TITLE: Protocol for the ‘Validated Entheses-based Reconstruction of Activity 2.0’ (VERA 2.0) Method: Semi-Automated Measurement of 3D Entheseal Changes
AUTHORS: Fotios Alexandros Karakostis

[DESCRIPTION]
This protocol introduces the "Validated Entheses-based Reconstruction of Activity 2.0" (VERA 2.0), an advanced iteration of the original VERA 1.0, previously developed by the author of this protocol (Karakostis, 2022; Karakostis & Harvati, 2021; Karakostis & Lorenzo, 2016). VERA 1.0 has become a valuable tool in anthropological studies for repeatably delineating, measuring, and analyzing 3D muscle attachment sites (surface areas), demonstrating that entheseal 3D surface variation can reflect habitual muscle coordination and physical activity. VERA 2.0 builds on this foundation with significant enhancements, offering a semi-automated protocol that greatly simplifies the 3D area quantification process. It requires only one initial set of manual steps – selecting a pre-specified broader region of the bone around the enthesis – after which the remaining procedure can be fully automated using a stored filter script (e.g., mlx file in the software Meshlab). After some practice, this semi-automation can potentially reduce the entire 3D area measurement time to less than 2 minutes per enthesis (see example video below), enabling the analysis of very large skeletal samples within a few working days. Unlike VERA 1.0, which measures the entire 3D surface area of bony structures associated with a muscle and/or ligament (e.g., the entire tubercle), VERA 2.0 identifies 3D surface deformations and irregularities within the footprint of the attaching soft tissue (i.e., “entheseal changes”), which can arguably be more clearly associated with direct muscle or ligament loading. Preliminary experimental analyses, which will be presented in the validation paper associated with this protocol, indicate that VERA 2.0 can effectively and repeatably identify activity-related differences, similar to VERA 1.0. 

This protocol provides a comprehensive, step-by-step guide for executing the VERA 2.0 workflow, covering both manual and automated components. It is designed to enable users to achieve measurement repeatability without necessarily requiring training from the developer, though direct contact with the author is encouraged for questions or explicit recommendations regarding particular entheses.

[BEFORE_START]

Please consider the following recommendations (1 and 2) before applying VERA 2.0:

1. When selecting the muscle or ligament attachment sites (entheses) for analysis in your study, the following must be kept in mind:

- VERA 2.0 is designed to identify projecting surface deformations and irregularities within entheseal areas, broadly referred to in the literature as “entheseal changes”. This makes VERA 2.0 particularly effective for detecting such changes in entheses that have the potential to exhibit these alterations at the sample level, even if not present in all individuals. Some key examples of such entheses include the occipital protuberance (cranium), the masseter muscle insertion (mandible), the costoclavicular ligament enthesis (clavicle), the deltoid muscle attachment (humerus), the biceps brachii enthesis (radius), various key muscle entheses of the hand (e.g., opponens pollicis, extensor pollicis brevis, flexor pollicis longus, flexor digitorum superficialis, opponens digiti minimi, and the origin sites of the interossei muscles), the iliac crest (pelvis), the greater trochanter and supracondylar line (femur), the tibial tuberosity (tibia), the calcaneal tuberosity (calcaneus), and multiple muscle entheses of the pedal metatarsals and phalanges. If there is insufficient bibliographic information on a particular enthesis and its potential changes, it is recommended to visually examine its morphology across your sample and assess whether any consistent irregularities or deformations occur that cannot be exclusively attributed to taphonomy (damage), injury, or inflammatory conditions unrelated to physical activity (e.g., systemic diseases). If an enthesis consistently shows smooth and unaltered surfaces across all individuals in your sample (i.e., no visible entheseal changes), then the traditional "VERA 1.0" approach, which focuses on overall 3D area measurement rather than within-enthesis surface changes, may be more suitable.

- When the aim of the study is to reconstruct habitual muscle or ligament use based on entheseal morphology, the presence of large pathological lesions (e.g., osteophytic or osteolytic) or taphonomic alterations within the enthesis that may likely be unrelated to physical activity can significantly impact the reliability of the measurements. If such alterations are visually identifiable and have a maximum diameter exceeding 4 mm (i.e., the threshold for defining third-level enthesopathies; see criteria in Mariotti et al., 2004), it is hereby recommended to exclude that enthesis from the sample before applying VERA 2.0. Alternatively, if the observer decides to retain the enthesis in the analysis – due to the unique importance of the specimen or the suspicion that the potential enthesopathy may be linked to activity-induced biomechanical stress – it is essential to clearly highlight the presence of these alterations in the study’s results and interpretations. In cases where such large pathological or taphonomic alterations are within VERA 2.0’s “broader selection region” but clearly not within the entheseal footprint region itself, users must ensure that the afflicted areas are manually excluded from the selection (refer to Step-Case 1, Step 11). Similarly, for all remaining lesions below the 4 mm threshold, follow the treatment guidelines provided in Step-Case 1, Step 11.


2. Please note that the delineation process detailed in this protocol is carried out using Meshlab v.2023.12 (ISTI-CNR, Pisa, Italy). While other software may be used as long as they support the same filtering algorithms, the specific steps outlined here may differ. The predominant portion of the VERA 2.0 workflow (Steps 13 to 23) can become fully automated by using a created Meshlab mlx filter script. For convenience, an example filter script is provided for download below, which can also potentially be reused in future studies. If necessary, after loading an existing script in Meshlab, its exact parameters can also be directly adjusted (using the process outlined in the paragraph below). Once the workflow has been applied to the first enthesis of a sample, all subsequent measurements of that enthesis in other individuals can be completed much faster by re-using such a script, potentially taking less than 2 minutes per individual enthesis (as demonstrated in the example delineation video provided further below). This allows for large samples to be analyzed in just a few days.

If observers wish to develop and use their own filter script in Meshlab, they are encouraged to save their filter script after completing the protocol manually for the first enthesis of their sample (Filters --> Show Current Filter Script --> Save Script). Then, for all other entheses in the sample, as long as the parameters remain unchanged, Steps 13 to 23 can be applied automatically with just a few clicks (Filters --> Show Current Filter Script --> Open Script --> Apply Script). Before running the script each time, it is advisable to quickly double-check that all parameters (and especially the parameter indicated in Step 21, which performs outlier removal based on a specific diameter threshold) are consistent by reviewing them in the "Edit Parameters" tab within the “Show Current Filter Script” window. To ensure consistency, it is strongly recommended to use the same exact script across all 3D models of the sample, avoiding discrepancies in the applied parameters.


Example Video and Reusable Meshlab Filter Script
The video below demonstrates the semi-automated VERA 2.0 approach for analyzing the insertion site of muscle opponens pollicis on a human right first metacarpal bone in under 2 minutes. The example delineation video (mp4) and the associated Meshlab filter script (mlx) can also be found here: Video and Filter Script Link. The 3D surface model (ply format) was created using an Artec Space Spider structured-light scanner (Artec Inc., Luxembourg) and has a resolution of 100 microns. For background information on the bone depicted in the video, see Karakostis and Hotz (2022).


 

  

Contents:
Part 1 – Technical requirements and recommendations
Part 2 – Selection of a broader bone area around the enthesis (manual component)
Part 3 – Mapping of projecting structures
Part 4 – Delineation and measurement of projecting structures


[GUIDELINES]
1. Some sections ("Parts") start with an introductory “Note” inserted within the first step right before the description of the step itself. 

2. Occasionally, important “Notes” are also included within steps, where necessary or useful.

3. Software options or commands are usually indicated within parenthesis (e.g., XYZ --> XYZ --> XYZ).

4. IMPORTANT: The entire analytical process of this protocol (Parts 1 to 4) is described in detail only for the insertion site of muscle opponens pollicis (OP) on the right first metacarpal bone. For Part 2, which focuses on the manual component of this protocol (i.e., the selection of a broader bone area around the enthesis), another eight examples are concisely provided in the form of eight Step-Cases (2-9). Nevertheless, Parts 3 and 4 of the protocol are only visible when selecting Step-Case 1 ("1. OP"). This is to avoid redundancy, given that Parts 3 and 4 are identical for any enthesis and can be fully automated using a Meshlab filter script (as shown in the provided video, below).

5. IMPORTANT: In Step 7 (Part 2), Step-Cases 1-5 are accessible via a drop-down menu located below the large "Explanatory Note." However, Step-Cases 6-9 will only become visible after selecting Step-Case 5 ("5. FDS") and scrolling down to find another drop-down menu. After viewing these additional example cases, you will need to re-select the first Step-Case ("1. OP") from the first drop-down menu to view Parts 3 and 4 of the protocol again.

[STEPS]
SECTION: Part 1 – Technical requirements and recommendations
2. Download and use the open-access software Meshlab Version 2023.12 or higher (https://www.meshlab.net/), since some steps of this protocol require functions not available in older versions of the software. If another 3D image processing software is used, please ensure that all steps of this protocol are available and that the same (or equivalent) 3D filtering algorithms are employed. Exact specifications on the software’s tools and functions can be found in Meshlab’s manual and associated references (available at https://www.meshlab.net/#references and https://www.meshlab.net/#support).  
SECTION: Part 1 – Technical requirements and recommendations
1. The 3D surface model to be analyzed can be developed using any technology (e.g., laser, structured light, computed tomography, or photogrammetry). The 3D model format used in this protocol is ply, but others are also compatible (e.g., obj or stl). Texture information is not required for VERA
SECTION: Part 1 – Technical requirements and recommendations
3. Confirm that the surface model’s resolution is adequate for virtually visualizing entheseal surface changes. Based on trial tests, a minimum resolution of 300 microns is recommended.
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4. Confirm that all the 3D models of your sample present the same resolution. If they do not, it is recommended to homogenize the resolution across specimens via downsampling those with a higher resolution. Different resolutions among specimens can lead to a different amount of entheseal changes identified and measured.
SECTION: Part 2 – Selection of a broader bone area around the enthesis (manual component)
5.   .. 
 

Open Meshlab and load the 3D surface model (e.g., either by selecting File --> Import Mesh --> 3D file selection --> Open, or by “drag & drop”). The 3D surface model is expected to appear (Fig. 1).

 

SECTION: Part 2 – Selection of a broader bone area around the enthesis (manual component)
6. 1. If texture is visible on the model (and you do not wish to remove it from the model prior to the analysis), deactivate it by moving your cursor to the right side of Meshlab (Fig. 2) and selecting “Off” next to “Texture Coord” and/or “User-def” next to “Color” (and choosing the grey color, which is the default).


 


 

SECTION: Part 2 – Selection of a broader bone area around the enthesis (manual component)
7. Using your cursor in Meshlab, rotate the first metacarpal from its initial random position (e.g., the one depicted in Fig. 1) to the specific orientation provided in Figure 3 below. The observer must view the first metacarpal from a fully lateral perspective, with the bone placed in an upright position, the distal end facing upward, and the proximal end downward. The characteristic distal-lateral ridge of the enthesis must be visible to the extent possible (as shown in Fig. 3).


 

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8. Colorize the bone surface using the “Discrete Curvatures” filter (Filters --> Colors creation and processing --> Discrete curvatures --> Apply) and maintaining the default option “Mean” under “Type”, to visualize surface curvature in the form of relative projections (in shades of blue) and depressions (shades of red) (Fig. 4). This facilitates the broader selection process indicated below, at Steps 9 to 12. 

 


 

SECTION: Part 2 – Selection of a broader bone area around the enthesis (manual component)
9. Select Meshlab's "Z-painting tool" by clicking the appropriate icon with the brush on the right side of Meshlab’s toolbar and then, in the window that pops up, click on the second (lower) brush that is used for manually selecting 3D surface areas. The user can adjust the size and shape of the brush to their convenience (Fig. 5). Areas can be selected by pointing the cursor to the desired location on the model’s surface and pressing the left mouse button. Respectively, areas can be de-selected by pressing the right mouse button.

 

SECTION: Part 2 – Selection of a broader bone area around the enthesis (manual component)
10. When using VERA 2.0 to analyze the insertion site of opponens pollicis on the first metacarpal, the user should always select the area indicated in Fig. 6, below. This is defined as the “broader selection region” (BSR) for that enthesis. In particular, it is advised that approximately the distal 4/5ths of the shaft must be selected (not considering the articular surfaces in this estimation), excluding the proximal 1/5th. The latter portion includes the tubercle that accommodates the distinct insertion site of abductor pollicis longus, whose distal extent can vary considerably among individuals but is not known to extend beyond 1/5th of the bone. Subsequently, the user must make sure to deselect the articular surface of the metacarpal head, including its rim (typically carrying an elongated blue-colored line, after the “Discrete Curvatures” filter is applied).


 

SECTION: Part 2 – Selection of a broader bone area around the enthesis (manual component)
11. Additionally, if the selected BSR encompasses clearly identifiable taphonomic alterations and/or pathological lesions (see example in Fig. 7A), these specific regions must also be manually deselected. This also includes alterations within the exact footprint of the tendon or ligament, unless the observer explicitly aims to investigate a potential link between potential enthesopathy and activity-induced stress in their study (in which case the presence of potential enthesopathies must be highlighted in that study’s results and interpretations). If that is the study's aim or intent, then this Step can be intentionally ignored. Otherwise, as explained above (in the "Before starting" section of this protocol), such artifacts must be removed, as they may be associated with taphonomic effects or various pathological reactions unrelated to lifetime muscle or ligament use. 

Nevertheless, it should be clarified that when such distinguishable alterations in the BSR are clearly isolated from other projections or depressions (thus tending to appear either as blue-colored "islands" or red-colored "pits" after applying the “Discrete Curvatures filter”) and their maximum diameter is less than 2 mm (see example in Fig. 7B), they do not necessarily need to be deselected manually, as a later step of the VERA 2.0 workflow (i.e., Step 21) will exclude them automatically. Therefore, such small and isolated structures on the bone surface can be safely ignored, greatly facilitating the manual selection stage required for VERA 2.0. In cases where it is not immediately clear whether an isolated structure surpasses the threshold of 2 mm, the maximum length of a suspected artifact can be quickly measured on the 3D model using the “Measuring Tool” of Meshlab (see examples in Fig. 7). Additionally, the user can easily double-check that such isolated structures did not make it to the final delineation, at the final step of the protocol (i.e., Step 24). If any of them finally did, the observer can choose to select them directly using the marker tool (or other available tools), delete their vertices, and re-measure the remaining delineated area (see steps below).

 

SECTION: Part 2 – Selection of a broader bone area around the enthesis (manual component)
12. Finally, the user must ensure that the selection (BSR) did not accidentally include remote and unrelated aspects of the bone. For this enthesis (opponens pollicis), the observer should first rotate the bone model palmarly (Fig. 8A) and ensure that the selection does not surpass the palmar aspect’s median (central) axis (indicated here by a black line). Subsequently, the user should view the bone from a dorsal perspective (Fig. 8B) and confirm that the selection does not surpass the dorsal aspect’s median (central) axis (indicated here by a black line). Additionally, in case a written label occurs within the selected area (e.g., indicating the specimen label) and this is captured by the 3D scanner, it is advised to manually deselect the labeled selection if possible (as was done in Fig. 8C). Even though the later steps of the automated part of the protocol will usually remove these markings before the final measurement (as long as the lines of the letters are not more than 2 mm in maximum length), it is safer to already manually exclude them at this stage (also see in the provided example delineation video of the "Before starting" section). Finally, the observer is encouraged to additionally observe the bone’s metacarpal head from a fully distal perspective, ensuring that the entire articular surface is clearly visible (to the extent possible). This allows for confirmation that no part of the distinguishable articular surface’s rim was accidentally selected (as demonstrated in the provided video).


 

SECTION: Part 2 – Selection of broader bone area around the enthesis (manual steps)
7. Case 2 Steps 7-12 (summary): In this example enthesis, the 3D surface model of the first metacarpal has to be manually rotated to obtain the SP shown in Fig. 9A. In particular, only the medial aspect of the palmar diaphysis must be clearly visible at the center of the viewpoint while the bone is placed in an upright position (distal up). As shown in Fig. 9A, after applying the “Discrete Curvatures” filter, the marker tool must be used to select a BSR that begins from the visible midshaft of the bone (distally) and expands proximally until the height of the most distal point of the proximal articular surface’s rim (which is indicated in the figure by the three lower black arrows). The rim of the proximal articular surface itself (typically represented by lines of blue, after applying the above filter) must not be included in the selection. Subsequently, the 3D model must be rotated twice to view its palmar-lateral and fully dorsal aspects (see Fig. 9B, left and right) and ensure that these two entire regions/aspects are entirely removed from the selection (with the right mouse button). This additional step leads to the final BSR highlighted in Fig. 9A, which covers most of the proximal half of the bone’s palmar-medial shaft, excluding the articular surface’s rim, the areas underneath it, and a very thin visible part along the bone’s palmar and dorsal aspects (indicated by the left black arrow).


 

SECTION: Part 2 – Selection of broader bone area around the enthesis (manual steps)
7. Case 3 Steps 7-12 (summary): For this enthesis, the 3D model of the proximal phalanx has to be viewed from a fully dorsal perspective (i.e., hereby defined as a viewpoint from which the dorsal basal dimension of the bone appears to be the widest), as shown in Fig. 10A. This is often a view in which the thumb’s phalangeal head tends to often appear slightly tilted (as shown in the example of Fig. 10A). After applying the “Discrete Curvatures” filter (maintaining the above SP), the entire proximal third of the visible dorsal aspect’s shaft (not considering the two articular surfaces when estimating total bone length) must be selected using the marker tool, excluding the rim of the articular surface (the lowest visible border of the SP) that is typically distinguishable by the presence of blue lines (as indicated by the two lower black arrows in Fig. 10A). Subsequently, the bone has to be rotated twice, palmar-laterally and palmar-medially (exactly as shown in Fig. 10B), and the user must ensure that the main bodies of the two tubercles (which correspond to other entheses of the thenar musculature, unrelated with the extensor pollicis brevis muscle) are removed from the selection (using the right mouse button). Consequently, as shown in Fig. 10A, the final selection before analysis will not include the two tubercles highlighted by the two upper black arrows in Fig. 10A, atensure the corners of the BSR.


 

SECTION: Part 2 – Selection of broader bone area around the enthesis (manual steps)
7. Case 4 Steps 7-12 (summary): For this enthesis, the 3D bone model must only be viewed from a full palmar perspective, from which the palmar mediolateral midshaft width appears to be the longest (as shown in Fig. 11). As with the other example entheses, the bone has to be placed in an upright position, with the distal apical tuft facing upward. Here, the roughly rectangular BSR must begin from the level of the apical tuft (i.e., a horizontal line linking the two most proximal points of the apical tuft, indicated by the upper black arrow in Fig. 11) and expand proximally until another oblique straight line that links the two most proximal points of the arch-shaped ridge, where the flexor pollicis longus is known to attach. In the example of Fig. 11, the two ends defining this line are indicated by the lowest two black arrows. Finally, to ensure that no medial and lateral borders of the palmar bone shaft are accidentally selected (even though they tend to be slightly visible from a palmar perspective), the observer must deselect an extremely thin line along the medial and the lateral borders of the selection (indicated by the remaining two black arrows in Fig. 11, pointing medially and laterally). It is important that the entire arch-shaped ridge (indicated by the lowest two black arrows) is finally included in the BSR.


 

SECTION: Part 2 – Selection of broader bone area around the enthesis (manual steps)
7. Case 5 Steps 7-12 (summary): This enthesis is associated with two palmar ridges (one medial and one lateral) on the shaft of intermediate phalanges (Fig. 12A). The 3D bone model must be viewed from a full palmar perspective (i.e., the palmar mediolateral width must be the longest possible), while the bone is placed in an upright position with its distal end facing upward (Fig. 12A). After enabling the “Discrete Curvatures” filter, the presence of the two flexor ridges is highlighted by a mixture of blue and green color shades on the visibly elevated areas. The entire spread of each of these two elevated regions must be included in the BSR, spanning from the neck of the phalanx distally (indicated by the two upper black arrows in both panels of Fig. 12) until the level of the two distinctive basal projections proximally (indicated in Fig. 12 by the lower two black arrows), which are adjacent to the articular surface rim. It is advised that these two basal projections are not included in the selection. Subsequently, the observer must rotate the bone twice (first palmar-medially and then palmar-laterally, as shown in the two images of Fig. 12B), to ensure that no considerable areas were accidentally selected dorsally to each distinguishable ridge (i.e., the elevated elongated surface). In each of the two images of panel B, the approximate dorsal limits of the two ridges are indicated by a black arrow.


 

SECTION: Part 2 – Selection of broader bone area around the enthesis (manual steps)
7.1. 
 


 




Case 6 Steps 7-12 (summary): For this enthesis, the 3D bone model must be viewed from a palmar-medial perspective (i.e., where the maximum width of the palmar-medial midshaft width appears to be the longest possible), as shown in Fig. 14A. As in the other examples/cases above, the bone must be placed again in an upright position, with the distal end (head) facing upward. The palmar median crest of the bone separating the palmar shaft into two components (medial and lateral) must not be directly visible in the SP (to the degree that this is possible), as indicated within the images of Fig. 14. The BSR must approximately include the middle 2/4ths of the total shaft length (not considering the articular surfaces from the estimation), thus excluding the large proximal tubercle acting as the insertion site of extensor carpi ulnaris as well as the neck of the metacarpal head (distally). Subsequently, as shown in Fig. 14A, the user must deselect a very thin line along the dorsal border (highlighted by the three left black arrows of Fig. 14A), to ensure that no dorsal aspects of the bone are selected. Optionally, rotating the bone dorsally (as it was previously done for the first metacarpal in Fig. 8) can further fully confirm that no dorsal aspects were accidentally selected. Finally, in case any small traces of the palmar median ridge are still visible within the BSR (see black arrow with text indication in Figs. 14A and 14B), these must be manually deselected too. Finally, the three lower black arrows in Fig. 14B show examples of minor “blue-colored” alterations with a diameter of less than 2 mm, which will be automatically excluded in the next steps of VERA 2.0 (Step 21), as long as any sizeable entheseal structures are also present in the first place (as explained in the above Note).


 

SECTION: Part 2 – Selection of broader bone area around the enthesis (manual steps)
7.1. Case 7 Steps 7-12 (summary): The 3D surface model of the clavicle should be viewed from an inferior-medial perspective, as shown in Fig. 15, with the medial end oriented upwards and the bone positioned upright. In this view, the broader selection region (BSR) should encompass the entire typically oval-shaped attachment surface area, which is usually outlined by lines and edges displayed in blue shades after applying the “Discrete Curvatures” filter (see Fig. 15). Ensure that you include all clearly connected blue lines and edges around the oval-shaped area. For example, in Fig. 15, the oblique blue-colored lines extending to the right should be included, as they are linked to the oval-shaped structure. However, the observer must exclude any projecting blue structures not connected to the outline (e.g., those indicated by the two black arrows in the figure). In cases where there are no lines or edges of considerable size within the attachment area (i.e., no projections of at least 5 mm in maximum length), or if the only present alterations appear to be taphonomic and/or pathological in nature (see instructions in Case 1 Step 11), it is recommended to record an area measurement of “0” (as in the previous example of the opponens digiti minimi), unless enthesopathies are of interest for the intended analysis/study.


 

SECTION: Part 2 – Selection of broader bone area around the enthesis (manual steps)
7.1. Case 8 Steps 7-12 (summary): In the 3D model of the humerus, the deltoid tuberosity should be viewed as shown in Fig. 16, from a lateral view, looking towards the proximal end of the bone, with the medial side of the humeral head facing upward and away. An oblique angle of approximately 45 degrees is recommended to better distinguish projecting lines and edges within the tuberosity, which will appear in blue shades after applying the “Discrete Curvatures” filter. The broader selection region (BSR) should encompass the entire elevated area of the deltoid tuberosity visible from this perspective, which often carries a combination of sharp blue lines and red depressions (Fig. 16). Any projections or depressions presenting taphonomic and/or pathological characteristics should be excluded using the marker tool and the right mouse button in Meshlab, following the previous recommendations (refer to Case 1, Step 11). 

In case that the elevation of the deltoid tuberosity also extends beyond the usual lateral aspect of the bone, you must additionally rotate the bone anteriorly and posteriorly along the same axis and perspective to include these elevated areas in the selection. If that happens, then ensure that these rotation steps are always applied consistently across all specimens in the sample (in the same order and direction) and that this procedure is documented in detail in the study.


 

7.1. Case 9 Steps 7-12 (summary): For this enthesis, the 3D model of the calcaneus should be viewed from a posterior upright perspective, with the superior aspect of the bone facing upward. The exact angle should be adjusted to minimize the visibility of all anterior structures of the bone, ideally revealing only the near-rectangular posterior aspect of the bone, as shown in Fig. 17. From this perspective, the broader selection region (BSR) must encompass the entire superior two-thirds of the visible area (Fig. 17A), excluding the inferior one-third. Additionally, the most superior border of this region (indicated by the four upper black arrows in the example of Fig. 17A) should be deselected. Subsequently, the bone should be rotated first laterally (Fig. 17B up) and then medially (Fig. 17B down). From these two angles, any areas showing distinguishable taphonomic or pathological alterations (examples marked in Fig. 17B by black arrows) should also be deselected, in line with the detailed recommendations of Case 1 Step 11.


 

SECTION: Part 3 – Mapping of projecting structures
14. Select the bone areas surrounding the BSR itself by inverting the current selection (Filters --> Selection --> Invert Selection) (Fig. 19).


 

SECTION: Part 3 – Mapping of projecting structures
13. 
 

Select the vertices corresponding to your BSR in Meshlab (Filters --> Selection --> Select Vertices from Faces) (Fig. 18). Subsequently, apply Laplacian smoothing on the selected area, (Filters --> 
Smoothing, Fairing and Deformation --> Laplacian Smooth --> Apply), maintaining Meshlab's default number of smoothing steps (3).

 


 

SECTION: Part 3 – Mapping of projecting structures
15. Crop the selected bone regions surrounding the BSR, using the “Delete Selected Vertices” tool located on the right side of Meshlab’s toolbar (Fig. 20).


 

SECTION: Part 3 – Mapping of projecting structures
16. To enhance the distinction of all smoothly projecting areas on the bone surface (i.e., blue-colored structures in the figure), apply colorization to the BSR surface based on computed principal curvature directions (Filters → Color Creation and Processing → Compute Curvature Principal Directions). In the pop-up panel, use the software’s default method "Quadric Fitting" and set the quality to "Min Curvature." This approach will colorize each vertex of the BSR according to the minimum observed curvature, which is ideal for capturing more subtle surface variations (Fig. 21). In case the filter fails to apply due to the presence of manifold edges, then these have to be first removed using the “Repair non Manifold Edges” option (Filters --> Cleaning and Repairing --> Repair non Manifold Edges --> Apply). 


 



 

SECTION: Part 3 – Mapping of projecting structures
17. To standardize all shades of blue in the BSR, equalize the color histogram to ensure uniform blue shades (Filters --> Color Creation and Processing --> Equalize Vertex Color --> Apply) (Fig. 22).


 

SECTION: Part 4 – Delineation and measurement of projecting structures
18. Select all projecting areas in the BSR, which should appear in blue after the previous colorization process (Fig. 23). Navigate to "Filters --> Selection --> Select Faces by Color". In the pop-up window, choose the blue color (#0000ff) and set “Variation from Blue or Value” to the maximum (1.0). Leave the remaining settings at their default values and click Apply. 

 


 

SECTION: Part 4 – Delineation and measurement of projecting structures
19. Invert the selection (as previously described in Step 14) to select all areas of the BSR except for the previously selected blue regions (Filters --> Selection --> Invert Selection) (Fig. 24).


 

SECTION: Part 4 – Delineation and measurement of projecting structures
20. Crop the selected regions surrounding the blue-colored projecting areas, using the “Delete Selected Vertices” tool located on the right side of Meshlab’s toolbar (Fig. 25).


 

SECTION: Part 4 – Delineation and measurement of projecting structures
21. In the remaining surface (now entirely blue), automatically detect and remove small and unconnected bone projections ("outliers" or "islands"), as these cannot typically be safely associated with the main entheseal changes observed (Filters --> Cleaning and Repairing --> Remove Isolated Pieces - wrt Diameter --> Apply). In the pop-up window, set the diameter threshold to 2 mm and then click Apply (Fig. 26). 

Note that the percentage (%) automatically indicated next to that value (2 mm) tended to be around 5% for the example human specimens analyzed for developing this protocol and its associated validation paper.


 


 



SECTION: Part 4 – Delineation and measurement of projecting structures
22. Select all remaining regions (Filters --> Selection --> Select All) (Fig. 27).


 

SECTION: Part 4 – Delineation and measurement of projecting structures
23. Measure the surface area of the final selected blue regions in square mm (provided the model is calibrated accordingly) by navigating to “Filters --> Quality Measure and Computations --> Compute Area/Perimeter of Selection”. The “Selection Surface Area” measurement will be displayed in the window at the bottom right corner of Meshlab (Fig. 28). The exact location of the measurement indication is also highlighted in the example delineation video provided in the "Before starting" section.


 

SECTION: Part 4 – Delineation and measurement of projecting structures
24. Optionally, you may project the delineated entheseal changes onto the bone 3D model of the bone by importing the original 3D model file (e.g., the ply file of the first metacarpal) into Meshlab. To overlay this model, you may "drag and drop" the file into the same workspace where the processed model is displayed, by holding down the left mouse button during the action. This overlay process, as illustrated in Fig. 29 as well as the example delineation video provided in the "Before starting" section, allows you to visualize the selected regions on top of the bone model. It provides an opportunity for final verification to ensure that the workflow was executed correctly and to check for any potentially irrelevant areas still selected (e.g., areas outside the defined BSR or regions with clear taphonomic or pathological damage). If additional adjustments are needed, such as eliminating erroneously selected areas using Meshlab’s selection tools, document the decision and reasoning clearly in your study’s Methods section (optimally using illustrations). Eight additional examples of this process are illustrated below in Fig. 30, corresponding to the eight example cases of BSR selection discussed in Part 2 of this protocol (Case-Steps 2-9).