Enhanced osteogenic and antibacterial properties of titanium implant surface modified with Zn-incorporated nanowires: Preclinical in vitro and in vivo investigations.

OBJECTIVE: This study aimed to synthesize zinc-incorporated nanowires structure modified titanium implant surface (Zn-NW-Ti) and explore its superior osteogenic and antibacterial properties in vitro and in vivo. MATERIALS AND METHODS: Zn-NW-Ti was synthesized via displacement reactions between zinc sulfate solutions and the titanium (Ti) surface, which was pretreated by hydrofluoric acid etching and hyperthermal alkalinization. The physicochemical properties of the Zn-NW-Ti surface were examined. Moreover, the biological effects of Zn-NW-Ti on MC3T3-E1 cells and its antibacterial property against oral pathogenic bacteria (Staphylococcus aureus, Porphyromonas gingivalis, and Actinobacillus actinomycetemcomitans) compared with sandblasted and acid-etched Ti (SLA-Ti) and nanowires modified Ti (NW-Ti) surface were assessed. Zn-NW-Ti and SLA-Ti modified implants were inserted into the anterior extraction socket of the rabbit mandible with or without exposure to the mixed bacterial solution (S. aureus, P. gingivalis, and A. actinomycetemcomitans) to investigate the osteointegration and antibacterial performance via radiographic and histomorphometric analysis. RESULTS: The Zn-NW-Ti surface was successfully prepared. The resultant titanium surface appeared as a nanowires structure with hydrophilicity, from which zinc ions were released in an effective concentration range. The Zn-NW-Ti surface performed better in facilitating the adhesion, proliferation, and differentiation of MC3T3-E1 cells while inhibiting the colonization of bacteria compared with SLA-Ti and NW-Ti surface. The Zn-NW-Ti implant exhibited enhanced osseointegration in vivo, which was attributed to increased osteogenic activity and reduced bacterial-induced inflammation compared with the SLA-Ti implant. CONCLUSIONS: The Zn-incorporated nanowires structure modified titanium implant surface exhibited improvements in osteogenic and antibacterial properties, which optimized osteointegration in comparison with SLA titanium implant surface.

Modulation of SEMA4D-modified titanium surface on M2 macrophage polarization via activation of Rho/ROCK-mediated lactate release of endothelial cells: In vitro and in vivo.

SEMA4D-modified titanium surfaces can indirectly regulate macrophages through endothelial cells to achieve an anti-inflammatory effect, which is beneficial for healing soft tissues around the gingival abutment. However, the mechanism of surface-induced cellular phenotypic changes in SEMA4D-modified titanium has not yet been elucidated. SEMA4D activates the RhoA signaling pathway in endothelial cells, which coordinates metabolism and cytoskeletal remodeling. This study hypothesized that endothelial cells inoculated on SEMA4D-modified titanium surfaces can direct M2 polarization of macrophages via metabolites. An indirect co-culture model of endothelial cells and macrophages was constructed in vitro, and specific inhibitors were employed. Subsequently, endothelial cell adhesion and migration, metabolic changes, Rho/ROCK1 expression, and inflammatory expression of macrophages were assessed via immunofluorescence microscopy, specific kits, qRT-PCR, and Western blotting. Moreover, an in vivo rat bilateral maxillary implant model was constructed to evaluate the soft tissue healing effect. The in vitro experiments showed that the SEMA4D group had stronger endothelial cell adhesion and migration, increased Rho/ROCK1 expression, and enhanced release of lactate. Additionally, decreased macrophage inflammatory expression was observed. In contrast, the inhibitor group partially suppressed lactate metabolism and motility, whereas increased inflammatory expression. The in vivo analyses indicated that the SEMA4D group had faster and better angiogenic and anti-inflammatory effects, especially in the early stage. In conclusion, via the Rho/ROCK1 signaling pathway, the SEMA4D-modified titanium surface promotes endothelial cell adhesion and migration and lactic acid release, then the paracrine lactic acid promotes the polarization of macrophages to M2, thus obtaining the dual effects of angiogenesis and anti-inflammation.

Genome-wide transcriptional responses of osteoblasts to different titanium surface topographies.

This is the first genome-wide transcriptional profiling study using RNA-sequencing to investigate osteoblast responses to different titanium surface topographies, specifically between machined, smooth and acid-etched, microrough surfaces. Rat femoral osteoblasts were cultured on machine-smooth and acid-etched microrough titanium disks. The culture system was validated through a series of assays confirming reduced osteoblast attachment, slower proliferation, and faster differentiation on microrough surfaces. RNA-sequencing analysis of osteoblasts at an early stage of culture revealed that gene expression was highly correlated (r = 0.975) between the two topographies, but 1.38 % genes were upregulated and 0.37 % were downregulated on microrough surfaces. Upregulated transcripts were enriched for immune system, plasma membrane, response to external stimulus, and positive regulation to stimulus processes. Structural mapping confirmed microrough surface-promoted gene sharing and networking in signaling pathways and immune system/responses. Target-specific pathway analysis revealed that Rho family G-protein signaling pathways and actin genes, responsible for the formation of stress fibers, cytoplasmic projections, and focal adhesion, were upregulated on microrough surfaces without upregulation of core genes triggered by cell-to-cell interactions. Furthermore, disulfide-linked or -targeted extracellular matrix (ECM) or membranous glycoproteins such as laminin, fibronectin, CD36, and thrombospondin were highly expressed on microrough surfaces. Finally, proliferating cell nuclear antigen (PCNA) and cyclin D1, whose co-expression reduces cell proliferation, were upregulated on microrough surfaces. Thus, osteoblasts on microrough surfaces were characterized by upregulation of genes related to a wide range of functions associated with the immune system, stress/stimulus responses, proliferation control, skeletal and cytoplasmic signaling, ECM-integrin receptor interactions, and ECM-membranous glycoprotein interactions, furthering our knowledge of the surface-dependent expression of osteoblastic biomarkers on titanium.

Efficient titanium surface modified using bifunctional chimeric peptides to prevent biofilm formation by multiple microorganisms.

It is still a challenge to prevent the formation of bacterial biofilms on the surfaces of oral implants. A chemical peptide with binding and antibacterial properties may be a promising agent if used to modify titanium (Ti) surfaces to inhibit biofilm formation. In this study, peptides were designed by linking the antimicrobial sequence derived from human ß-defensin-3 (hBD-3) to the Ti-binding peptide-1 (TBP-1) sequence by using a triple glycine (G) linker. The antimicrobial activity and biocompatibility characteristics of the chemical-peptide-modified Ti surface were then evaluated and the potential antibacterial mechanism was investigated. This study demonstrated that the chemical-peptide-modified surface exhibited satisfactory bactericidal activities against Streptococcus gordonii, Fusobacterium nucleatum, and Porphyromonas gingivalis. In addition to its potent bacteria-killing efficacy, the surface-immobilised chemical peptide also demonstrated excellent biocompatibility to L929 cells. Moreover, the disruption of the integrity of the bacterial membrane partially revealed the antibacterial mechanism of the peptide. This study demonstrated the potential of chemical-peptide-modified Ti surfaces for preventing the occurrence of peri-implant diseases, thereby providing a promising approach to improving the survival rate of oral implants.

Biofilm Removal from In Vitro Narrow Geometries Using Single and Dual Pulse Er:YAG Laser Photoacoustic Irrigation.

The disinfection and removal of biofilm from titanium dental implants remains a great challenge in oral medicine. Here we present results of novel photoacoustic irrigation laser modalities for biofilm removal in model geometries mimicking the peri-implant pocket. The efficacy of single pulse (Er:YAG-SSP) and dual pulse (Er:YAG-AutoSWEEPS) photoacoustic irrigation modalities were determined for Enterococcus faecalis biofilm decontamination from titanium surfaces in narrow cylindrical and square gap geometries. The density of bacteria as well as the number of live bacteria were determined prior and after different photoacoustic treatments. Both SSP and AutoSWEEPS photoacoustic irrigation techniques removed at least 92% of biofilm bacteria during the 10 s photoacoustic treatment. The effectiveness of cleaning was better in the narrow square gap geometry compared to the cylindrical geometry. The dual pulse Er:YAG-AutoSWEEPS photoacoustic irrigation showed better results compared to SSP modality. No chemical adjuvants were needed to boost the effectiveness of the photoacoustic irrigation in the saline solution. The results imply that photoacoustic irrigation is an efficient cleaning method for debridement and decontamination in narrow geometries and should be considered as a new therapeutic option for the treatment of peri-implant diseases.

Improving dental epithelial junction on dental implants with bioengineered peptides.

Introduction: The functionalization of titanium (Ti) and titanium alloys (Ti6Al4V) implant surfaces via material-specific peptides influence host/biomaterial interaction. The impact of using peptides as molecular linkers between cells and implant material to improve keratinocyte adhesion is reported. Results: The metal binding peptides (MBP-1, MBP-2) SVSVGMKPSPRP and WDPPTLKRPVSP were selected via phage display and combined with laminin-5 or E-cadherin epithelial cell specific peptides (CSP-1, CSP-2) to engineer four metal-cell specific peptides (MCSPs). Single-cell force spectroscopy and cell adhesion experiments were performed to select the most promising candidate. In vivo tests using the dental implant for rats showed that the selected bi functional peptide not only enabled stable cell adhesion on the trans-gingival part of the dental implant but also arrested the unwanted apical migration of epithelial cells. Conclusion: The results demonstrated the outstanding performance of the bioengineered peptide in improving epithelial adhesion to Ti based implants and pointed towards promising new opportunities for applications in clinical practice.

Osteogenic and anti-inflammatory effects of SLA titanium substrates doped with chitosan-stabilized selenium nanoparticles via a covalent coupling strategy.

Osseointegration is a prerequisite for the function of dental implants, and macrophage-dominated immune responses triggered by implantation determine the outcome of ultimate bone healing mediated by osteogenic cells. The present study aimed to develop a modified titanium (Ti) surface by covalently immobilizing chitosan-stabilized selenium nanoparticles (CS-SeNPs) to sandblasted, large grit, and acid-etched (SLA) Ti substrates and further explore its surface characteristics as well as osteogenic and anti-inflammatory activities in vitro. CS-SeNPs were successfully prepared by chemical synthesis and characterized their morphology, elemental composition, particle size, and Zeta potential. Subsequently, three different concentrations of CS-SeNPs were loaded to SLA Ti substrates (Ti-Se1, Ti-Se5, and Ti-Se10) using a covalent coupling strategy, and the SLA Ti surface (Ti-SLA) was used as a control. Scanning electron microscopy images revealed different amounts of CS-SeNPs, and the roughness and wettability of Ti surfaces were less susceptible to Ti substrate pretreatment and CS-SeNP immobilization. Besides, X-ray photoelectron spectroscopy analysis showed that CS-SeNPs were successfully anchored to Ti surfaces. The results of in vitro study showed that the four as-prepared Ti surfaces exhibited good biocompatibility, with Ti-Se1 and Ti-Se5 groups showing enhanced adhesion and differentiation of MC3T3-E1 cells compared with the Ti-SLA group. In addition, Ti-Se1, Ti-Se5, and Ti-Se10 surfaces modulated the secretion of pro-/anti-inflammatory cytokines by inhibiting the nuclear factor kappa B pathway in Raw 264.7 cells. In conclusion, doping SLA Ti substrates with a modest amount of CS-SeNPs (1-5 mM) may be a promising strategy to improve the osteogenic and anti-inflammatory activities of Ti implants.

Synergistic effects of enamel matrix derivatives and surface morphology of anodized titanium on osteogenic differentiation of bone marrow mesenchymal stem cells sheet.

OBJECTIVE: To explore the potential osteogenic induction mechanism of enamel matrix derivatives (EMDs) on bone marrow mesenchymal stem cell (BMSC) sheets with different titanium surface morphologies. METHODS: The BMSCs were inoculated on the surfaces of titanium alloys with different morphologies: anodic oxidation (AO), sand-blasted, large grit and acid-etched, and no treatment (control). The proliferation and osteogenic differentiation of BMSCs on the different surface morphologies were observed with the same concentration of EMDs. To further understand the osteogenic mechanism of EMDs on BMSC sheets with different morphologies, a real-time RT-PCR and a western blot were used to detect the overall levels of osteogenic genes and osteogenic proteins. Finally, to verify the osteogenic effect of BMSC sheets stimulated by EMDs in vivo, BMSC sheets with different morphologies were implanted into the subcutaneous tissue of the back of nude mice, and the bone formation was detected by HE staining. RESULTS: The EMDs and surface morphology in the AO group synergically increased the expression levels of osteogenic active factors (RUNX2, OSX and OCN) and enhanced the osteogenic differentiation effect of BMSCs. The in vivo experiments showed that the BMSC sheets in the AO group were rich in osteogenic active factors, and promoted the formation of ectopic bone tissue after implantation into the subcutaneous tissue of the back of nude mice. CONCLUSION: EMDs and AO morphology synergically enhance the secretion of bone osteogenic active factors of BMSCs and promote the formation of heterotopic bone.

Biological response of epithelial and connective tissue cells to titanium surfaces with different ranges of roughness: An in-vitro study.

OBJECTIVES: This study aimed to investigate how titanium (Ti) surface with different range roughness created by industrial machining influence the biological response of primary human gingival fibroblasts (HGFB) and keratinocytes (HGKC) in terms of cell proliferation and cytotoxicity. METHODS: Four Ti surfaces of different roughness ranges were investigated: smooth (S: 0.08-0.1 µm), minimally rough (MM: 0.3-0.5 µm), moderately rough (MR: 1.2-1.4 µm) and rough (R: 3.3-3.7 µm). Discs topography and surface roughness were evaluated by scanning electron microscopy (SEM) and non-contact profilometer. Both cell lines were cultured, expanded, and maintained according to their supplier's protocols. Cell proliferation and cytotoxicity were evaluated at days 1, 3, 5, and 10 using cell viability and cytotoxicity colorimetric assays. Data were analysed via two-way ANOVA, one-way ANOVA and Tukey's post hoc test (p = 0.05 for all tests). RESULTS: Both cell lines showed comparable initial proliferation activity of 70-86% for all the investigated roughnesses. HGKC showed better and higher proliferation % with S surface at all time points than all the other investigated surfaces which was significantly higher than MM at day 3 and higher than all the other investigated surfaces at day 5 and 10. On the other hand, HGFB exhibited the best proliferation with both MM and R surfaces with no significant differences from the other two surfaces (S and MR). Different surface roughnesses and exposure times showed significant effect on cell proliferation in both cell lines. Cytotoxicity for both cell lines was generally the highest on day 3, with the following order from highest to lowest: S (19.86%)> R> MR> MM for HGKC and MM (39.48%)> MR> S> R for HGFB. Different exposure times showed a significant effect on cell cytotoxicity in both cell lines and a significant effect of surface roughness in HGFB. SIGNIFICANCE: All investigated roughness levels were sufficiently biologically compatible with cells representative of the major population of the soft tissue surrounding dental implants. However, the S surface was most cytotoxic to HGKC, while the MM surface was most cytotoxic to HGFB cells.

Human Gingival Fibroblast Attachment to Smooth Titanium Disks with Different Surface Roughnesses.

Peri-implantitis is a significant problem associated with dental implants. It has been hypothesized that creating a soft-tissue seal around the implant neck prevents peri-implantitis. This study aims to clarify the effects of the surface smoothness of titanium disks on soft tissues. Thus, titanium disks were prepared through electrolytic composite polishing (ECP), sisal buffing (SB), hairline polishing (HP), and laser cutting (LC). The surface roughness values of seven items was measured. For ECP, SB, HP, and LC samples, the Ra values were 0.075, 0.217, 0.671, and 1.024 µm and the Sa values were 0.005, 0.115, 0.500, and 0.676, respectively, indicating that the surface roughness was remarkably lower with ECP. Moreover, the Wsk values for ECP, SB, HP, and LC were 0.521, 1.018, -0.678, and -0.558, respectively. The smooth surfaces produced by ECP and SB were biased toward the concave surface, whereas those produced by HP and LC were biased toward the convex surface. The Rku values for ECP, SB, HP, and LC were 2.984, 11.774, 14.182, and 26.232, respectively. Only the ECP exhibited a moderate bias peak and produced an extremely smooth surface. The contact angles in the cases of ECP, SB, HP, and LC were 60.1°, 66.3°, 68.4°, and 79.3°, respectively, indicating the hydrophobicity of the titanium disks. Human oral fibroblasts were then incubated on each disk for 24 and 48 h to measure cell attachment, and no significant differences were observed. The differences in Ra and Sa did not affect cell attachment. Therefore, by applying ECP to the abutment or implant neck, the cell attachment required for soft-tissue formation while preventing bacterial adhesion can be achieved.

Effects of Different Titanium Surface Treatments on Adhesion, Proliferation and Differentiation of Bone Cells: An In Vitro Study.

The objective of this study was to evaluate the impacts of different sandblasting procedures in acid etching of Ti6Al4V surfaces on osteoblast cell behavior, regarding various physicochemical and topographical parameters. Furthermore, differences in osteoblast cell behavior between cpTi and Ti6Al4V SA surfaces were evaluated. Sandblasting and subsequent acid etching of cpTi and Ti6Al4V discs was performed with Al2O3 grains of different sizes and with varying blasting pressures. The micro- and nano-roughness of the experimental SA surfaces were analyzed via confocal, atomic force and scanning electron microscopy. Surface free energy and friction coefficients were determined. hFOB 1.19 cells were seeded to evaluate adhesion, proliferation and osteoblastic differentiation for up to 12 d via crystal violet assays, MTT assays, ALP activity assays and Alizarin Red staining assays. Differences in blasting procedures had significant impacts on surface macro- and micro-topography. The crystal violet assay revealed a significant inverse relationship between blasting grain size and hFOB cell growth after 7 days. This trend was also visible in the Alizarin Red assays staining after 12 d: there was significantly higher biomineralization visible in the group that was sandblasted with smaller grains (F180) when compared to standard-grain-size groups (F70). SA samples treated with reduced blasting pressure exhibited lower hFOB adhesion and growth capabilities at initial (2 h) and later time points for up to 7 days, when compared to the standard SA surface, even though micro-roughness and other relevant surface parameters were similar. Overall, etched-only surfaces consistently exhibited equivalent or higher adhesion, proliferation and differentiation capabilities when compared to all other sandblasted and etched surfaces. No differences were found between cpTi and Ti6Al4V SA surfaces. Subtle modifications in the blasting protocol for Ti6Al4V SA surfaces significantly affect the proliferative and differentiation behavior of human osteoblasts. Surface roughness parameters are not sufficient to predict osteoblast behavior on etched Ti6Al4V surfaces.

In-Vitro Phenotypic Response of Human Osteoblasts to Different Degrees of Titanium Surface Roughness.

Objectives: This study aimed to investigate human osteoblast (HOB) responses towards different degrees of titanium (Ti) implant surface roughness. Methods: Four degrees of Ti surface roughness were investigated on a micrometer roughness scale: smooth (S: 0.08−0.1 µm), minimally rough (MM: 0.3−0.5 µm), moderately rough (MR: 1.2−1.4 µm), and rough (R: 3.3−3.7 µm). HOB cells were cultured, expanded, and maintained according to the supplier's protocol. Cell proliferation and cytotoxicity were assessed at day 1, 3, 5, and 10 using alamarBlue and lactate dehydrogenase colorimetric assays. Data were analyzed with one-way ANOVA, two-way ANOVA, and Tukey's post hoc test (p = 0.05 for all tests). Results: There was no significant difference in the cell proliferation or cytotoxicity of the HOB cells in contact with the different degrees of Ti surface roughness. There was, however, a significant time effect on cell proliferation (p < 0.0001) with different exposure durations for each roughness degree. Furthermore, a positive correlation (non-significant) between proliferation and cytotoxicity was observed for all investigated degrees of surface roughness. Conclusion: All investigated roughness degrees showed comparable HOB proliferation, with the MR surface presenting the highest percentage, followed by the R, MM, ad S, surfaces, respectively. The S surface showed the highest cytotoxic effect on HOBs; however, it did not reach the cytotoxic level suggested by the ISO for any medical device to be considered cytotoxic.

Effect Morphology and Surface Treatment of the Abutments of Dental Implants on the Dimension and Health of Peri-Implant Biological Space.

STATEMENT OF THE PROBLEM: The gingival configuration around implant abutments is of paramount importance for preserving the underlying marginal bone, and hence for the long-term success of dental implants. OBJECTIVE: The objective was to study, clinically and histologically, the effects of the change in the morphology of abutments connected to the endosseous implant, and of their surface treatment. In particular, the objective was to ascertain the effect of changing the shape of the transepithelial pillar and the treatment of its surface on the dimensions, quality and health of the components of the peri-implant biological space, such as the dimensions of the epithelial and connective tissues of the biological space, the concentration of inflammatory cells and the density of collagen fibers. METHODS: A clinical trial of 10 patients with a totally edentulous maxilla, who had four implants (IPX4010_GALIMPLANT®, Sarria, Spain) inserted in the area of the first and second molars on both sides with computer-guided implant surgery, was conducted with the final purpose of assessing the quality of the peri-implant soft tissue attachment around the transepithelial abutments which were employed (aesthetic machined (RM), aesthetic anodized (RA), slim machined (SM) and slim anodized (SA)). At 8 weeks and following the collection of the samples (removal of the implant-abutment assembly with its surrounding hard and soft tissue) and their processing for subsequent histological and histomorphometric analysis in order to study the dimensions, quality and health of the peri-implant soft tissue area, the variables previously mentioned were determined according to the aims of the study. By using appropriate diameter trephine in order to obtain a useful fringe of soft tissue around the transepithelial pillars, ANOVA and chi-square tests were performed. RESULTS: The SPSS statistical analysis ANOVA results revealed that the machined slim abutments have a better performance considering the variables analyzed with epithelial and connective attachment heights of 1.52 mm and 2.3 mm, respectively, and that connective density (density of collagen fibers) was high at 85.7% of the sample size affected by the design for the slim abutments and 92.9% of the high-density sample size affected by the surface treatment for the machined surface. CONCLUSIONS: All variables studied, despite the small sample size, showed the superiority of the slim machined abutment among the four groups.

A comparison of human dental pulp stem cell activity cultured on sandblasted titanium discs decontaminated with Er:YAG laser and air-powder abrasion: an in vitro study.

Decontamination of implant surfaces is important to the treatment of peri-implantitis. Er:YAG laser and air-powder abrasion system are regarded as the most effective means of decontamination of implant surfaces. The aim of this in vitro study was to compare the activity of human dental pulp stem cells (hDPSCs) cultured on decontaminated sandblasted titanium discs using Er:YAG laser irradiation and air-powder abrasion. Forty-five titanium discs were contaminated with Escherichia coli (E. coli) bacteria and fifteen titanium discs served as sterile control groups. Thirty contaminated titanium discs were decontaminated with Er:YAG laser or air-powder abrasion system and fifteen contaminated discs were used as contaminated control group. Afterwards, hDPSCs were seeded on all sixty experimental titanium discs. The effects of two decontamination tools on hDPSCs viability were evaluated by MTT assay. Alkaline phosphatase (ALP) activity assay, quantitative real-time PCR analysis and alizarin red staining method were performed to assess hDPSCs osteogenic differentiation. Scanning microscope electron (SEM) was also used to evaluate the effects of two different decontaminated methods on cellular morphology. Our study showed that decontamination using Er:YAG laser caused maximum cell viability. However, the ALP activity was not different in laser and air-abrasion groups. The significant expression of an osteoblastic marker and stronger Alizarin red staining were observed in laser irradiation groups. In addition, SEM observation indicated that grown cells were more stretched and more filopodia in Er:YAG-treated discs. In the present study, Er:YAG laser and air-powder abrasion improved the activity of the cells cultured on the decontaminated titanium discs. However, in comparison with air-powder abrasion, Er:YAG laser was more effective.

Efficiency of biofilm removal by combination of water jet and cold plasma: an in-vitro study.

BACKGROUND: Peri-implantitis therapy is a major problem in implantology. Because of challenging rough implant surface and implant geometry, microorganisms can hide and survive in implant microstructures and impede debridement. We developed a new water jet (WJ) device and a new cold atmospheric pressure plasma (CAP) device to overcome these problems and investigated aspects of efficacy in vitro and safety with the aim to create the prerequisites for a clinical pilot study with these medical devices. METHODS: We compared the efficiency of a single treatment with a WJ or curette and cotton swab (CC) without or with adjunctive use of CAP (WJ + CAP, CC + CAP) to remove biofilm in vitro from rough titanium discs. Treatment efficacy was evaluated by measuring turbidity up to 72 h for bacterial re-growth or spreading of osteoblast-like cells (MG-63) after 5 days with scanning electron microscopy. With respect to application safety, the WJ and CAP instruments were examined according to basic regulations for medical devices. RESULTS: After 96 h of incubation all WJ and CC treated disks were turbid but 67% of WJ + CAP and 46% CC + CAP treated specimens were still clear. The increase in turbidity after WJ treatment was delayed by about 20 h compared to CC treatment. In combination with CAP the cell coverage significantly increased to 82% (WJ + CAP) or 72% (CC + CAP), compared to single treatment 11% (WJ) or 10% (CC). CONCLUSION: The newly developed water jet device effectively removes biofilm from rough titanium surfaces in vitro and, in combination with the new CAP device, biologically acceptable surfaces allow osteoblasts to grow. WJ in combination with CAP leads to cleaner surfaces than the usage of curette and cotton swabs with or without subsequent plasma treatment. Our next step will be a clinical pilot study with these new devices to assess the clinical healing process.

Nanoporous Titanium Enriched with Calcium and Phosphorus Promotes Human Oral Osteoblast Bioactivity.

Implant surfaces are known to influence the osseointegration process; therefore, their modifications represent an important subject of investigation. On this basis, the purpose of this study was to evaluate the response of human oral osteoblasts (hOBs) to three different GR4 titanium discs: Machined, double-etched (Osteopore), and double-etched, surface-enriched with calcium and phosphorus (CaP) (Nanopore). The superficial topography was investigated with scanning electron microscopy (SEM) and the sessile drop technique. To test cellular response and osteoinductive properties, the following points were evaluated: (i) proliferation by MTS assay after 2 and 5 days; (ii) adhesion by multiphoton microscopy at day 2; (iii) the interaction with Ti discs by blue toluidine staining at day 5; (iv) alkaline phosphatase (ALP) activity by ALP assay after 14 days; (v) calcium deposition by alizarin red staining and by cetylpyridinium chloride after 14 days. The SEM analysis showed that Nanopore and Osteopore surfaces were characterized by the same micro-topography. Nanopore and Osteopore discs, compared to Machined, stimulated higher osteoblast proliferation and showed more osteoinductive properties by promoting the ALP activity and calcium deposition. In conclusion, the CaP treatment on DAE surfaces seemed to favor the oral osteoblast response, encouraging their use for in vivo applications.

Effect of a neodymium-doped yttrium aluminium garnet laser on the physicochemical properties of contaminated titanium surfaces and macrophage polarization.

AIM(S): The objective of this study was to evaluate the changes in the physical and chemical properties of titanium surfaces contaminated by a Nd:YAG laser with different levels of energy and the regulation of macrophage polarization. MATERIALS AND METHODS: The titanium specimens were divided into four groups. The blank control group consisted of the above-mentioned contaminated titanium specimens, and the conditioned control group consisted of sandblasted and acid-etched (SLA) titanium surfaces. The blank control and condition control groups were sealed and preserved in a sterile dark box. There were two experimental groups treated with the Nd:YAG laser-one with 0.5 W and the second with 1.0 W. Surface characteristics were evaluated using scanning electron microscopy, surface profilometry, and contact angle assays. The macrophage viability and proliferation of mouse RAW246.7 were analysed, and the macrophage surface markers, macrophage cytokines, and inflammatory and anti-inflammatory genes were expressed. RESULTS: The Nd:YAG laser increased the hydrophilicity and roughness of the titanium surface after decontamination. Fewer RAW264.7 cells were observed on the titanium surface after Nd:YAG decontamination than on the contaminated titanium surface expressing the M1-type macrophage marker CCR7, whereas more cells were observed after decontamination than on the contaminated titanium surface expressing the M2-type macrophage marker CD206. Following Nd:YAG laser treatment, the secretion of the inflammatory cytokines IL-1ß and TNF-α by RAW264.7 cells on the titanium surface was decreased, whereas the secretion of the anti-inflammatory cytokines IL-4 and IL-10 was increased. RAW264.7 cells cultured for 3 days on the titanium surface after Nd:YAG decontamination treatment expressed significantly reduced levels of the inflammation-related genes IL-1ß, TNF-α, IL-6 and iNOS. The expression of the anti-inflammatory genes Arg-1, IL-4, IL-10 and TGF-ß by RAW264.7 cells was significantly up-regulated after 3 days of incubation on the titanium surface after Nd:YAG decontamination treatment. CONCLUSION(S): The Nd:YAG laser increased the hydrophilicity and roughness of the titanium surface after decontamination, and this change inhibited M1-type macrophage polarization and promoted M2-type macrophage polarization.

Photoacoustic removal of Enterococcus faecalis biofilms from titanium surface with an Er:YAG laser using super short pulses.

Biofilms that grow on implant surfaces pose a great risk and challenge for the dental implant survival. In this work, we have applied Er:YAG photoacoustic irrigation using super short pulses (Er:YAG-SSP) to remove biofilms from the titanium surfaces in the non-contact mode. Mature Enterococcus faecalis biofilms were treated with saline solution, chlorhexidine, and hydrogen peroxide, or photoacoustically with Er:YAG-SSP for 10 or 60 s. The number of total and viable bacteria as well as biofilm surface coverage was determined prior and after different treatments. Er:YAG-SSP photoacoustic treatment significantly increases the biofilm removal rate compared to saline or chemically treated biofilms. Up to 92% of biofilm-covered surface can be cleaned in non-contact mode during 10 s without the use of abrasives or chemicals. In addition, Er:YAG-SSP photoacoustic irrigation significantly decreases the number of viable bacteria that remained on the titanium surface. Within the limitations of the present in vitro model, the ER:YAG-SSP seems to constitute an efficient therapeutic option for quick debridement and decontamination of titanium implants without using abrasives or chemicals.

Promoting the adhesion of human gingival epithelial cells on titanium surface by non-thermal atmospheric plasma irradiation.

OBJECTIVES: This work aimed to study the biological behavior of human gingival epithelial cells (HGECs) irradiated by non-thermal atmospheric plasma (NTAP) on a titanium surface. METHODS: Cultured HGECs (3⁃5 generations) with the best activity were digested and treated for varying times (0, 10, 20, 30, and 60 s) by NTAP and then seeded on the surface of a titanium disc. The HGECs were cultured in oral keratinocyte medium and 1% penicillin/streptomycin solution. The cells were kept in an atmosphere of 5% CO2 at 37 ℃ and incubated for different times (4, 12, 24, and 48 h; n=5). Cell counting kit-8 (CCK-8) was used to detect cell adhesion capacity. Scanning electron microscopy (SEM) was conducted to observe the morphology of cells on titanium plates. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were used to evaluate the gene expression of adhesion-related molecules, such as Laminin α3, Integrin ß4, and Plectin. RESULTS: The number of adhered cells increased at 0­20 s, whereas that gradually decreased at 20⁃60 s. Therefore, cell culture at the two time points showed that HGECs adhesion reached the maximum when NATP was irradiated for 20 s. Compared with the control group, more cells in the treatment group adhered to the titanium surface at each time point (P<0.05). Cells in the treatment group showed more irregular polygons, more protrusions and pseudopods, and a larger cell diffusion area on the titanium surface than those in the control group. qRT-PCR showed that the expression levels of Laminin α3, Integrin ß4, and Plectin adhesion-related genes on the titanium surface in the treatment group were higher than those in the control group at each culture time point (P<0.05). Western blot showed that the expression levels of Laminin α3, Integrin ß4, and Plectin adhesion-related proteins on the titanium surface were higher in the treatment group than in the control group at 4 and 12 h. CONCLUSIONS: After NTAP treatment, the results showed that 20 s of treatment time could maximize the number of adhered cells on the titanium surface; change the cell adhesion morphology; and significantly upregulate the expression of adhesion-related genes and proteins of Laminin α3, Integrin ß4, and Plectin. Furthermore, it could promote the biological sealing effect of HGECs on the titanium surface.

Efficacy of Er:YAG laser irradiation for decontamination and its effect on biocompatibility of different titanium surfaces.

BACKGROUND: Erbium yttrium-aluminum-garnet (Er:YAG) laser have been shown to be suitable for decontamination of titanium surfaces at a wide range of energy settings, however, high intensity of laser irradiation destroy titanium surface and low intensity cannot remove enough microbial biofilm. The aim of this study was to investigate the optimal energy setting of Er:YAG laser for decontamination of sandblasted/acid-etched (SLA) and hydroxyapatite (HA) titanium surfaces. MATERIAL AND METHODS: After supragingival biofilm construction in vivo, SLA and HA titanium discs were divided into three groups: blank control (BC, clean discs), experimental control (EC, contaminated discs) and experimental groups (EP, contaminated discs irradiated by Er:YAG laser at 40, 70, and 100 mJ/pulse). Scanning electron microscopy (SEM), live/dead bacterial fluorescent detection, and colony counting assay were used to detect the efficacy of laser decontamination. To investigate the effect of laser decontamination on titanium surface biocompatibility, MC3T3-E1 cell adhesion and proliferation activity were examined by SEM and CCK-8 assay. RESULTS: Er:YAG laser irradiation at 100 mJ/pulse removed 84.1% of bacteria from SLA titanium surface; laser irradiation at 70 and 100 mJ/pulse removed 76.4% and 77.85% of bacteria from HA titanium surface respectively. Laser irradiation improved MC3T3-E1 cell adhesion on both titanium surfaces. For SLA titanium discs, 100 mJ/pulse group displayed excellent cellular proliferation activity higher than that in BC group (P < 0.01). For HA titanium discs, 70 mJ/pulse group showed the highest activity comparable to BC group (P > 0.05). CONCLUSIONS: With regards to efficient microbial biofilm decontamination and biocompatibility maintenance, Er:YAG laser at 100 mJ/pulse and 70 mJ/pulse are considered as the optimal energy settings for SLA titanium and HA titanium surface respectively. This study provides theoretical basis for the clinical application of Er:YAG laser in the treatment of peri-implantitis.