Confocal Laser Scanning Microscopy of Alveolar Bone and Dentin in the Rat Mandible.

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  • ラット歯槽骨および象牙質の共焦点レーザ走査顕微鏡的観察

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To establish conditions for observing rat hard tissue by confocal laser scanning microscopy (CLSM), suitable specimens of bone and teeth were observed three-dimensionally after staining with various fluorescent labeling agents. Rats were injected subcutaneously, intramuscularly, intraperitoneally or intravenously with calcein. Two days later, the animals were euthanized and subjected to perfusion fixation. Their mandibles were dissected out and embedded in epoxy resin for preparation of undecalcified sections 500, μm in thickness. These specimens were examined by CLSM, using barrier filters for wavelengths of 535 nm (CH1) and 610nm (CH 2), at an excitation wavelength of 488nm. Rats were given calcein, tetracycline or alizarin red or combinations of these fluorescent dyes at various concentrations, then simultaneously treated and examined by CLSM. Calcein was easily detected using the CH 1 and CH 2 when administered at 1 or 2mg per 100g body weight. Alizarin red was detected using the CH 2 when administered at 4 or 8mg per 100g body weight. Tetracycline was detected using the CH 1 and CH 2 when administered at 4 or 8mg per 100g body weight. When calcein was injected intravenously, the labeling line was narrower and more strongly fluorescent than the labeling line after subcutaneous injection. The labeling line observed after intraperitoneal and intramuscular injection was intermediate in intensity between those after intravenous and subcutaneous injection. Double fluorescent staining with calcein at 2mg and alizarin red at 4mg per 100g body weight were found to be suitable for CLSM of mandibular specimens.<BR>Under these staining conditions, the dentin and outer zone of the supporting bone of the alveolar process showed a regular labeling pattern, whereas the alveolar bone proper adjoining the periodontal membrane displayed an irregular labeling pattern. Bone lacunae and their canaliculi were observable

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