Opsys mrtm 96 well microplate reader

The AlamarBlue^TM assay was used to assess the proliferation of osteoblasts over a period of 21 days on functionalized and non-functionalized PEEK disks. The fluorescence values were measured at a test wavelength of 570 nm and a reference wavelength of 630 nm using a microplate reader (Opsys MRTM 96-well microplate reader, Dynex Technologies, Chantilly, VA, USA). Each sample was measured in triplicate to ensure the accuracy of the results. Data were collected at 1, 4, 7, 14, and 21 days to monitor the temporal profile of osteoblast proliferation.

Calcium deposition was evaluated by Alizarin Red S staining on HOB cells. Alizarin red S is an anthraquinone dye that reacts with calcium and forms an Alizarin Red S/calcium complex that is birefringent. HOB cells were cultured for 21 days, and the disks were then washed in PBS, fixed with 10% formal saline for 15 min, and washed with deionized water three times. 2% Alizarin red S solution was added to the specimens for 15 min, followed by five washes in 50% ethanol. For qualitative assessment, images of the stained scaffolds were taken. A quantitative assessment was conducted using Lee et al. protocol [26 (link)]. The bound stain taken up by the cells on the disks was released using 1 mL of 10% cetylpyridium chloride (CPC) solution added to each well overnight (24 well plates). Aliquots (100 μL) of the supernatant were removed and read using a microplate reader (Opsys MRTM 96-well microplate reader, Dynex Technologies, Chantilly, VA, USA) at 570 nm. A standard curve, using known concentrations of alizarin red S in CPC solution, was used for the quantification. Cell mineralization results were expressed as μmol of calcium per well as 1 mole of Alizarin red S binds to 2 mol of calcium in an Alizarin red S-calcium complex [27 (link)].

To evaluate calcium deposition in HOB cells, alizarin red S staining was performed. alizarin red S is an anthraquinone dye that reacts with calcium, forming a birefringent alizarin red S/calcium complex. HOB cells were cultured for 21 days, after which the PEEK disks were washed in PBS, fixed in 10% formal saline for 15 min, and washed with deionized water thrice. The specimens were then treated with 2% alizarin red S solution for 15 min, followed by five washes in 50% ethanol.
The stained scaffolds were assessed using the protocol developed by Lee et al. [33 (link)]. To release the alizarin red S that had bound to the cells on the disks, 1 mL of 10% cetylpyridium chloride (CPC) solution was added to each well overnight in a 24-well plate. Aliquots (100 μL) of the supernatant were collected and measured using a microplate reader (Opsys MRTM 96-well microplate reader, Dynex Technologies, Chantilly, VA, USA) at 570 nm. A standard curve with known concentrations of alizarin red S in CPC solution was utilized for quantification. The results of cell mineralization were expressed as μmoles of calcium per well, as 1 mole of alizarin red S binds to 2 moles of calcium in an alizarin red S–calcium complex [34 (link)].