BIOLOGICAL PROPERTIES OF POLYMER-HAP COMPOSITE

MTS array as applying for mazan (CellTiter 96® Aqueous One Solution Cell Prolif­eration Assay, Promega, Madison, WI) used the novel tetrazolium compound (MTS) and the electron coupling reagent, phenazine metho sulfate (PMS). MTS is chemi­cally reduced by cells into formazan, which is soluble in tissue culture medium. The measurement of the absorbance of the formazan can be carried out using 96 well microplates at 492 nm. The assay measures dehydrogenase enzyme activity found in metabolically active cells.

The metabolic activity of cells was monitored using the MTS that is chemically reduced by metabolically active cells into formazan. The amount of formazan pro­duced is an indicator of the cell viability. The measurement of formazan absorbance was performed in 96 well plates after several days of incubation. Standard curves were prepared by diluting a series of cell suspensions from 15.7 cells/mL to 157,000 cells/ mL. An aliquot (1.0 mL) of each dilution was transferred to wells of a 24-well tissue culture plate in triplicate. Subsequently, 150 nL of the MTS solution was added to each suspension.

The plate was incubated at 37 °C in a humidified atmosphere containing 5% CO2 in the dark for 4 h, after which 1.0 mL of the kit’s solubilization/stop solution was added to each well. Plates was sealed and incubated overnight. Absorbance was read at 570 nm wavelength and also at 650 nm as a reference wavelength using the plate fluorimeter.

The scaffolds were sterilized by incubating with 70% ethanol for 30 min, seeded with human osteoblast-like cells (SaOS-2) and cultured for up to 14 days. The vi­ability of the osteoblasts seeded to the electrospun scaffolds was determined first by using the MTS assay (Fig. 2.13). The amount of formazan produced is proportional to number of living cells in culture since the chemical reduction of formazin to a colored product is dependent on the number of viable cells. The MTS viability assay demon­strated that the cells exposed to these scaffolds maintain the ability to proliferate for up to 14 days that the experiment lasted for.

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FIGURE 2.13 MTS cell viability assay testing results.44

To evaluate cell morphology on the scaffolds, samples were prepared for elec­tron microscopy (EM) staining. Samples were washed 2 times with PBS and fixed with 2.5% gluteraldeyde [Sigma] for 2 h then washed with PBS. The cell-scaffold constructs were then attached to aluminum stubs, sputter-coated with gold, and then examined under a LEO-Gemini Schottky FEG scanning electron microscope.

The cells exposed to the nano scaffolds interacted with multiple fibers. Anchoring sites for cell attachment to the fibers were visualized by SEM. The nanoclusters of HAp mineral were consistently located at the edge of cells, which provides additional evidence that they act as anchoring sites for cell attachment to the fibrous hybrids. This improvement in cell adhesion and growth are deemed to the biological role of HAp. Figure 2.14 shows the morphology of the cells cultured on the scaffolds for 1, 7 and 14 days. The size and number of the cells are both increasing with time.

Our studies suggest that the morphology and structure of the CA-HAp composite scaffolds play important roles in facilitating cell spreading and differentiation and en­hance apatite mineralization. Based on our observations, the electrospun CA scaffolds with nanosized HAp are considered as a promising candidate for bone tissue engineer­ing application.

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FIGURE 2.14 SEM images of cell morphologies on scaffold sculptured for up to 14 days. Yellow marks show the anchor age sites of cells. (a) CA-HAp day 1, (b) CA-HAp day 1 (high magnification), (c) CA-HAp day 7, (d) CA-HAp day 7 (high magnification), (e) CA-HAp day 14, (f) CA-HAp day 14 (high magnification).

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