| A firm functional soft tissue seal between the transmucosal part of implants and the surrounding soft tissues is significant to the long-term survival of the implant. To some degree, the integration of the implant neck and the surrounding soft tissue was dependent on a race between the host tissue cells, such as human gingival fibroblasts(HGFs), versus oral microorganisms, according to the theory of “the race for the surface”. If the cell win the competition, the soft tissue seal will form soon, vice versa. Therefore, the forming of a firm functional soft tissue seal was affected by two factors:the transmucosal part of implants should a) improve the peri-implant soft tissue interaction with the implant neck surfaces, that is, promote the biological behavior and function of HGFs, and b) have antimicrobial activity.In recent years, studies have found that microgroove surfaces can lead to the so-called “contact guidance”, and accelerated the cell proliferation and the expression of both genes and proteins in human gingival fibroblasts. Cationic antimicrobial peptides(AMPs) had been introduced into implant surfaces recently due to their broad-spectrum antimicrobial activity, non-selection of resistant mutants, and less of cytotoxicity, and these peptides showed antibacterial activity when immobilized onto titanium surfaces. We aimed to enhance the biological behavior of HGFs and bactericidal activity in one single coating/substrate system to form a firm functional soft tissue seal. Objective:In this study, GL13 k, a cationic antimicrobial peptides, was immobilized onto microgroove surfaces measuring 60 um in width by 10 um in depth via the method of silanization. The successful modification of GL13 K and the mechanical stability of the coatings were measured by the physical and chemical properties. In vitro antibacterial tests, the bactericidal activity was tested by the P.g. In vitro cytocompatibility tests, the adhesion and proliferation of HGFs were tested as well as the phenomenon of the contact guidance. Methods:The method of silanization was used to immobilize the antimicrobial peptide GL13 K, and the elements of surfaces, surface morphology, the roughness, and the water contact angles were confirmed by X-ray photoelectron spectroscopy(XPS), scanning electron microscopy(SEM), atomic force microscopy(AFM), and water contact angle measurement, respectively. The mechanical stability of the coatings was tested by ultrasonicating the different surfaces for 2 h, and the change of the FITC fluorescence signal intensity, element of the surfaces and the water contact angles was detected. To test the antibacterial properties of the modified surfaces, the antibacterial adhesion was measured by the acridine orange(AO) fluorescence staining, the live bacteria were quantitated by measuring CFUs, and the viability of the bacteria were measured by WST-8 staining. On the other way, the cell adhesion, the cell viability, the cell morphology and the phenomenon of the contact guidance were tested by DAPI fluorescence staining, the cell counting kit-8, scanning electron microscopy, and the immuno-fluorescence staining in vitro cytocompatibility tests, respectively. Results:(1) The increasing in N1 s and C1 s signals after the immobilization of the GL13 K peptide proved the modification of GL13 K. The average roughness increased from 1.71 ± 0.05 nm to 28.86 ± 4.48 nm after immobilization of GL13 K. The contact angle values for the surfaces increased from 60.68 ± 2.89? to 101.6 ± 6.75?. After 2h of ultrasonication in water, the silanized surfaces retained green signals largely similar to the non-ultrasonication ones, but the non-silanized surface lost most of its green fluorescence signal intensity. These demonstrated the mechanical stability of the coatings.(2) Antibacterial adhesion test: the number of the P. g was roughly the same on the surfaces whether modified with GL13 K or not at the three different time points, which demonstrated the coatings can’t prevent the adhesion of the P. g. Bactericidal activity test: the colony forming units of the GL13K-modified surfaces was markedly decreased( P < 0.001) compared with the pristine surfaces, including the smooth surfaces and the microgroove surfaces at the three time points. The metabolism test: the OD values of the P. g on the GL13K-modified surfaces were decreased(P<0.001), and the WST-8 results were the same with the CFUs’, being almost proportional to the CFUs.(3) Cell adhesion: the quantitative results showed that the numbers of cells attached to the three different surfaces were not significantly different at the 2 h time point. However, at the 4 h and 6 h time point, the cells attached to the treated and non-treated microgroove surfaces were significantly increased( P < 0.001) compared with the smooth surfaces. Moreover, significantly higher(P < 0.05) numbers of cells attached to the treated microgroove surfaces than to the non-treated microgroove surfaces at the 6 h time point. Cell viability: At all time points, the cells cultured on the treated microgroove surfaces displayed significantly higher(P < 0.001) viability than those grown on the microgroove surfaces, and even greater than on the smooth surfaces(P < 0.001). The cell morphology and the orientation of the cell growth: HGFs cultured on the treated and non-treated microgroove surfaces were mainly elongated and spindle-shaped and were aligned along the grooves. Meanwhile, the cells cultured on smooth surfaces had a less-defined morphology, some spindle-shaped and some polygon-shaped, and no consistent orientation. Conclusion:The antimicrobial peptide GL13 K was covalently conjugated onto the microgroove titanium surfaces using silane as a chemical linker. The successful immobilization of GL13 K was confirmed by the results of XPS, SEM, AFM, and water contact angles, as was the mechanical stability of the coating. The GL13K-coated microgroove surface had the property of bactericidal activity without preventing bacterial adhesion. In vitro cytocompatibility tests demonstrated that the GL13K-coated microgroove surfaces could promote the adhesion and proliferation of HGFs and guide the growth of the cells along the microgroove. As a result, this study provides an approach to develop a firm peri-implant soft tissue seal. |
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