Experimental Investigation Of Ultrasonic Debridement Combined With Polihexanide In Acute Local Osteomyelitis

BackgroundWar trauma and high-energy injury usually cause contaminated open fractures and lead to secondary infections easily.Early radical debridement is the key to successful treatment and the basis of other therapeutic methods.The widely used debridement method is sharp surgical resection and irrigation with isotonic sodium chloride solution or hydrogen peroxide and diluted povidone iodine,which have been used for many years.The effect of this kind of debridement is reliable for bacterial contaminated wound,but is questionable for the wounds in which the bacteria have been colonized and infection has occurred.In recent years,the debridement techniques have been developed and new instruments and new method,including high-pressure pulsatile lavage,low-pressure pulsatile lavage,and VersaJet System have been reported.But their damage to soft tissue and bone is questionable.There is still controversy about whether to use low mammalian toxicity antimicrobial substances or normal saline in irrigation during debridement in acute contaminated wounds.With a kind of unique energy release method(cavitation),low frequency ultrasonic debridement(LFUD)has been used in the treatment of various of acute and chronic trauma wound infection,but the mechanism of reduction of bioburden of infected tissue and the possibility of direct sterilization are not clear.All these need further research.Objectives1.To investigate bactericidal action of low frequency ultrasound and polihexanide(PHMB)to clear and destroy in vitro biofilm and planktonic Staphylococcus aureus alone and their synergistic effects;2.To develop a new reliable experimental local bone infection model of rabbit;3.To investigate the therapeutic effect of low frequency ultrasonic debridement combined with PHMB in rabbit proximal tibial infection after 48 h of staphylococcus aureus incubation.MethodsPart I(1)Plantonic Staphylococci aureus grown in Luria-Bertan liquid medium were exposed to low frequent ultrasound with different time grading(0 seconds,5 seconds,10 seconds,20 seconds,60 seconds,120 seconds,300 seconds,600 seconds).And after that the bacteria in LB liquid media were evaluated quantitatively by plate counting method and assessed qualitatively by turbidity observation of fluid medium culture.(2)Planktonic staphylococcus aureus cultured in LB fluid media were exposed to different concentration gradient media(1:100、1:200、1:500、1:100、1:1000、1:2000、1:5000、1:10000、1:20000、PBS solution)for one hour and perform quantitative detection of bacteria respectively.(3)Planktonic staphylococci aureus cultured in LB fluid media were exposed to low frequent ultrasound for 30 seconds and to different concentration gradient media(1:100、1:200、1:500、1:100、1:1000、1:2000、1:5000、1:10000、1:20000、PBS solution)for one hour and perform quantitative detection of bacteria respectively.(4)Bacterial biofilms of staphylococci aureus were developed at the bottom of cell culture plate by every other day fluid LB media exchange.Then the biofilms were exposed to different concentration gradient media(1:10,1:100,1:200,1:500,1:1000,1:2000,1:5000,、PBS control)for one hour.Then the quantitative determination of bacteria of each group was performed by plate counting method.(5)Bacterial biofilms of staphylococci aureus were developed at the bottom of cell culture plate by every other day fluid LB media exchange.Then the biofilms were exposed to low frequency ultrasonic irradiation for 30 seconds combining with different concentration gradient media(1:10,1:100,1:200,1:500,1:1000,1:2000,1:5000,、PBS control)for one hour.Then the quantitative determination of bacteria of each group was performed by plate counting method.(6)Bacterial biofilms of staphylococci aureus were developed at the bottom of cell culture plate by every other day fluid LB media exchange.The wells in the culture plate were divided into 4 groups and 9 wells in each group.The control group,US group,PHMB group and PHMB-US group were exposed to normal saline,ultrasonic treatment,0.04%PHMB,and ultrasonic treatment with0.04%PHMB,respectively.Then the wells were stained with crystal violet.The gray value of each wells’ photograph were determined and compared between groups.Part II(1)Inoculation fluid of staphylococcus aureus were prepared.Log10(CFU/ml)of bacterial normal saline suspension was 8.(2)The small pieces of bone harvested from rabbit were immerged in LB medium containing Staphylococcus aureus for 48 hours.The bacterial numbers of each bacteria-preloaded bone were determined by method of plate culture counting.The result of average counting of log10(CFU/piece)was 6.9±0.5。(3)20 rabbits were divided into two groups(Group A and Group B)randomly and 10 for each group.The rabbits in Group A and Group B were inoculated with bacterial suspension and biofilm preloaded bone respectively.The infection success rate,mortality,body temperature,CRP,presentation of wound,radiograph and histological examination were evaluated.Part III(1)Small pieces of bone harvested from two rabbits were implanted into LB medium containing Staphylococcus aureus for 48 hours.(2)50 male adult New Zealand rabbits were divided into five groups randomly.All the animals were inoculated with bacteria preloaded bone at the right proximal tibia.48 hours after the inoculation,rabbits in special groups were treated according to each treatment arrangements respectively.(3)Group NaCl underwent debridement followed by normal saline irrigation;group US underwent ultrasonic debridement combined by normal saline irrigation;group PHMB underwent debridement followed by 0.1% PHMB solution irrigation;group PHMB-US underwent ultrasonic debridement combined by 0.1% PHMB solution irrigation;group iodophor underwent debridement followed by 0.1% iodophor irrigation.(4)The body temperature and weight were recorded regularly.At the end point of this experiment,six weeks after treatment,wound appearances,radiograph appearances and pathological scores were compared between groups.ResultsPart I(1)The bacterial counts of planktonic bacteria were not significantly changed after exposure to different grading time of low frequency ultrasonic treatments.(2)For different dilution gradients of PHMB,In the diluted 1/200 groups,all the bacteria were killed.(3)After low frequency ultrasonic treatment combined with different dilution gradients of PHMB,the reduction of planktonic phenotype staphylococcus aureus showed significant concentration dependent.But this time,in the diluted 1/2000 groups,all the bactieria were killed.(4)After the treatment of 0.04%PHMB and ultrasonic treatment separately,the gray values of crystal violet of the two groups were higher than control group.And the combination of the 0.04%PHMB and ultrasonic treatment together,the increase of gray value were the highest.The variance analysis showed that there was a synergistic effect between 0.04%PHMB and ultrasonic treatment.Part II(1)Logarithm of the average number of bacteria loaded in the bone as inoculum was 6.9±0.5.(2)In group A,the postoperative body temperature fluctuation was greater than that of rabbits in group B.There were 3 animals died in group A,while all the animals in group B survived during 4 weeks of experiment.There was no statistically difference of body weight between the two groups.There were 5 animals in group A developed chronic osteomyelitis and 10 in group B.The X-ray manifestation of group A were intramedullary type,while that of group B were localized type.The CRP detection results in group A were higher than that in group B in the early postoperative period.Chronic infection can be found in the pathological examinations in both groups.Positive culture could be found in 5of 7 animals in group A,and all animals in Group B.Part IIIAll the animals survived.after incubation over 48 hours.There were one rabbit died in NaCl Group,PHMB Group,PHMB+US Group and,Iodophor Group each and two rabbits died in US Group.There were no significant differences of body temperature and weight after operation between groups.PHMB+US group was better than other groups in the local wound scores,radiography visual scores and pathological scores.Most scores between other groups were not significantly different.Conclusions1.Low frequency ultrasound has little effect on Staphylococcus aureus in vitro.2.PHMB has biocide effect on Staphylococcus aureus(both planktonic phenotype and biofilm).The higher concentration of it,the better bactericidal effect it has.But the biofilm of Staphylococcus aureus was more resistant to PHMB.3.For planktonic phenotype or biofilm phenotype of staphylococcus aureus,PHMB and low frequency ultrasound have synergistic effect in bactericidal action.4.Experimental osteomyelitis can be developed by incubation of biofilm preload bone.This method has the characteristics of high success rate,mild systemic symptoms and low mortality.It can simulate the human traumatic localized osteomyelitis.5.The application of ultrasound alone can not effectively control the infection of early localized bone infection.While ultrasonic treatment combined with PHMB is helpful to control early localized bone infection.