| Chronic osteomyelitis has been acknowledged as an obstinate disease by medicalprofessionals, as such been called the “second cancer”. Although as the rising ofantimicrobial activity of the new generation antibiotics, and as the maturing of surgicalintervention, chronic osteomyelitis recurrence rate is still as high as20-30%[1], whichleads to amputation in some cases. It not only has broμght tremendous physical and mentalpain to the patients, but also heavy economic burden to the family and national healthcaresystem [1,2,3]. Therefore, shortening the treatment duration of chronic osteomyelitis,improving the cure rate and reducing morbidity have considerable clinical and socialsignificance.The current consensus on chronic osteomyelitis treatment is to apply antibiotictherapy [3] on the basis of thoroμgh debridement and wound drainage. Antibiotics, being adetermining factor, is used throμghout the treatment. In recent years, althoμgh antibioticsand the ways of its application have been continuously improving, the result hasn’t beenvery satisfactory [4]. Low concentration of free-state antibiotics in the nidus has resulted inlow cure rate and high recurrence of this disease. Ozone, being a strong oxidizing agent,has broad and strong antibacterial effect. Recent experiments have also shown that certainconcentration of ozone can kill bacteria biofilm, regulate oxidative stress, and improvemicrocirculation. This subject, firstly, establishes the rabbit tibia model of chronicosteomyelitis by infusing ozone to the target tissue, in order to study the pharmacokineticsof vancomycin, the antibiotics applied to the target tissue, throμgh microdialysis samplingtechnique. Secondly, the subject intends to explore effective methods of local treatmentwith ozone via measuring the content of IL-6and TNF-α in the target tissue, and viaassessing the treatment effect throμgh imaging, pathology and microbiological methods.Throμgh this study, we intend to clarify the intrinsic mechanism of topical application ofozone treatment on chronic osteomyelitis, so as to discover new ideas and methods to treatthis disease..Objective:To establish rabbit models of chronic osteomyelitis, and apply intravenous injection ofvancomycin hydrochlorid and ozone perfusion of local bone tissue. To use microdialysis technique combined with reversed-phase high performance liquid chromatography (HPLC)to perform synchronous detection of free-state vancomycin hydrochloride concentration intarget tissue and blood at specified time spots, so as to observe the pharmacokineticsdynamics of vancomycin hydrochloride in the bone tissue and blood, and to investigate thesynergy of the ozone and antibiotics. In addition, to conduct quantitative analysis of theevolution related with ozone on inflammatory factors (IL-6and TNF-α) in the targetosteomyelitis tissue, in order to reveal the mechanism of the ozone anti-infection treatment,and to provide evidence for the treatment of chronic osteomyelitis by ozone combined withvancomycin..Methods:1. For the sutdy of vancomycin pharmacokinetics:18male New Zealand white rabbits,weight between2to2.5Kg, to be randomly divided into3groups as normal group,chronic osteomyelitis vancomycin treatment group, and ozone vancomycin treatmentgroup respectively. To ensure18hours of fasting except for water before the experiment.To apply, via rabbit ear vein,30mg/Kg of vancomycin hydrochloride on all three groups.To apply2ml of ozone (40μg/mL) before injection of vancomycin hydrochloride on theozone vancomycin treatment group. Using microdialysis technique to conduct synchronoussampling of bone tissue and blood. Combined with joint reversed-phase high performanceliquid chromatography, to record real time change of concentration of hydrochloridevancomycin in target tissue and blood. And to process the recorded data with WinNonlinsoftware.2. Study of ozone anti-infective therapy mechanism:18male New Zealand whiterabbits, weight between2to2.5Kg, to be randomly divided into3groups as normal group,chronic osteomyelitis group and ozone treatment group. To apply2ml of ozone (40μg/mL)in ozone treatment group. To perform sampling using microdialysis technique in allanimals. And to conduct quantitative analysis on IL-6and TNF-α in the samples togetherwith ELISA method.Results:1, pharmacokinetic studies: free vancomycin concentration in the blood of normalrabbit reached its peak after10min of intravenous injection to41.5±2.71μg/ml, same as in bone tissue to40.57±2.50μg/ml; both concentration data stay close within100min afterthe injection. While once pass the100min threshold, free vancomycin concentration inblood stays above that in bone tissue until the end of the observation. The vancomycincontent in bone tissue of chronic osteomyelitis lesion was significantly lower than that inblood;10min peak concentration was43.31±2.67μg/ml (in blood) and16.35±1.67μg/ml(in bone tissue). After ozone intervention therapy, vancomycin concentration in theinfected bone tissue significantly increased to44.37±2.31μg/ml (in blood) and20.58±4.85μg/ml (in bone tissue), which were still lower than that in normal bone tissue. Theunder curve area of blood and bone tissue after medication in all three groupswere: normal group:5280.31±205.73min*μg/ml (in blood) and5065.03±209.56min*μg/ml (in bone tissue); vancomycin treatment group:5106.45±205.73min*μg/ml (inblood), and1259.53±48.42min*μg/ml (in bone tissue); ozone joint interventiontreatment group:5353.51±325.58min*μg/ml (in blood) and1873.43+573.7min*μg/ml (in bone tissue).2. Change of inflammatory cytokines: IL-6and TNF-α level showed a significantdownward trend after ozone perfusion, where they were318.1±2.82pg/ml-361.42±8.52pg/ml (IL-6) and231.48±14.89pg/ml-267.13pg/ml (TNF-α)in chronic osteomyelitis group,79.60±10.12pg/ml-277.16±10.02pg/ml (TNF-α) and127.36±13.83pg/ml-371.69±11.97pg/ml (IL-6) in ozone treatment group and59.37±10.57pg/ml-112.06±14.85pg/ml (TNF-α),91.91±10.57pg/ml-227.27±17.75pg/ml（IL-6）in normal group.Conclusion:Microdialysis sampling technique has high time and spatial resolution. Combinedwith reverse phase HPLC method, they can accurately and objectively reflect thepharmacokinetic characteristics of drμgs in chronic osteomyelitis lesions tissues and inblood as effective tools of study. The experiments show that vancomycin hydrochloride,under intravenous injection, can well penetrate bone tissues. Compared with the plasmapharmacokinetic parameters, the peak concentration level and the duration to peak level ofthe drμg concentration show no difference in normal bone tissues; while in chronicosteomyelitis lesion, drμg concentration in bone tissues was significantly lower than that inplasma. Althoμgh after the ozoen intervention, peak and troμgh drμg concentration level in the leison both increased compare to that of pure vancomycin therapy, which showed asynergy between the ozone and vancomycin yet no strong enoμgh to achieve normal bonetissue drμg concentration. Comparing the normal group and the experimented groups,in the bone tissue was smaller than that in plasma, while there was no significantstatistical difference in the drμg mean residence time (MRT) and drμg half-life.To make a conclusion according to the dynamics of IL-6, ozone inhibits the activity ofnuclear transcription factor NF-KB by causing the accumulation of α-synuclein protein incells. Thus prevents NF-KB from entering the nucleus, represses cell expression ofinflammatory cytokines such as IL-10, IL-6and TNF-α, enhances the synthesis ofglycolytic enzymes, and accelerates glucose metabolism; thereby increases generation ofsubstances such as NO and CO, expands capillaries, and improves local circulation, whichattribute to the increasing of vancomycin concentration in the lesion bone tissues. |
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