The most critical application of nano-biomimetic platform in bone repair is to integrate progenitors, improve progenitor activity, and repair and reconstruct segmental bone defects more quickly[19–23]. In the changes of bone microenvironment, osteogenic differentiation is often accompanied by changes in the level of cell oxidation. In recent years, evidence has shown that the regulation of mitochondrial dynamics and function is necessary for the successful differentiation of bone marrow mesenchymal stem cells. In this paper, we review and provide a comprehensive view of the role of mitochondria in the differentiation of bone marrow mesenchymal stem cells. Mitochondria in bone marrow mesenchymal stem cells maintain a relatively low level of activity. After induction, mitochondrial DNA copy number, respiratory enzyme protein level, oxygen consumption, mitochondrial biosynthesis-related gene mRNA level and intracellular ATP content are increased. Studies have found that the regulation of mitochondrial ROS level affects differentiation and helps to determine the direction of differentiation [14]. Considering the above factors, we verified the determination of the level of oxidative stress in BMSCs by nanomaterials. The ROS level of BMSCs was detected by adding Pb-Fe-Cs to the culture medium. After 24 hours of culture, the effects of Pb-Fe-Cs on ROS levels after H202 treatment were observed by TEAC, GSH, SOD, and MDA kits (Fig. 3A). Data showed that compared with the PbS group and the sham group, the TEAC, GSH, and SOD levels in the Pb-Fe-Cs group were higher, while only the SOD value was lower than that of the other two groups, indicating that the material can alleviate the oxidative stress level of BMSCs cells and provide a good living environment for cells, which may be due to the activity of Pb-Fe-Cs similar to natural protease. Relevant studies have reported that BMSCs migration has a significant promoting effect on tissue repair[24], and related studies have shown that nanomaterials can better promote tissue regeneration by promoting cell migration. Therefore, we explored the effects of our synthesized nanomaterials on BMSCs migration (Fig. 3B). The results showed that in the cell scratch experiment, we observed cells at 24h and 48h, and found that many BMSCs were distributed at the edge and entered the exposed site, indicating that the BMSCs cells in the material group had strong migration ability, which may be due to the strong activity of the cells after reducing ROS by the nanomaterials. It is worth noting that after 48 hours of culture, the exposed leakage area of Pb-Fe-Cs was significantly lower than that of the control group and the PbS group. These results show that Pb-Fe-Cs is suitable for cell growth, proliferation, adhesion, and migration, which may be due to the hydrophilicity and conductivity of Pb-Fe-Cs.
As mentioned above, we demonstrated that the nano-bionic platform can improve the activity of progenitor cells, thereby faster repair and reconstruction of segmental bone defects.Relevant studies have shown that bone marrow mesenchymal stem cells have a unique role in bone repair.We prepared Pb-Fe-Cs to enhance the biological effect of the composite material, induce double differentiation of bone marrow mesenchymal cells, and achieve rapid bone repair [20–21]. More and more evidences have shown that.bionic nanomaterials can effectively promote bone formation induction differentiation.As Pb-Fe-Cs had a better effect on cell viability in the previous validation, we verified the effect of Pb-Fe-Cs on bone formation induction differentiation here.Firstly, bone marrow mesenchymal stem cells were cultured in the cell culture medium.Then, pre-configured osteogenic medium (ID), DMEM + 10%FBS, Basanti, glutamine and dexamethasone were added to promote the differentiation of bone marrow mesenchymal stem cells into osteoblasts by promoting the expression of mesenchymal stem cells differentiation-specific transcription factor (Cbfal mRNA) and osteogenic differentiation-specific transcription factor (Osterix mRNA).
On the 14th and 21st day, alkaline phosphatase (ALP) and alizarin red S (ARS) reagents were used for fixation staining to observe the cell morphology.As shown in Fig. 3C, compared with the ID group alone, the osteoblast differentiation of the material group was more obvious, which fully demonstrated that Pb-Fe-Cs had good cell viability and promoted the formation of osteoblasts. To further support our experimental results, we detected the expression levels of runt-related transcription factor 2 (Runx2) gene and osteoblast maturation marker OCN (Fig. 3D, 3E). The data showed that the osteoblast maturation markers associated with the material group were significantly increased compared with the other two groups. These results suggest that Pb-Fe-C has a role in promoting osteoblast differentiation induction, which may be related to its regulation of ROS and promotion of cell migration to maintain a healthy state of cells.