A total of 21 days after seeding into scaffolds, the cell numbers on each scaffold were determined by DNA assay as previously reported (Hoemann, 2004). Briefly, cell-seeded scaffolds were incubated in 1 mL of 4M guanidine hydrochloride (GuHCl) buffer supplemented with complete protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN, United States) for 48 h in a shaking bath at 4°C. The resulting mixture was centrifuged and aliquots (20 μl) of the supernatants were diluted with 1X TNE buffer (10 mM TRIS–HCl, 50 mM NaCl, 1 mM EDTA, pH 8.0) to fit into the standard curve and mixed with (90 μL) of Hoechst 33258 working solution (100 ng/ml, Thermo Fisher Scientific). DNA content was quantified spectro-fluorometrically using a T-Can multi-mode detection reader at a wavelength of 352 nm (emission wavelength of 461 nm) by correlating with a DNA standard curve which was generated by serial dilutions of calf thymus DNA (10 mg/ml). Adding equal volumes of TNE and Hoechst 33258 dye working solution was used as blank controls which were then subtracted from the corresponding samples. Detached cells after 24 h of seeding were collected and the DNA was quantified and subtracted from the DNA content of 5 × 105 cells and expressed as (attachment ratio).

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