Ionic Air Purifiers

Introduction
Nitrogen fixation is the reduction of N2 (atmospheric nitrogen) to NH3 (ammonia). Free living prokaryotes with the ability to fix atmospheric dinitrogen (diazotrophs) are ubiquitous in soil. But our knowledge of their ecological importance and their diversity remains incomplete. In natural ecosystems, biological N2 fixation is most important source of N. The capacity for nitrogen fixation is widespread among bacteria and archaea. The estimated contribution of free-living N-fixing prokaryotes to the N input of soil ranges from 0-60 kg/ha /year (Burgmann et al., 2003). Dinitrogen (N2)-fixing microorganisms (diazotrophs) play important roles in ocean biogeochemistry and plankton productivity (Church et al., 2005).

Nitrogen fixation can be an important source of nitrogen for biological productivity in the marine environment. Biological nitrogen fixation is catalyzed by the enzyme nitrogenase, which is possessed by diverse microorganisms representing virtually all phylogenetic groups. Interest in nitrogen fixation in the sea has usually been focused on rates of nitrogen fixation, but information on the types of species present with the capability for nitrogen fixation can be important for predicting nitrogen fixation rates in situ (Zehr et al., 1998).

Nitrogenase catalyzes the reduction of nitrogen gas to ammonium in an ATP-and reductant dependent reaction. It is one of the best characterized metalloenzyme and is an excellent model for elucidating metalloprotein assembly. Nitrogenase is composed of two oxygen-labile metallo protein; dinitrogenase and dinitrogenase reductase. Dinitrogenase is a 240-KDa alpha2-beta2 tetramer of the nifD and nifK gene products. Dinitrogenase reductase is a 60-KDa alpha2 dimer of the nifH gene products that contains a single 4Fe-4S center coordinated between the two subunits (Rubio et al., 2005). Understanding how fixed N regulates nitrogenase availability is necessary for devising strategies to increase the amount of ammonium synthesized by nitrogen fixing bacteria with the potential to be used in agriculture (Kennedy et al., 2004).

Molecular tools for detection and characterization of the nitrogenase (Nif) genes and immunoassays for nitrogenase protein can provide new information on the factors regulating the distribution and activity of diverse nitrogen fixing organisms in the marine environment. Amplification and characterization of NifH sequences has made it possible to identify the type(s) of organism responsible for nitrogen fixation, such as in aggregates of the cyanobacterium and Trichodesmium. Differences in nitrogen fixation patterns have been linked to genetic differences between Trichodesmium strains. Further development of these approaches will provide new and powerful ways to link the genetic potential for nitrogen fixation to nitrogen fixation rates in the ocean (Zehr et al., 1998)

Nitrogenase gene (NifH) sequences amplified directly from oceanic waters showed that the open ocean contains more diverse diazotrophic microbial populations and more diverse habitats for nitrogen fixers than previously observed by classical microbiological techniques (Zehr et al., 1998). Understanding how fixed N regulates nitrogenase availability is necessary for devising strategies to increase the amount of ammonium synthesized by nitrogen fixing bacteria with the potential to be used in agriculture (Kennedy et al., 2004).

The commercial history of biofertilizer began with the launch of “Nitrogin” by Nobbe and Hiltner; a laboratory culture of rhizobia in 1895, followed by the discovery of Azotobacter and then the blue green algae and a host of other microorganisms. Azotobacter is used as a biofertilizer in the cultivation of most crops. Azotobacter is an obligate aerobic diazotrophic soil-dwelling organism with a wide variety of metabolic capabilities, which include the ability to fix atmospheric nitrogen by converting it to ammonia. Azotobacter naturally, fixes atmospheric nitrogen in the rhizosphere. There are different strains of Azotobacter each has varied chemical, biological and other characters. However, some strains have higher nitrogen fixing ability than others (Burgmann et al., 2003). Besides, nitrogen fixation, Azotobacter also produces, Thiamin, Riboflavin, indol acetic acid and gibberellins. When Azotobacter is applied to seeds, seed germination is improved to a considerable extent, so also it controls plant diseases due to above substances produced by Azotobacter (Kader et al., 2002.)

This NifH gene has been largely studied by culture-independent approaches. These approaches provide a more complete picture of the diazotrophic community than culture-based approaches. Various techniques, such as PCR cloning, denaturing gradient gel electrophoresis, PCR-restriction fragment length polymorphism (RFLP), and fluorescently labeled terminal (FLT)-RFLP, have been used to analyze the composition of NifH gene pools in various environments. These studies found that the NifH gene is present in diverse environments: forest soil, the rhizosphere of native wetland species, such as Spartina, or of crop species, such as rice, aquatic or polar cyanobacteria, and the bacteria found in termite guts. All these studies described a large number of unknown sequences which correspond to diverse unidentified diazotrophs. Some NifH genes are characteristic of an ecological niche (Shaffer et al., 2000) evoked the possible relationship between the habitats of soil nitrogen-fixing bacteria and the structure of NifH gene pools (Poly et al., 2001).

Nitrogen fixation in A. vinelandii is complicated by the presence of three biochemically and genetically distant nitrogenase enzymes, each of which is synthesized under different conditions of metal supply. The regulation of conventional molybdenum nitrogenase, whose subunits are encoded by the Nif-HDK genes and which is similar to the enzyme purified from number of other nitrogen-fixing organisms. (Sabra et al., 2000). The Nif-HDK genes are located in a large cluster of nif genes, which includes, in order, NifHDKTYENXUSVWZMF (Bali et al., 1992). Molecular methods based on universal PCR detection of nifH marker genes have been successfully applied to describe diazotroph population in the environment (Burgmann et al., 2003).

Materials and Methods

Sample collection
Samples were collected in different locations of Rameshwaram marine region (Gulf of Mannar) at the depth of 1–5 m. The randomly collected samples in the sterile plastic bags (soil sample) and water sampling bottles (water sample) bottles were kept in an ice-cold box and transported safely to the lab for further analysis with in 12 hrs. The sample with media tubes were packed and transported safely to the laboratory.

Isolation of Azotobacter from water and sediment samples (Mary et al., 1985)
Different selective media were used for the isolation of Azotobacter sp from marine source as described previously. Azotobacter strains used for this study were maintained and cultured in Burk medium as previously described (Joerger et al., 1988). As the isolates are of marine origin, the media were prepared by the 3.5% sodium chloride (NaCl). Media used for the isolation of nitrogen fixing organism (Azotobacter) from marine sources were Jensen’s agar medium, Azotobacter agar medium, Burk’s Medium and marine agar medium.

Culture characteristics (Bagwell et al., 1988)
Gram-staining characteristics and cell morphologies were determined by standard methods(Gerhardt et al., 1981). Motility was observed in wet mount using phase contrast microscope. Preliminary physiological characterization such as catalase test, starch hydrolysis test were also carried out.

Extraction and purification of DNA (Kelly et al ., 1990)
Azotobacter genomic DNA was isolated as previously described (Robson et al., 1980). Linear DNA fragments were analyzed by electrophoresis in agarose gel in TEB buffer (Maniatis et al., 1982). The purity of the DNA was checked spectrophotometric method by using the formula OD at 260 nm/ OD at 280 nm (Wilfinger et al., 1997).

PCR amplification of the NifH gene fragment

Nitrogenase Fe protein genes (NifH) were amplified from Azotobacter sp derived genomic DNA, using the primer from OPERON diagnostic Ltd, USA. The samples were amplified by PCR in a mixture containing reaction buffer 5.0 µl, 10mM dNTP 1.0 µl, primer 1 (25 mer) 1.0 µl, primer 2 (24 mer) 1.0 µl, template DNA 1.0 µl, enzyme Taq polymerase 1.0 µl for 35 cycles ( 1 min at 94° C,1 min at 54° C and 1 min at 72° C) (Zehr et al., 1988).

Results and Discussion
Totally 100 samples were collected in marine region of both water and sediments in the intervals of approximately 20 days .Out of 70 marine water samples collected, all the 70 samples were showing the presence of Azotobacter, but only 23 marine sediments out of 30 were showing the presence of Azotobacter. These samples were processed through the commonly used procedures such as selective media. Gram’s staining, Phase contrast observation for motility, starch hydrolysis test and Catalase test for identification of free- living diazotrophic organism i.e Azotobacter from the above samples, and that can be processed, result shows that Azotobacter sp are motile, gram negative, catalase and starch hydrolysis positve.

The colony morphology of Azotobacter strains were varying during the isolation in the selective media. The colonies were very clear, large, mucoid, watery due drops like initially i.e. from the marine source. The mother culture was sub cultured in the same media; the colony morphology differs slightly i.e. small, and circular, convex in nature. All the isolated Azotobacter strains were numbered for the easy identification and convenience. From these isolates, well defined pure culture of Azotobacter strains (1, 16, 27, 82, 101, 103, 108, 115, 125, and 132) were selected for the nucleic acid analysis.

The reference (standard) cultures such as Azotobacter beijerinckii (123), A. chroococcum (446), and A. vinelandii (124) procured from MTCC, (Chandigarh, India) were also used along with marine isolates for the nucleic acid extraction and purification. The DNA of the selected strains was isolated and estimated OD at 260 nm, the value ranges from 0.141 to 0.177. The estimated value of the extracted DNA was ranging from 0.70 to 0.88.The purity of the DNA was analyzed by spectrophotometric method using OD at 260 and OD at 280 nm. The presence and purity of DNA was checked by OD at 260nm/ OD at 280 nm, the value ranges from 1.13 to 2.21. If the estimated value is 1.8 conforms the presence of pure DNA. If the estimated value is lesser /greater than 1.8 conforms the presence of DNA to protein / RNA contamination, according to respective values DNA was purified using the enzymes proteases and RNase. The purified form of the DNA was separated by the agarose gel electrophoresis for the comparison the banding pattern between the randomly selected marine samples and the standard strains. There is no substantial difference between the banding patterns of the chromosomal DNA on the gel as shown in the plates. This result confirms that molecular weight of chromosomal DNA in all strains is similar.

One µl of DNA was used as template in PCR. selected primers, primer1:5-GGAATTCCTGYGAYCCNAARGCCNA-3,
Primer2:5-CGGATCCGDNGCCATCATYTCNCC-3 procured from OPERON diagnostics LTD, USA, respectively was used to amplify a 324-bp region between sequence positions 336 and 660. All N2 fixers carry a NifH gene, which encodes the Fe protein of the nitrogenase (Poly et al., 2001). The results of the PCR products were compared on 2% agarose gel electrophoresis. Selective NifH primer from Anabaena sp strain PCC7120 was used for the amplification of the Azotobacter sp, the primers used in my study was exactly matching the Azotobacter genome. Free-living nitrogen-fixing prokaryotes (diazotrophs) are ubiquitous in soil and are phylogenetically and physiologically highly diverse. Molecular methods based on universal PCR detection of the NifH marker gene have been successfully applied to describe diazotroph populations in the environment. However, the use of highly degenerate primers and low-stringency amplification conditions render these methods prone to amplification bias, while less degenerate primer sets will not amplify all NifH genes (Bürgmann et al., 2003).

References

Bagwell, C.E., Piceno, Y.M., Lucas, A.M. and Lovell, C.R., 1988. Physiological diversity of the rhizosphere diazotroph assemblages of selected salt marsh grasses. Appl. Environ. Microbiol., 64(11): 4276-4282.
Bali, A., Gonzalo Blanco, Susan Hill, and Christina Kennedy 1992. Execretion of Ammonium by a NifL Mutant of Azotobacter vinelandii Fixing Nitrogen. Appl. Environ. Microbiol., 58(5 ): 1711-1718.
Bürgmann H, Franco Widmer, William Von Sigler, and Josef Zeyer. New Molecular Screening Tools for Analysis of Free-Living Diazotrophs in soil. Environmental Microbiology, Vol.70, No1 p.240 – 247.1999.
Bürgmann H, Manuel Pesaro, Franco Widmer and Josef Zeyer strategy for optimizing quality and quantity of DNA extracted from soil. Bacteriological Reviews, vol.36, No.2 p.295-341. 2003.
Church M J, Cindy M. Short, Bethany D. Jenkins, David M. Karl, and Jonathan P. Zehr, Temporal Patterns of Nitrogenase Gene (NifH) Expression in the Oligotrophic North Pacific Ocean’ Ocean Sciences Department, Environmental Microbiology, Vol 134, No.1 p.155-193, 1999.
Gerhard,P.,R.G.E.Murray,R.N.Costilow,E.W.Nester,W.A.Wood,N.R.Krieg,andG.B.phillips.1981.Manual of method for generalbacteriology.American SOCIETY FOR microbiology, Washington, D.C.
Joerger, R.D., Jacobson, M.R., Premakumar, R., Wolfinger, E.W. and Bishop, P.E., 1989. Nucleotide sequence and mutational analysis of the structural genes (anfHDGK) for the second alternative nitrogenase from Azotobacter vinelandii. J. Bacteriol., 171(2): 1075-1086.
Kader.M.A, Mian.M.H, Hoque.M.S. Effect or Azotobacter inoculant on the yield and nitrogen uptake by wheat. Department of soil science, Bangladesh Agricultural University, Mymensigh, Bangladesh. Vol 2(4) p 251-261,2002.
Kelly, M.J.S., Poole, R.K., Yates, M.G. and Kennedy, C., 1990. Cloning and mutagenesis of genes encoding the cytochrome bd terminal oxidase complex in Azotobacter vinelandii: mutants deficient in the cytochrome d complex are unable to fix nitrogen in air. J. Bacteriol., 172(10): 6010-6019.
Kennedy.C, R.K.Poole, M.G.Yates, and M.J.S.Kelly. Cloning and Mutagenesis of Genes Encoding the Cytochrome bd Terminal Oxidase Complex in Azotobacter vinelandii: Mutants Deficient in the Cytochrome d Complex are unable To Fix Nitrogen in Air.Journal of Bacteriology, Vol.172,No.10 p.6010-6019 . 1990
Maniatis,T.,E.F.Fritsch,and J.Sambrook.1982. Molecular cloning: a laboratory manual. Cold spring Harbour Laboratory,Cold Spring Harbor,N.Y
Mary. L. G and rita r. colwell, Enumeration ,isolation and characterization of N2 fixing bacteria from sea water . Department of microbiology, University of Maryland. Vol 50 no .2. 1985
Poly .F, Lionel Ranjard, Sylvie Nazaret, François Gourbière, and Lucile Jocteur Monrozier.. Comparison of NifH Gene Pools in Soils and Soil Microenvironments with Contrasting Properties in Applied and Environmental Microbiology Vol. 67 p. 2255-2262. 2001
Robson,R.L.,J.A.Chesshyre,C.Wheller,R.Jones,P.R.Woodley, and J.R.Postgate. 1984.Genomic size and complexity in Azotobacter chroococcum.J.Gen Microbial.130:1603-1612.
Rubio .L M, singer.S.W, and Ludden P.W. Purification and Characterization of NafY from Azotobacter vinelandii. Department of plant and microbiology,college of natural resources, University of California, California. 2005
Sabra, W., Zeng, A.P., Lunsdorf, H. and Deckwer, W.D., 2000. Effect of oxygen on formation and structure of Azotobaceter vinelandii alginate and its role in protecting nitrogenase. Appl. Environ. Microbiol., 66(9): 4037-4044.
Shaffer, B. T., F. Widmer, L. A. Porteous, and R. J. Seidler. Temporal and spatial distribution of the NifH gene of N2-fixing bacteria in forests and clear-cut in western Oregon. Microb. Ecol. 39 P.12-21. 2000.
Wilfinger W.,Mackey K. and Chomczynski P.1997 Effect of Ph and ionic strength on the spectrophotometric assessment of nucleic acid purity. Bio Techniques,22,474-481.
Zehr .J. P ,Mark T. Mellon, and Sabino Zani. New Nitrogen-Fixing Microorganisms Detected in Oligotrophic Oceans by Amplification of Nitrogenase (NifH) Genes. 1998.
Zehr .J. P. Sarah Braun, Yibu Chen and Mark Mellon Nitrogen fixation in the marine environment: relating genetic potential to nitrogenase activity. Department of Biology, Rensselaer Polytechnic Institute, Troy, NY 12180-3590, USA . 1999.

Introduction

Atmospheric air is made up of nitrogen (71%), oxygen (28%) and other gasses (1%) including ozone which is altered by processes related to altitude, temperature and air pollution.1,2 Ozone (O3) is a gas with a characteristic, penetrating odour that is present in small amounts in atmospheric air. Ozone molecules are composed of three oxygen atoms and present naturally in the upper layer of atmosphere in abundance as long as sun is shining. 1,3, 4 It protects living organisms by surrounding the earth at altitudes of 50,000 to 100,000 feet from the ultra-violet rays.1,2,6  As it falls downward to earth, being heavier than air, and combines with any pollutant it comes in contact, and cleans the air. This is Mother Earth’s natural way of self-cleaning.3

Medical Ozone is made when medical grade oxygen is electrically activated (using an Ozone Generator) to form ozone.3 It is a mixture of the purest oxygen and purest ozone. According to its application, the ozone concentration may vary between 1 and 100 µg/ml (0.05-5%). The ozone therapist determines the complete dosage according to the medical/dental indication and the patient’s condition.7, 8

Ozone is an unstable gas and it quickly gives up nascent oxygen molecule to form oxygen gas. The release of nascent oxygen has beneficial effects on every part and organ.1 It has been used in medical field since long back due to its extremely strong oxidant property that oxidises nearly all surfaces to the highest oxidation stage. It is used as a circulatory enhancement and stimulation of oxygen metabolism, disruption of tumor metabolism and to kill pathogens.1, 2 O3 is a powerful oxidant capable of interacting as metabolic & immune modulator as well as anti-microbial agent. Multiple microbiological & the biochemical studies justified that there are no doubts about the effectiveness of ozone in bacterial reduction.9 Among other things, ozone is used to purify drinking water and water in dental equipment and for sterilising instruments for medical use.10

 

HISTORY

The German chemist Christian Friedrich Schönbein (1840), of the University of Basel in Switzerland is regarded as the Father of ozone therapy.7,11 When he passed an electrical discharge through water, a gas strange smell was produced, which he called Ozone, derived from the Greek word ozein whichmeans odor.1,2,6,11 In 1857Joachim Hänsler, a German physicist and physician, along with German physician, Hans Wolff, developed the first ozone generator for medical use. Dr. C. Lender in 1870, for the first time applied O3 into medical field. He purified blood in test tubes by using O3.2,3,6 Later, O3 application gained as a popularity as a therapeutic procedure throughout Europe and America. In 1881, it was used as a disinfectant in the treatment of diphtheria.3 Dr. Charles Kenworthy,a Florida physician, in 1885, published his experiences with ozone in the Florida Medical Association Journal.5 In October 1893, Ousbaden, Holland became the first city to utilize a water treatment plant using ozone.3 In World War I and II it was used to treat wounded soldiers in the trenches.11, 12 In early 20th centuryFood and Drug Act, revised its use and effect in the field of medicine. They considered it as perfectly legal to use in US.  Since then, it was used in the treatment of then deadly diseases such as tuberculosis, pneumonia, diabetes etc. It was also used to cure wounds, gangrene and the effects of poisonous gas. As of 1929, more than 114 diseases were listed for treatment with oxygen/ ozone therapy.5A German dentist, Dr. E.A. Fisch, in 1950 was the first dentist to use ozone on regular basis in his dental practice in Zurich, Switzerland and published numerous papers on its application. He used ozone to treat Ernst Payr, a renowned Austrian surgeon, who then became an ozone enthusiast and began a line of research dedicated to its use in healthcare. At that time, ozone therapy was difficult and limited due to the lack of ozone-resistant materials, such as Nylon, Dacron and Teflon, until when ozone-resistant materials were manufactured.1,2, 5, 6Beginning of dental-application research with the approval of an Institutional Review Board for Human Research from Capital University of Integrative Medicine in Washington, D.C., started in 2001. The first formal lecture on oxygen/ozone therapy was given in 2001 at Capital University. Later on, extensive research continued with the publication of Dr. Siegfried Rilling’s and Dr. Renate Viebahn’s text, “The Use of Ozone in Medicine.” This text was a standard until 2002, when Dr. Velio Bocci published “Oxygen/Ozone Therapy – A Critical Review.”  This was followed by first dental-applications clinical seminar in Louisville, Ky., in 2003. In 2004, Professor Edward Lynch of Belfast, United Kingdom, contributed to and edited the book “Ozone: The Revolution in Dentistry.”5 Numerous researchers since that time have worked to elucidate the nature and actions of ozone. Mariniak and Delarive showed that it is an allotropic form of oxygen, and Mulliken and Dewar clarified its molecular structure.6, 13

 

BIOLOGICAL ACTIONS

There are several known actions of ozone on human body, such as immunostimulating and analgesic, antihypoxic and detoxicating, antimicrobial, bioenergetic and biosynthetic (activation of the metabolism of carbohydrates, proteins, lipids) etc.6

The antimicrobial effect of ozone as a result of its action on cells by damaging its cytoplasmic membrane due to ozonolysis of dual bonds and also ozone-induced modification of intracellular contents (oxidation of proteins loss of organelle function) because of secondary oxidants effects. This action is non-specific and selective to microbial cells; it does not damage human body cells because of their major antioxidative ability. Ozone is very efficient in antibiotics resistant strains. Its antimicrobial activity increases in liquid environment of the acidic pH. In viral infections the ozone action lies in the intolerance of infected cells to peroxides and change of activity of reverse transcriptase, which takes part in synthesis of viral proteins.6
Ozone influences cellular and humoral immune system. It stimulates proliferation of immunocompetent cells and synthesis of immunoglobulins. It also activates function of macrophages and increases sensitivity of micro-organisms to phagocytosis.6 When administered at low concentrations, the organisms own resistance is mobilized, i.e. ozone (re)activates the immune system. 8 As a response to this activation through ozone, the body’s immune cells produce special messengers called cytokines. These molecules in turn activate other immune cells, setting off a cascade of positive change throughout the immune system, which is stimulated to resist diseases. This means that the application of medical ozone is extremely useful for immune activation in patients with a low immune status and/or immune deficit.7 Ozone causes the synthesis of biologically active substances such as interleukins, leukotrienes and prostaglandins which is beneficial in reducing inflammation and wound healing.6
Ozone brings about the rise of pO2 in tissues and improves transportation of oxygen in blood, which results in change of cellular metabolism – activation of aerobic processes (glycolysis, Krebs cycle, β-oxidation of fatty acids) and use of energetic resources. It also prevents formation of erythrocytes aggregates and increases their contact surface for oxygen transportation. Its ability to stimulate the circulation is used in the treatment of circulatory disorders and makes it valuable in the revitalizing organic functions.6
Ozone causes secretion of vasodilators such as NO, which is responsible for dilatation of arterioles and venules.6
It activates mechanisms of protein synthesis, increases amount of ribosomes and mitochondria in cells. These changes on the cellular level explain elevation of functional activity and regeneration potential of tissues and organs.6

A high concentration of ozone kills bacteria very quickly and is thousand times more powerful than other bacterial killing agents.The average concentration of ozone used in treatments is 25 µgm of ozone per millilitre of oxygen/ozone gas mixture that translates into 0.25 parts of ozone to 99.75 parts of oxygen. Evidence-based research has shown that at this concentration, ozone effectively kills bacteria, fungi, viruses and parasites.5 As an antimicrobial agent, it is a powerful oxidizer at a dramatically lower concentration than chlorine with none of the toxic side effects. One molecule of ozone is equal to between 3,000 to 10,000 molecules of chlorine and it kills pathogenic organisms 3,500 times faster.5Studies have revealed that it only takes 10 sec to kill 99 % of bacteria, fungi and viruses.14 It can oxidize many organic compounds and it is a powerful germicide.15 Some of the other effects are circulatory enhancement, disruption of tumor metabolism and stimulation of oxygen metabolism. 16

 

 

goals of ozone therapy

Setting the standard-of-care and therapeutic goals are based on sound evidence-based science is critical. Therapeutic goals are inclusiveand not exclusive of standard of care. The goals of oxygen/ozone therapy are5:

Elimination of pathogens.
Restoration of proper oxygen metabolism.
Induction of a friendly ecologic environment.
Increased circulation.
Immune activation.
Simulation of the humoral anti-oxidant system.

 

Systems for generating ozone gas in dentistry: 1,2

Ultraviolet system: Produces low concentrations of ozone, used in aesthetics and for air purification.
Cold plasma system: used in air and water purification.
Corona discharge system: produces high concentrations of ozone. It is the most common system used in the medical/ dental field. It is easy to handle and it has a controlled ozone production rate.

 

Forms of application 7,8

·         As an infusion into infected jaw bone (cavitation).
·         As an infusion into the temporomandibular joint for the treatment of pain and inflammation.
·         As an irrigant during new root canal therapy to disinfect the involved tooth.
·         Adjunctive therapy with the use of Ozonated Olive Oil for periodontal disease.

  INDICATIONS

In dentistry, ozone has got its role in various dental treatment modalities. Ozone therapy presents great advantages when used as a support for conventional treatments.1,7

Prophylaxis and prevention of dental caries.
Remineralisation of pit and fissure, root and smooth surface caries.
Restoration of open cavitations along with conventional conservative measures.
Bleaching of discoloured root canal treated teeth.
Endodontic treatment.
Desensitization of extremely sensitive tooth necks.
Soft tissue pathoses.
The treatment of infected, badly healing wounds and inflammatory process.

 

OZONE THERAPY IN PROSTHODONTICS

Microbial plaque accumulating on the dentures is composed of several oral microorganisms, mainly C. albicans. Denture plaque control is essential for the prevention of denture stomatitis. In an attempt to solve this problem Arita et al. assessed the effect of ozonated water in combination with ultrasonication on C. albicans. Following exposure to flowing ozonated water (2 or 4 mg/l) for one minute they found no viable C. albicanssuggesting the application of ozonated water might be useful in reducing the number of C.albicanson denture bases.17

 

OZONE THERAPY IN SURGERY

The influence of ozonized water on the epithelial wound healing process in the oral cavity was observed by Filippi. It was found that ozonized water applied on the daily basis can accelerate the healing rate in oral mucosa. This effect can be seen in the first two postoperative days. The comparison with wounds without treatment shows that daily treatment with ozonized water accelerates the physiological healing rate.18 Patients treated with ozone got healed more quickly without the need for systemic medication when compared to the control group. This finding suggested ozonated oil might be effective in the treatment of alveolitis.19 Application of ozone therapy after tooth extraction and in case of post-extraction complications, was found quite useful.20

 

OZONE THERAPY IN ENDODONTICS

Current research has been focused on determining guidelines for ozone use. The principle line of study has evolved using the ozone to determine its effect on several kinds of caries (pit and fissure,21 noncavity22, and primary root caries21,23,24,25). The oxidative impact on this microbiota has been recognized in several studies; however, there is a divergence of opinion regarding the amount of time that ozone gas should be applied. One study suggests application of ozone gas for a period of 10 to 20 seconds resulted in 99% of the microorganisms being destroyed 24,25. Another report states 40 seconds of application was insufficient to decontaminate the area and failed to act on underlying infected dentin26. Interesting data shows ozone does not affect the sealing ability of the bonding-system. Ozone therapy was also used in prevention of dental caries in fissures of the first permanent molars in children.20 An interesting data shows ozone does not affect the sealing ability of the bonding-system 27.

Polymicrobial infections complicate cases of apical periodontitis. As a result, current research is focused on finding treatment solutions with a high antimicrobial effect while minimizing injury to the periapical tissues. The oxidative power of ozone characterizes it as an efficient antimicrobial and its indication for use in endodontic therapy seems quite appropriate. Its antimicrobial action has been demonstrated against bacterial strains such as: Mycobacteria, Staphylococcus, Streptococcus, Pseudomonas, Enterococcus and E. coli 31,32 S. aureus33, E. faecalis 34,35 and C. albicans17 using in vitro research models. The ozone showed effectiveness over most of the bacteria found in cases of pulp necrosis, but not when the bacteria are organized in biofilm20 in vitro studies.

Bleaching1

In root canal treated teeth, crown discolouration is a major aesthetic problem, especially in anterior teeth. Conventional walking bleaching requires much more time and results are not often satisfactory. Also, capping the tooth with ceramic crown is not always a good idea. But, now ozone has the answer to all these questions. After placing the bleaching agent into the inner of the tooth, the crown is irradiated with ozone for minimum of 3-4 minutes. This ozone treatment bleaches the tooth within minutes and gives the patient a happy and healthier-looking smile.

Desensitization of Sensitive Root Necks1

Quick and prompt relief from root sensitivity has been documented after ozone spray for 60 seconds followed by mineral wash onto the exposed dentine in a repetitive manner. This desensitization of dentine lasts for longer period of time. Smear layer present over the exposed root surface prevents the penetration of ionic Calcium and Fluorine deep into the dentinal tubules. Ozone removes this smear layer, opens up the dentinal tubules, broadens their diameter and then Calcium and Fluoride ions flow into the tubules easily, deeply and effectively to plug the dentinal tubules, preventing the fluid exchange through these tubules. Thus, ozone can effectively terminate the root sensitivity problem within seconds and also lasts longer than those by conventional methods.

 

OZONE THERAPY IN PERIODONTICS

Dental biofilm makes it difficult for antibiotics in targeting putative periodontal pathogens. Higher concentrations of antibiotics are required to kill these organisms which are inevitably associated with toxic adverse effect on the host microbial flora. The application of ozone therapy in periodontics showed promising results. Both gaseous and aqueous ozone are used as a substitute to mechanical debridement. Ozonated water (4mg/l) strongly inhibited the formation of dental plaque and reduced the number of sub gingival pathogens both gram positive and gram negative organisms. Gram negative bacteria, such as P. endodontalis and P. gingivalis were substantially more sensitive to ozonated water than gram positive oral streptococci and C. albicans in pure culture. Furthermore ozonated water had strong bactericidal activity against bacteria in plaque biofilm. In addition, ozonated water inhibited the accumulation of experimental dental plaque in vitro. Aqueous ozone was found to be more biocompatible than gaseous ozone. It resulted in toxic effect on human oral epithelial and fibroblast cells compared to antiseptics such as chlorehexidine digluconate, sodium hypochlorite and hydrogen peroxide during a 1-minute time period.28,29 Ozone gas found to be toxic to the human oral epithelial and gingival fibroblast cells and aqueous ozone was more biocompatible than gaseous ozone.30 The application of ozone therapy in  chronic gingival and periodontal diseases, showed subjective and objective improvement of their status, as well as patients with periodontal abscess, with no exudation was observed.20  

 

OZONE THERAPY IN STOMATOLOGY

Sechi et. al evaluated the effect of ozonized sunflower oil on different bacterial species isolated from different sites. Ozone proved to be effective against all bacteria when tested, while mycobacteria were shown to be the most susceptible to the oil.31 Macedo and Cardoso described a case report of the application of ozonated oil on herpes labialis and mandibular osteomyelitis and demonstrated faster healing times than conventional protocols.36

 

 

Ozone Toxicity

Ozone inhalation can be toxic to the pulmonary system and other organs. Complications caused by ozone therapy are infrequent at 0.0007 per application. Known side-effects are epiphora, upper respiratory irritation, rhinitis, cough, headache, occasional nausea, vomiting, shortness of breath, blood vessel swelling, poor circulation, heart problems and at a times stroke.4Because of ozone’s high oxidative power, all materials that come in contact with the gas must be ozone resistant, such as glass, silicon, and Teflon. However, in the event of ozone intoxication the patient must be placed in the supine position, and treated with vitamin E and n-acetylcysteine.2

 

CONTRAINDICATIONS 1,2,6

The following are contraindications for the use of ozone therapy:

Pregnancy.
Glucose-6-phosphate-dehydrogenase deficiency (favism).
Hyperthyroidism.
Severe anaemia.
Severe myasthenia.
Acute alcohol intoxication.
Recent myocardial infarction.
Haemorrhage from any organ.
Ozone allergy.
 
The Benefits of Ozone Dental TREATMENT 14

Ozone treatment is excellent as a preventative measure against future decay and dental work needed to correct the damage caused by bacteria. Benefits also include:

Eliminates the use of drills and fillings.
Eliminates the use of anaesthetics.
Kills 99% of bacteria in cavities.
Can whiten discoloured cavities.
Excellent for nervous or anxious patients.
Reduces inflammation and pain sensation.

 

CONCLUSION

Dentistry is changing as we are now using modern science to practice dentistry. The ozone therapy has been more beneficial than present conventional therapeutic modalities. This state of the art technology allows us to take a minimally invasive and conservative approach to dental treatment. The elucidation of molecular mechanisms of ozone further benefits practical application in dentistry. Treating patients with ozone therapy reduces the treatment time with a great deal of difference and it eliminates the bacterial count more precisely. The treatment is completely painless and increases the patients’ acceptability and compliance with minimal adverse effects.

 

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