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Polydopamine-assisted functionalization of heparin and vancomycin onto microarc-oxidized 3D printed porous Ti6Al4V for improved hemocompatibility, osteogenic and anti-infection potencies

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  • ReceivedOct 27, 2017
  • AcceptedJan 4, 2018
  • PublishedFeb 7, 2018

Abstract

Enhanced antiinfection activities, improved hemocompatibility and osteo-compatibility, and reinforced osseointegration are among the most important considerations in designing multifunctional orthopedic biomaterials. Hereby, anti-infective and osteogenic multifunctional 3D printed porous Ti6Al4V implant with excellent hemocompatibility was successfully designed and fabricated. In brief, osteogenic micro-arc oxidation (MAO) coatings with micro/nanoscale porous topography were generated in situ on 3D printed Ti6Al4V scaffolds, on which heparin and vancomycin were easily immobilized. The surface microstructure, morphology, and chemical compositions were characterized employing scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy (FTIR). High loading capacity and sustained vancomycin release profiles were revealed using high performance liquid chromatography (HPLC). Favorable antibacterial and antibiofilm performances against pathogenic Staphylococcus aureus (S. aureus) were validated in vitro through microbial viability assays, Live/Dead bacterial staining, and crystal violet staining. Human mesenchymal stem cells (hMSCs) were seeded on the scaffolds and their proliferation and viability were assessed using Cell Counting Kit and Live/Dead cell viability kit. Further, osteoblastic differentiation abilities were evaluated using alkaline phosphatase (ALP) activity as a hall marker. Additionally, the improved hemocompatibility of the heparinized scaffolds was confirmed by activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT). Overall, our results show that the surface-modified 3D printed porous Ti6Al4V possesses balanced antibacterial and osteogenic functions while exhibiting extra anticlotting effects, boding well for future application in customized functional reconstruction of intricate bone defects.


Funded by

the Ministry of Science and Technology of China(2016YFB1101501)


Acknowledgment

The authors acknowledge the Grant from Ministry of Science and Technology of China (2016YFB1101501) and research and financial support from the Beijing AKEC Medical Co., Ltd. and Medical Research Center of Peking University Third Hospital.


Interest statement

The authors declare no conflict of interest.


Contributions statement

Zhang T and Zhou W designed the research; Liu Z, Zheng Y, and Cheng Y supervised the project; Zhou W, Yan J and Fan D performed the experiments, Zhang T and Jia Z prepared the manuscript; Jia Z, Yin C, Wei Q, Cai H, Liu X and Zhou H contributed in discussion, language improvements, and proof reading; Yang X designed and fabricated the scaffolds.


Author information

Teng Zhang is currently a PhD student at Peking University Health Sciencce Center, under the supervision of Prof. Zhongjun Liu. His research interests include the 3D printed orthopaedic implants and its surface modification.


Zhongjun Liu is the Chair of the Department of Orthopaedic Surgery in Peking University Third Hospital, the Leader of Orthopaedic Innovating Team of Ministry of Education, China, the consultant of central health care, and the pioneer in clinical application of 3D printing spinal implants and related research.


Yufeng Zheng received his PhD in materials science from Harbin Institute of Technology, China, in 1998. Since 2004, he has been a full professor at Peking University in Beijing, China. His research focuses on the development of various new biomedical metallic materials (biodegradable Mg, Fe and Zn based alloys, β-Ti alloys with low elastic modulus, bulk metallic glass, ultra-fine grained metallic materials, etc.).


Supplement

Supplementary information

Supporting information is available in the online version of the paper.


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  • Figure 1

    SEM images (×1000 and ×5000 magnification) of the surface landscapes of TS (a), TS-M (b) and TS-M/P/V (c) scaffolds.

  • Figure 2

    Chemical composition of the samples: (a) XPS survey spectra of TS (1), TS-M (2), TS-M/P/H (3), and TS-M/P/V (4); (b, c) the high-resolution spectra of Ca 2p (b) and S 2p (c) for TS-M/P/V; (d, e) the high-resolution spectra of C 1s for TS-M/P/H (d) and TS-M/P/V (e); (f) representative FTIR spectrum of the TS-M/P/V scaffolds.

  • Figure 3

    Release kinetics of vancomycin from TS-M/P/V and TS-P/V.

  • Figure 4

    In vitro antibacterial activity against S. aureus on the surface (a) and in the surroundings (b) for 4 and 24 h (**p < 0.01).

  • Figure 5

    SEM images (×4000 and ×8000 magnification) of samples with adhered S. aureus: TS (a, b); TS-M (c, d) and TS-M/P/V (e, f). In (c–f), white arrows indicate the micro/nanoporous produced by MAO; black arrows indicate the S. aureus colonizing within the pores. In (e, f), red circles indicate the debris of disrupted S. aureus membrane.

  • Figure 6

    3D CLSM images of Live/Dead stained S. aureus on TS, TS-M and TS-M/P/V. Magnification is ×200 and the scale bar is 500 μm.

  • Figure 7

    The biomass including the bacteria and the components of biofilms for different groups (**p < 0.01) as visualized by crystal violet.

  • Figure 8

    Clotting time test of different samples by APTT, PT and TT (mean ± SD, n = 6, **p < 0.01).

  • Figure 9

    (a) Cell proliferation on the TS, TS-M and TS-M/P/V scaffolds (*p < 0.05). (b) ALP activity of cells on the TS, TS-M and TS-M/P/V scaffolds after 7 days of culture (*p < 0.05).

  • Figure 10

    Live/Dead staining results of the TS (a), TS-M (b) and TS-M/P/V (c) scaffolds after 14 days of cell culture. The right panels are magnified sections of the left. Live and dead cells appear green and red, respectively.

  • Figure 11

    SEM observations of hMSCs on the TS (a), TS-M (b) and TS-M/P/V (c) scaffolds after 14 days of culture. Arrows point to cells.

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