|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
PHAGOCYTES
From the Department of Pediatrics, Baylor College of
Medicine, Houston, TX.
Despite being inert and nontoxic, implanted biomaterials often
trigger adverse foreign body reactions such as inflammation, fibrosis,
infection, and thrombosis. With regard to the inflammatory responses to
biomaterial implants, it was previously found that a crucial
precedent event was the spontaneous adsorption and denaturation of
fibrinogen on implant surfaces. It was further found that interactions between the phagocyte integrin Mac-1 (CD11b/CD18) and one short sequence within the fibrinogen D domain ( In general, commonly used biomaterials are
physically and chemically stable, nonimmunogenic, and nontoxic. Despite
this, implanted and blood-contact biomaterials trigger a wide variety
of unwanted responses, including inflammation,1-5
thrombosis,6,7 infection,8,9 and
fibrosis.10-13 In many cases, these adverse responses are
associated with the rapid accumulation of large numbers of phagocytic
cells.2,3,14,15 Given the inert and nontoxic nature of
these materials, it is not clear how the host detects and then responds
to the presence of biomaterial implants.
Implanted materials very quickly acquire a layer of host proteins, well
before the arrival of inflammatory cells. Therefore, it is generally
believed that phagocytes interact with the spontaneously adsorbed
proteins rather than with the material itself. Consequently, we and
others have hypothesized that the species and state of adsorbed
proteins probably influence or absolutely dictate subsequent phagocyte
responses.14-18 To test this hypothesis, we had earlier implanted protein-precoated polyethylene terephthalate (PET) disks intraperitoneally in Swiss Webster mice and quantified the numbers of
adherent phagocytes to estimate the influence of surface protein type
on acute inflammatory responses.14,15 Our results
indicated that adsorbed fibrinogen is primarily responsible for the
accumulation of phagocytes on implant surfaces. In support of this,
implants precoated with serum or with hypofibrinogenemic plasma fail to exhibit phagocyte accumulation, although the addition of physiological concentrations of fibrinogen to these coatings fully restores their
proinflammatory properties. Perhaps the most direct supporting evidence
is that profoundly hypofibrinogenemic mice do not mount inflammatory
responses to implanted PET unless the material is precoated with
purified fibrinogen or the mouse peritoneal fluid is made to contain
fibrinogen before implantation.15
To determine which domain(s) of the very large (approximately
340 000 d) fibrinogen molecule might be important in triggering this
inflammatory response, we precoated implants with purified fragments of
fibrinogen and determined that the proinflammatory sequences reside
within the fibrinogen D30 domain.19 A peptide sequence, P1
( Despite this limited progress, the reasons that adsorbed, but not
solution-phase, fibrinogen is proinflammatory remained unknown. Therefore, we have investigated the events involved in the
surface-mediated conversion of fibrinogen to a proinflammatory state
and have attempted to determine whether the interactions of fibrinogen
with different biomaterials might explain why they trigger inflammatory
responses of varying severity. Overall, the results indicate that
fibrinogen adsorption to biomaterial surfaces exposes 2 normally occult
epitopes, P1 ( Materials
This study used 5 commonly used biomaterials.1
Polyethylene terephthalate ("Mylar"; PET) film (type A, 0.005-mm
thick) was obtained from Cadillac Plastic and Chemical (Birmingham,
MI).2 Low-density polyethylene (LDPE), a
well-characterized NHLBL reference material, was purchased from
Abiomed R&D (Danvers, MA).3 Films of polydimethyl siloxane
(PDMS) were obtained from Dow Corning (Midland, MI).4
Polyether urethane (PEU) 55D films were generously donated by Dr James
R. Keogh of Medtronic (Minneapolis, MN).5 Polyvinyl
chloride (PVC) films were kindly provided by Dr Yeong Hua Huang of
Sherwood-Davis & Geck (St Louis, MO).
Purification of fibrinogen and generation of proteolytic
fragments
Peptide synthesis and characterization Fibrinogen P1 ( 190-202), P2 (377-395), and scrambled
peptides were made by solid-phase synthesis by means of Fmoc
(9-fluorenylmethoxycarboxyl) chemistry in a peptide synthesizer
(model 431A) (Applied Biosystems, Foster City, CA). After synthesis,
the peptides were deprotected and cleaved from the resin with 95%
trifluoroacetic acid. The structure of the peptides was verified by
amino acid analysis and by molecular mass analysis with electrospray
mass spectrometry. Lyophilized crude peptides were purified by
preparative reverse-phase high-pressure liquid chromatography
on a C-18 column. Variant peptides of both P1 and P2 peptides were
synthesized with the addition of the residues K-Y (single-letter amino
acid codes) at the NH2 terminus to provide a site
for cross-linking to albumin. The amino acid sequences of P1 and P2 are
G(190)WTVFQKRLDGSV(202) and
Y(377)SMKKTTMKIIPFNRLTIG(395),19,23,24 respectively
(single-letter amino acid codes). Two scrambled control peptides were
produced on the basis of the constituent amino acids of P1 and P2
having the sequences FRLGWVQTSVDKG and RFSRLKGWTSVGDKITMSG, respectively.
Conjugation of the peptides to albumin The synthetic peptides, P1, P2, and the scrambled variants were chemically conjugated to human albumin by means of EDC. Earlier investigations indicate that the coupling of synthetic peptides to larger carrier proteins improves their interactions with cells.25-27 The conjugation of the peptides to albumin was done by means of a modified 2-step coupling procedure described earlier.19,28,29 Briefly, equal amounts (wt/wt) of the peptides and human albumin were solubilized in activation buffer (0.1 M MES, 0.5 M NaCl, pH 6.0). The reaction was started by addition of EDC (final concentration, 2 mM) and NHS (final concentration, 5 mM). After reaction at room temperature for 3 hours, the peptide-albumin complexes were dialyzed extensively against hypertonic phosphate-buffered saline (PBS) (100 mM sodium phosphate plus 0.8% NaCl, pH adjusted to 7.3) to remove excess linker and uncoupled peptides.Preparation of biomaterial disks Disks of 1.2 cm diameter were cut from the test materials PET, PVC, LDPE, PEU, and PDMS. The disks were stirred for longer than 96 hours in multiple changes of 70% ethanol to remove dust and sterilize the surface and were then stored in 100% ethanol. Prior to use, the disks were hydrated by immersion in sterile, pyrogen-free saline for at least 1 hour. To generate stable protein coatings, test surfaces were incubated with human albumin (1000 µg/mL), fibrinogen (200 µg/mL), or peptide-albumin (200 µg/mL) at room temperature on a rotary shaker (25 rpm) overnight (approximately 16 hours) under sterile conditions. Any unoccupied surface on the coated disks was blocked by subsequent incubation with unmodified human albumin (100 µg/mL) for 3 hours. Our recent results have demonstrated that, in such conditions, most surface-bound proteins (more than 60%) are irreversibly adsorbed (or denatured) onto the surfaces15,19,30 and will therefore remain on material surfaces for the duration of the experiments. These disks were directly used for in vitro measurements of exposed fibrinogen epitopes by enzyme-linked immunosorbent assays (ELISAs) and in vivo inflammatory responses in animal implantation experiments. Control studies employing 125I-labeled albumin indicated that the amounts of adsorbed protein were about 300 to 500 ng/cm2, roughly equivalent to a continuous monolayer.Preparation of fibrin-coated glass beads Fibrin-coated glass beads were produced by incubating 6 glass beads with 200 µL fibrinogen (4000 µg/mL in isotonic PBS; 50 mM sodium phosphate plus 100 mM NaCl, pH 7.4) in individual wells of a 48-well plate. Following the addition of fibrinogen, 0.5 µL (1 U/µL) thrombin was added to each well, and the glass beads were rolled in the wells to generate a homogeneous fibrin coating. Surface fibrin was then allowed to fully polymerize at 37°C for 5 minutes. After removal of the nonadherent fibrin, the glass beads were transferred to new wells for ELISA measurements of exposed fibrin epitopes as described below.Immunization and preparation of monoclonal antibody against P1 peptide An immunoglobulin-M monoclonal antibody (mAb) against P1 (mAb D8D8) was prepared according to procedures described by Kohler and Milstein.31 Briefly, female Balb/c mice were injected intraperitoneally with 200 µg bovine serum albumin (BSA)-conjugated synthetic P1 peptide (BSA-P1) in Freund complete adjuvant and twice more at 2-week intervals with 200 µg BSA-P1 in Freund incomplete adjuvant. An intraperitoneal injection of 200 µg BSA-P1 in 0.15 M NaCl was given 3 days before fusion. Spleen cells were harvested from immunized mice and fused with myeloma cells in the presence of 40% (wt/vol) polyethylene glycol 4000. Using an ELISA described below, we isolated a hybridoma cell line (D8D8) that produced monoclonal antibody against the P1 sequence.Measurement of the amounts of surface-exposed P1 and P2 peptides An ELISA procedure was developed to measure the extent of P1 and P2 exposure on various surfaces and fibrin(ogen) preparations. Test surfaces (disks or beads) precoated with different proteins, peptide-albumin conjugates, or fibrin preparations were incubated with the monoclonal antibodies (1:1000 dilution) in blocking buffer (isotonic PBS containing 0.5% BSA plus 0.05% Tween-20) at 37°C for 2 hours. After rinsing 3 times with washing solution (isotonic PBS containing 0.05% Tween-20), test surfaces were immersed in solutions of secondary antibody conjugated with horseradish peroxidase at 37°C for 2 hours. Unbound secondary antibodies were removed by washing 3 times in PBS, and test samples were transferred to new plates before color development. Color was developed by means of 1-Step Turbo TMB-ELISA (Pierce) and read at 450 nm with a Spectra Max 340 plate reader (Molecular Devices, Sunnyvale, CA).Implantation of biomaterial disks As an in vivo model for assessing the extent of inflammatory responses to various materials, sterile biomaterial disks were implanted intraperitoneally in male Swiss Webster mice (20 to 25 g body weight) (Taconic Farms, Germantown, NY) as described earlier.14,15 To reduce possible experimental variations due to age, different shipment batches, or even the season of the year, only mice from the same shipment were used in individual experiments, and control groups were included in every experiment for comparison. The values shown in all graphs represent the results of single implantation experiments using 5 animals per treatment. It should be noted that all experiments shown were performed at least 3 times with very similar results.Explantation of biomaterial disks Explantation was performed at 16 hours after implantation (earlier found to be the time of maximal phagocyte accumulation). After careful removal from the peritoneal cavity, explanted disks were gently washed with isotonic PBS. Adherent cells were then lysed by incubating the explanted disks with 0.6 mL of 1% (vol/vol) Triton X-100 for 1 hour (to release cytosolic and granular contents of adherent cells). The numbers of adherent phagocytes were then estimated by enzyme activities (myeloperoxidase for neutrophils [polymorphonuclear neutrophils, or PMNs] and nonspecific esterase for macrophages/monocytes [M s]).
Measurement of enzyme activities Myeloperoxidase (MPO), largely from PMNs, was measured by a guaiacol reaction.32 Our preliminary experiments showed that more than 95% of explant-associated peroxidase activity was from MPO as opposed to eosinophil peroxidase,15 so we did not perform separate assays for the latter. Control studies on purified PMNs from mice indicated that the MPO activity of mouse peripheral PMN is about 23 nU/cell.15Nonspecific esterase (NSE) is relatively restricted to
M Our earlier control experiments indicated that measured enzyme activity is a reliable indicator of numbers of surface-adherent phagocytes.14,15 Determinations of endotoxin contamination Subsamples of variously treated disks were assayed for endotoxin contamination by the chromogenic limulus amebocyte lysate test (BioWhittaker, Walkersville, MD). In no case was the reading above minimal detectable concentration (ie, no samples contained more than 0.01 ng endotoxin per square centimeter of material surface).Statistical analyses The significance of difference among surfaces coated with different proteins was assessed by means of a one-factor analysis of variance. A Bonferroni multiple comparison test was then used to compare pairwise means where the omnibus test was significant. Linear regression was used to analyze the correlations between biomaterial-mediated inflammatory responses and fibrinogen P1/P2 exposure.
The proinflammatory activity of adsorbed fibrinogen resides in the D domain Fibrinogen degradation products were purified, coated on PET disks, and then implanted in Swiss Webster mice for 16 hours. Quantification of the numbers of adherent phagocytes revealed, as previously reported,19 that the proinflammatory sequences reside within the fibrinogen D domain; PET disks coated with fibrinogen D30 and D100 but not fibrinogen E50fragments prompt the same degree
of inflammatory responses as disks coated with purified fibrinogen
(Table 1).
Both P1 and P2 motifs are responsible for triggering inflammatory responses to implanted biomaterials As previously mentioned, the phagocyte Mac-1 integrin mediates phagocyte adherence to fibrinogen-bearing material implants, and the proinflammatory activity of adsorbed fibrinogen resides in the D30 domain. Thus, the motifs that reside within D30 and interact with Mac-1 are likely to cause phagocyte accumulation on biomaterial implants. On the basis of available results, only P1 (190-202) and P2
(377-395) fulfill both criteria.23,24 Using an animal
implantation model, we tested the potency of both P1 and P2
sequences in triggering inflammatory responses to material implants in
vivo. The results (Figure 1) indicate
that these peptides are roughly equipotent with adsorbed fibrinogen in
triggering inflammatory responses. In contrast, PET disks coated with
the scrambled peptide-albumin conjugates had no such effect (Figure 1).
To evaluate whether the exposure of these P1 and P2 sequences might be
sufficient to explain inflammatory cell accumulation on implants,
fibrinogen-coated PET disks were incubated with the Fab portions of
antibodies against P1 or P2 and then implanted. The results indicate
that Fab blockade of either P1 (Figure 2) or P2 (not shown) significantly reduces the numbers of adherent phagocytes after implantation. It should be noted that both P1 and P2
sequencesdespite their distance in the linear amino acid sequenceare close together in the folded protein.24
Thus, Fab blockade of either epitope might hinder integrin access to
the other and help explain why either antibody almost totally prevents phagocyte accumulation. Overall, these results indicate that both P1
and P2 sequences participate in the accumulation of phagocytes on
biomaterial implants.
Fibrinogen-biomaterial interactions enhance the exposure of both P1 and P2 motifs Because soluble fibrinogen is not proinflammatory, it seemed likely that fibrinogen-biomaterial interactions might cause the unmasking of P1 and P2 motifs. Using monoclonal antibodies specifically against either P1 or P2 epitopes, we therefore measured the exposure of P1 and P2 on both surface-adsorbed and soluble fibrinogen. Indeed, using an ELISA assay, we find that fibrinogen-coated and plasma-coated PET surfaces display both epitopes (Table 2). Interestingly, the addition of large amounts (1000-fold excess; 1 mg soluble fibrinogen vs 1 µg adsorbed fibrinogen) of soluble fibrinogen to these assays did not affect the detection of either epitope (Table 2), suggesting that soluble fibrinogen does not expose either of these epitopes. On the other hand, the specificity of both antibodies is ensured by the fact that the addition of an excess of free peptides blocks the detection of P1 and P2 exposed by surface-bound fibrinogen (Table 2). Thus, adsorption of fibrinogen, whether in pure form or from plasma, to the surfaces of at least some kinds of biomaterials causes the simultaneous appearance of both P1 and P2 epitopes.
Material surface properties affect the extent of epitope exposure by adsorbed fibrinogen Material surface properties are known to influence fibrinogen adsorption and denaturation.18,20,36,37 The extent of conformational changes that occur in adsorbed fibrinogen might govern the degree of exposure of hidden epitopes such as P1 and P2. We therefore thought it likely that materials with different surface properties would vary in the extent to which they cause such neo-epitope exposure. Indeed, in a limited series of fibrinogen-coated biomaterials, we find that PET, PVC, and polyethylene (PE) are very effective in exposing both P1 and P2 epitopes. On the other hand, fibrinogen adsorbed to PEU and PDMS disks exhibits substantially less immunoreactive P1 or P2 (Figure 3).
The degree of P1 and P2 exposure correlates with the extent of biomaterial-mediated acute inflammatory responses As shown above, both P1- and P2-coated surfaces prompt strong inflammatory responses. Therefore, those surfaces that induce the greatest exposure of these epitopes might prompt maximal inflammatory cell accumulation in vivo. In support of this, we find large differences in the extent of inflammatory cell accumulation on implants composed of these different test surfaces in the following order: (1) PET, (2) PVC, (3) PE (4) PDMS and PEU (Figure 4). The possible importance of surface adsorption-mediated P1 and P2 exposure in this reaction is indicated by the significant correlation (P < .05) between the extent of P1/P2 exposure and the total numbers of adherent PMNs and Ms (Figure 5A-B). Overall, the
foregoing results support the proposition that biomaterial-mediated P1
and P2 exposure on adsorbed fibrinogen is required for the accumulation
of inflammatory cells on biomaterial implants.
Foreign body reactions may represent a variation on the theme of fibrin clot-mediated inflammatory and wound-healing responses The foregoing results suggested to us that phagocyte responses to fibrinogen adsorbed to biomaterial surfaces might be analogous to those normally triggered by the formation of fibrin clots. In other words, phagocytes might recognize fibrinogen adsorbed to biomaterials as fibrin clot and initiate a series of responses meant to ward off infection and initiate wound healing at a site of vascular injury. If fibrinogen adsorbed to biomaterial surfaces resembles fibrin, then fibrin should also display both P1 and P2 motifs. We therefore analyzed the appearance of the P1 and P2 neo-epitopes on fibrin coated on glass beads. Indeed, the results indicate that, following the conversion of fibrinogen to fibrin, substantial amounts of both P1 and P2 epitopes appear (Table 2).
Despite the increasing importance of implanted medical devices in the practice of medicine, the synthetic materials of which they are made often induce iatrogenic effects, such as acute and chronic inflammatory responses. Typically, biomaterial surfaces attract substantial numbers of adherent phagocytes. These implant-associated activated phagocytes are thought to be responsible for a variety of biomaterial-mediated adverse responses, including1 inflammation surrounding many types of implants1-3; implant degradation and "stress cracking"10,38-41; tissue fibrosis surrounding mammary prostheses, joint implants, and many other types of implants11-13,42-44; and device-centered infection.45-47 Such adverse responses to biomaterials may lead to implant failure and, in cases such as the oxidative degradation of pacemaker lead insulation,37 fatality. However, the mechanisms underlying biomaterial-mediated inflammatory responses remain largely unknown. In searching for the proximate trigger of inflammatory responses to
biomaterial implants, we earlier observed that, after implantation,
serum-coated, as well as albumin-coated, PET disks accumulate far fewer
phagocytes (PMNs and M In an attempt to determine the fibrinogen sequence(s) critical for attracting phagocytes, fibrinogen degradation products were produced with plasmin digestion and purified with gel-filtration chromatography. By implanting surfaces precoated with different fibrinogen degradation products and then measuring the numbers of adherent phagocytes, we found that the fibrinogen fragment D30 appeared to contain the proinflammatory sequence(s).19 We also found that the phagocyte integrin Mac-1 (CD11b/CD18) is required for phagocyte adhesion to fibrinogen-bearing implants, because both CD18 knockout and CD11b knockout mice fail to accumulate phagocytes on either untreated or fibrinogen-precoated PET implants.20,21 Thus, interactions between motifs within the fibrinogen D30 domain and the phagocyte Mac-1 integrin appear particularly important in the accumulation of phagocytes on implant surfaces and, therefore, in early biomaterial-mediated inflammatory responses. Two segments of D30, P1 ( It is remarkable that the simple adsorption of fibrinogen to
biomaterial surfaces evidently converts an abundant coagulation protein, fibrinogen, to a proinflammatory state. There is, however, some precedent for such a phenomenon. For example, binding of fibrinogen to platelet glycoprotein IIb-IIIa has been found to result
in conformational changes in both the occupied receptor and the bound
ligand.48-51 By the same token, the present results suggest that material-induced conformational changes of adsorbed fibrinogen are critical in the early phases of the foreign body reaction to biomaterials. This general concept is further supported by
several earlier observations. Adsorbed proteins, especially on
hydrophobic surfaces (which are typical of the majority of implanted
biomaterials), undergo conformational changes, becoming tightly
adherent and The inflammatory responses to adsorbed fibrinogen may derive from a resemblance between the surface denatured protein and fibrin, the latter being a well-known participant in inflammatory reactions. Indeed, fibrin formation is one of the earliest events following wounding and appears to be an important precedent to subsequent wound healing.67 Furthermore, like fibrinogen adsorbed to biomaterial surfaces, fibrin deposition has been associated with subsequent adverse reactions, including inflammatory conditions and fibrosis.68-70 Adsorbed fibrinogen in itself appears sufficient to prompt foreign body reactions. We have found that the in vivo modification of fibrinogen adsorbed to biomaterial surfaces (eg, by further coagulation or proteolytic attack) is not required for ensuing inflammatory responses, because the blockade of either coagulation or proteolysis (fibrinolysis) in vivo does not affect phagocyte accumulation on implanted biomaterials (L.T. et al, unpublished results, December 2000). Furthermore, on the basis of calorimetric determinations, the major conformational changes of both fibrin and surface-denatured fibrinogen occur within the D domain,71,72 which contains both P1 and P2 epitopes. Using monoclonal antibodies against either fibrinogen or fibrin, earlier studies have revealed that the D domains of fibrinogen and fibrin are immunologically distinct.73-75 The central region of the gamma chain is inaccessible to antibodies in native fibrinogen73,74 but hidden epitopes within it are exposed following platelet binding and/or enzyme degradation.75,76 Finally, and directly pertinent to the present results, we find that the critically important P1 and P2 epitopes are displayed on both fibrin and surface-bound fibrinogen but are occult in native fibrinogen. This is, to the best of our knowledge, the first solid evidence for a
critical opsonic activity of adsorbed fibrinogen in prompting
inflammatory reactions to biomaterial implants and, possibly, other
types of foreign bodies. Although the detailed mechanisms are still not
clear, on the basis of available information we believe that several
steps might participate in these reactions. First, after intrusion or
implantation, foreign bodies or biomaterials spontaneously acquire a
layer of adsorbed host proteins, including fibrinogen. Second,
interactions between adsorbed fibrinogen and the implant
surface If the foregoing is correct, then fibrinogen may spontaneously convert
to a fibrinlike conformation on foreign surfaces, such as those of
hydrophobic polymers. In this form, the adsorbed fibrinogen
Submitted June 12, 2000; accepted April 24, 2001.
Supported by grants R01-HL56187 and R01-HL60787 from the National Institutes of Health.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Liping Tang, Biomedical Engineering Program, Box 19138, University of Texas at Arlington, Arlington, TX 76019; e-mail: ltang{at}uta.edu.
1. Kottke-Marchant K, Anderson JM, Rabinovitch A. The platelet reactivity of vascular graft prostheses. Biomaterials. 1986;7:441-448[CrossRef][Medline] [Order article via Infotrieve]. 2. Kottke-Marchant K, Anderson JM, Umemura Y, Marchant RE. Effect of albumin coating on the in vitro blood compatibility of Dacron arterial prostheses. Biomaterials. 1989;10:147-155[CrossRef][Medline] [Order article via Infotrieve]. 3. Marchant RE, Miller KM, Anderson JM. In vivo biocompatibility studies, V: in vivo leukocyte interactions with Biomer. J Biomed Mater Res. 1984;18:1169-1190[CrossRef][Medline] [Order article via Infotrieve].
4.
Gordon M, Bullough PG.
Synovial and osseous inflammation in failed silicon-rubber prostheses.
J Bone Joint Surg Am.
1982;64:574-580 5. Kossovsky N, Millett D, Juma S, et al. In vivo characterization of the inflammatory properties of poly(tetrafluoroethylene) particulates. J Biomed Mater Res. 1991;25:1287-1301[CrossRef][Medline] [Order article via Infotrieve]. 6. Colman RW. Surface-mediated defense reactions: the plasma contact activation system. J Clin Invest. 1984;73:1249-1253. 7. Yates SG II, Nakagawa Y, Berger K, Sauvage LR. Surface thrombogenicity of arterial prostheses. Surg Gynecol Obstet. 1973;136:12-16[Medline] [Order article via Infotrieve]. 8. Hogt AH, Dankert J, Feijen J. Adhesion of coagulase-negative staphylococci to methacrylate polymers and copolymers. J Biomed Mater Res. 1986;20:533-545[CrossRef][Medline] [Order article via Infotrieve]. 9. Jansen B, Peters G, Pulverer G. Mechanisms and clinical relevance of bacterial adhesion to polymers. J Biomater Appl. 1988;2:520-543. 10. Picha GJ, Goldstein JA, Stohr E. Natural-Y Meme polyurethane versus smooth silicon: analysis of the soft tissue interaction from 3 days to 1 year in the rat animal model. Plast Reconstr Surg. 1990;85:903-916[CrossRef][Medline] [Order article via Infotrieve]. 11. Behling CA, Spector M. Quantitative characterization of cells at the interface of long-term implants of selected polymers. J Biomed Mater Res. 1986;20:653-666[CrossRef][Medline] [Order article via Infotrieve]. 12. Christenson L, Aebischer P, McMillan P, Galletti PM. Tissue reaction to intraperitoneal polymer implants: species difference and effects of corticoids and doxorubicin. J Biomed Mater Res. 1989;23:705-718[CrossRef][Medline] [Order article via Infotrieve]. 13. Domanskis EJ, Owsley JQ. Histological investigation of the etiology of capsule contracture following augmentation mammaplasty. Plast Reconstr Surg. 1976;58:689-693[Medline] [Order article via Infotrieve]. 14. Tang L, Lucas AH, Eaton JW. Inflammatory responses to implanted polymeric biomaterials: role of surface-adsorbed immunoglobulin G. J Lab Clin Med. 1993;122:292-300[Medline] [Order article via Infotrieve].
15.
Tang L, Eaton JW.
Fibrin(ogen) mediates acute inflammatory responses to biomaterials.
J Exp Med.
1993;178:2147-2156 16. Pitt WG, Park K, Cooper SL. Sequential protein adsorption and thrombus deposition on polymer biomaterials. J Colloid Interfacial Sci. 1986;111:343-362[CrossRef]. 17. Bohnert JL, Horbett TA. Changes in adsorbed fibrinogen and albumin interactions with polymers indicated by decreases in detergent elutability. J Colloid Interfacial Sci. 1986;111:363-777[CrossRef]. 18. Vroman L, Adams AL, Klings M, Fischer GC, Munoz PC, Solensky RP. Reactions of formed elements of blood with plasma proteins at interfaces. Ann N Y Acad Sci. 1977;283:65-75[CrossRef]. 19. Tang L, Ugarova TP, Plow EF, Eaton JW. Molecular determinants of acute inflammatory responses to biomaterials. J Clin Invest. 1996;97:1329-1334[Medline] [Order article via Infotrieve]. 20. Lu DR, Park K. Effect of surface hydrophobicity on the conformational changes of adsorbed fibrinogen. J Colloid Interfacial Sci. 1991;144:271-281[CrossRef]. 21. Tang L, Welty S, Smith CV, Eaton JW. Participation of adhesion molecules in inflammatory responses to biomaterials [abstract]. Trans Soc Biomater. 1997;23:261a. 22. Doolittle RF, Schubert D, Schwartz SA. Amino acid sequence studies on artiodactyl fibrinopeptides, I: dromedary camel, mule deer, and cape buffalo. Arch Biochem Biophys. 1967;118:456-467[CrossRef][Medline] [Order article via Infotrieve].
23.
Altieri DC, Plescia J, Plow EF.
The structural motif glycine 190-valine 202 of the fibrinogen gamma chain interacts with CD11b/CD18 integrin (alpha M beta 2, Mac-1) and promotes leukocyte adhesion.
J Biol Chem.
1993;268:1847-1853
24.
Ugarova TP, Solovjov DA, Zhang L, et al.
Identification of a novel recognition sequence for integrin
25.
Humphries M, Komoriya A, Akiyama K, Olden K, Yamada K.
Identification of two distinct regions of the type III connecting segment of human plasma fibronectin that promote cell type-specific adhesion.
J Biol Chem.
1987;262:6886-6892
26.
McCarthy JB, Skubitz A, Zhao Q, Yi X, Mickelson DJ, Furcht LT.
RGD-independent cell adhesion to the carboxyl-terminal heparin-binding fragment of fibronectin involves heparin-dependent and -independent activities.
J Cell Biol.
1990;110:777-787
27.
Haugen PKJ, McCarthy JB, Skubitz APN, Furcht LT, Letourneau PC.
Recognition of the A chain carboxy-terminal heparin binding region of fibronectin involves multiple sites: two contiguous sequences act independently to promote neural cell adhesion.
J Cell Biol.
1990;111:2733-2745 28. Grabarek Z, Gergely J. Zero-length crosslinking procedure with the use of active esters. Anal Biochem. 1990;185:131-135[CrossRef][Medline] [Order article via Infotrieve]. 29. Staros JV, Wright RW, Swingle DM. Enhancement by N-hydroxysulfosuccinimide of water-soluble carbodiimide-mediated coupling reactions. Anal Biochem. 1986;156:220-222[CrossRef][Medline] [Order article via Infotrieve]. 30. Tang L. Mechanisms of fibrinogen domains: biomaterial interactions. J Biomater Sci Polym Ed. 1998;9:1257-1266[Medline] [Order article via Infotrieve]. 31. Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 1975;256:495-497[CrossRef][Medline] [Order article via Infotrieve]. 32. Himmelhoch HS, Evans WH, Mage MG, Peterson EA. Purification of myeloperoxidase from the bone marrow of the guinea pig. Biochemistry. 1969;8:914-921[CrossRef][Medline] [Order article via Infotrieve]. 33. Yam L, Li C, Crosby W. Cytochemical identification of monocytes and granulocytes. Am J Clin Pathol. 1971;55:283-290 |
Top
Abstract
Introduction
-nicotinamide adenine dinucleotide, reduced form (NADH) (from
yeast), sodium pyruvate, Triton X-100 (octyl phenoxy
polyethoxyethanol), EDTA, glass beads (0.3 cm diameter),
2-(N-morpholino)ethane sulfonic acid (MES), N-hydroxysulfosuccinimide
(NHS), and complete and incomplete Freund adjuvant were obtained from
Sigma (St Louis, MO). We purchased
1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide hydrochloride (EDC) from
Pierce (Rockford, IL). Human 125I-fibrinogen was purchased
from Amersham Pharmacia Biotech UK (Buckinghamshire, England).
Monoclonal antibody (clone 4-2) against fibrinogen P2 sequence was
purchased from Accurate Chemical and Scientific (Westbury, NY).
70°C and thawed at 37°C before use.
-helices