Empirical Study On The Delayed Release In Vivo And In Vitro And The Treatment Of Infected Bone Defects Of Rabbits With GM/BG

ObjectiveTo study the effect of sustained release of GM/BG release system in vivo and in vitro, and its effect of anti-inflammatory and repair of bone defects, this is to provide a experimental basis for clinical prevention and treatment of infected bone defects in future.Methods(1)GM/BG delivery system was prepared by the ways of Kawanabe, etc. (2) GM/BG block was placed in a small ampoule with 3ml PBS solution. Then the small ampoule was placed in the temperature box at 37℃. All PBS solution was replaced every 2 days. The antibiotics concentration of the eluent was determined by HPLC, then the release curve of GM/BG was described. The release curve of GM/BG was fitted by Peppas equation, to explore the drug release mechanism. (3) Two GM/BG bloks and one BG blok were placed in the MH agar culture dish inoculated with staphylococcus aureus. Then the culture dishes were cultured at 35℃in 24h in attemperator. After measuring inhibition zone, the GM/BG bloks and BG bloks were removed and placed in the new the culture dishes to continue culturing. Subsequently the inhibition zones of the GM/BG bloks and BG bloks were measured each 3ds, and the culture dishes were replaced. This process was repeated until the inhibition zone disappear. And the curve with time was described based on the size of inhibition zone. (4) Take 6 New Zealand rabbits, regardless of sexes, with weights varying from 2kg to 2.5kg. The rabbits were made 10mm bone defects in the right proximal radial, then the GM/BG bloks were implanted into the bone defects. The drug concentrations of plasma and soft tissue at 0.5cm from the bone defects from rabbit were determined after 1,3,5,7,10,15,20 and 25 days.The curve of drug release in vivo was made according to the experimental results. (5) Twenty four New Zealand white rabbits regardless of sexes, with weights varying from 2kg to 2.5kg were randomly divided into experimental and control group in average.Firstly, the rabbits were made 3 * 5 * 10mm~3 rectangular bone windows in the right medial tibia.The bone windows were implanted with GM/BG in experimental group and with BG in control group.Then The criteria staphylococcus aureus were put in the bone windows.The general conditions of rabbits were observed postoperative.The rabbits were made X-ray examination at 4w and 8w, to observe the situation of osteomyelitis and the treatment of bone defect.The right tibia of the rabbits were taken out at 4w and 8w,with general observation,histological and bacterial culture examination.Results(1)The GM/BG has the capacity of drug release with long-term in vitro, up to 23 days. The drug concentrations were larger than the MIC of gentamicin (2ug/ml). According to the Peppas equation, the drug release mechanism of GM/BG consistented with the Fickian diffusion law. (2) The inhibition zone of GM/BG Continued 26 days. (3)The drug concentration of local tissue was 6.53±1.42ug/ml in the 20th day.It was still larger than the MIC of gentamicin 2ug/ml. But the plasma concentration was undetectable on the 3d day. (4) The experiment in the treatment of infected bone defects. After operation, 2 rabbits died in severe infection in the control group, but none of the rabbits died in the experimental group. General observation: at 4w and 8w, in the control group, the BG was wrapped around pus and inflammatory granulation tissue at bone defect, and was not fusion with bone tissue about; in the experimental group, the BG was gradual degradation, and was better integration with the surrounding bone. X-ray examination: At 4w, the phenomenon in the control group was periosteal reaction, bone hyperplasia, bone destruction, soft tissue mass, and sequestrum formation. In the experimental group, the density of BG faded and was absorbed gradually. And the BG was fused with the bone tissue together gradually.At 8w, the phenomenon in the control group was showed bone destruction, soft tissue mass, sequestrum. The density of BG faded, but it was not fused with the surrounding bone tissue together; In the experimental group, the density of the BG was further lighten and absorbed, and it was fused with the surrounding bone. The boundaries in bioglass and the surrounding bone was disappear. Pathological examination: at 4w, in the experimental group, it was observed that a small amount of inflammatory cells, fibrous tissue growthing in bioactive glass. In the control group, it was observed inflammatory cell infiltration and abscess formation, bioactive glass surrounded by purulent tissue, no cells and fibrous tissue growthing in BG. At 8w, in the experimental group, the BG was absorpted and the immature callus can be observed. In the control group, visible sequestrum can be still observed. The BG was wrapped inflammatory tissue. Bacterial culture examination: The results tested by chi square, was that the rate of positive bacterial culture in soft tissue and bone marrow cavity was significantly different betwee the experimental group and the control group at 4w and 8w. Bacteria being positive cocci by Gram, with hemolysis of the culture dish, were proved to be implanted staphylococcus aureus.Conclusions(1) GM/BG has a signaficent effect on delayed release in vitro , and it has a evident properties of bacteriostasis. (2) GM/BG has a good delayed release effect in vivo and high local drug concentration with the low concentration in body. Local administration is safe and effective. (3) GM/BG can repair the bone defect, and has the effect of anti-inflammatory. GM/BG can be used for prevention and treatment of infected bone defects in clinical. This can provide a experimental basis for clinical prevention and treatment of infected bone defects.