Isolation And Identification Of Human Odontoblast Cells

Dentin is the principal part of a tooth, unlike bone, under physiological condition, no absorption and reconstruction but mineralization course exists in dentin. Dentin formation is a consecutive process in whole life of a tooth and odontoblast cells are responsible for it through secretion and mineralization of dentin matrix. Thus, taking odontoblast cells as research target would help to better understand the mechanisms of dentinogenosis and to some extent provide valuable information in the formation of human mineralized tissues.Odontoblast cells, closely neighbor dentin, form a monolayer lining the periphery of dental pulp, and stretch cell processes into dentinal tubules. Moreover, odontoblast is a terminal differentiated cell, can not farther differentiate and proliferate in vivo, nor in vitro. Thus, it is impossible to obtain large number of pure odontoblast cells for research. Immortalized odontoblast-like cell lines have been established and provided a better source of odontoblast-like cell. However, owing to some plasmids transformed during immortalization process, influences on cell characteristics may occur. Therefore, to some extent, immortalized odontoblast-like cell lines are not good enough for studying the function and feature of odontoblast cells. Odontoblast cell isolated directly from freshly extracted tooth remains original characteristics, and is a better cell model for research.This study established a method for isolating odontoblasts from human tooth by using proteolytic enzymes. Cell morphology and odontoblast cell specific protein staining had been used for identification.This research consists of three parts:1. Isolation of human odontoblast cellsThis study isolated human odontoblast cells by means of tooth sectioning and enzyme digestion. Normal human permanent teeth were collected and sectioned into slices of l-2mm thick by manual, and then immersed with cold ECS. After repeated trials of different digestion condition and enzyme treatment, 3mg/ml collagenase IA and 0.25 mg/ml protease I incubated for about 45 minutes in a water bath at 38-38.5 were the best conditions for odontoblast cells isolation. Cells isolated in this experiment had typical columnar or cuboidal cell bodies and intact long processes.2. The identification of isolated odontoblast cellsThe isolated odontoblast cells were identified using the cell specific proteins staining experiment. The results showed that the isolated cells expressed proteins of collagen I, dentin matrix protein-l(DMP-l) and detin sialoprotein (DSP).and had typical and unique odontoblast-like cell configuration3. Image analysis on cytoskeleton of odontoblast cells with Laser confocal scaning microscopeThe spatial structure of F-actin in odontoblast cells was observed through Laser confocal scaning microscope combined with fluorescence double-labeled technique (F-actin marked by FITC-phalloidin and nucleic acid marked by PI). The results sho\yed that isolated odontoblast cells possessed unipolar figure. The filamentous F-actin existed mainly in cytoplasm, and distributed in bunches. The outline of nucleolus was clear.