Ann. Medit. Burns Club - vol. VII - n. 3 - September 1994

HEALING EFFECT OF RECOMBINED HUMAN/PIG SKIN ON DERMAL DEFECTS

Matou~kovd E, Ndmcovd D., Dvofdnkovd D., VogtovA D., KénigovA R.

SUMMARY. The healing effect of recombined human/pig skin (R11PS), composed of human keratinocytes growing on cell-free xenodermis, was studied. When R11PS was applied with the keratinocyte layer facing the wound, stimulation of healing of leg ulcers and deep-dermal wounds was observed. The structure of RHPS makes manipulation easier and more effective compared to cultured epidermal sheets.

As autologous skin is not available in massive burns and is often wasted in chronic skin defects, surgeons are still searching for an ideal skin substitute (1, 2). Cultured epidermis has been used to treat skin loss since 1981 (3), but a satisfactory dermo-epidermal skin substitute that is readily available in suf~ ficient quantities has still to be found. On the basis of long experience with the application of xenografts in bum treatment (4) and of experience with cultured epiderinis (5, 6) we have developed recombined human/pig skin (RBPS), a composite skin substitute which consists of human autologous or allogeneic keratinocytes cultivated on cell-free pig dermis (7). In this work we describe the healing effect of RBPS containing allogeneic keratinocytes on chronic leg ulcers and bums.

Methods

Epidermis and fibroblasts are removed from strips of sterile, living pig skin after trypsinization. The resulting dermis is macerated in water (to wash out the rest of the cells, salts and antibiotics), spread on the tissue culture dish and stuck to it by drying, forming a thin collagen film. Dry dermis behaves as a firm substrate for cell culture. The dishes containing dried dermis may be steril-ized with gamma-irradiation (not necessary when treated with antibiotics and fungicides) and preserved at room temperature. The dermis is pre-washed with the growth medium for several hours. Lethally irradiated 3T3 cells are then seeded on it in a fresh medium. Keratinocytes (2nd passage) are added the next day. This -method, based on the method of Rheinwald and Green (8), enables us to obtain more than 500 sq cm of RHPS from one sq cm of human split-skin graft. The 3T3 cells can be selectively washed off at any time of culture by differential trypsinization (Fig. 1).

Fig. 1 - Four-day-old colonies of keratinocytes growing on cell-free pig dermis. 3T3 cells were selectively washed off by trypsin. Fig. 2 - Vertical section of RHI'S composed of a keratinocyte layer '-rowing on cell-frce pig dermis.

At keratinocyte subconfluency or confluency, usually 711 days after seeding, the recombined skin (Fig. 2), which is very similar in consistency to normal skin, was peeled off without enzymatic release (Fig. 3) and placed on the skin defect (Fig. 4) with the keratinocyte layer facing the wound. It was then covered with a layer of vaseline gauze and several layers of gauze wetted with cholera-toxin-free (in the case of bums also serum-free) growth medium. The RBPS-covered defect was checked two to five days after application.

For treatment of leg ulcers RFIPS containing allogeneic keratinocytes was used. The healing effect depended on the size and cleanness of the wound.

Fig. 3 - RHPS peeled off without enzyinatic release. The struc-turc ofRIAPS is similar to that of normal skin. Fig. 4 - RHPS placed on a skin defect.

1. Small leg ulcers (up to 2 sq em). R11PS usually adhered to the wound in the course of two days. During this time the ulcer was covered either with full RInPS or just with a thin epithelial layer. In 11 out of 13 cases (85%) the ulcers healed in one to three weeks.
2. Medium leg ulcers (up to 20 sq em). These showed temporary RHPS "take" or only a stimulation of epithelialization. Four ulcers out of eight (50%) healed in four to six weeks after RHPS application. In the other cases pain decreased and the formation of granulation tissue was activated.
3. Large leg ulcers (three cases). Partial healing was observed in one case (50 sq em). In the two other cases (45 sq em and an extensive circular leg ulcer of about 200 sq em) only a temporary improvement of granulation tissue was observed.

The greatest obstacle to RBPS "take" was wound infection. The most successful method of leg ulcer clean~ing and disinfection was provided by the use of 5% salicyl acid cream.

R11PS with a subconfluent keratinocyte layer was applied on a leg ulcer 12 sq em in size (Fig. 5a). Rapid healing started from the edges of the wound after three days. The ulcer healed in two weeks and remained in a stable state (Fig. 5b).

RHPS was applied on a clean 4 sq em deep leg ulcer reaching to the bone. The wound filled up after seven days and only a narrow gap remained on the surface.

RHPS was applied in four patients. Dry application to the donor site was not successful (wetting is necessary). RHPS containing autologous and/or allogeneic keratinocytes applied in a patient with massive burns (80%) was dissolved.

Fig. 5a - Case 1. Leg ulcer before treatment with RHPS. Fig. 5b - Case 1. 6 months after R14PS aj)plication. The ulcer heal-ed in two weeks and remained stable.

Fig. 6a - Case 3. RHPS-treated burn area: a) 5 days after RHPS al)piication (4), xenografted area (44). Fig. 6b - Case 3. RHPS-treated bum area: b) 12 days after RHPS application (1), autografted area (4), as yet unhealed area - meshed autografts (~4).

A 20-year-old male with deep dermal burns on the leg (I I% BSA) was necrectomized and xenografted five days post-burn. Two days after necrectomy a 5 x 6 cm area of the wound was covered with RHPS containing allogeneic keratinocytes and wetted with the culture medium. The rest of the wound was cover-ed with xenografts. Two days later the xenografts were replaced by autografts, while RHPS remained untouched. Five days after RHPS application RHPS "take" and epithelialization of the treated area were apparent (Fig. 6a). Seven days after RHPS application a histological sample was taken from the RHPS grafted area (Fig. 7a). Dry RHPS started to peel off (Fig. 7b), but the wound underneath healed in two weeks (Fig. 6b).

A burn wound 60 sq cm in size was covered with R11PS containing allogeneic keratinocytes. In the course of five days 40% of the surface was epithelialized while the rest had to be autografted, probably because it did not contain any basal epidermal cells.

RHPS is a new type of skin substitute, combining the advantages of xenografts and cultured epidermal sheets. The antigenicity of pig skin is minimized by removing all potentially antigeneic elements (i.e. epidermis and other skin cells). Only a collagen matrix preserving the natural structure of the dermis is used as a firm substrate (dried and stuck to the bottom of the tissue culture dish) for keratinocyte culture. With the method used in this study - human keratinocytes cultured on cell-free pig dermis, applied with the keratinocyte layer facing the wound - RHPS had a remarkable healing action on skin defects. It also stimulated granulation tissue formation and the filling up of the base of small deep ulcers. When applied in a normal orientation (i.e. keratinocytes facing upwards), RHPS usually did not "take".
Histological samples show that RHPS did not remain in the wound, but in our opinion it is possible that epiderinal cells migrated into the wound and were later replaced by the patient's own skin cells.
In comparison to simple cultured epidermal sheets, recombined skin grafts have the following advantages:

1. suitable biomechanical properties (strength, elasticity, adhesiveness), similar to those of normal skin
2. provides protection of covered areas (both mechanical and against drying and infection)
3. possibility of mechanical removal from bottom of dish without enzymatic influence
4. same size as cultivate after detaching (cultured epider mis shrinks)
5. easy handling, including possibility of gradually cutting off small pieces
6. higher metabolic activity (evaluated from the lower mean pH)
7. presence of a dermal fibral structure, which serves:

• as a source of dermal elements
• as a deposit of stimulating factors contained in the medium which is absorbed in the dermis
• as a transfer substrate on which the keratinocytes multiply and can therefore be applied to the patient in any growth phase (in subconfluency after washing off 3T3 cells).

We are currently investigating the development of new generations of RHPS containing fibroblasts incorpo.rated in the dermal structure.

Fig. 7a - Vertical section of histological sample taken from RWStreated area 7 days post-application: a) healed young epidermis covered by a layer of RHPS. Fig. 7b - Vertical section of histological sample taken from RHI'S-treated area 7 days post-application: b) dry RHIS starting to peel off.

RESUME. Les auteurs ont étudié l'effet guérissant de la peau humaine/porcine recombinée (sigle anglais: RHPS), composée de kératinocytes humains qui croissaient sur le xénoderme libre de cellules. Quand la RHPS était appliquée avec la couche de kératinocyte face à la lésion il était possible d'observer la stimulation de la guérison des ulcères de la jambe et des brûlures du derme profond. La structure de la RHPS rend la manipulation plus facile et plus efficace par rapport aux lambeaux épidermiques cultivés.

BIBLIOGRAPHY

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