Effect Of Extracorporeal Shock Wave On Chondrocyte Apoptosis Of Rabbit Osteoarthritis Model And Activity Of Caspase-3

Objective: 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 reports.Additional,scientists have not pay much attention to the role of chondrocyte in development of OA.In order to demonstrate ESW as one uninvasion therapeutics in delay development of OA and provide theory foundation for clinical application,we do this research.The normal chondroycyte culture in vitro is incentived by difference levels of energy intensity and frequency of ESW.The purpose is to sieve the optimization energy intensity and frequency of ESW and to guide the ESW use in animal experiments.Then to establish OA model of rabbit knee which been exerted by already selected ESW and detect apoptosis of chondrocyte.Besides,demonstrate if ESW estists molecular metabolism to prevent inflammation, apoptosis of chondrocyte and promote repair of cartilage. Methods: Primary part:After attain normal chondrocyte of rabbit knee by enzymic method,the cell is incentived by various levels of energy and frequency of ESW.Sieve the opimization ESW parameter by CCK-8 and HE stain.Second part:establisment OA model of rabbit knee by ACLT.After the surgery,the rabbit knee of experimental group give ESW treatment.Rabbits been sacrificed at the period of 4 weeks and 8weeks.Morphology,tissue stain and caspase-3 were used for study.DAPI,TUNEL method and flow cytometry annexin V were used for the study of apoptosis.NO levels were measured in rabbit synovia.Experimental data above mentioned analysis by ANOVA and nonparameter rank sum test.A p value of <0.05 was considered significant analysis by SPSS13.0.Results: 1st,attain normal origin-degenration chondrocyte by enzymic digetion.The shape chondrocyte viewed by inversion microscope is edge sharpness and round,but the size has little difference.After 4 hours the cell begin to adherence and 24 hours there exist some enation on cell which become polygon.2 days later chondrocyte grow quickly ,in edge cell becomes fusiform and in middle cell becomes polygon shape like pave-stone.2nd,the reproductive activity of chondrocyte increased after incentived by ESW.HE stain show the treated chondrocyte has endochylema profusion,mang long foot process and big cell nucleus.Therefore,in control group the chondrocyte has low endochylema and small cell nucleus. 3rd,the reproductive activity of chondrocyte attain maxlmum at 600 vices and 1.5×105Pa energy of ESW.The cell activity is obvious decrease though the energy increase. At the fix energy level,the activity is determined by fix vices which attain maxlmum at 600 vices.4th,the rabbit knee appearance typical OA patho-change after ACLT.In 4 weeks experimental group,cartilage structure,cell proliferation and toluidine blue stain is normal. In 8weeks experimental group,the fiss attain shift layer,cell increasing,toluidine blue stain light decrease and tidal line isn't integrity.There is existed distinction in Morand and Markin scores.(p<0.01) 5th,in experimental groups the chondrocyte apoptosis rate is obviously lower than control groups.Apoptosis chondrocyte stain by DAPI appearance uneven blue fluorescent light and specificity TUNEL display viridis fluorescent light.These cell mainly exists in superficial and intermedium.The rate of chondrocyte apoptosis is 4.637±0.241%,10.240±0.760% in 4w,8w experimental groups detective by flow cytometry label annexin V-FITC/PI.The control groups apoptosis rate is 10.178±0.661%,17.465±0.621%.The dates exist statistics distinction(p<0.01). 6th,in each groups,the NO level of synovia in rabbit knees are cut down through weeks.The NO level exist statistics distinction in compare groups during 4 weeks and 8weeks (p<0.01).7th,the expression of caspase-3 exists disproportion in experimental group which mainly express in ecderon and little express in middle and stratum radiatum,the rate of masculine cell is 15.4%±2.9%,38.4%±2.9%.However,in control group the rate of masculin cell is 40.4%±2.9%,70.4%±2.9 ,which mainly exist in superficial lamella and middle layer. The rates in these groups exist statistics distinction.(p<0.01)Conclusion: As a kind of machine stress,ESW can predominance promote chondrocyte multiplication in certain energy intensity and frequency.The chondrocyte is confirmed as sensitiveness to stress which conforms energy dependency and additive effect.Besides,ESW can inhibit chondrocyte apoptosis by restrain expression of caspase-3 and level of NO,which can delay cartilage degeneration.As one of predominate therapy in treat OA,ESW has significant sense in clinic practice.