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Ex Vivo Prefabricated Rat Skin Flap Using Cell Sheets and an Arteriovenous Vascular Bundle.

Fujisawa D, Sekine H, Okano T, Sakurai H, Shimizu T - Plast Reconstr Surg Glob Open (2015)

Bottom Line: Recently, research on tissue-engineered skin substitutes have been active in plastic surgery, and significant development has been made in this area over the past several decades.These results show that we were able to produce native-like skin.We have succeeded in creating regenerative skin flap ex vivo that is similar to native skin, and this technique could be applied to create various tissues in the future.

View Article: PubMed Central - PubMed

Affiliation: Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, Tokyo, Japan; and Department of Plastic and Reconstructive Surgery, Tokyo Women's Medical University, Tokyo, Japan.

ABSTRACT

Background: Recently, research on tissue-engineered skin substitutes have been active in plastic surgery, and significant development has been made in this area over the past several decades. However, a regenerative skin flap has not been developed that could provide immediate blood flow after transplantation. Here, we make a regenerative skin flap ex vivo that is potentially suitable for microsurgical transplantation in future clinical applications.

Methods: In rats, for preparing a stable vascular carrier, a femoral vascular pedicle was sandwiched between collagen sponges and inserted into a porous chamber in the abdomen. The vascular bed was harvested 3 weeks later, and extracorporeal perfusion was performed. A green fluorescent protein positive epidermal cell sheet was placed on the vascular bed. After perfusion culture, the whole construct was harvested and fixed for morphological analyses.

Results: After approximately 10 days perfusion, the epidermal cell sheet cornified sufficiently. The desquamated corneum was positive for filaggrin. The basement membrane protein laminin 332 and type 4 collagen were deposited on the interface area between the vascular bed and the epidermal cell sheet. Moreover, an electron microscopic image showed anchoring junctions and keratohyalin granules. These results show that we were able to produce native-like skin.

Conclusions: We have succeeded in creating regenerative skin flap ex vivo that is similar to native skin, and this technique could be applied to create various tissues in the future.

No MeSH data available.


Related in: MedlinePlus

Perfused skin flap model at 10 days. A, Macroscopic appearance. B, Fluorescent observation. C, Cross-section of the harvested skin flap model was stained with hematoxylin and eosin. Asterisk indicates keratinized and cast-off epidermis. Double asterisk indicates the grafted epidermal cell sheet. Triple asterisk indicates the regenerated granulation tissue. D, Cross-section of the harvested skin flap model stained with anti-filaggrin antibody. Asterisk indicates keratinized and cast-off epidermis. E, Cross-section of the harvested skin flap model was stained with anti-Ki 67 antibody (red color). F, Cross-section of the harvested skin flap model stained with anti-laminin 332 antibody (red color). Nuclei were counterstained with DAPI (blue color).
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Figure 4: Perfused skin flap model at 10 days. A, Macroscopic appearance. B, Fluorescent observation. C, Cross-section of the harvested skin flap model was stained with hematoxylin and eosin. Asterisk indicates keratinized and cast-off epidermis. Double asterisk indicates the grafted epidermal cell sheet. Triple asterisk indicates the regenerated granulation tissue. D, Cross-section of the harvested skin flap model stained with anti-filaggrin antibody. Asterisk indicates keratinized and cast-off epidermis. E, Cross-section of the harvested skin flap model was stained with anti-Ki 67 antibody (red color). F, Cross-section of the harvested skin flap model stained with anti-laminin 332 antibody (red color). Nuclei were counterstained with DAPI (blue color).

Mentions: Keratinization increased with the duration of extracorporeal perfusion. After 10 days perfusion, the epidermal cell sheet cornified sufficiently (Figs. 4A–C). Intensity of green fluorescence was strong in the sites where epidermis keratinization was progressing (Fig. 4B). The desquamated corneum was positive for anti-filaggrin stain (Fig. 4D). In the basal cell of the epidermal cell sheet, Ki 67 was observed (Fig. 4E). Laminin 332 was deposited on the interface area of the vascular bed and the epidermal cell sheet (Fig. 4F). Observations with transmission electron microscopy showed the basement membrane and hemidesmosomes at the interface of the epidermal cell sheet and the tissue built from the collagen sponge (Fig. 5A). In the prickle cell layer, desmosomes were present (Fig. 5B). In the granular layer, keratohyalin granules were observed (Fig. 5C). These were similar to the structures observed in the native skin.


Ex Vivo Prefabricated Rat Skin Flap Using Cell Sheets and an Arteriovenous Vascular Bundle.

Fujisawa D, Sekine H, Okano T, Sakurai H, Shimizu T - Plast Reconstr Surg Glob Open (2015)

Perfused skin flap model at 10 days. A, Macroscopic appearance. B, Fluorescent observation. C, Cross-section of the harvested skin flap model was stained with hematoxylin and eosin. Asterisk indicates keratinized and cast-off epidermis. Double asterisk indicates the grafted epidermal cell sheet. Triple asterisk indicates the regenerated granulation tissue. D, Cross-section of the harvested skin flap model stained with anti-filaggrin antibody. Asterisk indicates keratinized and cast-off epidermis. E, Cross-section of the harvested skin flap model was stained with anti-Ki 67 antibody (red color). F, Cross-section of the harvested skin flap model stained with anti-laminin 332 antibody (red color). Nuclei were counterstained with DAPI (blue color).
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Related In: Results  -  Collection

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Figure 4: Perfused skin flap model at 10 days. A, Macroscopic appearance. B, Fluorescent observation. C, Cross-section of the harvested skin flap model was stained with hematoxylin and eosin. Asterisk indicates keratinized and cast-off epidermis. Double asterisk indicates the grafted epidermal cell sheet. Triple asterisk indicates the regenerated granulation tissue. D, Cross-section of the harvested skin flap model stained with anti-filaggrin antibody. Asterisk indicates keratinized and cast-off epidermis. E, Cross-section of the harvested skin flap model was stained with anti-Ki 67 antibody (red color). F, Cross-section of the harvested skin flap model stained with anti-laminin 332 antibody (red color). Nuclei were counterstained with DAPI (blue color).
Mentions: Keratinization increased with the duration of extracorporeal perfusion. After 10 days perfusion, the epidermal cell sheet cornified sufficiently (Figs. 4A–C). Intensity of green fluorescence was strong in the sites where epidermis keratinization was progressing (Fig. 4B). The desquamated corneum was positive for anti-filaggrin stain (Fig. 4D). In the basal cell of the epidermal cell sheet, Ki 67 was observed (Fig. 4E). Laminin 332 was deposited on the interface area of the vascular bed and the epidermal cell sheet (Fig. 4F). Observations with transmission electron microscopy showed the basement membrane and hemidesmosomes at the interface of the epidermal cell sheet and the tissue built from the collagen sponge (Fig. 5A). In the prickle cell layer, desmosomes were present (Fig. 5B). In the granular layer, keratohyalin granules were observed (Fig. 5C). These were similar to the structures observed in the native skin.

Bottom Line: Recently, research on tissue-engineered skin substitutes have been active in plastic surgery, and significant development has been made in this area over the past several decades.These results show that we were able to produce native-like skin.We have succeeded in creating regenerative skin flap ex vivo that is similar to native skin, and this technique could be applied to create various tissues in the future.

View Article: PubMed Central - PubMed

Affiliation: Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, Tokyo, Japan; and Department of Plastic and Reconstructive Surgery, Tokyo Women's Medical University, Tokyo, Japan.

ABSTRACT

Background: Recently, research on tissue-engineered skin substitutes have been active in plastic surgery, and significant development has been made in this area over the past several decades. However, a regenerative skin flap has not been developed that could provide immediate blood flow after transplantation. Here, we make a regenerative skin flap ex vivo that is potentially suitable for microsurgical transplantation in future clinical applications.

Methods: In rats, for preparing a stable vascular carrier, a femoral vascular pedicle was sandwiched between collagen sponges and inserted into a porous chamber in the abdomen. The vascular bed was harvested 3 weeks later, and extracorporeal perfusion was performed. A green fluorescent protein positive epidermal cell sheet was placed on the vascular bed. After perfusion culture, the whole construct was harvested and fixed for morphological analyses.

Results: After approximately 10 days perfusion, the epidermal cell sheet cornified sufficiently. The desquamated corneum was positive for filaggrin. The basement membrane protein laminin 332 and type 4 collagen were deposited on the interface area between the vascular bed and the epidermal cell sheet. Moreover, an electron microscopic image showed anchoring junctions and keratohyalin granules. These results show that we were able to produce native-like skin.

Conclusions: We have succeeded in creating regenerative skin flap ex vivo that is similar to native skin, and this technique could be applied to create various tissues in the future.

No MeSH data available.


Related in: MedlinePlus