Effect of different post-treatments on the bioactivity of alkali-treated Ti-5Si alloy.

BACKGROUND: As titanium (Ti) alloys are bioinert, various chemically-modified Ti surface has been developed to promote bioactivity and bone ingrowth. OBJECTIVE: In this study, various post treatments (water aging, hydrothermal, and heat treatments) were applied to NaOH-treated Ti-5Si to improve its bioactivity. METHODS: The bioactivity of surface-modified Ti-5Si was access by using the apatite formation ability of Ti-5Si surfaces soaking in a simulated body fluid (SBF). RESULTS: The results showed that the NaOH-treated surface formed a porous network structure composed of sodium titanate hydrogel, which was changed to sodium titanate after subsequent post treatments, whereas sodium titanate, anatase and rutile phases were found on the Ti-5Si surfaces after heat treatment. After immersion in SBF for 14 days, compact apatite layers were observed on the surfaces of all the Ti-5Si tested. The results of XRD and FTIR indicated that the apatite deposited on the Ti-5Si substrate with various surface modified conditions was carbonate-substituted hydroxyapatite. CONCLUSIONS: The apatite-forming ability of the surface of the Ti-5Si was excellent, even though Ti-5Si was not subjected to surface modifications. As a result, the bioactivity of Ti-5Si alloy was verified by the apatite-forming ability, making it suitable for use in orthopedic and dental implants.

Structure and Mechanical Properties of As-Cast Ti-5Sn-xMo Alloys.

Ti-5Sn-xMo (x = 0, 1, 3, 5, 7.5, 10, 12.5, 15, 17.5, and 20 wt %) alloys were designed and prepared for application as implant materials with superior mechanical properties. The results demonstrated that the crystal structure and mechanical properties of Ti-5Sn-xMo alloys are highly affected by their Mo content. The as-cast microstructures of Ti-5Sn-xMo alloys transformed in the sequence of phases α' → α″ → ß, and the morphologies of the alloys changed from a lath structure to an equiaxed structure as the Mo content increased. The α″-phase Ti-5Sn-7.5Mo (80 GPa) and ß-phase Ti-5Sn-10Mo (85 GPa) exhibited relatively low elastic moduli and had excellent elastic recovery angles of 27.4° and 37.8°, respectively. Furthermore, they exhibited high ductility and moderate strength, as evaluated using the three-point bending test. Search for a more suitable implant material by this study, Ti-5Sn-xMo alloys with 7.5 and 10 wt % Mo appear to be promising candidates because they demonstrate the optimal combined properties of microhardness, ductility, elastic modulus, and elastic recovery capability.

Microstructure and Characteristics of Calcium Phosphate Layers on Bioactive Oxide Surfaces of Air-Sintered Titanium Foams after Immersion in Simulated Body Fluid.

We propose a simple and low-cost process for the preparation of porous Ti foams through a sponge replication method using single-step air sintering at various temperatures. In this study, the apatite-forming ability of air-sintered Ti samples after 21 days of immersion in simulated body fluid (SBF) was investigated. The microstructures of the prepared Ca-P deposits were examined by X-ray diffraction (XRD), field-emission scanning electron microscopy (FE-SEM), Fourier transform infrared (FTIR) spectroscopy, and cross-sectional transmission electron microscopy (TEM). In contrast to the control sample sintered in vacuum, which was found to have the simple hexagonal α-Ti phase, the air-sintered samples contained only the rutile phase. High intensities of XRD peaks for rutile TiO2 were obtained with samples sintered at 1000 °C. Moreover, the air-sintered Ti samples had a greater apatite-forming ability than that of the Ti sample sintered in vacuum. Ti samples sintered at 900 and 1000 °C had large aggregated spheroidal particles on their surfaces after immersion in SBF for 21 days. Combined XRD, energy-dispersive X-ray spectroscopy, FTIR spectroscopy, and TEM results suggest that the calcium phosphate deposited on the rutile TiO2 surfaces consist of carbonated calcium-deficient hydroxyapatite instead of octacalcium phosphate.

Fabrication and characterization of porous Ti-7.5Mo alloy scaffolds for biomedical applications.

Porous titanium and titanium alloys are promising scaffolds for bone tissue engineering, since they have the potential to provide new bone tissue ingrowth abilities and low elastic modulus to match that of natural bone. In the present study, porous Ti-7.5Mo alloy scaffolds with various porosities from 30 to 75 % were successfully prepared through a space-holder sintering method. The yield strength and elastic modulus of a Ti-7.5Mo scaffold with a porosity of 50 % are 127 MPa and 4.2 GPa, respectively, being relatively comparable to the reported mechanical properties of natural bone. In addition, the porous Ti-7.5Mo alloy exhibited improved apatite-forming abilities after pretreatment (with NaOH or NaOH + water) and subsequent immersion in simulated body fluid (SBF) at 37 °C. After soaking in an SBF solution for 21 days, a dense apatite layer covered the inner and outer surfaces of the pretreated porous Ti-7.5Mo substrates, thereby providing favorable bioactive conditions for bone bonding and growth. The preliminary cell culturing result revealed that the porous Ti-7.5Mo alloy supported cell attachment.

Effects of near-field ultrasound stimulation on new bone formation and osseointegration of dental titanium implants in vitro and in vivo.

A near-field ultrasound stimulation system was designed for use in in vitro and in vivo trials. The intensity of ultrasound was studied to optimize the osseointegration of the dental titanium implant into the adjacent bone. MG63 osteoblast-like cells were seeded on commercial purity titanium (CP-Ti) plate, and then sonicated for 3 min/day at a frequency of 1 MHz and intensities of 0.05, 0.15 and 0.30 W/cm(2), using either pulsed or continuous ultrasound. Cells were analyzed to determine viability (inhibition of (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction) and alkaline phosphatase (ALP). Tissue culture was performed in vitro by placing a CP-Ti plate in a cultured rat neonatal calvarial defect in response to ultrasound stimulation. In the in vivo trial, screw-shaped CP-Ti implants were inserted into the metaphysis of rabbit tibia, and then stimulated by ultrasound for 10 min daily for 30 d. All samples were processed for histomorphometric evaluation and analyzed by image system. Color Doppler ultrasonography was inspected to evaluate the supply of blood flow. Pulsed ultrasound groups had higher MTT and ALP than control. Tissue culture indicated that pulsed ultrasound groups promoted cell migration and new bone regeneration more effectively than in the control. In animal study, blood flow and mature type I collagen fibers were more prevalent around titanium implants, and bone formation was accelerated in pulsed ultrasound groups. In conclusion, low-intensity pulsed ultrasound at 0.05-0.3 W/cm(2) may accelerate cell proliferation and promote the maturation of collagen fibers and support osteointegration.

Genotypic detection and molecular epidemiology of extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae in a regional hospital in central Taiwan.

This study was conducted to detect the genes encoding extended-spectrum beta-lactamases (ESBLs) and determine the epidemiological relatedness of 69 Escherichia coli and 33 Klebsiella pneumoniae isolates collected from a regional hospital in central Taiwan, mostly from inpatients (E. coli 87.0%; K. pneumoniae 88.0%). The phenotypes of these isolates were examined according to the combination disc method recommended by the Clinical and Laboratory Standards Institute. Most of the ESBL-producing E. coli and K. pneumoniae isolates (98.6% and 97%, respectively) could be detected using cefotaxime discs with and without clavulanate. Genotyping was performed by PCR with type-specific primers. CTX-M-14 type (53.6%) was the most prevalent ESBL among E. coli isolates while SHV type (57.6%) was the most dominant among K. pneumoniae isolates. Six E. coli and three K. pneumoniae isolates did not carry genes encoding ESBLs of types TEM, SHV, CTX-M-3, CTX-M-14, CMY-2 and DHA-1. The co-existence of two or more kinds of ESBL in a single isolate was common, occurring in 40.6% and 72.7% of E. coli and K. pneumoniae isolates, respectively. PFGE analysis revealed that ESBL producers isolated in this setting were genetically divergent.

Asymmetrically simultaneous synthesis of L-homophenylalanine and N6-protected-2-oxo-6-amino-hexanoic acid by engineered Escherichia coli aspartate aminotransferase.

L-Homophenylalanine (L-HPA) and N(6)-protected-2-oxo-6-amino-hexanoic acid (N(6)-protected-OAHA) can be used as building blocks for the manufacture of angiotensin-converting enzyme inhibitors. To synthesize L-HPA and N(6)-protected-OAHA simultaneously from 2-oxo-4-phenylbutanoic acid (OPBA) and N(6)-protected-L-lysine, several variants of Escherichia coli aspartate aminotransferase (AAT) were developed by site-directed mutagenesis and their catalytic activities were investigated. Three kinds of N(6)-protected-L-lysine were tested as potential amino donors for the bioconversion process. AAT variants of R292E/L18H and R292E/L18T exhibited specific activities of 0.70+/-0.01 U/mg protein and 0.67+/-0.02 U/mg protein to 2-amino-6-tert-butoxycarbonylamino-hexanoic acid (BOC-lysine) and 2-amino-6-(2,2,2-trifluoro-acetylamino)-hexanoic acid, respectively. E. coli cells expressing R292E/L18H variant were able to convert OPBA and BOC-lysine to L-HPA and 2-oxo-6-tert-butoxycarbonylamino-hexanoic acid (BOC-OAHA) with 96.2% yield in 8 h. This is the first report demonstrating a process for the simultaneous production of two useful building blocks, L-HPA and BOC-OAHA.

A novel hydantoinase process using recombinant Escherichia coli cells with dihydropyrimidinase and L-N-carbamoylase activities as biocatalyst for the production of L-homophenylalanine.

A dihydropyrimidinase gene (pydB) was cloned from the moderate thermophilic Brevibacillus agri NCHU1002 and expressed in Escherichia coli. The purified dihydropyrimidinase exhibited strict d-enantioselectivity for D,L-p-hydroxyphenylhydantoin and D,L-5-[2-(methylthio)ethyl]hydantoin, and non-enantiospecificity for D,L-homophenylalanylhydantoin (D,L-HPAH). The hydrolytic activity of PydB was enhanced notably by Mn2+, with a maximal activity at 60 degrees C and pH 8.0. This enzyme was completely thermostable at 50 degrees C for 20 days. A whole cell biocatalyst for the production of L-homophenylalanine (L-HPA) from D,L-HPAH by coexpression of the pydB gene and a thermostable L-N-carbamoylase gene from Bacillus kaustophilus CCRC11223 in E. coli JM109 was developed. The expression levels of dihydropyrimidinase and L-N-carbamoylase in the recombinant E. coli cells were estimated to be about 20% of the respective total soluble proteins. When 1% (w/v) isopropyl-beta-D-thiogalactopyranoside-induced cells were used as biocatalysts, a conversion yield of 49% for L-HPA with more than 99% ee could be reached in 16 h at pH 7.0 from 10mM D,L-HPAH. The cells can be reused for at least eight cycles at a conversion yield of more than 43%. Our results revealed that coexpression of pydB and lnc in E. coli might be a potential biocatalyst for L-HPA production.

Effects of unidirectional permeability in asymmetric poly(DL-lactic acid-co-glycolic acid) conduits on peripheral nerve regeneration: an in vitro and in vivo study.

The high outflow permeability of the nerve conduit used to emit the drained waste generated from the traumatized host nerve stump is critical in peripheral nerve regeneration. Our earlier studies have established that asymmetric conduits fulfill the basic requirements for use as nerve guide conduits. In this study, the inflow characteristics of optimal nerve conduits were further examined using in vivo and in vitro trials. Various asymmetric poly(DL-lactic acid-co-glycolic acid) (PLGA) conduits were controlled by modifying precipitation baths using 0, 20, and 95% isopropyl alcohol, with high-porosity (permeability), medium-porosity (high outflow and low inflow), and low-porosity (permeability), respectively. In the in vitro trial, the Schwann cells and fibroblasts were seeded on either side of the asymmetric PLGA films in a newly designed coculture system that simulated the repaired nerve conduit environment. The results of the directional permeable films indicated the statistically significant proliferation of Schwann cells and the inhibition of the division of fibroblasts in lactate dehydrogenase release and inhibition of 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide (MTT) reduction, compared with the other films. In the in vivo trial, the PLGA conduits seeded with Schwann cells were implanted into 10 mm right sciatic nerve defects in rats. After 6 weeks, implanted conduits were harvested. Histological examination verified that directional permeable conduits had markedly more A-type and B-type myelin fibers in the midconduit and distal nerve. In this work, the directional transport characteristics were established as an extremely important factor to the design and development of optimal nerve guide conduits in peripheral nerve regeneration.

Enantioselective synthesis of L-homophenylalanine by whole cells of recombinant Escherichia coli expressing L-aminoacylase and N-acylamino acid racemase genes from Deinococcus radiodurans BCRC12827.

L-Homophenylalanine (l-HPA) is a chiral unnatural amino acid used in the synthesis of angiotensin converting enzyme inhibitors and many pharmaceuticals. To develop a bioconversion process with dynamic resolution of N-acylamino acids for the l-HPA production, N-acylamino acid racemase (NAAAR) and l-aminoacylase (LAA) genes were cloned from Deinococcus radiodurans BCRC12827 and expressed in Escherichia coli XLIBlue. The recombinant enzymes were purified by nickel-chelate chromatography, and their biochemical properties were determined. The NAAAR had high racemization activity toward chiral N-acetyl-homophenylalanine (NAc-HPA). The LAA exhibited strict l-enantioselection to hydrolyze the NAc-l-HPA. A stirred glass vessel containing transformed E. coli cells expressing D. radiodurans NAAAR and LAA was used for the conversion of NAc-d-HPA to l-HPA. Unbalance activities of LAA and NAAAR were found in E. coli cell coexpressing laa and naaar genes, which resulted in the accumulation of an intermediate, NAc-l-HPA, in the early stage of conversion and a low productivity of 0.83 mmol l-HPA/L h. The results indicated that low activity of LAA present in the biomass is the rate-limiting factor in l-HPA production. In the case of two whole cells with separately expressed enzyme, the enzymatic activities of LAA and NAAAR could be balanced by changing the loading of individual cells. When the activities of two enzymes were fixed at 3600 U/L, 99.9% yield of l-HPA could be reached in 1 h, with a productivity of 10 mmol l-HPA/L h. The cells can be reused at least six cycles at a conversion yield of more than 96%. This is the first NAAAR/LAA process using NAc-HPA as substrate and recombinant whole cells containing Deinococcus enzymes as catalysts for the production of l-HPA to be reported.

Structure-stability-activity relationship in covalently cross-linked N-carbamoyl D-amino acid amidohydrolase and N-acylamino acid racemase.

N-Acylamino acid racemase (NAAAR) and N-carbamoyl-D-amino-acid amidohydrolase (D-NCAase) are important biocatalysts for producing enantiopure alpha-amino acids. NAAAR forms an octameric assembly and displays induced fit movements upon substrate binding, while D-NCAase is a tetramer that does not change conformation in the presence of a ligand. To investigate the effects of introducing potentially stabilizing S-S bridges in these different multimeric enzymes, cysteine residues predicted to form inter or intra-subunit disulfide bonds were introduced by site-directed mutagenesis. Inter-subunit S-S bonds were formed in two NAAAR variants (A68C-D72C and P60C-Y100C) and two d-NCAase variants (A302C and P295C-F304C). Intra-subunit S-S bonds were formed in two additional NAAAR variants (E149C-A182C and V265C). Crystal structures of NAAARs variants show limited deviations from the wild-type overall tertiary structure. An apo A68C-D72C subunit differs from the wild-type enzyme, in which it has an ordered lid loop, resembling ligand-bound NAAAR. The structures of A222C and A302C D-NCAases are nearly identical to the wild-type enzyme. All mutants with inter-subunit bridges had increases in thermostability. Compared with the wild-type enzyme, A68C-D72C NAAAR showed similar kcat/Km ratios, whereas mutant D-NCAases demonstrated increased kcat/Km ratios at high temperatures (A302C: 4.2-fold at 65 degrees C). Furthermore, molecular dynamic simulations reveal that A302C substantially sustains the fine-tuned catalytic site as temperature increases, achieving enhanced activity.

Asymmetrical synthesis of L-homophenylalanine using engineered Escherichia coli aspartate aminotransferase.

Site-directed mutagenesis was performed to change the substrate specificity of Escherichia coli aspartate aminotransferase (AAT). A double mutant, R292E/L18H, with a 12.9-fold increase in the specific activity toward L-lysine and 2-oxo-4-phenylbutanoic acid (OPBA) was identified. E. coli cells expressing this mutant enzyme could convert OPBA to L-homophenylalanine (L-HPA) with 97% yield and more than 99.9% ee using L-lysine as amino donor. The transamination product of L-lysine, 2-keto-6-aminocaproate, was cyclized nonenzymatically to form Delta(1)-piperideine 2-carboxylic acid in the reaction mixture. The low solubility of L-HPA and spontaneous cyclization of 2-keto-6-aminocaproate drove the reaction completely toward L-HPA production. This is the first aminotransferase process using L-lysine as inexpensive amino donor for the L-HPA production to be reported.

Structural basis for catalytic racemization and substrate specificity of an N-acylamino acid racemase homologue from Deinococcus radiodurans.

N-acylamino acid racemase (NAAAR) catalyzes the racemization of N-acylamino acids and can be used in concert with an aminoacylase to produce enantiopure alpha-amino acids, a process that has potential industrial applications. Here we have cloned and characterized an NAAAR homologue from a radiation-resistant ancient bacterium, Deinococcus radiodurans. The expressed NAAAR racemized various substrates at an optimal temperature of 60 degrees C and had Km values of 24.8 mM and 12.3 mM for N-acetyl-D-methionine and N-acetyl-L-methionine, respectively. The crystal structure of NAAAR was solved to 1.3 A resolution using multiwavelength anomalous dispersion (MAD) methods. The structure consists of a homooctamer in which each subunit has an architecture characteristic of enolases with a capping domain and a (beta/alpha)7 beta barrel domain. The NAAAR.Mg2+ and NAAAR.N-acetyl-L-glutamine.Mg2+ structures were also determined, allowing us to define the Lys170-Asp195-Glu220-Asp245-Lys269 framework for catalyzing 1,1-proton exchange of N-acylamino acids. Four subsites enclosing the substrate are identified: catalytic site, metal-binding site, side-chain-binding region, and a flexible lid region. The high conservation of catalytic and metal-binding sites in different enolases reflects the essentiality of a common catalytic platform, allowing these enzymes to robustly abstract alpha-protons of various carboxylate substrates efficiently. The other subsites involved in substrate recognition are less conserved, suggesting that divergent evolution has led to functionally distinct enzymes.

Mutational analysis of feedback inhibition and catalytic sites of prephenate dehydratase from Corynebacterium glutamicum.

Prephenate dehydratase is a key regulatory enzyme in the phenylalanine-specific pathway of Corynebacterium glutamicum. PCR-based random mutagenesis and functional complementation were used to screen for m-fluorophenylalanine ( mFP)-resistant mutants. Comparison of the amino acid sequence of the mutant prephenate dehydratases indicated that Ser-99 plays a role in the feedback regulation of the enzyme. When Ser-99 of the wild-type enzyme was replaced by Met, the specific activity of the mutant enzyme was 30% lower than that of the wild-type. The Ser99Met mutant was active in the presence of 50 microM phenylalanine, whereas the wild-type enzyme was not. The functional roles of the eight conserved residues of prephenate dehydratase were investigated by site-directed mutagenesis. Glu64Asp substitution reduced enzyme activity by 15%, with a 4.5- and 1.7-fold increase in Km and kcat values, respectively. Replacement of Thr-183 by either Ala or Tyr resulted in a complete loss of enzyme activity. Substitution of Arg-184 with Leu resulted in a 50% decrease of enzyme activity. The specific activity for Phe185Tyr was more than 96% lower than that of the wild-type, and the Km value was 26-fold higher. Alterations in the conserved Asp-76, Glu-89, His-115, and Arg-236 residues did not cause a significant change in the Km and kcat values. These results indicated that Glu-64, Thr-183, Arg-184, and Phe-185 residues might be involved in substrate binding and/or catalytic activity.