Programmed surface on poly(aryl-ether-ether-ketone) initiating immune mediation and fulfilling bone regeneration sequentially

Abstract

The immune responses are involved in every stage after implantation but the reported immune-regulated materials only work at the beginning without fully considering the different phases of bone healing. Here, poly(aryl-ether-ether-ketone) (PEEK) is coated with a programmed surface, which rapidly releases interleukin-10 (IL-10) in the first week and slowly delivers dexamethasone (DEX) up to 4 weeks. Owing to the synergistic effects of IL-10 and DEX, an aptly weak inflammation is triggered within the first week, followed by significant M2 polarization of macrophages and upregulation of the autophagy-related factors. The suitable immunomodulatory activities pave the way for osteogenesis and the steady release of DEX facilitates bone regeneration thereafter. The sequential immune-mediated process is also validated by an 8-week implementation on a rat model. This is the first attempt to construct implants by taking advantage of both immune-mediated modulation and sequential regulation spanning all bone regeneration phases, which provides insights into the fabrication of advanced biomaterials for tissue engineering and immunological therapeutics.

Keywords: bone regeneration; immune-mediated osteogenesis; poly(aryl-ether-ether-ketone); sequential release; surface modifications.

Graphical abstract

Figure 1

Sample fabrication and characterization (A) Flow chart showing the progress of sample fabrication. (B) SEM images showing the surface morphology of different samples. (C) Water contact angles of samples (n = 4). (D and E) (D) Survey XPS spectra as well as (E) atomic percentages determined on different samples. (F–I) High-resolution C 1s spectra of (F) P, (G) P-D, (H) P-DP, and (I) P-DPI samples. ∗p < 0.05 and ∗∗∗p < 0.001 compared with the P group, whereas ###p < 0.001 compared with the P-DPI group.

Figure 2

Release parameters of IL-10 and DEX of P-DPI samples in the solution containing lipase as a function of time (A) Cumulative released amounts. (B) Release velocities. (C) Percentages of cumulative release (symbols and dashed lines) as well as curves showing the first-order releasing kinetics (solid lines) (n = 4).

Figure 3

Biological response of macrophages stimulated by various samples (A) Macrophages viability test for 1, 3, and 5 days. (B and C) Secretion of (B) pro-inflammatory and (C) anti-inflammatory cytokines after cultivating macrophages for 1 and 3 days. Expression of (D) M1 and (E) M2 genes, and (F) autophagy-related genes after cultivating macrophages for 3 days. ∗∗p < 0.01 and ∗∗∗p < 0.001 compared with the P group, whereas ##p < 0.01 and ###p < 0.001 compared with the P-DPI group (n = 4).

Figure 4

In vitro performances of osteoblasts (A) Experimental design of conditioned culture and analysis. (B) Experimental design of direct culture and analysis. (C) ALP activity of osteoblasts in different groups after conditioned culture. (D) Viability of osteoblasts directly cultured on different samples for 1, 3, and 7 days. (E) ALP activity of osteoblasts directly cultured on different samples after osteogenic induction for 3 and 7 days. (F) Mineralization of osteoblasts directly cultured on different samples after osteogenic induction for 14 and 21 days. ∗∗p < 0.01 and ∗∗∗p < 0.001 compared with the P group, #p < 0.05, ##p < 0.01, and ###p < 0.001 compared with the P-DPI group, whereas &p < 0.05, &&p < 0.01, and &&&p < 0.001 by comparing RAW 264.7 (+) with RAW 264.7 (−) in each group (n = 4).

Figure 5

In vivo analysis of the inflammatory status (A) SEM images of macrophages on different samples after implantation for 7 days. (B) Immunofluorescent staining images of macrophages on different samples after implantation for 7 days. Red, green, and blue fluorescence reflect density for iNOS, CD163, and nuclei. (C) H&E staining images of peri-implant tissues after implantation for 7 days. The fibrous layers are marked by dashed lines. (D) Quantitative comparison of fibrous layer thickness after implantation for 1, 3, and 7 days. Scale bars, 100 μm (except those in the insets being equal to 10 μm). ∗∗p < 0.01 and ∗∗∗p < 0.001 compared with the P group, whereas ##p < 0.01 and ###p < 0.001 compared with the P-DPI group (n = 6).

Figure 6

In vivo analysis of bone regeneration after implantation for 8 weeks (A) 2D and reconstructed 3D micro-computed tomography (micro-CT) images of the bone with the implants inside. (B–D) Quantitative micro-CT data of (B) bone volume/total volume (BV/TV), (C) trabecular number (Tb.N), and (D) trabecular separation (Tb.Sp). (E) Formation of new bone labeled by sequential fluorescent staining (tissues labeled by alizarin red, tetracycline hydrochloride, and calcein show red, yellow, and green fluorescence, respectively). (F and G) Histological observation of peri-implant tissues after (F) Van Gieson and (G) H&E staining with new bone marked by dashed lines. Scale bars, 100 μm. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 compared with the P group, whereas ##p < 0.01 and ###p < 0.001 compared with the P-DPI group (n = 6).