Research About Inhibition Of TGF-β1/Smad Signaling Pathway To Promote Rotator Cuff Healing In The Bone- Tendon Junction

BackgroudIn orthopedic clinical practice, specialist doctors and patients show more and more attention to rotator cuff (RC) injury. The prior cadaveric and epidemiological study indicated that RC injury rate more than 50% of persons over the age of 65. With the aging of the population increase, RC disease in the future will gradually become a very prominent social health problem. The bone-tendon junction (BTJ) healing process can be affected by various factors after injury, including mechanical stress, inflammatory reaction and cytokines. Thus, BTJ healing has long been deemed as the weak link in the early healing of a joint injury. According to clinical data, the healing of RC is more likely to result in the scar healing after surgical repair. Many inflammatory factors and growth factors participate in this process. Normal RC injury healing process is divided into three stages:the inflammatory phase, the reparative phase and the reconstruction phase. In the first phase, the inflammatory cells migrate into the damaged parts under the action of chemokines and then release various cytokines and growth factors that can promote vascular ingrowth.These cytokines and growth factors include transforming growth factor-beita (TGF-β),platelet derived growth factor (PDGF), fibroblast growth factor (FGF),et al. In the reparative phase, the expression of such growth factors can be up-regulated and help promote cell proliferation and cell matrix deposition. While in the reconstruction phase, the massive fibrous tissue, newly-formed bone and tendon graft in the BTJ and its peripheral region become reconstructed and strengthened. This kind of incomplete reconstruction makes the biomechanical strength of RC far below its normal level and the possibility for re-tear recurrence of RC may increase. TGF-β1 has been proved a vital factor in BTJ healing according to prior study, thus scar healing may be inhibited by blocking TGF-β1/Smad signaling pathway.ObjectiveIn this study, we are to build the SD rats supraspinatus BTJ damage/repair model and perform in vivo study of TGF-β1 & Smad to analyze its distribution and correlation with scar formation in the RC healing process. At different times in healing and remodeling, we block TGF-β1/Smad signaling pathway using shRNA transfection towards TGF-β1/Smad protein family. Then we observe the changes of the cells and extracellular matrix and its biological and biomechanical variation after the blocking procedure and finally explore the regulating mechanism of signaling pathways in the RC BTJ healing, thus providing new ideas for clinical treatment.Methods1.Experimental design50 male SD rats (weighed 150-200g on average) were provided by the Second Military Medical University Animal Center ready for use.40 of them (randomly chosen) underwent the RC damage/repair simulated operation leaving another 10 SD rats untreated as the blank control group (No. (D).The operation group (containing 40 SD rats) in which each one received a simulated RC repair in its left forelimb were randomly divided into 4 groups and every one of the rat in those 4 groups got 3 injection in the BTJ area on the surgical limb at different time point (3d,5d,10d) after surgery. All of the animals were fed in the same environment and then every time 2 rats from each group were sacrificed at a time point after surgery (7d,14d,21d,28d). The specimen containing the whole shoulder joint were taken accordingly and then sent to undergo the biomechanical,histological and immunohistochemical test in order to estimate the efficacy of the RC healing. (NOTE:According to the different "medicine " injected into the rats, the 40 rats can be divided into 4 groups, naming②the NS(normal saline) contrast group; ③ the TGF-β1 blocking group; ④ the Samd2 blocking group; ⑤ the TGF-β1/Smad2 co-blocking group.2. Animal model-Simulated surgical repair of the supraspinatus in the rat left forelimbThe rat was anesthetized by abdominal injection using 10% chloral hydrate saline solution (0.4ml/500g in weight). Then we fixed the rat on a customized board in prone position by tying its four limbs on the board rim, the hair were cleaned and then the skin got disinfected with 1% medical iodine and covered with sterilized cloths. To begin with, we make a longitudinal incision (approximately 3-4cm long) and then separate the superficial fascia to expose the shoulder and upper arm muscles. Find the trapezius muscle attachment and gently open the muscle along the trapezius realm and we can easily see the supraspinatus tendon lying just under the trapezius. Carefully separate the whole supraspinatus to its greater tuberosity footprint (A tiny stripping of the deltoid may be needed to better expose the attachment point of the supraspinatus on the great tuberosity footprint). Then we strip the supraspinatus tendon off its attachment to the great tuberosity with blunt avulsion and make a oblique bone channel on the great tuberosity with a small towel clamp. We stich the supraspinatus on its original attachment point on the great tuberosity with a transosteoius knot-tying fixation and close the wound carfully. The rat was sent back into animal center to feed after the surgery.3.Inhibition of the TGF-β1/Smad2 signaling pathway(1) Design, synthesis and screening of siRNA specific to TGF-01, Smad2According to the principle of siRNA design and requirements, we design and synthesize three siRNA with fluorescent reporter gene markers for each gene based on the SD rat TGF beta 1, Smad2 gene sequence features; After liposome packaging, the siRNA were transfected into SD rats fibroblast in the same concentration and same time; Determine the best transfection concentration and time after screening with the help of Rt-PCR and Western Blot technology.(2) Effectiveness and evaluation of TGF beta 1/Smad2 related slow virusSynthesize effective shRNA plasmids according to the locus of siRNA sequences and package the corresponding slow virus. Then use the packaged slow virus to infect SD rat originated fibroblast and testify its inhibition efficiency toward target gene.(3) Inhibition injection in stagesTo inhibit the TGF-β1/Smad2 signaling pathway with packaged slow virus injection at different stages of BTJ healing and remodeling. The rat get injected with the virus in the left forelimb right on the BTJ area for three times right after the surgery (3d,5d and 10d postoperative injection).4.Specimen harvesting and makingWhen injection was done, there would be two rats from each group that were sacrificed for specimen harvesting at 4 different time point (say 7d,14d,21d,28d after surgery). The entire shoulder joint on the left forelimb including all RC tissue was preserved without irrelative muscle and soft tissue. The shoulder joint specimen were then sent to take undergo the biomechanical, histological and immunohistochemical test in order to estimate the efficacy of the RC healing. At the same time, specimen from the blank control group (Group No. ①) were tested accordingly. When biomechanical test was done, the used specimen can be re-utilized for HE staining and immunohistochemical test after decalcification and fixation, et al.5. Observation method and results evaluationLiving condition observation:At the very beginning, we compared their pre/post-operative survival condition at different time point before the rats were sacrificed for specimen collection. For example, the incision healing (presence of infection/inflammation) and the physical activity, et al.HE staining observation:All prepared, the specimen were sent to do the fixation, decalcifation, dehydration, washing, embedding, slicing and staining. Then we can see the nuclei dyed blue and the cytoplasm dyed reddish.Masson staining observation:Collagen fibers were dyed blue; muscle fibers, cytoplasm, cellulose, keratin and the red blood cell were dyed red, the nuclei were dyed blue and brown.6. Statistical analysisStudent-Newman-Keuls test (SNK-q test) was applied when it comes to biomechanical test at different time point in order to make clear whether there is a difference in BTJ healing between groups.Results1.The overall resultsOperation group VS nonoperation group:After surgery, the animal showed decreased activity and limited physical mobility which seriously affected its gait (claudication or dragged leg-moving). Wound splitting and even infection/fester could be found among the rats (with fairly high inflammatory rate up to 20%). No change were found in their eating habit but the rats tended to become more easily agitated after surgery. Results in the operation group were almost non-obviously statistical significant in animals’living condition and infection rate.2.Specimen staining resultsScar healing was found in group ②③④⑤;Scar healing in the BTJ showed better results in group ⑤ compared with group ②③④. Of all those groups, the group ⑤ exhibited the best healing and lest scar tissue in the BTJ which can be evaluated with naked eyes.While results like messy new collagen fibers and scar tissue can be found in group ②③④.The percentages of fibroblast content showed different results in each group: ①45%,②78%,③70%,④75% and ⑤50%.3.1mmunofluorescent test, Rt-PCR and Western Blot testAggrecan, Biglycan, Elastin and Tenascin-C were all increased in the cell matrix components in group③④⑤,and the increase in group ⑤ was most obvious.In the matrix enzyme change, MMP decreased in group③④⑤,but increased in group②.The results in TIMP were interestingly in contrast to that of MMP.Change about COX were different among groups which showed no statistical significance. And the content of transcription factor SOX9 showed a trend of increase among groups which also showed no significance in statistical analysis.Apoptosis related factors such as p53,NF-KB and heat shock protein exhibited a decreasing trend in group③④⑤ and group ⑤ decreased the most. The results totally contra those in group②.4.Biomechanical Test resultsThe overall trend of RC healing in the BTJ showed an enhanced terminal tension value. Statistical analysis used the SNK-q test method and showed that the best results lied in group⑤. According to the α= 0.05 level:statitics in group⑤howed great significance in every time point compared to that of group②③④,P<0.05;statistics in group①showed significance compared with group②③④ just in the first 3 weeks but none significance was found in the last week statistics,P>0.05; statistics between group ①and⑤ showed significance in the last week analysis rather than in the first 3 weeks,P <0.05; no statistically significant results were found among group②③④ in every time point,P>0.05.ConclusionSD rats unilateral supraspinatus damage repair model has been proved maneuverable, repeatable, stable and convenient in sample collection with high rate of success. It can be a good animal model for rotator cuff research and study; The HE staining technique is a powerful and commonly used method in observing BTJ tissue formation and its healing results; The Masson staining is a kind of special staining method in observing the collagen and its isoforms and finally estimating the healing outcome; The IHC, Rt-PCR and Western Blot technique are indispensible weapons in detecting the changes among proteins and enzymes and other different significant molecules along the TGF-beita/Smad signaling path. The whole study, to sum up, has found that BTJ can get better healing by inhibiting the TGF-beita/Smad signaling path way and it is more likely to regain an natural healing in the wound area with less scar tissue and enhanced anti-stress biomechanical characteristics. But inherent mechanism remains to be unveiled. Among those groups been treated with different inhibition methods, group ⑤ showed the best healing results, thus providing a new idea in clinical treatment toward rotator cuff diseases.