Gene expression analysis

To assess gene expression profiles, real time polymerase chain reaction (RT-PCR) was performed. Therefore, scaffolds were snap frozen in RNA Later (#160027967, Qiagen, Germany) and stored at −80°C. The total RNA extraction was carried out according to manufacturer’s protocol with RNAeasy Mini Kit (#74104, Qiagen, Germany). In the first step, 350 µl RLT buffer (10 μl β-Mercaptoethanol per 1 ml RLT buffer) and a stainless steel bead was added to the probes in each tube. Subsequently, a TissueLyser at 15 Hz was used for 30 s to homogenize the samples, which were then centrifuged for 3 min at 14,000 rpm. In the next step, the supernatant was purified by using the RNeasy Mini Kit pursuant according to manufacturer’s protocol. The measurement of RNA concentration and purity obtained was done using a nano-photometer (Implen, Germany). The subsequent cDNA synthesis was carried out per Transcriptor First Strand Kit (#4379012001, Roche, Switzerland) in a thermocycler (T-Professional Basic thermocycler, Biometra (Analytik Jena), Germany. Thereon, RT-PCR was conducted by using 5 µl of each cDNA sample and the InnuMIX DS Green Standard Kit (#012-18, Analytik Jena, Germany) in a Real-Time-Thermocycler (qTOWER ³ G, Analytik Jena, Germany). All primers used for this experiment were listed in Table 1 and designed with Sigma-Aldrich’s OligoArchitect™ Primer software. First, the denaturing was done at 95°C for 2 min, followed by amplification in 50 cycles of 95°C for 30 s, 60°C for 1 min, 68°C for 30 s. The relative gene expression was normalized to the corresponding HouseKeepingIndex, consisting of the average of GAPDH and HRPT1. Afterwards, the transcript levels were calculated as w = 2^−ΔΔCt, in which ΔΔCt = ΔInduction − ΔControl was calculated from the ac/ao-PCL- and uPCL-group.

Primer sequences.