Mussel inspired 3D elastomer enabled rapid calvarial bone regeneration through recruiting more osteoprogenitors from the dura mater

Abstract

Currently, the successful healing of critical-sized calvarial bone defects remains a considerable challenge. The immune response plays a key role in regulating bone regeneration after material grafting. Previous studies mainly focused on the relationship between macrophages and bone marrow mesenchymal stem cells (BMSCs), while dural cells were recently found to play a vital role in the calvarial bone healing. In this study, a series of 3D elastomers with different proportions of polycaprolactone (PCL) and poly(glycerol sebacate) (PGS) were fabricated, which were further supplemented with polydopamine (PDA) coating. The physicochemical properties of the PCL/PGS and PCL/PGS/PDA grafts were measured, and then they were implanted as filling materials for 8 mm calvarial bone defects. The results showed that a matched and effective PDA interface formed on a well-proportioned elastomer, which effectively modulated the polarization of M2 macrophages and promoted the recruitment of dural cells to achieve full-thickness bone repair through both intramembranous and endochondral ossification. Single-cell RNA sequencing analysis revealed the predominance of dural cells during bone healing and their close relationship with macrophages. The findings illustrated that the crosstalk between dural cells and macrophages determined the vertical full-thickness bone repair for the first time, which may be the new target for designing bone grafts for calvarial bone healing.

Keywords: 3D elastomer; bone healing; dural cells; macrophages; polydopamine.

Graphical abstract

Figure 1.

Characterization of 3D porous elastomers. (A) Schematic diagram of fabrication process. (B) SEM examination showed microstructures of 3D porous elastomers. (C) Surface porosity of 3D porous elastomers (n = 3). (D) XPS total spectrum of 3D porous elastomers. (E). XPS spectrum for N 1 s of the C0G100, C15G85 and C30G70 groups. (F). XPS spectrum for N 1 s of the C0G100-PDA, C15G85-PDA and C30G70-PDA groups. (G) Quantitative analysis of XPS (n = 3). (H) Tensile stress–strain curves of 3D porous elastomers. (I) Compressive stress–strain curves of 3D porous elastomers. (J) The hydrophilicity of 3D porous elastomers was detected by water contact angle. (K) Quantitative comparison of WCA (n = 3). Data are presented as mean ± SD. ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 2.

Biodegradability of 3D elastomers and the stability of PDA interface in vivo (A) H&E staining visualized degradation patterns of critical bone defects in the C0G100-PDA, C15G85-PDA and C30G70-PDA groups at 2, 4, 6 and 12 weeks. Quantitative comparison of remaining area of (B) PCL and (C) graft material (n = 3). Data are presented as mean ± SD. ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 3.

3D elastomer enabled rapid calvarial bone regeneration. (A) Schematic diagram of 3D porous elastomers’ application in rat calvarial defects. (B) 3D reconstruction and coronal/sagittal plane analysis of the defects in the C0G100-PDA, C15G85-PDA and C30G70-PDA groups at 4, 6 and 12 weeks. Quantitative analysis of (C) BV/TV and (D) Tb.N at 4, 6 and 12 weeks of the different groups (n = 5). Data are presented as mean ± SD. ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 4.

Examining of the bone regeneration. H&E staining showed the repair effects of critical bone defects at (A) 4, (B) 6 and (C) 12 weeks in the different groups. Masson’s trichrome staining assessed the collagen deposition of critical bone defects at (D) 4, (E) 6 and (F) 12 weeks in the different groups. (G) Quantitative comparison of new bone area (n = 5). Data are presented as mean ± SD. ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 5.

Early pro-healing microenvironment in calvarial bone defect. (A) The interior views of 3D porous elastomers after 2 weeks. Red circle marks boundary of the 8 mm rat calvarial defect. (B) Representative immunofluorescence showed M1 and M2 macrophage polarization in the C15G85, C0G100-PDA, C15G85-PDA and C30G70-PDA groups at 2 weeks. (C) Quantitative comparison of M1/M2 (n = 5). Data are presented as mean ± SD. ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 6.

Single-cell RNA seq analysis of the C15G85-PDA group at 2 weeks. (A) UMAP analysis atlas of C15G85-PDA graft remodeling at 2 weeks after implantation. (B) Specific cluster identification map of the cell type. (C) Feature plots showed the expression of specific marker genes in fibroblast subsets. (D) Specific cluster identification map of Fibroblast. (E) Feature plots showed the expression of specific marker genes in Dura-Arachnoid Fibroblast subsets. (F) Representative immunofluorescent staining of Alpl and Mgp in regenerative tissue at 2 weeks.

Figure 7.

Single-cell RNA seq analysis of the C15G85-PDA group at 2 weeks. (A) Differentiation trajectory inferred by RNA velocity of the C15G85-PDA group after 2 weeks of defect surgery. Red arrow indicates the differentiation direction. (a) and (b) represent two distinct cell differentiation trajectories. (B) Trajectory of differentiation from Cdhr1-Fibroblasts to Dura-Arachnoid Fibroblasts (a) and Nkrk2-Fibroblasts (b) lineages predicted by Monocle. Barplot showed the GO enrichment in (C) Dura-Arachnoid Fibroblasts, (D) Pial Fibroblasts and (E) Nkrk2-Fibroblasts subset.

Figure 8.

Macrophage depletion impaired the bone repair by C15G85-PDA graft. (A) Heatmap of cell–cell communication analysis based on CellPhoneDB. (B) Strength of ligand–receptor interactions among macrophages, Dura-Arachnoid Fibroblasts and Pial Fibroblasts pairs based on CellChat analysis. (C) Schematic diagram of the C15G85-PDA group loaded with PBS liposomes or clodronate liposomes into a rat calvarial defect. (D) Quantitative comparison of M1/M2 (n = 5). (E) Representative immunofluorescent of CD206, iNOS, Alpl and Mgp in regenerative tissue in the C15G85-PDA + PBS and C15G85-PDA + Clodronate at 2 weeks. (F) Quantitative comparison percentage of positive CD206 and iNOS cells (n = 5). (G) Quantitative comparison percentage of positive Alpl and Mgp cells (n = 5). (H) Micro-CT imaging analysis and (I) quantitative analysis of BV/TV and (J) Tb.N at 6 weeks in the C15G85-PDA + PBS and C15G85-PDA + Clodronate groups (n = 5). Edge width is proportional to the number of ligand–receptor pairs. Circle sizes are proportional to the number of cells per cluster. Data are presented as mean ± SD. ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Scheme 1.

Schematic diagram illustrates that the early enrichment of M2 macrophages and dural cells inside the bone defect mediated by 3D elastomer contributes to calvarial bone healing.