photochemically immobilized polymer coatings: effects on protein adsorption, cell adhesion, and...
TRANSCRIPT
This article was downloaded by: [Universitat Politècnica de València]On: 28 October 2014, At: 06:33Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Journal of Biomaterials Science,Polymer EditionPublication details, including instructions for authors andsubscription information:http://www.tandfonline.com/loi/tbsp20
Photochemically immobilizedpolymer coatings: effects onprotein adsorption, cell adhesion,and leukocyte activationKristin M. Defife a , Kris M. Hagen b , David L. Clapper c &James M. Anderson da Institute of Pathology, Case Western Reserve University,Cleveland, OH 44106, USAb SurModics, Inc. , Eden Prarie, MN 55344-3523, USAc SurModics, Inc. , Eden Prarie, MN 55344-3523, USAd Institute of Pathology, Case Western Reserve University,Cleveland, OH 44106, USA, Department of BiomedicalEngineering, Case Western Reserve University, Cleveland,OH 44106, USAPublished online: 02 Apr 2012.
To cite this article: Kristin M. Defife , Kris M. Hagen , David L. Clapper & James M.Anderson (1999) Photochemically immobilized polymer coatings: effects on proteinadsorption, cell adhesion, and leukocyte activation, Journal of Biomaterials Science,Polymer Edition, 10:10, 1063-1074, DOI: 10.1163/156856299X00685
To link to this article: http://dx.doi.org/10.1163/156856299X00685
PLEASE SCROLL DOWN FOR ARTICLE
Taylor & Francis makes every effort to ensure the accuracy of all the information(the “Content”) contained in the publications on our platform. However, Taylor& Francis, our agents, and our licensors make no representations or warrantieswhatsoever as to the accuracy, completeness, or suitability for any purposeof the Content. Any opinions and views expressed in this publication are theopinions and views of the authors, and are not the views of or endorsed byTaylor & Francis. The accuracy of the Content should not be relied upon andshould be independently verified with primary sources of information. Taylor andFrancis shall not be liable for any losses, actions, claims, proceedings, demands,
costs, expenses, damages, and other liabilities whatsoever or howsoever causedarising directly or indirectly in connection with, in relation to or arising out of theuse of the Content.
This article may be used for research, teaching, and private study purposes.Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expresslyforbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/terms-and-conditions
Dow
nloa
ded
by [
Uni
vers
itat P
olitè
cnic
a de
Val
ènci
a] a
t 06:
33 2
8 O
ctob
er 2
014
Photochemically immobilized polymer coatings: effects on
protein adsorption, cell adhesion, and leukocyte activation
KRISTIN M. DEFIFE1, KRIS M. HAGEN3, DAVID L. CLAPPER 3
and JAMES M. ANDERSON1.2.* 1 Institute of Pathology, Case Western Reserve University, Cleveland, OH 44106, USA 2 Department of Biomedical Engineering, Case Western Reserve University,
Cleveland, OH 44106, USA 3 SurModics, Inc. , Eden Prarie, MN 55344-3523, USA
Received 22 December 1998; accepted 26 March 1999
Abstract-Amphiphilic chains of 4-benzoylbenzoic acid moieties and polymer were photochemi- cally immobilized onto silicone rubber to ask whether the covalently coupled polymers would pas- sivate the silicone rubber by inhibiting protein adsorption and subsequent cell adhesion and acti- vation. Three groups of polymers were utilized: the hydrophilic synthetic polymers of polyacry- lamide, polyethylene glycol, and polyvinylpyrrolidone; the glycosaminoglycan, hyaluronic acid; and
poly(glycine-valine-glycine-valine-proline), a polypeptide derived from the sequence of clastin. Each coating variant decreased the adsorption of fibrinogen and immunoglobulin G compared to uncoated silicone rubber. All except the methoxy-polyethylene glycol coating nearly abolished fi- broblast growth, but none of the coating variants inhibited monocyte or polymorphonuclear leukocyte adhesion. Interleukin-1β, interleukin-1 receptor antagonist, and tumor necrosis factor-α secretion
by leukocytes were not statistically different between any of the coating variants and uncoated sil- icone rubber. However, the methoxy-polyethylene glycol and elastin-based polypeptide coatings, which supported the highest numbers of adherent monocytes, also elicited the lowest levels of pro- inflammatory cytokine secretion. When these in vitro data were collectively evaluated, the coating that most effectively passivated silicone rubber was the polypeptide derived from elastin.
Key words : Photochemical immobilization; silicone rubber; protein adsorption; fibroblast and
leukocyte adhesion; cytokine secretion.
INTRODUCTION
Protein adsorption is generally considered to be a critical initial step in the complex mechanisms of fibrin deposition and fibrous capsule development [1, 2], and
adequate inhibition of protein adsorption by a material surface may prevent both
*To whom correspondence should be addressed.
Dow
nloa
ded
by [
Uni
vers
itat P
olitè
cnic
a de
Val
ènci
a] a
t 06:
33 2
8 O
ctob
er 2
014
1064
of these detrimental processes. Serum proteins are more readily adsorbed by
hydrophobic polymers [3, 4], such as silicone rubber (SR), than by hydrophilic
polymers [5-7]. Moreover, it has been demonstrated that cells adhere well to
moderately hydrophobic or ionic surfaces but do not adhere well to hydrophilic, nonionic surfaces [6-8], a likely result of decreased adhesive protein adsorption. In
addition, inhibition of protein adsorption may result in reduced infection as protein
adsorption may mediate bacterial adhesion to biomaterials [9, 10]. Surface chemistry is one of the many characteristics of an implanted device that
influence the extent of the host's inflammatory response [ 1 ] and provide an avenue
for manipulation to achieve a more desirable response than possible from the un-
modified material. Using photochemical immobilization technology, a wide variety of molecules can be covalently coupled to biomaterials to modify the chemistry of
the surface [ 11 ]. Of particular interest are coating materials that decrease protein
adsorption, cell adhesion, thrombus formation, and/or fibrosis in an effort to effec-
tively passivate SR to inhibit fibrin deposition and fibrosis in vivo. Several hydrogels
suppress protein adsorption and/or improve biocompatibility when coated onto SR
and other biomaterials [5-7, 12-14]. Polyethylene glycol (PEG), polyacrylamide (PAAm), and polyvinylpyrrolidone (PVP) were the hydrogels selected for use in
this study. Hyaluronic acid (HA) was chosen as a naturally occurring extracellular
matrix glycosaminoglycan that has been reported to inhibit cell attachment and fi-
brosis [ 15, 16]. An elastomeric polypeptide based on the repeating oligopeptides
present in elastin, poly(glycine-valine-glycine-valine-glycine) (poly(GVGVP)), was included because poly(GVGVP), cross-linked into sheets, reduced cell attach-
ment and abdominal adhesion formation [ 17-19]. The goals of this study were to covalently couple each of these polymers to SR
using photochemical immobilization and to assay whether the modified surfaces
would alter responses that may be predictive of improved SR performance, includ-
ing : (i) fibrinogen (FGN) and immunoglobulin G (HGG) adsorption; (ii) fibroblast
growth; and (iii) leukocyte adhesion and activation.
MATERIALS AND METHODS
Materials
SR (Q7-4765) was purchased from Dow Coming (Midland, MI, USA). Methoxy
polyethylene glycol-amine (mPEG-amine) was purchased from Shearwater Poly- mers, Inc. (Huntsville, AL, USA). Monomers of PAAm and PVP were purchased from Aldrich Chemical Co. (Milwaukee, WI, USA). PVP polymer was purchased from BASF Corp. (Wyandotte, MI, USA), and PEG was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Average molecular weights (Mws) of poly- mers were as follows: PEG, 3350; mPEG-amine, 5000; and PVP, greater than one
million (Kollidon 90F). HA was purchased from Lifecore Biomedical (Chaska, MN,
USA). An elastin-based, synthetic polypeptide with 100 repeats, I (GVGVP) 100],
Dow
nloa
ded
by [
Uni
vers
itat P
olitè
cnic
a de
Val
ènci
a] a
t 06:
33 2
8 O
ctob
er 2
014
1065
was supplied in two forms: ( 1 ) cross-linked (CL-GVGVP) and (2) soluble GVGVP that was prepared with three lysine residues at the amino-terminal end (Bioelastics Research, Ltd., Birmingham, AL, USA). The remaining reagents that were used to
synthesize polymers and photocoupling reagents for this study were purchased from Aldrich Chemical Co. or other suppliers of reagent grade chemicals.
Syyzthe.si.s and photochemical application of coating reagent
Photoderivatized HA, PAAm, and PVP were large Mw polymers with photoreactive 4-benzoylbenzoic acid (BBA) moieties randomly distributed along the length of each molecule. BBA-HA, BBA-PAAm, and BBA-PVP were synthesized as described [ 11 ].
Two photoderivatized reagents had the BBA moieties clustered at one end of each molecule and were of lower MW than the aforementioned reagents. BBA-mPEG was synthesized by adding two BBA moieties to the amino group of mPEG-amine. BBA-GVGVP was synthesized by adding BBA moieties to the primary amines
present on lysine residues and the amino-terminal amine of soluble GVGVP. SR samples were extracted overnight with two changes of hexane to remove
silicone elastomers that were not incorporated during the vulcanization process. The BBA-PAAm, BBA-PVP, BBA-mPEG, and BBA-GVGVP reagents were ap- plied and illuminated as previously described to generate covalently immobilized
coatings [11]. Two-layer coatings, labeled BBA-PAAm/PEG, BBA-PAAm/PVP, and BBA-PAAm/BBA-HA, were generated by washing BBA-PAAm surfaces with deionized water (diH20) and applying and illuminating the second reagent (PEG, PVP or BBA-HA, respectively). Samples were sterilized by soaking in 70% ethanol for 20 min. The samples were then washed with sterile PBS and sterile diH20, dried with a proprietary stabilizing agent, and stored in sterile containers until use.
The surfaces were analyzed by measuring sessile drop contact angles with a
goniometer (Microview, Santa Ana, CA, USA) as described [20]. Replicates were obtained by measuring contact angles on six different areas of each coated sample. HA was visualized by staining with toluidine blue 0 (0.2% w/v in water), and PVP was stained with Congo Red (0.35% w/v in water).
Protein adsorption
Human fibrinogen (FGN; Sigma) and human immunoglobulin G (HGG; Sigma) were radiolabeled with tritium via reductive methylation as described [11]. The re- sultant specific activities were 1.8-3.2x 104 disintegrations per minute (DPM)/ J1g for each protein. For the experiments conducted with PBS, non-radiolabeled FGN and HGG were added at the approximate concentrations of each protein in plasma, 4 or 12 mgml-l, respectively [21]. Lyophilized citrated bovine plasma (Sigma), reconstituted to 100% with diH20, was used in some experiments.
Coated and uncoated SR disks were placed into polystyrene tissue culture plates, and 200 fil ( 1.6-6.3 x 106 DPM) of [3H]-HGG or [3H]-FGN in PBS or citrated
Dow
nloa
ded
by [
Uni
vers
itat P
olitè
cnic
a de
Val
ènci
a] a
t 06:
33 2
8 O
ctob
er 2
014
1066
bovine plasma were added. After an overnight incubation at room temperature, the
disks were washed four times in PBS with gentle agitation. The SR disks were
dissolved in Soluene-350 tissue solubilizer for 2 h at 55°C, and the activity was
counted in Hionic Fluor (each from Packard Instruments Co., Meriden, CT).
Leukocyte adhesion and cvtokine secretion
Human blood monocytes and PMNs were isolated from the venous blood of unmed-
icated donors by non-adherent, density centrifugation methods as described [22, 23] and were suspended in a medium of RPMI-1640 (GIBCO, Grand Island, NY, USA)
containing 20% autologous serum and antibiotic/ antimycotic mixture (GIBCO).
Monocytes and PMNs were added to culture plates containing the equilibrated (1 h
at 37°C in RPMI-1640) coated and uncoated SR disks and were allowed to adhere
for 1 h at 37°C in a humidified atmosphere of 95% air and 5% C02. Monocytes were
added at a concentration of 3 x 105 per well, and PMNs were added at a concentra-
tion of 3 x 106 per well. After the initial 1 h incubation of samples to be cultured
for 24 h, non-adherent cells were removed by aspirating the medium and rinsing the
wells with warmed (37°C) PBS containing calcium and magnesium. The remaining adherent leukocytes were covered with I ml per well of fresh medium.
After 1 and 24 h, supernatants were removed, and adherent cells were fixed for 5
min with methanol and air dried. Cells were stained with modified Wright stain, and
adhesion was quantified using Sigma Scan Pro (Jandel Scientific, San Rafael, CA,
USA). Monocyte adhesion was measured from five fields of view (0.13176 mm2 2
each), and PMN adhesion was measured from three fields of view (0.03375 mm2
each). The counts were averaged, converted to cells mm- , and are expressed as
mean + S.E.M. (n = 4). ELISA assays were performed on the supernatants for interleukin IL-1 I
receptor antagonist (IL-lra), and tumor necrosis factor (TNF)-a secretion according to the manufacturer's instructions (R & D Systems, Minneapolis, MN, USA). In addition, pro-inflammatory activation was estimated by calculating the ratio
of IL-l f3 to IL-1 ra. IL-1/5 and IL-1 ra compete for Type I IL-1 receptors with
the same avidity (Kd - 200 pM), and the unglycosylated forms of the proteins have similar Mws of approximately 17 kDa [24]. Results are presented as mean
(n = 4). '
Fibroblast growth '
Coated and uncoated SR disks, secured in polystyrene tissue culture plates with SR
rings, were equilibrated to 37°C by soaking for 2 h in minimum essential medium
(MEM) (Celox Laboratories, Inc., St. Paul, MN, USA). The disks and control tissue
culture wells were seeded with NRK-49F fibroblasts at a concentration of 5000
cells per well and cultured in MEM plus 5% fetal bovine serum until the cells in
the tissue culture wells reached 80-90% confluency as judged by phase contrast
Dow
nloa
ded
by [
Uni
vers
itat P
olitè
cnic
a de
Val
ènci
a] a
t 06:
33 2
8 O
ctob
er 2
014
1067
microscopy. The relative cell numbers present in each well were quantified with
MTT, a tetrazolium metabolic dye 26].
Statistical analyses
Unpaired Student's t-tests were performed using StatView 4.1 (Abacus Concepts,
Berkeley, CA, USA).
RESULTS
Surface analysis
All of the coatings were more hydrophilic than uncoated SR although BBA-mPEG
had a contact angle near that of uncoated SR (Table 1 ). BBA-PAAm, BBA-
PAAm/BBA-HA, and BBA-PVP were the most hydrophilic coatings. The low
variability of the contact angles measured from various locations on the samples indicates that the coatings were uniformly immobilized. This observation was also
supported by the uniform toluidine blue 0 staining of the BBA-PAAm/BBA-HA
coating and the uniform Congo Red staining of the BBA-PVP coating (results not
shown).
Proteirz czclsorption
Figures 1 and 2 show representative results for the adsorption of FGN and HGG to
coated and uncoated SR. When evaluated in PBS, coating variants containing BBA-
PAAm or BBA-PVP reduced FGN adsorption by 89-93% and HGG adsorption
by 80-88%. In contrast, coatings of BBA-GVGVP and BBA-mPEG were less
effective at inhibiting protein adsorption. Experiments for FGN in PBS were quite
reproducible, with several additional experiments producing results similar to those
shown in Fig. 1. However, a second experiment with HGG produced 50-60%
Table 1. Sessile drop water contact angles on coated and uncoated SR
" Replicates were obtained by measuring contact angles from six different areas of each coated sample.
Dow
nloa
ded
by [
Uni
vers
itat P
olitè
cnic
a de
Val
ènci
a] a
t 06:
33 2
8 O
ctob
er 2
014
1068
Figure 1. FGN adsorption to coated and uncoated SR. Results from one experiment that are expressed as a mean of three determinations ± S.E.M. BBA-GVGVP was not assayed.
inhibition by coatings that contained BBA-PAAm (BBA-PVP was not assayed) and
40 and 47% inhibition by BBA-mPEG and BBA-GVGVP, respectively. When evaluated in citrated plasma, both proteins showed considerable varia-
tion between experiments. For example, the three coatings (BBA-PAAm/PEG,
BBA-PAAm/PVP, and BBA-PAAm/BBA-HA) that inhibited FGN adsorption by 91-92% in Fig. l, produced 60-70% inhibition and intermediate levels of inhibi-
tion in additional experiments. With HGG, all of the coatings showed similar inhi-
bition within each experiment and significant variability between experiments. In
three such experiments, inhibition of adsorption ranged from 14-28% in one exper- iment (Fig. 2), 0-33% in a second experiment, and 53-71 % in a third experiment.
Leukocyte adhesion
None of the coating variants inhibited adhesion of either monocytes or PMNs s
compared to uncoated SR at either 1 or 24 h (Fig. 3). A 30-45% decrease
in adherent monocytes over the 24-h incubation period was observed for all
coating variants except for BBA-GVGVP which maintained 95% of the number of
monocytes adherent at 1 h. Robust PMN adhesion occurred after I h of culture, but
more than 80% of PMNs detached from the coating variants by 24 h. The remaining PMNs had very irregular shapes and appeared to have nuclear degeneration which
was not surprising considering the short life-span of these cells (not shown).
Leukocyte cytokine secretion
Surface-dependent monocyte activation, as measured by the concentrations of IL-
IL-lra, and TNF-a in culture supernatants, was not significantly different
Dow
nloa
ded
by [
Uni
vers
itat P
olitè
cnic
a de
Val
ènci
a] a
t 06:
33 2
8 O
ctob
er 2
014
1069
Figure 2. HGG adsorption to coated and uncoated SR. Results from one experiment that are expressed as a mean of three determinations S.E.M.
Figure 3. Monocyte (A) and PMN (B) adhesion to coated and uncoated SR after I and 24 h of culture. All 24 h results are significantly lower (p < 0.05) than 1 h results except for monocyte adhesion to BBA-GVGVP (ii = 4).
among all of the surfaces tested after 24 h of culture (Fig. 4). BBA-mPEG and
BBA-GVGVP not only supported adhesion of the highest numbers of monocytes at 24 h (Fig. 3) but also evoked the lowest levels of and TNF-a secretion,
suggesting that these surfaces elicited a lower pro-inflammatory response from the cells than the uncoated SR. The low ratios of to IL-lra calculated for the BBA-mPEG and BBA-GVGVP surfaces confirmed their designation as surfaces that do not strongly activate monocytes (Table 2).
Dow
nloa
ded
by [
Uni
vers
itat P
olitè
cnic
a de
Val
ènci
a] a
t 06:
33 2
8 O
ctob
er 2
014
1070
Figure 4. Supernatant cytokine concentrations from monocytes cultured on coated and uncoated SR for 24 h (n = 4).
Table 2. Pro-inflammatory activation of monocytes after 24 h of in vitro culture
The levels of IL-1 /3 and IL-lra measured from the supernatants of PMN cul-
tures at 24 h were near or below the minimum detectable concentrations of 1 and
14 pg ml-I, respectively, by ELISA assays (not shown). Two donors had unde-
tectable levels of IL-lra for all samples tested, and two had concentrations between
0 and 210 pg ml-l .Because the concentrations of IL-lra from the two donors that
produced measurable quantities were generally more than twelve-fold higher than
IL-1,8 and because the PMN from the other two donors produced undetectable lev-
els of both IL- I P and IL-lra, the surfaces were considered non-activating for PMNs
by these criteria. No detectable concentrations of TNF-a were measured from the
PMN supernatants at 24 h (the minimum detectable concentration was 4.4 pg ml-1 ).
Supernatants collected after 1 h of monocyte or PMN culture contained undetectable
levels of IL-lra, and TNF-a, indicating that the isolation procedure did not
activate the cells (not shown, n = 1).
Dow
nloa
ded
by [
Uni
vers
itat P
olitè
cnic
a de
Val
ènci
a] a
t 06:
33 2
8 O
ctob
er 2
014
1071
Table 3. Growth of NRK-49F fibroblast on coated SR"
" Representative experiment showing three determinations of relative cell number by the MTT assay.
t' % decrease in fibroblast growth on coated variants as compared to uncoated controls.
Fibroblasts growth
Fibroblast growth was inhibited by 93-99% on all coating variants except BBA-
mPEG, and a representative experiment is shown in Table 3. The BBA-GVGVP
coating was evaluated in a single experiment and inhibited fibroblast growth by 90% when compared to uncoated SR.
DISCUSSION
It is generally accepted that protein adsorption is an initial critical step follow-
ing biomaterial implantation that may ultimately lead to fibrosis and fibrin depo- sition [1, 2]. The results of these studies demonstrate that several coating variants
reduce in vitro protein adsorption and subsequent fibroblast growth and monocyte activation. All coating variants decreased adsorption of FGN and HGG, and all ex-
cept BBA-mPEG inhibited fibroblast growth by at least 90%. Others have demon-
strated that PEO and PAAtn coatings inhibit in vitro adsorption of blood proteins by 50-95% [5-7, 12, 13, 27, 28] and adhesion of fibroblasts by 90% [6, 7]. Biofilms of
HA esters [16] and CL-GVGVP [17, 18] have also been shown to decrease fibrob-
last adhesion. In the previous studies with CL-GVGVP, fibroblast adhesion was
inhibited by only 20-30% with serum-containing medium. Therefore, the 90% in-
hibition in the presence of serum by photochemically linked BBA-GVGVP in this
study represents a significant improvement in the ability of poly(GVGVP) to inhibit
fibroblast growth. Surface density of coatings is an important factor that influences prevention of
protein adsorption when hydrophilic coatings are applied to hydrophobic poly- mers [29]. Using this photochemical immobilization technology, amphiphilic chains, each of which has a hydrophilic backbone (the polymer chain) and hy-
drophobic groups (the BBA moieties), were immobilized with the BBA moi-
eties being oriented toward the SR. Theoretically, coatings of monolayer or greater
Dow
nloa
ded
by [
Uni
vers
itat P
olitè
cnic
a de
Val
ènci
a] a
t 06:
33 2
8 O
ctob
er 2
014
1072
should be achieved through adsorption of the BBA moieties when the amphiphiles in an aqueous solution are exposed to a hydrophobic polymer such as SR. Sub-
sequent illumination covalently bonds the BBA moieties to the SR, and a layer of tightly-packed hydrophilic polymer chains extending into the aqueous medium
would result. Indeed, photochemical immobilization of low Mw PEG in this study was more effective in reducing protein adsorption (by approximately 80%) than
other published techniques [6, 7] in which inhibition of FGN adsorption was 5-35%
when PEG of higher Mw was incorporated. Others have also found that protein ad-
sorption may be prevented by lower Mw PEG if the surface density is sufficiently
high [28 1. However, the surface density may have been too low to effectively inhibit
fibroblast growth. The grafting density and molecular weight of the mPEG may be
such that the polymer layer does not achieve tightly-packed alignment. In addition, the mobility of the SR chains may have prevented the mPEG from forming a stable
layer and may have ultimately buried or masked the surface coating.
Leukocyte adhesion was not significantly decreased by any of the coating variants
compared to uncoated SR at either I or 24 h of culture. The present results suggest that initial monocyte adhesion in vitro is largely independent of the culture surface
and is in agreement with our previous work with different surfaces [22, 30, 31 ]. It is not surprising that leukocytes adhered to all coating variants with equivalent success considering that the receptor-based adhesion mechanisms of monocytes,
macrophages, and PMNs possess the advantageous capabilities to bind to a variety of substrate chemistries [32-34].
Our leukocyte adhesion results agree with published reports demonstrating that
hydrophilic coating variants inhibit fibroblast adhesion but not inflammatory cell ad-
hesion. Inflammatory cells have been demonstrated to adhere in vivo to PEO-PET
polymers that inhibit protein adsorption and fibroblast adhesion in vitro [6, 35] and
to SR that had been graft polymerized with a variety of water-soluble monomers, such as acrylic acid and acrylamide [36]. Leukocyte adhesion was also not inhibited
by the BBA-GVGVP coating although poly(GVGVP) had been previously reported to inhibit fibroblast adhesion when cross-linked into sheets [17, 18] and presently shown to suppress protein adsorption and fibroblast adhesion when coated onto SR.
Interestingly, monocyte adhesion was reduced by only 5% on BBA-GVGVP after
24 h of culture compared to 1 h, and cytokine secretion from monocytes at 24 h was
among the lowest measured.
BBA-PAAm/BBA-HA also did not inhibit leukocyte adhesion or cytokine secre-
tion compared to uncoated SR. Monocytes and PMNs express an inducible HA-
binding receptor, CD44 [37, 38]. The ability of monocytic CD44 to bind HA is
induced in vitro by culture in the presence of serum [39] and by TNF-a [37]. Fur-
ther, ligation of monocytic CD44 by HA has been reported to induce release of IL-1 I
and TNF-a [40], which may account for monocyte cytokine release being among the highest on BBA-PAAm/BBA-HA. Fibroblasts are also CD44+, but growth was
inhibited by BBA-PAAm/BBA-HA. It has not been reported whether serum in-
Dow
nloa
ded
by [
Uni
vers
itat P
olitè
cnic
a de
Val
ènci
a] a
t 06:
33 2
8 O
ctob
er 2
014
1073
duces fibroblast CD44 binding to HA, and importantly, not all isoforms of CD44
bind HA [38, 39, 41, 42]. In summary, the photochemical immobilization of hydrogels, a glycosaminogly-
can, and an elastin-based polypeptide to SR reduced the adsorption of two proteins, FGN and HGG, that are important for cell-material interactions. Two coating vari-
ants, BBA-mPEG and BBA-GVGVP, elicited very low pro-inflammatory cytokine secretion from monocytes while none significantly inhibited the adhesion of mono-
cytes or PMNs. BBA-GVGVP was deemed to be the most potential passivating
coating based on our results which suggest that this surface does not activate in-
flammatory cells to the same degree as the other surfaces. In considering the criteria
for considering a surface passive, in vitro results may be misleading given the cur-
rent incomplete understanding of events that lead to fibrous capsule formation. In
this regard, the level of adherent leukocyte activation may be more important than
the amount of adsorbed protein and future animal implant studies are necessary to
correlate both these parameters with fibrous capsule formation.
Acknowledgements
This research was supported by the NIH grants HD 34318, HL 33849 and HL
55714, The Whitaker Foundation, The Center for Cardiovascular Biomaterials at
Case Western Reserve University, and SurModics, Inc.
REFERENCES
1. J. M. Anderson, Cardiovasc. Pathol. 2, 33S (1993). 2. T. A. Horbett, Cardiovasc. Pathol. 2, 137S (1993). 3. J. M. Anderson, N. P. Ziats, A. Azeez, M. R. Brunstedt, S. Stack and T. L. Bonfield, J. Biomater.
Sci. Polymer. Edn 7, 159 (1995). 4. D. J. Fabriziushoman and S. L. Cooper, J. Biomater. Sci. Polymer. Edn 3, 27 ( 1991). 5. J. H. Lee, P. Kopeckova, J. Kopecek and J. D. Andrade, Biomaterials 11, 455 (1990). 6. N. P. Desai and J. A. Hubbell, Biomaterial. 12, 144 (1991). 7. N. P. Desai and J. A. Hubbell, Biomed. Mater. Res. 25, 829 (1991). 8. T. Matsuda and T. Sugawara, J. Biomed. Mata Res. 32, 165 (1996). 9. A. L. Cheung and V. A. Fischetti, J. Infect. Dis. 161, 1177 (1990).
10. S. Nagaoka and H. Kawakami, ASAIO J. 41, M365 (1995). 11. R. A. Amos, A. B. Anderson, D. L. Clapper, P. H. Duquette, L. W. Duran, S. G. Hohle,
D. J. Sogard, M. J. Swanson and P. E. Guire, in: Encyclopedic Handbook of Biomaterials and Bioengineering: Part A: Materials, Vol. I, D. L. Wise, D. J. Trantolo, D. E. Altobelli, M. J. Yaszemski, J. D. Gresser and E. R. Schwartz (Eds), p. 895. Marcel Dekker, New York
( 1995). 12. S. Nagaoka, Trans. Am. Soc. Artif Intern. Organs 33, 76 (1987). 13. K. Fujimoto, Y. Takebayashi, H. Inoue and Y. Ikada, Polym. Sci., Part A Polym. Chem. 31, 1035
( 1993). 14. H. Mirzadeh, A. A. Katbab, M. T. Khorasani, R. P. Burford, E. Gorgin and A. Golestani,
Biomaterials 16, 641 (1995). 15. T. Matsuda, H. Miwa, M. J. Moghaddam and F. Iida, ASAIO J. 39, M327 (1993).
Dow
nloa
ded
by [
Uni
vers
itat P
olitè
cnic
a de
Val
ènci
a] a
t 06:
33 2
8 O
ctob
er 2
014
1074
16. R. Cortivo, P. Brun, A. Rastrelli and G. Abatangelo, Biomaterials 12, 727 (1991). 17. A. Nicol, D. C. Gowda and D. W. Urry, J. Biomed. Mater: Res. 26, 393 (1992). 18. A. Nicol, D. C. Gowda, T. M. Parker and D. W. Urry, J. Biomed. Mater. Res. 27, 801 (1993). 19. L. D. Hoban, M.Pierce, J. Quance, I. Hayward, A. McKee, D. C. Gowda, D. W. Urry and
T. Williams, J. Surg. Res. 56, 179 (1994). 20. S. A. Barenberg, B. Brozoski-Varnell, A. D. English, R. L. Hassel, R. E. Johnson, Jr., M. J. Kelley
and H. W. Starkweather, Jr., in: Techniques of Biocompatibility Testing, Vol. II, D. F. Williams (Ed.), p. 1. CRC Press, Boca Raton, FL (1986).
21. G. H. Grant and J. F. Kachmar, in: Fundamentals of Clinical Chemistry, N. W. Tietz (Ed.), p. 298. W. B. Saunders Company, Philadephia, PA (1982).
22. A. K. McNally and J. M. Anderson, Proc. Natl. Acad. Sci. USA 91, 101 19 (1994). 23. T. P. Stossel, T. D. Pollard, R. J. Mason and M. Vaughan, J. Clin. Invest. 50, 1745 (1971). 24. W. T. Arend, in: Clinical Immunology: Principles and Practice, Vol. II, R. R. Rich (Ed.), p. 2046.
Mosby-Year Book, Inc., St Louis, MO (1996). 25. L. M. Green, J. L. Reade and C. F. Ware, J. Immunol. Meth. 70, 257 (1984). 26. Bio-Rad technical bulletin, Bulletin 1202, Bio-Rad Laboratories (1985). 27. L. Litauski, L. Howard, L. Salvati and P. J. Tarcha, J. Biomed. Mater. Res. 35, 1 (1997). 28. J. H. Lee, B. J. Jeong and H. B. Lee, J. Biomed. Mater. Res. 34, 105 (1997). 29. T. McPherson, A. Kidane, I. Szleifer and K. Park, Langmuir 14, 176 ( 1998). 30. C. R. Jenney, K. M. DeFife, E. Colton and J. M. Anderson, J. Biomed. Mater. Res. 41, 171
( 1998). 31. C. R. Jenney and J. M. Anderson, J. Biomed. Mater. Res. 44, 206 (1999). 32. M. Patarroyo, R. Salcedo, J. Prieto, B. Åsjö. G. Skoglund, T. Anderson and C. G. Gahmberg. in:
Mononuclear Phagocytes: Biology of Monocytes and Macrophages, R. van Furth (Ed.), p. 92. Kluwer Academic Publishers, Dordrecht, The Netherlands (1992).
33. C. G. Gahmberg, M. Tolvanen and P. Kotovuori, Eur. J. Biochem. 245, 215 (1997). 34. I. Mellman, in: Mononuclear Phagocytes: Biology of Monocytes and Macrophages, R. van Furth
(Ed.), p. 169. Kluwer Academic Publishers, Dordrecht, The Netherlands (1992). 35. N. P. Desai and J. A. Hubbell, Biomaterials 13, 505 (1992). 36. T. Okada and Y. Ikada, J. Biomed. Mater. Res. 27, 1509 (1993). 37. M. C. Levesque and B. F. Haynes, J. Immunol. 159, 6184 (1997). 38. F. Pericle, G. Sconocchia, J. A. Titus and D. M. Segal, J. Immunol. 157, 4657 (1996). 39. M. C. Levesque and B. F. Haynes, J. Immunol. 156, 1557 (1996). 40. D. S. Webb, Y. Shimizu, G. A. van Seventer, S. Shaw and T. L. Gerrard, Science 249, 1295
( 1990). 41. J. Lesley, N. English, A. Perschl, J. Gregoroff and R. Hyman, J. Exp. Med. 182, 431 (1995). 42. A. Bartolazzi, A. Nocks, A. Aruffo, F. Spring and I. Stamenkovic, J. Cell Biol. 132, 1199 (1996).
Dow
nloa
ded
by [
Uni
vers
itat P
olitè
cnic
a de
Val
ènci
a] a
t 06:
33 2
8 O
ctob
er 2
014