The Investigation Of Different Roughed Sandblasted/Alumina/Acid Etched Titainium Surfaces

Background:With the development of dental implantology in recent years.implant surface treatment is always the emphasis of research work.And the target is obtaining a better osteointergration.When the interaction happens between implant and the host tissue,the first phase is reacting in the interface.And the influencing factors include the chemistry,surface topography,free energy, wettability, electric charge,etc.Cell adhesion is the basis of tissue reconstruction,a better adhesion would promote cell proliferation and differentiation.So, we need to make a thorough investigation of the adhesion,proliferaion and differentiation on material surface,to improve the interaction between cell and host tissue through surface characteristics Sandblasted/Large-grit/Acid etched(SLA) is widely used in commercial implants surface design.But,its unorientation and widely changed roughness result in the difficulty to evaluation between cell behavior and surface characteristics.Due to this, the chosen alumina particle sizes areΦ100～200μm,Φ200～300μm andΦ300～400μm,we expect an appropriate roughness and surface characteristics.Objective:This research is to investigate the relation between the surface properties of different coarsening Sandblasted/Alumina/Acid(SAA) etched titanium surface and compatibility of human osteoblastic sarcoma cells MG-63,to find the optimization of SAA and provide data basis.Methods:1. First, pure titainum disks are treated by AL2O3 blasting,and then acid etched by 10% HCl/H2SO4 40min.We define the groups by those particle size of AL2O3: group A isΦP100～200μm, group B isΦ200～300μm, and group C isΦ300～400μm.The control group is the grinding polished disk. The surface characterisitics were detemined by scanning electron microscopy(SEM)observation, light interferometer, OCA optical contact angle meter and X-ray photoelectron spectroscopy (XPS).2. After human osteoblastic sarcoma cells MG 63 were cultured on disks after 2h,4h,8h and Id, scanning electron microscopy (SEM) and laser confocal microscopy (LSCM) were used to observe the morphology of cells and F-actin architectonic change,after pretreatment.The cell adhesion amount were counted to comparethe adhesion rate.3. After human osteoblastic sarcoma cells MG 63 were cultured on disks after 4d,7d and 14d,MTS measured the proliferation by OD values. ALP activity were measured by ALP-ELISA kit after MG63 cultured on disks after 7d,15d and 21d. OPG and TGF-β1 were measured by ELISA kit after 7d.4. Each time-period above each group has six sample, each sample were measured three times. Data is discribed as x±s, statistical analysis software is SPSS 13.0. One way-ANOVA and Factorial analysis of variance were used.Result:1. Four groups by SEM observation present their respective characteristics. Control surface have the groove scratches with consistent,SAA surfaces has a complex microstructured surface with cavities,and micropit. Ra value is increased as the increasing sandblasting particle size. Wettability of SAA surfaces are better than control. XPS detection showed group B have more OH. 2. The surface of cell adhensios rate all rises up with time increased. At 2h, SAA is lower than control; at 4h, SAA is higher. The titanium slices after inoculated by scanning electron microscopy observation present adhesion process, and the group control has begun to spread at 2h, with elonged direction.At 4h,stretches are more significant on control, cells have obvious interactions between contact.At 2h SAA group occurred adhesion,with a spherical attachment on big hole area, forming starts stretch, that would present to the surface uplift edge out when fingerlike projection, at 1d,more widely and cell stretch, a relationship between them synapses group more stereo, rich in cytoplasm. Group B cells spread fast and stretching are broad range compared to A group and group C. After inoculated by fluorescence staining, the titanium slices under laser confocal microscopy observation for adhesion process of the cytoskeleton morphological characteristicsare like this:SAA group F-actins at 2h are gathered around the inner membranes; The control group F-actins are wheel shape arrangement.At 8h,each F-actins are not clear, but group B are visible of bunch shape structure. Along with the time development, F-actins of SAA group have more and more clearly multiple directions,more and extend, representing the SAA group of pleomorphic cells. F-actins of Group B have more obvious multiple differentiations than group A and group C.3. Compare four groups of MTS OD values as cell proliferation level. as time increases, proliferation activity increase. At 4d, group control was highest. At7d, four groups showed not significant difference; At 14d,thegroup control was lowest,group B was the highest. ALP-ELISA kit of surface testing osteoblast ALP activity, compares four groups of cell differentiation, including when that 15d level, SAA three groups reach peak, when 7d ALP group SAA three groups were a litter higher than the control, at 15d, group B> group A. Group B at 15d was achieved peak in different period and different treatment.OPG and TGF-β1 are almost the same as ALP at 7d.Conclusion:1.Different sizes of Al2O3 result in different surface of SAA microtopography, t the greater size Al2O3, the increased roughness.The static water contact angle of the three groups of SAA were less than the control group,the three have good wettity.B has more amount of OH.2. The adhesion rate increases over time, the controls was higher than the three groups of SAA at 2 hours.SEM showed the morphology about the cellular adhesive, stretch,differentiation and proliferation at different time and different roughness of SAA surface.CLSM also showed the distribution of cytoskeleton F-actin vary with time.The adhension rate would be higher when the increased roughness with the surface compared to the cell and molecular size.3. MTS showed that Cell proliferation activity was heighten vary with time, and the activity of cell proliferation on SAA surfacewas higher than the controls, especially the group B was higher than others. ALP test revealed that the ALP activity of SAA surface reached the peak at 15d,the group B also was higher than others. The ALP,OPG and TGF-β1 activities are almost the same at 7d.