Summary of

Report on the Negative Air Ion Generator ("Biogun")

Dr E Lynch,

Senior Lecturer, Department of Conservative Dentistry,

St Bartholomew's and the Royal London School of Medicine and Dentistry, London E1 2AD

Professor D Beighton,

Oral Microbiology, Royal College of Surgeons, Kings College School of Medicine and Dentistry, London SE5 9RW

INTRODUCTION

A negative air ion generator, known as the "Biogun", can deliver a concentrated stream of negative air ions. The generator acts by applying a high direct voltage to an electrode known as an emitter mounted in a probe. A stream of electrons is generated which bond to surrounding molecules of Oxygen forming the hydrated superoxide anion (O₂^-)(H₂O)n where n=4-8. This is understood to act as a nucleophile on the phospholipid bilayer which causes a de-esterification of the fatty acids and weakening the membrane of unicellular micro-organisms (Kellogg et al. 1979).

The negative air ion system has been shown to be effective in inhibiting the growth of micro-organisms implicated in the aetiology of dental caries and primary root caries in particular (Cousins et al. 1991, Burke et al. 1995a, Burke et al. 1995b) in vitro.

AIM

The aim of this study was to determine the efficacy of negative air ions to kill the micro-organisms implicated in the aetiology of primary root caries in vivo and to evaluate the clinical effects of exposure to negative air ions.

MATERIALS AND METHODS

Study Population

The study population was selected from regular dental patients of the St Bartholomew's and the Royal London School of Medicine and Dentistry. Patients with primary root caries lesions were selected. The procedure to be carried out was explained and written consent obtained.

 The criteria for inclusion in the study are that the participants:

 (a) be aged over 35 years

 (b) have one primary root caries lesion

 (c) be considered reliable for attendance for treatment

 (d) have given written, informed consent

Diagnostic Criteria

The following parameters was recorded at the dental examination:

1) Coronal caries (DMFT) according to WHO criteria (1987)

2) Partial denture wearing status.

3) Colour: Photographs of primary root caries were obtained and a four shade guide was developed. The colours were; yellow, light brown, dark brown and black. They were used as the standard when determining the colour of each lesion under investigation.

4) Dimensions: A periodontal probe marked at 1 mm intervals was used to determine the dimensions of each lesion. The maximum mesio/distal or bucco/palatal (width) and occluso-gingival (height) dimension was assessed. The product of these two values was used as an indicator of the size of the lesion. Furthermore  the minimum distance from the gingival margin of the lesion and the crest of the gingiva itself was measured.  An estimate of the amount of dentine that has been lost, cavitation or maximum loss of surface contour, was made by recording the greatest distance between the existing surface of the lesion and what was judged to have been the original root surface.

5)Texture: Three categories for the texture of each carious lesion were defined:

a)   'Hard': the caries was comparable to the remaining sound root dentine.

b)   'Leathery' caries will permit the penetration by a new No. 6 probe (Claudius Ash Sons & Co Limited, Potters Bar, Herts, UK) of the root tissue under moderate pressure and there was some resistance to withdrawal.

c)   'Soft' lesions will permit a new No. 6 probe to penetrate the root tissue with ease and with no resistance to withdrawal.

6) Perceived Treatment Need: This was defined relative to their clinical signs (Beighton et al 1993):

Leathery Debride: Leathery lesions that were judged to be shallow and the surface of the exposed sound dentine easily maintained plaque free.

Leathery Restore: all leathery lesions judged to be on root surfaces that were difficult to maintain plaque free.

Soft Restore: All soft lesions.

Samples for Microbial Analysis

The primary root caries lesions were sampled before and after application of the negative  air ions. The primary root caries lesions were first cleansed to remove plaque and any other material that might contaminate the sample of carious dentine. A hand held standard fine nylon fibre sterile toothbrush was used with sterile water as a lubricant to cleanse the surface but to avoid the removal of any carious surface dentine the surface were dried using a 3-in-1 syringe. A preimpression sample was taken. An impression of each lesion will then be taken using an addition-cured silicone (Extrude putty, Kerr, USA) by the standardised method which we have developed. The tooth will then be isolated using sterile cotton wool rolls and dried using a 3-in-1 syringe and a dry sterile cotton wool roll. A new size 2 stainless steel rosehead bur in a ten-in-one reduction head in a slow speed handpiece was used to remove the sample of carious dentine. Pressure comparable with that used in normal clinical practice was used. Each biopsy was then immediately placed in 1 ml of Fastidious Anaerobe Broth (FAB) (LabM, Bury Lancs, UK) and forwarded to the laboratory at King's College immediately for bacteriological investigation.

The Negative Air Ion System

A negative air ion generator, known as the "Biogun", can deliver a concentrated stream of negative air ions. The generator acts by applying a high direct voltage to an electrode known as an emitter mounted in a probe. The negative air ions have been shown to inhibit the growth of micro-organisms. The negative air ion system has been tested by the British Chiropody Association. No adverse effects have been reported and it has been shown to be effective in the management of verrucae, athlete's foot and hypergranulation (Lyall 1992, Stephens 1993).

 A stream of negative air ions was applied to each primary root caries lesion. The patient was earthed by wearing a wrist band with a metal button. The tip of the negative air ion generator was held at a distance of 5 mm from the surface of the lesion and a current of 60 mA applied for two minutes. The tip was moved slowly backwards and forwards over the area of the carious lesion to be treated. A further sample will then be taken from using the same technique as before the application of the negative air ions. The sample site was from an area distal to the initial sample site. The negative air ions were applied for a further two minutes and another sample was taken from another site distal to the previous sample site. Each sample was then immediately placed in 1 ml of Fastidious Anaerobe Broth (FAB) (LabM, Bury Lancs, UK) and forwarded to the laboratory within 30 minutes for bacteriological investigation. A further impression of each lesion was taken using an addition-cured silicone (Extrude putty, Kerr, USA). Given the sequence of sampling the pre-treatment site was the most mesially located site and the site treated for four minutes was the one located furthest distally.

Laboratory Methods

Sample Processing: To each 1 ml of FAB containing a biopsy of carious or negative air ion treated carious dentine sterile 3.5 to 4.5 mm diameter glass beads (BDH Limited, Poole, Dorset, UK) was added. They were vortexed for

15 seconds to facilitate the extraction of any micro-organisms from the carious dentine and disperse any aggregates. Then decimal dilution with FAB, 100 ml aliquots of these being spread as appropriate onto a range of culture media in plates.

Mutans Streptococci: (S mutans and S sobrinus) were enumerated on Mitis-Salivarius Agar (Difco Laboratories, Teddington, Surrey, UK) supplemented with 0.2 units per ml bacitracin and 15% (W/V) sucrose, MSB (Gold et al. 1973)  in an anaerobic chamber (Don Whitley, Shipley, West Yorkshire, UK)  at 37^oC for three days. The total number of organisms in each sample were determined by counting the colonies identified on each plate. Their identities were confirmed by subjecting up to five of these colonies to further examination by using a set of fermentation and enzymic tests. Tests for the production of acid from N-acetylglucosamine, arbutin and melibose, as well as the presence of a-galactosidase and a-glucosidase activities were useful in differentiating these species (Beighton et al. 1991).

Lactobacilli: were  grown on Lactobacillus Selective Agar LBS (Oxoid Limited, Basingstoke, Hampshire, UK) in an anaerobic chamber (Don Whitley, Shipley, West Yorkshire, UK)  at 37^oC for three days. The total number of organisms in each sample was determined by counting the typical colonies grown. They were confirmed as Gram-positive, catalase-negative and unbranched rods (Beighton et al. 1991b).

Yeasts: were grown on Sabouraud Dextrose Agar (Oxoid Limited, Basingstoke, Hampshire, UK) incubated in air at 37^oC for two days. The number of organisms were determined by counting the number of typical colonies on each plate. They were confirmed as large, ovoid Gram-positive, catalase positive cells (Beighton et al. 1991b).

Gram-positive pleomorphic rods (GPPRs): were grown of Fastidious Anaerobic Agar (LabM, Bury, Lancs, UK) supplemented with 5% (V/V) horse blood (FAA, LabM) in an anaerobic chamber (Don Whitley, Shipley, West Yorkshire, UK)  at 37^oC for seven days. The number of organisms including

Actinomyces spp were confirmed by examination of the Gram-stained smears of colonies on the Fastidious Anaerobic Agar  plates. The number of each colony type was counted and representatives of each examined so that the numbers of Gram-positive pleomorphic rods in each sample could be calculated. Because of the difficulties associated with the speciation of the Gram-positive pleomorphic rods, especially the identification of Actinomyces spp (Johnson et al. 1990) all Gram-positive pleomorphic rods were grouped together as a single taxon. From these plates the total numbers of all colony forming units (cfu) were also determined.

 The detection limit for mutans streptococci, yeasts and lactobacilli is 10 cfu per sample, and for the GPPR it is approximately 0.2% of the number of cfu present in a sample. If no colonies of a given taxon were recovered from a sample a value of zero was included in the analysis.

 Sample Volume

The impressions of the sample sites were scanned using a co-ordinate measuring machine. It was possible to superimpose the scans of the lesions before and after sample taking and so determine the volume, site and dimensions of the samples taken.

 Data Analysis

The total number of colony forming units (cfu) per sample biopsy were determined by translating the numbers of cfu grown on the FAA plates through the dilutions that were deployed to relate them to the total sample. These were transformed to log₁₀(colony count +1) to normalise the distributions of the individual colony counts. The numbers of each of the various categories of organisms per sample were determined in the same way and the proportions of all organisms in each sample also expressed as a percentage of the total colony count on the FAA plates. Means and standard errors of values will also be calculated, mean values being calculated by one way analysis of variance using Duncan's multiple range test, and distributions were analysed by the Chi squared statistic(s). The frequency of isolation of

individual taxa from each primary root caries lesion were recorded as zero if fewer than 10 cfu of that particular taxon are recovered from the biopsy. All statistical analyses were performed with the statistical suite of programmes: SPSS/PC + V3.0 (SPSS Inc., Chicago, Illinois, USA)

RESULTS

Sample Dimensions

The samples of carious tissue were taken from the gingival margin of the primary root carious lesions with the first sample being the most mesially located with further samples being located progressively further distally. The average dimensions of the samples of carious tissue were 0.583 mm width, 0.794 mm length and 0.152 mm depth. The average volume was 0.137 mm³. The consistency of the volumes of dentine removed indicate that the standardised sampling procedure enabled the removal of very similar volumes of dentine and confirm that, overall, the sampling procedure was valid.

 Microbial data.

The microbiological data are summarised in Table 4. The reductions in the numbers of yeasts, lactobacilli, mutans streptococci, Actinomyces (GPPR) and total microbial count were generally similar in both groups and after 3 and 6 months the microbial recoveries were not different from the samples taken at baseline.

Table 4. Microbial data

The data are for mean Log₁₀(CFU+1) +(SE) of the microflora involved.

Clinical Effect of Treatment

The carious lesions were categorised according to the Perceived Treatment Need. The changes in the clinical status of the perceived treatment needs of the lesions in the study is shown in Table 6. There was a significantly better clinical improvement in the test group compared to the control group. It can be seen that after three months while three of the lesions in the control group (8.8%) improved clinically fifteen of the lesions in the test group (41.7%) improved. Ten of the lesions in the control group  (29.4%) got worse clinically after three months while only two of the test lesions (5.6%) disimproved. After six months while two of the lesions in the control group (13.3%) improved clinically fifteen of the lesions in the test group (50.0%) improved. Seven of the lesions in the control group  (46.7%) got worse clinically after three months while only three of the test lesions (10.0%) got worse.

Table 6. Change in treatment need of primary root carious lesions

 Analysis of the data using Chi² analysis demonstrated that there was some significant improvement in the Test group compared to the control group at 3 months and also at 6 months, particularly in the lesions designated soft restore. Comparison of the leathery lesions indicated no significant difference at 3 months (c² = 2.54; ns) but at 6 months the difference was significant (c² = 4.94; p=0.026). For the leathery restore lesions the  differences at 3 and 6 months were not significantly different (c² = 4.67 and c² = 2.97, respectively). However for the soft restore lesions there was a significant difference between test and control at 3 months (c² = 6.4; p=0.011) while at 6 months the difference approached significance (c² = 3.59; p=0.058).

 Consideration of the treatment data as a whole indicates that 15 of 36 lesions in the Test group improved while 3 of 34 lesions in the Control group improved at 3 months while at 6 months 15 of 30 lesions in the test group exhibited improvement and only 2 of 15 in the Control group. Analysis of the distribution of these data indicate that the effect of exposure to the negative air ions was significant: (c² = 13.29; p=0.0012 and c² = 9.61; p=0.0082 at the 3 and 6 month interval, respectively).

Influence of NAI on the size of root caries lesions.

The size of the lesions at baseline and at the subsequent sampling times are shown in Table 9 while the changes in the size distribution of the lesions are shown in Table 10.

Table  10. Change in size of primary root carious lesions

A significantly greater proportion of the Test lesions exhibited a decrease in size compared to the control group at 3 months (c² = 8.16; p=0.0169) but this difference was no longer demonstrable after 6 months (c² = 5.16; p=0.076)

Influence of the NAI on the cavitation of root caries lesions.

The cavitation of lesions at baseline, 3 and 6 months following exposure to the negative air ions is shown in Table 11 while the changes in the measured cavitation of lesions is shown in Table 12.

   
Table  12. Change in cavitation of primary root carious lesions

At 3 months the changes in the cavitation approached significance (c² = 5.17; p=0.07) but was not significant after 6 months. Comparing the data at 3 months as those lesions which became shallower with the number that remained the same or got deeper the Test group was significantly better (c² = 3.94; p=0.047)

ACKNOWLEDGEMENT

This work arises from an invention by J H L Copus. The financial support from Dentron Ltd is also gratefully acknowledged.

REFERENCES

Beighton D, Hardie J, Whiley R. A scheme for the identification of viridans streptococci. Journal of Medical Microbiology 1991a;35:367-372.

Beighton D, Russell R R B, Whiley R. A simple biochemical scheme for the differentiation of streptococcus mutans and streptococcus sobrinus. Caries research 1991b;25:174-178.

Beighton D, Lynch E J R, Heath M R. A microbiological study of primary root caries lesions with different treatment needs. J Dent Res 1993;72:623-629.

Burke F M, Lynch E, Beighton D, Ludford R. Negative Air Ion effect on the viability of Actinomyces naeslundii isolated from active primary root-caries. Caries Res 1995;

Burke F M, Samarawickrama D Y D, Johnson N D, Beighton D, Lynch E. Use of negative air ion treatment on carious microflora. J Dent Res 1995;

Cousins D, Copus J, Wilson M. Microbicidal effects of negative air ions. J Dent Res 1991;70:709,315.

Gold O G, Jordan H V, van Houte J A. A selective medium for streptococcus mutans. Archives of Oral Biology 1973 ;18:1357-1364.

Johnson J L, Moore L V H, Kaneko B, Moore W E C. Actinomyces georgiae sp. nov. Actinomyces gerencseriae sp. nov., designation of two genospecies of Actinomyces naeslundii,  and the inclusion of A naeslundii serotypes II and III in A naeslundii genospecies 2. International Journal of Systematic Bacteriology 1990;40:273-286.

Kellogg E W , Yost M G, Barthakur N, Kreuger A P. Superoxide involvement in the bactericidal effects of negative air ions on Staphyloccus albus. Nature 1979;281:400-401.

Lyall A. A preliminary clinical trial with the Dentron Biogun. SMAE Journal 1992;52:6-8.

Lynch E J R. The diagnosis and management of primary root caries. PhD thesis, University of London 1994.

Stephens D.  Biogun versus verruca. SMAE Journal 1993;53:10-12.

Who Oral Health Surveys - Basic Methods, World Health Organisation, Geneva (1987).

Fitzgeorge R: Effect of negative air ion treatment on skin (Dentron ioniser).

Shakespeare P: Effect of Dentron "Biogun" on cultured human keratinocytes.

Shargawi JW, Drucker DB, Duxbury AJ: Effect of negative air ion (NAI) streams on Candida albicans. J Dent Res 1995;74: 887, 526.

Shargawi J, Drucker D, Duxbury A: Relationship between ozone, negative air ions and anti-candidal effects. J Dent Res 1996;75:350, 2664.

This summary was condensed by J H L Copus from an original open to inspection on request.

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