Reactive oxygen species inhibited by titanium oxide coatings Richard Suzuki,1,3 Julie Muyco,2,3 Joanna McKittrick,2,3 John A. Frangos1,3 1Department of Bioengineering, University of California, San Diego, La Jolla, California 92037 2Department of Mechanical and Aerospace Engineering, Material Science, and Engineering Program, University of California, San Diego, La Jolla, California 92037 3La Jolla Bioengineering Institute, 505 Coast Boulevard South, La Jolla, California 92037 Received 3 June 2002; revised 25 September 2002; accepted 30 September 2002 Abstract: Titanium is a successful biomaterial that pos- stimulated to produce superoxide and interleukin-6. Super- sesses good biocompatibility. It is covered by a surface layer oxide production was measured by the chemiluminescent of titanium dioxide, and this oxide may play a critical role in reaction with 2-methyl-6-[p-methoxyphenyl]-3,7-dihydro- inhibiting reactive oxygen species, such as peroxynitrite, imidazo[1,2-a]pyrazin-3-one (MCLA). Titanium dioxide- produced during the inflammatory response. In the present coated silicone exhibited a 55% decrease in superoxide com- study, titanium dioxide was coated onto silicone substrates pared to uncoated silicone and a 165% decrease in by radio-frequency sputtering. Silicone coating with tita- superoxide compared to uncoated polystyrene. Titanium nium dioxide enhanced the breakdown of peroxynitrite by dioxide-coated silicone inhibited IL-6 production by 77% 79%. At physiologic pH, the peroxynitrite donor 3-morpho- compared to uncoated silicone. These results show that the linosydnonimine-N-ethylcarbamide (SIN-1) was used to ni- anti-inflammatory properties of titanium dioxide can be trate 4-hydroxyphenylacetic acid (4-HPA) to form 4-hy- transferred to the surfaces of silicone substrates. © 2003 droxy-3-nitrophenyl acetic acid (NHPA). Titanium dioxide- Wiley Periodicals, Inc. J Biomed Mater Res 66A: 396–402, coated silicone inhibited the nitration of 4-HPA by 61% 2003 compared to aluminum oxide-coated silicone and 55% com- pared to uncoated silicone. J774A.1 mouse macrophages Key words: titanium dioxide; silicone; anti-inflammatory; were plated on oxide-coated silicone and polystyrene and oxide coating; peroxynitrite INTRODUCTION duces colonic inflammation in rats7 and has been dem- onstrated to be present in the inflamed guinea pig 8 Inflammatory response is part of a general pattern ileum. Peroxynitrite was found to be produced by of recovery and wound healing that leads to eventual acute inflammation from edema induced in the hind 9 acceptance of a foreign material placed in the body.1 paws of rats. This pattern of events typically leads to fibrotic encap- Clinical studies also provide evidence that per- sulation of the implant. Prolonged inflammatory re- oxynitrite is produced during inflammation. The sponses can have the consequence of more intense blood serum and synovial fluid from patients with the tissue reactions requiring extrusion of the implant.2 inflammatory joint disease rheumatoid arthritis were Ϫ The reactive oxygen species peroxynitrite (OONO ) found to contain 3-nitrotyrosine markers, indicating has been shown to play a role in inflammation. Per- peroxynitrite formation while body fluids from nor- ⅐Ϫ mal patients contained no detectable 3-nitrotyrosine. oxynitrite is formed from superoxide (O2 ) and nitric oxide (NO⅐),3 and it is a potent oxidant capable of a Similarly, no 3-nitrotyrosine markers were detected in wide range of reactions.4–6 Peroxynitrite directly in- body fluids from patients with osteoarthritis, a largely non-inflammatory joint disease.10 It is important to Correspondence to: J.A. Frangos @ Bioengineering Institute; note that it has been reported that 3-nitrotyrosine e-mail: [email protected] markers for peroxynitrite also have been observed at Contract grant sponsor: NIH; contract grant number: the interface membrane of hip implants suffering from EB00823 aseptic loosening, which is characterized by local in- Contract grant sponsor: NIH; contract grant number: 11,12 AR47032 flammation. Previously, it was shown that titanium dioxide is © 2003 Wiley Periodicals, Inc. capable of inhibiting the reactivity of peroxynitrite.13 OXIDE COATINGS ON SILICONE 397 Titanium dioxide was shown to enhance the break- Nitration of phenol (4-HPA) by peroxynitrite donor down of peroxynitrite and inhibit the nitration reac- tions of peroxynitrite at physiologic pH levels. Tita- Peroxynitrite has a half-life of 1.9 s at physiologic pH.16 The nium surfaces retained the ability to inhibit short half-life makes experiments difficult in this pH range. peroxynitrite even in the presence of 10% fetal bovine This problem was circumvented through the use of 3-morpho- serum, fibrinogen, and bicarbonate. linosydnonimine-N-ethylcarbamide (SIN-1) (Alexis Chemicals, Others have shown that a surface with the ability to San Diego, CA). SIN-1 slowly decomposes to release NO and superoxide at physiologic pH, which then react to form per- breakdown reactive oxygen species can improve the 4,15 biocompatibility of polymers. Polyethylene implants oxynitrite. Peroxynitrite is highly reactive and can nitrate phenolic coated with superoxide dismutase mimics showed a 15 residues, such as tyrosine. It nitrates 4-hydroxyphenylac- notable decrease in capsule thickness compared to 14 etic acid (4-HPA) to form 4-hydroxy-3-nitrophenyl acetic uncoated controls. These results indicate that super- acid (NHPA). NHPA absorbs at 432 nm, and its concentra- oxide, a precursor of peroxynitrite, plays a role in the tion was calculated by measuring the change of absorbance inflammatory response to biomaterial surfaces. at pH 6.0–6.5 and pH 10.0–10.5.17 The objective of this study was to determine if the Solutions of PBS buffer (Irvine Scientific, Irvine, CA) with ability to inhibit reactive inflammatory species also 0.5 mM of 4-HPA (Aldrich, Milwaukee, WI) were placed could be imparted to a polymer by coating the surface over coated silicone substrates in airtight containers. After with a thin film of titanium dioxide. Such coatings autoclaving, SIN-1 was added to yield a final concentration may lead to improvement in biocompatibility and mit- of5mM. A second oxide-coated silicone substrate was igation of the inflammatory response of implants. placed over the first sample, trapping the solution between the two samples. The containers were sealed and placed in the dark in 37°C incubators for 14 days. MATERIALS AND METHODS Surface interaction with superoxide from activated macrophages Sample preparation Mouse macrophages from the cell line J774A.1 (ATCC, Oxide-coated silicone elastomer samples were fabricated Manassas, VA) were plated on oxide-coated silicone sub- using radio frequency (RF) plasma magnetron sputtering. strates lining the bottoms of petri dishes. The cells grew to The silicone substrates were cut from sheets of non-rein- form a monolayer that adhered to the coated surfaces. Oxide forced, translucent silicone sheeting (SF Medical, Hudson, coatings at the thickness deposited were transparent, allow- MA) and were 1.5 mm in thickness. ing for spectrometry assays. Deposition rates of the oxide layer were calibrated using The macrophages were grown in DMEM on the oxide- film deposition on quartz substrates under conditions sim- coated silicone substrates contained in sterile petri dishes ilar to those on silicone substrates. The thickness of the oxide (Irvine Scientific, Irvine, CA) with 5% fetal calf serum (Hy- layer was measured with a Dektak IIa (Digital Instruments, clone) and 1 mM of sodium pyruvate (Aldrich). The cells Santa Barbara, CA). The typical range of thickness for oxide were stimulated to produce superoxide by addition of 15 coatings was 100–200 nm. ␮g/mL of phorbol 12-myristate 13-acetate (PMA). Superox- The continuity of the coatings was probed using energy- ide production was measured by the chemiluminescent re- dispersive X-ray spectroscopy (EDS) in a mapping function action with 2-methyl-6-[p-methoxyphenyl]-3,7-dihydroimi- (Oxford Instruments X-ray spectrometer, Concord, MA). El- dazo[1,2-␣]pyrazin-3-one (MCLA).18,19 MCLA is two orders emental information was obtained from points on the sur- of magnitude more specific for superoxide detection than face of the sample corresponding to the scanning electron lucigenin or luminol.20 microscope (SEM) image (Cambridge 360 SEM LEO Electron Macrophages were incubated in a solution of PBS with Microscopy, Thornwood, NY). No preferential location of MCLA (1.5 ␮M). The cells were stimulated with PMA (15 elements was seen, indicating there was no disruption of ␮g/mL) and placed in a receptacle with an attached photo- continuity of the coating within the resolution of the instru- multiplier tube and photon counter, allowing measurement ment. of the resulting chemiluminescence. The receptacle was lo- cated in a darkroom and was equipped with a temperature control system that maintained the cells at 37°C. Peroxynitrite degradation rates Cytokine production from activated macrophages Peroxynitrite was synthesized using a quenched-flow re- actor system.15 The breakdown of peroxynitrite over sub- Mouse J774A.1 macrophages were plated on silicone sub- strate samples was monitored by the decrease in absorbance strates with titanium dioxide coatings. Aluminum oxide- at 302 nm using a DU 640 Beckman spectrophotometer. coated silicone served as controls. The macrophages were 398 SUZUKI ET AL. coated silicone and a 55% decrease compared to un- coated silicone. Superoxide is a precursor to peroxynitrite and pro- duced by stimulated