Protocol for the ‘Validated Entheses-based Reconstruction of Activity 2.0’ (VERA 2.0) Method: Semi-Automated Measurement of 3D Entheseal Changes v1

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.