Cho M I, Matsuda N, Lin W L, Moshier A, Ramakrishnan P R
Department of Oral Biology, School of Dental Medicine, State University of New York, Buffalo 14214.
Calcif Tissue Int. 1992 May;50(5):459-67. doi: 10.1007/BF00296778.
The purposes of this study were to determine whether periodontal ligament (PDL) cells are capable of producing mineralized nodules in vitro and to analyze ultrastructural features of the nodules. Rat PDL cells were obtained from coagulum in the socket at 2 days after tooth extraction and cultured at confluence in standard medium containing Dulbecco's Modified Eagle's Medium supplemented with 10% FBS and antibiotics. To test mineralized nodule formation, cells were further cultured for an additional 3 weeks in the standard medium containing (1) ascorbic acid (50 micrograms/ml) and sodium beta-glycerophosphate (10 mM), (2) ascorbic acid, sodium beta-glycerophosphate, and dexamethasone (5 microM), or (3) ascorbic acid alone. Cells were then fixed in 2.5% glutaraldehyde, postfixed in 1% OsO4, and prepared for light and electron microscopy. Three-dimensional nodules containing mineralized matrices were formed only when the cells were cultured in the presence of ascorbic acid and dexamethasone. They were composed of multilayered fibroblasts (up to 13 layers), and highly organized collagen fibrils with 64 nm cross-banding patterns between the cell layers. The fibroblasts in the nodules exhibited an elongated shape with a high degree of cytoplasmic polarity throughout the nodule, and have the morphological features of PDL fibroblasts as seen in vivo. Mineral deposition with needle-like crystals was initiated on collagen fibrils located in intercellular spaces of the upper cell layers and became increasingly heavier towards the bottom half of the nodules. X-ray microanalysis and electron diffraction analysis confirmed that mineral deposition contained calcium and phosphate in the form of immature hydroxyapatite.(ABSTRACT TRUNCATED AT 250 WORDS)
本研究的目的是确定牙周膜（PDL）细胞在体外是否能够产生矿化结节，并分析这些结节的超微结构特征。大鼠PDL细胞于拔牙后2天从牙槽窝中的血凝块获取，并在含有补充了10%胎牛血清（FBS）和抗生素的杜氏改良 Eagle 培养基的标准培养基中汇合培养。为了测试矿化结节的形成，将细胞在含有（1）抗坏血酸（50微克/毫升）和β-甘油磷酸钠（10毫摩尔）、（2）抗坏血酸、β-甘油磷酸钠和地塞米松（5微摩尔）或（3）仅抗坏血酸的标准培养基中再培养3周。然后将细胞用2.5%戊二醛固定，再用1%四氧化锇后固定，并制备用于光镜和电镜观察。仅当细胞在抗坏血酸和地塞米松存在的情况下培养时才形成含有矿化基质的三维结节。它们由多层成纤维细胞（多达13层）组成，细胞层之间有高度有序的具有64纳米交叉带纹模式的胶原纤维。结节中的成纤维细胞呈现细长形状，在整个结节中具有高度的细胞质极性，并且具有在体内所见的PDL成纤维细胞的形态特征。针状晶体的矿物质沉积起始于位于上层细胞层细胞间隙中的胶原纤维上，并朝着结节的下半部分越来越重。X射线微分析和电子衍射分析证实矿物质沉积含有未成熟羟基磷灰石形式的钙和磷。（摘要截短至250字）
