Evaluation of alginate-coated ß-tricalcium phosphate fiber scaffold for cell culture.

Ex vivo tissue engineering is an effective therapeutic approach for the treatment of severe cartilage diseases that require tissue replenishment or replacement. This strategy demands scaffolds that are durable enough for long-term cell culture to form artificial tissue. Additionally, such scaffolds must be biocompatible to prevent the transplanted matrix from taking a toll on the patient's body. From the viewpoint of structure and bio-absorbability, a ß-tricalcium phosphate (ß-TCP) fiber scaffold (ßTFS) is expected to serve as a good scaffold for tissue engineering. However, the fragility and high solubility of ß-TCP fibers make this matrix unsuitable for long-term cell culture. To solve this problem, we developed an alginate-coated ß-TCP fiber scaffold (ßTFS-Alg). To assess cell proliferation and differentiation in the presence of ßTFS-Alg, we characterized ATDC5 cells, a chondrocyte-like cell line, when grown in this matrix. We found that alginate coated the surface of ßTFS fiber and suppressed the elution of Ca2+ from ß-TCP fibers. Due to the decreased solubility of ßTFS-Alg compared with ß-TCP, the former provided an improved scaffold for long-term cell culture. Additionally, we observed superior cell proliferation and upregulation of chondrogenesis marker genes in ATDC5 cells cultured in ßTFS-Alg. These results suggest that ßTFS-Alg is suitable for application in tissue culture.

Material Design of Porous Hydroxyapatite Ceramics via Inverse Analysis of an Estimation Model for Bone-Forming Ability Based on Machine Learning and Experimental Validation of Biological Hard Tissue Responses.

Hydroxyapatite and ß-tricalcium phosphate have been clinically applied as artificial bone materials due to their high biocompatibility. The development of artificial bones requires the verification of safety and efficacy through animal experiments; however, from the viewpoint of animal welfare, it is necessary to reduce the number of animal experiments. In this study, we utilized machine learning to construct a model that estimates the bone-forming ability of bioceramics from material fabrication conditions, material properties, and in vivo experimental conditions. We succeeded in constructing two models: 'Model 1', which predicts material properties from their fabrication conditions, and 'Model 2', which predicts the bone-formation rate from material properties and in vivo experimental conditions. The inclusion of full width at half maximum (FWHM) in the feature of Model 2 showed an improvement in accuracy. Furthermore, the results of the feature importance showed that the FWHMs were the most important. By an inverse analysis of the two models, we proposed candidates for material fabrication conditions to achieve target values of the bone-formation rate. Under the proposed conditions, the material properties of the fabricated material were consistent with the estimated material properties. Furthermore, a comparison between bone-formation rates after 12 weeks of implantation in the porcine tibia and the estimated bone-formation rate. This result showed that the actual bone-formation rates existed within the error range of the estimated bone-formation rates, indicating that machine learning consistently predicts the results of animal experiments using material fabrication conditions. We believe that these findings will lead to the establishment of alternative animal experiments to replace animal experiments in the development of artificial bones.

An ionic silver coating prevents implant-associated infection by anaerobic bacteria in vitro and in vivo in mice.

Currently, implants are utilized clinically for bone transplant procedures. However, if infectious osteomyelitis occurs at implant sites, removal of bacteria can be challenging. Moreover, altered blood flow at peri-implant infectious sites can create an anaerobic environment, making it more difficult to treat infection with antibiotics. Thus, it would be beneficial if implants could be modified to exhibit antibacterial activity, even in anaerobic conditions. Here, we show antibacterial activity of silver ions coated on titanium rods, even against the anaerobic bacteria Porphyromonas gingivalis (P. gingivalis), both in vitro and in vivo. Specifically, we implanted silver-coated or control uncoated titanium rods along with P. gingivalis in mouse femoral bone BM cavities and observed significantly inhibited P. gingivalis infection with silver-coated compared with non-coated rods, based on in vivo bio-imaging. Osteonecrosis by infectious osteomyelitis and elevation of the inflammatory factors C-reactive protein and IL-6 promoted by P. gingivalis s were also significantly reduced in the presence of silver-coated rods. Overall, our study indicates that silver ion coating of an implant represents a therapeutic option to prevent associated infection, even in anaerobic conditions or against anaerobic bacteria.

Preparation of antimicrobial calcium phosphate/protamine composite powders with fluoride ions using octacalcium phosphate.

Calcium phosphates are key biomaterials in dental treatment and bone regeneration. Biomaterials must exhibit antibacterial properties to prevent microbial infection in implantation frameworks. Previously, we developed various types of calcium phosphate powders (amorphous calcium phosphate, octacalcium phosphate (OCP), dicalcium phosphate anhydrate, and hydroxyapatite) with adsorbed protamine (which is a protein with antibacterial property) and confirmed their antibacterial property. In this study, as foundational research for the development of novel oral care materials, we synthesized calcium phosphate composite powders from three starting materials: i) OCP, which intercalates organic compounds, ii) protamine, which has antibacterial properties, and iii) F- ion, which promotes the formation of apatite crystals. Through investigating the preparation concentration of the F- ions and their loading into OCP, it was found that more F- ion could be loaded at higher concentrations regardless of the loading method. It was also observed that the higher the preparation concentration, the more the OCP converted to fluorapatite. The synthesized calcium phosphate composite powders were evaluated for biocompatibility through proliferation of MG-63 cells, with none of the powders exhibiting any growth inhibition. Antimicrobial tests showed that the calcium phosphate composite powders synthesized with protamine and F- ion by precipitation had enhanced antimicrobial properties than those synthesized by protamine adsorption. Thus, the calcium phosphate composite powder prepared from OCP, protamine, and F- ion forms the basis for promising antimicrobial biomaterials. Graphical abstract.

Adsorption of Inositol Phosphate on Hydroxyapatite Powder with High Specific Surface Area.

Chelate-setting calcium-phosphate cements (CPCs) have been developed using inositol phosphate (IP6) as a chelating agent. However, the compressive strength of the CPC fabricated from a commercially available hydroxyapatite (HAp) powder was approximately 10 MPa. In this study, we miniaturized HAp particles as a starting powder to improve the compressive strength of chelate-setting CPCs and examined the adsorption properties of IP6 onto HAp powders. An HAp powder with a specific surface area (SSA) higher than 200 m2/g (HApHS) was obtained by ultrasonic irradiation for 1 min in a wet synthesis process, greatly improving the SSA (214 m2/g) of the commercial powder without ultrasonic irradiation. The HApHS powder was found to be a B-type carbonate-containing HAp in which the phosphate groups in apatite were replaced by carbonate groups. Owing to the high SSA, the HApHS powder also showed high IP6 adsorption capacity. The adsorption phenomena of IP6 to our HApHS and commercially available Hap powders were found to follow the Freundlich and Langmuir models, respectively. We found that IP6 adsorbs on the HApHS powder by both physisorption and chemisorption. The fine HapHS powder with a high SSA is a novel raw powder material, expected to improve the compressive strength of chelate-setting CPCs.

Influence of Culture Period on Osteoblast Differentiation of Tissue-Engineered Bone Constructed by Apatite-Fiber Scaffolds Using Radial-Flow Bioreactor.

With the limitation of autografts, the development of alternative treatments for bone diseases to alleviate autograft-related complications is highly demanded. In this study, a tissue-engineered bone was formed by culturing rat bone marrow cells (RBMCs) onto porous apatite-fiber scaffolds (AFSs) with three-dimensional (3D) interconnected pores using a radial-flow bioreactor (RFB). Using the optimized flow rate, the effect of different culturing periods on the development of tissue-engineered bone was investigated. The 3D cell culture using RFB was performed for 0, 1 or 2 weeks in a standard medium followed by 0, 1 or 2 weeks in a differentiation medium. Osteoblast differentiation in the tissue-engineered bone was examined by alkaline phosphatase (ALP) and osteocalcin (OC) assays. Furthermore, the tissue-engineered bone was histologically examined by hematoxylin and eosin and alizarin red S stains. We found that the ALP activity and OC content of calcified cells tended to increase with the culture period, and the differentiation of tissue-engineered bone could be controlled by varying the culture period. In addition, the employment of RFB and AFSs provided a favorable 3D environment for cell growth and differentiation. Overall, these results provide valuable insights into the design of tissue-engineered bone for clinical applications.

Silver, silicon co-substituted hydroxyapatite modulates bacteria-cell competition for enhanced osteogenic function.

Combating bacteria while promoting tissue regeneration is an aim of highest priority for employing biomaterials in orthopedics that often embroiled with pre-operative contamination. Through simulating a surgical site infection environment and an infected implant site, we showcase the ability of a functionally modified hydroxyapatite, Ag,Si-HA that permits preferential adhesion of human bone marrow derived mesenchymal stem cells (BMSCs) over co-cultured bacterial pathogen,Pseudomonas aeruginosa, by displaying immediate suppression and killing of the bacteria present with minimum cytotoxicity for 28 d. And, at the same time, Ag,Si-HA stimulates BMSCs towards osteogenic differentiation despite being within the contaminated milieu. These findings provide well-defined requirements for incorporating antibacterial properties to biomaterials in managing pre-operative contamination. In addition, it highlights the dual positive attributes of Ag,Si-HA as an effective antibacterial biomaterial and at the same time, promotes bone tissue regeneration.

Bioresorbable porous ß-tricalcium phosphate chelate-setting cements with poly(lactic-co-glycolic acid) particles as pore-forming agent: fabrication, material properties, cytotoxicity, and in vivo evaluation.

Calcium-phosphate cements (CPCs) have been used as bone filling materials in orthopaedic surgery. However, CPCs are set using an acid-base reaction, and then change into stable hydroxyapatite (HAp) in a living body. Therefore, we developed bioresorbable chelate-setting ß-tricalcium phosphate (ß-TCP) cements based on surface modifications of inositol phosphate (IP6). In order to improve the bioresorbability, we fabricated IP6/ß-TCP cements hybridized with poly(lactic-co-glycolic acid) (PLGA) particles as a pore-forming agent. The compressive strengths of the cements with the amounts of 5 and 10 mass% PLGA particles were 23.2 and 22.8 MPa, respectively. There was no significant difference from cements without PLGA (23.4 MPa). The setting times of the cement specimens with PLGA particles (30 min) were a little longer than those without PLGA particles (26.3 min). The lack of cytotoxicity of the cement specimens was confirmed using osteoblast-like cells (MC3T3-E1). Cylindrical defects were made by drilling into the tibia of mini-pigs and injecting the prepared cement pastes into the defects. Twelve weeks after implantation the specimens were stained with toluidine blue and histologically evaluated. Histological evaluation of cement specimens with PLGA particles showed enhanced bioresorbability. Newly-formed bone was also observed inside cement specimens with PLGA particles. The IP6/ß-TCP cement specimens with PLGA particles had excellent material properties, such as injectability, compressive strength, high porosity, no cytotoxicity in vitro, bioresorption and bone formation abilities in vivo. Organic-inorganic hybridized CPCs are expected to be valuable as novel biodegradable paste-like artificial bone fillers.

Regulating size of silver nanoparticles on calcium carbonate via ultrasonic spray for effective antibacterial efficacy and sustained release.

Calcium carbonate is used as bone-filling material due to its good biocompatibility, bioactivity, and bioabsorbability, but the prevalence of infectious complications associated with calcium carbonate has created a persisting challenge in the treatment of bone defect. Therefore, this greatly necessitate the need to endow calcium carbonate with antibacterial properties. In this study, calcium carbonate powders loaded with silver nanoparticles (Ag-CaCO3) were prepared in attempt to serve as a novel antibacterial inorganic filler material. This objective was achieved using ultrasonic spray-pyrolysis (USSP) route to produce Ag-CaCO3 with 1, 5 and 10 mol% silver. The size of silver nanoparticles on CaCO3 microspheres could be regulated by adjusting silver concentration to facilitate effective release of Ag+ ions. This was demonstrated in Ag-CaCO3 (1), where the lowest silver content at 1 mol% achieved the highest Ag+ ions release over 28 days. This in turn gave rise to effective antibacterial efficiency against Staphylococcus aureus and Escherichia coli. Furthermore, CaCO3 (1) could also support osteoblast-like cells (MG-63) at a cell viability of 80%. Overall, this work extends the capabilities in employing USSP to produce inorganic filler materials with sustained antibacterial properties, bringing one step closer to the development of antibacterial products.

Zoledronic Acid-Loaded ß-TCP Inhibits Tumor Proliferation and Osteoclast Activation: Development of a Functional Bone Substitute for an Efficient Osteosarcoma Treatment.

Osteosarcoma has a poor survival rate due to relapse and metastasis. Zoledronic acid (ZOL), an anti-resorptive and anti-tumor agent, is used for treating osteosarcoma. Delivery of ZOL to the target region is difficult due to its high binding affinity to bone minerals. This study developed a novel treatment for osteosarcoma by delivering ZOL to the target region locally and sustainably. In this study, we fabricated a novel bone substitute by loading ZOL on ß-tricalcium phosphate (ß-TCP). The ZOL-loaded ß-TCP (ZOL/ß-TCP) would be expected to express the inhibitory effects via both bound-ZOL (bound to ß-TCP) and free-ZOL (release from ZOL/ß-TCP). To explore the ability to release ZOL from the ZOL/ß-TCP, the amount of released ZOL was measured. The released profile indicates that a small amount of ZOL was released, and most of it remained on the ß-TCP. Our data showed that ZOL/ß-TCP could successfully express the effects of ZOL via both bound-ZOL and free-ZOL. In addition, we examined the biological effects of bound/free-ZOL using osteosarcoma and osteoclasts (target cells). The results showed that two states of ZOL (bound/free) inhibit target cell activities. As a result, ZOL/ß-TCP is a promising candidate for application as a novel bone substitute.

An antibacterial coated polymer prevents biofilm formation and implant-associated infection.

To prevent infections associated with medical implants, various antimicrobial silver-coated implant materials have been developed. However, these materials do not always provide consistent antibacterial effects in vivo despite having dramatic antibacterial effects in vitro, probably because the antibacterial effects involve silver-ion-mediated reactive oxygen species generation. Additionally, the silver application process often requires extremely high temperatures, which damage non-metal implant materials. We recently developed a bacteria-resistant coating consisting of hydroxyapatite film on which ionic silver is immobilized via inositol hexaphosphate chelation, using a series of immersion and drying steps performed at low heat. Here we applied this coating to a polymer, polyetheretherketone (PEEK), and analyzed the properties and antibacterial activity of the coated polymer in vitro and in vivo. The ionic silver coating demonstrated significant bactericidal activity and prevented bacterial biofilm formation in vitro. Bio-imaging of a soft tissue infection mouse model in which a silver-coated PEEK plate was implanted revealed a dramatic absence of bacterial signals 10 days after inoculation. These animals also showed a strong reduction in histological features of infection, compared to the control animals. This innovative coating can be applied to complex structures for clinical use, and could prevent infections associated with a variety of plastic implants.

Investigation of the effects of urea cycle amino acids on the expression of ALB and CEBPB in the human hepatocellular carcinoma cell line FLC-4.

Cell lines are powerful tools for research into liver function at the molecular level. However, they are generally unsuitable for rigorously assessing the effects of amino acid composition, because many lines require serum-containing medium for their maintenance. Here, we aimed to investigate the effects of ornithine and arginine, which are included in the characteristic metabolic process in hepatocyte, on a human hepatoma-derived cell line (FLC-4) that can be cultured in serum-free medium. FLC-4 cells were cultured under the following three conditions: + ornithine/ - arginine, - ornithine/ - arginine, and -ornithine/ + arginine. Albumin expression evaluated by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay and showed no obvious differences based on the presence of ornithine or arginine. However, the mRNA levels of two liver-enriched transcription factors (CEBPB and HNF1A), which are involved in regulating albumin expression, were significantly higher in cells grown in medium-containing arginine than that in cells grown in ornithine-containing medium. Western blotting showed that the levels both activating and inhibitory C/EBPß isoforms were significantly increased in cells grown in arginine medium. Furthermore, we have found that depletion of both ornithine and arginine, the polyamine sources, in the medium did not cause polyamine deficiency. When ornithine and arginine were depleted, albumin production was significantly reduced at the mRNA level, CEBPB mRNA levels were increased, and the level of activating form of C/EBPß was increased. The results of this study suggest that in hepatocyte, these two amino acids might have different functions, and because of which they elicit disparate cellular responses.

Potential Application of Protamine for Antimicrobial Biomaterials in Bone Tissue Engineering.

Bacterial infection of biomaterials is a serious problem in the field of medical devices. It is urgently necessary to develop new biomaterials with bactericidal activity. Antimicrobial peptides and proteins (AMPs), alternative antibacterial agents, are expected to overcome the bacterial resistance. The aim of this study was to develop a new intelligent material in bone tissue engineering based on protamine-loaded hydroxyapatite (protamine/HAp) that uses AMPs rather than antibiotics. It was found that the adsorption of protamine to HAp followed the Langmuir adsorption model and was due to electrostatic and/or hydrophobic interactions. In vitro bacterial adhesion and growth on protamine/HAp was inhibited in a protamine dose-dependent manner. Adherent bacteria exhibited an aberrant morphology for high dosages of protamine/HAp, resulting in the formation of large aggregates and disintegration of the membrane. The released protamine from protamine/HAp also prevented the growth of planktonic bacteria in vitro. However, a high dosage of protamine from powders at loading concentrations over 1000 µg·mL-1 induced a cytotoxic effect in vitro, although those exhibited no apparent cytotoxicity in vivo. These data revealed that protamine/HAp (less than 1000 µg·mL-1) had both antimicrobial activity and biocompatibility and can be applied for bone substitutes in orthopedic fields.

Bactericidal and Bioresorbable Calcium Phosphate Cements Fabricated by Silver-Containing Tricalcium Phosphate Microspheres.

Bacterial adhesion to the calcium phosphate surface is a serious problem in surgery. To prevent bacterial infection, the development of calcium-phosphate cements (CPCs) with bactericidal properties is indispensable. The aim of this study was to fabricate antibacterial CPCs and evaluate their biological properties. Silver-containing tricalcium phosphate (Ag-TCP) microspheres consisting of α/ß-TCP phases were synthesized by an ultrasonic spray-pyrolysis technique. The powders prepared were mixed with the setting liquid to fabricate the CPCs. The resulting cements consisting of ß-TCP and hydroxyapatite had a porous structure and wash-out resistance. Additionally, silver and calcium ions could be released into the culture medium from Ag-TCP cements for a long time accompanied by the dissolution of TCP. These data showed the bioresorbability of the Ag-TCP cement. In vitro antibacterial evaluation demonstrated that both released and immobilized silver suppressed the growth of bacteria and prevented bacterial adhesion to the surface of CPCs. Furthermore, histological evaluation by implantation of Ag-TCP cements into rabbit tibiae exhibited abundant bone apposition on the cement without inflammatory responses. These results showed that Ag-TCP cement has a good antibacterial property and good biocompatibility. The present Ag-TCP cements are promising for bone tissue engineering and may be used as antibacterial biomaterials.

Fabrications of boron-containing apatite ceramics via ultrasonic spray-pyrolysis route and their responses to immunocytes.

Immunotherapy without side effects has been expected as a novel medical treatment for cancer. However, drugs such as cytokines typically used for immunotherapy are very expensive. Therefore, we propose the concept of immunoceramics that affect the immune system. Previous studies have shown that polymers including the phenylboronic acid group activate lymphocytes. This activation may be due to interaction between the sugar chains in cells and the OH group in B(OH)3 formed via dissociation of the BO2 group. In the present study, boron-containing apatite (BAp; Ca9.5+0.5x{(PO4)6-x(BO3)x}{(BO2)1-xOx} (0 ≤ x ≤ 1)) was successfully fabricated via the ultrasonic spray-pyrolysis (USSP) route. We examined the material properties of the BAp ceramics with an aim to application as immunoceramics and the responses of immune cells to the BAp ceramics. The crystalline phases of the BAp ceramics included the apatite phase and infrared (IR) absorption of BO2 and BO3 groups was detected in the BAp ceramics. The cellular response of immune cells derived from mice spleens to dense BAp ceramics was examined next. The proportion of helper T cells and killer T cells on BAp (x = 0.4) ceramics increased compared to that on hydroxyapatite (Ca10(PO4)6(OH)2; HAp) ceramics and on a control. These results indicate that BAp (x = 0.4) ceramics fabricated via the USSP route can be expected to act as immunoceramics that can affect the immune system.

Antimicrobial Activity of Protamine-Loaded Calcium Phosphates against Oral Bacteria.

Protamine is an antimicrobial peptide extracted from fish. In this study, we loaded protamine onto dicalcium phosphate anhydride (DCPA), a dental material. Protamine was loaded by stirring DCPA into a protamine solution. To explore the antimicrobial activity of the materials, we cultivated Streptococcus mutans on fabricated discs for 24 h. When S. mutans was cultivated on the discs under no sucrose conditions, the loaded protamine was not released, and the ratio of dead bacteria increased on the surface of P (125) DCPA (half of the saturated level of protamine (125 ppm protamine) was loaded). Aside from P (500) DCPA (saturated level of protamine was loaded), some protamine was released, and the number of planktonic bacteria in the supernatant decreased. Using medium containing 1% sucrose, the release of protamine was promoted from P (125) DCPA due to lowered pH. However, lowering of the pH decreased the antimicrobial activity of protamine. On the other hand, P (500) DCPA released protamine before the pH was lowered, and biofilm formation was inhibited. The loaded protamine expressed antimicrobial activity, both on the surface of the materials and in the surrounding environment. The interaction of loaded protamine with calcium phosphates could promote the application of protamine in the dental field.

Acceleration of Osteogenesis via Stimulation of Angiogenesis by Combination with Scaffold and Connective Tissue Growth Factor.

In bone regeneration, there are some important cellular biological processes, such as mineralization, cell organization, and differentiation. In particular, vascularization into regenerative tissues is a key step for the survival of cells and tissues. In this study, to fabricate biomimetic-engineered bone, including vascular networks, we focused on connective tissue growth factor (CTGF), a multifunctional protein which could regulate the extracellular matrix remodeling. By combination with CTGF and hydroxyapatite (HAp) ceramics (2D) or apatite-fiber scaffold (AFS, 3D), we have fabricated bioactive materials. The CTGF-loaded HAp ceramics could enhance the cellular attachment through interaction with integrin and promote actin cytoskeletal reorganization. CTGF-loaded HAp also enhanced the differentiation of osteoblasts by integrin-mediated activation of the signaling pathway. Under co-culture conditions, both osteoblasts and endothelial cells in the CTGF-loaded AFS were stimulated by CTGF, and each cell could penetrate the central region of the scaffold in vitro and in vivo. Direct cell-cell interaction would also improve the functionality of cells in bone formation. These results suggest that coupling between effective optimized scaffold and CTGF with multifunction could provide better mimicking natural bone by stimulation of angiogenesis.

Bio-artificial bone formation model with a radial-flow bioreactor for implant therapy-comparison between two cell culture carriers: porous hydroxyapatite and ß-tricalcium phosphate beads.

Bone grafting is necessary before dental implant treatment in patients with jaw bone defects. Currently, autologous bone grafting is a major burden on the patient. However, it is impossible to form a sufficient foundation for the implant with a bone-filling agent alone. It is, therefore, necessary to prepare hybrid artificial bone tissue containing osteoblasts and osteoclasts. In this study, mouse MC3T3-E1 pre-osteoblast cells and human embryonic-derived osteoblastic cell line hFOB1.19 were cultured in radial-flow bioreactors (RFB) to form three-dimensional artificial bone filled with porous beads of ß-tricalcium phosphate (ß-TCP) or hydroxyapatite (HA)-which are clinically used as bone-filling agents-as cell culture carriers. When circulation culturing was performed in the growth medium for the first 10-12 days, glucose consumption was increased in the cultures with HA beads in comparison to the cultures with ß-TCP beads. When cultured in the differentiation culture medium during the second half of the culture period, the glucose consumption decreased in the culture with HA beads. A DNA microarray analysis suggested that osteogenesis progressed fast in three-dimensional culture filled with HA beads and that partly differentiation into osteoblasts was prominent in cultures with ß-TCP beads. In the growth process of MC3T3-E1 cells, the vitamin A metabolism was also activated, the synthesis and degradation of retinoic acid was enhanced, and the metabolism of the same process decreased at the end of differentiation in three-dimensional cultures. Three-dimensional circulation culture in RFB is considered to be useful for the formation of hybrid bio-artificial bone tissue.

Elucidation of Racemization Process of Azaspirene Skeleton in Neutral Aqueous Media.

Azaspirene and related congeners, which possess various biological activities, have a unique spirocyclic core structure. However, there are few studies on the chemical properties of (-)-azaspirene, despite the fact that it may provide important insights into unveiling the biosynthetic pathway. Here, we report a nine-step chemical synthesis of an azaspirene analogue with a new finding that the natural (-)-azaspirene skeleton easily racemizes in neutral aqueous media.

On-site Determination of Arsenic, Selenium, and Chromium(VI) in Drinking Water Using a Solid-phase Extraction Disk/Handheld X-ray Fluorescence Spectrometer.

A rapid, simple technique combining disk solid-phase extraction and handheld X-ray fluorescence (XRF) spectrometry was developed for the on-site determination of As, Se, and Cr(VI) in drinking water. For the preconcentration of As, Se, and Cr(VI), a 50-mL aqueous sample was adjusted to pH 4, followed by passage through a Ti-loaded anion-exchange disk (Ti-AED). Both sides of the Ti-AED were coated with an adhesive cellophane tape prior to drying using a cordless hair iron, followed by a handheld XRF measurement. The Ti-AED adsorbed As(III), As(V), Se(IV), Se(VI), and Cr(VI) in water without requiring oxidation and reduction. The detection limits of As, Se, and Cr(VI) were all 1.0 µg L-1. Good recoveries were obtained by measuring certified reference materials, and spiking natural mineral water with As, Se, and Cr(VI). As the proposed method does not require a power supply or toxic reagents in any analytical step, it is suitable for the on-site determination of toxic elements in drinking water.