Development of fibrocartilage layers in Achilles tendon enthesis in rabbits

Objective: The details regarding the development of fibrocartilage layers in Achilles tendon (AT) enthesis are unknown. Therefore, we evaluated the development of fibrocartilage layers in AT enthesis using a rabbit model. Materials and Methods: Forty-eight male Japanese white rabbits were used in this study. Six of them were euthanized at different stages (day 1, and 1, 2, 4, 6, 8, 12, and 24 weeks of age). The proliferation, apoptosis, Sox9-positivity rates, and chondrocyte number were evaluated. Additionally, safranin O-stained glycosaminoglycan (GAG) areas, width of AT enthesis, and calcaneus length were assessed. All parameters were compared to those at 24 weeks of age. Results: The level of chondrocyte apoptosis was high from 1 to 8 weeks of age, and high expression level of Sox9 was maintained from day 1 to 6 weeks of age, which decreased gradually. Safranin O-stained GAG areas increased up to 12 weeks, calcaneus length increased up to 6 weeks, and the width of AT enthesis increased up to 1 week of age. Conclusion: The changes in chondrocyte and extracellular matrix were completed by 8 and 12 weeks of age, respectively. The development of fibrocartilage layers in AT enthesis was completed by 12 weeks of age. Our results contribute to the administration of appropriate treatments based on age and aid in the development of novel methods for regenerating AT enthesis.