Enhanced Periorbital Tissue Remodeling through Sequential Collagenase and Radiofrequency Treatment: A Quantitative Nanoscale Analysis in a Rabbit Model

Background: Despite the widespread use of collagenase and radiofrequency treatments in tissue remodeling procedures, the underlying physicochemical mechanisms of their sequential application and synergistic effects on dermal collagen remodeling remain poorly understood. Understanding these mechanisms is particularly important for periorbital rejuvenation, where the delicate eyelid and periocular tissues require optimized treatment protocols that maximize efficacy while ensuring safety. This study aimed to investigate the physicochemical effects of 0.01% collagenase pretreatment combined with radiofrequency thermal treatment on rabbit dermal collagen matrix—serving as a model for periorbital tissue—during the first 30 days after treatment. Methods: An experimental radiofrequency protocol based on clinical periorbital therapy (10–30 W power with multiple passages) was applied to 50 male white New Zealand rabbits following 0.01% collagenase pretreatment. Histological changes and surface characteristics were monitored using three histological staining methods (hematoxylin-eosin, Masson's trichrome, and picrosirius red) and two nanoscale surface analysis tools (scanning electron microscopy and atomic force microscopy) over 30 days. The collagen fibril structure, inflammatory response, myofibroblast density, and collagen matrix remodeling were evaluated to understand the tissue healing dynamics relevant to periorbital applications. Results: Pretreatment with 0.01% collagenase induced a controlled inflammatory response in the collagen fibril matrix, revealing locally degraded collagen matrix and swollen collagen fibrils with a random radial fibril structure. Subsequent 20 W-powered multiple-pass radiofrequency thermal stimulation enhanced the heat-mediated healing response, resulting in significant increases in myofibroblast density (1.7-fold enhancement, p < 0.05) and collagen density, as well as collagen fibril contraction (13.17 nm decrease). The combination treatment promoted new collagen synthesis more effectively than radiofrequency treatment alone, as evidenced by picrosirius red staining under polarized microscopy and atomic force microscopy measurements. Conclusions: The sequential application of collagenase and radiofrequency treatments accelerates the healing response through radiofrequency thermal stimulation acting on collagenase-induced controlled inflammation. This study provides the first quantitative evidence of enhanced collagen remodeling using nanoscale assessment technologies. While these findings demonstrate promising potential for periorbital and eyelid tissue rejuvenation in ophthalmic aesthetic applications, further clinical trials are required to determine the safety and efficacy of this combination therapy in human periocular tissues, particularly regarding eyelid function, ocular surface integrity, and proximity to sensitive ocular structures. Following appropriate clinical validation, this approach may offer ophthalmologists an enhanced non-invasive option for periorbital wrinkle improvement and skin tightness restoration.