Artificial gingival composed by regenerated epithelia and bone from stem cells

尽管口腔角质形成干细胞在组织动态平衡、伤口愈合和肿瘤形成中起着关键作用，但它们仍然很难被识别和表征。本研究的具体目的是描述使用磁性技术分离的口腔角质形成干细胞群体的特征。从角化的口腔粘膜中获得的口腔角质形成细胞用与增殖相关的标记CD71和α6SS4整合素进行磁分离。免疫荧光法检测不同干细胞标志物CD44H、Nestin、Nanog、Oct3/4、CD117的表达。同时检测α6ss4pos CD71neg组分形成口腔上皮细胞等效物的能力。经磁分离后获得3种不同的口腔角质形成细胞亚群：α6ss4pos CD71neg、α6ss4pos CD71pos和α6ss4neg。我们的α6ss4pos CD71neg干细胞部分Oct3/4、CD44H和细胞角蛋白19呈阳性，而Nanog、Nestin和CD117均不表达。同时，另外两个细胞组分α6ss4pos CD71pos和α6ss4neg对所有干细胞标志物均为阴性。此外，α6ss4pos CD71neg组分能够再生层次化和组织化良好的口腔上皮等效物。细胞角蛋白19和总蛋白在口腔上皮等效物中的分布反映了口腔牙龈上皮的体内情况。磁系统可能是获取口腔角质形成干细胞研究的重要工具。

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.