Osteogenesis assay kit

For adipogenic differentiation, cells were seeded into six-well plates with mouse mesenchymal stem cell adipogenic differentiation medium (Cyagen Biosciences, Guangzhou, China) according to the supplier’s instructions. After 3 weeks of differentiation, cells were washed with PBS and then fixed in 10% formalin for 20 minutes and stained with Oil-Red O (USA) solution for image acquisition. The intracellular Oil-Red O was extracted with isopropyl alcohol for quantification. The absorbance of the extracted Oil-Red O was measured at 490 nm using a Model 680 Microplate Reader (Bio-Rad). For osteogenic differentiation, cells were seeded into 24-well plates with mouse mesenchymal stem cell osteogenic differentiation medium (Cyagen Biosciences) according to the supplier’s instructions. After 3 weeks of differentiation, cells were washed with PBS and then fixed in 10% formalin for 20 minutes and stained with Alizarin Red (Sigma) solution for image acquisition. An osteogenesis assay kit (Millipore, Billerica, MA, USA) was applied to quantify the Alizarin Red according to supplier’s instructions.

Eight days following the establishment of osteogenesis, an ARS staining assay with an osteogenesis assay kit (Millipore, Billerica, MA) was employed to evaluate mineralization. In brief, cells were fixed using formalin for alizarin Red S staining. The dye was later extracted from stained cells using 10% acetic acid and absorbance at 405 nm was measured. All data were expressed after normalization to the control values.

The mineralization of cell-seeded constructs was analyzed by Alizarin red staining (ARS) using an osteogenesis assay kit (Millipore, Billerica, MA, USA) after 7 and 14 days of culture.
ARS was performed to assess the CaP content of the constructs with MG63 cells as well as the CaP content in the PCL-CaP composite material-based implants without cells. The assay procedure followed the instructions by the manufacturer, and the deposition of calcified matrix on the samples was documented with the 3D microscope Keyence VR-3100 (Keyence Deutschland GmbH, Neu-Isenburg, Germany).
For the quantitative analysis of the mineralization, ARS was extracted with 10% (w/v) cetylpyridium chloride (CPC) for 48 h on an orbital shaker. Alizarin red solutions from stained cell-laden scaffold and standards in a high range (2 mM, 1 mM, 500 mM, 250 mM, 125 mM, 62.5 mM, 31.3 mM, 0 mM) were prepared in ARS dilution buffer and added to a 96-well plate. The absorbance of the samples was measured at a wavelength of 405 nm in a microplate reader for quantification.

In vitro mineralization was evaluated using an Osteogenesis Assay Kit (#ECM815; Millipore, Billerica, MA, USA) 21 days after seeding ah-BM-MSCs in the presence (indirect contact) of PCL, PCL/CS, and PCL/CS/10%Van scaffolds. Briefly, the ah-BM-MSCs were fixed using 10% formaldehyde, stained using alizarin red stain solution, and visualized using an optical microscope (Axio Vert.A1, ZEISS, Jena, Germany) coupled to a digital camera (Axiocam 305 color, ZEISS, Germany). Then, the samples were treated following the manufacturer’s protocol to quantify matrix mineralization and the optical density (OD) was measured using a Spectramax iD3 plate reader (Molecular Devices, USA) at 405 nm.

The in vitro mineralization was evaluated by the specific binding of Alizarin Red Solution (ARS) staining to calcium deposits at 7 and 14 days after seeding using an Osteogenesis Assay Kit (#ECM815; Millipore, Billerica, MA, USA). Briefly, at each time period of the study, the ah-BM-MSCs were stained with ARS and visualized using an optical microscope (Motic AE2000, Shimadzu Corp., Kyoto, Japan). Then, to quantify matrix mineralization, the samples were treated following the manufacturer’s instructions, and the optical density (OD) at 405 nm was measured in a Synergy MX ultraviolet-visible (UV-Vis) spectrophotometer (Bio Tek Instruments Inc., Winooski, VT, USA).

Antibiotic-loaded ADSCs and ADSCs were analysed for their capacity for osteogenic differentiation using ALP staining and alizarin red histochemistry. To induce differentiation, cells were cultured in osteogenic medium composed of α-MEM (Wako Pure Chemical Industries) containing 10% FBS, 0.1 mM dexamethasone, 50 mM ascorbate-2-phosphate, 10 mM β-glycerophosphate, and 1% penicillin-streptomycin^{52 (link)}. Alkaline phosphatase (ALP) histochemistry was performed at 2 weeks after osteogenic induction culture. For ALP staining, cells were rinsed with PBS three times and fixed in 4% paraformaldehyde phosphate buffer (Wako) at room temperature for 5 min. They were then washed with deionized water. The fixed cells were incubated with 1-Step NBT/BCIP plus Suppressor Solution (Thermo Fisher Scientific) at 37 °C for 30 min, washed with deionized water, and observed both with the naked eye and under a light microscope (Biorevo BZ-9000; Keyence, Osaka, Japan). For alizarin red staining, cells were rinsed with PBS three times, fixed in 4% paraformaldehyde phosphate buffer, and stained using an Osteogenesis Assay Kit (ECM815; Millipore) per the manufacturer’s instructions.