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Cholesterol (Chol), hydrogen tetrachloroaurate (III) trihydrate (HAuCl4•3H2O) and silver nitrate (AgNO3) were purchased from Alfa Aesar. Doxorubicin (DOX), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS) and 2-mercapto-4-methyl-5-thiazoleacetic acid (MMTAA) were purchased from Sigma Aldrich. Methanol, chloroform and Triton X-100 were purchased from Aladdin Reagent Co, Ltd. Trisodium citrate (Na3C6H5O7•2H2O) was purchased from Sinopharm Chemical Reagent Co, Ltd. Phosphate buffered saline (PBS, 10mM, pH 7/4) was purchased from Nanjing KeyGen Biotechnology Co, Ltd. All the reagents were used as received. Deionized water (Millipore Milli-Q grade) with a resistivity of 18/2 MΩ cm-1 was used in all the experiments.


The levels of odontogenic-related proteins were detected before (0 day) and after ascorbic acid induction using Western blotting. Total cellular proteins were extracted from DFCSs and SHEDSs using the Total Protein Extraction Kit (KeyGEN (https://dkluchezar.ru/hack/?patch=1492), China). Aliquots of 20 μg of each cell lysate were separated on polyacrylamide gels and blotted onto a polyvinylidene fluoride (PVDF) membrane (Bio-Rad). Membranes were blocked with 5% milk for 1 h and then incubated with primary antibodies (COL-1, Arigo, China; Integrinβ1, Abcam, UK; DMP-1, Millipore, USA; ALP, Zenbio, China; OCN, Zenbio, China; GAPDH, Zenbio, China) overnight followed by the horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG antibodies (ZSGB-BIO, China). The labeled proteins were visualized using an ImageQuant LAS 4000 mini instrument (GE Healthcare).

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Cell survival and apoptosis were assessed by flowcytometry with (https://dkluchezar.ru/hack/?patch=1154) Annexin V-fluorescein isothiocyanate and propidiumiodide (KeyGEN (next) BioTECH) staining. Analysis of phosphatidylserine onthe outer leaflet of apoptotic cell membranes was performed usingAnnexin V-fluorescein isothiocyanate and propidium iodide toidentify apoptotic and necrotic cells, respectively. Briefly, thecells were collected with ethylenediaminetetraacetic acid-freetrypsin and washed with PBS three times. Then, the cells wereresuspended in 500 µl of HEPES buffer, mixed with 5µl of Annexin V-fluorescein isothiocyanate and 5 µlof propidium iodide, and incubated at room temperature for 5 min inthe dark. The cells were analyzed using a flow cytometer (BDBiosciences).

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