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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

Epidermal cell sheet derived from GFP-positive neonates. The cell sheets were harvested from the temperature-responsive cell culture inserts. A, The epidermal cell sheet was stained with hematoxylin and eosin. B, The epidermal cell sheet was stained with anti-laminin 332 antibody (the green color underneath the cell sheet; arrowheads). Nuclei were counterstained with DAPI (the blue color). C, The epidermal cell sheet was stained with anti-collagen type 4 antibody (the green color underneath the cell sheet; arrowheads). Nuclei were counterstained with DAPI (the blue color).
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Figure 1: Epidermal cell sheet derived from GFP-positive neonates. The cell sheets were harvested from the temperature-responsive cell culture inserts. A, The epidermal cell sheet was stained with hematoxylin and eosin. B, The epidermal cell sheet was stained with anti-laminin 332 antibody (the green color underneath the cell sheet; arrowheads). Nuclei were counterstained with DAPI (the blue color). C, The epidermal cell sheet was stained with anti-collagen type 4 antibody (the green color underneath the cell sheet; arrowheads). Nuclei were counterstained with DAPI (the blue color).

Mentions: After 7–8 days cultivation, epithelial cell sheets were harvested from thermoresponsive culture inserts (Fig. 1A). Adhesive basement membrane proteins were identified in the basal surface of the cell sheet (laminin 332, Fig. 1B; collagen 4, Fig. 1C).


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)

Epidermal cell sheet derived from GFP-positive neonates. The cell sheets were harvested from the temperature-responsive cell culture inserts. A, The epidermal cell sheet was stained with hematoxylin and eosin. B, The epidermal cell sheet was stained with anti-laminin 332 antibody (the green color underneath the cell sheet; arrowheads). Nuclei were counterstained with DAPI (the blue color). C, The epidermal cell sheet was stained with anti-collagen type 4 antibody (the green color underneath the cell sheet; arrowheads). Nuclei were counterstained with DAPI (the blue color).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4494494&req=5

Figure 1: Epidermal cell sheet derived from GFP-positive neonates. The cell sheets were harvested from the temperature-responsive cell culture inserts. A, The epidermal cell sheet was stained with hematoxylin and eosin. B, The epidermal cell sheet was stained with anti-laminin 332 antibody (the green color underneath the cell sheet; arrowheads). Nuclei were counterstained with DAPI (the blue color). C, The epidermal cell sheet was stained with anti-collagen type 4 antibody (the green color underneath the cell sheet; arrowheads). Nuclei were counterstained with DAPI (the blue color).
Mentions: After 7–8 days cultivation, epithelial cell sheets were harvested from thermoresponsive culture inserts (Fig. 1A). Adhesive basement membrane proteins were identified in the basal surface of the cell sheet (laminin 332, Fig. 1B; collagen 4, Fig. 1C).

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