RESUMEN
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.
RESUMEN
Objective To investigate the effect of different concenrtrations of basic fibroblast growth factor (bFGF) on proliferation of human dental pulp cell in vitro,and to find out the most effective concentration of bFGF. Methods Dental pulp cells were isolated from dental pulp tissue explants.The pulp cells were divided into 5 groups randomly,bFGF was added into each group until the ultimately concentrations were 0.1,1.0,10.0 and 100.0 ?g?L-1respectively while the group without bFGF as control group. The effects of bFGF on dental pulp cells were assayed by absorbency A and relative growth rate(RGR) with MTT colorimetric method. Results bFGF at concentrations of 1.0-100.0 ?g?L-1 promoted the cell proliferation (P0.05). Conclusion bFGF has the capability of promoting the proliferation of human dental pulp cells,and the smallest effective concentration is 1.0 ?g?L-1,the most strong cell proliferation takes place at bFGF concentration of 10.0 ?g?L-1.