Targeted Osteomyelitis Therapy With High Affinity Peptide Delivery System

Research BackgroundOsteomyelitis is a bacterial infection of bone characterized by progressive inflammatory bone destruction(osteolysis)with reactive bone formation and may involve a small portion or regions of any bone.The initial infection that causes osteomyelitis can be inoculated from the bloodstream of the pathogen to another part of the body,or directly through a wound or surgical wound.This results in an acute infection,usually lasting days or weeks,and may require antibiotics and/or surgical intervention.When the pathogenic microorganism continues for more than 10 days and causes further destruction of the bone,the infection is considered to be chronic osteomyelitis.Staphylococcus aureus(Sureus)is the single cause of acute and chronic osteomyelitis in children and adults,accounting for approximately 80%of these infections.Other microorganisms such as coagulase-negative staphylococci,streptococci,enterococci And M.tuberculosis may also cause osteomyelitis,but S.aureus is by far the most common bacterium,because virulence factors help it escape many host defenses.The number of bone infections has increased over the past few decades due to the increased number of prostheses and fracture fixation devices placed by orthopedic surgeons.Although improvements in surgical techniques and active antibiotic prophylaxis have reduced infection rates after orthopedic implantation to less than 5%,osteomyelitis remains a serious problem.Approximately 50%of clinical isolates are resistant S.aureus strains,such as methicillin-resistant Staphylococcus aureus(MRSA),which are commonly obtained in hospital and community settings.In addition,MRSA has surpassed HIV,becoming the deadliest pathogen in North America and continues to make the treatment of chronic osteomyelitis more difficult.Current estimates of MRSA infection around the prosthesis of the second revision surgery indicate that the reinfection rate is approximately 15%-25%.However,these numbers may actually be higher because they do not take into account the proportion of patients who are unable to undergo revision surgery due to persistent infections,and these patients are not allowed to re-implant the device.This suggests that there is a major need for alternative intervention strategies,particularly for immunocompromised individuals(ie,patients with diabetes or HIV infection),patients taking immunosuppressive drugs,and a combination of most undergoing total joint replacement.The elderly of the patient.There are approximately 112,000 infections associated with orthopaedic devices in the United States each year,and the cost per incident is approximately$15,000-70,000.Although the infection rates of joint prostheses and fracture fixation devices have been only 0.3-11%and 5-15%,respectively,over the past decade,these infections may result in amputation or even death.In addition,although unproven,the popularity of "Tender Minimally Invasive Surgery" for selective TJR,where very small incisions may lead to complications of prosthesis contact with the skin during implantation,is associated with a significant increase in the incidence of osteomyelitis.These infections require very expensive second-stage revision surgery,and recent reports indicate that the success rate may be as low as 63%.Currently,prophylactic treatment of MRSA infections that can be used to prevent patients undergoing TJR surgery is preoperative antibiotics,such as vancomycin.However,excessive use of these "final" antibiotics has led to the emergence of strains that are resistant to even our most effective antibiotics.Therefore,immunocompromised and elderly patients must have access to alternative intervention strategies that together account for the majority of the 1.5 million TJR per year in the United States.Therefore,a vaccine that reduces the incidence of MRSA by 50-80%can reduce the number one complication of joint replacement and open fracture repair surgery,and can also reduce the similar amount of medical burden.Based on more than 150 years of research,clear examples have emerged that explain the pathogenesis of microbes.This model is also applicable to osteomyelitis.The initial step in infection occurs when a single bacterium invades the body.At this point,the microorganism must respond to environmental changes and express a virulence gene that will help it resist innate immunity and provide it with an adhesin receptor attached to the host.Microorganisms also rely on the presence of host structures from necrotic tissue or foreign bodies such as implants.Successful completion of these steps results in an exponential growth phase that stops when nutrient depletion and/or adaptive immunity develops.After the exponential growth phase,bacteria are forced to persist under dormant growth conditions in a complex extracellular matrix called a biofilm.At this point the infection is now chronic and cannot be eradicated by drug or host immunization.Due to the importance of initial attachment,the focus of this field has been on cell surface adhesins,which specifically interact with extracellular matrix components,called MSCRAMMs(microbial surface components that recognize adhesion matrix molecules).In fact,most of the anti-S.aureus targeted therapies developed to date target MSCRAMM,which is important for colonization and invasion of host tissues.The purpose of these vaccines is to produce antibodies that block attachment to host tissues by binding to adhesins.Unfortunately,S.aureus has many adhesins,so inhibition of one adhesin may not be sufficient to prevent bacterial adhesion.The aim of this study was to find a new S.aureus-targeted binding site for S.aureus,and to clarify its binding mechanism,and to artificially synthesize short peptides to modify antibiotics such as vancomycin in order to reduce the dose of antibiotics in the treatment of osteomyelitis.,shorten the course of treatment,improve the efficacy and other results.Purpose1.Identify antigens used by osteomyelitis for targeted therapy in vivo.2.Obtain a specific binding site for the antigenic antibody.3.Follow-up treatments.Method1.Establishment of an implant-associated osteomyelitis mouse model We used 6-9 weeks old female SPF C57BL/6 wild type mice as an experimental subject.After anesthesia with ketamine(100 mg/kg)and xylazine(10 mg/kg),the fracture model was made after the middle tibia osteotomy.The infection was initiated by penetrating the tibial plateau perpendicular to the tibial plateau(the needle was pre-soaked with green-fluorescent S.aureus and dried for 20 minutes to form a biofilm).Carefully suture the skin to avoid additional environmental exposure.Once the mice recover from anesthesia,they will be returned to the standard isolation cage without additional treatment before the next experiment begins.2.Extraction of total protein from Staphylococcus aureus and screening and amplification of specific phage and short peptides.To identify potential protective antigens,pre-immune and convalescent sera were used as primary antibodies in Western blots of total S.aureus protein extracts.A significantly increased band of intensity was observed in the convalescent sera,indicating that these antigens are involved in a protective immune response in some way.3.In vovo experimentsIn vivo experiments in mice showed that the selected short peptides can specifically recognize S.aureus in the osteomyelitis model,but if S.aureus itself lacks GMD antigen,the selected short peptides are not recognized.Its ability to target the binding of MRS A at the lesion is shown.Result1,Successfully developed a mouse model of infection after fracture internal fixation.2,from the two-dimensional protein electrophoresis gel results,half of the area has color development,of which 138-kDa Staphylococcus aureus autolysin(GMD)is most obvious.We sequenced the phage with higher binding ability and screened 15 specific binding sequences.The specific binding number of TRAP2 and AMD1 was the highest specific antigen binding site.3,mouse in vivo results show that the selected short peptide can specifically identify S.aureus in the osteomyelitis model,but if S.aureus itself lacks GMD antigen,the selected short peptide is not recognized.Its ability to target the binding of MRSA at the lesion is shown.Conclusion1.The C57BL/6 mouse osteomyelitis model can be formed in about 14 days;2.S.aureus does have a highly conserved protective antigen(GMD)that is stably bound;3.Vancomycin modified by a polypeptide synthesized against its protective antigen binding site is more effective than normal therapy.