Selective laser melting porous metallic implants with immobilized silver nanoparticles kill and prevent biofilm formation by methicillin-resistant Staphylococcus aureus.

Implant-associated infection and limited longevity are two major challenges that orthopedic devices need to simultaneously address. Additively manufactured porous implants have recently shown tremendous promise in improving bone regeneration and osseointegration, but, as any conventional implant, are threatened by infection. In this study, we therefore used rational design and additive manufacturing in the form of selective laser melting (SLM) to fabricate porous titanium implants with interconnected pores, resulting in a 3.75 times larger surface area than corresponding solid implants. The SLM implants were biofunctionalized by embedding silver nanoparticles in an oxide surface layer grown using plasma electrolytic oxidation (PEO) in Ca/P-based electrolytes. The PEO layer of the SLM implants released silver ions for at least 28 days. X-ray diffraction analysis detected hydroxyapatite on the SLM PEO implants but not on the corresponding solid implants. In vitro and ex vivo assays showed strong antimicrobial activity of these novel SLM PEO silver-releasing implants, without any signs of cytotoxicity. The rationally designed SLM porous implants outperformed solid implants with similar dimensions undergoing the same biofunctionalization treatment. This included four times larger amount of released silver ions, two times larger zone of inhibition, and one additional order of magnitude of reduction in numbers of CFU in an ex vivo mouse infection model.

[1]  M. Usta,et al.  Characterization and formation of hydroxyapatite on Ti6Al4V coated by plasma electrolytic oxidation , 2013 .

[2]  S. Heo,et al.  Osseointegration of anodized titanium implants under different current voltages: a rabbit study. , 2007, Journal of oral rehabilitation.

[3]  Saulius Juodkazis,et al.  Bactericidal activity of black silicon , 2013, Nature Communications.

[4]  Elena P Ivanova,et al.  Antibacterial surfaces: the quest for a new generation of biomaterials. , 2013, Trends in biotechnology.

[5]  C. Sharma,et al.  Effect of calcium, zinc and magnesium on the attachment and spreading of osteoblast like cells onto ceramic matrices , 2007, Journal of materials science. Materials in medicine.

[6]  J. Duszczyk,et al.  In vitro cytotoxicity evaluation of porous TiO₂-Ag antibacterial coatings for human fetal osteoblasts. , 2012, Acta biomaterialia.

[7]  Kyunghee Choi,et al.  Silver nanoparticles induce cytotoxicity by a Trojan-horse type mechanism. , 2010, Toxicology in vitro : an international journal published in association with BIBRA.

[8]  Fan Yang,et al.  The future of biologic coatings for orthopaedic implants. , 2013, Biomaterials.

[9]  M. Riool,et al.  Host tissue as a niche for biomaterial-associated infection. , 2010, Future microbiology.

[10]  J. Duszczyk,et al.  An electron microscopical study on the growth of TiO2-Ag antibacterial coatings on Ti6Al7Nb biomedical alloy. , 2011, Acta biomaterialia.

[11]  Ralf Schumacher,et al.  Bone regeneration by the osteoconductivity of porous titanium implants manufactured by selective laser melting: a histological and micro computed tomography study in the rabbit. , 2013, Tissue engineering. Part A.

[12]  Dale A Pelletier,et al.  Relating nanomaterial properties and microbial toxicity. , 2013, Nanoscale.

[13]  B. Boyan,et al.  Implant osseointegration and the role of microroughness and nanostructures: lessons for spine implants. , 2014, Acta biomaterialia.

[14]  A. T. Te Velde,et al.  How honey kills bacteria , 2010, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[15]  M Epple,et al.  Uptake and intracellular distribution of silver nanoparticles in human mesenchymal stem cells. , 2011, Acta biomaterialia.

[16]  C. Vandenbroucke-Grauls,et al.  Tissue around catheters is a niche for bacteria associated with medical device infection , 2008, Critical care medicine.

[17]  Carla Renata Arciola,et al.  The significance of infection related to orthopedic devices and issues of antibiotic resistance. , 2006, Biomaterials.

[18]  K. Siebenrock,et al.  In vitro cytotoxicity of silver nanoparticles on osteoblasts and osteoclasts at antibacterial concentrations , 2013, Nanotoxicology.

[19]  H. Kim,et al.  Infection after prosthetic reconstruction in limb salvage surgery , 2002, International Orthopaedics.

[20]  Anima Nanda,et al.  Biosynthesis of silver nanoparticles from Staphylococcus aureus and its antimicrobial activity against MRSA and MRSE. , 2009, Nanomedicine : nanotechnology, biology, and medicine.

[21]  Y. Sul,et al.  The significance of the surface properties of oxidized titanium to the bone response: special emphasis on potential biochemical bonding of oxidized titanium implant. , 2003, Biomaterials.

[22]  M. von Walter,et al.  Structural, mechanical and in vitro characterization of individually structured Ti-6Al-4V produced by direct laser forming. , 2006, Biomaterials.

[23]  A. T. Te Velde,et al.  Two Major Medicinal Honeys Have Different Mechanisms of Bactericidal Activity , 2011, PloS one.

[24]  R. Müller,et al.  Hydroxyapatite particles maintain peri-implant bone mantle during osseointegration in osteoporotic bone. , 2009, Bone.

[25]  Pier Paolo Pompa,et al.  Nanosilver-based antibacterial drugs and devices: mechanisms, methodological drawbacks, and guidelines. , 2014, Chemical Society reviews.

[26]  T. Clyne,et al.  Cell adhesion to plasma electrolytic oxidation (PEO) titania coatings, assessed using a centrifuging technique. , 2011, Journal of the mechanical behavior of biomedical materials.

[27]  A. A. Zadpoor,et al.  Data on the surface morphology of additively manufactured Ti-6Al-4V implants during processing by plasma electrolytic oxidation , 2017, Data in brief.

[28]  Carla Renata Arciola,et al.  A review of the clinical implications of anti-infective biomaterials and infection-resistant surfaces. , 2013, Biomaterials.

[29]  Yoshiaki Toyama,et al.  Osseointegration of a hydroxyapatite-coated multilayered mesh stem. , 2004, Biomaterials.

[30]  B. Gao,et al.  Improved biological performance of microarc-oxidized low-modulus Ti-24Nb-4Zr-7.9Sn alloy. , 2009, Journal of biomedical materials research. Part B, Applied biomaterials.

[31]  A. A. Zadpoor,et al.  Selective laser melting‐produced porous titanium scaffolds regenerate bone in critical size cortical bone defects , 2013, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

[32]  H. Choe,et al.  Bioactivity evaluation of porous TiO2 surface formed on titanium in mixed electrolyte by spark anodization , 2013 .

[33]  Michiel Mulier,et al.  Bone regeneration performance of surface-treated porous titanium. , 2014, Biomaterials.

[34]  Marcus J Schultz,et al.  Biomaterial-Associated Infection: Locating the Finish Line in the Race for the Surface , 2012, Science Translational Medicine.

[35]  Chengtie Wu,et al.  Novel sphene coatings on Ti-6Al-4V for orthopedic implants using sol-gel method. , 2008, Acta biomaterialia.

[36]  M. Mildner,et al.  Nanoscalic silver possesses broad-spectrum antimicrobial activities and exhibits fewer toxicological side effects than silver sulfadiazine. , 2012, Nanomedicine : nanotechnology, biology, and medicine.

[37]  Amir A Zadpoor,et al.  Bone tissue regeneration: the role of scaffold geometry. , 2015, Biomaterials science.

[38]  E. Nkenke,et al.  Implants in bone: Part II. Research on implant osseointegration , 2014, Oral and Maxillofacial Surgery.

[39]  Yong Han,et al.  Formation mechanism of HA-based coatings by micro-arc oxidation , 2008 .

[40]  M. Laitinen,et al.  Orthopaedic Reconstruction of Complex Pelvic Bone Defects. Evaluation of Various Treatment Methods , 2013, Scandinavian journal of surgery : SJS : official organ for the Finnish Surgical Society and the Scandinavian Surgical Society.

[41]  Håvard Jenssen,et al.  Antimicrobial peptides on calcium phosphate-coated titanium for the prevention of implant-associated infections. , 2010, Biomaterials.

[42]  L. Visai,et al.  Decreased Bacterial Adhesion to Surface-Treated Titanium , 2005, The International journal of artificial organs.

[43]  M. Hande,et al.  Cytotoxicity and genotoxicity of silver nanoparticles in human cells. , 2009, ACS nano.

[44]  S. M. Ahmadi,et al.  Effects of bio-functionalizing surface treatments on the mechanical behavior of open porous titanium biomaterials. , 2014, Journal of the mechanical behavior of biomedical materials.

[45]  Milan Kolar,et al.  Silver colloid nanoparticles: synthesis, characterization, and their antibacterial activity. , 2006, The journal of physical chemistry. B.

[46]  Matthias Epple,et al.  Silver as antibacterial agent: ion, nanoparticle, and metal. , 2013, Angewandte Chemie.

[47]  Andreas F Widmer,et al.  Infections associated with orthopedic implants , 2006, Current opinion in infectious diseases.

[48]  L. Murr,et al.  Microstructure and mechanical behavior of Ti-6Al-4V produced by rapid-layer manufacturing, for biomedical applications. , 2009, Journal of the mechanical behavior of biomedical materials.

[49]  S. M. Ahmadi,et al.  Additively Manufactured Open-Cell Porous Biomaterials Made from Six Different Space-Filling Unit Cells: The Mechanical and Morphological Properties , 2015, Materials.

[50]  Irving M Shapiro,et al.  Immobilized antibiotics to prevent orthopaedic implant infections. , 2012, Advanced drug delivery reviews.

[51]  J. Hu,et al.  Effects of biomimetically and electrochemically deposited nano-hydroxyapatite coatings on osseointegration of porous titanium implants. , 2009, Oral surgery, oral medicine, oral pathology, oral radiology, and endodontics.

[52]  Ju-liang He,et al.  Plasma electrolytic oxidation of titanium and improvement in osseointegration. , 2013, Journal of biomedical materials research. Part B, Applied biomaterials.

[53]  Yoshinobu Watanabe,et al.  Bone regeneration in a massive rat femur defect through endochondral ossification achieved with chondrogenically differentiated MSCs in a degradable scaffold. , 2014, Biomaterials.

[54]  D. Pioletti,et al.  Effect of different Ti-6Al-4V surface treatments on osteoblasts behaviour. , 2002, Biomaterials.

[55]  D. Hungerford,et al.  Enhanced proliferation and osteocalcin production by human osteoblast-like MG63 cells on silicon nitride ceramic discs. , 1999, Biomaterials.

[56]  A. A. Zadpoor,et al.  Crystal structure and nanotopographical features on the surface of heat-treated and anodized porous titanium biomaterials produced using selective laser melting , 2014 .

[57]  Lingzhou Zhao,et al.  The osteogenic activity of strontium loaded titania nanotube arrays on titanium substrates. , 2013, Biomaterials.

[58]  S. Ahmadia,et al.  Mechanical behavior of regular open-cell porous biomaterials made of diamond lattice unit cells , 2014 .

[59]  Dae Hong Jeong,et al.  Antimicrobial effects of silver nanoparticles. , 2007, Nanomedicine : nanotechnology, biology, and medicine.

[60]  Yong Han,et al.  Initial osteoblast functions on Ti-5Zr-3Sn-5Mo-15Nb titanium alloy surfaces modified by microarc oxidation. , 2009, Journal of biomedical materials research. Part A.

[61]  Peter Fratzl,et al.  The effect of geometry on three-dimensional tissue growth , 2008, Journal of The Royal Society Interface.

[62]  H Weinans,et al.  Full regeneration of segmental bone defects using porous titanium implants loaded with BMP-2 containing fibrin gels. , 2015, European cells & materials.

[63]  Joanna Aizenberg,et al.  Bacteria pattern spontaneously on periodic nanostructure arrays. , 2010, Nano letters.

[64]  R. Singer,et al.  Cellular Ti-6Al-4V structures with interconnected macro porosity for bone implants fabricated by selective electron beam melting. , 2008, Acta biomaterialia.

[65]  F. Saltel,et al.  Dual effect of strontium ranelate: stimulation of osteoblast differentiation and inhibition of osteoclast formation and resorption in vitro. , 2008, Bone.

[66]  Miguel Castilho,et al.  Direct 3D powder printing of biphasic calcium phosphate scaffolds for substitution of complex bone defects , 2014, Biofabrication.

[67]  Anima Nanda,et al.  Extracellular synthesis of silver bionanoparticles from Aspergillus clavatus and its antimicrobial activity against MRSA and MRSE. , 2010, Colloids and surfaces. B, Biointerfaces.

[68]  Andrés J. García,et al.  Biomaterial strategies for engineering implants for enhanced osseointegration and bone repair. , 2015, Advanced drug delivery reviews.

[69]  Miss A.O. Penney (b) , 1974, The New Yale Book of Quotations.

[70]  W. Winkelmann,et al.  Characteristics and outcome of infections associated with tumor endoprostheses , 2006, Archives of Orthopaedic and Trauma Surgery.

[71]  H Van Oosterwyck,et al.  The effect of pore geometry on the in vitro biological behavior of human periosteum-derived cells seeded on selective laser-melted Ti6Al4V bone scaffolds. , 2012, Acta biomaterialia.

[72]  Jean-Pierre Kruth,et al.  Additively manufactured porous tantalum implants. , 2015, Acta biomaterialia.

[73]  H Weinans,et al.  Antibacterial Behavior of Additively Manufactured Porous Titanium with Nanotubular Surfaces Releasing Silver Ions. , 2016, ACS applied materials & interfaces.

[74]  G. Friedlaender,et al.  Malignant Bone Tumors: Limb Sparing Versus Amputation , 2003, The Journal of the American Academy of Orthopaedic Surgeons.

[75]  G. G. Stokes "J." , 1890, The New Yale Book of Quotations.

[76]  Haobo Pan,et al.  Enhanced osteoporotic bone regeneration by strontium-substituted calcium silicate bioactive ceramics. , 2013, Biomaterials.

[77]  W. V. van Wamel,et al.  Staphylococcus epidermidis originating from titanium implants infects surrounding tissue and immune cells. , 2014, Acta biomaterialia.

[78]  C. Beauchamp Salvaging the limb salvage: Management of complications following endoprosthetic reconstruction for tumours around the knee , 2008 .

[79]  Hongwei Ni,et al.  Antibacterial nano-structured titania coating incorporated with silver nanoparticles. , 2011, Biomaterials.

[80]  Lingzhou Zhao,et al.  Antibacterial coatings on titanium implants. , 2009, Journal of biomedical materials research. Part B, Applied biomaterials.

[81]  K. Neoh,et al.  Balancing osteoblast functions and bacterial adhesion on functionalized titanium surfaces. , 2012, Biomaterials.

[82]  G. Thompson,et al.  In vitro evaluation of cell proliferation and collagen synthesis on titanium following plasma electrolytic oxidation. , 2010, Journal of biomedical materials research. Part A.

[83]  Philip Kollmannsberger,et al.  How Linear Tension Converts to Curvature: Geometric Control of Bone Tissue Growth , 2012, PloS one.

[84]  A. M. Padovani,et al.  Plasma electrolytic oxidation coatings on γTiAl alloy for potential biomedical applications. , 2014, Journal of biomedical materials research. Part B, Applied biomaterials.

[85]  M. Rai,et al.  Silver nanoparticles as a new generation of antimicrobials. , 2009, Biotechnology advances.

[86]  Jurek Duszczyk,et al.  In vitro antibacterial activity of porous TiO2-Ag composite layers against methicillin-resistant Staphylococcus aureus. , 2009, Acta biomaterialia.

[87]  H. Luckarift,et al.  Hybrid antimicrobial enzyme and silver nanoparticle coatings for medical instruments. , 2009, ACS applied materials & interfaces.

[88]  S. Ivanovski,et al.  The effects of implant topography on osseointegration under estrogen deficiency induced osteoporotic conditions: Histomorphometric, transcriptional and ultrastructural analysis. , 2016, Acta biomaterialia.

[89]  Z. Gong,et al.  Toxicity of silver nanoparticles in zebrafish models , 2008, Nanotechnology.

[90]  王立平,et al.  Strontium enhances osteogenic differentiation of mesenchymal stem cells and in vivo bone formation by activating Wnt/catenin signaling , 2011 .

[91]  D. Kaplan,et al.  Porosity of 3D biomaterial scaffolds and osteogenesis. , 2005, Biomaterials.

[92]  Noam Eliaz,et al.  Enhanced osseointegration of grit-blasted, NaOH-treated and electrochemically hydroxyapatite-coated Ti-6Al-4V implants in rabbits. , 2009, Acta biomaterialia.