Engineering immunomodulatory and osteoinductive implant surfaces via mussel adhesion-mediated ion coordination and molecular clicking

Abstract

Immune response and new tissue formation are important aspects of tissue repair. However, only a single aspect is generally considered in previous biomedical interventions, and the synergistic effect is unclear. Here, a dual-effect coating with immobilized immunomodulatory metal ions (e.g., Zn²⁺) and osteoinductive growth factors (e.g., BMP-2 peptide) is designed via mussel adhesion-mediated ion coordination and molecular clicking strategy. Compared to the bare TiO₂ group, Zn²⁺ can increase M2 macrophage recruitment by up to 92.5% in vivo and upregulate the expression of M2 cytokine IL-10 by 84.5%; while the dual-effect of Zn²⁺ and BMP-2 peptide can increase M2 macrophages recruitment by up to 124.7% in vivo and upregulate the expression of M2 cytokine IL-10 by 171%. These benefits eventually significantly enhance bone-implant mechanical fixation (203.3 N) and new bone ingrowth (82.1%) compared to the bare TiO₂ (98.6 N and 45.1%, respectively). Taken together, the dual-effect coating can be utilized to synergistically modulate the osteoimmune microenvironment at the bone-implant interface, enhancing bone regeneration for successful implantation.

Fig. 1. Design strategy for engineering immunomodulatory and osteoinductive implant surfaces.

A Schematic illustration of the mussel-derived peptide for ion coordination and biomolecular click conjugation on a medical Ti screw. B In a bone implant model, the Zn²⁺ and BMP-2 peptide co-modified Ti screw shows osteoinductive and immunomodulatory dual functions in vivo, synergistically enhancing the interfacial osteogenesis and the intra-bone implant integration after implantation.

Fig. 2. Characterizations of clickable peptide and Zn²⁺ and BMP-2 peptide co-modified surface.

A, B The molecular structures of (DOPA)₆-PEG₅-DBCO and (2-Azido)-PEG₅-BMP-2. C, D ESI-MS spectra of the two synthetic peptides. E AFM images of different surfaces and (F) quantitation of the suface roughess with the different modified surfaces (n = 3 independent samples per group, by a one-way ANOVA with a Tukey’s post hoc test for multiple comparisons). Data are reported as mean ± SD, *p < 0.05, **p < 0.01). G The water contact angles of different surfaces and (H) the quantitative results (n = 3 independent samples per group, by a one-way ANOVA with a Tukey’s post hoc test for multiple comparisons. Data are reported as mean ± SD, **p < 0.01). I SEM-EDS elemental mapping for the Zn²⁺ and BMP-2 peptide co-modified surface (Zn/BMP-2) (scar bar = 5 μm, three independent experiments). J–L XPS analysis of the bare and modified TiO₂ surface (DBCO-TiO₂, Zn, BMP-2 and Zn/BMP-2. M Quantitative elemental analysis according to XPS. N Changes of N 1 s signal in the XPS spectrum of the Zn/BMP-2 surface after incubated in DMEM for 2 weeks. O Zn²⁺ release profiles of the Zn/BMP-² surface in PBS solution; red (left) and blue (right) represent the non-accumulative and accumulative Zn²⁺ release, respectively. Exact P values were given in the Source Data file.

Fig. 3. Biocompatibility properties of Zn²⁺ and BMP-2 co-modified TiO₂ surfaces.

A Live/Dead staining of BM-MSCs and RAW264.7 on the bare and modified TiO₂ surface (DBCO-TiO₂, Zn, BMP-2 and Zn/BMP-2) (scar bar = 50 μm, three independent experiments). B Scanning electron images of BM-MSCs on different surfaces (×500 scar bar = 100 μm and ×2000 scar bar = 25 μm, three independent experiments). C The cytoskeleton staining (FITC-phalloidin/DAPI) of BM-MSCs on different surfaces for 1 and 4 days (scar bar = 50 μm, three independent experiments). D Cell viability of BM-MSCs and RAW264.7 on different surfaces for 1 and 4 days (CCK-8) (D n = 3 independent samples per group, by a one-way ANOVA with a Tukey’s post hoc test for multiple comparisons). E Cell cytotoxicity of BM-MSCs and RAW264.7 on different surfaces for 24 h (E n = 3, independent samples per group, by a one-way ANOVA with a Tukey’s post hoc test for multiple comparisons). Data are reported as mean ± SD, *p < 0.05, **p < 0.01 compared with the bare TiO₂ surface; ^#p < 0.05, ^##p < 0.01 compared with the DBCO-TiO₂ surface; ^&p < 0.05, ^&&p < 0.01 compared with Zn surface. Exact P values were given in the Source Data file.

Fig. 4. Zn²⁺ and BMP-2 peptide co-modified TiO₂ surface regulate macrophages polarization in vitro.

A The illustration of experimental design; B The morphology of RAW246.7 was stimulated by LPS or without LPS; C The morphology of RAW264.7 was cultured on different surfaces (TiO₂, DBCO-TiO₂, Zn, BMP-2, and Zn/BMP-2 surface) (scar bar = 100 μm and 10 μm, three independent experiments) and (D) Quantitative results of pancake-like shape cells (M1) as a proportion of total cells (D n = 3 biologically independent samples per group, by a one-way ANOVA with a Tukey’s post hoc test for multiple comparisons); E TNF-α and (F) IL-10 cytokine secretion by ELISA (E–F n = 3 biologically independent samples per group, by a one-way ANOVA with a Tukey’s post hoc test for multiple comparisons). G, J Immunofluorescent staining results for RAW264.7 cultured on different surfaces: red (M1 marker: CD86 or iNOS and M2 marker: CD206 or Arg-1), green (F4/80, a monoclonal antibody specifically directing against the mouse macrophage), and blue (nuclei) (scar bar = 25 μm, three independent experiments); Corresponding percentage of double-positive macrophages M1 (H, I) and M2 (K, L) (H–I, K–L n = 3 biologically independent samples per group, by a one-way ANOVA with a Tukey’s post hoc test for multiple comparisons); M–R RT-PCR results of Tnf-α, Il-10, Ccr7, Cd206, Bmp-2, and Vegf respectively (M–R n = 3, biologically independent samples per group, by a one-way ANOVA with a Tukey’s post hoc test for multiple comparisons). Data are reported as mean ± SD, *p < 0.05, **p < 0.01. Exact P values were given in the Source Data file.

Fig. 5. Zn²⁺ and BMP-2 peptide co-modified TiO₂ surfaces enhance osteogenic differentiation in vitro.

A The illustration of experimental design; B, E ALP staining and (C, G) ARS staining of BM-MSCs cultured in osteogenic medium supplemented with MCM (B–C scar bar = 50 μm, three independent experiments); F ALP activity assay of the BM-MSCs on the different surfaces; D Images of the BM-MSCs after immunofluorescent staining:(green: OPN; red: cytoskeleton and blue: nuclei) (scar bar = 30 μm, three independent experiments) and (H) quantitative results; I–L Osteogenesis-related genes expression of the BM-MSCs cultured in MCM detected by RT-PCR (Alp, Runx2, Col1a1 and Opn). (E–L n = 3 biologically independent samples per group, by a one-way ANOVA with a Tukey’s post hoc test for multiple comparisons). Data are reported as mean ± SD, *p < 0.05, **p < 0.01. Exact P values were given in the Source Data file.

Fig. 6. Zn²⁺ and BMP-2 peptide co-modified Ti screws regulate macrophages polarization in vivo.

A H&E staining images of the peri-implant tissue (scar bar = 100 μm and 50 μm, three independent experiments) and quantified with (D) fibrous layers and (E) infiltration inflammatory cells; B Coimmunostaining images of the peri-implant tissue: green (M1 marker, CCR7 and M2 marker, CD206), red (CD68, rat macrophage-specific antigen marker), and blue (nuclei) with white arrows indicating the double-positive cells (scar bar = 50 μm and 20 μm, three independent experiments) and (F, G) Quantitative double-positive macrophages; C Images of immunohistochemical staining of IL-10 in the peri-implant tissue (scar bar = 100 μm and 50 μm, three independent experiments) and (H) quantification of IL-10 positive cells as a proportion of total cells. (D–H n = 3 biologically independent samples per group, by a one-way ANOVA with a Tukey’s post hoc test for multiple comparisons). Data are reported as mean ± SD, *p < 0.05, **p < 0.01 compared with the bare TiO₂ surface; ^#p < 0.05, ^##p < 0.01 compared with the DBCO-TiO₂ surface; ^&p < 0.05, ^&&p < 0.01 compared with BMP-2 surface. Exact P values were given in the Source Data file.

Fig. 7. Micro-CT and histological analysis on osseointegration between bone tissue and screw.

A Micro-CT 3D reconstructed images and (B) quantitatively evaluating the peri-implant bone generation according to the BMD, BV/TV, Tb.Sp, Tb.Th and Tb.N (B n = three biologically independent samples per group, by a one-way ANOVA with a Tukey’s post hoc test for multiple comparisons). C–E Calcein-Alizarin Red staining for the newly formed bone and (D) quantitative staining analysis; Van Gieson and bone implant contact (BIC). F Maximum fixation force in different groups determined by pull-out testing. G Schematic of the bone regeneration mechanism by enabling M2 phenotype switching and osteoinductivity (D–F n = 3 independent samples per group, by a one-way ANOVA with a Tukey’s post hoc test for multiple comparisons). Data are reported as mean ± SD; *p < 0.05, **p < 0.01. Exact P values were given in the Source Data file.