Spectra max plus 384 mk3

Cell proliferation was measured using a Cell Counting Kit-8 (CCK-8; Beyotime, China). BM-PCs were cultured on the Ti-6Al-4V control and the CaSiO₃, Zn-Ca 0.1, and Zn-Ca 0.3 coatings placed individually in a 24-well culture plate at a density of 1 × 10⁴ cells/cm² in growth medium. After 1, 4, 7 and 14 days, the CCK-8 stock solution (10% of the total volume) was added to each well of the 24-well plates and incubated for 4 h at 37 °C and 5% CO₂. Then, 100 μl of the solution was transferred to 96-well plates, and the absorbance was read at 450 nm by a microplate reader (SPECTRA MAX PLUS 384 MK3, Thermo Fisher Scientific, USA). Additionally, cell cytotoxicity was confirmed by a live/dead assay kit (Invitrogen, Carlsbad, CA, USA) after culturing for 48 h. The assay was performed according to the manufacturer’s instructions. Afterward, the stained cells were visualized using confocal laser scanning microscopy (CLSM, LeicaTCSSP5, Germany).

The stable p-nitrophenol phosphate substrate was used to quantify ALP activity. BM-PCs were cultured on the Ti-6Al-4V control and the CaSiO₃, Zn-Ca 0.1, and Zn-Ca0.3 coatings in osteogenic medium for 1, 7, 14, and 21 days. At each time point, the culture medium was removed, and the cells were washed with PBS and harvested in 1 ml of universal ALP buffer (100 mM citric acid, 100 mM KH₂PO₄, 100 mM sodium tetraborate.10H₂O, 100 mM Tris, and 100 mM KCl; pH 11). The cells were centrifuged at 3000 rpm for 5 min at 4 °C. The ALP activity in the supernatants was determined following the addition of p-nitrophenyl phosphate substrate, and the reaction was stopped using 100 µl of 0.1 N NaOH. The absorbance was read with a microplate reader (SPECTRA MAX PLUS 384 MK3, Thermo, USA) at a wavelength of 405 nm.
The levels of Col-I and OCN secreted into the culture medium were quantified using an enzyme-linked immunoassay (ELISA) kit (R&D Systems, Minneapolis, MN, USA) following the manufacturer’s instructions.

Osteogenesis was confirmed by Alizarin Red S staining. The cells were seeded on different coatings and cultured in osteogenic medium. At the indicated time point, the cells were fixed with 4% paraformaldehyde for 20 min at room temperature and stained with 0.1% Alizarin Red S (Beyotime, China) at room temperature for 30 min. Afterward, the cells were washed twice with PBS and air dried before the ARS staining was eluted with 5% perchloric acid (SCRC, Shanghai, China). The 100 μl solution from each well was then transferred to a 96-well plate, and the optical density (OD) was measured at 490 nm using a spectrophotometer (SPECTRA MAX PLUS 384 MK3, Thermo, USA).