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. 2024 Mar 3:25:101017.
doi: 10.1016/j.mtbio.2024.101017. eCollection 2024 Apr.

Immobilizing c(RGDfc) on the surface of metal-phenolic networks by thiol-click reaction for accelerating osteointegration of implant

Zeyu Shou  1   2 , Zhibiao Bai  1 , Kaiyuan Huo  3 , Shengwu Zheng  4 , Yizhe Shen  1 , Han Zhou  1 , Xiaojing Huang  1 , Hongming Meng  1 , Chenwei Xu  1 , Shaohao Wu  1 , Na Li  2 , Chun Chen  1   5   6
Affiliations

Immobilizing c(RGDfc) on the surface of metal-phenolic networks by thiol-click reaction for accelerating osteointegration of implant

Zeyu Shou et al. Mater Today Bio. .

Abstract

The limited osteointegration often leads to the failure of implant, which can be improved by fixing bioactive molecules onto the surface, such as arginyl-glycyl-aspartic acid (RGD): a cell adhesion motif. Metal-Phenolic Networks (MPNs) have garnered increasing attention from different disciplines in recent years due to their simple and rapid process for depositing on various substrates or particles with different shapes. However, the lack of cellular binding sites on MPNs greatly blocks its application in tissue engineering. In this study, we present a facile and efficient approach for producing PC/Fe@c(RGDfc) composite coatings through the conjugation of c(RGDfc) peptides onto the surface of PC/Fe-MPNs utilizing thiol-click reaction. By combined various techniques (ellipsometry, X-ray photoelectron spectroscopy, Liquid Chromatography-Mass Spectrometry, water contact angle, scanning electronic microscopy, atomic force microscopy) the physicochemical properties (composition, coating mechanism and process, modulus and hydrophilicity) of PC/Fe@c(RGDfc) surface were characterized in detail. In addition, the PC/Fe@c(RGDfc) coating exhibits the remarkable ability to positively modulate cellular attachment, proliferation, migration and promoted bone-implant integration in vivo, maintaining the inherent features of MPNs: anti-inflammatory, anti-oxidative properties, as well as multiple substrate deposition. This work contributes to engineering MPNs-based coatings with bioactive molecules by a facile and efficient thiol-click reaction, as an innovative perspective for future development of surface modification of implant materials.

Keywords: Arginyl-glycyl-aspartic (RGD); Implant surface; Metal-phenolic networks (MPNs); Osteointegration; Peptide immobilization.

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Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Image 1
Graphical abstract
Scheme 1
Scheme 1
(a) The structure of PC and its schematic diagram, (b) the structure of c(RGDfc) and its schematic diagram, (c) the construction of MPN and its oxidation in alkaline environment, (d) the reaction of the sulfhydryl group of cysteine on c(RGDfc) with MPN under alkaline conditions, (e) the schematic diagram of the construction of PC/Fe@c(RGDfc) composite coating on various substrates. Created with BioRender.com.
Fig. 1
Fig. 1
Characterization of PC/Fe@c(RGDfc). (a) The thickness of c(RGDfc) adsorbed on (PC/Fe)5-MPNs. (b) The total XPS spectra of the PC/Fe-MPNs and the PC/Fe@c(RGDfc) coatings, along with the elemental molar ratio obtained by XPS analysis in (c). (d) Zeta potential changes of coating surface resulting from c(RGDfc) adsorption at different concentrations on (PC/Fe)5-MPNs, while surface hydrophilicity changes of both types of composite coatings are shown in (e). (f) The variation of coating thickness with treatment time in PBS, 0.9%NaCl, DEME with or without 0.25% trypsin. (g) Optical photographs of PC/Fe-MPNs and PC/Fe@c(RGDfc) composite coatings on different substrates. (h) SEM photos and ImageJ roughness fitting of these coatings adhering to silicon substrate. N = 3, no significance noted as "ns," *p < 0.05, **p < 0.01, ***p < 0.001, using t-test. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig. 2
Fig. 2
Mechanism and Factors of the construction of c(RGDfc) peptide-grafted PC/Fe-MPNs substrates. N1s peaks of (a) c(RGDfc) and (b) the PC/Fe@c(RGDfc) composite coating. (c) LC-MS analysis of the interaction between c(RGDfc) and PC. (d) The thickness of c(RGDfc) adsorbed on (PC/Fe)5-MPNs at different PH values, (e) concentrations of c(RGDfc), and (f) after different deposition time. (g) Thickness changes of c(RGDfc) grafted on PC/Fe-MPNs formed with different cycles in the PC/Fe-MPNs fabrication process. (h) Schematic of the PC/Fe@c(RGDfc) coating construction mechanism, Created with BioRender.com. N = 3, no significance noted as "ns," *p < 0.05, **p < 0.01, ***p < 0.001, and #p < 0.05, ##p < 0.01 or ###p < 0.001 compared with the Control group, using t-test.
Fig. 3
Fig. 3
AFM images of PC/Fe-MPNs and PC/Fe@c(RGDfc) coatings in Liquid environment. (a–b) Surface roughness, (c–d) Young's modulus images and (e–f) Adhesion energy representative images and quantitative analysis. N = 3, no significance noted as "ns," *p < 0.05, **p < 0.01, ***p < 0.001, using t-test.
Fig. 4
Fig. 4
SEM images (a) and cell viability (b) of mBMSCs, HUVECs and NIH3T3 cultured on Ti, Ti-PC/Fe-MPNs, and Ti-PC/Fe@c(RGDfc). Cell cycle experiments in different cells on Ti, Ti-PC/Fe-MPNs, and Ti-PC/Fe@c(RGDfc). Scale bars in (a) are 200 nm. N = 3, no significance noted as "ns," *p < 0.05, **p < 0.01, ***p < 0.001, and #p < 0.05, ##p < 0.01 or ###p < 0.001 compared with the Control group, using t-test.
Fig. 5
Fig. 5
(a) Scratch test of mBMSCs, HUVECs and NIH3T3 cultured on Glass, PC/Fe-MPNs, and PC/Fe@c(RGDfc) under serum-free treatment with cell migration rate statistics (b). Scale bars in (a) are 100 μm. N = 3, no significance noted as "ns," *p < 0.05, **p < 0.01, ***p < 0.001, and #p < 0.05, ##p < 0.01 or ###p < 0.001 compared with the Control group, using t-test.
Fig. 6
Fig. 6
Adhesion of mBMSCs, HUVECs and NIH3T3 to Glass, PC/Fe-MPNs, and PC/Fe@c(RGDfc) coated substrates. (a) Fluorescence microscopy images of cells seeded on different surfaces for 2 and 4 h. (b) Cell number per square micrometer of the seeded cells. (c) Schematic representation of the cell-substrate adhesion ability assay after cell spreading. (d) Cell-substrate adhesion ability of cells seeded on different surfaces for 8 h. (e) Cell morphology after attachment of cells to different substrates. Scale bars in (a) are 50 μm, and in (e) are 40um. N = 3, no significance noted as "ns," *p < 0.05, **p < 0.01, ***p < 0.001, and #p < 0.05, ##p < 0.01 or ###p < 0.001 compared with the Control group, using t-test.
Fig. 7
Fig. 7
The intracellular ROS level of different cells cultured in coated or uncoated glass with or without treatment of H2O2 was detected by fluorescent staining. (a) The average fluorescence intensity of cells was detected by flow cytometry. (b) Comparison of data from (a). N = 3, no significance noted as "ns," *p < 0.05, **p < 0.01, ***p < 0.001, and #p < 0.05, ##p < 0.01 or ###p < 0.001 compared with the Control group, using t-test.
Fig. 8
Fig. 8
PC/Fe@c(RGDfc) coating promoted cell osteogenesis in vitro. Representative images of ALP staining of mBMSCs cultured on different coatings on days 3 and 7 of cell culture (a) and detection of ALP activity (b). Representative images of ARS staining (c) and quantification of calcium nodules (d) of mBMSCs cultured on different substrates for 14 and 21 days. LCSM images of mBMSCs cultured on different coatings for 7 d, and nuclei (blue) and collagen I(red) staining were performed. N = 3, no significance noted as "ns", ***p < 0.001 using t-test. N = 3, no significance noted as "ns," *p < 0.05, **p < 0.01, ***p < 0.001, and #p < 0.05, ##p < 0.01 or ###p < 0.001 compared with the Control group, using t-test. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig. 9
Fig. 9
Osteoinductive and osseointegration capacities of functionalized Ti-based materials in vivo. (a) Schematic diagram of the experiment in vivo. (b) Micro-CT was used to detect the quality of the regenerated bone around the implanted Ti rods containing different coatings after 8 weeks. (c) Comparison of the bone volume fraction (BV/TV %) of the implants of (b). (d) Comparison of the trabecular number (Tb. N 1/mm) of the implants of (b). (e) Representative histological images of Ti rob coated different coatings stained with toluidine blue. (f) The average histomorphometric values of bone-implant contact (BIC). Scale bars in (e) are 100 μm. N = 3, no significance noted as "ns", ***p < 0.001 using t-test. n = 3, no significance noted as "ns," *p < 0.05, **p < 0.01, ***p < 0.001, and #p < 0.05, ##p < 0.01 or ###p < 0.001 compared with the Control group, using t-test. (Schematic illustration created with BioRender.com). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
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