Evaluation of toxicity and biocompatibility of a novel Mg-Nd-Gd-Sr alloy in the osteoblastic cell

Evaluation of toxicity and biocompatibility of a novel Mg-Nd-Gd-Sr alloy in the osteoblastic cell | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Evaluation of toxicity and biocompatibility of a novel Mg-Nd-Gd-Sr alloy in the osteoblastic cell Yadong Xie, qinglin yang, Xiaorong Liu, ben xie, Xiaobo Zhang, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2553083/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Jul, 2023 Read the published version in Molecular Biology Reports → Version 1 posted 4 You are reading this latest preprint version Abstract Background We investigated the toxicity and biocompatibility of a novel Mg-3Nd-1Gd-0.3Sr-0.2Zn-0.4Zr (abbreviated to Mg-Nd-Gd-Sr) alloy in the osteoblastic cell line MC3T3-E1 as osteoblasts play an important role in bone repair and remodeling. Methods: We used cytotoxicity tests and evaluation of cell damage and apoptosis to investigate the effects of the Mg-Nd-Gd-Sr alloy on osteoblastic cells. Cell bioactivity, cell adhesion, cell proliferation, mineralization, ALP activity, and expression of BMP-2 and OPG by osteoblastic cells were also used to investigate the biocompatibility of Mg-Nd-Gd-Sr alloy. Results The results showed that the Mg-Nd-Gd-Sr alloy had no obvious cytotoxicity, and did not induce apoptosis or cause damage to MC3T3-E1 cells. Compared with the control group, the number of adherent cells within 12 hours was increased significantly in each experimental group (P < 0.05); the OD value of MC3T3-E1 cells was increased significantly in each experimental group on days 1 and 3 of culture (P < 0.05); the number of mineralized nodules formed in each experimental group was significantly increased (P < 0.05), and ALP activity was significantly increased in each experimental group (P < 0.05). RT-PCR results showed that the mRNA expression of BMP-2 and OPG was significantly higher in each experimental group compared with the control group (P < 0.05). Western blotting showed that the Mg-Nd-Gd-Sr alloy extract significantly increased the protein expression of BMP-2 and OPG compared with the control group (P < 0.05). Conclusions Our data indicated that the novel Mg-Nd-Gd-Sr-Zn-Zr alloy had no obvious cytotoxic effects, and did not cause apoptosis or damage to MC3T3-E1 cells; meanwhile it promoted cell adhesion, cell proliferation, mineralization, and ALP activity of osteoblasts. During this process, there was an increase in the expressions of BMP-2 and OPG mRNAs and proteins. Magnesium alloy Osteoblast MC3T3-E1 cells Damage Biocompatibility Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Introduction Biodegradable magnesium alloys are currently a hot topic in research into biomaterials. Compared with other metals, magnesium alloys can be degradable, and the degradation product, magnesium ions, can be fully excreted through the kidneys and intestines, thus avoiding secondary operations. Magnesium alloys are light in weight and have an elastic modulus equivalent to natural bone, thus reducing the probability of stress-shielding effects and helping fracture healing. Magnesium alloys have superior strength and extension compared with other metals. The fracture toughness of magnesium alloys is 15–40 MPa and the fracture toughness of natural bone is 36 MPa, both higher than that of ceramic biomaterials such as hydroxyapatite. Due to the biological activity of magnesium, ions degraded from magnesium alloys promote an osteogenic response and strengthen the contact between implant materials and bone tissue [1-4] . However, the clinical application of magnesium alloys is limited by their rapid degradation and uneven corrosion in physiological environments. In recent years, various methods have been used to strengthen the corrosion resistance of magnesium alloys in physiological environments, thereby solving problems such as excessive magnesium ion concentration, hydrogen generation, and local high pH values in the body due to the rapid degradation of magnesium alloys. An effective way to improve the properties of magnesium alloys is to add other suitable alloying elements, especially rare earth elements. When used to improve the corrosion resistance and mechanical properties of magnesium alloys, the elements that are added, such as Zn and Sr, have the ability to induce osteoblast differentiation, which can further promote fracture healing [5] . In our previous work we prepared a new type of Mg-3Nd-1Gd-0.3Sr-0.2Zn-0.4Zr (abbr.: Mg-Nd-Gd-Sr) alloy using the gravity casting method, in which Gd, Nd, Zr, Sr, and Zn were added at an appropriate ratio [6] . To date, there has been no report on the effect of Mg-Nd-Gd-Sr alloy on osteoblasts. Therefore, in this study we evaluated the biocompatibility of the new Mg-Nd-Gd-Sr alloy through cytotoxicity, cell viability, apoptosis and cell damage experiments. We also investigated the biological functions of the new Mg-Nd-Gd-Sr alloy on osteoblast adhesion, proliferation, and mineralization, and analyzed the mechanism via which the new Mg-Nd-Gd-Sr alloy affects the function of osteoblasts by analyzing the expression of bone morphogenetic protein-2 (BMP-2) and osteoprotegerin (OPG) at the protein level and BMP, OPG and Collagen type I (Col-I) at the mRNA level. The present study attempted to lay an experimental foundation for the clinical application of novel magnesium alloys as orthopedic implant materials. Materials And Methods 1.1 Materials The Mg-Nd-Gd-Sr alloy was obtained from the School of Materials Science and Engineering, Nanjing Institute of Technology. The composition of the Mg-Nd-Gd-Sr alloy is provided in Table 1 . Table 1 . Chemical composition of the Mg-Nd-Gd-Sr alloy MC3T3-E1 cells were provided by the Chinese Academy of Science Type Culture Collection (China). 1.2 Preparation of extracts The Mg-Nd-Gd-Sr alloy was prepared as disc-shaped samples with a diameter of 10 mm and a height of 2 mm, which were polished with metallographic emery paper to 1000 grits, followed by ultrasound washes in ethanol and distilled water. Prior to testing, samples were sterilized using ethylene oxide. Extracts were prepared using alpha-modified minimum essential medium (α-MEM) cell culture medium as the extraction medium, and with a ratio of surface area of samples to volume of extraction medium of 1.25 cm 2 /mL in a humidified incubator at 95% relative humidity and 5% CO 2 at 37°C for 24 hours [ 7 , 8 ] . After extraction, the extracts were diluted with α-MEM to make 25%, 50%, 75% and 100% groups. All the extracts were stored in a refrigerator at 4°C for use within 3 days. 1.3 Cytotoxicity testing The CCK-8 assay was used to evaluate the cytotoxicity of the Mg-Nd-Gd-Sr alloy to osteoblasts. Cytotoxicity tests were carried out by indirect contact, where the cells were cultured in extracts of 25%, 50%, 75% and 100% concentration. Simple α-MEM was chosen as the negative control and α-MEM containing 0.64% phenol was the positive control. After 1, 3 or 5 days in culture, 10 µL CCK-8 was added to each well and incubated at 37°C for 2 hours in the dark. The optical density (OD) at 490 nm was measured with a spectrophotometer (Wellscan MK3, Labsystems Diagnostics Oy, Vantaa, Finland). The cell relative growth rate (RGR) was calculated according to the following equation: RGR (%) = (OD t /OD n) ⋅ 100%, where ODt is the OD value of the tested group, and ODn is the OD value of the negative group. The cytotoxicity of the Mg-Nd-Gd-Sr alloy was evaluated according to the toxicity grading method (Table 2 ). Table 2 Cytotoxicity grading Cytotoxicity RGR(%) 0 Grade ≥ 100 1 Grade 75–99 2 Grade 50–74 3 Grade 25–49 4 Grade 1–24 5 Grade 0 Table 2 . Cytotoxicity grading 1.4 Apoptosis Apoptosis was detected by flow cytometry using annexin-V/FITC-PI assay. MC3T3-E1 cells that were growing well were seeded into 6-well plates and treated with different concentrations of Mg-Nd-Gd-Sr alloy extract or with cell culture medium without extract (control group). The cells were trypsinized at 1, 3, and 5 days of culture, rinsed thrice with PBS, then resuspended in the eluent. After the addition of Annexin V-FITC (5 µL) and PI (10 µL) reagents, the specimens were placed into an ice box and rapidly analyzed using flow cytometry. 1.5 Cell damage Analysis of reactive oxygen species (ROS) was used to evaluate any damage caused to osteoblasts by the Mg-Nd-Gd-Sr alloy extract. MC3T3-E1 cells that were growing well were seeded into 6-well plates and treated with different concentrations of Mg-Nd-Gd-Sr alloy extract or cell culture medium without extract (control group). On day 5 of culture, the cells were rinsed, covered with DCFH-DA medium (1 mL) containing a fluorescent probe, incubated at 37°C for 30 minutes, washed twice in PBS, and observed under a fluorescence microscope. 1.6 Cell bioactivity DAPI staining was used to analyze the bioactivity of MC3T3-E1 cells in the groups treated with different concentrations of extract or control medium. MC3T3-E1 cells were inoculated into 6-well cell culture plates, then different concentrations of Mg-Nd-Gd-Sr alloy extract or cell culture medium without extract (control group) were added. After 3 days of culture, the culture medium was discarded, the slides were washed thoroughly with PBS and DAPI was added for 5–10 minutes. Then the slides were rinsed under running water, and cell bioactivity was observed under a fluorescence microscope. 1.7 Cell adhesion MC3T3-E1 cells were cultured for 3 days then trypsinized and prepared into a cell suspension (1 mL). An aliquot of the cell suspension (100 µL) was diluted with 900 µL PBS, and 10,000 cells were counted using a hemocytometer. Cell suspensions containing 50,000, 25,000, 12,500, 6,250, and 3,125 cells were then obtained, seeded into 96-well plates and cultured for 4 hours. Then CCK-8 (10 µL per well) was added, cells were returned to culture for 2 hours, then the OD value was measured using a microplate reader. The OD values were used to create a standard adherence curve of MC3T3-E1 cells. MC3T3-E1 cells were cultured for 3 days then trypsinized and prepared into a cell suspension (1 mL). An aliquot of the cell suspension (100 µL) was diluted with 900 µL PBS, and 10,000 cells were counted using a hemocytometer. The cells were then inoculated into 96-well plates and treated with different Mg-Nd-Gd-Sr alloy extracts or the same volume of cell culture medium, three wells per group. After 2, 4, 6, 8, 10, or 12 hours of culture, the cell culture medium in each group was removed, and the cells were rinsed twice with PBS then cultured with CCK-8 (10 µL per well). Two hours later, the OD value was measured using a microplate reader. The number of adherent MC3T3-E1 cells in each group was calculated from the standard curve, and the cell adhesion curve in each group was drawn for statistical analysis. 1.8 Cell proliferation The proliferation of MC3T3-E1 cells was quantitatively assessed by CCK-8 assay. MC3T3-E1 cells that were growing well were inoculated into 96-well plates and treated with different concentration of Mg-Nd-Gd-Sr alloy extract or cell culture medium without extract (control group). After 1, 3, and 5 days of culture, CCK-8 (10 µL per well) was added and the cells were cultured for another 2 hours. The OD value was measured at 450 nm using a microplate reader. 1.9 Cell mineralization MC3T3-E1 cells that were growing well were inoculated into 6-well plates and treated with different concentrations of Mg-Nd-Gd-Sr alloy extract or cell culture medium without extract (control group). After 5 days of culture, osteogenic induction medium was added, and the cells were cultured for a further 21 days. Cultures were then fixed for 10 minutes, rinsed and stained using alizarin red. After 30 minutes, the cells were rinsed and observed under a light microscope. 1.10 Alkaline phosphatase(ALP) staining and ALP activity assay The differentiation of MC3T3-E1 cells was assessed by measuring alkaline phosphatase (ALP) activity. MC3T3-E1 cells that were growing well were inoculated into 6-well plates and treated with different concentrations of Mg-Nd-Gd-Sr alloy extract or cell culture medium without extract (control group). The cells were then fixed with the fixing solution, rinsed and stained in the dark with ALP staining solution for 15 minutes. After rinsing, the cells were counterstained using nuclear fast red for 3 minutes. Finally, the cells were rinsed and observed under a light microscope. After 1, 3, or 5 days of culture, MC3T3-E1 cells were lysed using 0.5% Triton X-100 solution at 4°C for 1 hour. A substrate developer was added, and the culture plate was shaken well on a shaker then placed in the incubator for 30 minutes until a full reaction was achieved. The reaction was terminated, and the OD value was measured using a microplate reader. 1.11 Expression of BMP-2 and OPG proteins Immunocytochemical staining was used to determine the expression of BMP-2 and OPG. MC3T3-E1 cells that were growing well were inoculated into 6-well plates containing slides and treated with different concentrations of Mg-Nd-Gd-Sr alloy extract or cell culture medium without extract (control group) for 3 days. The cells were rinsed three times with PBS, fixed with 4% paraformaldehyde for 15 minutes, and air dried for 5 minutes. After rinsing three times with PBS, the specimens were incubated with 0.5% Triton X-100 for 20 minutes, blocked with peroxidase, and incubated at room temperature for 10 minutes. After washing, the specimens were incubated with goat serum working solution at room temperature for 10 minutes, then after removal of the serum, the specimens were incubated with the primary antibody at 4°C overnight. On the second day, the specimens were incubated with the secondary antibody at room temperature for 15 minutes, then incubated with horseradish peroxidase-labeled streptomyces ovalbumin at room temperature for 15 minutes, and developed with DAB chromogenic solution for 10 minutes, counterstained with hematoxylin for 20 seconds, washed with tap water for 5 minutes, mounted with gum, and observed under a light microscope. 1.12 Expression of BMP-2 and OPG mRNA RT-PCR was used to analyze the mRNA expression of BMP-2 and OPG. MC3T3-E1 cells that were growing well were seeded into 24-well plates at a density of 1×10 4 /mL and treated with different concentrations of Mg-Nd-Gd-Sr alloy extract or cell culture medium without extract (control group). After 5 days of culture, the cells were collected and the mRNA expression of BMP-2 and OPG was detected using quantitative fluorescence PCR. 2.13 Statistical analysis The statistical software SPSS13.0 (SPSS Inc., Chicago, IL, USA) was used to analyze the data. Differences between the groups were compared using one-way analysis of variance (ANOVA). The statistical significance was defined as 0.05, and a P -value < 0.05 indicated a statistically-significant differences. Results 2.1 Cytotoxicity testing MC3T3-E1 cells were cultured in different concentrations of Mg-Nd-Gd-Sr alloy extract for 1, 3, and 5 days, and the OD values were measured using a microplate reader (Table 3 ). Table 3 OD values and RGR in each group ( n = 3 ) Group 1d 3d 5d OD (Mean ± SD) RGR (%) OD (Mean ± SD) RGR (%) OD (Mean ± SD) RGR (%) 0% group 0.365 ± 0.028 100 0.820 ± 0.1430 100 1.464 ± 0.1099 100 25% group 0.453 ± 0.068 124.1 1.065 ± 0.050 129.8 1.535 ± 0.236 104.8 50% group 0.540 ± 0.0386 147.9 1.291 ± 0.283 157.4 1.691 ± 0.317 115.5 75% group 0.557 ± 0.304 152.6 1.334 ± 0.166 162.7 1.528 ± 0.073 104.4 100% group 0.572 ± 0.009 156.7 1.404 ± 0.091 171.2 1.504 ± 0.091 102.7 Table 3 . OD values and RGR in each group (n = 3) At days 1 and 3, the OD values and RGR were increased significantly in each experimental group compared with the control group ( P < 0.05), and were also increased as the Mg-Nd-Gd-Sr alloy extract concentration increased. However by day 5 of culture, compared with the control group, the 25%, 75%, and 100% groups showed no significant increase in the OD values or RGR ( P > 0.05), whilst the OD values and RGR were the highest in the 50% group, which were significantly higher than those in the control group ( P < 0.05), and then showed a downward trend. At any time point, the RGR of MC3T3-E1 cells in any extract was greater than 100% (Fig. 1 ). Figure 1 . RGR of MC3T3-E1 cells in each group at the different time-points tested. Based on these results, the cytotoxicity of the Mg-Nd-Gd-Sr alloy was considered to be level 0, indicating that the Mg-Nd-Gd-Sr alloy had no obvious cytotoxic effect on MC3T3-E1 cells. 2.2 Apoptosis After 1, 3, or 5 days of culture with different concentrations of Mg-Nd-Gd-Sr alloy extract, apoptosis of MC3T3-E1 cells was analyzed by flow cytometry using Annexin-V/FITC-PI staining. Compared with the control group, no obvious apoptosis was observed in any experimental group ( P > 0.05). No obvious apoptosis was observed on days 1 or 3 of culture. On day 5, the apoptotic rate was increased to some extent in the 50%, 75%, and 100% extract groups compared with the control group ( P < 0.05), but it was still within the limits of acceptability (Fig. 2 ). Figure 2 . Apoptotic rate in each group on days 1, 3 and 5 of culture ( ∗ P < 0.05). 2.3 Cell damage After 5 days of culture with different concentrations of Mg-Nd-Gd-Sr alloy extract, MC3T3-E1 cells loaded with DCFH-DA probes were observed under a fluorescence microscope. Slight staining of the cells indicated a normal physiological metabolism, whilst cells with a stronger green fluorescence were damaged cells with increased reactive oxygen radicals. As the concentration of the Mg-Nd-Gd-Sr alloy extract increased, the number of damaged cells tended to increase (Fig. 3 ). Figure 3 . Reactive oxygen species of MC3T3-E1 cells in (A) control group, (B) 25% group, (C) 50% group, (D) 75% group, and (E) 100% group on day 5 of culture. 2.4 Cell bioactivity On day 3 of culture with different concentrations of Mg-Nd-Gd-Sr alloy extract, MC3T3-E1 cells were stained with DAPI and observed under a fluorescence microscope (Fig. 4 ). The nuclei of the MC3T3-E1 cells were stained an even blue and were equal in size, and the nuclear membrane was intact in each group. As the concentration of Mg-Nd-Gd-Sr alloy extract increased, the number of cells increased. Figure 4 . MC3T3-E1 cells stained with DAPI in (A) control group, (B) 25% group, (C) 50% group, (D) 75% group, and (E) 100% group on day 3 of culture. 2.5 Cell adhesion To study the influence of the Mg-Nd-Gd-Sr alloy on MC3T3-E1 cell adhesion, a standard adherence curve of MC3T3-E1 cells was drawn. With increasing time in culture, the number of adherent cells showed an increasing trend (Fig. 5 A). Subsequently, the cells were cultured with different concentrations of Mg-Nd-Gd-Sr alloy extract, and the OD value was measured by CCK-8 assay at different time-points to obtain the MC3T3-E1 cell adhesion curve (Fig. 5 B). Figure 5 . (A) Standard adherence curve of MC3T3-E1 cells, and (B) the number of adherent cells obtained from the cell adhesion curve in each group. Compared with the control group, within 12 hours, the number of adherent cells increased significantly in each experimental group ( P < 0.05). Moreover, the number of adherent cells showed an increasing trend in a concentration-dependent manner within 12 hours. 2.6 Cell proliferation The CCK-8 assay was used to investigate the proliferation of MC3T3-E1 cells cultured in different concentrations of Mg-Nd-Gd-Sr alloy extract. After 1, 3, or 5 days of culture, the OD value of MC3T3-E1 cells was measured. Compared with the control group, the OD value increased significantly in each experimental group on days 1 and 3 of culture ( P < 0.05; Fig. 6 ), and showed an increasing trend with the increase in concentration of Mg-Nd-Gd-Sr alloy extract. However, on day 5 of culture, the OD value did not change significantly in the 75% and 100% extract groups compared with the control group ( P > 0.05). Moreover, the cell proliferation peaked in the 50% extract group, which was significantly higher than that in the control group ( P < 0.05), but then reduced. Figure 6 . Cell proliferation of MC3T3-E1 cells in each group according to absorbance values on days 1 and 3 of culture ( * P < 0.05). 2.7 Cell mineralization After 21 days of culture with different concentrations of Mg-Nd-Gd-Sr alloy extract, MC3T3-E1 cells were stained with alizarin red and mineralized nodules were analyzed using Image-J software. Mineralized nodules of different shapes and sizes had formed in all groups by the end of 21 days of culture (Fig. 7 ). Figure 7 . Mineralized nodules formed in (A) control group, (B) 25% group, (C) 50% group, (D) 75% group, and (E) 100% group after 21 days of culture (alizarin red staining, magnification,×400). Compared with the control group, the number of mineralized nodules formed in each experimental group was significantly increased ( P < 0.05; Fig. 8 ), was highest in the 50% extract group, and then showed a downward trend. Figure 8 . The number of mineralized nodules formed in each group ( * P < 0.05). 2.8 Alkaline phosphatase assay Cells were cultured on slides in each group, and stained with ALP on day 3 of culture (Fig. 9 ). The cells in each group were positive for ALP, which was red in the nucleus. Figure 9 . MC3T3-E1 cells stained for ALP in (A) control group, (B) 25% group, (C) 50% group, (D) 75% group, and (E) 100% group on day 3 of culture. To study the influence of Mg-Nd-Gd-Sr alloy extract on the expression of ALP, a standard ALP curve of MC3T3-E1 cells was drawn. There was a certain relationship between OD value and ALP activity. An increased OD value indicated increased ALP activity (Fig. 10 A). The expression of ALP (OD value) was measured and the ALP activity in each group was calculated from the standard curve. ALP activity was significantly increased in each experimental group compared with the control group ( P < 0.05; Fig. 10 B). As the concentration of the Mg-Nd-Gd-Sr alloy extract increased, the activity of ALP also increased on days 1 and 3, and reached a peak in the 50% extract group on day 5, but then showed a downward trend. Figure 10 . (A) Standard ALP curve of MC3T3-E1 cells, and (B) activity of ALP obtained from the ALP curve in each group after culture for 1, 3, and 5 days. 2.9 Expression of BMP-2 and OPG Cells were cultured on slides in each group, stained immunocytochemically on day 3, and observed under the microscope. Compared with the control group, the expression of both BMP-2 (Fig. 11 ) and OPG (Fig. 12 ) was significantly increased in each experimental group on day 3 of culture ( P < 0.05), and showed an increasing tendency with the increase in the concentration of Mg-Nd-Gd-Sr alloy extract. Figure 11 . Expression of BMP-2 by MC3T3-E1 cells in (A) control group, (B) 25% group, (C) 50% group, (D) 75% group, and (E) 100% group on day 3 of culture (immunocytochemical staining). Figure 12 . Expression of OPG in MC3T3-E1 cells in (A) control group, (B) 25% group, (C) 50% group, (D) 75% group, and (E) 100% group on day 3 of culture (immunocytochemical staining). 2.10 The mRNA expression of BMP-2 and OPG Real-time fluorescence quantitative PCR was used to analyze the expression of BMP-2 and OPG mRNAs on day 5 of culture. The cells in each group continuously expressed BMP-2 and OPG mRNAs. Compared with the control group, the expression of BMP-2 and OPG mRNAs were both significantly increased in each experimental group ( P < 0.05; Fig. 13 ). Moreover, the expression of OPG mRNA reached a peak in the 75% extract group, whilst the expression of BMP-2 mRNA was highest in the 50% extract group. Figure 13 . The expression of BMP-2 and OPG mRNA in each group on day 5 of culture ( * P < 0.05). Discussion Although magnesium alloys have advantages as orthopedic implants [ 9 – 12 ] , magnesium alloy materials degrade too quickly in the human body and cannot yet meet the clinical requirements. The novel magnesium alloy Mg-Nd-Gd-Sr is prepared by gravity casting and has been confirmed to have good structure, mechanics and corrosion resistance [ 6 ] . However, it is necessary to clarify the effect of this novel alloy on osteoblast function before it can be used as an orthopedic implant material in clinical practice. The present study explored the effect of Mg-Nd-Gd-Sr alloy on the function of MC3T3-E1 osteoblastic cells. Biocompatibility of an implant is well reflected by in vitro cytotoxicity testing, as weaker cytotoxicity in vitro corresponds with weaker cytotoxicity in vivo [ 13 – 15 ] . In this study, CCK-8 was used for cytotoxicity analysis in MC3T3-E1 cells cultured with 25%, 50%, 75%, or 100% Mg-Nd-Gd-Sr alloy extract for 1, 3, or 5 days. The OD value of MC3T3-E1 cells was significantly increased after 1, 3, and 5 days of culture with different concentrations of Mg-Nd-Gd-Sr alloy extract. There was also a concentration-dependent increase in the OD value on days 1 and 3 of culture. However, on day 5 of culture, no significant increase was observed in the 25%, 75%, or 100% extract groups compared with the control group, and the OD value even showed a downward trend in the 75% and 100% extract groups but was still higher than that in the control group. Meanwhile, the OD value peaked in the 50% extract group. Results from the cytotoxicity test indicated that the Mg-Nd-Gd-Sr alloy extract had no toxic effect on MC3T3-E1 cells regardless of its concentration, and within a certain concentration range even promoted the proliferation of MC3T3-E1 cells. Previous research confirmed that osteoblasts exhibit better viability and differentiation when the magnesium ion concentration is at an appropriate level, while cell viability and differentiation are influenced when the magnesium concentration exceeds a certain limit. In this study, the magnesium alloy extract was added again on the 3rd day of culture, which could cause the magnesium ion concentration in the medium to exceed its critical limit, but MC3T3-E1 cells still proliferated in the medium containing high-concentration magnesium alloy extract. According to the ISO10993 standard [ 7 , 8 ] , the RGR of MC3T3-E1 cells was over 100% in all the different Mg-Nd-Gd-Sr alloy extract groups. This indicated that the Mg-Nd-Gd-Sr alloy extract had no obvious toxic effect on MC3T3-E1 cells, but instead promoted the proliferation of MC3T3-E1 cells. Early adhesion is a prerequisite for the future fate of osteoblasts, affecting cell proliferation and long-term survival. Research on cell adhesion is indispensable when studying the effect of Mg-Nd-Gd-Sr alloy on cell function [ 16 ] . Results from the cell adhesion experiment showed that the number of adherent cells was significantly higher in each experimental group than in the control group in the first 12 hours of culture, and moreover, cell adhesion showed an increasing trend with the increasing concentrations of Mg-Nd-Gd-Sr alloy extract. This may be related to the release of an appropriate level of magnesium ions from the degraded Mg-Nd-Gd-Sr alloy. Previous studies have shown that an appropriate concentration of magnesium ions is conducive to cell adhesion [ 17 ] . The results of our cell proliferation experiment also revealed that with time, the OD value increased significantly in each experimental group. On days 1 and 3 of culture, the OD value was increased significantly in each experimental group compared with the control group, and there was also a concentration-dependent increase in the OD value. This may be because the Mg-Nd-Gd-Sr alloy degrades and releases appropriate concentrations of magnesium ions, which then induce cell activation and promote cell proliferation by regulating the formation of osteoblast-related proteins [ 18 , 19 ] . The Mg-Nd-Gd-Sr alloy also contains other ions such as zinc and zirconium ions. Zinc ions are involved in early cell metabolism and promote cell proliferation [ 20 ] , while zirconium ions form ZrO 2 , creating a more suitable environment for cell adhesion and proliferation [ 21 ] . On day 5 of culture, the OD value of MC3T3-E1 cells was reduced in the 75% and 100% extract groups, indicating that the concentration of magnesium ions at this time has exceeded the optimum concentration. If the Mg-Nd-Gd-Sr alloy extract concentration continues to increase, the release of hydrogen will increase and the pH of the medium will continue to rise, which may have harmful and even fatal effects on cells [ 22 , 23 ] . Osseointegration of an orthopedic implant with the living bone is an orderly process. Bone marrow mesenchymal stem cells or osteoprogenitor cells are first recruited to the surface of the bone implant, and then a new bone matrix is formed after osteogenic differentiation and mineralization. Finally bone remodeling can be conducted [ 24 ] . Highly active alkaline phosphatase and calcified extracellular matrix are two representative indicators of the osteoblast phenotype, which indicate osteoblast maturation [ 25 ] . In this study, alizarin red staining was used to clarify the influence of Mg-Nd-Gd-Sr alloy extract on the mineralization of MC3T3-E1 cells. The number of mineralized nodules was significantly increased in each experimental group compared with the control group. The number of mineralized nodules was highest in the 50% extract group but decreased in the 75% and 100% extract groups, indicating that the Mg-Nd-Gd-Sr alloy extract promotes osteoblast mineralization and there is an optimal concentration. These findings indicated that some other ions such as strontium ions, as well as magnesium ions, promoted the mineralization of osteoblasts [ 26 ] . The biological function of implant materials was also evaluated based on their effects on apoptosis and cell damage. In this study, the Annexin V-FITC/PI assay was used to quantify apoptosis by flow cytometry, thereby evaluating the influence of the Mg-Nd-Gd-Sr alloy extract on MC3T3-E1 apoptosis. On days 1 and 3 of cell culture, there was no obvious apoptosis in any experimental group compared with the control group. On day 5 of cell culture, the apoptotic rate was significantly increased in the 50%, 75%, and 100% extract groups compared with the control group. This may be due to the fact that the concentration of Mg-Nd-Gd-Sr alloy extract increased after the culture medium was changed on day 3. However, excess magnesium ions can be metabolized through various channels in vitro. In summary, the Mg-Nd-Gd-Sr alloy does not promote apoptosis. However, a very high concentration of Mg-Nd-Gd-Sr alloy extract or poor conditions may have a certain apoptosis-inducing effect on cells. In this study, ROS production was used to evaluate the damage caused by Mg-Nd-Gd-Sr alloy extracts to osteoblasts. After 5 days of culture with Mg-Nd-Gd-Sr alloy extracts, the production of ROS was measured. The cells exhibited strong green fluorescence in each group. No significant difference was found in the proportion of cell damage between any of the experimental groups and the control group, indicating that the Mg-Nd-Gd-Sr alloy does not cause obvious cell damage. With increasing concentrations of the Mg-Nd-Gd-Sr alloy extract, the number of cells with strong green fluorescence showed an increasing trend, indicating that the Mg-Nd-Gd-Sr alloy extract at very high concentrations could damage cells under poor conditions. BMP is an important osteogenic factor that promotes bone formation and the differentiation of osteoblasts in the process of bone repair [ 27 ] . BMP-2, with its receptor, forms a heterologous receptor complex and induces intracellular signals in the SMAD complex, leading to the subsequent transcription of osteocalcin, osteopontin, and alkaline phosphatase. BMP-2 is involved in cell proliferation, differentiation, and apoptosis, thereby influencing the biological behaviors of the cells [28–32] . The ratio of OPG to RANKL is a key determinant of bone remodeling or bone resorption. When the ratio is > 1, the formation of osteoblasts is promoted; when the ratio is < 1, the formation of osteoclasts is promoted. Col-I is critical for hydroxyapatite deposition, has a good effect on cell adhesion, growth and function, and plays an important role in cell biological activity and osteogenicity [33, 34] . In this study, the expression of BMP-2 and OPG in MC3T3-E1 cells cultured with different concentrations of Mg-Nd-Gd-Sr alloy extract was detected by immunocytochemistry. The expression of BMP-2 and OPG in the cells increased significantly in each experimental group compared with the control group, and also showed a concentration-dependent increase. RT-PCR results showed that the expression of BMP-2, OPG, and Col-I mRNA in each experimental group was significantly increased compared with that in the control group. The expression of OPG and Col-I mRNA peaked in the 75% extract group, while the expression of BMP-2 mRNA was highest in the 50% extract group. This finding indicated that the Mg-Nd-Gd-Sr alloy extract promoted the expression of BMP-2, OPG, and Col-I mRNA in MC3T3-E1 cells. The increase in the expression of BMP-2 in MC3T3-E1 cells induced by the Mg-Nd-Gd-Sr alloy extract induced intracellular signals of the SMAD complex, upregulating the expression of osteogenesis-related genes and proteins, promoting adhesion, proliferation and mineralization of MC3T3-E1 cells, and ultimately influencing the cells’ biological behavior. Conclusion The Mg-Nd-Gd-Sr alloy had no obvious cytotoxicity and caused no apoptosis or damage in MC3T3-E1 cells; meanwhile it promoted cell adhesion, proliferation, and mineralization and had good biological functions. During this process, there was an increase in the expressions of BMP-2 and OPG mRNAs and BMP-2 and OPG proteins. This finding indicated that the Mg-Nd-Gd-Sr alloy extract upregulated the protein and mRNA expression of BMP-2 and OPG, thereby inducing intracellular signals and upregulating the expression of osteogenesis-related genes, ultimately promoting the adhesion, proliferation and mineralization of MC3T3-E1 cells. Declarations Funding This study was supported by the Scientific Research Projects of Gansu Province HealthIndustry(GSWSKY 2021-009); Scientific and Technology Research Projects of Lanzhou(2021-1-79):Natural Science Foundation of Gansu Province(20JR5RA369); and the National Natural Science Foundation ofChina(81960398). Competing interests The authors declared no potential conflcts of interest with respect to the research, author- ship and publication of this article. Availability of supporting data Data will be made available on request. Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors. Consent for publication Thisstudy was published with the informed consent of all authors. Authors'contributions Xie Yadong was responsible for the drafting of the article and the main experiments;Yang Qinglin and Xie Ben are responsible for some experiments and dataanalysis.Wang Yongping and others were responsible for experimental design and paper modification. Acknowledgements Thanks for thesupport of Health Industry Research Project of Gansu Province， Lanzhou Science and Technology Research Project, Natural Science Foundation of Gansu Province and National Natural Science Foundation of China. References Hou R, Victoria-Hernandez J, Jiang P, et al. In vitro evaluation of the ZX11 magnesium alloy as potential bone plate: Degradability and mechanical integrity. Acta Biomater, 2019, 97: 608-622. Dayaghi E, Bakhsheshi-Rad HR, Hamzah E, et al. Magnesium-zinc scaffold loaded with tetracycline for tissue engineering application: In vitro cell biology and antibacterial activity assessment. 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Cell adhesion on surface-functionalized magnesium. ACS Appl Mater Interfaces, 2016, 8(19): 11998-12006. Rude RK, Singer FR, Gruber HE. Skeletal and hormonal effects of magnesium deficiency. J Am Coll Nutr, 2009, 28(2): 131-141. Rubin H. Magnesium: The missing element in molecular views of cell proliferation control. Bioessays, 2005, 27(3): 311-320. Hernández-Escobar D, Champagne S, Yilmazer H, et al. Current status and perspectives of zinc-based absorbable alloys for biomedical applications. Acta Biomater, 2019, 97: 1-22. Zhao Y, Jamesh MI, Li WK, et al. Enhanced antimicrobial properties, cytocompatibility, and corrosion resistance of plasma-modified biodegradable magnesium alloys. Acta Biomater, 2014, 10(1): 544-556. Peron M, Bertolini R, Ghiotti A, et al. Enhancement of stress corrosion cracking of AZ31 magnesium alloy in simulated body fluid thanks to cryogenic machining. J Mech Behav Biomed Mater, 2020, 101: 103429. Amukarimi S, Mozafari M. 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BMP-2-releasing gelatin microspheres/PLGA scaffolds for bone repairment of X-ray-radiated rabbit radius defects. Artif Cells Nanomed Biotechnol, 2019, 47(1): 1662-1673. Kim K, Park J, Kim S. Bone morphogenetic protein-2 associated multiple growth factor delivery for bone tissue regeneration. J Pharm Investig, 2018, 48: 187–197. Rawadi G, Vayssière B, Dunn F, et al. BMP‐2 controls alkaline phosphatase expression and osteoblast mineralization by a Wnt autocrine loop. J Bone Miner Res, 2003, 18: 1842‐ 1853. Hashimi SM. Exogenous noggin binds the BMP-2 receptor and induces alkaline phosphatase activity in osteoblasts. J Cell Biochem, 2019, 120(8): 13237-13242. Yang B, Lin X, Yang C, et al. Sambucus Williamsii Hance promotes MC3T3-E1 cells proliferation and differentiation via BMP-2/Smad/p38/JNK/Runx2 signaling pathway. Phytother Res, 2015, 29(11): 1692-1699. Mushahary D, Wen C, Kumar JM, et al. Collagen type-I leads to in vivo matrix mineralization and secondary stabilization of Mg-Zr-Ca alloy implants. Colloids Surf B Biointerfaces, 2014, 122: 719-728. Slli YC，Worton L，Esteban L，et a1．Effects of continuous activation of vitamin D and Wnt response pathways on osteoblastic proliferation and differentiation. Bone, 2007, 41(1): 87-96. Cite Share Download PDF Status: Published Journal Publication published 05 Jul, 2023 Read the published version in Molecular Biology Reports → Version 1 posted Editorial decision: Major Revisions Needed 10 Apr, 2023 Reviewers agreed at journal 27 Feb, 2023 Editor assigned by journal 11 Feb, 2023 First submitted to journal 10 Feb, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2553083","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":179219453,"identity":"9a5a9e16-9792-41f2-8ff1-8735208c3776","order_by":0,"name":"Yadong Xie","email":"","orcid":"","institution":"Lanzhou University First Affiliated Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yadong","middleName":"","lastName":"Xie","suffix":""},{"id":179219454,"identity":"7beac5cf-40bf-4b69-a84c-44d15e26dbc7","order_by":1,"name":"qinglin 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(*\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05)\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-2553083/v1/d40b56277ef2690ee5e01856.png"},{"id":33638080,"identity":"9e706d22-5eaf-4624-af7b-eaa28a86fa20","added_by":"auto","created_at":"2023-03-01 16:54:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":203221,"visible":true,"origin":"","legend":"\u003cp\u003eApoptotic rate in each group on days 1, 3 and 5 of culture (*\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05)\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-2553083/v1/4d230c89bc97e60a6ea80c85.png"},{"id":33638775,"identity":"941262fc-606f-452e-a543-d07150206e44","added_by":"auto","created_at":"2023-03-01 17:02:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":13448037,"visible":true,"origin":"","legend":"\u003cp\u003eReactive oxygen species of MC3T3-E1 cells in (A) control group, (B) 25% group, (C) 50% group, (D) 75% group, and (E) 100% group on day 5 of culture\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-2553083/v1/e45bae60203c3f992876becf.png"},{"id":33638774,"identity":"15f6f065-1aaa-4497-a9d4-ee979d2a57d7","added_by":"auto","created_at":"2023-03-01 17:02:09","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":5277890,"visible":true,"origin":"","legend":"\u003cp\u003eMC3T3-E1 cells stained with DAPI in (A) control group, (B) 25% group, (C) 50% group, (D) 75% group, and (E) 100% group on day 3 of culture\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-2553083/v1/e8db5ca25ddf72def5e6bf98.png"},{"id":33638772,"identity":"757db7c5-0fff-4ec4-934f-13bb432ee31d","added_by":"auto","created_at":"2023-03-01 17:02:09","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":771936,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Standard adherence curve of MC3T3-E1 cells, and (B) the number of adherent cells obtained from the cell adhesion curve in each group\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-2553083/v1/b61575dbfc6e4971c809f5f2.png"},{"id":33638084,"identity":"0b02dcdf-c4d7-463d-b6c1-ea5412c72375","added_by":"auto","created_at":"2023-03-01 16:54:09","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":200003,"visible":true,"origin":"","legend":"\u003cp\u003eCell proliferation of MC3T3-E1 cells in each group according to absorbance values on days 1 and 3 of culture（*\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05）\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-2553083/v1/2efb5e2dc518d2cf2e035120.png"},{"id":33638088,"identity":"6dd514d4-810d-4036-a72e-8701afc8f1e6","added_by":"auto","created_at":"2023-03-01 16:54:09","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":10105642,"visible":true,"origin":"","legend":"\u003cp\u003eMineralized nodules formed in (A) control group, (B) 25% group,\u003c/p\u003e\n\u003cp\u003e(C) 50% group, (D) 75% group, and (E) 100% group after 21 days of\u003c/p\u003e\n\u003cp\u003eculture (alizarine red staining, Magnification,×400)\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-2553083/v1/9529e595a00a407ea015eab8.png"},{"id":33638773,"identity":"984e4f91-a6ac-44e6-b31c-7d1d13b03699","added_by":"auto","created_at":"2023-03-01 17:02:09","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":212686,"visible":true,"origin":"","legend":"\u003cp\u003eThe number of mineralized nodules formed in each group (*\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05)\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-2553083/v1/ec82bec63227af05fd8239d1.png"},{"id":33638090,"identity":"beb85451-6b69-4c52-a3e5-5e141233cbbe","added_by":"auto","created_at":"2023-03-01 16:54:10","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":23646793,"visible":true,"origin":"","legend":"\u003cp\u003eMC3T3-E1 cells stained for ALP in (A) control group, (B) 25% group, (C) 50% group, (D) 75% group, and (E) 100% group on day 3 of culture\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-2553083/v1/fa4cd2bff44ce91fcd23ddce.png"},{"id":33638086,"identity":"8eb84bc2-7346-43d9-acad-9098226e9867","added_by":"auto","created_at":"2023-03-01 16:54:09","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":700280,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Standard ALP curve of MC3T3-E1 cells, and (B) activity of ALP obtained from the ALP curve in each group after culture for 1, 3, and 5 days (*\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05)\u003c/p\u003e","description":"","filename":"floatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-2553083/v1/a1cc37dc4b8ffb12d4386519.png"},{"id":33638091,"identity":"1b74355a-ed0a-42b8-86c0-b0d5ea3bc856","added_by":"auto","created_at":"2023-03-01 16:54:10","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":19786289,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of BMP-2 by MC3T3-E1 cells in (A) control group, (B) 25% group, (C) 50% group, (D) 75% group, and (E) 100% group on day 3 of culture\u003c/p\u003e\n\u003cp\u003e(immunocytochemical staining)\u003c/p\u003e","description":"","filename":"floatimage11.png","url":"https://assets-eu.researchsquare.com/files/rs-2553083/v1/8b3eee1fb42c42104e5347c2.png"},{"id":33638089,"identity":"75fccd49-41b6-4071-94e3-5bdf7b2320e1","added_by":"auto","created_at":"2023-03-01 16:54:09","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":13218998,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of OPG in MC3T3-E1 cells in (A) control group, (B) 25% group, (C)\u003c/p\u003e\n\u003cp\u003e50% group, (D) 75% group, and (E) 100% group on day 3 of culture\u003c/p\u003e\n\u003cp\u003e(immunocytochemical staining)\u003c/p\u003e","description":"","filename":"floatimage12.png","url":"https://assets-eu.researchsquare.com/files/rs-2553083/v1/cfe6bdef0342f908bd017993.png"},{"id":33638082,"identity":"edebe987-d887-4583-9fe7-719855eb674a","added_by":"auto","created_at":"2023-03-01 16:54:09","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":59954,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eThe expression of (A) BMP-2 mRNA (B) and OPG mRNA in each group on\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eday 5 of culture\u003c/em\u003e (*\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05)\u003c/p\u003e","description":"","filename":"floatimage13.png","url":"https://assets-eu.researchsquare.com/files/rs-2553083/v1/2b6b4ddd4749d7ac380c8e82.png"},{"id":44733506,"identity":"859e1483-34b8-43fe-8771-00fa02696c26","added_by":"auto","created_at":"2023-10-16 22:06:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":12066894,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2553083/v1/9816f136-8711-4f0f-9b2f-aa35c0f73cb1.pdf"}],"financialInterests":"","formattedTitle":"Evaluation of toxicity and biocompatibility of a novel Mg-Nd-Gd-Sr alloy in the osteoblastic cell","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBiodegradable magnesium alloys are currently a hot topic in research into biomaterials. Compared with other metals,\u0026nbsp;magnesium alloys can be degradable, and the degradation product, magnesium ions, can be fully excreted through the kidneys and intestines, thus avoiding secondary operations. Magnesium alloys\u0026nbsp;are light in weight and have an elastic modulus equivalent to natural bone, thus reducing the probability of stress-shielding effects and helping fracture healing. Magnesium alloys have superior strength and extension compared with other metals. The fracture toughness of magnesium alloys is 15\u0026ndash;40 MPa and the fracture toughness of natural bone is 36 MPa, both higher than that of ceramic biomaterials such as hydroxyapatite. Due to the biological activity of magnesium, ions degraded from magnesium alloys promote an osteogenic response and strengthen the contact between implant materials and bone tissue \u003csup\u003e[1-4]\u003c/sup\u003e. However, the clinical application of magnesium alloys is limited by their rapid degradation and uneven corrosion in physiological environments.\u003c/p\u003e\n\u003cp\u003eIn recent years, various methods have been used to strengthen the corrosion resistance of magnesium alloys in physiological environments, thereby solving problems such as excessive magnesium ion concentration, hydrogen generation, and local high pH values in the body due to the rapid degradation of magnesium alloys. An effective way to improve the properties of magnesium alloys is to add other suitable alloying elements, especially rare earth elements. When used to improve the corrosion resistance and mechanical properties of magnesium alloys, the elements that are added, such as Zn and Sr, have the ability to induce osteoblast differentiation, which can further promote fracture healing \u003csup\u003e[5]\u003c/sup\u003e. In our previous work we prepared a new\u0026nbsp;type of\u0026nbsp;Mg-3Nd-1Gd-0.3Sr-0.2Zn-0.4Zr (abbr.: Mg-Nd-Gd-Sr)\u0026nbsp;alloy using the gravity casting method, in which Gd, Nd, Zr, Sr, and Zn were added at an appropriate ratio \u003csup\u003e[6]\u003c/sup\u003e. To date, there has been no report on the effect of Mg-Nd-Gd-Sr alloy on osteoblasts. Therefore, in this study we evaluated the biocompatibility of the new Mg-Nd-Gd-Sr alloy through cytotoxicity, cell viability, apoptosis and cell damage experiments. We also investigated the biological functions of the new Mg-Nd-Gd-Sr alloy on osteoblast adhesion, proliferation, and mineralization, and analyzed the mechanism via which the new Mg-Nd-Gd-Sr alloy affects the function of osteoblasts by analyzing the expression of bone morphogenetic protein-2 (BMP-2) and osteoprotegerin (OPG) at the protein level and BMP, OPG and Collagen type I (Col-I) at the mRNA level. The present study attempted to lay an experimental foundation for the clinical application of novel magnesium alloys as orthopedic implant materials.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv class=\"Section2\" id=\"Sec2\"\u003e\n \u003ch2\u003e1.1 Materials\u003c/h2\u003e\n \u003cp\u003eThe Mg-Nd-Gd-Sr alloy was obtained from the School of Materials Science and Engineering, Nanjing Institute of Technology. The composition of the Mg-Nd-Gd-Sr alloy is provided in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n \u003cp\u003e\u003cimg 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\"\u003e\u003c/p\u003e\n \u003cp\u003eTable\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. \u003cem\u003eChemical composition of the Mg-Nd-Gd-Sr alloy\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003eMC3T3-E1 cells were provided by the Chinese Academy of Science Type Culture Collection (China).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec3\"\u003e\n \u003ch2\u003e1.2 Preparation of extracts\u003c/h2\u003e\n \u003cp\u003eThe Mg-Nd-Gd-Sr alloy was prepared as disc-shaped samples with a diameter of 10 mm and a height of 2 mm, which were polished with metallographic emery paper to 1000 grits, followed by ultrasound washes in ethanol and distilled water. Prior to testing, samples were sterilized using ethylene oxide.\u003c/p\u003e\n \u003cp\u003eExtracts were prepared using alpha-modified minimum essential medium (\u0026alpha;-MEM) cell culture medium as the extraction medium, and with a ratio of surface area of samples to volume of extraction medium of 1.25 cm\u003csup\u003e2\u003c/sup\u003e/mL in a humidified incubator at 95% relative humidity and 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C for 24 hours \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. After extraction, the extracts were diluted with \u0026alpha;-MEM to make 25%, 50%, 75% and 100% groups. All the extracts were stored in a refrigerator at 4\u0026deg;C for use within 3 days.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec4\"\u003e\n \u003ch2\u003e1.3 Cytotoxicity testing\u003c/h2\u003e\n \u003cp\u003eThe CCK-8 assay was used to evaluate the cytotoxicity of the Mg-Nd-Gd-Sr alloy to osteoblasts. Cytotoxicity tests were carried out by indirect contact, where the cells were cultured in extracts of 25%, 50%, 75% and 100% concentration. Simple \u0026alpha;-MEM was chosen as the negative control and \u0026alpha;-MEM containing 0.64% phenol was the positive control.\u003c/p\u003e\n \u003cp\u003eAfter 1, 3 or 5 days in culture, 10 \u0026micro;L CCK-8 was added to each well and incubated at 37\u0026deg;C for 2 hours in the dark. The optical density (OD) at 490 nm was measured with a spectrophotometer (Wellscan MK3, Labsystems Diagnostics Oy, Vantaa, Finland). The cell relative growth rate (RGR) was calculated according to the following equation: RGR (%) = (OD t /OD n) \u0026sdot; 100%, where ODt is the OD value of the tested group, and ODn is the OD value of the negative group.\u003c/p\u003e\n \u003cp\u003eThe cytotoxicity of the Mg-Nd-Gd-Sr alloy was evaluated according to the toxicity grading method (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003e\u003c/p\u003e\u0026nbsp;\u003ctable border=\"1\" id=\"Tab2\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eCytotoxicity grading\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCytotoxicity\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRGR(%)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 Grade\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026ge;\u0026thinsp;100\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1 Grade\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e75\u0026ndash;99\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2 Grade\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50\u0026ndash;74\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3 Grade\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25\u0026ndash;49\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4 Grade\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u0026ndash;24\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5 Grade\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cp\u003eTable \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. \u003cem\u003eCytotoxicity grading\u003c/em\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec5\"\u003e\n \u003ch2\u003e1.4 Apoptosis\u003c/h2\u003e\n \u003cp\u003eApoptosis was detected by flow cytometry using annexin-V/FITC-PI assay. MC3T3-E1 cells that were growing well were seeded into 6-well plates and treated with different concentrations of Mg-Nd-Gd-Sr alloy extract or with cell culture medium without extract (control group). The cells were trypsinized at 1, 3, and 5 days of culture, rinsed thrice with PBS, then resuspended in the eluent. After the addition of Annexin V-FITC (5 \u0026micro;L) and PI (10 \u0026micro;L) reagents, the specimens were placed into an ice box and rapidly analyzed using flow cytometry.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec6\"\u003e\n \u003ch2\u003e1.5 Cell damage\u003c/h2\u003e\n \u003cp\u003eAnalysis of reactive oxygen species (ROS) was used to evaluate any damage caused to osteoblasts by the Mg-Nd-Gd-Sr alloy extract. MC3T3-E1 cells that were growing well were seeded into 6-well plates and treated with different concentrations of Mg-Nd-Gd-Sr alloy extract or cell culture medium without extract (control group). On day 5 of culture, the cells were rinsed, covered with DCFH-DA medium (1 mL) containing a fluorescent probe, incubated at 37\u0026deg;C for 30 minutes, washed twice in PBS, and observed under a fluorescence microscope.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec7\"\u003e\n \u003ch2\u003e1.6 Cell bioactivity\u003c/h2\u003e\n \u003cp\u003eDAPI staining was used to analyze the bioactivity of MC3T3-E1 cells in the groups treated with different concentrations of extract or control medium. MC3T3-E1 cells were inoculated into 6-well cell culture plates, then different concentrations of Mg-Nd-Gd-Sr alloy extract or cell culture medium without extract (control group) were added. After 3 days of culture, the culture medium was discarded, the slides were washed thoroughly with PBS and DAPI was added for 5\u0026ndash;10 minutes. Then the slides were rinsed under running water, and cell bioactivity was observed under a fluorescence microscope.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec8\"\u003e\n \u003ch2\u003e1.7 Cell adhesion\u003c/h2\u003e\n \u003cp\u003eMC3T3-E1 cells were cultured for 3 days then trypsinized and prepared into a cell suspension (1 mL). An aliquot of the cell suspension (100 \u0026micro;L) was diluted with 900 \u0026micro;L PBS, and 10,000 cells were counted using a hemocytometer. Cell suspensions containing 50,000, 25,000, 12,500, 6,250, and 3,125 cells were then obtained, seeded into 96-well plates and cultured for 4 hours. Then CCK-8 (10 \u0026micro;L per well) was added, cells were returned to culture for 2 hours, then the OD value was measured using a microplate reader. The OD values were used to create a standard adherence curve of MC3T3-E1 cells.\u003c/p\u003e\n \u003cp\u003eMC3T3-E1 cells were cultured for 3 days then trypsinized and prepared into a cell suspension (1 mL). An aliquot of the cell suspension (100 \u0026micro;L) was diluted with 900 \u0026micro;L PBS, and 10,000 cells were counted using a hemocytometer. The cells were then inoculated into 96-well plates and treated with different Mg-Nd-Gd-Sr alloy extracts or the same volume of cell culture medium, three wells per group. After 2, 4, 6, 8, 10, or 12 hours of culture, the cell culture medium in each group was removed, and the cells were rinsed twice with PBS then cultured with CCK-8 (10 \u0026micro;L per well). Two hours later, the OD value was measured using a microplate reader.\u003c/p\u003e\n \u003cp\u003eThe number of adherent MC3T3-E1 cells in each group was calculated from the standard curve, and the cell adhesion curve in each group was drawn for statistical analysis.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec9\"\u003e\n \u003ch2\u003e1.8 Cell proliferation\u003c/h2\u003e\n \u003cp\u003eThe proliferation of MC3T3-E1 cells was quantitatively assessed by CCK-8 assay. MC3T3-E1 cells that were growing well were inoculated into 96-well plates and treated with different concentration of Mg-Nd-Gd-Sr alloy extract or cell culture medium without extract (control group). After 1, 3, and 5 days of culture, CCK-8 (10 \u0026micro;L per well) was added and the cells were cultured for another 2 hours. The OD value was measured at 450 nm using a microplate reader.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec10\"\u003e\n \u003ch2\u003e1.9 Cell mineralization\u003c/h2\u003e\n \u003cp\u003eMC3T3-E1 cells that were growing well were inoculated into 6-well plates and treated with different concentrations of Mg-Nd-Gd-Sr alloy extract or cell culture medium without extract (control group). After 5 days of culture, osteogenic induction medium was added, and the cells were cultured for a further 21 days. Cultures were then fixed for 10 minutes, rinsed and stained using alizarin red. After 30 minutes, the cells were rinsed and observed under a light microscope.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec11\"\u003e\n \u003ch2\u003e1.10 Alkaline phosphatase(ALP) staining and ALP activity assay\u003c/h2\u003e\n \u003cp\u003eThe differentiation of MC3T3-E1 cells was assessed by measuring alkaline phosphatase (ALP) activity. MC3T3-E1 cells that were growing well were inoculated into 6-well plates and treated with different concentrations of Mg-Nd-Gd-Sr alloy extract or cell culture medium without extract (control group). The cells were then fixed with the fixing solution, rinsed and stained in the dark with ALP staining solution for 15 minutes. After rinsing, the cells were counterstained using nuclear fast red for 3 minutes. Finally, the cells were rinsed and observed under a light microscope.\u003c/p\u003e\n \u003cp\u003eAfter 1, 3, or 5 days of culture, MC3T3-E1 cells were lysed using 0.5% Triton X-100 solution at 4\u0026deg;C for 1 hour. A substrate developer was added, and the culture plate was shaken well on a shaker then placed in the incubator for 30 minutes until a full reaction was achieved. The reaction was terminated, and the OD value was measured using a microplate reader.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e1.11 Expression of BMP-2 and OPG proteins\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eImmunocytochemical staining was used to determine the expression of BMP-2 and OPG. MC3T3-E1 cells that were growing well were inoculated into 6-well plates containing slides and treated with different concentrations of Mg-Nd-Gd-Sr alloy extract or cell culture medium without extract (control group) for 3 days. The cells were rinsed three times with PBS, fixed with 4% paraformaldehyde for 15 minutes, and air dried for 5 minutes. After rinsing three times with PBS, the specimens were incubated with 0.5% Triton X-100 for 20 minutes, blocked with peroxidase, and incubated at room temperature for 10 minutes. After washing, the specimens were incubated with goat serum working solution at room temperature for 10 minutes, then after removal of the serum, the specimens were incubated with the primary antibody at 4\u0026deg;C overnight. On the second day, the specimens were incubated with the secondary antibody at room temperature for 15 minutes, then incubated with horseradish peroxidase-labeled streptomyces ovalbumin at room temperature for 15 minutes, and developed with DAB chromogenic solution for 10 minutes, counterstained with hematoxylin for 20 seconds, washed with tap water for 5 minutes, mounted with gum, and observed under a light microscope.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e1.12 Expression of BMP-2 and OPG mRNA\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eRT-PCR was used to analyze the mRNA expression of BMP-2 and OPG. MC3T3-E1 cells that were growing well were seeded into 24-well plates at a density of 1\u0026times;10\u003csup\u003e4\u003c/sup\u003e/mL and treated with different concentrations of Mg-Nd-Gd-Sr alloy extract or cell culture medium without extract (control group). After 5 days of culture, the cells were collected and the mRNA expression of BMP-2 and OPG was detected using quantitative fluorescence PCR.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e2.13 Statistical analysis\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eThe statistical software SPSS13.0 (SPSS Inc., Chicago, IL, USA) was used to analyze the data. Differences between the groups were compared using one-way analysis of variance (ANOVA). The statistical significance was defined as 0.05, and a \u003cem\u003eP\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 indicated a statistically-significant differences.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Cytotoxicity testing\u003c/h2\u003e \u003cp\u003eMC3T3-E1 cells were cultured in different concentrations of Mg-Nd-Gd-Sr alloy extract for 1, 3, and 5 days, and the OD values were measured using a microplate reader (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eOD values and RGR in each group ( n\u0026thinsp;=\u0026thinsp;3 )\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e1d\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e3d\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e5d\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOD (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRGR (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eOD (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRGR (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eOD (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eRGR (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0% group\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.365\u0026thinsp;\u0026plusmn;\u0026thinsp;0.028\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.820\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1430\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.464\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1099\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e25% group\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.453\u0026thinsp;\u0026plusmn;\u0026thinsp;0.068\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e124.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.065\u0026thinsp;\u0026plusmn;\u0026thinsp;0.050\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e129.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.535\u0026thinsp;\u0026plusmn;\u0026thinsp;0.236\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e104.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e50% group\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.540\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0386\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e147.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.291\u0026thinsp;\u0026plusmn;\u0026thinsp;0.283\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e157.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.691\u0026thinsp;\u0026plusmn;\u0026thinsp;0.317\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e115.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e75% group\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.557\u0026thinsp;\u0026plusmn;\u0026thinsp;0.304\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e152.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.334\u0026thinsp;\u0026plusmn;\u0026thinsp;0.166\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e162.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.528\u0026thinsp;\u0026plusmn;\u0026thinsp;0.073\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e104.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e100% group\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.572\u0026thinsp;\u0026plusmn;\u0026thinsp;0.009\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e156.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.404\u0026thinsp;\u0026plusmn;\u0026thinsp;0.091\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e171.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.504\u0026thinsp;\u0026plusmn;\u0026thinsp;0.091\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e102.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. \u003cem\u003eOD values and RGR in each group (n\u0026thinsp;=\u0026thinsp;3)\u003c/em\u003e\u003c/p\u003e \u003cp\u003eAt days 1 and 3, the OD values and RGR were increased significantly in each experimental group compared with the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and were also increased as the Mg-Nd-Gd-Sr alloy extract concentration increased. However by day 5 of culture, compared with the control group, the 25%, 75%, and 100% groups showed no significant increase in the OD values or RGR (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05), whilst the OD values and RGR were the highest in the 50% group, which were significantly higher than those in the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and then showed a downward trend. At any time point, the RGR of MC3T3-E1 cells in any extract was greater than 100% (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. \u003cem\u003eRGR of MC3T3-E1 cells in each group at the different time-points tested.\u003c/em\u003e\u003c/p\u003e \u003cp\u003eBased on these results, the cytotoxicity of the Mg-Nd-Gd-Sr alloy was considered to be level 0, indicating that the Mg-Nd-Gd-Sr alloy had no obvious cytotoxic effect on MC3T3-E1 cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Apoptosis\u003c/h2\u003e \u003cp\u003eAfter 1, 3, or 5 days of culture with different concentrations of Mg-Nd-Gd-Sr alloy extract, apoptosis of MC3T3-E1 cells was analyzed by flow cytometry using Annexin-V/FITC-PI staining. Compared with the control group, no obvious apoptosis was observed in any experimental group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). No obvious apoptosis was observed on days 1 or 3 of culture. On day 5, the apoptotic rate was increased to some extent in the 50%, 75%, and 100% extract groups compared with the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), but it was still within the limits of acceptability (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. \u003cem\u003eApoptotic rate in each group on days 1, 3 and 5 of culture (\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026lowast;\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Cell damage\u003c/h2\u003e \u003cp\u003eAfter 5 days of culture with different concentrations of Mg-Nd-Gd-Sr alloy extract, MC3T3-E1 cells loaded with DCFH-DA probes were observed under a fluorescence microscope. Slight staining of the cells indicated a normal physiological metabolism, whilst cells with a stronger green fluorescence were damaged cells with increased reactive oxygen radicals. As the concentration of the Mg-Nd-Gd-Sr alloy extract increased, the number of damaged cells tended to increase (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. \u003cem\u003eReactive oxygen species of MC3T3-E1 cells in (A) control group, (B) 25% group, (C) 50% group, (D) 75% group, and (E) 100% group on day 5 of culture.\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Cell bioactivity\u003c/h2\u003e \u003cp\u003eOn day 3 of culture with different concentrations of Mg-Nd-Gd-Sr alloy extract, MC3T3-E1 cells were stained with DAPI and observed under a fluorescence microscope (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The nuclei of the MC3T3-E1 cells were stained an even blue and were equal in size, and the nuclear membrane was intact in each group. As the concentration of Mg-Nd-Gd-Sr alloy extract increased, the number of cells increased.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. \u003cem\u003eMC3T3-E1 cells stained with DAPI in (A) control group, (B) 25% group, (C) 50% group, (D) 75% group, and (E) 100% group on day 3 of culture.\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Cell adhesion\u003c/h2\u003e \u003cp\u003eTo study the influence of the Mg-Nd-Gd-Sr alloy on MC3T3-E1 cell adhesion, a standard adherence curve of MC3T3-E1 cells was drawn. With increasing time in culture, the number of adherent cells showed an increasing trend (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSubsequently, the cells were cultured with different concentrations of Mg-Nd-Gd-Sr alloy extract, and the OD value was measured by CCK-8 assay at different time-points to obtain the MC3T3-E1 cell adhesion curve (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. \u003cem\u003e(A) Standard adherence curve of MC3T3-E1 cells, and (B) the number of adherent cells obtained from the cell adhesion curve in each group.\u003c/em\u003e\u003c/p\u003e \u003cp\u003eCompared with the control group, within 12 hours, the number of adherent cells increased significantly in each experimental group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Moreover, the number of adherent cells showed an increasing trend in a concentration-dependent manner within 12 hours.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Cell proliferation\u003c/h2\u003e \u003cp\u003eThe CCK-8 assay was used to investigate the proliferation of MC3T3-E1 cells cultured in different concentrations of Mg-Nd-Gd-Sr alloy extract. After 1, 3, or 5 days of culture, the OD value of MC3T3-E1 cells was measured. Compared with the control group, the OD value increased significantly in each experimental group on days 1 and 3 of culture (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), and showed an increasing trend with the increase in concentration of Mg-Nd-Gd-Sr alloy extract. However, on day 5 of culture, the OD value did not change significantly in the 75% and 100% extract groups compared with the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Moreover, the cell proliferation peaked in the 50% extract group, which was significantly higher than that in the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), but then reduced.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. \u003cem\u003eCell proliferation of MC3T3-E1 cells in each group according to absorbance values on days 1 and 3 of culture (\u003c/em\u003e\u003csup\u003e\u003cem\u003e*\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Cell mineralization\u003c/h2\u003e \u003cp\u003eAfter 21 days of culture with different concentrations of Mg-Nd-Gd-Sr alloy extract, MC3T3-E1 cells were stained with alizarin red and mineralized nodules were analyzed using Image-J software. Mineralized nodules of different shapes and sizes had formed in all groups by the end of 21 days of culture (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e. \u003cem\u003eMineralized nodules formed in (A) control group, (B) 25% group, (C) 50% group, (D) 75% group, and (E) 100% group after 21 days of culture (alizarin red staining, magnification,\u0026times;400).\u003c/em\u003e\u003c/p\u003e \u003cp\u003eCompared with the control group, the number of mineralized nodules formed in each experimental group was significantly increased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e), was highest in the 50% extract group, and then showed a downward trend.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e. \u003cem\u003eThe number of mineralized nodules formed in each group (\u003c/em\u003e\u003csup\u003e\u003cem\u003e*\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Alkaline phosphatase assay\u003c/h2\u003e \u003cp\u003eCells were cultured on slides in each group, and stained with ALP on day 3 of culture (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). The cells in each group were positive for ALP, which was red in the nucleus.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e. \u003cem\u003eMC3T3-E1 cells stained for ALP in (A) control group, (B) 25% group, (C) 50% group, (D) 75% group, and (E) 100% group on day 3 of culture.\u003c/em\u003e\u003c/p\u003e \u003cp\u003eTo study the influence of Mg-Nd-Gd-Sr alloy extract on the expression of ALP, a standard ALP curve of MC3T3-E1 cells was drawn. There was a certain relationship between OD value and ALP activity. An increased OD value indicated increased ALP activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe expression of ALP (OD value) was measured and the ALP activity in each group was calculated from the standard curve. ALP activity was significantly increased in each experimental group compared with the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eB). As the concentration of the Mg-Nd-Gd-Sr alloy extract increased, the activity of ALP also increased on days 1 and 3, and reached a peak in the 50% extract group on day 5, but then showed a downward trend.\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e. \u003cem\u003e(A) Standard ALP curve of MC3T3-E1 cells, and (B) activity of ALP obtained from the ALP curve in each group after culture for 1, 3, and 5 days.\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Expression of BMP-2 and OPG\u003c/h2\u003e \u003cp\u003eCells were cultured on slides in each group, stained immunocytochemically on day 3, and observed under the microscope. Compared with the control group, the expression of both BMP-2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e) and OPG (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003e) was significantly increased in each experimental group on day 3 of culture (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and showed an increasing tendency with the increase in the concentration of Mg-Nd-Gd-Sr alloy extract.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e. \u003cem\u003eExpression of BMP-2 by MC3T3-E1 cells in (A) control group, (B) 25% group, (C) 50% group, (D) 75% group, and (E) 100% group on day 3 of culture (immunocytochemical staining).\u003c/em\u003e\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003e. \u003cem\u003eExpression of OPG in MC3T3-E1 cells in (A) control group, (B) 25% group, (C) 50% group, (D) 75% group, and (E) 100% group on day 3 of culture (immunocytochemical staining).\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e2.10 The mRNA expression of BMP-2 and OPG\u003c/h2\u003e \u003cp\u003eReal-time fluorescence quantitative PCR was used to analyze the expression of BMP-2 and OPG mRNAs on day 5 of culture. The cells in each group continuously expressed BMP-2 and OPG mRNAs. Compared with the control group, the expression of BMP-2 and OPG mRNAs were both significantly increased in each experimental group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003e). Moreover, the expression of OPG mRNA reached a peak in the 75% extract group, whilst the expression of BMP-2 mRNA was highest in the 50% extract group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003e. \u003cem\u003eThe expression of BMP-2 and OPG mRNA in each group on day 5 of culture (\u003c/em\u003e\u003csup\u003e\u003cem\u003e*\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAlthough magnesium alloys have advantages as orthopedic implants \u003csup\u003e[\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e–\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e, magnesium alloy materials degrade too quickly in the human body and cannot yet meet the clinical requirements. The novel magnesium alloy Mg-Nd-Gd-Sr is prepared by gravity casting and has been confirmed to have good structure, mechanics and corrosion resistance \u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. However, it is necessary to clarify the effect of this novel alloy on osteoblast function before it can be used as an orthopedic implant material in clinical practice. The present study explored the effect of Mg-Nd-Gd-Sr alloy on the function of MC3T3-E1 osteoblastic cells.\u003c/p\u003e \u003cp\u003eBiocompatibility of an implant is well reflected by \u003cem\u003ein vitro\u003c/em\u003e cytotoxicity testing, as weaker cytotoxicity \u003cem\u003ein vitro\u003c/em\u003e corresponds with weaker cytotoxicity \u003cem\u003ein vivo\u003c/em\u003e \u003csup\u003e[\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e–\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. In this study, CCK-8 was used for cytotoxicity analysis in MC3T3-E1 cells cultured with 25%, 50%, 75%, or 100% Mg-Nd-Gd-Sr alloy extract for 1, 3, or 5 days. The OD value of MC3T3-E1 cells was significantly increased after 1, 3, and 5 days of culture with different concentrations of Mg-Nd-Gd-Sr alloy extract. There was also a concentration-dependent increase in the OD value on days 1 and 3 of culture. However, on day 5 of culture, no significant increase was observed in the 25%, 75%, or 100% extract groups compared with the control group, and the OD value even showed a downward trend in the 75% and 100% extract groups but was still higher than that in the control group. Meanwhile, the OD value peaked in the 50% extract group. Results from the cytotoxicity test indicated that the Mg-Nd-Gd-Sr alloy extract had no toxic effect on MC3T3-E1 cells regardless of its concentration, and within a certain concentration range even promoted the proliferation of MC3T3-E1 cells. Previous research confirmed that osteoblasts exhibit better viability and differentiation when the magnesium ion concentration is at an appropriate level, while cell viability and differentiation are influenced when the magnesium concentration exceeds a certain limit. In this study, the magnesium alloy extract was added again on the 3rd day of culture, which could cause the magnesium ion concentration in the medium to exceed its critical limit, but MC3T3-E1 cells still proliferated in the medium containing high-concentration magnesium alloy extract. According to the ISO10993 standard \u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e, the RGR of MC3T3-E1 cells was over 100% in all the different Mg-Nd-Gd-Sr alloy extract groups. This indicated that the Mg-Nd-Gd-Sr alloy extract had no obvious toxic effect on MC3T3-E1 cells, but instead promoted the proliferation of MC3T3-E1 cells.\u003c/p\u003e \u003cp\u003eEarly adhesion is a prerequisite for the future fate of osteoblasts, affecting cell proliferation and long-term survival. Research on cell adhesion is indispensable when studying the effect of Mg-Nd-Gd-Sr alloy on cell function \u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. Results from the cell adhesion experiment showed that the number of adherent cells was significantly higher in each experimental group than in the control group in the first 12 hours of culture, and moreover, cell adhesion showed an increasing trend with the increasing concentrations of Mg-Nd-Gd-Sr alloy extract. This may be related to the release of an appropriate level of magnesium ions from the degraded Mg-Nd-Gd-Sr alloy. Previous studies have shown that an appropriate concentration of magnesium ions is conducive to cell adhesion \u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. The results of our cell proliferation experiment also revealed that with time, the OD value increased significantly in each experimental group. On days 1 and 3 of culture, the OD value was increased significantly in each experimental group compared with the control group, and there was also a concentration-dependent increase in the OD value. This may be because the Mg-Nd-Gd-Sr alloy degrades and releases appropriate concentrations of magnesium ions, which then induce cell activation and promote cell proliferation by regulating the formation of osteoblast-related proteins \u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. The Mg-Nd-Gd-Sr alloy also contains other ions such as zinc and zirconium ions. Zinc ions are involved in early cell metabolism and promote cell proliferation \u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e, while zirconium ions form ZrO\u003csub\u003e2\u003c/sub\u003e, creating a more suitable environment for cell adhesion and proliferation \u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. On day 5 of culture, the OD value of MC3T3-E1 cells was reduced in the 75% and 100% extract groups, indicating that the concentration of magnesium ions at this time has exceeded the optimum concentration. If the Mg-Nd-Gd-Sr alloy extract concentration continues to increase, the release of hydrogen will increase and the pH of the medium will continue to rise, which may have harmful and even fatal effects on cells \u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOsseointegration of an orthopedic implant with the living bone is an orderly process. Bone marrow mesenchymal stem cells or osteoprogenitor cells are first recruited to the surface of the bone implant, and then a new bone matrix is formed after osteogenic differentiation and mineralization. Finally bone remodeling can be conducted \u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. Highly active alkaline phosphatase and calcified extracellular matrix are two representative indicators of the osteoblast phenotype, which indicate osteoblast maturation \u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. In this study, alizarin red staining was used to clarify the influence of Mg-Nd-Gd-Sr alloy extract on the mineralization of MC3T3-E1 cells. The number of mineralized nodules was significantly increased in each experimental group compared with the control group. The number of mineralized nodules was highest in the 50% extract group but decreased in the 75% and 100% extract groups, indicating that the Mg-Nd-Gd-Sr alloy extract promotes osteoblast mineralization and there is an optimal concentration. These findings indicated that some other ions such as strontium ions, as well as magnesium ions, promoted the mineralization of osteoblasts \u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe biological function of implant materials was also evaluated based on their effects on apoptosis and cell damage. In this study, the Annexin V-FITC/PI assay was used to quantify apoptosis by flow cytometry, thereby evaluating the influence of the Mg-Nd-Gd-Sr alloy extract on MC3T3-E1 apoptosis. On days 1 and 3 of cell culture, there was no obvious apoptosis in any experimental group compared with the control group. On day 5 of cell culture, the apoptotic rate was significantly increased in the 50%, 75%, and 100% extract groups compared with the control group. This may be due to the fact that the concentration of Mg-Nd-Gd-Sr alloy extract increased after the culture medium was changed on day 3. However, excess magnesium ions can be metabolized through various channels \u003cem\u003ein vitro.\u003c/em\u003e In summary, the Mg-Nd-Gd-Sr alloy does not promote apoptosis. However, a very high concentration of Mg-Nd-Gd-Sr alloy extract or poor conditions may have a certain apoptosis-inducing effect on cells. In this study, ROS production was used to evaluate the damage caused by Mg-Nd-Gd-Sr alloy extracts to osteoblasts. After 5 days of culture with Mg-Nd-Gd-Sr alloy extracts, the production of ROS was measured. The cells exhibited strong green fluorescence in each group. No significant difference was found in the proportion of cell damage between any of the experimental groups and the control group, indicating that the Mg-Nd-Gd-Sr alloy does not cause obvious cell damage. With increasing concentrations of the Mg-Nd-Gd-Sr alloy extract, the number of cells with strong green fluorescence showed an increasing trend, indicating that the Mg-Nd-Gd-Sr alloy extract at very high concentrations could damage cells under poor conditions.\u003c/p\u003e \u003cp\u003eBMP is an important osteogenic factor that promotes bone formation and the differentiation of osteoblasts in the process of bone repair \u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. BMP-2, with its receptor, forms a heterologous receptor complex and induces intracellular signals in the SMAD complex, leading to the subsequent transcription of osteocalcin, osteopontin, and alkaline phosphatase. BMP-2 is involved in cell proliferation, differentiation, and apoptosis, thereby influencing the biological behaviors of the cells \u003csup\u003e[28–32]\u003c/sup\u003e. The ratio of OPG to RANKL is a key determinant of bone remodeling or bone resorption. When the ratio is \u0026gt; 1, the formation of osteoblasts is promoted; when the ratio is \u0026lt; 1, the formation of osteoclasts is promoted. Col-I is critical for hydroxyapatite deposition, has a good effect on cell adhesion, growth and function, and plays an important role in cell biological activity and osteogenicity \u003csup\u003e[33, 34]\u003c/sup\u003e. In this study, the expression of BMP-2 and OPG in MC3T3-E1 cells cultured with different concentrations of Mg-Nd-Gd-Sr alloy extract was detected by immunocytochemistry. The expression of BMP-2 and OPG in the cells increased significantly in each experimental group compared with the control group, and also showed a concentration-dependent increase. RT-PCR results showed that the expression of BMP-2, OPG, and Col-I mRNA in each experimental group was significantly increased compared with that in the control group. The expression of OPG and Col-I mRNA peaked in the 75% extract group, while the expression of BMP-2 mRNA was highest in the 50% extract group. This finding indicated that the Mg-Nd-Gd-Sr alloy extract promoted the expression of BMP-2, OPG, and Col-I mRNA in MC3T3-E1 cells. The increase in the expression of BMP-2 in MC3T3-E1 cells induced by the Mg-Nd-Gd-Sr alloy extract induced intracellular signals of the SMAD complex, upregulating the expression of osteogenesis-related genes and proteins, promoting adhesion, proliferation and mineralization of MC3T3-E1 cells, and ultimately influencing the cells’ biological behavior.\u003c/p\u003e "},{"header":"Conclusion","content":"\u003cp\u003eThe Mg-Nd-Gd-Sr alloy had no obvious cytotoxicity and caused no apoptosis or damage in MC3T3-E1 cells; meanwhile it promoted cell adhesion, proliferation, and mineralization and had good biological functions. During this process, there was an increase in the expressions of BMP-2 and OPG mRNAs and BMP-2 and OPG proteins. This finding indicated that the Mg-Nd-Gd-Sr alloy extract upregulated the protein and mRNA expression of BMP-2 and OPG, thereby inducing intracellular signals and upregulating the expression of osteogenesis-related genes, ultimately promoting the adhesion, proliferation and mineralization of MC3T3-E1 cells.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Scientific Research Projects of Gansu Province HealthIndustry(GSWSKY 2021-009); Scientific and Technology Research Projects of Lanzhou(2021-1-79):Natural Science Foundation of Gansu Province(20JR5RA369); and the National Natural Science Foundation ofChina(81960398).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declared no potential conflcts of interest with respect to the research, author- ship and publication of this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of supporting data\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available on request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis article does not contain any studies with human participants or animals performed by any of the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThisstudy was published with the informed consent of all authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos;contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXie Yadong was responsible for the drafting of the article and the main\u003c/p\u003e\n\u003cp\u003eexperiments;Yang Qinglin and Xie Ben are responsible for some experiments and dataanalysis.Wang Yongping and others were responsible for experimental design\u003c/p\u003e\n\u003cp\u003eand paper modification.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThanks for thesupport of Health Industry Research Project of Gansu Province， Lanzhou Science and Technology Research Project, Natural Science Foundation of Gansu Province and National Natural Science Foundation of China.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHou R, Victoria-Hernandez J, Jiang P, et al. 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Bone, 2007, 41(1): 87-96.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Magnesium alloy, Osteoblast, MC3T3-E1 cells, Damage, Biocompatibility","lastPublishedDoi":"10.21203/rs.3.rs-2553083/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2553083/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eWe investigated the toxicity and biocompatibility of a novel Mg-3Nd-1Gd-0.3Sr-0.2Zn-0.4Zr (abbreviated to Mg-Nd-Gd-Sr) alloy in the osteoblastic cell line MC3T3-E1 as osteoblasts play an important role in bone repair and remodeling. Methods: We used cytotoxicity tests and evaluation of cell damage and apoptosis to investigate the effects of the Mg-Nd-Gd-Sr alloy on osteoblastic cells. Cell bioactivity, cell adhesion, cell proliferation, mineralization, ALP activity, and expression of BMP-2 and OPG by osteoblastic cells were also used to investigate the biocompatibility of Mg-Nd-Gd-Sr alloy.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe results showed that the Mg-Nd-Gd-Sr alloy had no obvious cytotoxicity, and did not induce apoptosis or cause damage to MC3T3-E1 cells. Compared with the control group, the number of adherent cells within 12 hours was increased significantly in each experimental group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05); the OD value of MC3T3-E1 cells was increased significantly in each experimental group on days 1 and 3 of culture (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05); the number of mineralized nodules formed in each experimental group was significantly increased (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and ALP activity was significantly increased in each experimental group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). RT-PCR results showed that the mRNA expression of BMP-2 and OPG was significantly higher in each experimental group compared with the control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Western blotting showed that the Mg-Nd-Gd-Sr alloy extract significantly increased the protein expression of BMP-2 and OPG compared with the control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eOur data indicated that the novel Mg-Nd-Gd-Sr-Zn-Zr alloy had no obvious cytotoxic effects, and did not cause apoptosis or damage to MC3T3-E1 cells; meanwhile it promoted cell adhesion, cell proliferation, mineralization, and ALP activity of osteoblasts. During this process, there was an increase in the expressions of BMP-2 and OPG mRNAs and proteins.\u003c/p\u003e","manuscriptTitle":"Evaluation of toxicity and biocompatibility of a novel Mg-Nd-Gd-Sr alloy in the osteoblastic cell","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-03-01 16:54:04","doi":"10.21203/rs.3.rs-2553083/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revisions Needed","date":"2023-04-10T06:07:28+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2023-02-27T09:32:18+00:00","index":0,"fulltext":""},{"type":"editorAssigned","content":"","date":"2023-02-11T15:06:33+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Biology Reports","date":"2023-02-11T04:28:21+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"00265991-70a4-4da1-8706-2b8e3a7447cb","owner":[],"postedDate":"March 1st, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-10-16T21:49:41+00:00","versionOfRecord":{"articleIdentity":"rs-2553083","link":"https://doi.org/10.1007/s11033-023-08637-5","journal":{"identity":"molecular-biology-reports","isVorOnly":false,"title":"Molecular Biology Reports"},"publishedOn":"2023-07-05 21:31:38","publishedOnDateReadable":"July 5th, 2023"},"versionCreatedAt":"2023-03-01 16:54:04","video":"","vorDoi":"10.1007/s11033-023-08637-5","vorDoiUrl":"https://doi.org/10.1007/s11033-023-08637-5","workflowStages":[]},"version":"v1","identity":"rs-2553083","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2553083","identity":"rs-2553083","version":["v1"]},"buildId":"-HB7Z8yhvgn0wM9Nzuekk","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}