Exosome-functionalized polyetheretherketone-based implant with immunomodulatory property for enhancing osseointegration

Abstract

The host immune response effecting on biomaterials is critical to determine implant fates and bone regeneration property. Bone marrow stem cells (BMSCs) derived exosomes (Exos) contain multiple biosignal molecules and have been demonstrated to exhibit immunomodulatory functions. Herein, we develop a BMSC-derived Exos-functionalized implant to accelerate bone integration by immunoregulation. BMSC-derived Exos were reversibly incorporated on tannic acid (TA) modified sulfonated polyetheretherketone (SPEEK) via the strong interaction of TA with biomacromolecules. The slowly released Exos from SPEEK can be phagocytosed by co-cultured cells, which could efficiently improve the biocompatibilities of SPEEK. In vitro results showed the Exos loaded SPEEK promoted macrophage M2 polarization via the NF-κB pathway to enhance BMSCs osteogenic differentiation. Further in vivo rat air-pouch model and rat femoral drilling model assessment of Exos loaded SPEEK revealed efficient macrophage M2 polarization, desirable new bone formation, and satisfactory osseointegration. Thus, BMSC-derived Exos-functionalized implant exerted osteoimmunomodulation effect to promote osteogenesis.

Keywords: BMSC-Derived exosomes; Bone regeneration; Macrophages polarization; Osteoimmunology; Polyetheretherketone.

Graphical abstract

Fig. 1

Characteristics of different samples. (a) Illustration of surface modification of PEEK. Fe³⁺ acts as an ionic cross-linker that can interact with up to three 3,4-dihydroxy-l-phenylalanine (DOPA) catechol functionalities to promote TA crosslinking. BMSC-derived Exos were reversibly bound to TA-SPEEK via hydrogen bond formation between phosphate groups in Exos phospholipid and polyphenol groups in the TA molecule. (b) FE-SEM images of PEEK, SPEEK, TA-SPEEK and Exo-TA-SPEEK. Scale bar represents 500 nm. (c) AFM images of PEEK, SPEEK, TA-SPEEK and Exo-TA-SPEEK. (d) 3D immunofluorescence images show an even distribution of Exos on the TA-SPEEK surface. Scale bar represents 200 μm. (e) The daily Exos released from SPEEK and TA-SPEEK within 14 days (n = 3). (f) Cumulative Exos release curve of SPEEK and TA-SPEEK up to 14 days (n = 3). (g) Immunofluorescence images show PKH26 labeled Exos were phagocytized by the BMSCs. Scale bar represents 200 μm. (h) Water contact angles of each sample (n = 3). ANOVA followed by Bonferroni's multiple comparison test was used for statistical analysis (*p < 0.05, **p < 0.01, ***p < 0.001).

Fig. 2

Biocompatibility of each sample. (a) Live/dead assay of BMSCs cultured on each sample surface for 1 day. The red staining indicated dead cells and green staining indicated live cells. Scale bar represents 100 μm. (b) Quantitative analysis of live/dead assay (n = 3). (c) CCK8 assay was used to evaluate the proliferation of BMSCs cultured on the surface of each sample for 1, 3 and 7 days (n = 5). (d) Immunofluorescence images showed the adhesion of BMSCs cultured on each sample after 3 days following culture. The green staining indicated cytoskeleton and the blue staining indicated nuclei. Scale bar represents 200 μm. (e) Quantitative analysis of cell spread area (n = 9). ANOVA followed by Bonferroni's multiple comparison test was used for statistical analysis (*p < 0.05, **p < 0.01, ***p < 0.001).

Fig. 3

In vitro RAW264.7 cells polarization. (a) An illustration of Exos loaded TA-SPEEK modulating macrophage polarization. (b) Immunofluorescence images show PKH26 labeled Exos phagocytized by RAW264.7 cells. Scale bar represents 50 μm (c) Effects of each sample on the expression of anti- and pro-inflammatory genes were evaluated by RT-qPCR (n = 3). (d) IBa-1 and Arg-1 immunofluorescent staining of RAW264.7 cells on each sample surface are shown three days following culture. IBa-1 was stained green, Arg-1 was stained red and nuclei was stained blue. Scale bar represents 50 μm. (e) Quantitative analysis of immunofluorescent staining (n = 3). (f) IBa-1 and iNOS immunofluorescent staining of RAW264.7 cells on each sample surface three days after culture. IBa-1 was stained green, iNOS was stained red and nuclei was stained blue. Scale bar represents 50 μm. (g) Quantitative analysis of immunofluorescent staining (n = 3). (h) Western blot analysis of Arg-1 and iNOS protein expression. (i) Quantitative analysis of Western blot (n = 3). (j) Western blot analysis of protein expression (IKBα, p- IKBα, P65, p-P65) of the NF-κB pathway. (k) Quantitative analysis of Western blot (n = 3). ANOVA followed by Bonferroni's multiple comparison test was used for statistical analysis (*p < 0.05, **p < 0.01, ***p < 0.001).

Fig. 4

Macrophage polarization effect on BMSCs osteogenesis. (a) A representative illustration of the co-culture system used. (b) ALP staining of BMSCs cultured for 7 days in a co-culture system. Scale bar represents 200 μm. (c) Quantitative analysis of ALP staining (n = 5). (d) ARS staining of BMSCs cultured for 7 days in the co-cultured system. Scale bar represents 200 μm. (e) Quantitative analysis of ARS staining (n = 5). (f) The expression of osteogenesis-related genes, such as Col I, RunX2, OCN, and OPN, was estimated by RT-qPCR after BMSCs co-culture for 7 days (n = 3). (g) RunX2 and OPN immunofluorescent staining of BMSCs was showed 7 days after being cultured in the co-culture system. RunX2 and OPN were stained red and the nuclei was stained blue. Scale bar represents 50 μm. (h) Quantitative analysis of immunofluorescent staining (n = 3). (i) The expression of RunX2 and OPN protein was evaluated by Western blot at 7 days. (j) Quantitative analysis of Western blot (n = 3). ANOVA followed by Bonferroni's multiple comparison test was used for statistical analysis (*p < 0.05, **p < 0.01, ***p < 0.001).

Fig. 5

Samples promoted osteogenic differentiation (a) ALP and ARS staining of BMSCs cultured for 7 days on the surface of the samples. (b) Quantitative analysis of ALP and ARS staining (n = 5). (c) Osteogenesis-related gene expression of BMSCs cultured on the surface of the samples at day 7 (Col I, RunX2, OCN, and OPN, n = 3). (d) RunX2 and OPN immunofluorescent staining of BMSCs was showed after 7 days cultured on the surface of the samples. RunX2 and OPN were stained red and the nuclei was stained blue. Scale bar represents 50 μm. (e) Quantitative analysis of immunofluorescent staining (n = 3). (f) The expression of RunX2 and OPN protein was evaluated by Western blot at 7 days. (g) Quantitative analysis of Western blot (n = 3). ANOVA followed by Bonferroni's multiple comparison test was used for statistical analysis (*p < 0.05, **p < 0.01, ***p < 0.001).

Fig. 6

Results of rat air-pouch model. (a) HE staining and Masson's trichrome staining of rat air-pouch skin. Scale bar represents 1 mm. (b) Quantitative analysis of the thickness of the fibrous layer (n = 3). (c) Immunofluorescent staining of Arg-1 and iNOS in air-pouch skin. Arg-1 and iNOS were stained red and the nuclei was stained blue. Scale bar represents 200 μm. (d) Quantitative analysis of immunofluorescent staining (n = 3). (e) The expression of Arg-1 and iNOS protein was evaluated by Western blot at 7 days. (f) Quantitative analysis of Western blot (n = 3). ANOVA followed by Bonferroni's multiple comparison test was used for statistical analysis (*p < 0.05, **p < 0.01, ***p < 0.001).

Fig. 7

In vivo new bone regeneration property of different implants. (a) An illustration of the rat femur surgical procedure. (b) General photos of the implantation site on the rat femur. Scale bar represents 2 mm. (c) Micro-CT images showing bone regeneration around the implants. The red arrows mark the new regeneration bone. (d) Reconstructed 3D images of samples and new bone. Yellow color indicates samples and red color indicates new regeneration bone. (e) Quantitative analysis of new bone 8 weeks after implantation (n = 3). (f) Histological images of methylene blue/acid fuchsin staining show the new regeneration bone around each sample 8 weeks post-operatively. Black arrow indicates collagen fiber formation. Red arrow indicates new bone formation. Scale bar represents 200 μm. ANOVA followed by Bonferroni's multiple comparison test was used for statistical analysis (*p < 0.05, **p < 0.01, ***p < 0.001).