Mohamed Jarraya

Viability of cryopreserved human skin allografts: effects of transport media and cryoprotectant.

Human skin allografts can be preserved by different methods. In our clinical practice, human skin allografts are harvested on multi-organ and tissue donors, transferred at +4°C in Ringer Lactate, cryopreserved with 15% Glycerol and held in the vapor phase of a liquid nitrogen freezer until delivery to the burn center. The aim of this experimental study was to evaluate the impact of transport medium and cryoprotectant on the viability of human skin allografts. For this purpose, we compared skin samples harvested from 19 multi-organ and tissue donors with two different transport media and two different cryoprotectants. Viability was assessed by the MTT assay after harvesting at laboratory reception, during storage (at +4°C) at day 2 and day 7, and after cryopreservation and thawing. Histopathological analysis was performed for each MTT assay. Results indicate that, when stored at +4°C, skin retains more viability with RPMI, whereas Glycerol and DMSO are equivalent cryoprotectants regardless of the transport medium. In conclusion, our protocol could be improved by the utilization of RPMI as transport medium.

Amniotic membrane grafts for the prevention of esophageal stricture after circumferential endoscopic submucosal dissection.

Background and Aims The prevention of esophageal strictures following circumferential mucosal resection remains a major clinical challenge. Human amniotic membrane (AM) is an easily available material, which is widely used in ophthalmology due to its wound healing, anti-inflammatory and anti-fibrotic properties. We studied the effect of AM grafts in the prevention of esophageal stricture after endoscopic submucosal dissection (ESD) in a swine model. Animals and Methods In this prospective, randomized controlled trial, 20 swine underwent a 5 cm-long circumferential ESD of the lower esophagus. In the AM Group (nu200a=u200a10), amniotic membrane grafts were placed on esophageal stents; a subgroup of 5 swine (AM 1 group) was sacrificed on day 14, whereas the other 5 animals (AM 2 group) were kept alive. The esophageal stent (ES) group (nu200a=u200a5) had ES placement alone after ESD. Another 5 animals served as a control group with only ESD. Results The prevalence of symptomatic strictures at day 14 was significantly reduced in the AM group and ES groups vs. the control group (33%, 40% and 100%, respectively, pu200a=u200a0.03); mean esophageal diameter was 5.8±3.6 mm, 6.8±3.3 mm, and 2.6±1.7 mm for AM, ES, and control groups, respectively. Median (range) esophageal fibrosis thickness was 0.87 mm (0.78–1.72), 1.19 mm (0.28–1.95), and 1.65 mm (0.7–1.79) for AM 1, ES, and control groups, respectively. All animals had developed esophageal strictures by day 35. Conclusions The anti-fibrotic effect of AM on esophageal wound healing after ESD delayed the development of esophageal stricture in our model. However, this benefit was of limited duration in the conditions of our study.

Technical note: comparison of the PrestoBlue and LDH release assays with the MTT assay for skin viability assessment

MTT assay is the gold standard for assessing skin sample viability but it is time-consuming. Here we compared the MTT test with two other assays for the assessment of skin viability. The MTT, PrestoBlue (colorimetric method) and LDH release assays were applied to fresh and cryopreserved skin. Skin viability was considered proportional to the optical density values of the relevant analytes. PrestoBlue did not reliably distinguish between fresh and cryopreserved skin. The LDH release assay did not allow us to establish a viability index. We recommend the MTT assay for assessing skin viability.

Viability and Efficacy of Coverage of Cryopreserved Human Skin Allografts in Mice

Human skin allografts are considered one of the best temporary biological coverages for severe burn patients. Human skin allografts can be either viable or nonviable depending on their preservation modalities. However, there is a debate about the use of viable versus nonviable skin for severe burn patients because there is no established correlation between viability and efficacy of coverage. The authors tried to correlate the viability of cryopreserved human skin allografts as assessed by the MTT assay, with efficacy of coverage, intensity of rejection at day 8, and delay of wound healing in a xenograft model using human fresh skin (FS) and cryopreserved skin (CPS) on murine recipients (n = 49). Cryopreserved grafts were less rejectable than fresh grafts, with statistically significant different delays (P = .0008). Mice that had received grafts healed with delays; the delays, whether associated with fresh grafts or cryopreserved grafts, were not statistically significant. On day 8 after the graft, the overall damage score for the tissue’s histological architectural integrity was higher for FS. Furthermore, flow cytometry analysis showed a significant increase in the number of CD4 and CD8 T-cells (P = .001) in the spleens of FS-grafted mice. These results confirm that the use of viable CPS does not change the potential for healing.

Cryopreserved human skin allografts: viability and delay of rejection in severely burned patients.

0305-4179/

Skin donors and human skin allografts: evaluation of an 11-year practice and discard in a referral tissue bank

36.00 # 2012 Elsevier Ltd and ISBI. All rights reserved. reproducibility and reliability have been successfully reported [1–3]. A debate exists regarding the importance of using viable, less viable or even non-viable allografts in order to guarantee full function of skin substitute. Currently, there is no established correlation between viability, efficacy of covery, intensity of rejection, and delay of healing in burned patients. Some practitioners consider viability essential for better engraftment or to improve tissue granulation, while others prefer a non-viable product for reasons of storage convenience and cost (storage at ambient temperature versus storage in b u r n s 3 8 ( 2 0 1 2 ) 6 1 4 – 6 1 8 616

Severe adult burn survivors. What information about skin allografts

The Saint Louis hospital tissue bank provides skin allografts to pediatric and adult burn units in the Paris area. The aim of this study was to analyze our activity during the last 11xa0years focusing on the reasons for skin discard. Skin is procured solely from the back of the body, which is divided into 10 zones that are harvested and processed separately. This retrospective study included all skin donors harvested between June 2002 and June 2013, representing a total of 336 donors and 2770 zones. The donors were multiorgan heart-beating donors in 91xa0% of cases (nxa0=xa0307). The main reason for discarding harvested skin was microbial contamination, detected in 99 donors (29xa0%). Most contaminants were of low pathogenicity. Other reasons for discard included positive serologic tests for 2 donors [17 zones (0.61xa0%)], unsuitable physical skin characteristics for 3 zones (0.11xa0%), the donor’s medical history for 53 zones (1.91xa0%), and technical issues with processing or distribution for 61 zones (2.2xa0%). In our experience, microbial contamination continues to be the main reason for discarding potential skin allografts. However, discards are limited by separate harvesting and processing of multiple zones in each donor.

Anti-HLA sensitization: should we abandon skin allografts for extensively burned patients?

During the acute phase of a severe burn, surgery is an emergency. In this situation, human skin allografts constitute an effective temporary skin substitute. However, information about the use of human tissue can not be given to the patients because most of the allografted patients are unconscious due to their injury. This study explored the restitution of information on skin donation to patients who have been skin allografted and who have survived their injury. A qualitative study was conducted due to the limited number of patients in ability to be interviewed according to our medical and psychological criteria. 12 patients who had been treated between 2002 and 2008 were interviewed. Our results show that 10 of them ignored that they had received skin allografts. One of the two patients who knew that they had received allografts knew that skin had been harvested from deceased donor. All patients expressed that there is no information that should not be delivered. They also expressed their relief to have had the opportunity to discuss their case and at being informed during their interview. Their own experience impacted their view in favor of organ and tissue donation.

Human skin allograft: storage medium at 4°C and viability.

• Origin of anti-HLA antibodies All 29 patients were transfused, and 14 of those who did not receive skin allografts developed HLA sensitization. The remaining 15 patients were both transfused and grafted: seven received glycerol-preserved allografts (GPSA), three received cryopreserved skin allografts (CPSA), and five received both types of allograft. They all developed HLA sensitization, except for one patient grafted with GPSA. A recent study of primary kidney transplant candidates showed a substantial risk of HLA sensitization among transfused patients (transfused with leukoreduced blood), compared to nontransfused patients [2]. The 100% rate of HLA sensitization observed by Duhamel et al. among nonallografted patients might be explained by massive and repeated transfusions (36 13 units of packed red blood cells per patient). As mentioned by the authors themselves, they did not determine whether HLA sensitization was due to skin allografting (15/29) or blood transfusion (29/29). • Contribution of allografts to HLA sensitization and quality of coverage Duhamel et al. found that GPSA seemed less likely than CPSA to induce HLA sensitization. They cited Cinamon et al. [3] and Richters et al. [4], but neither team provided information on the respective contributions of CPSA and GPSA to HLA sensitization. The quality of coverage is still debated [5]. Duhamel et al. referred to three studies: Dhenin et al. [6] reported their clinical experience with GPSA; Wachtel et al. [7] compared fresh allografts and two types of thawed CPCA in a clinical study; and Cinamon et al. [3] found that CPSA gave better coverage than GPSA in mice. None of these studies compared CPSA vs. GPSA in humans. A recent study by Kua et al. [8] showed a trend towards lower mortality and shorter hospital stays with CPSA vs. GPSA in severely burned patients. Nevertheless, human studies comparing the immune responses to CPSA and GPSA remain to be carried out. • Vascularized composite allotransplantation We fully agree that HLA sensitization must be avoided, when possible, to keep open the option for vascularized composite allotransplantation (VCA). But, in the acute phase of extensive burn injury, priority must be given to immediate survival and effective coverage. Predictive criteria could help to optimize the later use of VCA. A multiparameter scale needs to be established, including the percentage of total burn surface area, the affected body areas, and blood transfusion. A threshold value would be useful to judge whether the risk of sensitization is justified by the need for coverage: for example, concerning the recommended use of CPSA for wound-bed preparation before the application of cultured autologous keratinocytes [9]. The study by Duhamel et al. should further encourage the development of skin substitutes able to provide effective coverage during the acute phase of burn injury without causing HLA sensitization.

Cryopreserved human skin allografts: Efficacy and viability

We read with interest the article in press by Boekema et al. [1], in which the Authors aimed ‘‘to compare the effect of different storage solutions on the viability of SGG’S in time, based on metabolic activity and histology’’. They tested viability on skin pieces (3 T 5 cm) obtained from 8 living donors undergoing abdominoplasty, and 7 deceased donors. Pieces were submerged in three different storage solutions (0.9% NaCl, DMEM/ F12, and RPMI) at +4 8C, at days 0, 3, 7, 10, 14 and 21, and media were not refreshed during all the study. Viability was assessed on two or three punch-biopsy disks (4 mm) by MTT assay, and expressed as a viability index ‘‘using the following formula: (OD sample day x – OD negative control) divided by (OD sample day 0 – OD negative control)’’. Results showed a similar level of viability index with DMEM and NaCl, but lower than that obtained by storage in RPMI. We would like to draw your attention to several aspects. In terms of percentage of residual viability, the rates reported in this paper are very interesting given that they are higher than all those already published. When skin is stored at +4 8C, the viability decay is usually reported as being major during the first two days, irrespective of the storage medium [2–4]. Herein, first assessments of viability were performed at day 3. With RPMI, rate was near 95% at day 3, then 78% at day 7, more than 70% at day 14, and still near 50% at day 21. With RPMI, Gaucher et al. [4] found 87% at day 2 and 60% at day 7 without changing medium. Always with RPMI, Castagnoli et al. [3] reported less than 70% at day 2, near 50% at day 7, and almost 20% after day 15 despite changing medium every three days. As surprising as it may seem, the Authors chose not to measure the weight of each skin punch-biopsy, nor to take it into account for viability index calculation, because ‘‘variations in thickness and size was minimal and most of the activity is related to keratinocytes’’. However, in other studies the weight of the punch-biopsy was shown to be of high variability despite a standardized procedure of harvesting. These studies focused on the fact that it was desirable to take into account the weight value for viability index calculation [2–4], with a possible