In vitro and in vivo biocompatibility of a porcine cholecystic extracellular matrix (CECM) membrane for tissue regeneration.

Abstract

Background: Periodontal disease affects 3.5 billion people globally, resulting in annual treatment costs exceeding $54 billion. Guided tissue regeneration (GTR) membranes are essential for periodontal therapy, but commercially available options often suffer from limitations, including high cost, limited accessibility in resource-limited settings, and suboptimal mechanical properties. This study aimed to develop and characterize a novel porcine cholecystic extracellular matrix (CECM)-based GTR membrane and comprehensively evaluate its physicochemical properties, cytocompatibility, and in vivo biocompatibility compared to the commercially available Healiguide® membrane.

Methods: CECM membranes were fabricated through systematic decellularization, lyophilization, and ethylene oxide (ETO) sterilization of porcine gallbladders. Surface characterization was performed using scanning electron microscopy (SEM) with quantitative pore analysis, and biochemical composition was assessed via Fourier Transform Infrared Spectroscopy (FTIR). MTT assays were performed on L929 fibroblast cells to evaluate cytocompatibility. Wound healing capacity was assessed using scratch assays monitored over 72 h. In vivo biocompatibility was evaluated through subcutaneous implantation in Sprague-Dawley rats, with histological analysis performed at 1, 2, 3, and 4 weeks post-implantation.

Results: SEM analysis revealed that CECM membranes exhibited a heterogeneous, multilayered structure with larger average pore sizes compared to Healiguide® (18.2 ± 4.6 µm vs. 12.5 ± 3.2 µm, p < 0.05), facilitating enhanced cellular infiltration. FTIR confirmed the preserved integrity of collagen in both membranes, with CECM showing an enhanced glycoprotein content indicative of retained bioactive components. Cytocompatibility assessment demonstrated excellent cell viability for CECM, showing 97.4 ± 1.6%, 94.2 ± 1.8%, and 90.8 ± 1.4% viability at 20, 50, and 100 µg/mL CECM extracts, respectively. The scratch assay demonstrated superior wound healing capacity for CECM, with significantly enhanced wound closure at 72 h compared to Healiguide® (89.7 ± 6.1% vs. 79.4 ± 5.8%, p < 0.05). Subcutaneous implantation studies confirmed excellent in vivo biocompatibility, with CECM showing lower initial inflammatory response (inflammation score: 2.3 ± 0.5 vs 2.8 ± 0.6 at week 1, p < 0.05), enhanced vascularization (12.3 ± 2.1 vs 9.7 ± 1.8 vessels/hpf at week 3, p < 0.05), and superior tissue integration compared to commercial controls.

Conclusion: The porcine CECM membrane demonstrated favorable physicochemical properties, excellent cytocompatibility, enhanced wound healing potential, and superior tissue integration characteristics compared to commercial GTR membranes. These preliminary findings provide a strong scientific foundation supporting the development of the GTR membrane for periodontal regenerative therapy.