Surface Functionalization with Proanthocyanidins Provides an Anti-Oxidant Defense Mechanism That Improves the Long-Term Stability and Osteogenesis of Titanium Implants

Abstract

Purpose: Aseptic loosening is a major complication after total joint replacement. Reactive oxygen species generated by local tissue cells and liberated from implant surfaces have been suggested to cause implant failures. Surface modification of titanium (Ti)-based implants with proanthocyanidins (PAC) is a promising approach for the development of anti-oxidant defense mechanism to supplement the mechanical functions of Ti implants. In this study, a controlled PAC release system was fabricated on the surface of Ti substrates using the layer-by-layer (LBL) assembly.

Materials and methods: Polyethyleneimine (PEI) base layer was fabricated to enable layer-by-layer (LBL) deposition of hyaluronic acid/chitosan (HA/CS) multi-layers without or with the PAC. Surface topography and wettability of the fabricated HA/CS-PAC substrates were characterized by scanning electron microscopy (SEM), atomic force microscopy (AFM), Fourier-transform infrared spectroscopy (FTIR) and contact angle measurement. PAC release profiles were investigated using drug release assays. MC3T3-E1 pre-osteoblast cells were used to assess the osteo-inductive effects of HA/CS-PAC substrates under conditions H₂O₂-induced oxidative stress in vitro. A rat model of femoral intramedullary implantation evaluated the osseo-integration and osteo-inductive potential of the HA/CS-PAC coated Ti implants in vivo.

Results: SEM, AFM, FTIR and contact angle measurements verified the successful fabrication of Ti surfaces with multi-layered HA/CS-PAC coating. Drug release assays revealed controlled and sustained release of PAC over 14 days. In vitro, cell-based assays showed high tolerability and enhanced the osteogenic potential of MC3T3-E1 cells on HA/CS-PAC substrates when under conditions of H₂O₂-induced oxidative stress. In vivo evaluation of femoral bone 14 days after femoral intramedullary implantation confirmed the enhanced osteo-inductive potential of the HA/CS-PAC coated Ti implants.

Conclusion: Multi-layering of HA/CS-PAC coating onto Ti-based surfaces by the LBL deposition significantly enhances implant osseo-integration and promotes osteogenesis under conditions of oxidative stress. This study provides new insights for future applications in the field of joint arthroplasty.

Keywords: ROS; anti-oxidant; layer-by-layer methodology; osteogenesis; proanthocyanidins; surface modification of titanium.

Figure 1

Schematic diagram of sample preparation: the process of fabricating the HA/CS-PAC multilayer coatings on the PEI-primed Ti disk or rod surfaces. Abbreviations: CS, chitosan; HA, hyaluronic acid; PAC, proanthocyanidins; PEI, polyethyleneimine; Ti, titanium.

Figure 2

SEM and AFM images of different surfaces. Notes: SEM or AFM images of (A&G) Ti; (B) Ti-PEI, Ti after PEI priming; (C) Ti-PEI-HA, Ti-PEI after single HA coating; (D) Ti-PEI-HA-CS, Ti-PEI after HA-CS coating; (E&H) HA/CS, Ti-PEI coated with multilayer of HA-CS; (F&I) HA/CS-L, Ti-PEI coated with multilayer of HA-CS with PAC-low immobilization. Arrow indicates agglomerated PAC Abbreviations: CS, chitosan; HA, hyaluronic acid; PAC, proanthocyanidins; PEI, polyethyleneimine; Ti, titanium.

Figure 3

Surface chemical composition analyses by FTIR. Notes: FTIR wide scan spectra of (A) Ti; (B) Ti-PEI, Ti after PEI priming; (C) HA/CS, Ti-PEI coated with multilayer of HA-CS; (D) HA/CS-PAC, Ti-PEI coated with multilayer of HA-CS with PAC-low immobilization Abbreviations: CS, chitosan; HA, hyaluronic acid; PAC, proanthocyanidins; PEI, polyethyleneimine; Ti, titanium.

Figure 4

Contact angles of various samples. Notes: (A), Ti; (B), Ti-PEI; (C), HA/CS; (D), HA/CS-L; (E), HA/CS-M; (F), HA/CS-H. Data are expressed as mean±SD (n=3). *A statistical significance compared to the Ti group (P<0.05). ^#A statistical significance compared to the Ti-PEI group (P<0.05). Abbreviations: CS, chitosan; HA, hyaluronic acid; HA/CS-H, proanthocyanidins-high dose; HA/CS-L, proanthocyanidins–low dose; HA/CS-M, proanthocyanidins-middle dose.

Figure 5

In vitro release profile of HA/CS multilayer surfaces with different concentrations of PAC. Notes: (A) Cumulative release amount. (B) Cumulative release percentage. Data are expressed as mean ± SD (n=3). Abbreviations: CS, chitosan; HA, hyaluronic acid; HA/CS-H, proanthocyanidins-high dose; HA/CS-L, proanthocyanidins-low dose; HA/CS-M, proanthocyanidins-middle dose.

Figure 6

Antioxidant potential characterization. Notes: (A) antioxidant activity of different Ti substrates; (B&C) quantitative fluorescence intensity analysis based on images of D&E; (D&E) fluorescence images of intracellular ROS of osteoblasts grown onto different Ti substrates staining with DCFH-DA and DHE, respectively (Scale bar =100 μm). Data are expressed as mean ± SD (n=3). *A statistical significance compared to the Ti group (P<0.05). ^#A statistical significance compared to the HA/CS multilayer group (P<0.05). **p < 0.01 Abbreviations: CS, chitosan; HA, hyaluronic acid; HA/CS-H, proanthocyanidins-high dose; HA/CS-L, proanthocyanidins-low dose; HA/CS-M, proanthocyanidins-middle dose; NS, no significance.

Figure 7

In vitro ALP activity and osteogenic proliferation of MC3T3-E1 cells cultured on different surfaces. Notes: (A&B) The ALP activity (40x); (C) The CCK-8 assay. Data are expressed as mean ± SD (n=3). *A statistical significance compared to the Ti group (P<0.05). ^#A statistical significance compared to the HA/CS multilayer group (P<0.05). Abbreviations: CCK-8, cell counting kit-8; ALP, alkaline phosphatase; CS, chitosan; HA, hyaluronic acid; HA/CS-H, proanthocyanidins-high dose; HA/CS-L, proanthocyanidins-low dose; HA/CS-M, proanthocyanidins-middle dose.

Figure 8

PAC inhibit p53 pathway in the H₂O₂-induced apoptosis. Notes: (A): Western blot analysis of p53, Bax, and Bcl-2 in MC3T3-E1 cells post-treatment of H₂O₂ and PAC; (B, C & D): Percentage of p53, Bax, Bcl-2 in MC3T3-E1 cells post-treatment of H₂O₂ and PAC; Data are expressed as mean ± SD (n=3). *A statistical significance compared to the Ti group (P<0.05). ^#A statistical significance compared to the HA/CS multilayer group (P<0.05). Abbreviations: CS, chitosan; HA, hyaluronic acid; HA/CS-H, proanthocyanidins-high dose; HA/CS-L, proanthocyanidins-low dose; HA/CS-M, proanthocyanidins-middle dose.

Figure 9

Micro-CT reconstructed and quantitative analysis of distal femur at 2 weeks after implantation. Notes: (A) 3-D image of distal femur bone structure in both Ti rats and different concentration PAC-loaded of Ti rats. (B) Quantify analysis of bone volume per total volume (BV/TV), (C) trabecular number (Tb.N), (D) mean trabecular separation (Tb.Sp), (E) trabecular thickness (Tb.Th), (F) mean connective density (Conn.D), and (G) bone mineral density (BMD) for each group at 2 weeks. Data are expressed as mean ± SD (n=3), *A statistical significance compared to the Ti group (P<0.05). #A statistical significance compared to the HA/CS multilayer group (P<0.05). Abbreviations: HA/CS-H, proanthocyanidins-high dose; HA/CS-L, proanthocyanidins-low dose; HA/CS-M, proanthocyanidins-middle dose.

Figure 10

Histological analysis of the decalcification samples around Ti (A, B, E, F) and PAC-loaded (C, D, G, H) implants. Notes: (A-D) H&E staining; (E-H) Masson staining; (I) BF %. B, D, F and H depict zoomed areas of black box in A, C, E and G, respectively. Bars indicate 50 µm (A, C, E, G) and 20 µm (B, D, F, H). The black arrow denotes new bone formation; white arrow denotes osteoblast cell. Histological morphology analyses based on H&E staining after 2 weeks implantation was expressed as was BF % and shown in (I). Data are expressed as mean ± SD (n=3), *A statistical significance compared to the Ti group (P<0.05). Abbreviations: PAC, proanthocyanidins; Ti, titanium; BF%, bone formation percent.