In vitro and in vivo study of concentrated growth factor (CGF) mediating macrophage polarization in bone defect repair

Abstract

Concentrated growth factor (CGF) is widely applied in clinical practice, but whether it has bone promoting effects and its mechanism of action are still the focus of discussion. In this study, in vitro experiments demonstrate that CGF can promote the expression of Arg-1 in BMDM cells, facilitating their polarization towards the M2 macrophages and encouraging the secretion of IL-10 and VEGF-A. CGF modulates M1 macrophages by reducing the expression of iNOS, while enhancing Arg-1 expression, thereby converting them to M2 macrophages. This is accompanied by a decrease in the secretion of TNF- $\alpha$ and IL-1β, and an increase in the secretion of IL-10 and VEGF-A. Mechanistically, CGF promotes the phosphorylation of STAT3, which in turn induces M2 macrophage polarization, suggesting that the function of CGF-mediated macrophages may be associated with the STAT3 signaling pathway. Moreover, CGF-mediated macrophages were found to enhance osteoblast activity, increasing the expression of ALP, RUNX2, and BMP-2, and improving cell migration capabilities. In vivo experiments showed that CGF could early recruit M2 macrophages to the bone defect site, promoting the expression of bone formation-related proteins such as ALP and BMP-2, and accelerating bone tissue regeneration. In summary, our study demonstrates that CGF can induce bone repair and regeneration by promoting immune modulation and macrophage polarization.

Keywords: Bone regeneration; CGF; JAK/STAT; Macrophages.

Fig. 1

Surface characteristics of C GF and its effect on macrophages. A: CGF and its three-dimensional structure under electron microscopy; The proliferation of BMDM treated with different concentrations of CGF; B: ELISA method is used to detect the secretion of IL-1 $\mathrm{\beta }$、TNF-$\mathrm{\alpha }$、VEGF-A、IL-10 after CGF acts on M0 and M1 macrophages; C: Western blot detection of protein expression of Arg-1 and iNOS after CGF acts on M0 and M1 macrophages; D: Western blot detection of JAK/STAT pathway related protein expression after CGF acts on M0 and M1 macrophages, respectively (∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001. #, P < 0.05; ##, P < 0.01; ###, P < 0.001; ####, P < 0.0001. # compared with the control group, ns, P > 0.05,one-way ANOVA).

Fig. 2

Morphological observation of CGF on macrophage polarization. A: Morphological changes of macrophages under inverted microscope; B: Observation of Morphological Changes of Macrophages by Immunofluorescence Method (the scale represents 50 μm).

Fig. 3

The effect of C GF induced macrophage polarization on osteoblasts. A: The effect of CGF mediated M0 macrophages on the expression of osteogenic related proteins in MC3T3-E1 cells; B: The effect of CGF mediated M1 macrophages on the expression of osteogenic related proteins in MC3T3-E1 cells; C: Scratch assay to detect the effect of CGF induced macrophage polarization on the migration ability of MC3T3-E1 cells. (∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.00005, ns, P > 0.05).

Fig. 4

Immunofluorescence images of rat skull bone defects at 7 and 14 days post injury. A: Arg-1 immunofluorescence image on the 7th day. B: Arg-1 immunofluorescence image on the 14th day (ns, P > 0.05, ∗∗P < 0.01) (the scale represents 100 μm).

Fig. 5

Micro CT and immunohistochemical images of rat skull bone defects at 2 W, 4 W, and 8 W. A: Statistical analysis of Micro CT images and BV/TV, Tb. N values at 2 W, 4 W, and 8 W after skull bone defect in rats; B: Immunohistochemical staining images of ALP after 2 W, 4 W, and 8 W of skull bone defect in rats; C: Immunohistochemical staining images of BMP-2 after 2 W, 4 W, and 8 W of skull bone defect in rats; (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, ns, P > 0.05) (the scales represent 1 mm and 100 μm, respectively).