DECELLULARIZATION OF EQUINE AURICULAR CARTILAGE TO OBTAIN A CARTILAGINOUS BIOSCAFFOLD

Resumo

Introduction: Articular cartilage lesions represent a major challenge in medicine due to the low capacity for self -repair and progression to osteoarthritis, a chronic, progressive, and disabling disease. Data from the Brazilian Unified Health System (SUS) recorded 65,602 hospital admission authorizations for total knee arthroplasty, with a reported total expenditure of approximately R$ 271,297,010.60 between 2012 and 2021. In the search for effective and accessible therapeutic alternatives, tissue engineering has explored the use of acellular scaffolds derived from the extracellular matrix (ECM). In this context, equine auricular cartilage stands out as an abundant and promising raw material. The decellularization process eliminates the risk of immune rejection while preserving essential structural components that mimic native cartilage and stimulate functional tissue regeneration. Objectives: To establish a decellularization protocol for equine auricular cartilage and to evaluate the structural integrity of the ECM. Material and Methods: This is an in vitro (ex vivo) experimental study focused on tissue engineering. Equine auricular cartilage, donated by a commercial abattoir, was subjected to two protocols. Protocol 1 (P1) used fragments (1×1 cm) treated with SDS (1%) and Triton X-100 (1%) combined with ultrasonic vibration for up to 20 days. Protocol 2 (P2) used smaller fragments (0.5×0.5 cm) with trypsin-EDTA pretreatment, followed by exposure to SDS, NaOH (0.2 N), and Triton X-100 for up to 10 days, subdivided into groups with (P2G2) and without (P2G1) ultrasonic vibration. Monitoring included histological evaluation, genomic DNA quantification, and scanning electron microscopy (SEM). Results: P1 demonstrated a discrete cellular reduction but failed to achieve complete decellularization within 20 days. In P2, the decellularization kinetics were evaluated at 3, 5, and 10 days. Analyses demonstrated that complete cell removal was achieved in just 3 days (P2G1 and P2G2), maintaining the integrity of the tissue scaffold. Histology and SEM confirmed the absence of cell bodies and the presence of empty lacunae, with the cartilaginous surface remaining intact. Nevertheless, it was observed that prolonged exposure to chemical agents for 5 and 10 days resulted in progressive damage and degradation of the ECM architecture. Genomic DNA (gDNA) quantification confirmed the efficacy of P2 at 3 days, reducing the concentration from 198.6 ng/mg (native tissue) to 5.71 ng/mg (P2G1) and 14 ng/mg (P2G2), values significantly below the standard threshold of 50 ng/mg. Conclusion: P2G1 was established as a rapid, highly effective, and optimized method for the decellularization of equine auricular cartilage. It ensures the achievement of ideal standards for genomic DNA removal while preserving the main structural proteins of the ECM, rendering the tissue a promising scaffold for clinical applications in tissue engineering and regenerative medicine.