Intradiscal Injection Of Vancomycin Loaded PLGA Microsphere For MRSA Discitis

BackgroundDisctis also known as pyogenic disctis or intervertebral infection,it is the infection of the intervertebral disc and adjacent cartilage lesions.According to the pathogenesis,it can be divided as primary and secondary.The primary type is rare in clinic.The secondary type is more often secondary to intervertebral spinal puncture, angiography, surgery and other invasive operation or adjacent soft tissue infections.Infectious spondylodiscitis has been diagnosed with increasing frequency. This reflects expanding elderly and immunocompromised populations, the increasing use of invasive spinal procedures and development of imaging diagnosis technique. Currently, all the treatments for discitis are based on the application of antibiotics Because there is no blood vessel distribution in the intervertebral disc itself. Antibiotics enter the intervertebral space through the capillary plexus and cartilage end-plate, resulting in a lower drug concentration in the intra-intervertebral disc than the serum concentration. And because of the short half-life of the antibiotic, long-term repeated administration is required, resulting in higher health care costs, higher side-effect, longer hospitalization time.In order to solve this problem, researchers have begun to try the local application of antibiotics to deal with discitis.There are unoque advantages of local use of antibiotics to systemic application,and was gradually accepted by clinical practitioners. However, if a simple injection of antibiotics into the infection of space is used,the antibiotics will be washed away by the blood soon,and the antibacterial capacity will be weakend because of the decrease of the antibiotic concentration.Then repeated puncture and administration of antibiotics is needed.The irrigation administrated the antibiotic directly into the infected space,it's effectively treated the infection while avoided the systemic side effects.However,it's elevated the risk of retrograde infection. In recent years, sustained-release formulations of antibiotics in the treatment of acute and chronic osteomyelitis and other orthopedic clinical infections obtained a good experimental and clinical effects. Local injection of sustained-release antibiotics maintened effective drug concentration in the injection site after a long period of time and avoid systemic side effects, its drug release time and release rate can be prepared according to the requirement of treatment. So we hope to reduce the drug consumption,side-effect, medical cost and shorten the hospitalization time by intradiscal injection of a kind of sustain-released antibiotic.ObjectivesTo prepare the vancomycin hydrochloride (VA)-loaded poly lactic acid-glycolic acid copolymer (PLGA) microsphere by the multiple emulsion method, and to investigate it's general properties, such as the morphous of the microspheres, the particle diameter distributions, the drug loading rate, the drug encapsulation rate, the CH2C12 residue, and the drug release properties. And, we also used the vancomycin loaded PLGA microspheres for the treatment of rabbit Methicillin resistant S.aureus (MRSA) induced disctis, in order to investigate the therapeutic efficacy of vancomycin loaded microspheres, as the preliminary work for further clinic employment..Material and methodsPrepare the vancomycin hydrochloride-loaded poly lactic acid-glycolic acid copolymer microsphere by the multiple emulsion method, investigate the morphous of the microspheres and the particle diameter distributions by the scanning electron microscope and the particle size analyse apparatus;vestigate the durg loading rate, the drug encapsulation rate, and the durg release properties at the PBS solutions in vitro with HPLC method.Fifteen rabbits were classified randomly into two groups, experiment group(n=10) and control group(n=5) and were used to make discitis models. In the experiment group,The Methicillin resistant staphylococcus aureus was injected into the intervertebral disc of rabbits for inducing to occur the discitis. In the control group,the equal quantity normal saline was injected. All rabbits lumbar spine were examined by X-rays and Magnetic resonance inspector (MRI) respectively 2 and 4 weeks after the injection.Biopsies of nuclear pulpous were obtained from experimental intervertebral disc of lumbar spine for bacteria culture and pathological examination after 2 or 4 weeks. Serum C-reactive protein(CRP) and Erythrocyte sedimentation rate (ESR) were continuously monitored and recorded for all rabbitsRabbits with methicillin-resistant Staphylococcus aureus (MRSA) infective discitis were treated with VA-PLGA intra-discal injection. Meanwhile, VA intravenous injection, blank PLGA microspheres intra-discal injection and normal saline injection served as controls. Thirty days later, therapeutic effects were evaluated through X-ray radiophotography, histopathological, immunohistochemisty and bacteriological examination.ResultsThe morphology and the particle size distribution of the vancomycin microspheres were good, smooth wiehout accretion and with a narrow particle size distribution;The average drug loading was 20.08±0.53%, the average drug encapsulation rate was 60.20±1.61%, The CH2Cl2 residue was 52.69±1.34ppm, lower than the standard of CP 2005. The cumulative drug release ratio was 82.4±2.95% within thirty days. The vancomycin released by the microspheres within 30 days can maintain an therapeutic concentration to disctis.In the experiment group, The intervertebral disc inoculated with bacteria developed pathological evidence of discitis after two week.The levels of CRP and ESR increased obviously,The radiographic evidence of discitis has been observed by MRI, the bacteria culture was found positive, and the pathological evidence of discitis was found. Comparing with ESR,The peak of CRP occurred earlier, and the decline of the peak was faster and more regular. In the control group,There could not be found any sign of discitis,the bacteria culture was found negative. The method for the construction of this modle can easily manipulated,with highly succesful ration, similar pathological changes and easily duplicated,which was fit for surther pathology and pharmacodynamics work.The result of in vivo experiment showed that the inflammatory reaction in the VA-PLGA intra-discal injection group was milder than the intravenous injection group (P＜0.05), and the bacterial count was also significantly lower (1.02×103±1.22×103 CFU/g) than the intravenous injection group (7.51×104±7.16×104 CFU/g) (P＜0.05).CouculsionsThe vancomycin loaded PLGA microspheres prepared by double emulsion technical has a rather good pharmaceutic characteristic, which could maintain 30 days therapeutic effects in vitro.The spondylodiscitis model can be established by injecting Methicillin resistant staphylococcus aureus into the intervertebral disc of rabbits. The spondylodiscitis model was steady and the inflamaition was evident.it may be applied as an experimental model similar to the discitis of human on pathology,surgery and medication.MRI examination was a important and relative sensitive medical imaging methodt o find discitis. Spondylodiscitis should be reasonably considered if serm CRP levels are persisting elevated or consequential rise after spinal surgery.Comparing with ESR, CRP values show the advantage of good sensitivity and acuuracy in the nomalization time during the postoperative period. It should be used as the important and useful indicative sign in the prompt diagnosis of this disease.Intra-discal injection with vancomycin loaded PLGA sustained-release microspheres can effectively cure infective discitis, and the therapeutic effect is superior to that of intravenous injection.