| PART1The Effect of IL-1Receptor and OPG gene modification on weab-debris induced aseptic loosening and mechanism explorationBackgroundIn recent years with the people's living standards improvement a number of diseases have been caused by obesity and aging, such as the sever osteoarthritis, patients suffering with such advanced joint deformity that they lose the ability to act, seriously affecting the patient's quality of life. Accompanied by the development of artificial joints, joint replacement surgery, commonly used in treatment of severe joint disease,has solved the problem, However, aseptic prosthesis loosening, postoperative complication of joint replacement surgery occurres up to34%. joint revision surgery becomes necessary, difficult, not only the patients suffer with surgical pain caused by trauma due to arthroplasty, clinicians also requires the higher surgical technology. Based on joint replacement surgery extensively application and aseptic loosening as the most common complications of arthroplasty, more and more research focused intervention and treatment of aseptic loosening of the prosthesis.Due to the Special anatomy of joint, by systemic administration it is usually difficult to maintain a higher concentration of the drug around the joint, by the intra-articular injection it demands more aseptic operation, and the operation for invasive procedures make patients with greater suffering,also aseptic loosening of the prosthesis is a chronic disease, the treatment cycle is longer, and patients are often difficult to tolerate the treatment. How to adopt a mode of administration, long-term and efficient maintenance of the local drug concentration, but not caused by the systemic blood concentration increased,. Gene therapy provieds a good way by the gene transfection around the joint tissue, so that local tissue can continuely express therapeutic genes to achieve lasting and efficient drug concentration.The prosthesis Aseptic loosening has the complex pathological process, the specific mechanism has not yet been fully elucidated, but most studies have shown that periprosthetic wear debris plays an important role in the development in aseptic loosening. periprosthetic wear debris generated due to the mechanical activity, on the one hand wear debris itself as a foreign body to stimulate the body to produce inflammatory responses, on the other hand wear debris also produces mechanical injury to the surrounding soft tissue, the injured tissue will induce local inflammatory response. Due to local inflammatory factors release,such as IL-1and TNF and other inflammatory mediators, results in macrophage activation,whicn promote more inflammatory mediators released, and enhance phagocytosis of wear debris however, artificial joints are usually made of metallic materials, polymeric materials and ceramic materials,even the wear debreis of these materials are phagocyted by macrophages,they often can not be digested. so wear debris continue to induce the deterioration of the inflammation, and the inflammatory factors prompted a large number of osteoclastsgenesis, resulting in periprosthetic osteolysis increased, and further lead to prosthesis asptic loosening, with the increase in aseptic loosening, the mechanical activity results in more weardebris, the wear debris induces more severe inflammation and osteolysis. In summary, the entire pathological process presents a vicious circle.Above all we propose the hypothesis:inflammation and osteolysis are associated yet separated process in the development of prosthesis aseptic loosening. According to this hypothesis, we propose the combination of IL-1receptor antagonist and osteoprotegerin (OPG) gene therapy for aseptic loosening andfurther investigate the pathological mechanism of aseptic loosening.ObjectiveBy in vitro experiments combined with IL-1receptor antagonist and osteoprotegerin (OPG) gene therapy to inhibit osteoclastgenesis:1. to investigate the feasibility of combined anti-inflammatory and anti-osteolysis of prosthesis aseptic loosening2. to observe whether the combination group with half dose of the monotherapy group has a synergistic effect or not3. to further explore the mechanism of aseptic loosening pathogenesisMaterials and Methords1. Cell culture and in vitro gene transferThe RAW264.7cell line was purchased from the American Type Culture Collection, the size of the particles (2.3±0.168mm) was similar to the wear debris got from the periprosthetic tissue of patients revised for aseptic loosening. RAW cells at2×104ml-1were cultured in the presence of titanium particles (1×106ml-1) for72h before dividing into five groups:Group1was transduced with DFG-IL-1Ra (retroviral vectors code for IL-1receptor antagonist gene) by adding800ml of1×107pfu viral medium; Group2cells were transduced by AAV-GFP-OPG (Adeno-associated viral vectors encoding osteoprotegerin) at107particles ml-1; RAW cells in Group3were co-transduced sequentially with a half dose of AAV-OPG (0.4×104particles ml-1) and a half dose of DFG-IL1-Ra (0.4×104pfu), while the Group4RAW cells were transduced with107particles ml-1of AAV-LacZ as controls. Group5was control cells without viral infections. To achieve the highest concentration of the double gene expressions in the combination therapy group, the infection of AAV-OPG was introduced3days later after the retrovirus-IL-1Ra transduction. The culture media were changed every2days, pooled weekly and stored in-20℃for ELISA examination. All the cells were harvested periodically during the4-week cultivation.2. Enzyme-linked immunosorbent assayRelease of proinflammatory cytokines IL-lb and TNFa in the cell culture media following gene transduction was assessed using ELISA kits. The optical density was determined by an ELISA reader at405nm wavelength, and levels of cytokines were determined by regression analysis against a standard curve.3. TRAP stain for osteoclastsThe gene-modified RAW264.7cells were harvested for TRAP staining using a commercial kit. The cells in200ml suspension (1106cellsml1) were cytospun onto a histological slide followed by fixation for30s in buffered acetone in cold. The slide was incubated at371C for1h in0.1M acetate buffer (pH5.2), containing0.5mM naphthol AS-BI phosphoric acid,2.2mM FastGarnet GBC, and10mM sodium tartrate. The reaction was stopped by washing in several changes of distilled water. The presence of dark purple staining granules in the cytoplasm was considered as the specific criterion for identifying TRAP-positive cells.4. Real-time PCR for gene expression profileReal-time reverse transcriptase PCR was performed to assess the efficacy of anti-inflammation and anti-bone resorption after gene modifications. Total RNA from RAW cells after gene modifications was extracted in TRIzol reagent. cDNA was obtained by reverse transcription from0.5mg of total RNA in40ml of a reaction mixture containing1×PCR buffer,500mM each of nucleotide triphosphates,0.5Uml-1of ribonuclease inhibitor,2.5mM of random hexamers,5.5mM of MgC12, and1.25Uml-1of reverse transcriptase.The reaction mixture was incubated in a DNA Thermal Cycler at25℃for10min,48℃for5min, followed by95℃for5min. The expression of pro-inflammatory cytokines and mediators including IL-lb, RANK, c-Fos, TRAF6, JNK1, and CPK was determined using the ABI Prism7700sequence detector5. Statistical analysisStatistical analysis among groups was performed by one-way analysis of variance test with the Schafer formula for post hoc multiple comparisons. A P-value of0.05was considered as significant difference. Data are expressed as mean±s.e.m. Results1. The anti-inflammation effects of the combination gene therapyThe pre-osteoclastic mouse RAW264.7cells (2×104) were activated in vitro in the presence of titanium alloy particles (1×106ml-1), followed by virus-mediated gene transduction of IL-1receptor antagonist (DFG-IL-1Ra-neo) and osteoprotegerin (rAAV-GFP-OPG), individually or in combination. The combination group received one-half dosage of each individual viral vector (0.4×104pfu) to ensure transduction equivalence. RAW cells transduced with107particles ml-1of AAV-LacZ were used as nontherapeutic control. Enzyme-linked immunosorbent assay (ELISA)for IL-1in the culture media indicated significant inhibition of IL-1release in the IL-1Ra-treated group and the combination group at the first week following gene modification in comparison to LacZ controls, and the inhibition effect persisted through the3-week culture. During the later time period, all the gene therapy groups resulted in diminished IL-1expression levels, including OPG gene modification (with less efficiency). At the transcriptional level, real-time PCR on the RAW cell preparations at4weeks of culture post treatment revealed a significant decrease of IL-lb messenger RNA expression in all the therapeutic treatment groups, with best effect in the combination group.2. Effect of combination therapy on osteoclast differentiationRAW cells were harvested at4weeks after gene modifications. Tartrate-resistant acid phosphatase (TRAP) staining was performed to quantify mature osteoclasts. Significantly fewer TRAP-positive cells were present following OPG and OPGtIL-1Ra gene modifications, while IL-1Ra modification alone suggested less effectiveness. Although there were relatively fewer multinucleated cells in this type of cell line, acquirement of TRAPt characteristics suggests an indication of mature osteoclastic cells. Real-time PCR data further indicated that the double gene modification resulted in the most marked reduction of RANK messenger RNA expression levels within all treatment groups, thus revealing the influence of synergetic inhibition over the individual exogenous IL-1Ra or OPG gene transduction.3.The molecular exploration of the exogenous gene modifications on osteoclastogenesisReal-time PCR was performed to examine specific gene expression profiles on cells following the therapeutic gene modifications. Using gene expression data from the LacZ group as the baseline c-Fos was diminished54%,67%and88%in IL-1Ra, OPG, and double-gene groups, respectively, while TRAF6was inhibited25%,31%and44%, respectively. JNK1was inhibited in the combination group and IL-1Ra group to20%and16%, respectively, of the levels in the LacZ control, whereas no significant difference was found in the OPG group.Assessment of osteoclast marker CPK indicated that the combination gene therapy reduced its expression to more than50%compared with LacZ controls, more efficient than other two single gene modification groups.Conclusion1. IL-1Ra and OPG combined gene therapy can be effective anti-inflammatory, inhibits inflammatory cytokines IL-1release.2. IL-1Ra and OPG combined gene therapy significantly inhibited osteoclast differentiation.3IL-1Ra and OPG gene therapy had a better efficacy compared to single gene therapy group, suggesting the synergy effect of the combination gene therapy. PART2The Combination Gene Therapy Targetting on IL-1and RANKL for Weab-debris Induced Aseptic Loosening in A Murine Pin-ModelBackgroundThe prosthesis aseptic loosening is the most common long-term complications of joint replacement, in the literature it is reported up to34%of patients with aseptic loosening after joint replacement. The event of a prosthesis aseptic loosening, not only for patients suffering physical pain, but also the patient's condition is progressively increased with more weight-bearing activities, eventually it will lead to the loss of the walking ability again. The main treatment is to operate the joint revision surgery, Due to the surgical complexity, operating difficulties, higher cost, now it has become a research hotspot to explore a new treatment option to slow or prevent the occurrence of aseptic loosening.As to the research on the prosthesis aseptic loosening, the establishment of a suitable animal model has become an important content. There are reports in the literature to select the dogs, horses, sheep and other large animals used for model creation, those model has the advantages:similar to the human joint size, and suitable for long-term study, the disadvantages are high cost of the project and the limited amount of sample. Another model,air pounch mouse model, mimics intra-articular environment, which is easy to operate, but only suitable for short-term research, and shows only the pathological changes in acute phase.however, the Pin Model, in which titanium screw is implanted into mice knee, along with the injections of titanium particles that is similar to the wear debris in aseptic loosening, which successfully addresses the limited amount of sample, suitable for long-term research applications as well. the prosthesis aseptic loosening has complex pathological mechanism, histology shows the periprosthetic inflammatory pseudomembranous formation, inflammatory infiltration and periprosthetic osteolysis occurred. Most scholars believe that the wear debris leading to periprosthetic osteolysis is the main reason for aseptic loosening of the artificial joint. as a foreign body the periprosthetic wear debris generated to stimulate the local inflammatory response occurs, macrophages activated,and the large amounts of inflammatory factors such as IL-1TNF, IL-6released, as well as to stimulate the osteoblast, bonemarrow cells increasing the expression of receptor activator of expression of nuclear factor NF-KB of ligand(RANKL). Recent studies have found that OPG/RANKL/RANK system played an important role in the regulation of bone metabolic balance. the combination of RANKL and RANK which is expressed by osteoclast precursor cells, will start the osteoclastsgenesis, promoting the local osteolysis, however,osteoprotegerin (OPG) and RANKL are competitive combination of RANK, but OPG does not activate osteoclastogenesis, which play a role in protection of bone, so OPG is widely used in the research of osteoclast. during the repair process in the prosthesis-bone interface, a large number of granulation tissue formated and eventually fibrosis to form a pseudomembrane, bone loss and pseudomembranous formation further exacerbated the prosthesis aseptic loosening, and more wear particles generated, the pathological processes of inflammation and osteolysis aggravating a vicious cycle.This study has fully proposed that inflammation and osteolysis are associated yet separated pathological process in the asecptic loosening. in vitro experiments the combination gene therapy targetting on IL-1and RANKL dramatically inhibits inflammation and osteoclastogenesis.To investigate the efficacy of anti-inflammatory and anti-osteolysis treatment in the complex intra-articular environment in vivo, IL-1receptor antagonist and OPG gene therapy was induced in the murine model with the prosthesis aseptic loosening. Objective1. to establish the murine model with prosthesis aseptic loosening suitable for long-term study2. to observe the pathological changes of the prosthesis aseptic loosening3. To investigate the efficacy of anti-inflammatory and anti-osteolysis treatment in the complex intra-articular environment of Pin modelMaterials and Methords1.Establishment of the mouse pin-implantation model and in vivo gene transfer(s) BALB/c mice aged10--12weeks were quarantined in our local animal facility for2weeks before experimentation. Briefly, thetibial plateau of the mouse right knee was surgically exposed under strictsterile procedure, and a proximal6mm of the tibial intra-medullary canal through the center of the tibial plateau was reamed with a0.8mm dental drill. A titanium-alloy pin was then press-fit into the canal in a manner that the surface of the pin head became flush with the cartilaginous surface of the tibial plateau and did not interfere with the motion of the knee. Following the surgery, an X-ray was taken to confirm the correct position of the pin implantation. To mimic the prosthetic wear,10μL of a titaniumalloyparticle suspension (4×104particles of Ti--6Al--4V) was pipetted into the tibia canal before insertion of pin implant during surgery, and20μL of Ti particles were intra-articularly injected into the prosthetic joint every4weeks following surgery. Three weeks following the establishment of the knee-implant-failure murine model, the mice (n=24) were randomly divided into four groups:50and25μLof the DFG-IL-1Ra at the titer of10'pfu were injected into the prosthetic joint in the IL-1Ra group and the combination gene therapeutic group, respectively. Three days after the IL-1Ra gene transfer,50or25μL of the AAV-OPG at the titer of1×10'particle ml1was injected into the prosthetic joint of the mice in the OPG group or the combination group, respectively.50ml of AAC-LacZ (107particle ml-1) was injected into the prosthetic joint in the LacZ group as control. Mice were killed at8weeks after gene transduction for biomechanical and histological tests.2. micro-CT assessmentAll mice were scanned using a Scanco VivaCT40mCT scanner within a week of surgery to confirm the successful placement of the pin. A final scan was performed8weeks later at the time of killing the mice, for bone density measurements. Reconstruction and analysis were carried out using the manufacturer's supplied software. A region of interest was defined around the pin, and BMD, bone volume and total volume of the tibia were calculated.3. Biomechanical test of pin implantation in tibiaThe limb with tibia pin implantation was surgically harvested at the time of killing. All soft tissue around the prosthetic joint was carefully removed to expose the implanted pin surface and proximal tibia. Approximately10mm of the distal tibia was cemented into a custom-designed jig with dental cement. Proper vertical alignment of the tibia titanium implant to the loading axis of the BOSE ElectroForce testing system was achieved with a customer-designed fixture, and the actuator positions and loading data during the pulling test were recorded.4. Histological analysesFormalin-fixed prosthetic joints were decalcified in a solution containing22%formic acid and10%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. A computerized image analysis system with the Image-Pro Plus software package was used to quantify the histological data.5. Statistical analysisStatistical analysis among groups was performed by one-way analysis of variance test with the Schafer formula for post hoc multiple comparisons. A P-value<0.05was considered assignificant difference. Data are expressed as mean±s.e.m. Results1. Micro-computed tomography (mCT) assessment of bone mineraldensity (BMD) changes and osteolysisA Scanco in vivo mCT system was used to quantify peri-implant bone volume and BMD changes following gene modification treatments. mCT3-D reconstruction images of pin-implanted tibiae at8weeks post gene modifications. Focal bone osteolysis at the interface was remarkably diminished following the double gene therapy. Quantification of the bone volume/total volume was done, and the BMDs of the peri-implant bone specimens were also compared and are summarized. Therapeutic modifications significantly protected against the bone fraction volume and BMD loss in comparison with the LacZ controls.2. Biomechanical testing of the knee-implant tibia after combination gene therapy As peri-implant osteolysis weakens the stability of the pin implant, a pin pull-out test was performed to examine the implant's biomechanical stability after8weeks of gene modification treatments, with a customer-made fixture on a BOSE actuator;the force curve to dissociate the pin from the surrounding bone was recorded and analyzed. Although there were variations among the individual specimen's pullout force in the OPG gene modification group, all the pins in the double gene therapy group exhibited well-fixed mechanical stability that required significantly more force to dissociate the pin implants compared with the LacZ control group. However, the pullout test on the IL-1Ra transduction group did not show statistically significant difference from the LacZ controls(P=0.21).3.In vivo combination gene therapeutic effects on bone remodeling The mouse knee pin-implantation-failure model was used to evaluate the in vivo therapeutic efficacy of the gene modification therapies. Three weeks after titanium pin implantation into the proximal tibia and peri-implant titanium particles challenge, media containing DFG-IL-1Ra and AAV-GFP-OPG (individually or in combination) were injected intra-articularly into the implanted knee joint. Mice with AAV-LacZ viral vector injection were included as controls. Histological assessment of the proximal tibiae harvested8weeks following gene modifications exhibited ubiquitous peri-implant pseudo-membranes in the LacZ genetreated group, while dramatically thinner or disappearing periimplant soft tissue was exhibited in most of the harvested specimens following therapeutic gene modification(s). Quantitative measurement of the pseudo-membrane thickness using a computerized image analysis system confirmed that all therapeutic gene therapies, especially OPG and the double gene modification groups, significantly reversed the peri-implant pseudo-membrane formation and bone resorption compared with LacZ controls.Conclusion1. successfully created a murine model with the prosthesis aseptic loosening for long-term studies.2. in vivo the combination gene therapy of IL-1receptor antagonist and OPG the successfully suppress the local inflammation and osteolysis, increasing the stability of the implants.3. the combination gene therapy targetting on IL-1and RANKL for the treatment of the prosthesis aseptic loosening has a synergistic effect,and is better than single gene therapy group. |
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