The porosity was acquired using Mg powder particles, and during the alloy synthesis process, with heating, these particles were partially evaporated, promoting pore formation in the material. The main spacers used in metallurgy to provide porosity are carbamide, sodium chloride, ammonium hydrogen carbonate and Mg [15–17]. Among these spacers, Mg is still little explored as a spacer. Macropores of approximately 100 µm were acquired (Fig. 1), and it is believed that pores in the range of 100–400 µm are ideal for bone ingrowth . Other authors claim that size does not directly influence bone formation [18, 19]. Thus, studies related to the development of materials with different pore sizes and their interaction with cells and the in vivo environment are extremely important.
Several studies have reported positive effects of the use of Mg in Ti alloys due to their osteoconductive and biocompatible character [4, 14, 20], but little is known about the oxidative effect of this metal, especially in promising alloys such as Ti-Nb-Sn with a low elastic modulus in mesenchymal stem cells (MSCs).
The mechanical properties acquired for this material were promising in terms of elastic modulus, hardness and compressive strength when compared to materials commercially used in the biomedical sector. In the work by Elias et al., the mechanical properties of commercial Ti grades 2, 4 and 5 were investigated. The elastic moduli values were found in the range of 108 to 115 GPa. Hardness values ranged from 171 to 453 HV, and the Strength ranged from 310 to 932 MPa . For the Ti-Nb-Sn/Mg alloy obtained in the present work, the elastic modulus value was significantly lower (approximately 16 GPa) approaching the bone tissue modulus, which is in the range of 0.5 to 20 GPa depending on the type of bone and from the direction of the analysis [22–24]. The hardness found was 226 HV, which was higher among the values of Ti of different grades studied in the work of Elias et al. . The compressive strength of Ti-Nb-Sn alloy was approximately 325 MPa, compared to Ti grade 2 (362.6 MPa)  and with a value higher than that of cortical bone (41–115 MPa) [25–27].
Figure 2 shows that the BMMSCs fully adhered with a spindle-shaped morphology and sprawled in a short time when in contact with the surface metal. The same was demonstrated in 48 hours of cell culture grown on the surface of the alloy with similar chemical composition, suggesting that the obtained alloy has no adverse effects on the cells in question. [28, 29]. The interaction of the metal surface with integrins, which are extracellular matrix proteins, promotes better cell adhesion . According to Cavalcanti-Adam, the spreading process is related to the interaction with the metallic surface and the integrins present in the extracellular matrix . The biocompatibility of materials is related to cell behavior in contact with the materials . Cell attachment is presumably the most important stage of cell interaction with a material surface because cell behavior depends on signaling cascades initiated via the adhesion process  needed for other cellular activities, such as spreading, proliferation and biosynthesis. After initial attachment, the cells become flattened and finally fully spread  Thus, it is clear that the surface of the Ti-Nb-Sn alloy does not provide a harmful surface for eBMMSCs.
Notably, the presence of possible extracellular vesicles or apoptotic bodies released by the eBMMSCs was also notable. Extracellular vesicles tend to be homogeneous in size, between 0.1 and 1 µm, whereas apoptotic bodies are larger, varying from 1 to 5 µm. Such vesicles are considered an additional factor in the mechanism of intercellular communication, allowing cells to exchange proteins, lipids, genetic material and adhesion molecules . Therefore, extracellular vesicles may also have facilitated the adhesion process . According to other studies, extracellular vesicles derived from MSCs and immobilized on Ti surfaces promoted cell proliferation after 3 to 6 days, as shown in Figs. 5 and 6. Recent works have studied the release of apoptotic bodies by MSCs and their impact on bone homeostasis. Apoptotic body treatment is able to ameliorate the osteoporotic phenotype, suggesting the potential use of apoptotic bodies to treat osteoporosis. Apoptotic body treatment directly improves the function of osteogenic cells to enhance bone formation and indirectly inhibits osteoclast activity by upregulating mediators in MSCs related to osteoclast apoptosis . Wang et al. studied the immobilization of extracellular vesicles (exosomes) derived from MSCs adhered to the Ti surface. . These bodies rapidly promote MSC adhesion and proliferation. There are still few studies on the effect of exosomes, microvesicles or apoptotic bodies released by MSCs on biomaterial surfaces. Research in this field demonstrating how the alloying elements, microstructure and roughness of materials influence their increase or decrease can be carried out.
In Fig. 5, the presence of carbon in the elemental map confirms the presence of eBMMSCs, since it is the largest constituent of living matter. The presence of roughness both inside and around the pores may be able to promote bone internal cell growth in the pore region, providing not only anchoring for fixation but also a system capable of allowing stresses to be transferred from the implant to the tissue . In the same figure, note the difficulty of finding the adhered cells being molded according to the surface on which they were exposed. Many works have found that cells have the ability to mold and modify their geometry depending on the environment .
The early differentiation of cells treated with conditioned medium by biomaterial was proven due to the clear morphological change from fibroblastic to polygonal shape before treatment with conventional medium and calcium deposition. As one of the differentiation strategies based on the concept that bioactive biological clues can be added to the implant surface to promote the regenerative processes on its surface, one of the important roles of magnesium is observed, which would be able to stimulate osteoconductivity. Mg affects the activity of alkaline phosphatase (ALP), a marker of early osteogenic differentiation, and its activity improves in the presence of Mg particles. In our work, 2.2 µg/ml or 0.9 mM Mg ions were released in the medium. Studies have shown that high doses can hamper the osteointegrative process. Zhang et al. treated human bone marrow stem cells (hBMSCs) with different concentrations of Mg2+, and matrix mineralization was significantly inhibited in osteoinductive medium at concentrations equal to or above 1.3 mM. . Wang et al. demonstrated that concentrations of 1.8 mM and 3.8 mM Mg2+ significantly decreased calcium oscillation amplitude or frequency, while increasing the concentration of Mg2+ from 0.8 to 1.3 mM exhibited no effect on calcium oscillation frequency . The authors deduced that a high Mg2+ environment inhibits matrix mineralization by suppressing the calcium oscillation frequency in hBMSCs.
The effect of the culture medium identified on eBMMSC ROS (+) was notable after 7 days of analysis, as indicated in Fig. 9. Naturally, in the control group, the eBMMSCs ROS (+) increased. This effect was also demonstrated in the works by Tirza et al. . In the treated group, the number of eBMMSCs ROS (+) began to fall within 24 hours of treatment with the conditioned medium, up to 48 hours of analysis and up to 7 days remained constant. Some authors have reported that low extracellular Mg is linked to an increased generation of ROS in different kinds of cells .
Using human endometrial MSCs, Lyublinskaya et al. showed that intracellular basal ROS levels are positively correlated with the proliferative status of cell cultures. In fact, they observed that physiologically relevant levels of ROS are required for the initiation of human MSC proliferation and that low levels of ROS due to antioxidant treatment can block stem cell self-renewal .
Oxidative stress is a major factor impairing MSC function, resulting in decreased osteogenesis . It is clear that Mg has an effect on decreasing eBMMSCs (ROS) + and on osteogenic differentiation.