| Background:Osteomyelitis is characterized by extensive bone defects and severe infection.As we all know,we can’t promote bone healing until the infection is controlled.So it is particularly important to reduce infection.At present,the main clinical treatment for patients with infectious osteomyelitis is to implant antibiotic-loaded bone cement after thorough debridement.Remove bone cement and perform bone grafting after infection control.This treatment has cured some patients.But its non-degradability attracts the attention of scholars from all over the world.With the development of tissue engineering,a variety of novel biodegradable materials with antibacterial and osteogenic functions have been developed.In the past,our laboratory has invented anti-infective reconstituted bone xenograft(ARBX),It uses deproteinized bovine bone as the carrier to adsorb bovine bone morphogenetic protein(b BMP)into the pores of xenogeneic bone through negative pressure to prepare reconstituted bone xenograft(RBX),and then loads antibiotics on it through gelatin drug loading technology.ARBX has been successfully used in clinical treatment of infectious osteomyelitis,open fracture and other diseases.After the follow-up of patients,it can be proved that ARBX has achieved good therapeutic effect,which can inhibit bacterial infection and promote the growth of new bone.Now,bacterial resistance is becoming more and more serious.Clearly,gentamicin cannot cope with these bacteria.Therefore,we try to combine ARBX with antibiotic-loaded bone cement to treat osteomyelitis.ARBX avoids the disadvantages of antibiotic-loaded bone cement,such as non-degradability and non-osteogenic activity.We believe that it can become a treatment that can replace bone cement in the future.Purposes:1.The appropriate loading concentration of vancomycin was explored through cytotoxicity test,antibacterial test in vitro and osteogenesis test in vitro,and its antibiotic release time was measured in vivo.2.The rabbit femoral condylar osteomyelitis model infected by MRSA was constructed,and the model was scored according to the osteomyelitis scoring standard table.If the score was higher than 3,the model was considered successful.3.After successful modeling,the infected site was thoroughly debridement and implanted with a new ARBX,and then the therapeutic effect of the new ARBX on osteomyelitis caused by MRSA infection was verified by gross observation,imaging,histology and bacteriology.Methods:1.The vancomycin solution was heated in a water bath,and was divided into 35 ℃(control group),55 ℃,65 ℃ and 75 ℃ groups according to the heating temperature.Then,the K-B method was used to verify whether vancomycin can withstand continuous high temperature heating;A new ARBX with different vancomycin loading concentrations was constructed.The vancomycin concentrations were divided into 40mg/ml,50mg/ml and60mg/ml to explore the maximum drug loading concentration of vancomycin by gelatin loading and packaging method;According to the previous experiment,the maximum loading concentration of vancomycin for the new ARBX was determined to be 40mg/ml,and the concentration gradient was set as the maximum loading concentration,which was divided into 0mg/ml,10mg/ml,20mg/ml and 40mg/ml groups,respectively.The extracts of each group were extracted and co-cultured with mouse preosteoblasts(MC3T3-E1),The cytotoxicity test and PCR test were used to verify whether different vancomycin loading concentrations had cytotoxicity and whether they affected the activity of b BMP;According to the previous experiment,the loading concentration of vancomycin was determined to be 40mg/ml to construct a new ARBX,and then the loaded and non-loaded b BMP groups were set respectively.The extract of each group was extracted separately to verify whether b BMP had an effect on the activity of vancomycin by K-B method;The new ARBX was implanted into the muscle bag of rats,and the tissue around the implant was taken on the 1st,3rd,7th,14 th and 21 st days to detect the concentration of vancomycin,so as to determine the release of the new ARBX in vivo,and the concentration of vancomycin in blood was detected by extracting the heart blood of rats.2.Drill a hole at the highest point of the lateral femoral condyle of the rabbit,and inject a certain amount of sodium morrhuate and MRSA bacterial solution respectively to induce osteomyelitis of the femoral condyle.Conduct gross observation,X-ray examination and bacteriological examination 4 weeks after the operation,and score the model animal through the Norden osteomyelitis scoring table.A score of more than 3points is deemed to be successful in the construction of the model,and the next experiment can be carried out.3.The rabbits were randomly divided into three groups: group A: RBX group,group B: ARBX group and group C: new ARBX group.After thorough debridement and implantation of materials in each group of models,the osteogenesis and antibacterial ability of the materials were evaluated by gross observation,bacteriological examination,histological examination and imaging examination of the specimens at 8 weeks after operation.Results:1.After the vancomycin solution was heated in the water bath at 55 ℃,65 ℃ and75 ℃,the diameter of the bacteriostatic ring produced by the bacteriostatic ring method was not significantly different from that produced by the 35 ℃ water bath heating group(P>0.05),indicating that vancomycin can withstand the high temperature heating at 75 ℃and can adapt to the high temperature heating process of the gelatin wrapping method in this experiment;When the antibiotic loading concentration of gelatin coating method is50mg/ml and 60mg/ml,gelatin can not effectively wrap RBX,and when the loading concentration is 40mg/ml,a new ARBX with good structure can be constructed;The absorbance values measured by cck8 method after co-culture of the extracts of each group and MC3T3-E1 cells showed that there was no significant difference between the groups with vancomycin loading concentration of 10mg/ml,20mg/ml,40mg/ml and 0mg/ml(P>0.05),indicating that there was no cytotoxicity when vancomycin loading concentration of 40mg/ml;After co-culture with MC3T3-E1,the expression of COL1,OPN and RUNX2 were detected by PCR and statistically analyzed.The results showed that there was no significant difference between the groups with vancomycin loading concentration of 10mg/ml,20mg/ml,40mg/ml and 0mg/ml(P>0.05),indicating that the loading concentration of vancomycin at 40mg/ml did not affect the osteogenic activity of b BMP;There was no significant difference(P>0.05)in the diameter of the bacteriostatic ring between the new ARBX group loaded with b BMP and the new ARBX group not loaded with b BMP,indicating that b BMP did not affect the antibacterial activity of vancomycin;After the new ARBX was implanted into the muscle bag of rats,the drug concentration in the tissues around the implant was detected to be 641.31 on the 1st,3rd,7th,14 th and 21 st days respectively 238.42μg/ml、89.12μg/ml、56.21μg/ml、9.82μg/ml,and the concentration of vancomycin in the blood is extremely low,without organ toxicity.2.Thirty model animals were constructed,one died of anesthesia,two died of unexplained diarrhea,and one died of bacterial infection.The other animals survived.The X-ray examination results of living animals showed that soft tissue shadow increased,bone sclerosis and low density transparent absorption areas of different sizes.According to the Norden osteomyelitis scale,the degree of osteomyelitis of the living animals was scored,all of which were greater than 3 points.3.Norden osteomyelitis score of group C was lower than that of A and B,and there was significant difference(P<0.01);The BV/TV and BMD of group C were higher than those of group A and B,and there were significant differences(P<0.01);Bacteriological examination showed that the bacterial count in Group C was significantly lower than that in Group A and Group B(P<0.01).Conclusions:The antibacterial activity of vancomycin solution is not significantly affected at least75 ℃,indicating that vancomycin can withstand the heating process of gelatin drug loading technology,and the maximum drug loading concentration of vancomycin by gelatin drug loading technology is 40mg/ml.The new ARBX loaded with vancomycin at a concentration of 40mg/ml has no obvious cytotoxicity and the activity of vancomycin and b BMP does not affect each other.In vivo experiment proved that the new ARBX can release vancomycin locally and for a long time,and at the 21 st day,the concentration of vancomycin around the implant can still be detected to be 9.82 μg/ml,greater than the minimum inhibitory concentration of vancomycin 1μg/ml.Finally,the new ARBX can effectively treat chronic osteomyelitis caused by MRSA and promote osteogenesis while resisting infection.These results indicate that the new ARBX is expected to become an important method for clinical treatment of osteomyelitis in the future. |
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