Biotoxicity, Intramedullary Degradation, And Bacteriology Research Of Novel Degradable Implant Material JBDM Magnesium Alloy With Orthopedics

Part I Biotoxicity of a novel degradable medical material: JDBMmagnesium alloy【Objective】 JDBM is shortened form Jiao Da bio-magnesium series. It is designed and developed byLight Alloy Precision Forming National Engineering Research Center of Shanghai Jiaotong University.It’s a novel biodegradable high performance Mg-Nd-Zn-Zr based magnesium alloy series, with goodbiocompatibility, excellently matched strength and toughness, and almost even corrosion behavior. Thealloy systems are added in a small quantity of light rare earth element Nd as low alloying elements, whichhas a small cell toxicity. Nd can guarantee the magnesium alloy has good precipitation strengthening andsolid solution strengthening effect, and greatly improve the electrode potential of the alloy matrix, thenthe galvanic corrosion potential difference between matrix and the second phase decreases, in results, theuniform corrosion resistance of magnesium alloy improves. Zn is a trace element of human physiologicalneeds, and trace addition can improve the strength and plastic processing capacity of the alloy. Zr couldincrease Strong toughness and corrosion resistance of alloy as a grain refiner, and its biocompatibility inmagnesium alloy has been confirmed. The purpose of this study is to research cytotoxicity of Rare earthelements Nd in JDBM magnesium alloy to osteoblast cell MC3T3in vitro. And then study itsphysiological and pathological effects to Kunming mice bone and tissues nearby in vivo.【Methods】 Making leach liquor of Nd with aMEM culture according to the standard method, and thendilute to several different concentration gradient of1/2,1/4,1/8, as controls, making the leach liquor of Tialloy, WE43magnesium alloy, JDBM magnesium alloy and CaP coating JDBM magnesium alloy, andnormal training group as blank control, Ti alloy leach liquor group as negative control,0.64%phenolgroup as positive control. MC3T3-E1cells are cultured with aMEM culture containing10%Australianfetal bovine, observing the morphological changes and proliferation at different times. The activities of cells proliferations in different times were detected by CCK8method. Cells apoptosis in different timeswere detected by Annexin V PI double staining flow cytometry. Adding Nd particles into the culturemedium of cultured osteoblasts by quantity ladder. The minimum amount of Nd added in isapproximately equal to the amount of Nd released from JDBM implants. Observating the condition of Ndparticles into cells. Making the cell climbing glass pieces, and the morphological changes of cells wereobserved by HE staining.【Results】CCK8method was used to detect the cell proliferation activity. The basic solution of Nd leachliquor group has a great Cytotoxicity, as same as the group of0.64%phenol positive control group.Compared to the normal control group, Ti alloy leach liquor group, JDBM leach liquor group, WE43leach liquor group,1/4and1/8of Nd leach liquor group, P<0.00001. There are no statistically significantdifferences among the after several groups. The basic solution of Nd leach liquor group and0.64%phenol group each has statistically significant difference compared to1/2of Nd leach liquor group,P<0.01. We can know from the flow cytometry results that osteoblast cells apoptosis increasedsignificantly with the increase of the solution concentration and action time of Nd leach liquor. Fromexperiment of dynamic and static observation to that Nd particles get into cells, I find that Osteoblasts getNd particles in the surrounding environment into cells through active endocytosis phagocytosis. There isno significant effect on cell growth with less amount of Nd particles. A lot amount of Nd particles canlead to bone cell membrane rupture, until cell disintegration or death.【Conclusions】 The osteoblast cytotoxicity and effects on osteoblast cell apoptosis of Nd leach liquoreach decreases with the decrease of concentration. Trace Nd in JDBM has no obvious toxicity onosteoblast. Osteogenic cells uptake Nd particle in the surrounding environment through activeendocytosis. When the number of particles in cells arrive to a certain extent, osteogenic cells disintegrate.Trace Nd particles in JDBM have no obvious adverse effects on proliferation and morphology of bonecells. Part II Degradation and pathophysiology research of novel biodegradablemagnesium alloy JDBM intramedullary needle with rat femur【Objective】 The biodegradable JDBM magnesium alloy has been studied since its successfuldevelopment. The purpose of clinical front test is to demonstrate its biological properties, to improve itsperformance, and to fight for the application in clinical medical practice. We have done some research onJDBM magnesium alloy screw board system, gotten many important first-hand data, but have not donethe experimental studies about JDBM magnesium alloy intramedullary needle. The purpose of this studyis to test changes of pathophysiology and metabolism of rat’s for the first, and then to test the degradation behavior of JDBM magnesium alloy in the bone marrow cavity of the rat. So we can assess the medicalvalue of the material JDBM magnesium alloy by animal experimental study.【Methods】 JDBM magnesium alloy and control groups of Ti alloy, WE43magnesium alloy, CaP coatedJDBM magnesium alloy were made into intramedullary needle of1mm x30mm. After disinfection,asepsis operation has been done and the magnesium alloy intramedullary needle was implanted into thethe left femoral marrow cavity of the male SD rat. Each group has6rats.99mTc-MDP3-phase bone scanand SPECT-CT examination were done at1,3month. To know about the bone metabolic status after theneedle implantation of the rat femur intramedullary JDBM magnesium alloy by bone imagingradionuclide scan, and compared differences with the control side and control groups. X ray films weretaken in order to know about the position and degradation of intramedullary nail at1,3month. To detectthe changes of biochemical and electrolyte of rat blood at half of a month and1,3month. Hard tissuepathology slice of bone was done at month3and6. Pathological changes of rat femur were observed byhard tissue slice, and detect the degradation of intramedullary needle in different time, determine itsdegradation rate.【Results】 Rat femur radioactive marker uptake rate of JDBM intramedullary needle group wassignificantly higher than that of the contralateral femur and Ti alloy group, P <0.01. There is nodifference significant that JDBM intramedullary needle group compared with the WE43group, JDBMgroup and CaP coating JDBM group. Blood flow phase displays that magnesium alloy promotes localblood supply. No significant differences were detected in blood electrolyte test. Hard tissue pathologicalsection showed that JDBM, CaP coated JDBM group, WE43group has more new bone growth aroundintramedullary needle compared with Ti alloy. JDBM intramedullary needle corroded slowly in rat bonemarrow cavity during month1,3and6. JDBM intramedullary needle corroded less than20%of the masstill6months. While the CaP coated JDBM group was degraded more slowly. WE43group was degradedslightly faster than the JDBM group.【Conclusions】 JDBM intramedullary needle can promote the formation and metabolism of the bone,helps to repair the damaged bone tissue. JDBM magnesium alloy intramedullary nail in bone marrowcavity has a gradual degradation, but later degradation tends to slower. We wish it could present slowdegradation when we need its supporting role, and when no longer need the support role in the healingperiod, we wish it can has a completely degradation and disappear in controllable time. Part Novel degradable material JDBM magnesium alloy withstaphylococcal adhesion and biofilm model【Objective】 Staphylococcal infection is a knotty problem of orthopedics in the world. We reduce the incidence of infection by the application of various materials modification technology or research anddevelopment of new materials in generally. The pathogenic bacteria isolated by the orthopedics doctors ofour hospital are mainly staphylococcus epidermidis. The purpose of this study is to explore the effect ofsurface adhesion, growth and proliferation of staphylococcus with the novel degradable JDBMmagnesium alloy. To make the animal infection model of JDBM magnesium alloy implantation, and tostudy on the clinical application of new materials JDBM magnesium alloy.【Methods】 JDBM magnesium alloy, WE43magnesium alloy, Ti alloy each was made into1mm x10mm circular sheet, disinfecting them with ethylene oxide sterilization method. Our test using RP62A,ATCC12228strain Bacteria, etc. Turbidity instrument regulating bacterial concentration to about0.5Maxwell units (1x108/ml), using TSB medium. Take2ml bacteria TSB and put it into EP tube of4.5ml,then sterile alloy discs were added in and co-cultured for1to3days, make a repeat of cultured EP tubefor each kind of material. Absorbed TSB medium, washed metal piece with PBS for3times, then put2ml PBS and put the repeated EP tubes into ultrasonic concussion instrument and vibrated for5and10minutes with moderate power ultrasonic. The resulting liquid was diluted to102and104concentrationrespectively with PBS liquid. Take100ul of each diluted PBS liquid contained bacterium and scratchthem to blood plate, cultured for24hours and make bacterial count, observe the differences of theamount of adhesion bacteria. Thus we can study the effect of different metal sheet surface roughnesssurface on the adhesion of bacteria. As the animal test, we made JDBM, WE43alloy and Ti alloy into3mm x10mm screw, and the disinfection was use epoxy ethane fumigation method. Put the screw into thecommon New Zealand rabbit’s left femoral epicondyle with a strict aseptic operation. After operationwound healing, adjusted strain ST239, SF8300to0.5Maxwell unit (1x108/ml) with PBS liquid, andthen diluted bacterial concentration1x105/ml.0.5ml of each strain was injected into the experimentalrabbit tissues around femur near to implanted screw. Injection of saline as negative control. TakenX-ray photos at1st,3rd week after the bacteria was injected into rabbit tissue. Till the third week, X-rayphotos were taken and then the rabbits were killed, local tissue pathological examination was done. Thepartial secretion smear and Giemsa staining was done, and scratching bacteria onto blood agar cultureplate and cultured for one day and then smear examination were done after the culture ended. Bacterialcategory identification was done in the end.【Results】 JDBM, WE43, Ti alloy material smoothness is same at first, we found that there is nosignificant difference of the bacteria adhesion between RP62A and ATCC, and the bacteria count of theblood agar plate was in an order of magnitude. When we change the metal wafer smoothness of thesurface and do the experiment again, we found that more bacteria adhered to the metal sheet surface withlarge roughness. The amount of bacteria adhered to the surface has a positive correlation to the roughnessof the metal sheet surface. As animal experiment, when bacteria ST239and SF8300were injected intothe tissues near the rabbit femoral implants for1and3weeks, we found that there is a severe localreaction, and there are a lot of milk white cheese like substance emerged. Wright’s staining examination with direct smear or smear after cultured on blood agar plate was done, and a large number of bacteriawere found. After identification, we found that the bacteria caused the infection is the bacteria injected inthe test. While the saline injection rabbit has no significant local pus and secretions produced.【Conclusions】 There are no obvious difference about the amount of staphylococcus adhesion amongJDBM, WE43, Ti alloy material. But different surface roughness has an obvious influence on theadhesion of bacteria. Surface roughness increases, bacteria adhesion increase at the same time. Thematerial surface is smoother, the bacteria adhesion is less. We made the animal innfectionon biofilmmodel of prosthesis infection very easily by inject the bacterial ST239and SF8300of1X105concentration around the femoral implants of New Zealand white rabbits, while the injection of saline didnot cause obvious locality infection. The experimental results show that different bacterial species havedifferent infection ability to tissues near rabbit femur in the presence of a prosthesis implanted. Part IV JDBM and fibrinogen with internalization effect ofstaphylococcus on osteoblasts【Objective】 In orthopedics infection, a variety of pathogenic staphylococcus can enter bone cells, inwhich they grow and reproduce, produce toxins, destruct the cells, and cause cells damage, collapse andnecrosis in the end, or survive in the cells for a long time, as the result, the antibiotic cannot kill them.The purpose of this experiment is to study the difference of on the internalization role among clinicalStaphylococcus strains to the osteoblast cells, and study the effect of fibrinogen on the internalization ofthe test bacteria.【Methods】 MC3T3-E1osteoblast cells were cultured in the24well plate with DMEM containing10%Australia fetal bovine serum medium,1ml DMEM medium each hole, about5x104cells. Medium waschanged in two consecutive days, until cells growed to more than50%area. The concentration of23clinical strains and standard strains bacteria ATCC12593was adjusted to0.5Maxwell unit with DMEMcomplete medium containing fetal bovine serum through bacteria quantity turbidimetric instrument, theamount of bacteria is about1x108/ml. Absorbed off the medium inside the culture holes, each hole arerespectively added to1ml medium containing experimental bacteria and put the culture plate into thecarbon dioxide incubator and incubated for2hours at37℃. Then we washed the culture holescontaining the bacteria for three times with PBS. Each culture hole is added in1ml DMEM completemedium containing90ug/ml vancomycin, and put the plate into the carbon dioxide incubator andcultured for2hours at37℃. The culture holes were washed three times with PBS to remove the deadbacteria killed by vancomycin outside the cultured.0.1%Triton was added in and played role for10min,so cells disintegrated and the intracellular bacteria were released into the liquid of the culture wells. The liquid earned was diluted with PBS to102,104folds of concentration gradient dilution, and scratchedthem onto the blood agar culture plates and culture for one day at35℃, the plates were observed in thenext day if there has bacteria growth on them. If the bacteria were found on the blood agar culture plates,comparing the differences in magnitude and counting the amount of the bacteria. Repeated theexperiment for3times and compared if there were differences among the results of every times. As forthe effects of fibrinogen on bacteria internalization, MG63osteosarcoma cells were cultured with DMEMmedium containing10%Australian fetal bovine in the24well plate. When the cells developed to close tocover all of the bottom of the wells,9strains of pathogenic bacteria isolated from orthopedics clinicalinfection and ATCC12228, ATCC12598, RP62A were added to each well according to the concentrationof108/ml,1ml culture liquid containing bacteria strain every well. After3hours of culture, we absorbedthe culture liquid, and added1ml DMEM complete medium containing100ug/ml vancomycin, then weput the culture plate into carbon dioxide incubator and cultured the cells overnight at37℃. The next daywe absorbed the culture medium of every wells, and washed every culture well with PBS liquid for3times, and then added0.1%Triton prepared with PBS liquid to each well and let it play role for10min, socells disintegrated and the intracellular bacteria were released into the liquid of the culture wells. Theliquid earned was diluted with PBS liquid to102,104folds of concentration gradient dilution, andscratched them onto the blood agar culture plates and culture for one day at35℃, the plates wereobserved in the next day if there has bacteria growth on them, and the amount of the bacteria observedwere counted. We also research the effect of JDBM extract liquid on staphylococcus internalization,replaced the medium with JDBM, Ti, and WE43extract liquid.【Results】 The effects of internalization on osteoblast cells between every clinical bacteria strains areunequal, some bacteria have a very strong internalization effect on bone cells, and the other strains cannotbeen seen completely with the internalization effects of osteoblasts. Internalization effect ofstaphylococcus aureus is stronger in generally, and their virulence is bigger. Observed throughmicroscopic, osteoblasts with Staphylococcus aureus put in about0.5h, there are visible bacteria inosteoblasts. Let the bacteria play a role for a long time, we can see destruction or collapse of the cells. Ingenerally, internalization bacteria strains added into osteoblast cells cultured in wells plate for a certainperiod of time, added vancomycin into the wells for2h and then stopped the effect of the drug, the nextstop, washed them with PBS for3times to clear the remain of the antibacterial drug. The cleaning liquidobtained was scribed to the sheep blood agar culture plates and cultured for one day, a small amount ofbacteria may be seen sometimes. Our experimental results show that there are no obvious differenceswith the amount of internalization bacteria no matter if there is fibrinogen intervention. The extract liquidof JDBM, WE43and Ti has no obvious effects on staphylococcus internalization with osteoblasts.【Conclusions】 In generally, the virulence of staphylococcus aureus is stronger than Staphylococcusepidermidis. The SA has a strong invasiveness. More amount of bacteria can be seen in cells with the testof internalization experiments, and they grow in cells presenting a large of groups. While the virulence of Epidermal staphylococcal is weak, their invasion force is weak, their internalization effect is also weak,this present a scattered distribution in cells. Different clinical staphylococcus epidermidis strains havedifferent degree of internalization effect. Culturing together with osteogenic cells, after vancomycin playa role for2h and stopped the drug’s effect with cleaning for3times by PBS, some strains can be culturedout on the blood agar. Because the bacteria are evenly distributed in the space of the trend. Theoccurrence of this phenomenon may be due to bacteria associated with osteogenic cells through somesort of mechanism and the killing bacteria effect of vancomycin turn weaker in the result, or because thebacteria have a evenly distributed trend in the space, when vancomycin’s killing effect is stopped, theintracellular survival bacteria shift to the outside of the cell. Our experiment results show that fibrinogenor JDBM has no obvious influence on experimental bacterial internalization effect.