| BackgroudIn recent years, with the rapid development of traffic industry, transportation industry and construction industry and the arrival of the aging population in China, more and more patients with fracture need implant fixation materials. The implant fixation materials commonly used in clinical were made by titanium alloys, stainless steels and cobalt chromium alloys. As the permanent implant materials, those materials had many disadvantages such as easy to cause stress shelter, delaying the healing of fracture, need the second operation, inflammatory reaction causing by abrasive dust and toxic metal cation,and so on.Magnesium alloys, that 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, are the new class of biodegradable metal materials and has the potential of great medicinal value.Unfortunately, the rapid degradation rate of Mg alloy cann’t meet the requirement of rigid internal fixation, in addition, accompanied by the release of hydrogen gas upon degradation, limiting their clinical applications. Alloying can significantly modify the corrosion performance of magnesium alloy and now is the focus of the medical magnesium alloy for medical application.Some elements of magnesium alloy is harmful to human body. Aluminium element is toxic to neurons which is associated with dementia and alzheimer’s disease. Some rare earth elements have the liver toxicity such as praseodymium element, cerium element and yttrium element. Cerium element and zirconium element have cytotoxicity, Lithium element has the potential teratogenic effects. Thus, the design of the novel biomedical magnesium alloy system should be based on the alloy elements of non-toxic effect.Our previous studies suggested that Mg-1.0 wt.% Ca alloy had excellent biocompatibility and better corrosion resistance which would be the ideal biodegradable material in vitro, but it also showed that Mg-Ca alloy had the rapid degradation rate that could notmaintain enough mechanical intensity for a long time. Thus, the main problem is urgent to solve is that how to maintain the balance between the time of fracture healing and the time of keep certain inernsity after degradation. Strontium, one of the essential trace element and non-toxic element to human body, can promote osteoblast proliferation, differentiation and meanwhile inhibiting osteoclast activity.Many studies suggested Sr element could obviously refine the grain size of magnesium and improve its corrosion resistance. Thus, we proposed that added the Sr element in the Mg-1.0 wt.%Ca alloy to improve its corrosion resistance and enhance the mechanical intensity and meanwhile to promote osteoblast differentiation that shorten the time of fracture healing.Four different content Sr of Mg-1.0Ca-xwt.%Sr alloys(x=0.2,0.5,1.0,2.0) were prepared by materials science and engineering college of Peking University,and in vitro experiments had showed its excellent corrosion resistance.However,As the novel biodegradable metallic material for medical application, its detailed biological is still unclear.ObiectiveTo observe the effects of the four Mg-Ca-x Sr alloys on osteoblast growth, proliferation, differentiation, mineralization and collagen secretion and hemolysis. Based on the above result, to preliminary evaluate the biocompatibility and hemcompatibility of the four material, verify the safety and effectiveness of the Mg-Ca-Sr alloy as the novel biodegradable implant material and provide the basis for subsequent animal experiments and clinical application. Then, to further explore the Mg-Ca-Sr alloy mediated the osteogenetic differentiation signaling pathways.Methods1. Characterization of materials:were employed the ICP-AES, microscopy and scanning electron microscope(SEM), energy dispersive spectrum(EDS) and X-ray diffraction(XRD) to observe and analysis the microstructure and chemical composition of four Mg-Ca-x Sr alloys. The pH value of four Mg-Ca-x Sr alloy extracts was tested by pHS-2c digital pH meter and protein adsorption experiment was used to explore the protein adsorption capacity among the four Mg-Ca-x Sr alloys.2. the biocompatibility of materials:Extracts were prepared according to the IS010993-12 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 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 viability and cell proliferation was detection by MTT method. The hemolytic test was carried out to assess the blood compatibility. Based the above result to comprehensively evaluate the biological safety of four Mg-1.0Ca-x Sr alloys.3. Osteogenetic differentiation:The alkaline phosphatase (ALP) activities were determined by a colorimetric assay using an ALP reagent containing p-nitrophenyl phosphate (p-NPP) as the substrate, mineralized nodule formation was observed using Alizarin Red S staining and Collagen secretion was quantified by Sirius Red staining. The expressions of ALP, OPN, OCN, OSX, RUNX2 and BMP-2 genes were further evaluated using the real-time polymerase chain reaction (Real-time PCR).4. Osteogenesis signaling pathways:The protein expressions of p-ERK1/2, p-JNK and p-P38 related with MAPK signal pathway were detected by Western Blotting.Results1.1 Characterization of materials:1.1.1 The actual compositions of the Mg-1.0wt%-x wt.%Sr (x=0.2,0.5,1.0,2.0) alloys, that was determined by ICP-AES analysis, was the same with our design.1.1.2 The optical micrographs of the ternary Mg-1.0Ca-x wt% Sr(x=0.2,0.5,1.0,2.0) alloys are showed that with the increasing Sr content in the Mg-1Ca alloys, the grain showed a finer grain size, indicating that the addition of Sr can refine the grain size.1.1.3 SEM images were indicated that, with further increasing Sr content, more and coarser second phase particles were also presented in these alloys. It also showed that the second phase was mostly located at the boundaries of grain. EDS revealed that the grain boundaries of the Mg-l.OCa-x wt.%Sr alloys contained higher amounts of Sr and Ca.1.1.4 The XRD result showed that those Mg-Ca-x Sr alloys were mainly composed of α- Mg, Mg2Ca and Mg17Sr2. It is also noticeable that the Mg17Sr2 intermetallic phase increased with the increase of Sr.1.1.5 The Changes of pH value in the SBF after heat treated alloys soaked 7d showed that the pH value was gradually increased as the extension of soaking time and all the pH value is between 8 and 11.1.1.6 The early protein adsorption results showed that there is no obvious difference among four alloys.1.2 The biocompatibility of the materials1.2.1 The LDH levels of the Mg-1Ca-0.2Sr alloy, Mg-1Ca-0.5Sr alloy, Mg-1Ca-1.0Sr alloy,Mg-1Ca-2.0Sr alloy and control group were (906.9±105.1) U/L, (956.7±156.2) U/L, (906.0±123.9)U/L,(925.8±40.5)U/L and (967.0±106.9)U/L, respectively and there were no significant differences among the five groups (P>0.05). The result indicated that the four alloys were no cytotoxicity.1.2.2 The MC3T3-E1 cells cultured with the extract of four alloys for 72h was observed under a microscope. The images showed that adherent cells exhibited a clear outline and spindle morphology. Visible intracellular particles were observed in the cytoplasm and there were no significant cell lysis or death. The cell morphology toxicity was 0 grade.1.2.3 Cell proliferation at 1,4 and 7 days of incubation were measured by MTT. The results indicated that the OD value of four alloy groups and the control group gradually increased with the prolonged incubation time, and the difference was statistically significant(P<0.05). But there was no statistically difference among the four alloys at the different time point (P>0.05) and there is no difference between the four groups and the control group (P>0.05).1.2.4 Cell relative growth rate (RGR) was calculated based on the OD value of the cell proliferation. At the day l,the RGR value of Mg-1.0Ca-0.2Sr alloy,Mg-1.0Ca-0.5Sr alloy,Mg-1.0Ca-1.0Sr alloy and Mg-1.0Ca-2.0Sr alloy were 93.2%,88.3%,104.2% and 94.6%,respectively. At the day 4, the RGR value of Mg-1.0Ca-0.2Sr alloy, Mg-1.0Ca-0.5Sr alloy, Mg-1.0Ca-1.0Sr alloy and Mg-1.0Ca-2.0Sr alloy were 88.9%,82.1%,89.1%, and 101.5%, respectively. At the day 7,the RGR value of Mg-1.0Ca-0.2Sr alloy,Mg-1.0Ca-0.5Sr alloy, Mg-1.0Ca-1.0Sr alloy and Mg-1.0Ca-2.0Sr alloy were 91.8%,98.3%,93.7%and 104.5%, respectively. According to the table 2-2, the cytotoxic reaction of four alloys was 0 or 1 grade.1.2.5 The hemolytic test was carried out to assess the blood compatibility of the magnesium alloys and the HR value of Mg-1Ca-x wt.% Sr(x=0.2,0.5,1.0,2.0) is 0.21%,1.48%,1.05%,2.01%, respectively. According to ISO 10993-4 standard that the HR of the materials to be used in blood environment has to be less than 5%, the results indicated that the four samples had the good hemcompatibility.1.3 Osteogenic differentiation1.3.1 There were no significant differences among the four groups and the control group in the intracellular total protein synthesis. The ALP activity of the Mg-1.0Ca-0.5Sr, Mg-1.0Ca-1.0Sr and Mg-1.0Ca-2.0Sr were signicantly higher than control group (p<0.05), meanwhile, the ALP activity of the Mg-1.0Ca-2.0Sr alloy was obvious higher than the other three alloys(p<0.05).1.3.2 Extracellular matrix mineralization measured by Alizarin Red staining and the result showed that more extracellular matrix mineralization nodules were visibly increased in Mg-1.0Ca-2.0Sr alloy group. According to the quantitative results, it showed that the relative value of mineralized nodules cultured by Mg-1.0Ca-2.0Sr alloy extract significantly enhanced compared to the other three alloy (P< 0.05) and the control group(P<0.05), respectively. Thus,the results indicated that Mg-1.0Ca-2.0Sr alloy could significantly promote the osteogenesis mineralization.1.3.3 Collagen secretion was observed and quantified by the Sirius Red staining and the result showed that more and denser collagen was secreted by Mg-1.0Ca-2.0Sr alloy compared to the other three alloys and the control group. According to the quantitative results showed that collagen secreted and deposition inducing by Mg-1.0Ca-2.0Sr alloy significantly promoted compared to the other three alloys (P< 0.05) and the control group(P< 0.05), respectively. It indicated that Mg-1.0Ca-2.0Sr alloy could significantly promote extracellular matrix collagen secretion.1.3.4 The expressions of osteogenisis-related genes were evaluated using real-time PCR and the results indicated that the Mg-1.0Ca-2.0Sr alloy significantly induced the expression of osteogenetic differentiation related genes including RUNX2, OSX, OCN, BMP-2 and ALP (p<0.05), however inhibited the gene expression of OPN significantly (P<0.05).1.4. Osteogenetic differentiation of MAPK signaling pathwaysBy western blot analysis, phosphorylation of ERK1/2 (p-ERK1/2) was detected at 5 min after treatment of MC3T3-E1 cells with osteo-inductive medium. The increase of relative amount of p-ERK (p-ERK 1/2/ERK1/2) was noticed with time, and reached the peak value at around 30 min. The ratio of p-ERK1/2/ERK1/2 declined with the further increase in time upon 30 min, reaching almost the same level with pre-osteogenic induction (0 min). However, no detectable activation of JNK and P38 pathway could be observed at 5min,15min,30min and 60min.Thus, according to the result of all the above, we speculated that Mg-1.0Ca-2.0Sr may be involved in stimulating osteogenic differentiation of MC3T3-E1 cells via ERK1/2 pathway of MAPK signal pathway.Conclusion1.Those Mg-Ca-x Sr alloys were mainly composed of a-Mg, Mg2Ca and Mg17Sr2. It is also noticeable that the Mg17Sr2 intermetallic phase increased with the increase of Sr content. In addition, with the increasing Sr content in the alloys, the grain showed a finer grain size, indicating that the addition of Sr can refine the grain size.2.The early protein adsorption results showed that there is no obvious difference among four alloys.3.All the four alloys had non-cytotoxicity and showed good biocompatibility. These alloys all are the ideal implant materials.4.According to ISO 10993-4 standard that the HR of the four alloys has to be less than 5% and the results indicated that the four alloys had the good hemcompatibility.5.Mg-1.0Ca-2.0Sr alloy could promote cell osteogenic differentiation, mineralization and collagen secretion, showing excellent osteogenic induction.6.Mg-1.0Ca-2.0Sr alloy significantly induced the expression of osteogenetic differentiation related genes including RUNX2, OSX, OCN, BMP-2 and ALP, however inhibited the gene expression of OPN significantly.7.we speculated that Mg-1.OCa-2.0Sr alloy may be involved in stimulating osteogenic differentiation of MC3T3-E1 cells via ERK1/2 pathway of MAPK signal pathway.8.1n vivo the biocompatibility and osteogenesis differentiation of the Mg-Ca-Sr alloys were not carried out in this study, so further researches are required to validate the use of the Mg-Ca-Sr alloys as the novel biodegradable implant materials. |
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