Imidazole functionalized photo-crosslinked aliphatic polycarbonate drug-eluting coatings on zinc alloys for osteogenesis, angiogenesis, and bacteriostasis in bone regeneration

Abstract

Zinc (Zn) alloys have demonstrated significant potential in healing critical-sized bone defects. However, the clinical application of Zn alloys implants is still hindered by challenges including excessive release of zinc ions (Zn²⁺), particularly in the early stage of implantation, and absence of bio-functions related to complex bone repair processes. Herein, a biodegradable aliphatic polycarbonate drug-eluting coating was fabricated on zinc-lithium (Zn-Li) alloys to inhibit Zn²⁺ release and enhance the osteogenesis, angiogenesis, and bacteriostasis of Zn alloys. Specifically, the photo-curable aliphatic polycarbonates were co-assembled with simvastatin and deposited onto Zn alloys to produce a drug-loaded coating, which was crosslinked by subsequent UV light irradiation. During the 60 days long-term immersion test, the coating showed distinguished stable drug release and Zn²⁺ release inhibition properties. Benefiting from the regulated release of Zn²⁺ and simvastatin, the coating facilitated the adhesion, proliferation, and differentiation of MC3T3-E1 cells, as well as the migration and tube formation of EA.hy926 cells. Astonishingly, the coating also showed remarkable antibacterial properties against both S. aureus and E. coli. The in vivo rabbit critical-size femur bone defects model demonstrated that the drug-eluting coating could efficiently promote new bone formation and the expression of platelet endothelial cell adhesion molecule-1 (CD31) and osteocalcin (OCN). The enhancement of osteogenesis, angiogenesis, and bacteriostasis is achieved by precisely controlling of the released Zn²⁺ at an appropriate level, as well as the stable release profile of simvastatin. This tailored aliphatic polycarbonate drug-eluting coating provides significant potential for clinical applications of Zn alloys implants.

Keywords: Aliphatic polycarbonate; Angiogenesis; Drug-eluting coating; Osteogenesis; Zn alloy implant.

Graphical abstract

Scheme 1

Schematic illustration of the fabrication of photo-crosslinked aliphatic polycarbonate drug-eluting coating on Zn alloy. (A) Synthesis of the aliphatic polycarbonate poly(TMC-co-TMCI-co-TMCA) (PTMCIA). (B) Preparation of photo-crosslinked aliphatic polycarbonate drug-eluting coatings at the surface of Zn alloy. (C) The degradation and drug release process of polycarbonate coatings together with the osteogenesis, angiogenesis, and bacteriostasis of coated Zn alloy.

Fig. 1

(A) Schematic illustration of electrophoretic deposition of CP@SIM colloidal particles on a Zn alloys substrate. (B) Coating thickness for varying time at a constant voltage of 40 V and a colloidal concentration of 15 mg/mL (C) ATR-FTIR spectra of bare and CP@SIM coated Zn alloys before and after UV light irradiation. (D) Lap shear stress of PTMC, CP, and CP@SIM coating on Zn alloys. (E) SEM and WCAs images of bare, CP, and CP@SIM coated Zn alloys. (F) XPS wide-scan, (G) and (H) Zn 2p spectra of bare and CP, CP@SIM coating on Zn alloys, (I) Zn 2p spectra of CP coating on Zn alloys after 3 and 7 days immersion, and (J) N 1s, (K) C 1s spectra of PTMCF, CP, and CP@SIM coated Zn alloys.

Fig. 2

(A) Potentiodynamic polarization test of bare and coated Zn alloys in SBF (37 ± 0.5 °C) for 4 h. (B) SIM accumulates release curves for coated Zn alloys. (C) pH values, (D) Zn²⁺ concentrations, (E) surface SEM images, (F) digital photographs, and (G) cross-sectional SEM images of bare and coated Zn alloys after immersion for 60 days in SBF at 37 °C. (H) Fitted curve of CP@SIM coating thickness after immersing for 0 and, 15, 30, 45 days. (I) Schematic illustration of degradation and drug release process of coated Zn alloys.

Fig. 3

Viability and morphology of preosteoblast MC3T3-E1 on bare, CP coated, and CP@SIM coated Zn alloys. (A) Live/dead stained fluorescence images for MC3T3-E1 on different samples after culturing for 1 and 3 days. (B) Cell viability by CCK-8 and (C) LDH activity of MC3T3-E1 incubated in different extracts for 1 and 3 days. (D) Fluorescence images of MC3T3-E1 cultured on different sample surfaces for 3 days with action stained with FITC (green) and nuclei stained with DAPI (blue). (E) SEM images of MC3T3-E1 cultured on different sample surfaces for 3 days. (F) Total coverage area and (G) number of live cells. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 4

Osteogenic activity in vitro of the different Zn alloys. (A) Representative staining photographs and (B) quantification of ALP activity for MC3T3-E1 cultured on different samples for 7 days. (C) Representative ARS staining photograph and (D) quantification of calcium deposition for MC3T3-E1 cultured on different samples for 14 days. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 5

Angiogenic activity of the different Zn alloys samples. (A) Migration assay of EA.hy926 incubated with the extract of different samples after wounding. (B) Quantification statistical analysis of migration assay. (C) Tube formation assay of EA.hy926 in different extracts cultured on Matrigel. (D) Quantification statistical analysis of tube formation assay. (E) Quantification statistical analysis of in vivo CAM assay. (F) Representative photographs of in vivo CAM assay. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 6

Antibacterial activity of 316L SS and different Zn alloys samples against E. coli and S. aureus. (A) Photographs of agar plates of colonies of E. coli and S. aureus. The antibacterial rate of different samples against (B) E. coli and (C) S. aureus. (D) The SEM images of the morphological changes of E. coli and S. aureus after incubation with bare Zn alloy and Zn-CP@SIM samples. *p < 0.05, **p < 0.01.

Fig. 7

Results of the rabbit critical-size femur defect model. (A) 2D and 3D micro-CT images of the implants and surrounding bones. Quantification of (B) new bone volume and (C) new bone mineral density. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 8

Representative photographs of histological staining of H&E and Masson's trichrome, and immunohistochemical staining of OCN and CD31 after implanting 8 weeks. M = metal; T = tissue; FT = fibrous tissue; NB = newly formed bone; Black arrows = blood vessels.