A busca por técnicas alternativas para suprir a escassez de tecidos e órgãos danificados levou ao surgimento da engenharia de tecidos. Scaffolds biológicos criados a partir da matriz extracelular (MEC) de órgãos e tecidos tem sido uma promissora ferramenta aplicada para suprir esta necessidade. A matriz extracelular placentária descelularizada surge como uma potencial ferramenta para a produção de scaffolds biológicos para recelularização e implantação em áreas lesionadas. Para ser classificado como um scaffolds biológico ideal, a matriz extracelular deve ser acelular e ter preservada suas proteínas e características físicas para viabilizar a adesão celular. Neste contexto, desenvolvemos o scaffolds biológico descelularizado a partir de placentas caninas com 35 e 40 dias de gestação. A eficiência da descelularização foi confirmada pela ausência de conteúdo celular e quantidade de DNA remanescente. A arquitetura vascular e as proteínas da matriz extracelular, tais como, colágenos tipo I, III e IV, laminina e fibronectina, foram preservadas. Para o processo de recelularização, utilizamos células-tronco progenitoras endoteliais derivadas do saco vitelino canino (SVC) e células tronco mesenquimais (CTMs) derivadas de medula óssea canina (CMOC) e de polpa de dente canina (CPDC). O processo de recelularização em placas não aderentes por 7 e 14 dias, na presença do scaffolds placentário secos em ponto crítico auxiliou na eficiência da recelularização, comprovada por imunofluorescência e microscopia eletrônica de varredura, evidenciando a adesão das células no scaffolds e comprovando ser um promissor biomaterial para utilização na medicina regenerativa tecidual. / The search for alternative techniques to address the scarcity of damaged tissues and organs has led to the emergence of tissue engineering. Biological scaffolds created from the extracellular matrix (ECM) of organs and tissues have been a promising applied tool to meet this need. The decellularized placental extracellular matrix appears as a potential tool for the production of biological scaffolds for recellularization and implantation in injured areas. To be classified as an ideal biological scaffold, the extracellular matrix must be acellular and have preserved its proteins and physical characteristics to enable cell adhesion. In this context, we developed the biological scaffold decellularized from canine placentas with 35 and 40 days of gestation. The efficiency of the decellularization was confirmed by the absence of cellular content and amount of DNA remaining. Vascular architecture and extracellular matrix proteins, such as collagens type I, III and IV, laminin and fibronectin, have been preserved. For the process of recellularization, we used stem cells derived from the canine yolk sac (CYSC) and mesenchymal stem cells (MSCs) derived from canine bone marrow (CBMC) and canine dental pulp (CDPC). The process of recellularization in non-adherent plaques for 7 and 14 days in the presence of placental scaffold dried at a critical point assisted in the efficiency of the recellularization, evidenced by immunocytochemistry and scanning electron microscopy, evidencing the adhesion of the cells in the scaffold and proving to be a promising biomaterial for use in tissue regenerative medicine.

This research investigated gene expression in the canine placenta during the peripartum period. Previous studies have recognized molecular changes that occur in the placenta around the time of placental release in other species, but no study has looked at gene expression in the late gestation canine placenta. Of particular significance for this thesis work is the groundwork laid for future studies modeling placental abnormalities in dogs (e.g. subinvolution of placental sites) and humans (e.g. preeclampsia, placenta accreta). Despite years of research in multiple species, the exact mechanisms and processes regulating trophoblast invasion and placental release remain unclear. Therefore, the specific objective of this research was to characterize gene expression changes that occur during the peripartum period in the dog using microarray and real-time RT-PCR. Following total RNA isolation, the microarray analysis was performed by hybridizing total RNA to the Canine 2.0 Array (Affymetrix, Santa Clara, CA). Microarray analysis was carried out using the limma and affy packages through the Bioconductor software in the R statistical environment. Differential expression was defined as p ��� 0.05, FDR p ��� 0.10 and a log fold change of ��� 1.2. Following cDNA synthesis, real-time RT-PCR was performed using TaqMan primer and probes that were pre-made and pre-optimized for canine tissues (Applied Biosystems, Carlsbad, CA). Microarray analysis showed differential expression in 18 genes between pre-term and pre-labor sample groups, 38 genes that were differentially expressed between pre-term and parturient samples and no genes that were differentially expressed between pre-labor and parturient samples. Microarray analysis led to the identification of several candidate genes for closer investigation using real-time RT-PCR. These genes included MMP-1, MMP-2, MMP-9, TIMP-2, VEGF-A, Flt-1, CD44, DAG-1, IL-6 and CXCL10. All of these genes have been linked to trophoblast invasion or regression or placental release in a number of species including humans, cattle and rodents. Using real-time RT-PCR, there was a significant difference in MMP-9 mRNA expression in pre-term samples compared to pre- labor and parturient samples (p<0.05). However, there was no significant difference in mRNA expression of MMP-2, TIMP-2, VEGF-A, Flt-1 CD44, DAG-1, IL-6 or CXCL10. Future studies may focus on additional candidate genes identified by microarray that play a role in tissue remodeling at the end of canine gestation such as IL-8, EPHX2, PI3 and SERPINE1. / Graduation date: 2013