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. 2017 Mar-Apr;25(2):186-195.
doi: 10.1590/1678-77572016-0217.

Oral mucosa: an alternative epidermic cell source to develop autologous dermal-epidermal substitutes from diabetic subjects

Daniela Guzmán-Uribe  1   2 , Keila Neri Alvarado-Estrada  1 , Mauricio Pierdant-Pérez  2 , Bertha Torres-Álvarez  3 , Jesus Martin Sánchez-Aguilar  2 , Raúl Rosales-Ibáñez  1   4
Affiliations

Oral mucosa: an alternative epidermic cell source to develop autologous dermal-epidermal substitutes from diabetic subjects

Daniela Guzmán-Uribe et al. J Appl Oral Sci. 2017 Mar-Apr.

Abstract

Objective: The aim of this study was to obtain autologous dermal-epidermal skin substitutes from oral mucosa from diabetic subjects as a first step towards a possible clinical application for cases of diabetic foot.

Material and methods: Oral mucosa was obtained from diabetic and healthy subjects (n=20 per group). Epidermal cells were isolated and cultured using autologous fibrin to develop dermal-epidermal in vitro substitutes by the air-liquid technique with autologous human serum as a supplement media. Substitutes were immunocharacterized with collagen IV and cytokeratin 5-14 as specific markers. A Student´s t- test was performed to assess the differences between both groups.

Results: It was possible to isolate epidermal cells from the oral mucosa of diabetic and healthy subjects and develop autologous dermal-epidermal skin substitutes using autologous serum as a supplement. Differences in the expression of specific markers were observed and the cytokeratin 5-14 expression was lower in the diabetic substitutes, and the collagen IV expression was higher in the diabetic substitutes when compared with the healthy group, showing a significant difference.

Conclusion: Cells from oral mucosa could be an alternative and less invasive source for skin substitutes and wound healing. A difference in collagen production of diabetic cells suggests diabetic substitutes could improve diabetic wound healing. More research is needed to determine the crosstalk between components of these skin substitutes and damaged tissues.

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Figures

Figure 1
Figure 1. A. Air-liquid technique phase 1: Development of stroma with the fibrin/agarose gels. B. Specific culture medium placed in a Transwell System in accordance with the air-liquid technique
Figure 2
Figure 2. A. Fibroblast culture at confluence. B. Keratinocyte culture at confluence. C. Positive immunofluorescence for fibroblasts (anti-collagen-I). D. Positive immunofluorescence for keratinocytes (anti-cytokeratin 5-14)
Figure 3
Figure 3. Cell growth curve. Cell proliferation of the fibroblasts of diabetics subjects vs healthy subjects after 9 days of incubation. Diabetic subjects (green) had an increased cell proliferation since day 3 in comparison with healthy subjects (red)
Figure 4
Figure 4. A. Dermal-epidermal substitutes of healthy subjects observed under the inverted microscope (40X). A globular pattern is observed. B. Dermal-epidermal substitutes of diabetic subjects observed under the inverted microscope (40X). A fibrillar pattern is observed
Figure 5
Figure 5. Clinical appearance of the developed dermal-epidermal skin substitute
Figure 6
Figure 6. Dermal-epidermal skin substitute, immunostained, showing cell distribution. Red (Sytox, Invitrogen), green (Phalloidin, Invitrogen)
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