(LR-048) The Effects of Nintedanib on Orofacial Fibroblasts and Myoblasts in Muscle Regenration

Johannes Von den Hoff, PhD; Frank Wagener, PhD

Introduction: Following surgical interventions or acquired trauma affecting muscle tissue, fibrosis often inhibits muscle and skin regeneration, resulting in functional and aesthetic complications. For instance, fibrosis in the soft palate after cleft palate surgery impairs speech development in 10 to 30% of cleft patients. Nintedanib, an established antifibrotic drug for lung fibrosis, may be used to prevent orofacial fibrosis. This study aims to evaluate Nintedanib's potential for inhibiting the differentiation of myofibroblasts and its effect on the fusion of orofacial myoblasts into myotubes.

Methods: Rat gingival fibroblasts were isolated and cultured with TGF-β1 to induce myofibroblast differentiation. After two days of Nintedanib treatment, the expression of myofibroblast markers was assessed using RT-PCR. After four days of Nintedanib treatment, myofibroblast differentiation was further analyzed through immunofluorescence staining for α-SMA. In parallel, satellite cells (SCs), isolated from rat masseter muscles, were cultured to form myotubes. TGF-β1 was added to mimic fibrotic conditions, while Nintedanib was added to evaluate its effect on myotube formation. After two days of treatment, the gene expression of myoblast and myotube markers was assessed using RT-PCR. After four days of Nintedanib treatment, myotube formation from SCs was analyzed through immunofluorescence staining for MyHC.

Results: Adding 1 and 10 ng/ml TGF-β1 significantly increased the percentage of myofibroblasts from 12.8±6.8% (control) to 37.9±8.4% and 62.4±6.5%, respectively (p< 0.05). Although Nintedanib did not affect the percentage of myofibroblasts, it strongly decreased the total number of fibroblasts and myofibroblasts. Additionally, Nintedanib at a concentration of 2 μM markedly reduced the expression of Ki-67 in fibroblasts and myofibroblasts. In the SC cultures, 0.2 ng/ml TGF-β1 reduced the fusion index of SCs compared to the control group (28.5±6.4% vs 52.8±8.6%, p< 0.05). Treatment with 2 μM Nintedanib significantly increased the fusion index of SCs compared to the control (44.5±4.6% vs 27.6±2.0%, p< 0.05). Furthermore, MyoD and MyoG gene expression in SCs was also significantly promoted by Nintedanib at a concentration of 2 μM.

Discussion: In fibrotic conditions, Nintedanib promotes myotube formation while simultaneously inhibiting the proliferation of (myo)fibroblasts. This dual action stresses its potential as an anti-fibrotic therapy after orofacial surgery or traumatic injury affecting muscle tissue.