The Anti-inflammatory And Anti-inflammatory Bone Destruction Effect Of NBD Peptides In The Rabbit Osteomyelitis

Osteomyelitis becomes more and more common in clinic, In the traumatic fractures, metal particles brought by fixed intramedullary pin, steel and Extermal fixation metal stents produces a variety of inflammatory factor leading persistent inflammation and direct effect on fracture healing.The existence of long-term chronic inflammation caused by bacterial inflammation or other factors can cause delayed fracture healing and bone nonunion.There are about50million fracture patients in our country, the incidence of bone nonunion is about5%-10%, the most part of which are caused by open fractures. The pain, physical function and psychological barriers brought by fracture healing and bone nonunion has brought great torture to the patients, but also increase the economic burden of patients and society. And bone formation process is the contradictory unity of balanced process between the mutual antagonism of osteoblast and oateoclast activity, osteoblasts are differentiationed by mesenchymal cells, which is suitable for synthesis of matrix, on the contrary, osteoclasts are diffenentiationed by bone marrow mononuclear cells, which are suitable for the degradation of bone matrix. Fracture healing requires a lot of new osteoblast, so as to accelerate the synthesis and calcification of bone increasing bone mass and density. Chronic inflammation can stimulate bone resorption, so the research of blocking inflammation factor to restrain bone regeneration has great significance on guiding the clinic.Nuclear transcription factor NF-kB (nuclear factor kappa B) as an important transcription factor participate in regulating of gene expression of material such as the immune response n inflammatory reaction and apoptosis, Because the small molecule NBD peptides can block the combination of NF-kB essential modifier and IKKaand IKKy, the activation of IKK has been inhibited, preventing broken precursor cells from functional osteoclast so as to inhibiting inflammatory bone resorption, At present, the most research of NBD peptides are limited in the cellular level, althought some trails have proved that the NBD polypeptides can inhibit the osteoclast and promote the differentiation of osteoblast under the stimulation of inflammatory, there is little research on animal osteomyelitis model, which require further research.This experiment establish the tibia osteomyelitis model by injecting a certain amount of bacterial suspension into the rabbit tibia metaphyseal, aiming to study the anti-inflammatory and anti-inflammatory bone destruction effect of NBD peptides locally injected into the tibia metaphyseal in the animal inflammatory model.Chapter oneThe research on the establishment of experimental chronic osteomyelitis in a rabbitObjectives:To study the establishment of chronic osteomyelitis in a rabbit tibiaMethods:twenty-one New Zealand white rabbits were used for the creation of chronic osteomyelitis model. In brief, A2cm longitudinal incision was made from tubercle at the level of the proximal tibia. The medullary cavity was opened with a electric drill by inserting a lmm kirschner-wire through the lateral aspect of the tibia metaphysis.Thereafter, an12-gauge needle was inserted into the cavity.0.3ml bone marrow was extracted, Subsequently,0.1ml5%sodium morrhuate and0.6ml of0.5titer Staphylococcus aureus (ATCC25923) was injected into the cavity. The needle was still left in the medullary cavity. The animal were kept in individual cages and no antibiotics for prevention of bacteria. The experimental model was evaluated by clinical, radiologic, bacteriologic and histologic parameters after injection at the3th week.Results:①All rabbits were showed in poor mental health at early phase which drunk much water. Degrees of soft tissue swelling could be seen from the surgical area which presented purulent secretions and granulation tissue.②Three weeks after surgical process, the standardized lateral radiographs of tibia shows the bone destruction、sequestration formation and soft tissue swelling in the tibia metaphysic at3weeks,according to the Norden scoring,the average score was4.1±0.3.③Staphylococcus aureus could be found from surgical region④Pathological sections showed a great deal of inflammatory cell and sequestration presented the pathology of chronic osteomyelitis at the third week.Conclusion:The establishment of experimental chronic osteomyelitis can be achieved by injection of bacteria suspensions into the tibia metaphysis.Chapter twoThe anti-inflammatory effect of NBD peptides on the rabbit chronic osteomyelitis Objectives:This research was performed to analyzed the local injection of NBD peptides for the effect of anti-inflammatory in the rabbit chronic osteomyelitis.Methods:Twenty-one rabbits were used to establish the chronic osteomyelitis which was accomplished by locally injecting the bacteria suspension into tibia metaphysis, After the indentification of successful modeling, the animals were divided into three groups at random, the rabbits in the blank group received a cortical window (6mm) made in the anteromedial surface of the proximal tibia metaphysis, the rabbits in the control group were received the same cortical window and debridement, At the same time, the rabbits in the treat group were received the same window、debridement and NBD peptides (3mg/kg body weight was established as optimal dose for NBD), NBD peptides were locally injected into the window on first and third day of the experimental. Then all the window were filled with the sponge.In the procedure of debridement, the soft tissue debridement and curettage of the marrow cavity were completed sequentially. All infectious and necrotic tissues were removed according to the varying radiological severity of osteomyelitis. then the animals were subjected to repeated washing with normal saline, hydrogen peroxide, and chlorhexidine.In the process of experimental, the blood samples from the ear vein from all animals were collected every week for measurement of C-reactive protein (CRP) Erythrocyte sedimentation rate (ESR) and leucocyte count. At6weeks after successful model, the animals from each group were sacrificed after the treation operation and the sample were stained with hematoxylin and eosin (H&E) Result:①The CRP level of the three groups have significant difference at4th week, compared with the blank and control group (P<0.05), the CRP level of treatment group has significance decrease at the fifth week (P<0.05), there are no significant difference among groups in the ESR level at every week, however, there are significant difference in the leucocyte count level among groups at every week.②pathology show that there are many inflammatory cells and dead bone in the blank group, but it is hard to find dead bone and little inflammatory cells in the control group, however, there is no inflammatory cells in the treatment group, at the same time, the tibia medullary cavity was fill with a large number of fibershyperplasia.Conclusion:the experimental results show that there is certain anti-inflammatory effect on the rabbit chronic osteomyelitis by the locally injection of NBD peptides.Chapter3The anti-inflammatory bone destruction of NBD peptides in the rabbit osteomyelitis modelObjective:to study the anti-inflammatory bone destruction of NBD peptides in the rabbit chronic osteomyelitisMethod:twenty-one healthy New Zealand White rabbit were used to establish the chronic osteomyelitis which was accomplished by locally injecting the bacteria suspension into tibia metaphysis, After the indentification of successful modeling, the animals were divided into three groups at random, the rabbits in the blank group received a cortical window (6mm) made in the anteromedial surface of the proximal tibia metaphysis, the rabbits in the control group were received the same cortical window and debridement, At the same time, the rabbits in the treat group were received the same window、debridement and NBD peptides (3mg/kg body weight was established as optimal dose for NBD), NBD peptides were locally injected into the window on first and third day of the experimental. Then all the window were filled with the sponge. All the rabbits were performed the tibia X-ray observation at the forth and sixth week, then take lesions tibia to perform pathology staining and Micro-CT examination after the sacrifice at the sixth week.Result:①the reduction of bone window area were observed in the control and treatment group, the window were obvious in the control group②the Micro-CT three dimensional reconstruction show that the windows of treatment group were smaller than the blank and control group, and there is significant difference in the BV/TV③pathology of HE staining show that there were a large number of new osteoblast on the treatment group, the rest of the two groups has no obvious osteoblast, one of which were filled with inflammatory cells, the other filled with fibrous tissue.Conclusion:the experimental results show that there is certain anti-inflammatory bone destruction effect on the rabbit chronic osteomyelitis by the locally injection of NBD peptides.