Effect Of Extracorporeal Shock Wave On Cell Proliferation In Rabbit Articular Chondrocytes Cultured In Vitro

Objective:The damage of articular cartilage are an all too commonly encountered medicial problom in bone and joint surgery,which may be caused by trauma or evolve during the course of disedses such as osteoarthritis, osteochondritis and osteonecrosis.Articular cartilage has a limited capality to repair spontaneously after injury.The large defects at articular cartilage are often repaired by fibrocartilage rather than normal hyaline cartilage.The resulting fibrocartilage are different from hyaline cartilage either biochemically or biomechanically and it ultimately deteriorates.It generally was though that there was no effective clinical treatment that could restore a damaged cartilage surface and prevent the normal sequelae of degenerative jiont disease. Recently,though there are mang investigations to accelerate repairing of the damage of articular cartilage and improve the quality of repairing tissue,but no one satisfactory method and have a good long-term effect.Extracorporeal Shock Wave Therapy (ESWT) as one of original nonvirulence cure method in orthopedics, it exists many predominance in orthopaedic surgery and cure soft-tissue diseases than traditional surgery methods.In recent years, Extracorporeal Shock Wave(ESW) has significan curative effect in osteoarthritis(OA) treatment.However,its mechanism is unclearly in relation molecular biology reports.The normal chondroycyte culture in vitro is incentived by difference levels of energy intensity of ESW.The purpose is to approach the problems,which are estists molecular biology metabolism to enhance cartilage recovery effect by ESW,the best energy range that cartilage could well growth,if ESW has influence to proliferate of articular cartilage cell.Besides,demonstrate the feasibility that which made ESW to new uninvasion therapeutics to delay development OA and provide theory foundation for clinical application,we do this research.Methods:After attain normal chondrocyte of 20 rabbits knee,which 3 month old and breed variety were Newzerland rabbit by enzymic method,the cell is icultured 3 generation and drum dyeing by trypan blue exclusion methed to exame the vigour of cells. The 3 generation cells were dividatured to 4 terms and intervented by various level of energy of ESW. The cell were cultured 48h after intervented by ESW.Then it was known that if ESW has influence to proliferation active of rabbit keen chondrocye through trypan blue stain,inverted phase contrast microscope,texamined generation tendency with CCK-8 method,HE stain,ELISA detection which contain of bFGF, PCNA,CTGF,collagenⅡ,RT-PCR detection which bFGF, CTGF, collagenⅡ.Measurement datas showed by mean±S. Experimental data above mentioned analysis by ANOVA ,and the data of RT-PCR showed by chi-square criterion. A p value of <0.05 was considered significant. Data were analysised with statistics software SPSS13.0.Results:1st,cell proliferation through trypan blue staining:after trypan blue stained,dead cell were light blue and living cell were refused stain.The cytoactive of Group B was 95±3﹪(n=8),and vs Group A,Group C,Blank Control significant different was found(P<0.05).2nd,growth curve:ESW has predominace effect to normal rabbit keen chondrocyte, ESW on the normal rabbit knee cartilage cells significantly, the best frequency of 200 times the energy and intensity of 1.5×105Pa. ESW energy experimental group A and experimental group B at the slow growth period and the exponential growth phase than those in the experimental group and control group C increased (P <0.05). However, the experimental group B at the first 3 day time point compared with other three groups have significant difference (P <0.05). And early peak of cell proliferation, indicating that the appropriate energy ESW can obviously at the time and quantity on the promotion of cartilage cell proliferation.3rd,observed with inverted phase contrast microscope after stained by HE:we got the first generation normal rabbit knee chondrocytes by enzymic digest,and observe with inverted phase contrast microscope.It showed that the hypomenous chondrocytes appearanced edge sharpness round,nucelus were bright and distinctness which has refraction activity and optical activity.The size of cell were different.After 4 hours,bulks of cell begin adherence,cell shape got applanation and licency. After 24 hours, chondrocytes apprenced ecphyma,and the shape of cell were to polygon gradually.After 3 days,the cell got into exponential phase of growth,and connected to lamellar,ecphyma of border cells became longer,on the other hands the center, shorter and the shape were polygon.After 7 days, the shape of chondrocytes has changed to"pave road stone".After 8 days the amount of cell and endochylema content were increasing,a part of cell got cell aggregate or cartilage nodus.4th,detected by ELISA: The expression of bFGF, PCNA, CTGF,collagenⅡwere enhanced by examined in each term,and the dates exist statistics distinction (p<0.05). Especially,ESW could enhance the express of bFGF obviously. The expression of the growth factor of bFGF in term of 1.5×105Pa energy were 12.920±0.103,which was higher than other terms. This demonstration that ESW could enhance the proliferate of chondrocyte, first,through enhance the express of growth factors which could encourage generation to chondrocyte.5th,the expression detected by RT-PCR:The expression of bFGF, CTGF,collagenⅡwere enhanced by examined in Group B compared to Group Blank Control which different was found(P<0.05).The The expression of bFGF, CTGF not significant different was found between Group A and Group B.The result was conformity to the result of ELISA.Conclusion: 1.As a kind of machine stress,ESW can predominance promote chondrocyte multiplication in certain energy intensity. The chondrocyte is confirmed as sensitiveness to stress which conforms energy dependency effect.The result has provided theory foundation for animal experiment and scope of ESW parameters for using in cure OA. 2.We got a lot of cartilage cell in spuet time by intervented with ESW adequacy energy.It could provide sufficient phonetype stable cell to clinic experiment and seed cell of constitution engineering. 3.ESW could promote the express of bFGF,CTGF etal and this growth factor could promote the cell multiplication of chondrocyte in return.In all,as one of predominate therapy in treat OA,ESW has significant sense in clinic practice.