Quantitative reverse transcription polymerase chain reaction (RT-qPCR)

The samples for RT-qPCR were snap-frozen in liquid nitrogen on day 3, 7, 14 and 21 day and stored at −80°C until processed. Six scaffolds from one culture chamber were grouped into 3: 1st–2nd, 3rd–4th and 5th–6th scaffolds from the inlet, to examine variation within the group. Total RNA was extracted using a Maxwell^® 16 Cell LEV Total RNA Purification Kit (AS1280; Promega, USA) in accordance with the manufacturer’s protocol. Subsequently, reverse transcription was performed using a Transcription Kit (4368814; Applied Biosystems, USA). RT-qPCR was conducted with the StepOne™ real-time PCR system (4453320, Applied Biosystems, USA). The primers used are listed in table S1. Relative expression of each mRNA was calculated with the ΔΔCt method normalised by GAPDH. ³⁰ The data is represented as a mean value (±s.e.m) of three replicates.