The Effect Of NF-κB Inhibitors To Osteoclast Generation And Osteoclastogenesis

Artificial joint replacement surgery is the most common and effectivemethod at the end-stage of treatment,but with the extended using time,a loosenprosthesis happened with a rising rate. People always blame loosen prosthesis tobe a simply mechanical reasons , such as no closer paste between the twointerface of the bone and prosthesis,poor location of the prosthesis installation,bone cement fragmentation. In recent years,it was recognized that the mainlong-term wear and prosthesis or dissociation particles generated by the inducedbiological response addition to the factors mentioned above. Biological reactionprocess including:①wear particles generated ;②a foreign body reactionmembrane formation at the interface between prosthesis and bone ;③osteoclast osteolysis,resulting in prothesis bone mechanical properties of thesupport structure fell. Osteoclast especially the role of osteolysis was consideredsignificant. Therefore , To study the osteoclastolysis for prevention of boneprosthesis loosening is of great important values.RANKL/RANK/NF-κB is considered the most important signalingpathway mediated osteoclast differentiation and maturation. At Present the signal transduction pathway associated with RANKL mainly included four: NF-κB pathway,MAPK pathway,PI3K/Akt pathway and CN / NFAT pathway.NF-κB pathway which is considered play the most important role during theprocess of wear particles induced osteoclast activation. NF-κB inhibitorsinclude: PDTC (Pyrrolidine dithiocarbamate),NAC (N-acetylcysteine). Earlyresearch has proved that NAC and PDTC is specifically inhibited on theactivation of NF-κB. The mechanism of inhibiting NF-κB activation is due tothe inhibition of I-κB (binding with NF-κB in the cytoplasm of cells and preventthem into the nucleus) degradation,NF-κB can not turn into nucleus to bindwith the corresponding gene promoter,thereby can not initiate or regulate genetranscription of osteoclast differentiation and function.This study used a Polymethylmethacrylate (PMMA) particle induced a murinecalvarial osteolysis animal model system,through Micro-CT scanning three-dimensional reconstruction of a number of data ; after then we madehistochemistry slice , stained for HE and TRAP(tartrate-resistant acidphosphatase),and analysed NF-κB inhibitor function of osteoclastolysis and theimpact of local osteolysis in vivo. To illustrate osteolysis role of NF-κBinhibitors providing a new theoretical foundation on joint replacement surgery.Details are as follows:1. Animal model of osteolysis: murine calvarial model of the particle-inducedresorption was established process: Animals were anesthetized with a 2%sodium pentobarbital and administered intraperitoneal,infusion tape fixed limbsand head,Bromogeramine Tincture disinfected skin on head,made curettageperiosteal in a 0.5 cm long incision at the skull median sagittal on scalp. Thecarrier solution with PMMA particles(30 mg: 0.1ml)was then injected onto thesurface of skull,sutured head skin using 4-0 line. After surgery mice had been free in cage. Since daily from the next day injected drug intraperitoneal ,sacrifice all animals after two weeks.2. Scan specimens using Micro-CT,applicated eXplore MicroView analysisingsoftware for 3D image reconstruction,and analysis BMC,BMD,BVF,CA,CMT . All five datas indicated roughly the same overall trend. All analyticalresults show that bone mineral density and content of the PDTC,NAC groupwere higher than the control group of saline,the bone hardness of administrationgroups were significantly increased and corresponding improved in mechanicalproperties. BVF of two drug groups was significantly increased than the controlgroup , so bone resorption was inhibited by drug effection. CA and CMTanalysis also showed that the thickness and areas of cortical bone in drug groupswere increased significantly than the control group. These indicators haveproved that the drug significantly inhibited bone resorption. Description of thedrug inhibited particles-induced osteolysis significantly.3. Tissue preparation and staining3.1 HE staining , TRAP (tartrate-resistant acid phosphatase) staining. Allorganizations were measured using OsteoMeasure Analysis System,using 50×,100×,magnification microscope,shooting picture after the measurement of bonetissue analysis. Indicators include:①dissolved percentage of OA (Percentageof Osteolytic area),OA showed that the greater the more obvious effects ofosteolysis;②osteoclast number ON (Osteoclast number),that the greater ONosteoclast caused more greater osteolysis effect. The results showed that: OAlargest saline control group,showed that the most obvious role of osteolysis. OAof NAC group was more obvious than PDTC. Osteolysis of PBS control groupwas the weakest. Each group of osteolysis percentage was obvious statisticalsignificance (P <0.05),t-test analysis was differences between the two drug groups (P <0.05). PDTC have stronger inhibitory than the NAC on the effect ofosteolysis. Saline control group,the number of osteoclasts ON was the largest,PBS group was least,two drug group were middle,but by the t-test comparisonrevealed that compared the NAC with PDTC groups there was no significantdifference (P> 0.05).3.2 Immunohistochemical staining NF-κBp65 positive expression of the IOD(integral optical density).The more of NF-κBp65 content,the more greater ofIOD. Analysed of results showed that: Expression of NF-κBp65 at PBS controlgroup was weakest,saline control group was the strongest. One-way ANOVAanalysis showed that the four IOD significant difference between the values (P<0.05). PDTC group was weaker than NAC,compared the two drugs on NF-κBp65 inhibition effection,PDTC was stronger.Conclusion: NF-κB inhibitors inhibited the skull bone resorption in mice ,thereby protected the integrity of bone tissue. this experiment included Micro-CT scanning,preparation tissue slices and HE,TRAP,immunohistochemicalstaining after then analysed all of the data,compared of Inhibition of the twodrugs in mice skull bone resorption. Our experitment provide a theoretical basisfor clinical drug therapy bone resorption leading to prosthesis loosening afterjoint replacement.