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IN VITRO EVALUATION OF MESENCHYMAL STEM CELL BEHAVIOUR IN SCAFFOLDS CONTAINING DIFFERENT CONCENTRATIONS OF ALGINATE AND SODIUM CHLORIDE AS A STRATEGY IN REGENERATIVE MEDICINE
Alginate is a biomaterial widely used in the areas of odontology, pharmacy and food industry, and has been studied also due to its potential applications in regenerative medicine. It is a naturally occurring polymer obtained from different species of brown algae. In a certain pH range, with the addition of a bivalent ion such as calcium, it is capable of forming a hydrogel with properties similar to the extracellular matrix, becoming a potential tool in tissue engineering.
Objetivos - Metodologia - Resultados - Discussão dos Resultados/Objectives - Methodology - Results - Discussion of Results/Objetivos - Metodología - Resultados - Discusión de los resultados
The aim of this study was to evaluate which concentrations of calcium, alginate and mesenchymal stem cells (MSC) are ideal to the construction of scaffolds as a strategy in regenerative medicine. First, stem cells were treated only with calcium chloride, in order to analyse the toxicity of the excess of the ion to MSC. Thereafter, MSC, in a density of 400.000/well, were mixed to alginate 1% (w/v) and treated with different concentrations of calcium chloride (0; 25; 50; 75 and 100mM) to reticulate the biomaterial. As control, cells were cultivated as usual in the tissue culture plate. In another experiment, 400.000 cells/well were mixed to 0.5% and 1% alginate, and 50mM of calcium chloride were added. Ultimately, MSC, in the densities of 100.000, 200.000 and 400.000 per well were added to 1% alginate and treated with 50mM of calcium chloride. Experiments were performed with MSC obtained from human exfoliated deciduous teeth; cell viability was assessed via MTT assay and visualized with fluorescence microscopy after fluorescein diacetate and propidium iodide staining. As a measure of cytotoxicity, the lactate dehydrogenase enzyme (LDH) was dosed and free calcium was quantified. Results showed that after a few hours, even the smallest excess of calcium was toxic to the cells in the absence of alginate. Moreover, it was demonstrated that, after seven days, no significant statistical difference between scaffolds with 1% alginate, crosslinked with 50mM of calcium and 100.000 cells and the control wells (with the same concentration of MSC). However, in higher concentrations (especially 400.000/well) it was observed that cell viability was superior in the scaffolds. Furthermore, free calcium concentrations remained constant and LDH dosages corroborated with the data above. After seven days, the average absorbances of 400.000 cells in 1% alginate scaffolds with 25 and 50mM of calcium were superior to control; however, experiments have demonstrated that 25mM were not sufficient for completely reticulating alginate, giving rise to soft gels. In the same way, 1% alginate scaffolds showed better results in terms of viability when compared to control, and 0.5% alginate generated very malleable scaffolds, leading to the loss of a number of cells during manipulations.
Considerações Finais/Final considerations/Consideraciones finales
These results suggest that alginate provides a three-dimensional microenvironment which seems to favour survival of big quantities of cells, while the same amount of cells cultivated in the tissue culture plate may not have the same conditions. Besides that, 1% alginate and 50mM calcium chloride demonstrated the best results in mimicking, as far as possible, the characteristics of a natural extracellular matrix. Acknowledgments: MCTI, FINEP, CNPq and Stem Cell Research Institute (IPCT).
Palavras-chave/Key words/Palabras clave
Tissue engineering; regenerative medicine; mesenchymal stem cells; biomaterials; alginate; calcium.
Mesenchymal stem cells/adultas
CAROLINA LANDAU ALBRECHT, Natasha Maurmann, Patricia Pranke