The extracts were used for the in vitro tests. After 1, 3, 5, and 7-days of immersion, the Mg-Ti composite and pure Mg samples were immersed in an α-MEM medium containing 10% FBS for 72 h at 37°C in a humidified atmosphere of 5% CO₂. The immersion ratio was selected, as mentioned previously according to the standard ISO 10993. After filtrating with a filter (0.22 μm), the extracts were collected and then diluted six times with the α-MEM medium for in vitro tests.

MC3T3-E1 cells were chosen to test cell morphology, proliferation, apoptosis, and differentiation. The cells were cultured in α-MEM supplemented with 10% FBS and 1% penicillin and streptomycin in a humidified atmosphere at 37°C with 5% CO₂. When the monolayer reached sub-confluence, the cells were subcultured with 0.25% trypsin.

Cell-Counting Kit-8 (CCK-8, United States Everbright Inc., Silicon Valley, United States) assay was chosen to evaluate the effects of the Mg-Ti composite and pure Mg extracts on cell proliferation. Cells were seeded in 96-well plates at 3 × 10³ cells/well for 24 h. They were washed twice with PBS, and the medium was replaced by 100 μl extracts or a normal culture medium after 24 h of attachment. After 1, 2, and 3-days of culturing, 100 μl α-MEM with 10% CCK-8 was added after rinsing twice with PBS; then, the plate was incubated for 2 h at 37°C. The 450 nm optical density was measured by using a microplate reader (Infinite M200, Tecan, Austria). Three replicates were chosen per group.

To detect the effect of the Mg-Ti composite and pure Mg extracts on cell morphology, MC3T3-E1 cells were incubated on a 24-well cell culture plate with the diluted extracts at a density of 1 × 10⁴ per well for 4 and 24 h. At each time point, the cells were permeabilized with 0.1% Triton X-100 after washing with PBS three times. Then, the permeabilized cells were supplemented with 100 nmol/L rhodamine-phalloidin (Cytoskeleton, Inc., Denver, CO, United States) for 30 min in the dark at room temperature. After that, the cells were stained with DAPI for 2 min coupled with washing with PBS three times before observation with a fluorescence microscope (ZEISS, Germany).

An Annexin V-FITC/PI kit (United States Everbright Inc., Silicon Valley, United States) was used to detect the effects of the sample extracts on cell apoptosis quantified through the standard flow cytometry test, according to the manufacturer’s protocol. MC3T3-E1 cells were seeded in 12-well plates at 1 × 10⁵ cells/well in a 1 ml medium for 24 h. The medium was replaced by prepared extracts or a normal culture medium, respectively, for 1 and 3-days. At each time point, the cells were digested with 0.25% trypsin and collected for the stain after washing with PBS three times. The collected cells were re-suspended with 100 μl binding buffer and stained with Annexin V-FITC and propidium iodide (PI) for 15 min in the dark. Before the flow cytometry (BD, LSRFortessa, United States) test, 300 μl binding buffer was added to each sample and blended evenly.

MC3T3-E1 cells were seeded in 12-well plates at 1 × 10⁵ cells/well for 24 h. The culture medium was replaced with the prepared extracts, and the culture medium contained an osteogenesis-inducing component. The extracts were refreshed every 2-days. The activity of ALP was evaluated with an Alkaline Phosphatase Assay Kit, according to the manufacturer’s instructions (Beyotime, China) after culture for 7 and 14-days. The protein content was measured following the protocol of the BCA protein assay kit (Beyotime, China). The ALP activity test of all samples was normalized using the protein concentration.

To simulate the effect of 7-day degradation of Mg²⁺ production on MC3T3-E1 cells, we prepared α-MEM with varying Mg²⁺ concentrations. First, inductively coupled plasma mass spectrometry (7800 ICP-MS, Agilent, United States) was performed to detect the Mg²⁺ concentration of pure Mg and Mg-Ti composite extracts. Then, the sterilized Mg chloride solution was applied to elevate the α-MEM Mg²⁺ concentration, according to the result of the Mg²⁺ concentration test.

CCK-8 assay was performed to evaluate the effects of varying Mg²⁺ concentrations on MC3T3-E1 cells in cytotoxic and proliferation ability. The cell morphology was stained with phalloidin to observe the effect of varying Mg²⁺ concentrations on the intracellular F-actin cytoskeletal network for 4 and 24 h. The cell apoptosis rate of MC3T3-E1 cells incubated with varying Mg²⁺ concentrations was determined by Annexin V-FITC/PI double staining for the period of 1 and 3-days. To determine the effect of varying Mg²⁺ concentrations on the osteogenic differentiation ability, an ALP assay was carried out after incubation for 7-days and 14-days.

Figure 5 shows the Mg²⁺ concentration of pure Mg and the Mg-Ti composite extracts at different periods of immersion time. The Mg-1 day group exhibits the highest Mg²⁺ concentration (9.6 mM). With the immersion time prolonged, the Mg²⁺ concentration for the pure Mg groups decreases gradually and eventually stabilizes for 5-days (3 mM). For the Mg-Ti group, the Mg²⁺ concentration remains stable around 8 mM in the process of 7-days of degradation. Thereby, we selected a range of concentrations (2, 4, 6, 8, and 10 mM) to detect the effect of various Mg²⁺ concentrations on MC3T3-E1 cells.

The CCK-8 results (Figure 6) show that the cells proliferate well in the pure Mg and Mg-Ti groups during incubation for 3-days. It displays that the OD values of all the groups increase gradually over the incubation time. Within 3-days of culture, the Mg-3 day group displays higher proliferation than the control group (p < 0.05). The relative growth rates (RGR) of MC3T3-E1 cells are shown in Table. 1. The RGR is calculated by a formula, according to the standard United States Pharmacopeia. The cell viability results of all specimens are more than 75%, indicating no cytotoxicity of all groups through 3 days of culture. As shown in Figure 7, the effects of Mg²⁺ concentrations on the cell proliferation ability are observed. It can be seen that the OD values gradually increased over the incubation time. From 1-day to 3-days of culture, all groups showed no statistical difference within the RGR ranging from 0–1 (no toxicity).

The fluorescence staining results of the intracellular F-actin cytoskeletal network with rhodamine-phalloidin and DAPI are shown in Figure 8. At the period of 4 h, the cells show obvious spindle morphology with little filopodia in both pure Mg and Mg-Ti groups. With the incubation time prolonged to 24 h, the cells present a fibrous structure with apparent F-actin, filopodia, and lamellipodia observed in the Mg-Ti group. Compared with pure Mg, cells provide more area in the Mg-Ti group, which suggests that the initial attachment behavior of MC3T3-E1 cells is better. As shown in Figure 9, the addition of Mg²⁺ promotes the expansion of MC3T3-E1 cells. Moreover, the cell exhibits a better shape configuration and displays better adhesion in the 8-mM group.

The results of the cell apoptosis analysis are shown in Figure 10 and Figure 11. Apoptosis is an active biological mechanism leading to programmed cell death. The decrease in the apoptosis rate indicates an increase in the proliferative activity of MC3T3-E1 cells. Figure 10 demonstrates the cell apoptosis culture in pure Mg and Mg-Ti composite extracts with different degradation times. After seeding for 1-day and 3-days, the apoptosis of all groups including pure Mg and the Mg-Ti composite is less than 10%, which is an acceptable range. On day 1, the apoptosis of the Mg-Ti group is lower than that of the pure Mg group but is slightly higher than that of the control group. On day 3, the apoptosis rates of pure Mg and Mg-Ti groups are both lower than those of the control group (Table 2). The apoptosis results of MC3T3-E1 cells on varying Mg²⁺ concentrations within 3-days are shown in Figure 11. After 3-days of incubation, the addition of Mg²⁺ reduces the apoptosis of MC3T3-E1 cells with the 8 mM Mg²⁺ exhibiting minimum apoptosis.

The differentiation ability of MC3T3-E1 cells is used to describe the osteoblast maturation and can be assessed by an ALP test. Figure 12A shows that the ALP activity is promoted in the Mg-Ti composite and pure Mg groups on days 7 and 14. It can be found that the ALP content of both groups increased as the osteogenic induction time was extended. On days 7 and 14, the ALP content of the Mg-Ti group is significantly higher than that of the pure Mg group (p < 0.05). In the different immersion times of pure Mg and Mg-Ti groups, the results show little difference. Figure 12B shows the ALP activity of MC3T3-E1 cells incubated in a gradient Mg²⁺ concentration medium for 7-days and 14-days. It can be identified that the addition of Mg²⁺ to the medium promotes the ALP activity of cells. The ALP activity is the highest in the 8 mM Mg²⁺ medium within both groups.

The reactions between Mg and the biological environment have to be taken into account when Mg-based materials are used as orthopedic implants. In the degradation process, especially at its early stage, the reaction of Mg with water molecules in the aqueous environment leads to the formation of hydroxide ions (OH⁻), hydrogen gas (H₂), and a high concentration of Mg²⁺. These three factors affect the biocompatibility of Mg-based materials. However, the degradation rate of Mg-based materials in vivo is not easy to be simulated by extracts obtained from the current ISO 10993 standards because of the large differences between in vivo and in vitro conditions. In the study by Gao, Su and Qin (2021), the results suggested that the immersion degradation rate in vitro was 2–4 times higher than the degradation rate of the in vivo implantation experiment. Similarly, the 1–5 correlation factors of the degradation rate can be predicted between in vitro and in vivo when an appropriate medium was used for the in vitro immersion test (Witte et al., 2006). Sanchez et al. (2015) also reported that the degradation rate in vitro reaches up to nine times higher than the in vivo degradation. Therefore, Wang et al. (2015)suggested that the extract of Mg-based materials should be diluted 6–9 times to test biocompatibility in vitro, according to the selection of implantation.

In this work, the extracts of the samples were diluted six times to evaluate the Mg-Ti composite and pure Mg availabilities for cell morphology, proliferation, and formation of alkaline phosphatase. Then, the Mg²⁺ concentrations of the diluted extracts were measured. These values were used as the basis for preparing culture media with various Mg²⁺ concentrations. As shown in Figure 5, the Mg²⁺ concentration of diluted pure Mg extracts decreases gradually over time, yet that of the Mg-Ti composite remains stable around 8 mM in the process of 7-days of degradation.

In general, the primary requirement of orthopedic implants is to be non-toxic to cells. The CCK-8 method is most commonly used to detect cell proliferation in vitro. According to CCK8 results, it can be found that Mg-Ti groups exhibit a similar cell proliferation ability of osteoblasts compared to pure Mg groups in the process of 7-days of degradation. In addition, both pure Mg and Mg-Ti groups have positive effects on the proliferation ability of osteoblasts compared with the control group on days 1, 2, and 3, which indicated that all the extracts in this study are non-toxic and of excellent biocompatibility. Similar results are also obtained after 3 days of culture in media (0.8, 2, 4, 6, 8, and 10 mM Mg²⁺) where the cell proliferation rates of all groups have no significant difference, showing that Mg²⁺ is non-toxic to cells under 10 mM.

In addition, Mg²⁺ also exerts an effect on cellular morphology. An appropriate Mg²⁺ concentration promotes the formation of the actin filament bundle and the expansion of cells, which is beneficial to cell adhesion and motility. It is widely accepted that the F-actin cytoskeletal network is a key regulator of cellular shape and force generation in cell migration with crucial roles in maintaining the cellular shape and elasticity. F-actin staining results (Figure 9) show that cell morphology with more filopodia was observed in the group with an Mg concentration at 8 mM. In the report by Maier and Haraszti (2015), Mg²⁺ has a significant influence on the global structure of the actin filament bundles. The addition of Mg²⁺ contributes to the changes in the elasticity of the actin network even at low concentrations (2–12 mM). Moreover, the speed and extent of bundle formation increase when elevating the Mg²⁺ concentration from 1 to 5–10 mM (Hu and Kuhn 2012). These all suggest that Mg²⁺ around 8 mM is an appropriate concentration benefiting the integration of implants and surrounding tissues. Moreover, as shown in Figure 8, the cell morphology of the Mg-Ti composite is better than that of pure Mg. This may result from the cooperated effect of Mg²⁺ concentration and corrosion products caused by different degradation modes and rates.

Apoptosis, occurring over several hours, is well known as cell programmed death since death results from the cell itself, eliminating damaged cells (Matsui et al., 2017). The apoptosis rate is an essential index when characterizing the biocompatibility of orthopedic implant materials. In the process of apoptosis, mitochondria play an essential role, which is associated with intracellular Ca²⁺ overload. Larger amounts of Ca²⁺ influx result in intracellular Ca²⁺ concentration rise and accumulation in the mitochondria (Thor, Hartzell and Orrenius 1984). The accumulation of Ca²⁺ contributes to the opening of a high-conductance pore in the inner mitochondrial membrane (IMM), which has been termed mitochondrial permeability transition (MPT) (Douglas et al., 1979). Then, the osmotic swelling of the mitochondria and rupture of the mitochondrial membrane occur which results in the mitochondrial proteins, including cytochrome C, releasing into the cytosol (Lemasters et al., 1999). In the cytosol, an apoptosome complex is formed by cytochrome C together with apoptosis activating factor-1 (Apaf-1) and pro-caspase-9. Cell apoptosis can be triggered by the apoptosome complex through intrinsic or extrinsic apoptosis pathways.

In the experiment of incubating MC3T3-E1 cells with a series of culture media in increasing Mg²⁺ concentrations, the results of the apoptosis analysis implied that the apoptosis rate gradually decreased with the increase in the Mg²⁺ concentration. The minimum apoptosis was observed in the 8-mM Mg²⁺ concentration culture medium. The reasons may be as follows: on one hand, the elevation of extracellular Mg²⁺, as the well-known calcium competing ion, suppresses the increase in the intracellular Ca²⁺ concentration by competing with extracellular Ca²⁺ and inhibiting the release of intracellular calcium, thereby preventing cell damage and reducing apoptosis (Bojan et al., 2013). On the other hand, several in vitro studies implied that the elevation of intracellular Mg²⁺ was observed in the early phase of apoptosis (Zhang et al., 2005). However, in the research of Matsui et al. (2017), they developed a novel Mg²⁺ probe which achieved a long-term visualization of intracellular Mg²⁺ dynamics during apoptosis. The results showed that the increase in the Mg²⁺ concentration is associated with the decrease in the ATP concentration after apoptotic cell shrinkage, demonstrating that Mg-ATP is the main resource for the Mg²⁺ increase after cell shrinkage during apoptosis. Moreover, the same conclusion was drawn by Chien et al. (1999) that the elevation of cytosolic free Mg²⁺ was irrelevant to the extracellular Mg²⁺ concentration with the increase in intracellular Mg²⁺ derived from the mitochondria during the process of apoptosis. Together, Mg²⁺ concentration at an appropriate range tends to decrease the apoptosis rate of cells. A recent study has documented that the addition of Mg particles reduces the apoptosis caused by the Ti particles (Wang Y et al., 2020). However, the apoptosis results of experimental extracts were not completely consistent with the aforementioned trends. It might be attributed to the generation of other factors (pH, osmolality, and corrosion product) in the degradation process.

ALP, as the key factor governing the process of osteogenesis, is expressed in the early stage of bone development and used as an early marker of osteoblast differentiation (Hu et al., 2021; Yi et al., 2021). In the present study, the ALP activities of the Mg-Ti and pure Mg groups with different degradation times were both higher than those of the control group. In addition, the ALP activity gradually decreased over time, which was consistent with the Mg²⁺ concentration trend of pure Mg extracts. It showed that the extracts of Mg-based materials could enhance the ALP activity of cells and the enhancing efficiency was closely related to the Mg²⁺ concentration of extracts. Moreover, the ALP activity of Mg-Ti groups was higher than that of pure Mg groups at all degradation times, which indicated that the extracts of the Mg-Ti composite could better promote the osteogenic differentiation of cells compared with pure Mg. The difference in the ALP activity of the two materials might be attributed to the following aspects. On one hand, the Mg²⁺ concentration of Mg-Ti composite extracts remained at around 8 mM in 7-days of degradation, which is regarded as an appropriate range for cell osteogenic differentiation. This assumption was verified by the experimental result of MC3T3-E1 cells incubated with varying Mg²⁺ concentrations. As shown in Figure 12, the ALP activity of the 8-mM group was significantly higher than that of the other five groups with the difference being statistically significant. On the other hand, the Mg-Ti composite exhibited a slightly higher pH value of less than 8.5 than that of pure Mg in the process of degradation. It was reported that suitable alkalinity (pH 7.8–8.5) had a beneficial effect on osteogenesis (Liu et al., 2016; Wu et al., 2019). However, the 7-day extract groups showed a similar Mg²⁺ concentration to 5-days, whereas they exhibited a higher ALP activity. This phenomenon may result from the production of more calcium phosphate after 7-days of degradation, which improves the osteogenic ability of cells.

In summary, the extracts of the Mg-Ti composite exhibit good biocompatibility and excellent osteogenic activity in the process of 7-days of degradation. However, because of the difference in degradation rates in vitro and in vivo, animal studies need to be carried out in the future to determine the suitability of the Mg-Ti composite as an orthopedic implant.