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PHOSPHOLIPIDS IN AMELOGENESIS AND DENTINOGENESIS

Laboratoire de Biologie et Physiopathologie Crânio-Faciale EA 2496, Groupe Matrices Extracellulaires et Biominéralisation, Faculté de Chirurgie Dentaire–Université Paris V, 1, rue Maurice Arnoux, 92120 Montrouge, France;

View larger version (165K): [in a new window]   Figure 1. Rat incisor. Initial stage of enamel (E) formation. Terminal junctional complexes (arrowheads) are present between the cell bodies of secretory ameloblasts (A). Tomes' processes are located in spaces limited by interrod enamel. D, dentin. Uranyl acetate, lead citrate. x10,800.

View larger version (158K): [in a new window]   Figure 2. Rat incisor. Odontoblast cell body. Near the nucleus, the rough endoplasmic reticulum (RER), Golgi apparatus (Go), and lysosomes (Ly) are involved in synthesis and secretion of extracellular matrix components. Uranyl acetate, lead citrate. x27,000.

View larger version (154K): [in a new window]   Figure 3. Odontoblast process (OP) and its lateral branch. Electron-dense vesicles contain a material with an appearance of periodicity. A dense cytoskeleton, formed by intermediate and micro-filaments, is seen in the process. Predentin (Pd) surrounds the process. Uranyl acetate, lead citrate. x42,000.

View larger version (151K): [in a new window]   Figure 4. Lanthanum nitrate passes the terminal junctional complex located between the distal parts of odontoblast cell bodies, stains some matrix components in the predentin (Pd), and diffuses in metadentin (M). By contrast, dentin (D) is poorly stained. x54,000.

View larger version (141K): [in a new window]   Figure 5. Filipin-cholesterol complexes are seen along odontoblast cell processes in predentin (a). The density of filipin-cholesterol aggregates/µm2 is enhanced in dentin (b). The density is low along the Tomes' processes (c), and high for membrane remnants in the forming enamel (d). (a) x54,000, (b,c) x27,000, (d) x32,000.

View larger version (143K): [in a new window]   Figure 6. (a) Malachite green–glutaraldehyde (MGA) aggregates detected in the predentin (pd) of rat incisor. Aggregates appearing as branched filaments (arrowheads) are seen in the spaces located between collagen fibrils. D, dentin. Lead citrate. x81,000. (b) Higher magnification of MGA aggregates. Undemineralized section. x140,000. (c) Section floated on an EDTA solution. The staining is unchanged by the demineralization procedure in predentin (pd). In dentin (D), needle-like aggregates are seen along groups of electron-lucent mineralizing collagen fibrils. x110,000.

View larger version (152K): [in a new window]   Figure 7. (a) Visualization of phospholipids after rapid-freezing and malachite green-acrolein-osmium-tetroxide freeze-substitution fixation. Liposome-like structures are seen in intercollagen spaces in predentin (PD). Collagen fibrils (Co) appear as electron-lucent areas. X81,000. (b) Sample treated with methanol prior to MG-acrolein- osmium-tetroxide freeze-substitution. MGA aggregates have totally disappeared from predentin (PD) after phospholipid extraction. X140,000.

View larger version (106K): [in a new window]   Figure 8. (a) Dot blot showing interaction between iodoplatinate (IP) and malachite green-aldehyde (MGA) and major phospholipids (PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; SM, sphyngomyelin) (lanes 1 and 5). Lane 2 demonstrates that IP staining is not affected by pre-treatment with acetone (AC) but disappeared (lane 3) after pre-treatment with chloroform-methanol (Ch-m). Four amino-acid-bearing functions that may interfere with IP staining were unstained (lane 4). Lane 5 shows the staining obtained with MGA: PS and SM are stained, whereas only a thin rim is observed for PC, and PE is unstained. Dentin phosphoprotein (PP) is unstained. (b) The distribution of MGA-stained matrix components in rat incisor predentin (pd). Aggregates appear as granules and filaments in intercollagen spaces. X140,000. (c) IP-stained matrix components in rat incisor predentin. They are also located in intercollagen spaces. Collagen (Co) appears as electron-lucent areas. X140,000.

View larger version (174K): [in a new window]   Figure 9. Control (a) and suramin-treated (b) predentin (pd) and dentin (d) in a rat incisor, MGA staining. Accumulation of electron-dense amorphous material occurs in dentin (asterisk). Suramin is a mucopolysaccharidosis- and lipidosis-inducing pharmacological agent. X81,000.

View larger version (155K): [in a new window]   Figure 10. Control (a) and chloroquine-treated (b) predentin (pd) and dentin (d) of rat incisor. Metadentin (M) is thin in the control and enlarged when lipidosis was induced by the drug. Tannic acid staining. X13,500.

View larger version (129K): [in a new window]   Figure 11. Chloroquine induces defects during interrod enamel formation: lateral defects along a central core apparently normal (a) and irregular formation of interrod enamel (IR). A, ameloblasts. Tannic acid. (a,b) x13,500.

View larger version (153K): [in a new window]   Figure 12. MGA staining. Chloroquine-treated rat. Remnants of Tomes' processes are visualized (arrowheads) far away from the cell bodies inside rods (R) in the forming enamel. Interrod enamel (IR). x13,500.

View larger version (173K): [in a new window]   Figure 13. (a) A chloroquine-treated and MGA-stained rat incisor near the dentino-enamel junction. Tomes' processes remnants (asterisk) and some electron-dense amorphous material in dentin are MGA-positive. (b) Obtained after the same pharmacological treatment, but the section is stained by the IP reaction. Some crystal-like proteinaceous structures are stained, with or without demineralization of the sections, suggesting that some phospholipids are actually present in extracellular matrix. Dentin is unstained. x81,000.