Preparation and characterization of a novel skin substitute.

Autologous epidermal cell cultures (CEA) represent a possibility to treat extensive burn lesions, since they allow a significative surface expansion which cannot be achieved with other surgical techniques based on autologous grafting. Moreover currently available CEA preparations are difficult to handle and their take rate is unpredictable. This study aimed at producing and evaluating a new cutaneous biosubstitute made up of alloplastic acellular glycerolized dermis (AAGD) and CEA to overcome these difficulties. A procedure that maintained an intact basement membrane was developed, so as to promote adhesion and growth of CEA on AAGD. Keratinocytes were seeded onto AAGD and cultured up to 21 days. Viability tests and immunohistochemical analysis with specific markers were carried out at 7, 14, and 21 days, to evaluate keratinocyte adhesion, growth, and maturation. Our results support the hypothesis that this newly formed skin substitute could allow its permanent engraftment in clinical application.

Evaluation of donor skin viability: fresh and cryopreserved skin using tetrazolioum salt assay.

Cell viability assessment in allograft skin is an essential step to ensure a supply of good quality allograft skin for clinical repair of wounds. It is widely recognised that 'take' of allografts is strongly influenced grafted by tissue viability. The aim of this study was to set-up storage protocols that maintain high viability of the allograft after harvest, treatment and storage. In this study, the viability of post-mortem allografts (n=350) harvested from 35 different donors, was investigated using the MTT salt assay. The conditions of preparation and storage of the allograft included: 1. Fresh skin samples (about 12, 30, and 60h after harvesting). 2. The same specimens (stored at 4 and 37 degrees C) tested for at least 1 month. 3. Samples after cryopreservation and thawing. 4. Thawed specimens tested daily for at least 6 days. Parallel histomorphological analysis performed, under each of these conditions, showed a correlation between changes in structure and changes in viability as measured by the MTT quantitative assay. The viability index (VI) of skin is expressed as the ratio between the optical density (O.D.) produced in the MTT assay by the skin sample and its weight in grams. The percentage viability index is the ratio of the VI of the fresh sample (considered as 100% viability) and the value of specimens from the same harvest batch after storage or cryopreservation. The results indicated that samples tested within 12-30h from harvesting have an average viability index of about 75 with little variation. Samples tested within 60h have an average viability index of 40, showing a viability decrease of about 50%. A protocol to treat skin within a maximum of 30h was, therefore, set-up. The data suggested that skin stored at 37 degrees C, undergoes a viability increase during the first 2 days after harvesting. However, the viability under these conditions then decreased very quickly. After 6 days of preservation at this temperature the samples were no longer viable (PVI = 0). The tissue structure started to become damaged after 3 days. On the other hand, skin stored at 4 degrees C, showed a very slow viability decrease. After 15 days, viability was still almost 25% of the fresh sample. The tissue architecture showed no signs of damage under these conditions until day 7 from harvesting. MTT analysis was performed on the specimens cryopreserved with DMSO at 10%. These measurements were compared to viability assessment of the same fresh skin samples (considered as 100%) that were analysed within 30h from harvesting. The average PVI of thawed skin was 54% of the fresh sample. This result demonstrates that the viability of cryopreserved skin is comparable to the viability of fresh skin stored at 4 degrees C for 4 days. The PVI of thawed skin samples decreased dramatically within 24h, and had reached 0% within 6 days.