The Preparation, Characterization And Biological Activity Evaluation Of Biodegradable Magnesium Alloy In Vitro

In daily life, because of some unexpected circumstances, people often sufferfrom different kinds of bone fractures, and bone defects. The reasons for bone defectscould be prolonged disuse atrophy, cancer or trauma. No matter what reason makesimplants unable to obtain good initial stability, bone graft are aimed to solve thisproblem. Any bone materials should be fixed on the alveolar bone after theapplication, and should be suitable for internal fixation material of bone fracture. Atpresent, the commonly used clinical bone transplant materials and bone graftmaterials are mainly stainless steel, titanium alloy and absorbable polymer materials,etc. However, elastic modulus of stainless steel and titanium is largely different fromthat of the bone cortex, which could easily cause stress shelter effect. And, as a bonegraft material, stainless steel, and titanium alloy need a second operation, whichincreases the patient’s pain and costs. Absorbable polymer materials such aspoly(lactic-co-glycolic) acid (PLGA), poly (methyl methacrylate)(PMMA) has beenused in clinical, but there are still some shortcomings, such as bad biological activity,and poor mechanical performance, which can cause aseptic inflammation and fibrosisof the surrounding tissue. There are sone synthetic materials such as PRF, tissueengineering scaffold material, and so on. The materials above have their advantagesand disadvantages. Therefore, how to synthesize a kind of biological material withgood biocompatibility, certain mechanical properties, controllable degradationperformance has become a focus in the medical community.In recent years, magnesium and its alloys as a kind of metal matrix compositebiomaterial has attracted much attention. Magnesium is a chemically active metal,which can degradate within a short period in animal. Compared with other metal,magnesium’s elastic modulus and mechanical property is closer to the cortical bone.These physical properties make magnesium and its alloys possible to avoid stress shelter effect as bone graft and fixed material. As a result, the magnesium alloy isexpected to become the ideal new degradable biomedical metal materials. However,the biggest drawback of magnesium alloy is it degrades too fast, and produces a largeamount of hydrogen gas, which causes alkalization of the surrounding environment.Therefore, how to improve the degradation rate of magnesium alloys, and make itmatch that of osteogenesis in vivo now becomes one of the research focuses.This research investigated mechanical properties and the degradation ability ofmagnesium alloy material rich in Zn and HA in vitro, the effect of the alloy onpreosteoblasts (mice MC3T3-E1) proliferation, adhesion and differentiation. Thisexperiment also studied the influence of magnesium alloy material on the expressionof osteogenesis related genes by Real time PCR, and tested is effect on the expressionof RUNX2, FAK, p-FAK and integrin protein by Western Blot, in order to providetheoretical basis for its clinical studies in the future.Mg-4Zn-3HA alloy was prepared by powder metallurgy and hot extrusionmethod. Scanning electron microscopy (SEM) was applied to study thecharacterization of the surface of the material. Compression test was used to testmechanical properties of the material. The material had been immersed in simulatedbody fluid, for28days to detect the degradation of it and the change of pH value inthe fluid. Scanning electron microscopy (SEM) and energy spectrum quantitativeanalysis were utilized analyze material surface after degradation. X-ray diffraction(XRD) was used to analye the degradation products. Results showed that theMg-4Zn-3HA was porous, and its mechanical properties is closer to natural bonecompared with other implant materials, making Mg-4Zn-3HA alloy more suitable forimplant materials. During the28days pH values increased first and then went down,finally stabilized at about8.3.The corrosion rates were at the peak in the beginning,then decreased, and remained, stable with a also rise. The results of X-ray diffractionanalysis of degradation products showed a strong diffraction peaks of the Mg(OH)2,there were a number of other peaks, such as HA and Mg-Zn compounds. Materialdegradation after the scanning electron microscopy (SEM) and energy spectrum analysis showed that the material surface appear a lot of cracks and precipitation,degradation product rough surface porous. Spectrum analysis results showed that thesurface of the material corrosion products were rich in Mg, Ca, P and O.According to the ISO10993-5, leaching solution of material was prepared, andco-cultured with MC3T3-E1for1d,2d,3d, CCK-8cell cycle analisis and alkalinephosphatase detection were used to test cell toxicity, proliferation, and osteogenesisability. Scanning electron microscope was used to observe cells co-cultured withmaterial and to detect cell adhesion ability. Results showed that three differenttemperature treatments of magnesium alloy were non-toxic, processing temperature of300℃of magnesium alloy, have better biocompatibility compared with other twogroups. Leaching solution with different concentrations (100%,50%,10%) haddifferent effects on MC3T3-E1proliferation. The results showed that theconcentration of50%group had the most significant effect on the proliferation abilityof the cells. Flow cytometry cycle test results also confirmed that the magnesiumalloy could promoted cell proliferation. Scanning electron microscope found that thecells in the material surface had no obvious changes in cell morphology, cell stretchwas good, and filiform pseudopodia of cells had been spread to the surrounding cells.Alkaline phosphatase experiment showed the magnesium alloy materials had goodcapability for osteogenesis.250℃and300℃magnesium alloy groups’ intracellularALP activity was significantly higher than that of control group, and300℃treatmentgroup’s difference was most obvious.In molecular biology experiments, Real time PCR experiment results showedthat the relative expression of BSP in3and7days was significantly higher than thecontrol group,14days was lower than the control group. The relative expression ofOC was lower than the control group at testing time points, but there was a trend ofgrowth. Relative expression of OPN was significantly higher than control group onday3. On day7and14there was no significant difference with control group.Western Blot experiments results showed that the relative expression of RUNX2protein on day3and day14was higher than the control group, and on day14there was a significant difference. On day7, it was slightly lower than the control group;Relative expression of FAK protein was lower than the control group on day3, but onday7and day14was significantly higher than control group; Relative expression ofp-FAK protein awas significantly higher on day3and day7, but on day14, it wasslightly lower than the control group; Relative expression of integrin protein in threetest points were lower than the control group, but there was no significant differenceon day7.To sum up, Mg-4Zn-3HA alloy’s corrosion resistance had been improved. It hadgood performance in promoting cell proliferation and early osteodifferentiation, andhad good biocompatibility. This experiment provided a theoretical basis for theclinical application of magnesium alloys in the future. We have reason to believe thatrecent Mg-4Zn-3HA alloy can be an ideal medical biodegradable implant material.