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


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)


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.).


Supplementary information

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


[1] Yang F, Chen C, Zhou QR, et al. Laser beam melting 3D printing of Ti6Al4V based porous structured dental implants: fabrication, biocompatibility analysis and photoelastic study. Sci Rep, 2017, 7: 45360 CrossRef PubMed ADS Google Scholar

[2] Shah FA, Snis A, Matic A, et al. 3D printed Ti6Al4V implant surface promotes bone maturation and retains a higher density of less aged osteocytes at the bone-implant interface. Acta Biomater, 2016, 30: 357-367 CrossRef PubMed Google Scholar

[3] Medicrea International. French Surgeon Performs World’s First Spinal Fusion Surgery Using Customized 3-D Printed Spine Cages. Business Wire (English), 2014. Google Scholar

[4] Amin Yavari S, Loozen L, Paganelli FL, et al. Antibacterial behavior of additively manufactured porous titanium with nanotubular surfaces releasing silver ions. ACS Appl Mater Interfaces, 2016, 8: 17080-17089 CrossRef Google Scholar

[5] Jia Z, Xiu P, Xiong P, et al. Additively manufactured macroporous titanium with silver-releasing micro-/nanoporous surface for multipurpose infection control and bone repair—a proof of concept. ACS Appl Mater Interfaces, 2016, 8: 28495-28510 CrossRef Google Scholar

[6] Busscher HJ, van der Mei HC, Subbiahdoss G, et al. Biomaterial-associated infection: locating the finish line in the race for the surface. Sci Transl Med, 2012, 4: 153rv10-153rv10 CrossRef PubMed Google Scholar

[7] Saleh J, El-Othmani MM, Saleh KJ. Deep vein thrombosis and pulmonary embolism considerations in orthopedic surgery. Orthop Clin North Am, 2017, 48: 127-135 CrossRef PubMed Google Scholar

[8] Lv J, Jia Z, Li J, et al. Electron beam melting fabrication of porous Ti6Al4V scaffolds: cytocompatibility and osteogenesis. Adv Eng Mater, 2015, 17: 1391-1398 CrossRef Google Scholar

[9] Lopez-Heredia MA, Sohier J, Gaillard C, et al. Rapid prototyped porous titanium coated with calcium phosphate as a scaffold for bone tissue engineering. Biomaterials, 2008, 29: 2608-2615 CrossRef PubMed Google Scholar

[10] Xiu P, Jia Z, Lv J, et al. Tailored surface treatment of 3D printed porous Ti6Al4V by microarc oxidation for enhanced osseointegration via optimized bone in-growth patterns and interlocked bone/implant interface. ACS Appl Mater Interfaces, 2016, 8: 17964-17975 CrossRef Google Scholar

[11] Amin Yavari S, van der Stok J, Chai YC, et al. Bone regeneration performance of surface-treated porous titanium. Biomaterials, 2014, 35: 6172-6181 CrossRef PubMed Google Scholar

[12] Lee H, Jang TS, Song J, et al. Multi-scale porous Ti6Al4V scaffolds with enhanced strength and biocompatibility formed via dynamic freeze-casting coupled with micro-arc oxidation. Mater Lett, 2016, 185: 21-24 CrossRef Google Scholar

[13] Radtke A, Topolski A, Jędrzejewski T, et al. The bioactivity and photocatalytic properties of titania nanotube coatings produced with the use of the low-potential anodization of Ti6Al4V alloy surface. Nanomaterials, 2017, 7: 197 CrossRef PubMed Google Scholar

[14] Cao H, Liu X. Plasma-sprayed ceramic coatings for osseointegration. Int J Appl Ceram Technol, 2013, 10: 1-10 CrossRef Google Scholar

[15] Govindharajulu JP, Chen X, Li Y, et al. Chitosan-recombinamer layer-by-layer coatings for multifunctional implants. Int J Mol Sci, 2017, 18: 369 CrossRef PubMed Google Scholar

[16] Zhou L, LV GH, Mao FF, Yang SZ. Preparation of biomedical Ag incorporated hydroxyapatite/titania coatings on Ti6Al4V alloy by plasma electrolytic oxidation. Chin Phys, 2014, 3: 035205. Google Scholar

[17] Darouiche RO. Device-associated infections: a macroproblem that starts with microadherence. Clin Infect Dis, 2001, 33: 1567-1572 CrossRef PubMed Google Scholar

[18] Zimmerli W, Waldvogel FA, Vaudaux P, et al. Pathogenesis of foreign body infection: description and characteristics of an animal model. J Infect Dis, 1982, 146: 487-497 CrossRef Google Scholar

[19] Ceri H, Olson ME, Stremick C, et al. The Calgary Biofilm Device: New technology for rapid determination of antibiotic susceptibilities of bacterial biofilms. J Clin Microbiol, 1999, 6: 1771–1776. Google Scholar

[20] Ishibe T, Goto T, Kodama T, et al. Bone formation on apatite-coated titanium with incorporated BMP-2/heparin in vivo. Oral Surgery Oral Med Oral Pathol Oral Rad Endodontol, 2009, 108: 867-875 CrossRef PubMed Google Scholar

[21] Lee DW, Yun YP, Park K, et al. Gentamicin and bone morphogenic protein-2 (BMP-2)-delivering heparinized-titanium implant with enhanced antibacterial activity and osteointegration. Bone, 2012, 50: 974-982 CrossRef PubMed Google Scholar

[22] Kim SE, Song SH, Yun YP, et al. The effect of immobilization of heparin and bone morphogenic protein-2 (BMP-2) to titanium surfaces on inflammation and osteoblast function. Biomaterials, 2011, 32: 366-373 CrossRef PubMed Google Scholar

[23] Teixeira S, Yang L, Dijkstra PJ, et al. Heparinized hydroxyapatite/collagen three-dimensional scaffolds for tissue engineering. J Mater Sci-Mater Med, 2010, 21: 2385-2392 CrossRef PubMed Google Scholar

[24] Schroeder-Tefft JA, Bentz H, Estridge TD. Collagen and heparin matrices for growth factor delivery. J Control Release, 1997, 48: 29-33 CrossRef Google Scholar

[25] Meneghetti MCZ, Hughes AJ, Rudd TR, et al. Heparan sulfate and heparin interactions with proteins. J R Soc Interface, 2015, 12: 20150589 CrossRef PubMed Google Scholar

[26] Sofroniadou S, Revela I, Smirloglou D, et al. Linezolid versus vancomycin antibiotic lock solution for the prevention of nontunneled catheter-related blood stream infections in hemodialysis patients: a prospective randomized study. Seminars Dialysis, 2012, 25: 344-350 CrossRef PubMed Google Scholar

[27] Cesaro S, Cavaliere M, Spiller M, et al. A simplified method of antibiotic lock therapy for Broviac–Hickman catheters using a CLC 2000 connector device. Support Care Cancer, 2007, 15: 95-99 CrossRef PubMed Google Scholar

[28] Moore CL, Besarab A, Ajluni M, et al. Comparative effectiveness of two catheter locking solutions to reduce catheter-related bloodstream infection in hemodialysis patients. Clinical J Am Soc Nephrology, 2014, 9: 1232-1239 CrossRef PubMed Google Scholar

[29] Wu C, Han P, Liu X, et al. Mussel-inspired bioceramics with self-assembled Ca-P/polydopamine composite nanolayer: Preparation, formation mechanism, improved cellular bioactivity and osteogenic differentiation of bone marrow stromal cells. Acta Biomater, 2014, 10: 428-438 CrossRef PubMed Google Scholar

[30] Cheng C, Li S, Zhao W, et al. The hydrodynamic permeability and surface property of polyethersulfone ultrafiltration membranes with mussel-inspired polydopamine coatings. J Membrane Sci, 2012, 417-418: 228-236 CrossRef Google Scholar

[31] Lee H, Dellatore SM, Miller WM, et al. Mussel-inspired surface chemistry for multifunctional coatings. Science, 2007, 318: 426-430 CrossRef PubMed ADS Google Scholar

[32] Kang SM, Hwang NS, Yeom J, et al. One-step multipurpose surface functionalization by adhesive catecholamine. Adv Funct Mater, 2012, 22: 2949-2955 CrossRef PubMed Google Scholar

[33] Lee H, Rho J, Messersmith PB. Facile conjugation of biomolecules onto surfaces via mussel adhesive protein inspired coatings. Adv Mater, 2009, 21: 431-434 CrossRef PubMed Google Scholar

[34] Jia Z, Shi Y, Xiong P, et al. From solution to biointerface: graphene self-assemblies of varying lateral sizes and surface properties for biofilm control and osteodifferentiation. ACS Appl Mater Interfaces, 2016, 8: 17151-17165 CrossRef Google Scholar

[35] Jiang JH, Zhu LP, Li XL, et al. Surface modification of PE porous membranes based on the strong adhesion of polydopamine and covalent immobilization of heparin. J Membrane Sci, 2010, 364: 194-202 CrossRef Google Scholar

[36] Zhu LP, Yu JZ, Xu YY, et al. Surface modification of PVDF porous membranes via poly(DOPA) coating and heparin immobilization. Colloids Surfs B-Biointerfaces, 2009, 69: 152-155 CrossRef PubMed Google Scholar

[37] Lv J, Xiu P, Tan J, et al. Enhanced angiogenesis and osteogenesis in critical bone defects by the controlled release of BMP-2 and VEGF: implantation of electron beam melting-fabricated porous Ti6Al4 scaffolds incorporating growth factor-doped fibrin glue. Biomed Mater, 2015, 10: 035013 CrossRef PubMed ADS Google Scholar

[38] Del Frari D, Bour J, Ball V, et al. Degradation of polydopamine coatings by sodium hypochlorite: A process depending on the substrate and the film synthesis method. Polymer Degradation Stability, 2012, 97: 1844-1849 CrossRef Google Scholar

[39] Ma L, Cheng C, He C, et al. Substrate-independent robust and heparin-mimetic hydrogel thin film coating via combined LbL self-assembly and mussel-inspired post-cross-linking. ACS Appl Mater Interfaces, 2015, 7: 26050-26062 CrossRef Google Scholar

[40] Ordikhani F, Tamjid E, Simchi A. Characterization and antibacterial performance of electrodeposited chitosan–vancomycin composite coatings for prevention of implant-associated infections. Mater Sci Eng-C, 2014, 41: 240-248 CrossRef PubMed Google Scholar

[41] Liu T, Liu Y, Chen Y, et al. Immobilization of heparin/poly-l-lysine nanoparticles on dopamine-coated surface to create a heparin density gradient for selective direction of platelet and vascular cells behavior. Acta Biomater, 2014, 10: 1940-1954 CrossRef PubMed Google Scholar

[42] Liu Y, Zheng Y, Hayes B. Degradable, absorbable or resorbable—what is the best grammatical modifier for an implant that is eventually absorbed by the body?. Sci China Mater, 2017, 60: 377-391 CrossRef Google Scholar

[43] Yang J, Cai H, Lv J, et al. In vivo study of a self-stabilizing artificial vertebral body fabricated by electron beam melting. Spine, 2014, 39: E486-E492 CrossRef PubMed Google Scholar

[44] Xu N, Wei F, Liu X, et al. Reconstruction of the upper cervical spine using a personalized 3D-printed vertebral body in an adolescent with Ewing sarcoma. Spine, 2016, 41: E50-E54 CrossRef PubMed Google Scholar

[45] Zhang RF, Qiao LP, Qu B, et al. Biocompatibility of micro-arc oxidation coatings developed on Ti6Al4V alloy in a solution containing organic phosphate. Mater Lett, 2015, 153: 77-80 CrossRef Google Scholar

[46] Li G, Xie B, Pan C, et al. Facile conjugation of heparin onto titanium surfaces via dopamine inspired coatings for improving blood compatibility. J Wuhan Univ Technol-Mat Sci Edit, 2014, 29: 832-840 CrossRef Google Scholar

[47] Barradas AMC, Fernandes HAM, Groen N, et al. A calcium-induced signaling cascade leading to osteogenic differentiation of human bone marrow-derived mesenchymal stromal cells. Biomaterials, 2012, 33: 3205-3215 CrossRef PubMed Google Scholar

[48] Bouyer M, Guillot R, Lavaud J, et al. Surface delivery of tunable doses of BMP-2 from an adaptable polymeric scaffold induces volumetric bone regeneration. Biomaterials, 2016, 104: 168-181 CrossRef PubMed Google Scholar

[49] Inzana JA, Olvera D, Fuller SM, et al. 3D printing of composite calcium phosphate and collagen scaffolds for bone regeneration. Biomaterials, 2014, 35: 4026-4034 CrossRef PubMed Google Scholar

[50] Xu S, Song J, Zhu C, et al. Graphene oxide-encapsulated Ag nanoparticle-coated silk fibers with hierarchical coaxial cable structure fabricated by the molecule-directed self-assembly. Mater Lett, 2017, 188: 215-219 CrossRef Google Scholar

[51] Yang Y, Yang S, Wang Y, et al. Anti-infective efficacy, cytocompatibility and biocompatibility of a 3D-printed osteoconductive composite scaffold functionalized with quaternized chitosan. Acta Biomater, 2016, 46: 112-128 CrossRef PubMed Google Scholar

[52] Pérez-Anes A, Gargouri M, Laure W, et al. Bioinspired titanium drug eluting platforms based on a poly-β-cyclodextrin–chitosan layer-by-layer self-assembly targeting infections. ACS Appl Mater Interfaces, 2015, 7: 12882-12893 CrossRef Google Scholar

[53] Önder S. Surface modification of titanium using bsa-loaded chitosan and chitosan/gelatin polymers. J Med Biol Eng, 2016, 36: 661-667 CrossRef Google Scholar

  • 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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