Gene Therapy (2010) 17, 1262–1269 & 2010 Macmillan Publishers Limited All rights reserved 0969-7128/10 www.nature.com/gt ORIGINAL ARTICLE Cell-based osteoprotegerin therapy for debris-induced aseptic prosthetic loosening on a murine model L Zhang1,2, T-H Jia2, ACM Chong1, L Bai1,HYu1, W Gong2, PH Wooley1 and S-Y Yang1 1Orthopaedic Research Institute, Via Christi Regional Medical Center, Wichita, KS, USA and 2Department of Orthopaedic Surgery, Jinan Central Hospital, Shandong University School of Medicine, Jinan, China Exogenous osteoprotegerin (OPG) gene modification membranes existed ubiquitously at bone–implant interface appears a therapeutic strategy for osteolytic aseptic in control groups, whereas only observed sporadically in OPG loosening. The feasibility and efficacy of a cell-based OPG gene-modified groups. Tartrate-resistant acid phosphatase+ gene delivery approach were investigated using a murine osteoclasts and tumor necrosis factor a,interleukin-1b, model of knee prosthesis failure. A titanium pin was CD68+ expressing cells were significantly reduced in implanted into mouse proximal tibia to mimic a weight- periprosthetic tissues of OPG gene-modified mice. No bearing knee arthroplasty, followed by titanium particles transgene dissemination or tumorigenesis was detected in challenge to induce periprosthetic osteolysis. Mouse fibro- remote organs and tissues. Data suggest that cell-based blast-like synoviocytes were transduced in vitro with either ex vivo OPG gene therapy was comparable in efficacy with AAV-OPG or AAV-LacZ before transfused into the osteolytic in vivo local gene transfer technique to deliver functional prosthetic joint 3 weeks post surgery. Successful transgene therapeutic OPG activities, effectively halted the debris- expression at the local site was confirmed 4 weeks later after induced osteolysis and regained the implant stability in this killing. Biomechanical pullout test indicated a significant model. restoration of implant stability after the cell-based OPG Gene Therapy (2010) 17, 1262–1269; doi:10.1038/gt.2010.64; gene therapy. Histology revealed that inflammatory pseudo- published online 29 April 2010 Keywords: aseptic loosening; periprosthetic osteolysis; cell-based therapy; osteoprotegerin; implant stability Introduction around the implant and leads to osteolysis and aseptic loosening.6 Total joint replacement is a highly successful procedure The receptor activator of nuclear factor NF-kB ligand for the treatment of severe destructive arthritis. Although (RANKL)/osteoprotegerin (OPG) pathway has a key role failures of the surgery because of infections and errors in bone metabolism and osteolysis. RANKL is mainly have been greatly reduced in recent years, aseptic expressed by macrophages, osteoblasts, marrow stromal prosthetic loosening remains the most common compli- cells, and lymphocytes. RANKL binds to RANK, a cation of total joint replacement and represents a major receptor on the cell surface of osteoclasts and osteoclast problem for the long-term success and survival of precursors, resulting in proliferation, differentiation, and prostheses.1 Wear debris-induced inflammation and maturation of osteoclasts, which can subsequently osteoclastic resorption at the bone–implant interface are promote local osteolysis.7 In contrast, OPG, a secreted believed to have critical roles in the pathogenesis of protein with homology to members of the TNF super- aseptic loosening.2 It has been accepted that particles family, is considered to be a natural decoy receptor that generated during the mechanical wear of prosthetic profoundly modifies the effects of RANKL by inhibiting components are phagocytosed by macrophages and RANKL/RANK interaction.8,9 Mice genetically deficient other inflammatory cells, resulting in cellular activation for RANKL or RANK suffered severe osteopetrosis,10,11 and release of pro-inflammatory mediators and cyto- and OPG transgenic mice share the same pathology,12 kines such as interleukin-1 (IL-1b), tumor necrosis showing that RANKL and RANK are essential for factor (TNFa), and IL-6.3 These cytokines in turn osteoclast development and OPG is a potent negative provoke cellular proliferation, promote osteoclastogen- regulator for osteoclastogenesis. esis, and stimulate mature osteoclasts to absorb the On the basis of the anti-osteolytic nature of OPG, it adjacent bone.4,5 This process impacts bone remodeling appears to be a potential therapeutic agent to treat debris-associated periprosthetic bone resorption and aseptic loosening. However, the delivery of OPG to chronic periprosthetic osteolytic sites remains proble- Correspondence: Dr S-Y Yang, Orthopaedic Research Institute, Via matic. Current systemic anti-inflammation or anti- Christi Regional Medical Center, 929 N St Francis Street, Wichita, osteolysis therapies have several weaknesses including KS 67214, USA. E-mail: [email protected] high-dose requirements with modest efficiency, systemic Received 25 January 2010; revised 5 March 2010; accepted 5 March side effects, and the tendency for poor patient compli- 2010; published online 29 April 2010 ance.13 Gene therapy, though still in its infancy, provides OPG cell therapy for aseptic loosening L Zhang et al 1263 an attractive alternative to treat aseptic loosening as the Outcome of surgical procedures transfer of genes may facilitate the continuous release of A custom-made titanium pin was implanted into therapeutic proteins. Using a mouse calvaria model, mouse proximal tibia to form a weight-bearing Goater et al.14 were the first to examine the potential of knee implant. The mice tolerated surgery well and ex vivo OPG gene therapy with stably transfected ambulated with the implanted limbs within 3 days fibroblast-like synoviocytes (FLS) expressing OPG in after surgery. Injections of titanium particles and preventing wear debris-induced osteoclastogenesis. We FLS into the prosthetic knees appeared to exert no recently reported that in vivo gene transfer of OPG influence on their daily activity. The macroscopic effectively blocked osteoclastogenesis and reversed examination of the prosthetic joints at killing revealed periprosthetic bone resorption using a newly character- that the metal pins positioned in proximal tibiae with no ized mouse model of knee prosthesis failure.15 This study signs of scratching or inflammation on opposing articu- extended the investigation to evaluate the therapeutic late surfaces. There were no obvious surgical differences effects of a cell-based OPG gene modification by between the groups to the naked eye, except that a few delivering the stable OPG-transduced FLS into the failing pin implants from non-treated or LacZ-treated groups knee prosthesis, in comparison with the direct in vivo could be rotated or moved by hand because of loss of gene transfer approach. The biomechanical properties of the fixation. failing knee pin implantation after gene modification were also assessed. Transgene expression and dissemination RNA extracted from the treated joints and other organs/ tissues from animals with ex vivo OPG gene therapy were Results examined by conventional reverse transcription–poly- merase chain reaction (RT–PCR) with human OPG OPG and LacZ gene transduction efficacy in FLS primers. No positive PCR products were detected, except In preparation for the ex vivo gene therapy experiments, for the periprosthetic tissues where the ex vivo gene target cells stably expressing OPG and b-galactosidase transfer was conducted (Figure 1e), where results were (LacZ) were engineered by infecting the primary FLS similar to in vivo OPG treatment.15 All prosthetic joint with AAV-OPG-EGFP or AAV-LacZ, respectively. Fluor- samples with ex vivo LacZ gene transfer exhibited dark escent microscopy identified green fluorescent signals on blue coloration (Figure 1d). ELISA confirmed that OPG cells transduced with AAV-OPG-EGFP. X-gal stain was protein production in FLS-AAV-OPG culture medium used to trace LacZ gene-transduced cells. The transduc- was 4.20 ng per 72 h per 106 cells, where OPG levels in tion efficiencies in the FLS for both vectors were higher periprosthetic tissues from ex vivo OPG-treated groups than 90% at day 3 and were maintained for at least were 2.60 ng mgÀ1 total protein at killing, comparable 4 weeks (Figures 1a, b, and c). with levels observed during in vivo OPG treatment. Figure 1 Transgene expression in FLS cells. (a) Bright light microscopic appearance of mouse FLS cells 3 days after AAV-OPG-EGFP transduction; (b) fluorescent microscopy of the same cells for green fluorescent protein emission. (c, d) X-gal staining of in vitro transduction with AAV-LacZ on FLS cells (c) and a macroscopic joint sample 4 weeks after cell-based gene modification. (e) An agarose gel of conventional PCR products revealing OPG gene expression in tissues from FLS-AAV-OPG-treated mice: lane 1: DNA ladder; lane 2: liver; lane 3: lung; lane 4: lymph node; lane 5: muscle; lane 6: kidney; lane 7: spleen; lane 8: prosthetic tissue homogenate; lane 9: tissue with in vivo AAV-OPG transduction as positive control. (The color reproduction of this figure is available on the html full text version of the manuscript.) Gene Therapy OPG cell therapy for aseptic loosening L Zhang et al 1264 Immunohistochemistry analysis and tartrate-resistant acid phosphatase staining A profound accumulation of CD68+ macrophages, and TNFa and IL-1 expressing cells were observed at the interface between the pin and surrounding bone in sections from both FLS-AAV-LacZ and virus-free non- treated groups. In