Figure 4: miRNA transfection efficiencies.
From: Cell-free 3D scaffold with two-stage delivery of miRNA-26a to regenerate critical-sized bone defects
miR-26a and miR-26a agomir were labelled using Cy3 (orange red); the polymers (vectors) were labelled using FITC (green); and the cell nuclei were stained with DAPI (blue). To label lipofectamine 2000, NBD cholesterol (10% by weight) was added into lipoplex solution. The confocal images were taken after 2 days of transfection or 2 weeks after implantation. The transfection efficiency was determined by quantifying the fluorescence intensity and the miRNA expression level. (a) miRNA (60 pmol) was complexed with polymers at an N/P ratio of 10 and incubated at room temperature for 30 min, or with lipofectamine 2000 following the manufacturer’s protocol, and then co-cultured with osteoblasts in the presence of 10% FBS in vitro for 2 days. (b) The polymer/miRNA polyplexes were released from the PLGA microspheres immobilized on NF PLLA scaffolds and co-cultured with osteoblasts in vitro for 2 days. (c) The PLLA scaffolds with the immobilized PLGA microspheres containing the polymer/miRNA polyplexes or agomir were implanted on the back of nude mice in vivo for 2 weeks. *P<0.05; **P<0.01; #P<0.05; ##P<0.01. All experiments were carried out in triplicate. n=5 per group. Data are mean±s.d. Scale bar, 100 μm.
