- Email: [email protected]
Toxicity testing of wound dressing materials in vitro
Journal of Plastic Surgery (1995), 48, 23&235 0 1995 The British Association of Plastic Surgeons
British
Toxicity testing of wound dressingmaterials in vitro R. Dover*, W. R. Otto?, J. Nanchahalt
and D. J. Riches*
*Department of Anatomy, Queen Mary College, University of London t Histopathology Unit, Imperial Cancer Research Fund, London $ Department of Plastic Surgery, Mount Vernon Hospital, Northwood, Middlesex, UK SUMMARY. There is a bewildering array of dressing materials available for wound coverage. The choice of dressing is often by local custom or practical experience. We wished to investigate if different dressings varied in their ability to either stimulate or inhibit proliferative activity and differentiation in an in vitro test system. We have used a number of test systems for this study. Human dermal fibroblast and keratinocyte cultures were used to screen for proliferative and cytotoxic effects. A more complex “organotypic” method involving fibroblastimpregnated collagen gels overlaid with epidermal keratinocytes was used to investigate effects on differentiation. Dressings were selected from each of the major types available, from simple gauze to hydrocolloids. Of the dressings tested, some reduced cell growth rates but the majority showed no major effect on proliferation or differentiation. Of those displaying an effect, only one could be attributed to cytotoxicity.
The choice of dressing is an important decision in wound management. This choice is often governed by custom, habit or financial considerations rather than any solid scientific basis. The sheer number of dressings available is huge, and these can be divided into a number of types based on their composition (see review by Carver & Leighl). The simplest are gauzes, the most complex are biological skin substitutes. The market is competitive and financially rewarding and each manufacturer claims a range of benefits from use of their product. There are reports in the literature of independent clinical trials but there has been relatively little work looking for effects on in vitro systems. The in vitro method allows simultaneous testing of large numbers of dressings, something which is much more difficult to achieve in a clinical trial. The British Standard (BS5736 part 10 “Method of test for toxicity to cells in culture of extracts from medical devices”) for dressing materials requires a rather limited in vitro testing procedure. This involves soaking the dressing in a specified culture medium for between 24 and 72 hours and then preparing a range of dilutions of this extract. Then one of three specified cell lines (or any other cell fitting certain criteria) is exposed to the test agent for 24 hours and the cells fixed. The cells are stained and then scored for survival on a 5 point scale where: 0 is no cell death; 1 is 25 % death; 2 is 25-50 % death; 3 is 5&75 % ; 4 is greater than 75 % death. This method is rather basic and, although it is designed to protect the patient by detecting cytotoxicity, is somewhat crude and is incapable of detecting growth promoting or differentiation effects. The recommended cells are, respectively, L926 a mouse connective tissue line, MRC-5 human foetal lung fibroblasts and African Green Monkey kidney cells. Alternatively, any cell for which reproducible cytotoxic titres have been demonstrated could be used. Thus it is not a requirement to test the dressing materials on the target tissue.
To address this problem and to investigate any potential growth promoting effects we have used a range of in vitro tests. These all involve human cells obtained from the target tissue in vivo. We used methods with increasing complexity to study a range of effects. These included “organotypic” cultures composed of both epithelial and mesenchymal elements, which provide us with a more recognisable tissue histology and, to some degree, allow for potential synergistic effects between these elements. We also exposed the cells to the complete dressing rather than an extract. It seemed possible that in some cases the cells and the dressings might be able to interact to produce effects that might not be detectable using extracts alone. Such cultures cannot be considered as representative of the more complex wound bed but are a step closer to it. Methods Cell culture 3T3 cells. These were cultured in Dulbecco’s Modified
Eagles Medium (GIBCO) supplemented with 10% Newborn Calf serum (GIBCO) and Kanamycin (100 pg/ml) and Fungizone (1.25 pug/ml) (Squibb). These were cultured in Dulbecco’s Modified Eagles Medium supplemented with 10 % Foetal Calf serum (GIBCO) and Kanamycin and Fungizone.
Fibroblasts.
Keratinocytes. These were cultured in fibroblast medium supplemented with the following additions : Hydrocortisone (0.4 pg/ml), Insulin (5 pg/ml) Cholera Toxin (10-l’ M) (Sigma), Epidermal Growth Factor (10 rig/ml) (Advanced Protein Products). Keratinocytes were cultured on a 3T3 feeder layer prepared by irradiating freshly trypsinized 3T3s with 230
Toxicity
testing of wound dressing materials
Table 1 Product
name
Composition
Manufacturer
2nd Skin Bactigras
Polyethylene oxide gel Tulle gras/chlorhexidine
Fibrocol Geliperm dry Jelonet
Collagen/alginate Hydrocolloid Tulle gras/petroleum jelly Alginate/charcoal Alginate Polyurethane foam Polyurethane film
Spenco Smith & Nephew Medical Johnson & Johnson Geistlich Johnson & Johnson
Kaltocarb Kaltostat Lyofoam Nobecutane spray Opsite sheet
Polyurethane film
231
in vitro
Britcair BritcairSeton Healthcare Astra
Opsite spray
Polyurethane film
Silastic gel sheet Silicone n/a Spyrofoam Tegaderm Tegapore
Silicone gel
Smith & Nephew Medical Smith & Nephew Medical Dow Coming
Silicone coated viscose Polyurethane membrane Polyurethane film Woven nylon fabric
Johnson & Johnson Britcair 3M 3M
60 Gy X-rays. 3T3 cells were then plated at a density of 1.2 x lo4 cells/cm’. Keratinocytes were then plated with or on to the 3T3s at a range of densities. Normal human epidermal keratinocytes and fibroblasts were established from a punch biopsy from the inner aspect of the forearm of a volunteer. Keratinocytes were isolated by overnight incubation of the biopsy in 0.25 % trypsin at 4°C followed by peeling the epidermis which was then shaken in culture medium to release keratinocytes. The remaining dermis was cut into small explants and cells allowed to grow out for two weeks prior to harvesting. Both keratinocytes and fibroblasts were used for experiments between passage 2 and 10. For some tests (see below) pure keratinocytes were prepared by washing cultures vigorously with EDTA solution (2 % Ethylene Diamino Tetra Acetic Acid in PBS) to remove the feeder layer prior to trypsinisation. The released keratinocytes were then washed by centrifugation and plated without feeder cells at a density of lo5 cells per well in 24-well plates (Falcon). Dressings
Table 1 lists the dressings used in the study. These include examples of the major types of wound contact layers. In all cases, with the exception of the two spray dressings, the dressings were provided in sterile packaging. The dressings were cut under aseptic conditions into 5 x 5 mm squares for use. Instruments were changed and cleaned between each dressing to prevent cross-contamination by adhesives, etc. The most adhesive dressings (Opsite sheet and Tegaderm) posed some problems in handling. Small pieces readily self adhered and were discarded. For all the other sheet type dressings, 5 x 5 mm squares could be easily handled and applied to cultures or organotypic gels. The Silastic gel sheet was used in two different states: as provided without washing to remove surface material and following washing in soap flakes as recom-
mended by the manufacturer followed by extensive washing in phosphate buffered saline (PBS). Both spray dressings were applied to gels (see later) by placing the filter bearing the gel on a large sterile plastic dish and applying the spray in short (less than 1 s) bursts from a height of approximately 60 cm. This was the maximum height comfortably achievable within a tissue culture hood. The spray was left to dry for at least 30 s between applications in order to allow the solvent to evaporate. Used from this distance, the cover is not complete but this prevents physical damage from the force of the propellant gas and potential chemical damage from the solvent. Complete cover was achieved by between 5 and 8 applications. Growth assays
A number of different assay systems were used to investigate potential effects of the dressings. The first method was applied to cultures of either pure keratinocytes or fibroblasts growing in 24-well plates. Each dressing was added to quadruplicate wells for 3 days. Control wells received no other treatment. The dressings were carefully removed and the wells washed twice with PBS and frozen at - 20°C and stored prior to DNA assay. The DNA assay method of Rao and Otto’ was employed. This is a very sensitive fluorimetric assay based on Hoechst dye binding. This produces a measurement of the DNA content of each well, which correlates with cell number. All wells were scanned under phase microscopy prior to assay. All dressing materials were tested in at least three independent experiments. Protein content assays were also performed in parallel with the DNA assays. All cultures were pulsed for one hour with 1 @Zi/ml tritiated thymidine (3H-TdR) (Amersham International UK, 5 Ci/mmol) prior to termination. Aliquots of solubilised cells were then scintillation counted. The second and third assay methods involved organotypic gels. Briefly, collagen gels were prepared as described previously.3 Gels comprised 1 ml of collagen containing lo5 fibroblasts cast into a 24-well plate. Keratinocytes were added to the surface either 4-6 h later or 24 h later. Once keratinocytes were added to the gels, they were fed with keratinocyte culture medium as described above. Gels were maintained for 7-10 days prior to use. For some assays, gels were used in their submerged state and the dressing samples were applied to the overlying culture medium for 3 days prior to fixation. In other assays gels were raised to the air liquid interface. Briefly, this involved placing the gels on a sterile filter on a metal grid which raises the gel 24 mm off the base of the culture vessel. The level of the medium is then controlled so that the upper surface of the gel is above the liquid level and the gel is fed from below. Submerged gels were tested with spray dressings but this is technically difficult and results in the gel being effectively cut off from the outside atmosphere, and diffusion through the dressing may be limiting. Thus we used raised gels for testing both spray dressings and some sheet dressings. In this case,
232
British Journal of Plastic Surgery
cultured gels were left for 7 days post-raising before being assayed. Dressings were left in contact for 7 days before formalin fixation.
Results Microscopy
Visual inspection of the cells following treatment clearly showed differences in cell number for Bactigras and Kaltocarb exposed cultures, and the difference was clearest in the fibroblast cultures since these form monolayers. Kaltocarb treated wells were obvious as the medium became clear and lost its red colour,
Figure SComparison of DNA content following treatment of keratinocytes for 7 days, expressed as percentage of control. The bars show the mean and standard deviation for each group. The horizontal band shows the control’s standard deviation as a guide for comparison. N = 12. Stars indicate significance at p < 0.001.
Table 2 Product
Figure l--Comparison of DNA content following treatment of fibroblasts for 3 days, expressed as percentage of control. The bars show the mean and standard deviation for each group. The horizontal band shows the control’s standard deviation as a guide for comparison. N = 12. Stars indicate significance at p < 0.001.
Kaltostat Lyofoam Opsite Silastic Silicone n/a Spyrofoam Tegaderm Tegapore
Fibroblasts hY3
+ve NS NS NS +ve +ve +ve
Fibroblast hY7
Keratinocytes day 7
+ve -ve NS NS NS -ve +ve
NS +ve NS +ve NS -ve -ve
NS : not significant at p>O.OOl. +ve : significant at p
increase
in cell
decrease
in cell
presumably due to binding of the phenol red by charcoal incorporated in the dressing. DNA assays
Figure 24omparison of DNA content following treatment of fibroblasts for 7 days, expressed as percentage of control. The bars show the mean and standard deviation for each group. The horizontal band shows the control’s standard deviation as a guide for comparison. N = 12. Stars indicate significance at p < 0.001.
Data for all experiments was pooled and expressed as a percentage of the control value. Figure 1 shows the results for fibroblasts co-cultured with the dressings for 3 days, Figure 2 shows the results after 7 days. Figure 3 shows the effects on cultured keratinocytes treated for 7 days. t-Tests were performed comparing each dressing with its control, and those results significant at the p < 0.001 level are indicated on the figures. Table 2 summarises the results. From Table 2 it can
Toxicity testing of wound dressing materials in vitro
233
H Kaltocarb El Bactigras
F 0
Dressing Figure &This to dressing
graph shows changes material and a one hour
in tritiated thymidine incorporation pulse with isotype. N = 12.
into keratinocytes
compared
with controls
following
7 day exposure
I”
I @ L
A Figure SExample (B) treated
with
of the effects of dressing silastic sheet. (Haematoxylin
B
materials on submerged gels containing fibroblasts and Eosin, original magnification x 66).
be seen that the only consistent effects with time, and on both cell types, were those of Kaltocarb and Bactigras. These two dressings both reduce cell number, as determined by direct microscopic observation and by analysis of DNA per well. No other dressing produces a consistent effect. Fibrocol produces statistically significant effects on both cell types after longer exposures but further studies would be required to determine if these are biologically significant.
and overlain
with keratinocytes.
(A) control;
in DNA content, had no significant effect on protein
content. Thymidine assays
For both fibroblasts and keratinocytes, with the exception of Bactigras and Kaltocarb, no reproducible differences were found following any dressing treatment (data not shown). The results from Bactigras and Kaltocarb are shown in Figure 4, and thymidine incorporation was severely inhibited.
Protein assays
Composite collagen gels
These closely mirrored the pattern found with DNA content with minor variations (data not shown). The only reductions in protein content compared with control cultures were found for Bactigras and Kaltocarb. Fibrocol, which produced statistically significant increases but very small percentage changes
Figure 5 shows examples of the effects of dressing materials on submerged gels containing fibroblasts and overlaid with keratinocytes. All the sheet type dressings were tested, except Kaltocarb and Bactigras, as these had already been shown to be toxic or inhibitory. No detrimental changes were observed.
234
British Journal of Plastic Surgery
Figure &An example of the effect of spray dressings on composite collagen gels, which were raised to the air-liquid interface prior to treatment. (A) control; (B) treated with Opsite. (Haematoxylin and Eosin, original magnification x 66).
Composite collagen gels : spray dressings
Figure 6 shows the results of spray dressing (Opsite and Nobecutane) on composite collagen gels, which were raised to the air-liquid interface before treatment. Neither dressing appeared to affect the morphology or viability of the gels.
Discussion We have used a range of tests of increasing sophistication to examine the effects of commercially available wound dressings. The simplest tests were those performed on fibroblast cultures. These quickly revealed a decrease in DNA content of wells exposed to two of the dressings, Kaltocarb and Bactigras. By visual inspection, DNA content, protein content and by tritiated thymidine content these two dressings were found to reduce cell growth compared with controls. These findings were repeated using keratinocytes, where similar results were found. The DNA data for keratinocytes does not show such large inhibitions. This is probably due to the differentiated nature of these cultures. Cells which may be inhibited or killed can be trapped within the multilayer keratinocyte colonies. It is also possible that the treatment may have simply promoted differentiation thus changing the ratio of dividing to differentiating cells. Since cultured keratinocytes do not fully enucleate in submerged cultures, similar DNA levels might result. However, protein and tritiated thymidine incorporation assays both demonstrated a decrease when keratinocytes were treated with Kaltocarb or Bactigras. The other dressings did not show any consistent change in DNA or protein content nor in tritiated thymidine incorporation. Fibrocol produced a small effect on DNA content, which achieved statistical significance, but this change was not reflected in any other parameter measured. We believe that this effect is not biologically significant. In order to test for synergistic effects, requiring cooperation between fibroblasts and keratinocytes, assays were also performed on composite collagen gels cultures, No gross differences in histology were found following any dressing treatment.
Some dressing materials are not easily applicable to submerged cultures. Spray dressings are an example of this type. In these cases, composite cultures raised to the air-liquid interface were used. Under these conditions, a more mature epithelium is formed. Neither of the two spray dressings was found to affect the gross histology of such gels. However, spraying too close to the gels either physically dislodged the epithelium or allowed the solvent to remain on the cells for longer periods, effectively killing the epithelium. Of all the dressings tested, only two were found to inhibit cell replication, as evidenced by DNA content, protein content and thymidine incorporation. Kaltocarb and Bactigras are manufactured for use on infected wounds and are therefore atypical of dressing materials in general. Bactigras contains chlorhexidine which may be directly toxic to the cells, and previous work supports this view’ and our finding that keratinocytes may be less sensitive than fibroblasts. Kaltocarb contains charcoal which, as we describe, clearly absorbs the phenol red from the culture medium, and probably absorbs growth factors also. The effect may be indirect by reducing the mitogens available within the medium and thus reducing the growth rate of the ceils without direct toxicity. The British standard test/IS0 TC194 has been criticised previously4 as to its accuracy. We too were concerned that neither cells from the target organ nor the more complex organotypic culture models which are now available were required. van Luyn et al used a skin fibroblast line in methylcellulose gels as a test system.4 They found that some hydrocolloid dressings were toxic. Of those we have tested and were also tested by van Luyn et al., only Opsite produced a different result. In their experiments, van Luyn et al. found that Opsite sheet was slightly toxic. However, this was probably attributable to the adhesive which has been changed by the manufacturers since van Luyn’s observations. Interestingly, this group found that Lyofoam C, a charcoal containing polyurethane foam was “mildly toxic”. We would suggest that the carbon is partially inactivating growth factors in the medium and that the dressing may not be toxic, but that the effect is spurious and a tissue culture artefact. Rosdy and Clauss compared MRC5 fibroblasts with human skin keratinocytes in monolayer culture and
Toxicity
testing of wound
dressing materials
in vitro
found that when extracts of dressing materials were compared the results were inconsistent in 25% of cases, but when direct contact was allowed correlation was much closer.5 Their method involved media with a low calcium concentration and thus incomplete keratinocyte differentiation occurred. Their tests using MRC5 cells exposed confluent cells to the test agents and used a subjective four point scale. The use of confluent cultures does not allow the study of effects on proliferation. Counts of cell number were made for the keratinocyte cultures which were exposed for twice as long as the MRC5 cells to test agents. Of the dressings common to our study and theirs, similar results were found for Kaltostat. Rosdy and Clauss also noted Opsite sheet toxicity when applied directly but not when Tresent as an extract, but again this may be attributable to the older adhesive then in use. They also found that Lyofoam was toxic as an extract not only to keratinocytes but to both MRCS fibroblasts and keratinocytes when applied in direct contact. Our results are not directly comparable since we used high calcium, stratified, differentiating cultures. However, in repeated experiments we found no evidence of toxicity or growth inhibition of keratinocytes or fibroblasts when Lyofoam was present in the culture. We performed our experiments over a longer time scale than that of Rosdy and Clauss and, as we are able to measure changes in proliferation, any effects would be magnified in our experiments. The discrepancy between these two reports is difficult to explain. The manufacturer has informed us that no changes have been made to the product during the time between these two studies. We would suggest that a tiered array of testing methods such as we have used might be a preferable method to investigate potential dressing materials. Our method has several advantages : it uses cells from the target organ, it can test for some synergistic effects, it can be used for a wide variety of presentations of dressings, from powder, to spray to sheets. The disadvantage is that it is more labour intensive and complex to perform. However, by using simpler methods as a pre-screen it may be possible to target the more complex methods more effectively. Some keratinocyte-fibroblast co-cultures are commercially available and have been used with some success as predictors of skin irritancy.6 Clinical trials will always be required to test for problems such as the keratinocytes sticking to the dressing material.’
235
In this study, we have found only two dressings with detrimental effects on cell growth but these effects can easily be explained by the special nature of these particular dressings where “cleaning up” the wound is the first priority. Interestingly we found no evidence that any dressing increased cell proliferation (with the possible exception of Fibrocol) or protein content or had any gross effects on epithelial morphology. Acknowledgement This study was funded by the generous support of the Sir Jules Thorn charitable trust.
References 1. Carver N, Leigh IM. Synthetic dressings. Int J Dermatol 1992; 31: l&18. 2. Rao J, Otto WR, Fluorimetric DNA assay for cell growth estimation. Anal Biochem 1992; 207: 18692. 3. Nanchahal J, Otto WR, Dover R, Dhital SK. Cultured composite grafts: biological skin equivalents permitting massive expansion. Lancet 1989; ii: 191-3. 4. van Luyn MJA, van Wachem PB, Nieuwenhuis P, Jonkman MF. Cytotoxicity testing of wound dressings using methylcellulose cell culture. Biomaterials 1992; 13: 267-75. 5. Rosdy M, Clauss L-C. Cytotoxicity testing of wound dressings using normal human keratinocytes in culture. J Biomed Materials Res 1990; 24: 363377. 6. Osborne R, Perkins MA. An approach for development of alternative test methods based on mechanisms of skin irritation. Food Chem Toxicol 1994; 32: 13342. 7. Zheng HJ, Audus KL. Cytotoxic effects of chlorhexidine and nystatin on cultured hamster buccal epithelial-cells. Int J Pharmaceutics 1994; 101: 121-6.
The Authors Robin Dover BSc, PLD, Department of Anatomy, Queen Mary College, University of London and Histopathology Unit, Imperial Cancer Research Fund, London. William R. Otto, BSc, MSc, PhD, Histopathology Unit, Imperial Cancer Research Fund, London. Jagdeep Nanchalal, PhD, FRCS, Department of Plastic Surgery, Mount Vernon Hospital, Northwood, Middlesex. David J.Riches, BSc, PhD, MBBS, MRCSEng, LRCP, Department of Anatomy, Queen Mary College, University of London. Correspondence to Dr R. Dover, Histopathology Unit, Imperial Cancer Research Fund, 44 Lincoln’s Inn Fields, London WC2A 3PX. Paper received 16 June 1994. Accepted 14 December 1994, after revision.
British
Toxicity testing of wound dressingmaterials in vitro R. Dover*, W. R. Otto?, J. Nanchahalt
and D. J. Riches*
*Department of Anatomy, Queen Mary College, University of London t Histopathology Unit, Imperial Cancer Research Fund, London $ Department of Plastic Surgery, Mount Vernon Hospital, Northwood, Middlesex, UK SUMMARY. There is a bewildering array of dressing materials available for wound coverage. The choice of dressing is often by local custom or practical experience. We wished to investigate if different dressings varied in their ability to either stimulate or inhibit proliferative activity and differentiation in an in vitro test system. We have used a number of test systems for this study. Human dermal fibroblast and keratinocyte cultures were used to screen for proliferative and cytotoxic effects. A more complex “organotypic” method involving fibroblastimpregnated collagen gels overlaid with epidermal keratinocytes was used to investigate effects on differentiation. Dressings were selected from each of the major types available, from simple gauze to hydrocolloids. Of the dressings tested, some reduced cell growth rates but the majority showed no major effect on proliferation or differentiation. Of those displaying an effect, only one could be attributed to cytotoxicity.
The choice of dressing is an important decision in wound management. This choice is often governed by custom, habit or financial considerations rather than any solid scientific basis. The sheer number of dressings available is huge, and these can be divided into a number of types based on their composition (see review by Carver & Leighl). The simplest are gauzes, the most complex are biological skin substitutes. The market is competitive and financially rewarding and each manufacturer claims a range of benefits from use of their product. There are reports in the literature of independent clinical trials but there has been relatively little work looking for effects on in vitro systems. The in vitro method allows simultaneous testing of large numbers of dressings, something which is much more difficult to achieve in a clinical trial. The British Standard (BS5736 part 10 “Method of test for toxicity to cells in culture of extracts from medical devices”) for dressing materials requires a rather limited in vitro testing procedure. This involves soaking the dressing in a specified culture medium for between 24 and 72 hours and then preparing a range of dilutions of this extract. Then one of three specified cell lines (or any other cell fitting certain criteria) is exposed to the test agent for 24 hours and the cells fixed. The cells are stained and then scored for survival on a 5 point scale where: 0 is no cell death; 1 is 25 % death; 2 is 25-50 % death; 3 is 5&75 % ; 4 is greater than 75 % death. This method is rather basic and, although it is designed to protect the patient by detecting cytotoxicity, is somewhat crude and is incapable of detecting growth promoting or differentiation effects. The recommended cells are, respectively, L926 a mouse connective tissue line, MRC-5 human foetal lung fibroblasts and African Green Monkey kidney cells. Alternatively, any cell for which reproducible cytotoxic titres have been demonstrated could be used. Thus it is not a requirement to test the dressing materials on the target tissue.
To address this problem and to investigate any potential growth promoting effects we have used a range of in vitro tests. These all involve human cells obtained from the target tissue in vivo. We used methods with increasing complexity to study a range of effects. These included “organotypic” cultures composed of both epithelial and mesenchymal elements, which provide us with a more recognisable tissue histology and, to some degree, allow for potential synergistic effects between these elements. We also exposed the cells to the complete dressing rather than an extract. It seemed possible that in some cases the cells and the dressings might be able to interact to produce effects that might not be detectable using extracts alone. Such cultures cannot be considered as representative of the more complex wound bed but are a step closer to it. Methods Cell culture 3T3 cells. These were cultured in Dulbecco’s Modified
Eagles Medium (GIBCO) supplemented with 10% Newborn Calf serum (GIBCO) and Kanamycin (100 pg/ml) and Fungizone (1.25 pug/ml) (Squibb). These were cultured in Dulbecco’s Modified Eagles Medium supplemented with 10 % Foetal Calf serum (GIBCO) and Kanamycin and Fungizone.
Fibroblasts.
Keratinocytes. These were cultured in fibroblast medium supplemented with the following additions : Hydrocortisone (0.4 pg/ml), Insulin (5 pg/ml) Cholera Toxin (10-l’ M) (Sigma), Epidermal Growth Factor (10 rig/ml) (Advanced Protein Products). Keratinocytes were cultured on a 3T3 feeder layer prepared by irradiating freshly trypsinized 3T3s with 230
Toxicity
testing of wound dressing materials
Table 1 Product
name
Composition
Manufacturer
2nd Skin Bactigras
Polyethylene oxide gel Tulle gras/chlorhexidine
Fibrocol Geliperm dry Jelonet
Collagen/alginate Hydrocolloid Tulle gras/petroleum jelly Alginate/charcoal Alginate Polyurethane foam Polyurethane film
Spenco Smith & Nephew Medical Johnson & Johnson Geistlich Johnson & Johnson
Kaltocarb Kaltostat Lyofoam Nobecutane spray Opsite sheet
Polyurethane film
231
in vitro
Britcair BritcairSeton Healthcare Astra
Opsite spray
Polyurethane film
Silastic gel sheet Silicone n/a Spyrofoam Tegaderm Tegapore
Silicone gel
Smith & Nephew Medical Smith & Nephew Medical Dow Coming
Silicone coated viscose Polyurethane membrane Polyurethane film Woven nylon fabric
Johnson & Johnson Britcair 3M 3M
60 Gy X-rays. 3T3 cells were then plated at a density of 1.2 x lo4 cells/cm’. Keratinocytes were then plated with or on to the 3T3s at a range of densities. Normal human epidermal keratinocytes and fibroblasts were established from a punch biopsy from the inner aspect of the forearm of a volunteer. Keratinocytes were isolated by overnight incubation of the biopsy in 0.25 % trypsin at 4°C followed by peeling the epidermis which was then shaken in culture medium to release keratinocytes. The remaining dermis was cut into small explants and cells allowed to grow out for two weeks prior to harvesting. Both keratinocytes and fibroblasts were used for experiments between passage 2 and 10. For some tests (see below) pure keratinocytes were prepared by washing cultures vigorously with EDTA solution (2 % Ethylene Diamino Tetra Acetic Acid in PBS) to remove the feeder layer prior to trypsinisation. The released keratinocytes were then washed by centrifugation and plated without feeder cells at a density of lo5 cells per well in 24-well plates (Falcon). Dressings
Table 1 lists the dressings used in the study. These include examples of the major types of wound contact layers. In all cases, with the exception of the two spray dressings, the dressings were provided in sterile packaging. The dressings were cut under aseptic conditions into 5 x 5 mm squares for use. Instruments were changed and cleaned between each dressing to prevent cross-contamination by adhesives, etc. The most adhesive dressings (Opsite sheet and Tegaderm) posed some problems in handling. Small pieces readily self adhered and were discarded. For all the other sheet type dressings, 5 x 5 mm squares could be easily handled and applied to cultures or organotypic gels. The Silastic gel sheet was used in two different states: as provided without washing to remove surface material and following washing in soap flakes as recom-
mended by the manufacturer followed by extensive washing in phosphate buffered saline (PBS). Both spray dressings were applied to gels (see later) by placing the filter bearing the gel on a large sterile plastic dish and applying the spray in short (less than 1 s) bursts from a height of approximately 60 cm. This was the maximum height comfortably achievable within a tissue culture hood. The spray was left to dry for at least 30 s between applications in order to allow the solvent to evaporate. Used from this distance, the cover is not complete but this prevents physical damage from the force of the propellant gas and potential chemical damage from the solvent. Complete cover was achieved by between 5 and 8 applications. Growth assays
A number of different assay systems were used to investigate potential effects of the dressings. The first method was applied to cultures of either pure keratinocytes or fibroblasts growing in 24-well plates. Each dressing was added to quadruplicate wells for 3 days. Control wells received no other treatment. The dressings were carefully removed and the wells washed twice with PBS and frozen at - 20°C and stored prior to DNA assay. The DNA assay method of Rao and Otto’ was employed. This is a very sensitive fluorimetric assay based on Hoechst dye binding. This produces a measurement of the DNA content of each well, which correlates with cell number. All wells were scanned under phase microscopy prior to assay. All dressing materials were tested in at least three independent experiments. Protein content assays were also performed in parallel with the DNA assays. All cultures were pulsed for one hour with 1 @Zi/ml tritiated thymidine (3H-TdR) (Amersham International UK, 5 Ci/mmol) prior to termination. Aliquots of solubilised cells were then scintillation counted. The second and third assay methods involved organotypic gels. Briefly, collagen gels were prepared as described previously.3 Gels comprised 1 ml of collagen containing lo5 fibroblasts cast into a 24-well plate. Keratinocytes were added to the surface either 4-6 h later or 24 h later. Once keratinocytes were added to the gels, they were fed with keratinocyte culture medium as described above. Gels were maintained for 7-10 days prior to use. For some assays, gels were used in their submerged state and the dressing samples were applied to the overlying culture medium for 3 days prior to fixation. In other assays gels were raised to the air liquid interface. Briefly, this involved placing the gels on a sterile filter on a metal grid which raises the gel 24 mm off the base of the culture vessel. The level of the medium is then controlled so that the upper surface of the gel is above the liquid level and the gel is fed from below. Submerged gels were tested with spray dressings but this is technically difficult and results in the gel being effectively cut off from the outside atmosphere, and diffusion through the dressing may be limiting. Thus we used raised gels for testing both spray dressings and some sheet dressings. In this case,
232
British Journal of Plastic Surgery
cultured gels were left for 7 days post-raising before being assayed. Dressings were left in contact for 7 days before formalin fixation.
Results Microscopy
Visual inspection of the cells following treatment clearly showed differences in cell number for Bactigras and Kaltocarb exposed cultures, and the difference was clearest in the fibroblast cultures since these form monolayers. Kaltocarb treated wells were obvious as the medium became clear and lost its red colour,
Figure SComparison of DNA content following treatment of keratinocytes for 7 days, expressed as percentage of control. The bars show the mean and standard deviation for each group. The horizontal band shows the control’s standard deviation as a guide for comparison. N = 12. Stars indicate significance at p < 0.001.
Table 2 Product
Figure l--Comparison of DNA content following treatment of fibroblasts for 3 days, expressed as percentage of control. The bars show the mean and standard deviation for each group. The horizontal band shows the control’s standard deviation as a guide for comparison. N = 12. Stars indicate significance at p < 0.001.
Kaltostat Lyofoam Opsite Silastic Silicone n/a Spyrofoam Tegaderm Tegapore
Fibroblasts hY3
+ve NS NS NS +ve +ve +ve
Fibroblast hY7
Keratinocytes day 7
+ve -ve NS NS NS -ve +ve
NS +ve NS +ve NS -ve -ve
NS : not significant at p>O.OOl. +ve : significant at p
increase
in cell
decrease
in cell
presumably due to binding of the phenol red by charcoal incorporated in the dressing. DNA assays
Figure 24omparison of DNA content following treatment of fibroblasts for 7 days, expressed as percentage of control. The bars show the mean and standard deviation for each group. The horizontal band shows the control’s standard deviation as a guide for comparison. N = 12. Stars indicate significance at p < 0.001.
Data for all experiments was pooled and expressed as a percentage of the control value. Figure 1 shows the results for fibroblasts co-cultured with the dressings for 3 days, Figure 2 shows the results after 7 days. Figure 3 shows the effects on cultured keratinocytes treated for 7 days. t-Tests were performed comparing each dressing with its control, and those results significant at the p < 0.001 level are indicated on the figures. Table 2 summarises the results. From Table 2 it can
Toxicity testing of wound dressing materials in vitro
233
H Kaltocarb El Bactigras
F 0
Dressing Figure &This to dressing
graph shows changes material and a one hour
in tritiated thymidine incorporation pulse with isotype. N = 12.
into keratinocytes
compared
with controls
following
7 day exposure
I”
I @ L
A Figure SExample (B) treated
with
of the effects of dressing silastic sheet. (Haematoxylin
B
materials on submerged gels containing fibroblasts and Eosin, original magnification x 66).
be seen that the only consistent effects with time, and on both cell types, were those of Kaltocarb and Bactigras. These two dressings both reduce cell number, as determined by direct microscopic observation and by analysis of DNA per well. No other dressing produces a consistent effect. Fibrocol produces statistically significant effects on both cell types after longer exposures but further studies would be required to determine if these are biologically significant.
and overlain
with keratinocytes.
(A) control;
in DNA content, had no significant effect on protein
content. Thymidine assays
For both fibroblasts and keratinocytes, with the exception of Bactigras and Kaltocarb, no reproducible differences were found following any dressing treatment (data not shown). The results from Bactigras and Kaltocarb are shown in Figure 4, and thymidine incorporation was severely inhibited.
Protein assays
Composite collagen gels
These closely mirrored the pattern found with DNA content with minor variations (data not shown). The only reductions in protein content compared with control cultures were found for Bactigras and Kaltocarb. Fibrocol, which produced statistically significant increases but very small percentage changes
Figure 5 shows examples of the effects of dressing materials on submerged gels containing fibroblasts and overlaid with keratinocytes. All the sheet type dressings were tested, except Kaltocarb and Bactigras, as these had already been shown to be toxic or inhibitory. No detrimental changes were observed.
234
British Journal of Plastic Surgery
Figure &An example of the effect of spray dressings on composite collagen gels, which were raised to the air-liquid interface prior to treatment. (A) control; (B) treated with Opsite. (Haematoxylin and Eosin, original magnification x 66).
Composite collagen gels : spray dressings
Figure 6 shows the results of spray dressing (Opsite and Nobecutane) on composite collagen gels, which were raised to the air-liquid interface before treatment. Neither dressing appeared to affect the morphology or viability of the gels.
Discussion We have used a range of tests of increasing sophistication to examine the effects of commercially available wound dressings. The simplest tests were those performed on fibroblast cultures. These quickly revealed a decrease in DNA content of wells exposed to two of the dressings, Kaltocarb and Bactigras. By visual inspection, DNA content, protein content and by tritiated thymidine content these two dressings were found to reduce cell growth compared with controls. These findings were repeated using keratinocytes, where similar results were found. The DNA data for keratinocytes does not show such large inhibitions. This is probably due to the differentiated nature of these cultures. Cells which may be inhibited or killed can be trapped within the multilayer keratinocyte colonies. It is also possible that the treatment may have simply promoted differentiation thus changing the ratio of dividing to differentiating cells. Since cultured keratinocytes do not fully enucleate in submerged cultures, similar DNA levels might result. However, protein and tritiated thymidine incorporation assays both demonstrated a decrease when keratinocytes were treated with Kaltocarb or Bactigras. The other dressings did not show any consistent change in DNA or protein content nor in tritiated thymidine incorporation. Fibrocol produced a small effect on DNA content, which achieved statistical significance, but this change was not reflected in any other parameter measured. We believe that this effect is not biologically significant. In order to test for synergistic effects, requiring cooperation between fibroblasts and keratinocytes, assays were also performed on composite collagen gels cultures, No gross differences in histology were found following any dressing treatment.
Some dressing materials are not easily applicable to submerged cultures. Spray dressings are an example of this type. In these cases, composite cultures raised to the air-liquid interface were used. Under these conditions, a more mature epithelium is formed. Neither of the two spray dressings was found to affect the gross histology of such gels. However, spraying too close to the gels either physically dislodged the epithelium or allowed the solvent to remain on the cells for longer periods, effectively killing the epithelium. Of all the dressings tested, only two were found to inhibit cell replication, as evidenced by DNA content, protein content and thymidine incorporation. Kaltocarb and Bactigras are manufactured for use on infected wounds and are therefore atypical of dressing materials in general. Bactigras contains chlorhexidine which may be directly toxic to the cells, and previous work supports this view’ and our finding that keratinocytes may be less sensitive than fibroblasts. Kaltocarb contains charcoal which, as we describe, clearly absorbs the phenol red from the culture medium, and probably absorbs growth factors also. The effect may be indirect by reducing the mitogens available within the medium and thus reducing the growth rate of the ceils without direct toxicity. The British standard test/IS0 TC194 has been criticised previously4 as to its accuracy. We too were concerned that neither cells from the target organ nor the more complex organotypic culture models which are now available were required. van Luyn et al used a skin fibroblast line in methylcellulose gels as a test system.4 They found that some hydrocolloid dressings were toxic. Of those we have tested and were also tested by van Luyn et al., only Opsite produced a different result. In their experiments, van Luyn et al. found that Opsite sheet was slightly toxic. However, this was probably attributable to the adhesive which has been changed by the manufacturers since van Luyn’s observations. Interestingly, this group found that Lyofoam C, a charcoal containing polyurethane foam was “mildly toxic”. We would suggest that the carbon is partially inactivating growth factors in the medium and that the dressing may not be toxic, but that the effect is spurious and a tissue culture artefact. Rosdy and Clauss compared MRC5 fibroblasts with human skin keratinocytes in monolayer culture and
Toxicity
testing of wound
dressing materials
in vitro
found that when extracts of dressing materials were compared the results were inconsistent in 25% of cases, but when direct contact was allowed correlation was much closer.5 Their method involved media with a low calcium concentration and thus incomplete keratinocyte differentiation occurred. Their tests using MRC5 cells exposed confluent cells to the test agents and used a subjective four point scale. The use of confluent cultures does not allow the study of effects on proliferation. Counts of cell number were made for the keratinocyte cultures which were exposed for twice as long as the MRC5 cells to test agents. Of the dressings common to our study and theirs, similar results were found for Kaltostat. Rosdy and Clauss also noted Opsite sheet toxicity when applied directly but not when Tresent as an extract, but again this may be attributable to the older adhesive then in use. They also found that Lyofoam was toxic as an extract not only to keratinocytes but to both MRCS fibroblasts and keratinocytes when applied in direct contact. Our results are not directly comparable since we used high calcium, stratified, differentiating cultures. However, in repeated experiments we found no evidence of toxicity or growth inhibition of keratinocytes or fibroblasts when Lyofoam was present in the culture. We performed our experiments over a longer time scale than that of Rosdy and Clauss and, as we are able to measure changes in proliferation, any effects would be magnified in our experiments. The discrepancy between these two reports is difficult to explain. The manufacturer has informed us that no changes have been made to the product during the time between these two studies. We would suggest that a tiered array of testing methods such as we have used might be a preferable method to investigate potential dressing materials. Our method has several advantages : it uses cells from the target organ, it can test for some synergistic effects, it can be used for a wide variety of presentations of dressings, from powder, to spray to sheets. The disadvantage is that it is more labour intensive and complex to perform. However, by using simpler methods as a pre-screen it may be possible to target the more complex methods more effectively. Some keratinocyte-fibroblast co-cultures are commercially available and have been used with some success as predictors of skin irritancy.6 Clinical trials will always be required to test for problems such as the keratinocytes sticking to the dressing material.’
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In this study, we have found only two dressings with detrimental effects on cell growth but these effects can easily be explained by the special nature of these particular dressings where “cleaning up” the wound is the first priority. Interestingly we found no evidence that any dressing increased cell proliferation (with the possible exception of Fibrocol) or protein content or had any gross effects on epithelial morphology. Acknowledgement This study was funded by the generous support of the Sir Jules Thorn charitable trust.
References 1. Carver N, Leigh IM. Synthetic dressings. Int J Dermatol 1992; 31: l&18. 2. Rao J, Otto WR, Fluorimetric DNA assay for cell growth estimation. Anal Biochem 1992; 207: 18692. 3. Nanchahal J, Otto WR, Dover R, Dhital SK. Cultured composite grafts: biological skin equivalents permitting massive expansion. Lancet 1989; ii: 191-3. 4. van Luyn MJA, van Wachem PB, Nieuwenhuis P, Jonkman MF. Cytotoxicity testing of wound dressings using methylcellulose cell culture. Biomaterials 1992; 13: 267-75. 5. Rosdy M, Clauss L-C. Cytotoxicity testing of wound dressings using normal human keratinocytes in culture. J Biomed Materials Res 1990; 24: 363377. 6. Osborne R, Perkins MA. An approach for development of alternative test methods based on mechanisms of skin irritation. Food Chem Toxicol 1994; 32: 13342. 7. Zheng HJ, Audus KL. Cytotoxic effects of chlorhexidine and nystatin on cultured hamster buccal epithelial-cells. Int J Pharmaceutics 1994; 101: 121-6.
The Authors Robin Dover BSc, PLD, Department of Anatomy, Queen Mary College, University of London and Histopathology Unit, Imperial Cancer Research Fund, London. William R. Otto, BSc, MSc, PhD, Histopathology Unit, Imperial Cancer Research Fund, London. Jagdeep Nanchalal, PhD, FRCS, Department of Plastic Surgery, Mount Vernon Hospital, Northwood, Middlesex. David J.Riches, BSc, PhD, MBBS, MRCSEng, LRCP, Department of Anatomy, Queen Mary College, University of London. Correspondence to Dr R. Dover, Histopathology Unit, Imperial Cancer Research Fund, 44 Lincoln’s Inn Fields, London WC2A 3PX. Paper received 16 June 1994. Accepted 14 December 1994, after revision.