| ObjectiveThree antibacterial agents(antibiotics)including vancomycin,gentamicin sulfate and tigecycline were put into 10 % furan-based bone cement in order to overcome the lack of releasable antibacterial properties of the bone cement.The antibacterial properties,biocompatibility and mechanical properties of the obtained new furan-based bone cement loaded with antimicrobial agents were studied to provide experimental basis for clinical use.Methods(1)Experimental grouping and drug loading dose of each group: 10 %furan-based bone cement was mixed with vancomycin,gentamicin sulfate and tigecycline according to the ratio of 40 g:1 g(2.5 wt%),40 g:2 g(5 wt%),40 g:4 g(10 wt%)were mixed in the ratio,divided into vancomycin group(2.5 wt%,5 wt%,10 wt%),gentamicin group(2.5 wt%,5 wt%,10 wt%)and tigecycline group(2.5wt%,5 wt%,10 wt%),a total of 9 groups.Then supplement the corresponding control group according to the needs of the experiment.(2)Bone cement standard specimen and preparation of extraction solution: Mix corresponding doses of antibiotics with 10 % furan-based bone cement solid-phase powder according to the above groupings and prepare a disc-shaped bone cement standard specimen(Φ 6*3 mm)for antibacterial For performance experiments,a cylindrical bone cement standard specimen(Φ 6*12 mm)was prepared for mechanical performance experiments.Immerse the cylindrical specimen in cell culture medium or physiological saline,wherein the cell culture medium extract is used for cell proliferation and toxicity experiments,and the physiological saline extract is used for acute toxicity tests.(3)Antibacterial performance test: detect the antibacterial performance of each group of test pieces through drug sensitivity test,put each group of test pieces into Staphylococcus aureus or Escherichia coli MH agar medium,change the medium regularly every day,and observe continuously for 7 days,measure and record the diameter of the antibacterial zone around the specimen to judge the antibacterial performance of the new bone cement.(4)Biocompatibility test:(1).Cell proliferation and toxicity test(in vitro test): By the CCK8 method,the cell culture medium extracts of each group were co-cultured with MC3T3-E1 osteoblasts.Observe and record the morphology,OD value and relative proliferation rate of osteoblasts on the 1st,3rd and 5th day to judge the in vitro biocompatibility of the new bone cement;(2)Acute toxicity test(in vivo test):The physiological saline extracts of the three groups with the highest antibiotic content(10 wt%)were injected into the peritoneal cavity of C57 mice,and the general state and toxicity of the mice were observed before injection and on the 1st,2nd,and3 rd day after injection.On the third day,the mice in each group were sacrificed,and the liver and kidney samples were taken out for pathological observation to judge the in vivo biocompatibility of the new bone cement.(5)Mechanical properties experiment: The compressive strength of the 2.5 wt%group and 10 wt% group specimens was measured.Start the material testing machine to make a curve of deformation versus load for the cylindrical specimen.According to the ISO 5833 standard,whichever of the "failing strength","2% offset strength" and "upper yield strength" occurs first is considered to be the compressive strength.The compressive strength of each group of test pieces was recorded to judge the mechanical properties of the new bone cement.Result(1)In the drug sensitivity test for 7 consecutive days,no zone of inhibition was produced in the furan-based bone cement group of the blank control group,no zone of inhibition was produced in the vancomycin group against Escherichia coli,and the zone of inhibition was produced in the other groups,proving that All three antibiotics could be released from 10% furan-based bone cement.The diameter of the inhibition zone produced by each group of specimens was positively correlated with the amount of antibiotics added,and became larger with the increase of the amount of antibiotics added(P<0.05).The diameter of the inhibition zone of each group of test specimens was the largest on the first day(P<0.05),indicating that the release antibacterial performance was the strongest in the first 24 hours.Among them,the diameter of the bacteriostatic zone of gentamicin group and tigecycline group in Staphylococcus aureus was larger than that of Escherichia coli(P<0.05),indicating that these two antibiotics have stronger antibacterial properties against Staphylococcus aureus.On the first day,the gentamicin group had the largest inhibition zone diameter against Staphylococcus aureus,followed by the tigecycline group,and the vancomycin group was the smallest(P<0.05);The diameter of inhibition zone against Escherichia coli in the gentamicin group was smaller than that in the tigecycline group(P<0.05).From the 2nd day to the 7th day,the diameter of the inhibition zone of the gentamicin group against Staphylococcus aureus and Escherichia coli was larger than that of the tigecycline group(P<0.05).According to the time of each group of test pieces showing low antibacterial activity(<10 mm)against bacteria,the antibacterial cycle of the gentamicin group was the longest.(2)In the cell proliferation and toxicity test(in vitro test),the morphology of bone cells in each group was good during the 1st,3rd and 5th days;the OD value of each group increased with time(P<0.05),reflecting the Osteocytes were in a state of proliferation;the tigecycline group showed no cytotoxicity at each time;the vancomycin group and gentamicin group showed no cytotoxicity on the first day,and mainly showed mild cytotoxicity from the third day Cytotoxicity,all showed slight cytotoxicity on the 5th day.In the acute toxicity test(in vivo test),the mice in each group showed good general conditions before and within 3 days after injection of the extract,without obvious toxic reactions.On the third day,the mice were sacrificed to take liver and kidney samples for pathological observation.It was found that the liver and kidney cells and tissues in each group had no obvious degeneration or necrosis,and no toxic changes.(3)In the mechanical property test,the compressive strength of the specimens in the vancomycin group,gentamicin group and tigecycline group was negatively correlated with the amount of antibiotics added,and decreased with the increase of the amount of antibiotics added(P<0.05),and the compressive strength of the gentamicin group was the highest(P<0.05),indicating that gentamicin sulfate had the least effect on the mechanical properties of 10% furan-based bone cement.Conclusion(1)Vancomycin,gentamicin sulfate and tigecycline can be effectively released from 10% furan-based bone cement and play an antibacterial effect.(2)The antibacterial performance of furan-based bone cement loaded with antimicrobial agents was positively correlated with the amount of antibiotics added.When 4 g of antibiotics were added to 40 g of bone cement,the antibacterial performance was the strongest,2 g was in the middle,and 1 g was the weakest.Compared with the vancomycin group,the gentamicin group and the tigecycline group exhibited a wider antibacterial spectrum and stronger antibacterial performance,and the gentamicin group had the longest antibacterial cycle and the best comprehensive antibacterial performance.(3)Tegacycline-loaded furan-based bone cement has the least effect on the activity of osteoblasts,and has better biocompatibility than vancomycin-loaded or gentamicin sulfate-loaded furan-based bone cement.(4)The compressive strength of the furan-based bone cement loaded with antibacterial agent was negatively correlated with the addition amount of the three antibiotics,and the compressive strength of the bone cement group with 4 g of antibiotics added to 40 g of bone cement was significantly lower than that of the 1 g group.Among them,the gentamicin group had the highest compressive strength and the best mechanical properties. |
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