| Objective:PLGA/PLCA electrospun fiber membrane loaded with different antibiotics was prepared by emulsion electrospinning.The effect of guided bone regeneration(GBR)membrane on the repair of alveolar bone defects caused by periodontitis was observed through in vivo and in vitro experiments,and the PLGA/PLCA electrospun membrane with the best repair effect was screened.Method:1.A series of PLGA/PLCA electrospinning films loaded with metronidazole,doxycycline and minocycline were prepared by emulsion electrospinning technology.The morphology and structure of the electrospun films were observed by Scanning electron microscopy(SEM).The hydrophilicity of the electrospun films was compared by hydrostatic contact angle method.The crystallinity of the electrospun films was tested by differential scanning calorimetry(DSC),and the drug loading and drug release efficiency were tested by drug release experiments.2.Mouse osteoblast precursor cells(MC3T3-E1)were inoculated into PLGA/PLCA electrospun membranes for co culture.The OD value of MC3T3-E1(450nm)was detected by CCK-8 method on the 1st,4th and 7th day of the experiment;The osteogenic differentiation was detected by alkaline phosphatase(ALP)staining and alizarin red S(ARS)staining at the first,second and third weeks of induced osteogenic differentiation culture;Porphyromonas gingivalis was co cultured with each electrospun membrane.The diameter of the inhibition circle was measured on the 3rd and 21st days to evaluate the inhibitory effect of each electrospun membrane on the proliferation of Porphyromonas gingivalis.3.The rat alveolar bone defect model was established by drilling and grinding palatal alveolar bone and local injection of Porphyromonas gingivalis bacterial solution.At the same time,the series of PLGA/PLCA electrospun membranes was implanted into the bone defect,small animal CT was used to monitor the bone defect and repair of rats immediately,2,4,6 and 8 weeks after operation,and three-dimensional reconstruction was carried out with mimics medical 21.0 software to measure the volume of bone defect and the distance(DCE)from the enamel cementum boundary(CEJ)of maxillary first molar to the apical point of alveolar bone in the operation area;The gingival crevicular fluid of the operation area was collected for bacterial culture 5 days after operation,and the colony count was counted to evaluate the antibacterial effect of each electrospun membrane in the oral cavity of rats;After 5 days and 8 weeks after operation,the rats were killed in batches,and the maxillary specimens were collected for HE staining,Masson staining and TNF-α Immunohistochemical staining and other histological observations.Result:1.In this study,PLGA/PLCA electrospun fiber membranes loaded with metronidazole,doxycycline and minocycline were successfully prepared.SEM results showed that the pores on the surface of electrospun film were uniform and the diameter was the same;The results of hydrostatic contact angle experiment showed that the water contact angles of PLGA/PLCA electrospun membrane loaded with metronidazole,doxycycline and minocycline were 100.46±13.73,33.24± 1.93 and 95.62± 5.53 respectively,which were less than 116.46 ± 6.71 of pure PLGA/PLCA electrospun membrane;DSC results showed that the low temperature crystallization enthalpy of PLGA/PLCA electrospun membrane loaded with metronidazole,doxycycline and minocycline was higher than that of pure PLGA/PLCA electrospun membrane;The drug release results showed that the antibiotic loaded PLGA/PLCA electrospun membrane was relatively stable in 30 days,and could maintain the drug peak concentration for a long time.2.The results of CCK-8 experiment showed that the survival rate of MC3T3-E1 in minocycline loaded and pure PLGA/PLCA electrospun membrane group gradually increased,while the number of MC3T3-E1 cells in Doxycycline loaded and metronidazole loaded PLGA/PLCA electrospun membrane decreased.ALP and ARS staining results showed that better alkaline phosphatase blue stained particles and red calcified nodules could be observed in Metronidazole loaded,minocycline loaded and pure PLGA/PLCA electrospun membrane group;In vitro bacteriostatic circle experiment showed that the bacteriostatic diameters of PLGA/PLCA electrospun membrane loaded with metronidazole,doxycycline and minocycline on day 3 and day 21 were 38.36 ± 1.32mm,30.22 ± 0.98mm,27.63 ± 3.75mm and 34.53±5.24mm,22.96 ± 5.72mm and 24.61 ± 0.66mm respectively,indicating that PLGA/PLCA electrospun membrane loaded with different antibiotics inhibited the proliferation of Porphyromonas gingivalis(P<0.05).3.After 8 weeks,the bone defect volume of control group,pure PLGA/PLCA electrospun membrane group,metronidazole loaded,doxycycline loaded and minocycline loaded PLGA/PLCA electrospun membrane group were 3.33±0.50mm3,2.86 ± 0.17mm3,2.11±0.42mm3,2.85±0.35 mm3 and 2.08 ± 0.43mm3 respectively(P<0.05);The DCE of control group,pure PLGA/PLCA electrospun membrane group,metronidazole loaded,doxycycline loaded and minocycline loaded PLGA/PLCA electrospun membrane group were 2.11 ± 0.09mm,1.82± 0.18mm,1.69± 0.21mm,1.95± 0.20mm and 1.60± 0.24mm respectively(P<0.05).The bone defect volume and DCE of PLGA/PLCA electrospun membrane loaded with minocycline were the smallest;The results of bacterial culture in vivo showed that the average colony numbers of PLGA/PLCA electrospun membrane loaded with metronidazole,doxycycline and minocycline were 2.16 ± 0.51,1.95 ± 0.91 and 7.25 ± 2.26 respectively,which were less than 12.42± 1.06 in the control group,indicating that it also had antibacterial effect in the oral cavity(P<0.05).Histomorphological staining showed that connective tissue invaded discontinuous bone tissue in the control group,while no soft tissue invaded bone tissue in pure PLGA/PLCA electrospun membrane group,metronidazole loaded,doxycycline loaded and minocycline loaded PLGA/PLCA electrospun membrane group.Conclusion:In this study,PLGA/PLCA electrospun membranes loaded with different antibiotics were successfully prepared,which had good surface morphology,improved hydrophilic properties and good drug sustained-release ability.Cell experiments show that it has good biocompatibility and ability to promote bone differentiation;The results of bacteria in vivo and in vitro showed that each antibiotic loaded PLGA/PLCA electrospun membrane had antibacterial ability.The results of animal experiments show that PLGA/PLCA electrospun membrane loaded with antibiotics can not only inhibit inflammation,but also prevent soft tissue from invading bone defects and promote the repair of bone defects.Among them,PLGA/PLCA electrospun membrane loaded with minocycline has the best effect of bacteriostasis and repairing alveolar bone defects.It is a material with great potential to induce bone regeneration of bone defects caused by periodontitis. |
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