Formation of potential titanium antigens based on protein binding to titanium dioxide nanoparticles

Degradation products of titanium implants include free ions, organo-metallic complexes, and particles, ranging from nano to macro sizes. The biological effects, especially of nanoparticles, is yet unknown. The main objective of this study was to develop Ti-protein antigens in physiological solutions that can be used in testing of cellular responses. For this purpose, 0.1% TiO2 nanoparticles less than 100 nm were mixed with human serum albumin (HSA), 0.1% and 1%, in cell culture medium (DMEM, pH 7.2). The Ti concentrations in the resulting solutions were analyzed by inductively coupled plasma mass spectrometry. The stability of the nanoparticles in suspension was analyzed by UV-vis spectrophotometer and Dynamic Light Scattering. The concentration of Ti in suspension was dependent on the presence and concentration of HSA. Albumin prevented high aggregation rate of TiO2 nanoparticles in cell culture medium. It is shown that nano TiO2-protein stable aggregates can be produced under physiological conditions at high concentrations, and are candidates for use in cellular tests.     

Identification and characterization of a novel heparin‐binding peptide for promoting osteoblast adhesion and proliferation by screening an Escherichia coli cell surface display peptide library

Heparin/heparan sulfate (HS) plays a key role in cellular adhesion. In this study, we utilized a 12‐mer random Escherichia coli cell surface display library to identify the sequence, which binds to heparin. Isolated insert analysis revealed a novel heparin‐binding peptide sequence, VRRSKHGARKDR, designated as HBP12. Our analysis of the sequence alignment of heparin‐binding motifs known as the Cardin–Weintraub consensus (BBXB, where B is a basic residue) indicates that the HBP12 peptide sequence contains two consecutive heparin‐binding motifs (i.e. RRSK and RKDR). SPR‐based BIAcore technology demonstrated that the HBP12 peptide binds to heparin with high affinity (K_D = 191 nM ). The HBP12 peptide is found to bind the cell surface HS expressed by osteoblastic MC3T3 cells and promote HS‐dependent cell adhesion. Moreover, the surface‐immobilized HBP12 peptide on titanium substrates shows significant increases in the osteoblastic MC3T3‐E1 cell adhesion and proliferation. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

A comparison of two methods for determining titanium dioxide marker content in broiler digestibility studies

The use of inert markers in broiler diets eliminates the need to quantitatively evaluate feed intake and excreta output to determine diet digestibility, and enables nutrient uptake at specific points along the gastrointestinal tract to be examined. Titanium dioxide (TiO₂) is commonly used for this purpose and measured using a UV-spectrophotometric assay. Two experiments were conducted to observe whether an inductively coupled plasma optical emission spectrophotometer (ICP-OES) assay is able to replace the UV-spectroscopy assay for rapid analysis of TiO₂ in broiler feed and ileal digesta samples. In the first experiment, TiO₂ was added at 5 g/kg to 19 broiler diets. Ross 308 male broilers (n=452) fed these diets were involved in a series of digestion studies to determine ileal digesta recovery of TiO₂. In the second experiment, defined amounts of TiO₂ were added to ileal digesta samples from Ross 308 male broilers (n=176) and TiO₂ recoveries were determined. The feed and ileal samples from both experiments were analysed by both UV-spectroscopy and ICP-OES, and relatedness of the findings from the two assays was determined. Overall relatedness of the two assays was strong for determination of TiO₂ concentration in both the broiler diets and ileal digesta samples (r=0.908 and r=0.884, respectively). Overall recovery of supplemented TiO₂ was 97.62% by the UV-spectroscopy assay and 98.77% by the ICP-OES assay. The ICP-OES assay in this study was as accurate as spectrophotometric determination for the quantification of TiO₂ content. The ICP-OES method can also be used to analyse several elements within one assay, with a single preparation step, and thus the measurement of TiO₂ may be incorporated into the analysis of other minerals. Time and resources dedicated to determining diet digestibility in broilers could be minimised by using the ICP-OES assay to replace the UV-spectroscopy assay when measuring TiO₂ concentration.     

Photostabilized titanium dioxide and a fluorescent brightener as adjuvants for a nucleopolyhedrovirus

Titanium dioxide (TiO₂)reflects ultraviolet light, and so could beexpected to protect the occlusion bodies (OBs)of nucleopolyhedroviruses (NPVs) fromdegradation by sunlight. However, in thepresence of sunlight and water, TiO₂catalyzes the formation of hydrogen peroxide,which can degrade OBs. We tested microfineTiO₂ that had been photostabilized(particles were coated to prevent catalyticactivity), as a UV protectant for the OBs ofthe NPV of Helicoverpa zea (Boddie). Inthe absence of UV, activity of the OBs wasreduced by nonphotostabilized TiO₂ but wasunaffected by photostabilized TiO₂ or byzinc oxide (ZnO). None of these materialsinfluenced larval feeding rates. Undersimulated sunlight, photostabilizedTiO₂ protected the OBs to a greater degreethan did ZnO. Photostabilized TiO₂ wascompatible with a viral enhancer, thefluorescent brightener Blankophor HRS. Undersimulated sunlight, both materials increasedactivity of the OBs, relative to OBs withneither material, in a largely additive manner. In bioassays of foliage collected from fieldplots of lima bean plants sprayed with OBs withor without one or both of these materials,TiO₂ increased persistence of the OBs, butBlankophor HRS had no significant effect.     

Monitoring of biofilms grown on differentially structured metallic surfaces using confocal laser scanning microscopy

Imaging of biofilms on opaque surfaces is a challenge presented to researchers especially considering pathogenic bacteria, as those typically grow on living tissue, such as mucosa and bone. However, they can also grow on surfaces used in industrial applications such as food production, acting as a hindrance to the process. Thus, it is important to understand bacteria better in the environment they actually have relevance in. Stainless steel and titanium substrata were line structured and dotted surface topographies for titanium substrata were prepared to analyze their effects on biofilm formation of a constitutively green fluorescent protein (GFP)‐expressing Escherichia coli strain. The strain was batch cultivated in a custom built flow cell initially for 18 h, followed by continuous cultivation for 6 h. Confocal laser scanning microscopy (CLSM) was used to determine the biofilm topography. Biofilm growth of E. coli GFPmut2 was not affected by the type of metal substrate used; rather, attachment and growth were influenced by variable shapes of the microstructured titanium surfaces. In this work, biofilm cultivation in flow cells was coupled with the most widely used biofilm analytical technique (CLSM) to study the time course of growth of a GFP‐expressing biofilm on metallic surfaces without intermittent sampling or disturbing the natural development of the biofilm.     

In-Depth Characterization of the Clostridioides difficile Phosphoproteome to Identify Ser/Thr Kinase Substrates

Clostridioides difficile is the leading cause of postantibiotic diarrhea in adults. During infection, the bacterium must rapidly adapt to the host environment by using survival strategies. Protein phosphorylation is a reversible post-translational modification employed ubiquitously for signal transduction and cellular regulation. Hanks-type serine/threonine kinases (STKs) and serine/threonine phosphatases have emerged as important players in bacterial cell signaling and pathogenicity. C. difficile encodes two STKs (PrkC and CD2148) and one phosphatase. We optimized a titanium dioxide phosphopeptide enrichment approach to determine the phosphoproteome of C. difficile. We identified and quantified 2500 proteins representing 63% of the theoretical proteome. To identify STK and serine/threonine phosphatase targets, we then performed comparative large-scale phosphoproteomics of the WT strain and isogenic ΔprkC, CD2148, Δstp, and prkC CD2148 mutants. We detected 635 proteins containing phosphorylated peptides. We showed that PrkC is phosphorylated on multiple sites in vivo and autophosphorylates in vitro. We were unable to detect a phosphorylation for CD2148 in vivo, whereas this kinase was phosphorylated in vitro only in the presence of PrkC. Forty-one phosphoproteins were identified as phosphorylated under the control of CD2148, whereas 114 proteins were phosphorylated under the control of PrkC including 27 phosphoproteins more phosphorylated in the ?stp mutant. We also observed enrichment for phosphothreonine among the phosphopeptides more phosphorylated in the Δstp mutant. Both kinases targeted pathways required for metabolism, translation, and stress response, whereas cell division and peptidoglycan metabolism were more specifically controlled by PrkC-dependent phosphorylation in agreement with the phenotypes of the ΔprkC mutant. Using a combination of approaches, we confirmed that FtsK was phosphorylated in vivo under the control of PrkC and that Spo0A was a substrate of PrkC in vitro. This study provides a detailed mapping of kinase–substrate relationships in C. difficile, paving the way for the identification of new biomarkers and therapeutic targets.     

Characterization of a micro-roughened TiO2/ZrO2 coating： mechanical properties and HBMSC responses in vitro

Previous studies have shown phase to bioceramics can that using ZrO2 as a second significantly increase the bonding strength of plasma-sprayed composite material. In the present study, micro-roughened titanium dioxide/ zirconia （TiO2/ZrO2） （30 wt% ZrO2） coating and TiO2 coating were plasma-sprayed onto Ti plates. The microstructural characteristics and mechanical properties of both coatings were investigated. Furthermore, the biological behavior and osteogenic differentiation of human bone marrow mesenchymal stem cells （HBMSCs） on both TiO2/ZrO2 and TiO2 coatings were compared. The results indicated that the shear bond strength and microhardness of TiO2/ZrO2 coating were statistically higher than those of TiO2 coating. Scanning electron microscope observation revealed that more irregularly shaped protuberances and denser pores were formed on the surface of TiO2/ ZrO2 coating compared with those of TiO2 coating. Further comparative analysis of HBMSC proliferation and osteogenic differentiation on both coatings showed that significantly higher cellular alkaline phosphatase activity and expression levels of Runx2 and Osterix at day 10 after osteogenic culture were found on TiO2/ZrO2 coating compared with TiO2 coating, while no statistically significant difference in cell proliferation and extracellular calcium deposition was observed. The present study suggests that TiO2/ZrO2 coating may be favorable for dental implant applications.     

Immobilization of β-glucosidase on mercaptopropyl-functionalized mesoporous titanium dioxide

Mesoporous titanium dioxide (M-TiO₂) and mercaptopropyl-functionalized M-TiO₂ (SH-M-TiO₂) were prepared as carriers for immobilization of β-glucosidase (BG). Analyses suggested that in SH-M-TiO₂ the grafted mercaptopropyl groups attributed to about 3.5% of the total weight of the matrices. For the immobilization of BG, SH-M-TiO₂ or M-TiO₂ powder was added with free BG solution and stirred at 4 °C for 8 h, followed by centrifugation and washing. BG was effectively immobilized on SH-M-TiO₂ with an enzyme activity of 10.9 U/g, which was 92.8% of the total enzyme activity in the starting solution of free BG. BG immobilized on SH-M-TiO₂ (BG-SH-M-TiO₂) was more stable in pH, thermal, and storage tests than BG on M-TiO₂ or free BG in solution. The K_m and v_max of BG-SH-M-TiO₂ were similar to values for free BG in solution. Batch hydrolysis of cellobiose-containing substrate by BG-SH-M-TiO₂ was conducted. Conversion rates of cellobiose in 10 batches were consistently around 90%. Although the residual enzyme activity of BG-SH-M-TiO₂ decreased gradually, only a small amount of BG activity was detected in supernatants. This indicated effective adsorption of enzyme molecules to SH-M-TiO₂ matrices.     

可吸收棒与微型钛板在桡骨小头骨折治疗中的应用价值

目的:探讨可吸收棒与微型钛板在桡骨小头骨折治疗中的临床效果和应用价值的研究。方法:回顾性选取2014年1月-2017年1月于医院骨科诊治的桡骨小头骨折患者58例,根据不同的治疗方式分为两组,其中对照组28例患者采用微型钛板治疗;研究组30例患者采取可吸收棒治疗,患者术后均以石膏固定4周后,采用X射线复查恢复情况,统计两组患者的骨性愈合时间、肘关节HHS评分及并发症发生情况。结果:(1)两组患者在2年内骨折均愈合,研究组患者的骨性愈合时间(13.86±2.05)周明显少于对照组患者的骨性愈合时间(15.54±2.71)周(P0.05);(2)治疗后4周,研究组患者的肘关节HHS评分为(95.32±4.14)分明显高于对照组患者的(82.48±5.27)分(P均0.05);(3)研究组患者中仅有1例发生骨延迟愈合,对照组患者中2例肘关节活动范围缺失、2例骨折位移、1例肘关节骨关节炎,并发症发生率分别为3.33%和17.85%存在显著差异(P均0.05)。结论:桡骨小头骨折患者肘关节损伤严重,采用可吸收棒治疗能取得与微型钛板相似的治疗效果,并且患者的骨性愈合时间短、肘关节功能恢复较快,同时避免并发症的发生。