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Macrophage cell adhesion and inflammation cytokineson magnetostrictive nanowiresKristy M. Ainslie a; Eric M. Bachelder bc; Gaurav Sharma d; Craig A. Grimes de;Michael V. Pishko adfa Department of Chemical Engineering, The Pennsylvania State University,Bethesda, Marylandb National Institutes of Health, National Institute of Allergy and Infectious Diseases,Bethesda, Marylandc Department of Chemical Engineering, University of Nebraska, Lincoln, Nebraskad Materials Science and Engineering, The Pennsylvania State University,Pennsylvania, USAe Electrical EngineeringThe Pennsylvania State University, Pennsylvania, USAf Chemistry, The Pennsylvania State University, Pennsylvania, USA

Online Publication Date: 01 December 2007To cite this Article: Ainslie, Kristy M., Bachelder, Eric M., Sharma, Gaurav, Grimes, Craig A. and Pishko, Michael V.(2007) 'Macrophage cell adhesion and inflammation cytokines on magnetostrictive nanowires', Nanotoxicology, 1:4, 279 -290To link to this article: DOI: 10.1080/17435390701781142URL: http://dx.doi.org/10.1080/17435390701781142

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Macrophage cell adhesion and inflammation cytokines onmagnetostrictive nanowires

KRISTY M. AINSLIE1, ERIC M. BACHELDER2,3, GAURAV SHARMA4,

CRAIG A. GRIMES4,5, & MICHAEL V. PISHKO1,4,6

1Department of Chemical Engineering, The Pennsylvania State University, 2National Institutes of Health, National Institute

of Allergy and Infectious Diseases, Bethesda, Maryland, 3University of Nebraska, Department of Chemical Engineering,

Lincoln, Nebraska, Departments of 4Materials Science and Engineering, 5Electrical Engineering, 6Chemistry, The

Pennsylvania State University, Pennsylvania, USA

AbstractA common problem with medical implants is the biofouling response which can detrimentally damage implants orprevent the implant from function properly. This response is characterized by a thick, frequently avascular, layer ofproteins and cells over the implant. To study this problem, we have examined here the adhesion of macrophages andthe subsequent expression of inflammatory cytokines on nanowire arrays. We found that the cells on the nanowirestypically occupied less area and were more circular than on a flat surface of the same material as the nanowires ortissue culture polystyrene (TCPS) in both the presence and absence of fetal bovine serum. Furthermore, this differencewas amplified by pre-coating the surfaces with collagen. The smaller area and circular shape indicated that the cellswere not thriving on the surface. Since there was potentially a high amount of cell death on the material, andbiofouling is frequently characterized as a chronic inflammation, an eighteen cytokine Luminex† panel was performedon the supernatant from macrophages on nanowires, control wafers, and TCPS. As a positive control for inflammation,lipopolysaccharide (LPS) was added to macrophages on TCPS to estimate the maximum inflammation response of themacrophages. Our results indicated that the nanowire structure results in the up-regulation of production inmacrophages of inflammatory cytokines such as IL-1a, and IFN-g and the down-regulation of IL-6, compared tocontrol wafers. In addition, the nanostructure also increased the production of IL-10 which is known as an inhibitor ofinflammation. Our results showed that the nanoarchitecture can disrupt cell adhesion and may lead to an inflammatoryresponse.

Keywords: nanomaterials, biomaterials, biocompatibility

Introduction

As an implant, or foreign body, is introduced to the

body the first event at the biofluid-material barrier is

adsorption of proteins onto the surface of the

material. The adsorbed proteins serve as an anchor

to which cells and additional proteins adhere (Bala-

subramanian et al. 1999). The results of this protein

adsorption and subsequent cell adhesion can be

detrimental to the implant, such is the case of

biofouling, an adverse host response that results

in implant encapsulation with proteins and cells

(Padera & Colton 1996). The biofouling response is

characterized by several layers of macrophages and

large multinucleated cells known as Foreign Body

Giant Cells (FBGC). These FBGCs are generally

several fused macrophages and are surrounded by

collagen, and other extracellular matrix (ECM)

proteins (Anderson 1994). ECM proteins also serve

especially as anchors for these giant cells to form

(Ruoslahti & Pierschbacher 1987; Rosales & Juliano

1995). It is these giant cells that not only serve as a

mass transfer barrier if the implant is a biosensor,

but they also have been shown to release lysosomal

enzymes and reactive oxygen intermediates which

can degrade the surface of the implant (Abramson &

Gallin 1990; Zhao et al. 1990; Adams & Hamilton

1992).

The first step in cell adhesion to implanted

devices is the adsorption of proteins. Biomolecule

adsorption has been shown to be altered by the

nanoarchitecture of the surface in vitro (Lampin

et al. 1997; Luck et al. 1998; McFarland et al.

2000; Denis et al. 2002; Suh et al. 2004; Pallandre

et al. 2005) and in vivo (Brauker et al. 1995;

Rosengren et al. 1999; Rosengren & Bjursten

Correspondence: Michael V. Pishko, 3122 TAMU, Texas A & M University, College Station, TX 77845-3122, USA. Tel:

�1(979) 845 3348. Fax: �1(979) 862 3362. E-mail: mpishko@tamu.edu

Nanotoxicology, December 2007; 1(4): 279�290

ISSN 1743-5390 print/ISSN 1743-5404 online # 2007 Informa UK Ltd.

DOI: 10.1080/17435390701781142

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08 2003). Suh et al. showed a decrease in protein

adsorption on silica particles with decreasing pore

size from 45�2.2 nm (Suh et al. 2004). Similarly,

Luck et al. demonstrated that the adsorption of

serum proteins was reduced on polymeric nano-

particles ranging in size from 141�61 nm, respec-

tively (Luck et al. 1998). Lampin et al. concluded

that increasing surface roughness of poly(methyl

methacrylate) (PMMA) to 0.20�3.4 mm, thus

increasing hydrophobicity, resulted in increased

protein adsorption (Lampin et al. 1997). Further-

more, we have previously shown that protein

adsorption is significantly reduced, around an order

of magnitude, on the nanoarchitectured magnetos-

trictive wires (Ainslie et al. 2005).

By disrupting the adsorption of protein on the

surface the nanoarchitecture should also disrupt

the adsorption of cells, since protein adsorption is

the first step in cell attachment. Indeed several

researchers have shown that by altering the amount

of protein adsorbed to the surface, the amount or

success of the cells on the surface is altered also (Naji

& Harmand 1990; Martin et al. 1995; Altankov et al.

1996; Kanagaraja et al. 1996; Lampin et al. 1997;

Steele et al. 1997; Balcells & Edelman 2002; Lan

et al. 2005). Furthermore, Lampin et al. show that

cell adhesion increased with roughness, most likely

due to the change in fibronectin adsorption on the

surface. This was similarly seen by Martin et al. who

observed a reduced number of osteoblasts on

roughened titanium compared to a more smoothed

surface (Martin et al. 1995). Osteoblasts and fibro-

blast adhesion was also shown to be influenced by

the surface state of cobalt chromium based alloys by

Naji and Harmand (1990). Clearly protein adsorp-

tion and cell adhesion go hand-in-hand and can be

mutually altered as a result of surface architecture or

modifications.

The influence of architecture on protein adsorp-

tion led us to hypothesize that nanofabricated

surfaces would significantly inhibit the formation of

an avascular fibrous capsule. To explore this we have

created a nanowire array comprised of Fe-Co-Ni

ternary alloy, imaged with SEM in Figure 1. The

characterization of this array is outlined in the

author’s previous work (Sharma et al. 2004).

The diameter of the wires was 75 nm, which

approaches the dimensions of several proteins (e.g.,

immunoglobin G � 45�23.5 nm, fibronectin � 45�0.6 nm and albumin � 0.4�14 nm) (Zhang et al.

1998). The author’s previous work has highlighted

the attenuation of protein adsorption on the surface

of the nanowires (Ainslie et al. 2005). The wire

length is on the order of 10 microns, as are most

mammalian cells. To explore the hypothesis that cell

adhesion will be altered as a result of the nanowire

architecture we have studied the cell shape of

macrophages and cytokines released by those macro-

phages on the nanowire, control wafer (flat surfaces

of the same materials as nanowires) and tissue

culture polystyrene (TCPS).

Figure 1. Nanowire array as visualized with FE- SEM. The deposition conditions are 15 V AC 1000 Hz for 15 min. The alumina

membrane has been partially etched in 0.2 M NaOH solution for 35 min to expose standing nanowire array. The free standing nanowires

are 4 microns in length and have 75 nm diameter.

280 K. M. Ainslie et al.

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08 Materials and methods

Materials were purchased from Sigma (St Louis,

MO, USA; Dulbecco’s Modified Eagles Medium

(DMEM), Antibiotic-antimycotic, trypsin-EDTA

solution 0.25%, Type 1 collagen from rat tail,

lipopolysaccharide (LPS) from Escherichia coli

0127:B8, and Phosphate Buffered Solution (PBS;

0.15 M NaCl, 0.001 M KH2PO4, and 0.002 M

Na2HPO4 pH 7.4)), ATCC (Manasas, VA; RAW

264.7 mouse macrophages), Hyclone (Logan, UT;

Standard Fetal Bovine Serum [FBS]), and Molecu-

lar Probes (Eugene, OR; Cell Tracker Green

CMFDA).

Scanning electron microscopy

High resolution images were collected with a field

emission SEM (JEOL 6700F) after coating the

samples with gold.

Surface fabrication

The nanowires were developed in a method identical

to that outlined by Sharma et al. (2004). The control

(Fe-Co-Ni) wafers are flat surfaces comprised of the

same material as the nanowires, but lacking the

architecture of the wires. The electrolyte solution for

Fe-Co-Ni film deposition was the same as used for

nanowire deposition (14.055 g/l of CoSO4.7H2O,

52.5718 g/l of NiSO4.6H2O, 5.56 g/l of FeS-

O4.7H2O, and 24.7328 g/l of H3BO3). The deposi-

tion of the alloy was carried out at a current density

of 10 mA/cm2 for 30 min.

Cell culture

For both cell types, cell culture media was comprised

of 10% FBS and 1% antibiotic-antimycotic in

DMEM. The cells were maintained in T-75 poly-

styrene culture flasks at 5% COB2B and 378C and

subcultured by cell scraping. For each subculturing,

cells were assigned a passage number. For all

experiments presented here, passages 4�9 were used.

For cell adhesion and cytokine expression experi-

ments, cells were suspended in the media and

separated from that media through centrifugation.

The cells were either resuspended in media with

FBS (DMEM with 1% antibiotic-antimycotic and

10% FBS) or in FBS-free media (DMEM with 1%

antibiotic-antimycotic). The cells were diluted to a

concentration of 50,000 cells per a milliliter. They

were then seeded at 0.5 ml per a 24-well plate well.

For the desired cases, nanowires or control wafers

were placed at the bottom of the well with the

surface occupying the rectangular area of the circular

well. Experimental plates were maintained until

point of imaging wherein the surface for that

particular time point was removed from the incu-

bator and imaged.

For collagen pre-treated surfaces type 1 collagen

from rat tail was suspended in accordance with

manufacturer’s direction. The collagen was diluted

to a 0.1% solution in PBS and incubated with the

surfaces overnight in the refrigerator in a 24-well

plate. Surfaces were rinsed with fresh PBS and

warmed to 378C before cells were introduced. FBS

free media was used in conjunction with the collagen

experiments.

Cell imaging

Cells were imaged at discrete time points with

fluorescent dye Cell Tracker Green CMFDA†.

Cell tracker solution was prepared and incubated

with cells in accordance with manufacturer’s direc-

tions. Samples were washed in PBS prior to imaging.

The fluorphore laden cells were imaged with a

fluorescein isothiocyanate (FITC) filter on a Zeiss

Axiovert 200M. Cell number, area, and length were

calculated by manually tracing or measuring the cell

via AxioVision LE version 4.3 (Zeiss). Circularity is

calculated by Equation 1.

Circularity�Area

pL2

4

Equation 1

where area is the area of the cell and L is the length

of the cell.

Cytokine profile with Luminex

CytokineProfilerTM Testing Service from Upstate

(Lake Placid, NY, USA) was used to measure 18

cytokines using Luminex† technology. Briefly, by

using a Luminex† 100 instrument from the Lumi-

nex† Corporation (Austin, Texas) multiple cytokines

in a single sample can be detected at simultaneously.

This is achieved by using a combination of fluores-

cent microbeads and reporter molecules. The Lumi-

nex† 100 uses two lasers to detect the fluorescent

microbead and the reporter molecules. By using

standard curves for every cytokine, the cytokines in

the samples can be determined and quantified.

Activation of macrophages

In the Luminex† cytokine profile studies it was

necessary to show the cytokine profile of activated

macrophages as a positive control. As such we could

then determine if the nanowire or control wafer

surfaces were immunogenic. To fully activate macro-

phages, lipopolysaccharide (LPS) was used. LPS is a

Macrophage cell adhesion and inflammation cytokines on magnetostrictive nanowires 281

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08 major constituent of the cell wall of gram-negative

bacteria, is highly immunogenic, and is one of the

best activators of macrophages (Paul 1994). LPS

was added at 1 mg/ml to macrophages seeded on

TCPS to obtain the maximum production of in-

flammatory cytokines. By comparing the amount

and type of cytokines released by the LPS activated

macrophages to the nanowire or control wafer

cultured macrophages, it can be determined if

surfaces are immunostimulatory or not.

Statistical analysis

All graphical figures present the average of three or

more data points. The confidence interval of the

data set was determined with Microsoft Excel with

an alpha value of 0.05 (CI of 95%). The differences

between two groups of data were determined by a

Student’s t-test (Microsoft Excel; Redmond, WA,

USA). Differences among groups were determined

with an ANOVA (Minitab; State College, PA, USA).

A p-value less than 0.05 is considered significantly

different.

Results

Cell shape

The average area occupied by the macrophages on

the surfaces of interest is presented in Figure 2.

Macrophages on TCPS after 6 h occupied 309.79

27.6 mm2 with FBS present and 204.1916.7 mm2

without FBS, as represented by Figure 2a and 2b,

respectively. Similarly Figure 2 also shows macro-

phages occupied 294.5927.1, with FBS, and

251.6915.1, without FBS, mm2 on control wafers

(CW) after 6 h. While on the nanowires the

macrophages occupied 236.3920.1 mm2 with FBS

and 197.2915.7 without FBS. The area was related

to the circularity of the cells which is presented in

Figure 3. Figure 3 shows the circularity factor of

macrophages as 0.55290.077, with FBS, and

0.58690.057, without FBS, for the TCPS surfaces

after 6 h. As seen in Figure 3 the circularity factor of

macrophages at 6 h were 0.82990.065, with FBS,

and 0.74690.065, without FBS, for the control

wafer surface. The circularity factor of the macro-

phages on nanowires at 6 h was 0.88690.043 with

FBS and 0.89190.056 without FBS. The area and

circularity results demonstrate that cells attached to

the surface of the nanowire are considerably smaller

and more circular than cells on control wafers and

TCPS. Fluorescent micrographs supporting this

conclusion are given in Figure 5a and 5b. We can

thus conclude that the macrophages on these

nanowire surfaces did not spread and were not

thriving.

To examine the adhesion of macrophages under

conditions in which adhesion molecules were already

present on the surface, the surfaces were pretreated

with collagen (Figure 4 and Figure 5c). Here we

compared the control wafer and nanowire surface,

and found that macrophages on the control wafers

occupied 413.78935.47 mm2 as compared to the

162.89913.83 mm2 that the cells occupied on the

nanowires, after 6 h. The circularity of the cells on

the surfaces was greatly diverse also, in that on the

50

100

150

200

250

300

350

400(a)

(b)

0

50

100

150

200

250

Time (hrs)

CW NW TCPS

*

*

*

* *

*

* *

*

Cel

l Are

a (s

qu

are

mic

ron

s)

1 2 3 4 5 6

Cel

l Are

a (s

qu

are

mic

ron

s)

Figure 2. Cell area in square microns of macrophages with (a)

FBS, (b) without FBS. Data is presented as average plus or minus

a 95% confidence interval. Tissue culture polystyrene is repre-

sented as TCPS, CW stands for control wafers of Ni/Co/Fe which

are flat but comprised of the same material as the nanowires

(NW). An * indicates significance with respect to the area of the

macrophages on the control wafer flat surface. Cells with a small

area are less spread then cells with a larger area. The lack of

extensions on the cell could be indicative of cell viability, in that

cells that occupy less area are less viable. Initially the cells are

round and by 6 h, the cells occupy more area, especially on the flat

control wafer and tissue culture polystyrene surface. The cells on

the nanowire surface are significantly less spread over time in the

presences of FBS.

282 K. M. Ainslie et al.

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control wafers the circularity is 0.52190.086 com-

pared to the nanowires where the circularity is

0.82890.063. The collagen pretreatment seemed

to enhance the adhesion of cells to the flat control

wafer surface, but not significantly alter the adhesion

of the macrophages to the nanowires.

Inflammation cytokines

The results of Luminix† inflammation panel are

presented in Table I. These expression levels are

reported in picograms per milliliter. Bold values

indicate where the expression level was significant

with respect to the positive LPS control at that time

Cir

cula

rity

1 2 3 4 5 6

Cir

cula

rity

0.2

0.4

0.6

0.8

1

1.2

(b)

(a)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

Time (hrs)

CW NW TCPS

**

* *

* *

*

*

* * * * * * * *

Figure 3. Circularity of macrophages with (a) FBS, (b) without

FBS. Circularity is calculated via area divided by p multiplied by

the square of one-half the cell length. A value close to 1 represents

a perfect circle, and a rectangle would be 0.57 or less. Data is

presented as average plus or minus a 95% confidence interval. An

* represents significance with respect to the circularity of the cells

on the control surface. Tissue culture polystyrene is represented as

TCPS, CW stands for control wafers of Ni/Co/Fe which are flat

but comprised of the same material as the nanowires (NW).

Overtime, the graph represents a spreading of cells on the TCPS

surface in that the circularity generally goes down, especially in the

presence of FBS. The circularity of the cells on the control wafer

surface spread somewhat over time, however the cells on the

nanowire surface have a constant circularity and do not appear to

spread.

100

200

300

400

500

600

(b)

(a)

Cel

l Are

a (s

qu

are

mic

ron

s)

0

0.2

0.4

0.6

0.8

1

1 2 3 4 5 6

Time (hrs)

Cir

cula

rity

CW NW

*

*

**

* ** *

Figure 4. Cell area in square microns (a) and circularity (b) of

macrophages with collagen pretreatment. Circularity is calculated

via area divided by p multiplied by the square of one-half the cell

length. A value close to 1 represents a perfect circle, and a

rectangle would be 0.57 or less. Data is presented as average plus

or minus a 95% confidence interval. An * represents significance

with respect to the shape factor of the cells on the control surface.

Tissue culture polystyrene is represented as TCPS, CW stands for

control wafers of Ni/Co/Fe which are flat but comprised of the

same material as the nanowires (NW). Here an extracellular

matrix protein, collagen, was added to enhance cell adhesion.

Both figures conclude that the cells on the nanowire surface are

more circular and have less area than those on the control wafer.

The cells are more round and smaller, indicative of cell death or

apoptosis.

Macrophage cell adhesion and inflammation cytokines on magnetostrictive nanowires 283

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point. An * signifies significance with respect to the

expression of macrophages on the nanowires (NW).

A description of each cytokine is given in Table II.

Figure 6 shows a direct comparison between cyto-

kine expression levels between the nanowire and

control wafer surfaces.

Discussion

Cell number and shape

This study examined cell spreading in two ways:

First by monitoring its area; and second by noting

the shape of the cell and how circular it is. In general

Figure 5. Fluorescent micrographs of macrophages on the respective surfaces are presented (a) in the presences of FBS, (b) absence of FBS

and (c) with collage pretreatment. The micrographs were imaged with a 10� lens. Individual isolated cells were traced, at 40�, to obtain

area and circularity measurements. The lack of spreading and more circular nature of cells on the nanowire surface can be observed for all

the conditions.

284 K. M. Ainslie et al.

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the less spread a cell is, the less two dimensional area

it occupies. Linked to this is circularity which relates

how circular the cell is, the thought being that

circular cells are weakly adherent to the substrate.

If they find a viable surface on which to spread, they

will adhere and become less circular. A circular cell

(i.e., one that is spherical) will occupy the least

amount of area on a surface. In the absence of FBS

the cells appear to spread and adhere (Figure 2) less

over all and do not adhere as quickly on the TCPS

and NW surface. Although the area of the cell is

lowest at the one hour time point, more so for the

cultures without FBS, they are highest at the two

hour time point. Without FBS, the cells appear to

equilibrate to a smaller area at the three hour time

point and then spread, since all the average areas are

less at the three over six hour time point. In contrast,

the cells in the presence of FBS appear to shrink at

the five hour time point and then increase in area

again at the six hour time point. Similar trends for

the circularity are seen on the TCPS and CW

surface with respect to circularity in the FBS

Table I. Inflammation panel as measured with Lumix† for 18 cytokines. Table (a) is at the 24-hour time point and Table (b) is at the 48-

hour time point. Values are reported in picograms/milliliter and as average995% confidence interval. At least three samples were analyzed

for each surface at each time point and for DMEM. Where samples had one or more replicates detected below detection limits, the value is

reported as less than the maximum reported value or in the case of no reported value, the detection limit. Bold values indicate where the

value is significant with respect to the maximum or LPS value at that time point. The * denotes significance with respect to the nanowires

(NW). The DMEM values serve as background values for all cases and time points. Tissue culture polystyrene is represented as TCPS, CW

stands for control wafers of Ni/Co/Fe which are flat but comprised of the same material as the nanowires (NW). Except for chemokines

(MCP-1 and RANTES) and anti-inflammatory cytokines (IL-10 and IL-6), the cytokine expression of macrophages on the nanowire

surface is not significantly different from the LPS stimulated macrophages, indicating that the conditions are equally inflammatory. The

values of pro-inflammatory IFN-g, and IL-1a cytokine expression from macrophages on the control wafer surface are significantly lower at

the 48-hour time point compared to expression from macrophages on the nanowire surface, concluding that the flat surface is less

inflammatory than the nanowire surface.

(a) NW 24 hr CW 24 hr TCPS 24 hr LPS 24 hr DMEM

GM-CSF B228 B194 B216 B194 209957

IFNg 140925 B61 B145 94914 B70.9

IL-10 52950 B33 B83 324914 B34.6

IL-12p40 40915 4496 2799* 54931 B47.8

IL-12p70 65918 B58 B58 B48 B58

IL-13 87931 B122 4892* B76 4795*

IL-17 47939 B22 B49 17913 44925

IL-1a 1,4929368 98932* 27922* 308916 B15

IL-1b 115940 101970 112914 169977 133929

IL-2 490.2 B3 691* 492 490.0

IL-3 B8 B8 B8 B8 B8

IL-4 25913 32910 21917 31915 2590.1

IL-5 109926 1229114 80936 45913 121919

IL-6 55917 29995* 53919 1,8539323 1592*

KC B66 B66 B66 B66 B65

MCP-1 114932 138960 11294 813991 13195

RANTES 3599 B11 1393* 2,3189392 B51

TNFa B3 B3 B3 7719138 B3

(b) NW 48 hr CW 48 hr TCPS 48 hr LPS 48 hr

GM-CSF 86921 84938 71980 106917

IFNg 12399 62917* 50954* 60925

IL-10 61912 3595* 1999* 551947

IL-12p40 5194 B48 64931 B48

IL-12p70 53921 34923 B78 6391

IL-13 2697 19913 66930 33929

IL-17 B91 B96 38934 B14

IL-1a 931992 1409129* 37917* 757942

IL-1b 200910 230955 1899121 278935

IL-2 492 591 B6 792

IL-3 490.1 291 290 391

IL-4 32910 1295* 1696* 2895

IL-5 77915 7397 84931 67924

IL-6 127928 3259114* 189938 3,2659363

KC 1493 15910 38913 25911

MCP-1 73918 100935 1709141 2,7509161

RANTES 4398 34910 3293 3,9329347

TNFa B2 B3 B3 1,5939154

Macrophage cell adhesion and inflammation cytokines on magnetostrictive nanowires 285

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supplemented cultured. The circularity in the FBS

free cultures appears to deviate minimally with

respect to the individual surface at the different

time points. Overall FBS appears to alter the time

course of the spreading of the cell, with respect to

both area and circularity.

In both the presence and absence of FBS, the area

the cells occupied on the nanowire surface was

significantly less than on the control wafer surface,

as seen in Figure 2 after 6 h. Furthermore, the cells

were far more circular on the nanowire surface than

on the control wafer surface. This indicates that the

cells were not thriving on the nanostructured surface

and that this was not simply a property of the metal

oxide surface but rather the nanoarchitecture itself.

These results support the hypothesis that these

nanowire array might aid in the limiting of biofouling

on the surface of an implant.

The presence of FBS in the cell system is con-

founding. FBS has growth factors that stimulate cell

growth and proteins (cell adhesion molecules) that

can aid in cell adherence. As such, one must delineate

how growth factors or cell adhesion molecules

contributes to increase cell spreading and decrease

cell circularity. To potentially reduce the confound-

ing effects of FBS, the nanowire and control wafers

surfaces were pre-coated with 0.1% type 1 collagen

from rat tail. The aim of this experiment was to test

Table II. Cytokine definitions inflammation panel results in Table I. (Paul 1994; Al-Saffar et al. 1996).

Role(s)

GM-CSF Granulocyte/macrophage colony-

stimulating factor

A hematopoietic growth factor with a regulatory effect on the transformation of

immature macrophages into multinucleated giant cells (MNGC). Plays a role in

antibody stimulation.

IL-1 Interleukin-1 Major inflammatory mediator produced by activated macrophages. Increases local

blood flow, fever, release of other cytokines and enhanced expression of adhesion

molecules.

IL-10 Interleukin-10 Produced by macrophages and cytotoxic T-cells. Inhibits the production of

pro-inflammatory cytokines and interferences with macrophage-mediated antigen

presentation.

IL-12 Interleukin-12 Product of macrophages and B-cells that enhances the synthesis of IFN-g and

stimulates proliferation of NK, and T-cells.

IL-13 Interleukin-13 Produced by macrophages and cytotoxic T-cells. Inhibits the production of

pro-inflammatory cytokines and interferences with macrophage-mediated antigen

presentation. Aids in the differentiation of B-cells.

IL-17 Interleukin-17 Pro-inflammatory cytokine which induces stromal cell pro-inflammatory responses

and the production of hematopoietic cytokines.

IL-2 Interleukin-2 Produced by T-cells in response to antigenic or mitogenic stimulation. Also termed

a T-cell growth factor. Increases the growth and activity of macrophages, B-cells,

NK cells and other white blood cells.

IL-3 Interleukin-3 Produced in NK and T-cells to target mast and stem cells for growth and histamine

release.

IL-4 Interleukin-4 Participates in cell fusion related to granulomas and is anti-inflammatory. Also

related to allergic inflammation including stimulating of basophil development,

eosinophil chemotaxis, and expression of IgE receptors on B cells. Signals the

formation of giant or fused macrophages.

IL-5 Interleukin-5 Produced in T-helper cells and activates B-cells and the antibody response.

Proliferates and differentiates B-cells.

IL-6 Interleukin-6 Produced by T-cells, endothelial cells, monocytes and fibroblasts. Promotes

monocytes differentiations, increased number of platelets, and synthesis of

fibrinogen in T- and B-Cells and macrophages. Involved in the downregulation of

neutrophil superoxide production.

IL-8 Interleukin-8 Produced by monocytes, lymphocyte and Europhiles when stimulated with IL-1a,

IL-1b, or TNF. In neutrophils enhances chemotactic and degranulation response.

Induces an increase in the expression of cell surface adhesion molecules.

IFN-g Interferon-gamma Product of T- and natural killer (NK) cells. Increases generation of highly reactive

oxygen species (e.g., superoxide anion and hydrogen peroxide) and alters the cell

surface antigens of macrophages permitting them to engulf pathogens.

KC KC Potent neutrophil activator. Plays an important role in inflammation.

MCP-1 Monocyte chemotactic protein 1 Produced by most non-lymphocytes and plays roles in chronic inflammation,

activation of macrophages, humoral response and histamine release.

RANTES Regulated upon Activation, Normal T-cell

Expressed and Secreted

Produced in T-cells, endothelial cells and platelets. A T-cell product that promotes

mononuclear infiltrating (e.g., migration of macrophages into cancerous growth)

TNF Tumor Necrosis Factor Derived from activated macrophages. Is associated with the production of a fever

and promotes the stimulation of most other pro-inflammatory mediators. It is

primarily associated with the induction of cellular apoptosis.

286 K. M. Ainslie et al.

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that the presence of an adhesion promoting protein

aid in cell spreading without the presence of growth

factors. The macrophages on the control wafer sur-

face were much more spread than in the presence of

FBS, also they were much less circular. The differ-

ence between the area and circularity on the control

wafers and nanowires was more profound in the

presence of collagen. The macrophages on the

nanowires were slightly more circular, but occupied

roughly the same area as those on the control wafer

surface. There were two reasons for the potential

change of cell viability on the nanowires over the

control wafers. One, the disruption of cell adhesion

on the nanowires could be the result of the con-

formation of collagen on the surface i.e. collagen is

present in a conformation that the cells are unable to

attach. Secondly a lack of adsorbed collagen may

simply reduce cell adhesion and spreading. The

authors have previously shown that protein adsorp-

tion on the nanowires is attenuated by almost an

order of magnitude (Ainslie et al. 2005) It would

appear that despite the presence of ECM proteins,

the macrophages on the nanowires were not suffi-

ciently spread and much more circular than on the

control wafer surface. This trend was seen by the

authors with fibroblasts and endothelial cells also

(Ainslie et al. 2007). It would appear that the

nanowire surface disrupts the adherence of cells to

the surface, over a flat surface of equal material. To

look further into the ability of the surface to inhibit

the biofouling response, cytokines released from the

macrophages on these surfaces were studied.

Inflammation cytokine response

We examine cytokine release profiles on these

surfaces for two reasons. The first reason was that

an inflammatory response can occur when there is a

high amount of cell death on a surface (Matzinger

2002), which might result from rounded cells on a

surface. The second reason why we examined the

immune response is that biofouling response is

generally characterized as a chronic inflammatory

response. When an implant is placed in vivo, a high

percentage of cells bound to the surface are host

inflammatory cells Padera & Colton 1996). To

measure the cytokines produced with macrophages

cultured on nanowire surfaces we used Luminex†

Technology to measure a broad panel of expressed

cytokines. A description of the cytokines tested can

be seen in Table II. Cytokines released by macro-

phages can either stimulate (e.g., IL-1, IL-12,

1

10

100

1000

10000

IFNg IL-1a IL-4 IL-6 IL-10 RANTES

)L

m/smar

goci

p(n

oitcu

dor

Pe

nikoty

C

NW CW TCPS LPS

24 48

24 48

24 48

24 48

24 48

24 48

** ##

**

**##

** ##

* ###

* ###

# **###

*# # #

Figure 6. Cytokine data presented in Table I which highlights the cytokines that are statistically different when comparing the macrophages

on the surface of the nanowires and the other surfaces. IFN-g stands for IFN-g, IL-1a stands for IL-1a, tissue culture polystyrene is

represented as TCPS, and CW stands for control wafers of Ni/Co/Fe which are flat but comprised of the same material as the nanowires

(NW). The 24 and 48 indicators signal the data from 24- and 48-hour time points. Data is presented as average 995% confidence interval.

An * indicates significance with respect to the concentration of cytokine released from macrophages on the nanowires and a # represents

significance with respect to the LPS stimulated macrophage production. The production of pro-inflammatory cytokines IFN-g, and IL-1afrom the macrophages on the surface of the nanowires is not significantly different from LPS stimulated macrophages at any of the time

point, indicating that the conditions are equally inflammatory.

Macrophage cell adhesion and inflammation cytokines on magnetostrictive nanowires 287

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08 MCP-1 or TNF-a) or inhibit (e.g., IL-10, IL-4,

and IL-13) the inflammatory response.

IFN-g

As shown in Table I, the macrophages production of

IFN-g increased significantly on nanowire surfaces.

IFN-g is secreted by thymus derived lymphocytes

(T-cells), natural killer (NK) cells, and other im-

mune cells of the immune system. IFN-g secretion

by T-cells and NK cells is usually a strong indicator

of inflammation; however, IFN-g secretion by

macrophages has not been well studied and further

studies would need to be performed to show the

significance of the macrophages secreting IFN-gwhen cultured with the nanowires.

IL-1

IL-1 is an inflammatory cytokine of importance in

implant material pathologies. Ojo-Amaize et al. has

shown that women with breast implants have ele-

vated levels of IL-1, a common inflammatory

cytokine, circulating in their blood stream (Ojo-

Amaize et al. 1996). They concluded the IL-1 in the

bloodstream was due to a chronic inflammatory

response against the breast implant. In addition, it

has been shown that macrophages can cause bio-

corrosion on such metals as stainless steel and

implant alloys that contain cobolt (Lin & Bumgard-

ner 2004; Bailey et al. 2005). With this bio-corrosion

of metals there was a related increase in the

production of IL-1 b. We did not observe an increase

of production of IL-1 b on the control wafers or the

nanowires. Our results on the nanowires showed an

increase in IL-1a production compared to control

wafers, LPS, and TCPS at the 24- and 48-hour time

point. Even though IL-1 a and IL-1 b belong to the

same family of cytokines and have similar structure,

the regulation of production was completely differ-

ent. It is known that the release of IL-1a in vitro can

occur as a consequence of cell injury and cell death

(Paul 1994). It has been shown that 3T3 fibroblasts

cultured on the same nanowires used in this paper

resulted in significantly increased cell death, possibly

the result in the macrophages secreting high levels of

IL 1-a (Ainslie & Pishko 2007).

IL-4

At the 48-hour time point IL-4 was up-regulated on

the nanowire surface. The level of IL-4 expression in

the nanowires was higher than LPS, but not

significantly so. The up-regulation of IL-4 could be

of a concern in the formation of FBGC, which is of

concern in implanted devices. The formation of

these cells is sparked by the anchoring of ECM

proteins and the production of IL-4 and GM-CSF

(Jenney et al. 1998). Although IL-4 was up-regu-

lated, GM-CSF was not and actually less than the

negative control level in DMEM. This would

indicate that IL-4 production on the nanowire sur-

face was responsible for other cytokine actions and

not just the formation of FBGC. Like IL-10, the

presence of IL-4 could be that of an anti-inflamma-

tory response (Paul 1994).

IL-10

The amount of IL-10 produced by the macrophages

on the nanowires at the 48 hour time point was

significantly higher than the control wafers or TCPS.

This indicates that the nanoarchitecture seems to

lead to an up-regulation of anti-inflammatory IL-10.

IL-10 is a cytokine known to inhibit inflammatory

responses, especially in parts of the body that are

sensitive to inflammation such as the eye (Skelsey

et al. 2003) and the gut (Alpan et al. 2004). It has

been shown that nanofibrous PTFE (nPTFE) re-

sulted in the increased expression of IL-10 by

macrophages on the surface of this material (Ainslie

et al. 2007). This phenomenon that was found with

nPTFE may be the same phenomenon that was

found in the experiments described here.

IL-6

IL-6 seemed to be significantly up-regulated on the

control wafers over the nanowires or TCPS. Prior

research has shown IL-6 plays a key role in experi-

mental atherosclerosis. It is one of the key cytokines

that result in inflammation and plaque formation

(Schieffer et al. 2004). Also it has been shown that

IL-6 is secreted in vivo around copper implants

(Suska et al. 2005). Additionally, copper sulfate has

been shown to stimulate VEGF production in

keratinocytes (Sen et al. 2002). The nanowires

have also been shown to upregulate VEGF produc-

tion from 3T3 fibroblasts, equal to that of copper,

although no copper is in the alloy (Ainslie & Pishko

2007). Although no copper is present in the alloy

both IL-6 and VEGF were up-regulated. The IL-6

reduction in the nanowires may be a result of the

increase of IL-10. It has been shown that the

endogenous secretion of IL-10 by macrophages

results in the reduction of IL-6 (Wilson et al.

2005). This result can explain why the nanowires

have a much lower level of IL-6 compared to the

control wafers.

RANTES

RANTES is a chemokine and was elevated at the 24-

hour time point in nanowires compared to the

288 K. M. Ainslie et al.

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08 control wafers and then statistically equal to the

control wafers at the 48-hour time point. These

values at both time points were slightly higher than

the TCPS. Chemokines are a group of molecules

that are secreted by macrophages to recruit macro-

phages and other immune cells. Chemokines are

usually secreted at a site of inflammation to recruit

other cells to the site. The RANTES production is in

no way as high as the production by macrophages

stimulated by LPS, in fact it was roughly two orders

of magnitude less. Due to the small amount of

RANTES production and lack of significance at the

48-hour time point, the ability of the nanowires or

control wafers to recruit macrophages seems minor.

IL-13 and IL-2

Briefly, IL-13 and IL-2 were respectively up- and

down-regulated on the nanowire surface, compared

to TCPS, at the 12 hour time point. IL-13 typically

limits the production of inflammatory cytokines,

whereas IL-2 is termed a T-cell growth factor and

stimulates the activation of such cells. Since both of

these cytokines were no longer significant at the 48-

hour time points; their significance at the 24-hour

time point may be considered inconclusive.

MCP-1

The production of MCP-1 is significantly less on all

the surfaces compared to the LPS-stimulated control

at the 24- and 48-hour time points. Like RANTES,

MCP-1 is a chemokine that is linked with chronic

inflammation. The amount of MCP-1 expressed by

macrophages on the nanowires was on average the

lowest at the 48-hour time point, however it is not

significant. A reduced production of MCP-1 in vitro

may indicate that the surface does not stimulate a

chronic response associated with implants.

The potential for inflammation on nanowires

From the data presented with respect to IL-1a, IFN-

g, IL-10, and IL-6, in addition to the rest of the data

presented in Table II and Figure 6, one may

conclude the Ni-Co-Fe alloy that comprises the

nanowire and control wafer surface is immunogenic

and may yield an inflammatory response in vivo. The

nanoarchitecture of the surface appeared to affect

the production of cytokines, in particular the up-

regulation of IFN-g, IL-10, and IL-1a and down

regulation of IL-6. Further studies must be per-

formed to see if these nanowires would cause an

inflammatory response in vivo. The only other study

that includes the results of nanoarchitecture on

numerous inflammatory cytokine releases does not

compare a surface with and without nanoarchitec-

ture. It does, however, report that on nanofibrous

PTFE (nPTFE) there is not a significant up-regula-

tion of any of the 18 cytokines listed below after

48 h compared to TCPS (Ainslie et al. 2007, in

preparation).

Conclusion

This study compares a two Ni/Co/Fe surfaces, one

that is flat and one that is a nanowire array

comprised of nano- and micro-architecture. We

presented conclusive studies that show that na-

noarchitecture, in the form of a nanowire array,

disrupted cell adhesion by preventing cell adhesion

and spreading, and leads to the up-regulation of IL-

1a, IFN-g, IL-6 and IL-10 expression by adherent

macrophages. Furthermore, the architecture leads to

an inflammatory response above that of the flat

surface. The surface presented here has the ability to

prevent cell adhesion, as seen with biofouling, but

might create an inflammatory response in vivo.

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