| Objective:To construct?-tricalcium phosphate(TCP)bone tissue composite scaffolds loaded with vancomycin(VAN)/PLA microspheres and to study the characterization,drug release,mechanical properties,biocompatibility and vitro antibacterial properties of composite scaffolds loaded.To establish a rabbit model of chronic osteomyelitis with localized infectious bone defect and implant the composite stent to observe the effect of the stent in the animal's in vivo inhibition and induction of bone regeneration.The purpose is to prepare bone tissue engineering scaffolds that can effectively control infection and repair bone defects,and finally provide theoretical and experimental support for clinical application.Method:This experiment consists of four parts1.Optimization of preparation and drug release in vitro of VAN/PLGA microspheres by orthogonal design.Using the double-emulsion solvent evaporation method,the drug loading rate and entrapment efficiency of the microspheres were used as the main inspection criteria.The concentration of the different PLGA solutions,the concentration of the internal aqueous drug,the concentration of the external aqueous phase PVA,and the particle size of 100?m were used as the benchmarks.Four factors and three levels of orthogonal experiment were performed under the four process conditions of stirring speed.The characterization of optimized microspheres,drug release in vitro and antibacterial activity were measured.2.Construction and in vitro study of 3D printing porous?-TCP scaffold loaded VAN/PLGA Microsphere Composite Scaffold.Designing?-TCP scaffold model using CAD software,3D printing technology to make?-TCP scaffold,positively charged chitosan(CTS)wrapped scaffold,and loading negatively charged VAN/PLGA sustained-release microspheres on the scaffold by electrostatic adsorption,then microspheres and stents were again wrapped with CTS.The characteristics,mechanical properties,biocompatibility,drug release and antibacterial properties of composite stents were studied.3.Preparation of two different types of rabbit tibial osteomyelitis models.Different types of rabbit osteophymatic osteomyelitis models were prepared and compared in two ways.Methods:Forty-five New Zealand white rabbits were randomly divided into 5 groups:small hole control group(A1),small hole experimental group(A2),macroporous control group(B1),macroporous experimental group(B2),blank group;There were 5 blank groups and 10 other groups.In group A,a small hole method was used to drill a hole in the upper middle of the rabbit tibia with a 1 mm drill bit.Group A1 was injected with 100?l of normal saline.Group A2was infused with a suspension of Staphylococcus aureus(SA)(2*10~6cfu)in 100?l;group B used large.In the hole method,a 5-mm drill bit was used to drill a hole in the upper middle segment of the rabbit sacrum.The hole was partially filled with Folium leaf bio-paper.Group B1 was injected with 100?l physiological saline.Group B2 was infused with a suspension of SA(2*10~6cfu)100?l;After the groups A and B were injected with saline or bacterial fluid,they were closed with bone wax and the wound was closed.The blank group did nothing.4.Experimental study of?-TCP loaded VAN/PLGA microspheres in the treatment of infectious bone defects.The purpose of the experiment was to treat infectious bone defects.Based on the results obtained in the third part of the experiment,SA10~6cfu was implanted in the middle and upper humerus of 30 rabbits using the macropore method.After 4 weeks,28 rabbits were selected by X-ray examination to meet the next experiment.Divided into the following four groups:debridement group(A),debridement microsphere experimental group(B),debridement blank stent experimental group(C),debridement composite stent experimental group(D),each group of 6.At 1 month and 4 months after operation,local antibacterial and osteogenic ability of the material was observed by X-ray,CT,Micro CT,and histopathology.Result:1.Optimization of preparation and drug release in vitro of VAN/PLGA microspheres by orthogonal design.The optimum technological conditions are oil phase 9ml:PLGA500mg,Internal water phase 1ml:VAN300mg,External water phase:the concentration of PVA solution is 3%,Speed:1200r/min.After optimization,the microspheres were prepared:the drug loading rate was(17.40±1.87)%,the entrapment efficiency was(35.12±3.65)%,and the average particle size was(102±37)?m.The release rate of the microspheres at first days was(17.91±2.41)%,and there was a certain sudden release.After 2 days,the release rate of the microspheres decreased gradually,and the cumulative release rate of the microspheres slowed down at 12 days(58.78±1.54)%,and 1.41%was released on the average of 12 to 24 days,and the cumulative release(75.31±1.02)%in24 days.2.Construction and in vitro study of 3D printing porous?-TCP scaffold loaded VAN/PLGA Microsphere Composite ScaffoldThe volume of the blank stents was 6mm*6mm*5mm.400?m beam,500?m aperture,The actual porosity of the blank stents was(48.8±1.21)%;(centrifugation)composite scaffold(TCCS)porosity is(25.46±2.45)%;(static adsorption chitosan coating method)composite scaffold(EACS)porosity is(21.19±0.79)%.The maximum bearing capacity of EACS is 221.5N±9.7N by using universal material mechanics,and the support strength is about 6MPa(much higher than that of cancellous bone compression strength 2MPa).The co culture of bone marrow mesenchymal stem cells(BMSCs)in vitro showed that the scaffolds,microspheres and chitosan had no significant effect on the proliferation of BMSCs,and the number of cells attached to the three kinds of scaffolds increased with time.In vitro release and antibacterial experiments of scaffolds showed that:on the 24 days,the?-TCP bone scaffold loaded with VAN/PLGA microspheres still had good release rate and bacteriostatic effect.3.Preparation of two different types of rabbit tibial osteomyelitis models.After 30 days,the survival rate of the blank group and the control group(A1,B1)was 100%,the A2 group was 70%,and the B2 group was 100%.After operation,the body temperature and C reactive protein index of A2 and B2 group rabbits in experimental group increasedin varying degrees.The A2 group increased more significantly compared with the B2 group,and the calcitonin level decreased slightly.Molybdenum target X-ray examination:at 14 and 28 days after operation,the positive rate in group A2 was 72%and 100%,and the positive rate in group B2 was 89%and100%;CT examination:on the 14 day after operation,the positive rates in group A2and group B2 were all 100%,A2 group showed acute hematogenous osteomyelitis,and B2 group showed local bone infection signs;on the 28 day,A2 group showed osteosclerotic osteomyelitis,and B2 group showed local bone abscess.Pathological findings:neutrophil infiltration,thickening of periosteum,destruction and hyperplasia of bone cortex and trabecula and loss of bone structure integrity were observed on 30days after operation in group A2 and B2.After 30 days,group A2 and B2 had been killed and the local pus and necrotic bone tissues of the rabbit tibia were grinded and the culture of Staphylococcus aureus were all positive,and the blank group and the control group were all negative.4.Experimental study on the treatment of infected bone defects with?-TCP loaded VAN/PLGA microspheres in vivo.A local bone abscess type osteomyelitis model was prepared by macroporous method.By means of molybdenum target X-ray,CT,Micro CT and pathological section,the results showed that the simple debridement group was re infected after the operation,and the simple debridement could not eradicate the bacteria completely;although debridement and microsphere group control infection to some extent,but the repair of bone defect is mainly fibrous scar,bone regeneration rate is slow;in the simple stent group,because the scaffold has no anti infection ability,though it induces bone hyperplasia,it is finally unhealed because the infection is not controlled;only the scaffolds loaded with VAN microspheres obtained good results,which positively induced bone regeneration at the same time of infection control.New bone formation was observed in Micro CT material reconstruction and pathological results,showing that bone tissue was centered on material and formed new bone on the surface of the scaffolds.Conclusion:With the best process,VAN/PLGA sustained-release microspheres can be stably prepared with uniform particle size distribution of about 100?m;the osteomyelitis model of the local bone abscess can be prepared by macroporous method;?-TCP loaded VAN/PLGA microspheres composite scaffolds can be applied to treat local infected bone defects in rabbits;after implantation,the VAN can be released in all directions and long time in the local three-dimensional space of the infected bone defect to achieve the purpose of controlling infection in the lesion;At the same time,it can play a role in filling bone defects,inducing bone formation and accelerating bone tissue reconstruction.The results of this study have a deeper understanding of the pathological mechanism of osteomyelitis,and provide theoretical and experimental support for the future clinical treatment of osteomyelitis by implantation of sustained release antibiotic materials. |
PDF Full Text Request