| PART 1 DETERMINATION OF LEVOFLOXACIN CONCENTRATION IN RABBIT TIBIA AND BONE MARROWBY HPLCObjective:To establish a high performance liquid chromatography(HPLC) method for the determination of levofloxacin in the rabbit tibia and bone morrow;to evaluate the release characteristics of levofloxacin in nanoparticles contained levofloxacin degradation-controllable bone repair materials in rabbit local bone tissue and bone marrow.Methods:Different concentrations of levofloxacin were added in blank tibia and bone marrow respectively.Ciprofloxacin was used as an internal standard.Sepax BR-C18(250 mm×4.6 mm 5 mm)column was employed; the mobile phase was:0.01 mol/L potassium dihydrogen phosphate-methanol-0.5 mol/L tetrabutylammonium bromide(75: 25: 4); flow rate was 1.0 ml/min; column temperature was 40 ℃; detection wavelength was 290 nm. Evaluation indexes were linear range, recoveries, extraction recoveries, intra-day precision, inter-day precision, sample stability and accuracy.Results:When the tibia of levofloxacin concentrations in 0.05~300 mg/ml,good linear relationship was achieved(r=0.999);low, medium and high concentrations of three recoveries were(108.81±1.14)%,( 96.85±3.64)%,( 114.38±0.27)%; the extraction recoveries were(110.92±0.42)%,(110.48±0.97)%,(80.39±0.08)%;intra-day precision RSD were 1.30%, 0.23%, 5.51%; inter-day precision RSD were as follows: 7.09%, 3.71%, 2.55%, a good sample stability. When the bone marrow levofloxacin concentrations in 0.05~300 mg/ml, a good linear relationship was achieved(r= 0.999);low, medium and high concentrations of three recoveries were(91.67±3.73)%,(101.77±0.26)%,(87.72±0.72)%;the extraction recoveries were(102.97±0.81)%,(87±0.62)%,(99.97±1.15)%;intra-day precision RSD were 1.49%,1.06%,0.11%; inter-day recision RSD were as follows: 5.61%,2.56%,2.37%, a good sample stability.Conclusion:With the advantages of simple, sensitive, stable, high recovery rate, this method is applicable to the detection of distribution of levofloxacin in rabbit local bone tissue in a new bone repair materials.PART 2 LEVOFLOXAXIN LOADED MESOPOROUS SILICA MICROSPHERES/NANO-HYDROXYAPATITE/POLYURETHANE COMPOSITE SCAFFOLD FOR THE TREATMENT OFCHRONIC OSTEOMYELITIS WITH BONE DEFECTObjective:Evaluation of levofloxacin loaded mesoporous silica microspheres/ nano-hydroxyapatite/polyurethane composite scaffold for the treatment of chronic osteomyelitis in rabbit tibiaMethods:The bacteria was injected in the right tibia of 60 rabbitsanimal model of Chronic osteomyelitis was established 4 weeks later.Then all the rabbits were divided into five groups. Animals of group A only have debridement as a Blank control; 1mg Lev@ PMMA was implanted in Animals of group B; 5mg Lev@ PMMA was implanted in Animals of group C; 1mg Lev@ MSNs/n-HA/PU was implanted in Animals of group D; 5mg Lev@ MSNs/n-HA/PU was implanted in Animals of group E.At the time point of 1 week,3 weeks,6 weeks,12 weeks,gross pathology,X ray,Micro CT,histology was used to evaluate the efficacy of this new material for the treatment of chronic osteomyelitis.Evaluation of new bone formation induced by novel composite materials.Results:1.Gross pathology Rabbit Tibia of blank control group have classical performance of chronic osteomyelitis.Tibia severely damaged,there is sequestrum formation.The remaining four groups showed a combination between material and tibia bone.2.X-ray Tibial of blank control group was swelling, decreased bone density, significant bone destruction,cortical bone was "shell" like bulge,sequestrum formation,ill-defined,osteolytic lesions,cortical thinning.The material of 1mg Lev@PMMA group and 5mg Lev@PMMA group can be seen on X-ray.3.Micro CT Significant differences were observed between 5mg Lev@MSNs/n-HA/PU group and the other four groups in new bone formation(P<0.05);New bone formation of 1mg Lev@MSNs/n-HA/PU group is much higher than 1mg Lev@PMMA group and blank control(P<0.05).4.VG staining Little new trabecular bone were observerd in blank control group.New trabecular bone were found on the surface of material of 1mg Lev@PMMA. New trabecular bone were observerd in the interior material of 1mg Lev@MSNs/n-HA/PU group and 5mg Lev@MSNs/n-HA/PU group. Materials and trabecular bone are linked closely in 5mg Lev@MSNs/n-HA/PU group.5.HE staining Inflammatory cell accumulation could be seen in bone of blank control group.there is few inflammatory cells were observerd in bone of 1mg Lev @ PMMA group.the remaining three groups had no presence of inflammatory cells.Conclusion : The efficiency of Mesoporous silica microspheres/nano-hydroxyapatite/polyurethane composite scaffolds which loaded with 5mg levofloxaxin was much better than other groups for the treatment of chronic osteomyelitis. Our experiments supply a solution for the treatment of chronic osteomyelitis This composite scaffolds maybe used in clinical later. |
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