The Design And Fabrication Of Antibacterial Metal Implants And Its Effect Of Anti-infection

Infection is still one of the serious complications of orthopedic implants. Implantedmetallic foreign body are the risk factors leading to these infections, caused by a series ofin vivo reactions include the weakening of the macrophage function and passivation of thelocal immune system, which provide convenient conditions for the proliferation ofpathogens. In addition, adsorption of protein on the surface can promote bacterial adhesionto the implant to cause infection. Sterilization of the environment and personalcontamination is our most concerned in the past. However, the new strategy aim directly atpathogenesis of such infections, which reduce infection risk by surface modification of themetal implants.Systemic administration of antibiotics have the possible disadvantage of not beingsufficiently effective due to impaired blood circulation and scar tissue around the implants,also, bringing systemic toxic side effects. The study confirmed that once the bacteria isisolated in avascular bone tissue, systemic treatment of antibiotics is difficult to achievesatisfactory drug concentration locally. Moreover, once the pathogen attached to the boneor other biological material surface, occurrence of metabolic and phenotypic changes willlead to the drug resistance to the antibiotices and escaping of immune surveillance. On the contrary, the low concentration of antibiotics may provide the power to the strains of drugresistance selection. In order to improve the therapeutic effect of bone infection, local drugdelivery method have been widely investigated and developed. Controlled release systemfor antibiotics has been favored due to its continuity and adjustable release characteristics.On the basis of the research on low elastic beta titanium alloy and controlled drugrelease, we use a surface-adherent polydopamine film onto a micro-grooved surface,which will enable the gentamicin gelatine microsphere to combine to the micro-groove onthe material surface effectually, then forming an antibiotic system on the materialsurface. Through characterization, drug release and in vitro antibacterial experiments, weconfirmed that metal surface with the antibiotic drug delivery system had advantages suchas solid combination, continued and controlled drug release, obvious inhibitory effect invitro. In the present study, on the basis of preliminary groundwork, biocompatibility andanti-infection effect of the antibiotic release system will be explored in vitro and in vivo.ObjectiveOn the basis of pre-established antibiotic drug delivery system on the surface of thelow elasticity β-titanium alloy, the biocompatibility of the poly-DOPA coating areinvestigated in vitro by cell cultrue and in vivo by implantation to into mouse musclepouch; To investigate the anti-infection effect of antibiotics controlled release system onthe metal surface, a rabbit tibial osteomyelitis model has been established andimplantation of anti-infection metal implants into infected medullary cavity has beenadopted to study the effect of infection control, to aim at provide a theoretical andexperimental evidence for anti-infection research and development of metal materials.Methods1. Observation of cell biocompatibility of the dopamine modified titanium surface:To explore the biocompatibility of poly-dopamine coating, bone marrow stromal cellsfrom rabbits were seeded in poly-dopamine modified titanium surface, and then the MTTquantitative calculation and laser confocal microscope qualitative observation were usedto analyze cell adhesion, proliferation on the surface and compared with the uncoatedmaterial, observing the the biocompatibility changes after the coating modification. Calcein-AM and propidium iodide (PI) solution has been used to stain the live cells anddead cells respectively, in order to observe the cell growth and survival.2. Biocompatibility testing of composite materials in vivo:30Balb/c mouse wererandomly divided into three groups, group A, group B and group C were seperatelytreated with anti-infective metal implants, pure titanium alloy implants and incision-suturetreatment. After1,2,3,6weeks after operation, collected peripheral blood was tested byflow cytometry to detect changes in percentage of the T-lymphocyte subsets CD4and CD8,histological observation was conducted also, to evaluate the biocompatibility of thecomposite anti-infective material in vivo.3. Establishment an animal model of IAOMI:(1)48healthy rabbits were randomlydivided into five groups, the experimental group A, B, C, D(n=10), and the controlgroup(n=8). In the experimental groups,1ml volume of the serial dilutions of the bacteriaof S. aureus ATCC25923[10~5to10~8colony-forming units (CFU)/ml] were inoculatedinto the medullary cavity of the animals before a titanium cylinder (4.0cm length and0.25cm diameter) was put into. While in the control group, the same volume of saline wasinoculated and then the same size of titanium cylinder was implanted. The efficiency ofthe IAOMI model in rabbit was evaluated by clinical, radiologic, microbiologic andhistological parameters at the4th week after the operation, and then an optimalconcentration of the bacteria inoculated was chosen.4. Evaluation of the anti-infective effects with implantation of modified metalimplants into the rabbit infection model: New Zealand white rabbits were randomlydivided into2groups(A and B) after mesuring the body weight and body temperature. Theleft tibia were chosen as the experimental side, used to establish the infection model formaterial implantation. Group A was experimental group, insertion with the antibioticloaded metal material, while group B was the control group inserted with the metalmaterials without the antibiotics. The samples were harvested after the time of2weeks,4weeks and8weeks. All animals were detected by lateral X-ray at the tibia, aslo, thegeneral observation, bacterial culture and histological observation with HE staining atdifferent time points. Results1. Cell biocompatibility on the surface of the titanium alloy after modified bydopamine: The absorbance values for each time point in the two groups were assayed byMTT method, then, the values were statistically analyed by using the Wilcoxon rank sumtest. The results showed that there was no significantly difference for the number ofadhesive cells at the first day between the two groups. The number of the adhesive cells inuncoated group were slightly higher than that in the coated group at days3,5,7,9. Thedifference between the two groups was statistically significant ((P_a<0.01, Pb<0.05),which showed that the poly-dopamine coating have a negative impact about cellproliferation on the surface, but the impact limited to a low extent. Calcein green-stainedcells in the two groups confirmed that living cells grew well, apoptotic cells in the coatedgroup were more than the uncoated group, which was in accordance with the results ofMTT assay.2. Biocompatibility test of composite materials in vivo: The variance analysis of theresults of flow cytometry measurement showed that, after the first week, the CD4/CD8ratio of the coated group had no significant difference with the incision-suture group,while the CD4/CD8ratio of uncoated group was significantly lower than the other twogroups (P <0.01). The measurement results for the second and third weeks were similar tothe first week. At sixth week, CD4/CD8ratio were not significantly different between thethree groups. The histological observations showed that at the time point of1,2,3and6weeks, the uncoated group had a obvious fibrous tissue hyperplasia and moreinflammatory cell infiltration compared with the coated group. Fibrous tissue hyperplasiaand inflammatory cells infiltration were greatly deceased in the coated group with timeincreased. Obvious fibrous tissue hyperplasia and inflammatory cell infiltration still can beseen in the uncoated group at6weeks.3. Establishment of animal infection models: Clinical progression observed by thepostoperative swelling of the local soft tissue, and a significant increase in bodytemperature within the early two weeks and then had a decrease. There were varyingdegrees of periosteal reaction, osteolysis, sequestrum formation in the experimental groups at the4th week after the operations on radiology. Bacteriological culturing showed that theseverity of infection was aggravated in the experimental groups, with the increase of theconcentration of inoculums. The infection rate of group C and D was100%, and there wasthe highest survival rate in group C that was80%, which inoculated1.0×10~7(CFU)/mlbacterial, while there was the highest mortality in the group D which was70%.Intramedullary fibrosis and sequestrum formation indicated infection on histology at the4th week after the operations.4. Evaluation of the anti-infective effects with implantation of modified metal implantsinto the rabbit infection model: Postoperative X-ray follow-up to the experimental animalsshowed that periosteum reaction can be seen in the two groups at2weeks; Thisperiosteum reaction was aggravated in the group B at the time of4and8weeks,accompanyed with osteolysis destruction and sequestrum. Bacterial culture showed thatinoculation with bone tissue and implants had no bacterial colonies on the culture plates ingroup A, which indicating that the anti-infective metal implants in group A played asignificant bacteriostatic and bactericidal role. While the white colonies almost covered allthe petri dish in group B, which indicating that there were a large number of bacteriaexisting in the bone tissue and on the implant surface. The difference between the twogroups were significant. Histological observation showed that the marrow fat cellsarranged regularly with hematopoietic cells distributed in the cells’ gap in group A.Structure of ortical layer of was complete and tightly packed. Cells were also in a goodcondition, nuclei can be obviously observed. Infarction and fibrosis could be observed inthe medullary cavity in group B, plasma cells and monocytes were accumulated in themedullary cavity. Normal vacuole fat cell structure disappeared and occupied by a largenumber of pus. Necrotic bone fragments existed in the medullary cavity and cortical bone,and range of necrotic bone chip expanded with time increased.Conclusion1. Surface modification by using poly-dopamine coating to the titanium alloy willcause a slight negative impact on cell adhesion in vitro. The calcein-AM and PI stainingindicated that there was no obvious biological toxicity. Composite materials contain gelatin microsphere exhibit a good biocompatibility in vivo.2. An animal model of IAOMI had been successfully established，which was easilyprepared and had a high survival rate. It could be a good animal model for the study ofIAOMI.3. The antibiotic drug delivery system on titanium surface showed a goodanti-infection effect in the rabbit osteomyelitis model. Combined with good mechanicalcompatibility from low elastic titanium alloy and good biocompatibility from surfacemodification, this method is expected to providing a effective solutions for the preventionand treatment of IAOMI by achieving the two advantages of bone integration andsterilization.