: See Table 1 / Âge moyen par score pour chaque enthèse, illustrant la variabilité dans la relation entre âge et score. Abréviations : voir Tableau 1 (Table 3). Fine porosity also had an association with age for two of the entheses, the left infra- and supraspinatus and the right common flexor origin, with a maximum pseudo-R-square of 32%. The only other statistically significant association was for erosions in zone 2 [ER(Z2)] in the left infraand supraspinatus insertion with a pseudo-R-squared of only 21%. Using the whole data set, random forests support the ordinal regression by a minimal concordance between the true age and that predicted by the random forests (Fig. 2).</figcaption></figure>

Discussion

Anthropological research has focussed on two aspects of EC
presence: identifying occupations (activity-levels or tasks)
[1–2,6–7,38] and ageing [13–17,21–22]. To determine
whether it is possible to identify specific tasks or
movements or even broader patterns of activity, e.g. heavy
manual or nonmanual occupations, based on EC expression
requires the understanding of their aetiology. From an
anthropological perspective, it is easy to construct studies to
take into consideration biological sex and age, and these
studies have shown that this is necessary due to the increase
in EC presence with age [13–17,21–22]. Previous studies of
other methods have shown an increase in EC presence in
fibrocartilaginous entheses with age-at-death, particularly in
individuals aged over 50 [38], while others have shown an

increase with age that is more pronounced for mineralised
tissue forming changes, with less of an effect on osteolytic
changes [22].

The test of the Coimbra method to identify the effect of
age on a variety of skeletal changes in 2013 also showed that
the bone formation feature in either zone was most
obviously affected by age [16]. This updated study has
found similar results with bone formation most consistently
associated with age. Limited variability for many of the
other scores made the effect of age hard to trace. It is clear
that each enthesis is affected differently or has different
typical expressions of ECs, making an overall pattern hard
to determine. Fine porosity and textural change, in contrast
to bone formation, showed higher scores in the lower ages.
However, this was not completely consistent across all
entheses. This is very similar to the original paper, which
did not score textural change separately [16,26]. Textural
change was, in the original 2013 paper, scored as part of
bone formation, and this update shows the importance of
distinguishing between these, as the effect of age is very
different for both features [16].

This raises an important point: what is the aetiology of
these features? Enthesophytes are known to be associated
with traction [39,40], although others have found an
association with compression [41,42]. Research also
demonstrates that enthesophytes could be caused by wear
and tear or they have the effect of changing loading patterns
thus minimising stress-related damage [43]. Bone formation
in zone 2 has also been examined histologically with woven bone as well as bone formed through endochondral ossification identified [44]. The tidemark itself, the junction between the soft nonmineralised fibrocartilage and its mineralised form, has been found to be duplicated as it is in osteoarthritic joint cartilage, indicating that similar degenerative processes are at play [45]. While this latter change cannot be identified visually in skeletal remains, it is worth bearing in mind when considering the presence of other mineralised tissue formations.

Pores and fissures have been identified histologically and some of these, those larger than one millimetre in diameter, have sometimes been found to be cysts [44]. In others blood vessels (sometimes with accompanying nerves) penetrate from the bone marrow to the edge of the mineralised fibrocartilage providing a link between the bone marrow and the enthesis such that new tissue can be formed and damage can be healed (ibid.). These are probably what anthropologists identify as porosity. In some areas of the enthesis, there is no layer of cortical bone, just mineralised fibrocartilage on top of trabecular bone (ibid.), which is likely to be identified as porosity or as an erosion in our recording system. Only elderly cadaver specimens are available to the anatomist, so finding these changes in younger individuals in skeletal remains may help to determine whether these are a response to wear and tear or are present earlier. Large fissures and macropores are highly

visible on the rotator cuff entheses in adolescents during the development of the humeral head, and it is possible that some of the pores and erosions are left over from this period, but this requires further research.

Skeletal research, therefore, has a large role to play in identifying patterns of enthesis feature presence, which should improve our understanding of the causes of the changes described. What is clear from this specific piece of research is that there is a clear effect of increasing change presence with age for all changes, except fine porosity and textural change. However, it is unclear what this 'age effect' is. Ageing has numerous components and biomechanically can be considered in terms of normal age-related tissue degeneration, overuse (cumulative repetitive stress), cumulative one-off trauma or underuse injuries. The impact of these is harder to infer from skeletal remains than from in vivo studies. However, if normal age-related tissue degeneration were the cause, then left and right sides would be expected to show degeneration at the same rates. This study of asymmetry showed that most entheses and features had the same scores, indicating that the processes were occurring at the same rates in both sides (although it should be noted that this is also likely to be an effect of large numbers of zero scores). Where there were differences, there was a clear right side bias for all entheses, except the biceps

<table><caption>Table 3 Results of the ordinal regression / Résultats de la régression ordinaire</caption><thead><tr><th>Enthesis</th><th>Side</th><th>Feature</th><th>Estimate</th><th>Std. Error</th><th>z value</th><th>Pr(&gt;z)</th><th>Nagelkerke<br>pseudo R<br>squared</th><th>2013 paper<br>Nagelkerke<br>pseudo R<br>squared</th></tr></thead><tbody><tr><td rowspan="10">Infra-<br>and supraspinatus</td><td rowspan="5">Left</td><td>BFZ1</td><td>na</td><td>na</td><td>na</td><td>na</td><td>na</td><td>—</td></tr><tr><td>ERZ1</td><td>na</td><td>na</td><td>na</td><td>na</td><td>na</td><td>—</td></tr><tr><td>BFZ2</td><td>0.038</td><td>0.025</td><td>1.542</td><td>0.123</td><td>0.082</td><td>—</td></tr><tr><td>ERZ2</td><td>0.054</td><td>0.021</td><td>2.518</td><td>0.012 *</td><td>0.205</td><td>—</td></tr><tr><td>FPO</td><td>-0.038</td><td>0.018</td><td>-2.120</td><td>0.034 *</td><td>0.125</td><td>—</td></tr><tr><td rowspan="5">Right</td><td>MPO</td><td>0.027</td><td>0.031</td><td>0.866</td><td>0.386</td><td>0.033</td><td>—</td></tr><tr><td>CA</td><td>na</td><td>na</td><td>na</td><td>na</td><td>na</td><td>—</td></tr><tr><td>TC</td><td>na</td><td>na</td><td>na</td><td>na</td><td>na</td><td>—</td></tr><tr><td>BFZ1</td><td>na</td><td>na</td><td>na</td><td>na</td><td>na</td><td>—</td></tr><tr><td>ERZ1</td><td>na</td><td>na</td><td>na</td><td>na</td><td>na</td><td>—</td></tr></tbody></table>
<table><tbody><tr><td></td><td></td><td>BFZ2</td><td>0.027</td><td>0.031</td><td>0.866</td><td>0.386</td><td>0.033</td><td>-</td></tr><tr><td></td><td></td><td>ERZ2</td><td>0.003</td><td>0.016</td><td>0.183</td><td>0.855</td><td>0.001</td><td>-</td></tr><tr><td></td><td></td><td>FPO</td><td>0.001</td><td>0.016</td><td>0.088</td><td>0.930</td><td>0.000</td><td>-</td></tr><tr><td></td><td></td><td>MPO</td><td>na</td><td>na</td><td>na</td><td>na</td><td>na</td><td>-</td></tr><tr><td></td><td></td><td>CA</td><td>na</td><td>na</td><td>na</td><td>na</td><td>na</td><td>-</td></tr><tr><td></td><td></td><td>TC</td><td>na</td><td>na</td><td>na</td><td>na</td><td>na</td><td>-</td></tr><tr><td rowspan="10">Subscapularis</td><td rowspan="5">Left</td><td>BFZ1</td><td>0.069</td><td>0.019</td><td>3.627</td><td>2.87E-04 ***</td><td>0.338</td><td>0.22</td></tr><tr><td>ERZ1</td><td>0.057</td><td>0.035</td><td>1.639</td><td>0.101</td><td>0.120</td><td>na</td></tr><tr><td>BFZ2</td><td>0.057</td><td>0.016</td><td>3.650</td><td>2.62E-04 ***</td><td>0.336</td><td>0.04</td></tr><tr><td>ERZ2</td><td>0.023</td><td>0.016</td><td>1.407</td><td>0.159</td><td>0.054</td><td>na</td></tr><tr><td>FPO</td><td>-0.017</td><td>0.015</td><td>-1.104</td><td>0.269</td><td>0.034</td><td>0.02</td></tr><tr><td rowspan="5">Right</td><td>MPO</td><td>0.030</td><td>0.028</td><td>1.078</td><td>0.281</td><td>0.043</td><td>0.04</td></tr><tr><td>CA</td><td>na</td><td>na</td><td>na</td><td>na</td><td>na</td><td>na</td></tr><tr><td>TC</td><td>na</td><td>na</td><td>na</td><td>na</td><td>na</td><td>-</td></tr><tr><td>BFZ1</td><td>0.058</td><td>0.019</td><td>3.078</td><td>0.002 **</td><td>0.261</td><td>0.21</td></tr><tr><td>ERZ1</td><td>0.043</td><td>0.031</td><td>1.384</td><td>0.166</td><td>0.075</td><td>na</td></tr><tr><td rowspan="10">Common extensor origin</td><td rowspan="5">Left</td><td>BFZ2</td><td>0.076</td><td>0.019</td><td>3.999</td><td>6.37E-05 ***</td><td>0.436</td><td>0.16</td></tr><tr><td>ERZ2</td><td>0.040</td><td>0.019</td><td>2.119</td><td>0.034 *</td><td>0.130</td><td>0.09</td></tr><tr><td>FPO</td><td>0.022</td><td>0.016</td><td>1.348</td><td>0.178</td><td>0.050</td><td>0.08</td></tr><tr><td>MPO</td><td>na</td><td>na</td><td>na</td><td>na</td><td>na</td><td>0.16</td></tr><tr><td>CA</td><td>0.037</td><td>0.036</td><td>1.018</td><td>0.309</td><td>0.048</td><td>na</td></tr><tr><td rowspan="5">Right</td><td>BFZ1</td><td>0.070</td><td>0.037</td><td>1.891</td><td>0.059</td><td>0.171</td><td>0.41</td></tr><tr><td>ERZ1</td><td>na</td><td>na</td><td>na</td><td>na</td><td>na</td><td>na</td></tr><tr><td>BFZ2</td><td>na</td><td>na</td><td>na</td><td>na</td><td>na</td><td>0.31</td></tr><tr><td>ERZ2</td><td>0.021</td><td>0.034</td><td>0.628</td><td>0.530</td><td>0.017</td><td>na</td></tr><tr><td>FPO</td><td>na</td><td>na</td><td>na</td><td>na</td><td>na</td><td>na</td></tr><tr><td rowspan="5">Right</td><td>MPO</td><td>na</td><td>na</td><td>na</td><td>na</td><td>na</td><td>na</td></tr><tr><td>CA</td><td>na</td><td>na</td><td>na</td><td>na</td><td>na</td><td>na</td></tr><tr><td>TC</td><td>na</td><td>na</td><td>na</td><td>na</td><td>na</td><td>-</td></tr><tr><td>BFZ1</td><td>0.091</td><td>0.029</td><td>3.091</td><td>0.002 **</td><td>0.386</td><td>0.27</td></tr></tbody></table>
ERZ1 na na na na na na
BFZ2 0.068 0.026 2.624 0.009 ** 0.234 0.17
ERZ2 0.043 0.029 1.495 0.135 0.091 0.36
FPO -0.029 0.034 -0.866 0.386 0.031 na
(Suite page suivante)

<table><caption>Table 3 (suite)</caption><thead><tr><th>Enthesis</th><th>Side</th><th>Feature</th><th>Estimate</th><th>Std. Error</th><th>z value</th><th>Pr(&gt;z)</th><th>Nagelkerke pseudo R squared</th><th>2013 paper Nagelkerke pseudo R squared</th></tr></thead><tbody><tr><td rowspan="10">Common flexor na</td><td rowspan="3">Left na</td><td>MPO na</td><td>na</td><td>na</td><td>na</td><td>na</td><td>na</td><td>na</td></tr><tr><td>CA na</td><td>na</td><td>na</td><td>na</td><td>na</td><td>na</td><td>na</td></tr><tr><td>TC na</td><td>na</td><td>na</td><td>na</td><td>na</td><td>na</td><td>-</td></tr><tr><td rowspan="10">Right na</td><td>BFZ1 na</td><td>na</td><td>na</td><td>na</td><td>- origin</td><td>ERZ1 na</td><td>na</td></tr><tr><td>BFZ2 0.071</td><td>0.033</td><td>2.188</td><td>0.029 *</td><td>0.195</td><td>-</td><td>-</td></tr><tr><td>ERZ2 na</td><td>na</td><td>na</td><td>na</td><td>na</td><td>na</td><td>-</td></tr><tr><td>FPO na</td><td>na</td><td>na</td><td>na</td><td>na</td><td>na</td><td>-</td></tr><tr><td>MPO na</td><td>na</td><td>na</td><td>na</td><td>na</td><td>na</td><td>-</td></tr><tr><td>CA na</td><td>na</td><td>na</td><td>na</td><td>na</td><td>na</td><td>-</td></tr><tr><td>TC na</td><td>na</td><td>na</td><td>na</td><td>na</td><td>na</td><td>-</td></tr><tr><td>BFZ1 0.053</td><td>0.033</td><td>1.610</td><td>0.107</td><td>0.121</td><td>-</td><td>-</td></tr><tr><td>ERZ1 na</td><td>na</td><td>na</td><td>na</td><td>na</td><td>na</td><td>-</td></tr><tr><td>BFZ2 0.052</td><td>0.028</td><td>1.846</td><td>0.065</td><td>0.133</td><td>-</td><td>-</td></tr><tr><td>ERZ2 na</td><td>na</td><td>na</td><td>na</td><td>na</td><td>na</td><td>-</td></tr><tr><td>FPO -0.107</td><td>0.046</td><td>-2.329</td><td>0.020 *</td><td>0.322</td><td>-</td><td>-</td></tr><tr><td>MPO na</td><td>na</td><td>na</td><td>na</td><td>na</td><td>na</td><td>-</td></tr><tr><td>CA na</td><td>na</td><td>na</td><td>na</td><td>na</td><td>na</td><td>-</td></tr><tr><td>TC na</td><td>na</td><td>na</td><td>na</td><td>na</td><td>na</td><td>-</td></tr><tr><td rowspan="10">Biceps brachii</td><td rowspan="5">Left na</td><td>BFZ1 0.047</td><td>0.018</td><td>2.651</td><td>0.008 **</td><td>0.190</td><td>na</td><td>na</td></tr><tr><td>ERZ1 na</td><td>na</td><td>na</td><td>na</td><td>na</td><td>na</td><td>na</td></tr><tr><td>BFZ2 0.038</td><td>0.018</td><td>2.161</td><td>0.031 *</td><td>0.119</td><td>0.04 ERZ2</td><td>0.040</td></tr><tr><td>0.036 1.108</td><td>0.268</td><td>0.056 na</td><td></td><td></td><td></td><td></td></tr><tr><td>FPO -0.004</td><td>0.018</td><td>-0.223</td><td>0.823</td><td>0.001</td><td>0.10</td><td>0.10</td></tr><tr><td>MPO na</td><td>na</td><td>na</td><td>na</td><td>na</td><td>na</td><td>na</td></tr><tr><td>CA na</td><td>na</td><td>na</td><td>na</td><td>na</td><td>na</td><td>na</td></tr><tr><td>TC -0.033</td><td>0.019</td><td>-1.788</td><td>0.074</td><td>0.090</td><td>-</td><td>-</td></tr><tr><td rowspan="5">Right na</td><td>BFZ1 0.051</td><td>0.020</td><td>2.592</td><td>0.010 **</td><td>0.186</td><td>na</td><td>0.15</td></tr><tr><td>ERZ1 na</td><td>na</td><td>na</td><td>na</td><td>na</td><td>na</td><td>na</td></tr><tr><td>BFZ2 0.080</td><td>0.030</td><td>2.662</td><td>0.008 **</td><td>0.270</td><td>na</td><td>0.04</td></tr><tr><td>ERZ2 na</td><td>na</td><td>na</td><td>na</td><td>na</td><td>na</td><td>na</td></tr><tr><td>FPO 0.013</td><td>0.020</td><td>0.641</td><td>0.522</td><td>0.012</td><td>na</td><td>0.08</td></tr><tr><td>MPO na</td><td>na</td><td>na</td><td>na</td><td>na</td><td>na</td><td>na</td></tr><tr><td>CA na</td><td>na</td><td>na</td><td>na</td><td>na</td><td>na</td><td>na</td></tr></tbody></table>
NA indicates lack of variability, – indicates that the enthesis or feature was not tested, bold indicates a pseudo R-squared score > 0.29 and *indicates level of significance. The pseudo R-squared for this study is presented next to that from the 2013 paper [16]. Abbreviations: Table 1 / NA indique une absence de variabilité, – indique que cette enthèse ou ce changement n'a pas été testé, les valeurs en gras indiquent un score de pseudo R carré supérieur à 0,29 et * indique une valeur p inférieure à 0,05. Abréviations : voir tableaux 1

brachii insertion. A clear side difference may indicate the Comparing the results of this paper to the original method effects of use, but further research with a larger sample size paper shows some stark differences. Only three enthesis feais needed to explore which use effects are more likely to be tures demonstrated higher scores on the left side in the origicausing the changes, due to the small variations in this study nal paper; these were erosions at the biceps brachii insertion and the limited activity patterns. (both ER(Z1) and ER(Z2)) and FPO at the common extensor
<figure><img src="image_3.png" /><figcaption>Fig. 2 True age (in years) plotted against the random forest predicted age (in years). Line indicates concordance between the true and predicted age / Nuage de point suivant l'âge réel (en années) et l'âge prédit (en années) par les forêts aléatoires. La droite illustre une concordance parfaite</figcaption></figure>

origin [16]. While some of this may be due to a difference
in the method, it is also probable that population differences
and sampling effects play a role. However, biomechanical
differences may also explain these results and further
exploration of asymmetry is needed. In terms of the effect
of age, similar patterns are seen for all features (excluding
textural change which was not identified separately in the
original method). The effect of age was then, as now, found
to be present but was clearly not the only effect nor could
the underlying cause of the effect of ageing be identified.
Both studies suffer from the limited sample size available
for one occupational group. Future studies are needed using
larger samples with the same occupations to better
characterise asymmetry and the effect of age. To study the
cause of the age-related effects, large samples with multiple
occupations are required, and improved methods to
characterise and categorise occupations are also needed to
enable identification of overuse and underuse effects.

Conclusions

The aim of this paper is to present the results of asymmetry
and the effect of age on entheses and their features in a
single occupation category. The study shows that feature
expression varies by entheses with some features occurring
only rarely at some entheses, at least in this sample.

Asymmetry is rare, with predominantly higher scores on the
right side where it does occur. The effect of age is minimal
overall. Where there is an effect of age it tends to be towards
an increased score with age, except for fine porosity and
textural change, which often have a higher score in younger
individuals and may indicate a developmental origin for
these changes.

While there is an effect of age, it is unclear whether this
is from normal age-related tissue degeneration, cumulative
repetitive movement (overuse), cumulative one-off trauma
or underuse injuries. Further research with larger samples of
skeletal remains is needed to understand these effects.
Longitudinal in vivo studies are also required to improve our
understanding of the relationship between tendon
degeneration and ECs.

Acknowledgements This paper is based on a presentation at
the Working your Fingers to the Bone Conference, held in
July 2016 in Coimbra, Portugal.

The authors would like to express their thanks to the
Department of Life Sciences, University of Coimbra, for
providing access to the identified skeletal collection.
Charlotte Henderson is funded by Fundação para a Ciência
e a Tecnologia grant number SFRH/BPD/82559/2011.

Author's contribution

All authors contributed to the research design of the study.
Henderson collected the data and undertook all statistics
excluding the random forest tests, which were run by
Santos. All authors reviewed and critiqued the drafting,
contributing to the final version. The authors have no
conflicts of interest.

Conflict of interest: None.

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