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Titanium implants alter endothelial function and vasoconstriction via a protein kinase C-regulated pathway
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Acta Biomaterialia 5 (2009) 3258–3264 www.elsevier.com/locate/actabiomat
Titanium implants alter endothelial function and vasoconstriction via a protein kinase C-regulated pathway Rong Sen Yang a,1, Huei Ping Tzeng b,1, Feng Ming Ho c,1, Chia Chi Chuang b, Bo Lin Chen b, Chun-Fa Huang d, Ya-Wen Chen e, Ruei Ming Chen f, Shing Hwa Liu b,* a
Department of Orthopedics, College of Medicine, National Taiwan University, Taipei, Taiwan Institute of Toxicology, College of Medicine, National Taiwan University, Taipei 10051, Taiwan c Department of Biomedical Engineering, Chung Yuan Christian University and Department of Internal Medicine, Tao-Yuan General Hospital, Taoyan, Taiwan d Graduate Institute of Chinese Medical Science, College of Chinese Medicine, China Medical University, Taichung, Taiwan e Department of Physiology and Graduate Institute of Basic Medical Science, College of Medicine, China Medical University, Taichung, Taiwan f Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan b
Received 3 November 2008; received in revised form 21 April 2009; accepted 5 May 2009 Available online 13 May 2009
Abstract The application of titanium (Ti) alloy in joint prostheses is a good choice in orthopedic reconstruction. An elevated serum concentration of Ti has been shown in the patients with loosened knee prostheses. The precise actions of elevated Ti on the circulation remain unclear. In this study the maximal contractile responses elicited by phenylephrine in the aortas of rats 4 weeks after Ti alloy implantation and in cultured rat aortas treated with a soluble form of Ti for a period of 18 h were significantly decreased as compared with controls. Aortas isolated from rats with Ti alloy implants or aortas treated with a soluble form of Ti had enhanced protein expression of endothelial nitric oxide synthase (eNOS) and protein kinase C (PKC)-a and enhanced phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. Treatment of human umbilical vein endothelial cells (HUVECs) with a soluble form of Ti for 24 h dose-dependently increased eNOS protein expression. Short-term treatment of HUVECs with Ti for 1 h effectively enhanced the phosphorylation of eNOS, PKC (pan) and ERK1/2. PKC inhibitors RO320432 and chelerythrine effectively inhibited Ti-enhanced phosphorylation of eNOS and PKC (pan). These results indicate that Ti in the circulation may alter endothelial function and reduce vasoconstriction. Ó 2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. Keywords: Titanium alloy implants; eNOS; Aorta; Endothelial cells
1. Introduction Titanium (Ti) occurs widely in the natural environment and is detectable in many kinds of foods. The specific properties of resistance to corrosion and inertness allow many metallurgical applications of Ti in daily life. The introduction of Ti into orthopedic joint prostheses and the development of reconstructive techniques, as well as refinements in biomaterial science, have made total joint arthroplasty *
1
Corresponding author. Tel.: +886 2 23123456; fax: +886 2 23410217. E-mail address: [email protected] (S.H. Liu). These authors contributed equally to this work.
a breakthrough this century in the treatment of patients with severe arthritis. However, the wide use of joint prostheses has led to several post-operative complications, including regional osteolysis and loosening of the prostheses. These are difficult challenges to orthopedic surgeons. The dissemination of soluble metallic corrosion products and particulate metallic wear debris after long-term implantation has been shown to play an important role in prosthesis-related late complications [1,2]. It has been demonstrated that wear metal particles are disseminated in systemic organs such as the liver, spleen and para-aortic lymph nodes of patients with prostheses [3]. Metal particles generated at non-bearing surfaces in hip arthroplasty have
1742-7061/$ - see front matter Ó 2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.actbio.2009.05.006
R.S. Yang et al. / Acta Biomaterialia 5 (2009) 3258–3264
been found to accumulate in the liver and spleen [4]. Some studies have suggested that metals dissolve, circulate in the body fluid and accumulate in remote organs during total hip arthroplasty [1,5]. Kasai and colleagues have indicated that approximately one-third of patients with Ti alloy spinal implants analyzed exhibited abnormal serum or hair metal concentrations at a mean time of 5.1 years after surgery, and these metals might travel to distant organs after dissolution from spinal implants [6]. The long-term pathophysiological effects of metals accumulated in organs are unknown. In our previous studies we found that elevated blood levels of Ti in patients with loosened Ti–6Al–4V alloy prostheses [7] and in rats with Ti–6Al–4V alloy implants [8]. However, the precise action and mechanism of elevated concentrations of Ti on the circulation have not been well studied up to the present. Endothelial nitric oxide synthase (eNOS)-derived nitric oxide (NO) plays a key role in cardiovascular homeostasis [9,10]. The generation of NO by the vascular endothelium maintains a continuous vasodilator tone that is essential for the regulation of blood flow and blood pressure. The hypothesis of this study is that soluble forms of Ti may act on the circulation and further alter endothelial eNOS expression and function and blood vessel constriction. In the present study, therefore, we intend to explore the in vivo effects of Ti alloy implants on rat blood vessels and in vitro effects of soluble forms of Ti on organ cultures of isolated rat aortas and cultured human umbilical vein endothelial cells (HUVECs). We performed experiments to examine the effects of Ti on phenylephrine-induced blood vessel constriction and expression of eNOS and signaling proteins regulated by it in rat thoracic aortic rings. In addition, we investigated the cellular effects of Ti on the expression of eNOS and signaling proteins regulated by it in human endothelial cells.
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was dissolved in 1.25 mM sulfuric acid as a stock solution at a concentration of 10 mM and was adjusted to pH 7.0. 2.2. Rat thoracic aortic rings and vasoconstriction study
2. Materials and methods
Aortas were isolated from rats with titanium alloy implants and sham control rats under pentobarbital anesthesia. The vasoconstriction of aortic rings was measured by a method previously described [11]. Rings, 4–5 mm wide, of thoracic aortas were suspended between two hooks connected to a transducer (Grass FT.03) for the measurement of isometric force. Rings with an intact endothelium were used. The rings were suspended in 10 ml organ baths containing oxygenated (95% O2 + 5% CO2), warmed (37 °C) Krebs solution containing 118.3 mM NaCl, KCl 4.7, 2.5 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 25.0 mM NaHCO3 and 11.1 mM glucose. Basal tension was set at 1.0 g. The rings were washed three times for 20 min each time before a concentration–contractile response curve to phenylephrine (0.01–10 lM) was obtained. The tension was recorded through an isometric transducer on a Biopac MP 100 data acquisition system with analytic software (AcqKnowledge, Biopac Systems Inc., USA), the output of which was printed on a HP deskjet 500C. In some experiments aortic rings isolated from normal rats were cultured in organ culture Petri dishes (Falcon) with sterile Dulbeccomodified Eagle’s medium (DMEM) containing 10% fetal bovine serum and 1% antibiotic solution at 37 °C. After 18 h rings, treated or not with a soluble form of Ti were prepared for vasoconstriction experiments. For immunoblotting studies the rings were homogenized in buffer containing 20 mM HEPES, 0.25 M sucrose, 0.5 mM EDTA, 2 mM dithiothreitol, 1 mM phenylmethylsulphonyl fluoride (PMSF), 10 lg ml–1 leupeptin and 10 lg ml–1 aprotinin, pH 7.5, and centrifuged at 10,000 rpm for 20 min at 4 °C to remove debris.
2.1. The insertion of titanium alloy implants
2.3. Cell cultures
Male Wistar rats (200–250 g) were purchased from the Animal Center of the College of Medicine, National Taiwan University, Taipei, Taiwan. The Animal Research Committee of National Taiwan University, College of Medicine, approved the study in accordance with the guidelines for the care and use of laboratory animals. The insertion of titanium alloy implants, of a material similar to a clinical Ti alloy prosthesis, was done under pentobarbital anesthesia. The disc-shaped implant had a diameter of 5 mm and was 2 mm thick. All implants were rinsed in 70% ethanol in water and were then autoclaved. The implants were placed in the abdominal wall between the peritoneum and the rectus muscle. After 4 weeks the animals were killed under pentobarbital anesthesia to isolate the aortas. In some experiments titanium dioxide (TiO2, Aldrich) in sulfuric acid was prepared as a soluble form of Ti. TiO2
2.3.1. Human endothelial cells HUVECs were cultured as previously described [12,13]. Cells were seeded at a density of 1 105 per 75 cm2 flask in medium 199 (Gibco, Grand Island, NY), supplemented with 20 mM HEPES, 100 lg ml–1 endothelial cell growth substance (Collaborative Research Inc., Bedford, MA) and 20% fetal calf serum (Gibco). The cultures were maintained at 37 °C with a 5% CO2, 95% air atmosphere. Subcultures were performed using trypsin–EDTA. All media were filtered and supplemented with 5 U ml–1 heparin, 100 IU ml–1 penicillin and 0.1 mg ml–1 streptomycin. The medium was changed every 2 days. The endothelial cell monolayers were identified by the presence of factor VIII-related antigen (Histoset Kit, Immunolok, Carpinteria, CA) and the typical ‘‘cobblestone” appearance. Actively growing endothelial cells from passages 3–5 were used for the experiments.
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2.3.2. Mouse endothelial cells In some experiments mouse cerebral endothelial cells (CECs) were used. CECs were prepared from brain tissue following a previously described method [14]. Briefly, mouse cerebral cortex was removed, homogenized and filtered. After digestion with collagenase B, the homogenates were centrifuged in a 40% Percoll solution. The second band, containing microvessels, was collected and plated onto collagen-coated dishes. Mouse CECs were seeded in Dulbecco-modified Eagle’s medium supplemented with 10% heat-inactivated fetal bovine serum, L-glutamine, penicillin (100 IU ml–1) and streptomycin (100 lg ml–1) in 75 cm2 flasks at 37 °C in a humidified atmosphere of 5% CO2. 2.4. Western blot analysis A sample of 30–50 lg protein from cellular lysates was subjected to electrophoresis on 10% SDS–polyacrylamide gels. The samples were then electroblotted onto nitrocellulose paper. After blocking, the blots were incubated with anti-phospho-eNOS, anti-eNOS, anti-phosphoprotein kinase C (PKC) (pan), anti-PKC-a (Cell Signaling Technology, Danvers, MA), anti-phospho-extracellular signalregulated kinase (ERK) 1/2 and anti-ERK1/2 (BD Transduction Laboratories, Franklin Lakes, NJ) antibodies for 1 h in phosphate-buffered saline (PBS)/Tween 20 for 1 h, followed by two washes in PBS/Tween 20, and then incubated with horseradish peroxidase-conjugated secondary antibodies for 30 min. Enhanced chemiluminescence reagents were employed to depict the protein bands on membranes. a-Tubulin served as control for sample loading and integrity.
3. Results 3.1. Ti alters phenylephrine-elicited vasoconstriction in isolated aortic rings In intact endothelium aortic rings phenylephrine caused a concentration-dependent increase in contraction (Figs. 1 and 2). The maximal contractile responses elicited by phenylephrine in the aortas of rats 4 weeks after Ti alloy implantation were significantly decreased as compared with sham controls (Fig. 1). In an aorta tissue culture model treatment of rat aortas with a soluble form of Ti (10 and 30 lM) for a period of 18 h suppressed the maximal contractile response elicited by phenylephrine (Fig. 2). The acetylcholine (1 lM)-triggered relaxation of phenylephrine (10 lM)-elicited vasoconstriction was enhanced in Ti (30 lM)-treated aortas (from 24.51 ± 2.67% relaxation to 56.27 ± 3.14%, n = 3, P < 0.05) (Fig 2a). Moreover, the NOS inhibitor Nxnitro-L-arginine methyl ester (L-NAME, 0.1 mM) effec-
2.5. Cell viability assay Viability was measured using a Cell Titer 96TM AQueous cell viability assay kit (Promega, Madison, WI) as previously described [12]. This assay is based on cellular conversion of the colorimetric reagent MTS (3,4-(5-dimethylthiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt) in the presence of the electron coupling reagent phenazine methosulfate into soluble formazan by dehydrogenase enzymes found only in metabolically active, living cells. Formazan formation was measured as increased absorbance at 490 nm. 2.6. Statistics The values given in this article are means ± SEM. The significance of differences between the experimental groups and controls were assessed by one-way analysis of variance (ANOVA), followed by Dunnett’s test for each paired experiment. The differences were defined as significant at P < 0.05.
Fig. 1. Effect of Ti alloy implants on phenylephrine-elicited contraction in the aortic rings with an intact endothelium. The aortas were isolated from rats 4 weeks after Ti alloy implantation (open circles) or sham control rats (closed circles) and then contraction was measured in the presence of the indicated concentration of phenylephrine (0.01–10 lM). The isometric tensions were recorded (A). (B) The data expressed as percentage changes in tension in response to phenylephrine (10 lM). Data are presented as means ± SEM from five independent experiments. *P < 0.05 compared with controls.
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Fig. 2. Effect of Ti on phenylephrine-elicited contraction in organ culture of aortas. Each aortic ring was challenged with either vehicle (closed circles) or Ti (10 and 30 lM, closed triangles and open circles) for 18 h in organ culture conditions and then contraction was measured in the presence of the indicated concentration of phenylephrine (0.01–10 lM). Isometric tensions were recorded (A). In some experiments acetylcholine (1 lM) was added to induce vasorelaxation after treatment with 10 lM phenylephrine (A). (B) The data are expressed as percentage changes in tension in response to phenylephrine (10 lM). The NOS inhibitor L-NAME (0.1 mM) effectively reversed the inhibitory effect of Ti (30 lM) on phenylephrine-elicited vasoconstriction (insert in B). Data are presented as means ± SEM from four independent experiments. *P < 0.05 compared with the control.
tively reversed the inhibitory effect of Ti (30 lM) on phenylephrine-elicited vasoconstriction (insert in Fig. 2B). Ti by itself did not affect the basal tension of the aorta under these experimental conditions. 3.2. Ti alters the protein expression of eNOS and PKC-a and phosphorylation of ERK in aortas To investigate alterations in NO synthesis and its regulated signaling in the aortas of Ti alloy implanted rats or in vitro Ti-treated aortas, Western blot analysis was used to detect changes in protein expressions of eNOS and PKC-a and ERK phosphorylation. Ti alloy implantation for 4 weeks and treatment with a soluble form of Ti (30 lM) for 18 h enhanced the protein expressios of eNOS in the rat aortas (Fig. 3). PKC-a protein expression in the membrane fraction of aortas from Ti alloy implanted rats and 30 lM Ti-treated aortas was also increased (Fig. 3). Moreover, ERK phosphorylation was enhanced in the aortas of rats with Ti alloy implants and 30 lM Ti-treated aortas (Fig. 3).
Fig. 3. Effects of Ti alloy implants and a soluble form of Ti on eNOS and PKC-a protein expression and ERK1/2 phosphorylation in aortas. Aortas isolated from Ti alloy implanted rats and aortas treated with a soluble form of Ti (30 lM) were analyzed by Western blotting using anti-eNOS, anti-PKC-a, anti-phospho-ERK1/2 and anti-ERK1/2 antibodies. PKC-a protein expression in the membrane fraction of aortas was detected. a-Tubulin served as an internal control to verify equal protein loading. The data presented are representative of at least three independent experiments.
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3.3. Ti alters the protein expression of eNOS, PKC and ERK1/2 in cultured endothelial cells We next determined the effects of a soluble form of Ti on cultured endothelial cells. Treatment of HUVECs (Fig. 4A) and mouse CECs (Fig. 4B) with Ti (10–60 lM) for 24 h dose-dependently increased eNOS protein expression. Treatment with Ti (10–60 lM) for 48 h did not affect the cellular morphology or viability of HUVECs (Fig. 5). Short-term treatment of HUVECs with Ti (10 and 30 lM) for 1 h effectively enhanced the phosphorylation of eNOS, PKC (pan) and ERK1/2. Moreover, the NOS inhibitor L-NAME (0.1 mM) markedly reversed the increases in phosphorylation of eNOS and PKC (pan) induced by Ti, but did not affect ERK phosphorylation (Fig. 6A). The increased ERK and eNOS phosphorylation, but not PKC (pan) phosphorylation, induced by Ti could be reversed by the ERK inhibitor PD98059 (20 lM) (Fig. 6A). The PKC inhibitors RO320432 and chelerythrine (1 lM) could also reverse the increases in eNOS and
Fig. 5. Effects of Ti on cell viability of HUVECs. HUVECs were cultured with either vehicle or Ti (10–60 lM) for 48 h and then examined by light microscopy for cellular morphology (A) or assayed for cell viability using MTS (B). Data are presented as means ± SEM from four independent experiments. *P < 0.05 compared with the control.
PKC (pan) phosphorylation, but not the ERK1/2 phosphorylation, induced by Ti (Fig. 6B). 4. Discussion
Fig. 4. The effects of Ti on the expression of eNOS in HUVECs and mouse CECs. HUVECs (A) and CECs (B) were cultured with either vehicle or Ti (10–60 lM) for 24 h. (A) The proteins were separated by SDS–PAGE and analyzed by Western blotting using anti-eNOS antibody as described in Materials and methods. a-Tubulin served as an internal control to verify equal protein loading. All data are presented as means ± SEM from four independent experiments. *P < 0.05 compared with the control.
The results showed that Ti is capable of enhancing the expression of eNOS in rat aortas and human endothelial cells. The phosphorylation of eNOS, PKC (pan) and ERK1/2 proteins in rat aortas and human endothelial cells were also enhanced by Ti. PKC inhibitors could inhibit the increases in PKC phosphorylation and eNOS protein expression induced by Ti. These findings, therefore, indicate that Ti alloy implants can induce a PKC-activated eNOS/NO-mediated vasodilatation. With regard to the local reaction to wear particles, it is an important mechanism in the induction of a series of late complications in the long-term. Wide application of total joint replacement procedures in the treatment of patients with severe arthritis greatly increases exposure to metal ions in local tissues and body fluids [15–19]. These metals may be disseminated into body fluids. Furthermore, phagocytosed metal elements may be transported to the lymphoreticular systems and enter the circulatory system. It has been shown that fine Ti particles (1–3 lm), which are smaller than neutrophils (about 5 lm), were phagocytosed by these cells [20]. Metal elements in the blood may form complexes with serum proteins and be excreted in
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Fig. 6. Effects of Ti on the phosphorylation of eNOS, PKC and ERK1/2 in HUVECs. HUVECs were cultured with either vehicle or Ti (10 or 30 lM) in the presence or absence of the ERK inhibitor PD98059, the NOS inhibitor L-NAME (A) or the PKC inhibitors RO320432 and chelerythrine (B) for 1 h. The proteins were separated by SDS–PAGE and analyzed by Western blotting using anti-phospho-eNOS, anti-phosphoPKC (pan) and anti-phospho-ERK1/2 antibodies as described in Materials and methods. a-Tubulin and ERK1/2 served as controls for sample loading and integrity. The data presented are representative at least three independent experiments.
urine, stools, saliva or sweat or exhaled. Circulating metal ions may induce a lot of reactions. Jacobs and colleagues have stated that patients who underwent total hip arthroplasty and had hardware loosening exhibited Ti concentrations at least three times those of patients who underwent total hip arthroplasty and did not have hardware loosening [21]. Recent studies have shown that significantly higher serum Ti concentrations were observed in subjects with Ti alloy spinal instrumentation when compared with controls [6,22]. Our previous study has shown an elevated blood concentration of Ti elements in patients with loosening prostheses (319.6 ± 280.3 ppb, duration of implantation 2.2–9.4 years), as compared with normal people (9.3 ± 7.1 ppb) or patients with well-fixed prosthesis (17.4 ± 9.8 ppb) [7]. Such a high concentration of Ti may induce a series of local or systemic events. In the present work the blood concentration of elemental Ti in Ti alloy implanted (4 weeks) rats was 91.6 ± 17.1 ppb, in the clinically detectable range. A cell viability assay has shown that such concentrations (10–60 lM) do not cause cytotoxicity
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of human endothelial cells. This implies that the biocompatibility of Ti is good in this concentration range. Up to the present the precise action of Ti on the circulation remains unclear. We therefore investigated the effects of circulating Ti on blood vessel contractility and endothelial cell function in this study. The results show that the maximal contractile responses elicited by phenylephrine in the aortas of Ti alloy implanted rats and in cultured rat aortas treated with a soluble form of Ti were significantly decreased as compared with controls. These findings indicate that circulating Ti is capable of altering the contractile response of blood vessel. NO is well known as a signaling molecule in the vascular system. eNOS-derived NO plays an important role in cardiovascular homeostasis, angiogenesis and vascular remodeling [9,23–25]. The importance of phosphorylation in the regulation of eNOS activity and vascular relaxation has been emphasized by several studies [23,26–28]. Overexpression of PKC-a in primary cardiac mouse endothelial cells has been demonstrated to be associated with increased eNOS phosphorylation and increased NO production; the authors also found that PKC-a transduction in rat femoral arteries resulted in a significant increase in blood flow that was suppressed by treatment with the NOS inhibitor L-NAME [28]. Moreover, mitogen-activated protein kinase (MAPK)–ERK1/2 signaling has been shown to mediate the acute activation of eNOS to produce NO in uterine artery endothelial cells by estrogen; the authors further suggested that this pathway plays an important role in estrogen-induced, NO-mediated rapid uterine vasodilatation [29]. In the present work the contractile response elicited by phenylephrine were markedly decreased and eNOS and PKC-a protein expression and ERK1/2 phosphorylation were increased in the aortas of rats 4 weeks after Ti alloy implantation and in cultured rat aortas treated with a soluble form of Ti for a period of 18 h. Meanwhile, Ti treatment enhanced acetylcholineinduced vasorelaxation. The Ti-induced inhibitory effect on phenylephrine-elicited vasoconstriction could also be reversed by the NOS inhibitor L-NAME. Thus, these results indicate that Ti may possess the ability to induce endothelium-derived vasodilatation through alterations in eNOS-, PKC-a- and ERK1/2-related signals. Furthermore, in human endothelial cells the soluble form of Ti could also increase eNOS protein expression and enhance the phosphorylation of eNOS, PKC (pan) and ERK1/2, supporting the hypothesis that Ti can act on endothelial cells to induce endothelium-derived, vasodilatation-related signals. The results of pharmacological inhibitor studies also indicate that Ti affects endothelial function through PKC-a- or ERK1/2-regulated eNOS activation pathways. However, the ERK inhibitor PD98059 did not affect Tienhanced PKC phosphorylation; PKC inhibitors could also not affect Ti-enhanced ERK1/2 phosphorylation, implying that PKC and ERK may be two independent signals regulating eNOS activation in endothelial cells after Ti exposure.
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5. Conclusions In this study we have demonstrated that phenylephrineinduced contractile responses in the aortas of rats 4 weeks after Ti alloy implantation and in aortas treated with a soluble form of Ti were significantly decreased. Ti was capable of activating eNOS, PKC and ERK in rat aortas and human endothelial cells. These results indicate that Ti may alter endothelial function and reduce vasoconstriction. Further investigation is essential for the evaluation of the clinical significance of this biological effect induced by Ti. Acknowledgement This study was supported by a grant from the National Science Council of Taiwan (NSC92-2314-B-002-094). References [1] Dorr LD, Bloebaum R, Emmanual J, Meldnum R. Histologic, biochemical, and ion analysis of tissue and fluids retrieved during total hip arthroplasty. Clin Orthop Relat Res 1990;261:82–95. [2] Maloney Wj, Smith Rl. Periprosthetic osteolysis in total hip arthroplasty: the role of particulate wear debris. J Bone Joint Surg 1995;77A:1448–61. [3] Urban RM, Jacobs JJ, Tomlinson MJ, Gavrilovic J, Black J, Peoc’h M. Dissemination of wear particles to the liver, spleen, and abdominal lymph nodes of patients with hip or knee replacement. J Bone Joint Surg Am 2000;82:457–76. [4] Urban RM, Tomlinson MJ, Hall DJ, Jacobs JJ. Accumulation in liver and spleen of metal particles generated at nonbearing surfaces in hip arthroplasty. J Arthroplasty 2004;19(Suppl. 3):94–101. [5] Coleman RF, Herrington J, Scales JT. Concentration of wear products in hair, blood, and urine after total hip replacement. BMJ 1973;1:527–9. [6] Kasai Y, Iida R, Uchida A. Metal concentrations in the serum and hair of patients with titanium alloy spinal implants. Spine 2003;28: 1320–6. [7] Liu TK, Liu SH, Chang CH, Yang RS. Concentration of metal elements in the blood and urine in the patients with cementless total knee arthroplasty. Tohoku J Exp Med 1998;185:253–62. [8] Yang RS, Tsai KS, Liu SH. Titanium implants enhance pulmonary nitric oxide production and lung injury in rats exposed to endotoxin. J Biomed Mater Res A 2004;69:561–6. [9] Ignarro LJ, Cirino G, Casini A, Napoli C. Nitric oxide as a signaling molecule in the vascular system: an overview. J Cardiovasc Pharmacol 1999;34:879–86. [10] Feletou M, Tang EH, Vanhoutte PM. Nitric oxide the gatekeeper of endothelial vasomotor control. Front Biosci 2008;13:4198–217. [11] Tzeng HP, Yang RS, Ueng TH, Lin-Shiau SY, Liu SH. Motorcycle exhaust particulates enhance vasoconstriction in organ culture of rat aortas and involve reactive oxygen species. Toxicol Sci 2003;75:66–73. [12] Sheu ML, Ho FM, Yang RS, Chao KF, Lin WW, Lin-Shiau SY, et al. High glucose induces human endothelial cell apoptosis through
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Acta Biomaterialia 5 (2009) 3258–3264 www.elsevier.com/locate/actabiomat
Titanium implants alter endothelial function and vasoconstriction via a protein kinase C-regulated pathway Rong Sen Yang a,1, Huei Ping Tzeng b,1, Feng Ming Ho c,1, Chia Chi Chuang b, Bo Lin Chen b, Chun-Fa Huang d, Ya-Wen Chen e, Ruei Ming Chen f, Shing Hwa Liu b,* a
Department of Orthopedics, College of Medicine, National Taiwan University, Taipei, Taiwan Institute of Toxicology, College of Medicine, National Taiwan University, Taipei 10051, Taiwan c Department of Biomedical Engineering, Chung Yuan Christian University and Department of Internal Medicine, Tao-Yuan General Hospital, Taoyan, Taiwan d Graduate Institute of Chinese Medical Science, College of Chinese Medicine, China Medical University, Taichung, Taiwan e Department of Physiology and Graduate Institute of Basic Medical Science, College of Medicine, China Medical University, Taichung, Taiwan f Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan b
Received 3 November 2008; received in revised form 21 April 2009; accepted 5 May 2009 Available online 13 May 2009
Abstract The application of titanium (Ti) alloy in joint prostheses is a good choice in orthopedic reconstruction. An elevated serum concentration of Ti has been shown in the patients with loosened knee prostheses. The precise actions of elevated Ti on the circulation remain unclear. In this study the maximal contractile responses elicited by phenylephrine in the aortas of rats 4 weeks after Ti alloy implantation and in cultured rat aortas treated with a soluble form of Ti for a period of 18 h were significantly decreased as compared with controls. Aortas isolated from rats with Ti alloy implants or aortas treated with a soluble form of Ti had enhanced protein expression of endothelial nitric oxide synthase (eNOS) and protein kinase C (PKC)-a and enhanced phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. Treatment of human umbilical vein endothelial cells (HUVECs) with a soluble form of Ti for 24 h dose-dependently increased eNOS protein expression. Short-term treatment of HUVECs with Ti for 1 h effectively enhanced the phosphorylation of eNOS, PKC (pan) and ERK1/2. PKC inhibitors RO320432 and chelerythrine effectively inhibited Ti-enhanced phosphorylation of eNOS and PKC (pan). These results indicate that Ti in the circulation may alter endothelial function and reduce vasoconstriction. Ó 2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. Keywords: Titanium alloy implants; eNOS; Aorta; Endothelial cells
1. Introduction Titanium (Ti) occurs widely in the natural environment and is detectable in many kinds of foods. The specific properties of resistance to corrosion and inertness allow many metallurgical applications of Ti in daily life. The introduction of Ti into orthopedic joint prostheses and the development of reconstructive techniques, as well as refinements in biomaterial science, have made total joint arthroplasty *
1
Corresponding author. Tel.: +886 2 23123456; fax: +886 2 23410217. E-mail address: [email protected] (S.H. Liu). These authors contributed equally to this work.
a breakthrough this century in the treatment of patients with severe arthritis. However, the wide use of joint prostheses has led to several post-operative complications, including regional osteolysis and loosening of the prostheses. These are difficult challenges to orthopedic surgeons. The dissemination of soluble metallic corrosion products and particulate metallic wear debris after long-term implantation has been shown to play an important role in prosthesis-related late complications [1,2]. It has been demonstrated that wear metal particles are disseminated in systemic organs such as the liver, spleen and para-aortic lymph nodes of patients with prostheses [3]. Metal particles generated at non-bearing surfaces in hip arthroplasty have
1742-7061/$ - see front matter Ó 2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.actbio.2009.05.006
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been found to accumulate in the liver and spleen [4]. Some studies have suggested that metals dissolve, circulate in the body fluid and accumulate in remote organs during total hip arthroplasty [1,5]. Kasai and colleagues have indicated that approximately one-third of patients with Ti alloy spinal implants analyzed exhibited abnormal serum or hair metal concentrations at a mean time of 5.1 years after surgery, and these metals might travel to distant organs after dissolution from spinal implants [6]. The long-term pathophysiological effects of metals accumulated in organs are unknown. In our previous studies we found that elevated blood levels of Ti in patients with loosened Ti–6Al–4V alloy prostheses [7] and in rats with Ti–6Al–4V alloy implants [8]. However, the precise action and mechanism of elevated concentrations of Ti on the circulation have not been well studied up to the present. Endothelial nitric oxide synthase (eNOS)-derived nitric oxide (NO) plays a key role in cardiovascular homeostasis [9,10]. The generation of NO by the vascular endothelium maintains a continuous vasodilator tone that is essential for the regulation of blood flow and blood pressure. The hypothesis of this study is that soluble forms of Ti may act on the circulation and further alter endothelial eNOS expression and function and blood vessel constriction. In the present study, therefore, we intend to explore the in vivo effects of Ti alloy implants on rat blood vessels and in vitro effects of soluble forms of Ti on organ cultures of isolated rat aortas and cultured human umbilical vein endothelial cells (HUVECs). We performed experiments to examine the effects of Ti on phenylephrine-induced blood vessel constriction and expression of eNOS and signaling proteins regulated by it in rat thoracic aortic rings. In addition, we investigated the cellular effects of Ti on the expression of eNOS and signaling proteins regulated by it in human endothelial cells.
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was dissolved in 1.25 mM sulfuric acid as a stock solution at a concentration of 10 mM and was adjusted to pH 7.0. 2.2. Rat thoracic aortic rings and vasoconstriction study
2. Materials and methods
Aortas were isolated from rats with titanium alloy implants and sham control rats under pentobarbital anesthesia. The vasoconstriction of aortic rings was measured by a method previously described [11]. Rings, 4–5 mm wide, of thoracic aortas were suspended between two hooks connected to a transducer (Grass FT.03) for the measurement of isometric force. Rings with an intact endothelium were used. The rings were suspended in 10 ml organ baths containing oxygenated (95% O2 + 5% CO2), warmed (37 °C) Krebs solution containing 118.3 mM NaCl, KCl 4.7, 2.5 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 25.0 mM NaHCO3 and 11.1 mM glucose. Basal tension was set at 1.0 g. The rings were washed three times for 20 min each time before a concentration–contractile response curve to phenylephrine (0.01–10 lM) was obtained. The tension was recorded through an isometric transducer on a Biopac MP 100 data acquisition system with analytic software (AcqKnowledge, Biopac Systems Inc., USA), the output of which was printed on a HP deskjet 500C. In some experiments aortic rings isolated from normal rats were cultured in organ culture Petri dishes (Falcon) with sterile Dulbeccomodified Eagle’s medium (DMEM) containing 10% fetal bovine serum and 1% antibiotic solution at 37 °C. After 18 h rings, treated or not with a soluble form of Ti were prepared for vasoconstriction experiments. For immunoblotting studies the rings were homogenized in buffer containing 20 mM HEPES, 0.25 M sucrose, 0.5 mM EDTA, 2 mM dithiothreitol, 1 mM phenylmethylsulphonyl fluoride (PMSF), 10 lg ml–1 leupeptin and 10 lg ml–1 aprotinin, pH 7.5, and centrifuged at 10,000 rpm for 20 min at 4 °C to remove debris.
2.1. The insertion of titanium alloy implants
2.3. Cell cultures
Male Wistar rats (200–250 g) were purchased from the Animal Center of the College of Medicine, National Taiwan University, Taipei, Taiwan. The Animal Research Committee of National Taiwan University, College of Medicine, approved the study in accordance with the guidelines for the care and use of laboratory animals. The insertion of titanium alloy implants, of a material similar to a clinical Ti alloy prosthesis, was done under pentobarbital anesthesia. The disc-shaped implant had a diameter of 5 mm and was 2 mm thick. All implants were rinsed in 70% ethanol in water and were then autoclaved. The implants were placed in the abdominal wall between the peritoneum and the rectus muscle. After 4 weeks the animals were killed under pentobarbital anesthesia to isolate the aortas. In some experiments titanium dioxide (TiO2, Aldrich) in sulfuric acid was prepared as a soluble form of Ti. TiO2
2.3.1. Human endothelial cells HUVECs were cultured as previously described [12,13]. Cells were seeded at a density of 1 105 per 75 cm2 flask in medium 199 (Gibco, Grand Island, NY), supplemented with 20 mM HEPES, 100 lg ml–1 endothelial cell growth substance (Collaborative Research Inc., Bedford, MA) and 20% fetal calf serum (Gibco). The cultures were maintained at 37 °C with a 5% CO2, 95% air atmosphere. Subcultures were performed using trypsin–EDTA. All media were filtered and supplemented with 5 U ml–1 heparin, 100 IU ml–1 penicillin and 0.1 mg ml–1 streptomycin. The medium was changed every 2 days. The endothelial cell monolayers were identified by the presence of factor VIII-related antigen (Histoset Kit, Immunolok, Carpinteria, CA) and the typical ‘‘cobblestone” appearance. Actively growing endothelial cells from passages 3–5 were used for the experiments.
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2.3.2. Mouse endothelial cells In some experiments mouse cerebral endothelial cells (CECs) were used. CECs were prepared from brain tissue following a previously described method [14]. Briefly, mouse cerebral cortex was removed, homogenized and filtered. After digestion with collagenase B, the homogenates were centrifuged in a 40% Percoll solution. The second band, containing microvessels, was collected and plated onto collagen-coated dishes. Mouse CECs were seeded in Dulbecco-modified Eagle’s medium supplemented with 10% heat-inactivated fetal bovine serum, L-glutamine, penicillin (100 IU ml–1) and streptomycin (100 lg ml–1) in 75 cm2 flasks at 37 °C in a humidified atmosphere of 5% CO2. 2.4. Western blot analysis A sample of 30–50 lg protein from cellular lysates was subjected to electrophoresis on 10% SDS–polyacrylamide gels. The samples were then electroblotted onto nitrocellulose paper. After blocking, the blots were incubated with anti-phospho-eNOS, anti-eNOS, anti-phosphoprotein kinase C (PKC) (pan), anti-PKC-a (Cell Signaling Technology, Danvers, MA), anti-phospho-extracellular signalregulated kinase (ERK) 1/2 and anti-ERK1/2 (BD Transduction Laboratories, Franklin Lakes, NJ) antibodies for 1 h in phosphate-buffered saline (PBS)/Tween 20 for 1 h, followed by two washes in PBS/Tween 20, and then incubated with horseradish peroxidase-conjugated secondary antibodies for 30 min. Enhanced chemiluminescence reagents were employed to depict the protein bands on membranes. a-Tubulin served as control for sample loading and integrity.
3. Results 3.1. Ti alters phenylephrine-elicited vasoconstriction in isolated aortic rings In intact endothelium aortic rings phenylephrine caused a concentration-dependent increase in contraction (Figs. 1 and 2). The maximal contractile responses elicited by phenylephrine in the aortas of rats 4 weeks after Ti alloy implantation were significantly decreased as compared with sham controls (Fig. 1). In an aorta tissue culture model treatment of rat aortas with a soluble form of Ti (10 and 30 lM) for a period of 18 h suppressed the maximal contractile response elicited by phenylephrine (Fig. 2). The acetylcholine (1 lM)-triggered relaxation of phenylephrine (10 lM)-elicited vasoconstriction was enhanced in Ti (30 lM)-treated aortas (from 24.51 ± 2.67% relaxation to 56.27 ± 3.14%, n = 3, P < 0.05) (Fig 2a). Moreover, the NOS inhibitor Nxnitro-L-arginine methyl ester (L-NAME, 0.1 mM) effec-
2.5. Cell viability assay Viability was measured using a Cell Titer 96TM AQueous cell viability assay kit (Promega, Madison, WI) as previously described [12]. This assay is based on cellular conversion of the colorimetric reagent MTS (3,4-(5-dimethylthiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt) in the presence of the electron coupling reagent phenazine methosulfate into soluble formazan by dehydrogenase enzymes found only in metabolically active, living cells. Formazan formation was measured as increased absorbance at 490 nm. 2.6. Statistics The values given in this article are means ± SEM. The significance of differences between the experimental groups and controls were assessed by one-way analysis of variance (ANOVA), followed by Dunnett’s test for each paired experiment. The differences were defined as significant at P < 0.05.
Fig. 1. Effect of Ti alloy implants on phenylephrine-elicited contraction in the aortic rings with an intact endothelium. The aortas were isolated from rats 4 weeks after Ti alloy implantation (open circles) or sham control rats (closed circles) and then contraction was measured in the presence of the indicated concentration of phenylephrine (0.01–10 lM). The isometric tensions were recorded (A). (B) The data expressed as percentage changes in tension in response to phenylephrine (10 lM). Data are presented as means ± SEM from five independent experiments. *P < 0.05 compared with controls.
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Fig. 2. Effect of Ti on phenylephrine-elicited contraction in organ culture of aortas. Each aortic ring was challenged with either vehicle (closed circles) or Ti (10 and 30 lM, closed triangles and open circles) for 18 h in organ culture conditions and then contraction was measured in the presence of the indicated concentration of phenylephrine (0.01–10 lM). Isometric tensions were recorded (A). In some experiments acetylcholine (1 lM) was added to induce vasorelaxation after treatment with 10 lM phenylephrine (A). (B) The data are expressed as percentage changes in tension in response to phenylephrine (10 lM). The NOS inhibitor L-NAME (0.1 mM) effectively reversed the inhibitory effect of Ti (30 lM) on phenylephrine-elicited vasoconstriction (insert in B). Data are presented as means ± SEM from four independent experiments. *P < 0.05 compared with the control.
tively reversed the inhibitory effect of Ti (30 lM) on phenylephrine-elicited vasoconstriction (insert in Fig. 2B). Ti by itself did not affect the basal tension of the aorta under these experimental conditions. 3.2. Ti alters the protein expression of eNOS and PKC-a and phosphorylation of ERK in aortas To investigate alterations in NO synthesis and its regulated signaling in the aortas of Ti alloy implanted rats or in vitro Ti-treated aortas, Western blot analysis was used to detect changes in protein expressions of eNOS and PKC-a and ERK phosphorylation. Ti alloy implantation for 4 weeks and treatment with a soluble form of Ti (30 lM) for 18 h enhanced the protein expressios of eNOS in the rat aortas (Fig. 3). PKC-a protein expression in the membrane fraction of aortas from Ti alloy implanted rats and 30 lM Ti-treated aortas was also increased (Fig. 3). Moreover, ERK phosphorylation was enhanced in the aortas of rats with Ti alloy implants and 30 lM Ti-treated aortas (Fig. 3).
Fig. 3. Effects of Ti alloy implants and a soluble form of Ti on eNOS and PKC-a protein expression and ERK1/2 phosphorylation in aortas. Aortas isolated from Ti alloy implanted rats and aortas treated with a soluble form of Ti (30 lM) were analyzed by Western blotting using anti-eNOS, anti-PKC-a, anti-phospho-ERK1/2 and anti-ERK1/2 antibodies. PKC-a protein expression in the membrane fraction of aortas was detected. a-Tubulin served as an internal control to verify equal protein loading. The data presented are representative of at least three independent experiments.
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3.3. Ti alters the protein expression of eNOS, PKC and ERK1/2 in cultured endothelial cells We next determined the effects of a soluble form of Ti on cultured endothelial cells. Treatment of HUVECs (Fig. 4A) and mouse CECs (Fig. 4B) with Ti (10–60 lM) for 24 h dose-dependently increased eNOS protein expression. Treatment with Ti (10–60 lM) for 48 h did not affect the cellular morphology or viability of HUVECs (Fig. 5). Short-term treatment of HUVECs with Ti (10 and 30 lM) for 1 h effectively enhanced the phosphorylation of eNOS, PKC (pan) and ERK1/2. Moreover, the NOS inhibitor L-NAME (0.1 mM) markedly reversed the increases in phosphorylation of eNOS and PKC (pan) induced by Ti, but did not affect ERK phosphorylation (Fig. 6A). The increased ERK and eNOS phosphorylation, but not PKC (pan) phosphorylation, induced by Ti could be reversed by the ERK inhibitor PD98059 (20 lM) (Fig. 6A). The PKC inhibitors RO320432 and chelerythrine (1 lM) could also reverse the increases in eNOS and
Fig. 5. Effects of Ti on cell viability of HUVECs. HUVECs were cultured with either vehicle or Ti (10–60 lM) for 48 h and then examined by light microscopy for cellular morphology (A) or assayed for cell viability using MTS (B). Data are presented as means ± SEM from four independent experiments. *P < 0.05 compared with the control.
PKC (pan) phosphorylation, but not the ERK1/2 phosphorylation, induced by Ti (Fig. 6B). 4. Discussion
Fig. 4. The effects of Ti on the expression of eNOS in HUVECs and mouse CECs. HUVECs (A) and CECs (B) were cultured with either vehicle or Ti (10–60 lM) for 24 h. (A) The proteins were separated by SDS–PAGE and analyzed by Western blotting using anti-eNOS antibody as described in Materials and methods. a-Tubulin served as an internal control to verify equal protein loading. All data are presented as means ± SEM from four independent experiments. *P < 0.05 compared with the control.
The results showed that Ti is capable of enhancing the expression of eNOS in rat aortas and human endothelial cells. The phosphorylation of eNOS, PKC (pan) and ERK1/2 proteins in rat aortas and human endothelial cells were also enhanced by Ti. PKC inhibitors could inhibit the increases in PKC phosphorylation and eNOS protein expression induced by Ti. These findings, therefore, indicate that Ti alloy implants can induce a PKC-activated eNOS/NO-mediated vasodilatation. With regard to the local reaction to wear particles, it is an important mechanism in the induction of a series of late complications in the long-term. Wide application of total joint replacement procedures in the treatment of patients with severe arthritis greatly increases exposure to metal ions in local tissues and body fluids [15–19]. These metals may be disseminated into body fluids. Furthermore, phagocytosed metal elements may be transported to the lymphoreticular systems and enter the circulatory system. It has been shown that fine Ti particles (1–3 lm), which are smaller than neutrophils (about 5 lm), were phagocytosed by these cells [20]. Metal elements in the blood may form complexes with serum proteins and be excreted in
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Fig. 6. Effects of Ti on the phosphorylation of eNOS, PKC and ERK1/2 in HUVECs. HUVECs were cultured with either vehicle or Ti (10 or 30 lM) in the presence or absence of the ERK inhibitor PD98059, the NOS inhibitor L-NAME (A) or the PKC inhibitors RO320432 and chelerythrine (B) for 1 h. The proteins were separated by SDS–PAGE and analyzed by Western blotting using anti-phospho-eNOS, anti-phosphoPKC (pan) and anti-phospho-ERK1/2 antibodies as described in Materials and methods. a-Tubulin and ERK1/2 served as controls for sample loading and integrity. The data presented are representative at least three independent experiments.
urine, stools, saliva or sweat or exhaled. Circulating metal ions may induce a lot of reactions. Jacobs and colleagues have stated that patients who underwent total hip arthroplasty and had hardware loosening exhibited Ti concentrations at least three times those of patients who underwent total hip arthroplasty and did not have hardware loosening [21]. Recent studies have shown that significantly higher serum Ti concentrations were observed in subjects with Ti alloy spinal instrumentation when compared with controls [6,22]. Our previous study has shown an elevated blood concentration of Ti elements in patients with loosening prostheses (319.6 ± 280.3 ppb, duration of implantation 2.2–9.4 years), as compared with normal people (9.3 ± 7.1 ppb) or patients with well-fixed prosthesis (17.4 ± 9.8 ppb) [7]. Such a high concentration of Ti may induce a series of local or systemic events. In the present work the blood concentration of elemental Ti in Ti alloy implanted (4 weeks) rats was 91.6 ± 17.1 ppb, in the clinically detectable range. A cell viability assay has shown that such concentrations (10–60 lM) do not cause cytotoxicity
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of human endothelial cells. This implies that the biocompatibility of Ti is good in this concentration range. Up to the present the precise action of Ti on the circulation remains unclear. We therefore investigated the effects of circulating Ti on blood vessel contractility and endothelial cell function in this study. The results show that the maximal contractile responses elicited by phenylephrine in the aortas of Ti alloy implanted rats and in cultured rat aortas treated with a soluble form of Ti were significantly decreased as compared with controls. These findings indicate that circulating Ti is capable of altering the contractile response of blood vessel. NO is well known as a signaling molecule in the vascular system. eNOS-derived NO plays an important role in cardiovascular homeostasis, angiogenesis and vascular remodeling [9,23–25]. The importance of phosphorylation in the regulation of eNOS activity and vascular relaxation has been emphasized by several studies [23,26–28]. Overexpression of PKC-a in primary cardiac mouse endothelial cells has been demonstrated to be associated with increased eNOS phosphorylation and increased NO production; the authors also found that PKC-a transduction in rat femoral arteries resulted in a significant increase in blood flow that was suppressed by treatment with the NOS inhibitor L-NAME [28]. Moreover, mitogen-activated protein kinase (MAPK)–ERK1/2 signaling has been shown to mediate the acute activation of eNOS to produce NO in uterine artery endothelial cells by estrogen; the authors further suggested that this pathway plays an important role in estrogen-induced, NO-mediated rapid uterine vasodilatation [29]. In the present work the contractile response elicited by phenylephrine were markedly decreased and eNOS and PKC-a protein expression and ERK1/2 phosphorylation were increased in the aortas of rats 4 weeks after Ti alloy implantation and in cultured rat aortas treated with a soluble form of Ti for a period of 18 h. Meanwhile, Ti treatment enhanced acetylcholineinduced vasorelaxation. The Ti-induced inhibitory effect on phenylephrine-elicited vasoconstriction could also be reversed by the NOS inhibitor L-NAME. Thus, these results indicate that Ti may possess the ability to induce endothelium-derived vasodilatation through alterations in eNOS-, PKC-a- and ERK1/2-related signals. Furthermore, in human endothelial cells the soluble form of Ti could also increase eNOS protein expression and enhance the phosphorylation of eNOS, PKC (pan) and ERK1/2, supporting the hypothesis that Ti can act on endothelial cells to induce endothelium-derived, vasodilatation-related signals. The results of pharmacological inhibitor studies also indicate that Ti affects endothelial function through PKC-a- or ERK1/2-regulated eNOS activation pathways. However, the ERK inhibitor PD98059 did not affect Tienhanced PKC phosphorylation; PKC inhibitors could also not affect Ti-enhanced ERK1/2 phosphorylation, implying that PKC and ERK may be two independent signals regulating eNOS activation in endothelial cells after Ti exposure.
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5. Conclusions In this study we have demonstrated that phenylephrineinduced contractile responses in the aortas of rats 4 weeks after Ti alloy implantation and in aortas treated with a soluble form of Ti were significantly decreased. Ti was capable of activating eNOS, PKC and ERK in rat aortas and human endothelial cells. These results indicate that Ti may alter endothelial function and reduce vasoconstriction. Further investigation is essential for the evaluation of the clinical significance of this biological effect induced by Ti. Acknowledgement This study was supported by a grant from the National Science Council of Taiwan (NSC92-2314-B-002-094). References [1] Dorr LD, Bloebaum R, Emmanual J, Meldnum R. Histologic, biochemical, and ion analysis of tissue and fluids retrieved during total hip arthroplasty. Clin Orthop Relat Res 1990;261:82–95. [2] Maloney Wj, Smith Rl. Periprosthetic osteolysis in total hip arthroplasty: the role of particulate wear debris. J Bone Joint Surg 1995;77A:1448–61. [3] Urban RM, Jacobs JJ, Tomlinson MJ, Gavrilovic J, Black J, Peoc’h M. Dissemination of wear particles to the liver, spleen, and abdominal lymph nodes of patients with hip or knee replacement. J Bone Joint Surg Am 2000;82:457–76. [4] Urban RM, Tomlinson MJ, Hall DJ, Jacobs JJ. Accumulation in liver and spleen of metal particles generated at nonbearing surfaces in hip arthroplasty. J Arthroplasty 2004;19(Suppl. 3):94–101. [5] Coleman RF, Herrington J, Scales JT. Concentration of wear products in hair, blood, and urine after total hip replacement. BMJ 1973;1:527–9. [6] Kasai Y, Iida R, Uchida A. Metal concentrations in the serum and hair of patients with titanium alloy spinal implants. Spine 2003;28: 1320–6. [7] Liu TK, Liu SH, Chang CH, Yang RS. Concentration of metal elements in the blood and urine in the patients with cementless total knee arthroplasty. Tohoku J Exp Med 1998;185:253–62. [8] Yang RS, Tsai KS, Liu SH. Titanium implants enhance pulmonary nitric oxide production and lung injury in rats exposed to endotoxin. J Biomed Mater Res A 2004;69:561–6. [9] Ignarro LJ, Cirino G, Casini A, Napoli C. Nitric oxide as a signaling molecule in the vascular system: an overview. J Cardiovasc Pharmacol 1999;34:879–86. [10] Feletou M, Tang EH, Vanhoutte PM. Nitric oxide the gatekeeper of endothelial vasomotor control. Front Biosci 2008;13:4198–217. [11] Tzeng HP, Yang RS, Ueng TH, Lin-Shiau SY, Liu SH. Motorcycle exhaust particulates enhance vasoconstriction in organ culture of rat aortas and involve reactive oxygen species. Toxicol Sci 2003;75:66–73. [12] Sheu ML, Ho FM, Yang RS, Chao KF, Lin WW, Lin-Shiau SY, et al. High glucose induces human endothelial cell apoptosis through
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