Artificial gingival composed by regenerated epithelia and bone from stem cells

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Although oral keratinocyte stem cells play a key role in tissue homeostasis, wound healing, and neoplasia, they remain difficult to identify and characterize. The specific aim of the present study is to characterize an oral keratinocyte stem-cell population separated using a magnetic technique. Oral human keratinocytes obtained from keratinized oral mucosa were magnetically separated using a proliferation-related marker, CD71 and α6ss4 integrin. The expression of different stem cell markers: CD44H, Nestin,Nanog, Oct 3/4, CD117 was checked by immunofluorescence. The ability of α6ss4pos CD71neg fraction to form oral epithelial equivalents was also assayed. Three different oral keratinocyte subpopulations were obtained following magnetic separation: α6ss4pos CD71neg,α6ss4pos CD71pos and α6ss4neg. Our α6ss4pos CD71neg stem cell fraction was positive for Oct 3/4, CD44H and cytokeratin 19 while Nanog, Nestin and CD117 expression was absent. At the same time, the other two cell fractions α6ss4pos CD71pos and α6ss4neg were negative for all stem cell markers. Also, α6ss4pos CD71neg fraction was able to regenerate a well stratified and organized oral epithelial equivalent. The distribution of cytokeratin 19 and involucrin in the oral epithelial equivalent reflected the in vivo situation in oral gingival epithelium. It is also suggested that a magnetic system may be an important tool in acquiring oral keratinocyte stem cells for research.