Repeated exposure of the oral mucosa over 12 months with cold plasma is not carcinogenic in mice.

Peri-implantitis may result in the loss of dental implants. Cold atmospheric pressure plasma (CAP) was suggested to promote re-osseointegration, decrease antimicrobial burden, and support wound healing. However, the long-term risk assessment of CAP treatment in the oral cavity has not been addressed. Treatment with two different CAP devices was compared against UV radiation, carcinogen administration, and untreated conditions over 12 months. Histological analysis of 406 animals revealed that repeated CAP exposure did not foster non-invasive lesions or squamous cell carcinoma (SCCs). Carcinogen administration promoted non-invasive lesions and SCCs. Molecular analysis by a qPCR screening of 144 transcripts revealed distinct inflammatory profiles associated with each treatment regimen. Interestingly, CAP treatment of carcinogen-challenged mucosa did not promote but instead left unchanged or reduced the proportion of non-invasive lesions and SCC formation. In conclusion, repeated CAP exposure of murine oral mucosa was well tolerated, and carcinogenic effects did not occur, motivating CAP applications in patients for dental and implant treatments in the future.

Risk assessment of a cold argon plasma jet in respect to its mutagenicity.

Cold atmospheric pressure plasmas represent a favorable option for the treatment of heat sensitive materials and human or animal tissue. Beneficial effects have been documented in a variety of medical conditions, e.g., in the treatment of chronic wounds. It is assumed that the main mechanism of the plasma's efficacy is mediated by a stimulating dissipation of energy via radiation and/or chemical energy. Although no evidence on undesired side effects of a plasma treatment has yet been presented, skepticism toward the safety of the exposure to plasma is present. However, only little data regarding the mutagenic potential of this new treatment option is available. Accordingly, we investigated the mutagenic potential of an argon plasma jet (kinpen) using different testing systems in accordance with ISO norms and multiple cell lines: a HPRT1 mutation assay, a micronucleus formation assay, and a colony formation assay. Moderate plasma treatment up to 180 s did not increase genotoxicity in any assay or cell type investigated. We conclude that treatment with the argon plasma jet kinpen did not display a mutagenic potential under the test conditions applied and may from this perspective be regarded as safe for the use in biomedical applications.

Induction of proliferation of basal epidermal keratinocytes by cold atmospheric-pressure plasma.

BACKGROUND: Over the past few decades, new cold plasma sources have been developed that have the great advantage of operating at atmospheric pressure and at temperatures tolerable by biological material. New applications for these have emerged, especially in the field of dermatology. Recently it was demonstrated that cold atmospheric-pressure plasma positively influences healing of chronic wounds. The potential of cold plasma lies in its capacity to reduce bacterial load in the wound while at the same time stimulating skin cells and therefore promoting wound closure. In recent years, there have been great advances in the understanding of the molecular mechanisms triggered by cold plasma involving signalling pathways and gene regulation in cell culture. AIM: To investigate cold plasma-induced effects in ex vivo treated human skin biopsies. METHODS: Human skin tissue was exposed to cold plasma for different lengths of time, and analysed by immunofluorescence with respect to DNA damage, apoptosis, proliferation and differentiation markers. RESULTS: After cold plasma treatment, the epidermal integrity and keratin expression pattern remained unchanged. As expected, the results revealed an increase in apoptotic cells after 3 and 5 min of treatment. Strikingly, an induction of proliferating basal keratinocytes was detected after cold plasma exposure for 1 and 3 min. As these are the cells that regenerate the epidermis, this could indeed be beneficial for wound closure. CONCLUSION: We investigated the effect of cold plasma on human skin by detecting molecules for growth and apoptosis, and found that both processes are dependent on treatment time. Therefore, this approach offers promising results for further applications of cold plasma in clinical dermatology.

Skin decontamination by low-temperature atmospheric pressure plasma jet and dielectric barrier discharge plasma.

BACKGROUND: Over the past few years, plasma medicine has become an important field in medical science. Cold plasma has proven anti-inflammatory, antimicrobial and antineoplastic effects. AIM: To test the decontamination power of two cold plasma sources [low-temperature atmospheric pressure plasma jet (APPJ) and dielectric barrier discharge plasma (DBD)] in vivo on human fingertips. METHODS: After 3, 15, 30, 60, 90, 120, 150, 180, 210 and 240 s of spot treatment with the APPJ and DBD, the log reduction factors (RFs) of physiological (PF) and artificially (AF) contaminated flora (Staphylococcus epidermidis and Micrococcus luteus) were calculated. The bacterial load was determined after counting. Tolerance (paresthesia, pain and heat) was measured using a numerical rating scale. FINDINGS: Both plasma devices led to a significant reduction in PF and AF. The maximum log reduction factors for PF were 1.3 for the DBD at 210 s and 0.8 for the APPJ at 60 s. For AF, the maximum log reduction factors were 1.7 for the DBD at 90 s and 1.4 for the APPJ at 120 s. Treatment with both devices was well tolerated. CONCLUSION: Both the APPJ and DBD were highly effective in eradicating PF and AF from the fingertips of healthy volunteers. No plasma-resistant isolates were observed. Cold plasma appears to have potential for skin disinfection. For hand hygiene purposes, plasma exposure times would need to be reduced significantly by technical means.

Antimicrobial efficacy and potential application of a newly developed plasma-based ultraviolet irradiation facility.

A low-pressure mercury vapour discharge tube generating high-intensity ultraviolet (UV) resonance radiation at 254 nm was designed to achieve a nearly simultaneous all-round UV irradiation of products. Testing this 'universal homogeneous ultraviolet (UHUV) irradiation facility' with suspended Bacillus subtilis spores, resulted in a 10(6)-fold reduction in viable count within 30 s applying irradiation energy of 0.3 mW/cm(2). Moreover, this irradiation dose reduced the number of immobilized B. subtilis spores on several material surfaces (wood-free paper, aluminium foil, polystyrol, polypropylene, and polyethylene foil) 10(2)-10(4)-fold. To evaluate potential applications of this UHUV irradiation technique under more realistic conditions, dental hand pieces and orthodontic forceps were contaminated by a blood-saliva mix containing Staphylococcus aureus. Under these conditions, a reduction in viable count of 10(5)-10(6)-fold was achieved within 5-15 min, but higher irradiation energy levels up to 13 mW/cm(2) were necessary. Because of its construction, the shape of the newly developed UHUV irradiation device can be adapted to various shapes to achieve a fast and effective antimicrobial treatment.

Biosensors for in vivo glucose measurement: can we cross the experimental stage.

The development of in vivo working glucose sensors needs two decades, so far. The availability of long term functional implantable biosensors for continuous glucose measurings is a basic prerequisite for the individualized optimum insulin treatment of diabetics. Enzymatic electrochemical sensors are described which realize a functional stability over more than 2 years in vitro, however their function in vivo is limited due to certain bioincompatibility expressed by inflammation of the surrounding tissue, exudates, and immun reactions. The paper reflects an overview concerning different sensor covering materials used as more or less suitable diffusion membranes. From experimental studies in animals and human volunteers conclusions are drawn for further developmental steps of biosensors for in vivo use and for the applicability of glucose sensors for transient diagnostic purposes and as a basis for glucose controlled therapeutic measures. The results demonstrate that further progress aimed at long term biostability of implanted biosensors needs to solve technological problems and the serial production of sensors with really comparable qualities as a prerequisite for clinical trials.

Biosensor-controlled perfusion culture to estimate the viability of cells.

A perfusion cell culture is characterised by the continuous addition of fresh nutrient medium and the withdrawal of an equal volume of used medium, allowing the realisation of cell cultivation conditions that are approximated as closely as possible to the in vivo situation. The combination of a perfusion cell culture with an enzyme glucose biosensor allows the glucose consumption of the cell culture to be monitored continuously. The resulting biosensor-controlled perfusion cell culture is a complex biomonitoring system that is useful for checking the metabolic state of a perfusion cell culture continuously and non-invasively over several days. With this experimental setup, it has been possible to test detrimental external effects on living systems at early stages, in vitro, but under in vivo-like conditions.

Experimental data and theoretical considerations concerning the validity of the SAL concept to characterize non-thermal antimicrobial treatments.

For sterilization processes the pharmacopoeias demand a sterility assurance level (SAL) of 10(-6), i.e. a probability of not more than one viable microorganism among one million sterilized products. This SAL concept is based on the assumption that the inactivation of microorganisms by physical or chemical means generally follows first-order kinetics. In this paper it is demonstrated that this is not absolutely true for non-thermal antimicrobial processes. Using Bacillus subtilis spore test preparations the sporicidal efficacy of gamma and ultraviolet irradiation on the one hand as well as the treatment by glutaraldehyde and hydrogen peroxide containing solutions on the other hand was investigated. A range of mean spore contamination between 10(8) and 10(-2) spores per test item could be supported by experimental data. It was demonstrated that the antimicrobial treatment parameters which are sufficient to reduce a high spore burden were not valid for an adequate reduction of the remaining lower spore burden. It is concluded that any extrapolation of such experimental data to the SAL range as usual in the validation of sterilization process parameters may be not permitted. Possible theoretical explanations of the non-homogeneity of the spore inactivation by non-thermal methods as well as consequences for the safety evaluation of sterilization processes are discussed.

Reprocessing of thermosensitive materials--efficacy against bacterial spores and viruses.

To estimate process parameters for non-thermal methods of antimicrobial inactivation, the half-cycle method is very often used. However, the essential premise of this method of estimation, the independence of microbial inactivation kinetics from the microbial load, seems not to be true. Consequently, the attainment of the sterility assurance level as recommended by the pharmacopoeias by a process which has been validated using the half cycle method is not guaranteed. For the evaluation of such chemo-thermo disinfection processes, the quantification of remaining hepatitis B virus DNA (HBV-DNA) traces on the surface of instruments is a useful tool. An infection can be excluded if a decrease of the HBN3-DNA residues on the instruments below the minimum infective dose can be demonstrated. Using the signal amplification technique to detect HBV-DNA on instruments, the safety of an reprocessing procedures can be improved.

Aspects of the antimicrobial efficacy of grapefruit seed extract and its relation to preservative substances contained.

The antimicrobial efficacy as well as the content of preservative agents of six commercially available grapefruit seed extracts were examined. Five of the six extracts showed a high growth inhibiting activity against the test germs Bacillus subtilis SBUG 14, Micrococcus flavus SBUG 16, Staphylococcus aureus SBUG 11, Serratia marcescens SBUG 9, Escherichia coli SBUG 17, Proteus mirabilis SBUG 47, and Candida maltosa SBUG 700. In all of the antimicrobial active grapefruit seed extracts, the preservative benzethonium chloride was detected by thin layer chromatography. Additionally, three extracts contained the preserving substances triclosan and methyl parabene. In only one of the grapefruit seed extracts tested no preservative agent was found. However, with this extract as well as with several self-made extracts from seed and juiceless pulp of grapefruits (Citrus paradisi) no antimicrobial activity could be detected (standard serial broth dilution assay, agar diffusion test). Thus, it is concluded that the potent as well as nearly universal antimicrobial activity being attributed to grapefruit seed extract is merely due to the synthetic preservative agents contained within. Natural products with antimicrobial activity do not appear to be present.

[Synthesis, antimicrobial effects and adverse effects of new structures of urea, hydrogen peroxide and tensides]. / Synthese, antimikrobielle Wirksamkeit und Verträglichkeit neuer Strukturen von Harnstoff, Wasserstoffperoxid und Tensiden.

A substantial extension of the applicability of hydrogen peroxide will be reached by using occlusion compounds of anionic, amphoteric (dipolar ionic), cationic, nonionic, or polyfunctional tensides with hydrogen peroxide in urea. Occlusion compounds with 3 as well as with 4 components containing natural equivalent and antimicrobial effective tensides were synthesized and tested. By synthesis in an anhydrous system the content of active substances as well as the mixture ratio of the components may be adapted to the conditions necessary according to the appropriate purpose. Occlusion compounds are characterized by a broad and well-balanced spectrum of antimicrobial effectivity. Concentrations in the range of 0.1 to 0.4% are practicable to preserve pharmaceutical preparations or technical products, respectively. Several compounds show a sporicidal activity in concentrations between 0.3 and 0.6% and, with an appropriate action time (> 24 h) also a moderate effectivity against polioviruses. In the conventional cell culture, in the perfusion cell culture, and in the phytotoxicity test using cress seed, respectively, low biological activity of the occlusion compounds was found.

Clinical usefulness of the glucose concentration in the subcutaneous tissue--properties and pitfalls of electrochemical biosensors.

Biosensors are miniaturized analytical tools which comprise a biological detection element providing specificity to the analyte, and a physical transducer which guarantees an output signal, e.g. an electric current the size of which is proportional to the concentration of the analyte. They provide the unique possibility of continuous in vivo monitoring. Glucosensors were in fact the first biosensors under study. Among them, the most advanced devices are measuring amperometrically the hydrogen peroxide generated in a stoichiometric relation to the prevailing glucose concentration during glucose oxidase-mediated glucose oxidation. They proved useful in commercially available glucose analyzers and in experimental subcutaneous monitoring. Here it is shown (a) that under steady state conditions the s.c. glucose concentration is nearly identical to that in blood, (b) that s.c. inserted glucose electrodes do mirror the intracorporal glucose concentration both under hypo-, normo-, and hyperglycaemic conditions with a clinically relevant accuracy, (c) that even stable feedback control of intracorporal glucose concentration is possible employing s.c. glucosensor signal as an input to automated insulin pump controller, and (d) that stable function of s.c. sensor is usually accomplished over intervals up to one day but in some cases applications over up to ten days could be realized. The underlying problem consists in an insufficient functional biostability which is a function of biocompatibility and size of the sensor, of its sterility, and of the permanent skin penetration. The latter is still required to get the device in place, to keep it in function, and to make use of the data under any condition.(ABSTRACT TRUNCATED AT 250 WORDS)

Glucose oxidase electrodes: effect of hydrogen peroxide on enzyme activity?

This paper presents a simple procedure to assess the amount of hydrogen peroxide arising in amperometric glucose oxidase sensors. At an external glucose concentration of 30 mmol/l, which represents the linear range of calibration, a hydrogen peroxide concentration of approximately 0.18 mmol/l is generated. Taking into consideration that in medical applications the main range of interest would be within euglycaemia which is between 4 and 9 mmol/l, it is concluded that in the type of glucose electrode investigated, hydrogen peroxide produced during enzymatic glucose measurement does not appreciably damage the enzyme glucose oxidase.

Implantable glucose sensors: comparison between in vitro and in vivo kinetics.

This study was aimed at validating the in vitro estimated response characteristics of implanted glucose oxidase/H2O2 electrodes with respect to their in vivo function. Monoexponential non-linear regression analysis of sensor current vs. time curves in response to square alterations in glucose concentration gave response times T95 of between 1 and 5 min. Non-primed glucose infusions were applied to dogs with these electrodes implanted subcutaneously. The simultaneously monitored in vivo data were subjected to non-linear regression analysis. The time constants T of increases or decreases after starting or ending the glucose load were (mean +/- SEM) 53 +/- 10 and 26 +/- 4 min (significant difference, p less than 0.05) in sensor current, 28 +/- 8 and 15 +/- 2 min (NS) in whole blood, and 26 +/- 5 and 18 +/- 2 min (NS) in plasma. The in vivo kinetic patterns of sensors were not related to their in vitro response times. Non-linear regression analysis of in vitro responses of glucose sensors under clearly defined conditions is recommended as a basis for further studies. The physiological delay in the subcutaneous glucose system needs more attention in this field of research.

Automated feedback control of subcutaneous glucose concentration in diabetic dogs.

The subcutaneous tissue is generally considered as a potential site for the monitoring of intracorporal glucose concentration by means of implanted sensors. We studied the suitability of using the resulting signal from the interstitial glucose concentration as an input in a feedback-controlled system for insulin administration. Miniaturized glucose electrodes (amperometric glucose oxidase sensors for the measurement of hydrogen peroxide) were implanted in insulin-dependent diabetic dogs. The output of these sensors was fed into the controller of a bedside-type artificial B cell. Insulin was infused by the device intravenously on the basis of a proportional-differential algorithm. The glucose patterns were compared to identical experiments where feedback control was accomplished on the basis of paracorporal blood glucose measurement using the same algorithm. Normoglycaemia was restored and maintained in both sets of experiments and oral glucose loads were well compensated for. It is concluded that the apparent subcutaneous glucose concentration is appropriate as an input signal for an artificial B cell.

In situ calibration of implanted electrochemical glucose sensors.

A feasible and reliable method of in situ checking and calibration of implanted glucose sensors is required to compensate for alterations in the overall sensitivity of the "sensor plus subcutaneous fluid glucose compartment" system. In a study on nondiabetic dogs, the linear regression analysis of paired plasma glucose/sensor current data is validated as a potential basis of recalibration of intracorporal glucose sensors. These sensors were amperometric glucose oxidase/hydrogen peroxide electrodes of which the in vitro response time to square alterations in the ambient glucose concentration T95 was less than 5 min. The method presented may be incorporated into the data handling system of portable glucose monitors or of miniaturized artificial beta-cells. For its performance, no steady state glycaemia but a minimum alteration in the intracorporal glucose concentration is needed. The latter can be provided both by tests or by fluctuations as they occur spontaneously during the course of the day.

Oxygen tension at the subcutaneous implantation site of glucose sensors.

To elucidate potential influences of the average tissue pO2 on the function of implanted glucose sensors, non-miniaturized polarographic oxygen electrodes and glucose oxidase/H2O2 glucose electrodes were implanted in the subcutaneous tissue of spontaneously breathing normal and diabetic dogs. There was no appreciable run-in phenomenon of oxygen sensors but normally a pronounced initial decrease in current after implantation of glucose sensors. The subcutaneous pO2 amounted to an average of 7 kPa in air-breathing animals with no difference between normal and insulin-dependent diabetic dogs. It showed oscillations of approximately +/- 2 kPa but the mean was stable over the maximum duration of experiments of 16 h. Induced alterations of tissue pO2 between less than 2 and greater than 20 kPa (as verified by measurements of arterial pO2) were not followed by alterations in the current of nearby implanted glucose sensors. It is concluded that the frequently observed instabilities and losses in sensitivity of the system "implanted glucose sensor in situ + tissue glucose compartment" are not caused by alterations in tissue pO2.