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Spatial regulation and surface chemistry control of

monocyte/macrophage adhesion and foreign body giant

cell formation by photochemically micropatterned surfaces

Kristin M. DeFife,

Erica Colton,

Yasuhide Nakayama,

Takehisa Matsuda,

James M. Anderson

Institute of Pathology, Case Western Reserve University, Cleveland, Ohio 44125

Department of Bioengineering, National Cardiovascular Center Research Institute, Osaka 565, Japan

Received 14 August 1998; accepted 3 November 1998

Abstract: A long-standing goal of biomedical device devel-

opment has been the generation of specific, desired host

blood and tissue responses. An approach to meeting this

design criteria is precise surface modification that creates

micropatterns of distinct physicochemical character to direct

cell adhesion and behavior. For this study, poly(ethylene

terephthalate) films were coated with poly(benzyl N,N-

diethyldithiocarbamate-co-styrene) and sequentially ex-

posed to monomer solutions for photoirradiation. A photo-

mask was placed over different regions to generate micro-

patterned surfaces with graft polymer stripes of three

distinct ionic characters. Human monocytes were cultured

on these surfaces to ascertain whether adhesion and fusion

of monocytes/macrophages could be controlled. Nonionic

polyacrylamide greatly inhibited adhesion and induced

clumping of the few monocytes that did adhere. Macro-

phage adhesion and spreading led to high degrees of inter-

leukin-13 induced foreign body giant cell formation on both

the anionic poly(acrylic acid), sodium salt, and benzyl N,N-

diethyldithiocarbamate portions of the culture surface. In

spite of the highest observed levels of monocyte/macro-

phage adhesion on cationic poly(dimethylaminopropyl-

acrylamide), methiodide, the adherent cells were not com-

petent to undergo fusion to form foreign body giant cells.

These results suggest that inflammatory cell responses may

be spatially controlled in a manner that may be ultimately

exploited to improve the biocompatibility of medical de-

45, 148–154, 1999.

Key words: macrophage; foreign body giant cell; photo-

chemical micropattern; adhesion; interleukin-13

INTRODUCTION

The surface properties of functional biomedical de-

vices, such as advanced tissue-engineered materials

and microbiosensors, must control critical host re-

sponses during the foreign body reaction to implan-

tation.

Dimensionally precise surface control may be

employed to modulate regions of cellular behaviors,

such as adhesion, migration, proliferation, and activa-

tion, to create a desired pattern of response. Such sur-

face micropatterning mimics spatial control of cell be-

havior during tissue and organ development when

cell adhesion, motility, and activation are strictly con-

trolled.

2–4

To this end, photochemical techniques have been

developed to microprocess polymer surfaces to con-

tain clearly defined regions of chemically distinct

polymer.

5–7

Hydrophilic regions on hydrophobic sur-

faces and vice versa can be patterned with precision

on the order of microns via immobilization of benzyl

N,N-diethyldithiocarbamate onto a polymer and graft

copolymerization of monomers using a photomask

and UV irradiation.

5,6

Adhesion and orientation of a

variety of cell types, such as endothelial cells,

5–8

neural

cells,

and platelets,

10,11

have been controlled by these

methods. In general, low levels of cell adhesion oc-

curred on nonionic and hydrophilic (or very hydro-

phobic) surfaces. Cells preferentially adhered to mod-

erately hydrophobic or ionic surfaces.

An additional important biological design criteria is

the ability of these surfaces to affect the foreign body

reaction to implanted materials.

The inflammatory

and wound healing responses to implanted materials

are controlled by the extensive adhesive and secreto-

Correspondence to: Dr. J. M. Anderson

Contract grant sponsor: National Heart, Lung, and Blood

Institute, Devices and Technology Branch; Contract grant

number: HL 55714

Contract grant sponsor: The Whitaker Foundation

Contract grant sponsor: The Center for Cardiovascular

Biomaterials at Case Western Reserve University

Contract grant sponsor: Organization for Pharmaceutical

Safety and Research; Contract grant number: 97-15

ry capabilities of the monocyte-derived macro-

phage.

1,12–14

Moreover, persistent presence of the for-

eign material may support the cytokine-induced fu-

sion of macrophages to form multinucleated foreign

body giant cells (FBGC),

15–17

which can result in both

structural and functional failure of the implant. The

reactivity and versatility of inflammatory macro-

phages may impact the ability of a micropatterned

surface to control macrophage behavior in the same

manner as other cell types. Importantly, it is not

known if microprocessed copolymers can control det-

rimental FBGC formation.

In this study we utilized a hydrophobic polymer

surface that had three photograft copolymerized hy-

drophilic regions of distinct ionic character in conjunc-

tion with an in vitro cytokine-induced FBGC formation

protocol. The goals of this study were to elucidate the

effect of the micropatterned copolymers on critical

components of the development of the foreign body

reaction: human monocyte/macrophage adhesion, ad-

herent cell spreading, and macrophage fusion to form

FBGC.

MATERIALS AND METHODS

Culture surface preparation

Graft-polymerized samples were prepared with a custom-

designed, semiautomatic apparatus for laboratory-scale

mass production as described elsewhere.

5,18

Poly(ethylene

terephthalate) (PET) films were coated with poly(benzyl

N,N-diethyldithiocarbamate-co-styrene) (BDEDTC). Poly-

acrylamide (PAAm); sodium salt of poly(acrylic acid)

(PAANa); and methiodide of poly(dimethylaminopropyl-

acrylamide), (DMAPAAmMeI) were then photograft copo-

lymerized to the BDEDTC surface in an orientation shown

schematically in Figure 1. After sequential graft polymeriza-

tion, samples were cut into circles with a carbon dioxide

laser cutter. The advancing water contact angles were mea-

sured for each surface (Table I).

At least 1 day before the monocytes were to be cultured,

sample disks were immersed briefly in ethanol and placed

into sterile 24-well tissue culture polystyrene plates. Auto-

clave-sterilized silicone rings were used to secure the disks

in the bottom of the wells. Plates were wrapped in alumi-

num foil and stored in a sterile hood until use. Sample wells

were rinsed twice with sterile Dulbecco’s phosphate-

buffered saline (PBS; GIBCO, Grand Island, NY) before the

monocytes were added to the wells.

Monocyte isolation and culture

Human blood monocytes were isolated from the venous

blood of unmedicated donors by a nonadherent, density

centrifugation method.

Isolated monocytes were judged

>97% viable by Trypan Blue exclusion and >80% pure by

staining for nonspecific esterase and peroxidase. Monocytes

were suspended in a medium of RPMI-1640 (GIBCO) con-

taining 25% autologous serum and an antibiotic and anti-

mycotic mixture (GIBCO). Five × 10

monocytes in 0.5 mL of

medium were added to each sample well and were allowed

to adhere for2hat37°C in a humidified atmosphere of 95%

air and 5% CO

. Nonadherent cells were removed by aspi-

rating the medium and rinsing the wells with warmed

(37°C) PBS, and the remaining adherent monocytes were

covered with 1 mL per well of fresh medium. Cell results

termed day 0 were collected after this initial 2-h incubation.

On days 3 and 7 of incubation, the medium was replaced

with 25% heat-treated (56°C water bath for 1 h) autologous

serum in RPMI, and 10 ng/mL interleukin (IL)-13 (R & D

Systems, Minneapolis, MN) was added as indicated.

Samples were collected on days 0, 3, 7, and 10 by rinsing

the cultures twice with warmed (37°C) PBS and fixing for 5

min with methanol. Samples were stained with May–

Gru¨nwald/Giemsa as previously described

for light mi-

croscopic observation.

Evaluation of cell adhesion and FBGC formation

Cell adhesion was manually counted from three 40× ob-

jective fields for each condition, and results are expressed as

a percentage of the initial number of cultured cells (5 × 10

)

± the standard error of the mean (SEM, n = 3). Percent fusion

Figure 1. Photochemically microprocessed culture surface.

PET films were coated with poly(benzyl N,N-diethyldithio-

carbamate-co-styrene) (BDEDTC) and then photograft copo-

lymerized with polyacrylamide (PAAm); sodium salt of

poly(acrylic acid) (PAANa); and methiodide of poly(dimeth-

ylaminopropylacrylamide) (DMAPAAmMeI).

TABLE I

Evaluation of Photograft Copolymerized Polymer

Surface Chemistry

Polymer

Water Contact

Angle (°) Ionic Character

BDEDTC 83.4 ± 1.3 Nonionic

PAAm 31.6 ± 3.6 Nonionic

DMAPAAmMeI 29.2 ± 2.8 Cationic

PAANa 25.3 ± 3.3 Anionic

149MICROPATTERNED SURFACE CONTROL OF MC/M␪ ADHESION AND FBGC FORMATION

was determined as the number of nuclei in the FBGCs (cells

with >2 nuclei) divided by the number of nuclei contained in

all adherent cells counted.

Nuclei were counted in three

20× objective fields for each condition, and results are ex-

pressed as percent fusion ± SEM (n = 3). The total area of cell

surface coverage was measured from three 20× objective

fields for each culture condition using the morphometric

software SigmaScan Pro (Jandel Scientific Software, San Ra-

fael, CA). Surface coverage was determined as the area cov-

ered by adherent cells divided by the total surface area and

is expressed as the percent surface coverage ± SEM (n = 3).

The unpaired Student’s t test was used for all statistical

analyses (StatView, Abacus Concepts, Inc., Berkeley, CA).

RESULTS

Monocyte/macrophage adhesion is modulated by

surface micropatterning

Human monocytes were cultured on BDEDTC-

coated PET with photochemically linked micropat-

terns of distinct ionic character (Fig. 1, Table I) to ask

whether surface-dependent patterning of monocyte/

macrophage adhesion could be achieved. Initial

monocyte adhesion was significantly affected by the

chemistry of the micropatterned surface (Fig. 2).

DMAPAAmMeI supported the highest degree of

monocyte adhesion after2hofculture, and adhesion

to PAAm was significantly lower than to any of the

other surfaces (p < 0.05). A culture time-dependent

decrease in adhesion was observed on all surfaces (p <

0.05) except for PAAm, which had a low degree of

initial adhesion. In general, monocyte/macrophage

adhesion to BDEDTC, PAANa, and DMAPAAmMeI

was not statistically different.

It was surprising that DMAPAAmMeI consistently

supported the most cell adhesion because it qualita-

tively appeared to support less adhesion than PAANa

and BDEDTC (Fig. 3). Monocytes/macrophages on

PAANa and BDEDTC appeared denser than those on

DMAPAAmMeI. In order to address this question, the

total surface area covered by the adherent cells was

measured. As shown in Figure 4, cell surface coverage

on PAANa and BDEDTC increased significantly with

culture time (p < 0.05) whereas surface coverage on

DMAPAAmMeI remained fairly constant for the en-

tire culture period. Surface coverage on PAAm was

always significantly lower than other surfaces, which

is consistent with the low degree of cell adhesion

(p < 0.05). By day 3 of culture, the monocytes/macro-

phages on PAANa were spreading, but on BDEDTC

the adherent cells were not contributing to an increase

in surface coverage, indicating that monocyte to mac-

rophage phenotypic development was delayed on this

surface compared to PAANa. By day 7 of culture, sur-

face coverage on BDEDTC and PAANa was equiva-

lent.

When surface coverage was expressed as a function

of cell adhesion, very different trends became appar-

ent among the micropatterned surfaces (Fig. 5). Very

few cells adhered to PAAm, and those that were pres-

ent clumped together and did not spread (Fig. 3). This

resulted in a very tight line on the plot in which no

points were statistically different. DMAPAAmMeI

provided the most linear trend (Fig. 5). The decrease in

cell number was fairly linear (Fig. 2), and the surface

coverage was fairly consistent (Fig. 4). The cells pro-

gressed to a macrophage morphology as judged by an

increase in size; however, these monocytes/macro-

phages became spindly and elongated (Fig. 3). Mono-

cytes on PAANa matured into macrophages, and ad-

herent cells spread to cover almost 90% of the avail-

able surface area; so even with fewer adherent cells

than DMAPAAmMeI, the surface coverage was

greater (Fig. 5). The trend on BDEDTC is similar to

PAANa and is not shown for clarity.

Macrophage competency to participate in FBGC

formation is controlled by surface micropatterns

IL-13 was added to the monocyte/macrophage cul-

tures to determine the effect of micropatterned sur-

faces on macrophage fusion to form FBGCs. Too few

cells adhered to PAAm to support macrophage fusion,

Figure 2. Micropattern-dependent monocyte/macrophage

adhesion. Human monocytes were cultured on BDEDTC-

coated PET films with three polymeric micropatterns of dis-

tinct ionic character. On the indicated days, cultures were

fixed and stained with May–Gru¨nwald/Giemsa to count the

numbers of adherent monocytes/macrophages. Percent ad-

hesion is expressed as mean ± SEM.

150 D

EFIFE ET AL.

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