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APICAL PAPILLA STEM CELLS: CHANGES IN PROTOCOL ISOLATION AND NEW DISCOVERIES
Dental apical papilla is an important source of stem cells and the establishment of a protocol to preserve the stem cell niche is very important for the development of effective therapies.
Objetivos - Metodologia - Resultados - Discussão dos Resultados/Objectives - Methodology - Results - Discussion of Results/Objetivos - Metodología - Resultados - Discusión de los resultados
Therefore, the objective of this study was to establish an innovative propose to stem cell from human apical papilla (SCAPs) isolation aiming to preserve the stem cell niche in in vitro environment. Ten healthy donors aged 13–16 years were invited to donate third molar teeth and the apical papilla region was collected. To evaluate the influence of enzymatic digestion in cell migration, explants were submitted to different concentrations of Type I Collagenase (Col), Dispase (Dis) and TrypLE™ Express. The enzymes were neutralized with DMEM/F12 supplemented with fetal bovine serum (FBS). Stem cell markers (CD90, CD44, CD146, p75, STRO-1, CD14, and CD19) were analyzed by flow cytometry. Proliferation and migratory capacity of SCAPs were assessed in a three-dimensional (3D) culture system of Type I Collagen gel matrix. To investigate the influence of the serum supplementation medium, SCAPs were maintained in DMEM/F12 supplemented with (1) 10% fetal bovine serum, (2) 10 % human serum (HS), or (3) 2% HS plus 1% insulin-transferrin-selenite media (ITS). Cell migration from explants incubated in Col 0.5 mg/ml began at 7 days with complete extracellular matrix (ECM) preservation. In TrypLE group, the cell migration was observed at 14 days with complete ECM preservation until 21 days. Col 3.0 mg/mL and Col/Dis caused the complete ECM disruption at 21 days and 14 days respectively. Cells expressed positively for CD90, CD44, p75 and CD146, and negatively for STRO-1, CD14 and CD19. Development of 3D culture system was able to support papilla niche, with cells migrating continuously after consecutives passages of the same explant, suggesting the presence of cell niches. HS and HS plus ITS groups presented an innate ability to form spheroid-like clusters with fibroblast- or neuro-like features. Supplementation with HS plus ITS decreased p75, but not CD90 expression. In conclusion, low concentration of digestive enzymes preserved extracellular matrix and may retain stem cell niches in dental papilla tissue. The 3D culture system was able to support papilla stem cell niche and induced striking differences in cell shape, proliferation and migration. HS and HS plus ITS supplementation changed morphological and phenotypic characteristics of SCAPs.
Considerações Finais/Final considerations/Consideraciones finales
Taken together, this study provides that processing methods should be designed to maintain a biologic scaffold composed of apical papilla and that SCAPs display cell-intrinsic differences in response to microenvironmental factors.
Palavras-chave/Key words/Palabras clave
dental apical papilla, mesenchymal stem cells, adult stem cells, human serum, insulin-transferrin-selenite media, ITS, 3D culture, niche stem cells.
Mesenchymal stem cells/adultas
LARISSA MATUDA MACEDO, Lorrany Aparecida Gomes Silva, Artur Christian Garcia da Silva, Marize Campos Valadares, Eliana Martins Lima, Gregory Thomas Kitten, Carlos Henrique de Castro, Elisandra Gava