Electron Microscopic Analysis
For electron microscopic examination, scar tissue samples from control and experimental groups, were fixed in 2.5% glutaraldehyde
solution in phosphate buffer, pH 7.4 for four hours, and post-fixed for one hour in 1% osmium tetroxide solution, dehydrated in ethanol, treated with propylene oxide and embedded
in Araldite. After heat polymerization, sections were cut using an ultramicrotome. Semi-thin sections were stained with methylene blue-azure II and examined using an Olympus BX-40 microscope. Ultra-thin sections were double-stained with uranyl
acetate and lead citrate, and examined in a Zeiss EM 900 transmission electron microscope.
We observed immunoreactivity to TGF-β in the fibroblasts, adipose cells, and muscle cells, to fibronectin on the extracellular
compartments of dermal layer of the skin, and to laminin in the basal lamina component of epithelium, adipose cells, and muscle cells. In the dermal layer of the scar samples, we also observed increased immunoreactivities of laminin, fibronectin, and TGF-β in the control group; moderate immunoreactivities
in the heparin group and allantoin group; mild immunoreactivity in the Contractubex gel group (Figure 1). The intensities of immunohistochemical scores were shown in Table 1. It was observed that the immunoreactivities of TGF-β, fibronectin, and laminin had increased in control group. On the other hand, the results were statistically significant in Contractubex group (P<.001), heparin group (P<0.05), and allantoin
In Group 1, the semi-thin tissue sections revealed a thickened epidermis, active fibroblasts, and granulation-like connective tissue in the dermis. There were intercellular separations in the granular layer and significant separation between the cells of the stratum corneum, however, normal dermal structures had not yet formed. In Group 2, appearance of keratinocytes forming
the epidermis was close to normal, and papillary layer of dermis occupies a large area in semi-thin sections. In Group 3, normal structure in stratum granulosum and stratum corneum could be seen. There was granulation-like connective tissue, consisting of loosely-structured collagen fibers and vessels in the papillary dermis. In Group 4, epidermal keratinocytes were rather normal in shape; separations between stratum granulosum
and stratum corneum were not as large as in the control group; and dermal papillary layer consists of collagen bundles rich in vessels (Figure 2).
Electron microscopy examination of the scar tissue samples of Group 1 revealed cells that had not completed keratinization,
lost their intercellular bindings, and been separated from the epidermis. In the wound-healing area, the modified cells of stratum corneum had not completed their keratinization and degenerating cells were observed. The scar tissues of Group 2 exhibited the thinnest epidermis of the four groups, with normal-appearing epidermal cells that had completed keratinization,
normal-appearing keratinocytes forming the epidermis, papillary wavy structures in the epidermo-dermal junction, and a dermal papillary layer occupying a large area (Figure 3).
The scar tissues of Group 3 revealed intercellular bindings with a firm appearance among cells of stratum corneum and stratum
granulosum; separations only in the cells of the outer layer of stratum corneum; continuing keratinization in lower cells of the stratum corneum; desmosomes still connected to the intact epidermis; epidermal keratinocytes that showed keratinization; and normal-appearing cells of the granular and corneal layers. In the dermal area close to epidermis, granular tissue has transformed
into papillary dermis. Beneath the epidermis, formation of connective tissue consisting of loosely-structured collagen fibers and blood vessels could be seen, but this area was not as wide as in the applied mixture group (Group 2) (Figures 2, 3).
The scar tissues of Group 4 demonstrated normal-appearing keratinocytes forming the epidermis; separations between the cells of stratum corneum and stratum granulosum that were not as large as in the control group (Group 1). A dermal papillar
layer that consisted of well-vascularized collagen bundles; and granulation tissue that had almost transformed into dermal connective tissue. Throughout the stratum corneum, cells were tightly spaced and intercellular binding units were evident, but some cells had not completed keratinization (Figures 2, 3).
Wound healing is a complex pathophysiological process involving
interplay of several cellular and biochemical processes. It includes the interaction of inflammation, re-epithelialization, angiogenesis, granulation tissue formation and collagen deposition.
14 This process is driven in part by a complex mixture of growth factors and cytokines, which are released coordinately into the wounds.15 Cytokines play important roles in the evolution
of granulation tissue through recruitment of inflammatory leukocytes and stimulation of fibroblasts and epithelial cells. In case of any impairment in the normal reparative process, delayed
healing or excessive fibrosis may occur. Overhealing or excessive fibrosis of wounds is observed in fibroproliferative disorders such as keloids and hypertrophic scars. Keloids and hypertrophic scars are considered to be atypical manifestations of the wound healing process following trauma to the skin. These scars consist of excessive dense fibrous tissue growing in all directions,
resulting in a prominent elevation above the skin.16
Transforming growth factor-β (TGF-β) is known to be the most potent growth factor involved in wound healing throughout the body.17 It is synthesized by several types of cells in vivo and in