Real-Time Vital Mineralization Detection and Quantification during In Vitro Osteoblast Differentiation

Abstract

BackgroundIn vitro studies of osteoblasts traditionally use Alizarin Red as a golden standard for the detection and quantification of mineralization, which is a marker of osteoblast differentiation. However, this method presents a number of drawbacks, including the need to fix cells, which prevents additional measurements. Years ago, Calcein Green was proposed as an alternative to Alizarin Red, with the advantage to be directly detectable in live cells. However, the protocol was still time-consuming, and it never managed to replace Alizarin Red. Now, with more efficient imaging systems, we present a protocol using Calcein Green which provides significant advantages.ResultsThe osteoblast mineralization was efficiently detected and accurately quantified in real time at any desired time point across the entire differentiation period, with a minimum time expenditure.ConclusionsThe combination of Calcein Green and the real-time imaging station IncuCyte ZOOM can efficiently replace the Alizarin Red method, and allows very accurate and time-saving assessment of the level and the dynamics of matrix mineralization.Electronic supplementary materialThe online version of this article (10.1186/s12575-018-0079-4) contains supplementary material, which is available to authorized users.

Synthetic Biodegradable Aliphatic Polyester Nanocomposites Reinforced with Nanohydroxyapatite and/or Graphene Oxide for Bone Tissue Engineering Applications

“…Calcium deposition can be characterized by staining with Alizarin red S dye, and inspection in the phase-contrast microscope. For quantitative analysis, the deposit can be extracted and quantified by a colorimetric assay [256].…”

CRISPR‐Cas9–Mediated Genome Editing Confirms EPDR1 as an Effector Gene at the BMD GWAS‐Implicated ‘STARD3NL’ Locus

“…Our previous RNAi targeting of EPDR1 in hMSCderived osteoblasts resulted in lower levels of both alkaline phosphatase staining (ALP) and Alizarin red S staining (ARS) (9) . ALP activity is necessary for hydroxyapatite deposition and mineralization (16) , while ARS stains for the deposition of calcium (17) . However, while hFOB1.19 cells undergo osteoblast differentiation, they do not produce an appreciable amount of calcium for detection by ARS (18) .…”

Calcein Binding to Assess Mineralization in Hydrogel Microspheres

“…The in vitro mineralization formed in osteoprogenitor cell cultures is typically assessed by von Kossa, Alizarin Red S, or, to a lesser extent, calcein staining [ 1 , 2 ]. All methods are used to observe the presence of calcium phosphate (hydroxyapatite) in osteoprogenitor cell cultures.…”

“…For instance, hydrogels are increasingly being used to microencapsulate cells. In osteoprogenitor monolayers, qualitative comparisons of Alizarin Red S and calcein stain have been demonstrated to show comparable staining patterns of calcium phosphate generated by the cells [ 2 ]. In microencapsulated osteoprogenitors, we observed that this was not the case.…”

“…The von Kossa procedure was not assessed because in preliminary tests it had resulted in opaque hydrogels that had been formerly transparent. Calcein staining has repeatedly been proposed as a replacement for Alizarin Red S stain when assessing cultured osteoblasts, but it has yet to be widely adopted [ 1 , 2 ]. We have demonstrated that for assessing mineralization by microencapsulated osteoblasts, calcein stain is a superior method than Alizarin Red S stain.…”