Recombinant human bone morphogenetic protein-2 (rhBMP-2) induced macrophage biphasic polarization regulated by dexamethasone in vivo

Abstract

Objectives: To evaluate macrophage polarization dynamics in vivo after implantation of recombinant human bone morphogenetic protein-2 (rhBMP-2) incorporated biomaterials, with a focus on dose-dependent effects and polarization modulation strategies.

Methods: A murine dorsal subcutaneous implantation model was utilized to analyze macrophage responses to varying concentrations of rhBMP-2-loaded biomaterials with or without dexamethasone (Dex). Polarization patterns were assessed through phenotypic characterization and cytokine expression profiling.

Results: Elevated rhBMP-2 concentrations amplified macrophage polarization activities, and concurrent activation of M1 and M2 polarization was observed accompanied by enhanced expression of both pro-inflammatory (M1-associated) and anti-inflammatory (M2-associated) cytokines. Dexamethasone co-administration effectively attenuated pro-inflammatory polarization patterns induced by high-dose rhBMP-2 implants while preserving regenerative cytokine expression.

Conclusions: Optimized rhBMP-2 dosage facilitates a balanced macrophage polarization state, creating a pro-regenerative microenvironment through coordinated inflammatory resolution and tissue remodeling signals. For clinical applications requiring high rhBMP-2 doses, concurrent short-term anti-inflammatory therapy (e.g., dexamethasone) is recommended to mitigate excessive M1 polarization without compromising osteoinductive capacity.

Keywords: BMP-2; Macrophage; bio-material; dexamethasone; polarization.

Figure 1

Representative histological images of HE-stained explanted sponges from each experimental group are presented here, clearly indicating the regions colonized with recruited infiltrating cells (Left panel, Scale bar: 50 μm). These images provide a visual representation of cellular infiltration and colonization within the sponges over the course of the experiment. Additionally, a quantitative analysis of cell numbers in the corresponding exudates collected from the mouse dorsal subcutaneous area at 2, 4, and 7 days after implantation is shown (Right panel, A-C). This analysis offers a numerical assessment of the cellular response to the implanted sponges over time.

Figure 2

To evaluate the dynamic polarization of macrophages in response to biomaterial implantation, the expression of cell surface markers on macrophages isolated from inflammatory exudates in the mouse dorsal subcutaneous area was analyzed using dual-color immunofluorescence staining for CCR7 (a classical M1 macrophage marker) and CD206 (a prototypical M2 macrophage marker). Representative flow cytometry dot plots are presented, illustrating the co-expression patterns of CCR7 (vertical axis) and CD206 (horizontal axis) antigens across experimental groups. The percentage of F4/80-positive macrophages exhibiting CCR7⁺ (pro-inflammatory M1 phenotype), CD206⁺ (anti-inflammatory M2 phenotype), or dual-positive subpopulations was quantified to assess macrophage polarization states under different treatment conditions. A. Control: absorbable gelatin sponges as blank control. B. LBMP: sponges loaded with lower dose rhBMP-2. C. HBMP: sponges loaded with higher dose rhBMP-2. D. HBMP+DEX: sponges loaded with higher dose of rhBMP-2 and Dex.

Figure 3

Representative immunofluorescent images of the explanted absorbable gelatin sponges, both with and without the addition of rhBMP-2 and dexamethasone, were captured at 2, 4, and 7 days post-implantation (A-C). In these images, F4/80-positive cells are stained with a green fluorophore, indicating the presence of macrophages. CCR7-positive cells are stained with a red fluorophore, while CD206-positive cells are also stained with a red fluorophore, distinguishing between different macrophage subpopulations. The nuclei of the cells are revealed by DAPI staining, which appears blue. The arrows in the images point out examples of cells that exhibit double staining: F4/80+/CCR7+ cells (appearing yellow) represent M1 macrophages, which are typically pro-inflammatory. Conversely, F4/80+/CD206+ cells (also appearing yellow) represent M2 macrophages, which are generally anti-inflammatory and involved in tissue repair and healing (Scale bar: 50 μm).

Figure 4

Gene expression profiles of pro-inflammatory cytokines interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and anti-inflammatory cytokine interleukin-10 (IL-10) along with vascular endothelial growth factor-A (VEGF-A) in inflammatory cells colonizing explanted sponges were quantitatively analyzed across experimental groups using real-time PCR. Statistically significant differences between groups are denoted as *P < 0.05 and **P < 0.01. A. IL-1β expression levels. B. IL-6 expression levels. C. TNF-α expression levels. D. IL-10 expression levels. E. VEGF-A expression levels.

Figure 5

Cytokine production profile of inflammatory cells isolated from recovered exudates. The concentrations of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and vascular endothelial growth factor-A (VEGF-A) in cell culture supernatants were quantified using enzyme-linked immunosorbent assay (ELISA). see (A-C). Statistically significant differences between groups are indicated as *P < 0.05 and **P < 0.01.

Figure 6

Schematic illustration depicting macrophage response mechanisms to varying concentrations of rhBMP-2 alone and in combination with dexamethasone (DEX).