| ObjectiveChronic osteomyelitis is a common orthopedic disease,which is difficult to treat and prone to recurrence,and its treatment problem is always a difficult problem in the clinic.At present,the clinical application of high-dose antibiotics for chronic osteomyelitis patients,but the effect is not ideal,and is prone to toxic side effects,drug resistance,pathogenic bacteria mutation and other problems.Ozonated water has a strong bactericidal effect,and negative pressure suction can create a closed healing environment,so ozone antibacterial and negative pressure suction therapy have good prospects for application in the treatment of chronic osteomyelitis.In this study,we investigated the effect and mechanism of ozone-water infusion and negative pressure suction in the treatment of chronic osteomyelitis by constructing a rat femur model of chronic osteomyelitis and ozone-water sterilization experiment,which is expected to provide reference for the formulation of clinical treatmentplan.MethodsThis study was divided into three parts.The first part is the experiment of constructing the model of chronic osteomyelitis,applying saline group(group A)and three concentrations of Staphylococcus aureus bacterial suspension 0.1mL3×105cfu/mL(group B),0.1mL3×106cfu/mL(group C),0.1mL3×107cfu/mL(group D)to construct the rat femur chronic osteomyelitis The model was used to explore the method of constructing chronic osteomyelitis by comparing the general conditions,inflammatory factors tumor necrosis factor(TNF-α),interleukin 1β(IL-1β),interleukin 6(IL-6)levels,imaging scores,anatomical observation of the femur,and histological analysis of the four groups,in preparation for further therapeutic experiments.In the second part,ozonated water bactericidal experiments were conducted to investigate the killing function of ozonated water on common pathogenic bacteria of osteomyelitis by observing the changes of ozonated water concentration under different temperature conditions,neutralizer experiments and the bactericidal function of ozonated water at different concentrations on S.aureus,P.aeruginosa and Escherichia coli,taking three common pathogenic strains of osteomyelitis as examples.The third part is the ozone water infusion of negative ozone water.The third part is the investigation of the effect and mechanism of ozonated water perfusion negative pressure suction in the treatment of chronic osteomyelitis in the femur of rats,with blank group,model group,control group and study group.The rats in the blank group were not modeled and did not take any treatment measures,and were free to ingest food and water;the model group was treated with saline for wound irrigation on the basis of chronic osteomyelitis of the femur;the control group was treated with gentamicin injection for perfusion of negative pressure suction on the basis of chronic osteomyelitis of the femur;the study group was treated with ozonated water for perfusion of negative pressure suction on the basis of chronic osteomyelitis of the femur.By comparing the gross condition,inflammatory factor expression level,trauma p H,imaging examination score,femoral anatomy observation,bacterial culture,histological detection,ERK,p38MAPK,JNK protein level of the four groups,the effect and molecular mechanism of ozonated water perfusion negative pressure suction treatment for chronic osteomyelitis were comprehensivelyevaluated.Results(1)No chronic infection occurred in group A rats after the application of saline,and in group B after the application of 0.1 mL of 3×105 cfu/mLSA infection,the inflammatory response was quickly diluted,the levels of TNF-α,IL-1β,IL-6 were low,the imaging score level of the inflammatory response score observed by the naked eye was low,the infection rate was 66.67%,and the results of histological analysis showed that the chronic infection was mild and could not meet the chronic osteomyelitis model construction needs.In group C,after applying 0.1mL of 3×106cfu/mLSA infection,the inflammatory response was maintained for a longer period of time,the general condition,imaging score,sarcoid score,and histological score were all high,the infection rate was100.00%,and no death occurred,which met the need of chronic osteomyelitis model construction.In group D,after applying 0.1 mL of 3×105 cfu/mLSA infection,the chronic osteomyelitis model was successfully constructed with an infection rate of100.00%,but the degree of infection was too severe and the mortality rate was high(33.33%),which could not meet the experimental needs.(2)The stability of ozonated water is temperature-dependent,and as the temperature increases,the ozonated water concentration decays faster and the half-life decreases.Application concentration1.0mg/L ozone water 2min,or application concentration 2.0mg/L ozone water 1min,can completely kill Staphylococcus aureus(SA);application concentration 1.0mg/L ozone water 2min,or 2.0mg/L ozone water 30s,can completely kill Pseudomonas aeruginosa;application concentration 1.0mg/L or 2.0mg/L ozone water for 30s,which can completely kill E.coli.(3)After treatment,the rats in the blank group showed good growth,the model group showed obvious chronic osteomyelitis,and the rats in the control and research groups showed improvement in chronic osteomyelitis,with the most obvious improvement in the research group.After 1 week and 2 weeks of treatment,the comparison of TNF-α,IL-1β,IL-6,trauma p H,imaging score and infection rate of the four groups of rats were:blank grou P<study grou P<control grou P<model group,and the difference was significant(P<0.05);femoral anatomical score and histological analysis showed that the blank group had no inflammatory reaction,the model group had severe inflammatory reaction,and the control and model groups had inflammatory reaction were improved,among which the improvement was more obvious in the study group,and the inflammatory response in the study group was lighter than that in the control group.After 2 weeks of treatment,the comparison of ERK,p38MAPK,and JNK protein expression in the model group,control group,and study group was blank grou P<study grou P<control grou P<model group,and the difference was significant(P<0.05).Conclusion(1)Apply 5%sodium morrhuate and 0.1ml3×10~6 CFU/ml Staphylococcus aureus suspension can construct an ideal rat model of chronic osteomyelitis of femur,simulate the condition,and provide a strong guarantee for further clinical treatment research.Gross observation,imaging examination,femoral anatomical observation,bacterial culture and histological analysis are helpful to accurately evaluate the construction of rat femoral chronic osteomyelitis.(2)Ozone water is unstable,and its concentration will be affected by experimental temperature and time.Therefore,when using ozone water for experiments and treatment,attention should be paid to the application of fresh ozone water,and the application temperature of ozone water should be reduced as far as possible.Under room temperature conditions,it should be applied within 20min of ozone water.In addition,this experiment verified that ozone water can kill the three main pathogens of chronic osteomyelitis,and the killing effect of 2.0mg/l ozone water for1min on the three main pathogens of osteomyelitis can reach 100%.(3)The treatment of chronic osteomyelitis of the rat femur with ozonated water infusion negative pressure suction can effectively control the infection,inhibit the inflammatory response,,and its effect is better,which can be an effective method for the treatment of chronic osteomyelitis.The possible molecular mechanism of ozone-water perfusion negative pressure suction therapy is down-regulation of ERK,p38MAPK,and JNK protein expression,thus reducing the inflammatory response. |
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