| BackgroundRotator cuff injury is a kind of common joint sports injury disease,which has a high incidence,but the rate of re-laceration after rotator cuff surgery is still high.At present,international studies on various factors(biological factors,surgical methods,etc.)promoting the healing of rotator cuff have not found a very effective method.Radial extracorporeal shock wave treatment of skeletal muscle diseases has been widely used all over the world.Compared with traditional surgical treatment,it has many advantages,such as non-invasiveness,simple operation,significant curative effect,low risk and low consumption,etc.However,there are few studies on the mechanism of shock wave in the treatment of rotator cuff injury and no uniform standards for the animal models used to study the mechanism.ObjectiveTo explore the effect of radial extracororeal shock wave on the repair of supraspinatus injury in rabbit and its related mechanism,and provide relevant theoretical basis for clinical treatment of rotator cuff tendonitis.MethodsThe animal model of rotator cuff injury repair was constructed to screen the optimal extracorporeal shock wave treatment parameters,to evaluate the effect of shockwave treatment on rotator cuff injury in rabbits,and to explore the internal mechanism through stem cell experiment.This study can be divided into four major parts.1.Establishment of rotator cuff injury model in rabbits:2~3 months New Zealand rabbits were given general anesthesia and put on the surgical desk in prone position.The supraspinatus muscle of the left foreleg was exposed and isolated,and the supraspinatus muscle was passivated at the greater tuberosity of the humerus.Supraspinatus muscle was fixed by supraspinatus suture,closed incision layer by layer,antibiotics were applied during and after surgery,animals were kept under standard conditions,and artificial manipulation rehabilitation began 2 weeks after surgery.At each time node after modeling(4w,6w,8w and 12 w after surgery),the data were collected from multiple perspectives of biology and biomechanics,and the success rate and stability of the model were analyzed 2.Setting of shock wave optimization parameters:The repair model of unilateral supraspinatus(left shoulder)injury was established according to the single tunnel modeling method selected in the first part of the study.60 rabbits were randomly divided into 4 groups(N=15)and treated with extracorporeal shock wave at 2w postoperatively(with an interval of 2 days).Then,the animals were sacrificed at 4w,8w and 12 w after the operation for ink perfusion test and pathological section staining analysis(HE and collagen staining analysis).The optimal treatment parameters were selected through the final comparison results.3.Evaluation of the efficacy of shockwave in the treatment of rotator cuff injuries in rabbits: 40 healthy 8-week old rabbits were selected and randomly divided into 2 groups : the model building group(N=20)and the ESWT group(N=20),the healthy limbs were used as the blank control(N=40).After routine modeling and feeding procedures,annimals in the model building group began to undergo shock wave therapy 2 weeks after surgery(the treatment regimen was the same as step 2).From the beginning of week 4 to the end of week 12,animals were sacrificed at different time node to retain tissue for subsequent tests.General observation of animal survival status and changes was performed,including food intake,weight,gait,infection rate,mortality,and so on.Biological test,including pathological section,Western Blot,ink perfusion test for neovascularization.Imaging examination,including X-ray,B-mode ultrasound,MRI plain scan;Biomechanical test of terminal resistance strength of the supraspinatus muscle were all undertaken at the same time.To comprehensively evaluate the effect of shockwave therapy and its effect on rotator cuff healing,and preliminarily explore the changes of relative signal pathways and molecular proteins during tendon repair;4.Study on the mechanism of shock wave promoting the healing of rotator cuff tendon: A.The rotator cuff tissues were extracted from the rabbits,and the mesenchymal stem cells and tendon cells were cultured in vitro.Shock waves of different energy Settings were directly applied to the cell culture system to observe the expression of tendinous genes and proteins,cell activity,cell proliferation and migration.B.Smad3 gene of mouse embryonic mesenchymal stem cells(C3H10)was knocked out and co-cultured with TT-D6 mouse tendon cells.Three pairs of fluorescent reporter genes labeled si RNA were designed and synthesized according to the Smad3 gene sequence characteristics of C3H10 cells.The liposomes encapsulated si RNA and transfected C3H10 cells of mice at the same concentration and at the same time.RT-PCR and WB were used to screen the highly specific si RNA to determine the optimal transfection concentration and time.The co-culture model of C3H10 cells and TT-D6 cells was established by using the Transwell chamber to verify the effect of Smad3 knockdown on TT-D6 tendon cells.Flow cytometry,ELISA and RT-PCR were used to evaluate the expression of cell proliferation,collagen synthesis and tendon markers.Results1.The repair model of rabbit supraspinatus acute injury is stable and reliable with high repeatability,operative convenience and relatively high cost performance;2.Compared with the model of double tunnel repair,the method of establishing the humeral greater tubercle single-tunnel repair of the supraspinatus muscle is more advantageous,which takes less time in the molding operation and is convenient for taking materials;3.Shock wave therapy can significantly promote the healing of rotator cuff injuries in rabbits,improve tissue fibers,scars,tissue morphology,angiogenesis,and promote tendon repair and tissue remodeling.In addition,research shows that the optimal energy flow density of shock wave therapy in this study was 0.20 m J/mm2;4.The shock wave can promote the healing process of the rabbit rotator cuff,inhibit the expression of Smad3 and up-regulate the expression of Scleraxis;5.The Smad3 down-regulated C3H10 cells co-cultured with tendon cells can significantly enhance the mobility of tendon cells,promote the up-regulation of Scleraxis expression,and up-regulate the expression of type I collagen and Tenascin C.Previous studies have found that this change inhibits scar formation,improves the repair of tissue structure,and thus enhances the terminal tensile strength of the healing tissue.ConclusionRadial extracorporeal shock wave has definite therapeutic effect on rotator cuff injury in rabbits.The single-tunnel repair model of the supraspinatus muscle in rabbit is a reliable animal model for studying the mechanism of shock wave therapy.Extracorporeal shock wave can inhibit the expression of Smad3 and may possibly activate the mesenchymal stem cells and enhance tendon cell activity and act on the Scx gene to up-regulate the expression of Tenasin C and type I collagen,enhance the migration activity and proliferation ability of the tendon cells,and finally achieve the effect of promoting the healing of the injury of the supraspinatus in rabbits. |
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