Experimental Study Of Titanium Modified By Ammonia Plasma Ion Implantation On Osteoblast-like Cell Activity

Objective: To prepare titanium subjected the modification of plasmaimmersion ion implantation technique using the ammonia, and to determine thecharacterization of the modified titanium layer. Method: Titanium containing nitrogenfunctional group was prepared by PIII technique using ammonia gas. The surfacemorphology of the modified layer and pure titanium was observed by scanning electronmicroscopy (SEM). The chemical composition of NH3PIII-treated substrates and puretitanium was characterized by X-ray photoelectron (XPS). And then the chemical contentof elements in pure titanium and modified titanium was calculated using XPSPEAKsoftware. The hydrophilicity of titanium in both groups was measured by sessile drop usingthe Contact Angel Meter. And the surface energy was determined using an AutomaticContact Angel Meter with two polar liquids and one non-polar liquid. Result: Nosignificant difference of surface morphology was found between Ti and NH3-TiO2. Ti, O,C elements exist in both surfaces whereas the N element only existing in the NH3-Tisurface. And the chemical content of N is significantly high. The measured contact anglesfor unmodified Ti were higher than those of NH3–Ti. The corresponding surface energy ofunmodified Ti was lower than that of NH3–Ti. Conclusion: The ion implantationimmersion using NH3did not break the surface morphology of titanium. The N-functionalgroups can be implanted into the titanium using PIII technology. The modified Ti canobtain better hydrophilcity. NH3–Ti is worthy of further investigation for artificial jointmaterials applications. Objective: To investigate the influences of NH3-implanted Ti surface onadhesion and proliferation of MC3T3-E1cells. Method: Osteoblastic cells were seededdirectly on pure Ti plates as Control group and modified Ti plates as Experiment group.After6,18and24hours of culture, cells were collected for detection. MTT assay wereused to measure the number of attached cells; Cell spreading and density was observed byscanning electron microscopy (SEM)12hours after cell seeded; The viable cell number ofboth groups was determined by OD values using CCK-8kit1,4and7days after cellseeded, meanwhile they were directly counted using arcidine orange stain. Result: Theresult of MTT indicated that after18and24hours culture, cells number attached onNH3-Ti increased significantly compared with that on pure Ti. The SEM after12hourscells culture showed that cells spread well and had a moderate density on both groups;After1day culture, both the CCK-8and arcidine orange stain assay showed that viablecells number had no difference between two groups. After4and7days culture, the cellsnumber on NH3-Ti surface was significantly higher than that on pure Ti (P<0.05).Conclusion: It was showed that the novel Ti surface containing N-functional groups usingPIII were able to accelate the adhesion and late growth of MC3T3-E1cells, whichsuggesting that the NH3-Ti is worthy of further investigation for artificial joint materialsapplications. Objective: To explore the influences of Ti prepared by ammonia ionimmersion implantation on differentiation of MC3T3-E1cells. Method: Osteoblastic cellswere seeded directly on pure Ti plates as Control group and modified Ti plates asExperiment group. After1,4,7days of cultivation respectively, cells were collected fordetection. ALP activity of both group were detected. After3,6,9days of cultivation, ALP, type I collagen, BSP and osteocalcin were determined as the gene expression of markers ofosteoblastic differentiation. Result: With the progression of culture time, ALP activity andALP gene expression increased in both groups. ALP activity of NH3-Ti was statisticallyhigher than that of Ti (p<0.05)1and4days after cell seed. ALP gene expression of NH3-Tiwas statistically higher than that of Ti (p<0.05) on day3and6. Gene expression of COL-Iincreased with time. On day6and9, COL-I gene expression of NH3-Ti was statisticallyhigher than that of Ti (p<0.05). Gene expression of BSP and OC in both group increasedon day6then decreased on day9, While the mRNA levels of BSP showed no significantdifferences in both groups in the whole period (p>0.05). The gene expression of OC ofNH3-Ti was significantly higher than that of Ti6days after cell seed (p<0.05). Conclusion:Ti modified by ammonia ion implantation had no effect on late differentiation, whereas itwas able to accelerate the early differentiation of MC3T3-E1cells and promote theexpression of typical gene in proliferation period of differentiation phase. Objective: To explore the the factors in Wnt signal pathway which promotedthe osteoblast-like cell activity on Ti surface modified by ammnia ion implantation.Method: Osteoblast-like cells were cultured on NH3-Ti and pure Ti. After1,4,7,15daysof culture, cells were collected for determination. Gene expression of Wnt-1, β-catenin,LRP5, Frizzled-2and Runx2were measured by real-time PCR. After1,412and24hoursof culture, the gene expression of Tcf-1and DKK-1were determined by RT-PCR. Result:Wnt-1expression increased in both groups on day1,4and7. Gene expression of Wnt-1inNH3-Ti group was statistically higher than that of Ti on day1and4(p<0.05). Geneexpression of β-catenin and LRP5increased in the whole period. On day4,7and15β-catenin and LRP5gene expression of NH3-Ti was statistically higher than that of Ti(p<0.05). The gene expression of Fzd-2of NH3-Ti was significantly higher than that of Ti4and7days after cell seed (p<0.05). On day1and4, Runx2gene expression of NH3-Tiwas statistically higher than that of Ti (p<0.05). The gene expression of Tcf-1and DKK-1showed no significant differences in both groups1,4,12and24hours after cell seed(p>0.05). Conclusion: The enhanced cell activity on NH3-Ti surface may be mediated by Wnt signal pathway by accelerating the proliferationand differentiation of MC3T3-E1osteoblast-like cells.