Integrity of proteins in human saliva after sterilization by gamma irradiation

Microbial contamination of whole human saliva is unwanted for certain in vitro applications, e.g., when utilizing it as a growth substratum for biofilm experiments. The aim of this investigation was to test gamma irradiation for its suitability to sterilize saliva and to investigate the treatment's influence on the composition and integrity of salivary proteins in comparison to filter sterilization. For inhibition of bacterial growth by gamma irradiation, a sterility assurance level of 10(-6) was determined to be reached at a dose of 3.5 kGy. At this dose, the integrity of proteins, as measured by fluorescence, circular dichroism, and gel electrophoretic banding pattern, and the enzymatic activities of salivary amylase and lysozyme were virtually unchanged. Filtration reduced the total protein concentration to about half of its original value and decreased lysozyme activity to about 10%. It can be concluded that irradiation is suitable for sterilizing whole saliva in its native form.     

The influence of antimicrobial treatments on the cytocompatibility of polyurethane biosensor membranes

The cytocompatibility of polyurethane membranes was tested following ultraviolet or gamma irradiation as well as treatment with hydrogen peroxide or glutaraldehyde containing solutions. Despite the fact that all of the methods had been recommended for antimicrobial treatment of glucose biosensors, the treatments investigated significantly influenced cytocompatibility characteristics. Cytotoxicity of membrane eluates was not observed following irradiation treatments. This was also the case when the membranes were repeatedly washed following chemical treatment. Cell growth upon the membranes was stimulated to a different extent after gamma and UV irradiation as well as following hydrogen peroxide treatments. Residues of an urea-based hydrogen peroxide inclusion compound caused a restriction in cell growth upon the membranes as was similarly observed with 2 and 4% glutaraldehyde solutions acting over 2 and 4 h, respectively. It is concluded that cytocompatibility in vitro reflecting the host response against a biomaterial in vivo does not only depend upon the material itself but also upon antimicrobial treatments which could have consequences for its bioperformance characteristics.     

Sterilization of Allograft Bone: is 25 kGy the Gold Standard for Gamma Irradiation?

For several decades, a dose of 25 kGy of gamma irradiation has been recommended for terminal sterilization of medical products, including bone allografts. Practically, the application of a given gamma dose varies from tissue bank to tissue bank. While many banks use 25 kGy, some have adopted a higher dose, while some choose lower doses, and others do not use irradiation for terminal sterilization. A revolution in quality control in the tissue banking industry has occurred in line with development of quality assurance standards. These have resulted in significant reductions in the risk of contamination by microorganisms of final graft products. In light of these developments, there is sufficient rationale to re-establish a new standard dose, sufficient enough to sterilize allograft bone, while minimizing the adverse effects of gamma radiation on tissue properties. Using valid modifications, several authors have applied ISO standards to establish a radiation dose for bone allografts that is specific to systems employed in bone banking. These standards, and their verification, suggest that the actual dose could be significantly reduced from 25 kGy, while maintaining a valid sterility assurance level (SAL) of 10⁻⁶. The current paper reviews the methods that have been used to develop radiation doses for terminal sterilization of medical products, and the current trend for selection of a specific dose for tissue banks.     

Ultrastructural changes in Cryptosporidium parvum oocysts by gamma irradiation

Cryptosporidium parvum is known as one of the most highly resistant parasites to gamma irradiation. To morphologically have an insight on the radioresistance of this parasite, ultrastructural changes in C. parvum sporozoites were observed after gamma irradiation using various doses (1, 5, 10, and 25 kGy) following a range of post-irradiation incubation times (10 kGy for 6, 12, 24, 48, 72, and 96 hr). The ultrastructures of C. parvum oocysts changed remarkably after a 10-kGy irradiation. Nuclear membrane changes and degranulation of dense granules were observed with high doses over 10 kGy, and morphological changes in micronemes and rhoptries were observed with very high doses over 25 kGy. Oocyst walls were not affected by irradiation, whereas the internal structures of sporozoites degenerated completely 96 hr post-irradiation using a dose of 10 kGy. From this study, morphological evidence of radioresistance of C. parvum has been supplemented.     

Induction of DNA damage in mammalian cells by hydrogen peroxide generated by glucose oxidase immobilized in agarose slides

A new model system has been developed to study the influence of reactive oxygen species on isolated mammalian cells in conjunction with the comet assay. The glucose-glucose oxidase system was used as a hydrogen peroxide generating source. The level of DNA damage was assessed in the splenocytes and the cells of bone marrow of mouse and in human leukocytes both in untreated cells and in cells treated with hydrogen peroxide generated by glucose oxidase using the alkaline comet assay in vitro. Various options for the location of the enzyme in the slides have been studied: in the layer with the cells, in the layer above the cells, or in solution on the surface of the slides. The option where glucose oxidase was in the upper layer of 0.5% agarose over the layer of the cells was optimal. It provided separation of the enzyme from the cells and avoided obstruction to the hydrogen peroxide exposure. For the whole blood study, the content of endogenous glucose must be taken into account. This approach can be used to study the level of DNA damage induced in vitro and for the detection of DNA repair, thereby expanding the possibilities of the method, while the experiments are conducted under controlled conditions.     

Effect and aftereffect of gamma radiation pretreatment on enzymatic hydrolysis of wheat straw

Irradiation pretreatment of wheat straw was carried out at different doses by using Co-60 gamma radiation. The weight loss and fragility of wheat straw after irradiation, the combination effect of irradiation and mechanical crushing on enzymatic hydrolysis of wheat straw as well as the aftereffect of irradiation were examined. It is shown that irradiation can cause significant breakdown of the structure of wheat straw. The weight loss of wheat straw increased and the size distribution after crushing moved to fine particles at elevated irradiation doses. The glucose yield of enzymatic hydrolysis of wheat straw increased with increasing doses and achieved the maximum (13.40%) at 500 kGy. A synergistic effect between irradiation and crushing was observed, with a glucose yield of 10.24% at a dose of 500 kGy with powder of 140 mesh. The aftereffect of irradiation had important impact on enzymatic hydrolysis of wheat straw. The aftereffect (at 22nd day) of 400 kGy irradiation accounted for 20.0% of the initial effect for glucose production, and the aftereffects of 50, 100, 200 (at 9th day) and 300 kGy (at 20th day) accounted for 12.9%, 14.9%, 8.9% and 9.1%, respectively, for reducing sugar production.     

A flexible and wearable glucose sensor based on functional polymers with soft-MEMS techniques

A novel biosensor for glucose measurement using functional polymers was fabricated and tested. The biosensor utilizes the physical and chemical functions of hydrophobic polydimethyl siloxane (PDMS) and hydrophilic 2-methacryloyloxyethyl phosphorylcholine (MPC) copolymerized with dodecyl methacrylate (DMA). The glucose sensor was constructed by immobilizing glucose oxidase (GOD) onto a flexible hydrogen peroxide electrode (Pt working electrode and Ag/AgCl counter/reference electrode). The electrodes were fabricated using microelectromechanical systems (MEMS) techniques onto those functional polymers. The sensor showed novel functions of flexibility and it was stretchable so that the sensor could normally work when it was released after expanding to 120% longer than that of normal length. Also, basic characteristics of the sensor were evaluated. The output current of the hydrogen peroxide electrode was linearly related to the hydrogen peroxide concentration in a range of 0.20-2.50 mmol/l, with a correlation coefficient of 0.998. GOD was then immobilized onto the surface of the sensor using MPC polymer. In this case, the current output of the glucose sensor related to the glucose level over a range of 0.06-2.00 mmol/l, with a correlation coefficient of 0.997. The calibration range includes the reported concentration of tear glucose in normal human subject (0.14 mmol/l).     

Validation and Substantiation of 25 kGy as Sterilization Dose for Lyophilized Human Amnion Membrane

The validation and substantiation of sterilization dose for lyophilized human amnion membrane by gamma irradiation delivered by Co⁶⁰ source were investigated. The validation experiments were conducted according to ISO 13409 method B. A total of 120 human amnion membranes were collected. Of these, 10 membranes were used for estimation of bioburden and 20 membranes were used for the individual sterility test at verification dose. The average bioburden per product unit with sample item portion (SIP = 1) for lyophilized human amnion membrane was 572 cfu. The verification dose experiments were done at dose of 8.1 kGy and the results of sterility tests showed that human amnion membrane got one positive. Consequently, the sterilization dose of 25 kGy was confirmed and substantiated.     

Inactivation of enveloped and non-enveloped viruses on seeded human tissues by gamma irradiation

Human tissue allografts are widely used in a variety of clinical applications with over 1.5 million implants annually in the US alone. Since the 1990s, most clinically available allografts have been disinfected to minimize risk of disease transmission. Additional safety assurance can be provided by terminal sterilization using low dose gamma irradiation. The impact of such irradiation processing at low temperatures on viruses was the subject of this study. In particular, both human tendon and cortical bone samples were seeded with a designed array of viruses and the ability of gamma irradiation to inactivate those viruses was tested. The irradiation exposures for the samples packed in dry ice were 11.6-12.9 kGy for tendon and 11.6-12.3 kGy for bone, respectively. The viruses, virus types, and log reductions on seeded tendon and bone tissue, respectively, were as follows: Human Immunodeficiency Virus (RNA, enveloped), >2.90 and >3.20; Porcine Parvovirus (DNA, non-enveloped), 1.90 and 1.58; Pseudorabies Virus (DNA, enveloped), 3.80 and 3.79; Bovine Viral Diarrhea Virus (RNA, enveloped), 2.57 and 4.56; and Hepatitis A Virus (RNA, non-enveloped), 2.54 and 2.49, respectively. While proper donor screening, aseptic technique, and current disinfection practices all help reduce the risk of viral transmission from human allograft tissues, data presented here indicate that terminal sterilization using a low temperature, low dose gamma irradiation process inactivates both enveloped and non-enveloped viruses containing either DNA or RNA, thus providing additional assurance of safety from viral transmission.     

Effect of gamma irradiation on mechanical properties of human cortical bone: influence of different processing methods

The secondary sterilisation by irradiation reduces the risk of infectious disease transmission with tissue allografts. Achieving sterility of bone tissue grafts compromises its biomechanical properties. There are several factors, including dose and temperature of irradiation, as well as processing conditions, that may influence mechanical properties of a bone graft. The purpose of this study was to evaluate the effect of gamma irradiation with doses of 25 or 35?kGy, performed on dry ice or at ambient temperature, on mechanical properties of non-defatted or defatted compact bone grafts. Left and right femurs from six male cadaveric donors aged from 46 to 54?years, were transversely cut into slices of 10?mm height, parallel to the longitudinal axis of the bone. Compact bone rings were assigned to the eight experimental groups according to the different processing method (defatted or non-defatted), as well as gamma irradiation dose (25 or 35?kGy) and temperature conditions of irradiation (ambient temperature or dry ice). Axial compression testing was performed with a material testing machine. Results obtained for elastic and plastic regions of stress-strain curves examined by univariate analysis are described. Based on multivariate analysis it was found that defatting of bone rings had no significant effect on any mechanical parameter studied, whereas irradiation with both doses decreased significantly the ultimate strain and its derivative toughness. The elastic limit and resilience were significantly increased by irradiation with the dose 25?kGy, but not 35?kGy, when the time of irradiation was longer. Additionally, irradiation at ambient temperature decreased maximum load, elastic limit, resilience, and ultimate stress. As strain in the elastic region was not affected, decreased elastic limit resulted in lower resilience. The opposite phenomenon was observed in the plastic region, where in spite of the lower ultimate stress, the toughness was increased due to the increase in the ultimate strain. The results of our study suggest that there may be an association between mechanical properties of bone tissue grafts and the damage process of collagen structure during gamma irradiation. This collagen damage in cortical bone allografts containing water does not depends on the temperature of irradiation or defatting during processing if dose of gamma irradiation does not exceed 35?kGy.     

Validating a Low Dose Gamma Irradiation Process for Sterilizing Allografts Using ISO 11137 Method 2B

This paper describes the validation of an allograft sterilization method specifically designed for the processing methods used at AlloSource in Centennial, CO. The methods used for this validation followed ISO Standard 11137, Method 2B. Three hundred allografts, collected from three defined production batches were dosed using a series of five incremental doses, beginning at 1 kGy and increasing by 1 kGy until 5 kGy was achieved. Following sterilization dosing, each allograft test article was analyzed using a sterility test to identify any viable microorganisms. The number of positive sterility samples was used to calculate the verification dose (1.27 kGy), which was then verified by an additional batch of 100 allografts. The results from this validation indicate that sterility (10⁻⁶ SAL) on human allograft tissue using gamma ⁶⁰Co radiation can be achieved when a dose of at least 9.2 kGy is employed.     

Evaluating in vitro and in vivo the interference of ascorbate and acetaminophen on glucose detection by a needle-type glucose sensor.

The aim of this work was to assess, in vitro and in vivo, the interference of ascorbate and acetaminophen on glucose measurements by a needle-type glucose sensor detecting hydrogen peroxide generated during the enzymatic oxidation of glucose, and to ascertain whether the protection against interference by the membranes used in the construction of the electrode is feasible. The oxidation of ascorbate and acetaminophen on a platinum electrode set at a 650 mV potential yielded a current representing 75 +/- 5% and 25 +/- 6% of that generated by the oxidation of an equimolar concentration of hydrogen peroxide, respectively. The bias introduced by the presence of 100 mumol l-1 ascorbate on the reading of 5 mmol l-1 glucose by the complete sensor (electrode + membranes) would be minimal (approximately 0.4 mmol l-1). By contrast, the bias introduced by 200 mumol l-1 of acetaminophen (a plasma concentration easily reached in clinical practice) was about 7 mmol l-1. The sensor was implanted subcutaneously in anaesthetized rats (n = 3). Using the current generated in the presence of a plasma acetaminophen concentration of about 200 mumol l-1 for glucose monitoring would lead to a major underestimation (approx. 6 mmol l-1) of subcutaneous glucose concentrations.     

An enzyme-chromogenic surface plasmon resonance biosensor probe for hydrogen peroxide determination using a modified Trinder's reagent

An absorption-based surface plasmon resonance (SPR(Abs)) biosensor probe has been developed for simple and reproducible measurements of hydrogen peroxide using a modified Trinder's reagent (a chromogenic reagent). The reagent enabled the determination of the hydrogen peroxide concentration by the development of deep color dyes (lambda(max)=630nm) through the oxidative coupling reaction with N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline sodium salt monohydrate (MAOS; C(13)H(20)NNaO(4)S.H(2)O) and 4-aminoantipyrine (4-AA) in the presence of hydrogen peroxide and horseradish peroxidase (HRP). In the present study, urea as an adduct of hydrogen peroxide for color development could be omitted from the measurement solution. The measurement solution containing 5mM hydrogen peroxide was deeply colored at a high absorbance value calculated as 46.7cm(-1) and was directly applied to the SPR(Abs) biosensing without dilution. The measurement was simply performed by dropping the measurement solution onto the surface of the SPR sensor probe, and the SPR(Abs) biosensor response to hydrogen peroxide was obtained as a reflectivity change in the SPR spectrum. After investigation of the pH profiles in the SPR(Abs) biosensor probe, a linear calibration curve was obtained between 1.0 and 50mM hydrogen peroxide (r=0.991, six points, average of relative standard deviation; 0.152%, n=3) with a detection limit of 0.5mM. To examine the applicability of this SPR(Abs) biosensor probe, 20mM glucose detection using glucose oxidase was also confirmed without influence of the refractive index in the measurement solution. Thus, the SPR(Abs) biosensor probe employing the modified Trinder's reagent demonstrated applicability to other analyte biosensing tools.     

Effect of gamma irradiation on microbial load,aflatoxins and phytochemicals present in Trigonella foenum-graecum

The effects of gamma irradiation on microbial load, total aflatoxins and phytoconstituents content of Trigonella foenum-graecum have been studied. Gamma irradiation at a dose of 2.5 kGy resulted in 2 log reduction of the total aerobic microbial count. A complete sterilization was, however, observed at 10 kGy. The total aflatoxin level decreased gradually with increase in gamma irradiation dose as compared to its un-irradiated counterparts, whereas the high performance liquid chromatography (HPLC) profile showed no change in the levels of phytochemicals up to the gamma irradiation dose of 10 kGy. HPLC profiles, however, differed in peak areas, and retention times of the components. These results suggest that gamma irradiation at a dose of 5.0 kGy was very effective for microbial decontamination because it did not adversely affect the active components of T. foenum-graecum.     

Optimal gamma-ray dose and irradiation conditions for producing low-molecular-weight chitosan that retains its chemical structure

This study focuses on the optimal conditions for gamma irradiation to reduce the molecular weight of chitosan but still retain its chemical structure. Chitosan was irradiated under various conditions, i.e. flake solid state (condition 1), flake dispersed in water (condition 2), flake dispersed in 0.05, 0.1, 1 and 2% aqueous K(2)S(2)O(8) solution (conditions 3a, 3b, 3c and 3d, respectively), flake dispersed in 0.5, 1 and 2% aqueous H(2)O(2) solution (conditions 4a, 4b and 4c, respectively), and chitosan acetic acid solution (condition 5). Comparative studies were done using three types of chitosans with molecular weights of the order of 10(5) Da with degrees of deacetylation of 0.80, 0.85 and 0.90%. For all conditions, after irradiation, there were two regions of molecular weight reduction. A severe degradation occurred in the first region with decreases in the molecular weight of 80% for radiation doses up to 50 kGy for conditions 1, 2 and 3 (3a-3c) and 20 kGy for condition 4. In the second region, a slow degradation occurred, which resembled a plateau stage. The results for conditions 3d and 5 were the most dramatic, since the primary structure of chitosan was changed after the irradiation. The degradation of chitosan by gamma rays was found to be most effective for the amorphous structure. The retention of the structure of chitosan after gamma irradiation makes it possible to produce a low-molecular-weight chitosan that retains its functionality, as demonstrated by its activity in the coupling reaction with N,N'-carbonyldiimidazole.     

A combination of gamma irradiation and CaCl2 immersion for a pectin-based biodegradable film

Using citrus pectin a biodegradable film was prepared by a combination treatment of gamma irradiation (0, 10, 20, and 30 kGy) and CaCl₂ immersion (0, 5, and 10%) cross-linking. The tensile strength of the pectin-based film was the highest in the 5% CaCl₂ treatment at 20 kGy of an irradiation dose. The tensile strength of the film with CaCl₂ was generally higher than that of the non-CaCl₂ treatment. The elongation at break and water vapor permeability were the lowest at a CaCl₂ of 5% among the irradiated treatments. The total organic carbon content produced from the Paenibacillus polymyxa and Pseudomonas aeruginosa showed that the film of the 20 kGy-irradiated film was lower than those of the 0, 10, and 30 kGy-irradiated films. In conclusion, irradiation of the film casting solution at 20 kGy combined with a 5% CaCl₂ immersion resulted in film with improved mechanical properties and biodegradability.     

Effect of monopropylene glycol and gamma irradiation on Yarrowia lipolytica lipase stabilization

This work investigated the effects of monopropylene glycol, protease inhibitor, and gamma irradiation on Yarrowia lipolytica lipase stability during storage. Enzyme liquid stabilization was achieved by addition of monopropylene glycol (MPG) at respective concentrations of 50, 75, and 90%, the protease inhibitors (P2714 and P8215) at 0.1%, and the gamma irradiation with 10kGy, 15kGy, and 25kGy doses. The results showed that monopropylene glycol limited the microorganism growth and decreased the enzymatic activity at high concentration (up to 50%), at two temperatures (20 and 4 degrees C). Enzyme stored at 20 degrees C lost its activity by 80% after two months. This loss was attributed to the protease's effect. At this temperature, the protease's activities have been limited by the specific inhibitors. The gamma irradiations improve microbial safety of liquid enzyme.     

In vitro testing of a simply constructed, highly stable glucose sensor suitable for implantation in diabetic patients

We have constructed and tested in vitro a potentially implantable, needle-type amperometric enzyme electrode which is suitable for continuous monitoring of glucose concentrations in diabetic patients. The major requirements of stability during operation and ease of manufacture have been met with a sensor design which involves a simple dip-coating procedure for applying to a platinum base electrode an inner membrane of glucose oxidase immobilised in polyhydroxyethyl methacrylate (pHEMA), and an outer membrane composed of a pHEMA/polyurethane mixture. Sensors were operated at 700 mV for detection of hydrogen peroxide. Calibration curves for the sensor were linear to at least 20 mM glucose and were unaffected by a reduction in PO2 from 20 to 5 kPa. During continuous operation in 5 mM buffered glucose solutions in vitro, sensors suffered no significant loss of response over periods of up to 60 h. Such electrodes are, therefore, useful for development as in vivo glucose sensors.     

Electrochemical biosensing utilizing synergic action of carbon nanotubes and platinum nanowires prepared by template synthesis

Platinum nanowires (PtNWs) prepared by electrodeposition method with the help of porous anodic aluminum oxide (AAO) templates have been solubilized in chitosan (CHIT) together with carbon nantubes (CNTs) to form a PtNW-CNT-CHIT organic-inorganic system. The resulting PtNW-CNT-CHIT material brings capabilities for utilizing synergic action of PtNWs and CNTs to facilitate electron-transfer process in electrochemical sensor design. The PtNW-CNT-CHIT film modified electrode offered a significant decrease in the overvoltage for the hydrogen peroxide and showed to be excellent amperometric sensors for hydrogen peroxide at -0.1 V over a wide range of concentrations, and the sensitivity is 260 microAmM-1cm-2. As an application example, by linking glucose oxidase (GOx), an amplified biosensor toward glucose was prepared. The glucose biosensor exhibits a selective determination of glucose at -0.1 V with a linear response range of 5 x 10(-6) to 1.5 x 10(-2)M with a correlation coefficient of 0.997, and response time <10s. The high sensitivity of the glucose biosensor is up to 30 microAmM-1cm-2 and the detection limit was 3 microM. The biosensor displays rapid response and expanded linear response range, and excellent repeatability and stability.     

Continuous glucose monitoring and control in a rotating wall perfused bioreactor

A glucose control system consisting of a single in-line glucose sensor, concentrated glucose solution, and computer hardware and software were developed. The system was applied to continuously control glucose concentrations of a perfusion medium in a rotating wall perfused vessel (RWPV) bioreactor culturing BHK-21 cells. The custom-made glucose sensor was based on a hydrogen peroxide electrode. The sensor continuously and accurately measured the glucose concentration of GTSF-2 medium in the RWPV bioreactor during cell culture. Three sets of two-point calibrations were applied to the glucose sensor during the 55-day cell culture. The system first controlled the glucose concentration in perfusing medium between 4.2 and 5.6 mM for 36 days and then at different glucose levels for 19 days. A stock solution with a high glucose concentration (266 mM) was used as the glucose injection solution. The standard error of prediction (SEP) for glucose measurement by the sensor, compared to measurement by the Beckman glucose analyzer, was +/-0.4 mM for 55 days.