._
267
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Cytotoxicity testing of wound dressings using me~ylcellulose cell culture M&A. van Luyn, P.B. van Wachem and P. Nieuwenhuis De~rtment of ~istofogy and Cefl Bfofogy, Section of Siomaterials Oostersingel6912, 9713 EZ Groningen, The Netherlands
Research,
Universi~
of GroffingeR,
M.F. Jonkman Department
of Dermatology,
University
Hospital,
Oostersingel
59, 9713 EZ Groningen,
The Netherlands
Wound dressings may induce cytotoxic effects. in this study, we check several, mostly commercially available, wound dressings for cytotoxicity. We used our previously described, newly developed and highly sensitive 7 d methyiceiluiose cell culture with fibrobiasts as the test system. Cytotoxicity is assessed by monitoring ceil growth inhibition, supported by cell morphological evaluation using light and transmission electron microscopy. We tested conventional wound dressings, polyurethane-based films, composites, hydrocolloids and a collagen-based dressing. it was shown that only 5 out of 36 wound dressings did not induce cytotoxic effects. All 5 hydrocoiioids were found to inhibit ceil growth (>70%), while cells had strongly deviant morphologies. The remaining wound dressings showed medium cytotoxic effects, with cell growth inhibition, which varied from low (?15%), medium-low (425%) to medium-high (lt50%). Measurable cytotoxic effects of dressings detected in vitro are likely to interfere with wound healing when applied in vivo. The results are discussed in view of the clinical uses with contaminated wounds, impaired epitheiiaiization or hypergranuiation. Keywords:
Cyfotoxici~,
wound dressings,
~ethy/ce/lutose
Received 15 July 1991; revised 3 October 1991; accepted 20 October 1991
The cytotoxicity of biomaterials can be tested in vitro using various culture systems’-“. Liquid culture systems may detect cytotoxicity of a material either by culture of cells with extracts or with the material itself. In the latter instance, renewing the medium will remove any cytotoxic products released. The agar overlay test is a short-term semisolid culture system in which the possible cytotoxicity of biomaterials is identified only by the presence of cell-free zones. We developed a culture test system, called the methylcellulose (MC) cell culture”, which combines high sensitivity of testing with very detailed testing. High sensitivity is obtained by the ability to evaluate cells for a period of 7 d without refreshing the culture medium. Detailed testing is obtained by the ability to measure cell growth by accurate cell counting and evaluate cell morphology at both the light microscopical (LM) and transmission electron microscopical (TEM) level. With MC cell culture, we studied a collagen wound dressing, which was evaluated as moderately cytotoxic (k50% inhibition of cell growth and deviant cell Correspondence
to Dr M.J.A. van Luyn. .-.-
0
1992 6u~erwort~-Heinemann 0142-9612/92/050267-09
Ltd
morphology) *I. The cytotoxic effects of the collagen wound dressing were confirmed by in viva studiesi2~ 13,in which we used TEM. We used MC cell culture to check 16 different wound dressings for possibIe ~~otoxicity. Six groups of wound dressings were used, i.e. conventional wound dressings, such as paraffin gauze dressing (group A), synthetic wound dressings, such as polyurethane-based films (groups B and C), composites (group D) and hydrogels [group E); the collagen wound dressing previously mentioned (group F) was used as a reference. All wound dressings are commercially available with the exception of one, which is a type of polyurethane-based film co-developed in the Department of Polymer Chemistry, University of Groningen, The Netherlands (group B)14. With MC cell culture, it was shown that only 5 out of 16 wound dressings are evaluated as non-cytotoxic. Another group of 5 wound dressings can be regarded as highly cytotoxic, and the remaining wound dressings induced medium cytotoxicity. The relevance of these results is discussed in relation with the fact that, in general, wound dressings are supposed to induce granulation tissue formation and re-epithelializationX5* 16, _____-_ Biomaterials
1992,
Vol.
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Cytotoxicity of wound dressings: M.J.A.
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MATERIALS AND METHODS Materials Culture conditions Human skin fibroblasts (HF) from the established cellline PK 84 were routinely cultured in RPM1 1640 medium (Gibco Biocult Co., Paisley, UK), supplemented with 10% fetal calf serum (FCS), 2 mM/ml glutamine (Glut) (Merck, Darmstadt, Germany), penicillin (Pen) and streptomycin (Strep), both 100 units/ml (Gibco). The cells were incubated at 37°C in air containing 5% CO,, A stock gel of MC, using Methocel~ high viscosity (3000-5000 mPa) from Fluka, Bio Chemica, Buchs, Switzerland, was prepared according to the method of Iscove and Schreier”, with Iscove’s modification of Dulbecco’s medium (IMDM) [ICN Biomedicals Inc., Costa Mesa, CA, USA) to a final concentration of 2.25%. Materials tested All wound dressings, their manufacturers and compositions are listed in Table 3, The wound dressings are arranged in groups of the material’s composition. The 6 groups of wound dressings (A-F) were used. One type of wound dressing was not commercially available: polyetheru~thane (PEU, no. 4) was codeveloped in the Department of Polymer Chemistry, University of Groningen, The Netherlands14. It is made from Tecoflex” EG80A [Thermedics Inc., Woburn, MA, USA) and has a thickness of l5pm, which swells up to 80-1OO~m when wet. PEU is composed of many noninterconnected microcavities of < 5pm diameter” to achieve a high water vapour permeabilitylg. It was sterilized by y-irradiation (2.5 Mrad] (Gammaster, Ede, The Netherlands).
vaanLuyn et al.
The collagen wound dressing (no. 16)lf-13, the processed dermal layer of sheep skin cross-linked with hexamethylenediisocyanate, is commercially available, but was kindly provided directly by the Zuid Nederlandse Zeemlederfabriek, Oosterhout, The Netherlands. It was sterilized by ~-irradiation j.25 Mrad). All wound dressings were punched into discs of 10 mm diameter, under sterile conditions.
Methods
After washing twice with phosphate buffered saline [PBS) (NPBI, Emmer-Compascuum, The Netherlands], human fibroblasts were harvested from routine culture using 0.25% (w/v) trypsin in Ca/Mg-free Hanks’ Salt Solution (Gibco). Thereafter cells were centrifuged and ~suspended in fresh IMDM, HFfgel mixtures were made by gently and thorougly mixing 30% [v/v) HF/IMDM with 50% (v/v) MC stock gel and 20% (v/v) FCS”. Volumes of Pen, Strep and Glut, added to IMDM, had been adjusted in order to obtain the same final concentration in the culture gel as described for the routine culture medium, For all cultures, a final volume of 4.0 ml culture gel containing 5 X lo4 HF’*, was placed into each well of a six-well tissue-culture plate (Greiner B.V., Alphen a/d Rijn, The Netherlands) using a plastic syringe. Since each well has a surface area of 10 cm’, the final seeding density was 5 X lo3 HFfcm’. Thereafter, cultures were incubated at 37’C in air containing 5% CO,. All cultures were set up in triplicate. Except for the three wells for the control culture, after 24 h two discs of each type of wound dressing were put on top of the HF/gel mixture in
Table 1 List of dressings tested and their compositions Commercial name
Manufacturer
Composition
:
Jelonet Adaptic
Smith & Nephew Ltd, Hull, UK Johnson & Johnson Inc., New Brunswick, USA
White petrolatum gauze Viscose 100% plus emulsion white petrolatum plus water
B
3 4
Omiderm PEU
Omikron Scientific Ltd, Nevogot, Israel University of Groningen, Department of Polymer Chemistry, The Netherlands
Non-adhesive acrylated polyurethane film Non-adhesive polyetherurethane film
c
5
Tegaderm
3M Health Care Inc., St Paul, MN, USA
6
Opsite
Smith & Nephew Ltd, Hull, UK
Polyurethane film plus adhesive acrylate layer Polyurethane film plus adhesive vinyiether layer
li 9 10
Epigard Cutinova plus Syspurderm Lyofoam C
Parke-Davis & Co., Inc., Detroit, Ml, USA Beiersdorf AG, Hamburg, Germany Paul Hartman A.G., Heidenheim, Germany Ultra Laboratories Ltd, Sittingbourne, UK
Polyurethane foam plus polypropylene film Hydrogel plus polyurethane fOam Polyurethane soft foam Viscose fibres plus activated carbon plus polyurethane foam
11 12 13
Biofilm Comfeel Duoderm E
Hydrocoiloid dressing Hydrocolloid dressing Hydrocolloid dressing
14
Ulcer dressing
15
Cutinova hydro
Biotrol Pharma SPA, Paris, France Coloplast AS, Espergaerde, Denmark ConvaTec-Squibb Inc., Greensborough, USA Johnson & Johnson Inc., New Brunswick, USA Beiersdorf AG, Hamburg, Germany
16
Collagen dressing
Zuid Nederlandse Z~mlede~abriek, Oosterhout, The Netherlands
Hexamethylen~iisocyanate collagen dressing
Group
No.
A
D
E
F
Biomaterials 1992, Vol. 13 NO. 5
Hydrocolloid dressing Hydroactive polyurethane dressing cross-linked
Cytotoxicity of wound dressings: M.J.A.
Figure 1 A six-wells tissue culture plate, at day 7 after the start of cell culture. Upon seeding, cells had migrated and adhered to the bottom of the wells. After 1 d, two discs of Comfeel (top row) and Cutinova hydro (bottom row) per well were put on top of the gel. At day 7, Cutinova especially had swollen and the gel had increased in viscosity.
each of three wells of a test culture and the immersed just below the gel surface [Figure 3).
discs
Cell counts At 7 d after starting the cultures, discs and gel were removed without damaging the cells, using a 5 ml syringe. The cell layer remaining on the bottom of each well was extensively washed three to four times with 4 ml PBS to remove the gel completely. Subsequently, cells were trypsinized, resuspended and counted in a Biirker counting chamber. The mean cell number (X103/cm2 f s.d.) was calculated, and from these numbers the cell proliferation inhibition index (CPII), expressed as a percentage of the cell number in control culture, was calculated, using the following relationship:
CPII(%) = 100% -
mean cell number of test culture mean cell number [ of control
Microscopy
culture
269
van Luyn et al.
X 100%
1
During the 7 d test period LM evaluation of the cells was performed without removing the culture gel or discs. At day 7 in situ photography with the phase-contrast inverted light microscope was done after removing discs and washing off the culture gel when the cells were still covered with PBS. For TEM, trypsinized cells were pooled from the three wells, washed with PBS and centrifuged. The resulting pellets were fixed with 2% glutaraldehyde in PBS at 4°C and cut into small pieces of about 2 X 2 mm. These were in PBS”, post-fixed in 1% OsO,, 1.5% K,Fe(CN), dehydrated in graded alcohols and embedded in Epon 812. Ultrathin sections were stained with uranyl acetate and lead citrate and examined with a Philips EM 201 transmission electron microscope, operated at 40 kV. RESULTS
subsequently adhered and proliferated. After 24 h, two discs of each wound dressing were placed on top of the culture gel in each of the three wells. The CPII of day 7 are shown in Table 2 and Figure 2 for each type of wound dressing. The results can be arranged in groups of wound dressings inducing no (OW), low (+15%], medium-low (f25%), medium-high (? 50%) or high (>70%) inhibition, It can be seen that this arrangement does not exactly correspond to the arrangement of groups according to material composition. With LM, the control culture showed a confluent multilayer of well-spread HF at day 7. Some illuminated particles were present within cells (Figure 3s). TEM of the trypsinized cells showed that these particles in fact represent lipid droplets. Furthermore, both welldeveloped rough endoplasmic reticulum (RER) and pseudopodia were observed’ (Figure 3b). Five dressings, i.e. Jelonet, Omiderm, PEU, Tegaderm and Epigard (nos 1, 8, 4, 5 and 7) induced no significant inhibitions in cell growth. The cells of these cultures had, at both the LM and the TEM level, normal morphologies similar to the cells of the control culture. The polyurethane foams Cutinova plus, Syspurderm
Table2 Dressings proliferation Group
No.
A
tested
their
inhibition
:
Jelonet Adaptic
0.7 + 1.8 24.5 f 2.1
3 4
Omiderm PEU
-0.5 + 3.2 0.6 f 4.6
:
Tegaderm Opsite
0.3 + 3.6 29.1 + 4.6
: 9 10
Epigard Cutinova plus Syspurderm Lyofoam C
-1.5 13.1 16.1 13.7
* + f +
3.4 1.2 1.3 2.9
E
11 12 13 14 15
Biofilm Comfeel Duoderm Ulcer dressing Cutinova hydro
71.5 89.3 92.9 95.3 99.6
f + f f +
2.3 1.2 2.5 1.2 0.3
F
16
Collagen dressing 51.5 k 5.3
B C D
Degree of +50,
of
cell
Commercial name Inhibition CPII (%) + s.d.
cytotoxicity
medium-high:
CPII >70,
(%):
+O,
no;
+15.
low;
+25,
medium-low;
high.
Epigard Omiderm Tegaderm PEU Jelonet Cutlnova plus Lyofoam C Syspurderm Adaptlc Opsite Collagen-dressing Biofilm
Comfeel Duoderm Ulcer dressing Cutinova hydro -23
0 Cell
HF, seeded in culture gel to a final density of 5 X lo3 HF/cm’, migrated towards the bottom of the wells and
and
20
proliferation
40
60
lnhlbitlon
80
Index
100
120
(W) ?s.d.
Figure 2 The cell proliferation inhibition indices (CPII) at day 7 for each type of wound dressing. Biomaterials
1992. Vol. 13 No. 5
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of wound
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M.J.A. van Luyn et al.
low inhibition with CPIIs of 24.5 and 29.1% respectively. Adaptic induced easily macroscopically recognizable cell-free zones. Cells near the cell-free zone showed impaired spreading and an increase in lipid droplets (Figure sa), but in general the cell morphology was normal. This was confirmed by TEM, which showed cells with many pseudopodia, increased inclusions of cell remnants and small lipid droplets (Figure 5b). A nonconfluent cell layer with impaired cell spreading, but without a cell-free zone, was observed with Opsite (Figure 6a]. At the TEM level, the cells had rather rounded membranes and somewhat degenerated morphologies (Figure 6b). The collagen dressing, which was taken as a reference material, induced evident cell-free zones, a medium-high CPII of approximately 50% and concomitant deviant cell morphology. To sum up, deviances in morphology were the presence of proportionally larger cells, poorly adhering cells, the increased presence of lipid droplets, the reduction in the amount and dilatation of RER, many inclusions with remnants of dead cells and a decrease in pseudopodia. The hydroactive wound dressings of group E all had high CPIIs ranging 70-100%. They sometimes swelled markedly [Figure 2) when one compares diameters of discs of Comfeel (no. 12) and of Cutinova hydro [no. 15). This may result in a change of viscosity of the culture gel, which makes it more difficult to remove. Since cell growth was extensively inhibited, cell-free zones were not observed. The few cells in cultures with Duoderm, Ulcer dressing and Cutinova hydro (nos 13, 14 and 15) were found as detached cells, either singly or in small strings (shown for Cutinova hydro, no. 15, Figure 7). TEM preparations of these few cells could not be made. The cells of cultures with Biofilm (no. 11) and Comfeel (no. 12) were found in small clusters which still showed rather good adhesion (shown for Comfeel, Figure f?a). TEM of the trypsinized cells of Comfeel and Biofilm showed many small lipid accumulations, a reduction in the amount of RER and many pseudopodia (shown for Comfeel, Figure 8b).
Figure 3 Human fibroblasts (HF) at day 7, cultured for 6 d in the presence of Epigard discs. a, Light micrograph of a multilayer of completely spread cells (cell morphology is similar to that of the control culture). Original magnification X720. b, Transmission electron microscopy (TEM) micrograph of trypsinized human fibroblasts (HF). Cells have many pseudopodia, well-developed rough endoplasmic reticulum (RER) (arrows) and some small lipid droplets (L). Original magnification X4665.
DISCUSSION
and Lyofoam C (nos 8, 9 and lo), resulted in low inhibitions with a CPII of approximately 15%. Small cellfree zones were vaguely recognized macroscopically, but with LM clearly recognized when discs were removed. Close to the cell-free zones, cells showed decreased adherence with elongated or spider-like cell structures and illuminated cell edges and an increase in lipid droplets, as evaluated with both LM and TEM. Cells had more normal morphologies with increasing distance from the cell-free zones. The cells grown in the presence of Syspurderm had many dark particles, which were not extensively found in any other culture [Figure 4s). TEM revealed that these particles represent low electrondense multilocular lipid droplets (Figure 4b) and/or compact myelin bodies [Figure 4~). Adaptic (no. 2) and Opsite (no. 6) induced a medium-
The MC cell culture accurately shows that only 5 out of 16 wound dressings are non-cytotoxic (nos 1, 3, 4, 5 and 71, but 5 wound dressings are highly cytotoxic [nos 11,12, 13,14 and l5), and the remaining wound dressings induced an intermediate cytotoxicity (nos 2, 6, 8, 9, 10 and 16). The five non-cytotoxic wound dressings include petrolatum gauze (Jelonet, no. 1) and four types of polyurethane-based dressings. According to BSI standards, Jelonet (no. l), Omiderm (no. 3) and Tegaderm (no. 5) had already been found to be non-cytotoxicl*‘. Since the MC cell culture” is a more sensitive test system than any other yet available’-“, these five wound dressings may be considered as truly biocompatible. It is encouraging that the polyurethane film PEU turned out to be among this group. PEU was compared with Jelonet in a clinical trial and applied on skin donor sites”. Healing rates of the two wound dressings were similar, but PEU was favoured by patients since it reduced
Biomaterials 1992, Vol.
13 No. 5
Cytotoxicity
of wound
dressings:
M.J.A.
van Luyn et al.
Figure 4 Human fibroblasts (HF) at day 7, cultured for 6 d in the presence of Syspurderm discs. a, Light micrograph of a bilayer of human fibroblasts (HF) showing many illuminated and dark particles (arrows) in the cytoplasm. Original magnification X720. b, Transmission electron microscopy (TEM) micrograph of trypsinized human fibroblasts (HF) containing many small lipid droplets and remnants of dead cells (R). Original magnification X10752. c, Transmission electron microscopy micrograph of trypsinized human fibroblasts (HF) containing electron-dense lipid droplets and several myeline bodies (arrows), possibly representing the dark particles observed with light microscopy (LM). The amount of rough endoplasmic reticulum (RER) present is comparable with that in cells of the control culture. Original magnification X7142.
271
pain’l. PEU is now being investigated as an artificial skin, by combining it with a microporous biodegradable bottom layer, and first results with full-thickness wounds in pigs are promising”. The group with low or medium-low cytotoxic effects includes a synthetic petrolatum gauze (Adaptic), an adhesive polyurethane film (Opsite) and composites with a foam-layer (Cutinova plus, Syspurderm and Lyofoam C). Of these, Adaptic had already been found to be cytotoxic’v ‘. Opsite appears to have passed the BSI standard test, since its extraction media were not cytotoxic” ‘. However, our statement of Opsite having medium-low cytotoxic effects agrees with results of Rosdy and Clauss’, who tested Opsite in a direct contact test with either fibroblasts or keratinocytes and observed cytotoxic effects, but no inhibition of cell proliferation. Opsite did not induce cell-free zones in MC cell culture. A tentative explanation might be that cytotoxic products were released at a slow rate, thereby diffusing throughout the culture gel. This explains why cytotoxicity was not detected by BSI methods’* ‘. Unlike Rosdy and Clauss’, we did not observe a change in pH of the culture gel, and therefore cannot agree with the suggestion that increased acidity was responsible for the cytotoxic effects. The cytotoxic effects of Opsite may result from the adhesive vinylether layer, since Tegaderm which is said to have the same polyurethane film as Opsite, but a different (acrylate) adhesive layer, did not show cytotoxic effects. It seems that the manufacturer of Opsite recently changed the composition of the adhesive layer, Rosdy and Clauss’ also showed that Syspurderm and Lyofoam C had cytotoxic effects on fibroblasts when in direct contact, but not via their extract media, thus performing in accordance with the BSI standard test. However, the extract media were cytotoxic for normal human keratinocytes’ and it was concluded, in line with this study, that both Syspurderm and Lyofoam C induce low-level cytotoxic effects. It was suggested that the common constituent, polyurethane foam, might be responsible for the cytotoxicity’. If so, it does not explain why Epigard, which also contains a polyurethane foamlayer, is not cytotoxic. A medium-high cytotoxicity with a CPII of approximately 50% was previously found for the collagen wound dressing*’ and confirmed in this study. We had tested the collagen wound dressing because of contradictory results concerning collagens, processed with the same cross-linking agent hexamethylenediisocyanate3’ 23,24 (C. Metselaar, Personal Communication). With the BSI test method’, the collagen wound dressing would have been evaluated as non-cytotoxic” (C. Metselaar, Personal Communication). By testing also in MC cell culture, apart from the collagen wound dressing, its extracts and the extracted collagen, we could even differentiate between primary and secondary cytotoxic effects”. Primary cytotoxic effects are defined as being due to direct leakage of cytotoxic products, whilst secondary cytotoxic effects are due to cell-biomaterial interactions. Hypothetically, collagenase produced by proliferating fibroblasts diffuses through the culture gel, cleaves the collagen molecules of the collagen, resulting in cleavage products, which induce secondary cytotoxicity. We could relate these results with the cross-links obtained during cross-linking 25. The intracellular accumulation of Biomaterials
1992.
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Figure 5 Human fibroblasts (HF) at day 7, cultured for 6 d in the presence of Adaptic discs. a, Light micrograph of detaching cells at the edge of the cell-free zone with many illuminated particles in the cytoplasm. Original magnification X720. b, Transmission electron microscopy (TEM) micrograph of trypsinized human fibroblasts (HF). Many pseudopodia are observed. Furthermore, cells had many small, low electron-dense lipid particles (arrows) and several inclusions of cell remnants (R) in the cytoplasm. Original magnification x4665.
Figure 6 Human fibroblasts (HF) at day 7, cultured for 6 d in the presence of Opsite discs. a, Light micrograph of a nonconfluent cell layer. Impaired cell spreading and small illuminated particles are observed. Original magnification X720. b, Transmission electron microscopy (TEM) micrograph of trypsinized human fibroblasts (HF). Cells showed rather rounded cell membranes. Several inclusions of cell remnants (R) and some lipid droplets (L) were observed in the cytoplasm. In general, normal amounts of rough endoplasmic reticulum (RER), sometimes with fusions (arrows) were present in the (degenerated) cells. Original magnification X4665.
the lipid and the inclusion of remnants of dead cells are indications of a rather slow release of primary and secondary cytotoxic products, at first allowing cell proliferation, but thereafter inducing cell degeneration, followed by cell death and phagocytosis. At the moment, we are in the process of removing cytotoxic products from the collagen wound dressing by extensive washing, and/or choosing other processing methods or coating techniques. Preliminary results showed that washing removes primary cytotoxic productsz5, whilst crosslinking with non-toxic agents may prevent secondary cytotoxic effects, both therefore markedly reducing the cell growth inhibitory effects. The group of hydroactive wound dressings, with CPII
>70%, induced high cytotoxicity. These materials, except for Ulcer dressing, induced hardening of the culture gel. This may hinder the culture conditions, e.g. O,/CO, exchange, which is however unlikely, since for example agar, in the agar overlay test culture used extensively5-‘, has an even harder consistency, which does not interfere with the culture conditions. That O,/ CO, exchange is not hindered, is also indicated by the small cell clusters with rather good cell morphology found with Biofilm and Comfeel, which we also classified as highly cytotoxic. Although passing the BSI standard test’,’ because their extracts did not induce cytotoxic effects, the cytotoxic effects induced by Biofilm and Comfeel agree with the results obtained with the direct
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Cytotoxicity
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dressings:
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273
Figure 7 Light micrograph of human fibroblasts (HF) at day 7, cultured for 6 d in the presence of Cutinova discs. Illuminated, detaching cells, singly or in small strings or cell fragments were observed. Original magnification X720.
contact test of Rosdy and Clauss’. Another indication of the highly cytotoxic effect of the whole group of hydroactive wound dressings was the deviant morphology of most of the cultured cells, e.g. lipid degeneration of cells. To analyse hydroactive dressings and the cytotoxic and hardening effects further, we separated the two layers of Duoderm E, i.e. the hydrocolloid layer and the polyurethane top layer, and tested them separately in MC cell culture. Both inhibition of cell growth and hardening of the gel was completely induced by the hydrocolloid layer, whilst normal cell growth was observed with the polyurethane layer (results not shown). We suggest that, as with the other hydrocolloid wound dressings, the hydrocolloid layer is the layer inducing cytotoxicity. This layer contains substances such as gelatin, polyisobutylene, carbox~ethylcellulose and karaya gum, any one or any combination of which might be responsible for the observed cytotoxic effects. In the case of the hydroactive wound dressing Cutinova Hydro, the polyurethane matrix may be responsible for cytotoxicity. Since the hydroactive wound dressings were applied on the culture gel after 24 h when normal cells were present, and these cells had degenerated at day 7, our study indicates that the cytotoxic products interfere directly with the cells. This means that normal cells have been triggered at the metabolism level. This triggering may be so intensive that the disturbed metabolism, observed as degenerative effects, results in cell death. Cytotoxic products may also interfere at the level of p~liferation, i.e. cells may keep their normal mo~hology, but stop proliferating. Other levels, such as the interference of cytotoxic wound dressing products with release of cellular attachment molecules, as suggested by Rosdy and Clauss’, are possible as well. Cytotoxicity of wound dressings is bound to interfere with in viva tissue regeneration. The use of noncytotoxic wound dressings with the same characteristics is therefore recommended. Factors, such as the type of wound [acute or chronic, wet, dry, contaminated and the size) and the condition of the patient, play important roles”~ 27. In case of acute wounds we can think of one
Figure 8 Human fibroblasts (HF) at day 7, cultured for 6 d in the presence of Comfeel discs. a, Light micrograph of clusters of cells with rather good cell adhesion and with some illuminated particles. Original magnification X720. b, Transmission electron microscopy (TEM) micrograph of trypsinized cells, showing many pseudopodia. Many lipid droplets (L), sometimes observed as vacuoles due to lipid extraction by the embedding procedure, and a reduction in the amount of rough endoplasmic reticulum (RER) were observed. Original magnification X7142.
possibility in favour of choosing a [low) cytotoxic dressing, the contaminated wound, in which the presence of cytotoxic substances might have an antibacterial sideeffect26.28, This must indeed be a side-effect since the manufacturers do not mention the addition of any antiseptic or antibiotic substances to the dressings investigated in this study. Another case, in which low or medium-low cytotoxic dressings might be favoured, is the chronic wound, in which the presence of cytotoxic substances might activate the wound-healing reaction by the increase of growth factors and subsequent epithelializationzg3 30. For the activation of wound healing, we suggest that highly cytotoxic wound dressings, such as the group of hydrocolloid wound dressings, may eventually result in Biomaterials
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Cytotoxicity of wound dressings: M.J.A. van Luyn et al.
formation of hypergranulation tissue as a consequence of hyperactivation, resulting from an increase of growth factors from giant cell formation, immunogenic reactions and/or hyperproliferation of fibroblasts. Hypergranulation may be caused by the cytotoxic effects of these wound dressings and as a result of occlusive effects”* 3*. In contrast to h~e~ctivation, the in vivu application of wound dressings which cause medium or high cell growth inhibition (i.e. cell death) in vitro, may, however, result in impaired epithelialization3’. In conclusion, the MC cell culture is an accurate cytotoxicity test system and can, up to a certain limit, function as an in v&v wound healing/d~ssing model. Fibroblasts, MC culture gel and wound dressing may mimic circumstances of the tissue and the jelly clot formed, especially under a permeable wound dressing, such as PEU14. Our results cast doubts on the BSI and IS0 standard tests (recently IS0 has taken over the BSI standards, ISO/ TC 194). As to the clinical implications of our results, one may wonder, whether some wound dressings should be used on epithelializing wounds. Use of low-level, or at most medium-low, cytotoxic wound dressings may be tolerated in special cases, such as the contaminated acute or the chronic wound, which needs wound-healing activation. ACKNOWLEDGEMENTS
11
12
13
14
15 16
17
18
19
The authors wish to thank Mr D. Huizinga, Mr P. van der Sijde and Mr H.R.A. Meibog from our laboratory for the photography.
20
REFERENCES 1 2
3
4
5
British Standard Institute
(BSI), Cytotoxicity Standard Test for Medical Devices No. 5736, Part 10, 1988 Rosdy, M. and Clauss, L.C., Cytotoxicity testing of wound dressings using normal human keratinocytes in culture, J. Biomed. Meter. Res. 1990, 24, 363-377 Ulreich, J.B. and Chvapil, M., A quantitative microassay for in-vitro toxicity testing of biomaterials, I, Biomed. Mater. Res. 1981, 15, 913-922 Cooke, A., Olivier, R.F, and Edward, M., An in vitro cytotoxicity study of aldehyde-treated pig dermal collagen, Br. j. Exp, pathol. 1983,64, 172 Taylor, J.A., Abodeely, R.A. and Fuson, R.L., Rapid screening of biomedical polymers by two methods of tissue culture, Trans. Am. Sot. Artif. Intern, Organs 1973,
19,175178 6
7
8
9
10
1992,
Vol.
22
23
24
Guess, W.L., Rosenbluth, S.A., Schmidt, B. and Autian, J.. Agar diffusion method for toxicity screening of plastics on cultured cell monolayers, I. Pharm. Sci. 1985, 45, 1545-1547 Hensten-Pettersen, A. and Helgeland, K., Evaluation of biologic effects of dental materials using for different cell culture techniques, Scaod. 1, Dent. Res. 1977,85,291-296 Autian, J., App~aches to toxicity evaluation of biomaterials with emphasis on preliminary acute toxicity tests, Adv. Mod. Toxicol, New Concept Saf. 1979, Eval-I, 119-149 Johnson, H.J., Northup, S.J., Seagraves, P.A., Atallah, M., Lin, L. and Darby, T.D., Biocompatibility test procedures for biomaterials evaluation in vitro. II. Objective methods of toxicity assessment, Biomed. Mater. Res. 1985,19,489-508 Adams, A.M., Soames, J.V. and Searle, R.F., An in vitro
Biomaterials
21
13 NO.
5
25
26 27
28
29
model for the biological evaluation of dental materials using human periodontal ligament cells, Cytotechnology 1988,1,179-182 van Luyn, M.J.A., van Wachem, P.B., Olde Damink, L., Ten Hoopen, H., Feijen, J. and Nieuwenhuis, P., Methylcellulose cell culture as a new cytotoxicity test system for biomaterials,~. Mater. Sci.: Mater. Med. 1991,2,142-148 van Wachem, P.B., van Luyn, M.J.A., Koerten, H.K., Olde Damink, L., Ten Hoopen, H., Feijen, J. and Nieuwenhuis, P., In vivo degradation of dermal sheep collagen evaluated with transmission electron microscopy, Biomateriak 1991, 12, 215-223 van Wachem, P.B., van Luyn, M.J.A., Olde Damink, L., Dijkstra, P-J., Feijen, J. and Nieuwenhuis, P., Tissue interactions with dermal sheep collagen implants; a transmission electron microscopical evaluation, Cells and Materials 1991, 1,251-263 Jonkman, M.F. and Bruin, P.A., A new high water vapor permeable polyetherurethane film dressing, I. Biomater. Appl. 1990, 5, 3-19 Lawrence, J.C., Wound dressings, Pha~. update 1987, 147-150 Thomas, S., Loveless, P. and Hay, N.P., Comparative review of the properties of six semipermeable film dressings, Pharmaceut. J. 1988, 785-788 Iscove, N.N. and Schreier, H., Clonal Growth of Cells in Semisolid or Viscous Medium, Immunofogjca~ Methods Academic Press, New York, 1979 Bruin, P., Jonkman, M.F., Meijer, H. J. and Pennings, A. J.. A new porous polyetherurethane wound covering, J. Biomed. Mater. Res. 1990, 24, 217-228 Erasmus, M.E. and Jonkman, M.F., Water vapour permeance a meaningful incasure for water vapour pe~eability of wound coverings, Burns 1989, 15, 371-375 Hulstaert, C.E., Kalicharan, D. and Hardonk, M.J., Cytochemical demonstration of phophatases in rat liver by a cerium-based method in combination with osmium tetroxide and potassium ferrocyanide postfixation, Histochemistry 1983, 78, 71-79 Jonkman, M.F., Coenen, J.M.F.H., Bruin, P., Pennings, A.J. and Klasen, H.J., Poly(ether urethane) wound covering with high water vapour permeability compared with conventional tull gras on split-skin donor sites, Burns 1989, 15, 211-218 Coenen, J.M.F.H., Jonkman, M.F., Nieuwenhuis, P., Klasen, H.J* and Pennings, A.J., Artificial skin: A new porous biodeg~dable polymeric template for neodermis regeneration, Abstract, 8th Int. Congress on Burn Injuries New Delhi, India, 13-14 November 1990 Chvapil, M., Considerations on manufacturing principles of a synthetic burn dressing: a review, 1, Biomed. Mater. Res. 198218, 245-263 Chvapil, M., Speer, D.P., Mora, W. and Eskelson, CD., Effect of tanning agent on tissue reaction to tissue implanted collagen sponge, J. Surg. Res. 1983, 35, 402-409 van Luyn, M.J.A., van Wachem, P.B., Olde Damink, L., Dijkstra, P.J., Feijen, J. and Nieuwenhuis, P., Relations between in vitro cytotoxicity and crosslinked dermal sheep collagen, f. Biomed. Mater. Res. (in press) Falanga, V., Occlusive wound dressings, Why, when, which?, Arch. Dermatol. 1988, 124, 872-877 Varghese, M.C., Balin, A.K., Carter, M. and Caldwell, D., Local environment of chronic wounds under synthetic dressings, Arch. Dermatol. 1986, 122, 52-57 Holland, K.T., Davis, W., Ingham, E. and Gowland, G., A comparison of the in vitro antibacterial and complement activating effect of ‘Opsite’ and ‘Tegaderm’ dressings, f. Hosp. Infection 1964, 5, 323-328 Alper, J-C., Tibbetts, L.L. and Sarazen, A.A., The in vitro
Cytotoxicity of wound dressings: M.J.A. van Luyn et al.
30
response of fibroblasts to the fluid that accumulates under a vapor-permeable membrane, J. Invest. Dermatol. 1985, 84, 513-515 Madden, M.R., Finkelstein, J.L., Nolan, E,, StaianoCoico, L., Goodwin, C.W. and Hefton, J.M., Analysis of epithelialization under semi-occlusive dressings, J. Trauma 1988,28,1091
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Chvapil, M., Chvapil, T.A. and Owen, J.A., Comparative study of four wound dressings on epithelization of pa~ial-thickness wounds in pigs, J. Tramna 1987,27(3), 278-282 Zitelli, J.A., Delayed wound healing with adhesive wound dressing, J. Dermatol. Surg. Oncol. 1984, 10, 709-710
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Biomaterials
1992, Vol. 13 No. 5
267
._
Cytotoxicity testing of wound dressings using me~ylcellulose cell culture M&A. van Luyn, P.B. van Wachem and P. Nieuwenhuis De~rtment of ~istofogy and Cefl Bfofogy, Section of Siomaterials Oostersingel6912, 9713 EZ Groningen, The Netherlands
Research,
Universi~
of GroffingeR,
M.F. Jonkman Department
of Dermatology,
University
Hospital,
Oostersingel
59, 9713 EZ Groningen,
The Netherlands
Wound dressings may induce cytotoxic effects. in this study, we check several, mostly commercially available, wound dressings for cytotoxicity. We used our previously described, newly developed and highly sensitive 7 d methyiceiluiose cell culture with fibrobiasts as the test system. Cytotoxicity is assessed by monitoring ceil growth inhibition, supported by cell morphological evaluation using light and transmission electron microscopy. We tested conventional wound dressings, polyurethane-based films, composites, hydrocolloids and a collagen-based dressing. it was shown that only 5 out of 36 wound dressings did not induce cytotoxic effects. All 5 hydrocoiioids were found to inhibit ceil growth (>70%), while cells had strongly deviant morphologies. The remaining wound dressings showed medium cytotoxic effects, with cell growth inhibition, which varied from low (?15%), medium-low (425%) to medium-high (lt50%). Measurable cytotoxic effects of dressings detected in vitro are likely to interfere with wound healing when applied in vivo. The results are discussed in view of the clinical uses with contaminated wounds, impaired epitheiiaiization or hypergranuiation. Keywords:
Cyfotoxici~,
wound dressings,
~ethy/ce/lutose
Received 15 July 1991; revised 3 October 1991; accepted 20 October 1991
The cytotoxicity of biomaterials can be tested in vitro using various culture systems’-“. Liquid culture systems may detect cytotoxicity of a material either by culture of cells with extracts or with the material itself. In the latter instance, renewing the medium will remove any cytotoxic products released. The agar overlay test is a short-term semisolid culture system in which the possible cytotoxicity of biomaterials is identified only by the presence of cell-free zones. We developed a culture test system, called the methylcellulose (MC) cell culture”, which combines high sensitivity of testing with very detailed testing. High sensitivity is obtained by the ability to evaluate cells for a period of 7 d without refreshing the culture medium. Detailed testing is obtained by the ability to measure cell growth by accurate cell counting and evaluate cell morphology at both the light microscopical (LM) and transmission electron microscopical (TEM) level. With MC cell culture, we studied a collagen wound dressing, which was evaluated as moderately cytotoxic (k50% inhibition of cell growth and deviant cell Correspondence
to Dr M.J.A. van Luyn. .-.-
0
1992 6u~erwort~-Heinemann 0142-9612/92/050267-09
Ltd
morphology) *I. The cytotoxic effects of the collagen wound dressing were confirmed by in viva studiesi2~ 13,in which we used TEM. We used MC cell culture to check 16 different wound dressings for possibIe ~~otoxicity. Six groups of wound dressings were used, i.e. conventional wound dressings, such as paraffin gauze dressing (group A), synthetic wound dressings, such as polyurethane-based films (groups B and C), composites (group D) and hydrogels [group E); the collagen wound dressing previously mentioned (group F) was used as a reference. All wound dressings are commercially available with the exception of one, which is a type of polyurethane-based film co-developed in the Department of Polymer Chemistry, University of Groningen, The Netherlands (group B)14. With MC cell culture, it was shown that only 5 out of 16 wound dressings are evaluated as non-cytotoxic. Another group of 5 wound dressings can be regarded as highly cytotoxic, and the remaining wound dressings induced medium cytotoxicity. The relevance of these results is discussed in relation with the fact that, in general, wound dressings are supposed to induce granulation tissue formation and re-epithelializationX5* 16, _____-_ Biomaterials
1992,
Vol.
13 No. 5
Cytotoxicity of wound dressings: M.J.A.
268
-
MATERIALS AND METHODS Materials Culture conditions Human skin fibroblasts (HF) from the established cellline PK 84 were routinely cultured in RPM1 1640 medium (Gibco Biocult Co., Paisley, UK), supplemented with 10% fetal calf serum (FCS), 2 mM/ml glutamine (Glut) (Merck, Darmstadt, Germany), penicillin (Pen) and streptomycin (Strep), both 100 units/ml (Gibco). The cells were incubated at 37°C in air containing 5% CO,, A stock gel of MC, using Methocel~ high viscosity (3000-5000 mPa) from Fluka, Bio Chemica, Buchs, Switzerland, was prepared according to the method of Iscove and Schreier”, with Iscove’s modification of Dulbecco’s medium (IMDM) [ICN Biomedicals Inc., Costa Mesa, CA, USA) to a final concentration of 2.25%. Materials tested All wound dressings, their manufacturers and compositions are listed in Table 3, The wound dressings are arranged in groups of the material’s composition. The 6 groups of wound dressings (A-F) were used. One type of wound dressing was not commercially available: polyetheru~thane (PEU, no. 4) was codeveloped in the Department of Polymer Chemistry, University of Groningen, The Netherlands14. It is made from Tecoflex” EG80A [Thermedics Inc., Woburn, MA, USA) and has a thickness of l5pm, which swells up to 80-1OO~m when wet. PEU is composed of many noninterconnected microcavities of < 5pm diameter” to achieve a high water vapour permeabilitylg. It was sterilized by y-irradiation (2.5 Mrad] (Gammaster, Ede, The Netherlands).
vaanLuyn et al.
The collagen wound dressing (no. 16)lf-13, the processed dermal layer of sheep skin cross-linked with hexamethylenediisocyanate, is commercially available, but was kindly provided directly by the Zuid Nederlandse Zeemlederfabriek, Oosterhout, The Netherlands. It was sterilized by ~-irradiation j.25 Mrad). All wound dressings were punched into discs of 10 mm diameter, under sterile conditions.
Methods
After washing twice with phosphate buffered saline [PBS) (NPBI, Emmer-Compascuum, The Netherlands], human fibroblasts were harvested from routine culture using 0.25% (w/v) trypsin in Ca/Mg-free Hanks’ Salt Solution (Gibco). Thereafter cells were centrifuged and ~suspended in fresh IMDM, HFfgel mixtures were made by gently and thorougly mixing 30% [v/v) HF/IMDM with 50% (v/v) MC stock gel and 20% (v/v) FCS”. Volumes of Pen, Strep and Glut, added to IMDM, had been adjusted in order to obtain the same final concentration in the culture gel as described for the routine culture medium, For all cultures, a final volume of 4.0 ml culture gel containing 5 X lo4 HF’*, was placed into each well of a six-well tissue-culture plate (Greiner B.V., Alphen a/d Rijn, The Netherlands) using a plastic syringe. Since each well has a surface area of 10 cm’, the final seeding density was 5 X lo3 HFfcm’. Thereafter, cultures were incubated at 37’C in air containing 5% CO,. All cultures were set up in triplicate. Except for the three wells for the control culture, after 24 h two discs of each type of wound dressing were put on top of the HF/gel mixture in
Table 1 List of dressings tested and their compositions Commercial name
Manufacturer
Composition
:
Jelonet Adaptic
Smith & Nephew Ltd, Hull, UK Johnson & Johnson Inc., New Brunswick, USA
White petrolatum gauze Viscose 100% plus emulsion white petrolatum plus water
B
3 4
Omiderm PEU
Omikron Scientific Ltd, Nevogot, Israel University of Groningen, Department of Polymer Chemistry, The Netherlands
Non-adhesive acrylated polyurethane film Non-adhesive polyetherurethane film
c
5
Tegaderm
3M Health Care Inc., St Paul, MN, USA
6
Opsite
Smith & Nephew Ltd, Hull, UK
Polyurethane film plus adhesive acrylate layer Polyurethane film plus adhesive vinyiether layer
li 9 10
Epigard Cutinova plus Syspurderm Lyofoam C
Parke-Davis & Co., Inc., Detroit, Ml, USA Beiersdorf AG, Hamburg, Germany Paul Hartman A.G., Heidenheim, Germany Ultra Laboratories Ltd, Sittingbourne, UK
Polyurethane foam plus polypropylene film Hydrogel plus polyurethane fOam Polyurethane soft foam Viscose fibres plus activated carbon plus polyurethane foam
11 12 13
Biofilm Comfeel Duoderm E
Hydrocoiloid dressing Hydrocolloid dressing Hydrocolloid dressing
14
Ulcer dressing
15
Cutinova hydro
Biotrol Pharma SPA, Paris, France Coloplast AS, Espergaerde, Denmark ConvaTec-Squibb Inc., Greensborough, USA Johnson & Johnson Inc., New Brunswick, USA Beiersdorf AG, Hamburg, Germany
16
Collagen dressing
Zuid Nederlandse Z~mlede~abriek, Oosterhout, The Netherlands
Hexamethylen~iisocyanate collagen dressing
Group
No.
A
D
E
F
Biomaterials 1992, Vol. 13 NO. 5
Hydrocolloid dressing Hydroactive polyurethane dressing cross-linked
Cytotoxicity of wound dressings: M.J.A.
Figure 1 A six-wells tissue culture plate, at day 7 after the start of cell culture. Upon seeding, cells had migrated and adhered to the bottom of the wells. After 1 d, two discs of Comfeel (top row) and Cutinova hydro (bottom row) per well were put on top of the gel. At day 7, Cutinova especially had swollen and the gel had increased in viscosity.
each of three wells of a test culture and the immersed just below the gel surface [Figure 3).
discs
Cell counts At 7 d after starting the cultures, discs and gel were removed without damaging the cells, using a 5 ml syringe. The cell layer remaining on the bottom of each well was extensively washed three to four times with 4 ml PBS to remove the gel completely. Subsequently, cells were trypsinized, resuspended and counted in a Biirker counting chamber. The mean cell number (X103/cm2 f s.d.) was calculated, and from these numbers the cell proliferation inhibition index (CPII), expressed as a percentage of the cell number in control culture, was calculated, using the following relationship:
CPII(%) = 100% -
mean cell number of test culture mean cell number [ of control
Microscopy
culture
269
van Luyn et al.
X 100%
1
During the 7 d test period LM evaluation of the cells was performed without removing the culture gel or discs. At day 7 in situ photography with the phase-contrast inverted light microscope was done after removing discs and washing off the culture gel when the cells were still covered with PBS. For TEM, trypsinized cells were pooled from the three wells, washed with PBS and centrifuged. The resulting pellets were fixed with 2% glutaraldehyde in PBS at 4°C and cut into small pieces of about 2 X 2 mm. These were in PBS”, post-fixed in 1% OsO,, 1.5% K,Fe(CN), dehydrated in graded alcohols and embedded in Epon 812. Ultrathin sections were stained with uranyl acetate and lead citrate and examined with a Philips EM 201 transmission electron microscope, operated at 40 kV. RESULTS
subsequently adhered and proliferated. After 24 h, two discs of each wound dressing were placed on top of the culture gel in each of the three wells. The CPII of day 7 are shown in Table 2 and Figure 2 for each type of wound dressing. The results can be arranged in groups of wound dressings inducing no (OW), low (+15%], medium-low (f25%), medium-high (? 50%) or high (>70%) inhibition, It can be seen that this arrangement does not exactly correspond to the arrangement of groups according to material composition. With LM, the control culture showed a confluent multilayer of well-spread HF at day 7. Some illuminated particles were present within cells (Figure 3s). TEM of the trypsinized cells showed that these particles in fact represent lipid droplets. Furthermore, both welldeveloped rough endoplasmic reticulum (RER) and pseudopodia were observed’ (Figure 3b). Five dressings, i.e. Jelonet, Omiderm, PEU, Tegaderm and Epigard (nos 1, 8, 4, 5 and 7) induced no significant inhibitions in cell growth. The cells of these cultures had, at both the LM and the TEM level, normal morphologies similar to the cells of the control culture. The polyurethane foams Cutinova plus, Syspurderm
Table2 Dressings proliferation Group
No.
A
tested
their
inhibition
:
Jelonet Adaptic
0.7 + 1.8 24.5 f 2.1
3 4
Omiderm PEU
-0.5 + 3.2 0.6 f 4.6
:
Tegaderm Opsite
0.3 + 3.6 29.1 + 4.6
: 9 10
Epigard Cutinova plus Syspurderm Lyofoam C
-1.5 13.1 16.1 13.7
* + f +
3.4 1.2 1.3 2.9
E
11 12 13 14 15
Biofilm Comfeel Duoderm Ulcer dressing Cutinova hydro
71.5 89.3 92.9 95.3 99.6
f + f f +
2.3 1.2 2.5 1.2 0.3
F
16
Collagen dressing 51.5 k 5.3
B C D
Degree of +50,
of
cell
Commercial name Inhibition CPII (%) + s.d.
cytotoxicity
medium-high:
CPII >70,
(%):
+O,
no;
+15.
low;
+25,
medium-low;
high.
Epigard Omiderm Tegaderm PEU Jelonet Cutlnova plus Lyofoam C Syspurderm Adaptlc Opsite Collagen-dressing Biofilm
Comfeel Duoderm Ulcer dressing Cutinova hydro -23
0 Cell
HF, seeded in culture gel to a final density of 5 X lo3 HF/cm’, migrated towards the bottom of the wells and
and
20
proliferation
40
60
lnhlbitlon
80
Index
100
120
(W) ?s.d.
Figure 2 The cell proliferation inhibition indices (CPII) at day 7 for each type of wound dressing. Biomaterials
1992. Vol. 13 No. 5
270
Cytotoxicity
of wound
dressings:
M.J.A. van Luyn et al.
low inhibition with CPIIs of 24.5 and 29.1% respectively. Adaptic induced easily macroscopically recognizable cell-free zones. Cells near the cell-free zone showed impaired spreading and an increase in lipid droplets (Figure sa), but in general the cell morphology was normal. This was confirmed by TEM, which showed cells with many pseudopodia, increased inclusions of cell remnants and small lipid droplets (Figure 5b). A nonconfluent cell layer with impaired cell spreading, but without a cell-free zone, was observed with Opsite (Figure 6a]. At the TEM level, the cells had rather rounded membranes and somewhat degenerated morphologies (Figure 6b). The collagen dressing, which was taken as a reference material, induced evident cell-free zones, a medium-high CPII of approximately 50% and concomitant deviant cell morphology. To sum up, deviances in morphology were the presence of proportionally larger cells, poorly adhering cells, the increased presence of lipid droplets, the reduction in the amount and dilatation of RER, many inclusions with remnants of dead cells and a decrease in pseudopodia. The hydroactive wound dressings of group E all had high CPIIs ranging 70-100%. They sometimes swelled markedly [Figure 2) when one compares diameters of discs of Comfeel (no. 12) and of Cutinova hydro [no. 15). This may result in a change of viscosity of the culture gel, which makes it more difficult to remove. Since cell growth was extensively inhibited, cell-free zones were not observed. The few cells in cultures with Duoderm, Ulcer dressing and Cutinova hydro (nos 13, 14 and 15) were found as detached cells, either singly or in small strings (shown for Cutinova hydro, no. 15, Figure 7). TEM preparations of these few cells could not be made. The cells of cultures with Biofilm (no. 11) and Comfeel (no. 12) were found in small clusters which still showed rather good adhesion (shown for Comfeel, Figure f?a). TEM of the trypsinized cells of Comfeel and Biofilm showed many small lipid accumulations, a reduction in the amount of RER and many pseudopodia (shown for Comfeel, Figure 8b).
Figure 3 Human fibroblasts (HF) at day 7, cultured for 6 d in the presence of Epigard discs. a, Light micrograph of a multilayer of completely spread cells (cell morphology is similar to that of the control culture). Original magnification X720. b, Transmission electron microscopy (TEM) micrograph of trypsinized human fibroblasts (HF). Cells have many pseudopodia, well-developed rough endoplasmic reticulum (RER) (arrows) and some small lipid droplets (L). Original magnification X4665.
DISCUSSION
and Lyofoam C (nos 8, 9 and lo), resulted in low inhibitions with a CPII of approximately 15%. Small cellfree zones were vaguely recognized macroscopically, but with LM clearly recognized when discs were removed. Close to the cell-free zones, cells showed decreased adherence with elongated or spider-like cell structures and illuminated cell edges and an increase in lipid droplets, as evaluated with both LM and TEM. Cells had more normal morphologies with increasing distance from the cell-free zones. The cells grown in the presence of Syspurderm had many dark particles, which were not extensively found in any other culture [Figure 4s). TEM revealed that these particles represent low electrondense multilocular lipid droplets (Figure 4b) and/or compact myelin bodies [Figure 4~). Adaptic (no. 2) and Opsite (no. 6) induced a medium-
The MC cell culture accurately shows that only 5 out of 16 wound dressings are non-cytotoxic (nos 1, 3, 4, 5 and 71, but 5 wound dressings are highly cytotoxic [nos 11,12, 13,14 and l5), and the remaining wound dressings induced an intermediate cytotoxicity (nos 2, 6, 8, 9, 10 and 16). The five non-cytotoxic wound dressings include petrolatum gauze (Jelonet, no. 1) and four types of polyurethane-based dressings. According to BSI standards, Jelonet (no. l), Omiderm (no. 3) and Tegaderm (no. 5) had already been found to be non-cytotoxicl*‘. Since the MC cell culture” is a more sensitive test system than any other yet available’-“, these five wound dressings may be considered as truly biocompatible. It is encouraging that the polyurethane film PEU turned out to be among this group. PEU was compared with Jelonet in a clinical trial and applied on skin donor sites”. Healing rates of the two wound dressings were similar, but PEU was favoured by patients since it reduced
Biomaterials 1992, Vol.
13 No. 5
Cytotoxicity
of wound
dressings:
M.J.A.
van Luyn et al.
Figure 4 Human fibroblasts (HF) at day 7, cultured for 6 d in the presence of Syspurderm discs. a, Light micrograph of a bilayer of human fibroblasts (HF) showing many illuminated and dark particles (arrows) in the cytoplasm. Original magnification X720. b, Transmission electron microscopy (TEM) micrograph of trypsinized human fibroblasts (HF) containing many small lipid droplets and remnants of dead cells (R). Original magnification X10752. c, Transmission electron microscopy micrograph of trypsinized human fibroblasts (HF) containing electron-dense lipid droplets and several myeline bodies (arrows), possibly representing the dark particles observed with light microscopy (LM). The amount of rough endoplasmic reticulum (RER) present is comparable with that in cells of the control culture. Original magnification X7142.
271
pain’l. PEU is now being investigated as an artificial skin, by combining it with a microporous biodegradable bottom layer, and first results with full-thickness wounds in pigs are promising”. The group with low or medium-low cytotoxic effects includes a synthetic petrolatum gauze (Adaptic), an adhesive polyurethane film (Opsite) and composites with a foam-layer (Cutinova plus, Syspurderm and Lyofoam C). Of these, Adaptic had already been found to be cytotoxic’v ‘. Opsite appears to have passed the BSI standard test, since its extraction media were not cytotoxic” ‘. However, our statement of Opsite having medium-low cytotoxic effects agrees with results of Rosdy and Clauss’, who tested Opsite in a direct contact test with either fibroblasts or keratinocytes and observed cytotoxic effects, but no inhibition of cell proliferation. Opsite did not induce cell-free zones in MC cell culture. A tentative explanation might be that cytotoxic products were released at a slow rate, thereby diffusing throughout the culture gel. This explains why cytotoxicity was not detected by BSI methods’* ‘. Unlike Rosdy and Clauss’, we did not observe a change in pH of the culture gel, and therefore cannot agree with the suggestion that increased acidity was responsible for the cytotoxic effects. The cytotoxic effects of Opsite may result from the adhesive vinylether layer, since Tegaderm which is said to have the same polyurethane film as Opsite, but a different (acrylate) adhesive layer, did not show cytotoxic effects. It seems that the manufacturer of Opsite recently changed the composition of the adhesive layer, Rosdy and Clauss’ also showed that Syspurderm and Lyofoam C had cytotoxic effects on fibroblasts when in direct contact, but not via their extract media, thus performing in accordance with the BSI standard test. However, the extract media were cytotoxic for normal human keratinocytes’ and it was concluded, in line with this study, that both Syspurderm and Lyofoam C induce low-level cytotoxic effects. It was suggested that the common constituent, polyurethane foam, might be responsible for the cytotoxicity’. If so, it does not explain why Epigard, which also contains a polyurethane foamlayer, is not cytotoxic. A medium-high cytotoxicity with a CPII of approximately 50% was previously found for the collagen wound dressing*’ and confirmed in this study. We had tested the collagen wound dressing because of contradictory results concerning collagens, processed with the same cross-linking agent hexamethylenediisocyanate3’ 23,24 (C. Metselaar, Personal Communication). With the BSI test method’, the collagen wound dressing would have been evaluated as non-cytotoxic” (C. Metselaar, Personal Communication). By testing also in MC cell culture, apart from the collagen wound dressing, its extracts and the extracted collagen, we could even differentiate between primary and secondary cytotoxic effects”. Primary cytotoxic effects are defined as being due to direct leakage of cytotoxic products, whilst secondary cytotoxic effects are due to cell-biomaterial interactions. Hypothetically, collagenase produced by proliferating fibroblasts diffuses through the culture gel, cleaves the collagen molecules of the collagen, resulting in cleavage products, which induce secondary cytotoxicity. We could relate these results with the cross-links obtained during cross-linking 25. The intracellular accumulation of Biomaterials
1992.
Vol. 13 No. 5
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Cytotoxicity
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dressings:
M.J.A. van Luyn et al.
Figure 5 Human fibroblasts (HF) at day 7, cultured for 6 d in the presence of Adaptic discs. a, Light micrograph of detaching cells at the edge of the cell-free zone with many illuminated particles in the cytoplasm. Original magnification X720. b, Transmission electron microscopy (TEM) micrograph of trypsinized human fibroblasts (HF). Many pseudopodia are observed. Furthermore, cells had many small, low electron-dense lipid particles (arrows) and several inclusions of cell remnants (R) in the cytoplasm. Original magnification x4665.
Figure 6 Human fibroblasts (HF) at day 7, cultured for 6 d in the presence of Opsite discs. a, Light micrograph of a nonconfluent cell layer. Impaired cell spreading and small illuminated particles are observed. Original magnification X720. b, Transmission electron microscopy (TEM) micrograph of trypsinized human fibroblasts (HF). Cells showed rather rounded cell membranes. Several inclusions of cell remnants (R) and some lipid droplets (L) were observed in the cytoplasm. In general, normal amounts of rough endoplasmic reticulum (RER), sometimes with fusions (arrows) were present in the (degenerated) cells. Original magnification X4665.
the lipid and the inclusion of remnants of dead cells are indications of a rather slow release of primary and secondary cytotoxic products, at first allowing cell proliferation, but thereafter inducing cell degeneration, followed by cell death and phagocytosis. At the moment, we are in the process of removing cytotoxic products from the collagen wound dressing by extensive washing, and/or choosing other processing methods or coating techniques. Preliminary results showed that washing removes primary cytotoxic productsz5, whilst crosslinking with non-toxic agents may prevent secondary cytotoxic effects, both therefore markedly reducing the cell growth inhibitory effects. The group of hydroactive wound dressings, with CPII
>70%, induced high cytotoxicity. These materials, except for Ulcer dressing, induced hardening of the culture gel. This may hinder the culture conditions, e.g. O,/CO, exchange, which is however unlikely, since for example agar, in the agar overlay test culture used extensively5-‘, has an even harder consistency, which does not interfere with the culture conditions. That O,/ CO, exchange is not hindered, is also indicated by the small cell clusters with rather good cell morphology found with Biofilm and Comfeel, which we also classified as highly cytotoxic. Although passing the BSI standard test’,’ because their extracts did not induce cytotoxic effects, the cytotoxic effects induced by Biofilm and Comfeel agree with the results obtained with the direct
Biomaterials
1992. Vol. 13 No. 5
Cytotoxicity
of wound
dressings:
M.J.A. van Luyn et al.
273
Figure 7 Light micrograph of human fibroblasts (HF) at day 7, cultured for 6 d in the presence of Cutinova discs. Illuminated, detaching cells, singly or in small strings or cell fragments were observed. Original magnification X720.
contact test of Rosdy and Clauss’. Another indication of the highly cytotoxic effect of the whole group of hydroactive wound dressings was the deviant morphology of most of the cultured cells, e.g. lipid degeneration of cells. To analyse hydroactive dressings and the cytotoxic and hardening effects further, we separated the two layers of Duoderm E, i.e. the hydrocolloid layer and the polyurethane top layer, and tested them separately in MC cell culture. Both inhibition of cell growth and hardening of the gel was completely induced by the hydrocolloid layer, whilst normal cell growth was observed with the polyurethane layer (results not shown). We suggest that, as with the other hydrocolloid wound dressings, the hydrocolloid layer is the layer inducing cytotoxicity. This layer contains substances such as gelatin, polyisobutylene, carbox~ethylcellulose and karaya gum, any one or any combination of which might be responsible for the observed cytotoxic effects. In the case of the hydroactive wound dressing Cutinova Hydro, the polyurethane matrix may be responsible for cytotoxicity. Since the hydroactive wound dressings were applied on the culture gel after 24 h when normal cells were present, and these cells had degenerated at day 7, our study indicates that the cytotoxic products interfere directly with the cells. This means that normal cells have been triggered at the metabolism level. This triggering may be so intensive that the disturbed metabolism, observed as degenerative effects, results in cell death. Cytotoxic products may also interfere at the level of p~liferation, i.e. cells may keep their normal mo~hology, but stop proliferating. Other levels, such as the interference of cytotoxic wound dressing products with release of cellular attachment molecules, as suggested by Rosdy and Clauss’, are possible as well. Cytotoxicity of wound dressings is bound to interfere with in viva tissue regeneration. The use of noncytotoxic wound dressings with the same characteristics is therefore recommended. Factors, such as the type of wound [acute or chronic, wet, dry, contaminated and the size) and the condition of the patient, play important roles”~ 27. In case of acute wounds we can think of one
Figure 8 Human fibroblasts (HF) at day 7, cultured for 6 d in the presence of Comfeel discs. a, Light micrograph of clusters of cells with rather good cell adhesion and with some illuminated particles. Original magnification X720. b, Transmission electron microscopy (TEM) micrograph of trypsinized cells, showing many pseudopodia. Many lipid droplets (L), sometimes observed as vacuoles due to lipid extraction by the embedding procedure, and a reduction in the amount of rough endoplasmic reticulum (RER) were observed. Original magnification X7142.
possibility in favour of choosing a [low) cytotoxic dressing, the contaminated wound, in which the presence of cytotoxic substances might have an antibacterial sideeffect26.28, This must indeed be a side-effect since the manufacturers do not mention the addition of any antiseptic or antibiotic substances to the dressings investigated in this study. Another case, in which low or medium-low cytotoxic dressings might be favoured, is the chronic wound, in which the presence of cytotoxic substances might activate the wound-healing reaction by the increase of growth factors and subsequent epithelializationzg3 30. For the activation of wound healing, we suggest that highly cytotoxic wound dressings, such as the group of hydrocolloid wound dressings, may eventually result in Biomaterials
1992, Vol. 13 No. 5
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Cytotoxicity of wound dressings: M.J.A. van Luyn et al.
formation of hypergranulation tissue as a consequence of hyperactivation, resulting from an increase of growth factors from giant cell formation, immunogenic reactions and/or hyperproliferation of fibroblasts. Hypergranulation may be caused by the cytotoxic effects of these wound dressings and as a result of occlusive effects”* 3*. In contrast to h~e~ctivation, the in vivu application of wound dressings which cause medium or high cell growth inhibition (i.e. cell death) in vitro, may, however, result in impaired epithelialization3’. In conclusion, the MC cell culture is an accurate cytotoxicity test system and can, up to a certain limit, function as an in v&v wound healing/d~ssing model. Fibroblasts, MC culture gel and wound dressing may mimic circumstances of the tissue and the jelly clot formed, especially under a permeable wound dressing, such as PEU14. Our results cast doubts on the BSI and IS0 standard tests (recently IS0 has taken over the BSI standards, ISO/ TC 194). As to the clinical implications of our results, one may wonder, whether some wound dressings should be used on epithelializing wounds. Use of low-level, or at most medium-low, cytotoxic wound dressings may be tolerated in special cases, such as the contaminated acute or the chronic wound, which needs wound-healing activation. ACKNOWLEDGEMENTS
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The authors wish to thank Mr D. Huizinga, Mr P. van der Sijde and Mr H.R.A. Meibog from our laboratory for the photography.
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REFERENCES 1 2
3
4
5
British Standard Institute
(BSI), Cytotoxicity Standard Test for Medical Devices No. 5736, Part 10, 1988 Rosdy, M. and Clauss, L.C., Cytotoxicity testing of wound dressings using normal human keratinocytes in culture, J. Biomed. Meter. Res. 1990, 24, 363-377 Ulreich, J.B. and Chvapil, M., A quantitative microassay for in-vitro toxicity testing of biomaterials, I, Biomed. Mater. Res. 1981, 15, 913-922 Cooke, A., Olivier, R.F, and Edward, M., An in vitro cytotoxicity study of aldehyde-treated pig dermal collagen, Br. j. Exp, pathol. 1983,64, 172 Taylor, J.A., Abodeely, R.A. and Fuson, R.L., Rapid screening of biomedical polymers by two methods of tissue culture, Trans. Am. Sot. Artif. Intern, Organs 1973,
19,175178 6
7
8
9
10
1992,
Vol.
22
23
24
Guess, W.L., Rosenbluth, S.A., Schmidt, B. and Autian, J.. Agar diffusion method for toxicity screening of plastics on cultured cell monolayers, I. Pharm. Sci. 1985, 45, 1545-1547 Hensten-Pettersen, A. and Helgeland, K., Evaluation of biologic effects of dental materials using for different cell culture techniques, Scaod. 1, Dent. Res. 1977,85,291-296 Autian, J., App~aches to toxicity evaluation of biomaterials with emphasis on preliminary acute toxicity tests, Adv. Mod. Toxicol, New Concept Saf. 1979, Eval-I, 119-149 Johnson, H.J., Northup, S.J., Seagraves, P.A., Atallah, M., Lin, L. and Darby, T.D., Biocompatibility test procedures for biomaterials evaluation in vitro. II. Objective methods of toxicity assessment, Biomed. Mater. Res. 1985,19,489-508 Adams, A.M., Soames, J.V. and Searle, R.F., An in vitro
Biomaterials
21
13 NO.
5
25
26 27
28
29
model for the biological evaluation of dental materials using human periodontal ligament cells, Cytotechnology 1988,1,179-182 van Luyn, M.J.A., van Wachem, P.B., Olde Damink, L., Ten Hoopen, H., Feijen, J. and Nieuwenhuis, P., Methylcellulose cell culture as a new cytotoxicity test system for biomaterials,~. Mater. Sci.: Mater. Med. 1991,2,142-148 van Wachem, P.B., van Luyn, M.J.A., Koerten, H.K., Olde Damink, L., Ten Hoopen, H., Feijen, J. and Nieuwenhuis, P., In vivo degradation of dermal sheep collagen evaluated with transmission electron microscopy, Biomateriak 1991, 12, 215-223 van Wachem, P.B., van Luyn, M.J.A., Olde Damink, L., Dijkstra, P-J., Feijen, J. and Nieuwenhuis, P., Tissue interactions with dermal sheep collagen implants; a transmission electron microscopical evaluation, Cells and Materials 1991, 1,251-263 Jonkman, M.F. and Bruin, P.A., A new high water vapor permeable polyetherurethane film dressing, I. Biomater. Appl. 1990, 5, 3-19 Lawrence, J.C., Wound dressings, Pha~. update 1987, 147-150 Thomas, S., Loveless, P. and Hay, N.P., Comparative review of the properties of six semipermeable film dressings, Pharmaceut. J. 1988, 785-788 Iscove, N.N. and Schreier, H., Clonal Growth of Cells in Semisolid or Viscous Medium, Immunofogjca~ Methods Academic Press, New York, 1979 Bruin, P., Jonkman, M.F., Meijer, H. J. and Pennings, A. J.. A new porous polyetherurethane wound covering, J. Biomed. Mater. Res. 1990, 24, 217-228 Erasmus, M.E. and Jonkman, M.F., Water vapour permeance a meaningful incasure for water vapour pe~eability of wound coverings, Burns 1989, 15, 371-375 Hulstaert, C.E., Kalicharan, D. and Hardonk, M.J., Cytochemical demonstration of phophatases in rat liver by a cerium-based method in combination with osmium tetroxide and potassium ferrocyanide postfixation, Histochemistry 1983, 78, 71-79 Jonkman, M.F., Coenen, J.M.F.H., Bruin, P., Pennings, A.J. and Klasen, H.J., Poly(ether urethane) wound covering with high water vapour permeability compared with conventional tull gras on split-skin donor sites, Burns 1989, 15, 211-218 Coenen, J.M.F.H., Jonkman, M.F., Nieuwenhuis, P., Klasen, H.J* and Pennings, A.J., Artificial skin: A new porous biodeg~dable polymeric template for neodermis regeneration, Abstract, 8th Int. Congress on Burn Injuries New Delhi, India, 13-14 November 1990 Chvapil, M., Considerations on manufacturing principles of a synthetic burn dressing: a review, 1, Biomed. Mater. Res. 198218, 245-263 Chvapil, M., Speer, D.P., Mora, W. and Eskelson, CD., Effect of tanning agent on tissue reaction to tissue implanted collagen sponge, J. Surg. Res. 1983, 35, 402-409 van Luyn, M.J.A., van Wachem, P.B., Olde Damink, L., Dijkstra, P.J., Feijen, J. and Nieuwenhuis, P., Relations between in vitro cytotoxicity and crosslinked dermal sheep collagen, f. Biomed. Mater. Res. (in press) Falanga, V., Occlusive wound dressings, Why, when, which?, Arch. Dermatol. 1988, 124, 872-877 Varghese, M.C., Balin, A.K., Carter, M. and Caldwell, D., Local environment of chronic wounds under synthetic dressings, Arch. Dermatol. 1986, 122, 52-57 Holland, K.T., Davis, W., Ingham, E. and Gowland, G., A comparison of the in vitro antibacterial and complement activating effect of ‘Opsite’ and ‘Tegaderm’ dressings, f. Hosp. Infection 1964, 5, 323-328 Alper, J-C., Tibbetts, L.L. and Sarazen, A.A., The in vitro
Cytotoxicity of wound dressings: M.J.A. van Luyn et al.
30
response of fibroblasts to the fluid that accumulates under a vapor-permeable membrane, J. Invest. Dermatol. 1985, 84, 513-515 Madden, M.R., Finkelstein, J.L., Nolan, E,, StaianoCoico, L., Goodwin, C.W. and Hefton, J.M., Analysis of epithelialization under semi-occlusive dressings, J. Trauma 1988,28,1091
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Chvapil, M., Chvapil, T.A. and Owen, J.A., Comparative study of four wound dressings on epithelization of pa~ial-thickness wounds in pigs, J. Tramna 1987,27(3), 278-282 Zitelli, J.A., Delayed wound healing with adhesive wound dressing, J. Dermatol. Surg. Oncol. 1984, 10, 709-710
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1992, Vol. 13 No. 5
