Effects Of Tobacco On Human Gingival Fibroblats Attached To Titanium

Objective:The study aimed to apply the smokeless tobacco extract (ST) and nicotine to Human Gingival Fibroblasts(HGFs) to titanium in vitro. And then observed the effect of ST and nicotine on the HGFs attachment, morphology, structure, proliferation and collagen synthesis, in the hope that we could investigate the potential impact of ST in the destruction of Ti-implants.Methods:Part1: HGFs was obtained from explants of human normal gingival tissues by using the tissue-explant technique. The cellular growth conditions were observed under inverted phase-contrast microscope. The third passage of HGFs was applied to be indentified by immunochemistry of vimentin and cytokeratin. Hematocytomete was used to determine the numbers of cells and the mean of cell numbers was calculated for drawing the growth curve of human gingival fibroblasts.Part2: The titanium disks with 1mm in thickness and 10mm in diameter were manually finished by grit papers. Surface topography was observed by reflex light microscope and SEM. Parameter Ra was used to describe the surface roughness.Part3: Round disks of titanium were placed in 24 well plates. HGFs which contain 0, 0.01, 0.1, 1, 5, 10, 25g/L ST and 0.03, 0.15, 0.3g/L nicotine respectively were implanted on the surface of titanium. The titanium disks were washed by PBS to remove non-adherent cells after cells were cultured for 2h, 24h respectively. At the points of 2h, the numbers of HGFs attached to titanium were measured by MTT. At the points of 24h, the shapes of HGFs attached to titanium were measured by a computer analysis system.HGFs were implanted on the surface of titanium for 24h and then cultured in DMEM containing 0, 0.01, 0.1, 1, 10g/L ST and 0.03g/L nicotine respectively. The titanium disks were washed by PBS to remove non-adherent cells after cells were cultured for 1d, 4d, 7d and 10d respectively and the numbers of HGFs attached to titanium were measured by MTT.Part4: HGFs were implanted on the surface of titanium for 24h and then cultured in DMEM containing 0, 0.01, 0.1, 1, 5, 10,25g/L ST and 0.03,0.15,0.3g/L nicotine respectively. After 2d, the cell culture supernatants were collected. Hydroxyproline chromatometry was performed to detect the level of collagen.Result:1. Emigration of HGFs cultured with tissue-explant method occurred earliest at the sixth day. Most cells were fusiform with large body. The cells covered full of the floor of 25 cm~2 cell culture flask about two to three weeks of primary culture. The immunochemistry study showed that vimentin was expressed in HGFs while cytokeratin failed to be detected, which indicated that HGFs originated from mesoblastma. It is a S-shaped growth curve for the cultured human gingival fibroblasts. The cell number declined a little after the first 24 h cultivation, and then it increased gradually. From day 6, the cells entered the plateau phase, and grew slowly.2. The titanium finished surface showed regular structure, with narrow chimb and blunt ridge. The parameter of Ra of the titanium plates is.0.1022±0.00766μm..3. The results of MTT at 2 hours assay showed that both ST and nicotine inhibited attachment of HGFs in all groups in a dose-dependent manner. There was no significant difference between control group and 0.01, 0.1, 1 g/L ST and 0.03g/L nicotine groups (P > 0.05). While cell attachment was significantly higher on control group than 5, 10, 25g/L ST and 0.15, 0.3g/L nicotine groups (P < 0.05).HGFs attached to the titanium surfaces after 24 hours culture were flat, spindle-shaped along the groove. ST and nicotine inhibited area of HGFs in all groups in a dose-dependent manner. As dose increase, the cells on titanium surface were less spread and showed round shape.The results of MTT at 1d, 4d, 7d, 10d assay showed that ST and nicotine inhibited proliferation of HGFs in all groups in a dose-dependent manner. The ST absolutely inhibited proliferation of HGFs in 10 g/L ST group.4. ST and nicotine reduced the synthesis of collagen of HGFs in a dose-dependent manner.Conclusion:1. It had a high success rate for primary culture of HGFs, which can provide enough cells for the research.2. The finished titanium plates demonstrated regular structure. The surface roughness fitted for the research.3. ST and nicotine inhibited attachment, spreading shape and proliferation of HGFs in all groups in a dose-dependent manner.4. ST and nicotine reduced the synthesis of collagen of HGFs in a dose-dependent manner.