*Corresponding author. Tel.: 001 608 265 8171.

Biomaterials 20 (1999) 573—588

Effects of synthetic micro- and nano-structured surfaceson cell behavior

R.G. Flemming!, C.J. Murphy!, G.A. Abrams!, S.L. Goodman", P.F. Nealey#,*!Department of Surgical Sciences, School of Veterinary Medicine, UW-Madison, 2015 Linden Drive West, Madison, WI 53706, USA

"Center for Biomaterials MC-1615, University of Conn. Health Center, Farmington, CT 06030, USA#Department of Chemical Engineering, School of Engineering, UW-Madison, 1415 Engineering Drive, Madison, WI, USA

Received 10 June 1998; accepted 30 September 1998

Abstract

Topographical cues, independent of biochemistry, generated by the extracellular matrix may have significant effects upon cellularbehavior. Studies have documented that substratum topography has direct effects on the ability of cells to orient themselves, migrate,and produce organized cytoskeletal arrangements. Basement membranes are composed of extracellular matrix proteins and foundthroughout the vertebrate body, serving as substrata for overlying cellular structures. The topography of basement membranes isa complex meshwork of pores, fibers, ridges, and other features of nanometer sized dimensions. Synthetic surfaces with topographicalfeatures have been shown to influence cell behavior. These facts lead to the hypothesis that the topography of the basement membraneplays an important role in regulating cellular behavior in a manner distinct from that of the chemistry of the basement membrane.This paper describes the topography of the basement membrane and reviews the fabrication of synthetic micro- and nano-structuredsurfaces and the effects of such textured surfaces on cell behavior. ( 1999 Elsevier Science Ltd. All rights reserved

Keywords: Basement membranes; Cell behavior

1. Introduction

Fundamental knowledge of cell—substrate interactionsis important for tissue engineering, in the development ofmedical implants, and the production of pharmaceu-ticals. Cell—substrate interaction may also explain differ-ences in cell behavior in vivo and in vitro. To gain insightinto these interactions, a logical approach is to investi-gate the substrates on which cells attach and grow inliving systems.

Basement membranes are found throughout the verte-brate body and serve as substrata for overlying cellularstructures. Basement membranes consist of extracellularmatrix (ECM) components, including fibrous collagen,hyaluronic acid, proteoglycans, laminin, and fibronectin.The effects of the surface chemistry on cell and tissuefunction has been explored extensively in the past fewdecades. Hyaluronic acid, for example, has been shownto inhibit cell—cell adhesion and promote cell migration,

and laminin can prevent cell migration [1, 2]. Fibronec-tin has been observed to allow greater translocation ofcells than does laminin [3], and excess ECM has beenshown to inhibit endothelial cell replication by causingincreased cell—ECM adhesion and cytoskeletal re-arrangements [4]. One well-understood mechanism inwhich components of the basement membrane modulatecell behavior is the activation of plasma membrane integ-rin receptors, such as RGD that bind to ligands on thebasement membrane [5—7]. In addition to mediating cellattachment, integrin receptors also act as signaling mol-ecules, activating intracellular pathways important in cellgrowth and survival [8—11]. In the cornea, componentsof the basement membrane have been shown to influencethe distribution of cytoskeletal elements and of their ownintegrin receptors [12, 13] as well as modulating prolifer-ation [14], migration [15, 16], and differentiation [17].

The mechanical and tensile properties of the basementmembrane also influence fundamental cell behaviors.Cells can sense restraining forces and respond bystrengthening cytoskeletal linkages [18]. The strength ofthese integrin—cytoskeleton links depend on both the

0142-9612/99/$ — see front matter ( 1999 Elsevier Science Ltd. All rights reserved.PII: S 0 1 4 2 - 9 6 1 2 ( 9 8 ) 0 0 2 0 9 - 9

Fig. 1. Scanning electron micrograph of a corneal epithelial basementmembrane of Macaque monkey. After the corneal epithelium wasstripped, the cornea was fixed in 2% glutaraldehyde, dried by thecritical point method, and imaged on Hitachi S-900 scanning electronmicroscope (Bar"1 lm).

matrix rigidity as well as its biochemical composition[18]. When cells bind to ligands on the basement mem-brane, the cell receptors can act as mechanochemicaltransducers, activating signal transduction pathwaysand modulating gene expression [19]. Fibroblasts at-tached to strained collagen matrix produced more tenas-cin and collagen XII than those attached to a morerelaxed matrix [19]. Resilience and deformability alsoinfluences in vitro migration and morphology of somecells [3].

In addition to biochemical and mechanical properties,basement membranes possess a complex, three-dimen-sional topography consisting of nanometer sized features.Physical topography is known to affect cell behavior. Forreviews on the subject of the influence of substratumtopography on cells, see Curtis and Clark [20], vonRecum and van Kooten [21], Curtis and Wilkinson [22],Singhvi et al. [23], and Clark [24]. Paul Weiss, amongothers, pioneered the field of ‘contact guidance’ duringthe 1930s, 1940s and 1950s [25]. Rosenberg claimed, asearly as 1962 and 1963, that nanometer sized featuresinfluenced cells [26, 27]. Despite recognition of the im-portance of substratum topography, relatively little isknown about the effects of topographical features ofnanometer scale on cell behavior. The purpose of thispaper is to: (1) describe the nanometer scale topographyof basement membranes, (2) review the techniques usedto fabricate synthetic micro- and nano-structured surfa-ces, and (3) discuss the effects of micro- and nano-struc-tured synthetic surfaces on cellular behavior.

2. Topography of the basement membrane

Basement membranes are comprised of a complexmixture of pores, ridges, and fibers which have sizes in thenanometer range. A scanning electron micrographof a corneal epithelial basement membrane is shown inFig. 1. Abrams and coworkers used scanning electronmicroscopy, transmission electron microscopy, andatomic force microscopy to measure the sizes of featureson the surface of the corneal epithelial basement mem-brane of the Macaque monkey [28]. The average featureheights were between 147 and 191 nm and the averagefiber width was 77 nm [28, 29]. They found that poresmade up 15% of the total surface area of the membraneand had an average diameter of 72 nm [28]. Similarfeatures were observed on the human corneal epithelialbasement membrane [30] and on MatrigelTM, a commer-cially available basement membrane matrix [31].

Shirato et al. studied the glomerular basement mem-brane of the rat kidney and observed a meshwork offibrils 5—9 nm thick and pores 11—30 nm wide on thelamina rara interna and 6—11 nm fibrils and 10—24 nmpores on the lamina rara externa [32]. Yamasaki andcoworkers examined the pores and fibers present in

bovine glomerular and tubular basement membranes[33]. They found pore diameters near 10 nm in theglomerular membranes and pores near 12 nm in dia-meter in the tubular membranes. Fibrous strands wereseen to have widths of 3—15 nm. Another study byHironaka et al. found that the glomerular, tubular, andBowman’s capsule basement membranes of the rat kid-ney all had mean fiber diameters of 6—7 nm while havingmean pore diameters of 9.7, 14.1, and 13.1 nm, respective-ly [34].

3. Fabrication of micro- and nano-structured surfaces

A list of the fabrication strategies employed to createsynthetic substrates with topography is given in Table 1.The majority of the studies used photolithography toproduce features with controlled dimensions and specificshapes. Among the first to employ photolithography tocreate controlled features for the study of cell behaviorwas Brunette et al. [35].

A simplified schematic diagram of the photolitho-graphic process is shown in Fig. 2. A substrate is coatedwith a thin polymeric film called a resist. The resist isexposed to light through a mask such that the lightirradiates only selected regions of the resist. A photo-chemical reaction in the irridiated regions renders thoseregions either more soluble or less soluble in a solventcalled a developer. Hence, immersion in the developeryields either a positive-tone image of the mask, or a

574 R.G. Flemming et al. / Biomaterials 20 (1999) 573—588

Tab

le1

The

effec

tof

text

ured

surfac

eson

cell

beh

avio

r

Fea

ture

Fab

rica

tion

Mat

eria

lFea

ture

Fea

ture

Cel

lty

pe

Cel

lula

reff

ect

Ref

.ty

pete

chniq

ue

dim

ensions

freq

uen

cyst

udi

ed

Gro

oves

Photo

litho

grap

hy

and

reac

tive

ion

etch

ing,

UV

and

glow

disch

arge

trea

tmen

t

PD

MS

cast

ofsilic

on

origi

nal

Squar

egr

oove

s2,

5,10

lmw

idth

0.5

lmde

pth

Equa

lgr

oov

ean

dridge

wid

thR

atder

mal

fibro

bla

sts

2,5

lmgr

oove

sin

duc

edst

ronge

ror

ienta

tion

than

10lm

groov

es;gr

ow

thlo

wer

on

UV

trea

ted

surfac

eth

anon

glow

disch

arge

trea

ted

surfac

e

[62]

Photo

litho

grap

hy

and

reac

tive

ion

etch

ing

Quar

tz0.

5,5,

10,25

lmw

idth

0.5,

5lm

dep

thEqua

lgr

oov

ean

dridge

wid

thM

urine

P38

8D1

mac

rophag

eC

ells

spre

adfa

ster

on

shal

low

groove

s,but

elon

gate

dfa

ster

on

deep

ergr

oov

es;m

ore

elong

atio

non

wid

ergr

oove

s;orien

tation

dep

enden

ton

dep

thdu

ring

firs

t30

min

;60

%m

ore

F-a

ctin

ince

lls,

ingr

oov

es;L

PS-a

ctiv

atio

nen

hance

dorien

tation

[98]

Photo

litho

grap

hy

and

reac

tive

ion

etch

ing

Quar

tzSq

uar

egr

oove

s0.

98—4.

01lm

wid

th1.

12—1

.17

lmde

pth

Gro

ove

and

ridg

ew

idth

sim

ilar

Mes

ench

ymal

tiss

ue

cells

Cel

lsm

igra

ted

along

groo

ves;

cells

bec

ame

high

lypo

larize

d;hig

hest

alig

nm

enton

wid

estre

pea

tsp

acin

g

[97]

Photo

litho

grap

hy

and

anisot

ropi

cet

chin

g,gl

owdisch

arge

Titan

ium

coat

edsilic

on

V-s

hape

d15

lmw

idth

3lm

dep

th

Equa

lgr

oov

ean

dridge

wid

thPorc

ine

epithel

ial

cells

Cel

lsorien

ted

indirec

tion

ofgr

oov

es;ac

tin

fila

men

tsan

dm

icro

tubule

sal

igne

dal

ong

wal

lsan

ded

ges;

singl

ece

llssh

ow

edle

ssva

riab

ility

ofal

igne

dcy

tosk

elet

alar

rang

emen

tsth

ance

llcl

ust

ers;

no

sign

ifica

nt

ellip

tica

lm

orp

hol

ogy

[100

]

Photo

litho

grap

hy

and

reac

tive

ion

etch

ing

Epox

yre

plic

aof

silic

on

origi

nal

Squar

egr

oove

s0.

5lm

wid

th1

lmdep

th

Equa

lgr

oov

ean

dridge

wid

thH

um

angi

ngi

val

fibro

bla

sts

Cel

lssh

ow

edst

rong

alig

nm

ent

toto

pog

raph

y;ce

llsbr

idge

dor

confo

rmed

tofe

ature

s[6

8]

Photo

litho

grap

hy

and

reac

tive

ion

etch

ing

Epox

yre

plic

aof

silic

on

origi

nal

Squar

egr

oove

s0.

5lm

wid

th1

lmdep

th

Equa

lgr

oov

ean

dridge

wid

thH

um

angi

ngi

val

fibro

bla

sts

Cel

lsgr

ewm

ost

lyin

mon

ola

yers

;so

me

cells

exte

nded

pro

cess

esin

togr

oove

s;in

ner

corn

ers

of

groov

esno

tocc

upi

edby

cellul

arpro

cess

es;s

om

ece

llsbridg

edgr

oov

es;cy

tosk

elet

alel

emen

tsorien

ted

para

llelto

long

axis

ofgr

oov

es

[69]

Photo

litho

grap

hy

and

anisot

ropi

cet

chin

gTitan

ium

coat

edep

oxy

replic

aof

silic

on

origi

nal

Squar

ean

dV

-sha

ped

groov

es,30

lmre

peat

spac

ing

with

3,10

,or

22lm

dept

hor

7an

d39

lmre

pea

tsp

acin

gw

ith

3or

10lm

dep

th

Reg

ular

spac

ing,

but

uneq

ual

groov

ean

dridge

wid

th

Rat

par

ieta

lim

plan

tm

odel

groov

esor

iente

dho

rizo

nta

lly

or

vert

ical

lyto

long

axis

ofim

pla

nt

Endo

thel

ialce

llat

tach

men

tob

serv

edon

smooth

and

3,10

lmgr

oove

s;en

doth

elia

lce

llsbridg

ed22

lmhorizo

ntal

groo

ves;

fibr

obla

sts

enca

psu

late

dsm

ooth

and

3,10

lmhor

izonta

lgr

oove

s;fibro

blas

tsin

sert

edob

liquel

yin

to22

lmhorizo

nta

lgr

oov

es;

epithel

ialdow

ngro

wth

grea

test

on

vert

ical

and

smoo

thsu

rfac

ew

hile

leas

ton

10,22

lmhor

izonta

lgr

oov

es

[60]

Photo

litho

grap

hy

and

anisot

ropi

cet

chin

gTitan

ium

coat

edsilic

on,

epox

yre

plic

as,p

hot

o-

resist

Squar

ean

dV

-sha

ped

groov

es0.

5—60

lmdep

thre

pea

tsp

acin

g30

—220

lm

Reg

ular

spac

ing,

but

groo

vean

dridge

wid

thno

tlis

ted

Porc

ine

per

iodo

nta

llig

amen

tep

ithe

lial

cells

Cel

lsorien

ted

byal

lgr

oov

es;h

ighe

storien

tation

onsm

alle

stre

peat

spac

ing;

som

ece

llscr

oss

edridge

sor

desc

ended

into

groo

ves;

groo

ves

direc

ted

mig

ra-

tion

ofce

lls;

0.5

lmdee

pgr

oove

sle

sseff

ective

than

deep

ergr

oove

sat

direc

ting

cells

;ce

llob

serv

edto

hav

ela

mel

lae

and

filio

podi

aben

din

gar

ound

edge

s

[76]

(con

tinu

edon

nextpa

ge)

R.G. Flemming et al. / Biomaterials 20 (1999) 573—588 575

Tab

le1

(con

tinu

ed)

Fea

ture

Fab

rica

tion

Mat

eria

lFea

ture

Fea

ture

Cel

lty

pe

Cel

lula

reff

ect

Ref

.ty

pete

chniq

ue

dim

ensions

freq

uen

cyst

udi

ed

Photo

litho

grap

hy

and

reac

tive

ion

etch

ing

Quar

tz5,

10,25

lmw

idth

0.5,

1,2,

5lm

depth

Spac

ing

notlis

ted

BH

Kce

llsF-a

ctin

conde

nsa

tions

obs

erve

dat

topog

raphi

cdi

scontinui

ties

;con

dens

atio

nsoften

atrigh

tan

gles

togr

oov

eed

gew

ith

per

iodi

city

of0.

6lm

;vi

ncu

linor

ganiz

atio

nsim

ilar

toth

atofac

tin;m

icro

tubul

esob

serv

edaf

ter

30m

in;co

lcem

idin

crea

sed

spre

adin

gan

dre

duce

dorien

tation

and

elong

atio

n;cy

toch

alas

inD

reduc

edsp

read

ing,

elonga

tion,

and

orien

tation;

taxo

lre

duce

del

ong

atio

n

[99]

Photo

litho

grap

hy

and

reac

tive

ion

etch

ing

Poly

styr

ene

cast

ofsilic

on

origi

nal

0.5

lmw

idth

0.5,

5.0

lmdep

thR

adia

lar

ray

of

groov

es5

lmlo

ng

at1°

inte

rval

s

Spra

gue-

Daw

ley

rat

cala

varial

cells

Multip

lela

yer

pro

tein

adso

rption

from

seru

m;c

ells

grew

toco

nflue

nce

in4

days

and

pro

duce

dE

CM

afte

r7

days

[61]

Cut

ting

with

diam

ond

or

tung

sten

Poly

styr

ene,

epox

yre

plic

as2,

10lm

wid

thde

pth

not

liste

d5—

30lm

repe

atsp

acin

gC

hick

hea

rtfib

robla

sts,

murine

epithe

lialce

lls

75%

ofce

lls

alig

ned

on

5lm

groove

s;60

%ofce

llsal

igned

on

30lm

groove

s;cy

topl

asm

icex

tensions

not

rela

ted

tosu

rfac

efe

atur

es;al

ignm

ent

ofce

llsnot

guid

edby

lam

ella

eor

filo

pod

ia;c

ells

brid

ged

2an

d10

lmgr

oov

esw

ithout

touch

ing

surfac

e

[70]

Photo

litho

grap

hy

and

anisot

ropi

cet

chin

gSi

licon

dioxi

de

0.5

lmw

idth

1lm

dep

thEqua

lgr

oov

ean

dridge

wid

thH

um

anfib

robla

sts,

ging

ival

kera

tinocy

tes,

neut

roph

ils,

mono

cyte

s,m

acro

phag

es

100%

offib

robla

sts

and

20%

ofm

acro

pha

ges

alig

ned

;no

orien

tation

oral

ignm

ent

obse

rved

with

kera

tinocy

tes

or

neu

troph

ils;so

me

mac

roph

ages

exte

nded

pro

cess

espa

rallel

tolo

ng

axis

ofgr

oov

esaf

ter

2h

[101

]

Photo

litho

grap

hy

and

anisot

ropi

cet

chin

gTitan

ium

coat

edsilic

on,

epox

yre

plic

as,p

hot

ore

sist

Squar

ean

dV

-sha

ped

maj

or

groo

ves

5—12

0lm

deep

(wid

thnot

list

ed),

min

or

groo

ves

2lm

dee

pon

floor

at54

°to

maj

or

groov

es

5—80

lmre

peat

spac

ing

Hum

angi

ngi

val

fibro

bla

sts

Alig

nm

entobs

erve

din

groo

ves

and

on

flat

ridge

s;ce

llsorien

ted

pref

eren

tial

lyto

maj

orgr

oove

s;m

inor

groov

esca

use

dorien

tation

ofce

llsin

abse

nce

of

maj

or

groo

ves

or

whe

ndi

scon

tinuity

existe

din

maj

or

groo

vepat

tern

[59]

Photo

litho

grap

hy

follow

edby

glow

disch

arge

PD

MS

cast

ofsilic

on

origi

nal

2.0,

5.0,

10.0

lm

wid

th0.

5lm

depth

Equa

lgr

oov

ean

dridge

wid

thR

atder

mal

fibro

bla

sts

Cel

lson

2an

d5

lmgr

oove

sw

ere

elon

gate

dan

dal

igned

par

alle

lto

groov

es;c

ells

on10

lmgr

oove

sw

ere

sim

ilar

toth

ose

on

smoot

hsu

bst

rate

[63]

Photo

litho

grap

hy

and

anisot

ropi

cet

chin

g,gl

owdisch

arge

Titan

ium

-coa

ted

silic

on

V-s

hape

d,3

lmdep

thw

idth

not

liste

d6—

10lm

repe

atsp

acin

gH

um

angi

ngi

val

fibro

bla

sts

Cel

lsel

onga

ted

and

orien

ted

along

groo

ves;

cell

heig

ht1.

5-fo

ldgr

eate

ron

groove

s;fib

ronec

tin

mR

NA

and

secr

eted

fibr

onec

tin

incr

ease

din

cells

ongr

oov

es;G

APD

mR

NA

notaff

ecte

d;ha

lf-liv

esof

fibro

nec

tin

mR

NA

alte

red;

2-fo

ldin

crea

sein

fibro

nec

tin

asse

mbl

edin

toE

CM

[95]

Photo

litho

grap

hy

and

reac

tive

ion

etch

ing

Quar

tz1.

65—8

.96

lmw

idth

0.69

lmdep

th3.

0—32

.0lm

repea

tsp

acin

gC

hick

hea

rtfib

robla

sts

Rid

gew

idth

mor

eim

por

tant

than

groove

wid

thin

dete

rmin

ing

cell

alig

nm

ent;

alig

nm

ent

ofce

llsin

vers

ely

pro

por

tion

alto

ridge

wid

th

[96]

576 R.G. Flemming et al. / Biomaterials 20 (1999) 573—588

Photo

litho

grap

hy,

anisot

ropi

cet

chin

gTi-co

ated

silic

onV

-sha

ped

groo

ves

70,13

0,16

5lm

wid

th80

,14

0,17

5lm

repea

tsp

acin

gH

um

angi

ngi

val

cells

,por

cine

epithe

lialce

lls

Cel

lsfrom

susp

ension

alig

ned

tolo

ng

axis

of

groov

es;ep

ithel

ialce

llsdid

not

ben

dar

ound

ridge

sbe

twee

nce

lls;gr

oove

sca

used

alig

nm

entofm

igra

tion

ofex

plan

ted

cells

;m

ultila

yering

ofep

ithel

ialce

llsw

ithin

and

alon

ggr

oove

s

[35]

Photo

litho

grap

hy

and

anisot

ropi

cet

chin

gEpox

yre

plic

aof

silic

on

origi

nal

V-s

hape

dgr

oove

s17

lmw

idth

10lm

dep

th

22lm

ridge

wid

thPorc

ine

per

iodo

nta

llig

amen

tep

ithe

lial

cells

,rat

pariet

alim

plan

tm

odel

Epithel

ialce

lls

atta

ched

togr

oove

dsu

rfac

esm

ore

than

tosm

oot

hsu

rfac

esan

dw

ere

orien

ted

bygr

oov

es;sh

orte

rle

ngth

epithe

lialat

tach

men

tan

dlo

nge

rco

nnec

tive

tiss

ue

atta

chm

ent

ingr

oov

edpar

tsof

impl

antco

mpar

edto

smoot

hpar

ts;g

roove

sim

pede

dep

ithe

lialdo

wngr

ow

thon

impla

nts

[72]

Photo

litho

grap

hy

and

anisot

ropi

cet

chin

gTi-co

ated

silic

onV

-sha

ped

groo

ves

3lm

dep

th6—

10lm

repe

atsp

acin

gH

um

angi

ngi

val

fibro

bla

sts

Cel

lsorien

ted

along

groo

ves

by16

h;ce

llson

groove

ssh

ow

edal

tere

dm

atrix

met

allo

pro

tein

ase-

2m

RN

Atim

e-co

urs

eex

pres

sion

and

leve

lsco

mpar

edto

cells

onsm

ooth

Tior

tiss

ue

culture

pla

stic

[102

]

Photo

litho

grap

hy

and

anisot

ropi

cet

chin

gTi-co

ated

silic

onV

-sha

ped

15lm

wid

th3

lmdep

th

Gro

ove

and

ridg

ew

idth

equa

lH

um

angi

ngi

val

fibro

bla

sts

Mic

rotu

bul

esw

ere

the

first

elem

ent

tobe

com

eal

igned

;m

icro

tubu

les

alig

ned

atbot

tom

ofgr

oove

saf

ter

20m

in;ac

tin

obse

rved

first

atw

all-ridg

eed

ges

afte

r40

—60

min

;af

ter

3h

am

ajority

ofce

llsex

hibi

ted

alig

ned

foca

lco

ntac

ts

[103

]

Photo

litho

grap

hy

and

reac

tive

ion

etch

ing,

glow

disch

arge

trea

tmen

t

PD

MS

cast

ofsilic

on

origi

nal

2,5,

10lm

wid

th0.

5lm

depth

Gro

ove

and

ridg

ew

idth

equa

lR

atder

mal

fibro

bla

sts

Mic

rofil

amen

tsan

dvi

ncu

linag

greg

ates

orien

ted

alon

g2

lmgr

oove

saf

ter

1,3,

5,an

d7

day

s,bu

tw

asle

ssor

iente

don

5an

d10

lmgr

oov

es;vi

ncu

linlo

cate

dprim

arily

on

surfac

eridge

s;bov

ine

and

endo-

gene

ousfibro

nect

inan

dvi

trone

ctin

wer

eor

iente

dal

ong

groov

es;gr

oov

e-sp

anni

ng

fila

men

tsal

soob

serv

ed

[73]

Ele

ctro

n-bea

mlit

hog

raph

yan

dw

etet

chin

g,gl

owdisch

arge

trea

tmen

t

PD

MS

cast

ofsilic

on

origi

nal

Squar

egr

oove

s1

lmw

idth

1lm

dep

th

Gro

ove

sse

par

ated

by4

lm-w

ide

ridge

s

Hum

angi

ngi

val

fibro

bla

sts

Vin

culin

-positive

atta

chm

entsite

sob

serv

ed;ce

llsal

igned

togr

oove

sin

PD

MS,w

hic

hhad

been

mad

ehy

drop

hilic

by

glow

disc

har

getr

eatm

ent;

foca

lad

hesion

cont

acts

also

alig

ned

togr

oove

s

[56]

Photo

litho

grap

hy

and

wet

etch

ing

PD

MS

cast

ofsilic

on

origi

nal

2,5,

10lm

wid

th0.

5lm

depth

Gro

ove

and

ridg

ew

idth

equa

lH

um

ansk

infib

robla

sts

Cel

lson

smoo

thP

DM

Sen

tere

dS

phas

eof

cell

cycl

esfa

ster

than

cells

on

text

ured

PD

MS;

cells

on

10lm

text

ure

prol

ifera

ted

less

than

thos

eon

2an

d5

lmte

xture

s

[74]

Cut

ting

with

diam

ond

Seru

m-c

oate

dgl

ass

2lm

wid

th2

lmdep

thSp

acin

gnot

liste

dH

um

anne

utr

oph

ille

ukoc

ytes

Whe

nce

llsm

ovin

gac

ross

plan

eofgl

ass

enco

un-

tere

da

groove

,they

wer

ehig

hly

likel

yto

mig

rate

alon

ggr

oov

era

ther

than

cros

sit

[104

]

Photo

litho

grap

hy

and

reac

tive

ion

etch

ing,

glow

disch

arge

trea

tmen

t

PD

MS

cast

ofsilic

on

oxi

deor

igin

al

Squar

egr

oove

s1.

0—10

.0lm

wid

th0.

45,1.

00lm

dept

h

1.0—

10.0

lmridg

ew

idth

Rat

der

mal

fibro

bla

sts

Cel

lsorien

ted

and

elong

ated

along

groo

ves

with

ridge

wid

ths

4.0

lmor

less

;pro

trus

ions

cont

acting

ridge

sobse

rved

on

orien

ted

cells

;ce

llsra

ndo

mly

orie

nted

and

wer

em

ore

circ

ula

ron

groov

esw

ith

ridge

sm

ore

than

4.0

lm

wid

e;gr

oov

ew

idth

and

dept

hdid

not

affec

tce

llsize

,sha

pe,

ororien

tation

[64]

(con

tinu

edon

nextpa

ge)

R.G. Flemming et al. / Biomaterials 20 (1999) 573—588 577

Tab

le1

(con

tinu

ed)

Fea

ture

Fab

rica

tion

Mat

eria

lFea

ture

Fea

ture

Cel

lty

pe

Cel

lula

reff

ect

Ref

.ty

pete

chniq

ue

dim

ensions

freq

uen

cyst

udi

ed

Photo

litho

grap

hy

and

reac

tive

ion

etch

ing

Quar

tzan

dpr

otei

n-co

ated

quar

tzSq

uar

egr

oove

s2,

10lm

wid

th30

–282

nmde

pth

Equa

lgr

oov

ean

dridge

wid

thP38

8D1

mac

rophag

es,ra

tpe

rito

neal

mac

rophag

es

Spre

adar

eaof

cells

on

282

nm

deep

groov

esw

astw

ice

that

ofce

lls

onpl

ain

subst

rate

;de

gree

of

orie

nta

tion

ofce

llsin

crea

sed

with

incr

easing

depth

and

dec

reas

ing

wid

thofgr

oove

s;ce

llson

groo

ves

had

incr

ease

dnum

ber

ofpro

trusions

exte

ndin

gpe

rpen

dic

ular

togr

oov

es;gr

oov

esca

use

din

crea

sein

F-a

ctin

;F

-act

inan

dvi

ncu

linac

cum

ula

ted

along

groov

e/ridge

bound

arie

s;ce

llson

groo

vessh

ow

edhi

gher

phag

ocy

tic

activi

ty

[80]

Photo

litho

grap

hy

and

reac

tive

ion

etch

ing

PM

MA

2,3,

6,12

lmw

idth

0.2,

0.56

,1.

10,1.

9lm

dept

hEqua

lgr

oov

ean

dridge

wid

thBH

Kce

lls,M

DC

Kce

lls,c

hic

kem

bry

oce

rebra

lne

uro

n

Alig

nm

entofB

HK

cells

incr

ease

dw

ith

dep

thbut

decr

ease

dw

ith

incr

easing

wid

th;w

idth

had

noeff

ect

onM

DC

Kce

lls;al

ignm

ent

ofM

DC

Kce

lls

incr

ease

dw

ith

dep

th;re

spons

eofM

DC

Kce

llsdep

ended

on

whe

ther

orno

tce

llsw

ere

isol

ated

;al

ignm

entofch

ick

embry

oce

rebr

alneu

rons

also

incr

ease

dw

ith

depth

[78]

Las

erholo

grap

hic

tech

niq

ue

used

tode

fine

mas

ksfo

rX

-ray

lithog

raph

yan

dre

active

ion

etch

ing

Quar

tzan

dpo

ly- L

-ly

sine

-coa

ted

quar

tz13

0nm

wid

th10

0,21

0,40

0nm

dept

hEqua

lgr

oov

ean

dridge

wid

thBH

K,M

DC

K,

chic

kem

bry

oce

rebra

lne

uro

ns

BH

Kce

llsal

igned

on

allgr

oove

pat

tern

s,bu

tde

gree

ofal

ignm

ent

incr

ease

dw

ith

incr

easing

dep

th;

MD

CK

alig

ned

and

elong

ated

togr

oove

s,but

only

elon

gation

incr

ease

dw

ith

dep

th;M

DC

Kce

llsin

group

san

dch

ick

embry

oce

rebr

alneu

rons

not

affec

ted

bygr

oov

es

[79]

Las

erholo

grap

hic

tech

niq

ue

used

tode

fine

mas

ks

for

X-r

aylith

ogr

aphy

and

reac

tive

ion

etch

ing

Poly

-D-lys

ine-

coat

edch

rom

e-pl

ated

quar

tz

0.13

–4.

01lm

wid

th0.

1–1.

17lm

dept

h0.

13–8

.0lm

spac

ing

Rat

optic

ner

veol

igode

ndr

ocy

tes,

optic

ner

veas

trocy

tes,

hippo

cam

pal

cere

bel

lar

neu

rons

Olig

oden

droc

ytes

wer

ehi

ghly

alig

ned

by

feat

ure

sas

smal

las

100

nmdep

than

d26

0nm

repe

atsp

acin

g:as

trocy

tes

wer

eal

soal

igned

while

hippo

cam

pal

and

cere

bel

lar

neu

ron

cells

wer

enot

;ol

igod

endr

ocyt

essh

ow

edlit

tle

hig

h-o

rder

F-a

ctin

netw

ork

s;al

igned

astr

ocy

tes

show

edex

tensive

arra

nge

men

tofac

tin

stre

ssfib

ers;

max

imum

olig

ode

ndro

cyte

alig

nm

ent

induc

edby

pat

tern

corr

espo

ndi

ngto

dia

met

erof

axon

in7

day

optic

ner

ve

[81]

Ele

ctro

n-bea

mlit

hog

raph

y,w

etet

chin

g,an

dre

active

ion

etch

ing

Quar

tz,po

ly-L

-ly

sine

-coa

ted

quar

tzan

dpo

lyst

yren

ere

plic

as

Squar

egr

oove

s1,

2,4

lm

wid

th14

–110

0nm

dept

h

Spac

ing

not

liste

dEm

bryo

nic

Xen

opus

spin

alco

rdne

uron

s,ra

thi

ppoca

mpal

neur

ons

Xen

opus

neu

rite

sgr

ewpar

alle

lto

allgr

oov

esize

s;hi

ppo

cam

pal

neurite

sgr

ewper

pendi

cula

rto

narr

ow,

shal

low

groov

esan

dpar

alle

lto

wid

e,de

epgr

oov

es;

Xen

opus

neu

rite

sem

erge

dfrom

som

are

gion

spar

alle

lto

groov

es;ra

thip

poca

mpa

lpr

esum

ptive

axon

sem

erge

dpe

rpen

dic

ular

togr

oove

s,bu

tpre

sum

ptive

dend

rite

sem

erge

dpa

ralle

lto

groove

s;neu

rite

stu

rned

toal

ign

togr

oove

s

[58]

Ele

ctro

n-bea

mlit

hog

raph

y,w

etet

chin

g,an

dre

active

ion

etch

ing

Quar

tz,po

ly-L

-ly

sine

-coa

ted

quar

tzan

dpol

ysty

rene

replic

as

Squar

egr

oove

s1,

2,4

lm

wid

th14

–110

0nm

dept

h

Spac

ing

not

liste

dEm

bryo

nic

Xen

opus

spin

alco

rdne

uron

s,ra

thi

ppoca

mpal

neur

ons

Orien

tation

ofX

enop

usan

dhip

poca

mpa

lneu

rite

sw

asuna

ffec

ted

bycy

toch

alas

inB

,whic

hel

imin

ated

filopo

dia

;tax

olan

dnoc

odaz

ole

disr

upte

dhip

po-

cam

pal

mic

rotu

bule

s,bu

tdid

not

affec

tor

ienta

tion

ortu

rnin

gto

war

dgr

oov

es;pe

rpen

dic

ula

ral

ignm

ent

of

[57]

578 R.G. Flemming et al. / Biomaterials 20 (1999) 573—588

hippo

cam

palne

urite

sw

asno

tin

hib

ited

by

seve

ral

calc

ium

chan

nel

,Gpro

tein

,pro

tein

kinas

ean

dpr

otei

nty

rosine

kin

ase

inhi

bito

rs;s

om

eca

lciu

mch

annel

and

pro

tein

kin

ase

inhib

itor

sdid

inhib

ital

ignm

ent

Gro

oves

and

pits

Photo

litho

grap

hy

and

anisot

ropi

cet

chin

gTitan

ium

-coa

ted

epox

yre

plic

asof

silic

on

origi

nal

V-s

hape

dgr

oove

s35

—165

lmw

idth

30,60

,and

120

lmdep

thV

-sha

ped

pits

35—2

70lm

wid

than

d30

,60

,12

0lm

depth

Rep

eatsp

acin

g40

—175

lmfo

rgr

oov

esan

d40

—280

lmfo

rpits,

uneq

ual

groov

ean

dridge

wid

th

Rat

par

ieta

lbo

ne

impl

antm

odel

Min

eral

izat

ion

occ

urre

dof

ten

ongr

oov

edor

pitt

edsu

rfac

es,but

rare

lyon

smooth

contr

olsu

rfac

es;

freq

uency

offo

rmat

ion

ofbo

nel

ike

foci

incr

ease

dde

crea

sed

asgr

oove

dept

hin

crea

sed;freq

uen

cyof

min

eral

izat

ion

incr

ease

das

dept

hofpit

incr

ease

d;bo

nel

ike

foci

orien

ted

along

long

axis

ofgr

oove

s

[66]

Gro

oves

and

chem

ical

patter

n

Photo

litho

grap

hy

and

reac

tive

ion

etch

ing,

sila

niza

tion

Am

ino-

sila

nean

dm

ethyl

-sila

ne-

coat

edqu

artz

Squar

egr

oove

s2.

5,6,

12.5

,25

,50

lmw

idth

0.1,

0.5,

1.0,

3.0,

6.0

lmde

pth

Gro

ove

and

ridg

ew

idth

equa

l,sila

netr

acks

equal

BH

Kce

llsC

ells

alig

ned

mos

tto

25lm

amin

osila

netr

acks

and

5lm

wid

e,6

lmdee

pgr

oove

s;st

ress

fiber

san

dvi

nculin

alig

ned

with

adhes

ive

trac

ksan

dgr

oove

san

dridg

es;al

ignm

ent

incr

ease

dw

hen

adhe

sive

trac

ks

and

groov

espa

ralle

l;ce

llsal

igne

dto

adhe

sive

trac

ksw

hich

wer

eper

pendic

ula

rto

groo

ves;

F-a

ctin

orie

nted

tobo

thad

hes

ive

cues

and

topog

raphi

ccu

esw

ithi

nsa

me

cell

on

the

subst

rate

sw

ith

3an

d6

lmdep

th;

adhe

sive

cues

dom

inan

t

[86]

Photo

litho

grap

hy

anisot

ropi

cet

chin

g,pol

ymer

mic

rom

old

ing,

trea

tmen

tw

ith

alka

net

hiols

Ti,

Au-

coat

edpo

lyure

than

etr

eate

dw

ith

fibro

nec

tin,al

kan

eth

iols

V-s

hape

dgr

oove

s25

,50

lmw

idth

dept

hnot

liste

d

Gro

ove

and

ridg

ew

idth

equa

lBovi

neca

pilla

ryen

doth

elia

lce

llsC

ells

adhe

red

tore

gions

coat

edw

ith

fibro

nec

tin,

whic

had

sorb

edto

regi

ons

sila

niz

edw

ith

met

hylbu

tno

ttr

i(eth

ylen

egl

ycol

)-te

rmin

ated

sila

nes

;ce

llsat

tach

edto

eith

ergr

oove

sor

ridg

es,d

epen

din

gon

whi

chposs

esse

dth

em

ethy

l-te

rmin

ated

sila

nean

dfib

ronec

tin

coat

ings

[77]

Rid

ges

Photo

litho

grap

hy

and

reac

tive

ion

etch

ing

Poly

styr

ene

cast

of

silic

on

origi

nal

0.5—

100.

0lm

wid

th0.

03–5

.0lm

heig

ht0.

5—62

lmbe

twee

nridge

sº

rom

yces

appe

ndic

ulat

usfu

ngu

s

Max

imum

cell

diff

eren

tiat

ion

obs

erve

dfo

rridg

esor

plat

eaus

0.5

lm

high

;ridg

eshig

her

than

1.0

lmor

smal

ler

than

0.25

lm

wer

enot

effec

tive

sign

als;

ridg

esp

acin

gof0.

5—6.

7lm

cause

dhig

hde

gree

ofor

ienta

tion

ofth

efu

ngus

[65]

Eva

por

ativ

eco

atin

gSi

licon

oxid

eon

poly

styr

ene

4lm

wid

th50

nmhe

ight

Rad

ialar

ray

Murine

neur

obla

stom

ace

lls

Cel

lsad

here

dto

lines

and

pro

cess

esal

igne

dal

ong

the

lines

;pro

cess

esgr

ewin

bip

ola

rm

anne

r[9

0]

Step

sPhoto

litho

grap

hy

and

reac

tive

ion

etch

ing

PM

MA

1—18

lmst

eps

Spac

ing

not

liste

dBH

Kce

lls,ch

ick

embry

oni

cne

ural

,ch

ick

hear

tfib

robla

st,r

abbit

neut

roph

ils

Cel

lsex

hib

ited

dec

reas

ein

freq

uen

cyof

cros

sing

step

san

din

crea

sed

alig

nmen

tat

step

sw

ith

incr

easing

step

heig

htre

gard

less

ofdi

rect

ion

ofap

pro

ach;

rabbit

neut

roph

ilssh

owed

twic

eth

ecr

ossing

freq

uenc

yov

er5

lmst

eps

asdid

the

other

cells

;pre

senc

eofad

hesive

differ

ence

resu

lted

inde

crea

sein

freq

uen

cyre

sulted

inde

crea

sein

freq

uen

cyof

asce

nt

only

for

step

heig

hts

of1

and

3lm

[105

]

Wav

esSol

ution

pol

ymer

izat

ion

PD

MS

gels

ofva

ryin

gso

ftne

ssSo

fter

gels

had

smal

ler

wav

esw

hile

hard

gelhad

larg

erw

aves

3,4,

15lm

periodic

ity

Hum

ande

rmal

fibro

bla

sts

and

kera

tinocy

tes

Fib

robl

asts

prolife

rate

deq

ual

lyon

allsu

bst

rate

s;ke

ratinocy

tes

spre

adm

ore

and

secr

eted

mor

eEC

Mon

soft

gels

than

on

har

dge

l

[111

]

(con

tinu

edon

nextpa

ge)

R.G. Flemming et al. / Biomaterials 20 (1999) 573—588 579

Tab

le1

(con

tinu

ed)

Fea

ture

Fab

rica

tion

Mat

eria

lFea

ture

Fea

ture

Cel

lty

pe

Cel

lula

reff

ect

Ref

.ty

pete

chniq

ue

dim

ensions

freq

uen

cyst

udi

ed

Wel

lsan

dno

des

Photo

litho

grap

hy

and

etch

ing

PD

MS

replic

asof

silic

on

origi

nal

2,5

lmdia

met

erro

und

nodes

,0.

38an

d0.

46lm

high

,re

spec

tive

ly8

lmro

und

wel

l,0.

57lm

dee

p

4,10

,19

lmce

nter

-to-c

ente

rsp

acin

gfo

rth

e2,

5,8

lm

feat

ure

s,re

spec

tive

ly

Rab

bit

impla

nt

mode

lm

urine

mac

rophag

es

2an

d5

lmte

xture

dim

pla

nts

had

few

erm

onon

ucl

ear

cells

and

thin

ner

fibro

usca

psul

esth

andi

dsm

ooth

and

8lm

text

ured

impla

nts;

cells

on

smoot

hP

DM

Sw

ere

roun

dw

ith

few

pseu

dopo

ds,bu

tce

llson

2an

d5

lmte

xture

sw

ere

elon

gate

dw

ith

pse

udopo

ds

[75]

Photo

litho

grap

hy

and

etch

ing

PD

MS

cast

ofsilic

on

origi

nal

2,5,

8lm

diam

eter

variab

lesp

acin

g2,

5,10

lmco

nsta

ntsp

acin

g

Var

iable

spac

ing

orco

nst

ant

spac

ing

of20

.4lm

Murine

per

itone

alm

acro

phag

esC

ells

on

5lm

text

ure

shad

smal

lest

dim

ensions

whi

lece

llson

smoo

thsilico

ne

and

glas

shad

larg

est

dim

ensions

;mitoch

ondr

ialac

tivi

tyhig

hest

once

llson

5an

d8

lmva

riab

lepitch

surfac

esan

don

poly

sty-

rene;

PM

A-s

tim

ula

ted

cells

on

smal

ler

text

ures

wer

ele

ssac

tive

than

unst

imul

ated

cells

[71]

Photo

litho

grap

hy

and

etch

ing

PD

MS

cast

ofsilic

on

origi

nal

Squar

enod

esor

wel

ls2,

5,10

lmdia

met

erD

epth

or

hei

ght

of0.

5lm

ATC

Chu

man

abdo

men

fibro

blas

tsC

ells

on

2an

d5

lmnodes

show

edin

crea

sed

rate

ofpr

olifer

atio

nan

din

crea

sed

cell

den

sity

com

par

edto

cells

on

2an

d5

lmw

ells;10

lmnode

san

dw

ells

did

notdiff

erst

atistica

llyfrom

smoo

thsu

rfac

es

[67]

Las

erm

odifi

cation

Poly

carb

onat

e,po

lyet

herim

ide

Squar

enod

es7,

25,or

50lm

wid

th0.

5,1.

5,2.

5lm

hei

ght

Unifo

rmsq

uar

ear

ray

Hum

anne

utr

oph

ils,

fibro

bla

sts

None

ofth

ete

xture

dsu

rfac

essign

ifica

ntly

stim

ula

ted

neut

roph

ilm

ovem

entco

mpar

edto

chem

ical

stim

ul-

ator

s,al

thoug

hne

utr

ophi

lmov

emen

twas

grea

teron

som

eof

the

text

ure

dsu

rfac

esth

anon

anun

text

ured

surfac

e;no

effec

tson

fibro

blas

tor

ient

atio

n,sp

read

ing,

orel

onga

tion

[106

]

Pill

ars

and

pore

sLas

erab

lation

use

din

conju

nct

ion

with

mas

ksm

ade

by

elec

tron-b

eam

lithog

raph

y,re

active

ion

etch

ing

PM

MA

,PE

T,

poly

styr

ene

Circu

lar

pill

ars

and

pore

s1,

5,10

,50

lmdia

met

erU

nifo

rmar

ray

Hum

anos

teobla

sts

and

amnio

tic

epithe

lialce

lls

Cel

lsen

gulfe

dpill

ars

or

stre

tche

dbe

twee

nad

jace

nt1

and

5lm

pill

ars;

cells

atta

ched

toed

ges

ofpo

res,

espe

cial

lyon

10lm

pore

s;te

xture

caus

edin

crea

sein

cell

adhes

ion

onal

lm

ater

ials

butPM

MA

;gr

eate

stin

crea

sein

adhe

sion

was

on

50lm

PE

Tpill

ars;

10lm

pore

sca

use

d5%

incr

ease

inre

sist

ance

tosh

ear

forc

e

[107

]

Por

esM

icro

poro

usfil

ter:

Nyl

ondi

p-c

oate

dw

ith

PV

C/P

AN

copoly

mer

Unco

ated

and

silic

on

coat

edfil

ters

0.2–

10lm

diam

eter

dept

hnot

liste

dSp

acin

gnot

liste

dIn

vivo

cani

nem

ode

lN

on-

adher

ent,

cont

ract

ing

caps

ule

sar

ound

impl

ants

with

pore

ssm

alle

rth

an0.

5lm

;im

pla

nts

with

1.4—

1.9

lmpo

res

show

edad

here

ntca

psul

esbut

no

inflam

mat

ory

cells

;por

esbi

gger

than

3.3

lmw

ere

infiltra

ted

with

infla

mm

ator

ytiss

ue;

pore

s1—

2lm

allo

wed

for

fibro

blas

tat

tach

men

t

[84]

Sphe

res

Par

ticl

ese

ttlin

gPoly

(NIP

AM

)pa

rtic

les

onpo

lyst

yren

esu

rfac

e

0.86

—0.6

3lm

diam

eter

whe

nte

mper

atur

era

ised

from

25to

37°C

2Dhe

xago

nal

latt

ice,

0.96

lm

avg.

dista

nce

betw

een

sphe

rece

nter

s

Neu

trop

hil-

like

induc

edH

L-6

0ce

llsC

ells

loos

ely

adher

edbu

tdi

dnot

spre

adon

sphe

re-

coat

edsu

rfac

ean

dco

uld

roll

easily

;exc

ess

active

oxyg

enre

leas

edw

hen

tem

pera

ture

was

incr

ease

don

spher

e-co

ated

surfac

e,bu

tnot

on

pol

y(N

IPA

M)gr

afte

dsu

rfac

e[108

]

Cyl

inde

rsFib

er-o

ptic

light

condui

t-fu

sed

quar

tzcy

lindr

ical

fibe

rspl

aced

onag

arose

-cov

ered

cove

rslip

s

Fuse

dquar

tz12

—13

or25

lmra

dii

Spac

ing

not

liste

dPrim

ary

mou

seem

bry

ofib

robl

asts

and

rat

epithe

lial

cell

lines

Cel

lsin

the

pola

riza

tion

stag

eofsp

read

ing

with

stra

ight

actin

bun

dle

sbe

cam

eel

onga

ted,

orien

ted

alon

gcy

linder

,and

resist

edben

ding

around

cylin

ders

;ce

llsin

the

radi

alst

age

ofsp

read

ing

with

circ

ula

rac

tin

bundle

sor

cells

with

no

actin

bun

dle

ste

nded

tobe

ndar

oun

d

[109

]

580 R.G. Flemming et al. / Biomaterials 20 (1999) 573—588

cylin

der

and

exhi

bited

less

elonga

tion

and

orien

tation

tolo

ng

axis

ofcy

linder

Gen

eral

roug

hnes

sR

eact

ive

ion

etch

ing

follow

edby

pho

tolith

ogr

aphy

and

isot

ropic

wet

etch

ing

Are

asofro

ugh

er,

reac

tive

ion

etch

edsilic

on

and

smoo

ther

,w

etet

ched

silic

on

Rea

ctiv

eio

net

ched

feat

ures

:57

nmav

g.di

amet

er,

230

nmhe

ight

wet

etch

edfe

ature

s:11

5nm

peak

-to-

valle

yro

ughn

ess,

depr

ession

s10

0–25

0nm

inwid

th

Rea

ctiv

eio

net

ched

feat

ure

s:13

7/lm

2su

rfac

ede

nsity

wet

etch

edfe

ature

s:27

/lm

2

surfac

ede

nsity

Tra

nsfo

rmed

rat

astr

ocy

tes,

prim

ary

rat

cort

ical

astr

ocy

tes

Tra

nsfo

rmed

cells

atta

ched

pref

eren

tial

lyto

wet

-et

ched

regi

ons

rath

erth

anre

active

ion

etch

edco

lum

nar

stru

ctur

es;t

rans

form

edce

llson

wet

-etc

hed

area

ssp

read

inep

ithel

ial-lik

em

anner

and

wer

esm

oot

h;

tran

sfor

med

cells

on

colu

mna

rre

gions

wer

ero

und

ed,

loose

lyat

tach

ed,an

dex

hibited

com

ple

xsu

rfac

epr

oje

ctio

ns;

tran

sform

edce

llspr

efer

red

area

sex

pose

dto

incr

easing

amoun

tsofw

etet

chin

g;pr

imar

yce

llspr

efer

red

colu

mna

rst

ruct

ure

sofre

active

ion

etch

edar

eas

and

did

not

spre

adon

wet

etch

edar

eas

[87]

Aci

dw

ashin

g,el

ectr

opol

ishin

g,sa

ndbl

asting

,pl

asm

a-sp

raye

dT

i

Titan

ium

1—2

lmpi

ts,1

lmpits,

10lm

crat

ers

10—2

0gl

obu

les

and

shar

pfe

atur

esof

(0.

1lm

Ran

dom

MG

63os

teobla

stEle

ctro

polis

hed

surfac

ehad

mor

ece

llsw

hile

TI-

plas

ma-

spra

yed

had

less

than

TC

PS;sa

ndbl

aste

dsu

rfac

eshad

the

sam

eas

TC

PS;

thym

idin

ein

corp

ora

tion

inve

rsel

yre

late

dto

rough

ness

;pr

ote

ogl

ycan

synt

hes

isde

crea

sed

on

allsu

rfac

es;al

kal

ine

phos

phat

ase

produ

ctio

ndec

reas

edw

ith

incr

easing

roug

hne

ssex

cept

onco

arse

blas

ted

Ti;

corr

elat

ion

obse

rved

betw

een

rough

nes

san

dR

NA

and

CD

Ppro

duc

tion

[83]

Alu

min

aem

ulsion

polish

ing,

grin

din

gw

ith

SiC

pap

er

Ti,

Ti/A

l/V

allo

y,TiT

aal

loy

0.04

,0.

36,an

d1.

36lm

peak

-to-

valle

yhe

ight

sR

andom

Hum

angi

ngi

val

fibro

bla

sts

Cel

lsal

igne

dto

grin

din

gm

arks

:10%

ofce

llsorien

ted

onsu

rfac

ew

ith

0.04

lm

rough

nes

s,60

%on

0.36

lmro

ughn

ess,

and

72%

on1.

36lm

roug

hne

ss

[89]

Ele

ctro

polis

hing,

sandbl

asting

,ac

idet

chin

g

Titan

ium

0.14

,0.

41an

d0.

80lm

peak

-to-

valle

yhe

ight

sfo

rel

ectr

o-po

lish

ed,et

ched

,an

dsa

ndb

last

edT

i,re

spec

tive

ly

Ran

dom

Hum

angi

ngi

val

fibro

bla

sts

Cel

lson

smoo

th,e

lect

ropolis

hed

surfac

essh

owed

flat

morp

holo

gyan

dgr

ewin

laye

rs;ce

llson

etch

edT

im

igra

ted

alon

girre

gula

rgr

oove

s;ce

llson

sand

bla

sted

Tigr

ewin

clust

ers;

round

and

flatce

llsfo

und

on

etch

edan

dsa

ndbl

aste

dT

i;ac

tin

bundl

esan

dvi

nculin

-co

ntai

ning

foca

ladh

esio

nsobse

rved

insp

read

ing

cells

onel

ectr

opol

ished

and

etch

edTi,

butnot

insp

read

ing

cells

on

sand

blas

ted

Ti

[88]

San

dbl

asting

with

diff

eren

tgr

ain

size

san

dai

rpre

ssure

s

PM

MA

Sand

grai

nsize

sof50

,12

5,an

d25

0lm

pro

duc

edpe

ak-t

o-va

lley

heig

hts

from

0.07

to3.

34lm

Ran

dom

Chi

ckem

bryo

vasc

ula

ran

dco

rnea

lce

lls

Surfac

ero

ugh

nes

sw

ashig

hest

for

surfac

essa

nd-

blas

ted

with

larg

est

size

grai

ns;m

igra

tion

area

ofce

llsin

crea

sed

2-fo

ldfo

rva

scul

arce

llsan

d3-

fold

for

corn

eal

cells

on

rough

surfac

esco

mpar

edto

smooth

;ce

llad

hesion

incr

ease

dw

ith

surfac

ero

ughn

ess

[91]

Indu

strial

polis

hing,

sandbl

asting

,pl

asm

a-sp

rayi

ng

with

Ti-6A

l-4V

Titan

ium

/alu

mi-

nium

/van

adiu

mal

loy

Smoo

th,ro

ugh

,por

ous

-co

ated

surfac

es10

0—10

00lm

pore

son

poro

us-

coat

edsu

rfac

es

Ran

dom

Chi

ckem

bryo

nic

calv

aria

los

teob

last

sC

ells

adhe

red

tosu

rfac

es,u

sing

cellul

arpro

cess

esto

brid

geun

even

area

s;EC

Msy

nth

esis

and

min

eral

izat

ion

wer

een

han

ced

onro

ugh

and

por

ous

tita

nium

surfac

es

[110

]

Scr

atch

ing

with

glas

sro

dPoly

styr

ene

and

H2S

O4-t

reat

edpo

lyst

yren

e

Dim

ension

snot

liste

dR

andom

Murine

per

itone

alm

acro

phag

esfib

robla

sts

Mac

roph

ages

accu

mula

ted

pref

eren

tial

lyon

rough

ened

surfac

esw

hile

fibro

bla

sts

pref

erre

dsm

oot

hsu

rfac

es

[112

]

(con

tinu

edon

nextpa

ge)

R.G. Flemming et al. / Biomaterials 20 (1999) 573—588 581

Tab

le1

(con

tinu

ed)

Fea

ture

Fab

rica

tion

Mat

eria

lFea

ture

Fea

ture

Cel

lty

pe

Cel

lula

reff

ect

Ref

.ty

pete

chniq

ue

dim

ensions

freq

uen

cyst

udi

ed

Poly

mer

solu

tion

cast

ing

Nitro

-cel

lulo

se,

PV

DF

Smoo

than

dro

ugh

surfac

es,

feat

ure

size

notlist

edR

ando

mR

atsc

iatic

ner

veim

plan

tm

odel

Tissu

est

rips

brid

ged

ner

vest

umps

inal

lofth

ero

ugh

and

inso

me

ofth

esm

ooth

nitro

cellu

lose

and

PV

DF

tube

impl

ants

;bel

l-sh

aped

tiss

ue

adher

edto

rough

tube

impl

ants

;fre

e-flo

atin

gner

veca

bles

,co

ntai

ning

mye

linat

edan

dunm

yelin

ated

axon

san

dSch

wan

nce

llsgr

ouped

inm

icro

fasc

icle

san

dsu

rroun

ded

byan

epin

eurial

laye

rob

serv

edin

smoo

thtu

bes

;m

acro

-ph

ages

com

prise

din

itia

lce

llla

yer

on

rough

poly

mer

s;ep

ineu

rial

laye

rth

inner

on

rough

PV

DF

than

on

rough

nitr

oce

llulo

se,s

moot

hP

VD

Fsh

ow

edm

ore

mye

linat

edax

ons

than

did

smoo

thni

troce

llulo

se

[113

]

Pro

tein

trac

ksPhoto

litho

grap

hy

follow

edby

sila

niza

tion

and

lam

inin

coat

ing

Quar

tz,

hydr

ophob

icsila

ne,

lam

inin

2,3,

6,12

,25

lmw

idth

,th

ickn

ess

notlis

ted

Fea

ture

and

spac

ing

equal

,al

so2

lm

trac

ksse

par

ated

by

50lm

Chi

ckem

bryo

neur

ons,

murine

dors

alro

otga

ngl

iane

urons

Smal

ler

spac

ing

cause

dde

crea

sed

guid

ance

;isola

ted

2lm

trac

ks

stro

ngly

guid

edne

urite

exte

nsion

while

2lm

repe

attr

acks

did

not;

grow

thco

nes

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582 R.G. Flemming et al. / Biomaterials 20 (1999) 573—588

Fig. 3. Schematic diagram of the pattern transfer process. Regions of the substrate not protected by resist are etched isotropically or anisotropically.Three examples of possible etching results are shown: anisotropic, reactive ion etching of silicon substrate to yield square grooves, anisotropic, wetetching of silicon [1 0 0] in KOH to yield V-shaped grooves, and isotropic, wet etching of glass in HF (that undercuts the resist). After the etching iscomplete, the remaining resist is removed.

Fig. 2. Schematic diagram of the photolithographic process. A substra-te coated with a thin polymeric resist in irradiated through a mask.Depending on the type of resist, the irradiated regions become eithermore soluble or less soluble in a developer. Immersion in the developeryields either a positive-tone or negative-tone image of the mask.

negative-tone image of the mask. Due to diffraction lim-itations, the smallest lateral, or width, dimension that canbe achieved by photolithography without using phase-shift masks are of the same size scale as the wavelength ofthe light source used to make them. Current state-of-the-art photolithography employs UV light of wavelength248 nm and is able to achieve features with lateral dimen-sions of 220—250 nm.

The regions of the substrate not covered by resist canthen be etched isotropically or anisotropically, as shownin Fig. 3, to transfer the pattern in the resist to theunderlying substrate. A common topographical featureinvestigated by many researchers was grooves, structuresmade by etching the substrate using a photoresist pat-terned in the shape of lines. Depending on the type of

etchant used, grooves can be constructed to have crosssections of square wave, V-shape, or truncated V-shape[36, 37]. A few examples of different shapes of etchedstructures are depicted in Fig. 3. Repeat spacing is con-sidered the distance from the beginning of a feature to thebeginning of the next feature. A groove and its associatedridge would thus be considered one repeat unit. The etchdepth and the lateral, or width dimensions are alsoshown in Fig. 3. After the etching is complete, the remain-ing resist is removed. The final product is a three-dimen-sional positive or negative relief image duplicating theopaque and transparent regions of the light mask.

One problem encountered with some of the etchingprocesses is undercutting of the resist, yielding shapeswith different dimensions than the patterned resist andwith curved sides. In addition, some etchants produceroughened surfaces. These roughened surfaces sometimessuperimpose random nanometer sized features on top ofthe intended topography [38, 39]. The effects of roughen-ed surfaces on cell behavior have yet to be addressed inthe literature. For a more complete discussion of thelithographic and pattern transfer processes, the reader isdirected elsewhere [36, 37].

Other techniques used to produce features with con-trolled dimensions include glancing angle deposition[40, 41], laser ablation [42, 43], laser deposition [44],replica molding of X-ray lithography masters [45, 46],imprint lithography [47, 48], microcontact printing andetching [49, 50], and ink-jet printing [51, 52]. Some ofthese techniques are capable of producing nanometerscale features. Electron-beam lithography, for example, isthe most developed, high-resolution lithographic tech-nique known, and has been used to fabricate featuresas small as 50 nm over large areas [53—55]. Thesetechniques, for the most part, are not represented inTable 1 because no data has been reported in the literature

R.G. Flemming et al. / Biomaterials 20 (1999) 573—588 583

concerning the behavior of cells upon surfaces createdby these techniques. Cell studies have been conductedon surfaces created by electron-beam lithography, but inthese studies the lateral, or width dimension was micronscale [56—58].

In some of the studies summarized in Table 1, thematerial on which cells were cultured was quartz, silicondioxide, or titanium-coated silicon. In other studies poly-meric replicas of the textured surfaces were created bycasting or embossing techniques [56—77]. For example,the fifth entry in Table 1 lists the fabrication technique asphotolithography and reactive ion etching, and the mate-rial on which the cells were cultured as an epoxy replicaof the silicon original. This means that the original pat-tern was created in silicon, which was used to makea negative polymeric cast, which, in turn, was used tocreate a final, positive replica. In some cases, such as inthe first entry, the negative cast was simply used in thecell assays. If the entry lists a polymer ‘cast’ in the‘Material’ column, then the cells were cultured on a nega-tive replica of the original surface. If the entry lists a poly-mer ‘replica’, then cells were cultured on a positive rep-lica. Polymers used for making such casts and replicasinclude PDMS, epoxy, and polystyrene. Other polymersused in studies include PMMA, PET, polycarbonate,polyetherimide, poly (NIPAM), nitrocellulose, and PVDF.

Most of the studies to date address the effects ofsurface features with dimensions of 0.5 lm and greater.The relevance of these studies to topography found onbasement membranes is unclear since membrane topo-graphy is comprised of much smaller nanometer scalefeatures, as discussed in the previous section. A few inves-tigators have fabricated structures with control over atleast one dimensional less than 500 nm [57, 58, 64, 65, 75,78—89]. These studies are highlighted in bold type inTable 1. In most of those studies, the lateral dimension ofthe features was greater than 0.5 lm, and the depthdimension was less than 0.5 lm. The depth dimension iseasily controlled in the etching process. Using reactiveion etching, Webb et al. [81] and Clark et al. [79]achieved grooves with depths of 100 nm. In anotherstudy, Clark et al. fabricated grooves with depths of200 nm, but with lateral dimensions of 2—12 lm on a sur-face of PMMA using photolithography (for patterningthe lateral dimensions) and reactive ion etching (to con-trol the etch depth) [78]. Wojciak-Stothard and cowork-ers used photolithography and reactive ion etching toproduce silicon grooves 2—10 lm in width and withdepths of 30—282 nm [80]. Several series of polystyreneridges with heights of 30 nm and widths of 0.5—100 lmwere made by Hoch et al. via electron-beam lithographyand reactive ion etching [65]. Grooves as shallows as14 nm and 1, 2, or 4 lm wide were achieved by Rajniceket al. [57, 58]. Britland and coworkers also used photoli-thography followed by reactive ion etching to producegrooves of 100 nm in depth, but 2.5—50 lm in width [86].

Cooper et al. were able to create radial arrays of groovesonly 50 nm in height [90]. Schmidt and von Recumreport round nodes only 380—460 nm in height [75].

Only two studies were found in which cell behaviorwas investigated on surfaces with features of controlledshape and size, and lateral dimensions less than 0.5 lm.Webb et al. [81] and Clark et al. [79] both used a laserholographic method developed for fabrication of masksfor X-ray lithography to create patterns of grooves inquartz with widths of 130 nm and depths of 100—400 and100—1170 nm, respectively.

Other methods have been used to fabricate surfaceswith nanoscale features, but with no direct control overthe topographical shapes and dimensions. Turner et al.used reactive ion etching to produce columnar structureswith average diameters of 57 nm [87]. Martin et al.sandblasted then acid-etched titanium surfaces to createfine and coarse structures with sharp features (100 nmas well as larger pits and craters [83]. Kononen et al.,Lampin et al., and Eisenbarth et al. have used sandblast-ing [88, 91] and sanding [89], respectively, to producefeatures less than 0.5 lm in size. Polymer-coated micro-porous filters [84] and fibronectin-coated glass [82] havealso been used as textured surfaces and have been foundto have features as small as 200 nm. Goodman et al.replicated the surface topography of extracellular matrixvia poly(methylmethacrylate) polymerization casting,which produced an inverse replica, followed by poly-urethane solution casting, which yielded the final positivereplica [85]. The microtextured surface produced by thismethod contained micron and nanometer scale topo-graphical features. Sims and Albrecht have also achievedsome success in replicating the surface structure of fibril-lar collagen, but interactions of cells with such surfaceshave not been reported [92].

Protein tracks have been used as guidance cues forcells. Laminin tracks have been patterned by adsorptionto hydrophobic silane tracks, which had been patternedusing photolithography [93, 94]. In another study, fibersof fibronectin as small as 200 nm in diameter were ad-hered to glass [82].

4. Effects of micro- and nano-structured surfaceson cell behavior

Studies of the interactions between substrate topogra-phy and cells have encompassed a wide variety of celltypes and substratum features including grooves, ridges,steps, pores, wells, nodes, and adsorbed protein fibers.Table 1 summarizes the literature, providing a list offeature type, fabrication technique employed, substratummaterial, feature size and spacing, cell type studied, andthe cellular effect generated by the surface features.

Grooves are the most common feature type employedin the study of the effects of surface structure on cells

584 R.G. Flemming et al. / Biomaterials 20 (1999) 573—588

[35, 56—64, 68—70, 72—74, 76, 78—81, 90, 95—104]. Typi-cally, the grooves are arrayed in regular, repeating pat-terns, often with equal groove and ridge width. The crosssections of the groove are often of the square wave,V-shape, or truncated V-shape [59, 80, 95].

In general, investigations of grooved surfaces revealedthat cells aligned to the long axis of the grooves [35,56—64, 68—70, 72—74, 76, 78—81, 95—104], often with or-ganization of actin and other cytoskeletal elements in anorientation parallel to the grooves [56, 69, 73, 80, 81, 99,100, 103]. The organization of cytoskeletal elements wasobserved to occur in some cases with actin and micro-tubules aligned along walls and edges [69, 98, 100]. Woj-ciak-Stothard et al. noted that F-actin condensationsappeared at topographic discontinuities, often at rightangles to groove edges [98]. They also observed thatmicrotubules formed after 30 min and that vinculin or-ganization was similar to that of actin. In addition, somecells were observed to have lamellae and filipodia be-nding around edges. Similar results were obtained byOakley and Brunette, who found that microtubules werethe first element to align to grooves, followed by actin[103]. Many studies found that the depth of grooves wasmore important than their width in determining cellorientation [78—80, 96]. Orientation often increased withincreasing depth, but decreased with increasing groovewidth. Repeat spacing also played a role, with orientationdecreasing at higher repeat spacing [76, 96]. In otherwords, as ridge width or groove width increased, theorientation phenomena of cells on grooves diminished.Dunn and Brown reported that ridge width is a moreimportant factor than groove width [96].

Certain hierarchical effects have been encountered aswell. Brunnette exposed cells to a pattern of major andminor grooves, in which the minor grooves were placedon the floor of the major grooves at an angle of 54° to themajor grooves [59]. Brunnette found that the cellsoriented preferentially to the major grooves, althoughcells did align to the minor grooves when major grooveswere not present or when a discontinuity existed in thepattern of major grooves. Britland et al. investigated thebehavior of cells upon grooves that were overlaid withadhesive tracks of silane and observed that the cellsresponded to both the topographical and adhesive cues,but that the adhesive cues had the dominant effect[86].

Although most cell types studied exhibited alignmentto grooves, some did not align. One study conducted byMeyle et al. found that, while 100% of fibroblasts alignedto grooves, alignment was observed in only 20% ofmacrophages and not at all in keratinocytes or neuro-phils [101]. Webb et al. noticed that oligodendrocytesand astrocytes were aligned by grooves, but that hippo-campal and cerebellar neuron cells were not aligned [81].In addition, although the oligodendrocytes were aligned,they exhibited little high-order F-actin organization. On

the other hand, the aligned astrocytes showed extensiveorganization of actin stress fibers. Rajnicek and McCraigobserved that Xenopus neurites grew parallel to grooves,but that rat hippocampal neurites grew perpendicular togrooves [57].

When ridges are placed far apart, relative to cellulardimensions, they may be considered individually as stepsor cliffs which the cell must encounter and traverse. Onlya few studies are available in the literature that deal withthis type of substratum topography: the study by Hochet al., which investigated the effect of ridges on funguscells [65], and the study by Clark et al., in which severaldifferent cell types were exposed to steps of varyingheights [105]. As might be expected, cell alignment in-creased with increasing step height.

There are some studies in the literature investiga-ting the behavior of cells on other synthetic features,including wells and nodes [67, 71, 75, 106, 107], pores[84, 107], spheres [108], and cylinders [109]. Green andcoworkers found that nodes of 2 and 5 lm resulted inincreased cell proliferation compared to 10 lm nodes andsmooth surfaces [67]. Cambell and von Recum examinedthe effects of pore size and hydrophobicity in their studyinvolving a canine in vivo implant model [84]. They usednylon mesh coated with polyvinylchloride/polyacrylonit-rile with or without an additional silicone coating andfound that pore size played a larger role than materialhydrophobicity in determining tissue response, withpores of 1—2 lm allowing for direct fibroblast attachment[84]. Fujimoto et al. investigated the behavior of cells onspheres and observed that cells responded to a change insphere size produced by an increase in temperature[108]. The cells released excess active oxygen whensphere diameters shrunk as a result of the temperaturebeing increased from 25 to 37°C.

There were several studies in the literature in whichtextured surfaces were created by techniques which yieldtopographical features that were less definable than thosepreviously discussed, but nevertheless equally interesting.The behavior of cells on sandblasted surfaces has beenstudied, although the observed trends seem less clearthan those on controlled morphologies, such as grooves.In general, adhesion, migration areas, and ECM produc-tion were greater on rougher surfaces, or those surfacessandblasted with larger grain sizes [83, 91, 110]. Turneret al. compared cell behavior on reactive ion and wetetched surfaces and found that primary cells behaved ina manner opposite that of a transformed cell line [87].The primary cells preferred the narrow, columnar struc-tures created by the reactive ion etching, but the trans-formed cells preferred the smoother, wet etched surfaces.

A few studies were found in which protein tracks wereemployed as guidance cues for several cell types, includ-ing neural cells [82, 93, 94]. Isolated tracks were found toprovide stronger guidance than repeated tracks [94].One interesting result from these studies was that cells

R.G. Flemming et al. / Biomaterials 20 (1999) 573—588 585

remained aligned to laminin tracks even when an ortho-gonal DC field was applied [93].

Lastly, Goodman et al. used polymer casting to repli-cate the topographical features of the extracellular matrix[85]. Goodman and coworkers observed that endothelialcells cultured on the ECM textured replicas spread fasterand had appearance more like cells in their native arteriesthan did cells grown on untextured surfaces [85].

5. Conclusions

Topographical cues, independent of biochemistry, gen-erated by the ECM may have significant effects uponcellular behavior. Clearly, substratum topography hasdirect effects on the abilities of cells to orient themselves,migrate, and produce organized cytoskeletal arrange-ments, as documented by the studies summarized here.However, the relevance of such studies to the behavior ofcells adhering to basement membranes is unclear sincethe topographical features of basement membranes havebeen shown to possess much smaller features in thenanometer size range [28—34]. The fact that basementmembranes are composed of unique and intricate topo-graphies into which cells adhere and extend processes,coupled with the fact that topographical features havebeen shown to influence cell behavior, leads to the hy-pothesis that the topography of the basement membraneis important in regulating cellular behavior in a mannerdistinct from that of the chemistry of the basement mem-brane.

It is therefore concluded, that, in order to fully under-stand the role that substrate topography plays in theregulation of cell behavior, smaller, denser, nanometerscale features must be fabricated. Techniques such aslaser holography and X-ray lithography offer the clearestway to synthesize topographical features with controlledlateral dimensions in the nanometer size range. Sincereactive ion etching is already a proven method for con-trolling etch depth, a coupling of etching techniques withstate-of-the-art lithographic techniques to create denser,nanometer scale features is encouraged in order to morefully understand the effects of substrate topography oncellular behavior.

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