| BackgroudNowadays, the implantation of the implants into the desired place may lead to bacterial infections on the material surface or along the bone/material interface despite total disinfection prior to the surgeries.These infections were often caused by bacterial adherence. After the bacterial proliferation periods, the infections associated with the implants can hardly be cured by traditional systemic antibiotic therapy due to the formation of biofilms. The biofilms can also protect the bacteria against the human defense system.It was reported that the infection rate inorthopedic and trauma surgeries for open fracture could be up to about 1% and 12-53%, respectively.Implant infections also lead to implant failure, a secondary surgery, osteomyelitis, member amputation, or even death. Although sterile operation and antibiotics were adopted in clinical situations, post-surgical infections are still some of the intractable problems.Hence, there is a pressing need for the development of biocompatible implants with antibacterial properties.Mg is one of the most reactive metals and, thus, susceptible to rapid dissolution, especially in chloride-containing aqueous solutions.Therefore, Mg and its alloys can degrade gradually in the body fluid environment after the bone healing procedure, which can eliminate the need for a second operation for implant removal. The biodegradability of Mg and its alloys can also eliminate the complications associated with the long-standing presence of implants in the body and the impact on computed tomography (CT) as well as other imaging examinations. Furthermore, Mg alloys have a high specific strength and a modulus that is comparable with that of human natural bones, thereby the Mg and its alloys would reduce the risk of the stress shielding effect in orthopedic applications.Additionally, as an essential metal element, Mg is rich in the human body and the degradation products from Mg are expected to be nontoxic to the surrounding tissue, due to the homeostatic control mechanisms. Once thesematerials degrade within the body, the degradation products are able to be metabolized and absorbed by the body.However, Mg usually exhibits a fast degradation rate before bone healing, which can lead to premature loss of the mechanical integrity and excess hydrogen evolution of the implant, resulting in the failure of the implantation.Fortunately, the degradation behavior of Mg can be tailored by the alloying elements.Calcium (Ca) is one of the most abundant metal elements in the human body, and is also the major component of human bones. Meanwhile, Ca can increase the corrosion resistance of pure Mg. It is reported that strontium (Sr) has the potential to stimulate bone formation and Sr ranelate has shown to be effective for treating osteoporosis by increasing bone mineral density and bone strength.Furthermore, it has been verified that adding a small amount of Ca and Sr into Mg-based alloys can improve the mechanical properties of pure Mg by the grain refinement mechanism.Berglund et al. further found that low amounts of alloying elements enhanced the corrosion resistance properties, with an optimal composition of 1wt% Ca and 0.5 wt% Sr. The ternary alloy exhibited a higher compressive strength than that of binary Mg-Ca alloys with similar Ca contents, and also showed negligible toxicity.On the other hand, multifunctional biodegradable metals are in great need for biomedical applications. It is known that Zinc (Zn) is an essential mineral component for hundreds of biological enzymes and transcription factors and is widely associated with the normal function or the structure of more than 300 proteins. Moreover, many studies demonstrated that Zn exhibits an antibacterial effect on Staphylococcus aureus (S. aureus). Thus, the addition of Zn in the Mg-Ca-Sr based alloys may produce a biomaterial with antibacterial properties.Herein, in this study, we want to combine the antibacterial property of Zn with the ternary Mg alloys (Mg-1.0Ca-0.5Sr) and fabricate the quaternary Mg-0Ca-0.5Sr-(2,4,6 wt%)Zn alloys, while the Mg-lCa-0.5Sr and Ti-6A1-4V alloys were selected as control. S. aureus was selected to conduct the in vitro antibacterialassays. The cytotoxicity of the alloys was also evaluated in vitro by indirect MTT assays. The antibacterial property coupled with biodegradability of the prepared Mg-Ca-Sr-Zn alloys would provide a new type of multifunctional orthopedic biomaterial.ObiectiveTo observe the effects of the four Mg-Ca-Sr-Zn alloys on osteoblast growth, proliferation. Based on the above result, to preliminary evaluate the antibacterial ability of the four material, verify the biocompatibility and antibacterial effect of the Mg-Ca-Sr-Zn alloy as the novel biodegradable implant material and provide the basis for subsequent animal experiments and clinical application.Methods1. Characterization of materials:Ion concentration of the alloy extracts were determined by ICP-AES; surface morphology of the material were observed by SEM; material grain boundaries and chemical composition of the material were measured by Energy dispersive spectroscopy and X-ray diffraction; pH and osmolality of alloy extracts from Hank’s and PBS buffer solution were tested by pH meter (Sartorius, PB-10) and Micro-Sample Osmometer (Fiske 210); hydrogen release rate of alloys immersed in Hank’s were determined by a homemade device,According to the above methods to achieve a comprehensive evaluation and comparion of corrosion resistance and mechanical properties. Of different alloys.2. the biocompatibility of materials.Extracts were prepared according to the ISO 10993-12 standard (sample surface area/extraction medium=1.25cm2/ml), respectively. The a-MEM medium supplemented with 10%FBS was used as the control group. the cell morphology cultured with different alloys extracts were observed by a microscopy; using LDH assay to evaluate the toxicity of different alloys to cells; the impact of different alloys extracts to the cell proliferation was detected by MTT method; using Live/Dead assay to observe the cell viability cultured with different alloy extracts; Based the above result to comprehensively evaluate the biological safety of four Mg-1.0Ca-0.5Sr-xZn(x=,2,4,6) alloys.3. Antibacterial properties of materials:we use the indirect contact to evaluate antibacterial properties of different materials extracts on Staphylococcus aureus, and draw a curve according to antibacterial activity of different time piont; we carry out the direct contact assay to evaluate the antibacterial properties of material on Staphylococcus aureus; Live/Dead assay to evaluate the effect of different alloys on Staphylococcus aureus viability and observe the live/dead bacterial numbers; DAPI staining assay and electron microscopy to observe the number, shape of bacterial on,the surface of alloys and evaluate the adhesion properties of different materials,Based the above result to assess and compare antibacterial properties of four different magnesium alloys on Staphylococcus aureus.Results1.1 Characterization of materials:1.1.1 Components and elements of Mg-1.0wt% -0.5Sr-x wt.% Zn (x=0,2,4,6) were determined by ICP-AES, and the result was the same with our design.1.1.2 The SEM of the Mg-1.0wt% -0.5Sr-x wt.% Zn (x=0,2,4,6) alloys are showed that with the increasing Zn content in the Mg-1Ca-0.5Sr alloys, the grain showed a finer grain size, indicating that the addition of Zn can refine the grain size; the result of the EDS showed a large number of Ca and Zn among the grain boundary of Mg-1.0wt%-0.5Sr-x wt.% Zn(x= 0,2,4,6) alloys, indicating that calcium and zinc mainly act as a form of a second phase in the grain boundaries of the alloys.1.1.3 The XRD result showed that those Mg-Ca-Sr-Zn alloys were mainly composed of α-Mg, Mg2Ca,CaZn3,Ca2Mg5Zn13,Ca2Mg6 Zn3, Ca2Mg6Zn3,Mg17Sr2. It is also noticeable that Ca2Mg6Zn3 of the intermetallic phase increased with the increase of Zn1.1.5 The result of pH analyzer and Micro-Sample Osmometer (Fiske 210) showed that pH of materials immersed in Hank’s liquid is gradually increasing with the immersion time, pH of the zinc addition magnesium alloys tend to keep steady finally (pH values were 8.75-10.75), while pH of the ternary Mg-Ca-Sr alloy are increasing and a steady trend has not been seen during the entire experiment. On the other hand, significant difference between pH and osmotic pressure of the four different materials extract which are diluted by the PBS solution were not observed except for the pH of Mg-1Ca-0.5Sr showed a slight significant difference compared with other materials groups but all of them maintained within the desired range of bacterial activity.1.1.6 Immersion and release hydrogen experiments showed that after zinc added into the magnesium alloys, the corrosion resistance and mechanical properties have been improved.1.2 The biocompatibility of the materials1.2.1 The LDH toxic test results show that after co-cultured with the MC3T3-E1 cells for 24h, no significant difference among the zinc content of the magnesium alloy material group were observed when compared with the control group. The result indicated that all of the magnesium alloys have no cytotoxicity.1.2.2 Four magnesium materials extract groups and the control group cultured with MC3T3-E1 cell for 2,4 days, no significant difference between the groups have been observed, and in line with standard 0, when cultured with six days, compared with the control group, cell proliferation rate of magnesium alloy groups have declined and Mg-1Ca-0.5Sr-6Zn and Mg-1Ca-0.5Sr group show a statistical difference but both in line with standard 0-1 grade.1.2.3 The result of Live/Dead assays show that cells cultuled with different alloy extracts act a good adherent and show a normal morphology, red dead cells have not been observed obviously.1.3 The antibacterial properties of materials1.3.1 The result of different materials extracts immersed at different time points co-cultured with the bacterial for 24 hours found that with the immersing time increasing, the antibacterial properties of Zn-containing magnesium alloy material exhibit a gradually increasing trend. After co-culture with the 72 hours antibacterial rate of the three Zn-containing magnesium alloy material were close to 100%, Zn content was positively correlated with the strength of the antibacterial properties of magnesium alloys and magnesium alloy materials without Zn also showed a relatively slight antibacterial properties, and the antibacterial properties during the whole process showed no significant change.1.3.2 Materials and bacterial co-culture after 6 hours showed:Four magnesium alloy material surface bacteria Jie strong antibacterial properties, calcein staining showed that:the co-culture of bacteria magnesium alloy material surface almost died; DAPE experiments show:surface of the anti-bacterial adhesion and Zn contents were positively correlated; electron microscopy results showed that:the material after co-cultured with almost no change in the form of bacteria, the number relative to the control group decreased significantly.1.3.3 Bacterial co-cultured for 4 hours and materials calcein/PI, DAPI and SEM results showed that the four magnesium alloys have good good antibacterial and anti-adhesive properties.Conclusion1. Four magnesium alloy by Mg2Ca, CaZn3, Ca2Mg5Zn13, Ca2Mg6 Zn3, Ca2Mg6Zn3, Mg17Sr these compounds, but with the increase in the Zn content in the material, the content appears Ca2Mg6Zn3 increases with increasing addition of Zn content, the grain size of the material surface to be significantly refined;2. The four materials have a good biocompatible and can be ideal implant materials3.Mg-1.0Ca-0.5Sr-6Zn alloy better than the other three kinds of materials can have better corrosion resistance and antibacterial properties;4. The magnesium alloy body of calcium, strontium, zinc and antibacterial mechanism of biological safety issues not covered in this and other tests, and thus its ability to become the kind of novel biodegradable implants pending further post-test verification. |
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