Huxma 90021

PDLSCs at passage 2 were cultured on specific dECMs and no-ECM at a density of 1 × 10⁶ cells per well in 6-well plates at 80% confluence. For osteogenesis assays, the adherent cells were cultured in osteoinductive differentiation medium (Cyagen, HUXMA-90021) 43 (link). The medium was changed every two days. After osteogenic induction, the formation of mineralized nodules was identified using alizarin red staining at day14 and ALP staining at day 7.

After 14 days of osteogenic induction, alizarin red staining was used to evaluate calcium mineralization. Briefly, cells were fixed with 4% paraformaldehyde for 15 min at room temperature, washed with PBS and stained with alizarin red (Cyagen, HUXMA-90021) for 20 min at room temperature. Cells were washed with PBS, images of mineralized nodules were acquired with an inverted microscope, and absorbance was quantitatively measured at 560 nm for statistical analysis.

To induce osteogenic differentiation, hBMSCs were cultured in a commercially available osteogenic differentiation medium (catalog no. HUXMA-90021; Cyagen Biotechnology Co. Ltd., Taicang, China). On day 21, cells were stained with Alizarin Red in accordance with the manufacturer's protocol. To induce adipogenic differentiation, hBMSCs were cultured in a commercially available adipogenic differentiation medium (catalog no. HUXMA-90031; Cyagen Biotechnology Co. Ltd.). On day 21, cells were stained for 30 min with Oil Red O, diluted 3:2 with distilled water and filtered. To induce chondrogenic differentiation, hBMSCs were cultured in a commercially available chondrogenic differentiation medium purchased from Cyagen (catalog no. HUXMA-90041; Cyagen Biotechnology Co. Ltd.). On day 28, cells were stained with 1 mg/ml Alcian blue for 30 min. Cells were observed under a Nikon Eclipse E200 light microscope (Nikon Corporation, Tokyo, Japan).

MC3T3-E1 subclone 14 was inoculated in a 12-well plate. After the cells adhered, they were replaced with osteogenic induction medium (HUXMA-90021; Cyagen, USA) and cultured for 3 weeks. The medium was changed once every 3 days. Pre-stained cells were washed three times with PBS (10010023; Thermo Fisher Scientific, USA) for 5 min each time to remove the residual culture medium, fixed with 95% alcohol at room temperature for 15 min, and washed with PBS three times for 5 min each time to remove excess alcohol. 1 mL of 0.5% alizarin red staining reagent was added to the well, stained at room temperature for 15 min, and washed with PBS 3–5 times for 5 min each time to remove excess staining solution. After drying, they were visualized under an inverted microscope (Olympus, BX53; Melville, NY, USA). For alizarin red staining (ARS) staining quantification, 500 μL/well (6-well plates) 10% (w/v) of cetylpyridinium chloride (Sigma, 1104006) was added to the samples and incubated for 10 min. Then 200 μL/well dissolved solution was used to measure absorbance at 562 nm by utilizing a microplate spectrophotometer (BioTek Instruments). The staining experiment was repeated three times, n = 10. Data are presented as mean ± SD (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; # no significance).