REGENERATION OF ORAL MUCOSA IN A PORCINE MODEL THROUGH TISSUE ENGINEERING AND BIOPRINTING

Resumo

Introduction: Extensive oral mucosa lesions resulting from surgical resections, trauma, chronic diseases, or complications of antineoplastic therapies impair essential functions such as mastication, speech, and swallowing, in addition to representing a major reconstructive challenge. Although autogenous grafting is still considered the gold standard for repair ing these defects, its limitations, including donor -site morbidity and limited tissue availability, reinforce the need for alternative therapies. In this context, biomimetic tissues associated with the use of decellularized extracellular matrix have emerge d as a promising strategy for the development of oral mucosa substitutes. Objective: The present study proposes the development of a 3D bioprinted construct using a hybrid bioink composed of decellularized porcine buccal mucosa extracellular matrix, alginate, and GelMA, followed by recellularization with human fibroblasts and keratinocytes, aiming to obtain an oral mucosa model. Methodology: To achieve this, tissue decellularization was performed. Briefly, 1 cm² porcine oral mucosa fragments were treated wi th 0.5% and 1% sodium dodecyl sulfate (SDS) for 4 days under continuous agitation (100 rpm), with daily solution replacement. Detergent removal was subsequently carried out by washing in 1× PBS for 24 hours, with solution changes every 3 hours. Decellulari zed tissues were characterized by histology (Hematoxylin and Eosin, Picrosirius Red, Alcian Blue, and Masson’s Trichrome), residual DNA quantification, immunohistochemical analysis (collagen IV, vimentin, and fibronectin), and scanning electron microscopy to evaluate cell removal efficiency and extracellular matrix preservation. Results: Partial results demonstrated that the 0.5% SDS concentration showed superior performance, promoting effective decellularization and greater preservation of extracellular ma trix components compared with the 1% concentration. The efficiency of the process was confirmed by DNA extraction and quantification, with mean values of 10.47 ng/mg of tissue in the 0.5% group and 11.10 ng/mg in the 1% group. These values are consistent w ith the decellularization criteria described in the literature, which establish residual content below 50 ng/mg of tissue. In the immunohistochemical analysis, vimentin staining in the decellularized tissues was not satisfactory, whereas collagen IV and fibronectin indicated good preservation after removal of the cellular content from the matrix. Furthermore, histological and ultrastructural analyses revealed changes compatible with the decellularization process, such as preservation of tissue architecture and pore opening. Conclusion: Based on these findings, the decellularization process was considered successful at both concentrations, with 0.5% SDS being selected due to its superior preservation profile. Thus, the results demonstrate the feasibility of p rocessing the decellularized extracellular matrix to be digested and combined with GelMA and alginate polymers, exploring its potential in 3D bioprinting techniques for the development of formulations that better mimic the oral mucosa microenvironment and provide ideal conditions for cell survival and proliferation.