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Clinical outcomes of bone allograft procedures may be improved by modifying the surface of the graft with an osteoconductive biopolymeric coating. In this comparative in vitro study, we evaluated the dimensional stability, mechanical strength, hydrophilicity, and water uptake of biodegradable foams of poly(propylene fumarate) (PPF) and poly(d,l-lactic-co glycolic acid) (PLGA) when applied as surface coatings to cortical bone. Cortical bone samples were divided into four groups: Type I, untreated bone; Type II, laser-perforated bone; Type III, partially demineralized bone; and Type IV, laser-perforated and partially demineralized bone. Results show that PPF wets easily, achieving 12.5% wt/wt in 30 min. Compressive tests on the PPF foam material showed that the compressive strength was 6.8 MPa prior to in vitro incubation but then gradually reduced to 1.9 MPa at 8 weeks. Push-out and pulloff strength tests showed that initially both PPF and PLGA foam coatings had comparable adherence strengths to the cortical bone samples (100-150 N). When additional geometrical surface alteration by perforation and demineralization of the bony substrate was employed, in vitro adherence of the PPF foam coating was further increased to 120 N, demonstrating a statistically significant improvement of push-out strength throughout the entire 8-week observation period (p<0.0002 for all four data points). The pore geometry of PPF-foam coatings changed little over the 2-month evaluation period. In comparison, PLGA foam coating around the cortical bone samples rapidly lost structure with a decrease of 67% in strength seen after 1-week in vitro incubation. These new types of bone allografts may be particularly useful where the use of other replacement materials is not feasible or practical.

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Regeneration of skeletal tissues has been recognized as a new means for reconstruction of skeletal defects. We investigated the feasibility of an injectable and expandable porous implant system for in situ regeneration of bone. Therefore, a composite biodegradable foaming cement based on poly(propylene fumarate) was injected into a critical size defect made in the rat tibia. Animals were divided into two groups comparing the foam in the experimental group against sham-operated animals having a drill hole but no implant in the control group. Eight animals were included in each group. Animals were sacrificed at 1, 3, and 7 weeks postoperatively. Implantation sites were then evaluated with histologic and histomorphometric methods. Results of this study showed that defects did not heal in sham-operated animals. In the experimental group, metaphyseal and cortical defects healed within the first postoperative week by formation of immature woven bone. At the site of the cortical drill hole defect, healing was noted to progress to complete closure by formation of mature bone. Histomorphometry corroborated these findings and showed that metaphyseal bone remodeling peaked at 1 week postoperatively and then decreased as healing of the cortical defect progressed. This suggests that near-complete restoration of the original state of the tibial bone occurred in this animal model supporting the concept of in situ bone regeneration by application of engineered biodegradable porous scaffolds. () ()

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The influence of moisture on the survival, movement and degradation activity of a 2,4-D degrading bacterium, Burkholderia cepacia strain BRI6001L, genetically engineered to contain bioluminescent and lactose utilization genes, was studied in unsaturated soil columns. The distance traveled by BRI6001L was dependent on the clay content of the soil, higher clay contents being responsible for higher filtration coefficients. Long term survival, in excess of one year, was attributed to strain BRI6001L's ability to survive dry conditions. Changes in the 2,4-D biodegradation rate showed a better correlation with the BRI6001L population density than with the total viable bacterial population. At moisture levels between field capacity and 40% moisture (-33 kPa to -100 kPa) 2,4-D degradation was attributed mainly to BRI6001L. At moisture levels between 6 and 15%, 2,4-D disappearance was attributed to the indigenous microbial population, with no degradation occurring at moisture levels below 6%. Returning the moisture to above 40% led to an increase of 4 orders of magnitude in the BRI6001L population density and to a 10-fold increase in the 2,4-D degradation rate. The ability to monitor a specific microbial population using reporter genes has demonstrated the importance of controlling moisture levels for maximizing biodegradation rates in unsaturated soil environments.

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Cyclodextrins, macrocyclic carbohydrates with apolar internal cavities, can form complexes with, and solubilize many normally water-insoluble compounds. Ferrocene and its derivatives, tetrathiafulvalene and tetramethylbenzidine, can function as redox mediators, but are insoluble in water; when they are complexed with cyclodextrins, they can be used in enzymatic assays and in the construction of mediated biosensors. In addition, the solubilization of polynuclear aromatic hydrocarbons (PAHs), including the potent carcinogen benzo[a]pyrene, by cyclodextrins has enabled the detection of these important environmental contaminants.

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3,3',5,5'-Tetramethylbenzidine (TMB), a hydrophobic and noncarcinogenic chromogen with a high absorption coefficient widely used in solid-phase assays involving labeled horseradish peroxidase was rendered soluble (up to 40 mM) and more stable for at least 2 months at 22-24 degrees C by forming a water-soluble inclusion complex with 2-hydroxypropyl-beta-cyclodextrin (hp-beta-CyD). Cyclic voltammetry and absorbency measurement were employed to characterize the TMB-hp-beta-CyD complex. Well-defined cyclic voltammograms of TMB exhibited two oxidation waves which merged into a single wave with increasing hp-beta-CyD concentrations. Cyclic voltammetry was then used to examine the effect of complexation with hp-beta-CyD on the oxidation potential of TMB and provided evidence of a 1:1 complex between TMB and the cyclodextrin molecule with a formation constant of 1.6 M-1. Enzyme assays for D-glucose, lactate, and glutamate were performed by coupling the TMB-hp-beta-CyD/horseradish peroxidase system to the respective oxidase enzymes with the formation of either a blue (absorption coefficient of 35,800 M-1 cm-1 at 650 nm) or a yellow color (absorption coefficient of 67,300 M-1 cm-1 at 450 nm) as an indication of the metabolite concentration. These assays possessed a sensitivity limit below 10 microM and the results obtained were in excellent agreement with standard enzymatic assays when tested in various food and clinical samples.

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A chemiluminescence fiber optic system coupled to flow injection analysis (FIA) and ion exchange chromatography has been developed for determining glucose in blood and urine. Immobilized glucose oxidase acted on beta-D-glucose to produce hydrogen peroxide, which was then reacted with luminol in the presence of ferricyanide to produce a light signal. Endogenous ascorbic acid and uric acid present in urine or blood samples were effectively retained by an upstream acetate anion exchanger. In addition, acetaminophen could also be adsorbed by this ion exchanger. The detection system exhibited a sensitivity of 1.315 +/- 0.044 RU microM-1 for glucose with a minimum detection level of 1 microM. When applied for the determination of urinary and blood glucose levels, the results obtained compared well with those of the reference hexokinase assay. Immobilized glucose oxidase was reused for over 500 analyses without losing its original activity. A conservative estimate for the reuse of the acetate ion exchange column was about 100 analyses.

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Together with flow injection analysis (FIA), a chemiluminescence (CL) fiber optic biosensor system has been developed for determining glutamine in animal cell cultures. Glutaminase (GAH) and glutamate oxidase (GLO) were onto separate porous aminopropyl glass beads via glutaraldehyde activation and packed to form an enzyme column. These two enzymes acted in sequence on glutamine to produce hydrogen peroxide, which was then reacted with luminol in the presence of ferricyanide to produce a light signal. An anion exchanger was introduced on-line to eliminate interfering endogenous glutamate in view of its negative charge at pH above 3.22 (isoelectric pH). Among several resins tested, the acetate form was most effective, and this type of ion exchanger also effectively adsorbed uric acid, acetaminophen, and aspartic acid.There was an excellent linear relationship between the CL response and standard glutamine concentration in the range 1 to 100 muM. A complete analysis could be performed in 2 min, including sampling and washing with a good reproducibility (+/- 4.4%). Both the bi-enzymic and ion exchange columns were useful for at least 500 analyses when the biosensor system was applied for the glutamine determination in murine hybridoma cell cultures and insect cell cultures. The values obtained compared well with those of HPLC, thus validating the applicability of the CL fiber optic system.

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A chemiluminescence fiber-optic biosensor system has been developed for determining glutamine in hybridoma cell cultures producing monoclonal antibodies against viral surface antigens. Glutaminase and glutamate oxidase (GLO) were immobilized onto aminopropyl glass beads via glutaraldehyde activation separately and packed in a column. Two separate columns containing immobilized GLO and catalase were placed upstream to eliminate endogenous glutamate. In the presence of ferricyanide, luminol reacted with hydrogen peroxide released from the enzymatic reactions to produce a chemiluminescence (CL) light signal which was detected and quantitated with a fiber-optic system. In combination with flow injection analysis it was possible to process samples virtually identically, thus avoiding difficulties in reproducing the CL signal. There was an excellent linear relationship between the CL response and standard glutamine concentration in the range 10(-6) to 10(-3) M. A complete analysis could be performed in 2 min including sampling and washing. Each immobilized enzyme column was stable for at least 300 repeated analyses without any loss of activity. When the biosensor system was used for the determination of glutamine in spent mammalian cell cultures, the values obtained compared well with those of high-performance liquid chromatography, thus validating the applicability of the CL fiber-optic system.

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An amperometric biosensor has been developed for monitoring glutamine in the pulsed-batch cultivation of murine hybridoma cells. Glutamine oxidase was cross-linked with bovine serum albumin (BSA) via glutaraldehyde activation and deposited on a preactivated nylon membrane. Glutaminase was then immobilized on the protein layer and the resulting membrane was attached to the sensing area of a hydrogen peroxide probe (platinum vs silver/silver chloride polarized at +0.7 V). An orthogonal test was performed to optimize the activity of the membrane for glutamine with respect to the concentrations of glutamate oxidase, BSA, glutaminase and glutaraldehyde. There was an excellent linear relationship between the biosensor's response and glutamine in the range 0.1-3 mM. The determination of glutamine could be performed in 2 min and each membrane was reused for at least 300 consecutive analyses. The data obtained also agreed well with those high-performance liquid chromatography, thus validating the applicability of the biosensor.

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Although successful in reducing urea levels, the use of oral microcapsules containing a urease-silica adduct and a zirconium phosphate ion exchanger result in a number of problems, including a negative calcium balance. In this study, it is demonstrated that the use of microcapsules containing a urease-zeolite preparation may be a potential route to urea removal. The use of zeolite ion exchangers, and zeolite W in particular, can alleviate the problems encountered with zirconium phosphate. Unlike zirconium phosphate, zeolite W is nonselective toward calcium ions and is stable at the high pH found in the intestinal tract. Zeolite W, when present in the sodium form, has a high ammonium capacity of 3.6 mEq NH4+/g zeolite under simulated intestinal conditions; its reactivity to ammonium is also higher. The application of enzyme envelopes to zeolite particles is a novel immobilization procedure that does not involve the use of colloidal silica and can reduce the amount of ingested material by as much as 25%. The current in vitro study shows that cellulose acetate butyrate microcapsules, containing a urease-zeolite preparation, remove up to 80% of urea in less than 1 hour. These microcapsules can be dried and retain activity when sealed in a jar at 4 degrees C.

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