An In Vitro Biological Evaluation On The Percutaneous Sites Of MAO-treated Titanium Implants

Among all the implant materials, pure titanium is singled for its greatbiocompatibility and mechanical properties. After its application in the clinicuse in the past decades, titanium has been selected as one of the best implantmaterials in the medical field. However, the healing time of the titaniumimplant-to-tissue integration is very inefficient, which has broughtoverwhelming troubles to the clinicians and patients. To increase the speed ofimplant-to-tissue integration efficiently, a series of surface treatment techniquescame out in the past twenty years, such as microarc oxidation (MAO),simulated body fluid (SBF), titanium plasma sprayed (TPS) etc. Literatureshave demonstrated that these surface treatments can enhance theimplant-to-tissue integration by improving the surface structure andbiocompatibility of the implant materials. Nevertheless, few researches wereconducted to investigate these surface treatments integrated to the percutaneoussites of the implants. Clinical data revealed that15%-20%rehabilitation patients suffered peri-implantitis due to the percutaneous problems, whichcould lead to osteomyelitis and soft tissue damage and even implant failure.Therefore, the reaction of percutaneous site of the implant to the body tissue isan essential factor in the implant success.ObjectivesMicroarc oxidation has been considered to be one of the most effectivesurface treatments to improve the osseointegration of the implants. However,the influences of such treatment on the skin epithelium interface still gained lessattention in present literatures. Aimed at evaluating the biological properties ofthe percutaneous sites of the MAO treated titanium (MAO_Ti) implants, thebehavior of human skin epithelial cell (HSEC) and the Staphylococcus aureus(S. aureus) on the MAO_Ti and the untreated titanium (Un_Ti) surfaces wereevaluated using in vitro models. Meanwhile, the susceptibility of differentimplant surface treatments to bacterial adhesion were evaluated in across-sectional study, including Ti-based sandblasting (Ti_SB), Ti-basedmicroarc oxidation (Ti_MAO) and Ti-based simulated body fluid coating(Ti_SBF). Furthermore, to efficiently lower the risk of peri-implantitis, theMAO technique was further optimized and the surface properties wereimproved. Finally, a pilot animal experiment was conducted to investigate thesoft tissue inflammation of prcutaneous implants and further verify the effect ofNaCl impregnated MAO_Ti implants. We hope that these data can providebasic information for further clinical study on the MAO treated titaniumimplant to soft tissue integration.Methods and Results Firstly, the in vitro study described the visualization and quantification ofS.aureus adhering to the three Ti-based surface coatings. Overall results showedthat Ti_SB, Ti_MAO and Ti_SBF were much more susceptible to S. aureusthan Ti surfaces. Counting results confirmed that there was no statisticallysignificant difference between the amount of bacteria on Ti_SB and Ti_MAOsurfaces, while bacteria on Ti_SBF surfaces were significantly more comparedto the other two treated surfaces. Data showed that surface roughnesses of thethree Ti-based treatments were generally higher than the pure Ti group. It isreported that surface roughness can be divided into three levels depending onthe scale of the features: macro-, micro-and nano-sized topologies. Ti_SB,Ti_MAO, and Ti_SBF surface profiles can be categorized into the micro level,which is defined for topographical features as being in the roughness range of1-10μm. Numerous studies have shown that surface roughness in this rangeresulted in greater bone-to-implant contact and higher resistance to torqueremoval than other macro-/nano-types of surface topography and also titaniumimplants with smoother surfaces. However, a major risk with increase in surfaceroughness may be an increase in peri-implantitis. Our study demonstrated theview that roughening a surface can enhance bacteria adhesion due to theincrease in surface area.In case of the XRD pattern, Ti_SB spectra showed that there still somealumina (Al2O3) existed on the SB surfaces. Al2O3, frequently used as a blastingmaterial is often embedded into the implant surface and residue remains evenafter ultrasonic cleaning, acid passivation and sterilization, as it is insoluble inacid and is thus hard to remove from the titanium surface. It is reported that thepresence of some chemicals, such as aluminium, on the surface could influence bacterial adhesion. However, surface chemical analysis in this study showed avery low concentration of aluminium after the sandblasting. Moreover, S.aureus counting results exhibited no significant less due to the existence ofremained alumina.Secondly, the study examined the biological behavior of HSEC andS.aureus on the percutaneous site of MAO_Ti implant surface. Our resultsdemonstrated that MAO structure did not well support the growth, spreading,attachment, and proliferation of HSEC. What’s more, MAO_Ti surface wasmuch more susceptible to S. aureus. According to our data from present study,the MAO treatment seemed not a good choice for the percutaneous site ofimplant. From the growth data of cells, we can see that the attachment andproliferation of HSEC improved faster on the Un_Ti surface than on the MAOsurface with time increasing. As expected, cell contacts increased cell spreading,but surprisingly, this phenomenon cannot be found on the MAO treated surface.It seemed that MAO treated surface in some degree inhibited the growth of theHSEC. The SEM images also showed that cells were much more spread withclearer cell contacts and cell nucleus on the Un_Ti surface while on the MAOsurface cells were small and fusiform, which indicated that HSEC on the Un_Tisurface kept in a much better state of viability. Moreover, the images confirmedthat in general there were more proliferated cells in doublets and clusters on theUn_Ti surface while on the MAO treated surface almost all cells were isolatedfrom each other. Thus, both the counting results and the SEM images indicatedthat the growth, attachment and proliferation of HSEC could be betterconducted on the Un_Ti surface, while on the MAO treated surface, cell growthand proliferation seemed to be inhibited in a certain degree. Meanwhile, the SEM images as well as the counting results of bacteria onthe MAO_Ti and Un_Ti surfaces revealed that the multiplication of the S.aureus was more obvious on the MAO treated surfaces than on the Un_Tisurface as time increased. Especially at24h, bacteria observed on the MAOtreated surfaces were almost confluent, suggesting that the bacteria furtherproliferated and attached, tending to form a layer of bacterial film. It is knownthat once S. aureus adhere to metal surfaces they form biofilms that can bedifficult to treat clinically because the bacteria are protected from phagocytosisand antibiotics, which explains the need to prevent the initial bacterial adhesion.A possible solution is to modify the implant surface by using an antimicrobialor protein-resistant coating. Furthermore, an interesting phenomenon of thestudy was that bacteria were more liable to spread and multiply in the pores ofMAO surface where were less accessible to cells. Occasionally we could alsofind, on the deficient and rough places of the Un_Ti surface, S. aureus tended toaccumulate more than the relatively flat places. These findings along with theSEM results of the MAO treated crateriform-like surface supported the viewthat roughening a surface can enhance bacteria adhesion due to the increase insurface area.Thirdly, this in vitro study investigated the effect of10%NaClimpregnated MAO treated titanium (NaCl_MAO_Ti) surfaces on the resistanceof bacteria adherence. The results revealed that the NaCl_MAO_Ti surfacesignificantly prevented the adhesion and multiplication of S. aureus. Thecounting results revealed the changes in the number of adhered bacteria on thetwo different surfaces with time increasing, and SEM images further confirmedthe results. Although there was no statistical difference in the number of adhered bacteria between MAO_Ti and NaCl_MAO_Ti groups during the first2to4hours, the total amount of bacteria on the NaCl_MAO_Ti surface hadstarted to drop at4h time point. After24h, the number of bacteria on theNaCl_MAO_Ti surface was radically decreased compared to what on theMAO_Ti surface.Impregnated with10%NaCl, the surface roughness of MAO_Ti slightlyincreased compared to the control group, but there was no statistical differencebetween two groups. Therefore, the subtle changes in the surface roughness oftwo types of surfaces should not significantly affect the results of the bacteriaadherence. When incubated in the bacterial culture solution, the sodiumchloride coating was dissolved. These findings were similar to those of Ewaldet al, who inferred that once inside the human body, the NaCl coating would berinsed off the implant and enable the body cells to interact with the implantsurface directly. Moreover, previous studies have shown that highly hydrophilicsurfaces were more desirable than hydrophobic ones due to their affinity tobiological fluids, cells and tissues. Our results found that the hydrophilicity ofNaCl_MAO_Ti surface was higher than the control surface, which indicatedthat the NaCl_MAO_Ti surface might display better biocompatibility to thebody cells.Finally, the animal experiment investigated the peri-implant soft tissueinflammatory reaction and the changes of microorganisms during the wholeimplantation. It was found that S. aureus was dominant in the early stage of theinfection, while in the late infection, the proportion of G-streptococci increasedas that of S.aureus decreased. The soft tissue reaction to the three different implants showed that the MAO_Ti implants had the worst inflammation, whileNaCl impregnated MAO_Ti and Un_Ti implants were much better.Furthermore, the peri-implant soft tissue inflammatory reaction together withthe detection results of inflammatory secretion suggested that there could be acorrelation between the G-streptococci infection and the progress ofperi-implantitis.ConclusionsAnalysis of our data, taken together, MAO treated titanium surface did notwell support the growth, adhesion and proliferation of HSEC. Additionally, itwas much more susceptible to S. aureus when compared with untreatedtitanium (Un_Ti) surface. Besides, the number of S. aureus adhering to thethree Ti-based treatments was significantly more than that on the pure Tisurfaces. Such an observation revealed that the Ti_SB, Ti_MAO, and Ti_SBFsurface coatings encouraged S. aureus adhesion, and could lead to higherinfection rates in vivo, while pure Ti surface showed advantages in preventingbacterial adhesion and lowering the rate of infection. Furthermore, NaClimpregnated MAO_Ti surface may efficiently lower the risk of peri-implantitisand hopefully it could facilitate the implementation of MAO coating techniqueto the clinic trials in the near future.Animal experiment revealed that NaCl impregnated MAO_Ti implantscould achieve a good antibiotic function. At the early stage of theperi-implantitis, S. aureus was the dominant bacteria, while at the late stage, theproportion of G-streptococci increased and became the major portion.Furthermore, the peri-implant soft tissue inflammatory reaction suggested that there could be a correlation between G-streptococci infection and the progressof peri-implantitis.