479

Printed in Great Britain

Bums (1992) 18, (6), 479-485

Cytotoxicity evaluation of antiseptics and antibiotics on cultured human fibroblasts and keratinocytes 0. Damour, Laboratoire

S. Zhi Hua, F. Lasne, M. Villain, I’. Rousselle

des Substituts

and C. Collombel

Cutanes, Pavillon 15, HBpital Edouard Herriot, Lyon, France

Infection is thegreatest problem in bum patients ana’ fopical antimicrobial agents must be chosen with great care, especially when cultured skin is grafted. We examined the cytotoxic effect of six antiseptics and six antibiotics commonly used on cultured human @rob&s and kerafinocytes. Cultured cells were exposed for 15 min to Hibitant? (chlorhexidine), Bisepfi&@ (chlorhexidine + benzalkonium chloride + benzylic alcohol), Benzalkonium Chloride, Yellow Betadine (polyvidone-iodine + nonoxinol), Betadine Scrub (polyvidone-iodine + quaferna y ammonium) and Green Betadine (polyvidone-iodine) and uiabilify ~a.5 determined using ihe MTT test. At therapeutic concentrations all the antiseptics are cytotoxic for fibroblasts and keratinocytes. Additionally the cells were exposed for 48 h to uancomycin, colistin, amikacin, imipeneme, pefloxmin, piperacillin and cell viability was determined using the MTT test. The concentration.5of antibiotics corresponding to the plasma peak obtained after therapeutic appliction were not cytotoxic to the tested cells. The CD50 was much higher than the MlC from 725 to 875 times for keratinocytes and from 1400 to 5900 times for fibroblasts). These data suggest that commonly applied antiseptics must not be used before grafting cultured skin grafts. After grafting any infection can be controlled with topical applications of appropriafe antibiotics.

Introduction In extensive bums it is necessary to cover the wound as soon as possible. Infection is one of the greatest problems because it often leads to death and always impedes the take of grafts. Usually skin replacement in definitive reconstruction requires skin autografts as split-thickness meshed or unmeshed autografts. In bums over SO per cent of the body surface, adequate donor areas are rare. For this reason surgeons and researchers are trying to make skin substitutes for definitive wound cover. Different alternatives have been proposed using keratinocytes as cultured sheets (Rheinwald and Green, 1975; Gallico et al., 1984), gels contracted by fibroblasts with keratinocytes cultured on their surface (Bell et al., 1979, 1983), collagen GAG matrix crosslinked by glutaraldehyde (Yannas and Burke, 1980; Burke et al., 1981) or by chitosane (Collombel et al., 1987), collagen GAG-matrix with autologous fibroblasts cultured in vitro within it, and autologous keratinocytes on its surface (Boyce and Hansbrough, 1988; Shahabeddin et al., 1990). Several antimicrobial products have been tested in vivo with evaluation of the rate of epithelialization of the wound using animals (Geronemus et al., 1979) or human wound 0 1992 Butterworth-Heinemann 0305-4179/92/060479-07

Ltd

healing (Lineaweaver et al., 1985; Leyden and Bartelt, 1987). In vitro tests have also been done after applying the agent directly to cultured fibroblasts (Leaper and Brennan, 1986) or keratinocytes (Cooper et al., 1990) or both of those together for quantitative assessment of their cytotoxicity (Tatnall et al., 1990). If the cytotoxic effect of antiseptics is now proven (Lineaweaver et al., 1985; Leyden and Bartelt, 1987; Tatnall et al., 1990), topical antibiotics have shown no adverse effect on wound healing (Geronemus et al., 1979; Leyden and Bartelt, 1987) or on keratinocytes (Cooper et al., 1990). Only a few antibiotics have been tested on human fibroblasts and keratinocytes (Cooper et al., 1990). Considering our usual use of cultured epidermis, it appeared essential for us to test the antimicrobial agents available in our department on keratinocyte cultures. Moreover, penicillin and streptomycin, which are the usual antibiotics added to the medium for cultured cells, are inefficient on skin biopsies contaminated by resistant organisms. The use of appropriate antibiotics at efficient concentrations is of a great interest. This is also true for cryopreserved allogenic skin used for temporary cover and conservation media for living organs. The3-(4,S-dimethylthiazol-2-y1)-2,S-diphenyltetrazolium (MTT) test has been used because it is less time consuming than the cell counting method. It allows the processing of large number of samples (Mossman, 1983; Hansen et al., 1989) and is well correlated to the counting method.

Materials

and methods

Cell culture conditions For these studies we used human fibroblasts and keratinocytes. The cells were isolated from different donors to determine the range of concentrations covering CDSO, the obtained results did not show significant variability. The definitive experimentation reported here used cells from the same donor for all the antimicrobial agents. Human foreskin fibroblasts (HFF) derived from explants were subcultured in fibroblast tissue culture medium (FTCM): Dulbecco’s modified Eagle’s medium (DMEM) (ICN Laboratories, Irvine, UK) supplemented with 10 per cent fetal calf serum (FCS) (PAA, Linz, Austria), 100 U/ml penicillin, 100 pg/ml streptomycin and 0.5 pg/ml amphotericin B (Gibco Laboratories, Grand Island, NY,

Burns (1992)Vol. 18/No.6

480

USA). The HFF were generally and fifth passage.

used between

the second

Normal human keratinocytes (NHK) were isolated from adult skin specimens (plastic surgery) by standard trypsinization overnight at + 4°C in 0.25 per cent trypsin. Cells were cultured in serum-free MCDB 153 medium (Molecular, Cellular and Developmental Biology) containing 0.15 mM Ca2+ (KBM Clonetics Corp., CA., USA) supplemented with 5 pg/ml insulin (Sigma, St Louis, MO, USA), long/ml epidermal growth factor (a generous gift of Dr C. G. Nascimento, Chiron Corp., CA., USA), 0.5 pg/ml hydrocortisone (Sigma), 0.5per cent (v/v) bovine pituitary extract prepared in our laboratory according to Boyce and Ham (1985) and penicillin-streptomycin-Amphotericin B as described above for HFF. Cultures were incubated at 37”C, in an atmosphere of 5 per cent CO,/95 per cent air. The medium was changed 3 times per week. Subconfluent primary cultures were harvested and subcultured in the same medium. NHK were generally used after the second passage for the MTT test. For cytotoxicity tests, 1 x 10’ cells (keratinocytes and fibroblasts) per well corresponding to 5 x lo4 cells/cm’ were seeded into 24 well plates and cultured for 24 h at 3 7°C in air containing 5 per cent CO, in order to allow the cells to reach subconfluence before exposure to the antimicrobial agents. Cellular density was determined to be in the linear part of the dose-response curve and to have a good sensitivity (Sagnes, 1991). Both keratinocytes and fibroblasts were tested against each antimicrobial agent. Exposure to antiseptic agents Six antiseptic agents solutions were tested: Hibitane hexidine), Biseptine (chlorhexidine + benzalkonium

(chlorchlo-

ride + benzylic alcohol), Benzalkonium Chloride, Yellow Betadine (polyvidone-iodine + nonoxinol), Betadine Scrub (polyvidone-iodine + quaternary ammonium) and Green Betadine (polyvidone-iodine). The composition of the different agents and the clinical and experimental concentrations are reported in TableI with minimal bactericidal concentration (MBC) (Girardo et al., 1989). The agents were all freshly prepared prior to use and serially diluted in phosphate buffered saline (PBS). Antiseptic ointments were discarded because of incompatibility with the following procedures. The cells were washed twice and exposed to the dilutions of the antiseptic agents for 15 min, which corresponds to the clinical exposure time to the antiseptic. Eight cultures were set up for each concentration. Controls were treated with PBS only. Exposure to antibiotic agents Six antibiotic agents were chosen depending on the organisms frequently found on extensive bum patients, vancomytin, colistin, amikacin, pefloxacin, imipenem and piperacillin. The experimental concentrations and minimal inhibitive concentrations (MIC) of the antibiotic agents are reported in Table II (Acar et al., 1990). As a first step the antibiotics were tested at serial dilutions ranging from MIC to therapeutic concentrations. As a second step, the antibiotics were tested at higher concentrations, as reported in TableII, to obtain cytotoxicity effects. The exposure time was 48 h since a clinical antibiotic treatment lasts at least as long as this. The cells were washed twice and 2 ml of medium (FTCM or keratinocytes tissue culture medium (KTCM)) containing the antibiotics at different concentrations were added to each well. The cells were incubated at 37°C in air containing 5 per

Table I. Antiseptics tested with the experimental concentrations, clinical concentrations (CC) and minimal bactericidal concentrations WBC) Clinical concentration:

Test dose Composition

of the agents

Hibitane”’ 5% Chlorhexidine: digluconate vehicle: octylcresylpolyoxyethylenic acetate, degluconolactone, water

&g/m0

2500-l

00-50-25-l

2.5-8.3

200-50-25-l

Benzalkonium chloride 0.025 g%

20-5-2.5-l

CC: 2500 MBC: 1 O-25

6.5-12.5

CC: 250 MBC: 1 CC: 1-25

.65-l .25

Benzylic alcohol 4 ml vehicle : water 250-25-l

chloride

Yellow Betadine? (Sarget) Polyvidone iodine 10 g% Nonoxinol 9,0.25 g% vehicle : water, pH 5 Betadine Scrub”: (Sarget) Polyvidone iodine 4 g% Ammonium salt of sulphuric alkylphenoxypolyethylene 1 g%, lauric acid and diethanolamine vehicle : water, pH 5 Green Betadine? (Sarget) Polyvidone iodine 10% vehicle : water, pH 5

cc: 100-5000 MBC: 8-62.5

alcohol (3.14% p/v) linalyle

Biseptine? (Nicholas) Chlorhexidine gluconate, 0.25 9%

Benzalkonium vehicle : water

CC, MBC

(pg/ml)

2000-l

ethan

ester

cc: 200-I 0 000 MBC: 15-l 25

2.5-6.25-3.1

000-500-250-l

25

200-80-40-25-I 6.6 50-20-l O-6.25-4.1 5

CC: 100 000 pg/ml on healthy skin CC: 30 000 pg/ml on wound bed MBC: 125-l 000 CC: 1333 pg/ml on wound bed MBC : 125-l 000

2000-I

000-500-250-100-50

MBC: 125-I 000

Damour et al.: Cytotoxic

effect of antiseptics and antibiotics on cultured skin cell

cent CO, for 48 h. Eight cultures were set up for each concentration. Controls were treated with medium only. Cytotoxicity

assay using the MTT test

The cell viability remaining in each well was evaluated using a calorimetric assay based on the tetrazolium salt 3(4,5dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) according to Mossman (1983) modified by Hansen et al. (1989) This compound is a yellow tetrazolium salt which is reduced by mitochondrial enzymes in viable cells to an insoluble blue formazan product. After contact with antiseptic agents or antibiotic solutions, these antimicrobial agents were removed and the cells were washed twice in PBS. Two millilitres of DMEM without phenol-red containing MTT (I mg/ml) were added to each well. After 2 h incubation at 37°C in 5 per cent CO,, the unreacted dye was removed and 2ml of dimethyl sulphoxide (DMSO) were added to each well to solubilize the intracellular blue formazan product. The optical density of each well was read using a spectrophotometer at 550 nm. The percentage of viability compared to control for each concentration was used to establish dose-response curves. CD50 value was defined as the concentration allowing SO per cent survival of cells and was determined after log dose-response transformation and linear regression.

Results The dose-response

curves obtained

using HFF and NHK

481

cultures exposed to chlorhexidine gluconate, benzalkonium chloride, and polyvidone chloride are respectively shown in Figwe I. Each point corresponds to the means of eight replicate tests with a coefficient of variation of < 10 per cent. The CD50 values are shown in Table111 with their 95 per cent confidence limits. These results show that all agents are cytotoxic on both HFF and NHK. However there is a significant difference between the susceptibility of fibroblasts and keratinocytes versus these antiseptics (PC 0.005) determined by pairwise comparison using Student’s t test. A comparison between the CD50 values related to MBC allows the classification of the agents according to the degree of the cytotoxic effect: benzalkonium chloride > Betadine Scrub’\ > Green and Yellow Betadine > Hibitane > Biseptine\“, as shown in Table 111. On the other hand no cytotoxicity effects were found from all the tested antibiotics on these two types of skin cells when they were exposed to concentrations ranging from MIC to plasma therapeutic concentrations. At higher concentrations, it was possible to determine CDSO’S. The dose-response curves obtained using HFF and NHK exposed to each antibiotic are shown in Figti~e2. Each point corresponds to the mean of eight replicate tests with a coefficient of variation < 10 per cent. The CD50 values are reported in TableIV with their 95 per cent confidence limits. Each CD50 is much higher than the MIC (from 125 to 875 times) for keratinocytes and (from 1400 to 5900 times) 120

?

100

20

0

200

100

a

300 0

Dose (vg/ml)

C

b

o

IO

'0

1000

2000 Dose

3000

(uglml)

Figure 1. Dose-response curves of antimicrobial agents. a. Dose-response curves of chlorhexidine contained in Hibitane and in Biseptine” on HFF and on NHK. Points represent the mean of eight replicate tests with coefficient of variation < 10 per cent. L, Hibitane/F; +, Biseptine”‘/F; X, Hibitane’“‘/K; 0, Biseptine@/ K. b, Dose-response curves of benzalkonium chloride in pure aqueous solutions and in Biseptine ’ on HFF and on NHK. Points represent the mean of eight replicate tests with a coefficient of variation < 10 per cent. 3, Cl-benzalkonium/F; +, Cl-biseptine/ F; X, Cl-benzalkonium/k; 0, Cl-Biseptine/K. c, Dose-response curve of polyvidone-iodine in three agents: Betadine Scrub, Yellow Green Betadine

60

40

0

I

I

I

I

I

I

1

2

3

4

5

6

a

Antibiotic

concentration

0

(mglml)

I

I

I

I

10

20

30

40

Dose (mgiml)

Figure 2. Dose-response curves of each antibiotic. Points represent the mean of eight replicate tests with coefficient of variation < 10 per cent. a, On HFF. 0, Vancomycin/K; l , Colimycine/K; A, Amikin/K; 0, Tienam/K; x , Peflacine/K. b, On NHK. 0, Vancomycin/F; +

Colimycine/F; A Am&n/F; 0, Tienam/F; x ,-Peflacine/F. caused by proteins (Afnor, 1981, 1982-83). It is not possible to compare the different antimicrobial agents using CD50 alone because the minimal bactericidal concentration of antiseptics or minimal inhibitive concentration of antibiotics are different for each of them. So the ratio CD50/MBC and CDSo/MIC permits their classification; the higher this ratio the higher the concentrations can be raised beyond MBC or MIC for clinical applications without a risk of cytotoxicity. We obtained the following ranking order of cytotoxicity: benzalkonium chloride > Betadine Scrub% > Green and Yellow Betadine@ > Hibitanea > Biseptine@ as shown in Table III. Biseptine is less cytotoxic than Hibitane only by comparing values of CD50/MBC. In addition, other studies have shown that chlorhexidine gluconate is less cytotoxic than sodium hypochlorite and hydrogen peroxide on fibroblasts and keratinocytes (Lineaweaver et al., 1985). By comparing the CD50 of active molecules, we can Table IV. Comparison

conclude that the cytotoxicities of either chlorhexidine alone or Biseptine that also contains chlorhexidine are not significantly different. Nevertheless, Biseptine is the less cytotoxic antiseptic for the same efficiency, if we compare CD5O/MBC. In fact, Biseptine contains three compounds which enhance the bacteriological effect. Its broad spectrum of activity is Gram positive, Gram negative, mycobacterium and Candida albicans. Comparing the dose-response curve of polyvidone iodine (Figure IC), we notice that Green and Yellow Betadine are less cytotoxic than Betadine Scrub. We also notice that addition of an ‘ammonium IV’ strongly increases the CD50 of the polyvidone iodine and brings its ratio CD5O/MBC value closer to that of benzalkonium chloride. Nevertheless, addition of nonoxinol had no effect at all. We observed that human fibroblasts are more susceptible than human keratinocytes to all antimicrobial agents. The differences in cell membrane composition might be the reason, considering

of CD50 means and CDSOIMIC for six antibiotics tested on basal keratinocytes Keratinocytes

Fibroblasts

CD50 Agent Vancocine” Vancomycin

1g

Colimycine” Colistin methane

-33.33

pg/eq.

1000000

UI

and fibroblasts

CD50

(mg/W

CDSO/MIC

(m&-W

CDSO/MIC

3.5 (3.1-4)

875

(23.6) (23-24.5)

5900

160

5.9 (5.2-6.5)

5900

1.62 (1.55-I .80)

202.5

8.2 (7.6-8.8)

1025

2.75 (2.45-3.10)

687.5

5.6 (5.1-6.1)

1400

125

(2.433.5)

3000

0.160 (0.148-0.175)

base

Amiklin” Amikacin Tienam”

500 mg

lmipbnem

500 mg

Cilastatin 500 mg Peflacine” Pefloxacin mesylate 400 mg Piperilline” Piperacillin Means are reported

0.125 (0.11 o-o.1

40) 2875

(242-832) with 95% confidence

limits in parenthesis:

n= 8.

40

5000

484

that, in vivo, keratinocytes have a function of protection. However, this argument will remain an hypothesis as long as the culture media used in these studies are different (the fibroblast medium contains 10 per cent FCS). Moreover polyvidone iodine should be used cautiously in severely burned patients and should be monitored with care because of substantial elevations of serum free iodine due to absorption of iodine (Lavelle et al., 1975). Concerning the antibiotics, we observed that concentrations located between MBC and plasma peak obtained after therapeutic injection did not cause any cytotoxicity on tested skin cells. Much higher concentrations were then used to determine the CD50 of each antibiotic. Interpretations of in vitro effects are difficult to compare with in vivo effects. However, we can expect that the in vitro cytotoxic effect will be higher on monolayer cultured cells due to lack of the function of protection normally expressed in vivo. Moreover, the susceptibility of molecules is total in vitro, whereas it is only partial in vivo because of pharmacokinetic mechanisms. The binding of antibiotics to proteins alters their antibacterial effect which depends on the free drug (Merrikin et al., 1983; Dudley et al., 1990). Only a single cell density has been used for these studies (1 x 10’ cells/well corresponding to 5 x lo4 cells/cm’). The chosen test is a test of cytotoxicity (Sagnes, 1991). The incubation time of the antibiotic has been increased from 2 to 48 h because of their weak cytotoxicity, and also because of their long-term use in bum treatment compared to the rapid use of antiseptics. The effect observed with high levels of antibiotics on skin cells seems more likely to be due to cytotoxic effects than to growth inhibition since the tests were performed using subconfluent cells which are not in an exponential stage of growth. Other published reports have tested different antibiotics, and no cytotoxicity was found. Our results are in agreement with theirs. Lineaweaver et al. (1985) found no cytotoxicity of bacitracin, kanamycin and neomycin on fibroblasts. Cooper et al. (1989) reported that polysporin, neosporin (neomycin 40 pg/ml, polymyxin B sulphate) at concentrations < 200 U/ml and gentamicin sulphate at a concentration of CO.1 pg/ml have no effect on the growth of cultured keratinocytes. Antibiotics could be used at defined concentrations in treated cultured grafts especially susceptible to infection. The use of antibiotics as external topical antimicrobial agents is much debated because of development of bacterial resistance. However the topical concentration can be much higher than the systemic one. The toxicological problems, like nephrotoxicity or ototoxicity for instance, are avoided especially for non-diffusible antibiotics like colimycin. In this study, we have tested the cytotoxicity of the most commonly used antibiotics in our bum centre. The choice of the appropriate antibiotic is dependent on each patient and the infecting organisms. Considerations of broad spectrum of activity, diffusibility and low selection of resistant organisms, will ensure agreement with clinicians and bacteriologists on how to keep cultured skin substitutes free from infection. The possible systemic absorption of the antibiotics will be assessed by blood determinations in future studies. In conclusion, antiseptic agents with a broad spectrum of activity can be used during. a short period of time followed by extensive washing just ,before grafting, and repeated at each dressing, if necessary. After grafting any infection can be controlled with topical application of an appropriate

Burns (1992) Vol. IS/No.

6

antibiotic. The antibiotics should be chosen with considerable care in agreement with bacteriologists and clinicians to avoid development of resistance.

Acknowledgements We thank M. E. Reverdy and B. Linat for their bacteriological assistance. We thank also I’. Y. Gueugniaud and F. Durand who helped us to choose the antiseptics and antibiotics, and M. C. Guimaraes for typing the manuscript. This work was supported partly by DRET no. 90226, INSERM no. 8869001, CNRS and Hospices Civils de Lyon.

References Acar J., Bergogne-Berezin E., Chabert Y. et al. (1990) Communique 1990 du ComitC de I’Antibiogramme de la Societe Francaise de Microbiologic. Path. Biol. 38, 749. Association Francaise de Normalisation (AFNOR) (1981) In: Antiseptiques et desinfectants, pp. 13-27, Paris, l’ere ed., AFNOR. Association Francaise de Normalisation (AFNOR) (1982-83) T72-101 Secteur Medical, T72-103 Secteur Agroalimentaire, Paris, AFNOR. Bell F., Ivarssen B. and Merrill C. (1979) Production of a tissue-like structure by contraction of collagen lattices by human fibroblasts of different proliferation potential in vitro. Prec. Nnfl. Acad. Sci. USA 76, 1274. Bell F., Sher S., Hull B. et al. (1983) The reconstitution of living skin. 1. Invest. Dermatol. 81, 29. Boyce S. T. and Ham R. G. (1985) Cultivation, frozen storage and clonal growth of normal human epidermal keratinocytes in serum free media. 1. Tissue CL&. Melh. 9, 83. Boyce S. T. and Hansbrough J. F. (1988) Biologic attachment, growth, and differentiation of cultured human epidermal keratinocytes on a graftable collagen and chondroitin-6-sulfate substrate. Surgey 103, 421. Burke J, F., Yannas I. V., Quinby W. C. et al. (1981) Successful use of a physiologically acceptable artificial skin in the treatment of extensive bum injury. Ann. Surg. 194,413. Collombel C., Damour O., Gagnieu C. et al. (1987) Peau artificielle et pro&de de fabrication. Brevet d’invention Francais no. 8708752, 15 Juin 1987. Brevet d’invention Europeen no. 884201948. Cooper M. L., Boyce S. T., Hansbrough J. F. et al. (1990) Cytotoxicity to cultured human epidermal keratinocytes of topical antimicrobial agents. 1. Surg. Res. 48, 150. Dudley M. N., Blaser J., Gilbert D. et al. (1990) Significance of ‘extravascular’ protein binding for antimicrobial pharmacodynamics in an in vitro capillary model of infection. Antimicrob. Agents Chemother 34, 98. Gallico G. G., O’Connor N. E., Compton C. L. et al. (1984) Permanent coverage of large bum wounds with autologous cultured human epithelium. N. Engl. J Med. 311, 448. Geronemus R. G., Mertz P. M. and Eaglstein W. H. (1979) Wound healing. The effects of topical antimicrobial agents. Arch. Demafol. 115, 1311. Girardo P., Reverdy M. E., Martra A. et al. (1989) Determination de la concentration minimale bactericide de 3 antiseptiques et 1 desinfectant sur 580 souches de bacilles Gram negatif d’origine hospital&e par une micromethode. Pafh. Biol. 37, 605. Hansen M. B., Nielsen S. E. and Berg K. (1989) Re-examination and further development of a precise and rapid dye method for

Damour et al.: Cytotoxic effect of antiseptics and antibiotics on cultured skin cells

measuring cell growth and cell kill. 1. Immctnol. Meth. 119,203. Lavelle K. J., Doedens D. J., Leit S. A. et al. (1975) Iodine absorption in bum patients treated topically with povidoneiodine. C/in. Pham. Tker. 17,355. Leaper D. J. and Brennan S. S. (1986) Let’s have a re-think about the use of antiseptic for verrous ulcer. In: Negus D. and Jantet G. (eds), Pklebology ‘85. London: Libbey, p. 580. Leyden J. J. and Barteit N. M. (1987) Comparison of topical antibiotic ointments, and antiseptics for the treatment of human blister wounds contaminated with Staphylococcus aureus. 1. Fum. Prucf. 24, 601. Lineaweaver W., Howard R., Saucy D. et al. (1985) Topical antimicrobial toxicity. Arch. Sttrg. 120, 257. Merrikin D. J., Briant J. and Rolinson G. N. (1983) Effect of protein binding on antibiotic activity in vivo. J. Antimicrob. Ckemofker. 11, 233. Mossman T. (1983) Rapid calorimetric assay for cellular growth and survival; application to proliferation and cytotoxicity assays. 1. Immunol. Mefk. 65, 55. Rheinwald J. G. and Green H. (1975) Serial cultivation of strains of

485

human epidermal keratinocytes: the formation of strains of keratinizing colonies from single cells. Cells 6, 331. Sagnes I. (1991) Etude de la cytocompatibilite du chitosane vis-a-vis des fibroblastes et des keratinocytes humains: utilisation dun test au MTT. Phese DiplBme d’Etat de Docteur en Pharmacie. Shahabeddin L., Berthod F., Damour 0. et al. (1990) Characterisation of skin reconstructed on a chitosan-crosslinkedcollagen-glycosaminoglycan matrix. Skin Phnmacol. 3, 107. Tatnall F. M., Leigh I. and Gibson J. R. (1990) Cytotoxic effect of antiseptics on cultured cells. Skin Phrrnacol. 3, 157. Yannas I. V., and Burke J. F. (1980) Design of an art&Cal skin: I. Basic design principles. 1. Biomed. Mater. Res. 14, 65. Paper accepted

23 June 1992.

Correspondence should be addressed fo: Dr Odile Damour, Laboratoire des Substituts Cutanes, Pavillon 15, Hbpital Edouard Herriot, 5 Place d’Arsonva1, 69437 Lyon, CEDEX 03, France.

G. Whitaker International Burns Prize - Palermo (Italy) under the patronage of the Authorities of the Sicilian Region for 1993 By law n.57 of 14 June 1983 the Sicilian Regional Assembly authorised the President of the Region to grant the “Giuseppe Whitaker Foundation”, a non profit making organization under the patronage of the Accademia dei Lineci, with its seat in Palermo, an annual contribution for the establishment of a “G. Whitaker International Burns Prize” aimed at recognizing the activity of the most qualified experts from all countries in the field of burns pathology and treatment. The amount of the prize is fixed at twenty million Italian Lire. The prize will be awarded every year by the month of June at the Palermo seat of the G. Whitaker Foundation. The Adjudicating Committee is composed of the President of the Foundation, the President of the Sicilian Region, the Representative of the Accademia dei Lincei within the G. Whitaker Foundation, the Dean of the Faculty of Medicine and Surgery of Palermo University, the President of the Italian Society of Plastic Surgery, three experts in the field of prevention, pathology, therapy and functional recovery of burns, and a legal expert nominated in agreement with the President of the Region as a guarantee of the respect for the scientific purpose which the legislators intended to achieve when establishing the prize. Anyone who considers himself to be qualified to compete for the award may send by 3 1 January 1993 his detailed curriculum vitae to: Dr Michele Masellis M.D., Secretary-Member of the Scientifr Committee G. Whitaker Foundation, Via Dante 167-90141 Palenno, Italy.

Cytotoxicity evaluation of antiseptics and antibiotics on cultured human fibroblasts and keratinocytes.

Infection is the greatest problem in burn patients and topical antimicrobial agents must be chosen with great care, especially when cultured skin is g...
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