[Porous matrix and primary-cell culture: a shared concept for skin and cornea tissue engineering]. / Matrice poreuse et culture de cellules primaires : un même concept pour la reconstruction cutanée et cornéenne.

Skin and cornea both feature an epithelium firmly anchored to its underlying connective compartment: dermis for skin and stroma for cornea. A breakthrough in tissue engineering occurred in 1975 when skin stem cells were successfully amplified in culture by Rheinwald and Green. Since 1981, they are used in the clinical arena as cultured epidermal autografts for the treatment of patients with extensive burns. A similar technique has been later adapted to the amplification of limbal-epithelial cells. The basal layer of the limbal epithelium is located in a transitional zone between the cornea and the conjunctiva and contains the stem cell population of the corneal epithelium called limbal-stem cells (LSC). These cells maintain the proper renewal of the corneal epithelium by generating transit-amplifying cells that migrate from the basal layer of the limbus towards the basal layer of the cornea. Tissue-engineering protocols enable the reconstruction of three-dimensional (3D) complex tissues comprising both an epithelium and its underlying connective tissue. Our in vitro reconstruction model is based on the combined use of cells and of a natural collagen-based biodegradable polymer to produce the connective-tissue compartment. This porous substrate acts as a scaffold for fibroblasts, thereby, producing a living dermal/stromal equivalent, which once epithelialized results into a reconstructed skin/hemicornea. This paper presents the reconstruction of surface epithelia for the treatment of pathological conditions of skin and cornea and the development of 3D tissue-engineered substitutes based on a collagen-GAG-chitosan matrix for the regeneration of skin and cornea.

Use of allogenic epidermal sheets for difficult wound healing: selection and testing of relevant growth factors.

The clinical interest of using allogenic epidermal sheets (AES) has largely been shown [1,2,3]. As well as covering, they also stimulate healing, by simultaneously secreting numerous growth factors (GFs), although little is known on their mechanism of action. Our objectives were to: (a) devise a test for the efficacy of AES release, (b) select keratinocyte-secreting strains and optimal culture conditions. Three GFs were selected: IL-1alpha, IL-8 and VEGF. Three different keratinocyte strains were cultured for 3 and 6 days after confluence for 3 passages. Assays were performed after 3 h and 24 h+3 h after dispase treatment (AES conservation for 24 h then change of medium and sampling after 3 h). AES were found to secrete GFs in DMEM and the amounts were greater when cultured for 6 rather than 3 days after confluence. Each strain had different secretory patterns depending on passage and time in culture, this variability being explained by inter-individual heterogeneity.

Use of an artificial dermis (Integra) for the reconstruction of extensive burn scars in children. About 22 grafts.

We used an artificial dermis (Integra) for the reconstruction of extensive burn scars in children. Integra was initially developed for the primary coverage of acute burns, but several authors report good experiences with Integra for reconstructive surgery. We present a group of 10 children who underwent Integra grafting at 22 different operational sites. Five children received several grafts with Integra. On average, 260 cm(2) per session were grafted. We compared the surface of Integra on the day of grafting and then again on the evaluation day to measure the secondary retraction of the grafts. Complications (infection of Integra, failure of the epidermal graft) were observed in 5 cases. At the final evaluation, 20 grafts were visible. The surface of the Integra graft represented less than 50% of the initial surface in 7 cases, 51-75% in 5 cases and more than 76% in 8 cases. The disadvantages of Integra in reconstructive surgery are that two operative procedures are necessary and the recurrence of contraction seems to be more significant than with full thickness auto grafts. However, Integra has many advantages: the immediate availability of large quantities, the simplicity and reliability of the technique, the pliability and the cosmetic aspect of the resulting coverage. In light of these preliminary results, Integra appears to offer a new alternative for the reconstruction of extensive burn scars in children.

Reconstructive surgery using an artificial dermis (Integra): results with 39 grafts.

Integra was initially developed for the primary coverage of acute burns. It acts as a network for dermal reconstruction. An epidermal graft overlay is necessary after 3 weeks to achieve the in vivo reconstruction of a full-thickness skin equivalent. The quality of the functional and aesthetic results achieved led us to evaluate the potential of Integra in the treatment of burn scars and for general reconstructive surgery. We present a series of 31 patients who underwent Integra grafting for reconstructive surgery at a total of 39 operational sites. The average area grafted per procedure was 267 cm(2). Complications (silicone detachment, failure of the graft, haematoma) were observed in nine cases. The length of follow-up ranged from 0.5 year to 4 years. Two patients (two sites) were lost to follow-up; the final results in the remaining patients were considered to be good in 28 cases, average in six cases and poor in three cases. The disadvantages of using Integra in reconstructive surgery are the necessity of two operations, the risks of infection under the silicone layer, of the silicone becoming detached and of recurrence of contraction. On the other hand, Integra has many advantages including its immediate availability, the availability of large quantities, the simplicity and reliability of the technique, and the pliability and the cosmetic appearance of the resulting cover. In the light of these preliminary results, Integra appears as a new alternative to full-thickness skin grafting, skin expansion and even skin flaps for reconstructive surgery.

Grafting of large pieces of human reconstructed skin in a porcine model.

Present techniques can save about 25% of patients burnt over more than 90% of their body surface. However, problems of functional and aesthetic repair arise, which are often resolved only by major therapeutic procedures. Current advances in skin substitutes permit the cultivation, from a skin biopsy, of large surfaces of in vitro human reconstructed skin (HRS). Our model, obtained by the co-culture of fibroblasts and keratinocytes on a dermal substrate composed of collagen-glycosaminoglycan-chitosan, reproduces, in vitro, a tissue close to human skin, which could play a role in reconstructive surgery. The objectives of this experiment were to assess whether it is possible to perform large HRS grafts and to evaluate the preliminary cosmetic results. We used four immunosuppressed female pigs. Full-thickness skin resections of 50-100 cm(2)were performed on the dorsa of the animals. The defects were grafted with between one and six pieces of HRS under tied-over dressings. At day 14, we found a soft and smooth surface of good transparent healthy pink skin, which was very easy to distinguish from the surrounding tissues. The junctions between different pieces of living skin were not visible. Immunohistological studies with specific anti-human keratin 14 antibodies confirmed the graft take: 7 days after grafting the human epidermis was attached to the living dermis and showed good organisation with a basal cell layer and suprabasal cells; 28 days after grafting the human epidermis seemed to be replaced by pig epidermis. This study highlights the possibility of grafting large surfaces with HRS using a routine operating technique.