In group III, there was complete separation of the epidermal cells from the basement membrane. Collagen fibers were observed to be arranged in the same way as the control group. The papillary dermis was found to be composed of fine interlacing bundles. However, the reticular dermis was observed to be composed of coarse, branching, wavy bundles of collagen fibers. The dermis showed loss of most of the cellular components. However, some fields showed the presence of nuclear debris in between the collagen bundles (Fig. 3). Group III showed a highly significant decrease in the number of cells/hpf in both the papillary and the reticular dermis as compared with groups I and II (P < 0.001) (Table 1).
In group I, Mallory's trichrome-stained sections showed fine interlacing collagen bundles in the papillary dermis. The reticular dermis was composed of coarse, wavy collagen bundles running in different directions. Most of the bundles appeared to be of uniform diameter. There was a narrow zone of condensed collagen fibers just beneath the epidermis (Fig. 4).
Mallory's trichrome-stained sections of groups II and III showed a normal arrangement of collagen fibers in the dermis as the papillary dermis was composed of fine interlacing bundles and the reticular dermis was composed of coarse, wavy collagen bundles. The narrow zone of condensed collagen fibers just beneath the dermoepidermal junction was observed to be preserved; however, distortion of this zone was seen occasionally (Fig. 5a and b).
In aldehyde fuchsin-stained sections of group I, elastic fibers in the papillary dermis appeared in the form of a fine interlacing network of fibers. Very thin fibers appeared to be extending and perpendicular to the dermoepidermal junction, named the oxytalan fibers, and thicker transverse fibers, named the elaunin fibers, deep to the oxytalan fibers (Fig. 6).
In groups II and III, aldehyde fuchsin-stained sections showed preservation of the elastic fibers. In the papillary dermis, the oxytalan fibers appeared in the form of thin fibers extending and perpendicular to the dermoepidermal junction. In some areas, there were fewer oxytalan fibers that appeared kinked. The elaunin fibers appeared thick and arranged transversely deep to the oxytalan fibers (Fig. 7a and b).
In group I, immunohistochemical stain for the detection of laminin in the basement membrane showed a positive brownish reaction in the epidermal basement membrane and the basement membrane of the endothelial cells lining blood vessels (Fig. 8).
Immunohistochemical stain of laminin in groups II and III showed a positive brownish reaction in the epidermal basement membrane and the basement membrane of the endothelial cells lining blood vessels, indicating preservation of the laminin content and therefore preservation of the basement membrane (Fig. 9a and b).
Skin defects are one of the common medical problems that need to be treated using skin substitutes. Dermal substitutes are generally classified into three types. The first type utilizes a synthetic matrix consisting of a cross-linked bovine collagen matrix acting as a dermal substitute covered with a silicone membrane on its exposed surface or cultured keratinocytes. Neovascularization of this dermal substitute had been observed, but the resultant tissue resembles granulation tissue rather than a normal dermis .
The second type is made of a cryopreserved split-thickness cadaver skin allograft. Several days after grafting, the epidermis is removed by abrasion. The dermis of the allograft provides a dermal bed on which cultured epidermal sheets can then be placed. However, immunological reactions with the epithelial components of these dermal homograft and the potential risks associated with infectious pathogens have not been resolved .
The third type is an ADM. ADM is a native dermal matrix in which all the cells have been destroyed depending on the specific methods of its preparation. It is derived from full-thickness skin treated to remove epithelial components (keratinocytes, sweat glands, and sebaceous glands) and dermal cellular components (fibroblasts, vascular endothelium, and smooth muscle) .
The most acceptable substitute is ADM, derived from full or split-thickness skin treated to remove epithelial and dermal components . Therefore, this study was designed to prepare ADMs using two different methods and to evaluate their benefits as dermal substitutes.
In group II, the skin specimens were subjected to three repeated freeze–thaw cycles at 12-h intervals. Then skin specimens were soaked in PBS till the epidermis was separated from the dermis. The same procedure was followed by other investigators who prepared ADM by exposing skin specimens to rapid freeze–thaw cycles, which led to cell devitalization. Subsequently, skin specimens were incubated in sterile PBS for 1 week. After 1 week, the epidermis was separated from the dermis .
In the current experiment, group II showed a highly significant decrease in the number of cells/hpf in both the papillary and the reticular dermis as compared with the control group (P < 0.001). However, occasional nuclear fragments were observed in the dermis. In agreement with this result, other investigators have reported that the freeze–thawing technique is generally insufficient to achieve complete decellularization and must be combined with an additional treatment. Moreover, they reported that the mechanism of action of the freeze–thawing technique involved disruption of the cell membranes and release of the cell contents, which facilitate subsequent rinsing and removal of the cell contents from the ECM. Therefore, they also recommended using another method of decellularization after freezing to remove the cellular material from the tissue as freezing is an effective method of cell lysis . In contrast, a previous study has shown that the treatment of porcine skin with 0.05% trypsin and repeated freeze–thaw cycles was an effective decellularization method .
To achieve efficient decellularization, we modified the technique used in group II. Therefore, in group III, ADMs were prepared using the same technique as that used in group II but with additional exposure to γ irradiation for further decellularization.
In group III, there was loss of most of the cellular components in the dermis as there was a highly significant decrease in the number of cells/hpf in the papillary and the reticular dermis as compared with the control group and group II (P < 0.001). This was in agreement with some authors who used the same technique for ADM preparation and reported that fibroblasts were not detectable in ADM after 2 weeks of PBS soaking. Moreover, endothelial cells were not detectable after 4 weeks of soaking, after which the dermis was acellular by light microscopy .
Radiation works by damaging the DNA of exposed tissue, leading to cellular death. This DNA damage is caused by one of two types of energy: photon or charged particle. This damage is either direct or indirect ionization of the atoms that make up the DNA chain. Indirect ionization occurs as a result of the ionization of water, forming free radicals, notably hydroxyl radicals, which then damage the DNA . Direct damage to cell DNA occurs through high linear energy transfer charged particles such as proton, boron, carbon, or neon ions, which exert an antitumor effect. These particles act mostly through direct energy transfer, usually causing double-stranded DNA breaks .
In both groups II and III, collagen fibers showed a normal arrangement as the papillary dermis was composed of fine interlacing bundles and the reticular dermis was composed of coarse, wavy collagen bundles. Different combination techniques for ADM preparation (e.g. the Dispase–Triton technique and the NaCl–SDS technique) showed similar results as the basic dermal architecture of collagen bundles meshwork remained unaltered .
The elastic fibers in both the groups were preserved but in some areas they appeared fewer, had thickened, and had lost their regular parallel arrangement. Similar results were obtained by some authors, who observed that elastic fibers were largely absent in the papillary dermis. In the reticular dermis, the elastic fibers were somewhat fragmented and less numerous in ADM than in normal skin .
In terms of immunohistochemical staining of laminin, group II showed a positive immune reaction at the epidermal basement membrane and basement membrane of the endothelial cells lining blood vessels, which is important for the adherence, outgrowth, and differentiation of keratinocytes . This was in agreement with a previous study, in which preservation of the basement membrane was observed after the same decellularization procedure . In addition, some authors found that the basement membrane was preserved when porcine skin was treated with repeated freeze–thaw cycles and 0.05% trypsin .
The additional use of γ irradiation in group III resulted in the preservation of laminin in the epidermal basement membrane and the basement membrane of the endothelial cells lining blood vessels. These findings were in agreement with the result of some investigators who used the same procedure in ADM preparation . Moreover, a previous study found that γ-ray exposure preserves the basement membrane structure. The study showed normal thickness of basal laminae of the testis in irradiated rats with normal laminin, type IV collagen, and heparan sulfate proteoglycan within the basal laminae .
The addition of radiation to the freeze–thawing technique is recommended in the preparation of ADM for efficient decellularization.
Conflicts of interest
There is no conflict of interest to declare.
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Keywords:© 2012 The Egyptian Journal of Histology
acellular dermal matrix; freeze–thawing; laminin