3D-printed bredigite scaffolds with ordered arrangement structures promote bone regeneration by inducing macrophage polarization in onlay grafts

Abstract

Bone tissue engineering scaffolds may provide a potential strategy for onlay bone grafts for oral implants. For determining the fate of scaffold biomaterials and osteogenesis effects, the host immune response is crucial. In the present study, bredigite (BRT) bioceramic scaffolds with an ordered arrangement structure (BRT-O) and a random morphology (BRT-R) were fabricated. The physicochemical properties of scaffolds were first characterized by scanning electron microscopy, mechanical test and micro-Fourier transform infrared spectroscopy. In addition, their osteogenic and immunomodulatory properties in an onlay grafting model were investigated. In vitro, the BRT-O scaffolds facilitated the macrophage polarization towards a pro-regenerative M2 phenotype, which subsequently facilitated the migration and osteogenic differentiation of bone marrow-derived mesenchymal stem cells. In vivo, an onlay grafting model was successfully established in the cranium of rabbits. In addition, the BRT-O scaffolds grafted on rabbit cranium promoted bone regeneration and CD68 + CD206 + M2 macrophage polarization. In conclusion, the 3D-printed BRT-O scaffold presents as a promising scaffold biomaterial for onlay grafts by regulating the local immune microenvironment.

Keywords: Bone regeneration; Bone scaffold; Onlay graft; Oral implant; Osteoimmunomodulation.

Fig. 1

The characterization of bredigite scaffolds. (A) The schematic and scanning electron microscopy images of the BRT-R and BRT-O scaffolds. (B) The micro-FTIR spectra and mappings of the scaffolds. (C) The compressive strength of the scaffolds. (D) Weight loss (%) of the scaffolds after soaking in buffer for different time periods. (E) The pH value change after soaking the scaffolds at different time points. The (F) Ca, (G) Mg and (H) Si concentration in Tris buffer at different time points. The data were expressed as mean ± standard deviation (SD) (n = 3). ^***p < 0.001 vs. BRT-R. Scale bar: A, 1 mm and 5 μm

Fig. 2

Scaffolds polarized the macrophage phenotypes in vitro. (A and B) Flow cytometry analysis of RAW264.7 cells that were seeded on different scaffolds for three days. (C) The qRT-PCR analysis of the macrophage polarization-related gene expression at day three. (D) The enzyme-linked immunosorbent assay analysis of cytokines in the supernatants of RAW264.7 cells cultured on different scaffolds on day three. (E) The morphology of macrophages seeded on different scaffolds visualized by scanning electron microscopy. (F) The immunofluorescent staining of macrophages seeded on different scaffolds on day three with CD68 (green), CD206 or iNOS (red), and nuclei (blue). The data were expressed as mean ± standard deviation (SD) (n = 3). The cells without scaffolds were set as a control group. Ns, no significance; ^**p < 0.01 vs. the control group; ^***p < 0.001 vs. the control group; ^###p < 0.01 vs. the BRT-R group. Scale bars: E, 5 μm; F, 50 μm

Fig. 3

Polarized macrophages by BRT-O scaffolds promoted the osteogenic differentiation of BMSCSs in vitro. (A) CCK-8 analysis of BMSCSs after 1, 3 and 7 days of co-culture with different conditioned media (CM). (B) The qRT-PCR analysis of the relative mRNA expression levels of RUNX2 and BMP2 in BMSCSs on day seven. (C) Western blot results for the RUNX2 expression levels in BMSCSs on day seven. (D) The transwell assay of BMSCSs induced by different CM. (E) The quantification analysis of cell migration. (F) Representative images of the wound healing capacities of BMSCSs after co-culture with different CM for the indicated time periods. (G) Quantitative analysis of the wound healing. (H) ARS staining of BMSCSs on day 21. (I) Semi-quantification analysis of ARS. The data were expressed as mean ± standard deviation (SD) (n = 3). Ns, no significance; ^**p < 0.01 vs. the control group; ^***p < 0.001 vs. the control group; ^#p < 0.05 vs. the BRT-R group; ^###p < 0.01 vs. the BRT-R group. Scale bars: D, 100 μm; F, 200 μm; H, 100 μm

Fig. 4

The BRT-O scaffold promoted bone regeneration in the onlay grafting model of rabbit cranium. (A) Images of the surgical procedure, and specimens of the different groups at six and 16 weeks after surgery. (B) Representative micro-CT images of different groups at six and 16 weeks, post-surgery. Representative images for the (C) H&E and (D) toluidine blue staining of samples collected at six and 16 weeks, post-surgery (A, auto graft; S, scaffold; ^*, new bone; OB, old bone). (E) Immunohistochemical staining for RUNX2 and macrophage polarization pan marker CD68, M1 marker iNOS, and M2 marker CD206 in the onlay grafting area. Quantitative results for (F) BV/TV and (G) BMD by micro-CT. (H) Quantitative results for the new bone (%) in the scaffold area. (I) Quantification of positively stained cells. The data were expressed as mean ± standard deviation (SD) (n = 3). Ns, no significance; ^**p < 0.01 vs. the control group; ^***p < 0.001 vs. the control group; ^#p < 0.05 vs. the BRT-R group; ^###p < 0.01 vs. the BRT-R group. Scale bars: B, 1 mm; E, 500 μm and 200 μm; F, 300 μm and 200 μm; H, 500 μm