Cell-based osteoprotegerin therapy for debris-induced aseptic prosthetic loosening on a murine model

Abstract

Exogenous osteoprotegerin (OPG) gene modification appears a therapeutic strategy for osteolytic aseptic loosening. The feasibility and efficacy of a cell-based OPG gene delivery approach were investigated using a murine model of knee prosthesis failure. A titanium pin was implanted into mouse proximal tibia to mimic a weight-bearing knee arthroplasty, followed by titanium particles challenge to induce periprosthetic osteolysis. Mouse fibroblast-like synoviocytes were transduced in vitro with either AAV-OPG or AAV-LacZ before transfused into the osteolytic prosthetic joint 3 weeks post surgery. Successful transgene expression at the local site was confirmed 4 weeks later after killing. Biomechanical pullout test indicated a significant restoration of implant stability after the cell-based OPG gene therapy. Histology revealed that inflammatory pseudo-membranes existed ubiquitously at bone-implant interface in control groups, whereas only observed sporadically in OPG gene-modified groups. Tartrate-resistant acid phosphatase+osteoclasts and tumor necrosis factor α, interleukin-1β, CD68+ expressing cells were significantly reduced in periprosthetic tissues of OPG gene-modified mice. No transgene dissemination or tumorigenesis was detected in remote organs and tissues. Data suggest that cell-based ex vivo OPG gene therapy was comparable in efficacy with in vivo local gene transfer technique to deliver functional therapeutic OPG activities, effectively halted the debris-induced osteolysis and regained the implant stability in this model.

Figure 1

Transgene expression in FLS cells. Panel A: bright light microscopic appearance of mouse FLS cells 3 days after AAV-OPG-EGFP transduction; Panel B illustrates fluorescent microscopy of the same cells for green fluorescent protein emission. Panels C and D show X-gal staining of in vitro transduction with AAV-LacZ on FLS cells (1C) and a macroscopic joint sample 4 weeks following cell-based gene modification. Panel E illustrates an agarose gel of conventional PCR products revealing OPG gene expression in tissues from FLS-AAV-OPG treated mice: Lane 1: DNA ladder; 2 – liver, 3 – lung, 4 – lymph node, 5 – muscle, 6 – kidney, 7 – spleen; Lane 8: prosthetic tissue homogenate; Lane 9: tissue with in vivo AAV-OPG transduction as positive control.

Figure 2

Summary of maximum pulling force required to pull out the implants at 4 weeks following treatments (* p<0.05). The insert illustrates an example trace of pulling forces applied to extract the pin implant from the surrounding bone.

Figure 3

Histological appearance of the pin-implanted tibiae at 4 weeks following in vivo and ex vivo gene modifications: (A) in vivo AAV-OPG treated; (B) cell-based FLS-AAV-OPG therapy; (C) in vivo AAV-LacZ injected; (D) FLS-AAV-LacZ transfused; (E) virus-free non-treated control. Panel (F) illustrates the comparison of periprosthetic membrane thickness among the five groups (** p< 0.01).

Figure 4

Quantitive bone collagen changes determined by integrated optical density (IOD) of Trichrome staining quantified using a computerized image analysis system (Left plot, ** p<0.01). Plot on the right side summarizes the numbers of TRAP+ cells in periprosthetic membranes among groups (** p<0.01).

Figure 5

IHC stained periprosthetic tissue sections to detect mouse IL-1β (upper panels), TNFα (middle panels) and CD68 (lower panels).

Figure 6

Implant sample mounted on the MTS 858 for the pull-out test. The insert shows the pin head captured between the blades of the holding jig.