Adeno-Associated Virus-Mediated Osteoprotegerin Gene Transfer Protects Against Wear Debris Induced Osteolysis In A Murine Pin Model

BackgroundTotal joint replacement is a highly successful and common procedure in the treatment of end stage arthritis. However, as many as 34% of the total joint replacement components will loosen and eventually fail because of aseptic loosening, which has become the major complication of this procedure.While failures due to infection, fracture, and dislocation have become relatively rare, osteolysis-associated aseptic loosening (AL) has become more common and important. Although the precise pathogeneses of aseptic loosening is unclear, cumulative evidence indicates that particulate biomaterial debris generated from the mechanical wear of prosthetic components plays a critical role. It is generally accepted that wear debris provokes diverse biological tissue responses, including vascularized granulomatous tissue formation along the implant-to-bone interface, inflammatory cell (macrophages, lymphocytes) influx, bone resorption, and finally osteolysis and loss of prosthesis fixation.At present the main therapy for debris associated loosening include: inhibit the bone absorption, accelerate the synthesis of new bone and improve the material of artificial joint.However, treatment of the debris associated loosening process is highly problematic, partially due to the difficulty of administering and maintaining effective dosages of therapeutic agents at the site of loosening. When anti-inflammation or anti-osteolysis drugs are administered systemically, their effects at the bone- implant interface rely on vascular perfusion, suggesting that relatively high systemic levels are required to achieve anti-inflammatory activity at the site of loosening process. High systemic drug levels often induce adverse side affects and subsequent poor patient compliance. Gene therapy, though still in its infancy, provides an attractive alternative to overcome this difficulty.To investigate the molecular and biomechanical mechanisms underlying aseptic loosening, and to explore therapeutic intervention to prevent or treat the complication, the development of an animal model that closely mimics human joint arthroplasty and reflects the characteristics of aseptic loosening is an essential prerequisite. Currently, most animal models of joint arthroplasty involve large animals such as sheep and dogs, while the small animal models (rats or mice) are often restricted to short-term studies due to the difficulty of implanting hardware.Objective1. Establish the long-term pin mouse model of debris-associated bone resorption and joint prosthesis failure2. Characterize the biomechanical and pathological aspects of the loosening process.3. Exam the feasibility of gene therapy as a potential alternative avenue to control this common complication.Method1. Establish the Pin-model use mouseFifty-four mice aged 10-12 weeks, weighted 20g, quarantined in cages for 2 weeks prior to experimentation. All mice weighed at least 20g, divided into three gourps: stable group, Ti-pariticle group and gene exame group, at the start of the experiment. Titanium-alloy pins of the same specification were kindly produced by Stryker Orthopaedic Inc. particle size 0.53μm with a range of 0.1-4.1μm. Adeno-associated virus codingβ-galactosidase (AAV-LacZ) was obtained from the Gene Core facility at University North Carolina, Chapel Hill. Under aseptic condition, the proximal tibia condyle was exposed a cavity for the implant was reamed. A titanium pin was press-fitted into the canal in a manner. For the evaluation of debris, mice were injected with 10μl of titanium suspension into the tibia canal before the insertion of the pin, and 20μl of debris particles was then injected intraarticularly to the prosthetic joint every month following surgery. The control implantation mice symmetrically received intraarticular injection of particle-free PBS. The mice were sacrificed at 2, 4, 12, and 24 weeks for biomechanical, histological, and molecular evaluation.2. Micro-Computerized Tomography (Micro CT) ScansAll mice were scanned immediately following surgery using Micro CT system to confirm the proper position of pin implantation. Following acquisition and reconstruction, the image data were collectd every 4 weeks post operation and analyzed using GEHC MicroView1 software to generate isosurfaces of the region of ' interest (ROI) and to calculate the bone mineral densities (BMD) of the tibia bone surrounding the titanium pin.3. Interfacial Shear Strength TestFollowing sacrifice, the mouse limb with the implant intact was removed by disarticulating the knee joint. All soft tissue around the prosthetic joint was carefully removed to expose the implanted pin surface and proximal tibia. A custom aluminum fixture was designed to align the long axis of the orthopedic implant with the loading axis of the Instron model 8841 (Canton, MA). The implant was pulled out of the bone at a rate of 2.0 mm/min. Actuator position and load was recorded.4. In Vivo Exogenous TransferAdeno-associated virus coding for the LacZ gene (AAV-LacZ) was used to examine the feasibility of in vivo gene transfer using this model. At 4 weeks after pinimplantation surgery, 50μl of sterile culture medium containing 10~8IU/ml of AAV-LacZ was injected into each prosthetic joint of five mice. The prosthetic joints of control mice received vehicle culture medium without virus. All mice were sacrificed 7 days after in vivo gene transfer. X-gal staining was performed on the prosthetic joint using procedures according instruction. 5. Histological and Immunohistochemical (IHC) AnalysesFormalin-fixed prosthetic joints were decalcified with formic acid/sodium citrate before paraffin-embedding. The sections were stained with hematoxylin and eosin to examine new bone formation or bone erosion around the prosthetic pin, and to evaluate debris-associated inflammation, including periprosthetic tissue formation and cellular infiltration. Modified trichrome staining was performed to examine bone collagen changes. Immunohistochemical stains were carried out to evaluate pro-inflammatory cytokines and mediators (IL-1, TNF and CD68) of osteoclastogenesis in periprosthetic tissues. X-gal stain was employed to trace LacZ gene transduced Cells.RESULTS1. Operative OutcomeThe mice tolerated the surgical procedure well and ambulated with the implanted limb within 3 days after surgery. Injections of titanium particles appeared to exert no influence on daily activity in comparison to the animals without particle injections. The macroscopic examination of the prosthetic joints during sacrifice revealed metal pins positioning in proximal tibiae, and no scratch nor inflammation on the opposing articulate surfaces. There were no obvious structural differences between stable and particle-stimulated implantation groups by naked eyes.2. Micro CT EvaluationMicro CT scans indicated that the implants were well fixed with no obvious migration up to 6 months after surgery without particle challenge. However, titanium particle injection induced marked periprosthetic bone resorption illustrates typical debris-associated aseptic loosening on CT imaging. Images with Ti alloy pin implantation provided good evidence of stable BMD between pin-implanted limbs immediately postoperative and the limbs at 24 weeks months after implantation in the absence of particle challenge. In contrast, a significant BMD loss was observed obviously in debris-injected implanted joints since 12 weeks.3. Implant Stability Tested by Pullout Test The average interfacial shear strength against pulling at 24 weeks was 4.5±0.43N on the stable implant and there was no statistical difference between 4, 12 and 24 weeks after surgery. However, the introduction of titanium particles significantly decreased the implant stability, with only 0.44±0.31N at 24 weeks of pulling force required to dissociate the implant from the bone.4. Histological AnalysesThe histological appearances of the pin prosthesis model clearly revealed that the implantation of pins without particles resulted in a stable condition and irregular new bone formation, with bone collagen content well preserved. However, an extensive periprosthetic soft membrane developed in the implanted joints exposed to monthly particle injections. Further, the periprosthetic bones stimulated with titanium particles exhibited much fainter blue color staining using Modified Trichrome staining, when compared to the staining seen using sections from stable implants. Using a computerized image analysis system, the IOD of Trichrome staining in debris-induced bone resorption group averaged 35%±3.5% loss at 6 months after pin implantation, in comparison with debrisfree stable implant group (p<0.05). The time study of pin-implantation with monthly titanium particle injections to mimic debris-associated loosening process indicated a continuous inflammatory cellular infiltration and periprosthetic membrane formation, starting at 4 weeks following surgery. The cellularity and thickness of the periprosthetic membranes correlated with the amount of debris accumulated and length of time of debris stimulation (p<0.05). Immunohistochemical assessment using antimouse cytokine antibodies revealed a profound accumulation of TNF and IL-1 expressing cells in the particle-stimulated sections. CD68+ macrophages were also present in marked aggregations in particle-stimulated periprosthetic membranes.5. Feasibility of In Vivo Gene Transfer in the ModelX-gal staining revealed that a direct single injection of AAV-LacZ into the implanted joint resulted in strong transgene expression, indicated by strong blue coloration in the synovial membranes and periprosthetic tissue. In contrast, the joints receiving virus-free medium injections remained negative using this stain. CONLUSION1. We have established a murine model to represent knee prosthesis failure due to wear debris stimulation.2. The time study of pin-implantation with monthly titanium particle injections to mimic debris-associated loosening process indicated a continuous inflammatory cellular infiltration and periprosthetic membrane formation.3. The possibility of in vivo gene transfer in this kind of model. BACKGROUNDTotal joint replacement is a highly successful and common procedure in the treatment of end stage arthritis. However, as many as 34% of the total joint replacement components will loosen and eventually fail because of aseptic loosening, which has become the major complication of this procedure. How to prevent or treat this disease becomes the important issue in the implant field.Among the most important factors that may contribute to loosening is the adverse tissue response to particulate wear debris. Titanium-alloy has been broadly used in total joint prosthesis, and the Titanium-alloy components removed at revision surgery regularly show wear. Histologic evaluation of tissues from failed primary arthroplasties showed that particulate polyethylene is the most common debris found in periprosthetic tissue. It has been accepted that particles generated by mechanical wear of the prosthesis are phagocytosed by macrophages, resulting in cellular activation and release of proinflammatory mediators and cytokines, such as interleukin-1 (IL-1), tumor necrosis factor (TNF), and IL-6. These mediators, in turn, induce local chronic inflammation with activation and recruitment of osteoclasts to the bone-implant interface. This affects bone remodeling around the implant and leads to osteolysis and aseptic loosening.While many different cytokines contribute to this process, studies have shown that the osteoclast differentiation factor (also called receptor activator of nuclear factor-κB ligand [RANKL]) is 1 of the only 2 essential mediators to promote osteoclastogenesis. It binds to its membrane-bond signaling receptor, RANK, and stimulates osteoclast differentiation and maturation. Recently, a soluble protein osteoprotegerin (OPG) was identified in many types of cells and proved a natural "decoy" receptor that competed for RANKL with RANK and blunted its effects of osteoclastogenesis. Mice genetically deficient for RANKL or RANK suffered severe osteopetrosis, whereas OPG transgenic mice expressed the same pathology, demonstrating that RANKL and RANK are essential for osteoclast development, and OPG is a potent negative regulator for osteoclastogenesis.Based on the anti-osteolytic nature of OPG, it may be a potential therapeutic agent to treat debrisassociated periprosthetic bone resorption and aseptic loosening. While it is difficult, by conventional therapy, to administer sufficient OPG to osteolytic sites around the prosthetic joint, gene therapy provides an elegant solution to the delivery problem.OBJECTIVE1. Examine AAV-OPG gene transfer to protect against wear debris induced osteolysis in a murine Pin-model of bone resorption2. Examine The feasible effects of AAV-OPG gene therapy3. Debate the mechanism of gene therapyMETHOD1. Establish the Pin-model use mouseFifty-four mice aged 10-12 weeks, weighted 20g, divided into three groups: AAV-OPG, Ti group and Stable group, quarantined in cages for 2 weeks prior to experimentation. Titanium-alloy pins and Particle used in this experiment as part one. AAV-OPG was obtained as part one mentioned.Under aseptic condition, the proximal tibia condyle was exposed a cavity for the implant was reamed. A titanium pin was press-fitted into the canal in a manner. For the evaluation of debris, mice were injected with 10μl of titanium suspension into the tibia canal before the insertion of the pin, and 20μl of debris particles was then injected intraarticularly to the prosthetic joint every 4 weeks following surgery. AAV-OPG was injected into the jointraarticular after 1 week of surgery. The control implantation mice symmetrically received intraarticular injection of particle or virus-free PBS. The mice were sacrificed at 2, 4 and 12 weeks for biomechanical, histological, and molecular evaluation.2. Micro-Computerized Tomography (Micro CT) ScansAll mice were scanned immediately following surgery using MicroCT system to confirm the proper position of pin implantation. Following acquisition and reconstruction, the image data were analyzed using GEHC MicroView1 software to generate isosurfaces of the region of interest (ROI) and to calculate the bone mineral densities (BMD) of the tibia bone surrounding the titanium pin. The following data were collected per 4 weeks after operaton.3. Interfacial Shear Strength TestFollowing sacrifice, the mouse limb with the implant intact was removed by disarticulating the knee joint. All soft tissue around the prosthetic joint was carefully removed to expose the implanted pin surface and proximal tibia. A custom aluminum fixture was designed to align the long axis of the orthopedic implant with the loading axis of the Instron model 8841 (Canton, MA). The implant was pulled out of the bone at a rate of 2.0 mm/min. Actuator position and load was recorded.4. Histological and Immunohistochemical (IHC) AnalysesFormalin-fixed prosthetic joints were decalcified with formic acid/sodium citrate before paraffin-embedding. The sections were stained with hematoxylin and eosin to examine new bone formation or bone erosion around the prosthetic pin, and to evaluate debris-associated inflammation, including periprosthetic tissue formation and cellular infiltration. Modified trichrome staining was performed to examine bone collagen changes. Histochemical tatrate-resistant acid Phosphatase (TRAP) staining performed to localize the osteclast-like cells in the Pin-model of bone-implant surrounding. The presence of dark purple staining granules in the cytoplasm was a specific criterion for counting TRAP-positive cells. Immunohistochemical stains were carried out to evaluate pro-inflammatory cytokines and mediators (IL-1, TNF and CD68) of osteoclastogenesis in periprosthetic tissues.5. Molecular and immunologic analysis-ELISA Enzyme-linked immunosorbent assays (ELISA) were conducted on the supernatants of the tabular homogenates to examine OPG-transgene production. Tests were performed using the standardized protocol previously described.6. RT-PCR to analysis the expression of OPGReal-time reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to assess the influence of gene transfer on osteoclastogenesis. Gene expression of OPG was examined. Total RNA from homogenates was extracted following the manufacturer's instructions. The cDNA was reverse transcribed. Real-time PCR was performed according to the manufacturer's instructions.RESULTS1. Operative OutcomeThe mice tolerated the surgical procedure well and ambulated with the implanted limb within 3 days after surgery. Injections of titanium particles appeared to exert no influence on daily activity in comparison to the animals without particle injections. The macroscopic examination of the prosthetic joints during sacrifice revealed metal pins positioning in proximal tibiae, and no scratch nor inflammation on the opposing articulate surfaces. There were no obvious structural differences between stable and particle-stimulated implantation groups by naked eyes.2. MicroCT EvaluationMicroCT scans indicated that the implants were well fixed with no obvious migration up to 12 weeks after surgery without particle challenge. However, titanium particle injection induced marked periprosthetic bone resorption without AAV-OPG injection illustrates gene therapy can protect debris-associated aseptic loosening (p<0.05).3. Implant Stability Tested by Pullout TestThe average interfacial shear strength against pulling was 4.6±0.31N on the stable implant and 4.3±0.5N in the AAV-OPG group. There was no statistical difference between 2, 4, and 12 weeks after surgery. However, the introduction of titanium particles significantly decreased the implant stability in group Ti group, with only 0.44±0.31N of pulling force required to dissociate the implant from the bone (p<0.05).4. Histological AnalysesThe histological appearances of the pin prosthesis model clearly revealed that the implantation of pins without particles or AAV-OPG therapy resulted in a stable condition and irregular new bone formation, with bone collagen content well preserved. However, an extensive periprosthetic soft membrane developed in the implanted joints exposed to monthly particle injections in Ti group.Further, the periprosthetic bones stimulated with titanium particles exhibited much fainter blue color staining using Modified Trichrome staining, when compared to the staining seen using sections from stable implants and gene therapy. Using a computerized image analysis system, the IOD of Trichrome staining in debris-induced bone resorption group averaged 33%±2.5% loss at 12 weeks after pin implantation, in comparison with debris free stable implant or AAV-OPG group (p<0.05).The TRAP staining was conducted to identify osteoclast-like cells in the model. A typical TRAP-staining photomicrograph revealing that dark brown TRAP-positive cells accumulate along the bone surface in the non OPG therapy group with debris challenge, while fewer cells could be found in AAV-OPG therapy group.Immunohistochemical assessment using antimouse cytokine antibodies revealed a profound accumulation of TNF and IL-1 expressing cells in the particle-stimulated sections. CD68+ macrophages were also present in marked aggregations in particle-stimulated periprosthetic membranes.5. Molecular and immunologic analysis ELISAEnzyme-linked immunosorbent assays (ELISAs) were conducted on the supernatants of the tabular homogenates to examine OPG-transgene production. The OPG protein level is higher in OPG therapy group than the other groups illustrated the succeed transduction and expression of AAV-OPG.6. RT-PCRGene expression of OPG was examined and is higher expressed in AAV-OPG therapy group. CONLUSION1. The long term murine model of knee joint implant loosening is a successful model for evaluates the bone resorption to screen therapeutic approaches to debris-associated osteolysis.2. Gene transfer using AAV-OPG appears to be a feasible and effective candidate to treat or prevent wear debris-associated ostelysis and aseptic loosening.3. Further studies are under to help us understand the transductive efficacy and long-term effects of the AAV-OPG gene transfer and related therapeutic mechanisms and safety concerns.