Abstract: BACKGROUND: The immune microenvironment is closely associated with the osteogenic differentiation of stem cells, especially the polarization state of macrophages plays a key regulatory role in bone tissue regeneration. Interleukin-4, as an important immunomodulatory cytokine, holds promise for promoting bone defect repair by modulating the local immune milieu.
OBJECTIVE: To investigate the effects of different concentrations of interleukin-4 on the proliferation and osteogenic polarization of adipose-derived mesenchymal stem cells and macrophages, and to analyze the regulatory effects of interleukin-4 on the osteogenic differentiation of adipose-derived mesenchymal stem cells at different time points in order to elucidate the mechanisms of immunomodulation in tissue-engineered bone repair.
METHODS: Adipose-derived mesenchymal stem cells were co-cultured with macrophages in 12-well plates, and interleukin-4 at different concentrations (0, 30, and 60 μg/L) was administered to assess the effects of interleukin-4 on the adhesion, proliferation, osteogenic differentiation, and macrophage polarization of adipose-derived mesenchymal stem cells. Phalloidin staining was used to observe cell morphology and adhesion, while cell proliferation activity was evaluated using the CCK-8 assay. RT-qPCR was performed to detect the mRNA expression levels of the M1 macrophage marker CCR7 and the M2 marker CD206, as well as osteogenic gene expression. Osteocalcin protein expression was detected by immunofluorescence, and osteogenic differentiation was assessed by alkaline phosphatase and Alizarin red staining. In the time-dependent experiment, the optimal concentration of interleukin-4 was delivered at different time points (days 2, 4, 6, and 8) to analyze the effects of interleukin-4 on macrophage polarization and osteogenic gene expression in adipose-derived mesenchymal stem cells.
RESULTS AND CONCLUSION: (1) Interleukin-4 at 30 μg/L markedly promoted macrophage polarization toward the M2 phenotype and enhanced the proliferation and osteogenic differentiation of adipose-derived mesenchymal stem cells. (2) Compared with the blank control group, alkaline phosphatase and alizarin red staining were significantly enhanced in the 30 μg/L interleukin-4 group, and the expression of osteoblast-related genes (alkaline phosphatase, type I collagen, osteocalcin, Runt-related transcription factor 2) and osteoblastic protein (osteocalcin) was the highest (P < 0.05). (3) In the culture system, the addition of interleukin-4 on days 4-8 improved the polarization state of macrophages and enhanced osteogenic differentiation, suggesting that the immunomodulatory effect mediated by interleukin-4 was time-dependent. (4) These findings indicate that appropriate concentration and timing of interleukin-4 delivery enhance osteogenic differentiation of adipose-derived mesenchymal stem cells by balancing M1 and M2 macrophages, highlighting the key role of osteoimmunomodulation in tissue-engineered bone repair.

Key words: ">adipose-derived mesenchymal stem cells, macrophage polarization, interleukin-4, immune microenvironment, cell co-culture, cell adhesion, cell proliferation, osteogenic differentiation