| BackgroundBone-Tendon Junction (BTJ) injury is quite common among people. But there are nowell-elucidated mechanisms for its repair after rotator cuff injury. Any overly aggressiverehabilitation could contribute to the failure of rotator cuff repair, especially during thehealing period of bone and tendon. Therefore, an animal model that can simulate humanBTJ injury and healing process is needed, so that we can further explore new methods thatcan promote rotator cuff healing fast and firmly. In this study, we are to design a ratmodel on supraspinatus muscle tendon injury and healing based on the characteristics ofhuman rotator cuff damage.ObjectiveIn our study, we have designed a new animal model on supraspinatus BTJ injury andhealing, trying to simulate the histologic healing process in BTJ injury,thus providing atheroretical and experimental basis for the healing mechanisms of supraspinatus muscleBTJ injury.Methods1、Experimental design40female SD rats (provide by The Animal Center of Second Military MedicalUniversity),weighed(180-200g)35of them experiment supraspinatus surgery while another5act as control groupdon’t take surgery.10rats were killed respectively at7,14,21day after surgeries, and werecut at supraspinatus distal tendon and the connecting humerus bone to form complete BTJsamples for histological and immunohistochemical observation.2、Animal surgery10%choloral hydrate injection400mg/kg anesthetize by intraperitoneal injection.Take the prone position after anesthesia and placed in sterile desk. In the left shoulderscapular region, shoulder blade under the edge of the incision2-3cm rampant skin incision,gap along the teres major and latissimus dorsi muscle and oblique sharp dissertion toscapula, reveal the next segment in the scapula, the scapula the outside of the lower edge ofthe supraspinatus muscle and tendon part are slightly visible. Find along the tendon part ofthe left humerus connection part and reveal the humeral head. Cut SD rats one-side shoulder supraspinatus tendon from the bone-tendon junction. Use towel clamp to formtwo parallel bone tunnel in the greater tuberosity footprint where original supraspinatusattaches and use nonabsorbale tendon sutures follow through the bone tunnel, make surethe supraspinatus tendon was sutured in place and a SD rat BTJ injury and healing model isformed.3、Sample collecting and making10rats were killed respectively at7,14,21day after surgery,and were cut atsupraspinatus distal tendon and the connecting humerus bone to form complete BTJsamples for histological and immunohistochemical observation. One control group ratshould be made into sample in the same way. We performed HE staining, observing andcomparing at different time points by BTJ healing process on the basis of scar tissueformation and turnover characteristics, together with the distribution of fibrous cartilageand difference with normal BTJ. We compared the immunohistochemical distributionresults by the measurement of TGF-beta1、AR-Smads(including Smad2and Smad3)between BTJ samples and normal BTJ using microscope(x400).4、Observation methods and results evaluationWe compared the immunohistochemical distribution results by the measurement ofTGF-beta1、AR-Smads(including Smad2and Smad3) between BTJ samples and normalBTJ using microscope(x400) and randomly select5high power field in each sample slice.Using secondary scoring method to determine the results:(Staining intensity grading),ayellowish to1points; yellow or deep yellow to2points; brown to3points.(Positive cellgrading), positive cell <10%to0points, positive cell10%-25%to1points, positive cell25%-50%to2points, positive cell50%-75%to3points, positive cell>75%to4points.Two results gather together, the total points<2is (-);2-3points is (+);4-5points is (++);6-7points is (+++)5、Statistical analysisUse Chi-square test to analyze the positive rate of TGF-beta1、AR-Smads at differenttime point and find the significance of the difference in TGF-beta1、AR-Smads expressionat different time point.Results1、samples observation (from supraspinatus distal tendon and the connecting humerusbone) indicate that: control group: normal supraspinatus tendon connection with the normal humerus bone tendon can be seen;7d\14d\21d surgery group: scar tissueproliferation can be seen around the supraspinatus tendon and humeral junction and thescope of scar tissue is gradually increasing over time.2、HE staining observation: clear layers of muscle tendon fibers, cartilage, and bonetissues were confirmd, cells were arranged trimly.7d\14d surgery group: masscollagenoblasts could be seen in microscopic view, cartilage cells could not be found.21dsurgery group: several scattered cartilage cells could be found at BTJ.3、Histological and immunohistochemical observation. Statistical significance couldbe found in all time points after surgery compared with control group.(P<0.05).TGF-beta1positive rate：7d group is20%,14d group is60%,21d group is50%.Statistical significancecould be found between7d and14d group(P<0.05),no statistical significance could befound between other groups.AR-Smads positive rate:7d group is30%,14d group is70%,21d is60%, Statistical significance could be obviously found between7d and14dgroup(P<0.05),but there is no significant difference between14d and21d group(P>0.05).ConclusionStudy shows that there are many highly expressed growth factors in the early phase ofBTJ healing, and scar tissue formation has been closely associated with the presence of theTGF-beita1.In a ACL reconstruction rat model,we can find the macrophages(which is themain body that secrete TGF-beta1) accumulating just4days after surgery and will last3-4weeks.In our animal model on supraspinatus muscle BTJ injury and healing, we find thatTGF-beta1accumulates in the Bone-Tendon Junction in the early healing process,reachingits peak10days after the surgery, which accidently coincides with the peak of scar tissueformation. TGF-beta1is highly expressed14days after surgery and will last for another7days, which precisely coincides with the peak of scar tissueproliferation.AR-Smads(including Smad2and Smad3) can also be seen in high expression14days after surgery, which coincides with the peak of scar tissue formation. In thisstudy,we prove that TGF-beta1and AR-Smads are key factors that prote BTJ healing. |
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