Cell Tissue Bank (2014) 15:429–433 DOI 10.1007/s10561-013-9407-8
ORIGINAL PAPER
Structural changes of skin and amnion grafts for transplantation purposes following different doses of irradiation H. Mra´zova´ • J. Koller • G. Fujerı´kova´ P. Baba´l
•
Received: 20 August 2013 / Accepted: 9 November 2013 / Published online: 21 November 2013 Springer Science+Business Media Dordrecht 2013
Abstract 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, H. Mra´zova´ (&) G. Fujerı´kova´ P. Baba´l Department of Pathology, Comenius University, Bratislava, Slovak Republic e-mail:
[email protected] J. Koller Department of Burns and Reconstruction Surgery, Faculty of Medicine, Comenius University, Bratislava, Slovak Republic
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. Keywords Skin Xenografts Allografts Amnion Sterilization Irradiation
Introduction Temporary coverage by skin substitutes is important for correct and faster healing of burns and other skin defects where permanent coverage is not possible. A broad spectrum of biological and synthetic materials are used for the purpose of acceleration of wound healing, lowering of pain and serving as physiological barrier decreasing losses of water, minerals and proteins through the open wound surfaces (Ge et al. 2011). In order to increase the safety of biological materials for the purpose of transplantation, an important step following their preparation is sterilization to prevent transfer of infectious diseases. Sterilization is supposed to eliminate all living microorganisms
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including bacterial spores and to provide the sterility assurance level (SAL) of 10-6. This can be reached by exposure of the tissue transplants to physical factors, such as hot air, saturated water vapors, ionizing radiation, plasma, or chemical substances like peracetic acid, ethylene oxide, glutaraldehyde, formaldehyde, etc…) (Matthews et al. 1994; USP 30-NF 2007). The procured tissues are during processing in tissue establishments exposed to cocktails of antibiotics, which by themselves are not sufficient for thorough decontamination. Consequently the tissue grafts intended for clinical use should undergo sterilization with either by chemical methods or by ionizing irradiation. For chemical sterilization mostly ethylene oxide, propylene glycol and peracetic acid are used (Ghosh et al. 1997; Lomas et al. 2004; Rooney et al. 2008). The use of peracetic acid with glycerol or propylene glycol appears to be the most gentle method among the sterilizations of biological materials. In such combinations their cytotoxicity and production of proinflammatory cytokines is reduced when compared with the use of glycerol or propylene glycol alone (Lomas et al. 2004). To avoid toxicity of the used chemicals, it appears more suitable to use sterilization by irradiation. This was used for the first time and patented in 1921 for the purpose of sterilization (Boyd 2002). Beta, gamma and X rays may be used, but gamma rays are preferred because of their excellent penetrance into tissues, no increase of temperature, no harmful residues, reduction of the antigenicity of tissues and little biological and mechanical changes (Bolgiani 2003). Thanks to its penetrance, the tissues can be sterilized in their packages, which eliminates repeated microbial contamination (Bourroul et al. 2002). The most common sources of gamma rays are cobalt (60Co) or cesium (137Cs). The internationally recommended dose for sterilization of medical devices is 25 kGy. For sterilization of tissues it is recommended to estimate first the bioburden of the tissue and thereafter, according to its result, calculate the appropriate irradiation dose assuring SAL 10-6. The main effect of irradiation is ionization of water molecules with formation of H3O? and OH-. Hydroxyl radicals seriously damage the DNA of microorganisms and have strong oxidative effects as well. Bacterial spores are more resistant ¨ zer because of their low water content (Berk and O 1999). Less sensitive to gamma radiation are also prions and some viruses, the concentration of which will be at
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routinely used dose of 25 kGy only reduced (Ge et al. 2011). The sensitivity of pathogens to gamma radiation may be modified by several factors, such as temperature, presence of oxygen and water. For example, elimination of HIV in deeply frozen plasma (-80 C) is effective by irradiation with the dose of 50–100 kGy, which on the other hand leads to denaturation of collagen fibers in tissues (Ge et al. 2011; Hiemstra et al. 1991). At the temperature of 15 C there is reduction of HIV already at the dose of 25 kGy (Hiemstra et al. 1991). Another problem represents contamination of tissues by endotoxins of devitalized Gram negative bacterial bodies. For endotoxin inactivation doses above 50 kGy are required (Kairiyama et al. 2009). The present study was then conducted to assess the impact of ionizing radiation on three types of biological tissues and the impact of different doses on cells and extracellular matrix.
Materials and methods Three different types of biological tissue grafts (porcine skin xenografts, human skin allografts and human amnion grafts) were frozen following procurement by standard operating procedures of Central Tissue Bank, kept at -80 C and divided in five groups: a control (not irradiated) and 4 groups were exposed to gamma radiation at 12.5, 25, 35, 50 kGy, delivered by commercial 60 Co irradiation source. The tissues following thawing were fixed in 10 % formalin and processed by routine paraffin embedding technique. Histological slices 5 lm thick were stained with hematoxylin and eosin, alcian blue (ALC) at pH 2.5 (mucin staining), orcein (elastic fibers), PAS reaction (Periodic Acid Schiff reaction—neutral saccharides staining) and metenamine silver (collagen fibers staining). The slides were mounted in acryl medium and covered with cover slips (Table 1). The slides were evaluated by light microscopy in Eclipse 9i40 microscope (Nikon, Japan) with focus on fibrilar structures.
Results Each staining documented a different type of morphological changes in the structure of the irradiated
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Table 1 Postirradiation changes in three types of biological tissues Xenograft
Allograft
Amnion
Vacuolar degeneration of epithelial cells
???(12,5 kGy)
?(35 kGy)
-
Nuclear chromatin—condensation
?(12,5 kGy)
?(35 kGy)
???(12,5 kGy)
Disruption and loss of basement membrane density Decomposition of fine elastic fibers of papillary dermis
?(25 kGy) ??(12,5 kGy)
?(25 kGy) ?(35 kGy)
??(25 kGy) -
Fig. 1 Elastic fibers in dermis: Network of fine elastic fibers (arrows) in the subepidermal dermis in control porcine (Xe–C) and human (Al–C) skin; loss of the fine elastic fibers after
irradiation with 12.5 kGy and 35 kGy in the porcine xenograft (Xe-12.5) and human allograft (Al-35), respectively. Orcein staining, bar = 25 lm
tissues. Basic staining with hematoxylin and eosin showed hydropic (vacuolar) degeneration of the cells of epidermis in xenografts starting with the dose of 12.5 kGy, in human skin after the dose of 35 kGy. Orcein staining for elastic fibers disclosed damage and decomposition of fine elastic fibers in the papillary dermis of xenografts after the dose of 12.5 kGy and in allografts after 35 kGy (Fig. 1). Impregnation with silver salts (metenamine silver) detected condensation of nuclear chromatin in cells of epidermis and of the amniotic membrane already at the dose of 12.5 kGy, in xenografts appeared changes of nuclei by the dose of 12.5 kGy and in allografts by the dose of 35 kGy (Fig. 2). An additional change was observed in the basement membrane, which was mainly in the form of loosening of density and disruption of the fibers of
collagen. These changes in the amniotic membrane could be observed at the dose of 25 kGy, getting very clear at 50 kGy (Fig. 2). Stainings by alcian blue and PAS reaction did not uncover any additional changes in the irradiated tissues.
Discussion Biological materials used for transplantation purposes should be sterilized before use, unless they are procured and processed by aseptic techniques. Sterilization methods should aim to preserve their original tissue structure as much as it would be possible. Since the beginning of the 20th century there have been attempts to develop an optimal way of tissue processing that lead
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Fig. 2 Collagen of the basement membrane detected with silver salts impregnation is well detectable (arrows) in the control tissues (Xe–C, Al–C, Am–C), undergoes decline after irradiation with 25 kGy in porcine skin xenograft (Xe-25), human skin allograft (Al-25) and fragmentation in the amniotic
membrane (Am-25); picnosis of nuclei with condensation of nuclear chromatin is prominent in the amniotic membrane after irradiation (empty arrowhead). Impregnation with metenamine silver, bar = 50 lm
to development of a whole spectrum of sterilization methods (Bourroul et al. 2002). Approaches that included increased temperature proved to be unsuitable for treatment of biological materials. The best accepted method was the sterilization of allografts with ethyleneoxide. It is a volatile substance that does not change the biomechanical properties of tissues but leaves cytotoxic and cancerogenic residues. These residues have to be removed before application (Nakheon et al. 2007) In contrast to that way of sterilization the application of ionizing radiation does not lead to creation of any toxic residues (Nakheon et al. 2007). The study by Rooney et al. (Rooney et al. 2008) evaluated deeply frozen skin allografts mounted in four different concentrations of glycerol (20, 50 a 85 %) with consequent irradiation with the dose of 25 kGy
gamma rays. All specimens were kept at the temperature of -80 C except for the one in 85 % glycerol that was irradiated at the temperature between 30 and 40 C. Only the deep frozen tissue specimens mounted in 20 % glycerol remained without histological, cytotoxic or physical changes (Rooney et al. 2008). Glycerol at high concentrations (85 %) seems to be a suitable preservation medium that preserves the tissue structures by the way of equal distribution of water to all cells. It does not have any negative effect on collagen and elastic fibers of the dermis and it showed to be bacteriostatic. The last mentioned effect of glycerol, however, starts to appear after three to 4 weeks of treatment (Marshall et al. 1995; Richters et al. 1996). This period can be shortened by consequent irradiation with ionizing radiation. Despite of
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the expectations for gamma radiation to be a gentle method for biological materials sterilization, it does not leave the tissues without changes. Irradiation, in addition to structural changes, causes devitalization of viable tissue grafts. The most affected tissue structure is the collagen type I. There is reduction of single collagen fibers and formation of abnormal fascicles (Tzaphlidou 2002). The effects of gamma radiation are direct and indirect, based on the water content. The absence of water may even lead to breaking of collagen polypeptide chains, which is defined as the direct effect. With the presence of water in irradiated tissue the gamma irradiation leads to hydroxyl radicals formation that causes cross linking of collagen fibers as an indirect effect (radiation 2012). There has been proved already, that ionizing irradiation for sterilization purposes does change to certain extent some of the properties of the temporary biological skin substitutes. The most important changes include loss of graft viability, irradiation dose dependent impact on their structural integrity and tensile strength, as well as damage or inactivation of important biological substances (such as cytokines and growth factors) contained in the grafts (Rooney et al. 2008). However, many other beneficial properties of the grafts including their barrier functions preventing wound contamination and decreasing both discharge and evaporative water loss from open wounds substantiate their use in situations where temporary coverage of open wounds is indicated (Ge et al. 2011). Radiation sterilized grafts can offer less expensive but still beneficial alternatives for wound coverage materials in health care systems with limited resources. Our results showed that the ionizing radiation in relation to its dose imposes structural changes in the irradiated materials, especially in the epidermis. Skin xenografts and amnion grafts show degenerative changes of epithelial cells already at the lowest studied dose of 12.5 kGy while in the skin allografts similar changes were observed at the dose of 35 kGy. In the papillary dermis there are changes of the extracellular matrix. These changes are crucial as it might affect the use of biological materials in their clinical application. For this reason in the future we will focus our attention to a more detailed specification of extracellular matrix structural modification, with the use of immunohistochemical techniques targeting particular components of the connective tissue.
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