The Treatment Of Chronic Osteomyelitis With Bone Defect By Liposomal Gentamicin-impregnated Substitution Of Bone

Both bacterial biofilms and floating bacteria are two forms of bacterial growth. The bacterial biofilms can formed as surface-bound organisms begin to actively replicate (and die) and extracellular components generated by attached bacteria interact with organic and inorganic molecules in the immediate environment to create the glycocalyx. Biofilm organisms are far more resistant to antimicrobial agents than organisms in suspension. In some extreme cases, the concentrations of antibiotics required to achieve bactericidal activity against adherent organisms can be three to four orders of magnitude higher than for planktonic bacteria, depending on the species-drug combination, which is the main reason of the recrudescence of chronic osteomyelitis. Once bacteria have irreversibly attached to a surface of bone, dead bone, internal fixation or artificial joint, the process of biofilm maturation begins, and the infection of bone is recurrent attacks, which bring more pain to the patients. Polymethylmethacrylate(PMMA) and Osteoset T can delayed release antibiotics in the infected bone, and improve the healing rate. However, PMMA need to be taken out after operation. At the same time, the cure rate of implantation of Osteoset T after debridement is similar to debridement alone. In addition, it is indicated that high concentration of gentamicin can inhibit the proliferation of osteoblast and endothelial cell, which can inhibit the union of fracture.Liposomes are the vesicles which are formed from the arrangement of phospholipid during the dispersion in the water. Liposomes can encapsule antibiotics and are devided to cation, anion and neutral liposomes. It is indicated that the inhibited effect of the penicillin encapsulated in the cation liposomes to the Staphylococcus aureus(S. aureus) biofilms is much better than the penicillin alone, and the mechanism is related to the negative charge of the surface of the S. aureus biofilms. On the other hand, liposomes can make the drug gathered in the bone marrow, which make it possible that infection of bone is eliminated by intravenous injection of antibiotics impregnated in the liposomes. At the same time, liposomes can reduce the toxic effect of high concentration antibiotics to the osteoblast and the endothelial cell significantly. However, the reticuloendothelial system (RES) of human body can eliminate the liposomes in the blood, which induces the half life period of the liposomes and weakens the anti-infection ability of the liposomal antibiotics. Due to the delayed release of high concentration antibiotics of antibiotics delayed released system in the area of bone infection, the weaken of RES to the liposomal antibiotics can be solved by impregnating the liposomal antibiotics to the carrier of antibiotics delayed released system. However, it is unknow whether the anti-biofilm ability of the liposomal antibiotics released from the antibiotics delayed released system is affected. The cation liposomal gentamicin was prepared in our reasearch firstly. Then the cation liposomal gentamicin was impregnated in allogeneic cortical bone of rabbit or calcium sulfate, and the release experiment was carried out. The anti-biofilm effect of cation liposomal gentamicin released from the substitution of bone was explored. During the preparation of the mode of chronic bone infection with bone defect, the affection of the injected dose of S. aureus ATCC29213 to the formation of chronic osteomy elitis with bone defect. In addition, the treatment of injection of three kinds of liposomal gentamicin to the chronic bone infection with bone defect of rabbit was also done.The study includes four parts. Part one: the affection of the injected dose of S. aureus ATCC29213 to the formation of chronic bone infection with bone defect of rabbit. Part two: the treatment of intravenous injection of liposomal gentamicin to the chronic bone infection with bone defect of rabbit. Part three: the preparation of cation liposomal getamicin antimicrobial bone and the effect of anti-biofilm in vitro. Part four: the treatment of cation liposomal antimicrobial bone to the chronic tibial infection with bone defect of rabbit.Main experiments show below:Part one: the affection of the injected dose of S. aureus to the formation of chronic bone infection with bone defect of rabbit.Methods:1 S. aureus(ATCC 29213) with total number of colony from 6×10¹ to 6×10⁶ was prepared. The bacteria were injected in the tibia after an area of bone defect was made. 2 The inoculated lesions were assessed for changes in macroscopic observation, radiological and bacteriological parameters at 3 weeks after infection.Main results:Obvious infection sign can be seen in the E and F groups due to the macroscopic observation, radiological and bacteriological parameters. There is significant difference between both E, F groups and other groups. However there is no difference between E and F group.Part two: the treatment of intravenous injection of liposomal gentamicin to the chronic bone infection with bone defect of rabbit.Methods:1 The preparation of different liposomal gentamicinThree liposomal compositions were mixed in a round-bottomed flask and dried to a thin lipid film with a rotary evaporator. The lipids were then hydrated by agitation in aqueous solution of gentamicin. Then the lipid suspensions were sonicated for 40 s in an ultrasonic bath. Unencapsulated gentamicin was removed by centrifugation. The final pellets were resuspended in distilled water.2 The preparation of chronic tibial infection with bone defect of rabbit.After anesthetized, the tibial skin of rabbit was cut off, and the tibia was exposed. The bone defect was made and sealed with bone wax. The ATCC 29213 was injected in the bone defect. Treatment was done till the infection formed through the macroscopic and radiological observation.3 The treatment of liposomal gentamicin to the chronic tibial infection with bone defect.The treatment with injection of liposomal gentamicin began after the chronic tibial infection with bone defect of rabbit was made successfully. The rabbit was executed at 3m after the treatment and the macroscopic observation, radiological and bacteriological examinations were done.Main results:The bacterial culture of blood was posive in A group, and all negative in other groups. The bacterial culture of tibia was posive in A, C, E groups, 4 negative in B group, and 2 negative in D group. Part three: Preparation of liposomal gentamicin-impregnated allogeneic cortical bone and the effect of antin-biofilm in vitro.Methods:1 Preparation of the cation liposomal antimicrobial boneThe preparation of the cation liposomal gentamicin is the same with part two. The metaphysic of tibia and femur was obtained, defated in mixature of chloroform and methanol, deproteinized in H₂O₂, and decalcificated in HC1. The production was observed by scanning electron microscope. The allogeneic cortical bone and calcium sulfate were impregnated in the cation liposomal gentamicin, evaporation to dryness, and sterilized by irradiation of ⁶⁰Co.2 Release experiment of cation liposomal antimicrobial bone in vitroSpecimens of liposomal gentamicin-impregnated allogeneic cortical bone and CS were immersed in dd H₂O on a shaking device. The water was replaced at different time points. The replaced water at different time points was added Triton X-100. After rupturing the liposomes, gentamicin concentrations were measured using an AxSym System.3 Release experiment and regrowth assayLiposomal gentamicin released at eight time points during the release experiment was chosen to detect the effectiveness of anti-biofilm which was assessed from the rate of regrowth of bacteria from the biofilms after exposure to the cation liposomal gentamicin, relative to regrowth after exposure to an equivalent concentration of free drug or liposomal gentamicin prepared freshly. After exposure of the biofilms to the test samples the plate wells were washed with PBS and growth medium was added and the absorbance of the growth medium was measure at 30 min intervals at 630nm over a period of 24h. The plate reader was programmed to read the optical density vs. time profile and determine the time to reach maximum growth rate(Dod/dt)max. These times were compared with those from growth of biofilms exposed to only nutrient broth(positive controls).Main results:The combination of liposomal gentamicin and allogeneic cortical bone showed initial burst-release of active liposomal gentamicin and had continuous-release. Liposomal gentamicin released from allogeneic cortical bone had the same anti-biofilm activity with the liposomal gentamicin prepared freshly. Meanwhile, both agents were more effective relative to free gentamicin at low drug concentration.Part four: The treatment of osteomyelitis with bone defect by liposomal gentamicin-impregnated allogeneic cortical bone or calcium sulfateMethods:1 The preparation of the cation liposomal antimicrobial bone and the chronic tibial infection with bone defect was the same with part three.2 After the preparation of the chronic tibial infection, rabbits were treated with intravenous injection of liposomal gentamicin or debridement with implantion of the cation liposomal antimicrobial bone. At 2w after treatment, radiological and bacteriological examinations were done.Main results:Therapeutic trials with antibiotics given intravenously revealed that free gentamicin for 14 days was ineffective in sterilizing bone. Treatment with cation liposomal gentamicin for 14 days resulted in recovery of 33.3% of treated animals, which was lower slightly than the result treated with implantation of gentamicin-impregnated calcium sulfate(66.7%). Complete sterilization of bone tissues on cultures (100% cure) was obtained only in the group of liposomal gentamicin anti microbial bone treated for 14 days.Conclusions:1. Development of significant histological and radiological signs of infected bone defect required an inoculum of at least 6×10⁵ CFU/5 ul.2. Cationic liposomes change the pharmacokinetics of gentamicin which can improve the concentration of gentamicin in the bone marrow. Cationic liposomal gentamicin was most effect to cure the chronic osteomyelitis with bone defect among the three liposomal gentamicin.3. The anti-biofilm effciency of liposomal gentamicin was not affected by the impregnating process. Compared with free drug, liposomal gentamicin is very effective in inhibiting the regrowth of cells from S. aureus biofilms at low drug concentration. 4. The new drug delivery system was effective in preventing biofilm infection in a contaminated defect, and it could also be used clinically for bacterial infections in the conditions like plaque formation or in arresting biofilm formation in the implanted devices or dead bone of osteomyelitis.