Identification and Dynamics of Proteins Adhering To the Surface of Medical Silicones in Vivo and in Vitro Aleksandar Backovic,† Hong-Lei Huang,‡ Barbara Del Frari,§ Hildegunde Piza,§ Lukas A. Huber,‡ and Georg Wick*,† Division for Experimental Pathophysiology and Immunology, Biocenter, Innsbruck Medical University, Fritz-Pregl Str 3/IV, A6020 Innsbruck, Austria, Division of Cell Biology, Biocenter, Innsbruck Medical University, Innsbruck, Austria, and Department of Plastic and Reconstructive Surgery, University Clinic, Innsbruck Medical University, Innsbruck, Austria Received July 27, 2006
Abstract: Silicone has been used in medical practice as a paradigmatic implant material for decades despite significant detrimental side effects. Our targeted proteomics approach was aimed at identification of the proteins adsorbed to the surface of silicone because they have been characterized as key components in the onset and perpetuation of local immune reactions to silicone. The composition of the proteinacious film, the dynamics of protein deposition, and protein modifications after adsorption were analyzed both in vivo and in vitro. Differential analysis of protein deposition was performed, followed by protein identification with mass spectrometry, database matching, and Western blots. Thus far, we have identified the 30 most abundant proteins deposited on the surface of silicone, the largest known inventory of such proteins so far. Structural and extracellular matrix proteins predominated, followed by mediators of host defense, metabolism, transport, and stress related proteins. In addition, several biochemical modifications of fibronectin, vitronectin, and heat shock protein 60 were detected. Our analyses also revealed previously undetected proteins deposited on the surface of silicone. As tentative initiators and/or modulators of the response to silicone, they are therefore valuable candidates for prognosis and therapy. Keywords: silicone • biomaterials • biocompatibility • fibrosis • immune • protein adhesion
Introduction Because of its assumed high degree of biocompatibility, silicone is the most widely used medical implant material despite its detrimental local1-4 and systemic5-7 effects in many patients. Although meta-analyses have failed to provide a definitive correlation between silicone implants and an increased risk of autoimmune diseases,8,9 unequivocal strong * To whom correspondence should be addressed. E-mail:
[email protected]. † Division for Experimental Pathophysiology and Immunology. ‡ Division of Cell Biology. § Department of Plastic and Reconstructive Surgery.
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local immune reactions have been detected in peri-implant connective tissue capsules.10,11 The chronic proliferative state of inflammation of the cells in the capsule entails dense collagenous fibrosis at various times after implantation.12 On a systemic level, several studies successfully correlated plasma autoantibody concentrations with clinical symptoms like capsule contraction, pain, and fibromyalgia13,14 in patients with various types of silicone implants. It is believed that the systemic side effects reflect local immunological changes, but this functional relationship has never been defined. The women undergoing breast augmentation are a unique and large group of patients where most of the epidemiological studies on silicone side effects have been conducted. We adapted this concept as a paradigm for reactions to other types of silicone implants as well. A thin proteinacious film forms on the implanted silicone surface almost immediately after implantation.15 However, in biological systems, flow conditions, protein conformational changes, competitive adsorption, initiating steps in blood clotting, and complement activation strongly influence the formation of the protein film on artificial surfaces (“Vroman effect”).16 Because the proteins adsorbed to the surface of silicone implants have been characterized as key components in the onset and perpetuation of local immune reactions to silicone,17 it is important to determine the extent to which the hydrophobic silicone surface promotes protein degradation and unfolding18 and how the exposure of cryptic antigens and/or formation of “altered self” leads to systemic immune effects and local fibrotic processes. Deplorably, the composition of the proteinacious film on the surface of silicone implants is poorly understood, especially in vivo. This study extended our earlier work related to the immunogenic properties of medical silicone materials previously considered to be biologically inert. Proteins eluted from silicone implants ex vivo were analyzed and compared to the in vitro system developed in our laboratory. Standardized silicone surfaces were incubated with serum or wound bed fluid, and proteins eluted and analyzed at different time points. This approach allowed us to determine the time required for protein deposition and establishment of a steady state on the surface of silicone. Proteins eluted from both explanted and in vitro incubated SMIs were compared and identified. This specifically targeted proteomics screen yielded results that 10.1021/pr0603755 CCC: $37.00
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technical notes illuminated the protein composition on the surface of materials previously considered to be inert.
Methods Study Design. Twenty-three healthy women undergoing breast augmentation for cosmetic reasons were included in the study. All patients involved in the analysis gave informed consent to participate in the study approved by the Ethical Committee of the Innsbruck Medical University (study number AN2218). Thirteen patients were removing or replacing silicone implants due to fibrotic complications (ages 31-63 years, median 41, average implant duration 6 years), and 10 control patients were undergoing primary breast augmentation (ages 28-51 years, median 37 years). Patients undergoing implant replacement were divided in 2 groups according to the implant duration, i.e., less than 5 years (8 patients, ages 29-63 years, median 35) and more than 5 years (5 patients, ages 38-54 years, median 51). SMIs were collected from all patients, as well as sera that were aliquoted and frozen at -80 °C until use. Ex vivo Protein Isolation. If not stated otherwise, chemicals were purchased from Sigma-Aldrich (Vienna, Austria). Proteins were prepared in urea lysis buffer (7 M urea, 2 M thiourea (both from Calbiochem, Bad Soden, Germany), 30 mM TRIS, 4% CHAPS, pH 8.5) if they were going to be analyzed by 2D electrophoresis. For Western blot analysis, proteins were prepared in SDS buffer (2% SDS, 100 mM DTT, 50 mM TrisCl, 10% Glycerol, pH 9). Protease inhibitor cocktail composed of pepstatin, leupeptin, and aprotinin was added in final concentration of 1 µg/mL. Resolyte buffer and IPG strips were from Amersham Bioscienses (Uppsala, Sweeden). Thirteen explanted SMIs were obtained from the Clinic of Plastic and Reconstructive Surgery, Innsbruck Medical University. Nine SMIs were gel, and 4 were saline-filled. Eleven SMIs were rough and 2 were smooth surfaced. Only intact implants without silicone “gel bleeding” were considered for analysis and taken immediately after surgery. The fibrotic capsule was removed in the surgical theater, and SMIs were transported to the laboratory in PBS supplemented with protease inhibitors and on ice. Explanted SMIs were washed with PBS (3 × 15 min) and water (5 × 10 min) at 4 °C. After washing, proteins were eluted in a buffer appropriate for the downstream analysis, and protein concentration was measured by the bicinchoninic acid protein assay (Pierce Biotechnology, Rockford, IL,USA). In vitro Protein Adhesion to Silicone. Being the most commonly used silicone for medical devices, unrestricted medical silicone type MED1511 (Nusil, Waldbrun, Germany) was tested foremost. Fifty µL per well of unpolymerized silicone was added to 96 well flat bottom polystyrol plates (Cat No. 655180, Greiner: Frickenhausen, Germany), which were centrifuged (10 min at 300 g) to ensure even distribution. After silicone curing (3 days at 37 °C and 98% humidity), plates were sterilized for 120 s, under 200 mJoules of UV light (GsGene UV chamber, Biorad, Hercules, CA), and incubated with wound bed fluid (WBF) supplemented with protease inhibitors for 4 days. WBF is plasma-like liquid that extravasates during and after the surgery, surrounding the implant for a longer period of time after the implantation. It is easily obtained through postoperational catheters. Although the protein composition of WBF resembles that of the serum to over 90%, it is also supplemented with other proteins, such as heat shock proteins, structural cellular proteins, and degraded proteins from necrotic cells. Initial data were obtained by incubating silicone with human serum, but because the incubation with WBF
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reflects the in vivo situation more accurately, it was chosen further on as our method of choice. After the incubation with WBF, plates were washed (10 × 15 min) in PBS and (5 × 10 min) distilled H2O at 4 °C, and the proteins were eluted. Protein Analysis. Proteins eluted in vitro and ex vivo were primarily analyzed after separation by two-dimensional gel electrophoresis (2D electrophoresis).19 For the first dimension separation, proteins were dissolved in urea lysis buffer. Prior to loading, 200 µg of total protein was supplemented with rehydration buffer (7 M Urea, 2 M Thiourea, 4% CHAPS, 18 mM DTE) to a final volume of 350 µL. Resolyte buffer was added and loaded proteins on 18 cm IPG strip, pI 3-11. Proteins were focused for a total of 55 000 Vhrs (IPGphor, Amersham Biosciences). After isoelectric focusing, proteins on strips were reduced (2% DTT, 15 min) and subsequently alkylated (2.5% IAA, 15 min). Second dimension electrophoresis was performed under reducing conditions, in 20 × 24 cm gradient 9-16% polyacrylamide gels at 16 °C. After 2D electrophoresis, gels were fixed, and silver staining was performed using Farmer’s reducer procedure.20 Oxidized proteins were detected with the Oxiblot protein oxidation detection kit (Chemicon, Germany). In brief, carbonyl groups in the protein side chains were derivatized to 2,4-dinitrophenylhydrazones by reaction with 2,4-dinitrophenylhydrazine. After the derivatization, samples were run on 12% SDS-PAGE gels, blotted onto nitrocellulose membranes, and probed with antibodies against dinitrophenylhydrazone moiety of the proteins. Silver stained protein spot patterns were matched and compared to the ExPasy database of 2D gels (http://us.expasy. org/ch2d/) using the “Melanie” software (version 4, Swiss Institute of Bioinformatics, Geneva, Switzerland).21 Spots of interest were manually excised, proteins were digested with trypsin (Promega, Madison, WI, USA), and peptides were analyzed by Mass spectrometry (Ultraflex TM TOF/TOF, BrukerDaltonics, Germany). Gel digestion was performed as described by Shevchenko et al.22 Samples analyzed by peptide mass fingerprint (PMF) from MALDI-TOF were additionally analyzed using LIFT-TOF/ TOF MS/MS from the same target. A maximum of three precursor ions per sample were chosen for MS/MS analysis. PMF was measured with reflector mode, followed by MALDITOF/TOF measurement with LIFT mode. PMF spectra were interpreted with the “Mascot” and “Profound” software. A probability score higher than 64 for “Mascot” and 1.65 for “Profound” was used as a criterion for correct identification. Polyacrylamide gel electrophoresis (PAGE) was used to complement and confirm the data obtained from mass spectroscopy and database search. Gels were either silver stained or blotted to nitrocellulose membrane for Western blot detection. Primary antibodies against most of the proteins and appropriate conjugates were obtained from DAKO, polyclonal rabbit antibodies against Myeloid Related Protein (MRP) 8 and MRP14, the latter a kind gift from Dr. Claus Kerkhoff (Institute of Experimental Dermatology, Muenster, Germany), and affinity chromatography purified monoclonal antibodies against human HSP60 from clone II-1323 were produced in our laboratory. Cy-Dye Labeling. Cy-dye- based differential in gel electrophoresis (DIGE, Amersham Biosciences Upsala, Sweeden) was performed when differential expression of proteins was being investigated. Fifty µg of each sample were alternately labeled with 100 pmol of Cy3 and Cy5 to avoid dye- binding bias. Twenty-five µg of every sample was pooled and labeled with Cy2 control dye as an internal standard. Cy3- and Cy5-labeled Journal of Proteome Research • Vol. 6, No. 1, 2007 377
technical notes
New Proteins Identified Adhering to the Medical Silicones
Figure 1. Relative protein amount eluted from SMIs ex vivo. Protein amount eluted from 13 explanted SMIs and normalized per surface area. In the group of women carrying SMIs more than 5 years, significantly more protein is deposited on the surface of the implants, as determined by student’s test (* - p < 0.05).
samples were combined and mixed with Cy2-pooled internal standard, complemented with rehydration buffer up to 350 µL, and loaded on IPG nonlinear strips pI 3-11. Second dimension electrophoresis was performed as described above, and gels were immediately scanned with the fluorescent laser scanner “Typhoon 9210” (Amersham Biosciences) at a resolution of 100 µm. Statistics. Student’s T-test and one-way ANOVA statistical analyses were applied in most experiments. P-values were calculated with Excel (Microsoft). Distribution and statistical analysis of protein patterns was performed by integrated statistical routines in “Melanie” and “Decyder”.
Results Ex vivo Eluted Proteins. The amount and composition of proteins deposited in vivo onto the surface of explanted SMIs was analyzed. Because implants had surfaces between 162 and 363 cm2, all protein amounts were normalized per surface area. No difference in protein deposition was observed between gelor saline-filled SMIs, and no correlation in relative protein amount was found with regard to implant size or surface texture (smooth or rough) of the implant (data not shown). However, in implants over 5 years duration, the amount of protein deposited on the surface was significantly higher than those in place for shorter periods (Figure 1). Dynamics of In Vitro Protein Adsorption. To examine the dynamics and composition of proteins adhering to silicone in vitro, 96-well microtiter plates were coated with silicone type MED1511 and incubated with WBF for various time periods. The amount of protein deposited per surface unit of silicone at different time points was analyzed with Bradford protein assay (Figure 2). After approximately 50 h in the dark at 37 °C and 95% humidity, protein amounts deposited on the silicone surfaces remained constant, averaging 7.8 µg per cm2. Despite sterile conditions and the addition of protease inhibitors, spontaneous protein degradation was usually noticed after approximately 130 h of incubation: sera and WBF turned turbid, a gelatinous film of protein fragments formed on the bottom of the wells, and the amount of proteins eluted 378
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increased up to 1 mg per cm2. Because of the metabolic equilibrium and immune system surveillance present in vivo, these late events may not reflect the in vivo situation. Therefore, we analyzed proteins deposited from 50 h to the occurrence of spontaneous protein degradation (∼130 h). Comparison of Protein Deposited on Silicone Surfaces In Vivo and In Vitro. To determine whether our in vitro model corresponded to the situation ex vivo, we compared the two systems by 1D and 2D gel electrophoresis by the DIGE method. To avoid color bias, two fluorescent dyes (Cy3 and Cy5) were used for alternating sample labeling, and the third dye (Cy2) was used as an internal standard. Protein spot patterns were analyzed with the “Decyder” software. The amount of proteins deposited was considered different if the spot volume ratio between samples exceeded two standard deviations of total spot volume distribution, and present only in one system if the volume ratio exceeded 5 times. We detected a total of 184 protein spots. Of those, 163 spots were present in the same amounts in both systems, 13 proteins bound more strongly in the ex vivo, and 5 in the in vitro system, and only a small number of spots were unique to either system (Figure 3). The spots present exclusively in the in vivo system were identified by MS and proven to be mainly intracellular proteins, like NADH hydrogenase subunit IVb and Uracil DNA glycosylase (data not shown). Proteins Deposited on the Surface of Silicone In Vivo and In Vitro. Having defined the difference between proteins adhering to silicone in vivo and in vitro, we focused our attention on identifying the proteins emerging in both systems either by MS analysis of tryptic digest fragments or by comparison to the ExPasy database and confirmed these results by Western blots. Because WBF composition is more than 90% identical to the composition of serum, the database comparison with serum proteins proved to be an efficient and cost- effective method for initial screens of protein content. For MS analysis, the 30 most abundant spots were selected, as detected by silver staining. After the identity of these proteins was confirmed in at least two of three detection systems, they were assigned to 1 of 3 main functional groups: extracellular matrix proteins, mediators of host defense, and extracellular transport related proteins (Table 1). Data on protein identification are given in supplement Figures 1 and 2. It is important to emphasize that we observed protein fragmentation of HSP 60, fibronectin, and vitronectin upon their deposition in vivo and in vitro manifested in Western blots as protein smears or additional bands (Supplement Figure 1, Supporting Information).
Discussion The problem of protein adherence to silicone is important from a theoretical and biotechnological viewpoint and is of great practical medical relevance. Our current analyses revealed previously undetected proteins deposited on the surface of silicone. As tentative initiators and/or modulators of the response to silicone, they are therefore valuable candidates for disease prevention, therapy, and a parameter to the assessment of biocompatibility. Our studies on ex vivo eluted proteins showed up to 80 µg/ cm2 from various explanted SMIs. Women carrying implants for over 5 years had substantially more proteins deposited on the surface of their implants than those wearing implants less than 5 years. The proteins found on long-term implants proved
technical notes
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Figure 2. Dynamics of protein adhesion to silicone in vitro. (A) Protein amount eluted from silicone-coated microtiter plate and expressed per square centimeter. Ninety-six-well flat-bottom microtiter plates were coated with silicone and inclubated with WBF in triplicates. Proteins were eluted with 1% SDS at the indicated time points, and protein concentration in eluates was measured and expressed as mean value ( standard deviation. The remaining eluates were loaded onto 12% SDS-PAGE gels and silver stained after the run. In the upper part of the graph, a selection of silver-stained bands with eluted proteins resolved is given. The evaluation of the protein degradation was performed densitometrically upon scanning on a Typhoon (Amersham Biosciences, Upsala, Sweden) confocal laser scanner. (B) Proteins eluted in the same experiment were blotted onto a nitrocellulose membrane, and oxidized proteins were detected using an antibody against derivatized oxidized groups. The white flairs at later time points are caused by local exhaustion of the substrate for the secondary antibody used for visualization.
to be primarily intracellular proteins, reflecting strong attachment of capsular cells to the SMI surface. We also constructed an in vitro system of protein deposition that successfully models in vivo protein deposition on silicone implants in both composition and amount. After reaching the plateau of protein deposition, an average of 7.8 µg of proteins per cm2of implanted surface was eluted in vitro, an amount equivalent to the average eluted from SMIs of less than 5 years duration (8.5 µg/cm2). Comparing protein profiles from both systems with DIGE, 184 protein spots were detected, with only minor differences between the two systems. It should, however, be noted that the 184 spots included different post-translational modifications of the same proteins, and that the actual number of proteins might be considerably smaller. The 2D-DIGE system is an excellent analytical tool, but has several drawbacks such as impaired resolution in the low molecular weight region resulting in poor accuracy of spot picking and, due to high
background, frequent failures in MS analysis of low abundant protein spots. Consequently, we supplemented those data with MS analysis of proteins excised from silver stained gels. The major advantage of the in vitro system is that it allows for standardized and reproducible analysis of the proteinacious film without interference from cells and fibrotic lesions formed in the later stages of fibrosis in vivo. It allowed us to follow the dynamics of protein adhesion, and to make a detailed inventory of proteins deposited. These findings are concordant with similar work performed by other groups.24 Although a protein film forms on the silicone surface immediately after implantation, it requires a longer time to reach a steady state. We confirmed and extended previous research on this topic and found that the minimal equilibration time for medical silicones is in the range of 50 h. Knowing this time frame is important to investigations of proinflammatory properties of the adhering Journal of Proteome Research • Vol. 6, No. 1, 2007 379
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New Proteins Identified Adhering to the Medical Silicones
Table 1. Proteins Detected Deposited on the Surface of Silicone In Vivo and In Vitroa MW [D]
protein name
pI
identified byb
acession numberc
Mediators of host defense
Figure 3. Spot distribution in differential analysis (DIGE) of proteins eluted in vivo and in vitro. To compare protein deposition in vivo and in vitro, differential analysis of proteins eluted from both systems and labeled by fluorescent dyes was performed. Dyes are excited at different wave lengths, and pictures obtained from different dyes are merged into one image and analyzed by “Decyder” statistical software. Statistical analysis from three independent experiments showed 89% identity (without two standard deviations) in protein deposition in vivo in comparison to in vitro conditions. A cutoff for unique expression was set at a difference higher than 5 fold. With these cutoffs, 11 spots were present exclusively in vivo and 3 exclusively in vitro.
proteins and to deriving therapies effective in the early, presymptomatic stages of fibrosis development. We have previously shown the abundant presence of HSP60 in the fibrotic capsule around the implant.12 This is not surprising, because expression of HSP60 has been demonstrated at the site of various forms of stress, including mechanical stress, in several diseases such as rheumatoid arthritis and atherosclerosis.25,26 The HSP60 detected on the surface of silicone most likely reflects the response of tissue surrounding SMIs to mechanical stress under physiological conditions. Whether there are pathophysiolgical consequences of this phenomenon has yet to be determined. Because of their structural properties, ECM proteins are particularly susceptible to modification upon adsorption to silicone. They are abundant in hydrophobic domains that are easily exposed upon adhesion and begin to act like “crystallization foci” for other extra-cellular matrix proteins.27 These characteristics are tightly connected to the innate and adaptive immune responses, either directly (as shown by activation of macrophages and fibroblasts), or through surface-mediated protein modifications that are then recognized as foreign by the immune system. Thus far, exposure of cryptic domains upon adhesion has been shown only for fibronectin and laminin,28 whereas the reactions of other ECM proteins remain unclear. The presence of matrix metalloproteinase 2 (MMP2) is indicative of prominent remodeling processes in the protein layer, a hypothesis supported by protein fragmentation of several ECM proteins detected in Western blots. A second group of proteins found on the surface of silicone proved to be directly involved in the immune response. Proteins involved in both innate (complement components, 380
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immunoglobulin A light chain heavy chain
28 826 5.38 WB, DB 54 474 6.86
P99003
immunoglobulin G light chain heavy chain
28 826 5.38 WB, DB 54 474 6.86
P99007 P99006
complement C2 precursor
83 267 7.23 MS
gi|67611
complement C1s light chain heavy chain
27 651 7.00 WB 47 253 4.56
n/a
C reactive protein 23 028 5.28 WB myeloid related protein 8 10 835 6.51 WB myeloid related protein 14 13 242 5.71 WB alpha1-microglobulin/ 33 544 4.86 DB bikunin precursor HT018 27 866 8.88 MS integrin b4 202 392 5.82 MS bullous pemphigoid antigen 245 470 6.2 MS Structural/Extracellular matrix proteins actin 41 321 5.29 WB, MS fibronectin 259 544 5.38 WB vitronectin 75 000 5.50 WB fibrinogen 94 973 5.70 WB collagen I 94 738 9.29 MS, WB collagen IV 121 155 6.88 WB collagen VII 293 608 5.92 MS, WB procollagen III 170 455 5.60 WB laminin 62 928 5.22 WB matrix metalloproteinase 2 62 400 5.02 WB Transport proteins apolipoprotein A1 23 601 5.48 MS, DB transthyretin albumin alpha2-macroglobulin precursor haptoglobin 1 hemoglobin R chain β chain HSP 60 PR02619 fibroblast growth factor 11
35 463 5.52 DB 66 472 5.67 MS, WB 190 040 5.33 DB
n/a n/a n/a P02760 gi|9963853 gi|21361207 gi|179519 gi|15277503 n/a n/a n/a n/a n/a gi|627406 n/a gi|27804346 n/a P02647 gi|178777 P02766 gi|28592 P01023
17 109 5.68 DB
P00738
15 126 15 867 Other 57 356 69 366 25 009
8.73 MS 6.81
gi|13195586 gi|55635219
5.7 WB 6.81 MS 9.92 MS
n/a gi|11493459 gi|20160215
a Subheadings are the different Ontologies. Proteins eluted both under in vivo and in vitro conditions were identified either by Western blots (WB), SwissProt 2D database matching analysis (DB), or Mass spectrometry (MS). b Details on protein identification are given in the Supporting Information. c Accession number is given only if the proteins were identified by DB and/ or MS. In the case where proteins were detected only with Western blots, accession number is not applicable (n/a).
C-reactive protein) and adaptive immune responses (immunoglobulins, R1-microglobulin) were detected. Although macrophages and other professional antigen presenting cells can be stimulated through adhered immunoglobulins and complement components, the acute foreign body reaction is avoided because the process of protein modification is slow and allows other metabolic and immune regulatory mechanisms to act. Over the course of years, the balance between pro- and antiinflammatory stimuli shifts toward inflammation and fibrosis, the time point at which the first local clinical symptoms occur. A regulatory network that receives opposing signals from several directions leads to a break in tolerance and subsequent systemic effects.29 Although we did not find any profibrinogenic cytokines (such as TGF beta, IL4 or IL 14) deposited on the surface of silicone, they are certainly produced by various
technical notes mononuclear cells upon reaction with proteinacious film on the surface of silicone. The causative relation between autoimmune disease development and silicone implants is still a matter of debate. This paper cannot yet resolve this issue, but it shows that silicone promotes at least the adhesion of altered self-proteins, which in turn may trigger an autoimmune response of the immune system. Abbreviations: SMI, silicone mammary implants; DIGE, differential in-gel electrophoresis; PBS, phosphate buffered saline; 2D, two-dimensional; PMF, peptide mass fingerprint; PAGE, polyacrylamide gel electrophoresis; HSP, heat skock protein; WBF, wound bed fluid.
Acknowledgment. This project has been supported by grants from Kompetenzzentrum Medizin Tirol (KMT), and the Lore and Udo Saldow Foundation. Work in the Huber Laboratory is supported by the Austrian Proteomics Platform (APP) within the Austrian Genome Program (GEN-AU), Vienna, Austria. We thank Prof. Dr. Guenther Bonn for access to the mass-spectrometry equipment, Dr. Claus Kerkhoff for the antibodies to MRP8 andMRP14 provided, M. K. OcchipintiBender for editorial assistance, and Christina Mayerl for technical and logistic support. Special thanks go to Dr. Blair Henderson for creative advice on experimental designs and critical reading of the manuscript. Supporting Information Available: Supplementary Figures 1 and 2. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Siggelkow, W.; Faridi, A.; Spiritus, K.; Klinge, U.; Rath, W.; Klosterhalfen, B. Histological analysis of silicone breast implant capsules and correlation with capsular contracture. Biomaterials 2003, 24 (6), 1101-1109. (2) Vermeulen, R. C.; Scholte, H. R. Rupture of silicone gel breast implants and symptoms of pain and fatigue. J. Rheumatol. 2003, 30 (10), 2263-2267. (3) Shanklin, D. R.; Stevens, M. V.; Hall, M. F.; Smalley, D. L. Environmental immunogens and T-cell-mediated responses in fibromyalgia: evidence for immune dysregulation and determinants of granuloma formation. Exp. Mol. Pathol. 2000, 69 (2), 102-118. (4) Goldblum, R. M.; Pelley, R. P.; O’Donell, A. A.; Pyron, D.; Heggers, J. P. Antibodies to silicone elastomers and reactions to ventriculoperitoneal shunts. Lancet 1992, 340 (8818), 510-513. (5) Bridges, A. J.; Conley, C.; Wang, G.; Burns, D. E.; Vasey, F. B. A clinical and immunologic evaluation of women with silicone breast implants and symptoms of rheumatic disease. Ann. Intern. Med. 1993, 118 (12), 929-936. (6) Varga, J.; Schumacher, H. R.; Jimenez, S. A. Systemic sclerosis after augmentation mammoplasty with silicone implants. Cancer Res. 1989, 111 (5), 377-383. (7) Zazgornik, J.; Piza, H.; Kaiser, W.; Bettelheim, P.; Steiner, G.; Smolen, J.; Biesenbach, G.; Maschek, W. Autoimmune reactions in patients with silicone breast implants. Wiener Klinische Wochenschrift 1996, 108 (24), 781-787. (8) Janowsky, E. C.; Kupper, L. L.; Hulka, B. S. Meta-analyses of the relation between silicone breast implants and the risk of connective-tissue diseases. N. Engl. J. Med. 2000, 342 (11), 781-790. (9) Sanchez-Guerrero, J.; Colditz, G. A.; Karlson, E. W.; Hunter, D. J.; Speizer, F. E.; Liang, M. H. Silicone Breast Implants and the Risk of Connective-Tissue Diseases and Symptoms. N. Engl. J. Med. 1995, 332 (25), 1666-1670.
Backovic et al. (10) Wick, G.; Wagner, R.; Klima, G.; Wilflingseder, P. Immunohistochemical Analysis of the Connective Tissue Capsule Formation and Constriction Arround Mammary Silicone Prostheses. In Cellular, Molecular and Genetic Approaches to Immunodiagnosis and Immunotherapy; Kano, K., Mori, S., Sugisaki, T., Torisu, M., Eds.; University of Tokyo Press: Tokyo, 1987; pp 231-241. (11) Gabbiani, G.; Hirschel, B. J.; Ryan, G. B.; Statkov, P. R.; Majno, G. Granulation tissue as a contractile organ. A study of structure and function. J. Exp. Med. 1972, 135 (4), 719-734. (12) Wolfram, D.; Rainer, C.; Niederegger, H.; Piza, H.; Wick, G. Cellular and molecular composition of fibrous capsules formed around silicone breast implants with special focus on local immune reactions. J. Autoimmunity 2004, 23 (1), 81-91. (13) Tenenbaum, S. A.; Rice, J. C.; Espinoza, L. R.; Cuellar, M. L.; Plymale, D. R.; Sander, D. M.; Williamson, L. L.; Haislip, A. M.; Gluck, O. S.; Tesser, J. R. Use of antipolymer antibody assay in recipients of silicone breast implants. Lancet 1997, 349 (9050), 449-454. (14) Wolf, L. E.; Lappe, M.; Peterson, R. D.; Ezrailson, E. G. Human immune response to polydimethylsiloxane (silicone): screening studies in a breast implant population. FASEB J. 1993, 7 (13), 1265-1268. (15) Elwing, H. Protein absorption and ellipsometry in biomaterial research. Biomaterials 1998, 19 (4-5), 397-406. (16) Vroman, L.; Adams, A. L.; Fischer, G. C.; Munoz, P. C. Interaction of High Molecular-Weight Kininogen, Factor-Xii, and Fibrinogen in Plasma at Interfaces. Blood 1980, 55 (1), 156-159. (17) Tang, L.; Eaton, J. W. Natural responses to unnatural materials: A molecular mechanism for foreign body reactions. Mol. Med. 1999, 5 (6), 351-358. (18) Bao, G.; Suresh, S. Cell and molecular mechanics of biological materials. Nat. Mater. 2003, 2 (11), 715-725. (19) Anderson, L.; Anderson, N. G. High resolution two-dimensional electrophoresis of human plasma proteins. Proc. Natl. Acad. Sci. U.S.A. 1977, 74 (12), 5421-5425. (20) Wray, W.; Boulikas, T.; Wray, V. P.; Hancock, R. Silver staining of proteins in polyacrylamide gels. Anal. Biochem. 1981, 118 (1), 197-203. (21) Hughes, G. J.; Frutiger, S.; Paquet, N.; Ravier, F.; Pasquali, C.; Sanchez, J. C.; James, R.; Tissot, J. D.; Bjellqvist, B.; Hochstrasser, D. F. Plasma protein map: an update by microsequencing. Electrophoresis 1992, 13 (9-10), 707-714. (22) Shevchenko, A.; Wilm, M.; Vorm, O.; Mann, M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal. Chem. 1996, 68 (5), 850-858. (23) Xu, Q.; Schett, G.; Seitz, C. S.; Hu, Y.; Gupta, R. S.; Wick, G. Surface staining and cytotoxic activity of heat-shock protein 60 antibody in stressed aortic endothelial cells. Circ. Res. 1994, 75 (6), 10781085. (24) Kim, J. K.; Scott, E. A.; Elbert, D. L. Proteomic analysis of protein adsorption: Serum amyloid P adsorbs to materials and promotes leukocyte adhesion. J. Biomed. Mater. Res. Part A 2005, 75A (1), 199-209. (25) Wick, G.; Knoflach, M.; Xu, Q. B. Autoimmune and inflammatory mechanisms in atherosclerosis. Annu. Rev. Immunol. 2004, 22 361-403. (26) Van Eden, W. Heat Shock Proteins and Inflamation; Birkha¨user Verlag: Basel, 2003. (27) Zhong, C.; Chrzanowska-Wodnicka, M.; Brown, J.; Shaub, A.; Belkin, A. M.; Burridge, K. Rho-mediated contractility exposes a cryptic site in fibronectin and induces fibronectin matrix assembly. J. Cell Biol. 1998, 141 (2), 539-551. (28) Faisal Khan, K. M.; Laurie, G. W.; McCaffrey, T. A.; Falcone, D. J. Exposure of cryptic domains in the alpha 1-chain of laminin-1 by elastase stimulates macrophages urokinase and matrix metalloproteinase-9 expression. J. Biol. Chem. 2002, 277 (16), 1377813786. (29) Langer, R.; Tirrell, D. A. Designing materials for biology and medicine Nature 2004, 428 (6982), 487-492.
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