ARTICLE IN PRESS

0142-9612/$ - se

doi:10.1016/j.bi

�CorrespondE-mail addr

Biomaterials 27 (2006) 5230–5241

www.elsevier.com/locate/biomaterials

The effect of topographic characteristics on cell migration velocity

Jean-Pierre Kaisera,�, Andreas Reinmannb, Arie Bruininka

aMaterials – Biology Interactions Laboratory, Materials Science and Technology (Empa), Lerchenfeldstrasse 5, CH-9014 St. Gallen, SwitzerlandbHochschule fur Technik und Informatik, Quellgasse 21, CH-2501 Biel, Switzerland

Received 1 May 2006; accepted 2 June 2006

Available online 30 June 2006

Abstract

The migration of cells on structured surfaces is known to be affected by its surface topography. Although the effects of topography

have been extensively investigated the crucial parameters determining the cell–surface reaction are largely unknown. The present study

was performed to describe and to define the role of groove/elevation (ridge) dimensions at the micrometre scale on fibroblast cell

migration by correlating cell shape, migration angle a, cell orientation b and velocity with these dimensions. For this a quantitative

method was developed. We could show that the surface structures significantly influenced migration direction a, cell orientation b and

mean velocity, as well as migration speed in the directions parallel and perpendicular to the grooves/elevations in a surface structure

dependant way. Cell migration velocity parallel, respectively, perpendicular to the structures was significantly affected by the geometries

and dimensions of the substratum. Surface structures were not able to significantly affect distribution patterns of cell shapes. Overall, it

could be shown that differently structured surfaces influenced the cells but no crucial feature could be clearly identified, suggesting that

the reaction of the surface structure might be far more complex than generally is assumed.

r 2006 Elsevier Ltd. All rights reserved.

Keywords: Cell migration; Cell orientation; Cell shape; Image analysis; Fibroblast

1. Introduction

Cell migration plays a key role in normal physiologicalprocesses, such as embryogenesis and morphogenesis, andalso in healing processes. Cell behaviour is influenced bythe substrate characteristics such as chemical and structuralproperties. Knowledge on the critical features influencingcell–surface interactions is sparse [1]. The observation andmonitoring of cell behaviour such as cell shape, cellmigration direction (termed migration angle a) and cellorientation b is of key importance to understand themechanisms behind cell migration. The mechanisms anddifferential responses of cells to various substrate topo-graphy types and dimensions remain largely unknown [2].The understanding of the mechanisms behind cell–surfaceinteractions including migration is therefore of keyimportance to make statements about which the criticalsurface features are that influence cell behaviour.

e front matter r 2006 Elsevier Ltd. All rights reserved.

omaterials.2006.06.002

ing author. Tel.: +4171 274 76 89; fax: +4171 274 76 94.

ess: jean-pierre.kaiser@empa.ch (J.-P. Kaiser).

So far in only a few studies, cell migration has beencompared with a defined set of differently structuredsurfaces under the same test conditions. Each of thesestudies covered only a limited number of aspects.Furthermore, the observation period was short or limitedto only one time point, i.e., the time point at which the cellswere fixated and stained. In the case of micro-machined,grooved surfaces it is known that surface topographyinduces, on the one hand, an alignment of the cells togrooves and on the other hand, steer the cells to migratepredominantly along these grooves. Elongation andorientation of the cells increases with the groove depth[3,4]. When the grooves or ridges are above the cell size, cellorientation b is not considerably affected, although at theedges of the grooves cells may align. With the reduction ofthe groove/ridge width at or below the width of the cells,effects on orientation become more pronounced [5]. Notonly the depth of the grooves but also the width seem toplay an important role in the cell reaction [3]. Regardingmigration velocity, Berry and his team showed that surfacestructure may significantly affect cells [6]. In the present

ARTICLE IN PRESS

Table 1

Description of the structures

Shape Structure Z (mm) X (mm) W (mm) Y (mm)

A — — — —

B 5 5 5 —

C 5 5 25 —

D 10 10 5 —

E 22 22 18 —

F 5 11 24 6

G 10 16 19 6

H 22 28 12 6

I 5 35 5 30

J 10 40 10 30

K 22 52 18 30

J.-P. Kaiser et al. / Biomaterials 27 (2006) 5230–5241 5231

study, dimensions of grooves and ridges were chosen thatcan also be reproduced industrially by micro-machiningand that are known to affect cells, i.e. groove depth/ridgeheight of 5–22 mm, and spacing of 5–30 mm. In a previousstudy, we could show that structures in the range between 1and 50 mm differently affect the cell morphology [7].Furthermore, several teams have shown that by increasingthe dimensions of the grooves in the micrometre range, theeffects on cell migration and functionality also becomemore pronounced [5,8,9]. Most studies investigating theeffects of surface topography on cells used surfacechemistries other than titanium [2,4,10–15]. Cell behaviouron coated polymer surfaces may be different, since thechemistry of the surfaces has major effects on cellbehaviour [16,17]. Since surface chemistry greatly affectscell responses [18] in the present study a commonly usedbiocompatible titanium alloy (Ti4Al6V) was used assubstrate.

Different kinds of approaches were chosen so far toanalyse cell shape and migration of living cells. Partlystatements were made only on the basis of the interpreta-tion of single microscopic pictures [10,15,16]. Others usedmore mathematical- and statistical-based approaches. Forinstance, Lo et al. [19] investigated only the velocity ofselected cells with time-lapse images with custom madesoftware. Berry et al. [6] determined the migrationdistances of the cells by measuring the migration distancesof the selected cells frame by frame. Both methods arerelatively time-consuming, enabling the analysis of only asmall number of cells. Recently, we described a set-up andanalysis system enabling the ‘‘long-term’’ monitoring andanalysis of a very large quantity of cells at the same time[20]. This approach allows qualitative and quantitative

statements. So far no reports are available describing theeffects of differently structured surfaces, which cover theaspects cell shape, cell orientation b, migration direction aand migration velocity simultaneously, including a statis-tical analysis thereof.In the present study, we sought to answer the following

questions: (i) Is the cell shape, as defined by length/widthratio pattern, and cell orientation b affected by differentmicro-grooves/ridges characteristics with dimensionsabove 5 mm? (ii) Is the migration velocity and migrationdirection a modified by these groove/ridge characteristics?(iii) Does cell orientation b determine the cell migrationdirection a? (iv) Does cell migration direction a influencecell migration velocity? To answer these questions, 10differently structured surfaces with micro-grooves/eleva-tions in various micrometre dimensions and unstructuredplane surfaces as a reference substrate were prepared(Table 1). Biological effects, i.e. effects on migrationdirection a, cell orientation b, cell shape and migrationvelocity were assessed using 3T3 fibroblasts. Fibroblastswere chosen because of their extensive migration activityand their known reactivity on surface structures [21–23].Grooves and elevations were the base of the present studysince this kind of structure represents the simplest type ofrepeated surface structuring after plane surfaces.

2. Materials and methods

2.1. Substrata

The substrata used consisted of passivated Ti4Al6V disks of 16mm

diameter. XPS analysis revealed a surface ion composition of 24.3% O,

4.09% Al, 68.57% Ti and 3.02% V. The top surface of each disk consists

ARTICLE IN PRESSJ.-P. Kaiser et al. / Biomaterials 27 (2006) 5230–52415232

of 11 differently structured areas, one of them being plane and

unstructured. The Ra value of the surface was 0.20mm. The 10 differently

structured areas could be grouped into three classes of surface structures.

The first class (structures B–E) consisted of V-shaped grooves with plane

tracks (ridges) between the grooves. The second class (structure F–H)

consisted of U-shaped grooves with plane tracks (ridges) between the

grooves and plane tracks on the bottom of each groove. The third class

(structure I–K) consisted of elevations with plane tracks between two

elevations and plane tracks (ridges) on the top of each elevation. The

geometry and dimensions of the different structures are summarized in

Table 1. The structures had sizes in the ranges of 5–30mm, sizes which can

be reproduced industrially by micro-machining in large quantities. In

previous studies it had been shown that not only structures in the sub-

micrometre range were affecting cell behaviour, but also structures in the

range above the cell diameter, i.e. of 50mm and above had severe effects on

cell orientation and cell morphology [7].

2.2. Cell cultures

In the present study, 3T3 Mouse Swiss Albino embryo fibroblasts were

used, which were obtained from the European Collection of Cell Cultures,

Salisbury, UK. The mean length and width of these cells on unstructured

plain Ti4Al6V surface was 39.9 and 26.5mm, respectively. Cell labelling

and migration monitoring was carried out as previously described [20].

Briefly, the 3T3-fibroblasts were adapted to a carbonate-free, HEPES

buffered (25mM) Dulbecco’s modified Eagle medium (DMEM). Cells were

harvested using trypsin-EDTA and labelled with a fluorescent lipophilic

dye (DiI) (1,1-dioctadecyl-3,3,30,30,-tetramethylindocarbocyanin perchlor-

at). Of the cell suspension, 2.5ml containing 5� 104 DiI labelled cells were

seeded on the structured discs. After an incubation period of 15 h at cell

culture conditions, enabling attachment of cells, non-adherent cells were

removed. Thereafter, the structured sample was transferred into a flow

chamber containing culture medium. The monitoring of cell migration was

started after an equilibrium period of 3 h at 37 1C. The whole observation

of the cell behaviour occurred at a temperature of 37 1C and lasted 24 h.

The cell migration monitoring was performed by taking pictures (Fig. 1A

and B) of the culture at the same location every 15min. From the resulting

pictures, the migration trajectories (Fig. 1C and D) and by that migration

angle a, migration velocity and also cell orientation b and cell shape were

estimated using special image analysis software. Between two subsequent

pictures, the dislocation of each cell centre was estimated by representing a

‘‘migration vector’’. The migration vector of the cells between two

subsequent pictures was expressed as a dot in the XY-diagram (Fig. 1E

and F). It is characterized by its angle (migration angle a) and length

(migrated distance within 15min as measure of migration velocity). The

possible value of the migration angles a is between 01 and 3601. These

values were clustered into 18 ranges of 201 and the frequency of occurrence

of each migration angle a within a certain cluster was counted (Fig. 1G

and H). The grooves/elevations were orientated in the direction 01/1801

relative to the X-axis. The cell orientation b, expressed as angle of the

longest diameter across a cell relative to the X-axis within each picture was

also calculated. The obtained values were then clustered into nine ranges

of 201 and the frequency of occurrence counted (Fig. 1I and J).

2.3. Statistics

The experiments were repeated independently three times. In each

experiment, cell migration was analysed on all structures for an

observation period of 24 h. For each experiment, cells that could be

exactly allocated during the whole experiment (giving result to 96 data

points each), 10 migrating cells were randomly selected for statistical

analysis. Additionally, for statistical analysis, the respective frequency of

each migrating cell (96 data points) was estimated. Significant effects were

determined using the ANOVA two factorial Bonferroni/Dunn test with

experiment number and surface structure as factors. Differences were

considered at po0.05. With the w2-test, it could be evaluated to what

extent the squares of the differences between mean values and expected

values divided by the expected values were significantly higher than the

critical w2-value.

3. Results

Cell migration has been compared with a set ofdifferently structured surfaces on fibroblast cell behaviourunder the same conditions. In the present study, dimen-sions of grooves and elevations were chosen, which areknown to affect cells, i.e. groove depth/ridge height of5–22 mm, and spacing of 5–30 mm. So it was possible tocompare cell behaviour such as migration direction (termedmigration angle a), cell orientation b and migration. Evenif the groove/elevation dimensions were within the rangescomparable to the sizes of the 3T3 fibroblasts, cellbehaviour on structured surfaces was significantly influ-enced by these structures. In time-lapse experiments it wasobserved that the 3T3 fibroblast cells used the edgesbetween the grooves and the ridges, respectively, the edgesbetween the plane tracks and the elevations. As a result cellorientation b, and migration direction a values were foundto be correspondingly affected. Beside the size of thestructures also the elevation density had significantinfluence on the cell behaviour.

3.1. Effects of cell collisions on cell migration

In our study cells migrating in the opposite direction onneighbouring grooves on the structured surfaces B and D,structures with practically no space between the grooves,were crossing without affecting each others migratingcharacteristics. Only in the few cases, when cells weremigrating towards each other on exactly the same groove,generally one cell was changing its migration directionwhereas the other continued its migration in the samedirection. Only in very few cases it was observed that bothcells changed their migration direction. It happened onlyseldom that two cells were migrating on exactly the samegroove in the opposite direction on these highly groovedstructures. Therefore, the few cases where cells changedtheir migration direction, because they were migrating onexactly the same groove in the opposite directions wereneglect able. On structures C and E–H the cells were usingthe larger areas between the grooves, respectively, thelarger areas of the walls of the deeper grooves formigration. The width of the ridges, respectively, the wallsof the grooves were large enough that two 3T3 fibroblastcells were able to cross without affecting the cell migratingin the opposite direction. It happened very seldom that acell changed its migration direction after approachinganother cell migrating in the opposite direction on the sametrack. A similar behaviour was found for 3T3 fibroblast cellmigration on the structures I–K. The cells were migratingon the flat areas between the elevations along the edges.The areas between the elevations were large enough thattwo cells could cross without disrupting each other. 3T3fibroblast cells migrating on the plane surface A changed

ARTICLE IN PRESS

Fig. 1. Analysis of cell behaviour. The data analysis of two surfaces of one representative experiment is shown (plane surface A, and structured surface E).

One representative picture of a series made of DiI-labelled 3T3 fibroblast cells on the plane surface (A) and on the structured surface (B). The covered trails

(trajectories) of the cells migrating on a plane surface (C) and structured surface (D) over an observation period of 24 h was drawn. The migration vector

of the cells between two subsequent pictures expressed as a dot in the XY-diagram (E: plane surface and F: structured surface). The frequency of cells

migrating within a certain angle range of 201 each on a plane surface (G) and structured surface (H). The data point is placed in the centre of the angle

range. The frequency of occurrence of each cell orientation b within 18 angle ranges of 101 each on plane surface (I) and structured surface (J).

J.-P. Kaiser et al. / Biomaterials 27 (2006) 5230–5241 5233

ARTICLE IN PRESS

Fig. 1. (Continued)

J.-P. Kaiser et al. / Biomaterials 27 (2006) 5230–52415234

migration direction without stopping when two cells wereapproaching each other. A cell–cell collision between two3T3 fibroblasts on the plane surface was never observed.

3.2. Migration angle a

On the structured surfaces, cell migration occurredpreferably in the two directions parallel to the grooves/elevations resulting in, a significantly increased frequencyof cell migration parallel to the grooves/elevations (range07101 and 1807101) (Fig. 1H). Between 30% and 44% ofall cell dislocations occurred in these two angle ranges. Ifcell migration is random, a value of 11% could be expected(Table 2). Of all tested structured surfaces the cellmigration frequency parallel to the structures was higheston surface E, whereas on surfaces C, F and I, structureswith flat grooves, respectively, low elevations, the lowestfrequencies of cells migrating in this direction were found.The frequency of cells migrating perpendicular to thegrooves/elevations (ranges 907101 and 2707101) was just

inversely correlated to the frequency of cells migratingparallel to the structures. The highest number of cellsmigrating perpendicular to the structures was found onstructures with flat grooves/elevations. These were thestructures where among the structured surfaces the lowestfrequency of migrating cells in direction parallel to thegrooves/elevations was observed. In analogy, data analysisrevealed that the frequency of cell migration angles on aplane surface was homogenously distributed within the 18angle ranges of 201 (Fig. 1G). The covered trails of the cellson the plane surface were random (Fig. 1C). Employing thew2-test, it could be shown that the cells migrating on theplane surface (surface A) had no preferred direction.

3.3. Cell orientation b

On structured surfaces cells significantly preferredorientating themselves parallel to the axis of the grooves/elevations within a sector 7101. Cell orientation occurredalso on larger grooves/elevations with dimensions up to

ARTICLE IN PRESS

Table 2

Frequency of cells migrating parallel, respectively, perpendicular to the grooves/elevations within a sector of 7101

Structure Percentage of cells migrating parallel to the

structures within a sector 7101

Percentage of cells migrating perpendicular to the

structures within a sector 7101

A 14.7171.07 13.9171.86

(BCDEFGHIJK) (BCDEFGHIJK)

B 36.0273.49 5.4471.24

(A) (A)

C 31.2575.56 8.1172.65

(AE) (A)

D 37.1674.08 6.1371.04

(A) (A)

E 44.1973.99 3.9270.57

(ACFI) (AI)

F 32.8374.16 7.3572.15

(AE) (A)

G 37.9474.93 5.2970.99

(A) (A)

H 36.9373.97 5.3671.10

(A) (A)

I 30.4575.90 6.7171.68

(AE) (A)

J 37.5873.16 5.1471.39

(A) (A)

K 36.7675.64 6.2872.01

(A) (A)

Calculated (%) 11.11 11.11

(no preference)

Data represent mean7SEM over mean values of three independent experiments. In brackets: significant values different from the indicated structure

(p40.05).

J.-P. Kaiser et al. / Biomaterials 27 (2006) 5230–5241 5235

30 mm. Among all structured surfaces structures C and I,structures with shallow grooves/elevations (5 mm) and largeplane sections between the grooves/elevations exhibited thelowest number of cells orientated parallel to the structures(Table 3). Cells orientated perpendicular to the structureswere found with higher frequency on structures (C, E, G),being structures with large plane sections between thegrooves/elevations, respectively, on structures (H, K)characterized by deep grooves/elevations and large walls.

3.4. Correlation between cell orientation b and cell

migration angle a

For all surfaces, the correlation between cell orientationb and migration angle a was estimated (Table 4). In thecase of structured surfaces, cells that were orientatedparallel and those that were orientated perpendicular to thegroove/ridge direction were migrating preferentially paral-lel to the grooves. The percentage of cells that weremigrating in direction parallel to the grooves/elevationswas significantly higher in the case of cells orientatedparallel to the grooves/elevations (41–55% instead of16–37%). On surface E, the highest percentage of cellswas found to migrate parallel to the groove directionindependent from the cell orientation b. In general, it wasfound that parallel orientated cells migrating with higherfrequency in direction parallel to the structures in case the

dimension (depth/height) of the grooves/elevations was30 mm. On the plane surface A a frequency of 23% ofparallel orientated cells within a sector of 201 weremigrating in a sector with latitude of 7101 parallel to thegrooves/elevations. Furthermore, a frequency of 15% ofperpendicular orientated cells within a sector of 201 weremigrating in a sector with latitude of 7101 perpendicularto the groove direction. In case no correlation is present avalue of 11% is expected. These results clearly demonstratethat cell migration did not necessarily occur in directionparallel to the cell orientation but that the chance is nearlydoubled. A detailed analysis of cells on the structuredsurfaces revealed that there was no significant correlationbetween cell orientation b and cell migration angle a (datanot shown).

3.5. Cell shape

The cell shape was expressed as length/width ratio. Themost prominent cell length/width ratio range on all 11surfaces tested was significantly the type with a length/width ratio of 1.1–1.5. No significant difference in thefrequencies of a certain type of cell shape was found amongthe different structured surfaces. The frequency of roundcells (length/width ratio 1.0–1.1) was slightly higher on theplane surface (A), than the amount of cells with this shapefound on the structured surfaces. On the plane surface

ARTICLE IN PRESS

Table 3

Frequency of cells orientated parallel, respectively, perpendicular to the grooves/elevations within a sector of 7101

Structure Percentage of cells orientated parallel to the

structures within a sector 7101

Percentage of cells orientated perpendicular to the

structures within a sector 7101

A 9.8470.95 9.4170.40

(BCDEFGHIJK) (—)

B 38.3474.83 6.1672.36

(A) (—)

C 27.3076.26 8.8973.24

(AGH) (—)

D 42.5274.59 5.8671.78

(A) (—)

E 40.7173.32 9.4673.92

(A) (—)

F 38.9573.57 7.0271.20

(A) (—)

G 44.6876.78 11.7773.82

(AC) (—)

H 43.6971.90 8.9474.25

(AC) (—)

I 30.8579.23 7.4473.45

(A) (—)

J 38.1575.23 6.2072.07

(A) (—)

K 40.9176.97 8.9172.92

(A) (—)

Calculated % 11.11 11.11

(no preference)

Data represent mean7SEM over mean values of three independent experiments. In brackets: significant values different from the indicated structure

(p40.05).

J.-P. Kaiser et al. / Biomaterials 27 (2006) 5230–52415236

20.1% of the cells showed a circular shape, whereas thefrequency of this type of cell shape on the structuredsurfaces varied between 7.4% and 17.1% (data not shown).

3.6. Migration velocity

Each cell changed its migrating velocity continuouslyduring the observation period. Of all 11 surfaces tested,cells migrated most frequently with a velocity of2.070.5 mm/15min (data not shown). The migrationvelocity pattern was largely the same on all surfaces. Themean cell migration velocity was, however, different on thevarious surface structures (Table 5). Cells migrating onsurface E and F, being structures with large plane tracksbetween the grooves, respectively, on surfaces I and J,being structures with large plane areas between theelevations exhibited the highest mean migration velocity.The lowest mean migration velocity was found on surfaceD, a structure with 10 mm deep grooves and very narrowtracks between the grooves.

Cells migrating parallel to the grooves/elevations (7101)increased their migration velocity by a factor of 1.2–1.4compared to the overall mean migration velocity of thatsurface. Cells migrating perpendicular to structures (7101)migrated with a significantly reduced migration velocitycompared to mean overall migration velocity of thatstructure. Depending on the surface structures cells

exhibited a migration velocity in direction parallel to thegrooves/elevations which was 1.5–2.6 times faster than themigration velocity of cells migrating perpendicular (7101)to the structures.

4. Discussion

In the present study, the effects of the different surfacescharacterized by differently sized ridges and grooves onfibroblast cell behaviour was assessed simultaneously underthe same conditions over a period of 24 h by time lapsemicroscopy. The in-depth computer analysis of theresulting pictures allows us to conclude that migrationangle a, cell orientation b, cell shape and migration velocityare differently affected by the surface structure.To mimic the in vivo situation the experiments were

performed under cell density conditions where cell–cellcontact during cell migration was possible. Cell behaviourmight be different under single cell conditions. However, ithad been reported by Damji et al. [24] that fibroblast cellconfrontations on structured substrata resulted in mostcases in a lateral deflection in cases where two cells contacteach other. In line with our findings it was reported thatafter fibroblast–fibroblast collision on grooved surfaces inmost of the time cells moved to the sides of the grooves andcontinued in their original directions. In their study contactinhibition after fibroblast cell collision occurred only in

ARTICLE IN PRESS

Table 4

Correlation of cell orientation b and migration angle a

Structure Frequency in (%)

Percentage of cells orientated parallel and

migrating parallel to the structures within

a sector 7101

Percentage of cells orientated

perpendicular and migrating

perpendicular to the structures within a

sector 7101

Percentage of cells orientated

perpendicular and migrating parallel to

the structures within a sector 7101

A 22.9673.96 15.3471.51 10.8270.15

(BCDEFGHIJK) (GIJK) (—)

B 49.0474.40 7.4471.73 20.2372.75

(A) (—) (—)

C 43.5374.36 9.5472.81 19.8974.06

(A) (—) (—)

D 50.2273.99 11.7173.27 25.2571.03

(A) (—) (—)

E 54.5477.84 6.3570.39 37.1374.46

(AFG) (—) (—)

F 41.4071.35 8.0774.45 23.3274.51

(AE) (—) (—)

G 43.8576.50 6.8870.94 26.5573.41

(AE) (A) (—)

H 47.6374.83 5.5871.90 20.4471.94

(A) (—) (—)

I 43.6175.21 7.4872.15 19.8073.97

(A) (A) (—)

J 50.3973.26 4.0772.05 15.8874.10

(A) (A) (—)

K 52.0074.33 7.1871.66 21.3375.82

(A) (A) (—)

Data represent mean7SEM over mean values of three independent experiments. In brackets: significant values different from the indicated structure

(p40.05).

Table 5

Mean cell migration velocity and velocity of cells migrating parallel (7101) and perpendicular (7101) to the grooves/elevations on different structures

Structure Velocity of cells migrating on a certain surface (mm/15min)

All cells Cells migrating parallel to the grooves/

elevations within a sector 7101

Cells migrating perpendicular to the

grooves/elevations within a sector 7101

A 5.0470.82 5.1870.81 4.8970.79

(I) (EIK) (BCJK)

B 4.8270.41 6.3470.50� 2.9970.44�

(EFI) (I) (AI)

C 4.8770.47 6.2770.67� 2.7370.45�

(EFI) (I) (AFI)

D 4.5270.36 6.1370.30� 3.5270.26�

(EFIJ) (EI) (—)

E 5.2370.38 6.7470.50� 3.1770.54�

(BCDHIK) (AD) (—)

F 5.3770.16 6.5470.08� 4.3670.10�

(BCDGHK) (—) (CJ)

G 4.9970.37 6.3270.49� 3.5470.33�

(FI) (I) (—)

H 4.7370.16 6.1370.10� 3.0670.38�

(EFIJ) (I) (—)

I 5.6270.25 7.2470.28� 4.6870.20�

(ABCDEGHJK) (ABCDGH) (BCJK)

J 5.0870.63 6.5770.51� 2.4870.12�

(DHI) (—) (AFI)

K 4.7870.19 6.6470.21� 2.9070.33�

(EFI) (A) (AI)

Data represent mean7SEM over mean values of three independent experiments. In brackets: significant values different from the indicated structure

(p40.05).�Significant values different from the mean cell migration velocity observed on a certain structure.

J.-P. Kaiser et al. / Biomaterials 27 (2006) 5230–5241 5237

ARTICLE IN PRESSJ.-P. Kaiser et al. / Biomaterials 27 (2006) 5230–52415238

11% of all cases. In the few cases that contact inhibitioncells ceased movement, it was seen that they rested for aperiod and continued migrating thereafter. Also we couldnot find clearcut differences in cell migration characteristicsas a result of cell collisions.

4.1. Cell migration angle a

The effect of the structures on the migration angle a wasmuch more prominent compared to the effect on cellorientation b. Cells migrating on structured surfacespreferred a migration angle in the direction parallel tothe grooves/elevations. Similar observations had beenmade by Lenhert et al. [25]. Structures with a depth or aheight of 10 or 22 mm, respectively, showed a slightly higherfrequency of 3T3 fibroblast cells with a migration angle a inthe direction parallel to the structures, when compared tostructures that were only 5 mm deep/high (surfaces C, F, I).Of the grooved surfaces deep V-shaped grooves (surface E)as well as surfaces with a higher density of shallow grooves(surface B and D) were most efficient in influencingmigration direction of cells. Dalton et al. [26] reportedthat the depth of the grooves is a crucial factor in directingthe migration of cells. Structure E, a surface with 22 mmdeep grooves, had the largest influence in directing cellmigration. However, all other structured surfaces, even thestructure B, C, F and I with grooves/elevations of 5 mmdepths/heights, were quite effective in directing migratingcells. These observations were different from the findingsreported by Li et al. [11], who investigated the behaviour ofbovine aortic endothelial cells migrating on cell-adhesiveand non-adhesive stripes of various widths. Cells that wereforced to move on the narrowest strips (15 mm) migratedpersistently in the direction of the strips, whereas cells onthe 30 and 60 mm strips were less persistent in direction andchanged migration direction frequently.

4.2. Cell orientation b

It is generally accepted that cells are able to orientatethemselves in the direction of narrow (nm–mm) periodicallygrooved substrata [2,14]. To which degree groovedstructures with a size above 5 mm are able to affect cellorientation b remain, however, controversial [10,17,24]. Inour study 3T3 fibroblastic cells plated on structuredsurfaces with dimensions in the range of 5–30 mmsignificantly preferred to orientate themselves in the sectorsparallel to the grooves/elevations along the edges, for allstructures tested, even for structures where the tracks had awidth of 30 mm, representing roughly the main diameter ofthe cells. Only minor differences between the various typesof structures were found (Table 3). On structures withshallow grooves and large tracks between the grooves(surface C) the lowest frequency of parallel orientated cellsamong all structured surfaces was observed. Perpendicularorientated cells were found with higher frequency onstructures with large ridges (surfaces C, E, G), respectively,

on structures with large flat areas between the elevations,e.g. surface K. These data indicate that an effect on cellorientation b was present although no effect on cell shape(length to width ratio) was found. This suggests that thelatter two characteristics act as independent parameters.Our data regarding alignment are similar to thosedescribed for osteoblasts on 150 nm deep grooved surfacesand of primary human corneal epithelial cells contactinggrooved surfaces with a 400 nm sized pitch, including thefrequency of alignment [14,25]. The frequency of alignmentis, however clearly lower than that described by Diehl et al.[2] for transformed human corneal epithelium cells ongrooved surfaces with a pitch of 0.4–4 mm. In this case, theyachieved around 80% alignment within 101 in direction ofthe grooves. Teixiera et al. [14] chose SiO2 and Diehl et al.[2] polyurethane as the surface, suggesting that surfacematerial composition (chemistry and/or rigidity) may playa crucial role in how cells react on surface structures. Thelatter hypothesis is supported by findings of differentbehaviour on various surfaces [18] including degree ofrigidity. This is the reason why we used Ti4Al6V assubstrate, a biocompatible material commonly used inmedical applications.

4.3. Correlation between cell orientation b and cell

migration angle a

Our observations regarding cell orientation b andmigration angle a of cells migrating on a plane surfaceindicate that cells were not necessarily migrating in thedirection of their orientation (Table 4) and even that cellorientation b plays only a minor role, if at all, with respectto cell migration angle a. Similar observations, i.e. thatorientation of the cell gives a causal clue on the migrationdirection have also been made by Oakley and Brunette [16].On structured surfaces, however, the grooves/elevationsmight trigger a strong correlation between cell orientationb and migration angle a if only cells oriented parallel to thegrooves/elevations were taken into account. The latter wasmost prominent on V-grooved surface E giving rise to morethan 54% of the cells migrating in the direction of their cellorientation within latitude of 7101. As already observed inthe behaviour of 3T3 fibroblast cell orientation, surface E,a surface with deep V-shaped grooves (22 mm) showed thehighest amount of parallel orientated cells which weremigrating in direction parallel to the structures. Onsurfaces with shallow structures and large flat tracksbetween the structures (surface C, F, G, I) the lowestfrequency of parallel orientated cells migrating parallel tothe structured surfaces was observed. Since also of the cellsorientated perpendicularly to the grooves/elevations, mostcells migrated parallel to the grooves, it can be concludedthat the latter correlation between cell orientation b andmigration angle a is not a causal one. Thus our resultsshow that 3T3 fibroblast cells that were orientatedperpendicular to the structures were predominantly forcedby the structures to migrate in the direction parallel to the

ARTICLE IN PRESSJ.-P. Kaiser et al. / Biomaterials 27 (2006) 5230–5241 5239

structures and, if at all, only marginally by their cellorientation b. Similar observations had been reported forosteoblast like cells [25]. Cells seeded on a polystyrenesurface with 50 nm deep grooves were significantly migrat-ing in direction parallel to the grooves even the cellorientation was not always the same as the groovedirection.

Regarding cell shape of 3T3 fibroblast cells our analysisshowed that structured surfaces did not necessary alter therelative number of elongated cells. In addition, nodifferences in the frequency of a given length/width ratiocould be detected among the various surface structures.This is in line with other reports about fibroblast spreadingbehaviour [27–29] showing that the cell shape on planarsubstrates and on narrow linear strips of 15 mm width weresimilar. Concerning the preferential location of the cells onthe differently structured surfaces the 3T3 fibroblast cellsspread along the grooves and were located on the top of theridges, inside the grooves and their walls. Similar observa-tions were made by Wojciak-Stothard et al. [30] for babyhamster kidney and Walboomers et al. [31] for dermalfibroblasts. Thus fibroblasts spread on the top of the ridgesand inside the grooves and their walls keeping their length/width ratio. In contrast to fibroblasts, epithelial cells didnot maintain similar cell lengths, when cultivated ongrooved surfaces [28]. Epithelial cells by that squeezethemselves into the grooves of structured surfaces. Thesedata suggest that the reaction of cells on surface structuresis not the same for all cells but is cell type dependant.

4.4. Migration velocity

If and to what degree grooves in the micrometredimensions are able to accelerate migration speed stronglydepends on the investigated cell type and, in the case of celllines, on the strain used [21,32]. Even with the same celltype, different results have been obtained. In a study usingendothelial cells and chemically structured surfaces (15 mmwide cell adhesive separated by cell repellent stripes) astrong increase in migration velocity was found in one case[11] and in another case not [12]. Which surface features areable to speed up the cells are still unknown and may also becell type dependant [21].

The mean velocity of the 3T3 fibroblast cells migratingon the different structured surfaces in the present study wasdepending from the surface geometry and the sizes of thestructures. Cells migrating on the plane surface were by farnot migrating with the lowest mean velocity (Table 5). Forall 10 tested structured surfaces the 3T3 fibroblast cellsestablished a significant higher migration velocity parallelto the structures and a significant lower migration velocityperpendicular to the structures compared to the meanvelocity of all 3T3 fibroblast cells migrating on that certainsurface. 3T3 fibroblast cells migrating on surface I showedthe highest mean velocity as well as the highest velocitiesparallel and perpendicular to the structures among the 10differently structured surfaces. Obviously this structure

with shallow elevations (5 mm) and large flat tracks betweenthe elevations were supporting an effective cell migration.The increased main migration speed on this surface was innearly all cases significantly higher than the velocities onthe other 10 surfaces (Table 5). Structuring of the surfaceresulted in an increase in cell migration velocity parallel tothe grooves/elevations by roughly 1.3 times compared tothe overall main velocity on that certain surface. Similarobservation had been reported by Curtis et al. [21]. Thisgroup reported that epitena (synovia) cells increased theirmigration speed on grooved surfaces compared to flatsurfaces. In their study the velocity increase was dependingon the groove width. The epitena cells increased theirvelocity on a grooved surface with 10 mm wide grooves by afactor of 1.8 and on 5 mm wide grooves even by a factor 5.5.3T3 fibroblast exhibited on the surfaces B and C,representing comparable surfaces with the same geometrybut different sizes of the ridges, no significant difference inmigration velocities (Table 5). The different sizes of ridgewidths were not influencing the velocity of the 3T3fibroblast cells. Grooved structures enhanced the rate ofmovement of epitena cells compared to plane surfaces [21].For the 3T3 fibroblasts it was different. It had beendemonstrated that the mean velocities were not necessarilyenhanced when cells were migrating on structured surfaces.Depending on the surface geometry and the sizes of thestructures cell migration velocity could be influenced in apositive as well as in a negative way.The comparison of the migration velocities between 3T3

fibroblast cells migrating parallel to the structures (7101)and cells migrating perpendicular to the structures (7101)revealed that the migration velocity of perpendicularmigrating cells was significantly lower than the migrationvelocity of cells migrating within a sector 7101 parallel tothe structures. The differences between the velocities indirection parallel and perpendicular to the structuresdepended on the surfaces structure. On surface E adifference between the velocity parallel and the velocityperpendicular to the structures varied by a factor 2.1,whereas for structure F only a factor of 1.5 was estimated.Similar observations have been reported by others. Lenhertet al. [25] reported that osteoblast-like cells (calf periost)migrated on a 150 nm deep grooved surface 1.5 times fasterin direction parallel to the grooves than in directionperpendicular to the grooves. Dalton et al. [26] observedthat bovine corneal epithelial tissues migrated on groovedsurfaces with a groove depth of 5 mm approximately 20times faster in direction parallel to the grooves comparedwith the velocity exceeded perpendicular the grooves. Li etal. [11] reported that bovine aortic endothelial cellscultured on collagen strips with a width of 15 mm migratedapproximately 4.6 times faster in the direction parallel tothe strips than in the direction perpendicular to the strips.These results clearly demonstrate that the migrationvelocity is not only dependant on the surface geometryand the dimensions of the surface structures but also onwhich cell type is used.

ARTICLE IN PRESSJ.-P. Kaiser et al. / Biomaterials 27 (2006) 5230–52415240

Cells with a nearly round shape (shape with a length/width-ratio below 1.5) exhibited on most surfaces asignificantly lower migration velocity than the cells with amore elongated shape. This is in line with findings of Webbet al. [1] who reported that elongated shape primaryneonatal mouse dermal fibroblasts plated on a collagensurface exhibited a higher migration velocity than cellsplated on other surfaces, where the cells had a rather roundshape. However, in our study practically no significantdifferences were observed between the different elongatedcells (shape with a length/width-ratio of 1.5 or higher). Thedegree of cell elongation was obviously not a criticalparameter influencing the migration velocity. It musthowever be noted that in our study 3T3 fibroblast cellswith similar round shape exceeded different velocities onstructures with different geometries and sizes. Primaryneonatal mouse dermal fibroblasts have been reported tobehave similarly. Cells with a more or less round shapeexceeded on different surfaces quite different migrationvelocities [33].

5. Conclusion

To elucidate the role of surface structuring in cellmigration behaviour, surfaces with grooves and/or ridgesof different dimensions and spacing were biologicallyevaluated. The main findings were that cell orientation b,cell migration angle a and migration velocity parallel,respectively, perpendicular to the grooves/elevations weresignificantly different on structured surfaces compared tothe plane surface. No relationship could be found betweencell orientation b and migration direction a. Regardingmigration velocity (overall mean velocity, as well asmigration velocity parallel and perpendicular) among allthe tested structured surfaces, the structure with shallowelevations and large flat tracks between the elevationssupported most effectively cell migration.

References

[1] Webb K, Hlady V, Tresco PA. Relationship among cell attachment,

spreading, cytoskeletal organization, and migration rate for ancho-

rage-dependent cells on model surfaces. J Biomed Mater Res

2000;49:362–8.

[2] Diehl KA, Foley JD, Nealey PF, Murphy CJ. Nanoscale topography

modulates corneal epithelial cell migration. J Biomed Mater Res A

2005.

[3] Curtis A, Wilkinson C. Topographical control of cells. Biomaterials

1997;18:1573–83.

[4] Clark P, Conolly P, Curtis A. Topographical control of cell

behaviour: II. Multiple grooved substrata. Development 1990;108:

635–44.

[5] Clark P, Connolly P, Curtis AS, Dow JA, Wilkinson CD.

Topographical control of cell behaviour: I. Simple step cues.

Development 1987;99:439–48.

[6] Berry CC, Campbell G, Spadiccino A, Robertson M, Curtis ASG.

The influence of microscale topography on fibroblast attachment and

motility. Biomaterials 2004;25:5781–8.

[7] Bruinink A, Kaiser J-P, Meyer DC. Effect of biomaterial surface

morphologies on bone marrow cell performance. Adv Eng Mater

2005;7:411–8.

[8] Brunette DM. Spreading and orientation of epithelial cells on

grooved substrata. Exp Cell Res 1986;167:203–17.

[9] Palmaz JC, Benson A, Sprague EA. Influence of surface topography

on endothelialization of intravascular metallic material. J Vasc Interv

Radiol 1999;10:439–44.

[10] Duncan AC, Weisbuch F, Rouais F, Lazare S, Baquey C. Laser

microfabricated model surfaces for controlled cell growth. Biosens

Bioelectron 2002;17:413–26.

[11] Li S, Bhatia S, Hu YL, Shiu YT, Li YS, Usami S, et al. Effects of

morphological patterning on endothelial cell migration. Biorheology

2001;38:101–8.

[12] Matsuda T, Sugawara T. Control of cell adhesion, migration, and

orientation on photochemically microprocessed surfaces. J Biomed

Mater Res 1996;32:165–73.

[13] Meyle J, Gultig K, Nisch W. Variation in contact guidance by

human cells on a microstrucutred surface. J Biomed Mater Res

1995;29:81–8.

[14] Teixeira AI, Abrams GA, Bertics PJ, Murphy CJ, Nealey PF.

Epithelial contact guidance on well-defined micro- and nanostruc-

tured substrates. J Cell Sci 2003;116:1881–92.

[15] Zhu B, Zhang Q, Lu Q, Xu Y, Yin J, Hu J, et al. Nanotopographical

guidance of C6 glioma cell alignment and oriented growth.

Biomaterials 2004;25:4215–23.

[16] Oakley C, Brunette DM. Response of single, pairs, and clusters of

epithelial cells to substratum topography. Biochem Cell Biol 1995;

73:473–89.

[17] Qu J, Chehroudi B, Brunette DM. The use of micromachined

surfaces to investigate the cell behavioural factors essential to

osseointegration. Oral Dis 1996;2:102–15.

[18] Walboomers XF, Croes HJE, Ginsel LA, Jansen JA. Contact

guidance of rat fibroblasts on various implant materials. J Biomed

Mater Res 1999;47:204–12.

[19] Lo CM, Wang HB, Dembo M, Wang YL. Cell movement is

guided by the rigidity of the substrate. Biophys J 2000;79:

144–52.

[20] Kaiser J-P, Bruinink A. Investigating cell–material interactions by

monitoring and analysing cell migration. J Mater Sci: Mater Med

2004;15:429–35.

[21] Curtis A, Wilkinson C, Wojciak-Stothard B. Accelerated cell

movement. Cell Eng 1995;1:35–8.

[22] Curtis AS. The effects of topographic and mechanical pro-

perties of materials on cell behavior. Crit Rev Biocomp 1990;5:

343–62.

[23] Curtis A, Wilkinson C. New depths in cell behaviour: re-

actions of cells to nanotopography. Biochem Soc Symp 1999;

65:15–26.

[24] Damji A, Weston L, Brunette DM. Directed confrontations between

fibroblasts and epithelial cells on micromachined grooved substrata.

Exp Cell Res 1996;228:114–24.

[25] Lenhert S, Meier MB, Meyer U, Chi L, Wiesmann HP. Osteoblast

alignment, elongation and migration on grooved polystyrene surfaces

patterned by Langmuir–Blodgett lithography. Biomaterials 2005;

26:563–70.

[26] Dalton BA, Walboomers XF, Dziegielewski M, Evans MD, Taylor S,

Jansen JA, et al. Modulation of epithelial tissue and cell migration by

microgrooves. J Biomed Mater Res 2001;56:195–207.

[27] den Braber ET, de Ruijter JE, Smits HTJ, Ginsel LA, von Recum

AF, Jansen JA. Quantitative analysis of cell proliferation and

orientation on substrata with uniform parallel surface micro-grooves.

Biomaterials 1996;17:1093–9.

[28] Levina EM, Kharitonova MA, Rovensky YA, Vasiliev JM.

Cytoskeletal control of fibroblast length: experiments with linear

strips of substrate. J Cell Sci 2001;114:4335–41.

[29] Dunn GA, Zicha D. Dynamics of fibroblast spreading. J Cell Sci

1995;108:1239–49.

ARTICLE IN PRESSJ.-P. Kaiser et al. / Biomaterials 27 (2006) 5230–5241 5241

[30] Wojciak-Stothard B, Curtis AS, MonaghanW, McGrath M, Sommer

I, Wilkinson CD. Role of the cytoskeleton in the reaction of

fibroblasts to multiple grooved substrata. Cell Motil Cytoskel

1995;31:147–58.

[31] Walboomers XF, Ginsel LA, Jansen JA. Early spreading events of

fibroblasts on microgrooved substrates. J Biomed Mater Res 2000;51:

529–34.

[32] Dunn GA, Brown AF. Alignment of fibroblasts on grooved surfaces

described by a simple geometric transformation. J Cell Sci

1986;83:313–40.

[33] Poole K, Khairy K, Friedrichs J, Franz C, Cisneros DA, Howard J,

et al. Molecular-scale topographic cues induce the orientation and

directional movement of fibroblasts on two-dimensional collagen

surfaces. J Mol Biol 2005;349:380–6.