In Vitro Mineralization Assay: A Colorimetric Method to Quantify Osteoblast Calcium Deposits on Bone Graft Substitute by Alizarin Red S Staining and Extraction

During bone regeneration, osteoblasts - bone-forming cells - are recruited to the fractured site, where they proliferate and secrete osteoid - a collagenous matrix. After a maturation period, the osteoblasts mineralize the osteoid by regulated deposition of calcium and phosphate ions, initiating new bone synthesis.

To quantify the degree of osteoblast mineralization in vitro, obtain primary osteoblast suspension in suitable media. Seed the suspension into a multi-well plate containing porous bone-graft substitute discs and incubate. 

The discs serve as a scaffold, allowing cell adhesion and proliferation.

Replace spent media with fresh media containing osteogenic supplements. Incubate for a prolonged duration. The osteogenic supplements stimulate the osteoblasts to produce and subsequently mineralize the collagenous matrix.

Post-fixation, stain the mineralized matrix with alizarin red S, ARS, solution. ARS, a negatively charged dye, binds to positively charged calcium ions in the matrix, forming a red-colored complex. Remove excess stain. Image the plate to detect red calcium deposits, suggestive of osteoblast mineralization. 

Next, add cetylpyridinium chloride, CPC, solution. CPC, a positively charged surfactant, in micellar form, complexes via its head groups with oppositely charged ARS bound to calcium ions in the matrix, facilitating dye extraction into the solution.

Collect and centrifuge the solution. Transfer the supernatant containing CPC-dye complexes into a multi-well plate and measure absorbance using a microplate reader.

The absorbance of the CPC-dye complexes indicates the amount of calcium deposits in the matrix, an estimation of the degree of osteoblast mineralization.