Itaya T, Kagami H, Okada K, Yamawaki A, Narita Y, Inoue M, Sumita Y, Ueda M
Department of Oral and Maxillofacial Surgery, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan.
J Periodontal Res. 2009 Aug;44(4):425-33. doi: 10.1111/j.1600-0765.2008.01137.x.
Although periodontal ligament-derived cells are expected to be a useful source of cells for periodontal tissue engineering, the characteristic changes of primary cultured cells have not been well studied. Therefore, the aim of this study was to investigate the characteristics of periodontal ligament-derived cells and their changes during passage.
Human periodontal ligament tissue was obtained from extracted third molars. Cells were subcultured until passage 6 and the cell characteristics from early to late passages were evaluated using immunofluorescence microscopy, alkaline phosphatase activity analyses, reverse transcription-polymerase chain reaction and quantitative real-time polymerase chain reaction. To examine the function of periodontal ligament-derived cells further, cells were transplanted into the renal subcapsule of an immunocompromised rat.
Immunofluorescence results showed relatively uniform expression of MSX-2 and osteonectin from passage 1 until passage 6. The STRO-1-positive fraction was 33.5% at passage 0, which was reduced to 14.7% at passage 3. Cultured cells at passage 1 expressed mRNA for collagen type I, collagen type XII, Runx2, alkaline phosphatase, osteonectin, osteopontin, scleraxis, tenomodulin, Msx2, GDF5 and GDF7 genes, but not for bone sialoprotein. The level of mRNA expression from tenomodulin and collagen type XII genes decreased after passage 3. Alkaline phosphatase activity decreased in cells at later passages. Osteogenic induction of periodontal ligament-derived cells resulted in a down-regulation of the tenomodulin gene. Transplanted cells from both early and late passages produced dense collagen fiber bundles without calcified tissue.
Cultured periodontal ligament-derived cells were a morphologically homogeneous population, although expression of STRO-1 was limited in primary culture. Cultured cells showed de-differentiation during passage for both osteogenesis- and tendo/ligamentogenesis-related genes.
尽管牙周膜来源的细胞有望成为牙周组织工程中有用的细胞来源，但原代培养细胞的特征变化尚未得到充分研究。因此，本研究的目的是探讨牙周膜来源细胞的特征及其传代过程中的变化。
从拔除的第三磨牙获取人牙周膜组织。细胞传代至第6代，并使用免疫荧光显微镜、碱性磷酸酶活性分析、逆转录-聚合酶链反应和定量实时聚合酶链反应评估从早期到晚期传代的细胞特征。为了进一步研究牙周膜来源细胞的功能，将细胞移植到免疫缺陷大鼠的肾被膜下。
免疫荧光结果显示，从第1代到第6代，MSX-2和骨连接蛋白的表达相对均匀。第0代时STRO-1阳性比例为33.5%，第3代时降至14.7%。第1代培养细胞表达I型胶原、XII型胶原、Runx2、碱性磷酸酶、骨连接蛋白、骨桥蛋白、硬骨素、肌腱调节蛋白、Msx2、GDF5和GDF7基因的mRNA，但不表达骨唾液蛋白。第3代后，肌腱调节蛋白和XII型胶原基因的mRNA表达水平下降。晚期传代细胞的碱性磷酸酶活性降低。牙周膜来源细胞的成骨诱导导致肌腱调节蛋白基因下调。早期和晚期传代的移植细胞均产生致密的胶原纤维束，无钙化组织。
培养的牙周膜来源细胞在形态上是均匀的群体，尽管STRO-1在原代培养中的表达有限。培养细胞在传代过程中，与成骨和腱/韧带生成相关的基因均表现出去分化。
