In vitro and invivo detection of microbial gene expression in bioactivated scaffolds seeded with cyanobacteria.

Abstract

Dermal replacement materials bioactivated with cyanobacteria have shown promising potential for wound regeneration. To date, extraction of cyanobacteria RNA from seeded scaffolds has not been described. The aim of this study was to develop a method to isolate total RNA from bioactivated scaffolds and to propose a new approach in determining living bacteria based on real-time PCR. Transgenic Synechococcus sp. PCC 7002 (tSyn7002) were seeded in liquid cultures or scaffolds for dermal regeneration invitro and invivo for 7 days. RNA was extracted with a 260/280 ratio of ≥2. The small subunit of the 30S ribosome in prokaryotes (16S) and RNAse P protein (rnpA) were validated as reference transcripts for PCR analysis. Gene expression patterns differed invitro and invivo. Expression of 16S was significantly upregulated in scaffolds invitro, as compared to liquid cultures, whilst rnpA expression was comparable. In vivo, both 16S and rnpA showed reduced expression compared to invitro (16S: invivo Ct value 13.21 ± 0.32, invitro 12.44 ± 0.42; rnpA invivo Ct value 19.87 ± 0.41, invitro 17.75 ± 1.41). Overall, the results demonstrate rnpA and 16S expression after 7 days of implantation invitro and invivo, proving the presence of living bacteria embedded in scaffolds using qPCR.