Annals of Burns and Fire Disasters - vol. XVI - n. 2 - June 2003

TOPICAL ANTISEPSIS AND TRANSPLANT OF KERATINOCYTE CULTURES IN SEVERELY BURNED PATIENTS

Di Lonardo A., Maggio G., Gargiuoli F., Dioguardi D.

Department of Innovative Technological Applications in Surgery,
Institute of Plastic Reconstructive Surgery and Burns Centre, Bari University, Italy

SUMMARY. In the light of experimental trials conducted on the cytotoxicity in vitro of some of the most frequently used topical antiseptics in the treatment of burn patients, we report our experience in the use of an antiseptic association consisting of silver sulphadiazine and gentamicin in various concentrations. This particular association has shown itself to be very effective as regards common bacteria that colonize burn areas. It also possesses very low cytotoxicity. This feature has favoured its use for antiseptic prophylaxis in burn areas reconstructed with autologous cultures of keratinocytes. The clinical results indicated that the use of topical antiseptics possessing low cytotoxicity led to a significant increase in the survival of keratinocytes compared with results using the antiseptic solutions frequently used in routine clinical practice.

Introduction

The possibility of keratinocytes surviving when transplanted onto large raw areas is linked to the presence of a dermal interface. For this reason the Cuono technique is standard practice in severely burned patients. This technique uses allogeneic skin de-epidermized before the positioning of keratinocytes.

Infection is another important factor that can interfere with the survival of transplanted keratinocytes. In order to minimize the effects of infection, it is necessary - after the application of keratinocytes - to counteract infection by using effective antiseptics possessing low cytotoxicity.

To identify antiseptics that were clinically effective and possessed low cytotoxicity, a clinical and microbiological multi-centre survey was carried out in 11 Italian burns centres in 1993-1994.

Altogether 95 patients were involved (mean age, 39.3 yr), admitted within 24 h post-burn with a burn surface greater than 20% body surface area (BSA) (mean BSA, 35.6%).

Three main aims were pursued:

• to identify the pathogenic agents most frequently encountered in the lesions;
• to select the most effective topical antiseptics against isolated bacterial strains;
• and to assess, for each antiseptic selected, the minimum inhibiting concentrations (MIC) in pathogenic strains and its cytotoxicity towards in vitro cultivated keratinocyte colonies.

A total number of 315 biopsies and 319 surface swabs were taken in the burn areas.

The bacterial strains isolated in the lesions were mainly (82% of the biopsies) Staphylococcus aureus (44.6%) and Pseudomonas aeruginosa (37.6%). Other strains were much rarer: Acinetobacter (4.8%), Enterococcus (3.2%), C. albicans (2.0%), E. coli (1.6%), and Klebsiella (1.2%).

Maximum attention clearly had to be paid to the control of infection from S. aureus and P. aeruginosa, which together represented the majority of strains encountered.

The following substances were used to assess the in vitro antimicrobial effectiveness of the topical antiseptics most widely used in clinical practice against these two pathogens:

• chlorhexidine
• silver sulphadiazine
• gentamicin
• rifamycin
• gentamicin + silver sulphadiazine
• merbromin

With regard to S. aureus, Table I gives the percentage of sensitivity to topical antiseptics on the 195 strains isolated:

With regard to the 67 strains of P. aeruginosa isolated from burn wounds, the sensitivity to topical antiseptics is reported in Table II:

The next phase of the study was to assess the cytolesive effects of the same topical antiseptics on cell cultures of human keratinocytes grown in vitro, in order to define their cytotoxicity. It was thus possible to identify the substances that combined high antimicrobial efficiency with low toxic effect.

We tested chlorhexidine, silver sulphadiazine, gentamicin, and rifamycin, starting at standard clinical concentrations and, with scalar reductions, reaching MIC. The substances were added to the growth media of confluent keratinocyte cultures, and incubated for 24 h. After 24 h the cells were trypsinized and replated in order to test their colony forming efficiency, which is a basic parameter of epidermic cell viability.

Results

• At clinically used concentrations, all four substances tested proved to be highly cytotoxic after 24 h of incubation.
• Chlorhexidine had the highest toxicity levels even at the lowest useful concentration (10 mg/ml) only 1 h after exposure.
• Rifamycin also proved to be toxic, provoking cell death after 12 h of exposure to the MIC (100 mg/ml).
• Gentamicin was tolerated by epithelial cells up to a maximum concentration of 50 mg/ml.
• Silver sulphadiazine used at a dose of 1 mg/ml was well tolerated, while 12 days of exposure at a dose of 10 mg/ml reduced the vitality of keratinocytes only by 20%.
• After 12 days, the association of gentamicin 50 + silver sulphadiazine 10 determined cell survival at a rate of 75%, with an antibacterial effectiveness of 80%.

These results suggest that the association of gentamicin 50 + silver sulphadiazine 10 combines an excellent antibacterial effect with low cytotoxity. For this reason, since 1994 we have made wide use of this combination in our burns centre in the topical treatment of burn areas reconstructed using keratinocyte autologous cell cultures. The clinical effectiveness of the antiseptic association was confirmed by a comparison of the percentage of survival of keratinocytes transplanted in two different groups of patients:

• Group A: eight patients (of whom four were treated with allogeneic dermis) subjected to topical antisepsis with gentamicin-silver sulphadiazine 50/10 intra-operatively and at 3 and 7 days;
• Group B: eight patients (of whom four were treated with allogeneic dermis) subjected to topical antisepsis with traditional antiseptics (betadine, chlorhexidine, etc.) with the same techniques and times

Table III shows the percentage take of keratinocytes in the two groups of patients. It can be seen that the survival of cells cultivated in vitro was notably affected by the degree of toxicity of the antiseptic with which they came into contact as also by the presence or absence of an suitable dermal interface.

Conclusions

Topical antiseptics are among the main clinical aids for the care and prevention of infection in burned skin area. The selection of the most appropriate antiseptic on each separate occasion is however a problem because as yet no product possessing both high bactericide capacity and low cytotoxicity has been found, and studies conducted in recent years have shown that none of the most widely used clinical antiseptics, at microbiologically active concentrations, is tolerated by keratinocytes grown in vitro. The association of gentamicin + silver sulphadiazine 50/10 mg/ml has proved to the best compromise between microbiological effectiveness and cytotoxicity in the topical treatment of keratinocyte-grafted areas. In our experience, this antiseptic association increased the percentage take of keratinocytes without ever leading to the appearance of resistant strains and without any evident side effects for the patient.

RESUME. Sur la base des évaluations expérimentales conduites sur la cytotoxicité in vitro de certains antiseptiques topiques fréquemment utilisés dans le traitement des patients brûlés, les Auteurs décrivent leur expérience avec l’emploi d’une association antiseptique composée de sulphadiazine argentée et gentamicine en concentrations diverses. Cette association en particulier s’est démontrée très efficace contre les bactéries communes qui colonisent les zones brûlées mais qui possèdent une cytotoxicité basse. Cet aspect a suggéré son emploi dans la prophylaxie antiseptique dans les zones brûlées reconstruites avec des cultures autologues de kératinocytes. Les résultats cliniques ont mis en évidence que l’emploi d’antiseptiques topiques ayant une cytotoxicité basse a déterminé un incrément significatif dans la survie des kératinocytes par rapport à ce qui s’est vérifié avec les solutions antiseptiques les plus communes dans l’emploi clinique.

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