Methods: Human healthy retained deciduous incisors without caries and untreated were collected in this study. Odontoclasts were obtained and isolated from the resorpted root surface of the deciduous teeth using the method of enzyme digestion. To identify odontoclasts, HE and TRAP staining were used to observe the hismorphology of the cultured cells and the resorption lacunas formed by odontoclasts in the human teeth slices was viewed by scanning electron microscopy (SEM). The expression of MT-MMP and MMP12 in the cultured cells was detected by the method of immunohistochemistry.
Results: 1.The isolated and cultured cells appear to be characteristic pseudopodia, such as oblong, ellipse, pan-fried egg, and so on, with different number of cell nuclei varied from one to dozens. The cytoplasm was full of numerous granules and vesicles. 2. In HE staining, most of cultured cells appeared as multinucleated giant cells with blue cell nuclei and red cytoplasm. TRAP staining indicated that many specific TRAP particles could be seen in the cultured cells, with claret-red cytoplasm and fluorescent blue nuclei. 3.The Spherical or oval resorption lacuna formed by odontoclasts could be observed on the surface of human teeth hard tissue by SEM. 4.The expression of MT-MMP and MMP12 was observed in the cytoplasm of cultured multinucleated cells.
Conclusions: Odontoclasts were successfully isolated, cultured from fresh primary teeth by the method of enzyme digestion, which can be identified by the histomorphological observation and resorption function investigation. The presence of MMP-2 and TIMP-1 can be confirmed detected in cultured odontoclasts by immunocytochemical staining.
Keywords: Cell culture, Enzymes, Resorption and odontoclasts; isolation