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| Author(s): |
Carolina Scanavez Martins
Total Authors: 1
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| Document type: | Master's Dissertation |
| Press: | Ribeirão Preto. |
| Institution: | Universidade de São Paulo (USP). Faculdade de Odontologia de Ribeirão Preto (PCARP/BC) |
| Defense date: | 2012-08-03 |
| Examining board members: |
Paulo Tambasco de Oliveira;
Daniela Bazan Palioto Bulle;
Luana Marotta Reis de Vasconcellos
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| Advisor: | Paulo Tambasco de Oliveira |
| Abstract | |
Bioactive glasses and glass-ceramics have been successfully applied in various therapeutic strategies to promote the formation of mineralized matrix in bone defects. The exposure of these materials to biological fluids results in chemical and topographical modifications that may affect the interactions of cells with the biomaterial surface, with potential effects on cytoskeletal protein expression and/or organization and cell spreading. The aim of the present study was to evaluate whether changes in the labelling pattern for the cytoskeletal proteins actin and tubulin in osteogenic cells cultured on bioactive Bioglass® 45S5 and Biosilicate® are due to altered mRNA and protein expression levels. Osteogenic cells were obtained by enzymatic digestion of newborn Wistar rat calvarial bone and plated on Bioglass® 45S5, Biosilicate® and borosilicate (bioinert control) for periods of up to 7 days. The following parameters were assayed: i) qualitative epifluorescence analysis of actin and tubulin distribution; ii) quantitative mRNA expression for actin and tubulin by real time polymerase chain reaction (real time PCR); iii) quantitative actin and tubulin expression by enzymelinked immunoabsorbent assay (ELISA), and iv) qualitative analysis of cell morphology by scanning electron microscopy (SEM). At days 3 and 7, cells grown on borosilicate showed typical actin and tubulin labeling patterns of adherent and spread cells on flat, rigid substrates, whereas those on Bioglass® 45S5 and Biosilicate® showed dark areas devoid of fluorescent signals for the cytoskeletal proteins. At the same time points, cultures grown on the bioactive materials showed significant changes in mRNA expression for actin and tubulin, although only slight differences in the amount of actin and tubulin were detected compared with borosilicate. Moreover, a positive correlation between mRNA and protein expression levels as well as a correspondence between epifluorescence imaging and the quantitative data were only detected for cultures grown on borosilicate. SEM analysis revealed that cells cultured on bioactive surfaces were partly or totally covered with material accumulations, whose characteristics resembled the ones for the substrate topography, and which, in some cases, prevented the visualization of the cell limits. In conclusion, Bioglass® 45S5 and Biosilicate® affect actin and tubulin mRNA levels, but not the corresponding protein expression, in osteogenic cell cultures. Thus, the observed changes in the labeling pattern for these proteins should be attributed, at least in part, to the accumulation of materials on the cell surface, likely due to substrate reactions that take place on Bioglass® 45S5 and Biosilicate® when exposed to the cell culture medium. (AU) | |
| FAPESP's process: | 10/04063-9 - Expression of the cytoskeletal proteins actin and tubulin in osteogenic cells cultured on bioactive glass-based surfaces |
| Grantee: | Carolina Scanavez Martins |
| Support Opportunities: | Scholarships in Brazil - Master |