This article discusses the process of osteoblast mineralization during bone regeneration. It outlines the methods to quantify mineralization in vitro using primary osteoblasts and a porous bone-graft substitute.
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.
Transfer the stained beta-tricalcium phosphate disks to a new well, and scan the plates with a flatbed scanner to record mineralization.
After image capture, add 250 microliters of 10% cetylpyridinium chloride solution and shake for 15 minutes to extract the Alizarin red S dye. Next, transfer the solution from the wells to individual 1.5-milliliter tubes, and centrifuge at 17,000 times g for 5 minutes at room temperature.
Dilute the extract with 10% cetylpyridinium chloride solution at a dilution ratio of 1:10 to 1:20 in a 1.5-milliliter tube. Then, transfer 300 microliters of each diluted sample into the wells of the 96-well plate. Include two blank wells containing only 10% cetylpyridinium chloride.
Next, prepare seven Alizarin red S reference standards ranging from 4 to 400 micromolar by diluting 40 millimolar Alizarin red S staining solution with 10% cetylpyridinium chloride solution to generate a standard curve.
Load 300 microliters of each standard into the plate. Lastly, read the absorbance of the samples, blanks, and reference standards at 520 nanometers.
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