Mc3t3 e1 subclone 14

MC3T3-E1, a murine osteoblastic cell line, was purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences (MC3T3-E1 Subclone 14). Cells were cultured in MEM alpha modification medium (α-MEM) (HyClone) supplemented with 10% fetal bovine serum (FBS) (Excell) and 1% penicillin and streptomycin (Beyotime) in a humidified incubator with 5% CO₂ at 37° C. For osteoblast induction, cells were cultured in OBM after cells reached 90% confluence. OBM was prepared with α-MEM medium supplemented with 10% FBS, 10 mM β-glycerophosphate, 50 μg/mL α-ascorbic acid, and 0.1 μM dexamethasone, and the medium was changed every 3 days. For the induction of inflammation, lyophilized LPS powder (Sigma, L2630, Escherichia coli O111:B4) was diluted with cell culture medium to 1 mg/mL for storage and further diluted to the working concentration. FK866 (Beyotime, SD7257) was diluted with cell culture medium to 1 nM. EX527 (Beyotime, SC0281), a selective Sirt1 inhibitor, was diluted with cell culture medium to 30 μM. NMN (Sigma, N3501), the NAD⁺ precursor, was diluted with cell culture medium to 100 μM. SRT 1720 (Beyotime, SC0267), a selective Sirt1 agonist, was diluted with cell culture medium to 5 μM.

Gelatin Porcine Skin, MA and Irgacure 2959 were purchased from Sigma Aldrich. The reagents such as absolute alcohol (purity ≥99.7%), Calcium Nitrate (CaNO₃) silver nitrate (AgNO₃) and Trisodium Phosphate (Na₃PO₄) were analytical grade and purchased from Tianjin Kaitong Chemical Reagent Co., Ltd., China. The Dulbecco’s modified eagle medium (DMEM) and fetal bovine serum (FBS) were obtained from Sigma Aldrich. Sangon Biotech (Shanghai) Co., Ltd. Supplied trypsin-ethylenediaminetetraacetic acid and phosphate buffer saline (pH 7.4). All of other reagents were analytical grade and used as received without further purification. Mouse embryo osteoblast precursor cells (MC3T3-E1) subclone 14 pre-osteoblasts were sourced from the Shanghai Cell Bank of the Chinese Academy of Sciences.