Scalise A., Pierangeli M., Di Benedetto G., Sperti V., Bertani A.

Burn Department of Plastic and Reconstructive Surgery, Ancona Medical School, Italy

SUMMARY. This paper presents the multiple possible uses of a new strategy in wound healing. This novel technique consists of the separate cultivation in vitro of autologous fibroblasts and keratinocytes obtained from a cutaneous biopsy from the patient. The biopsy technique permits the resolution of pathologies frequently encountered in plastic surgery such as burns, ulcers in diabetic patients, ulcers in patients with venous insufficiency, and ulcers due to traumas.

Introduction

Autologous keratinocyte and fibroblast grafts on hyaluronic acid derivative scaffolds represent a breakthrough in the various techniques for the healing of wounds that have persisted even as long as 25 years. The first attempts using autologous keratinocytes (Figs. 1-4) cultured in vitro go back to 1979,1 while tissue repairing with allogenic and autologous tissues of various origin dates from 1968.2 The great step forward that accelerated progress towards the products that are now available came about 15 years ago.3 The final results of the evolution of these studies have shown that if we are to obtain a correct proliferation of the dermis and/or epidermis it is necessary that dermal and epidermal cellular components should exist simultaneously.4

A further concept is to cultivate autologous fibroblasts and keratinocytes on hyaluronic acid derivative based scaffolds. The use of such scaffolds with a three-dimensional network suitable for fibroblast growth and a lamina suitable for keratinocyte growth permits the adhesion, proliferation, and organization of the cells. The proliferating and growing cells on these scaffolds secrete extracellular matrix components.5,6

We based our clinical experience on this new concept and obtained effective solutions for problems caused by severe substance loss of acute or chronic origin.

Materials and methods

We enrolled in our study patients presenting extensive deep dermal wounds with irregular margins that did not tend to heal spontaneously (Figs. 5-7). We intentionally included pathologies of various aetiologies in order to test the grafts in patients in a variety of general metabolic conditions and wound conditions. Five patients in each of the following pathological groups were enrolled: burns, ulcers in diabetic patients (Figs. 8-10), vascular ulcers, and post-traumatic ulcers. The average age of the patients was 51.4 yr (range, 23 months to 72 yr). All patients were treated with a common protocol regime (Table I).

On the sixteenth day the autologous fibroblasts grown on Hyalograft 3D (Fidia Advanced Biopolymers, srl - Abano Terme, Italy) were grafted directly onto the wound bed. This dermal-like graft was allowed to take and to stimulate the formation of a well-vascularized neodermis capable of receiving the keratinocyte graft. The autologous keratinocytes grown on Laserskin (Fidia Advanced Biopolymers, srl - Abano Terme, Italy) were applied 7 days after application of the fibroblasts.

Discussion

In recent years great progress has been made in the understanding of the basic mechanisms of interaction and proliferation of the diverse cutaneous cellular components involved in wound healing repair processes. The most recent advance in biopolymer research has identified different matrixes composed of a derivative of hyaluronic acid that permit the adhesion and proliferation of fibroblasts and keratinocytes.

The present good standard of tissue repair obtained with the clinical application of the grafts in the first patients to be treated prompted us to establish a protocol, in association with the company that produces the grafts, that could resolve typical pathologies in the field of reconstruction and thus extend the indications beyond that of burns to include also vascular, post-traumatic, and diabetic ulcers.

The results obtained have enabled us to heal difficult wounds in each of the four pathological groups treated in our study. Complete healing was achieved in 30-45 days in 14 of the 16 patients treated. Healing was obtained at later time points in the other two patients treated (complications in the take of the keratinocyte graft in a patient with a severe vascular pathology and previous amputation of the other limb, and in a diabetic patient with low compliance to hypoglycaemic treatment). The average follow-up in these patients lasted 9 months. The donor site also healed with good results, as is characteristic of this particular technique.

Conclusions

Consistent and reliable results were obtained using the two-step autologous graft composed of a fibroblast and keratinocyte graft both delivered and allowed to proliferate on two distinct matrixes, consisting entirely of a derivative of hyaluronic acid, and used in patients with wounds of various aetiology (burns, ulcers in diabetic patients, vascular ulcers, and post-traumatic ulcers). A particularly appreciable aspect was the short time required to achieve complete closure of difficult-to-heal wounds, due to the fact that the biopsy taken to obtain the fibroblasts and keratinocytes for the grafts was minimal and thus permitted the donor site to heal with good scarring results. The particular protocols for the biopsy and the graft make this the technique of first choice for wounds in paediatric (Figs. 11-14) and female patients.

A further noteworthy feature was that the operating room was not necessary for all patients either for the biopsy or for the grafts.

The advantage of the small size of the biopsy (approximately 1 x 1 cm) needed to obtain the autologous grafts is particularly appreciated by the patients and the clinicians, who have to take scarring results into consideration prior to opting for the most appropriate procedure.

RESUME. Les Auteurs décrivent les nombreuses possibilités d’emploi d’une nouvelle stratégie dans la guérison des lésions. Cette technique originale consiste en la culture séparée in vitro des fibroblastes autologues et des kératinocytes obtenus d’une biopsie cutanée du patient. Cette technique de biopsie permet la solution de diverses pathologies fréquentes dans la chirurgie plastique comme les brûlures, les ulcères dans les patients diabétiques, les ulcères dans les patients atteints d’insuffisance veineuse, et les ulcères causées par les traumatismes.

BIBLIOGRAPHY

1. Green H., Kehinde O., Thomas J.: Growth of human epidermal cells into multiple epithelia suitable for grafting. Proc. Natl. Acad. Sci. USA, 76: 5665-8, 1979.
2. Karasek M.A.: Growth and differentiation of transplanted epithelial cell cultures. J. Invest. Dermatol., 51: 247-52, 1968.
3. Zambruno G., Santantonio M.L., Giannetti A.: Colture di cheratinociti umani normali. G. Ital. Dermatol. Venerol., 125: 59, 1990.
4. Sato T., Kirimura Y., Moti Y.: The co-culture of dermal fibroblasts with human keratinocytes induces increased prostaglandin E2 production and cyclo-oxygenase2 activity in fibroblasts. J. Invest. Dermatol., 109: 334-9, 1997.
5. Zacchi V., Soranzo C., Cortivo R., Radice M., Brun P., Abatangelo A.: In vitro engineering of human skin-like tissue. J. Biomed. Mater. Res. 40: 187-94, 1988.
6. Carver N., Leigh I.: Keratinocyte grafts and skin equivalents. Int. J. Dermat., 30: 540-7, 1991.