PMC:4548005 / 10373-26715 JSONTXT

{"target":"http://pubannotation.org/docs/sourcedb/PMC/sourceid/4548005","sourcedb":"PMC","sourceid":"4548005","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4548005","text":"Results\n\nI. Normal process of skin development\nWe observed nine skin preparations of 0, 2, 4, 7, 15, 16, 18, and 21 days after birth using the electron microscope.\nThe 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\nFig. 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).\nAbout 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\nFig. 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).\nThe 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\nFig. 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).\n\nII Skin full-thickness excision wound healing process\nSkin 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.\n\nII-A. Wound healing process of an opossum 1 day after birth\n\nWound healing process 1 day after injury\nCells 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\nFig. 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.).\n\nWound healing process 3 days after injury\nThe 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.\n\nWound healing process 6 days after injury\nAn 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).\nAs noted above, healing of a full-thickness excision wound in the opossum 1 day after birth was not complete 1 day after injury.\n\nII-B Wound healing process of an opossum 15 days after birth\n\nWound healing process 1 day after injury\nThe 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\nFig. 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.\n\nWound healing process 3 days after injury\nNo 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).\n\nWound healing process 6 days after injury\nMost 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.\nWhen 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.","divisions":[{"label":"title","span":{"begin":0,"end":7}},{"label":"sec","span":{"begin":9,"end":6154}},{"label":"title","span":{"begin":9,"end":46}},{"label":"p","span":{"begin":47,"end":163}},{"label":"p","span":{"begin":164,"end":2321}},{"label":"figure","span":{"begin":478,"end":1297}},{"label":"label","span":{"begin":478,"end":485}},{"label":"caption","span":{"begin":486,"end":1297}},{"label":"p","span":{"begin":486,"end":1297}},{"label":"p","span":{"begin":2322,"end":4596}},{"label":"figure","span":{"begin":3004,"end":3691}},{"label":"label","span":{"begin":3004,"end":3011}},{"label":"caption","span":{"begin":3012,"end":3691}},{"label":"p","span":{"begin":3012,"end":3691}},{"label":"p","span":{"begin":4597,"end":6154}},{"label":"figure","span":{"begin":5026,"end":5637}},{"label":"label","span":{"begin":5026,"end":5033}},{"label":"caption","span":{"begin":5034,"end":5637}},{"label":"p","span":{"begin":5034,"end":5637}},{"label":"sec","span":{"begin":6156,"end":6701}},{"label":"title","span":{"begin":6156,"end":6209}},{"label":"p","span":{"begin":6210,"end":6701}},{"label":"sec","span":{"begin":6703,"end":10674}},{"label":"title","span":{"begin":6703,"end":6762}},{"label":"sec","span":{"begin":6764,"end":8578}},{"label":"title","span":{"begin":6764,"end":6804}},{"label":"p","span":{"begin":6805,"end":8578}},{"label":"figure","span":{"begin":7294,"end":8576}},{"label":"label","span":{"begin":7294,"end":7301}},{"label":"caption","span":{"begin":7302,"end":8576}},{"label":"p","span":{"begin":7302,"end":8576}},{"label":"sec","span":{"begin":8580,"end":9994}},{"label":"title","span":{"begin":8580,"end":8621}},{"label":"p","span":{"begin":8622,"end":9994}},{"label":"sec","span":{"begin":9996,"end":10674}},{"label":"title","span":{"begin":9996,"end":10037}},{"label":"p","span":{"begin":10038,"end":10545}},{"label":"p","span":{"begin":10546,"end":10674}},{"label":"title","span":{"begin":10676,"end":10736}},{"label":"sec","span":{"begin":10738,"end":13502}},{"label":"title","span":{"begin":10738,"end":10778}},{"label":"p","span":{"begin":10779,"end":13502}},{"label":"figure","span":{"begin":11099,"end":13062}},{"label":"label","span":{"begin":11099,"end":11106}},{"label":"caption","span":{"begin":11107,"end":13062}},{"label":"p","span":{"begin":11107,"end":13062}},{"label":"sec","span":{"begin":13504,"end":14422}},{"label":"title","span":{"begin":13504,"end":13545}},{"label":"p","span":{"begin":13546,"end":14422}},{"label":"title","span":{"begin":14424,"end":14465}},{"label":"p","span":{"begin":14466,"end":15617}}],"tracks":[]}