Biomedical Engineering Reference
In-Depth Information
2.3 Cell differentiation
Mesenchymal stem cells were cultivated in T75 flasks with control medium (DMEM +
10%SBF) and chondrogenic medium for 1, 3, 6 and 9 weeks. To verify to differentiation
process immunofluorescence was perfomed using antibodies for collagen II, CD54,
CD90,CD45 and CD73, osteocalcin (Zuk et al, 2002 and Huang et al, 2006).
2.4 Immuno-fluorescence
Mesenchymal stem cells (1x10
5
) cultivate in normal medium and chondrogenic medium for
1, 3, 6 and 9 weeks were used for immunofluorescence. To conduct this experiment, we used
cells from eGFP rats. Due to green GFP fluorescence, these cells were able to fluoresce under
confocal microscopy (green) and we used others markers with red fluorescence. Initially,
when we used to identify chondrogenic differentiation, we used markers from msenchymal
stem cells (CD90, CD54and CD73), hematopoietic stem cells (CD45), cartilage-specific (type
II collagen) and bone-specific (osteopontin). Cells were seeded in glass laminules. By 48 h,
all cells were fixed with formaldehyde (2%) and stained with each antibody. Each well was
washed with cold PBS 0,15M and the secondary antibodies (polyclonal anti-rat IgG made in
rabbit, Molecular Probes) were applie and the cells were observed in confocal microscope
(Soliman et al, 2008).
2.5 3D scaffolds
We developed two kinds of 3D scaffolds with similar chitosan (85% deacetylated, Sigma)
and gelatin (Sigma) ratios. The difference between scaffolds is the reticulate use:
glutaradehyde (0,1%, Sigma) or genipin (0,1%, Challenge Bioproducts). In order to solubilise
chitosan we diluted it in acetic acidic (0,5mM) and gelatin was diluted in water. Misture of
chitosan to gelatine was performed maintaining the ratio of 3 parts of chitosan solution to 1
part gelatin solution. Immediately, we shook these solutions and distributed 1mL/well in 24
wells plate. These plates were shaken in mechanic shaker overnight, protect from light. On
the next day we added 1mL reticulate to each well. After 60min, these plates were frozen at
-20°C overnight and then were transferred to -80°C. Plates were freeze-drying for 8 hours.
The cilinders (3D scaffolds) were sterililzed (120°C) and used for cell culture (Guo et al, 2006
and Yamane et al, 2005). The scaffolds were analyzed at the Department of Metallurgical
and Materials Engineering, Federal University of Minas Gerais. The matrices were covered
with gold (Sputter Coater - SPI Supplies) for 90 sec at 13mA. The images were obtained by
means of scanning electron microscope (JEOL 6360LV), at 15kV and 750mA, to qualitatively
assess the pore interconnectivity and size.
2.6 Cell and 3D scaffold
GFP cells were used because of the need to trace these cells; they were processed the same
way as the MSC characterized by IMF. These cells were cultured in a 3D scaffold with
chondrogenic medium for 3 weeks and surgically grafted in the rat subcutaneous dorsal
region. After 6 weeks, all animals were dead and the samples were analyzed by IMF to
verify presence of collagen type II. This assay was conducted because of the need to know
whether differentiated cells can dedifferentiate after implantation (Janune et al, 2006).
2.7 Statiscal analyzes
All data are presented as an average ± standard deviation (SD). To test the significance of
the observed differences between the study groups, a statistical evaluation was carried
