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The Egyptian Journal of Histology:
doi: 10.1097/01.EHX.0000413855.94491.c7
Original articles

Preparation of an acellular dermal matrix using the freeze–thawing technique with and without γ irradiation

Abd Eltawab, Salwa Mohameda; Kousha, Faten Sobhya; Abd Elaziz, Abd Elaziz Hanfyb; Moussa, Manal Hassana; Elgawad Elseba, Sara Abda

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aDepartments of Histology, Faculty of Medicine, Ain Shams University, Cairo, Egypt

bDepartments of Plastic Surgery, Faculty of Medicine, Ain Shams University, Cairo, Egypt

Correspondence to Manal Hassan Mossa, Department of Histology, Faculty of Medicine, Ain Shams University, Cairo, Egypt Tel: +20 114 342 4291; e-mail: doctor_206@hotmail.com

Received November 6, 2011

Accepted January 8, 2012

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Abstract

Introduction: An acellular dermal matrix (ADM) is a dermal substitute in which the skin is treated to remove epithelial and dermal cellular components.

Aim of the work: To compare the histological and immunohistochemical structures of ADMs prepared using the freeze–thawing technique with or without γ irradiation.

Materials and methods: Twenty-one human skin specimens were used and divided into three equal groups: group I (control group), group II, in which skin specimens were subjected to three repeated freeze–thawing cycles, and group III, in which skin specimens were subjected to three repeated freeze–thawing cycles and subsequent exposure to 5000 rad γ irradiation. Skin specimens from the previous groups were examined histologically and immunohistochemically for laminin. A morphometric study was carried out for the determination of the number of cells per high-power field (hpf) in both the papillary and the reticular dermis.

Results: Both methods of ADM preparation resulted in extensive extraction of cellular components with preservation of the basic dermal architecture as there was a highly significant decrease in the number of cells/hpf in both layers of the dermis in groups II and III as compared with the control group (P < 0.001). However, there was further decellularization in group III as there was a highly significant decrease in the number of cells/hpf in both the papillary and the reticular dermis in group III as compared with group II (P < 0.001). Immunohistochemical stain of laminin revealed preservation of the epidermal basement membrane in groups II and III.

Conclusion: A combination of irradiation and a freeze–thawing technique is recommended in the preparation of ADM for efficient decellularization.

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Introduction

The dermis is a dense fibrillary connective tissue, closely related to the epidermis, and protects it. The dermis is conventionally divided into two parts: the papillary dermis and the reticular dermis. The papillary dermis is characterized by thin, haphazardly arranged collagen fibers, delicate branching elastic fibers, and numerous spindle-shaped fibroblasts. The reticular dermis is composed of collagen fibers arranged in coarse wavy bundles, networks of coarse wiry elastic fibers, and a few fibroblasts in between the blood vessels [1].

The connective tissue of the skin is composed mostly of collagen and elastin. Collagen makes up 70–80% of the dry weight of the skin and provides the dermis with its mechanical and structural integrity. Elastin is a minor component of the dermis, but it serves an important function of providing the elasticity of the skin and accounts for 2–4% of the extracellular matrix [2].

Several lines of evidence have shown that the dermis plays an important role in the treatment of skin defects. The dermis enhances the take of the thin and fragile cultured epidermal sheets as it accelerates the growth of keratinocytes, and prevents the formation and shrinkage of scars in the wounded areas without inducing any clinically detectable immune response [3].

Skin substitutes are classified as epidermal, dermal, and composite (both epidermal and dermal) [4].

In the recent years, a number of attempts have been made to produce a dermal bed suitable for a thin split thickness skin graft or cultured epidermal grafts, but none have been entirely satisfactory. Dermal substitutes should have three fundamental properties: very low antigenicity, the capacity for rapid vascularization, and stability as a dermal template [5].

To decrease the antigenicity of a dermal substitute, a decellularization protocol is needed. This decellularization protocol is essential for the efficient removal of all cellular and nuclear material while minimizing any adverse effect on the composition, biological activity, and mechanical integrity of the remaining extracellular matrix. A decellularization protocol generally involves lysis of the cell membrane using physical treatments, followed by the separation of cellular components from the ECM (extracellular matrix) using enzymatic treatments, solubilization of cytoplasmic and nuclear cellular components using detergents, and finally removal of cellular debris from the tissue [6].

Therefore, the aim of this study is to compare the histological and immunohistochemical structures of acellular dermal matrices (ADMs) prepared using the freeze–thawing technique alone or a combination of freeze–thawing and γ-irradiation techniques.

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Materials and methods

Skin specimens

The current study was performed using 21 human skin specimens divided into three equal groups. Normal adult human thin skin specimens of average size 3 × 3 cm were obtained from the discarded skin of patients undergoing breast reduction after obtaining written consent from the patients.

In group I (control group), the structure of normal skin specimens was studied.

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Preparation of acellular dermal matrices in group II

ADMs in group II were prepared according to the Gulsun and Robert method [7]. Skin specimens were placed in sterile tissue culture dishes and rinsed in PBS (Lonza Bioproduct, Belgium). The skin specimens were wrapped in sterile gauze and soaked in PBS in sterile tissue culture dishes and then subjected to three repeated freeze–thawing cycles at 12-h intervals. The freezing temperature was −20°C and the thawing temperature was 21°C (room temperature). The skin specimens were then soaked in PBS in sterile tissue culture dishes till the epidermis began to separate from the dermis, usually within 4–7 days. The epidermis was then stripped off using a sterile scalpel.

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Preparation of acellular dermal matrices in group III

ADMs in group III were prepared according to the Krejci et al. [8] method. Skin specimens were treated as in group I, but after the freeze–thawing steps, skin specimens were subjected to a single dose of γ irradiation (5000 rad) over 1 min. The specimens were then soaked in PBS in sterile tissue culture dishes till the epidermis began to separate from the dermis, usually within 4–7 days. The epidermis was then stripped off using a sterile scalpel.

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Processing of the skin specimens

The specimens were fixed in buffered formalin for 48h, dehydrated in ascending grades of alcohol, and then the specimens were cleared in xylene. After that, they were embedded in paraffin and paraffin blocks were obtained. Serial sections of 5μm thickness were cut and stained with histological and immunohistochemical stains for the detection of laminin in the basement membranes.

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Immunohistochemical staining

Paraffin sections were deparaffinized and rehydrated down to distilled water. The slides were covered by H2O2 (10%) for 15min. Antigen retrieval was carried out by immersing the sections in a preheated citrate buffer solution (pH 6) for 10–20min. Then the slides were incubated in normal blocking goat serum for 10min. The slides were covered by the primary antibody for laminin overnight, washed with PBS, and blotted. The slides were covered by the secondary biotinilated anti-mouse antibody and incubated for 30min, washed well, and blotted. Then the slides were covered by a streptavidin horseradish peroxidase conjugate for 15min. Color was developed using 3,3-diaminobenzidine. The slides were washed well and counterstained with hematoxylin.

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Morphometric and statistical study

A morphometric study was carried out for the determination of the number of cells per high-power field (hpf) in both the papillary and the reticular dermis. Measurements were carried out in 5 hpf of each specimen and data were described as mean ± SD. One-way analysis of variance and the post-hoc Dunnett T3 test were used to compare the parameters in the different groups with each other.

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Results

Histological examination of H&E-stained sections of the control group showed the skin to be composed of two layers: epidermis and dermis. The dermis is the connective tissue layer below the epidermis. The epidermis is composed of stratified squamous keratinized epithelium. The dermis was observed to have two layers: a superficial papillary layer and a deep reticular layer. The papillary layer was observed to be composed of dermal papillae and the adjacent connective tissue. The papillary layer was composed of fine interlacing bundles of collagen fibers. The reticular layer appeared be composed of coarse wavy bundles of collagen fibers (Fig. 1).

Figure 1
Figure 1
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In group II, there was complete separation of the epidermal cells from the basement membrane. Collagen fibers showed a normal arrangement as the papillary dermis was observed to be composed of fine interlacing bundles and the reticular dermis was observed to be composed of coarse, wavy bundles of collagen fibers. Occasional basophilic nuclear staining was found in the dermis in the form of nuclear fragments (Fig. 2). Group II showed a highly significant decrease in the number of cells/hpf in both the papillary and the reticular dermis as compared with group I (P < 0.001) (Table 1).

Figure 2
Figure 2
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Table 1
Table 1
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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).

Figure 3
Figure 3
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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).

Figure 4
Figure 4
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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).

Figure 5
Figure 5
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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).

Figure 6
Figure 6
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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).

Figure 7
Figure 7
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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).

Figure 8
Figure 8
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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).

Figure 9
Figure 9
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Discussion

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 [4].

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 [9].

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) [5].

The most acceptable substitute is ADM, derived from full or split-thickness skin treated to remove epithelial and dermal components [5]. 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 [7].

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 [6]. 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 [10].

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 [8].

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 [11]. 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 [12].

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 [13].

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 [13].

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 [14]. This was in agreement with a previous study, in which preservation of the basement membrane was observed after the same decellularization procedure [7]. 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 [10].

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 [8]. 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 [15].

The addition of radiation to the freeze–thawing technique is recommended in the preparation of ADM for efficient decellularization.

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Acknowledgements
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Conflicts of interest

There is no conflict of interest to declare.

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Keywords:

acellular dermal matrix; freeze–thawing; laminin

© 2012 The Egyptian Journal of Histology

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