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

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. We used cytotoxicity tests 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. The results showed that the Mg-Nd-Gd-Sr alloy had no obvious cytotoxicity, and did not induce apoptosis to MC3T3-E1 cells. Compared with the control group, the number of adherent cells within 12 h 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). Our data indicated that the novel Mg-Nd-Gd-Sr-Zn-Zr alloy had no obvious cytotoxic effects, and did not cause apoptosis 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.

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  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][2][3][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 and apoptosis 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
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. MC3T3-E1 cells were provided by the Chinese Academy of Science Type Culture Collection (China).

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 h [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.

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 h 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).

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.

Cell number
DAPI staining was used to analyze the number 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 min. Then the slides were rinsed under running water, and cell number was observed under a fluorescence microscope.

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, 6250, and 3125 cells were then obtained, seeded into 96-well plates and cultured for 4 h. Then CCK-8 (10 μL per well) was added, cells were returned to culture for 2 h, 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 h 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.

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 h. The OD value was measured at 450 nm using a microplate reader.

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 min, rinsed and stained using alizarin red. After 30 min, the cells were rinsed and observed under a light microscope.

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 min. After rinsing, the cells were counterstained using nuclear fast red for 3 min. 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 h. A substrate developer was added, and the culture plate was shaken well on a shaker then placed in the incubator for 30 min until a full reaction was achieved. The reaction was terminated, and the OD value was measured using a microplate reader.

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 min, and air dried for 5 min. After rinsing three times with PBS, the specimens were incubated with 0.5% Triton X-100 for 20 min, blocked with peroxidase, and incubated at room temperature for 10 min. After washing, the specimens were incubated with goat serum working solution at room temperature for 10 min, 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 min, then incubated with horseradish peroxidase-labeled streptomyces ovalbumin at room temperature for 15 min, and developed with DAB chromogenic solution for 10 min, counterstained with hematoxylin for 20 s, washed with tap water for 5 min, mounted with gum, and observed under a light microscope.

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.

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.

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).
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).
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.

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).

Cell number
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. 3). 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.

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. 4A). 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. 4B).
Compared with the control group, within 12 h, 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 h.

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. 5), 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.

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. 6).
Compared with the control group, the number of mineralized nodules formed in each experimental group was significantly increased (P < 0.05; Fig. 7), was highest in the 50% extract group, and then showed a downward trend.

Alkaline phosphatase assay
Cells were cultured on slides in each group, and stained with ALP on day 3 of culture (Fig. 8). The cells in each group were positive for ALP, which was red in the nucleus.
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. 9A).
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  Fig. 9B). 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.

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. 10) and OPG (Fig. 11) 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.

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. 12). 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.

Discussion
Although magnesium alloys have advantages as orthopedic implants [9][10][11][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][14][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 h 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 ZrO2, 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. 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 apoptosisinducing effect on cells.
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][29][30][31]. 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 [30,31]. 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 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.