Title : The effects of botulinum toxin type a and ascorbic acid on elastic cartilage regeneration: A comparison of single and combined experimental applications
Abstract:
Investigation of agents that may enhance tissue regeneration in elastic cartilage damage is important for the development of regenerative therapies. Botulinum toxin type A (BTXA) is known for its ability to induce muscle paralysis, but also because it can modulate the inflammatory response and enhance fibroblast activity. Ascorbic acid (AA) supports collagen biosynthesis and possesses potent antioxidant properties. However, no experimental study has been found in the literature comparing the effects of these two agents on elastic cartilage regeneration or evaluating the potential synergistic effect of their combination. The aim of this study is to evaluate the regenerative effects of BTXA and AA, administered separately or in combination, in an induced model of elastic cartilage damage in rabbits, using histochemical and immunohistochemical methods. The study was ethically approved by the Mugla S?tk? Koçman University Animal Experiments Local Ethics Committee (MUDEM-HADYEK; 2025/01-01/25, 11/02/2025) and supported by The Scientific and Technological Research Council of Turkey (TÜB?TAK; Project No. 125S270). The study utilised 20 six-month-old female New Zealand white rabbits. Three identical elastic cartilage wound models, each 2 mm in diameter, were created in the rabbits’ ears, and the rabbits were randomly divided into four groups: control, BTXA, AA and BTXA+AA. The experiment was terminated on day 30, and the tissues were examined. In histochemical assessments, haematoxylin and eosin, orcein, Masson’s trichrome and safranin-O/fast green stains were used; in immunohistochemical analyses, anti-TGF-β3 and anti-Ki-67 antibodies were applied, and the samples were examined using a light microscope. All groups were assessed in terms of epimorphic regeneration score, elastic fibre regeneration score and proportion of safranin-O-positive areas and H-Score. The epimorphic regeneration score was significantly higher in the BTXA group compared with the control and AA groups (p<.05; p<.001). In the BTXA+AA group, it was significantly higher than in the control, BTXA and AA groups (p<.001). It was found that the elastic fibre regeneration score in the BTXA group was higher than in the control and AA groups (p<.001). In the safranin-O/fast green staining, the percentage of safranin-O-positive area in the BTXA+AA group was significantly higher than in the control and AA groups (p<.001). The Ki-67 score was significantly higher in the BTXA group compared with the AA group (p<.05). The Ki-67 H-score was significantly higher in the BTXA+AA group compared with all other groups (p<.001, p<.01 and p<.001). TGF-β3 immunoreactivity was significantly increased in the BTXA group compared with the control group (p<.001). It was also significantly higher in the AA group compared with the control group (p<.05). The findings show that BTXA induces the regeneration of elastic cartilage. It also shows that AA contributes to tissue repair by supporting regenerative pathways, but that it cannot provide a sufficient level of regeneration on its own. However, the combination of BTXA and AA demonstrated the most pronounced regenerative effect in terms of epimorphic cartilage regeneration, matrix organisation and cellular proliferation. These results suggest that the combination of BTXA and AA may represent a promising new approach to the treatment of elastic cartilage damage.
