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PLACENTA-DERIVED MESENCHYMAL STEM CELLS CULTURE ON PMMA SCAFFOLDS AS A 3D PUTATIVE MODEL OF HEMATOPOIETIC STEM CELLS NICHE IN VITRO
The umbilical cord blood (UCB) have practical and ethical advantage over classical sources of hematopoietic stem/progenitor cells (HSPCs) for transplantation . Nevertheless, the small volume of blood available in the vascular networks of umbilical cord blood and placenta compromises its clinical use for transplantation into adults due to lower counts of HSPCs . One challenge of tissue engineering is reproducing the biological bone marrow microenvironment where the HSPCs naturally expand . The use of mesenchymal stem cells (MSCs) associated with three-dimensional (3D) scaffolds is a good approach because they represent keystone players in the marrow niche that modulates the behavior of HSPCs through biochemical signals that positively favors it symmetric divisions .
Objetivos - Metodologia - Resultados - Discussão dos Resultados/Objectives - Methodology - Results - Discussion of Results/Objetivos - Metodología - Resultados - Discusión de los resultados
The objective of this study was to evaluate the interaction and morphology of human placenta-derived mesenchymal stem cells (P-MSCs) in a 3D culture system based on poly(methyl methacrylate) (PMMA) scaffolds. For this purpose, full-term health human placentas were collected in the Department of Obstetrics from the University Hospital of the Federal University of Santa Catarina. All procedures were approved by the Human Being Research Ethics Committee of the University (Protocol Number 198/03). Cells were isolated based on their culture plastic adhesion from the chorionic villi of the human placenta which compose one of the HSPCs niche during embryonic development. As described in previous work, P-MSCs were morphologically and functionally characterized in vitro, having fibroblast-like morphology, and exhibiting ability to differentiate to adipogenic and osteogenic mesodermal linages. We also checked the cell ability to form colonies when cultured at low density. In addition, porous poly(methyl methacrylate) (PMMA) scaffolds were fabricated using a modification of the commonly used salt-fusion particulate leeching method with the polymerization of methyl methacrylate monomer mixed with 2,2′-Azobis(2-methylpropionitrile (AIBN). Stereomicroscope and scanning electron microscopy (SEM) analysis revealed test groups with typical spongy 3D structure with well-interconnected macropores and porosities ranging from 71,71% to 75,14%. These biomaterials correlate with bone trabeculae. We also observed micro-topographies pattern on the wall surfaces of all produced biomaterials. Finally, it was possible to culture the P-MSCs in the PMMA scaffolds. SEM analysis revealed that P-MSCs were firmly attached to the PMMA scaffolds and they were individually distributed or forming interconnections and clusters. The cells showed multiple morphologies with elongated or strained shape. Confocal microscopy analysis showed the presence of long and parallel actin bundles.
Considerações Finais/Final considerations/Consideraciones finales
Based on our results we concluded that this 3D culture system of P-MSCs in PMMA scaffolds may be an attractive model for ex-vivo expansion of HSPCs in association with P-MSCs as stromal elements of the niche.
Palavras-chave/Key words/Palabras clave
Mesenchymal stem cells, human placenta, PMMA scaffolds, hematopoietic stem cells niche.
Mesenchymal stem cells/adultas
RODRIGO PEREZ LUCAS, Marcio Ferreira Dutra, Rogerio Gargioni, Luiz Fernando Belchior, Bruna de Freitas Caetano, Marcio Alvarez-Silva