Divergent Macroscopic and Microscopic Patterns in Bony-Fibrous Entheses Revealed by a Multidisciplinary Approach

Introduction: Entheseal changes are influenced by factors such as physical activity, age, sex, and genetics. While macroscopic and 3D geometric morphometric approaches have been used to study EC, the relationship between macroscopic appearance and microstructure remains still poorly understood. Material and Methods: M. deltoideus enthesis was analyzed in nine adult male humeri from Santa María de la Soledad ossuary in Almansa (12th–18th centuries, Spain). Macroscopic robusticity was recorded using Mariotti method to evaluate entheseal changes, while microanatomical analysis combined m-CT scanning and histological thin sections. Mineralized area (% Min. Ar.) was quantified using Fiji-ImageJ, and spatial distribution of non-mineralized cavities was mapped using GIS software. A Kruskal-Wallis test was performed in RStudio. Results: The value and distribution of the percentage of mineralized area (% Min. Ar.) depend on the degree of robusticity. Grade 3 entheses showed lower and more variable mineralization, with a valley-shaped distribution. Grades 1 and 2 entheses had a flat-distributed % Min. Ar. whose mean was over 90%. Histological analysis revealed secondary osteon dominance, while spatial analysis showed concentration of bone cavities within the entheses development areas. Conclusion: The study suggests that macroscopic EC grades may not represent successive phases of bone remodeling and adaptation to mechanical loading. Bone growth of entheses with degrees 1 and 2 is gradual, with a compact and well-organized internal anatomy, whereas entheses with degree 3 may result from extreme mechanical stress that triggers intense bone (re)modeling. Micro-CT and histology provided complementary insights, supporting non-destructive methods for future research.