| BackgroundHeterotopic Ossification are pathological bone formation in the organization, trauma can occur Heterotopic Ossification in many parts of the body. It is neither a simple calcium salt deposition, nor different from the new bone formation in the fracture healing.When Heterotopic Ossification attack the patient, it will limit the corresponding joints and dysfunction. Heterotopic Ossification will seriously affect the quality of life and ability to work for the patients. With the widely application of the artificial joint replacement and the increase in the number of high-energy trauma caused by traffic injury, a marked increase to heterotopic ossification’s morbidity. The incidence of artificial hip replacement (THA) postoperative about 0.6-90%, the majority of reported about 53%.patients after open reduction and internal fixation of acetabulum fracture incidence of more than 60% and after spinal cord injury incidence of about 20-25%, roughly 18-35% obviously restricted joint activities. The incidence of heterotopic ossification after traumatic brain injury is about 10-20%, of which about 10% severe restricted joint activities. The incidence of heterotopic ossification is commonly burn patient 1-3%.Research has shown when patient absence of preventive treatment, the incidence of heterotopic ossification is as high as 60%-75%. The incidence of traumatic heterotopic ossification has a tendency to rise in recent years, and get quite seriously. There are a variety of measures to prevent the attack of the heterotopic ossification, such as radiation therapy, nonsteroidal anti-inflammatory drugs (NSAIDS) and two phosphate, etc. Among them, nonsteroidal anti-inflammatory drugs often used after surgery. However, traditional have gastrointestinal side effects. The effects limiting its application and bringing pain to patients., at the same nonsteroidal anti-inflammatory drugs time also to have interfere with the normal distribution (such as fracture healing and bone ingrowth after prosthesis implantation) risk, there is still a high incidence after treatment.MMPs family is a kind of highly conservative gene polymorphisms of zinc and calcium ions. It depend on cutting proteolytic enzyme family. MMPs by neutrophils, macrophages and microglia, radial glial cells, neurons and secrete vascular endothelial cells. We know that MMPs expression by the early gene and the regulation of cytokines and growth factors. Many cytokines to induce the expression of MMPs, such as interleukin 1, interleukin-6, platelet derivation growth factor, vascular endothelial growth factor, transforming growth factor-B, fibroblast growth factor, tumor necrosis factor-a and so on, the inhibitory effect of interferon B. MMPs except in transcription regulation, as in its active enzyme activation and inhibition of enzyme activity link under control, such as different between MMPs may also have activation, proteolytic enzyme activity of MMPs are nonspecific and specific inhibitors inhibit, including nonspecific inhibitor to a2 macroglobulin, al-antitrypsin, Obama sting (batimastat BB-1101,BB-94),etc.Matrix metalloproteinases 9 is a kind of saccharification protease, involved in endothelial cells, fibroblasts and migration of epithelial and stromal cutin cell nucleus in matrix degradation, tissue remodeling, cell migration, angiogenesis and wound healing, bone growth, such as play a key role in the pathophysiological process, is one of the most important enzyme in cartilage matrix degradation, is the extracellular matrix (ECM) reconstruction of early biomarkers. Expression of MMP-9 cells found in bone marrow, skeletal muscle in hypoxia environment can make the MMP-9 greatly enhanced, at the same time in vitro experiments have shown that mesenchymal stem cells to increase the growth of the capillary network, and is accompanied by the rise of MMP-2 and MMP-9. Other scholars believe that the formation of heterotopic ossification after THA can be caused by ischemia-reperfusion injury in the process of operation, result in connective tissue oxygen cooperation with problems that cause ectopic bone formation.Matrix metalloproteinase-2 can be expressed in the cytoplasm of osteoblast and part of osteoclast, also may carry on the degradation of bone matrix, and inhibit bone absorption and promote bone repair. Osteoblast MMP-2 protein secreted in the process of change can reflect the basic situation of the bone metabolism, if MMP-2 protein levels in serum and bone converting speed is faster.Metal protease inhibitors widely distributed in body fluids and tissues, its source is mainly macrophages and connective tissue, as well as fiber cells and osteoblasts secretion, can remain relatively between MMPs and TIMPs specificity and the balance of dynamic function. When the pathological conditions, the dynamic balance between MMPS and TIMPS is broken, can make the extracellular matrix decomposition, a corresponding pathological changes. Research has shown that TIMP-1 the function of the protein can be selective inhibition of MMP-9 and TIMP-2 the function of the protein can be selective inhibition of MMP-2 protein, protein TIMPs in extracellular matrix metabolism process of MMP protein played a negative regulatory role, prevent MMP activation and the effect of the degradation of extracellular matrix, the extent of the damage influence protein decomposition and injury time.Recent research suggests that MMP-9 protein can be used to predict early emergence of heterotopic ossification, this research through the use of brain trauma model of rats with induced rat Achilles clamps model of heterotopic ossification, comprehensive comparison and MMP-9 belong to gelatin gelatin enzyme enzyme protein A (MMP-2), and the change of its inhibitors TIMP-1. Through the blood samples using ELISA method to detect the contents of protein, local tissue HE sliced, immunohistochemical staining and rt-pcr gene expression. Analysis of MMP-9 protein heterotopic ossification in the specificity of the early prediction function and relationship between changes in the secretion of local tissue and to understand the early stages of heterotopic ossification local tissue protein involved in the change process, as the research provided a reference for the pathogenesis of heterotopic ossification.Chapter I:DYNAMIC CHANGES OF MATRIX METALLOPROTEINASE 9 IN HETEROTOPIC OSSIFICATION OF RAT MODELObjective:In recent years, HO rat model with Achilles tenotomy is used frequently and is more suitable for studies of traumatic HO. matrix metalloproteinase 9 is a kind of Saccharification protease, secreted by mesenchymal cells, macrophages, neutrophils, capillary endothelial cells. MMP-9 process play a key role in matrix degradation, tissue remodeling, cell migration, angiogenesis and wound healing, bone growth and pathophysiological.To observing the expression of MMP-9 in heterotopic ossification of the early trauma rat model. We want to explore the mechanism of MMP-9 in heterotopic ossification. Explore the MMP-9 to predict the value of heterotopic ossification occurred.Methods:A total of 132 male Sprague Dawley rats, aged 4-4.5 weeks, weighing (135.0 ± 6.5) g, were randomly divided into experimental group and control group (n=66). In experimental group, the Achilles tendon was cut off and clamped to prepare heterotopic ossification model; in control group, only Achilles tendon was exposed by making a incision. The general condition of the rats was observed after operation; at 2,3,4,5,6,7, and 8 days after operation, the Achilles tendon tissue was harvested for gross observation, histological observation, and immune histochemical staining observation; the serum and Achilles tendon tissue were harvested to detect the expressions of MMP-9 protein and mRNA by ELISA and RT-PCR. The X-ray films at 5 and 10 weeks and histological examination at 10 weeks after operation were used to observe heterotopic ossification.Results:All rats survived to the end of the experiment. The Achilles tendon had no significant change in control group at each time point, showing normal tendon structure. In experimental group, the hardness of Achilles tendon tissue gradually increased with the time; there were a large number of irregular connective tissue and cartilage cells; and immune histochemical staining for MMP-9 was positive results. The MMP-9 protein and mRNA expression levels of experimental group were significantly higher than those of the control group at each time point (P< 0.05). MMP-9 protein and mRNA expression levels of experimental group showed an increasing tendency (P< 0.05). According to the results of X-ray films and histological observation, heterotopic ossification occurred at 10 weeks after operation in experimental group, but no heterotopic ossification was observed in control group.Conclusion:In early heterotopic ossification of rat Achilles tendon, the expression of MMP-9 increases significantly, indicating that it has referencesignificance in predicting heterotopic ossification.Chapter Ⅱ:DYNAMIC CHANGES OF MMP-9,MMP-2,TIMP-1 IN HETEROTOPIC OSSIFICATION OF RAT MODELObjective:In recent years, the rat brain trauma model of free fall is a relatively mature brain trauma model.HO rat model with Achilles tenotomy is used frequently and is more suitable for studies of traumatic HO. We are using these two models with each other to compare the merger brain trauma, brain trauma with the influence of heterotopic ossification. At the same time analyzing the curves of MMP- 9, MMP-2 and TIMP-1 protein on early heterotopic ossification.To observing the expression of MMP-9, MMP-2 and TIMP-1 in heterotopic ossification of the early trauma rat model. We want to explore the mechanism of MMP-9, MMP-2 and TIMP - 1 in heterotopic ossification.Methods:A total of 264 male Sprague Dawley rats, aged 4-4.5 weeks, weighing (135.0 ± 6.5) g, were randomly divided into four groups (n=66). Brain trauma with Achilles tendon clamped shut off group (group A) and brain trauma merge the control group (group B), Achilles tendon clamps to cut off the group (group C), trauma in the control group (group D). The general condition of the rats was observed after operation; at 1,2,3,4,5,6, and 7 days after operation, the Achilles tendon tissue was harvested for gross observation, histological observation, and immune histochemical staining observation; the serum and Achilles tendon tissue were harvested to detect the expressions of MMP-9, MMP-2 and TIMP-1 protein and mRNA by ELISA and RT-PCR. The X-ray films at 5 and 10 weeks and histological examination at 10 weeks after operation were used to observe heterotopic ossification.Results:All rats survived to the end of the experiment. Rats in group A and group B become move less, eat less, weight loss and other symptoms. Four groups of rats decreases the activity on the right limb, after 2 to 3 weeks the activity returned to normal. Group A after five weeks X-ray slice observation, highlighting the shadow 100% (10/10),10 weeks postoperatively highlighted shadow.100%(10/10). Group B of rats after 5 weeks, highlighting the shadow 40% (4/10),10 weeks postoperatively highlighted shadow 80% (8/10). Group C rats after five weeks X-ray slice observation, highlighting the shadow 80% (8/10),10 weeks postoperatively highlighted shadow. 100%(10/10). Group D rats after five weeks X-ray slice observation, highlighting the shadow 0% (0/10),10 weeks postoperatively highlighted shadow.0% (0/10). Four groups of animal models in serum levels of MMP-9, MMP-2 and TIMP-1 protein content and its gene expression in elevated (P< 0.05). A, B, C group of rats serum levels of MMP-9 protein content were higher than in group D rats (P< 0.05), group A and group C rats after 5-7 days MMP-9 protein content were higher than group B in rats (P< 0.05). Experiments in group A, B, C amount of MMP-2 gene expression in rats were higher than in group D (P< 0.05). A, B, C group, all TIMP-1 protein in rats rats increased in the D group (P< 0.05). A, C, two groups of Achilles tendon clamps in rats, TIMP-1 gene expression quantity obviously higher compared with B and D two group (P< 0.05). Since 5 days postoperatively, MMP-9 groups of rats sensitivity curve group D area is 1.0 (P= 0.001) in the rat.TIMP-1, MMP-9 protein and protein were positively correlated (P< 0.05), A, B, C group rats all have no relationshipConclusion:1. Brain trauma can cause rat MMP-9 and MMP-2 in the early postoperative rise;2. Brain trauma can make have not reached the degree of heterotopic ossification occurred of heterotopic ossification of pathological changes, to improve the chances of developing the heterotopic ossification and the severity of the heterotopic ossification;3. MMP-9, MMP-2 and TIMP-1 the three kinds of proteins on the pathogenesis of ectopic ossification process and all play an important role in the process of wound healing;4. MMP-9 protein content in serum may be related to the severity of the heterotopic ossification was positively, and early prediction of heterotopic ossification occurred significance;MMP-9 protein content in serum 5. Aftersurgery5 to 7 days in the diagnosis of ectopic ossification of the sensitivity of the highest;6. TIM-1 protein mainly related to the local tissue damage degree, major in local tissue protection;7. The generation of heterotopic ossification and MMP-9 and TIMP-1 protein balance disorders related to the expression of MMP-9 and TIMP-1 disorder lead to pathological extracellular matrix degradation. |
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