Basic Research On The Treatment Of Vancomycin-PCL Membrane By Electrospinning Combined With Bone Graft For Infective Segement Bone Defect

Infective segement bone defect of long bone is still a challenge for orthopeadics,even though lots of methods available nowadays, including free fibular graft,bone transport,Papineau technique(open bone graft),Masquelet technique, anti-infective reconstitued bone xenograft and so on. There are controversies about the most effective method for this challenge. Guided bone regeneration(GBR), which was extended from the phenomenon of guided tissue regeneration(GTR) discovered by dentist. Based on the GBR, we developed a resorbable drug-release membrane with micro-poms by connecting Vancomycin and PCL through electrospinning. We armed to explore a simple and effective method for the treatment of infective segement bone defect using this material by in vitro and in vivo experiments.This study included material preparing, in vitro and in vivo experiments. We are going to explore the feasibility and eflfectivity of this membrane in the treatment of infective segement bone defect of rabbit femur through macro and micro molecular markers and radiological study.Parti The prepare of a resorbable vancomycin- PCL membrane by electrospinning and rabbit allogeneic bone graft.Objective: To prepare a resorbable vancomycin- PCL membrane by electrospinning and rabbit allogeneic bone graft for the repair of infective segement bone defect and acquire ideal electrospinning conditions.Methods: DMP and acetone were mixed together at room temperature, with a ratio of7: 3. Then 15wt% Poly s-caprolactone were added at the temperature of 80 °C in impermeable condition by magnetism for 2h. Membranes with lO^m diameter porus were developed through adjusting voltage and receive distance. The vancomycin powder was then mixed with the fore mentioned PCL solution in impermeablecondition by magnetism for 8h,with a ratio of 1: 20. The resorbable vancomycinPCL membrane was then developed under the condition acquired and subjected to electron microscope. Bone tissue from pelvic and extremities of healthy male Japanese rabbits were harvested. The subsequent bony stock was processed according to human allogenic bone processing technique to form rabbit allogenic bone.Results: The membrane was in good shape and characteristics when made under room temperature and normal pressure, with humidity of 40%-70%, voltage of 15 KV and receive distance of 10 cm. The allogenic bone stock processed by human allogenic bone processing technique was in good shape and quality.Conclusion:①Resorbable vancomycin- PCL membrane,with lOjiin diameter or smaller poms,could be developed through electrospinning.②The allogenic bone graft could be made according to human allogenic bone processing technique.Part II The study of the drug released in vitro, biocompatibility and migration of fibroblast of the resorbable vancomycin- PCL membrane by electrospinning.Objective: To examine the drug released in vitro, biocompatibility and migration of fibroblast of the resorbable vancomycin- PCL membrane through cell experiments.Methdods:① Cytotoxicity examinationFive pieces of resorbable vancomycin- PCL membrane(Icmxlcm) was put in a-MEM solution with 10% FBS under 37 "C for 5 days to get leach liquor. The subsequent leach liquor were added to the cells of 7F2 osteoblasts seeded in 24-well plate, while the positive control well was added with completed culture media and the blank well was added with completed culture media without cells. The cells werecultured for Oh,48 h and 96 h. Then the culture media were replaced by 270 ^iL fresh culture media and 30 jiLSOjiL Alamar Blue® reagent in the dark. The fluorescence at590 nm was examined 4h later.② The influence of vancomycin- PCL membrane on the growth of osteoblast under fluorescence microscope.The resorbable vancomycin- PCL membrane(Icmxlcm) was incubated in20ug/mL collagenase I at 37 °C overnight. Cell suspensions of 7F2 osteoblasts were added to the surface of the membrane, with 200,000 cell per well in 1000 pL,and then cultured for Id and 4d. After taht, the cells were fixed with 4% paraform and incubated in 0.1% Triton X-100,followed by incubation in TRITC-phalloidin overnight. Then the cells were incubated with l|ig/mL DAPI in dark and observed under inversion fluorescence microscope after washing.③ The in intra drug release of the resorbable vancomycin- PCL membrane by electrospinningThe resorbable vancomycin- PCL membrane(2.5cmx2.5cm) was incubated in100 ml PBS at 37°C on the swing bed. One ml incubation solution was harvest at Ih、12h、ld、2d、3d、4d、5d、6d、7d、8d、9<i、lOd、lid、12d、13d andl4 dand centrifugated.The corresponding fluorescence of the consequent supernatant at 538 nm was examined,④ The influence of the resorbable vancomycin- PCL membrane by electrospinning on the migration of fibroblast examined by Transwell method.The resorbable vancomycin- PCL membrane(Icmxlcm) was incubated in20ug/mL collagenase I at 37 "C overnight then placed on the top of filter mambrane in the wells of Transwell. Cell suspension of 3T3-L1 fibroblasts were adjusted to 5X 10^cells/mL. 200 \iL cell suspension was added to the surface of the membrane, while the lower chamber was added with SOO^L culture media containing 10% FBS. The cells were cultured for 24 h,48h and 72 h and then fixed in methanol and stained with0.1%crystal violet. The cells on the top of the membrane were removed gently and the migration cells were detected by inversion fluorescence microscope.Results:①The resorbable vancomycin- PCL membrane by electrospinning have no obvious cytotoxicity to osteoblasts and no influence on the proliferation of osteoblasts.②In vitro drug release data showed that the vancomycin peaked at 24 h with a rapid release and attenuated afterward.③Transwell method showed that no fibroblasts migrated at the first 24 h. But72 h later, there were a few cells migration.Conclusion:①The resorbable vancomycin- PCL membrane by electrospinning had good biocompatibility and safty.②The resorbable vancomycin- PCL membrane by electrospinning possessed good drug release characteristics.③The resorbable vancomycin- PCL membrane by electrospinning had a little influence on fibroblast migration.Part III The study of the resorbable vancomycin- PCL membrane by electrospinning combined with rabbit allograft in the treatment of Infective segement bone defect of femur.Objective; To explore the feasibility and effectivity of the resorbable vancomycinPCL membrane by electrospinning combined with rabbit allograft in the treatment of infective segement bone defect of femur.Methods: Animal model establishment and treatment: 24 rabbit infective segement bone defect of femur were created and treated with micro-external fixation. The rabbits were then divided into control group and experiment group. The wounds received debridement and then were treated by following methods: in experiment group,the bone defect were treated with the resorbable vancomycin- PCL membrane by electrospinning combined with rabbit allograft; in control group 1,the bone defectwere treated with the rabbit allograft alone; in control group 2,the bone defect were treated with the resorbable vancomycin- PCL membrane by electrospinning alone.(1)The granulation tissue in the center of the wound at OD and 12 D were harvested and preserved accordingly. And the macro view the the wound was recorded.(2) Bacterial count was used to reveal the model developed and the infection of the wound.(3) HE staining and immunohistostaining were applied to show the inflammatory cell infiltration and new capillaries formamtion.(4)Radiography examination were taken at 4w,8w and 12 w, and the bone healing time and rate were recorded.Results:①The wound at OD was moist and hyperemia, with a few purulent secretion.While at 12 w,the purulent secretion was less in experiment group and control group 2.The granulation tissue in experiment group was fresher and better than control group1.②Bacterial count revealed that all the wound had staphylococcus aureus,with a number more than lxl05cfu/ml, which indicated the success of animal model. While at 12 w, the bacterial in the center of the wound was statistically less in experiment group and control group 2.③At 12 w,the inflammation cells were statistically less in experiment group and control group 2, HE staining showed that the inflammatory cell infiltration in the bone defect area was less in experiment group and control group 2 and the bone formation was better in experiment group and control group 2,④Radiography study showed that callus formation at the bone defect site was better in experiment group,and bone healing rate was higher in experiment group at4 w, 8w and 12 w.Conclusion:①The resorbable vancomycin- PCL membrane by electrospinning can control the infection of the infective bone defect wound.②The resorbable vancomycin- PCL membrane by electrospinning combined with rabbit allograft can successfully repair the infective segement bone defect.Part IV Clinical study of commercial resorbable membrane combined bone graft for bone defect in long bone of extremities.Objective: To study the clinical efficiency of commercial resorbable membrane combined bone graft for bone defect in long bone of extremities.Methods: Ninteen patients with bone defect of in long bone of extremities were recruited,including 18 male and 6 female. The ages were from 16-65 years,with an average of 34 years. The range of defective areas was from 1.5cm-5.0cm. After debrid,intramedullary nail or plate fixation were applied. Bone defect was treated by autogenic bone graft wrapped by resorbable membrane(Fibrillar?, Tthicon LLC,USA). Anibiotics was given routinely. The wound were checked every two days.ESR and CRP were check if any kind of infection was suspectedResults: All patients had 汪 good short term clinical result,with normal body temperature. Increased levels of ESR and CRP were detected,with CRP decreasing to normal shortly and ESR decreasing to normal slowly. Seventeen wounds healed uneventfully without any kind of wound infection. Two wound had a little exudation,with negative culture and delayed healing.All patients were followed for 12-18 months,with an average of 14.3 months.Seventeen patients got bone healing, ranging from 4-8 months,with an average of 5.3months. One patient who did not get bone healing 8 months after the surgery healed 4months after a second bone graft operation(without commercial resorbable membrane). No infeaction was abserved. The function and outlook was excellent.Conclusion: Good clinical effects could be achieved using commercial resorbable membrane combined bone graft for bone defect in long bone of extremities.