Proliferation and differentiation of adult human dental pulp cells cultured by tissue explant method / 中国组织工程研究

ABSTRACT

BACKGROUND: Human pulp tissue has been known to be less, and exhibit poor tolerance to enzymatic digestion and less adherent cells after step-by-step digestion of trypsin and collagenase, thereby often leading to a failure of passage. Only several kinds of dental pulp cells with poor activity can be obtained by the tissue explant-collagenase digestion. OBJECTIVE: To investigate human dental pulp cells cultured in vitro by tissue explant method. DESIGN, TIME AND SETTING: A cytological observation was performed at Heping Campus and School of Stomatology, Jilin University from 2005 to 2007. MATERIALS Healthy young human teeth extracted for orthodontic correction or impaction. METHODS: Pulp tissue from the third molar teeth was collected, cut into small blocks with a size of 1.0 mm×1.0 mm×0.5 mm under the infiltration of small amount of Dulbecco's modified eagle's medium, and then transferred into a 6-well plate containing culture medium for incubation in a 5% CO2 and saturated humidity atmosphere at 37 ℃. During the process of incubation, pulp tissue was adjusted at a density of 3-6 blocks/well, with an equal spacing of 0.5 cm and the 6-well plate was kept inverted. Three hours later, the 6-well plate was turned over to make tissue blocks adhering to the plate wall. Culture was continued after addition of 2 mL of culture medium. Culture medium was renewed every 4-6 days. After 6-15 days, cells emigrated from the edge of tissue blocks and call outgrowth appeared around each tissue block. When cells closed to confluency, a digestion procedure of 2.0-3.0 minutes (0.25% trypsin and 0.02% ethylenadiamine tetraacetic acid) was followed by passage culture at a proportion of 1 (2-3) in 25 mL of culture flasks. Purified fibroblast-like cells were gradually obtained from primarily cultured cells by repeated digestion and passage. MAIN OUTCOME MEASURES: Cellular morphology was identified by immunohistochemistry; secreted dental pulp cells were determined using alkaline phosphatase activity; the growth curves of human pulp tissue cells were depicted by MTT assay. RESULTS: Under an inverted phase contrast microscope, the obtained dental pulp cells were primarily typical fibroblasts with a long-shuttled appearance, well-rounded call body, uniform cytoplasm, round or oval nucleus, and clear nucleolus. Immunohistochemistry results showed call surface vimentin-positive, pan cytokeratin-negative, and alkaline phosphatase-posltive These cells were decreased after culturing 1 day, were slightly increased after 2 days, entered the logarithmic growth period and were markedly increased after 4 days, entered a platform period after 8 days, and began to decrease again after 9 days. The whole growth curve of cells appeared in "S" shape.CONCLUSION: The dental pulp cells isolated from human pulp tissue by tissue explant method can effectively proliferate end retain a poody differentiated state in vitro.