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

Published: April 30, 2023

Abstract

Source: Kampleitner, C. et al. Biological Compatibility Profile on Biomaterials for Bone Regeneration. J. Vis. Exp. (2018)

In this video, we describe a method for the detection of osteoblast-secreted calcium deposits using an anionic dye, Alizarin Red S. The cultured osteoblasts are first stained and then the dye is recovered for colorimetric quantification of calcium deposits or the degree of osteoblast mineralization.

Protocol

1. Osteoblast mineralization assay Pre-incubate 14 mm diameter β-tricalcium phosphate (β-TCP) disks in 1 mL of bone growth medium (BGM) in a 24-well suspension culture plate at 37 °C and 5% CO₂ for 24 h before adding the osteoblasts (OBs). Use standard tissue culture plates for controls and suspension culture plates for the biomaterial samples to avoid cell attachment to the plastic. Aspirate the pre-incubation medium and then resuspend primary mouse OBs in BGM. Add 8.8 x 10&#83…

Divulgaciones

The authors have nothing to disclose.

Materials

β-tricalcium phosphate disks (β-TCP, 14 mm) Disks were sintered in a muffle furnace at a temperature of 1150 °C. Samples were UV-irradiated (15 min for each side) before using in cell cultures
Alizarin Red S Sigma-Aldrich A5533
Bone growth medium (BGM) α-MEM + 10% heat-inactivated FBS + 1% penicillin/streptomycin
Formalin solution neutral buffered 10% Sigma-Aldrich HT501128
Hexadecylpyrdinium (cetylpyridinium) chloride monohydrate Sigma-Aldrich C9002
L-Ascorbic acid 2-phosphate sesquimagnesium salt hydrate Sigma-Aldrich A8960
MEM alpha medium (α-MEM) Gibco Life Technologies  11900-073
Osteogenic mineralization medium (MM) α-MEM + 10% heat-inactivated FBS + 1% penicillin-streptomycin + 50 µg/mL ascorbic acid + 5 mM β-glycerophosphate
Penicillin-streptomycin   Sigma-Aldrich P4333
Phosphate-buffered saline 1x (PBS) pH 7.4 Gibco, Thermo Fisher Scientific  10010023
β-Glycerophosphate disodium salt hydrate Sigma-Aldrich G5422
Ultrapure water: Cell culture water pyrogen free VWR L0970-500
96-well transparent plate Greiner bio-one 655001
24-well suspension culture plate Greiner bio-one 662102
24-well tissue culture plate Greiner bio-one 662160
Centrifuge: VWR Mega Star 600R VWR
CO2-Incubator: HeraCell 240 Thermo Scientific
Flatbed scanner: Epson Perfection 1200 Photo Epson
Infinite M200 Pro microplate reader Tecan
Microcentrifuge tube: Eppendorf tube safe-lock 1.5 mL VWR 21008-959
Shaking incubator GFL3032 GFL  3032
Single-use pipet: serum pipet Greiner bio-one 612301

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In Vitro Mineralization Assay: A Colorimetric Method to Quantify Osteoblast Calcium Deposits on Bone Graft Substitute by Alizarin Red S Staining and Extraction. J. Vis. Exp. (Pending Publication), e21167, doi: (2023).

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