A Time-Dependent Evaluation of Chitosan and Therapeutic Ultrasound Treatments on Achilles Tendon Healing: Assessing Their Effects on Tendon Repair, Collagen Synthesis, and Cellular Regeneration

Abstract

Objective This study aimed to evaluate the time-dependent effects of chitosan and therapeutic ultrasound both individually and in combination on Achilles tendon healing in a rat model. Methods A total of 156 adult male Wistar albino rats were randomly assigned to five groups: Control (n = 12), Injury (n = 36), Chitosan (n = 36), Ultrasound (n = 36), and Ultrasound + Chitosan (n = 36). Achilles tendon injuries were surgically induced and repaired. Chitosan solution (> 70% deacetylated) was applied topically, while therapeutic ultrasound (1 MHz, 1 W/cm(2), 5 min/day, 5 days/week) was administered. Healing was assessed at 2, 4, and 6 weeks using functional walking tests (AFI), histological and immunohistochemical staining (type III collagen and tenascin-C), and transmission electron microscopy. Results The combined treatment group (Ultrasound + Chitosan) demonstrated the most significant improvements in functional recovery and tissue remodeling. By week 6, this group exhibited well-organized collagen fibers, increased tenocyte proliferation, and reduced expression of type III collagen and tenascin-C indicating mature tendon regeneration. In contrast, groups treated with chitosan or ultrasound alone showed moderate improvement. H-SCORE and TEM analyses supported these findings, revealing synergistic effects in collagen synthesis and fiber architecture in the combination group. Conclusion Chitosan and therapeutic ultrasound, when applied together, produce synergistic effects that accelerate and enhance Achilles tendon healing more effectively than either treatment alone. These findings suggest that this combined approach may serve as a promising strategy for tendon repair and regenerative therapy.