Magnetofection of miR-21 promoted by electromagnetic field and iron oxide nanoparticles via the p38 MAPK pathway contributes to osteogenesis and angiogenesis for intervertebral fusion

Abstract

Background: Magnetofection-mediated gene delivery shows great therapeutic potential through the regulation of the direction and degree of differentiation. Lumbar degenerative disc disease (DDD) is a serious global orthopaedic problem. However, even though intervertebral fusion is the gold standard for the treatment of DDD, its therapeutic effect is unsatisfactory. Here, we described a novel magnetofection system for delivering therapeutic miRNAs to promote osteogenesis and angiogenesis in patients with lumbar DDD.

Results: Co-stimulation with electromagnetic field (EMF) and iron oxide nanoparticles (IONPs) enhanced magnetofection efficiency significantly. Moreover, in vitro, magnetofection of miR-21 into bone marrow mesenchymal stem cells (BMSCs) and human umbilical endothelial cells (HUVECs) influenced their cellular behaviour and promoted osteogenesis and angiogenesis. Then, gene-edited seed cells were planted onto polycaprolactone (PCL) and hydroxyapatite (HA) scaffolds (PCL/HA scaffolds) and evolved into the ideal tissue-engineered bone to promote intervertebral fusion. Finally, our results showed that EMF and polyethyleneimine (PEI)@IONPs were enhancing transfection efficiency by activating the p38 MAPK pathway.

Conclusion: Our findings illustrate that a magnetofection system for delivering miR-21 into BMSCs and HUVECs promoted osteogenesis and angiogenesis in vitro and in vivo and that magnetofection transfection efficiency improved significantly under the co-stimulation of EMF and IONPs. Moreover, it relied on the activation of p38 MAPK pathway. This magnetofection system could be a promising therapeutic approach for various orthopaedic diseases.

Keywords: Bone tissue engineering; Electromagnetic field; Gene therapy; Iron oxide nanoparticles; Magnetofection.

Fig. 1

Design and characterization of PEI@IONPs and corresponding magnetofection complexes systems. A Schematic illustration of synthesis process of magnetofection complexes systems (miR-PEI@IONPs) using polyethyleneimine (PEI)-coated iron oxide nanoparticles (IONPs) to deliver miRNAs. B–D Zeta potential (B), diameter (C) and morphology (D) of the PEI@IONPs. E Transmitting electron microscope images indicating the internalisation of IONPs by seed cells. The image on the right is a magnification of the marked box on the left. F Cell counting kit-8 results of seed cells treated with various conditions (IONPs concentration: 0, 10, 25, 50, 100 µg/mL and EMF intensity: 0, 1, 2 or 5 mT)

Fig. 2

Different transfection efficiencies under various magnetofection conditions. A Fluorescence microscope images showing different transfection efficiencies according to different distant ratios of PEI@IONPs:miRNAs [5, 10, 20, or 30, with or without electromagnetic field (EMF) stimulation]. C Semi-quantifications of mean fluorescence intensity under those various conditions. B, D Transfection efficiency under various treatments [PBS, lipofectamine 2000 (lipo 2000), EMF, PEI@INPs, EMF + PEI@IONPs] and corresponding mean fluorescence intensity. E Relative miRNA expression levels of miR-21 under various treatments (PBS, lipo 2000, EMF, PEI@INPs, EMF + PEI@IONPs). *p < 0.05, **p < 0.01, ***p < 0.001 compared to the PBS group; ^#p < 0.05, ^##p < 0.01 compared to the lipo 2000 group

Fig. 3

Magnetofection of miR-21 promoted osteogenesis. A, B Alizarin red S (ARS) staining pictures 14 days after different treatments and the corresponding semi-quantification analysis. C Quantification analysis of alkaline phosphatase (ALP) activity 7 days after treatment. D–G Western Blotting analysis of Col 1, OCN, and OPN as well as corresponding semi-quantifications of protein expression levels of Col 1 (E), OCN (F) and OPN (G). H–J Relative mRNA expression levels of Col 1 (H), OCN (I), and OPN (J). K, L Immunofluorescence images of OCN (K) and Runx2 (L) after various treatments (PBS, lipofectamine 2000 (lipo 2000), electromagnetic field (EMF), PEI@INPs, EMF + PEI@IONPs). OCN/Runx2 was labelled with Cy3 (red), and nuclei were labelled with 4′,6-diamidino-2-phenylindole (DAPI, blue). *p < 0.05, **p < 0.01, ***p < 0.001 compared to the PBS group; ^#p < 0.05, ^##p < 0.01 compared to the lipo 2000 group

Fig. 4

Magnetofection of miR-21 promoted angiogenesis. A Assessment of the migratory activity of human umbilical endothelial cells (HUVECs) at 24 h by scratch wound assay; the red dashed lines are the edges of the cell migration. B Assessment of the cell migration rate of HUVECs by transwell assay. C Tube formation assay of HUVECs. D–G Quantitative analysis of wound size recovery rate (D), migration rate (E), total tube length (F) and branch points (G). *p < 0.05, **p < 0.01, ***p < 0.001 was compared to the PBS group; ^#p < 0.05, ^##p < 0.01 was compared to the lipo 2000 group. H, I Immunofluorescence images of CD31 (H) and vWF (I) after various treatments. CD31 and vWF was labelled with Cy3 (red), and nucleus were labelled with DAPI (blue)

Fig. 5

Characterization of polycaprolactone/hydroxyapatite (PCL/HA) scaffolds before and after cell seeding. A Gross morphology of PCL/HA scaffolds. B Scanning electron microscope (SEM) images showing the macroporous structure of PCL/HA scaffolds at different magnifications (500, 200, 100, 50 μm). The top row shows scaffolds before cell seeding, while the bottom one shows them after cell seeding. C Confocal microscope pictures showing cell morphology and distribution. F-action was marked in red and nuclei were marked in blue. D–F Porosity (D), compression strength (E), and modulus of elasticity (F) of the PCL/HA scaffolds

Fig. 6

Radiographic assessment (X-ray and micro-CT) of bone regeneration. A X-ray images showing the progression of intervertebral fusion 2, 4, 8, and 12 weeks after surgery for the blank, scaffold, scaffold-cell, and scaffold-cell-miR groups. B Three-dimensional images reconstructed by micro-CT showing intervertebral fusion conditions under different treatments (blank, scaffold, scaffold-cell, and scaffold-cell-miR groups). The top row shows the sagittal plane; middle, coronal; and bottom, transverse. C, D Quantification analysis of bone volume relative to total volume (BV/TV) (C), and bone mineral density (BMD) (D). *p < 0.05, **p < 0.01, ***p < 0.001 compared to the blank group. ^#p < 0.05, ^##p < 0.01 compared to the scaffold-cell group

Fig. 7

Histological verifications [haematoxylin and eosin (HE), Masson, and immunohistochemical staining analysis] of bone regeneration. A, C HE and Masson staining images detecting the intervertebral fusion condition (A), and corresponding new bone area fraction analysis (C). B, D–F Immunohistochemical staining of the osteogenic markers alkaline phosphatase (ALP), Col 1, and OCN (B) and corresponding quantification of ALP (+) (D), Col 1(+) (E), and OCN (+) (F) cells

Fig. 8

Electromagnetic field (EMF) enhanced transfection efficiency through the activation of p38 MAPK pathway. A, B Western blotting bands and corresponding protein expression levels of phosphorylated p38, p38, tau, and HAP27 (p-p38, p38, p-tau, p-HSP27, respectively). *p < 0.05, **p < 0.01, ***p < 0.001 compared to the control group. ^#p < 0.05, ^##p < 0.01 compared to the EMF + PEI@IONPs + SB202190 group. C Fluorescence microscope images showing different transfection efficiencies under different treatments (control, EMF + PEI@IONPs, and EMF + PEI@IONPs + SB202190). D, E Immunofluorescence images of Runx2 (D) and CD31 (E) after various treatments (control, EMF + PEI@IONPs, and EMF + PEI@IONPs + SB202190). Runx2/CD31 was labelled with Cy3 (red), and nucleus were labelled with 4′,6-diamidino-2-phenylindole (DAPI, blue)