| Previous studies have demonstrated that the human systems, including musculoskeletal system, cardiovascular system, immune system, nerve-endocrine system and self-generate physiological coherence system, were affected in the space microgravity. For example, the microgravity induces the occurrence of bone loss, amyotrophy, cardiovascular dysfunction, decreases in orthostatic tolerance and cognitive function. So what effect could be caused for periodontal tissue in microgravity environment ? In this study, three dimensional clinostat(TDC)was employed to study the effect of simulated weightless environment on human periodontal ligament fibroblasts ( hPDLFs).ObjectiveTo investigate the structure and function of hPDLFs under simulated weightlessness environment .Then the periodontal tolerance of some individuals in such special environment, such as the astronauts and the space tourists, was analyzed preliminarily. Method:Passage 4-7 hPDLF cells were used in the experiments. The hPDLFs were seeded in 20ml flask and pre-cultured for 24h, and were divided into rotary groups and control groups randomly. Through setting the random speed at 4-10 r/min, TDC device was employed to simulate the weightlessness environment, which microgravity level is between 10-3-10-4 g. Rotary groups were fixed on TDC and rotated for 48h, 72h, 96h respectively, and three control groups were also cultured normally at three time points, then the following assays wereperformed:1 cell morphologic detection:HE staining and immunofluorescence method(rhodamine-phalloidin)were used to analyze the differences of morphology and cytoskeleton between rotary and static groups.2 cell functional detection:Cell counting, MTT assay, flow cytometry, Hoechst 33258 staining and RT-PCR were employed to analyze the differences of cell proliferation, apoptosis and the expression of collagen typeⅠbetween rotary and static groups.Result1 Effects of TDC simulated weightlessness on cell morphologyHE staining: After cultured for 48h, the morphology of hPDLF cells were observed under microscope and detected with HE staining method . In both rotary and static groups, the cells appeared with a spindle or polygonal shape .The intact nucleis were in the middle of cells.The number of cytoplasmic particles increased. Intracellular vacuoles, nuclear bifurcate , nuclear disintegrate were not observed. However, the nuclear area is smaller in the rotary group than in the control by 12% (P<0.05). After exposed continuously to TDC for 72h and 96h, the differences in the cell morphology and nuclear area between rotary and static groups disappeared gradually.Immunofluorescence method(rhodamine-phalloidin):After cultured for 48h, intracellular cytoskeleton stucture of hPDLF cells were observed, the cells appeared disorganized microfilament structure as compared with their controls. After exposed continuously to TDC for 72h and 96h, the clear and well-organized stress fiber was observed in hPDLF cells of rotary groups.2 Influences of TDC simulated weightlessness on cell functionsCell counting: After cultured for 48h and 72h by using TDC, the cell number was not affected as compared with control(P>0.05), when hPDLFs were exposed to simulated weightlessness for 96h, we observed that there was a clearly increase in cell number (P<0.01).MTT assay: After cultured for 48h, the cell viability was not affected as compared with control(P>0.05), when hPDLFs were exposed to simulated weightlessness for 72h and 96h, we observed that there was a clearly increase in cell viability (P<0.01).Cell cycle: After exposed to TDC for 48h and 72h, percentage of cells arrested in S phase was not affected as compared with control(P>0.05), when hPDLFs were exposed to simulated weightlessness for 96h, we observed that there was a clearly increase in percentage of cells arrested in S phase (P<0.05).Hoechst 33258 staining: After exposed to TDC for 48h,72h and 96h, the apoptosis was not affected as compared with control.RT-PCR:After exposed to TDC for 48h and 72h, the RT-PCR results showed that the transcriptional expression of collagen typeⅠin hPDLF cells was not affected as compared with control(P>0.05), After cultured for 96h, we observed that there was a clearly increase in the expression of collagen typeⅠas compared with static controls (P<0.05).ConclusionAfter exposed to TDC for 48h,the morphology of hPDLF cells were affected,such as decreased nuclear area, disorganized microfilament structure. But it does not appear cell cracking,apoptosis,cell viability decline. However, the morphology, structure, viability , prolifertion and collagen typeⅠof hPDLFs were gradually recovered after 72h. This results suggested that hPDLF cells were tolerable to exposure to the simulated microgravity environment. However, we still need to investigate deeply how the TDC-induced enhancement in hPDLFs' viability and functions afffects the structure of periodonal tissue. |
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