Aseptic loosening of orthopaedic implants secondary to wear debris-induced
osteolysis is a serious problem. Osteoprotegerin (OPG) is a natural
decoy protein that inhibits osteoclast activation and bone resorption.
This study investigated whether gene therapy using a recombinant
adeno-associated viral vector that expresses OPG can inhibit wear
A recombinant adeno-associated virus (rAAV) vector co-expressing
OPG (rAAV-OPG-IRES-EGFP) was generated. A control vector expressing
b-galactosidase (rAAV-LacZ) was also prepared. In vitro validation
experiments were performed to determine rAAV-OPG-IRES-EGFP transduction
efficiency, OPG expression level and function in bone wafer, and
The effect of rAAV-OPG-IRES-EGFP in vivo gene therapy on wear
debris-induced osteolysis was then evaluated in a mouse calvarial
model in which a single intramuscular injection of the vector was
administered prior to the introduction of the wear debris. The effects
of the rAAV-OPG-IRES-EGFP gene therapy on wear debris-induced osteoclastogenesis
and bone resorption were determined by histomorphometry on day 10.
In vitro experiments revealed that 100% of human embryonic kidney
293 cells were transduced at a multiplicity of infection of 1000
with both rAAV-OPG-IRES-EGFP and rAAV-LacZ. At a rAAV-OPG-IRES-EGFP
multiplicity of infection of 1000, an OPG concentration of 135 ng/mL
of culture media was achieved after four days. Using a bone-wafer
assay for osteoclast activity, we found that treatment with rAAV-OPG-IRES-EGFP
reduced resorption sevenfold compared with parathyroid hormone-stimulated
controls and elevenfold compared with rAAV-LacZ controls. Furthermore,
a seventeenfold decrease in RANKL and macrophage colony-stimulating
factor-induced splenocyte osteoclastogenesis was observed in co-cultures
containing rAAV-OPG-IRES-EGFP-infected fibroblasts.
In vivo administration of rAAV-OPG-IRES-EGFP resulted in detectable
transduction of myocytes at the injection site and a significant
increase in expression of serum OPG levels by the second day (p < 0.05).
Maximal concentrations were obtained on day 6 and then leveled off
throughout the observation period. In contrast, serum OPG could
not be detected in the sham-treated, uninfected titanium-stimulated,
or rAAV-LacZ-infected mice. In the control mice, titanium implantation resulted
in a threefold increase in the mean number of osteoclasts adjacent
to the sagittal suture as well as a twofold increase in the mean
area of soft tissue in the sagittal suture compared with the sham-treated
mice. In contrast, osteoclast numbers remained at basal levels,
and the area of soft tissue in the sagittal suture was markedly
reduced in titanium-implanted animals that received rAAV-OPG-IRES-EGFP treatment,
demonstrating a complete inhibition of osteolysis in response to
A single intramuscular injection of the rAAV-OPG-IRES-EGFP vector
can efficiently transduce myocytes to produce high levels of OPG.
The OPG effectively inhibits wear debris-induced osteoclastogenesis
Currently, there is no approved drug therapy to prevent or inhibit
periprosthetic osteolysis. Although preclinical studies have identified
potential drug therapies (i.e., bisphosphonates), there is no evidence
that these drugs can effectively treat aseptic loosening in patients.
This is the first evidence that in vivo OPG gene therapy can be
used to prevent wear debris-induced osteolysis.