Human Skin Allografts Research Articles

Structural changes of skin and amnion grafts for transplantation purposes following different doses of irradiation

An important part of the preparation of biological material for transplantation is sterilization. The aim of our study was to assess the impact of ionizing radiation on three types of biological tissues and the impact of different doses on cells and extracellular matrix. Three types of frozen tissues (porcine skin xenografts, human skin allografts and human amnion) were divided into five groups, control and groups according to the dose of radiation to which these samples were exposed (12.5, 25, 35 and 50 kGy). The tissue samples were fixed by formalin, processed by routine paraffin technique and stained with hematoxylin and eosin, alcian blue at pH 2.5, orcein, periodic acid schiff reaction and silver impregnation. The staining with hematoxylin and eosin showed hydropic degeneration of the cells of epidermis in xenografts by the dose of 12.5 kGy, in human skin it was observed by the dose of 35 kGy. The staining for elastic fibers revealed damage of fine elastic fibers in the xenografts dermis by the dose of 12.5 kGy, in the allografts by 35 kGy. Another change was the disintegration of basement membrane of epithelium, especially in the human amnion at the dose of 50 kGy. The silver impregnation visualized nuclear chromatin condensation mainly in human amnion at the dose of 12.5 kGy. Our results have shown that the porcine xenografts and human amnion were more sensitive to irradiation than the human skin. In the next phase of the project we will focus at more detailed changes in the tissues using immunohistochemical techniques.

Severe adult burn survivors. What information about skin allografts?

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.

Evaluation of the Biocompatibility of a Bilayer Chitosan Skin Regenerating Template, Human Skin Allograft, and Integra Implants in Rats

Introduction. Chitosan is a nontoxic, biocompatible, and biodegradable polymer obtained from chitin by N-deacetylation using strong alkali. Chitosan in a form of a bilayer skin regenerating template can act as a scaffold for regeneration. Integra is a two-layer skin regeneration system, constructed of a matrix of crosslinked fibers that acts as a scaffold for regenerating dermal skin cells. Human skin allografts (HSAs) are the “gold standard” for temporary coverage of clean burn wounds. Objectives. The objective of this study was to conduct in-vivo, preclinical biocompatibility evaluations of Integra, HSA, and Chitosan skin regenerating template (SRT). Methods. Paravertebral subcutaneous pockets were created for the implantation of test materials. Implants were retrieved after 4, 7, 14, 21, and 28 days. Slides of sections through the implants were examined to determine biocompatibility. Results. Chitosan SRT and Integra showed similar inflammatory patterns. HSA showed a higher inflammatory reaction initially which then reduced to levels similar to Integra and Chitosan SRT. Chitosan SRT and Integra also shared similar angiogenesis levels. Towards the end, all implants were degraded with decreased tissue response. Conclusion. Integra, Chitosan SRT, and HSA have been shown to be biocompatible. Integra and Chitosan SRT seem to illicit similar tissue responses.

Glycerol treatment as recovery procedure for cryopreserved human skin allografts positive for bacteria and fungi

Human donor skin allografts are suitable and much used temporary biological (burn) wound dressings. They prepare the excised wound bed for final autografting and form an excellent substrate for revascularisation and for the formation of granulation tissue. Two preservation methods, glycerol preservation and cryopreservation, are commonly used by tissue banks for the long-term storage of skin grafts. The burn surgeons of the Queen Astrid Military Hospital preferentially use partly viable cryopreserved skin allografts. After mandatory 14-day bacterial and mycological culture, however, approximately 15% of the cryopreserved skin allografts cannot be released from quarantine because of positive culture. To maximize the use of our scarce and precious donor skin, we developed a glycerolisation-based recovery method for these culture positive cryopreserved allografts. The inactivation and preservation method, described in this paper, allowed for an efficient inactivation of the colonising bacteria and fungi, with the exception of spore-formers, and did not influence the structural and functional aspects of the skin allografts.

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.

Ex vivo-expanded human regulatory T cells prevent the rejection of skin allografts in a humanized mouse model.

Composite tissue transplantation effectively reconstructs the most complex defects, but its use is limited because of harmful immunosuppression and the high susceptibility of skin to rejection. Development of tolerance is an ideal solution, and protocols using regulatory T cells (Tregs) to achieve this have been promising in experimental animal models. The aim of this study was to investigate the ability of human Tregs to regulate immune responses to a human skin allograft in vivo. We isolated and expanded naturally occurring CD127loCD25+CD4+ human Tregs from peripheral blood mononuclear cells (PBMCs) and examined their phenotype and suppressive activity in vitro. Using a clinically relevant chimeric humanized mouse system, we transplanted mice with human skin grafts followed by allogeneic populations of PBMCs with or without Tregs derived from the same PBMC donor. Ex vivo-expanded Tregs maintain the appropriate Treg markers and retain suppressive activity against allostimulated and polyclonally stimulated autologous PBMCs in vitro. Mice receiving allogeneic PBMCs alone consistently reject human skin grafts, whereas those also receiving Tregs display stable long-term human skin transplant survival along with a reduction in the CD8+ human cellular graft infiltrate. We show for the first time the unique ability of human Tregs to prevent the rejection of a skin allograft in vivo, highlighting the therapeutic potential of these cells clinically.

A Retrospective Clinical Study of 188 Consecutive Patients to Examine the Effectiveness of a Biologically Active Cryopreserved Human Skin Allograft (TheraSkin ®) on the Treatment of Diabetic Foot Ulcers and Venous Leg Ulcers

A biologically active human skin allograft, currently distributed under the brand name TheraSkin(®), was examined for safety and efficacy in the treatment of venous leg ulcers (VLUs) and diabetic foot ulcers (DFUs). The objective was to determine if TheraSkin could serve as a safe and effective alternative to bioengineered skin substitutes such as Apligraf(®) and Dermagraft(®). The authors conducted a retrospective study of 214 consecutive patients seen at the Inova Wound Center (Mt Vernon, Virginia), with either a DFU or a VLU. After excluding patients who did not meet the study criteria, the final eligible cohort consisted of 188 subjects, with 134 VLUs and 54 DFUs. Multivariate logistic regression was used to evaluate the relationship between baseline wound size and the proportion of healed wounds after 12 and 20 weeks from initial allograft application. The authors found that by the 12th week, DFUs closed 60.38% of the time and VLUs closed 60.77% of the time. After 20 weeks, the number of closed DFUs increased to 74.1% and the number of VLUs increased to 74.6%. The mean wound size in the DFU group was 6.2 cm(2) (±11.8) and 11.8 cm(2) (±22.5) in the VLU group. The mean number of TheraSkin allografts required ranged from 1 to 8, with an average of 2.03 (±1.47) at the 12-week point and an average of 3.23 (±2.77) at the 20-week point. Multivariate logistic regression was used to calculate the odds of wound healing by week 12 and week 20 in each group. The authors also analyzed adverse events and found TheraSkin to be noncontributory to any adverse events, verifying the safety of TheraSkin in this study population. In this study, TheraSkin has been shown to be highly effective for the treatment of both VLUs and DFUs with an acceptable safety profile.

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.

Human skin allografts as a useful adjunct in the treatment of purpura fulminans

The aim of this report is to discuss the role of human skin allografts in surgical coverage procedures for patients with purpura fulminans. We describe cases of purpura fulminans in three adults and one infant treated at our burns unit between October 2006 and January 2008. The application of cryopreserved human skin allografts allowed us to obtain immediate wound closure after necrosis excision and enabled our team to subsequently perform autografts on favourable graft recipient sites. Recourse to human skin allografts must be considered a pertinent therapeutic option in patients with purpura fulminans.

Short- and long-term bacterial inhibiting effect of high concentrations of glycerol used in the preservation of skin allografts

Human skin allografts are important in the treatment of severe burns. Transplantation of skin allografts can cause bacterial transmission. Glycerol in higher concentrations is an appropriate storage medium for allograft cadaver skin and has been attributed an antimicrobial effect. We investigated this effect in more detail. First, the minimal inhibitory concentration of glycerol was determined for 13 bacteria and 1 yeast. This gives an indication about an immediate (20 h of incubation) antibacterial effect of glycerol. Second, effect of glycerol in the long-term was studied. Therefore, the survival time was determined for 11 different bacteria suspended in different concentrations of glycerol (50% and 85%) and incubated at three temperatures (4, 24, and 36 °C). The minimal inhibitory concentration exceeded 256 μg/mL, thus glycerol had no direct inhibitory effect. In contrast, a long-term antimicrobial effect was present and more pronounced at higher concentrations of glycerol and higher temperatures of incubation. The mean survival time of Pseudomonas aeruginosa strains in glycerol 85% at 24 °C was 2.6 days, 14.7 days for the tested staphylococci and 29.6 days for three vegetative Bacillus species. In conclusion, microbial safety of glycerol-preserved skin can be increased by preserving skin allografts for some weeks at room temperature.

Properties of Sterilized Human Skin Allografts by Gamma-Irradiation

The most commonly used sterilization method of human skin allografts is ethylene oxide (EO). EO gas, however, can change the biomechanical properties of the grafts and produce toxic residues which are harmful to the recipient. The heating method cannot be applied for sterilization of soft tissues because of damage due to heat. The purpose of this study was to determine whether it is possible to apply gamma-irradiation for sterilization of soft tissues as well as for hard tissues.

Assessment of organ culture for the conservation of human skin allografts

Human skin allografts are used in the treatment of severe burns and their preservation is therefore critical for optimal clinical benefit. Current preservation methods, such as 4 degrees C storage or cryopreservation, cannot prevent the decrease of tissue viability. The aim of this study was to assess viability and function of skin allografts in a new skin organ culture model, allowing conservation parameters as close as possible to physiological conditions: 32 degrees C, air-liquid interface and physiological skin tension. Twelve skin samples, harvested from 6 living surgical donors, were conserved 35 days in two conditions: conservation at 4 degrees C and organ culture. Viability and function of skin samples were investigated at Day 0, 7, 14, 21, 28 and 35 using cell culture methods (trypan blue exclusion, Colony Forming Efficiency and Growth Rate), histopathological and histoenzymological studies (Ki67 immunostaining). In the two conditions, fibroblast and keratinocyte viability was progressively affected by storage, with a significant decrease observed after 35 days. No statistical difference could be observed between the two conditions. The two methods were also comparable regarding alterations of fibroblast and keratinocyte culture parameters, which were respectively significantly reduced at Day 7 and 21, compared to fresh skin. By contrast, histopathological and histoenzymological studies revealed a better preservation of skin architecture and proliferative potential at 4 degrees C, as compared to organ culture. These results indicate that skin organ culture does not provide significant advantages for skin allograft preservation. However, its potential use as an experimental model to study skin physiology and wound healing should be further evaluated.

In vitro and in vivo characteristics of frozen/thawed neonatal pig split-skin strips: A novel biologically active dressing for areas of severe, acute or chronic skin loss

The recurrent shortage of human skin autografts or allografts used to close extensive wounds has rekindled the search for feasible alternatives. In the past, adult pig skin was a popular biological dressing, yet doubts regarding its benefits have induced most people to abandon its use. Here we investigated the aptness of neonatal pig split-skin (NPSS) strips to be used as a novel kind of temporary dressing for areas of skin loss. NPSS strips are able to be prepared in bulk amounts, stored at -80 degrees C for up to six months, and recovered by swift thawing at 45 degrees C with no change in histological structure. When set into organ cultures in vitro for up to three weeks, these frozen/thawed NPSS strips exhibited both a skin-typical energy-linked metabolism (i.e., a predominant consumption of L-glutamine instead of glucose), and an enduring ability to secrete cytokines/chemokines such as IL-1alpha, IL-6, GM-CSF, and TNF-alpha; all features alike in quantitative terms to those exhibited by freshly prepared NPSS strips directly set into culture. Moreover, once applied as temporary dressings onto deep burn wounds in vivo, frozen/thawed NPSS strips produced, for at least seven days, porcine IL-1alpha, IL-6, GM-CSF, TNF-alpha, and TGF-beta; the cytokines/chemokines importantly involved in wound healing. Hence, frozen/thawed NPSS strips not only are capable of closing extensive areas of skin loss, but even release several cytokines/chemokines beneficial to tissue regeneration and repair.

Flow Cytometric Viability Assessment and Transmission Electron Microscopic Morphological Study of Bacteria in Glycerol

Human cadaveric skin allografts are used in the treatment of burns and can be preserved in glycerol at high concentrations. Previously, glycerol has been attributed some antimicrobial effect. In an experimental set-up, we aimed at investigating this effect of prolonged incubation of bacteria in 85% glycerol. Staphylococcus epidermidis, Staphylococcus aureus, Pseudomonas aeruginosa, and Bacillus subtilis were incubated in 85% glycerol. The influence of duration of incubation and temperature on ultrastructure and viability were investigated. Unstressed cultures served as controls. Survival was studied after 24-36 h and 10 days incubation in 85% glycerol at 4 degrees C and 36 degrees C with transmission electron microscopy (TEM) and flow cytometry using viability stains indicating membrane damage (SYTO9, propidium iodide) or esterase activity (carboxyfluorescein diacetate). TEM clearly demonstrated variability in morphological changes of bacteria suggesting different mechanisms of damage. Viability stains supported these findings with faster declining viable cell populations in 85% glycerol at 36 degrees C compared with 4 degrees C. Both methods demonstrated that Gram-negative species were more susceptible than Gram-positive species. In conclusion, 85% glycerol may have some additional antimicrobial effect. Temperature is an important factor herein and Gram-negatives are most susceptible. The latter finding probably reflects the difference in cell wall composition between Gram-positive and Gram-negative bacteria.

Memory T Cells and Their Costimulators in Human Allograft Injury

Both CD4(+) and CD8(+) human memory but not naive T cells respond to allogeneic human dermal microvascular endothelial cells (HDMEC) in vitro by secreting cytokines and by proliferating. Several recently identified costimulators, namely, 4-1BB ligand, ICOS ligand, and OX40 ligand, are up-regulated on cultured HDMEC in response to TNF or coculture with allogeneic T cells. Blockade of these costimulators each partially reduces IFN-gamma and IL-2 secretion and proliferation of previously resting memory T cells. The effects of these costimulators are overlapping but not identical. Memory but not naive T cells are the principal effectors of microvascular injury in human skin allografts following adoptive transfer into immunodeficient mice. Furthermore, blocking 4-1BB ligand, ICOS ligand, or OX40 ligand in this model reduces human skin allograft injury and T cell effector molecule expression. These data demonstrate that human memory T cells respond to microvascular endothelial cells and can injure allografts in vivo without priming. Furthermore, several recently described costimulators contribute to these processes.

Performance and safety of skin allografts

Skin preservation for transplantation began almost 200 years ago with the pioneering work of Baronio (cited by in Ref. 2). Since that time, hundreds of papers have been published on the preservation of skin for later application in wound treatment. This interest stems from the excellent clinical results obtained with skin as a permanent autograft or temporary allograft for wound cover, coupled with the relative ease of preservation and storage methods. The general recognition of the need for human skin allografts has stimulated the establishment of banking facilities and research to improve the methods for harvesting, processing, storage, and subsequent evaluation of transplantation performance.

Mechanism of the immunosuppressive effect in vivo of novel immunosuppressive drug β-SQAG9, which inhibits the response of the CD62L + T-cell subset

Introduction We synthesized sulfo-glycolipid, β-SQAG9 (designate □ β-SQAG9 liposome, because it efficiently forms a liposome structure) that possessed immunosuppressive effects such as inhibition of T-cell responses in human allogeneic MLR and skin allograft survival in rats, and bound to CD62L (L-selectin) in vitro. In this study, we further investigated the immunosuppressive mechanism in vivo by β-SQAG9 liposome in a skin-allografted rat model. Methods ACI rats (RT1 a) were grafted skin of LEW rats (RT1 1) treated with PBS or β-SQAG9 liposome IV once a day for 7 days. Subsequently, we investigated the population of T cells and CD62L + T-cell subset in the spleen, axillary lymph nodes (ALNs), and peripheral blood of skin-allografted rats by two-color flow cytometry. Results Five of 11 (45.5%) rats that were treated with 50 mg/kg β-SQAG9 liposome showed graft survival and another showed moderate rejection in graft. The CD62L + T-cell subset population in ALNs of β-SQAG9 liposome-treated rats decreased in a dose-dependent manner. No significant difference in the T-cell population was observed between the β-SQAG9 and control groups. These data suggest that β-SQAG9 could bind to the CD62L + T-cell subset in vivo as well as in vitro and affect T-cell migration, which might lead to T-cell tolerance in vivo.

Treatment with β-SQAG9 prevents rat hepatic ischemia-reperfusion injury

Background Ischemia-reperfusion (I/R) injury occurs in various situations, including transplantation, trauma, and shock. We previously reported that the synthetic β-SQDG (18:0), which was derived from sulfoquinovosyl diacylglycerol of the sea urchin, possessed immunosuppressive effects, such as inhibition of T-cell responses in human allogenic human mixed lymphocyte reactions (MLR) and skin allograft survival in rats. β-SQAG9 was synthesized from β-SQDG (18:0) to improve structural stability in aqueous solution with the same biological activities to bind to CD62L (L-selectin) and CD62P (P-selectin) in vitro. We hypothesized that β-SQAG9 might attenuate leukocyte rolling on the endothelium and neutrophil infiltration in which L-selectin and P-selectin are key molecules. We investigated the protective effect of β-SQAG9 against hepatic I/R injury. Methods Male Lewis rats were divided into 6 groups: sham, control, and treatment. Rats in the control, and the treatment groups were subjected to hepatic ischemia for 30 minutes. They were injected with PBS or β-SQAG9 at doses of 5, 10, 25, and 50 mg/kg into the penile vein immediately before reperfusion. To assess the damage to the hepatic parenchyma, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) were measured and histological evaluation was performed at 6 hours after reperfusion. Results In the group treated with β-SQAG9 at a dose of 10 mg/kg, AST, ALT, and LDH were significantly reduced, and the amount of neutrophil infiltration also was significantly reduced. Conclusions Our data suggest that SQAG-9 (10 mg/kg) reduces the warm hepatic I/R injury.

Inhibition of CD62L + T-cell response in vitro via a novel sulfo-glycolipid, β-SQAG9 liposome that binds to CD62L molecule on the cell surface

We previously reported that synthetic sulfo-glycolipid, 3- O-(6-deoxy-6-sulfono-β- d-glucopyranosyl)-1,2-di- O-acylglycerol (β-SQDG(18:0)) which was deduced from sulfonoquinovosyl-diacylglycerols of sea urchin possessed immunosuppressive effects, such as human mixed lymphocyte reaction (MLR) and skin allograft in rat, and that these effects were caused by contact inhibition between T-cells and antigen presenting cells (APCs). Here, we investigated the mechanism of these immunosuppressive effects on human MLR by β-SQAG9 which had been newly synthesized from β-SQDG(18:0) to improve structural stability in water solution. CD62L + T-cells in peripheral blood predominantly respond to APCs, and β-SQAG9 inhibited the response of CD62L + T-cell subset in human allogeneic MLR. Surprisingly, it was demonstrated that β-SQAG9 bound to L- and P-selectin (CD62L and P) molecule in vitro. Meanwhile, β-SQAG9 efficiently formed liposome structure and bound to L-selectin on the cell surface of CD62L + T-cell subset but might not be incorporated into the cells. Because the immunosuppressive effects of β-SQAG9 disappeared when β-SQAG9 liposome was changed to soluble form by detergent, the liposome structure of β-SQAG9 was presumed to be essential for these effects. These findings suggested β-SQAG9 to be a novel drug with a unique immunosuppressive action.

Skin graft of double transgenic pigs of N-acetylglucosaminyltransferase III (GnT-III) and DAF (CD55) genes survived in cynomolgus monkey for 31 days

Background Our previous study reported that cynomolgus monkey did not hyperacutely reject a skin xenograft from a N-acetylglucosaminyltransferase III (GnT-III) transgenic pig. In the present study, we reported on the survival time of skin xenografts in GnT-III, DAF (CD55), and double (D/G) transgenic pigs, and the effect of FK506 thereon. Material and methods Skin from GnT-III, DAF and D/G transgenic pigs were transplanted to cynomolgus monkeys. Under general anesthesia, full thickness skin defects (1.5×1.5 cm each) were made on the back of the monkey. Pig abdominal skin, obtained using an electric dermatome, was cut into pieces and transplanted onto the monkey wounds and fixed. In addition, skins of GnT-III and D/G pigs were also transplanted to cynomolgus monkeys that had been treated intramuscularly with FK506 at a dose of 0.5 mg/kg/day for 14 days after transplantation. Grafts were observed and photographed each day and skin graft biopsies were done on days 3, 5, 7, 10, 11, 14, 21, 28 and 31 after transplantation. Graft rejection was assessed histologically, based on our previous criteria for skin allografts. Results Even in the immuno-suppressive drug free condition, skin xenografts of GnT-III, DAF and D/G transgenic pigs were not hyperacutely rejected in early phase after transplantation by the cynomolgus monkey. The pattern of these xenograft rejections was histologically similar to those for rat allograft rejections. Most of the GnT-III, DAF and D/G pig skin grafts remained nearly intact up to day 5, but slight lymphocyte infiltration was noted on day 7 (grade 1). On day 9, while the GnT-III skin showed moderate lymphocyte and eosinophilic infiltration, the DAF and D/G pig skin grafts showed complete epidermal separation (grade 3). On the other hand, in the case of cynomolgus monkeys treated with FK506, the GnT-III skin showed complete epidermal separation (grade 3) on day 21. In addition, one of the D/G skin graft was intact on day 21 and moderate lymphocyte infiltration and intraepidermal blister formation (grade 1) was finally seen on day 31. Conclusion Our data show the possibility that both the DAF and GnT-III double transgenic pig skin xenografts can be used in place of human skin allografts in cases of severe burns.