**4. Titanium research**

**Figure 4** shows that the morphology of stem cells treated with a chemotherapeutic agent of doxorubicin at 10 μg/mL on Days 1, 3, 5, and 7. A cell viability analysis of the stem cells was performed on Days 1, 3, 5, and 7. WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5- (2,4-disulfophenyl)-2H tetrazolium, monosodium salt] (CCK-8; Dojindo, Tokyo, Japan) was added to the cultures, and the spheres were incubated for 1 h at 37°C. Viable cells were identified by the assay, which relies on the ability of mitochondrial dehydrogenases to oxidize

142 Cell Culture

**Figure 4.** Morphology of the stem cells in growth media: (a) untreated group on Day 1 (original magnification 200×); (b) chemotherapeutic group of doxorubicin at 10 μg/mL on Day 1 (original magnification 200×); (c) untreated group on Day 3 (original magnification 200×); (d) chemotherapeutic group of doxorubicin at 10 μg/mL on Day 3 (original magnification 200×); (e) untreated group on Day 5 (original magnification 200×); (f) chemotherapeutic group of doxorubicin at 10 μg/mL on Day 5 (original magnification 200×); (g) untreated group on Day 7 (original magnification 200×); (h) chemotherapeutic group of doxorubicin at 10 μg/mL on Day 7 (original magnification 200×); and (i) cellular viability of the stem cells on

Days 1, 3, 5, and 7 using CCK-8. The bar indicates 200 μm.

**Figure 5** shows the morphology of the stem cell culture on modified titanium discs. Machined titanium discs measuring 10 mm in diameter and 2 mm in thickness were used. The stem cells were plated at a density of 1.0 × 10<sup>5</sup> cells/well on 24-well plates containing titanium discs and cultured. Each implant disc was fixed with 4% paraformaldehyde at room temperature for 30 min. Permeabilization was performed with 0.1% Triton X-100/Dulbecco's phosphate-buffered saline for 2 min and blocking solution consisting of 0.2 μm filtered 1% bovine serum albumin/ Dulbecco's phosphate-buffered saline for 30 min. Actin filaments were stained with rhodamineconjugated phalloidin (Molecular Probes, Eugene, OR), and the nuclei were counterstained with 4′,6-diamidino-2-phenylindole. The cells were observed using a confocal laser microscope (LSM5 Pascal, Zeiss, Jena, Germany) at a magnification of 200×. The cells attached to the titanium discs showed well-organized actin cytoskeletons with blue nuclei with confocal microscopy.

**Figure 5.** The morphology of stem cells culture on modified titanium discs. (a) Disc with limited number of cells (objective lens 20×) and (b) disc with higher number of cells (objective lens 20×).
