Biomimetic periosteum combining BMP-2-loaded M2 macrophage-derived exosomes for enhanced bone defect repair

Abstract

Bone defect repair continues to present a significant clinical challenge due to the limitations of traditional grafting techniques and the complexity involved in establishing a conducive regenerative microenvironment. In this study, we described the development of a multifunctional biomimetic periosteum based on electrospun gelatin methacryloyl (GelMA) membranes functionalized with bone morphogenetic protein-2 (BMP-2)-loaded M2 macrophage-derived exosomes. This engineered periosteum replicated the structural orientation and functional properties of natural periosteum, thereby providing a synergistic approach to promoting bone regeneration. Our findings indicated that the biomimetic periosteum served as a biocompatible scaffold that supported cell adhesion, proliferation, and differentiation. The incorporation of M2 macrophage-derived exosomes facilitated the creation of an anti-inflammatory immune microenvironment by polarizing macrophages towards the M2 phenotype, while the sustained release of BMP-2 enhances osteogenic differentiation and mineralization. In vivo experiments using a rat cranial defect model demonstrated that the BMP-2@Exo-GelMA membrane significantly accelerated bone defect repair, achieving superior outcomes in new bone formation and vascularization compared to control groups. This study underscored the potential of integrating immunomodulatory and osteoinductive strategies to develop next-generation biomaterials for bone tissue engineering. The biomimetic periosteum represented a promising therapeutic approach for addressing critical-sized bone defects and advancing clinical practices in bone regeneration.

Keywords: BMP; biomimetic periosteum; bone defect; exosomes; macrophage.

FIGURE 1

Schematic diagram of biomimetic periosteum promoting bone regeneration.

FIGURE 2

Characterization of exosomes and biomimetic periosteum. (A) The representative TEM images of exosomes. (B) The size of exosomes. (C) Western blot analysis of the exosomes specific protein markers. (D) The representative TEM images of GelMA and Exo-GelMA. (E) FTIR of GelMA and Exo-GelMA. (F) The stress-strain curve of GelMA and Exo-GelMA. (G) The release rate of exosomes.

FIGURE 3

Biocompatibility of biomimetic periosteum. (A) Cytoskeletal staining of BMSCs seeded on the surface of biomimetic periosteum after 24 h (B) SEM images of cell morphology on the surface of biomimetic periosteum after 24 h. (C) Live/dead staining of BMSCs seeded on the surface of biomimetic periosteum after 24 h (D) CCK-8 assay indicating the proliferation rate of BMSCs after 1, 3 and 5 days. (*P < 0.05). OD, optical density.

FIGURE 4

The effect of biomimetic periosteum on the polarization of macrophages in vitro. (A) Fluorescence microscope images of immunofluorescent staining of CD86 after 24 h. (B) Fluorescence microscope images of immunofluorescent staining of CD206 after 24 h (C) Il1β, Tnf, Il10 and Tgfβ1 gene expression of macrophages evaluated by qRT-PCR after 24 h *P < 0.05.

FIGURE 5

Osteogenic differentiation of BMSCs. (A) The represent images of ALP staining after 7 days. (B) ALP activity of BMSCs after 7 days. (C,D) The represent images and quantitative analysis of ARS after 7 days. (E) The expression of osteogenesis-related genes after 7 days *P < 0.05.

FIGURE 6

The evaluation of bone defect regeneration in vivo. (A) The new bone formation in the bone defects evaluated by Micro-CT imaging and 3D reconstruction at 4 and 8 weeks. (B) Quantitative analysis of BV/TV and BMD in defect area at 4 and 8 weeks. (C) Representative HE staining of the defect area. *P < 0.05.

FIGURE 7

Immunohistochemical analysis. (A) Immunohistochemical staining for TNF-α and TGF-β in the defect area at 4 weeks. (B) Quantitative analysis of TNF-α and TGF-β‐positive area. (C) Immunohistochemical staining for COL1A1 and CD31 in the defect area at 4 and 8 weeks. (D,E) Quantitative analysis of COL1A1 and CD31‐positive area at 4 and 8 weeks *P < 0.05.