This study investigates the transformation of valve interstitial cells (VICs) into osteoblasts and the subsequent calcification of aortic valves. The methodology includes culturing VICs and measuring calcium concentrations to understand the calcification process.
Aortic valve leaflets contain a heterogeneous population of valve interstitial cells, or VICs, distributed throughout the extracellular matrix layers. Upon stimulation, VICs transform into osteoblasts ― bone matrix-forming cells.
Mature osteoblasts secrete collagen and bone-matrix proteins. These proteins help osteoblasts attach to the matrix. Finally, intracellular calcium ions enter the matrix milieu and, along with phosphates, deposit as mineralized nodules, leading to aortic valve calcification.
To assess the calcium concentration in calcified aortic valves, begin with a culture of VICs in a six-well plate. Discard the culture medium, add an osteogenic medium into the well, and incubate.
During incubation, insulin in the osteogenic medium stimulates the VICs to differentiate into osteogenic phenotypes. These osteoblasts secrete bone-matrix proteins and ascorbic acid in the osteogenic medium which induces them to form a collagen-rich extracellular matrix.
Finally, the osteoblasts' intracellular calcium and the medium's phosphates form mineralized crystals in the medium.
Remove the medium, and add the hydrochloric acid solution. Incubate to solubilize the calcium ions into the solution. Dispense the acidic medium containing calcium into the well of a multi-well plate.
Add a calcium-sensitive dye, and incubate. The dye molecules bind to the calcium ions in the acidic medium, forming a purple-colored dye-calcium complex.
Measure the absorbance of the complex, which is proportional to the calcium concentration in the sample.
Before beginning the assay, clean the biosafety hood with 70% ethanol, and warm the DMEM medium to 37 degrees Celsius. Next, seed 100,000 cells per condition into 6-well plates in complete DMEM, and culture at 37 degrees Celsius.
After 24 hours, replace the supernatant medium with the calcifying medium, and incubate the cells for 7 days at 37 degrees Celsius, replacing the medium on the third day.
After 7 days, measure the calcium concentration in a 96-well plate by using the Arsenazo III reagent, and recording the absorbance at 650 nanometers.
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