Objective: To fabricate TiO₂ nanotube material functionalized by antimicrobial peptide LL-37, and to explore its effects on biological behaviors such as adhesion and migration of human keratinocytes (HaCaT) and its antibacterial properties. Methods: The TiO₂ nanotube array (NT) was constructed on the surface of polished titanium (PT) by anodization, and the antimicrobial peptide LL-37 was loaded on the surface of TiO₂ nanotube (LL-37/NT) by physical adsorption. Three samples were selected by simple random sampling in each group. Surface morphology, roughness, hydrophilicity and release characteristics of LL-37 of the samples were analyzed with a field emission scanning electron microscope, an atomic force microscope, a contact angle measuring device and a microplate absorbance reader. HaCaT cells were respectively cultured on the surface of three groups of titanium samples. Each group had 3 replicates. The morphology of cell was observed by field emission scanning electron microscope. The number of cell adhesion was observed by cellular immunofluorescence staining. Cell counting kit-8 (CCK-8) assay was used to detect cell proliferation. Wound scratch assay was used to observe the migration of HaCaT. The above experiments were used to evaluate the effect of each group on the biological behavior of HaCaT cells. To evaluate their antibacterial effects, Porphyromonas gingivalis (Pg) was respectively inoculated on the surface of three groups of titanium samples. Each group had 3 replicates. The morphology of bacteria was observed by field emission scanning electron microscope. Bacterial viability was determined by live/dead bacterial staining. Results: A uniform array of nanotubes could be seen on the surface of titanium samples in LL-37/NT group, and the top of the tube was covered with granular LL-37. Compared with PT group [the roughness was (2.30±0.18) nm, the contact angle was 71.8°±1.7°], the roughness [(20.40±3.10) and (19.10±4.11) nm] and hydrophilicity (the contact angles were 22.4°±3.1° and 25.3°±2.2°, respectively) of titanium samples increased in NT and LL-37/NT group (P<0.001). The results of in vitro release test showed that the release of antimicrobial peptide LL-37 was characterized by early sudden release (1-4 h) and long-term (1-7 d) slow release. With the immunofluorescence, more cell attachment was found on NT and LL-37/NT than that on PT at the first 0.5 and 2.0 h of culture (P<0.05). The results of CCK-8 showed that there was no significant difference in the proliferation of cells among groups at 1, 3 and 5 days after culture. Wound scratch assay showed that compared with PT and NT group, the cell moved fastest on the surface of titanium samples in LL-37/NT group at 24 h of culture [(96.4±4.9)%] (F=35.55, P<0.001). A monolayer cells could be formed and filled with the scratch in 24 h at LL-37/NT group. The results of bacterial test in vitro showed that compared with the PT group, the bacterial morphology in the NT and LL-37/NT groups was significantly wrinkled, and obvious bacterial rupture could be seen on the surface of titanium samples in LL-37/NT group. The results of bacteria staining showed that the green fluorescence intensity of titanium samples in LL-37/NT group was the lowest in all groups (F=66.54,P<0.001). Conclusions: LL-37/NT is beneficial to the adhesion and migration of HaCaT cells and has excellent antibacterial properties, this provides a new strategy for the optimal design of implant neck materials.
Humans ; Titanium/chemistry* ; Antimicrobial Peptides ; Cathelicidins ; Sincalide ; Anti-Bacterial Agents/pharmacology* ; Nanotubes/chemistry* ; Dental Materials ; Bacteria ; Keratinocytes ; Surface Properties

OBJECTIVE: To review antibacterial/osteogenesis dual-functional surface modification strategy of titanium-based implants, so as to provide reference for subsequent research. METHODS: The related research literature on antibacterial/osteogenesis dual-functional surface modification strategy of titanium-based implants in recent years was reviewed, and the research progress was summarized based on different kinds of antibacterial substances and osteogenic active substances. RESULTS: At present, the antibacterial/osteogenesis dual-functional surface modification strategy of titanium-based implants includes: ① Combined coating strategy of antibiotics and osteogenic active substances. It is characterized in that antibiotics can be directly released around titanium-based implants, which can improve the bioavailability of drugs and reduce systemic toxicity. ② Combined coating strategy of antimicrobial peptides and osteogenic active substances. The antibacterial peptides have a wide antibacterial spectrum, and bacteria are not easy to produce drug resistance to them. ③ Combined coating strategy of inorganic antibacterial agent and osteogenic active substances. Metal ions or metal nanoparticles antibacterial agents have broad-spectrum antibacterial properties and various antibacterial mechanisms, but their high-dose application usually has cytotoxicity, so they are often combined with substances that osteogenic activity to reduce or eliminate cytotoxicity. In addition, inorganic coatings such as silicon nitride, calcium silicate, and graphene also have good antibacterial and osteogenic properties. ④ Combined coating strategy of metal organic frameworks/osteogenic active substances. The high specific surface area and porosity of metal organic frameworks can effectively package and transport antibacterial substances and bioactive molecules. ⑤ Combined coating strategy of organic substances/osteogenic active substancecs. Quaternary ammonium compounds, polyethylene glycol, N-haloamine, and other organic compounds have good antibacterial properties, and are often combined with hydroxyapatite and other substances that osteogenic activity. CONCLUSION The factors that affect the antibacterial and osteogenesis properties of titanium-based implants mainly include the structure and types of antibacterial substances, the structure and types of osteogenesis substances, and the coating process. At present, there is a lack of clinical verification of various strategies for antibacterial/osteogenesis dual-functional surface modification of titanium-based implants. The optimal combination, ratio, dose-effect mechanism, and corresponding coating preparation process of antibacterial substances and bone-active substances are needed to be constantly studied and improved.
Anti-Bacterial Agents/pharmacology* ; Coated Materials, Biocompatible/chemistry* ; Metal-Organic Frameworks/pharmacology* ; Osteogenesis ; Surface Properties ; Titanium/pharmacology* ; Prostheses and Implants

The poor mechanical property and vulnerability to bacterial infections are the main problems in clinic for dental restoration resins. Based on this problem, the purpose of this study is to synthesize silver-titanium dioxide (Ag-TiO₂) nanoparticles with good photocatalytic properties, and add them to the composite resin to improve the mechanical properties and photocatalytic antibacterial capability of the resin. The microstructure and chemical composition of Ag-TiO₂ nanoparticles and composite resins were characterized. The results indicated that Ag existed in both metallic and silver oxide state in the Ag-TiO₂, and Ag-TiO₂ nanoparticles were uniformly dispersed in the resins. The results of mechanical experiments suggested that the mechanical properties of the composite resin were significantly improved due to the incorporation of Ag-TiO₂ nanoparticles. The antibacterial results indicated that the Ag-TiO₂ nanoparticle-filled composite resins exhibited excellent antibacterial activities under 660 nm light irradiation for 10 min due to the photocatalysis, and the Ag-TiO₂ nanoparticle-filled composite resins could also exhibit excellent antibacterial activities after contact with bacteria for 24 h without light irradiation because of the release of Ag ions. In summary, this study provides a new antibacterial idea for the field of dental composite resins.
Anti-Bacterial Agents/pharmacology* ; Composite Resins ; Metal Nanoparticles/chemistry* ; Nanoparticles ; Titanium/pharmacology*

Epithelial attachment via the basal lamina on the tooth surface provides an important structural defence mechanism against bacterial invasion in combating periodontal disease. However, when considering dental implants, strong epithelial attachment does not exist throughout the titanium-soft tissue interface, making soft tissues more susceptible to peri-implant disease. This study introduced a novel synthetic peptide (A10) to enhance epithelial attachment. A10 was identified from a bacterial peptide display library and synthesized. A10 and protease-activated receptor 4-activating peptide (PAR4-AP, positive control) were immobilized on commercially pure titanium. The peptide-treated titanium showed high epithelial cell migration ability during incubation in platelet-rich plasma. We confirmed the development of dense and expanded BL (stained by Ln5) with pericellular junctions (stained by ZO1) on the peptide-treated titanium surface. In an adhesion assay of epithelial cells on A10-treated titanium, PAR4-AP-treated titanium, bovine root and non-treated titanium, A10-treated titanium and PAR4-AP-treated titanium showed significantly stronger adhesion than non-treated titanium. PAR4-AP-treated titanium showed significantly higher inflammatory cytokine release than non-treated titanium. There was no significant difference in inflammatory cytokine release between A10-treated and non-treated titanium. These results indicated that A10 could induce the adhesion and migration of epithelial cells with low inflammatory cytokine release. This novel peptide has a potentially useful application that could improve clinical outcomes with titanium implants and abutments by reducing or preventing peri-implant disease.
Amino Acid Sequence ; Animals ; Benzeneacetamides ; chemical synthesis ; pharmacology ; Cattle ; Cell Adhesion ; drug effects ; Cell Movement ; drug effects ; Cells, Cultured ; Cytokines ; metabolism ; Dental Implants ; Enzyme-Linked Immunosorbent Assay ; Epithelial Attachment ; drug effects ; Epithelial Cells ; cytology ; metabolism ; Microscopy, Confocal ; Microscopy, Electron, Scanning ; Piperidones ; chemical synthesis ; pharmacology ; Platelet-Rich Plasma ; Receptors, Thrombin ; Surface Properties ; Titanium ; chemistry

To evaluate the effect of laser solid forming (LSF) of porous titanium on receptor activator of NF-κB ligand (RANKL)/osteoprorotegerin (OPG) expression and osteoblast cells growth.
 Methods: The DMEM and sterile saline were used for porous titanium extract. The osteoblast cells were cultured in the extract while equal amount of  DMEM and sterile saline were added to the control group. The growth of the cells were observed under an inverted phase contrast microscope. MTT was used to detect the growth inhibitory rates. The adhesion capacity of osteoblasts were measured. The growth in the material surface was examined by the electron microscope, and the expressions of RANKL and OPG were determined by Westen blot.
 Results: At the first day, the osteoblast proliferation rate was significantly different (P<0.05), at the fourth and seventh day, the osteoblast proliferation rate was not significantly affected in the LSF group (P>0.05); at each time point, the osteoblast proliferation rate were significantly different between the two groups (P<0.05). Compared with the control group, RANKL and OPG protein expression were not significantly different (P>0.05). The laser solid forming of porous titanium showed well bone compatibility.
 Conclusion: The porous titanium did not affect osteoblast proliferation due to its well bone compatibility. It did not affect the OPG/RANKL/RANK-axis system of bone metabolism, exibiting a wide applicable prospect for tissue engineering.
Biocompatible Materials ; chemistry ; Cell Adhesion ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Culture Media ; chemistry ; Ligands ; Osteoblasts ; drug effects ; Osteogenesis ; drug effects ; Osteoprotegerin ; metabolism ; Porosity ; Receptor Activator of Nuclear Factor-kappa B ; metabolism ; Tissue Engineering ; instrumentation ; Tissue Scaffolds ; chemistry ; Titanium ; pharmacology

Titanium and its alloys are routinely used as biomedical implants and are usually considered to be corrosion resistant under physiological conditions. However, during inflammation, chemical modifications of the peri-implant environment including acidification occur. In addition certain biomolecules including lipopolysaccharide (LPS), a component of Gram-negative bacterial cell walls and driver of inflammation have been shown to interact strongly with Ti and modify its corrosion resistance. Gram-negative microbes are abundant in biofilms which form on dental implants. The objective was to investigate the influence of LPS on the corrosion properties of relevant biomedical Ti substrates as a function of environmental acidity. Inductively coupled plasma mass spectrometry was used to quantify Ti dissolution following immersion testing in physiological saline for three common biomedical grades of Ti (ASTM Grade 2, Grade 4 and Grade 5). Complementary electrochemical tests including anodic and cathodic polarisation experiments and potentiostatic measurements were also conducted. All three Ti alloys were observed to behave similarly and ion release was sensitive to pH of the immersion solution. However, LPS significantly inhibited Ti release under the most acidic conditions (pH 2), which may develop in localized corrosion sites, but promoted dissolution at pH 4-7, which would be more commonly encountered physiologically. The observed pattern of sensitivity to environmental acidity of the effect of LPS on Ti corrosion has not previously been reported. LPS is found extensively on the surfaces of skin and mucosal penetrating Ti implants and the findings are therefore relevant when considering the chemical stability of Ti implant surfaces in vivo.
Acids ; chemistry ; Corrosion ; Electrodes ; Hydrogen-Ion Concentration ; Lipopolysaccharides ; pharmacology ; Materials Testing ; Microscopy, Electron, Scanning ; Titanium ; chemistry

The aim of this study was to investigate the inhibitory effect of heparin/fibronectin (Hep/Fn) complexes on neointimal hyperplasia following endovascular intervention. Hep/Fn complexes were immobilized onto titanium (Ti) surfaces, with subsequent X-ray photoelectron spectroscopy (XPS), Toluidine Blue O (TBO) and immunohistochemistry methods were used to characterize surface properties. Smooth muscle cell (SMC) cultures were used to evaluate the effect of Hep/Fn complexes on SMC proliferation. Results showed that Hep/Fn complexes successfully immobilized onto Ti surfaces and resulted in an inhibition of SMC proliferation. This study suggests that Hep/Fn surface-immobilized biomaterials develop as a new generation of biomaterials to prevent neointimal hyperplasia, particularly for use in cardiovascular implants.
Biocompatible Materials ; Cell Proliferation ; drug effects ; physiology ; Cells, Cultured ; Fibronectins ; chemistry ; pharmacology ; Heparin ; chemistry ; pharmacology ; Humans ; Immobilized Proteins ; chemistry ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; drug effects ; physiology ; Surface Properties ; Titanium ; chemistry ; Umbilical Arteries

OBJECTIVETo construct a multiple-scale organized implant surface with super-hydrophilicity.

METHODSThe SiC paper polished titanium disc was sandblasted and treated with HF/HNO₃ and HCl/H₂SO₄, then acid-etched with H₂SO₄/H₂O₂. The physicochemical properties of the surfaces were characterized by scanning electron microscope, static state contact angle and X-ray diffraction. MC3T3-E1 cells were used to evaluate the effects of the surface on the cell adhesion, proliferation and differentiation.

RESULTSThe acid-etching process with a mixture of H₂SO₄/H₂O₂ superimposed the nano-scale structure on the micro-scale texture. The multiple-scale implant surface promoted its hydrophilicity and was more favorable to the responses of osteoprogenitor cells, characterized by increased DNA content, enhanced ALP activity and promoted OC production.

CONCLUSIONA multiple-scale implant surface with super-hydrophilicity has been constructed in this study, which facilitates cell proliferation and adhesion.

3T3 Cells ; Animals ; Cell Adhesion ; drug effects ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Dental Etching ; Dental Implants ; Hydrophobic and Hydrophilic Interactions ; Mice ; Surface Properties ; Titanium ; chemistry ; pharmacology

OBJECTIVETo study the effects of ultraviolet ray (UV)-irradiation on the surface characteristic and antibacterial activity of TiO(2) nanotubes with different diameters.

METHODSTiO(2) nanotubes with different diameters were fabricated on polished pure titanium (PT) samples by anodization at 5, 10 and 20 V with PT as control. The samples were exposed to UV-irradiation for 24 h, then the characteristic and antibacterial activity were analyzed and evaluated. The surface topograph was observed by field emission scanning electron microscope (FE-SEM). Contract angle measurements were carried out with three liquids. Staphylococcus aureus (Sa) were used to evaluate the antibacterial activity of samples with the film contact method. The bacterial morphology was observed by FE-SEM. The bacterial adhesion and cell membrane injury were evaluated by fluorescent staining analysis under laser scanning confocal microscope (LSCM).

RESULTSAfter the TiO(2) nanotubes with different diameters were exposed to UV-irradiation, no change was observed in its surface topograph. With the increase of the diameters of nanotubes, each contract angle of nanotubes decreased, and bacterial FIt and dead/live ratio were also increased. We found 20 V FIt was the biggest (26.550 ± 2.940) and ranks the highest ratio of death/live (0.728 ± 0.091) among the others (P < 0.05).

CONCLUSIONSThe UV-irradiation can decrease the contract angle of TiO(2) nanotubes and promote the Sa adhesion on nanotubes. Meanwhile, the antibacterial activity of TiO(2) nanotubes with different diameters was remarkably enhanced by UV-irradiation. Nanotubes anodized at 20 V showed the best antibacterial activity.

Anti-Bacterial Agents ; chemistry ; pharmacology ; radiation effects ; Bacterial Adhesion ; Microscopy, Electron, Scanning ; Nanotubes ; chemistry ; radiation effects ; Staphylococcus aureus ; drug effects ; Surface Properties ; Titanium ; chemistry ; pharmacology ; radiation effects ; Ultraviolet Rays

OBJECTIVETo investigate the effect of minocycline hydrochloride ointment on cell attachment and proliferation on titanium disks.

METHODSCommercially pure (grade 4) machined titanium discs with three different kinds of surfaces (smooth, acid-etched and sandblasted combined with acid-etched) were treated with minocycline ointment for 1 week, and then cleaned in ultrasonic cleanser for 10 minutes. Surface properties were examined by scanning electron microscope (SEM) and roughness tester before and after the treatment. Surface roughness was compared by paired t test. MG-63 (human osteoblast-like osteosarcoma cell) cells were seeded on these three kinds of discs with or without minocycline treatment, and methl thiazolyl tetrazolium (MTT) was performed to investigate the attachment in the 1st day and proliferation in the 4th and 7th day. Data were analyzed by double factor analysis of variance.

RESULTSSurface roughness before and after minocycline application was as follows, Smooth: (0.093 ± 0.025) µm, (0.086 ± 0.026) µm; Acid-etched: (1.100 ± 0.095) µm, (1.009 ± 0.196) µm; Sandblasted combined with acid-etched: (2.837 ± 0.283) µm, (2.968 ± 0.206) µm. No significant changes in roughness were found before and after minocycline application (P values were 0.118, 0.436 and 0.692). SEM examination revealed as similar surface configuration after minocycline application as before, except for some remnant of the minocycline ointment in acid-etched and sandblasted combined acid-etched groups. In MTT test, the growth of MG-63 cells in the 1 st, 4th day and 7th day was not different between groups with and without minocycline application (P values were 0.450, 0.848 and 0.835), and among three groups of different surface (P values were 0.184, 0.579 and 0.331).

CONCLUSIONSMinocycline hydrochloride ointment did not affect the surface configuration, surface roughness or the properties for cell attachment and proliferation of titanium discs.

Acid Etching, Dental ; Bone Neoplasms ; pathology ; Cell Adhesion ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Humans ; Microscopy, Electron, Scanning ; Minocycline ; administration & dosage ; pharmacology ; Ointments ; Osteoblasts ; pathology ; Osteosarcoma ; pathology ; Surface Properties ; Titanium ; chemistry