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Ultrastructural analysis between fetal and adult wound healing process of marsupial opossum skin Abstract The opossum delivers a newborn baby equivalent to tremature fetus state by postpregnancy. The peculiarity is advantageous for studies of fetus, because operations to take out fetus from the uterus of a mother are not necessary. When mammalian skin is wounded by full-thickness excision, fetal and adult wound healing processes differ. Fetal-type wound healing does not leave a scar. However, studies of how the fetal wound healing process differs in detail from the adult type are not advanced. We first observed the normal skin development of the gray short-tailed opossum (Monodelphis domestica) using an electron microscope. As for normal skin, an epidermis became multi-layered, and thickened from birth through to 7 days after birth. The quantity of extracellular matrix of the dermis increased thereafter, and several types of cells were found in the dermis. To examine the wound healing, we used material from a 1 day-old newborn baby, and from another 15 days after birth, and compared the wound healing style morphologically. Differences in the constitution of cells and fine structures of the skin were observed, it was obviously suggested that change in the wound healing style from fetal-type to adult-type occurred between 1 to 15 days after birth. Introduction When skin is wounded, the wound healing process begins almost immediately, as a self-defense action. Detailed reports of the wound healing process have found that inflammatory reaction occurs just after injury, followed by the process of granulation. These wound healing processes are called scarring [7]. The wound healing process in which healing produces a scar is called an adult-type wound, and this healing style is generic with the adult skin. It is rare that a fetus in the womb of a mother can suffer a wound. However, should this occur, fetal wound healing is faster than adult-type wound healing, and granulation and scarring are not observed. This wound healing style is called fetal-type wound healing, and the processes involved differ from the adult type. It has been found that the healing style in the rat switches from fetal-type to adult-type during pregnancy, at 16 to 18 days after conception [3, 9, 10]. Adult-type wound healing has been described as follows. When full-thickness excision wounds are made on the skin of adult mammals, a clot formed by the blood-clotting reaction covers the wounded area, and temporarily shielding the wound from the external environment. Next, cells of the inflammation system such as neutrophils or macrophages move into the corium of the wounded area and multiply. These cells discharge cytokine and other growth factors, and inflammatory reaction occurs in the wounded area. Mast cells play a major role in this inflammatory reaction [16]. As a result, fibroblasts increase in number in the corium of the wounded area. Granulation tissue, which is a transient connective tissue, is formed by fibroblasts discharging abundant extracellular matrixes such as fibronectin or collagen. Granulation tissue is constrictive tissue containing myofibroblasts, and shrinks by the vulnerary last healing process, but leaves a scar. The myofibroblasts disappear from granulation tissue by apoptosis, after the granulation tissue shrinks [6, 8]. The type and expression intension of the proteins in human skin change in each developmental stage, from the fetus to the adult [4]. Furthermore, the type and expression level of the protein in each healing process or in each area vary in the human skin [5]. These reports suggest that the types and expression level of the proteins and the cellular quantity or activity that are associated with them during wound healing may be characteristic structures and functions in adult-type wound healing. Reports suggest that organization of a scar causes differences in the network and accumulation speed of collagen [11, 12]. Fetal-type wound healing is thought to differ from the adult-type wound healing process, but studies of fetal-type wound healing are not yet advanced, because of the difficulty of handling fetal tissues. However, a few morphological studies of mammalian fetal-type wound healing suggest that the proteins and form of connection change at the cellular level [15]. In studies of fetal-type wound healing, the handling of pregnant animals and fetal tissue is accompanied by various difficulties. In animals such as the rat or mouse, there is large risk to both the pregnant mother and the fetus, during surgery to access the womb and embryo-lemma. In contrast, the opossum is an advantageous animal for observation and post-natal surgery, because opossum birth occurs at a development stage which seems to be equivalent to a mammalian fetus. In stages of development of the skin of the opossum, the keratinocyte in each stage of development constitutes a keratinized layer, basement membrane, and basal cells which are distributed in the intermediate part of the layer, and the membrane shows a remarkable histological change [2]. It has been reported that cell constitution around full-thickness excision wounds differs conspicuously from that in the normal skin of the opossum [1]. Inflammatory reaction did not occur during the wound healing process of the skin of a newborn opossum one day after birth. However, inflammatory reaction similar to that occurring in adult-type wound healing was observed in an infant opossum 15 days after birth [14]. Armstrong and Ferguson (1995) also reported that wound healing style in a newborn opossum switched its wound healing style from fetal-type style to adult-type at about 9 days after birth. Consequently, in the early stages the newborn opossum can be used as a model for fetal-type wound healing. In this study, we analyze the process of the histological development of opossum skin by electron microscopy, in an effort to make a model for normal skin development in the opossum. Firstly, as a marker of the change in fine structure during normal development, we observed the keratinocyte, fibroblasts and other cells in an electron microscope, and examined change of these cellular forms during the development. Next, we aim to discuss how the fine structures of these cells changed by the processes of fetal-type and adult-type wound healing. We used a newborn (1 day) baby for the fetal-type wound healing model, and an infant 15 days after birth for the adult-type wound healing model, and review the morphological difference in detail. Structural changes of cells observed during these processes must be closely associated with variations in type and quantity of proteins during the wound healing process. We believe that analysis of fetal-type wound healing and comparison with adult-type healing style contribute significantly to future wound healing study. Materials and Methods The gray short-tailed opossum (Monodelphis domestica), which is the experimental animal of this study, belongs to the marsupials, as do the kangaroo and koala, but the bag-shaped marsupium is not present, and a portion of the skin of the hypogastric region becomes a substitute for the marsupium. Because the opossum cannot rear a fetus in the uterus, because the placenta is unripe, the animal delivers a newborn baby equivalent to a premature infant state by postpregnancy, equivalent to about 14 days after conception in mouse [13]. Opossums are also advantageous for the experimental animal because of production a large number of young. The opossums used in our study were obtained as adults from Nippon University Matsudo Faculty of Dentistry, and crossbred in our laboratory to obtained newborn infants. For materials of the normal coetaneous development, we used material from babies of 0, 1, 2, 4, 7, 15, 16, 18 and 21 days after birth. These babies are considered to be equal after the conception about 14, 15, 16, 18, 21, 29, 30, 32 and 35 days respectively. We used a newborn baby (1 day after birth) to examine fetal-type wound healing, and a 15 day-old baby for adult-type wound healing material. Because newborn babies cannot be kept away from the marsupium after surgery for 0 day to 15 days after birth, we used a Muromachi “Small Animal Anesthetizer”. Mothers were anesthetized with isoflurane steam, and the babies in the marsupium anesthetized by exposure to ice. All animal experiments were performed in accordance with the guidelines for use of experimental animals established by Shimane University. Peeling normal skins Skins of each stage of development were peeled from two points of a linear symmetry area after shaving. These skins were trimmed to about 2 mm × 2 mm after washing with a prefixative solution. Prefixative solution containing 1.5% glutaraldehyde and 1.5% paraformaldehyde in 0.1 M sodium cacodylate buffer (pH 7.4). Peeling experimental skins Skin full-thickness excision wounds 1 mm in diameter were made babies 1 day and 15 days after birth, using a dermal punch at two points of a linear symmetry area after shaving. After 1, 3 and 6 days, we peeled the skin of the wounded areas as a comparison between fetal-type wound healing and the adult-type. The peeled skins were first cut into 5 mm × 5 mm pieces, and then trimmed to about 2 mm × 2 mm around the wound, after washing with a prefixative solution. Fixation, dehydration, embedding and sectioning The specimens prepared as above were fixed with a prefixative solution for 90 min. After prefixation, they were then washed two or three times with 0.1 M sodium cacodylate buffer (pH 7.4). They were then fixed again with a postfixative solution containing 1% osmium tetroxide in 0.1 M phosphate buffer (pH 7.4) for 120 min. Each specimen was dehydrated in a series of 80, 90, 95 and 100% ethanol for 10 min, and then embedded in epoxy resin. The embedded preparations were hardened in an incubator, and cut into ultra-thin sections using a diamond knife. The sections were cut across the center of the wounded area, and were cut to observe from the keratinized to the corium in succession. Sections were stained with a saturated solution of uranyl acetate and a silver nitrate solution by Reynold’s solution, and observed using a JEM-1010 type electron microscope. Photographs were taken at magnifications of 800, 1,200, 3,000 and 6,000 ×. Results I. Normal process of skin development We observed nine skin preparations of 0, 2, 4, 7, 15, 16, 18, and 21 days after birth using the electron microscope. The skin of the newborn opossum at 0 day after birth was clearly divided into an epidermis and a dermis. Three types of cells were apparent. One or two layers of flat keratinocytes containing keratohyaline granules were arranged at the outermost part of the epidermis, forming a keratinized layer (Figs. 1A and 1B Fig. 1. A. Electron micrograph of a keratinized layer of an opossum baby 0 day after birth: Many keratohyaline granules are included in the cells (arrow). Scale bar 1 µm. B. Electron micrograph of an epidermis and a dermis of a baby 0 day after birth: border between the epidermis and the dermis is clear, and two types of fibroblasts are observed within the dermis. OF; oval or polygonal fibroblast, SF; slim fibroblast. Scale bar 10 µm. C. Electron micrograph of a fibroblast and the neighboring intercellular space from a baby 0 day after birth. The fibroblast contains compact packed endoplasmic reticula in its cytoplasm. Bundles of Collagen fibers are observed at the space. Scale bar 1 µm. D. Enlarged view of the bundle which marked in Fig. 1C. The bundle is composed of some collagen fibers. Scale bar 0.25 µm.). Two or three layers of squamous cells with projections were arranged just beneath part of the keratinized layer. A layer of basal cells was distributed in lower part of the epidermis. Therefore, it was evident that in the skin at this stage, the epidermis consisted of 5–8 layers of several types of cells. The epidermis of this stage was composed of thick gathering of some types of cells, and was divided into a definite lower dermis, but typical basal cells were not observed. However, cells that seemed to be basal cells were distributed in an irregular line between the epidermis and dermis (Fig. 1B). Two types of fibroblasts were observed in the dermis, judging from their fine structures. Both fibroblast types bore well-developed endoplasmic reticular systems, suggesting they were in an active stage to create much protein, such as protocollagen. Bundles of collagen fiber were distributed between the fibroblasts in a disordered but connected arrangement, forming a three-dimensional network (Figs. 1C and 1D). About 8 layers of differing cells constructed the epidermis of the opossum specimen 2 days after birth. An outmost layer of keratinocytes, which contained keratohyaline granules, was the same thickness as in the previous stage, but the inner layer of keratinocytes had thickened. These observations suggest that a keratinized layer was complete from this stage. The squamous cells became larger and thinner, and were piled upon each other with their projections. Increasing numbers of squamous cells caused the thickness a squamous layer to grow compared to the previous stage. Basal cells arranged in a layer at the base of the epidermis divided an epidermis and a dermis (Fig. 2A Fig. 2. A. Electron micrograph of an epidermis of a baby of 2 days after birth. Squamous cells show platy shape, and basal cells appear polygonal. The basal cells have many projections at an apical and both sides of the cell. SC; squamous cell, BC; basal cell. scale bar 1 µm B. Electron micrograph of a dermis of a baby 2 days after birth. Most fibroblasts are oval or polygonal. OF; oval or polygonal type fibroblast, SF; slim fibroblast. Scale bar 5 µm. C. Electron micrograph of fibroblasts from a baby 2 days after birth. Fibroblast has well-developed endoplasmic reticula in its cytoplasm. Fibers are thick, and are gathered into bundles in the intracellular space. Scale bar 1 µm.). The basal cells of this stage had shapes typical of this cell type. The basal cells were tall, and contacted a basement membrane at their base. The cell lacked projections at the base, but featured many projections at their apical portions, binding them to each other, or to neighboring squamous cells (Fig. 2A). In the dermis, the slim type of fibroblast decreased in number, and the oval or polygonal type increased. The intercellular space in the dermis began to expand in proportion with the increase of cell number. However, with the expansion of the intercellular space and the increase in cell number, overall cell count per unit area did not differ from the previous stage (Fig. 2B), with cell count per unit area of about 16 cells/40 µm × 40 µm. Endoplasmic reticula were well-developed in fibroblasts, and thin fibers in intercellular space were thickened and gathered into bundles (Fig. 2C). The preparations from specimens of 4, 7, 15 and 18 day age did not show any new characteristics compared to that 2 days after birth. Consequently, we tried to observe other notable developmental processes using the electron microscope. Other processes have previously been reported previously from light microscope observations [1]. Capillaries were first observed in the dermis of the specimen taken 4 days after birth (Fig. 3A Fig. 3. A. Electron micrograph of a capillary in a baby 4 days after birth: A large blood corpuscle is found within the vessel. The corpuscle has a large nucleus. Scale bar 1µm. B. Electron micrograph of collagen fiber bundles in the dermis of a baby 4 days after birth. The bundles do not show the characteristic striated pattern which is caused by polymerization of type I or III collagen. Scale bar 1µm. C. Electron micrograph of a developing follicle in a baby 7 days after birth. Anlage cells of the follicle are arranged cylindrically, and another type of cell surrounds the circumference. Scale bar 10µm.). Although fibers in this dermis did not show the characteristic striated pattern which is caused by polymerization of collagen, thick bundles of fibers were observed. A follicle appeared in the specimen taken 7 days after birth. It seemed that the follicle differentiated by the following process: Anlage cells of the follicle were arranged in a concentric circle with compact desmosomes, another type of cells surrounded the anlage cell group, and the large group of cells then developed into a follicle (Fig. 3C). II Skin full-thickness excision wound healing process Skin full-thickness excision wounds with diameter of 1 mm were made in opossum at 1 day and 15 days after birth, using a dermal punch, to observe details of the wound healing process. Skin was peeled from the wounds 1, 3 and 6 days after the initial wounding, and these parts then fixed to observe the wound healing processes under an electron microscope. Observation of those preparations must reveal whether the wound healing follows the same process between 1 day and 15 days after birth. II-A. Wound healing process of an opossum 1 day after birth Wound healing process 1 day after injury Cells which construct an epidermis such as a well developed squamous cells (keratinocytes) or pre-developed squamous cells were observed in the excised base of the wound, but no other types of cells were seen. Newly-formed groups of cells stretched toward the center of the excised wound, but the origin of these cells was unknown. These cells seemed to be still in the process of development, because their external form was almost oval, and they were filled with cell organelle (Fig. 4A Fig. 4. A. An electron micrograph of the wound area one day after surgery on a one-day old baby. Unknown cells formed from the wound wall show undeveloped aspect. The original squamous cells in an epidermis are shown in the lower right. Scale bar 5 µm. B. Electron micrograph of the basal part of an epidermis, 3 days after injury in a baby 1 day after birth. The basal cells are seemingly well- differentiated, and a basal membrane has appeared (arrows). BC; basal cell. Scale bar 2 µm. C. Electron micrograph of a dermis, 3 days after injury in a baby 1 day after birth. Two types of fibroblast are evident. Slim fibroblasts are mainly distributed in the lower part. OF; oval or polygonal fibroblast, SF; slim fibroblast. Scale bar 5 µm. D. Electron micrograph of an epidermis 6 days after injury of a baby 1 day after birth. An epidermis becomes thick as squamous cells have increased in number. The basal cells in this case resemble squamous cells. BC; basal cell, SC; squamous cell. Scale bar 10 µm. E. Electron micrograph of an epidermis and dermis 6 days after injury of a baby 1 day after birth: Distinction between basal and squamous cells is clear. Most fibroblasts are oval or polygonal. BC; basal cell, SC; squamous cell, OF; oval or polygonal fibroblast. Scale bar 2µm.). Wound healing process 3 days after injury The wound was completely closed, a scab had formed, and a keratinized layer had differentiated. An epidermis constructed of about nine layers and two layers of keratinocyte containing keratohyaline were observed at the outermost part of the epidermis. Squamous cells had long projections, and their external forms were oval. These cells seemed to be basal cells arranged at the base of an epidermis. A basement membrane still in the process of development was also observed (Fig. 4B, arrows). Two types of fibroblasts were observed in the dermis at this stage, with oval or polygonal fibroblasts distributed in the upper part near the epidermis, and slim fibroblasts distributed in the lower part (Fig. 4C). The slim fibroblasts were aligned parallel to the basement membrane. The oval or polygonal type fibroblasts were larger than those present 3 days after birth during normal development. The dermis of the wound healing was thinner than that of the dermis of normal skin during healing, and the density of fibroblasts was low. The dermis showed low electron density due to the presence of many intercellular spaces. Other cell components such as follicles, capillaries, or fat cells were not observed in this stage (Fig. 4C). These observations closely resembled the appearance of normal skin one day after birth, except for the differing distribution of fibroblasts. Wound healing process 6 days after injury An epidermis was constructed of 6–7 layers of cells, including 1–2 keratinized layers. The intercellular spaces of squamous cells seemed wider than those of normal skin. Electron density of the squamous cells differed depending on the area in which they were distributed (Fig. 4D). The basement membrane was not clear, but the presence of many basal cells was confirmed (Fig. 4E). Oval or polygonal fibroblasts accounted for the majority in the dermis, and slim fibroblasts were few at this stage (Fig. 4E). As noted above, healing of a full-thickness excision wound in the opossum 1 day after birth was not complete 1 day after injury. II-B Wound healing process of an opossum 15 days after birth Wound healing process 1 day after injury The wounded area was filled with scab, and vesicles and a homogeneous framework was distributed beneath the scab. The origin of the vesicles and the framework was unknown, but both were presumably secreted by cells neighboring the wall of the wound. The framework contained abundant small vesicles and granules (Fig. 5A Fig. 5. A. Electron micrograph of a scab and its vicinity in a 15-day old baby 1 day after injury. The cell component is not confirmed, and vesicles are distributed over the scab. Cells contacting the scab are of unknown origin. SCAB; scab. Scale bar 2µm. B. Electron micrograph of the wall of a wound in a 15-day old baby, 1 day after injury. Cells which seem to be extending from the wound area are distributed at the wound wall. The place in the upper region where squamous cells are not distributed is the edge of the wound (arrow). Scale bar 2 µm. C. Electron micrograph of a scab and an epidermis in a 15 day-old baby, 3 days after injury. Keratinocyte cells or layers are not observed, but the epidermis covers the wound. SCAB; scab Scale bar 2 µm. D. Electron micrograph of a dermis in a 15 day-old baby, 3 days after injury. Bundles of fibers and fibroblasts are densely distributed, and the intercellular space is small. OF; oval or polygonal fibroblast, SF; slim fibroblast. Scale bar 2 µm. E. Electron micrograph of an epidermis in a 15 day-old baby, 6 days after injury. Large squamous cells are arranged in layers. Basal cells are clearly distinguishable from squamous cells. BC; basal cell, SC; squamous cell. Scale bar 10 µm. F. Electron micrograph of a dermis in a 15 day-old baby, 6 days after injury. Oval or polygonal fibroblasts are mainly distributed in the upper part of the dermis. OF; oval or polygonal type fibroblast, SF; slim type fibroblast. Scale bar 10 µm. G. Electron micrograph of a fibroblast in a 15 day-old baby, 6 days after injury. Abundant endoplasmic reticula are distributed in the cytoplasm. Collagen fibers are gathered into bundles in the intercellular space. Scale bar 1 µm. H. Electron micrograph of a granulation-like tissue in a 15 day-old baby, 6 days after injury. The tissue was observed in a dermis and showed a compact packing group of many fibroblasts and some macrophage in it. MF; macrophage Scale bar 2 µm.). Fibroblasts contacting muscle cells in the dermis were observed at the base of the wound, and we suppose that these fibroblasts were not newly-differentiated, but migrated from the wounded wall. No other cells were observed in the dermis of this stage. The wound margin was occupied by groups of epidermal cells, which were stratified into layers (Fig. 5B). These cells are also considered to have originated from the surrounding tissue. Wound healing process 3 days after injury No keratinized layer of keratinocytes or individual keratinocytes were present beneath the scab. An epidermis completely covered the wound, which seemed to be completely closed. Four or five layers of squamous cells were observed in the epidermis, but typical basal cells were not distinguishable, and the basal membrane was not clear (Fig. 5C). Two types of fibroblasts (oval or polygonal type and a slim type) were distributed in the dermis. The slim fibroblasts were distributed throughout the dermis. The oval or polygonal fibroblasts were few, and were distributed between the slim fibroblasts. The slim fibroblasts were arranged parallel to the skin surface. The fibroblasts were abundant in the dermis, and the intercellular spaces were small. Collagen fibers in the intercellular spaces were thin, and ran in various directions without forming thick bundles (Fig. 5D). Wound healing process 6 days after injury Most of the scab had loosened and fallen away, and only a small fragment of the scab remained. Two layers of keratinocytes covered the wound. Large squamous cells with long projections were arranged in 6 layers, and the intercellular space in the epidermis seemed to be large. Basal cells were distributed in one or two layers close against a dermic layer (Fig. 5E). Two types of fibroblasts were observed in a dermis. The oval or polygonal fibroblasts were similar in size to those in a normal skin, but the slim type seemed to have increased in thickness. The oval and polygonal fibroblasts were mainly distributed over the epidermis side of the dermis, and the slim fibroblasts were mainly concentrated in the lower part. The intercellular spaces in the dermis were large in the upper region near the epidermis, and became small in the lower region (Fig. 5F). The collagen fibers became thick, and were gathered into bundles running in differing directions (Fig. 5G). At the lower region of the wound, granulation-like tissue was observed (Fig. 5H). The tissue showed compact packing gathering of many fibroblasts and some broken fibroblasts in it. When the full-thickness excision wound was made in an opossum infant 15 days after birth, the wound was not closed 1 day after injury. Neighboring epidermal cells seemed to move to the wound wall, as the first step of wound healing. The origin of this cell group was unknown. The wound was closed 3 days after injury, but the density of fibroblasts was low at this stage compared that of a normal skin, even though the epidermis was thicker. Six days after injury, an epidermis had developed into about 9 layers, and the density of fibroblasts neared that of a normal skin at this stage. The granulation-like tissue in which active/broken fibroblasts were gathered into a group was observed at the lower region of the wound. Discussion Observation of normal skin development of an opossum by a light microscope showed skin thickness on 0 day after birth was 14.0 µm, and thickness gradually increased to 43.0µm by 7 days [1]. Thickness then tended to decrease from 7 days onward. These observations agree with a previous report that skin thickness increased by temporary hyperplasia during the development [1, 2]. When normal cutaneous constitution was observed under an electron microscope, an epidermal and a dermis were distinguishable, and two types of fibroblast were confirmed in the dermis 0 days after birth. Furthermore, squamous cells and basal cells were definitely present in the epidermis 1 day after birth. These observations showed that a basic skin structure had already formed at this stage. Skin development is then thought to proceed in the following steps: collagen fibers, which are the main extracellular matrix, are produced in abundance, and each cell within the epidermis and dermis multiply or enlarge. Cells of all types constitute a skin that has developed by 3 days after birth, and capillaries form 4 days after birth, so it seems that the skin was completed systematically. Therefore, it is thought that the skin of the newborn baby opossum switches to adult-type in 3 or 4 days. Based on these results and the report that the opossum gives birth 14 days after conception [13], switch to adult-type skin in the opossum will be completed 17–18 days after conception. This suggestion agrees with a previous report that wound healing in the rat changes from fetal-type to adult-type 16–18 days after conception [9]. The skin thickens, and appearance of capillaries may be related to formation of follicles, because these appear 7 days after birth as the skin thickens. The capillaries then begin to extend in the dermis. In addition, the skin of an infant 15 days after birth already showed activation of fat cells, and development of a vascular system similar to that of an adult skin. The skin at 15 days was thus completed the same as an adult, hence suggesting that wound healing style shifts from fetal-type to adult-type 15 days after birth. When examining the wound healing process of a baby 1 day after birth, we observed that the full-thickness excision wound had yet not closed, and cells from the wound margin had migrated into the wound center at one day after injury. Such migration of cells has been suggested from light microscope examination [1], and is confirmed in its details by our electron microscope observations. It has been suggested that actins led wound healing of an insect embryo [15], but we did not observe actin around the full-thickness excision wounds in the opossum. The wound healing of an opossum baby of 1 day after birth was complete 3 days after injury. At the stage, keratinocytes showed similar features to that of a normal skin of the same stage, and the epidermis contained large intercellular spaces and squamous cells maintaining adhesion by the desmosome. These fine structural observations are consistent with reports that tissue around the wound in the rat migrates to the central area, filling the excised part of the wound [3, 9, 10]. In addition, at 1 day after injury, the intercellular spaces of squamous cells in the epidermis were large, and granulation tissue was not observed. Oval or polygonal fibroblasts seemed to be actively synthesizing protein. These observations suggest that wound healing advances after completion of the wound choke, and that healing then progresses to achieve the construction of a normal skin. The process of wound healing observed in this study seems to be a similar process to that in a previous report which identified fetal wound healing style in the opossum 1 day after birth [14]. Therefore, our observations of a process of wound healing in a baby of 1 day after birth are appropriate for tracing the process the fetal-type wound healing. Histological analysis of the wound healing process in an infant 15 days after birth showed that one day after the full- thickness excision wound was made; the wound area was not filled with cells. Instead, it filled with structure of unknown source resembling granulation tissue under a scab. Cell migration into the wound center, as observed in the wound healing process of the opossum 1 day after birth, was not seen. An imperfect keratinized layer was formed under the scab three days after injury, and the basement membrane in the epidermis was undifferentiated at that stage. The wound healing style observed in our study resembled to that of human adult-type, as reported for healing of burns [5]. Consequently, it is evident that 15 days after birth the opossum infant had grown to the stage showing an adult-type wound healing style. Six days after injury, the wound had closed, and basal cells showed systematic arrangement. It was notable that the nucleus of squamous cells of an epidermis showed activated features comparable with those of the normal skin at the same stage, and the cells were enlarged. The intercellular space in the epidermis also became wider. In the dermis, intercellular matrix density was low, but fibroblasts were abundant, and the dermis increased in thickness. Many oval or polygonal fibroblasts were also observed, which seemed to be in an activated state, and creating proteins in the cells. Our observations of a wound healing process the 15 day-old opossum infant were quite similar to the previously reported light microscope observations of the process of an adult-type wound healing style [1]. But, the granulation-like tissue in which many active/broken fibroblasts were gathered into a group was observed at the lower region of the wound in our observations, although these structures had not repotted in the previous study by light microscope [1]. From the above results, it concluded that the wound healing style of the opossum is fetal-style one day after birth, and switches to an adult style by 15 days after birth. Although the switch time of 9 days after birth has been suggested in a previous report [2], we could not confirm the exact switch time, but the switch should be occurred during a few days between one day after birth and 15 days after birth from this electron microscope observations. Differentiation between fetal-type and adult-type wound healing can be made by observations of the following characteristic fine structures of each style: 1) whether vesicles and framework structures of unknown source are present; 2) whether cells from the wound margin region migrate into the wound center; 3) differences of abundance and period of activation of squamous cells in the epidermis; 4) differences of abundance and period of activation of a fibroblasts; and 5) presence of capillaries and follicles in the dermis; 6) whether granulation –like tissue are present. The authors thank Dr. A. Matsuno (Emeritus Prof. of Shimane Univ.) for his encouraging advice during of this work and Dr. R. Barry (Assoc. Prof. of Shimane Univ.) for his critical reading of this manuscript.