Roles, mechanism of action, and potential applications of sulfur-oxidizing bacteria for environmental bioremediation.

Sulfur (S) is a crucial component in the environment and living organisms. This work is the first attempt to provide an overview and critical discussion on the roles, mechanisms, and environmental applications of sulfur-oxidizing bacteria (SOB). The findings reveal that key enzymes of SOB embarked on oxidation of sulfide, sulfite, thiosulfate, and elemental S. Conversion of reduced S compounds was oxidatively catalyzed by various enzymes (e.g. sulfide: quinone oxidoreductase, flavocytochrome c-sulfide dehydrogenase, dissimilatory sulfite reductase, heterodisulfide reductase-like proteins). Environmental applications of SOB discussed include detoxifying hydrogen sulfide, soil bioremediation, and wastewater treatment. SOB producing S0 engaged in biological S soil amendments (e.g. saline-alkali soil remediation, the oxidation of sulfide-bearing minerals). Biotreatment of H2S using SOB occurred under both aerobic and anaerobic conditions. Sulfide, nitrate, and sulfamethoxazole were removed through SOB suspension cultures and S0-based carriers. Finally, this work presented future perspectives on SOB development, including S0 recovery, SOB enrichment, field measurement and identification of sulfur compounds, and the development of mathematical simulation.

Fabrication of porous chitosan-polyvinyl pyrrolidone scaffolds from a quaternary system via phase separation.

Three-dimensional porous chitosan-polyvinyl pyrrolidone (PVP) scaffolds were fabricated for tissue engineering applications via liquid-liquid or liquid-solid phase separation. A mixture of an acidic aqueous solution with butanol as a non-solvent and a chitosan-PVP quaternary system were freeze-dried. We then studied the homogenous open pore structure and the minute pore distribution in order to improve the mass transfer and cell seeding efficiency while also obtaining the optimal ratio of PVP to provide high interconnectivity and to improve the open-pore structure. The properties of the porous chitosan-PVP scaffolds - including the microstructure, chemical release, water absorption properties, and cell proliferation tests were studied - and the results were compared against those obtained from conventional scaffolds. chitosan-PVP scaffolds with a porosity of over 70% were obtained, and the pore morphology on the surface and within the porous scaffolds showed the presence of homogenous open pores with excellent interconnectivity. As the PVP content increased, main pores (50-100 µm) and minute pores (4-10 µm) could be clearly observed. Also, the porous scaffold showed an improved efficiency for cell adhesion after the cells were cultured for 4 h. After 72 h, the cultured cells presented an increase in the cell proliferation and on the porous scaffolds. These results strongly suggest that the porous chitosan-PVP scaffolds can be widely used in tissue engineering, including for biopatches and artificial skin applications.

Enhanced biocompatibility and adhesive properties by aromatic amino acid-modified allyl 2-cyanoacrylate-based bio-glue.

Cyanoacrylates have numerous advantages, including that they can be applied quickly during first aid and can provide good cosmetic outcomes, but they also have limitations in that they have a low bond strength and local tissue toxicity. Consequently, they are primarily used only in urgent applications. To improve both the biocompatibility and the mechanical properties of cyanoacrylate, allyl 2-cyanoacrylate (AC) was prepolymerized and mixed with a dopamine co-initiator. Various properties of prepolymerized AC (PAC)/dopamine mixtures were tested using mouse fibroblast cell (L-929), including their bond strength, setting time, crystallization intensity, and cytotoxicity. Enhanced mechanical properties and biocompatibility were confirmed, and a cytotoxicity test was used to determine the optimal conditions for prepolymerization of AC to be 130°C for 60min. A combination of 5mg of dopamine in 5ml of PAC achieved a high bond strength with cytotoxicity of the dopamine/PAC at approximately 1.5 times lower than that of PAC. These results indicate that dopamine/PAC materials can be extensively used as advanced bio-glues in various applications.

Preparation and evaluation of PEO-coated materials for a microchannel hemodialyzer.

The marked increase in surface-to-volume ratio associated with microscale devices for hemodialysis leads to problems with hemocompatibility and blood flow distribution that are more challenging to manage than those encountered at the conventional scale. In this work stable surface modifications with pendant polyethylene oxide (PEO) chains were produced on polydimethylsiloxane (PDMS), polycarbonate microchannel, and polyacrylonitrile membrane materials used in construction of microchannel hemodialyzer test articles. PEO layers were prepared by radiolytic grafting of PEO-polybutadiene-PEO (PEO-PB-PEO) triblock polymers to the material surfaces. Protein repulsion was evaluated by measurement of surface-bound enzyme activity following contact of uncoated and PEO-coated surfaces with ß-galactosidase. Protein adsorption was decreased on PEO-coated polycarbonate and PDMS materials to about 20% of the level recorded on the uncoated materials. Neither the triblocks nor the irradiation process was observed to have any effect on protein interaction with the polyacrylonitrile membrane, or its permeability to urea. This approach holds promise as a means for in situ application of safe, efficacious coatings to microfluidic devices for blood processing that will ensure good hemocompatibility and blood flow distribution, with no adverse effects on mass transfer.

Association of collagen with calcium phosphate promoted osteogenic responses of osteoblast-like MG63 cells.

In this investigation, the effects of the association of the collagen (COLL) molecules with the calcium phosphate (CaP) film were examined with respect to both the physicochemical properties of the CaP films and the osteoblast responses, such as the adhesion, proliferation, differentiation, and mineralization. The COLL pre-adsorbed CaP film (CaPA) exhibited significant changes in the surface morphology compared to the COLL incorporated CaP film (CaPC). The adhesions of the osteoblast-like MG63 cells were similar on the CaPC or CaPA films. However, the proliferation of the MG63 cells on CaPC was comparable to CaP but considerably different than CaPA. The differentiation of the MG63 cells was greatly improved on CaPC and CaPA compared to CaP and more pronounced on CaPA. The presence of COLL within or on the CaP films significantly modulated the expression of the phenotypic genes, including osteopontin (OPN), alkaline phosphatase (ALP), and the transforming growth factor-ß (TGF-ß). The expression patterns of these genes elucidated that COLL that was present within or on the CaP film supported the osteoblast proliferation and differentiation. These positive effects were stronger for CaPA than CaPC. The bone-like nodules formed on all of the specimens. However, the mineralization of CaPC and CaPA was significantly higher than CaP, indicating that the association of CaP with COLL promoted the mineral deposition. Therefore, the association of the COLL molecules with the CaP film induced positive effects on the biomineralization. Overall, the incorporation of COLL efficiently enhanced the osteoblast responses of CaP. This system can be utilized in a drug delivery system using calcium phosphate. Although the incorporation effects were slightly higher for the osteoblast responses of CaPA than CaPC, CaPC can be used when the longer drug release times are desirable.

Effect of surface modification on the in vitro calcium phosphate growth on the surface of poly(methyl methacrylate) and bioactivity.

Poly(methyl methacrylate) (PMMA) is a biocompatible polymer widely used for bone substitutes. Its surface properties, however, are not favorable for the induction of biological apatite which can be directly related to natural bone formation. In this study, the surface of PMMA was modified by NaOH treatment or sequential treatments with ethanol (EtOH) and NaOH. Results displayed that surface hydrophilicity was improved for increasing treatment time and NaOH concentration. Field-emission scanning electron microscope (FE-SEM) displayed that in vitro formation of calcium phosphate (CaP) coating was significantly promoted by the surface modifications. X-ray photon spectroscopy (XPS) examination elucidated that the films prepared on PMMA consisted of calcium and phosphorus and their values for Ca/P ratio were closed to octacalcium phosphate (OCP). Fourier transform infrared (FT-IR) spectra of the film coated on PMMA revealed a band characteristic of phosphate groups confirming that CaP films were formed and their characteristics were dependent on the surface properties of PMMA. Cellular assay demonstrated that the adhesion of osteoblast-like MG63 cells was significantly promoted on CaP-coated PMMA. Proliferation assay showed that CaP films appeared not to exert any cytotoxic effects on the growth of MG63 cells.

A new method for the preparation of bioactive calcium phosphate films hybridized with 1alpha,25-dihydroxyvitamin D3.

The primary goal of this investigation was to develop a calcium phosphate film hybridized with 1alpha,25-dihydroxyvitamin D(3) for the improvement of osteoconductivity of bone substitutes. The hybrid films (hCaP) were prepared at the different concentrations of 1 x 10(-10), 1 x 10(-8), and 1 x 10(-6) M designated as hCaPL, hCaPM, and hCaPH, respectively. The change of the hormone concentration during the preparation of the hybrid films did not cause significant variations on the physical properties of hCaPs, i.e. surface morphology and roughness. On the other hand, X-ray photon spectroscope (XPS) measurements revealed that the concentration change affected the chemical composition of the hybrid films. Recruitment of osteoblast-like MG-63 cells was considerably improved on hCaPs compared to tissue culture plate (TCP). However, cell proliferation on hCaPs was substantially suppressed and inversely proportional to the hormone concentration used. It was observed that bone-like nodules which consisted of bead-like components and well-developed matrix were rapidly formed on hCaPs. Masson's trichrome and safranin-O stainings elucidated that the bead-like components were MG-63 cells. Safranin-O staining showed that proteoglycan was produced actively. These results indicate that the cells cultured on hCaPs were strongly stimulated by the hormone to produce proteoglycan which can be considered as an induction of premature bone formation. The number of the nodules was increased with hormone concentration and most pronounced at the hCaPH. Gene expression patterns of alkaline phosphatase (ALP), transforming growth factor-beta (TGF-beta), and osteopontin (OPN) were strongly modulated by hybridized the hormone. For ALP and OPN, gene expressions were activated earlier on hCaPs than untreated calcium phosphate (CaP) confirming the effect of the hybridization was substantial. The TGF-beta gene expression was immediately activated after seeding but difference between samples was not significant suggesting that the gene expression was modulated not by the hormone hybridization but by CaP itself. As a result, hybridization of 1,25(OH)(2)D(3) with CaP can be a potentially strong candidate to promote osteoconductivity of implant materials.

Competitive adsorption of bacteriophage T4 lysozyme stability variants at hydrophilic glass surfaces.

The competitive adsorption behavior exhibited by the wild-type T4 lysozyme and two of its structural stability variants was studied by 125I radioisotope labeling. The mutant lysozymes were produced by substitution of the isoleucine residue at position 3 in the wild type with a tryptophan residue, resulting in a protein with lower structural stability, or with a cysteine residue, resulting in a protein with higher structural stability. Adsorption kinetics were recorded for binary protein mixtures in contact with a clean glass surface, in which one variant had been radiolabeled and the other had not. All pair permutations were tested. The kinetic data show that in instances in which exchange reactions between adsorbed protein and dissolved protein occur, they occur such that more stable variants are removed from the surface by less stable variants. The less stable proteins thus exhibited an advantage in competitive adsorption over the more stable proteins, in these tests.

Osteoblastic cell response to thin film of poorly crystalline calcium phosphate apatite formed at low temperatures.

The response of osteoblastic cells to a thin film of poorly crystalline calcium phosphate apatite crystals (PCA) was examined in vitro. The PCA thin film was prepared on polystyrene culture dishes using highly metastable calcium phosphate ion solution at low temperatures. The PCA thin film was formed through fusion and transformation of granular calcium phosphate particles, which had initially formed on the surface, into a film of calcium phosphate apatite crystal. The PCA thin film was used for cell culture without additional surface treatment. The osteoblastic cell behaviors including adhesion, proliferation, expression of the marker genes, and calcified matrix formation were examined on the PCA thin film using primary osteoblasts or MC3T3-E1 cells. The cells were well attached and had spread in a slender shape over the PCA thin film. The extent of cell proliferation on the PCA thin film is as much as on the plain dishes. In addition, a much larger number of calcified nodules had formed on the PCA thin film than on the plain dish. The expression of the marker genes such as alkaline phosphatase, osteocalcin, osteopontin, osteonectin was apparent. These results demonstrate that the osteoblasts exhibit a full spectrum of cellular activity including the adequate differentiation on the PCA thin film. Therefore, a PCA thin film can be used as a coating material for biomaterials where the surface is not adequate for inducing the full activity of bone cells.

MG63 osteoblastic cell adhesion to the hydrophobic surface precoated with recombinant osteopontin fragments.

The hydrophobicity of biomaterials has been recognized as a limitation to the adequate function of anchorage-dependent cells when hydrophobic biomaterials are used for tissue engineering. This is due to flawed solid-state signals from cell adhesion. In this study, a recombinant osteopontin (rOPN17-169) fragment containing the cell adhesion motifs was expressed in E. coli and was precoated on the hydrophobic surface prior to osteoblastic MG63 cell culture. Precoating the hydrophobic surface with rOPN17-169 improved osteoblastic cell adhesion, which was blocked by soluble RGDS. The adhesion of MG63 cells to rOPN17-169 pre-coated surface-activated mitogen-activated protein kinases (MAPK) such as extracellular signal-receptor kinase 1/2, p38, and c-Jun N-terminal kinase (JNK). In addition, p38 MAPK was activated in response to a soluble factor of transforming growth factor-beta in the cells adhered to the hydrophobic surface via rOPN17-169. This suggests that rOPN17-169 precoated on the hydrophobic surface can allow osteoblastic cells to generate adhesion signals sufficient for cell adhesion, MAPK activation, and the cytokine activation of osteoblastic cells.

Effect of electrostatic interaction on the adsorption of globular proteins on octacalcium phosphate crystal film.

The electrostatic effect on the adsorption of globular proteins, such as bovine serum albumin (BSA), hen egg white lysozyme (LZM), and beta-lactoglobulin (beta-Lg), on octacalcium phosphate (OCP)-like crystal thin films was investigated. A poorly crystalline thin film was synthesized on a tissue culture polystyrene (TCP) surface and used as a model surface in this study. The solution pH clearly affected the electrostatic properties of both proteins and surface. The adsorbed amounts obtained at quasi-steady state were readily related to the solution pH for each protein. The adsorption rate is fast during the initial period and levels off gradually. The maximum adsorbed mass occurred at pH 7 for BSA and at pH 9 for LZM. beta-Lg adsorbed similar amounts at pHs lower than 9, but the adsorbed mass decreased at pHs higher than 9 where electrostatic repulsion exists. The pH values where the maximum adsorbed mass occurred may be considered as the conditions where electrostatic attraction is most favorable. The adsorbed mass of beta-Lg was the greatest among the proteins of interest while BSA adsorbed the least despite its greater molecular mass. LZM falls into the intermediate region. According to these observations, BSA has undergone conformational changes that prevent further adsorption to a greater extent than the others. A simple relationship between the adsorption rate and the electrostatic properties was not established. However, the order of magnitude of the adsorption rate at the initial period tends to be the same as that of maximum adsorbed mass for each protein.

Concentration effects on adsorption of bacteriophage T4 lysozyme stability variants to silica.

The adsorption kinetics and dodeceyltrimethylammonium bromide-mediated elution of the wild type and two structural stability mutants of bacteriophage T4 lysozyme were recorded in situ, at silica surfaces. Experiments were performed at different solution concentrations, ranging from 0.01 to 1.0 mg/ml. Plateau values of adsorbed mass generally increased with increasing solution concentration, with the adsorbed layer being only partially eluted by buffer. Treatment with surfactant removed more of the adsorbed protein in each case, with the remaining adsorbed mass varying little with concentration. Comparison of the data to an adsorption mechanism allowing for three adsorbed states, distinguished by binding strength, showed that the fraction of adsorbed molecules present in the most tightly bound state (state 3) decreased as adsorption occurred from solutions of increasing concentration. However, the absolute amounts of state 3 molecules present in each case were less dependent on solution concentration. Adsorption of T4 lysozyme into state 3 is suggested to occur early in the adsorption process and continue until some critical surface concentration is reached. Beyond this critical value of adsorbed mass, adsorption is suggested to progress with adoption of more loosely bound states.