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Title: Influence of amniotic membrane scaffold on the expression of adhesion molecules in stromal stem cells
Introduction: Among the various biological materials used as scaffold, the amniotic membrane (AM) is one of greater availability, being a promising matrix for the delivery of cells. The association of the amniotic membrane with stromal stem cells has demonstrated a potential for tissue regeneration, requiring a better understanding of the interaction of this biological matrix with stromal stem cells and its possible influence on cell adhesion and migration. For this purpose, the expression of genes involved in these cellular processes was evaluated by quantitative PCR to clarify probable mechanisms of influence of the membrane on the cells.
Objetivos - Metodologia - Resultados - Discussão dos Resultados/Objectives - Methodology - Results - Discussion of Results/Objetivos - Metodología - Resultados - Discusión de los resultados
Objectives: To characterize the human amniotic membrane in vitro as a scaffold for human adipose derived stromal stem cells (ADSCs) evaluating the expression of genes involved with cell adhesion and migration.
Methodology: Cell culture of ADSCs was performed and then immunophenotyped by flow cytometry. The cells were divided in two groups: one cultured with the decellularized amniotic membrane and one of conventional cell culture. Total RNA was extracted from the two groups for the production of complementary DNA and polymerase chain reaction (PCR) was performed by qualitative and quantitative methods for beta-actin, TATA box, glyceraldehyde-3-phosphate dehydrogenase, fibronectin, N-cadherin and vimentin. The PCR results were analyzed by the 2-ΔΔCt method.
Results: The cells demonstrated mesenchymal characteristics and through quantitative PCR they suggested TBP and ACTB as normalizing genes suitable for genetic analysis. The results obtained from real time RT-qPCR for adhesion and proliferation genes presented an important variability and for this reason we didn't perform statistical analysis. We believe that the biological diversity of patients and other technical issues could affect the expression of these genes. More independent cell cultures are needed to provide more robust results. However, it can be observed that there was a slight increase in fibronectin and N-cadherin expressions, suggesting ADSCs in contact with AM can improve cellular adhesion.
Discussion: The possibility of using the amniotic membrane as a dermal substitute associating it with stem cells aims at faster recovery of damaged tissues and opens space for questions about the interaction of this scaffold with the cells and the relation of both biological materials with the bed of the wound. The expression of bioactive molecules by the stromal cells is influenced by the environment and can generate responses or suppress signals according to external stimuli which, in the case of the amniotic membrane, may be involved in its structure rich in collagen, elastin, laminin and fibronectin. The cell adhesion properties in scaffolds for tissue engineering are among the most critical characteristics that may affect its efficiency for application in regenerative medicine, hence a superior expression of N-cadherin, vimentin and fibronectin in the group of ADSCs grown with the AM can indicate it as a scaffold that makes stem cell therapy more suitable for clinical application as a dermal substitute.
Considerações Finais/Final considerations/Consideraciones finales
Final considerations: The study allowed us to consider the amniotic membrane as a suitable scaffold for stromal stem cells derived from adipose tissue and it may be suggested that it is involved in up-regulation of cell adhesion genes when co-cultured.
Palavras-chave/Key words/Palabras clave
Key words: human adipose-derived stem cells, amniotic membrane, cell adhesion
Mesenchymal stem cells/adultas
SAMLAI VEDOVATTO, Ana Paula Santin Bertoni, Aline Francielle Damo Souza, Márcia Rosângela Wink