| Background:At present,implant denture has been widely used for the restoration of dentition defect and edentulous for its advantages of good retention, avoidance of injury, protection of adjacent teeth.But there are lacks of periodontal ligaments surround the dental implant,so it will be lack of the similar perception as natural teeth in theory. However, scholars found that the implant denture patients had sense of implant.In the 1990 s, "osseoperception" was proposed,which was considered to be a special body sensing function in the case of lack of periodontal proprioceptors. As this sense threshold is high,it’s difficult for patients to sense the excessive bite force,which may result in the absorption of the bone around dental implant, temporomandibular joint disease or even breakage of the implant,at last the implantation is failed. Therefore,the research of peri-implant nerve regeneration has great significance.Objective:This study focus on the effects of three implant surfaces(M,S,E) on biological behaviors of nerve cells, in order to provide objective basis for investigating the mechanism of nerve regeneration and a theoretical reference for peri-implant nerve regeneration.Methods:Round titanium specimens were treated by different ways to get mechanical machining surface group(M),sand-blasted and acid-etching surface group(S), the electrolytic etching surface group(E). After preparation, using scanning electron microscope(SEM) to observe the characteristics of different implant surface morphology. Inoculated RSC96 cells on different pure titanium specimens, MTT test was done on the 1st,3rd,5th days.And on the 4th and 7th days, gene expressions relative level of BDNF and NGF were analysed by Real-time PCR.At last, Dorsal root ginglion neurons were extracted and identified by Nissl stain, then co-cultured on the specimens,testing their adhesion by SEM.Using SPSS17.0statistical packages for statistical analysis.Results:1.Under the scanning electron microscope(SEM), M, S, E specimens had significant difference about topography.There was lack of obvious spatial structure on M specimen, with the parallel stripes ravines; On the S specimen, there were a lot of sag nest holes and cracks, with irregular shapes, size and shades, the parameter of circular pores were about from 400 nm to 5um with some contacting pores,and the edge was sharp,at the same time, there were residual spray sands on the surface; E specimen appeared as a surface with uniform micro holes,and the diameters of them were 20-50 um and the height of were 5-12 um,additionally,on the bottom of the holes, there were full of 20-30 nm processes.2.Immunofluorescence staining showed that RSC96 cells could adhere to different pure titanium specimens.On the M surface,the number of adhering cells are fewer and the cells mainly appeared circular morphology,and part of them were spindle-shape cells with few short processes;On the S surface,the number was bigger than M group,a few were circular while others were spindle-shape;Cells on the E surface extended better, with the largest number and the longer pseudopodia, almost all were with longer spindle-shape.3.At the 1st day, the OD value in the MTT assay was E > S > M group with statistical differences between E and M group,as well as S and M group,however,there was the no difference between E and M group; At the 3rd and5 th days, there was almost no difference among groups even though with the OD value of E > S > M group.4.Real-time PCR analysis showed that, on different pure titanium specimens,the relative quantity of BDNF and NGF gene expressions on both 4th and 7th days were E > S > M group.And there was no statistical differences among the three for the relative quantity of BDNF gene expression on the 4th day; on the 7th day,the statistical differences were observed between E and M group,as well as E and S group,whereas,the relative quantity was similar between S and M group.As for the NGF gene,among the three,on the 4th day,there was statistical difference only between E and M surface;And on the 7th day,there were statistical differences between E and M group,S and M group, the former with apparent significance.Whereas,the relative quantity was similar between S and M group.5.The dorsal root ganglion neurons were successfully extracted,identified,and then were testified that they could adhere to different pure titanium specimen surfaces,surviving and growing.And on the M surface,the neurons were oval,a small amount of the neurons were with extremely short pseudopodia without neural processes;On the S group,flat cells with neural processes and short pseudopodia grasping the wall of pores;However,group E with more apparent neural processes and longer pseudopodia grasping the wall of pores was much better than group S and M with more contacts among cells.Conclusions:1.Through electrolytic etching technology,the rough surface of pure titanium specimen formed,with 20-50 um micro-structured aperture and 20-30 nm processes,and the heights of the holes were about 5-12 um.2. Compared with the control group, the electrolytic etching pure titanium surface can apparently promote early adhesion of RSC96 cells,also the early stage of the cell proliferation,as well as the gene expressions of neurotrophic factors in RSC96 cells.3.The dorsal root ganglion neurons were successfully extracted and identified,which could adhere to different pure titanium specimen surfaces,surviving, growing,and group E were much better than group S and M,with more contacts among cells. |
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