Rhdkk1

Bone marrow stromal cells and OBs were treated with OB differentiation medium containing 50 μg·mL⁻¹ ascorbic acid and 10 mm β‐glycerophosphate for 0, 7, or 14 days 23. BMSCs, OBs, OCYs, or 8‐week‐old male mice femoral bones were treated with 100 ng·mL⁻¹ recombinant human BMP2 (R&D Systems, Minneapolis, MN, USA), several concentrations (0, 10, 20, 50, 100, or 200 ng·mL⁻¹) of rhWnt3a (R&D Systems), 100 ng·mL⁻¹ rhDkk1 (R&D Systems), 100 ng·mL⁻¹ rhWnt1 (R&D Systems), 100 ng·mL⁻¹ rhWnt5a (R&D Systems), 10 μm KCl (Sigma Aldrich Chemicals, St. Louis, MO, USA), or 10 μm LiCl (Sigma Aldrich Chemicals) for 1 day. HEK293T cells were maintained and cultured as reported previously 24.

Healthy premolars were extracted from patients (n = 12, 14–18 years of age, details please see Supplementary Information) with written consent signed by parents during orthodontic treatment in the West China Stomatology Hospital. All experiments were conducted in accordance with the ethics protocol approved by the Committee of Ethics of the Sichuan University. Periodontal ligaments, collected from the middle third of the root, were cultured in alpha-Modified Eagle’s Medium (α-MEM; Hyclone, USA) supplemented with 10% fetal bovine serum (FBS) (Hyclone, USA), 1% penicillin/streptomycin (Solarbio, China) in a humidified atmosphere at 37 °C with 5% CO₂ as previously described2 (link). Cell culture medium was refreshed every two days and all experiments were carried out with passage 3–4 cells. For the HG treatment, D-Glucose (Sigma-Aldrich, USA) was added to the culture medium (30 mM final concentration) for the described time intervals51 (link)52 (link). 5-aza-dC (Selleck Chemicals, UH, USA) was added to the culture (1 μM final concentration) for the described time intervals. rhDKK1 (R&D, Wiesbaden, Germany) was added to the culture (100 ng/ml final concentration)53 (link) for 2d to block the Wnt signaling pathway.