RESUMO
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.
Assuntos
Transplante de Células/métodos , Diabetes Mellitus Tipo 2 , Células Epidérmicas , Células Epiteliais/transplante , Mucosa Bucal/citologia , Pele Artificial , Adulto , Idoso , Materiais Biocompatíveis , Estudos de Casos e Controles , Técnicas de Cultura de Células , Proliferação de Células , Células Cultivadas , Colágeno/análise , Diabetes Mellitus Tipo 2/terapia , Feminino , Fibroblastos , Humanos , Queratinócitos/citologia , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Úlcera Cutânea/terapia , Fatores de Tempo , Transplante Autólogo , CicatrizaçãoRESUMO
Abstract Oral mucosa has been highlighted as a suitable source of epidermal cells due to its intrinsic characteristics such as its higher proliferation rate and its obtainability. Diabetic ulcers have a worldwide prevalence that is variable (1%-11%), meanwhile treatment of this has been proven ineffective. Tissue-engineered skin plays an important role in wound care focusing on strategies such autologous dermal-epidermal substitutes. 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.
Assuntos
Humanos , Masculino , Feminino , Adulto , Pessoa de Meia-Idade , Idoso , Pele Artificial , Transplante de Células/métodos , Diabetes Mellitus Tipo 2 , Epiderme/citologia , Células Epiteliais/transplante , Mucosa Bucal/citologia , Úlcera Cutânea/terapia , Fatores de Tempo , Transplante Autólogo , Cicatrização , Materiais Biocompatíveis , Estudos de Casos e Controles , Queratinócitos/citologia , Células Cultivadas , Reprodutibilidade dos Testes , Colágeno/análise , Técnicas de Cultura de Células , Proliferação de Células , Diabetes Mellitus Tipo 2/terapia , FibroblastosRESUMO
Odontogenesis is extensively studied in animal models but less understood in human. In early amelogenesis, amelogenin constitutes 90% of enamel organic matrix, which is degraded by enamelysin and replaced by hydroxyapatite crystals. Here, amelogenin and enamelysin distribution changes during amelogenesis were shown by co-localization experiments by confocal microscopy. Early bell stage showed more amelogenin labeling than enamelysin, as free immune-reactive granular patches towards basal membrane between ameloblast and odontoblast. Increased amelogenin expression and secretion towards extracellular matrix formation region was found. Enamelysin distribution was perinuclear in early bell stage. During late bell stage, a decreasing amelogenin labeling in contrast with enamelysin increasing along the cells was found, suggesting specific temporal amelogenin degradation. Enamelysin was located initially around nuclei and later was found in all the ameloblast and stellate reticulum cytoplasm. Amelogenin was observed inside ameloblast, stellate reticulum, and intermediate stratum cells in the enamel as well as in the newly formed dentin extracellular matrix. In contrast, in dentin more amelogenin than enamelysin was found located close to the periphery.
