title: Lentivirus-mediated short hairpin RNA interference targeting TNF-alpha in macrophages inhibits particle-induced inflammation and osteolysis in vitro and in vivo.
abstract: 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.
  
  
  
  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.
  
  
  
  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.
  
  
  
  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.
authors.affiliation: Department of Orthopaedic Surgery, The Memorial Hospital of Sun Yat-Sen University, 107 Yanjiangxi Road, 510120, Guangzhou, China.
authors.forename: Chu-Qiang
authors.initials: CQ
authors.lastname: Qin
authors.affiliation: Department of Orthopaedic Surgery, The Memorial Hospital of Sun Yat-Sen University, 107 Yanjiangxi Road, 510120, Guangzhou, China.
authors.forename: Dong-Sheng
authors.initials: DS
authors.lastname: Huang
authors.affiliation: Department of Orthopaedic Surgery, The Memorial Hospital of Sun Yat-Sen University, 107 Yanjiangxi Road, 510120, Guangzhou, China.
authors.forename: Chi
authors.initials: C
authors.lastname: Zhang
authors.affiliation: Department of Orthopaedic Surgery, The Memorial Hospital of Sun Yat-Sen University, 107 Yanjiangxi Road, 510120, Guangzhou, China.
authors.forename: Bin
authors.initials: B
authors.lastname: Song
authors.affiliation: Department of Orthopaedic Surgery, The Memorial Hospital of Sun Yat-Sen University, 107 Yanjiangxi Road, 510120, Guangzhou, China.
authors.forename: Jian-Bin
authors.initials: JB
authors.lastname: Huang
authors.affiliation: Department of Orthopaedic Surgery, The Memorial Hospital of Sun Yat-Sen University, 107 Yanjiangxi Road, 510120, Guangzhou, China. dingyue36@126.com.
authors.forename: Yue
authors.initials: Y
authors.lastname: Ding
mesh_terms: D000818:Animals; D002516:Ceramics / Q000009:adverse effects; D004195:Disease Models, Animal; D055785:Gene Knockdown Techniques; D005822:Genetic Vectors / Q000627:therapeutic use; D006801:Humans; D007249:Inflammation / Q000209:etiology / Q000628:therapy*; D007595:Joint Prosthesis / Q000009:adverse effects*; D016086:Lentivirus / Q000235:genetics; D008264:Macrophages / Q000378:metabolism*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D010014:Osteolysis / Q000209:etiology / Q000628:therapy*; D010316:Particle Size; D011475:Prosthesis Failure; D000067996:RAW 264.7 Cells; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D000067492:RNAi Therapeutics / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction; D012886:Skull; D014025:Titanium / Q000009:adverse effects; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics*
chemical_list: D012333:RNA, Messenger; D034741:RNA, Small Interfering; D014409:Tumor Necrosis Factor-alpha; D014025:Titanium
keywords: Hip arthroplasty; Lentivirus; Periprosthetic osteolysis; RNA interference; Wear particle