RNA expression analysis in dental pulp cells cultured by cell sheets

Abstract

Tissue Engineering is a multidisciplinary science that aims to develop technology needed to replacement or repair of absent or compromised organs and tissues.Three-dimensional structure is essential for tissue and organs behavior since cell fate is regulated by signals from extracellular matrix environment that leads them to proliferation, differentiation, apoptosis or migration.Autologous cells are important Tissue Engineering sources due to avoiding immunological incompatibility, however, expansion and purification processing protocols has not been established, furthermore, the maintenance of properties expressed in origin tissue while they are expanded in culture is still a challenge. The interaction between tissue and organs cells occurs through receptors, often on surface proteins which interact to their extracellular ligands like glycans, growth factors, proteins and neighboring cells. In many cases the connect triggers specific signaling pathways interfering on cell fate.The cell transcriptome involves protein synthesis and regulation through RNAm transcription, alternative splicing and microRNA, these transcriptomes dramatically changes in response to environment and physiological condition, modifying cellular phenotype.The cell culture intends to ensure in vitro growth and proliferation, however, traditional culture methods use proteolytic enzymes, as trypsin, which destroys the cell-cell interaction, surface proteins and extracellular matrix, these structures has a crucial role in cell signaling. Studies indicate receptors immediate interaction with cytoskeleton, nucleus, nucleolus and cell adhesion influence in growth factors translocation to the nucleus.Cell sheets cultures (CS) were developed to allow cell detachment, after cultured cells growth and confluence, without proteolytic enzymes. These cells remains connected to each other as a membrane, maintaining the extracellular matrix, surface proteins and cell-cell interactions. In CS technology the well-plates are coated with thermoresponsive polymers which at 32 ° C allow the release of the connected cells.On regenerative medicine fields, several experiments using CS technology revealed promising results. One study transplanted CS cardiomyocytes for myocardial reconstruction, these cells presented synchronized pulse, suggesting a morphological and electrical cell-cell connection. Another experiment transplanted oral epithelium CS in esophagus ulcerations and obtained complete healing in less time. The CS technology proved to be also profitable in periodontal tissue regeneration when CS ligamentary formed an immature cementun and periodontal tissue six weeks after transplantation. Mesenchymal cells extracted from human dental pulp demonstrate regenerative therapies potential to present great capacity for in vitro differentiation into a variety sort of tissues, however, the culture cells artificial microenvironment designed to proliferate a specific cell line and the cellular structure modifications by proteolytic enzymes, changes the signaling dynamic, cellular transcriptome and therefore the fate and interest phenotype.Presented the aforementioned context, the evaluation of culture methods which maintain cell structures, transcripts stability, preventing unwanted cellular mutation and reprogramming are essential when the final purpose is the safe clinic cell implantation to develop biological substitutes. (AU)