Biofunctionalization Of Ti O₂ Nanotubes With Peptide LL-37 Improves The Biological Behavior Of Human Gingival Fibroblast And Antibacterial Effect

With the development of medical technology,implant prosthesis has gradually become the first choice for patients with dentition defect.After implantation,whether the titanium implant can form a stable combination with oral soft and hard tissue is the key to the desired prognosis of implant.Among them,the complex structure comprised of gingiva and the cervical abutment of implant is largely exposed to the complicated and bacteria-rich oral environment,and forms the first barrier of the biological interface of the titanium implant,which is exceedingly important for the long-term stability of the osseointegration of implant.Therefore,Enhancing the biological activity of the interface of gingival-abutment complex and endowing it with great antibacterial properties is of great significance to promote the soft tissue sealing in the neck of implant and improve the success rate of implantation.Antimicrobial peptides are kinds of small molecular peptides with broad-spectrum antibacterial effects and are usually encoded by specific genes of organisms.Compared with traditional antimicrobial agents such as antibiotic and silver ion,antimicrobial peptides have the advantages of low drug resistance,low allergenicity and low toxicity,leading to a broad application prospect.Among them,LL-37,as the only member of the Cathelicidin family’s antimicrobial peptides of human,not only has antibacterial effects,but also plays an important role in cell recruitment,immunomodulation and wound healing.So it is expected to be an ideal alternative for the functional modification of the implant abutment.In the previous study of our research group,a well-organized and highly ordered nanotube array was constructed on the surface of titanium by anodic oxidation.which is similar to the honeycomb with an open top and closed bottom.The nanotube array can act as a drug reservoir and provide a great platform for the loading of LL-37.Therefore,this study intends to fabricate the bio-functionalized TiO₂nanotubes with LL-37 by means of dopamine mediated coating method,and to explore its effects on the biological behaviors of human gingival fibroblasts（HGFs）,including adhesion,migration and proliferation,and the inhibition of Porphyromonas gingivalis（Pg）and Streptococcus mutans（Sm）,which provides a new idea for the optimal design of the neck materials of implant.Objective:With the help of dopamine mediated coating method,the antimicrobial peptide LL-37was loaded into the nanotubes on the smooth titanium surface to fabricate the bio-functionalized TiO₂nanotubes with LL-37;to explore the effects of bio-functionalized TiO₂nanotubes with LL-37 on the biological behaviors of human gingival fibroblasts（HGFs）,including adhesion,migration and proliferation;to verify the antibacterial effects of bio-functionalized TiO₂nanotubes with LL-37 on Porphyromonas gingivalis and Streptococcus mutans.Methods:Part 1:Preparation and characterization of bio-functionalized TiO₂nanotubes with LL-37TiO₂nanotube arrays（NT）were fabricated on the surface of polished titanium（P）by anodic oxidation,and then the antimicrobial peptide LL-37 with different concentrations（25μg/m L and 100μg/m L）were loaded into TiO₂nanotubes by means of dopamine mediated coating（PD@NT）,which was labeled as LL-37-PD@NT（25）and LL-37-PD@NT（100）,respectively.Surface morphology,roughness,element composition and hydrophilicity of the samples were analyzed with a field emission scanning electron microscope（FE-SEM）,an atomic force microscope（AFM）,X-ray photoelectron spectrometer（XPS）and a contact angle measuring device.The loading and release characteristics of LL-37 were determined by FITC fluorescence labeling experiment and a microplate absorbance reader.Part 2:Effect of bio-functionalized TiO₂nanotubes with LL-37 on the biological behaviors of HGFsHGFs were cultured on the surface of titanium samples of different groups,respectively.The numbers and cellular morphology of HGFs were observed by cellular immunofluorescence staining,confocal laser scanning microscope（CLSM）and FE-SEM.The migration and proliferation of HGFs were examinated by scratch test,Transwell chamber assay,and MTT colorimetric assay.The above experiments were aimed to estimate the bio-compatibility of LL-37-PD@NT.Part3:Detection of antibacterial effects of bio-functionalized TiO₂nanotubes with LL-37Porphyromonas gingivalis（Pg）and Streptococcus mutans（Sm）were inoculated on the surface of titanium samples of different groups,respectively.The bacterial adhesion number and morphology were observed by plate coating and FE-SEM.Bacterial viability and the ratio of live/dead bacteria were determined by live/dead bacterial staining.The above experiments were aimed to evaluate the antibacterial effects of LL-37-PD@NT.Results:Part 1:Preparation and characterization of bio-functionalized TiO₂nanotubes with LL-371.The result of SEM demonstrated that the surface of the material in group P was smooth and the group NT had an evenly arranged tubular of which the diameter ranged from 80nm to 100nm.The PD@NT group had the same tubular structure as the NT group,but the wall became thicker and the diameter of the tube became significantly smaller.There is no significant difference of surface morphology between the LL-37-PD@NT group and the PD@NT group.2.The contact angle measurement results illustrated that:P group>LL-37-PD@NT group>PD@NT group>NT group;AFM obtained:the roughness of NT,PD@NT and LL-37-PD@NT group is greater than that of P group.3.In XPS results,the disappearance of Ti peak,the appearance of N peak and C-N peak in PD@NT group indicated the dopamine coating was successfully constructed;the enhancement of N peak and N element content in LL-37PD@NT group indicated that LL-37 was loaded successfully.The enhancement of N,C-N and C=O peak,the increased content of N element in LL-37PD@NT group indicated that LL-37 was loaded successfully.4.The results of loading test of fluorescently labeled antimicrobial peptides showed that the fluorescence intensity of the surface of samples in LL-37-PD@NT（100）group was higher than that in LL-37-PD@NT（25）group,indicating that the loading capacity of antimicrobial peptide increased with the immersion concentration.5.The results of in vitro test showed that the release of antimicrobial peptide LL-37was characterized by early sudden release（1-24h）and long-term slow release（2-8d）.The cumulative release of antimicrobial peptides on the surface of LL-37-PD@NT（25）and LL-37-PD@NT（100）groups was 3.6μg and 8.8μg at 24h,respectively.The release of antimicrobial peptides was still detected after 8 days.Part 2:Effect of bio-functionalized TiO₂nanotubes with LL-37 on biological behavior of HGFs1.The nuclei of HGFs in each group were stained by cellular immunofluorescence.The results of CLSM revealed that the early adhesion number of HGFs in NT group was significantly lower than that in P group,while in PD@NT,LL-37-PD@NT（25）and LL-37-PD@NT（100）groups the number were increased,especially at 2 h.The morphology of HGFs was observed by SEM.It was found that the spread area of HGFS in PD@NT,LL-37-PD@NT（25）and LL-37-PD@NT（100）groups was significantly larger than that in P group at 4h,and the pseudopods were slender.Cytoskeleton protein of HGFs in each group was stained by immunofluorescence.The results of CLSM showed that the cell aspect ratio of HGFs cells in LL-37-PD@NT（25）and LL-37-PD@NT（100）groups increased significantly after 24h of culture.The HGFs showed a bipolar extension trend compared with other groups.2.Wound scratch assay demonstrated that LL-37-PD@NT（25）group has the largest number of HGFs moving out and the shortest scratch healing time.The results of transwell trial indicated that the number of HGFs migrating to the lower chamber was the highest in LL-37-PD@NT（100）group.3.MTT colorimetric assay indicated that compared with group P,the proliferative ability of HGFs in group NT was significantly decreased,while in PD@NT,LL-37-PD@NT（25）and LL-37-PD@NT（100）groups the proliferative ability were significantly increased.There was no significant difference in PD@NT,LL-37-PD@NT（25）and LL-37-PD@NT（100）groups.Part3:Detection of antibacterial effects of bio-functionalized TiO₂nanotubes with LL-371.The results of plate coating indicated that the sequence of colony formation of Porphyromonas gingivalis and Streptococcus mutans on the surface of each group was as follows:P group>NT group>PD@NT group>LL-37-PD@NT（25）group>LL-37-PD@NT（100）group.2.The images of SEM showed that compared with P,NT and PD@NT groups,the morphology of Porphyromonas gingivals and Streptococcus mutans in LL-37-PD@NT（25）and LL-37-PD@NT（100）groups had changed obviously,the membrane shrank,the contour collapsed,and the connections between bacteria were sparse.The bacteria were even broken in LL-37-PD@NT（100）group.3.The results of live/dead bacterial staining illustrated that compared with P,NT and PD@NT groups,the number of live bacteria on the surface of samples in LL-37-PD@NT（25）and LL-37-PD@NT（100）groups decreased significantly,and the ratio of live/dead bacteria in LL-37-PD@NT（100）group was the lowest.Conclusion:With the help of dopamine mediated coating method,the antimicrobial peptide LL-37can be successfully loaded on the surface of titanium with nanotube topography.The bio-functionalized TiO₂nanotubes with LL-37 has good biological activity,which not only promotes the early adhesion,migration and proliferation of HGFs,but also inhibits the growth of Porphyromonas gingivalis and Streptococcus mutans.It is beneficial to enhance the soft tissue sealing of the neck of implant,providing a new idea for the optimal design of the biological interface in"gingival-abutment complex".