Fibroblastand epithelialcell ~~rac~ons with s~face-treated implant materials J.A. Jason, J.P.C.M.va,n der Warden and K. de clot ~e~a~~eff~ of 3jomater~als. University of Leiden, R~~sbo~ge~eg 10, 2333 A.4 Letden, The Netherlands (Received 9 October 1989; accepted 8 November

1989)

The basic mechanisms underlying skin-implant interactions were investigated by means of in vitro experiments with fibroblast and epithelial cell cultures. Cells were cultured on various implant materials treated with radio frequency glow discharge. The possible influence of this treatment on the morphological mode of cell attachment, as well as cell behaviour in attachment and growth rate were investigated. The results demonstrate that there are significant behavioural differences between fibroblasts and epithelial

Keywords: Implants, wound healing, cell-material

interactions

Wound healing is a complex phenomenon, involving biochemical as well as temporal aspects’. Normal wound healing is disturbed in the presence of a foreign object, such as an implanted artificial material. In the case of a percutaneous implant altered wound healing can influence the implant-skin interactions, which are critical for the longterm prognosis of these implants. For example, the formation of a stable skin-implant junction can be hampered, which will result in the ingress of bacteria and debris into the tissues or the ~ngrowth of epidermis along the implant surface. Two events, either alone or in conjunction with each other, are important for percutaneous device-skin interface healing: (1) epithelial healing processes around the implants; (2) connective tissue healing processes around the implants. In view of this, knowledge of the behaviour of fibroblasts and epithelial cells at the implant-tissue interface is essential for the understanding of the regenerative processes involved in wound healing. The influence of implant materials on the cellular events in wound healing can be examined by in vitro investigations. Cell culture experiments enable study of the behaviour of specific types of living cells in a well-controlled environment2. One of the parameters used to assess the interactions of cells with foreign surfaces is cellular adhesion, which in this context is defined in terms of abashment, spreading and growth. Although cells are able to adhere and grow on different kinds of non-cellular materials, it has been known for over 20 years that certain surface features of the substratum can influence the interactions between cells and substrate. Many studies Corresoondence to Or J.A. Jansen 0 199 1 %u~e~o~h-Heinemann

have shown that cellular adhesion, tends to correlate with the free energy of the substratum surface3-lo. This has led to the development of new methods, such as glow discharge, to modify the surface characteristics of materials in favour of the cellular behaviour5,“. As part of our research on wound healing processes around percutaneous implants’2-‘4, we reported in previous publications the effect of different surface treatments on attachment and growth of epithelial cells on implant surfaces’5-‘7. In all these studies no significant relation between epithelial cell behaviour and surface treatment was found. The objective of the present study was to obtain more information about the influence of surface features on the biological adhesiveness of various implant materials. ln vitro experiments with fibroblasts and epithelial cell cultures were performed to evaluate cell-substrate interactions in a quantitative and qualitative manner. For the quantitative study, fibroblasts and epithelial cells were cultured on the implant material. The influence of the bare substrate material and the use of radio frequency glow discharge (RFGD) on cell attachment and cell growth was determined using three different methods. For the qualitative study, epithelial cells were cultured on the same implant materials as used for the quantitative study and the morphology of the cell-implant interface was determined by transmission electron microscopy.

MATERIALS

AND

METHODS

Materials The materials used were 13 mm round tissue coverslips (ThermanoxB, Lux), as received or coated (vacuum evapo-

Ltd. 0142-9612/91/010025-07 f3iomaterials f991,

Vof 12 January

25

Surface treatment and cell behaviour: J.A Jansen et al.

ration) with titanium or carbon films. The coverslips were untreated or treated by radio frequency glow discharge. The coated coverslips were sterilized with 70% ethanol (for 30 min at room temperature, subsequently air drying) or treated with RFGD. A compact electrodeless glow discharge apparatus was used (Harrick PDC-3XG). The specimens were placed inside the Pyrex sample tube of the apparatus. When a vacuum of 6.7 Pa was achieved, the system was flushed with argon at a vacuum pressure of 20 Pa. Then the radio frequency field was turned on and the substrates were exposed to the glow discharge for 5 min. Directly after the RFGD treatment, the specimens were used for the cell culture experiments. Contact angle measurements demonstrated that RFGD increases the wettability of all surfaces (Table 7). Figure I

Phase contrast micrograph of a culture of rat dermal fibroblasts.

Figure 2 cells.

Phase contrast micrograph of a culture of rat palatal epithelial

Cell culture Rat dermal fibroblasts (RDF) and rat palatal epithelial (RPE) cells were used (Figures 1 and 2). The fibroblasts were isolated and subcultured using a dissociation method’*. Skin sections were cut into small fragments, which were immersed in a solution of trypsin/collagenase in Ca- and Mgfree phosphate buffered saline (PBS), and incubated for 45 min at 37°C with constant agitation. The suspension of detached cells and tissue fragments was then filtered and centrifuged. After centrifugation the cells were resuspended in culture medium (Ham-F1 0, Gibco) containing 10% fetal calf serum, 50pg/ml gentamycin and 2.5 pug/ml amphotericin. Cells were grown in 25 cm* tissue culture polyin an styrene flasks (Nunclon@, Denmark) at 37°C atmosphere of humidified air containing 5% COz. Every 7 d cells were subdivided by trypsinization (0.25% trypsin (Difco) and 0.02% EDTA (Baker) in Hepes buffered saline). Seventh generation cells were used for all the experiments. The epithelial cells used were a gift of Dr A. Arenholt, University of Arhus, Arhus, Denmark. They were cultured in Eagle’s MEM medium (Gibco), supplemented with 10% fetal calf serum, 100 units/ml penicillin, 1 OOpg/ml streptomycin, and 2.5 ,eg/ml amphotericin in a 5% COJair atmosphere at 34°C. lmmunohistochemical staining for cytokeratinslg demonstrated that the cell cultures were 100% epithelial. Before the experiments, cells were detached by trypsinization and suspended in culture medium supplemented with fetal calf serum.

Cell attachment Different test substrates were positioned on the bottom of 24-well plates (Greiner@), and 0.5 ml of trypsinized fibroblasts (1 .l X 1 O5 cells per ml) were added to each substrate. The cultures were incubated at 37°C in 5% COz/air. Since pilot studies had shown, that the maximum attachment percentages of RDFs to tissue culture coverslips (Thermanox) was obtained after 4 hand that cell proliferation starts between approximately 12 and 24 h, the cultures

were incubated for 6 h. After incubation, non-attached cells were aspirated with a pipette and the substrates were rinsed twice with PBS. The non-attached cells were counted immediately, using a Coulter counter (aperture current 2, threshold control 9; Coulter Electronics Ltd, Dunstable, UK). The percentage of attached cells was determined using the equation: % attachment =

No. seeded cells - No. unattached cells No. of seeded cells

x 100

Two runs of experiments were carried out. In each run all materials were tested in quintriple. To determine the influence of the untreated materials on cell attachment, cell attachment percentages were calculated considering that 100% cell attachment had occurred on untreated coverslips. To elucidate the influence of surface treatment on cell attachment, attachment percentages were determined considering that 100% cell attachment had occurred on the untreated material.

Cell growth Table I surfaces

Contact angles of radio frequency glow discharge (RFGD) treated

Material

Thermanox” Titanium Carbon

26

Biomaterials

Contact angle (degrees) Before RFGD

After RFGD

21 20 44

4 5 6

199 1. Vol 12 Januaw

Counting the attachedcells with a Coultercounter. RDF cell suspensions, containing 1 .I X 1 O5 cells per ml, were seeded on the substrates as described before and incubated at 37°C. After 6 h of incubation, the non-attached cells were removed by phosphate buffer rinses. The number of attached cells to the various substrates was then determined by counting the number of unattached cells. Fresh culture medium was added and the cultures were incubated for 3 d.

Surface

At

the

end

discarded buffer.

of the

Incubation

The attached

percentages

were

The

separate were

Migration

rate measurements

nique

are based

and was

study16 for the determination surface

treatments

RDF

cell

cells/ml)

assay.

experiment

all the

the

rings

were

discarded

and the dishes

attached

cells. This resulted

an area of 0.196 the

cultures

incubation

were

ml at approx.

coverage

system.

Migration

rate

percentages

for

blue calorimetric at 34°C was

to

was added and end

of the For the

were

of each

all

the

1 1 1A

carried test

out.

surface.

materials

were

are based on the average of

expenments.

x IO” cells/ml)

incubated

the non-

constrained

the areas of cellular

pieces

The results presented

Methylene

medium

test

assay”

was seeded

“. RPE cell suspension

on the test substrates

rrr 5% CO,/arr.

removed

and

After 3 d of incubation,

and the cultures

were

the

washed

PBS. The cells were then frxed with 4% formaldehyde

in PBS buffer

(pH 8.5), 0.07

at

and

4”C,

washed

stained

with

with 1%

0.01

(w/v)

methylene

borate buffer for 30 min. Cultures

M

four times

with

borate

buffer

borate

M

until the remaining

off and allowed

ethanol

and HCI were added to each substrate.

for

each

solution

photometer was

the

elution

performed. were

tested

to dry. To elute the dye, 0.5

obtained

(Zeiss,

was

M403)

measured

at 667.5

solution.

of radio frequency in Tab/e 3.

One-way revealed

In Tab/e

dye was ml of 1 : 1

Absorbance

presented ratio

relative

are shown

coefficient

(r) is -0.9820, modification material

all the investigated

9 and

11 d at 34°C

In 5%

materials

period,

the non-attached

cells were

buffer rinses. The attached glutaraldehyde/tannic

After removed

the cultures alcohol

Growth

rate

relative

growth

materials

were

for 24 h at 60°C. Reichert

dehydrated embedded

fixative.

through in situ

Ultrathin

sections,

as well as the cells attached ultramicrotome

RFGD

The results

of the

correlation correlation

that an energetical

characteristics produces

of the a greater

attachment.

of RDF

cells.

Table

5 shows

rate of RDF cells on the various

as determined

Table 2

Relative mater/a/s

by the cell counting

attachment

of

rat

dermal

the

substrate

and the fence

f/hroblast

Marenal

cells

Relative

Thermanox’

to

WNOUS

attachment

(%)

100

Tltamum

alcohol

126.5

alcohol

110

Values

in parentheses

represent

Table 3

Relatwe

standard

attachment

frequency

glow

of rat

(7 8) 5 (16.3)

devlattons

dermal

fibroblast

(RFGD)

treated

discharge

Relative

attachment

(I%)

119

RFGD alcohol

100

RFGD

117.5

Tltanlum Carbon

alcohol

100

by phosphate

Carbon

RFGD

120

cc//s

cultured

substrate

Contact

100

Thermanox”

the Incubation

with

solution

rinsed with

0~0~

After

a graded by covering

with a layer of Spurr resin. The specimens strates

of the surface

evaluation

substrate

Titanium

for 3.4,5,7,

acid/paraformaldehyde

in PBS buffered

and finally

before

on

mater&

angle

(degrees)

(RFGD)

treatment

21 (2.8)

4 20

(0 7)

5 44

(21.2)

6

cells were fixed in situ with 2.5%

PBS for 30 mm at room temperature, then post-frxed

was added to the

were incubated

CO,/air.

are is the

Cell growth

Thermanox’

and the cultures

and

a negative

This implies

by the RFGD treatment

of fibroblast

procedure

percentage

3. The computed

indicating

stronger

test

were

assay

test substrates

RFGD treatment.

substrate increase

after

and

regression

in a spectro-

of experiments

X 1 O5 cells/ml)

RFGD treatment linear

to

the possible

angle is the ratio of the contact

in Figure

the two variables.

VENOUS radjo

(4.3

a simple

between

the

RDF cells attach

To examine

attachment

after and before

Matenal

RPE cell suspension

more

relation between

the ratio contact

analysis

between

The data used for the regression

of the

treatment

to the various materials.

in Tab/e 4. The ratio attachment

treatment; angle

that

materials.

cell attachment,

was applied.

Carbon

In octuple.

Morphology

3 show

of a definite

fibroblast

on the

of data in Tab/e 2,

(P > 0.05)

of fibroblasts

than to untreated

discharge

(ANOVA)

differences

percentages

data

treated

glow

analysis variance

no significant

attachment

measurements

in

nm. The blank control

Four runs

In each experiment

blue

were then rinsed

washed

were

electron

with

for

30 mm

sections

are given in Tab/e 2, and those regarding

are compiled

existence

7 h was

with a HP 87/9

Two runs of experiments

the two separate

(1.7

and digitized

four

After

photographed.

of the cellular outgrowth,

X lo5

medium

3 d. At the

were

The

transmission

of the cell attachment

materials

the influence

The

(diameter

to remove

medium

for

the cultures

run included

calculated.

rinsed

the

in RDF cultures

Fresh culture

were outlined

graphic

were

results

tech-

4.5

rings

removed,

incubated

period

determinatron

Each

cm’.

‘fence’

the ‘fence’

0.5 cm), placed in the centre of the test substrates. of incubation,

The

untreated

in polystyrene

citrate. 301

et al

of

rate of RPE cells.

(0.5

inoculated

lead

Philips

Jansen

Cell attachment

average

of the effect of several different

suspension

was

a

JA

RESULTS

used by us in an earlier

on the growth

and

with

hehawour

was based

on the

from

acetate

cell

microscope.

cell attachment

of RDFs by a modified

IS adapted

et a/.”

uranyl

and

Cell growth

in quadruplicate.

Thus method

of Hasson

with

investigated

by trypsinization.

This approach

In each

was

phosphate

counter.

the cell attachment

experiments. tested

medium

with

considering

presented

materials

technique.

detached

to be 100%.

regarding

results

the

rinsed

using a Coulter

calculated

after 6 h of incubation on our findings

were

ceils were

The cells were counted

three

period,

and the substrates

treatment

Values

in parentheses

represent

standard

devlatlons

PBS and rinsing, series

of

the cells

Table 4 of the

Relation substrates

between and

radio frequency

relative

rat dermal

flow discharge fibroblast

Material

Ratto attachment

(%)

Thermanox”

119

0.1’)

Tltanlum

1.17

0.2!