Alizarin red

To analyze bone mineralization, calcein and alizarin red fluorochromes, which bind calcium in the mineralized areas of the bone, were injected into the DP and DP + PTH groups [21 (link)]. After placement of the ligatures around the teeth as described above, the DP and DP + PTH groups were given an intraperitoneal injection of calcein (10 mg/kg, Sigma-Aldrich, St Louis, MO, USA) on days 10 and 27, and alizarin red (20 mg/kg, Sigma-Aldrich, St Louis, MO, USA) on day 20, respectively. On day 30 after placement of the ligatures, the right mandibles and tibiae were fixed in 10% neutral-buffered formalin and dehydrated in a graded series of ethanol solutions. The molar regions of the mandibles were embedded in methyl methacrylate-based resin and cut into 200-μm-thick sections (Struers, Germany). The sections were polished to obtain 50-μm slices using a hard tissue grinding system (EXAKT, Germany). calcein and alizarin red labeling was observed in alveolar bone within the furcation of the first molars and trabecular bone under the epiphyseal growth plate of the tibiae by confocal laser scanning microscopy (LSM 700, Zeiss, Göttingen, Germany).

As cells reached 80–90% confluence, DPSCs were seeded into 6-well plates at a density of 1 × 10⁵ cells/well. The complete culture medium was replaced with the OriCell osteogenic induction differentiation medium (Cyagen, China) to continue the culture. The medium was changed every 3 days and mineralization induction was terminated after 21 days. On the 7th day, the supernatant was discarded, and the DPSCs were fixed with 4% paraformaldehyde (Lianke Bio, China) for 15 min and stained with alkaline phosphatase (ALP, Sigma-Aldrich, USA). Mineral deposits in DPSCs were stained with alizarin red to evaluate odontoblastic differentiation. On the 14th day, the DPSCs were fixed with 4% paraformaldehyde and stained with 0.2% alizarin red (Sigma-Aldrich, USA) for 20 min. The ALP-positive area and alizarin red-positive area were analyzed under a stereoscopic microscope (Zeiss, Germany).