| BackgroundAt present, the conventional medical metallic materials are most commonly used for bone fracture fixation. However, the main disadvantage of these materials is the need to remove the implant by a second surgery. In addition, the conventional medical metals do not match the mechanical properties of human bone tissue and are easy to produce "stress shielding", leading to complications such as nonunion or refracture. Although biodegradable polymers, whicn can advoid a second surgery, are used as a substitute of conventional medical metals, they also show some disadvantages, such as fast or low degradation rate, insufficient mechanical properties, poor biocompatibility and causing inflammation.In recent years, magnesium alloy has been concerned widely because it is a new class of biodegradable metal material, possessing excellent mechanical properties with high strength, appropriate density and elastic modulus similar to those of natural bones which can effectively reduce the "stress sheltering" for other metallic bone implants. Unfortunately, the rapid degradation rate of magnesium alloy can not meet the requirement of rigid internal fixation, in addition, accompanied by the release of hydrogen gas upon degradation, limiting their clinical applications. Surface modification can significantly improve the corrosion resistance of magnesium alloys and is the focus of magnesium alloys research for medical applicaiton. Micro-arc oxidation (MAO) is an emerging surface modification technology which had been widely used for surface modification of magnesium alloys due to its low cost and simplicity in operation. In addition, the MAO process can produce a compact, hard coating which possesses excellent abrasive and corrosion resistance and good bonding with the substrate compared with other coating techniques.A micro-arc oxidation coated magnesium alloy ZK60was prepared by the Institute of Metal Research, Chinese Academy of Sciences, and in vitro experiments had proved its good corrosion resistance. However, as a new class of biodegradable metallic material for medical application, its detailed biological is still unclear.ObjectivesTo observe the effects of MAO coated ZK60alloy on cell proliferation, adhesion, osteogenic differentiation, mineralization and hemolysis. To preliminarily evaluate the biocompatibility and bioactivity of the material, validate the safety and effectiveness of MAO coated ZK60alloy as a new class of biodegradable orthopeadic implant material, and provide a foundation for the later in vitro experiments and clinical applications.Methods1. Surface characterization and biocompatibility of the materialThe surface microstructure, phase and chemical compositions of the coating were determined by a scanning electron microscopy (SEM), energy dispersive spectrum (EDS) analysis and X-ray diffractometer. Cell experiments were carried out by both indirect and direct contacts and divided into four groups, which were bare alloy group, coating group, titanium alloy group and negative control group. Extracts were prepared respectively with the surface area of extraction medium ratio1.25cm2/ml according to IS010993-12standard. The H-DMEM medium supplemented with10%FBS was used in the negative control group. The extracts were used as culture medium and the cell morphology was observed by a microscopy. The lactate dehydrogenase (LDH) activity assay was used as an index of the cytotoxicity. The CCK-8kit was used to determine the cell viability. Cell morphology on the surface of different samples was observed by a SEM. Protein adsorption of the coating surface was assayed, then4’,6’-diamidino-2-phenylindole (DAPI) and Calcein-AM/EthD-1staining of cells were carried out to observe early cell adhesion and growth status.2. Cell osteogenic differentiationThe experiments were carried out by indirect contact and divided into bare alloy group, coating group, titanium alloy group and negative control group. The intracellular alkaline phosphatase (ALP) activities were determined using a p-nitrophenylphosphate (p-NPP). Alizarin red was used to stain mineralized nodules and quantitative analysis using real-time quantitative PCR (QRT-PCR) to detect intracellular osteogenesis-related gene expressions.3. Hemolysis testThe experiment was divided into bare alloy group, coating group, negative control group and positive control group. The hemolysis test was carried out according to the national medical silicone materials biological evaluation experimental methods GB/T16175-2008. The hemolysis was visually observed and the hemolytic ratio was calculated.Results1. Surface characterization and biocompatibility of the material1.1The surface characterization by SEM showed that a rough and porous surface morphology is formed on the coating, with pore diameter of around1μm as well as some microcracks. The cross-section observation shows that the MAO coating formed on ZK60is relatively compact and exhibits a good bonding with the substrate, and the thickness is about20μm. EDS analysis on a area of the coating surface indicates that the coating layer is mainly composed of O, Mg, Si and a small amount of Zn, F, K. The XRD pattern of the MAO coating displays that both Mg2SiO4(forsterite) and MgO (periclase) exist in the coating.1.2Cells cultured in the extracts of each group showed good condition under a microscopy. Adherent cells exhibited a clear outline and spindle morphology. Most of the cells connected with each other by pseudopodias. Visible intracellular particles were observed and there were no significant cell lysis or death. The cell morphology toxicity was graded as0.1.3The LDH levels of the bare alloy group, coating group, titanium group and control group were (1101.5±165.2) U/L,(1195±123.5) U/L,(1126.5±158.5) U/L and (1158.5±90.3)U/L respectively, and there were no significant differences between the four groups (P>0.05)1.4Cell viability assay indicated that the OD value of each group gradually increased with the prolonged incubation time, and the difference was statistically significant within the group at different time points (P<0.05). At day1, cells cultured in the extracts of both the naked alloy and the coating groups showed relatively lower absorbance in comparison with the control group, and the differences were statistically significant (P<0.05). The relative growth rates were90%and87%respectively. Cytotoxicity evaluation was graded as1. However, at day3and day5, the OD values between groups were no significant differences (P>0.05), and the relative growth rates of experimental materials were around100%, cytoxicity evaluation were graded as0.1.5More cells were observed on the surfaces of the coating group and the titanium alloy group by a SEM, but only a few cells were observed attaching on the surface of bare alloy group. Cells on the coating group surface were polygonal, elongated and thicker, with fine pseudopodia extension and connection, exhibiting a better cell shape and adhesion than titanium alloy group. Cells on the surface of the bare alloy group were observed to maintain a spindle-like, thin morphology and poor extension.1.6Protein adsorption in the coating group was (152.7±6.3) μg/ml, which was significantly higher than (96.3±3.9)μg/ml of bare alloy group and (96.1±8.7)μg/ml of control group (P<0.05). There was no significant difference between bare alloy group and titanium alloy group (P>0.05).1.7The result of DAPI staining showed that the adherent cell numbers on the sample surface of each group gradually increased with the prolonged incubation time, and the difference was statistically significant within the group at different time points (P<0.05). At each time interval, the adherent cell numbers on the sample surface of coating group were dramatically larger than bare alloy group and titanium alloy group. At60min, there was no obvious difference between the bare alloy group and titanium alloy group. However, cell number in the bare alloy group was significantly lower than that in the titanium alloy group after incubation for30and120min (PO.01)1.8The result of live/dead staining showed that there were few cells observed on the surface of the bare alloy group, on which most cells exhibited rounded and poor morphology. In contrast, cells on the surfaces of coating group and titanium alloy group were almost green staining with good shape and extending pseudopodias to adhere on the surface of the materials. Even better cell stretch was observed in the coating group compared with the titanium group.2. Cell osteogenic differentiation2.1There were no significant differences among the different groups in the intracellular total protein synthesis. The ALP activity in coating group and bare alloy group were higher than titanium alloy group and control group (P<0.05). And the ALP activity in coating group was higher than the bare alloy group (P<0.05).2.2Mineralized nodules stained with alizarin red in each group showed scarlet in appearance with varying degree. The staining was deepest and most extensive in the coating group, followed by the bare alloy group. And there was no significant difference in appearance between the titanium group and control group. The result of quantitative analysis showed that the OD values of the bare alloy group and coating group were0.218±0.008and0.299±0.036respectively, which were significantly higer than0.109±0.001and0.112±0.006of the titanium alloy group and control group (P<0.01). And the coating group was higher than the bare alloy group (P<0.05). There was no significant difference between the titanium group and control group (P>0.05). The degree of cell mineralization was as follows:coating group>bare alloy group>titanium alloy group/control group.2.3The result of osteogenesis-related gene expressions assay indicated that there was no significant difference in the expressions of the ALP, OPN, OCN and CO I between the titanium alloy group and control group (P>0.05). The ALP mRNA expression levels of the bare alloy group and coating group were2.155±0.339and 3.990±0.181respectively, which were significantly higher than the titanium alloy group and control group (P<0.05). The coating group was higher than the bare alloy group (P<0.05). The result was consistent with the ALP activity assay above. The OPN mRNA expression levels of the bare alloy group and coating group were1.799±0.066and1.838±0.038respectively, which were significantly higher than the titanium alloy group and control group (P<0.05). And there was no significant difference between bare alloy group and coating group (P>0.05). The OCN mRNA and CO I mRNA expression levels of the bare alloy group and coating group were significantly higher than the titanium alloy group and control group (P<0.05). And the coating group was higher than the bare alloy group (P<0.05).3. Hemolysis testThe supernatant in the centrifuge tube of the coating group was colorless and transparent in appearance, which was almost the same with the negative control group, indicating no significant hemolysis. The supernatant of bare alloy group was pale red, indicating obvious hemolysis. The supernatant of the positive control group was rose, meaning the red blood cells completely dissolved. The hemolytic ratio of the bare alloy group was28.7%, which was significantly higher than the national standard (5%). The hemolytic ratio of the coating group was only1.04%, and the OD value showed no statistically significant difference compared with the negative control group (P>0.05), indicating that the MAO coated magnesium alloy conformed to the hemolytic standard and would not cause obvious hemolysis.Conclusions1. A rough, porous coating could be prepared on the magnesium alloy surface by a micro-arc oxidation (MAO) technique.2. Both the bare ZK60alloy and the MAO coated alloy were non-cytotoxicity. Cell affinity of ZK60alloy was significantly improved by the MAO coating treatment which promoted cell adhesion and growth and showed good biocompatibility.3. Both the bare ZK60alloy and the MAO coated alloy could promote cell osteogenic differentiation, mineralization and osteogenesis-related gene expressions, showing good osteogenic induction. And the coated alloy even presented a higher activity due to the release of silicate from the coating.4. Bare ZK60alloy showed hemolytic effect and the hemolytic ratio was reduced by MAO coating treatment to meet the requirement of a biological material. The MAO coated alloy exhibited good blood compatibility.5. The MAO coating technique significantly improved the biocompatibility and bioactivity of magnesium alloys, which was an ideal magnesium alloy surface modification method.6. In vivo degradation properties and biocompatibility of the materials were not carried out in this study, so further researches are required to validate the use of the MAO coated ZK60alloy as a new class of biodegradable orthopeadic implant material. |
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