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Objectives and questions: Successful anterior cruciate ligament (ACL) reconstruction critically depends on biological tendon-bone healing within the osseous drill tunnels. Although available human data are largely derived from revision specimens, comprehensive immunohistological characterization of the tendon–bone adaptation zone remains limited. The cellular and molecular characteristics of the tendon-bone adaptation zone in humans are therefore incompletely understood. The aim of this study was to immunohistologically characterize the tendon-bone interface after ACL reconstruction with hamstring autografts in human revision specimens, with particular emphasis on osteogenic cell populations, angiogenesis, and remodelling activity.

Material and methods: Ten well-preserved tendon-bone interface specimens were selected from patients undergoing revision ACL reconstruction due to graft failure. Specimens were harvested by over-reaming the original femoral and/or tibial drill tunnels at the time of revision surgery, performed between 5.4 month and 7,97 years after primary ACL reconstruction. All samples were processed for immunohistochemical analysis using markers for osteogenic induction (bone morphogenetic protein-2 (BMP-2)), osteogenesis inhibition (bone morphogenetic protein-3 (BMP-3)), osteoblast differentiation (bone-specific alkaline phosphatase-1 (BAP-1)), osteoclast activity (tartrate-resistant acid phosphatase (TRAP+)), and angiogenesis (vascular endothelial growth factor-2 (VEGF-2)). All specimens were evaluated independently by two observers. Qualitative assessment focused on cellular localization and regional distribution within the adaptation zone.

Results: All specimens demonstrated a distinct tendon–bone adaptation zone characterized by fibrovascular tissue, newly formed bone, and Sharpey-like collagen fibers extending from bone into the graft. BMP-2 and BAP-1 showed pronounced expression in osteoprogenitor cells, preosteoblasts, and osteoblasts concentrated toward the bone front, indicating active osteogenic differentiation. VEGF-2 expression was localized predominantly to endothelial cells of newly formed microvessels, suggesting ongoing angiogenesis. BMP-3 expression was present but less pronounced, while TRAP osteoclasts were identified in bone resorption zones, confirming continuous remodelling activity.

Discussion and conclusions: Human revision ACL specimens demonstrate persistent osteogenic differentiation, angiogenesis, and balanced bone remodelling at the tendon-bone interface over a wide postoperative time span. These findings indicate that the adaptation zone remains a dynamic biological compartment and that active osteoneogenesis contributes to graft anchorage via Sharpey-like fibers, even in failed reconstructions.