The Effects Of Storage Media For Avulsed Teeth On Biological Characteristics Of Periodontal Ligament Fibroblasts

AIM: The instant and effective storage of dislocation tooth is very important forpreventing periodontal ligament dehydration, which can directly influence the successful rate of tooth replantation. The following factors are responsible for periodontal healing (the sequence was arranged from the strongest to the weakest):the root development stage, the extraoral storage time, instant replantation and the moist storage time. The teeth should be dipped into physiological medium as soon as possible. The screening of storage medium is one major content of tooth replantation investigation, there is little research on the selection of storage medium domestically, moreover, there were few reports on medium selection by measuring the activity of the humanperiodontal ligament cells in vitro. The present study were to culture the human periodontal ligament cells and to compare the biological activity of cells which were dipped into six different medium for half an hour, one hour ,one and an half hour and two hours respectively.Methods:1. The culture of human periodontal ligament cellsThe premolars without caries and gingivititis were extracted for orthodonicreasons from 13-19 year old patients. After extraction, the periodontal ligamentwere scraped from middle 1/3 root.According to tissue attachment method, the cells were cultured in DMEM whichcontained 10% fetal calf serum, and incubated in 37℃, 5%CO₂ conditions. Afterreached confluence, the cells were digested with 2.5 g / L trypsin and subcultured,and the passage 8 cells were used for the following experiments.2.Cell proliferation activityThe well growth passage 8 cells were digested regularly and seeded into four96-well plates at the concentration of 2×10⁵/m l , 100 μ l per well. Afterincubated in DMEM for 24 hours and the majority of cells were attached onto the wall under the microscope , then the liquid and unattached cells were abandoned,after washed with PBS for three times, the different storage medium were added.(1) Different solutions were added to the first plate for half an hour, and there were 8 groups, 8 wells in each group. The first group was DMEM, the second was HBSS, the third was 0.9%saline, the fourth was saliva, the fifth was milk, the sixth was tap water; and the control group were DMEM which contained 10% FCS, 100μ l per well. The blank group contained no solution.(2) Different solutions were added into the second, the third and the fourth plate for one hour, one hour and an half and two hours respectively, and the rest treatment was the same as the that of the first plate.Then the changes of the cell proliferation activity were measured by MTT method.Results:1. At half an hour, there was no significant difference between groups( P>0.05).2. At 1 hour, the difference between groupB1 and groupB4, groupB1 and group B5 were significant (P<0.05),and the cell activity of groupB6 was significantly lower than that of groupB1 (P<0.01)。3.At one hour and an half, there was significant difference between groupC3 and group C6, group C1 and group C5 (P<0.05) 。 Moreover, the difference betweengroup CI andC 2: groupC land C3: group C2 and C4: group C2 and C5; group C2 andC6; group C3 and C4; group C3 andC 5; groupC4andC 6; group C5 and C6; group Cland C6 were significant (/><0.01>*respectively. 4.At 2 hours, there was significant difference between groupDland groupD2, group D2and group D3 (P<0.05^?, and there were significant difference between group DlandD3; DlandD6; D2andD6; D3andD4; D3andD5; D4andD6 group; D5andD6 group (P<0.01)??oConclusion: There was no significant difference among the different media when stored within half an hour. With the elongation of the time, then at one hour, DMEM culture medium exhibited its superiority and there was significant difference when compared with saliva, milk and tap water, among those media, the amount of vital cells was the least for tap water group which indicated that tap water wasn't an ideal medium for tooth storage. At one hour and an half, the amount of vital cells in each group decreased, the difference between DMEM and tap water, saliva and tap water were significant, and the MTT value of tap water got close to that of blank control .At 2 hour, there was significant difference between DMEM and tap water. Among the six storage media, the best was DMEM, and tap water was not the first selected medium.