Lentivirus-mediated short hairpin RNA interference targeting TNF-alpha in macrophages inhibits particle-induced inflammation and osteolysis in vitro and in vivo

Abstract

Background: Aseptic loosening is a significant impediment to joint implant longevity. Prosthetic wear particles are postulated to play a central role in the onset and progression of periprosthetic osteolysis, leading to aseptic loosening of the prosthesis.

Methods: We investigated the inhibitory effects of a lentivirus-mediated short hairpin RNA that targets the TNF-alpha gene on the particle-induced inflammatory and osteolytic changes via macrophages both in vitro and in vivo. An siRNA sequence targeting the mouse TNF-alpha gene from four candidates, transcribed in vitro, was screened and identified. A lentivirus vector expressing short hairpin RNA (shRNA) was then constructed in order to facilitate efficient expression of TNF-alpha-siRNA. Lentivirus-mediated shRNA was transduced into cells of the mouse macrophage line RAW 264.7. Ceramic and titanium particles were introduced 24 h after lentivirus transduction to stimulate cells. TNF-alpha expression, represented by both mRNA and protein levels, was quantified with real-time PCR and ELISA at all time intervals. Lentivirus-mediated shRNA suspension was locally administered into the murine calvarial model, followed by local injection of particles. A multi-slice spiral CT scan was used to evaluate the osteolysis of the calvaria by detecting the width of the cranial sutures.

Results: Macrophages developed pseudopods when co-cultured with particles. Lentivirus-mediated shRNA was shown to effectively inhibit the expression of TNF-alpha at both the mRNA and protein levels in RAW 264.7. The multi-slice spiral CT scan showed that the lentivirus-mediated shRNA significantly suppressed osteolysis of mouse calvaria.

Conclusions: Our investigation highlighted the results that lentivirus-mediated shRNA targeting the TNF-alpha gene successfully inhibited particle-induced inflammatory and osteolytic changes both in vitro and in vivo. Therefore, lentivirus-mediated gene therapy may provide a novel therapeutic approach to aseptic joint loosening.

Keywords: Hip arthroplasty; Lentivirus; Periprosthetic osteolysis; RNA interference; Wear particle.

Fig. 1

Measurement of cranial suture on orthogonal MPR images. The thin-slice images were reformatted with the MPR technique. The orthogonal sagittal (a), axial (b), and coronal planes (c) of the murine head were obtained. On the orthogonal coronal image (d), the largest cranial suture width (between the two arrows) was determined and measured

Fig. 2

Macrophage and particles under scanning electron microscopy. a Non-stimulated macrophage alone (8000×), b Macrophages stimulated with titanium particles (0.82 ± 0.12 μm) (5500×)

Fig. 3

Transfection efficiency of lentivirus-mediated shRNA interference determined by fluorescence microscopy analysis. a and c transfected macrophage cells under light microscopy of shRNA-LV and NC-LV group (20×); b and d transfected macrophage cells under fluorescence microscopy of the same visual field of shRNA-LV and NC-LV group (20×)

Fig. 4

Multiple comparison tests of TNF-alpha mRNA levels at different time points. mRNA levels of TNF-alpha in transfected macrophage cells at 3 h after particle stimulation. *p < 0.05 When compared to the control group; *p < 0.05 when compared to the Particle + TNF-LV group. a Titanium particles treatment; b Ceramic particles treatment

Fig. 5

Multiple comparison tests of TNF-alpha protein levels for different time points. Protein levels of TNF-alpha in transfected macrophage cells at 24 h after particle stimulation. *p < 0.05 When compared to the control group; *p < 0.05 when compared to the Particle + TNF-LV group. a Titanium particles treatment; b Ceramic particles treatment

Fig. 6

The width of the murine cranial suture was measured on the obtained orthogonal coronal images by 64-slice spiral CT. Significant reduction of cranial suture width was revealed in the Particle + TNF-LV group compared with the Particle and Particle + N.C-LV groups (P < 0.01). There was no difference between the Particle and Particle + N.C-LV groups in this parameter (P > 0.05)