UV-Curing Assisted Direct Ink Writing of Dense, Crack-Free, and High-Performance Zirconia-Based Composites With Aligned Alumina Platelets.

Additive manufacturing (AM) of high-performance structural ceramic components with comparative strength and toughness as conventionally manufactured ceramics remains challenging. Here, a UV-curing approach is integrated in direct ink writing (DIW), taking advantage from DIW to enable an easy use of high solid-loading pastes and multi-layered materials with compositional changes; while, avoiding drying problems. UV-curable opaque zirconia-based slurries with a solid loading of 51 vol% are developed to fabricate dense and crack-free alumina-toughened zirconia (ATZ) containing 3 wt% alumina platelets. Importantly, a non-reactive diluent is added to relieve polymerization-induced internal stresses, avoid subsequent warping and cracking, and facilitate the de-binding. For the first time, UV-curing assisted DIW-printed ceramic after sintering reveals even better mechanical properties than that processed by a conventional pressing. This is attributed to the aligned alumina platelets, enhancing crack deflection and improving the fracture toughness from 6.8 ± 0.3 MPa m0.5 (compacted) to 7.4 ± 0.3 MPa m0.5 (DIW). The four-point bending strength of the DIW ATZ (1009 ± 93 MPa) is also higher than that of the conventionally manufactured equivalent (861 ± 68 MPa). Besides homogeneous ceramic, laminate structures are demonstrated. This work provides a valuable hybrid approach to additively manufacture tough and strong ceramic components.

Does speed-sintering affect the optical and mechanical properties of yttria-stabilized zirconia? A systematic review and meta-analysis of in-vitro studies.

Zirconia restorations are increasingly popular in dental treatment. Yttria-stabilized zirconia (YSZ) needs to be sintered for clinical applications and novel speed-sintering protocols are being developed for chairside treatments. Whether the properties of speed-sintered YSZ meet clinical requirements, however, remains unclear. Therefore, we conducted a systematic review and meta-analysis on the influence of speed-sintering on the optical and mechanical properties of dental YSZ according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) 2020 guidelines. A literature search was conducted using PubMed, Embase, and Web of Science databases for relevant articles published between January 1, 2010 and February 28, 2022 in English, Chinese, or Japanese. After full-text evaluation and quality assessment, 26 articles were selected. Meta-analysis revealed that speed-sintering does not significantly affect the CIEDE2000-based translucency parameter, contrast ratio, three-point flexural strength, biaxial flexural strength, or fracture toughness of YSZ (p < 0.01) compared to conventional sintering. However, the CIELab-based translucency parameter of conventionally sintered YSZ is higher than that of speed-sintered YSZ. The descriptive analysis indicated that speed-sintering does not affect the hardness of YSZ compared to that of conventionally sintered YSZ. The results indicate that speed-sintering is suitable for preparing YSZ for dental restorations.

Layer characteristics in strength-gradient multilayered yttria-stabilized zirconia.

OBJECTIVES: To investigate crystallography, translucency, phase content, microstructure and flexural strength of two commercial strength-gradient multilayered dental zirconia grades. METHODS: Two zirconia grades, i.e., KATANA Zirconia YML (Kuraray Noritake; referred to as "YML"; composed of four layers: enamel, body 1-3) and IPS e.max ZirCAD Prime (Ivoclar Vivadent; referred to as "Prime"; composed of three layers: enamel, transition, body) were investigated. Fully sintered square-shaped zirconia specimens from each layer were prepared. Microstructure, chemical composition, translucency parameter and zirconia-phase composition of each layer were characterized. Four-point and biaxial flexural strength of each layer was measured using fully sintered bar- and square-shaped specimens. Square-shaped samples were used to measure strength across the layers. RESULTS: For both multilayer zirconia grades, the 'enamel' layer contains a higher amount of c-ZrO2, which resulted in higher translucency but lower flexural strength than the 'body' layers. The characteristic 4-point flexural strength of the YML 'body 2' (923 MPa) and 'body 3' (911 MPa) layers, and of the Prime 'body' (989 MPa) layer were comparable and higher than for the YML 'enamel' (634 MPa), Prime 'transition' (693 MPa) and 'enamel' (535 MPa) layers. The biaxial strength of specimens sectioned across the layers was in-between that of the 'enamel' and 'body' layers for both YML and Prime, implying the interfaces did not form a weak link. SIGNIFICANCE: The difference in yttria content affects the phase composition and mechanical properties of each layer of the multi-layer zirconia. The strength-gradient approach allowed to integrate monoliths with irreconcilable properties.

3D printing and milling accuracy influence full-contour zirconia crown adaptation.

OBJECTIVES: To correlate trueness and cement-space characteristics of crowns milled chairside and in the laboratory with those of inkjet 3D-printed crowns, and to assess whether 3D-printing accuracy meets the clinical standard. METHODS: Thirty crowns were either (1) milled using a chairside Cerec MCXL unit from Cerec Zirconia Mono L (Dentsply Sirona), (2) milled using a LX-O 5-axis (Matsuura Machinery) industrial machine from Initial Zirconia HT (GC), or (3) 3D-printed using an inkjet Carmel 1400 (Xjet) printer (n = 10). Crown trueness determined by comparing the original CAD with each visible-light digitized crown was correlated with the 3D cement-space characteristics recorded by micro-CT. Statistics involved Kruskal-Wallis testing and Spearman correlation. RESULTS: Crown trueness at the intaglio marginal area positively correlated with the marginal and axial cement-space characteristics. 3D-printing revealed data in-between those of the two milling systems with undercut values being not statistically different from those recorded for chairside milling and a low overcut level that was statistically similar to that obtained by laboratory milling. Laboratory milling revealed a significantly better marginal accuracy with a consequently lower cement-space thickness. A higher overcut level was recorded for the chairside-milled crowns in the marginal/occlusal thirds, resulting in the significantly highest occlusal cement-space thickness and cement-volume percentage with a cement thickness above 120 µm (limit considered as clinically acceptable). No statistical difference in trueness was found for the external crown dimensions. SIGNIFICANCE: The 3D-printed zirconia crowns provided sufficient manufacturing accuracy for clinical use. Accurate milling and printing of the crown's intaglio marginal area is primordial.

Fracture analysis of one/two-piece clinically failed zirconia dental implants.

OBJECTIVES: Analyzing factors that may have led to fracture of zirconia implants by macro/micro-fractography. METHODS: Six one-piece and ten two-piece full-ceramic zirconia implants from two manufacturers, Z-Systems and CeraRoot, were retrieved after clinical failure. The time-to-failure ranged from 3 to 49 months. Optical and scanning electron microscopy (SEM) were used to analyze the fracture planes at the macro- and microscopic level. Treatment planning, surgical protocol, fracture-origin location and characteristic fracture features were assessed. RESULTS: The fracture of all implants seemed to have been primarily due to overload in bending mode, while the fracture-initiation sites varied for the one- and two-piece implants. The fracture of all one-piece implants originated in the constriction region between two threads in the endosseous implant part. For two-piece implants, the abutment neck, internal abutment-implant connections and inner threads were found to be the main fracture-initiation sites. Surface defects at the root area for one-piece implants and damages at the abutment surface for two-piece implants were connected to the fracture origins. Importantly, the clinical failures of implants were often found to result from combined effects related to patient aspects, treatment planning/protocols, a high bending moment at the weakest link, implant-surface conditions and specific implant designs. SIGNIFICANCE: This study provided information to be considered for future optimization of treatment planning and the surgical protocol for zirconia implants. Optimization of the surface conditions and the zirconia-starting powder were also suggested.

3D printed zirconia dental implants with integrated directional surface pores combine mechanical strength with favorable osteoblast response.

Dental implants need to combine mechanical strength with promoted osseointegration. Currently used subtractive manufacturing techniques require a multi-step process to obtain a rough surface topography that stimulates osseointegration. Advantageously, additive manufacturing (AM) enables direct implant shaping with unique geometries and surface topographies. In this study, zirconia implants with integrated lamellar surface topography were additively manufactured by nano-particle ink-jetting. The ISO-14801 fracture load of as-sintered implants (516±39 N) resisted fatigue in 5-55 °C water thermo-cycling (631±134 N). Remarkably, simultaneous mechanical fatigue and hydrothermal aging at 90 °C significantly increased the implant strength to 909±280 N due to compressive stress generated at the seamless transition of the 30-40 µm thick, rough and porous surface layer to the dense implant core. This unique surface structure induced an elongated osteoblast morphology with uniform cell orientation and allowed for osteoblast proliferation, long-term attachment and matrix mineralization. In conclusion, the developed AM zirconia implants not only provided high long-term mechanical resistance thanks to the dense core along with compressive stress induced at the transition zone, but also generated a favorable osteoblast response owing to the integrated directional surface pores. STATEMENT OF SIGNIFICANCE: Zirconia ceramics are becoming the material of choice for metal-free dental implants, however significant efforts are required to obtain a rough/porous surface for enhanced osseointegration, along with the risk of surface delamination and/or microstructure variation. In this study, we addressed the challenge by additively manufacturing implants that seamlessly combine dense core with a porous surface layer. For the first time, a unique surface with a directional lamellar pore morphology was additively obtained. This AM implant also provided strength as strong as conventionally manufactured zirconia implants before and after long-term fatigue. Favorable osteoblast response was proved by in-vitro cell investigation. This work demonstrated the opportunity to AM fabricate novel ceramic implants that can simultaneously meet the mechanical and biological functionality requirements.

Mechanical properties-translucency-microstructure relationships in commercial monolayer and multilayer monolithic zirconia ceramics.

OBJECTIVES: To evaluate the phase composition, microstructure, optical properties and mechanical properties of eight commercially available multilayer and monolayer monolithic dental zirconias. METHODS: Five commercial 3Y-TZP (GC ST, GC HT [GC, Tokyo Japan]; Katana ML, Katana HT [Kuraray Noritake] and Lava Plus [3M Oral Care]) and three Y-PSZ (Katana STML, Katana UTML [Kuraray Noritake]; GC UHT [GC, Tokyo Japan]) zirconia ceramic grades were cut in plate-shaped specimens, sintered according to the manufacturer's instructions and mirror polished. The zirconia chemical composition was determined using X-ray fluorescence (XRF), phase composition was characterized using X-ray diffraction (XRD), while the grain size was measured using scanning electron microscopy (SEM). The translucency Parameter (TP) and Contrast Ratio (CR) were measured with a spectrophotometer (n = 10/group). The indentation fracture toughness (n = 10), Vickers hardness (n = 10) and biaxial strength (n = 20) of the sintered ceramics were assessed. The stress distribution during biaxial testing was assessed by Finite element analysis (FEA). Statistical analysis involved one-way ANOVA and post-hoc Tukey's HSD test and Pearson correlation test (α = 0.05). RESULTS: FEA showed that the stress distribution in plate shape specimens was the same as for disks, rationalizing the use of plates for biaxial strength testing. As expected, higher quantities of Y2O3 were related to a higher cubic ZrO2 phase content and lower tetragonality t-ZrO2, which improved translucency but diminished flexural strength and toughness. While there was no significant correlation between grain size and other material properties, addition of pigments to the zirconia grade statistically negatively affected hardness. CONCLUSION: Even though an improvement in strength and translucency could be recorded for the last Y-TZP generation, future research still needs to strive for combined improvement of optical properties and mechanical reliability of zirconia ceramics.

Additively Manufactured Zirconia for Dental Applications.

We aimed to assess the crystallography, microstructure and flexural strength of zirconia-based ceramics made by stereolithography (SLA). Two additively manufactured 3 mol% yttria-stabilized tetragonal zirconia polycrystals (3Y-TZP: LithaCon 3Y 230, Lithoz; 3D Mix zirconia, 3DCeram Sinto) and one alumina-toughened zirconia (ATZ: 3D Mix ATZ, 3DCeram Sinto) were compared to subtractively manufactured 3Y-TZP (control: LAVA Plus, 3M Oral Care). Crystallographic analysis was conducted by X-ray diffraction. Top surfaces and cross-sections of the subsurface microstructure were characterized using scanning electron microscopy (SEM). Biaxial flexural strength was statistically compared using Weibull analysis. The additively and subtractively manufactured zirconia grades revealed a similar phase composition. The residual porosity of the SLA 3Y-TZPs and ATZ was comparable to that of subtractively manufactured 3Y-TZP. Weibull analysis revealed that the additively manufactured LithaCon 3Y 230 (Lithoz) had a significantly lower biaxial flexural strength than 3D Mix ATZ (3D Ceram Sinto). The biaxial flexural strength of the subtractively manufactured LAVA Plus (3M Oral Care) was in between those of the additively manufactured 3Y-TZPs, with the additively manufactured ATZ significantly outperforming the subtractively manufactured 3Y-TZP. Additively manufactured 3Y-TZP showed comparable crystallography, microstructure and flexural strength as the subtractively manufactured zirconia, thus potentially being a good option for dental implants.

Mechanical properties, aging stability and translucency of speed-sintered zirconia for chairside restorations.

OBJECTIVE: To evaluate the performance of zirconia ceramics sintered in a speed sintering induction furnace by comprehensive understanding of their optical and mechanical properties, microstructure, phase composition and aging stability, in comparison to ceramics sintered in a conventional furnace. METHODS: Speed sintered (SS) Katana STMLSS (Kuraray Noritake) (total thermal cycle/sintering time/dwell temperature: 30min/16min/1560°C) and CEREC Zirconia (CEREC ZrSS) (Dentsply Sirona) (15min/2min/1578°C) were compared to conventionally sintered (CS) Katana STMLCS (6.8h/2h/1550°C) and inCoris TZICS (4h/2h/1510°C). The translucency parameter (TP) and contrast ratio (CR) were measured with a spectrophotometer. The chemical composition of the materials was determined by XRF and phase composition was characterized using XRD. Hydrothermal aging behavior was evaluated by measuring the tetragonal-to-monoclinic ZrO2 phase transformation after accelerated hydrothermal aging in steam at 134°C. The indentation fracture toughness, Vickers hardness and biaxial strength of the sintered ceramics were assessed. RESULTS: Speed and conventionally sintered zirconia revealed similar density, microstructure, average strength and hydrothermal aging stability. Both Katana STMLSS/CS 5Y-PSZ ceramics were characterized with a higher content of cubic phase (≈53wt%), which resulted in a higher amount of Y2O3 in the remaining tetragonal ZrO2 phases compared to the 3Y-TZP CEREC ZrSS and inCoris TZICS (8 and 20wt%, respectively). The sintering program did not affect the hydrothermal aging behavior of Katana STMLSS and CEREC ZrSS. TP of Katana STMLSS (TP≈32) was not affected by speed sintering, while the translucency of CEREC ZrSS (TP=14) was significantly reduced. Hardness, fracture toughness and Weibull characteristic strength of Katana STMLSS and CEREC ZrSS also reached the optimal level, but speed sintering substantially lowered their mechanical reliability. SIGNIFICANCE: Speed sintering of 3Y-TZP and 5Y-PSZ in a speed sintering induction oven appeared suitable for clinical applications. However, further studies should focus on improving of translucency and mechanical reliability of the speed-sintered zirconia ceramics.

High-translucent yttria-stabilized zirconia ceramics are wear-resistant and antagonist-friendly.

OBJECTIVES: To evaluate two-body wear of three zirconia ceramics stabilized with 3, 4 and 5mol% yttria and to compare their wear behavior with that of a lithium-disilicate glass-ceramic. METHODS: Sixteen rectangular-shaped specimens made from three grades of zirconia ceramics and a lithium-disilicate glass-ceramic were polished and dynamically loaded in a chewing simulator (2kg vertical load, 2.1Hz) under water at 90°C for 1.2×106 cycles (about 7 days) in the ball-on-plate mode against steatite antagonists. Surface roughness was measured before and after wear testing. Wear tracks were scanned with a non-contact 3D profilometer and super-impositions were used to determine wear loss of the antagonists. Wear surfaces were imaged by SEM. XRD and micro-Raman spectroscopy were used to characterize phase transformation and stress status in the worn and unworn areas of the zirconia ceramics. RESULTS: Independent of fracture toughness, strength and aging-susceptibility, the three zirconia ceramics showed a similar and limited amount of wear (∼10µm in depth) and were more wear-resistant than the lithium-disilicate glass-ceramic (∼880µm in depth). Abrasive wear without obvious cracks was observed for all investigated zirconias, whereas the glass-ceramic with a lower fatigue threshold and high susceptibility to surface dissolution exhibited significant abrasion, fatigue and corrosion wear. All three zirconia ceramics yielded a lower antagonist wear than the glass-ceramic and no significant differences were found between the zirconia ceramics. SIGNIFICANCE: In the context of this study, high-translucent zirconia ceramics stabilized with a higher yttria content, recently introduced in the dental field, were as wear-resistant and antagonist-friendly as conventional high-strength zirconia and suitable for monolithic restorations.

An antibiofilm coating of 5-aryl-2-aminoimidazole covalently attached to a titanium surface.

Biofilms, especially those formed by Staphylococcus aureus, play a key role in the development of orthopedic implant infections. Eradication of these infections is challenging due to the elevated tolerance of biofilm cells against antimicrobial agents. In this study, we developed an antibiofilm coating consisting of 5-(4-bromophenyl)-N-cyclopentyl-1-octyl-1H-imidazol-2-amine, designated as LC0024, covalently bound to a titanium implant surface (LC0024-Ti). We showed in vitro that the LC0024-Ti surface reduces biofilm formation of S. aureus in a specific manner without reducing the planktonic cells above the biofilm, as evaluated by plate counting and fluorescence microscopy. The advantage of compounds that only inhibit biofilm formation without affecting the viability of the planktonic cells, is that reduced development of bacterial resistance is expected. To determine the antibiofilm activity of LC0024-Ti surfaces in vivo, a biomaterial-associated murine infection model was used. The results indicated a significant reduction in S. aureus biofilm formation (up to 96%) on the LC0024-Ti substrates compared to pristine titanium controls. Additionally, we found that the LC0024-Ti substrates did not affect the attachment and proliferation of human cells involved in osseointegration and bone repair. In summary, our results emphasize the clinical potential of covalent coatings of LC0024 on titanium implant surfaces to reduce the risk of orthopedic implant infections. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1908-1919, 2019.

* Harnessing the Osteogenicity of In Vitro Stem Cell-Derived Mineralized Extracellular Matrix as 3D Biotemplate to Guide Bone Regeneration.

Advanced biomaterials that are capable of guiding robust bone regeneration are highly demanded for translational therapy of bone defects or bone augmentation in clinics. One of the strategic approaches is to produce tissue engineering (TE) constructs that mediate bone regeneration by recapitulating the natural bone formation or healing process. In this study, we aimed at producing devitalized mineralized carriers with augmented bone forming capacity via a modified culture protocol (i.e., culture conditions with high calcium and/or phosphate concentrations) that first promotes cell growth and, subsequently, mineralized extracellular matrix (ECM) deposition by human periosteum-derived osteoprogenitor cells (hPDCs) on additive manufactured three-dimensional (3D) porous titanium (Ti)-based scaffolds. Qualitative and quantitative analysis was performed to characterize the physicochemical properties of the produced devitalized mineralized carriers, as well as their effects as carriers on in vitro cell growth and osteochondrogenic differentiation of hPDCs under a perfusion bioreactor culture set-up. The results showed that the modified culture protocol was useful to produce devitalized mineralized carriers with different amount, distribution, composition, and morphology of mineralized matrix that resembled hydroxyapatite, and exhibited different Ca2+ release kinetics, distinct human bone morphogenetic protein (hBMP)-2, human vascular endothelial growth factor (hVEGF) proteins, and collagen contents. The produced devitalized mineralized carriers supported 3D growth of hPDCs, with minor osteochondrogenic differentiation effects under the perfusion bioreactor culture condition. Subcutaneous implantation of hPDC-seeded devitalized mineralized carriers in athymic nude rats showed nearly five-fold augmentation in the ectopic bone-forming capacity, with no bone induction obtained for unseeded, devitalized mineralized carriers and plain Ti scaffolds. Implantation of devitalized mineralized carriers in critical-sized calvarial defects resulted in encouraging defect bridging as compared with limited defect bridging by plain Ti scaffolds or in empty defects. This defect bridging was not enhanced by implanting hPDC-seeded devitalized mineralized carriers. In conclusion, the investigated modified culture protocol was useful to produce devitalized mineralized carriers with augmented bone-forming capacity, which potentially could aid bone repair or augmentation in clinics.

The antifungal caspofungin increases fluoroquinolone activity against Staphylococcus aureus biofilms by inhibiting N-acetylglucosamine transferase.

Biofilms play a major role in Staphylococcus aureus pathogenicity but respond poorly to antibiotics. Here, we show that the antifungal caspofungin improves the activity of fluoroquinolones (moxifloxacin, delafloxacin) against S. aureus biofilms grown in vitro (96-well plates or catheters) and in vivo (murine model of implanted catheters). The degree of synergy among different clinical isolates is inversely proportional to the expression level of ica operon, the products of which synthesize poly-N-acetyl-glucosamine polymers, a major constituent of biofilm matrix. In vitro, caspofungin inhibits the activity of IcaA, which shares homology with ß-1-3-glucan synthase (caspofungin's pharmacological target in fungi). This inhibition destructures the matrix, reduces the concentration and polymerization of exopolysaccharides in biofilms, and increases fluoroquinolone penetration inside biofilms. Our study identifies a bacterial target for caspofungin and indicates that IcaA inhibitors could potentially be useful in the treatment of biofilm-related infections.

Strength, toughness and aging stability of highly-translucent Y-TZP ceramics for dental restorations.

OBJECTIVE: The aim was to evaluate the optical properties, mechanical properties and aging stability of yttria-stabilized zirconia with different compositions, highlighting the influence of the alumina addition, Y2O3 content and La2O3 doping on the translucency. METHODS: Five different Y-TZP zirconia powders (3 commercially available and 2 experimentally modified) were sintered under the same conditions and characterized by X-ray diffraction with Rietveld analysis and scanning electron microscopy (SEM). Translucency (n=6/group) was measured with a color meter, allowing to calculate the translucency parameter (TP) and the contrast ratio (CR). Mechanical properties were appraised with four-point bending strength (n=10), single edge V-notched beam (SEVNB) fracture toughness (n=8) and Vickers hardness (n=10). The aging stability was evaluated by measuring the tetragonal to monoclinic transformation (n=3) after accelerated hydrothermal aging in steam at 134°C, and the transformation curves were fitted by the Mehl-Avrami-Johnson (MAJ) equation. Data were analyzed by one-way ANOVA, followed by Tukey's HSD test (α=0.05). RESULTS: Lowering the alumina content below 0.25wt.% avoided the formation of alumina particles and therefore increased the translucency of 3Y-TZP ceramics, but the hydrothermal aging stability was reduced. A higher yttria content (5mol%) introduced about 50% cubic zirconia phase and gave rise to the most translucent and aging-resistant Y-TZP ceramics, but the fracture toughness and strength were considerably sacrificed. 0.2mol% La2O3 doping of 3Y-TZP tailored the grain boundary chemistry and significantly improved the aging resistance and translucency. Although the translucency improvement by La2O3 doping was less effective than for introducing a substantial amount of cubic zirconia, this strategy was able to maintain the mechanical properties of typical 3Y-TZP ceramics. SIGNIFICANCE: Three different approaches were compared to improve the translucency of 3Y-TZP ceramics.

Antibacterial activity of a new broad-spectrum antibiotic covalently bound to titanium surfaces.

Biofilm-associated infections, particularly those caused by Staphylococcus aureus, are a major cause of implant failure. Covalent coupling of broad-spectrum antimicrobials to implants is a promising approach to reduce the risk of infections. In this study, we developed titanium substrates on which the recently discovered antibacterial agent SPI031, a N-alkylated 3, 6-dihalogenocarbazol 1-(sec-butylamino)-3-(3,6-dichloro-9H-carbazol-9-yl)propan-2-ol, was covalently linked (SPI031-Ti). We found that SPI031-Ti substrates prevent biofilm formation of S. aureus and Pseudomonas aeruginosa in vitro, as quantified by plate counting and fluorescence microscopy. To test the effectiveness of SPI031-Ti substrates in vivo, we used an adapted in vivo biomaterial-associated infection model in mice in which SPI031-Ti substrates were implanted subcutaneously and subsequently inoculated with S. aureus. Using this model, we found a significant reduction in biofilm formation (up to 98%) on SPI031-Ti substrates compared to control substrates. Finally, we demonstrated that the functionalization of the titanium surfaces with SPI031 did not influence the adhesion and proliferation of human cells important for osseointegration and bone repair. In conclusion, these data demonstrate the clinical potential of SPI031 to be used as an antibacterial coating for implants, thereby reducing the incidence of implant-associated infections. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:2191-2198, 2016.

Covalent immobilization of antimicrobial agents on titanium prevents Staphylococcus aureus and Candida albicans colonization and biofilm formation.

OBJECTIVES: Biofilm-associated implant infections represent a serious public health problem. Covalent immobilization of antimicrobial agents on titanium (Ti), thereby inhibiting biofilm formation of microbial pathogens, is a solution to this problem. METHODS: Vancomycin (VAN) and caspofungin (CAS) were covalently bound on Ti substrates using an improved processing technique adapted to large-scale coating of implants. Resistance of the VAN-coated Ti (VAN-Ti) and CAS-coated Ti (CAS-Ti) substrates against in vitro biofilm formation of the bacterium Staphylococcus aureus and the fungal pathogen Candida albicans was determined by plate counting and visualized by confocal laser scanning microscopy. The efficacy of the coated Ti substrates was also tested in vivo using an adapted biomaterial-associated murine infection model in which control-Ti, VAN-Ti or CAS-Ti substrates were implanted subcutaneously and subsequently challenged with the respective pathogens. The osseointegration potential of VAN-Ti and CAS-Ti was examined in vitro using human bone marrow-derived stromal cells, and for VAN-Ti also in a rat osseointegration model. RESULTS: In vitro biofilm formation of S. aureus and C. albicans on VAN-Ti and CAS-Ti substrates, respectively, was significantly reduced compared with biofilm formation on control-Ti. In vivo, we observed over 99.9% reduction in biofilm formation of S. aureus on VAN-Ti substrates and 89% reduction in biofilm formation of C. albicans on CAS-Ti substrates, compared with control-Ti substrates. The coated substrates supported osseointegration in vitro and in vivo. CONCLUSIONS: These data demonstrate the clinical potential of covalently bound VAN and CAS on Ti to reduce microbial biofilm formation without jeopardizing osseointegration.

Fungal ß-1,3-glucan increases ofloxacin tolerance of Escherichia coli in a polymicrobial E. coli/Candida albicans biofilm.

In the past, biofilm-related research has focused mainly on axenic biofilms. However, in nature, biofilms are often composed of multiple species, and the resulting polymicrobial interactions influence industrially and clinically relevant outcomes such as performance and drug resistance. In this study, we show that Escherichia coli does not affect Candida albicans tolerance to amphotericin or caspofungin in an E. coli/C. albicans biofilm. In contrast, ofloxacin tolerance of E. coli is significantly increased in a polymicrobial E. coli/C. albicans biofilm compared to its tolerance in an axenic E. coli biofilm. The increased ofloxacin tolerance of E. coli is mainly biofilm specific, as ofloxacin tolerance of E. coli is less pronounced in polymicrobial E. coli/C. albicans planktonic cultures. Moreover, we found that ofloxacin tolerance of E. coli decreased significantly when E. coli/C. albicans biofilms were treated with matrix-degrading enzymes such as the ß-1,3-glucan-degrading enzyme lyticase. In line with a role for ß-1,3-glucan in mediating ofloxacin tolerance of E. coli in a biofilm, we found that ofloxacin tolerance of E. coli increased even more in E. coli/C. albicans biofilms consisting of a high-ß-1,3-glucan-producing C. albicans mutant. In addition, exogenous addition of laminarin, a polysaccharide composed mainly of poly-ß-1,3-glucan, to an E. coli biofilm also resulted in increased ofloxacin tolerance. All these data indicate that ß-1,3-glucan from C. albicans increases ofloxacin tolerance of E. coli in an E. coli/C. albicans biofilm.

Highly-translucent, strong and aging-resistant 3Y-TZP ceramics for dental restoration by grain boundary segregation.

Latest trends in dental restorative ceramics involve the development of full-contour 3Y-TZP ceramics which can avoid chipping of veneering porcelains. Among the challenges are the low translucency and the hydrothermal stability of 3Y-TZP ceramics. In this work, different trivalent oxides (Al2O3, Sc2O3, Nd2O3 and La2O3) were selected to dope 3Y-TZP ceramics. Results show that dopant segregation was a key factor to design hydrothermally stable and high-translucent 3Y-TZP ceramics and the cation dopant radius could be used as a controlling parameter. A large trivalent dopant, oversized as compared to Zr(4+), exhibiting strong segregation at the ZrO2 grain boundary was preferred. The introduction of 0.2 mol% La2O3 in conventional 0.1-0.25 wt.% Al2O3-doped 3Y-TZP resulted in an excellent combination of high translucency and superior hydrothermal stability, while retaining excellent mechanical properties.

Novel anti-infective implant substrates: controlled release of antibiofilm compounds from mesoporous silica-containing macroporous titanium.

Bone implants with open porosity enable fast osseointegration, but also present an increased risk of biofilm-associated infections. We design a novel implant material consisting of a mesoporous SiO2 diffusion barrier (pore diameter: 6.4 nm) with controlled drug release functionality integrated in a macroporous Ti load-bearing structure (fully interconnected open porosity: 30%; pore window size: 0.5-2.0 µm). Using an in vitro tool consisting of Ti/SiO2 disks in an insert set-up, through which molecules can diffuse from feed side to release side, a continuous release without initial burst effect of the antibiofilm compound toremifene is sustained for at least 9 days, while release concentrations (up to 17 µM daily) increase with feed concentrations (up to 4mM). Toremifene diffusivity through the SiO2 phase into H2O is estimated around 10(-13)m(2)/s, suggesting configurational diffusion through mesopores. Candida albicans biofilm growth on the toremifene-release side is significantly inhibited, establishing a proof-of-concept for the drug delivery functionality of mesoporous SiO2 incorporated into a high-strength macroporous Ti carrier. Next-generation implants made of this composite material and equipped with an internal reservoir (feed side) can yield long-term controlled release of antibiofilm compounds, effectively treating infections on the implant surface (release side) over a prolonged time.

In vivo Candida glabrata biofilm development on foreign bodies in a rat subcutaneous model.

OBJECTIVES: Biofilm studies have been mostly dedicated to the major human fungal pathogen Candida albicans, whereas much less is known about this virulence factor in Candida glabrata, certainly under in vivo conditions. This study provides a deeper understanding of the biofilm development of C. glabrata, its architecture and susceptibility profile to fluconazole and echinocandins. METHODS: In vitro and in vivo C. glabrata biofilms were developed inside serum-coated triple-lumen catheters placed in 24-well polystyrene plates or implanted subcutaneously in the back of a rat, respectively. Scanning electron microscopy and confocal scanning laser microscopy were used to visualize the biofilm architecture. Quantitative real-time PCR was used to demonstrate the expression profile of EPA1, EPA3, EPA6 and AWP1-AWP7 during in vivo biofilm formation. RESULTS: Mature biofilms were observed within the first 48 h and the amount of biofilm reached its maximum by 6 days. Architecturally, mature C. glabrata biofilms consisted of a thick network of yeast cells embedded in an extracellular matrix. Moreover, in vivo biofilms were susceptible to echinocandin drugs, whereas fluconazole remained ineffective. Gene expression profiling revealed that EPA3, EPA6, AWP2, AWP3 and AWP5 were up-regulated in in vivo biofilms compared with in vitro biofilms. CONCLUSIONS: C. glabrata is a unique microorganism, which, despite the lack of transition to the hyphal form, formed thick biofilms inside foreign bodies in vivo. To our knowledge, this is the first study that has described in vivo C. glabrata biofilm development and its architectural changes in detail and provides an insight into the susceptibility profile, as well as the gene expression machinery, of biofilm-associated infections.