The Preliminary Study About Epidemiology Of Nonunion And Expression Of ADAMTS-7 In Rat Femoral Nonunion Model

PART I Preliminary Study about Epidemiology of Aseptic NonunionBackgroundsWith the development of transportation and construction, the number of fractured patients remained high. At present, the treatment of the fractures has made great progress. However, as one of the most common and important complications from the fractures, the occurrence of nonunion is still very common. The incidence was reported about 5-10%. For hundreds of years, the causes and mechanism of nonunion have not interrupted.Nonunion is defined that the bone has not healed about six to nine months after fractures, and there are no signs of healing of fracture with continuous observation for 3 months. There are two important elements to judge the nonunion. First, the time must reach six to nine months after fractures. Second, the fracture gaps or callus has no obvious progress compared with last time in X-ray for three months. The both conditions are necessary. So in the clinics, we should consider more factors in the diagnosis of the nonunion.However, the studies about the epidemiology of nonunion were rarely reported. In 1978, Okhotsky reported 197 cases of patients with nonunion and it should be the earliest research on epidemiology of nonunion. Later, Rosen, and Muller also reported it respectively. In our country, in 1986, Professor Liu Yi first analyzed 520 cases of nonunion and delayed union. The results were different from that reported abroad. This shows that due to differences in national conditions, we should have our own epidemiological analysis on diseases in order to conduct better diagnosis and treatment for the Chinese patients.In China, Yingze Zhang studied better in the orthopedic epidemiology. His team has studied much about the epidemiology of some fractures and the pathogenesis characteristics, distribution, diagnosis, and treatment etc. As a result, it promoted the development of the orthopedics powerfully. So, we decided to carry on the preliminary epidemiological analysis on nonunion to summarize its characteristics, types and treatment method. We believe that it can provide technical reference on the diagnosis and treatment of nonunion.ObjectiveTo understand the preliminary epidemiology of nonunion in Shandong Provincial Hospital between January 2006 and December 2006 including characteristics of patients, the causes, types of nonunion, and the diagnosis and treatment. It can provide a solid foundation for the epidemiology about nonunion in Shandong province even in the nation.MethodsAll of the jobs were conducted by experienced doctors who received the professional training. All the patients were come from the database of Shandong Provincial Hospital. All the patients with nonunion problems admitted to Shandong Provincial Hospital from January 2006 to December 2015 were collected. The patients’age, gender, information of fractures (including time, location, open or closed, treatment and so on), information about nonunion (signs, time, locations, types and so on), diagnosis and treatment were recorded in detail. All the information were inputted into Excel 2007. Then analyze the patient’s age, gender, location and types of nonunion, and treatment of nonunion with SPSS 16.0. And p<0.05 were considered the results was statistically significant.Results1. General information:the data shows that, there were about 654 cases with nonunion admitted to our hospital between January 2006 and December 2015. There were 478 males and 76 females with ratio of 2.72:1. The average age was 39.9 (ranged,18 to 83). Nonunion was distributed in the range of 21 to 50 (73.1%). There is no significant statistical difference between right or left side. The male young and middle-aged patients were most common.2. Analysis of nonunion:About the first fractures:traffic accident was the most common causes of fractures. The closed fractures were the most common (458 cases). Most of the fractures were single part injury (472 cases). The plates and screws were main methods in the primary treatment of the fractures (396 cases); and intramedullary nail were performed in 108 cases. The locations of nonunion were seen most in the femur with 268 cases and tibia with 226 cases. About the types of nonunion:the hypertrophic nonunion was developed in 378 cases, and atrophic nonunion in 276 cases. The ratio was 1.37:1. The main methods of treatment of the nonunion were bone graft and the internal fixation change. We choose the intramedullary nail as the main internal fixation in 220 cases and plates in 164 cases. The ilium grafting accounts for 62.7% and 360 degree surround.3. Nonunion in different parts:the nonunion has its own characteristics in different parts. In femur, the nonunion was seen commonly in male and the primary fractures were closed. The initial treatment is used steel plate in priority. There were 166 cases with hypertrophic nonunion and 102 cases with atrophic nonunion. The treatment of nonunion was mainly with intramedullary nails. The nonunion in tibia was similar to the femur except that the primary fractures were open and the initial treatment was plate and external fixation. The treatment of nonunion in humerus, radius or ulna was mainly with plates.Conclusions1. The aseptic nonunion was mainly developed in the young and middle-aged male patients. The occupation was mainly distributed in farmers and workers.2. The high-energy damage was common in the primary fractures, which caused the severe soft tissue injuries. The plate was common seen in the initial treatment that can damage the blood supply easily, and that can cause the nonunion.3. The location of nonunion was found more in the long bones. The order was as follows: femur> tibia> humerus> radius and ulna. The nonunion in different bone has its own characteristic. The proportion in the genders was correlated with the ages.4. Two types of nonunion were similar in the numbers and the proportion in the genders was correlated with the ages. The hypertrophic nonunion was mainly distributed in femur and atrophic nonunion in tibia.5. The treatment of nonunion was different and intramedullary nail in femur and tibia, while plate in humerus, radius and ulna. The ilium grafting is the first choice and 360 degree surround the fractures is best.PART II The Treatment of Infected Nonunion by Bone TransportBackgroundsInfected nonunion is defined as nonunion with infection after 6 to 8 months. It is mainly caused by postoperative infection after internal fixation of open fractures or closed fractures, and chronic osteomyelitis. These patients were performed surgery for many times, and often lead to many conditions such as bone infection, deformity, defects, osteoporosis, joint stiffness, limb length inequality, soft tissue defects, scarring. This makes it difficult to treat and it is a great challenge to the orthopedic surgeons.The goals of the treatment of infected nonunion are as follows:First, the healing of bone and soft tissue; Second, correct angulations and shortening deformity; Third, the recovery of joint activities; Fourth, The healing of fractures; Fifth, the eradication of infection. The traditional method is first of all, to control the infection, including drainage, transfer of skin flap, antibiotic chain and so on. Then we can treat the nonunion. This method cost long time and the effect is unsatisfactory. So some patients may eventually choose amputation.Distraction osteogenesis was discovered by Ilizarov and finally the law of tension stress was formed, which is the essential theory of the bone transport. With the development of the law of tension stress, the treatment of the infected nonunion entered a new period. The advantage of the distraction osteogenesis is it can correct the bone defect and shortening, and even the soft tissue defects simultaneously. The patients can take exercises early, which can avoid the stiffness of the joint. And now, the bone transport has been the golden standard in treatment of the infected nonunion.ObjectiveTo assess the results of bone transport in the treatment of the infected nonunion by retrospective analysis the cases in the past five years.MethodsCollect the cases of the patients with infected nonunion in the past five years and choose the 194 patients treated with the bone transport to analysis the results.The treatment of these patients included the steps as follows:the preoperative plan including the ESR, CRP, bacterial culture, the preoperative imaging and the choice of the external fixator. Thorough debridement. The wound can be sutured or left open for the gradually close in the process of bone transport. External fixator was divided into two types: the Ilizarov circular external fixator and Orthofix monolateral external fixator. We should install the fixator according to the length of the infected bone and the position of the osteotomy. The metaphyses was often selected to be the position of the osteotomy. And the method was simple, minimally invasive and with good results. The post operative management was critical. We should observe the swelling, feeling, motion and blood supply of the limb. The transport can be performed after 7 to 10 days with speed of 1mm/d,4 parts. Early exercise should be taken.The results were assessed by the ASAMI criterion.Results194 cases were included in the study with male vs. female 1.20:1.The average age was 39.6 years old and the peak period of disease was from 31 to 40 years old. The causes of injury included the accident in 95 cases, the falling in 63 cases and the crush in 36 cases. The location of the nonunion was tibia in 135 cases, the femur in 42 cases and humerus or ulna and radius in 17 cases. Open fractures were seen in 123 cases in the initial injury and closed in 71 cases. The open fractures were classificated according to Gustilo classification criterion: Gustilo I in 14 cases, Gustilo II in 39 cases, GustiloIII in 70 cases. The external fixation was the main initial treatment in 98 cases, plate in 61 cases and intramedullary nail in 35 cases. The skin defects were seen in 49 cases and sinus tract formation in 78 cases. The results of bacterial culture showed that the positive rate was low (41.7%) and the Staphylococcus aureus was the main pathogenic bacteria (69.8%).In the 194 cases, the Ilizarov circular external fixator was used in 113 cases and Orthofix monolateral external fixator in 81 cases.186 cases were followed up and 113 cases have healed in average 13.8 months. The external fixator was weared for about 21 months. Average healing index was 1.78 months/cm. According to the ASAMI criterion, the healing results, excellent in 82 cases, good in 23 cases, fair in 6 cases and bad in 2 cases, the rate of excellent and good was 92.9%. The functional results, excellent in 76 cases, good in 23 cases, fair in 11 cases and bad in 3 cases, the rate of excellent and good was 87.6%. In all the patients,23 developed pin infection; 5 mineralization in advance; 3 intense pains in the process of transport; 12 skin retraction; 8 malalignment; 6 stiffness of joints, In all the patients, the infection was controlled successfully and the bone healed.ConclusionsThe bone transport is a good method in treatment of the infected nonunion. It is simple and effective, and worth popularizing.PART Ⅲ The Building of Rat Nonunion Model and the Expression of ADAMTS-7 in the Nonunion ModelsBackgroundsNonunion is termination of normal healing process. The bone will not heal if no active measures are taken. Nonunion is not only brought great pain and dysfunction to the patients, but also increased the economic burden of patients and society. There is no effective way to treat the nonunion. Therefore, the molecular biology mechanism of bone nonunion occurred should be further studied.ADAMTS (a disintegrin and metalloproteinase domain with thrombospondin motifs) family has been found for many years. It was originally found in 1997. This type of metalloproteinase occupies a large proportion in ECM (extracellular matrix). Members of ADAMTS family have important functions in multiple physiological processes, such as in the processing of collagen, the degradation of proteoglycans, inhibition of angiogenesis, in the balance of coagulation, etc. ADAMTS-7 belongs to ADAMTS family, and it is a kind of new metal protease. In 2009, X.H. Bai reported the function of ADAMTS-7 about controlling cartilage formation and cartilage calcification. This is the first time in the world to reveal the role of ADAMTS-7 in the production of cartilage and cartilage ossification. ADAMTS-7 may also directly combine and degrade COMP (cartilage oligomeric matrix protein) protein, which can cause arthritis.Researches show that:in the development of cartilage in embryonic mice, the expression of ADAMTS-7 in cartilage cells of are increased gradually; As a downstream molecules of PTHrP (parathyroid hormone related peptide), ADAMTS-7 plays a negative regulatory role in cartilage formation, and through the degradation of GEP (granulin-epithelin precursor), it significantly inhibits the growth of bone and calcification, which shows that ADAMTS-7 is likely to be involved in the occurrence of nonunion and play an important role. But no studies about the biological functions of ADAMTS-7, especially its role in nonunion occurred were reported in the world.ObjectivesTo build the rat nonunion models, and detect the expression of ADAMTS-7 in the process of nonunion occurrence. To provide a reference to explore the molecular mechanism of ADAMTS-7 for nonunion and to local import ADAMTS-7 purified protein to treat nonunion.Methods1. Groups of experiment rats40 adult Wistar rat were classified into two groups randomly:nonunion group (Experimental group) and fractured group (control group). The specimen was collected on 3rd day,7th day,14th day,28th day and 56th day.2. The building of rat fractured and nonunion model and the assessment of the nonunion of the femur.Use the methods reported by Bonnarens and Einhor to make transverse fracture of femur. The nonunion was built by burning the periosteum. The assessment of the nonunion of the femur was used the methods of general observation, X-ray and histopathologic examination.3. Detection of ADAMTS-7 expression in nonunion model in different time points by immunohistochemistry.Decalcification of the femur was performed and paraffin section was made. Follow the steps of immunohistochemical staining:antigen repair and immunohistochemical staining (SABC method). Use immunohistochemical method to observe fracture healing and expression of ADAMTS-7 in different periods and localization.4. Detection of ADAMTS-7 expression in nonunion model in different time points by Western-blot method.Using Western-blot method (electrophoresis, transfer, antibody binding) to observe the fracture healing and expression of ADAMTS-7 in different periods and localization in the protein level.5. Detection of ADAMTS-7 expression in nonunion model in different time points by Real-time PCR method.mRNA was isolated and was analysis by real-time PCR to detect the expression of ADAMTS-7 in the nonunion model in different time points。Results1. Building the rat nonunion model:the nonunion was developed after 8 weeks from the fracture. And this was proven by the general observation, X-ray, and histopathologic examination. We found the activity of the fractured ends and sclerosis in the general observation. In the X-ray, the sign was that the fracture line was clear and there were no callus formation. The tissue filled in the fractured ends and no bony callus was found.2. The expression of ADAMTS-7 in different time points in the nonunion models: according to the methods of immunohistochemistry, western-blot and Real-time PCR, we found that the expression of ADAMTS-7 was different in different time point. Before 3rd day, the expression was low and from 7th day, the expression increased remarkably, and remained high from then on. However, in the control group, the expression of ADAMTS-7 remained low in every time point.Conclusions1. The rat nonunion model can be built by burning periosteum and fixating by kirschner wire. The method was simple and the success rate was high.2. The expression of ADAMTS-7 was high in the process of the nonunion and it may be upregulated modulator of nonunion.3. ADAMTS-7 can inhibit the growth and differentiation of cartilage to block endochondral ossification and that can cause the nonunion.4. We can observe the process of bone healing dynamically by detecting the expression of ADAMTS-7 levels so that we can prognose the results of the fractures.