Minocycline-Modified Pure Titanium Has Good Antibacterial Properties and Improves the Osteogenesis of Bone Marrow Stem Cells

Background : The development of an ideal implant material with appropriate antibacterial properties and that improves osteogenesis is essential for the guidance of new bone formation in orthopedic and tooth implant surgeries. In this study, we developed minocycline-modified pure titanium. Methods: We exploited the chemistry of polydopamine (PD) for the coating of minocycline. PD was coated on pure titanium, on which minocycline was subsequently immobilized under certain conditions. Minocycline coating was verified by characterizing the surface chemical composition of the coated Ti sheet and was quantitatively measured by fluorescamine assay. Results: The minocycline-coated pure titanium showed a lower bacterial adhesion rate and supported the spread of the osteogenesis differentiation of human mesenchymal stem cells (hMSCs). A remarkable increase in alkaline phosphatase activity and calcium deposition was found when hMSCs were cultured on minocycline-coated pure titanium for 28 days. Conclusions: The minocycline-coated pure titanium may be optimized as clinically applicable bioactive materials for implant and bone materials.


Background
Pure titanium is widely used in surgical implant, orthopedic fixation, and tooth implant materials because of their good biocompatibility and corrosion resistance [1][2][3] . Implants are surrounded by cells, cytokines, and other parts of the marrow cavity with healing progress; parts of bone marrow stromal stem cells in the marrow cavity gradually differentiate into osteoblasts, which induce osteogenesis in the implant surface, form new bone tissue, and promote osteosynthesis [4][5][6][7] .
However, infections on and around titanium implants remain a difficult problem. Implant-associated infection may lead to implant failure due to the formation of a bacterial surface biofilm [8][9][10] .
Therefore, the antibacterial surface modification of Ti implants is important to prevent initial bacterial adhesion and promote bone bonding performance [11][12][13][14] . Minocycline is a second-generation semisynthetic tetracycline broad-spectrum antibiotics, and its antibacterial spectrum is similar to that of tetracycline with a high degree of lipophilicity, strong tissue penetration, and good antibacterial effect. Minocycline is widely used in the clinical treatment of acne, non-gonococcal urethritis, and 3 periodontitis. It specifically binds to the 305 subunit of ribosome and prevents aminophthalein tRNA from entering the site. Thus, it prevents the extension of the peptide chain, inhibits the protein synthesis of the pathogen, and plays an antibacterial role. The drug is bacteriostatic at low concentration and has bactericidal effect at high concentration. Its antibacterial spectrum includes Gram-positive and -negative bacteria, such as Rickettsia, Mycoplasma, Chlamydia, Spirochetes, Mycobacterium, Plasmodium falciparum, and Toxoplasma [15][16][17][18] .
In this study, we developed a minocycline-coated pure titanium as an antibacterial material and a guidance material for bone regeneration. We used polydopamine (PD) as the coating medium for minocycline coating [19,20] . The objectives of this study were to evaluate the antibacterial properties of minocycline-coated pure titanium and to investigate its effects on the viability and osteogenic differentiation of human mesenchymal stem cells (hMSCs).

Characterization of MHCI-Ti
The amount of minocycline coated on Ti sheets increased with increasing concentration. Approximately 1.9 mg minocycline was immobilized on 1.0 cm 2 substrate reacted with 200 mg/mL of minocycline and was remarkably greater than that immobilized in the 100 mg/mL group (Fig. 1A).
SEM images of the surface of Ti and MHCI-Ti showed similar morphology (Fig. 1B). The surface chemical composition of the fibers was analyzed through XPS. As shown in Fig. 1a, the intensity of the N1s (399 eV) peak on the surface of MHCI-Ti was higher than that on Ti. The appearance of N1s and Ti-O-N peaks indicated that PD and minocycline were successfully coated (Fig. 1C).  Figs. 2A-D). However, the attached bacteria on Ti surface ( Fig. 2A) were relatively more compared with those on MHCI-Ti (Figs. 2B-D). CLSM revealed an obviously vital (green) bacterial layer on surface of Ti and a dead (red) microorganism layer on the surface of MHCI-Ti (Fig. 3A), and MHCI-Ti (200 mg/mL group) was wholly red in color and showed the highest antibacterial properties among the samples (Fig. 3C).

Viability and osteogenic differentiation of hMSCs on polished Ti and MHCI-Ti
The cell morphology of hMSCs cultured on different substrates for 1 day in osteogenic medium was characterized by SEM. The hMSCs showed good cell adhesion and spreading at day 1 (Fig. 4A). MHCI-Ti was more biocompatible, as evaluated by the comparable viability levels at days 1, 4, and 7 (Fig.   4B). ALP activity and the amount of calcium were measured to examine the osteogenic differentiation of hMSCs (Figs. 4C and D). The ALP activity of hMSCs cultured on MHCI-Ti was remarkably higher than that on Ti at days 21 and 28. The amount of mineralized calcium in hMSCs cultured on MHCI-Ti was considerably higher compared with that on Ti at days 21 and 28.

Discussion
Titanium implants are widely used in clinical orthopedic materials, dental implants are one of 7 them [21] .However, the use of titanium implants can lead to implant-related infections, leading to the loosening and even falling off of implants. The internal environment of oral cavity is especially suitable for the colonization and reproduction of pathogenic microorganisms, which leads to the decrease of bone binding ability and healing speed of bone tissue around dental implants, and the destruction of stability of surrounding soft tissue, which further affects the curative effect of implant repair.The stability around the implant depends on the stability of the surrounding bone tissue and soft tissue, especially the stability of bone tissue, which mainly depends on the binding process of the surrounding bone tissue [22] .Therefore, the bone tissue absorption around the implant is closely related to the repair effect.To solve this problem, the implant was modified by surface functionalization to achieve antibacterial and enhance the ability of bone bonding.In this study, we developed a minocycline-coated pure titanium as an antibacterial material and a guidance material for bone regeneration. The minocycline-coated pure titanium showed a lower bacterial adhesion rate and supported the spread of the osteogenesis differentiation of human mesenchymal stem cells (hMSCs). A remarkable increase in alkaline phosphatase activity and calcium deposition was found when hMSCs were cultured on minocycline-coated pure titanium for 28 days. We believe that this material is an ideal material for bone repair and dental implant. Further research should focus on the bone-binding and antibacterial properties of the material in vivo

Conclusions
We developed minocycline-coated titanium through a simple coating method. We obtained the chemical composition of the surface of titanium with minocycline coating via XPS. Minocycline-coated titanium reduced bacterial survival and enhanced the osteogenic differentiation of hMSCs. Therefore, we conclude that minocycline-coated titanium can be used as tooth implant and bone fixation materials.