Research On Promoting The Healing Of Rotator Cuff In The Bone-Tendon Junction By Inhibiting Transcription Factor LEF1 In BMSCs

BackgroundRotator cuff injury is one of the main causes of shoulder pain and affects normal activity.At present,the best method for the treatment of rotator cuff injury is arthroscopic repair.However,due to the particularity of rotator cuff anatomic structure,the healing of tendon bone interface（bone-tendon junction,BTJ）determines the prognosis of patients.Current studies have shown that tendon and bone healing after surgery is generally carried out in the form of scar healing,it is difficult to achieve the original physiological structure of rotator cuff healing.This healing method of scar formation makes the biomechanical strength of rotator cuff tissue obviously weaker than the normal structure,which becomes an obstacle to early functional exercise after operation,and may increase the probability of rotator cuff re-injury in the future.LEF1 is a transcription factor in Wnt pathway and a member of TCF/LEF transcription factor family.It plays an important role in the regulation of cell differentiation by Wnt pathway and can affect other signaling pathways.Therefore,this study is intended to 1.SD rat bone marrow mesenchymal stem cells（Bone Mesenchymal Stem Cells,BMSCs）were extracted and lentivirus transfection was used to inhibit the expression of LEF1 to study the effect of transcription factor LEF1 on the differentiation of MSCs.2.The model of supraspinatus muscle injury and repair in SD rats was established.BMSCs,of SD rats was infected with lentivirus inhibiting LEF1,and gelatin sponge mixed with transfected BMSCs was sewn into BTJ,to observe the tissue changes of tendon and the healing of BTJ.Then observe the effect of this transcription factor on the healing of rotator cuff.It may lays a foundation for clinical application.ObjectiveThe main purposes of this study are:1.SD rat bone marrow mesenchymal stem cells were extracted and transfected with lentivirus to inhibit the expression and effect of LEF1,to study the effect of transcription factor LEF1 on the differentiation of MSCs.2.The model of supraspinatus muscle injury and repair in SD rats was established.BMSCs,of SD rats infected with lentivirus inhibiting LEF1 was sewn with gelatin sponge mixed with transfected BMSCs into rotator cuff injury BTJ,to observe the tissue changes of tendon and bone healing after operation,and the effect of the transcription factor on the healing of rotator cuff.Methods1.Regulate transcription factor LEF1 and observe its effect on BMSCs in rats.（1）The BMSCs of SD rats was extracted from SD rats born 7 days ago（provided by Animal Experimental Center of Naval military Medical University）.After 75%alcohol drowning,the femur of lower limbs was taken and the culture medium was absorbed by 1ml syringe.The femoral bone marrow was washed out of the medullary cavity for culture.（2）Design,synthesis and screening of siRNA for LEF1:according to the principle and requirement of siRNA design,three pairs of siRNA were designed.The siRNA sequence with the highest inhibition efficiency was selected.Package lentiviruses with the selected one.Western Blot technique was used to observe the expression of Sox9 protein after transfection.2.Animal experiment designForty 4-week-old male SD rats（provided by Animal Experimental Center of Naval military Medical University）weighing about 100¹50g were divided into1E_A LEF1inhibition group,_A2E_ABMSCs group,_A3E_A gelatin sponge control group and _A4E_A control group.The left supraspinatus rotator cuff injury models were made in all the groups.Gelatin sponge transfected with lentivirus inhibited LEF1 expression was sewn into rotator cuff in group_A1E _A,gelatin sponge mixed with common BMSCs was sewn into rotator cuff in group_A2E_A,only gelatin sponge was sewn in group_A3E_A,and nothing was sewn in group_A4E_A.5 SD rats were killed on the 14th and 28th day after operation.the supraspinatus muscle specimens of the left upper limb were taken for histological examination to test the biomechanics and observe the healing of rotator cuff.3.Establishment of rotator cuff injury repair model in SD rats.SD rats were anesthetized by intraperitoneal injection of 2%sodium pentobarbital solution.After completing the anesthesia,the skin was disinfected.The skin was cut longitudinally along the longitudinal axis of the upper limb.The blunt avulsion of the supraspinatus muscle was at the stop of the greater tibia.A tunnel was established on the tibia with a towel clamp.The round needle with the non-absorbable suture passed 2mm away from the end of the supraspinatus muscle tissue,then passed through the gelatin sponge block mixed with the transfected BMSCs,and then passed the non-absorbable suture through the bone tunnel with a round needle.The muscle tendon was sutured and fixed on the avulsion end point of the humerus,and the gelatin sponge was fixed together with the contact point of the supraspinatus muscle and the large nodule of the tibia.The model was completed.4.specimen preparationOn the 14th and 28th day after operation,5 SD rats in each group were killed.The local specimens of left upper limb and supraspinatus muscle were taken for biomechanical examination and histological observation,and the healing condition of rotator cuff was evaluated.The scapula and the humerus connected to it were taken as a complete partial biomechanical test.After biomechanical examination,the large nodule footprint tissue（about 5mm）was taken for decalcification and fixation,and histological examination was carried out to observe the tissue type of tendon and bone healing in each group.5.observation methods and determination of resultsObservation of survival state:morphology,adhesion and macroscopical view of cells in petri dish,whether contaminated,survival condition of SD rats after operation,such as animal activity,surgical incision healing（whether there was infection or other inflammatory reaction）,limb activity and so on.HE staining:after fixation,decalcification,dehydration,clarity,embedding,section staining and so on,the nucleus was blue and the cytoplasm was light red.6.statistical analysisStatistical analysis was performed by SPSS 22.0.At the same time point,biomechanical pairwise statistical analysis was performed using SNK-q（Student-Newman-Keuls）test to compare whether there was statistical difference between the groups,whether BMSCs have an effect on the healing of the TBJ.The independent sample t test was used to compare groups at different time points of 14d and 28dResults1.The results of RT-PCR and Western-Blot.（1）According to the principle of siRNA design,three sequences were designed,among which siRNA-1 had the best inhibition effect on LEF1;（2）The expression of Sox9 in transfected BMSCs was higher than that in non-transfected cells by Western Blot.2.The overall results.Postoperative activity of the upper limbs of the rats was decreased,and limb movement was limited.Infection occurred in 2 cases,which were removed and new rats were supplemented.No significant change in diet was observed.The animals showed irritability after operation.No significant difference was found in any other aspects.3.Biomechanical test results.At the same time,according toα=0.05 level:On the 14th day,there was no significant difference in biomechanics between groups,p>0.05;on the 28th day,there were statistics difference between _A1E_A and _A2E_A3E_A4E_Agroups,p<0.05.There was no statistically significant difference between the groups_A2E_A3E_A4E_A,p>0.05.Group_A1E_A had a larger strength than group _A2E _A3_EA4_EA.The mean strength of the same group at different time points was statistically different,according toα=0.05.The strength at 28d was greater than 14d,indicating that the overall biomechanical strength of the rotator cuff showed time-dependent improving.4.Tissue section results.（1）The rotator cuff healed in all the groups,which finally formed scar healing;（2）The final healing of group _A1E_Awas relatively good,and more type 2 collagen fibers were observed.In _A2E_A3E_A4E_Agroups,there were more scars and a less amount of type 2 collagen in BTJ,and there was no significant difference in sections.ConclusionThe construction technology of supraspinatus muscle injury repair model in SD rats is mature,the modeling is convenient,and the survival rate of model animals is high.RT-PCR and Western-Blot can detect the role of signaling pathway in the repair of rotator cuff injury.It is suggested that the signaling pathway may play a role in the healing process of tendon and bone,and HE staining is a common method of tissue section,making observing the tissue of tendon and bone junction possible.According to this study,by inhibiting the expression of transcription factor LEF1,BMSCs can be induced to differentiate into chondrocytes at a certain extent,and the addition of lentivirus transfected BMSCs to the repair site of rotator cuff injury can promote the healing of BTJ in a short period of time,enhancing the biomechanical function of the healing site and making functional exercise in the early stage after operation possible.This study is helpful to provide a new possible method for the clinical treatment of rotator cuff injury.