Annals of Burns and Fire Disasters - vol. XVIII - n. 4 - December 2005

TRANSPLANTATION OF COMPOSITE SKIN CONTAINING KERATINOCYTES CULTURED ON A FIBROBLAST-CONDITIONED ACELLULAR DERMAL MATRIX

Xiao S.-C., Ben D.-F., Yang J., Tang H.-T., Wang G.-Q., Yang Y., Yu W.-R., Xia Z.-F.

Burns Centre, Changhai Hospital, Shanghai, People’s Republic of China

SUMMARY. To evaluate the role of fibroblasts in composite skin reconstructed in vitro, four different types of composite skin (A, B, C, and D) were prepared. Human keratinocytes were seeded onto the epidermal side of an acellular dermal matrix (ADM) in type A. Keratinocytes were seeded onto the epidermal side of an ADM and human fibroblasts onto the dermal side in type B. Both keratinocytes and fibroblasts were seeded onto the epidermal side in type C. Type D consisted of fibroblasts on both sides of the ADM and keratinocytes on the epidermal side. The adherence of keratinocytes to the ADM was observed. The composite skin was then transplanted onto full-thickness skin defect wounds in nude mice. Results showed that the adherence of keratinocytes to the ADM was improved when fibroblasts were pre-seeded onto the epidermal side of the ADM. The composite skin was able to close full-thickness skin defect wounds. The take rates were respectively 44.1 ± 7.8%, 47.3 ± 5.4%, 75.2 ± 8.8%, and 81.2 ± 8.1% for types A, B, C, and D. The take rates of types C and D were significantly higher than those of types A and D. There was no significant difference in take rate between types C and D. In conclusion, composite skin consisting of keratinocytes cultured on a fibroblast-conditioned ADM was a good skin substitute.

Introduction

Fibroblasts play an important role in the regeneration of new skin tissue. Previous studies have shown that the presence of fibroblasts in a skin substitute stimulated epidermal differentiation and dermal regeneration. It is assumed that the fibroblasts accelerate the growth of tissue cells by secreting several growth factors and extracellular matrix.1 In this study, we investigated the effects of living fibroblasts seeded in a skin substitute on keratinocyte proliferation, adherence, and survival in a nude mice wound model.

Materials and methods

Preparation of an acellular dermal matrix (ADM) and composite skins

ADM was prepared from pigs by treatment of Dispase II (Gibco BRL Life Technologies, inc., USA) and Triton X-100 (Sigma Chemical Co., St Louis, MO, USA).2 Human keratinocytes and fibroblasts were previously derived from foreskin from circumcision. The secondary and tertiary cultures of keratinocytes and fibroblasts were used to reconstruct the composite skin.

Four types of composite skin were prepared (Fig. 1): type A = human keratinocytes were seeded (5 ¥ 105/cm2) on the epidermal side of ADM; type B = human fibroblasts (2 ¥ 105/cm2) were pre-seeded onto the dermal side of ADM for two days and the keratinocytes were then seeded onto the epidermal side; type c = keratinocytes were seeded on the epidermal side of ADM pre-seeded with fibroblasts (2 ¥ 104/cm2) two days before; type D = fibroblasts were seeded onto both sides (epidermal side seeded with 2 ¥ 104/cm2, dermal side with 2 ¥ 105/cm2) two days after seeding. The keratinocytes were seeded onto the epidermal side. The composite skin was cultured in vitro, and biopsies were obtained at intervals of 1, 3, 5, 7, and 14 days for routine histological examination.

Transplantation of composite skin

Sixty-four nude mice were divided equally into four groups (groups A, B, C, and D). Full-thickness wounds 1.5 cm in diameter were created on the dorsal area of each animal. Composite skin in the culture dish for one week was placed on the wound, with the dermal side of ADM in contact with the wound. The skin-graft chamber was inserted into the wounds to prevent epithelial migration from the wound edge. Skin biopsies were taken from the mice for examination on days 7 and 14.

Two weeks after graft placement, the wound was examined to determine the borders of the graft and areas of necrosis. The percentage of graft survival was determined using the paper template technique.3 Percentage graft take was quantified as follows:

Statistical analysis

Data were expressed as the mean ± SEM. P values less than 0.05 were considered significant. Non-paired t-tests were used to compare control versus test group in the graft rate data. Tukey’s test was used to identify statistically significant differences between specific intergroup mean values.

Results

Effect of fibroblasts on adherence of keratinocytes to ADM

Keratinocytes reached confluence after culturing in vitro for one week. Histological observation showed that keratinocyte growth in one layer on the surface of ADM. However, the adherence of keratinocytes to ADM in types A and B was too weak to fix on the surface of ADM: the cell layer often became detached from ADM during section procedures in histological observation. Keratinocytes in types C and D appeared to be integrated onto the surface of ADM. Dermal-epidermal integration was sufficient to allow transplantation without detachment of the epidermis.

Take rate of composite skins in nude mice

The skin-graft chamber prevented migration of epithelium from the wound edge. The take rates were respectively 44.1 ± 7.8%, 47.3 ± 5.4%, 75.2 ± 8.8%, and 81.2 ± 8.1% for types A, B, C, and D. The take rate of types C and D were significantly higher than those of types A and D. There was no significant difference in take rate between types C and D (Table I).

Discussion and conclusion

Keratinocytes showed different growth characteristics in different dermal substitutes. A previous study4 showed that keratinocytes seeded onto the surface of acellular reticular dermis did not attach and spread, but formed small islands of rounded cells. Human fibroblasts could achieve a similar role as 3T3 cells in the initiation of keratinocyte colonization/proliferation on ADM. Seeding cultured dermal fibroblasts on acellular reticular dermis resulted in both colonization of the surface and invasion of the dermis. Dermal-epidermal integration was sufficient to allow meshing without detachment of the epidermis. In the present study, keratinocytes were seeded onto the surface of ADM, which was pre-seeded with fibroblasts. Dermal-epidermal integration was sufficient to allow transplantation without detachment of the epidermis. However, the adherence of keratinocytes to ADM without fibroblasts was too weak to fix on the surface. Our observations confirmed that ADM pre-seeded with fibroblasts became a better dermal substitute for keratinocyte attachment and growth. It seems likely that fibroblasts promote keratinocyte growth by modifying the surface of acellular dermis.

Lamme5 demonstrated that proliferative fibroblasts released a large amount of collagen, fibronectin, and growth factors in living dermal substitute and that proteins enhanced the incorporation of the cultured graft onto the recipient wound bed. On the other hand, cultured fibroblasts seeded in an artificial dermal substitute survived and proliferated after being transplanted onto the wound. Seeded fibroblasts could reduce subcutaneous fibroblastic cell migration and/or proliferation into the wounds. Our study confirmed the above point: the take rates of types C and D were significantly higher than those of types A and B. In conclusion, composite skin consisting of keratinocytes cultured on a fibroblast-conditioned ADM could be a good skin substitute in severely burned patients.

RESUME. Pour évaluer le rôle des fibroblastes dans la peau composite reconstruite in vitro, les Auteurs ont préparé quatre différents types (A, B, C et D) de peau composite. Des kératinocytes humains ont été ensemencés du côté épidermique d’une matrice dermique acellulaire (MDA) dans le type A. Dans le type B, des kératinocytes ont été ensemencés du côté épidermique de la MDA et des fibroblastes humains du côté dermique. Des kératinocytes et à la fois des fibroblastes ont été ensemencés du côté épidermique dans le type C. Le type D était composé de fibroblastes de tous les deux côtés de l’MDA et de kératinocytes du côté épidermique. Les Auteurs ont observé l’adhérence des kératinocytes à l’MDA. La peau composite a été ensuite transplantée sur des lésions cutanées à toute épaisseur dans des souris nues. Les résultats ont démontré que l’adhérence des kératinocytes à l’MDA s’améliorait quand les fibroblastes étaient pré-ensemencés du côté épidermique de l’MDA. La peau composite était capable de fermer les lésions cutanées à toute épaisseur. Les taux de la prise de la greffe étaient respectivement 44,1 ± 7,8%, 47,3 ± 5,4%, 75,2 ± 8,8% et 81,2 ± 8,1% dans les quatre types A, B, C et D. Les taux de prise des types C et D étaient significativement plus élevés par rapport à ceux des types A et D. Les Auteurs n’ont observé aucune différence significative dans le taux de prise entre les types C et D. Ils concluent que la peau composite composée de kératinocytes cultivées sur l’MDA conditionné par des fibroblastes constituait un bon substitut cutané.

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