cytotoxicity testing of wound dressings using normal human keratinocytes in culture

Cytotoxicity testing of wound dressings using normal human keratinocytes in culture

Martin Rosdy and Louis-Christian Clauss Biolaboratory, QA department, Dow Corning European Health Care France S.A. Route des cretes-B. P. 43 Sophia Antipolis-Les Bouillides 06561 Valbonne Cedex. France

Comparative cytotoxicity testing of 16 wound dressings of different composition show that normal human keratinocytes (NHK) growing on a fibroblastic feeder layer are as sensitive to toxic materials by direct contact as the confluent MRC5 fibroblasts used for standard cell culture cytotoxicity testing, and slightly more sensitive when extracts of the dressings were tested. After direct contact with each of the cell types, we found effects due to 12 dressing samples (75%), but the extracts of only 6 of them induced changes in cell shape or cell death on NHK, and 4 of them on MRC5 cells. In order to assess the compatibility of these dressings with a pure population

of epidermal cells, the cell type responsible for reepidermization of healing wounds, we then tested the sensitivity, both to dressing samples and extracts, of normal human keratinocytes (NHK) grown in chemically defined medium and without a feeder layer: The results show epidermal cytocompatibility of 10 dressing extracts, while 6 others induced cytopathic effects. Three of these extracts specifically damaged epidermal cells and inhibited their proliferation. When comparing the sensitivities of NHK (in defined medium) and MRC5 cells, we observed complete correlation for 75% of the dressings by extract testing and in 94% of the cases after direct contact.

INTRODUCTION

During the past years, the biocompatibility testing of medical materials and devices has evolved toward the development and use of specific cytocompatibility test systems. Cell or tissue culture techniques are now avail- a b P 6 for rapid and meaningful evaluation and control of the compatibility of medical devices with the cells or tissues with which they will be in contact. These developments are important because of the high costs of classical animal testing which, in addition, often gives results not easily transposable to the clinical situation in humans.

We report here a study of wound dressings supposed to permit reepidermization of superficial wounds and which therefore should not interfere with the proliferation and migration of epidermal In order to assess their compatibility with epidermal cells, we exposed samples of these dressings and also their respective extraction medium to normal human keratinocytes in culture.

First we have studied 16 wound dressings of different compositions with regard to their cytotoxicity by direct contact to MRC5 embryonic fibroblastic

Journal of Biomedical Materials Research, Vol. 24, 363-377 (1990) 0 1990 John Wiley & Sons, Inc. CCC 0021-9304/90/030363-15$04.00

364 ROSDY AND CLAUSS

cells. Extraction media of the same dressings were also tested on MRC5 cells, using the B.S.I. standard procedure for cytotoxicity testing of medical devices.'

Second, equivalent dressing samples were extracted in epidermal cell culture medium; both these extracts and 1-cm2 samples were placed in direct contact with normal human keratinocytes (NHK: the cell-type allowing reepidermization of the healing wound in vivo) cultured on a feeder layer of mouse 3T3 fibroblasts.10-" Sensitivity of these cells to toxic dressing material was comparable to the one of MRC5 cells.

But the similar sensitivities to toxic dressing material of confluent MRCS fibroblasts and colonies of keratinocytes grown on a feeder layer might have been due to an inhibition of the keratinocyte-growth-supporting properties of the feeder-layer fibroblasts. To check if the normal human keratinocytes themselves are as sensitive to the tested material as the MRC5 fibroblasts, a pure keratinocyte culture system was needed. Therefore, a chemically defined media culture system developed by Ham and coworker^,^^-'^ was optimized for the growth of normal human adult keratinocytes directly on plastic culture dishes, without a feeder layer. Addition- ally, this allowed us to eliminate possible cross reactions with unknown factors of the fetal calf serum and, moreover, to simplify the culture method employed for this epidermal cytotoxicity test.

MATERIALS AND METHODS

Cell cultures

MRCS human embryonic lung fibroblasts (passage 28) were purchased at Biom&ieux, Lyon, France. Culture dishes 35 mm in diameter (Falcon) were inoculated with lo5 cells/cm2 and grown to confluency in BME medium containing 10% fetal calf serum and antibiotics (Boehringer, France).

3T3 mouse fibroblasts were treated 3 hours at confluency with 0.33 mg/mL Mitomycine C (Sigma Chemicals, France) and then inoculated in 35-mm- diameter culture dishes (Falcon, France) at a density of 2 X lo4 cells/cm2 to form a feeder layer for the epidermal cells. The normal human keratinocytes (NHK) were isolated from dermatologic surgery skin samples using the 0.25% trypsin dermis-epidermis separation method' and subcultured at a density of lo4 tells/cm2 on the 3T3 feeder layer in DME/F12 (1 : 1) medium containing 10% fetal calf serum, antibiotics, hydrocortisone (5 x M), choleratoxin (lo-'* M), insuIin (5 ,ug/mL) (Sigma, France) and EGF (10 ng/mL) (Collaborative Research, MA, USA).

Normal human keratinocytes were subcultured at lo4 cells/cm2 in a chemically defined medium (MCDB 1.53) which was entirely prepared in the laboratory, according to the procedure of Peehl and Ham? it was supple- mented with an amino-acid stock s o l u t i ~ n , ~ ~ - ' ~ insulin (5 pg/mL), hydrocortisone (5 X M), epidermal growth factor (10 ng/mL), ethanolamine

CYTOTOXICITY OF WOUND DRESSINGS 365

M), phosphoethanolamine M), antibiotics and, for stock cultures only, bovine pituitary extract (70 pg/mL). The calcium concentration was adjusted to lop4 M (= low calcium concentration).

All stock cultures were checked for absence of microplasma using the Hoechst stain method.

All chemicals and salts for MCDB 153 were purchased at Sigma Chemicals (France). Trace elements were from Fluka (Buchs, CH) and Merck (Darm- stadt, Germany).

Test procedures

Direcf contact

Sixteen wound dressings of different composition (see Table I) were cut aseptically in l-cm’ pieces, and deposited (one per dish) directly on the cells in the dishes containing 2 mL of fresh culture medium. When floating, they were charged with similar pieces of negative control material (Silicone M D X 4 4515) to be in direct contact with the cells. Pieces of natural latex from catheters were used as positive control material.

Extraction method

The quantity of any substances extracted from the dressings is related to the surface area with which the extractant is brought into contact both during exposure to a wound in vivo and during experimental extraction. There- fore, although the dressing materials have different weight/mass ratio, we extracted 6-cm2 samples of each dressing tested in 12 mL sterile culture medium at 37°C for 24 h. This corresponds to an extraction ratio of 0.5 cm2/mL extractant.g The extraction media were then checked for neutral pH and if necessary, adjusted using 10 mM HC1 or 10 mM NaOH solutions. Two milliliters of each extract was deposited on the cells in each of two dishes.

MRC5 cells were used for testing when confluent, and NHK were used on feeder layer or in defined medium when colonies had not fewer than 100 cells. Tests were performed in duplicate for each experiment. After exposure to the extraction liquids or directly to the dressing material, the shape of the cells in each dish was evaluated under the inverted microscope using the following scale by comparison with the control dishes:

0 = No change noted. 1 = Slight but clear change in shape without decrease of cell density. 2 = Considerable alterations in cell shape. Slight decrease of cell density. 3 = Loss of cell shape. More than 50% of cells detached. 4 = All cells are dead, lysed or detached.

TABL

E I

Lis

t of

Tes

ted

Dre

ssin

gs a

nd T

heir

Com

posi

tions

Com

mer

cial

nam

e N

o.

(Man

ufac

ture

r)

Lot N

o.

Com

posi

tion

1

Gra

nufle

x (S

quib

bs S

urgi

care

) 3U

166

Poly

uret

hane

foam

+ hy

droc

ollo

ld

parti

cles

t h

ydro

phob

ic p

olym

er

2 K

alto

stat

(Cai

r)

8614

.4.8

C

alci

um a

lgin

ate

fiber

s 100

%

nonw

oven

3 St

eris

eal S

orbs

an (N

I Med

ical

) 26

.06.

87

Alg

inat

e Fi

bers

4 A

dapt

ic (J

ohns

on +

John

son)

99

305

Vis

cose

100

% +

emul

sion

va

selin

e +

wat

er

5 O

psite

(Sm

ith +

Nep

hew

) 44

0484

Po

lyur

etha

ne f

oam

6 Sy

spur

derm

(H

artm

an)

0306

08 8

0 Po

lyur

etha

ne s

oft f

oam

7 Te

gade

rm (3

M)

8 Ly

ofoa

m 2

726

(Ultr

a La

bs)

9 C

orad

em (R

evlo

n H

ealth

)

10

Epig

ard

(Par

ke +

Dav

is)

11

Com

feel

3210

(Col

opla

st)

12

Tran

sige

n (S

mith

+ N

ephe

w)

0385

52

V04

9109

3

Poly

uret

hane

film

Visc

ose

Fibe

rs +

activ

ated

car

bon

+ po

lyur

etha

ne f

oam

Mod

ified

pol

yure

than

e fo

am

Poly

uret

hane

foa

m

+ po

lypr

opyl

ene

film

Car

boxy

met

hyl c

ellu

lose

+

poly

uret

hane

film

Poly

uret

hane

film

+ in

ert

nonw

oven

mat

eria

l + ad

hesi

ve

w

m

m P

TABLE

I.

(con

tinue

d)

13

Foam

She

etin

g Si

last

ic (D

ow C

orni

ng)

KO

5603

9 Po

lysi

loxa

ne fo

am

14

Gel

She

etin

g Si

last

ic (D

ow C

omin

g)

VL0

2601

3 Si

licon

e ge

l

15

Jelo

net (

Smith

+ N

ephe

w)

2932

3 Pa

raff

in g

auze

16

Om

ider

m (

Om

ikro

n Sc

ient

ific)

HS

KOOS

-164

Poly

uret

hane

film

17

Posi

tive

cont

rol

Nat

ural

late

x (c

athe

ter)

18

Neg

ativ

e co

ntro

l Si

licon

e MD

X4-

4515

(Dow

Cor

ning

)


Exposure to MRC5 cells was for a period of 24 h, while tests involving keratinocytes lasted 48 h to permit measurement of significant effects on cell proliferation.

Cell counting

After 48 h exposure to NHK in defined medium, the dressing samples were removed with forceps, the media or extracts aspirated, cells were rinsed twice in phosphate buffered saline (PBS) and detached by 1 mL trypsin 0.05%-EDTA 0.02%. Each cell suspension was added entirely to 10 mL isotonic solution and cells in 0.5 mL were counted twice by a Coulter Counter (Coultronics, France). The means of cell numbers in duplicate dishes were calculated.

RESULTS

Test on MRC5 fibroblastic cells

Twelve of the 16 dressing samples tested induced morphological changes when placed directly on MRC5 fibroblastic cells, but the corresponding extraction media induced cell alterations only in 4 cases (Kaltostat, Adaptic, Transigen, Silastic foam); the other 12 dressing extracts were not cytotoxic on MRC5 fibroblasts (Table 11).

Test on NHK with fibroblastic feeder-layer

Comparable results were obtained when samples of the same dressings were placed in direct contact with normal human keratinocytes growing on a fibroblastic feeder layer in serum containing medium. Six of the corresponding extracts induced alterations of the epidermal cells, including the 4 which were cytotoxic in the classical MRC5 test. The two additional dressings, extracts of which induced cytotoxicity only on epidermal cells, are both composed of modifjed polyurethane foams: Syspurderm and Cora- derm (Table 11).

To check whether this greater sensitivity was intrinsic to the epidermal cells or due to the longer exposure of the extracts to the cell layer, we extracted samples of these two dressings during 48 h and exposed them 48 h to MRC5 cells: still no cytopathic effects were observable, suggesting specific sensitivity of the epidermal cells to certain substances extractable from these dressings.

Test on NHK in defined medium

To test this hypothesis, samples of the same 16 dressings and their respective extracts were placed in direct contact with colonies of growing normal

0

TABL

E I1

3 2 9

Cyt

otox

icity

Tes

t Res

ults

on

MR

C5

Cel

ls a

nd o

n N

orm

al H

uman

Ker

atin

ocyt

es (N

HK

) Gro

wn

on a

Fib

robl

astic

Fee

der

Laye

r 0

Dir

ect C

onta

ct

Ext

ract

Med

ium

E <

Te

st o

n M

RC

5 Te

st o

n N

HK

on

Test

on

MR

C5

Test

on

NH

K o

n N

o.

Com

mer

cial

Nam

e C

ells

Fe

eder

Lay

er

Cel

ls

Feed

er L

ayer

1 2 3 4 5 6 7 8 9 10

11

12

13

14

Gra

nufl

ex

Kal

tost

at

Ster

isea

l Sor

bsan

Ada

ptic

Ops

ite

Sysp

urde

rm

Teg

ader

m

Lyo

foam

Cor

ader

m

Epi

gard

Com

feel

Tra

nsig

en

Sila

stic

foam

Sila

stic

gel

2 4 1 4 3 1

0 0 4 0 2 0 2 0

2 3

3 2

2 1

4 4

2 3

1 2

0 0

1 3

4 4

1 1

2 2

1

0

2 1

0 0

4 0

2 4

1 0

4 2

4 0

2 0

0 0

2 0

3 0

1 0

2 0

1 2

1

1

0 0

0 0

4 3

0 0

2 4

0 0

0 2

0 0

0 0

0 4

0 0

0 0

2 4

1 1

0 0

0 3 0 4 0 2 0 0 4 0 0 4 1

0

TABL

E 11

. (c

ontin

ued)

15

Jelo

net

00

0

0

00

16

Om

ider

m

00

0

0

00

17

Posit

ive

cont

rol

33

3

3

33

18

Neg

ativ

e co

ntro

l 0

0

00

0

0

00

00

33

00

Note

. 0 =

No

chan

ge n

oted

; 1

= s

light

but

cle

ar c

hang

e in

sha

pe w

ithou

t dec

reas

e of

cel

l den

sity

; 2 =

Con

side

rabl

e al

tera

tions

in

cell

shap

e, s

light

dec

reas

e of

cell

dens

ity; 3

= lo

ss o

f ce

ll sh

ape,

mor

e th

an 5

0% o

f ce

lls d

etac

hed;

4 =

All

cells

are

dea

d, l

ysed

or

deta

ched

.

P


human adult keratinocytes: As described,*-** the chemically defined culture medium used suppresses growth of fibroblastic cells, and melanocytes are only detected in primary cultures; the test was performed on cultures at the third passage, containing a pure keratinocyte population of cells.

I) The sensitivity to direct dressing contact of NHK grown in chemically defined medium was comparable to that of confluent MRC5 cells or NHK cells growing on a fibroblastic feeder-layer in serum containing medium (see Tables II, III). Effects ranged from changes in cell shape and morphology, cytoplasmic granulations, to decrease in cell density and cell death (Fig. 1).

11) The extracts of six dressing samples induced clear effects on epidermal cells, indicating greater sensitivity of normal keratinocytes, compared to the MRC5 fibroblasts, to 3 of the 16 dressings tested: Lyofoam, Syspurderm, and Coraderm, and lesser sensitivity to Silastic foam dressing. To check if these effects correlated with growth arrest of the epidermal cells, the number of cells remaining attached to the culture dish after rinsing with saline, before and after the 48-h incubation period (see Table 111) were counted: Cy- totoxicity induced by direct dressing contact or extracts (Adaptic, Syspur- derm, Coraderm) correlated with a significant decrease in the number of attached cells, whereas three cytotoxic extracts provoked just growth arrest of the keratinocytes (Kaltostat, Lyofoam, Transigen).

In presence of each of the noncytotoxic extracts or dressing samples the cell number increased between two to three fold during the 48 h incubation (Table 111).

DISCUSSION

In summary, the 16 dressings tested can be classified in four groups:

Group 1

Effects on Effects on MRC5 fibroblasts epidermal cells

direct extract direct extract Dressing

N" name contact medium contact medium

7 Tegaderm no no no no 14 Silastic gel no no no no 15 Jelonet no no no no 16 Omiderm no no no no

These dressings gave negative results in all tests performed; they can clearly be stated as non cytotoxic according to the test methods proposed by the BSI (9), ASTM, and AFNOR; moreover they are compatible with proliferating human normal epidermal cells in culture.


Figure 1. Shape of normal human keratinocytes cultured in defined medium after 48 h exposure to dressing extracts (original magnification x 100). A) Notation 0 (Silastic gel sheeting Q7-9119), B) Notation 2 (Coraderm), C) Notation 4 (Syspurderm).


Figure 1. (continued)

Group 2

Effects on Effects on MRC5 fibroblasts epidermal cells

direct extract direct extract N" name contact medium contact medium

Dressing

2 Kaltostat Yes Yes Yes Yes 4 Adaptic Yes Yes Yes Yes

12 Transigen Yes Yes Yes Yes 13 Silasticfoam Yes Yes Yes no

These dressings induced positive results on both cell types. They have to be considered as cytotoxic devices according to the standard test methods proposed by the BSI, ASTM, and AFNOR. Their cytotoxicity cannot be correlated with one or several specific biomaterials, because these four dressings are composed of very different materials (see Table I).

Group 3

Cytopathic effects Cytopathic effects on epidermal cells on MRC5 fibroblasts

Dressing direct extract direct extract

N" name contact medium contact medium

1 Granuflex Yes 3 Sorbsan Yes

10 Epigard Yes 11 Comfeel yes

5 Opsite yes

no no no no no

no no no no no


TABLE I11 Cytotoxicity Test Results on NHK Grown in Defined Medium

Direct contact Extract Medium

Cytotoxicity Cell NB/Dish Cytotoxicity Cell NB/Dish No. Commercial Name Notation 0-4 % of Control Notation 0-4 % of Control

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

Granuflex

Kaltostat

Steriseal Sorbsan

Adaptic

Opsite

Syspurderm

Tegaderm

Lyofoam

Coraderm

Epigard

Comfeel

Transigen

Silastic foam

Silastic gel

Jelonet

Omiderm

Positive control

Negative control

3

3

1

4

3

2

0

1

1

0

1

1

1

0

0

0

3

0

- 3

2 3.5

1 57.0

4 5.3

4 7.6

3 22.4

0 76.0

1 66.7

1 47.6

0 47.6

1 52.4

1 52.4

1 47.6

0 133.0

0 81.0

0 90.0

3 9.5

0 100.0

Percentage of cells present before the 48 h incubation (mean of four dishes)

45.0

0

2

0

3

0

4

0

1

2

0

0

1

0

0

0

0

3

0

- 0

2 46.7

0 83.0

3 9.8

0 70.0

4 9.0

0

1 44.3

2 6.7

0 105.0

0 87.7

1 31.2

0 95.0

0 98.4

0 96.0

0 112.3

3 14.3

0 100.0

45.0

-

Note. 0 = No change noted; 1 = slight but clear change in shape without decrease of cell density; 2 = Considerable alterations in cell shape, slight decrease of cell density; 3 = Loss of cell shape, more than 50% of cells detached; 4 = All cells are dead, lysed or detached.

These five dressings induced cytopathic effects by direct contact, but their extraction media were not cytotoxic, thus passing the BSI standard test. Two dressing samples (Sorbsan/Opsite) significantly increased the pH of the extractant, therefore the relative acidity induced during direct contact


exposure could be responsible for the effects observed. By rectdying the pH of the extraction medium, we found that cytotoxicity by direct contact was due exclusively to excessive acidity.

Group 4

Cytopathic effects Cytopathic effects on epidermal cells on MRC5 fibroblasts

direct extract direct extract Dressing

medium contact medium N" name contact

6 Sy spurderm Yes no Yes Yes 8 Lyofoam Yes no Yes Yes 9 Coraderm Yes no Yes Yes

Three dressings induced cytopathic effects on both cell types by direct contact, but their respective extraction media gave positive results only on epidermal cells. This specific toxicity to epidermal cells could be due to their common constituant (polyurethane foam). In the direct contact method, 1 cm2 of dressing material is deposited on a monolayer of MRC5 cells or on colonies of proliferating epidermal cells in 2 mL of culture medium: the dressinghedium ratio is therefore the same as for the extraction method, thus the only difference remaining is the actual presence of the dressing samples in the culture dish. Therefore no molecu€e present in the extract medium is responsible for the cytotoxicity observed only after direct contact.

It is well known that growth of cells in culture is very sensitive to attachment molecules such as collagen and vitronectin present in the culture medium or secreted by the cells t h e m s e l v e ~ . ~ ~ ~ ~ Epidermal growth factor and related factors were recently reported to be active in epidermal cell migration in vifro",26 and during wound healing in vivo. 272e It could be argued that the presence of a dressing sample in the culture dish interferes with the deposition of such factors on the surface of the dish: by releasing com- petitive molecules (which during extraction adhere to the walls of the extraction vials and are therefore not present in the extracts when tested next day), the dressing materials might disturb the attachmenthurface character- istics of the culture substrate.

Both phenomena, the possible interference with deposition of attachment molecules or the increased acidity, could disturb the migration and proliferation of epidermal cells in vivo during reepidermization of a healing or superficial wound; indeed, the same factors which are necessarily present or secreted in the culture dish to allow attachment and growth of epidermal cells, are also directly involved in the migration of epidermal cells in vivo during wound healing.24*29*30 Therefore, more specific parameters such as attachment factor deposition, detailed extracellular matrix composition, interference with cell migration and dilution of acidity by wound fluids, for example, should be studied to assess epidermal cytocompatibility of dressing materials. We propose to use the described epidermal cell culture method (NHK in defined medium) for such cytocompatibility studies.


Only the four dressings of the second group can be assumed to be clearly cytotoxic, while the three dressings of the fourth group might just not be intended for reepidermization of healing or superficial wounds. Obviously, these indications have to be confirmed by in vivo reepidermization test data.

After screening 16 dressings regarding their compatibility with proliferating normal human keratinocytes in culture, we suggest possible interference of certain dressings with the proliferation and attachment/migration mechanisms of epidermal cells during the reepidermization process.

Finally, comparing cell sensitivities of the NHK test method and of the standard MRC5 cytotoxicity test procedure,' we found complete correlation in 75% of the cases when tests are conducted on extracts, and for 94% of the dressings when tested by direct contact.

The authors wish to thank Nigel McWilliam for providing the dressing samples and Christine Ferrere for typing the manuscript.

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(Stockh), 65, 379-384 (1985).

Received September 15, 1988 Accepted September 6, 1989

cytotoxicity testing of wound dressings using normal human keratinocytes in culture

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