DECELLULARIZATION OF THE BOVINE MAMMARY GLAND: STRUCTURAL AND BIOCHEMICAL CHARACTERIZATION OF THE EXTRACELLULAR MATRIX FOR TISSUE ENGINEERING APPLICATIONS

Resumo

Tissue engineering based on decellularized extracellular matrix (dECM) has emerged as a promising strategy for reconstructing biomimetic microenvironments capable of preserving the structural architecture and biochemical signs of native tissues. The bovine mammary gland represents a highly relevant experimental model due to its morphofunctional similarity to human mammary tissue, exhibiting substantial potential for the development of bioactive scaffolds applicable to three -dimensional culture systems and r egenerative medicine. The present study aimed to standardize, optimize, and validate a decellularization protocol for the bovine mammary gland, as well as to perform an integrated structural and biochemical characterization of the resulting dECM. Bovine ma mmary gland fragments (1 × 0.5 cm) were subjected to three decellularization protocols employing different sodium dodecyl sulfate (SDS) concentrations (0.1%, 0.5%, and 1%), followed by treatment with 1% Triton X -100 under constant agitation for 12 days (80 rpm). Decellularization efficiency was assessed through macroscopic and histological analyses, DAPI immunofluorescence, genomic DNA quantification, scanning electron microscopy (SEM), and immunohistochemical (IHC) evaluation of key extracellular matrix pr oteins. dECM biocompatibility was investigated using an indirect cytotoxicity assay with extracts derived from wet and lyophilized dECM fragments previously sterilized through graded ethanol exposure (70%, 80%, 90%, and 100%) followed by ultraviolet irradi ation. The protocol employing 1% SDS combined with 1% Triton X -100 demonstrated superior cellular removal efficiency, yielding translucent dECM fragments with extensive nuclear depletion confirmed by DAPI and hematoxylin-eosin staining, as well as a marked reduction in genomic DNA content (4.14 ng/mg), below the established immunogenicity threshold of 50 ng/mg tissue. Simultaneously, preservation of extracellular matrix architecture was observed, including maintenance of collagen fibers (types I, III, and I V), fibronectin, and elastin, as demonstrated by IHC analyses, whereas Alcian Blue staining revealed a partial reduction in glycosaminoglycan content. SEM analysis confirmed preservation of the native three -dimensional ultrastructure, characterized by a po rous fibrillar network potentially favorable for nutrient diffusion in three -dimensional cell culture applications. Biocompatibility assays demonstrated fibroblastic cell adhesion and proliferation, suggesting the absence of cytotoxic residuals and preserv ation of matrix associated bioactive properties supporting cell -matrix interactions. Collectively, these findings demonstrate that the optimized protocol employing 1% SDS and 1% Triton X -100 enabled the generation of an architecturally preserved and biolog ically functional dECM with high biomimetic potential for subsequent recellularization and hydrogel production, supporting future applications in 3D bioprinting models of the mammary gland and contributing to advances in lactation physiology research and translational regenerative medicine.