Standard curve of calf thymus dna

For biochemical analyses (n = 3), specimens were digested in a pre-prepared papain solution containing 0.5 M EDTA, 0.05 M cysteine hydrochloride, and 1.0 mg·mL⁻¹ papain enzyme (Sigma, St. Louis, MO, USA) at 60 °C for 12 h. The aliquots of the sample digestion were used for the measurements of DNA and proteoglycan contents. DNA content was measured using a fluorescence assay. Sample digestion was kept at 37 °C for 20 min with 200.0 μL of Hoechst 33258 working solution at a concentration of 2.0 μg·mL⁻¹. The fluorescence was read at 360 nm for excitation and 460 nm for emission. The DNA content was normalized with a standard curve of calf thymus DNA (Sigma, St Louis, MO, USA). Total glycosaminoglycan (GAG) content was determined using a 1,9-dimethylmethylene blue (DMMB; Sigma, St. Louis, MO, USA) dye-binding assay with chondroitin-6-sulfate from shark (Sigma, St. Louis, MO, USA) as a standard. Briefly, 20.0 μL of sample was mixed with 200.0 μL of DMMB reagent, and absorbance was read at 525 nm.

To detect the cell proliferation during in vitro osteogenic induction, cell seeded constructs were cultured in osteogenic differentiation medium (RBXMX-90021; Cyagen Biosciences Inc., Guangzhou, China) for 14 days. At 1, 7, and 14 days, The cell-scaffold constructs were collected and washed gentle in cold PBS (4 °C) before lysis in RIPA buffer (Beyotime, Jiangsu, China). The aliquots of the sample digestion were used for DNA content measurements. DNA content was measured using Hoechst 33258 working solution (2 μg/mL) for fluorometric anaylysis. The fluorescence was read at 360 nm for excitation and 460 nm for emission. The DNA content was normalized with a standard curve of calf thymus DNA (Sigma, St Louis, Missouri, USA).
Part of the lysis solution was centrifuged, and the supernatant was used for ALP activity with p-nitrophenyl phosphate assay. The ALP activity were detected following manufacturer’s instructions (BioVision, CA, USA). The ALP activity of the test samples was calculated as umol of converted p-nitrophenol/min, and then normalized to the detected DNA content. The final ALP activity was expressed as U/mg DNA.