Malka Chaouat

How long can cryopreserved skin be stored to maintain adequate graft performance

Skin graft preservation for the purpose of delayed application is still a basic tool in burn treatment and plastic and reconstructive surgery. As the demand for skin allografts has increased the responsibility for processing, storage and evaluation of graft performance of preserved skin has become an important issue of banking organizations. The present experiments were undertaken to determine how long can cryopreserved cadaveric skin be stored to maintain adequate graft performance? We applied a mouse recipient model, developed by us: Human cadaveric skin cryopreserved and stored for 5,6 or 7 years was grafted on Balb/c mice, and primary take was evaluated by gross observation and predetermined histologic criteria after 7 days. The results demonstrate that graft performance of cryopreserved skin decreased with time, as reflected in the lower percent of samples with high score of separate histologic criteria after prolonged storage. Nevertheless, paired comparison analysis between cryopreserved and fresh skin indicated that this decrease was not significant for storage of 5 years; whereas it was highly significant for 6 years of storage. Linear regression analysis indicated that there was no correlation between the score of the histologic criteria and storage period for upto 65 months. These results are in line with the paired comparison analysis. We feel that our in vivo model and analysis may be used as an evaluation procedure for transplantation performance of banked skin.

Cryopreserved cadaveric allografts for treatment of unexcised partial thickness flame burns: clinical experience with 12 patients

Partial thickness burns (PTB) usually heal within 3 weeks. Prevention of infection and desiccation of the wounds are crucial for optimal healing. Early tangential excision of the burn eschar and allografting prevent deepening of the burns, and are therefore advocated for treatment with the best functional and aesthetic results. For superficial partial thickness burns (SPTB) conservative use of topical antimicrobial agents with frequent dressing changes are implemented. We compared the conservative treatment for PTBs and SPTBs to grafting cryopreserved cadaveric allografts with no prior excision. Twelve patients with flame PTB areas were allografted after mechanical debridement without excision of the burn wounds. The allografts were cadaveric skin cryopreserved by programmed freezing and stored at -180 degrees C for 30-48 months. Matching burns for depth and area were treated with silver sulfadiazine (SSD) one to two times daily until healing or debridement and grafting were required. It was found that 80 per cent of the cryopreserved allografts adhered well and 76 per cent of the treated areas healed within 21 days, whereas only 40 per cent of the SSD-treated burns healed within 21 days. Partial thickness burns can be treated successfully with viable human allografts (cryopreserved cadaveric skin) with no prior surgical excision. The burn wounds heal well within 3 weeks. For deep partial thickness burns (DPTB) treatment with allografts has no advantage if they have not been previously excised.

Tyrphostins suppress the growth of psoriatic keratinocytes

Abstract Tyrosine kinase inhibitors of the tyrphostin family which block EGF receptor kinase are reported to arrest the growth of psoriatic keratinocytes in vitro. Three tyrphostins with the potency ratio AG555 >> AG18 >> AG814 were found to arrest growth with no adverse cytotoxic effects. The potency ratio to inhibit keratinocyte proliferation follows their potency to inhibit EGF receptor kinase activity in vitro. These compounds represent novel leads for the therapy of psoriasis.

Superoxide dismutase (SOD) for mustard gas burns.

Mustard gas (MS) has been used in chemical warfare since World War I. The blistering skin lesions are slow to heal. Secondary inflammation might occur, as well as damage to organs distant from the original wound. Presently there is no specific antidote for burns and poisoning by MS. This study examined treatment modalities with free oxygen radical scavengers, copper-zinc, and manganese superoxide dismutase (SOD), for MS skin burns in an experimental guinea pig model. Each of the SOD compounds reduced dramatically burn lesion area when administered intraperitoneally/intralesionally (i.p./i.l.) before wound infliction. The protective action of the SODs was also evident in the significantly higher histopathological score of biopsies obtained on day 7 from local tissue, caused with the lower dose of MS. When the SOD compounds were administered i.p. 1 hour after burn infliction, and repeated daily for 7 days, no protective effect could be detected under the present experimental conditions.

A simplified testing system to evaluate performance after transplantation of human skin preserved in glycerol or in liquid nitrogen.

We have designed and tested a mouse recipient model for evaluation and direct comparison of skin-preservation procedures. Glycerolized skin was compared with cryopreserved and fresh cadaveric skin. Human skin samples were grafted on Balb/c mice, and primary take was evaluated after 4 and 7 days. The results demonstrate that although all grafted specimens were initially accepted as indicated by gross observations, histologic differences were evident and significant. Cryopreserved skin grafts performed better than did glycerolized skin even after a transplantation period as short as 4 days; this difference became even more pronounced after 7 days. Both methods of preservation provided a less successful product than did fresh viable cadaveric skin. However, for very short periods of grafting the performance of glycerolized skin might be considered adequate (79% compared with cryopreserved). Because the present study used an immunocompetent xenogeneic model, it is possible that the period of adequate allogeneic grafting will be prolonged in patients who are immunosuppressed. The present model is simple to use, requires a minimum of maintenance and expertise, and has inherent clinical relevance, because it is concerned primarily with the clinical performance of the skin. Thus it may be used as a quality control test for banked skin.

Transplantation Performance of Human Skin Cryopreserved by Programmed or Stepwise Freezing and Stored at −80° C or −180° C

We developed a mouse recipient model that was used to evaluate and compare four cryopreservation procedures for human cadaveric skin stored for two time periods. Skin specimens were identically processed and preserved by programmed (1 degree C/min), or stepwise freezing, and stored at -180 degrees C or -80 degrees C for periods of 1 month and 6 to 10 months. Samples were grafted on Balb/c mice, and primary take was evaluated after 7 days. The results indicate that although all grafted specimens were initially accepted, as indicated by gross observations, histologic differences were evident and significant. The study groups were analyzed for the effect of method and skin sample variety; the effect of freezing procedure and temperature level; time effect (storage period); and advantage of method 1 (programmed freezing at -180 degrees C) over the other methods. The significance (p value) was determined for separate histologic criteria and average skin score or quality. The overall results indicate that average score of skin preserved by method 1 is highest for both storage periods. This method has an almost significant advantage (p = 0.057) over the others on quality of skin stored for 1 month, and a highly significant advantage (p = 0.007) on graft adherence of skin stored for 6 to 10 months. The effect of method and samples variety on the separate histologic criteria and average score of skin is not always significant. However, an interaction factor (between method and samples) has a highly significant effect (p < 0.001) on almost all of the histologic criteria and average skin score. The effects of freezing method is significant only on average skin score, for 1 month of storage; whereas temperature effect is seldom significant. Evaluating the effects of time, samples, and the interaction factor (between time and samples) indicated that the interaction factor is highly significant (p < 0.001). Time and samples effects are rarely significant. Thus the quality of the final product-the cryopreserved skin-is determined by many factors, and quite often they interact. Highly significant is the combined effect, or interaction factor, of sample variability with method of cryopreservation or with storage period.

Comparison of topical iodine and silver sulfadiazine as therapies against sulfur mustard burns in a pig model.

Sulfur mustard (SM) is a powerful vesicant used as an agent of chemical warfare. The severity of lesions incurred after exposure to SM reiterated the need for an efficient and rapid neutralizing agent against SM. Previous studies have shown that postexposure treatment with iodine is effective against SM lesions in rodents. In the current study we used the pig model to emulate SM‐induced burn lesions, and observed the immediate effect of a single dose of iodine formulation treatment on these burns. SSD, a common agent recommended for use in both chemical and thermal burns was used as control. Results indicated that 1.27 mg of SM caused deep lesions and histopathological changes in the pig skin as scored in the biopsies obtained. A single application of an iodine formulation 20 minutes from exposure to SM exhibited no protective action on the skin as evident in the biopsies obtained 1, 3, 5, 10, and 21 days after treatment. SSD treatment induced the least protective action. The SSD‐treated wounds also took the longest to heal. Attempts to neutralize the SM action with iodine compounds were not successful in the pig model. Currently, other compounds are being investigated. Attention must be drawn to the adverse effect of SSD on SM‐induced wounds. Further studies must be initiated to elucidate this phenomenon.

Phosphorous pentachloride chemical burn - a slowly healing injury

A 51-year-old chemical engineer sustained phosphorous pentachloride partial skin thickness burns over 20 per cent of his body surface area. Although macroscopically and microscopically the wound seemed to be superficial, the course of clinical healing of this injury was very slow and painful. Retrospectively this burn should have been treated by early excision and grafting.

Cyclosporin A treatment failed to extend skin allograft survival in two burn patients

Prolongation of skin allograft survival by immunosuppression of burn casualties has been reported sporadically during the past two decades. Recently cyclosporin A (CycA) has been used effectively for such an indication. We report here two paediatric patients with extensive burns (85-95 per cent BSA) treated with fresh, family-related skin allografts that were rejected during CycA treatment after 14-18 days. One of these children survived while the other died with candida sepsis.

Plasminogen activator activity in differentiating rat myoblasts

SummaryPrimary cultures of skeletal muscle cells secrete plasminogen activator (PA) activity to the conditioning medium and display membrane-bound PA. Growth of these cells in culture in presence of 10-7 M dexamethasone resulted in a marked reduction of the membranal and secreted PA activity. The hormone also reduced cytosolic creative phosphokinase (CPK) activity and cytosolic protein content. However, cell viability and their ability to undergo fusion were uneffected. The extent of hormone-induced reduction in PA activity depended on the time and extend of exposure. Maximal suppression was obtained by exposing the cells to dexamethasone during the first 4 days of culture. The medium conditioned with dexamethasone-treated cells did not inhibit plasmin, endogenous PA or exogenous PA. Exposure of the conditioned medium from hormone-treated cells to sodium dodecyl sulphate (SDS) or trypsin restored the activity to values observed in media from cells not exposed to the hormone. Acidification of the medium failed to reactivate the enzyme. The myogenic cell line L-8 also displayed membrane-bound PA activity, which was of a comparable magnitude in both fusing and non-fusing L-8 cells. However, in contrast to the primary cultures, exposure of L-8 cells to dexamethasone had no effect on their PA activity whether studied under conditions which allowed or prohibited fusion. The present findings imply that PA has no conducive role in the process of fusion associated with maturation of skeletal muscle cells.