Wen-Lang Lin

In vitro formation of mineralized nodules by periodontal ligament cells from the rat

SummaryThe 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 Dulbeccos Modified Eagles 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 μg/ml) and sodium β-glycerophosphate (10 mM), (2) ascorbic acid, sodium β-glycerophosphate, and dexamethasone (5 μM), or (3) ascorbic acid alone. Cells were then fixed in 2.5% glutaraldehyde, postfixed in 1% OsO4, and prepared for light and electron microscopy. Threedimensional 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. These nodules contained neither osteoblasts nor osteocytes, and have their own morphological organization and characteristics which differ from those formed by bone cells in culture. Therefore, these data suggest that PDL cells are capable of forming mineralized tissue in vitro with the morphological characteristics different from bone mineralized nodules.

Synthesis of noncollagenous extracellular matrix proteins during development of mineralized nodules by rat periodontal ligament cells In vitro

To characterize the mineralized nodules produced by rat periodontal ligament (PDL) cells in vitro, we have studied the synthesis and distribution of mineralized tissue proteins at various stages of nodule formation. PDL cells were obtained from coagulum in the socket at 2 days after tooth extraction and cultured in Dulbeccos Modified Eagles Medium (DMEM) containing 10% fetal bovine serum and antibiotics. Confluent cells were grown in the presence of ascorbic acid (50 μg/ml), dexamethasone (5 μM), and β-glycerophosphate (10 mM) for 3 weeks. Four stages showing distinct morphological characteristics during development of mineralized nodules were identified. Protein synthesis and deposition of proteins into the matrix were studied during these stages by metabolic labeling with [35S]methionine for 24 hours. Large quantities of SPARC (secreted protein, acidic and rich in cysteine) were synthesized by confluent cells but decreased during the progress of mineralized nodule formation. Two forms of osteopontin (OPN) (67 kDa and 61 kDa) were synthesized in comparable quantities by confluent cells; OPN and bone sialoprotein (BSP) were induced by dexamethasone and represented the major proteins in the mineralized matrix. The 67 kDa form of OPN was the predominant species in the mineralized matrix. Both OPN and BSP were localized by immunogold electron microscopy on globular as well as fused electron-dense structures at sites of tissue mineralization.

Evidence for up-regulation of epidermal growth-factor receptors on rat periodontal ligament fibroblastic cells associated with stabilization of phenotype in vitro

This study sought to understand the role of epidermal growth factor receptor (EGF-R) in periodontal ligament (PDL) fibroblasts. Rat PDL fibroblastic cells and ROS 17/2.8 cells (highly differentiated osteoblastic osteosarcoma cells) were cultured and treated with transforming growth factor-alpha (TGF-alpha), EGF, dexamethasone (Dex) or a combination of EGF and Dex. Alkaline phosphatase (ALP) activity, an early differentiation marker for mineralized tissue-forming cells, was measured using p-nitrophenylphosphate as a substrate. For Scatchard analysis of [125I]-EGF binding, cells were incubated in Dulbeccos modified Eagles medium containing 0.2% bovine serum albumin and 0-64 ng/ml of [125I]-EGF for 4 h at 4 degrees C. Also, the synthesis of EGF-R protein and the expression of mRNA for EGF-R were measured by immunoprecipitation and Northern blot analysis, respectively. Untreated PDL fibroblastic cells showed a gradual increase in spontaneous ALP activity from 32.4 U/10(6) cells at 2 days to 49.6 U/10(6) cells at 7 days of culture. ALP activity was further increased to 70.8 U/10(6) cells at 7 days after treatment with Dex, whereas EGF treatment reduced it to 19.4 U/10(6) cells. Culture of PDL fibroblastic cells in the presence of a combination of Dex and EGF decreased the Dex-induced ALP activity from 70.8 U to 41.8 U/10(6) cells at 7 days. A similar inhibitory effect on ALP activity was found after treatment with TGF-alpha. In contrast, ROS cells maintained a high ALP activity (1748 U/10(6) cells) throughout culture, unaffected by EGF. Scatchard analysis demonstrated that PDL fibroblastic cells have both high- and low-affinity forms of EGF-R, while ROS cells did not have any detectable EGF-R. Treatment of PDL cells with Dex for 2 days decreased the synthesis of EGF-R protein, the expression of EGF-R mRNA and the number of EGF-R. In contrast, EGF treatment increased the expression of EGF-R mRNA. These data suggest that PDL fibroblastic cells express numerous EGF-R, but the number decreases during their differentiation into mineralized tissue-forming cells under the influence of Dex. Thus, EGF-R may function in the stabilization of phenotype in PDL fibroblastic cells.

Down-Regulation of Osteoblastic Cell Differentiation by Epidermal Growth Factor Receptor

Abstract The role of epidermal growth factor receptors (EGF-R) in osteogenic cell differentiation was investigated using preosteoblastic MC3T3-E1 (MC3T3) cells and osteoblast-like ROS 17/2.8 (ROS) cells. When cultured in the presence of β-glycerophosphate (GP) and ascorbic acid (AA), MC3T3 cells underwent spontaneous differentiation into osteoblasts which was confirmed as they expressed osteoblast markers such as alkaline phosphatase (ALP), bone sialoprotein (BSP) and osteocalcin (OC). Interestingly, the number of EGF-binding sites decreased during their differentiation into osteoblasts, and the osteogenic protein-1 (OP-1) treatment, which accelerated their differentiation, lowered the number of EGF-binding sites even further. On the other hand, ROS cells with high expression levels of osteoblast markers and no EGF-R, after being transfected with human EGF-R cDNA (EROS cells), expressed numerous EGF-binding sites as well as EGF-R mRNA and protein; in the process, they ceased to express osteoblast markers, indicating their dedifferentiation into osteoprogenitor cells. Both MC3T3 and EROS cells showed increased cell growth in response to EGF, whereas ROS cells did not. These results imply that the EGF/EGF-R system in osteogenic cells has a crucial function in osteoblast phenotype suppression and osteogenic cell proliferation.

Interleukin-1β—Induced Release of Matrix Proteins into Culture Media Causes Inhibition of Mineralization of Nodules Formed by Periodontal Ligament Cells In Vitro

Abstract. The mechanism by which interleukin-1β (IL-1) inhibits the formation of mineralized tissue nodules by periodontal ligament (PDL) cells in vitro was investigated through the processes of morphological analysis, immunoprecipitation, and Northern blot analysis. PDL cells were obtained from a 2-day-old coagulum in tooth socket and cultured in Dulbeccos Modified Eagle Medium (DMEM) containing 10% fetal bone serum (FBS) and antibiotics. Confluent cells were grown for up to 3 weeks in the presence of ascorbic acid (AA), β-glycerophosphate (GP), and dexamethasone (Dex), or IL-1. PDL cells cultured in the presence of GP and AA did not differentiate, but those treated with Dex, GP, and AA (Dex group) underwent differentiation, showing four stages (confluent, multilayer, nodule, and mineralization) of disparate morphological characteristics. In contrast, the cells treated with IL-1, Dex, GP, and AA (IL-1 group) did form multilayers but failed to form mineralized nodules. Electron microscopy demonstrated that the Dex-induced mineralized nodules contain multilayers of fibroblastic cells, numerous collagen fibrils, and dense globular as well as fused electron dense patches that are associated with numerous apatite crystals. The nodule-like structures in the IL-1 group were also comprised of multilayered fibroblastic cells, but they contained only a small number of collagen fibrils, and no dense globular or fused patches. Von Kossa staining confirmed the presence of numerous mineralized nodules in the Dex group and their scarceness in the IL-1 group. Northern blot analysis of IL-1-treated cells, however, revealed the presence of mRNAs for type I collagen (Col I), secreted protein, acidic and rich in cysteine (SPARC), osteopontin (OPN), alkaline phosphatase (ALP), bone sialoprotein (BSP), and osteocalcin (OC), whose expression patterns and levels were comparable to those of the Dex group. Immunoprecipitation analysis of OPN and BSP in the cell/matrix layers and the culture media after [35S]-methionine labeling showed their deposition primarily in the mineralized nodules of the Dex group, and their release into the media in the IL-1 group. Immunogold labeling demonstrated the location of OPN and BSP in mineralized nodules of the Dex group, but no significant labeling occurred in the nodule-like structures from the IL-1 group. Interestingly, IL-1 treatment increased the expression of collagenase mRNA by sevenfold, compared with that of the Dex group. These data suggest that the IL-1-induced formation of unmineralized nodules by PDL cells results not so much from the downregulated formation of matrix proteins, which plays a crucial role in the mineralization process, as from their release into the culture media. Finally, collagenase synthesis upregulated by IL-1 may be involved in this process.