BOVINE ENDOMETRIAL DECM -BASED BIOMIMETIC SCAFFOLD FOR LPS - INDUCED INFLAMMATORY RESPONSE

Resumo

Introduction: Endometritis is a uterine disorder of major relevance in veterinary medicine, particularly in large female animals, because it directly compromises fertility and generates economic losses in animal production systems. Characterized by inflammation of the endometrium, this condition can impair embryo implantation and pregnancy maintenance. However, current experimental models still have limitations in reproducing the structural, cellular, and functional complexity of the endometrial microenviro nment, restricting a deeper understanding of endometrial inflammatory responses. In this context, tissue bioengineering has emerged as a promising strategy for developing more representative in vitro models. The use of decellularized extracellular matrix ( dECM) is particularly relevant because it may preserve key structural and biochemical components, such as collagens, glycoproteins, and proteoglycans, which regulate cell adhesion, proliferation, phenotype maintenance, and cell -matrix interactions. Hypothe sis: A biomimetic three -dimensional scaffold incorporating decellularized bovine endometrial extracellular matrix will support the adhesion, viability, proliferation, and phenotype maintenance of bovine endometrial epithelial and stromal cells. Furthermore , after lipopolysaccharide stimulation, this model will reproduce selected molecular features of endometrial inflammation through the induction of pro- inflammatory cytokines. Objective: To develop a biomimetic three - dimensional endometrial model using de cellularized extracellular matrix derived from bovine endometrium incorporated into a gel -based scaffold, and to evaluate its cellular behavior and inflammatory responsiveness after lipopolysaccharide stimulation. Materials and Methods: Bovine endometrial tissue will be decellularized to remove cellular and nuclear components while preserving the architecture and biochemical composition of the extracellular matrix. The resulting dECM will be characterized by histological analysis, genomic DNA quantification , scanning electron microscopy, and immunohistochemical evaluation of specific extracellular matrix proteins. Subsequently, the dECM will be processed and incorporated into a three-dimensional gel to generate a biomimetic scaffold suitable for endometrial cell culture. The dECM -based gel scaffold will be recellularized with bovine endometrial epithelial and stromal cells, allowing the construction of a model that better represents the endometrial microenvironment. Cell adhesion, viability, proliferation, ph enotype maintenance, and cell -matrix interactions will be evaluated. Gene expression analysis by PCR will be used to assess endometrial phenotype and functional behavior. To evaluate inflammatory responsiveness, lipopolysaccharide will be used as an inducer of pro -inflammatory cytokine synthesis. This model will not aim to reproduce the complete inflammatory stage of endometritis, but rather to establish an in vitro platform capable of modeling selected molecular features of an LPS-induced endometrial inflammatory response. The production of TNF-α, IL-1β, and IL-6 will be analyzed by PCR and ELISA. Quantitative data will be analyzed according to the experimental design. Comparisons between two groups will be performed using Student’s t-test, whereas comparisons among three or more groups will be analyzed by one - way ANOVA followed by Tukey’s post hoc test, when applicable. Statistical significance will be set at p < 0.05. Expected Outcomes: The developed model is expected to reproduce key structural and cellular features of the bovine endometrial microenvironment and respond to LPS stimulation by inducing pro-inflammatory cytokines. Therefore, this biomimetic model may provide a relevant platform for studying epithelial -stromal interactions, cell -matrix respon ses, and inflammatory mechanisms associated with bovine endometrial inflammation.