An ultrastructure analysis of the developing human anterior cruciate ligament tibial enthesis

This study aimed to investigate the ultrastructural anatomy of the developing ACL tibial enthesis. We hypothesized that enthesis architecture would progressively mature and remodel, eventually resembling that of the adult by the early postnatal stage. Five fresh-frozen human pediatric cadaveric knees aged 1-36 months underwent anatomical dissection to harvest the ACL insertion and underlying tibial chondroepiphysis. The samples were prepared for scanning electron microscopy (SEM) to examine the ultrastructural anatomy of the enthesis and underwent histological staining for circular polarized light (CPL) and light microscopy imaging. SEM analysis of the 1- and 8-month-old samples revealed a shallow interdigitation between the dense fibrous (ligamentous) tissue and unmineralized chondrogenic tissues, with a minimal transition zone. By 11-month, a more complex transition zone was present. By age 19- and 36-month-old, a progressively more complex and defined fibrocartilage zone was observed. CPL analysis revealed distinct collagen fiber continuity, alignment, and organization changes over time. By 19 and 36 months, the samples exhibited complex fiber arrangements and a progression toward uniform fiber orientation. Similarly, histological analysis demonstrated progressive remodeling of the enthesis with increasing age. Our results suggest that the ACL enthesis of the developing knee begins to mimic that of an adult as early as 19 months of age, as a more complex transition between ligamentous and chondro-epiphyseal tissue can be appreciated. We hypothesize that the observed changes are likely due to mechanical loading of the enthesis with the onset of weightbearing. Future investigations of ACL reconstruction and repair will benefit from improved understanding of the chondro-epiphyseal/ACL regions.