Travia G.,* Palmisano P.A.,* Cervelli V.,** Esposito G.,* Casciani C.U.**

** Centro Grandi Ustionati, Ospedale S. Eugenio, Rome, Italy
** Scuola di Specializzazione in Chirurgia Plastica e Ricostruttiva, Università di Tor Vergata, Rome

SUMMARY. This experimental study examined the use of semisynthetic, autologous, engineered and biocompatible skin substitutes in burn patients. These biomaterials are used as dermal and epidermal grafts in extensive or deep burns in order to improve healing by means of a repair process that is as physiological as possible. The latest techniques of bioengineering help to improve therapy, long-term prognosis, and the quality of life of seriously burned patients.

Introduction

In the last few years, progress in the field of tissue engineering - the science that studies the creation of artificial and semiartificial organs and tissues - has led to the production of numerous biomaterials of particular interest in burns therapy. These materials are created out of biological molecules that are normally present in human tissue. They are often chemically modified and are capable of acting as a simple temporary cover or as a support for the growth of autologous cells previously removed from the patient.1-3 These methods also arouse interest because they eliminate the need to use the non-autologous materials - allografts or xenografts - that are employed as a temporary cover in most of the other techniques currently available.

The object of this experimental study is the use of semisynthetic, autologous, engineered and biocompatible skin substitutes. These biomaterials are used as dermal and epidermal grafts in extensive or deep burns in order to improve healing through a repair process that is as physiological as possible. Thanks to the latest techniques of bioengineering, we can improve therapy, long-term prognosis, and the quality of life of seriously burned patients.

Patients, materials, and methods

The patients selected for treatment with the bioengineered autologous tissues included patients with acute burns.

The patients’ age range was from 2 to 63 years. There were deep skin lesions in 20-55% of the body surface area. Every patient received standard intensive care. Infections were treated by specific antibiotic therapy. Either parenteral or enteral nourishment was used according to need.

We compared the results obtained in three patients treated in 1999 only using keratinocytes cultured on a support of fibrin glue with the results obtained in three other patients treated more recently with cultured autologous keratinocytes grafted onto a lesion pre-treated with cultured autologous fibroblasts (dermal reconstruction).

The selected patients underwent normal clinical procedures in order to assess the degree of their burns, and patients presenting deep dermal third-degree burns were deemed suitable for the treatment.

All patients included in the study, in addition to their individual surgical treatment, were subjected to skin biopsy (area, 1-2 cm2; thickness, 0.8 mm), which was sent on to a laboratory specializing in the creation of bioengineered tissues (Laboratori TISSUEtech™).

Preparation of cultured tissues

The skin biopsy was processed in order to achieve epidermal-dermal separation and to isolate respectively fibroblasts and keratinocytes. In the two cell types, the culture phase was initiated, which was necessary in order to increase the number of cells available. This procedure required 8-12 days.

The second phase was the seeding of cells on two biomaterial supports (Hyalograft 3D™ and Laserskin®). This procedure also lasted about eight days.

The supports used both consisted of benzilic esters of hyaluronic acid. This is the most elementary of the anionic glycosaminoglycans (AGAG) and is composed of repeated disaccharidic units of acid-D-glucuronic and N-acetylglucosamine.

The two supports differ in their conformation and characteristic features:

1. Hyalograft 3D™ is a dermal substitute consisting of autologous fibroblasts on a three-dimensional matrix of HYAFF®, a biomaterial derived from hyaluronic acid.
2. Laserskin® autograft is an epidermal substitute consisting of autologous keratinocytes on a laser-microperforated membrane of HYAFF®.

Some 16-21 days after initiation of the Hyalograft 3D™ procedure, it was ready for grafting and was returned to the hospital. Seven days were allowed for it to take. It was possible to apply Laserskin® autograft, or thin autologous grafts, on the newly-formed granulation tissue.

The two groups of patients are described in Table I.

Group 1: three patients with acute burns grafted with autologous keratinocytes cultured on fibrin glue.

Group 2: three patients with acute burns treated with reconstruction of the dermis with cultured autologous fibroblasts and grafts of cultured autologous keratinocytes (Hyalograft 3D™ and Laserskin®.

Surgical protocol

Group 1. When the patients’ skin biopsy was received, early tangential escharectomy of the deep lesions was performed using a manual dermotome. It was necessary to medicate the lesions for about 16 days, until the bioengineered tissues became available. Various synthetic and semisynthetic skin substitutes were used. When the cultured tissues were received, a sufficient number of sheets of cultured keratinocytes were grafted to cover the burned areas completely. The grafted areas were covered with degreased petrolatum gauze and wrapped in sterile bandages. The external medication was changed and the grafts monitored every 2-3 days. In these patients, in the 7-10 days post-graft, it was not possible to use any type of disinfectant solution because of the great delicacy of the keratinocytes. The dressings were finally removed on day 7 and an assessment was made of the keratinocyte take and the state of the lesions.

Group 2. Also the patients in this group were subjected to early tangential escharectomy of their lesions by manual dermatome, and synthetic and semisynthetic skin substitutes were used as a temporary covering for the lesions in the meantime. As soon as they became available, a sufficient number of cultured fibroblast sheets were grafted to cover the lesions entirely. The graft areas were dressed and checked every 2-3 days and the external medication was changed. Fibroblast take and the state of the lesions were assessed. After 7 days the patients were again subjected to surgery, when keratinocyte sheets were grafted onto the newly formed granulation tissue. In this phase, the lesion areas presented partial integration of

the grafted biomaterial (HYAFF®), which presented macroscopic evidence of colliquation in some areas and was partially incorporated granulation tissue in others (Fig. 1). It was not possible to demonstrate whether the fibroblasts cultured on the sheets of hyaluronic acid had populated and synthesized the neodermis, at least in part, but histological tests revealed fragments of HYAFF® in the process of degradation (Fig. 2).

Before take of the keratinocytes, the receiving areas were disinfected with chlorhexidine solutions and then washed in copious amounts of sterile physiological solution. The graft areas were covered in degreased petrolatum gauze and wrapped in sterile bandages. The dressing was changed every 2-3 days and the state of grafts was monitored. It was not possible to use any type of disinfectant solution in these patients in the first 7-10 days post-surgery because of the delicacy of the keratinocytes. The dressings were finally removed after 10-12 days and an assessment was made of keratinocyte take and the state of the lesions.

The clinical results in all the patients was assessed, the following aspects being considered:

• rate of take
• rate of take of cultured autologous keratinocytes
• incidence of infections
• incidence and degree of scar hypertrophy
• incidence and degree of scar contracture

Results and discussion

The clinical results obtained in the patients are summarized in Table II. As can be seen, the exclusive use of keratinocyte cultures was seriously hampered by the incidence of factors that interfered with take, e.g. bacterial attack, a tendency towards weak take at the base of the lesions, and elevated vulnerability to mechanical trauma and damage by overaggressive disinfectants. Despite all our care not to damage the autologous keratinocytes and to preserve their viability, we found that in most cases the low take rate was due to bacterial contamination at the base of the lesion (as confirmed in the literature.

With regard to the onset and gravity of long-term sequelae, the use of keratinocytes alone offers no great advantages in relation to the occurrence of hypertrophy and scar contracture. However, as already remarked, if techniques involving reconstruction of the dermis are used (e.g. the Cuono technique) before the keratinocyte sheets are grafted, the results are often very good. In our experience, the results obtained in Group 1 were unsatisfactory, confirming the need to reconstruct valid dermal tissue before using skin grafts.

Although the cases are few in number and not univocal, we can regard Group 1 as the control group of Group 2, in which the patients were treated with dermal reconstruction by the use of sheets of autologous fibroblasts. In these patients we observed a percentage of keratinocyte take that was quite good and in line with results reported in the literature by other centres that use techniques of dermal reconstruction before proceeding to keratinocyte grafting (Fig. 3).

This method could however prove to be a valid alternative to techniques using non-autologous material if ever - for particular reasons related to public health - it were not feasible to make use of homologous or even heterologous material.

Several aspects of the methods we followed merit further study. In particular, with regard to optimization of the procedure, the problem of the waiting time between grafting of the dermis and grafting of epidermis or keratinocytes remains unresolved. Also, the thickness of the sheet of hyaluronic acid that holds the fibroblasts can be increased or decreased according to need. Another aspect that needs perfecting is the way to treat the base of the lesion before positioning of the epithelial covering.

The most important question still remains, i.e. that of establishing the real advantage of the presence of autologous fibroblasts in the dermal substitute that we used, above all considering the intrinsic properties of the “support” adopted. It has to be borne in mind that hyaluronic acid is itself involved in the scarless repair observed in foetal lesions. Physiologically, elevated local concentrations of hyaluronic acid play a very important role in the repair of lesions. This glycosaminoglycan is a mediator capable of modulating the inflammatory response and neoangiogenesis.

The solution of this problem would obviate the waiting time necessary for the realization of the autologous cell cultures and also solve problems related to the high cost of producing these biomaterials, which considerably reduces any wide-scale use.

Fig. 3 - Patient M.A. Extensive electric burns in 43% TBSA. a) Condition after escharectomy and preparation of wound bed with heterologous skin; b) Hyalograft 3D implant, intra-operative condition; c) grafting of sheets of cultured keratinocytes, intra-operative condition; d) condition one month post-graft.

RESUME. Les Auteurs de cette étude expérimentale ont examiné l’emploi, dans les patients brûlés, des remplacements de la peau autologues sémisynthétiques, biocompatibles et manipulés. Ces biomatériaux sont usés comme des greffes dermiques et épidermiques dans les brûlures étendues ou profondes pour améliorer la guérison à travers un mécanisme de réparation le plus physiologique possible. Les techniques les plus modernes de la bioingénierie contribuent à l’amélioration de la thérapie, du pronostic à long terme et la qualité de vie des grands brûlés.

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