The scaffolds with cells at different time points were lysed using M-PER Mammalian Protein Extraction Reagent (78501, Thermo Scientific) supplemented with protease inhibitor and phosphatase inhibitor cocktail (1:100) (Thermofisher, 78446). The lysed cell solution was centrifuged (4 °C, 14,000×g, 10 min), and the extracted protein supernatant was stored at −20 °C for further measurement of total protein and CK quantification. CK activity was calculated corresponding to the manufacturer’s procedure (MAK116, Sigma Aldrich). Briefly, 10 µL of protein extract was added to a 96-well plate, and 100 mL of reconstituted reagent and incubated at 37 °C. The incubated samples on 96-well plate was measured at 340 nm at 20 and 40 min using a microplate reader (TECAN, infinite M200pro). The final CK activity was normalised by total protein content, which was determined by BCA protein assay (23225, Thermo Scientific). CK activity was calculated using the absorbance of 20 and 40 min per the protocol-instructed formula, as shown in Eq. (1 ) below27 (link). Data were analysed using GraphPad Prism (software version 9.5.0). Statistical significance was determined through one-way ANOVA with Tukey’s multiple comparisons tests.
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