The Initial Biocompatibility Research Of Ca-P Coating On Magnesium Alloy(AZ60)

ObjectMagnesium alloys have been recently proven as effectivebiodegradable orthopedic implants. However, the in vivo corrosion ofmagnesium alloys critically hinders the use of these materials asbiodegradable implant materials. During corrosion, the rapid degradationof magnesium alloys leads to subcutaneous bubbles from hydrogenevolution and an increase in the pH of body fluids and blood by localalkalization. Therefore, controlling the degradation rate and mechanicalintegrity of these alloys in the physiological environment is the key totheir applications.Several strategies have been reported to overcome the low corrosionresistance and regulate the biocorrosion rate of magnesium alloys. Onestrategy is the addition of other elements such as Mn, Zn, and Al asalloying elements to develop different alloys in the past decades. Inrecent years, Ca and rare-earth elements have been used to producebinary magnesium alloys that show good corrosion resistance but limitedbone response. Another strategy for effectively reducing the corrosion ofmagnesium alloys is surface modification. Calcium phosphate (Ca-P)coating is recognized as one of the most biocompatible materials for bone replacement and regeneration and is widely used as a bioactivecoating in clinical orthopedic implants such as titanium alloys.Recently, Ca-P coatings have been used to protect magnesium alloysfrom fast corrosion. For example, Ca-P coating on AZ30Mg andMg-Mn-Zn alloys reportedly improves corrosion resistance [10,11].In the present study, Ca-P coating was prepared on an Mg-Al alloy(AZ60) by a two-step chemical process without pretreatment. Then, thecytotoxicity in vitro was evaluated by CCK-8test, ALP activity test, andapoptosis test. The samples were implanted into the femoral shaft ofrabbits. Analysis and evaluation of the degradation in vivo wereconducted based on corrosion measurements and micro-computedtomography (CT) technology.3. MethodThe test of corrosion resistance in SBF solutionImmerse the samples of magnesium alloys in SBF solution,the ratio ofsample surface area and SBF solution volume is1cm2/20mL,thetemperature is at37±1°C. Then observe the changes of pH value and Mgion concentration.The cytotoxicity test in vitroThe experiment is divided into3groups:A group is uncoatedmagnesium alloy; B gruoup is Ca-P coated magnesium alloy; C group isnormal control group. MC3T3-E1osteoblast cells are cultured in96pores plate withsample soaked solution,A group is the solution which the sample ofuncoated magnesium alloy soaked in DMEM; B group is the solutionwhich the sample of Ca-P coated magnesium alloy soaked in DMEM;Cgroup is the solution of DMEM. Observe cell growth after cultured in1d,3d,5d and7d. CCK-8test the OD value in450nm and calculate thecytotoxicity in sample soaked solution.MC3T3-E1osteoblast cells are cultured in24pores plate with samplesoaked solution,A group is the solution which the sample of uncoatedmagnesium alloy soaked in DMEM; B group is the solution which thesample of Ca-P coated magnesium alloy soaked in DMEM;C group isthe solution of DMEM. After the cells cultured5d, the cell culture slideis removed out the plate.The cells are fixed with75%alcohol, stainedwith HE, and observed the cell’ growth with microscope.MC3T3-E1osteoblast cells are cultured in24pores plate with samplesoaked solution,A group is the solution which the sample of uncoatedmagnesium alloy soaked in DMEM; B group is the solution which thesample of Ca-P coated magnesium alloy soaked in DMEM;C group isthe solution of DMEM. After the cells cultured5d,p-NPP test the ODvalue in410nm, observe the cells’ ALP activity and evaluate theirproliferation and differentiation.MC3T3-E1osteoblast cells are cultured in6pores plate with samples directly.A group is the sample of uncoated magnesium alloy;B group isthe sample of Ca-P coated magnesium alloy;C group is only DMEM.After the cells cultured5d, the samples are removed from the plate.Collecting cells and double staining with Annexin V/PI, then observe thecell apoptosis with flow cytomytry.The experiment of samples biocompatibility in vivoThe experiment is divided into3groups, A group is the group ofuncoated magnesium alloy; B group is the group of Ca-P coatedmagnesium alloy; C group is the group of stainless steel.All animal experiments were conducted based on the animal welfarerequirements of ISO10993-2:2006. A total of36adult New Zealandrabbits with a body weight of2.5kg to3.0kg were used. The rabbits wererandomly divided into three groups. All rabbits were anesthetized with0.5pentobarbital sodium solution for surgery. After predrilling with a2.8mm hand operated drill, the samples were implanted into bothfemoral shafts of rabbits in the experimental groups. After surgery, therabbits’behavior, eating, activity, healing and body weight change wereobserved.After1month、2month、3month，each time12rabbits were sacrificed.The sacrificed rabbit’s femoral shaft were removed. Then the specimenswere observed the sample degradation and the new bone around thesample by Micro-CT scanning. After that, the specimens were conducted with hard tissue section, stained with Ponceau S staining solution, andobserved the changes in bone tissue.ResultThe corrosion resistance in SBF solutionThe corrosion degree of the coated and uncoated samples wasevaluated based on the changes in magnesium ion concentration and pHvalue in SBF. The result shows the change in magnesium ionconcentration. Uncoated magnesium alloy in SBF degraded faster, andthe magnesium ion concentration in SBF increased more quickly thancoated magnesium alloy (p<0.05).The test result shows the variation of pH value in SBF after differentimmersion period. In the uncoated magnesium group, the pH valueincreased greatly and reached10.7after one day’s immersion.Subsequently, the pH value kept on increasing slowly. While in thecoated magnesium alloy group, the pH value reached the peak of about9.7after3days and then descended.The tests of cytotoxicityCell proliferation is analyzed by viability observation of osteoblastMC3T3-E1cells using CCK-8assay for different culture periods. Asshown in Fig.3, the cell viability was no significant difference betweenthe coated magnesium alloy group and the negative control group. Butthe cell viability of uncoated magnesium alloy group was lower as compared with negative control group (p<0.05). The difference in thecell proliferation between different extract mediums was shown in Fig.4.The cells morphology in uncoated magnesium alloy group had obviousdifference with the negative control group and coated magnesium alloygroup, which showed significant reduction in both the number and thedensity of cells in uncoated magnesium alloy group and abnormal shapeand nucleus of the cellsThe ALP activities of osteoblast MC3T3-E1cells in extract mediumwere measured by using aBCA Protein Assay Kit (Sigma, USA). Asexhibited in Fig5, compared with the negative control group, the ALPactivity of the cells in uncoated magnesium alloy group was observeddecreasing much evidently (p<0.05), while the ALP activity of the cellsin coated magnesium alloy group was the same as the control group.The apoptosis and death of osteoblast MC3T3-E1cells were detectedusing flow cytometry with Annexin V-FITC/propidium iodide doublestaining of cells. Typical figures for the flow cytometric analysis areshowed in Fig.6. The cell apoptosis rate shows that after culturingdirectly with samples5days, the number of cells in uncoatedmagnesium alloy group reduced rapidly and the apoptosis rate reachedto30.8%. While in coated magnesium alloy group, the cells apoptosisrate was about17.82%, which was lower than the negative control19.33%. Biocompatibility tests in vivoAll rabbits awoke2h after surgery. All rabbits healed well, and nodifference was observed between the two groups. No obvioussubcutaneous emphysema was observed in the operated sites for allrabbits. Two weeks later, a rabbit in the uncoated Mg alloy groupappeared weakness and low activity. After the rabbit was sacrificed, ahuge mass was observed in the soft tissue of the executed site. Anoyster-white caseous substance was contained in the capsule (Fig.3).The rabbits’ weights were recorded in different periods. We observedthat the mean weight increased in the Ca-P-coated Mg alloy group,whereas the mean weight decreased in the uncoated Mg alloy groupThe corrosion mass loss of the samples is shown in Fig.5. The meanmass loss after1month was0.12and0.21g for the Ca-P-coated anduncoated groups, respectively. No significant difference was observedbetween the two groups. One month after surgery, the mass lossgradually increased in the uncoated Mg alloy group; Three months aftersurgery, the mean mass loss in the uncoated Mg alloy group increased to1.42g. By contrast, the mean mass loss was0.23g in the Ca-P-coatedgroup. The difference in corrosion weight loss between the two groupswas not obvious.The result illustrates the micro-CT reconstruction images of the rabbitfemora containing Mg implants1and3months after surgery. We observed that the surface morphologies of the samples in both groupsslightly degraded1month after surgery. Although the surface wassmooth in the uncoated magnesium alloy group, a few corrosion pitswere observed (red arrow in Fig.6). With increased implantation time,the uncoated samples exhibited more serious corrosion than theCa-P-coated samples after3months.The3D micro-CT reconstruction images showed more obviousdegradation in the two groups of the samples than the2D reconstructionimages. Fig.8shows that the calculated corrosion rate of the uncoatedsamples was approximately three times as high as that of theCa-P-coated samples.The spicemens were conducted hard tissue section, and stained inPonceau staining solution. Then the bone trabecula was observed. In Band C groups, the trabecula was arranged orderly、compactly and thick,while in A group, the trabecula was messy、few and scattered, Abundantamorphous mess was also observed in the gap between sample and bonetissue.ConclutionThe Ca-P coating prevent the degradation of magnesium alloy.There is not obviously cytotoxicity of Ca-P coated magnesium alloy invitro.In vivo, the Ca-P coating has a good corrosion resistance and biocompatibility.The experiments provide the theoretical and practical basis forbiocompatibility of Ca-P coating.Ca-P coated magnesium alloy(AZ60) is possible for a biodegradatedmaterial in orthopedics.