Deciphering the heterogeneity of calcified structure in different entheses via high-resolution characterization techniques

Enthesis is a specialized structure where tendons anchor to bone, enabling efficient transmission of force from tendon to bone. There are numerous entheses present throughout the body, bearing different tensile loads. However, our understanding of different entheses remains limited, particularly in calcified structure at the microscale that undertakes more significant mechanical tasks. Here, we employed multiple high-resolution techniques and proteomics to decipher the heterogeneity of three entheses at supraspinatus, patellar tendon and Achilles tendon in Sprague-Dawley rats. Our findings revealed that although these three entheses had consistent overall composition, the characteristics of lacunae analysis showed the enthesis of supraspinatus displayed the most circular lacunae, while the enthesis of patellar tendon exhibited the most flattened lacunae. In contrast, Achilles tendon enthesis exhibited smaller, fewer, and more circular lacunae. Meanwhile, the heterogeneity might be regulated by Fibronectin-1 (FN1), Versican (VCAN), and three enriched pathways related to the formation of cartilage extracellular matrix networks. Furthermore, despite their narrower widths of the transition region at calcification front, both the supraspinatus and Achilles tendon entheses exhibited more graded features based on the presence of ACP, compared to the enthesis of patellar tendon. The heterogeneity might be regulated by Ectonucleotide pyrophosphatase/phosphodiesterase-1 (ENPP1), Secreted phosphoprotein-1 (SPP1), and two enriched pathways related to endochondral bone morphogenesis. In summary, we interpreted the heterogeneity of calcified structure in these three entheses, which helped us to understand how different entheses adapt to various tensile loads, thereby providing valuable insights into the design of diverse entheses grafts and promoting future clinical treatment. STATEMENT OF SIGNIFICANCE: The enthesis plays a crucial role in load transferring between tendon and bone. Moreover, the entheses at different locations bear distinct tensile loads, implying their potential heterogeneity. Regrettably, current understanding of heterogeneity across entheses is limited, as most studies focus on a single site. Therefore, this study employs multiple high-resolution techniques to investigate three typical entheses of Sprague-Dawley rats, aiming to identify their heterogeneity at calcified fibrocartilage and calcification front. Meanwhile, proteomics analysis also reveals differential proteins and enriched pathways related to the formation of the observed heterogeneity. These discoveries contribute to promoting the understanding of how entheses adapt to different tensile loads, thereby furnishing compelling substantiation for the advancement of the design of different entheses grafts.