Saliha Saad

-rinse formulations on oral malodour

Keywords: Immunology and Microbiology, Mouth-rinses oral malodour Oral malodour, mouth rinse, volatile sulphur compounds, hydrogen sulphide, zinc, chlorhexidine, organoleptic, Halimeter, OralChroma, anaerobic bacteria

Comparative effects of various commercially available mouth

A significant source of oral malodour is from organisms on the surface of the tongue with microbes inhabiting the tongue biofilm being responsible for approximately 80% of cases of oral malodour (Yeagaki and Sanada, 1992;[START_REF] Rosenberg | Experiences on an Israeli malodour clinic[END_REF][START_REF] Van Den Broek | A review of the current literature on management of halitosis[END_REF]. The particular papillary surface of the tongue with its large number of crypts and fissures allows it to harbour a high number of bacteria in a relatively anaerobic environment [START_REF] Tonzetich | Production and origin of oral malodour: a review of mechanisms and methods of analysis[END_REF]. The extremely diverse microflora particularly Gram-negative anaerobes possess enzymes that allow biotransformations of sulphur-substrates (cysteine, methionine and glutathione) into volatile sulphur compounds (VSC) [START_REF] Kleinberg | Oral malodour[END_REF]Westbay, 1990: Scully et al., 1997). The main VSC in oral malodour is believed to be hydrogen sulphide (H 2 S), although methyl mercaptan (CH 3 SH) [START_REF] Tonzetich | Direct gas chromatographic analysis of sulphur compounds in mouth air in man[END_REF][START_REF] Yaegaki | Volatile sulfur compounds in mouth air from clinically healthy subjects and patients with periodontal disease[END_REF] and dimethyl sulphide (CH 3 ) 2 S may also play a role [START_REF] Quirynen | Management of oral malodour[END_REF][START_REF] Suarez | Morning breath odor: influence of treatments on sulfur gases[END_REF]. In addition to producing bad breath, VSC produced by periodontopathogens in the gingival crevice have been implicated in the aetiology of periodontal disease resulting in tooth loss if left untreated (Shapiro et al., 1977;[START_REF] Radcliff | The relationship between oral malodour, gingivitis and periodontitis, a review[END_REF]. Other volatile organic compounds (VOC) contribute to an unknown extent to oral malodour and they are thought to include indole, amines and acids [START_REF] Kostlec | Salivary volatiles as indicators of periodontitis[END_REF][START_REF] Goldberg | Cadaverine as a putative component of oral malodour[END_REF][START_REF] Radcliff | The relationship between oral malodour, gingivitis and periodontitis, a review[END_REF].

Numerous mouthwashes are available for use as part of a daily oral hygiene routine. The formulations contain actives that may inhibit microbial growth, enzymatic reactions or may react directly with VSC to reduce their levels in the breath. In addition, these formulations may include flavour-compounds, which can mask the effects of odiferous compounds.

Certain metal ions, in particular zinc (Zn), are well known to reduce or inhibit the formation of VSC [START_REF] Tonzetich | Direct gas chromatographic analysis of sulphur compounds in mouth air in man[END_REF][START_REF] Yaegaki | The effect of zinc chloride mouthwash on the production of oral malodour, the degradations of salivary cellular elements and proteins[END_REF][START_REF] Young | The oral anti-volatile sulphur compound effects of zinc salts and their stability constants[END_REF] as do certain antibacterial agents such as chlorhexidine (CHX) and cetylpyridinium chloride (CPC) with a subsequent reduction in oral malodour [START_REF] Denton | Chlorhexidine Disinfection, Sterilization and Preservation[END_REF][START_REF] Loe | The effect of mouthrinses and topical applications of chlorhexidine on the development of dental plaque and gingivitis in man[END_REF][START_REF] Lang | Toothbrush frequency as it is related to plaque development and gingival health[END_REF][START_REF] Grossman | A comparative clinical study of extrinsic tooth stain removal with two electric toothbrushes (Braun D7and D9) and a manual brush[END_REF][START_REF] Young | The oral anti-volatile sulphur compound effects of zinc salts and their stability constants[END_REF][START_REF] Winkel | Clinical effects of a new mouthrinse containing chlorhexidine, cetylpyridinium chloride and zinclactate on oral halitosis A dual-center, double blind placebo-controlled study[END_REF][START_REF] Roldan | The effect of a new mouthrinse containing chlorhexidine, cetylpyridinium chloride and zinc lactate on the microflora of oral halitosis patients: a dual-centre, double-blind placebocontrolled study[END_REF]. The combination of low concentrations of Zn and CHX seems to be particularly effective [START_REF] Young | The oral anti-volatile sulphur compound effects of zinc salts and their stability constants[END_REF][START_REF] Winkel | Clinical effects of a new mouthrinse containing chlorhexidine, cetylpyridinium chloride and zinclactate on oral halitosis A dual-center, double blind placebo-controlled study[END_REF]. More evidence is emerging for the efficacy of this combination including double-blind comparisons with several widely used formulations against halitosis. The studies have in the main used gas chromatography (GC) to measure VSC [START_REF] Tonzetich | Gas chromatographic method for trapping and detection of volatile organic compounds from human mouth air[END_REF][START_REF] Yaegaki | Volatile sulfur compounds in mouth air from clinically healthy subjects and patients with periodontal disease[END_REF][START_REF] Rosenberg | Clinical assessment of bad breath: Current concepts[END_REF].

Two common approaches to assessing oral malodour include halimetry and organoleptic measurements by a trained odour judge [START_REF] Rosenberg | Reproducibility and sensivity of oral malodour measurements with a portable sulphide monitor[END_REF][START_REF] Boever | Relationship between volatile sulfur compounds, BANA-hydrolyzing bacteria and gingival health in patients with and without complaints of oral malodor[END_REF]. More recently another instrument has been commonly employed; a portable GC system (OralChroma®). Organoleptic assessments by a trained judge have been shown to correlate with halimetry [START_REF] Rosenberg | Reproducibility and sensivity of oral malodour measurements with a portable sulphide monitor[END_REF][START_REF] Boever | Relationship between volatile sulfur compounds, BANA-hydrolyzing bacteria and gingival health in patients with and without complaints of oral malodor[END_REF] but the relationship between these measurements and Oral Chroma have not been widely studied. preparations and a negative control using both organoleptic measures and a halimeter. A secondary aim was to compare these results with those obtained using an OralChroma®.

The hypothesis to be tested in this study was that the combination of Zn and CHX in low concentrations is at least as effective as a selection of other currently used antibacterial agents/mouth rinses against malodour and VSC concentrations. By testing the active formulations against a negative control (water), information could be gained as to the efficacy of test compounds in terms of their immediate (within 30 minutes) and intermediate (3 hour) effects.

Materials and methods

Human subjects

Fourteen volunteers from the University of the West of England were selected from a database of volunteers previously screened for inclusion in malodour trials. The panel included 8 female and 6 male with a mean age at onset of 39 years (range 23 -64). They were all healthy adults with no sign of oral disease.

Study design

The study was double-blind and neither judge, technician or panellists knew which product was administered for all test days. Test days were one week apart. Each subject was randomly assigned a label 1 to 14. The mouthwashes were assigned letters A to F. All products were dispensed into 15 ml volumes by an independent technical member of staff. The volunteers rinsed for 2 minutes for each mouthwash. Each subject received all test products in random order thereby acting as their own control.

Eligibility criteria included informed consent and availability at the specified study intervals and sampling times plus a baseline organoleptic malodour score of >2 on each study morning. Exclusion criteria included; medical history of infectious diseases (e.g. hepatitis, HIV, tuberculosis); obvious gingival inflammation, active or severe caries, gingivitis or advanced periodontitis and oral thrush; antibiotic medication within 1 month prior to the start of the trial or during the trial period; consumption of medicated sweets containing antimicrobial agents; changes in oral hygiene practices during the trial; consumption of foods associated with oral malodour (such as garlic, spices or alcohol) on the day prior to, and on the sampling day; wearing of strongly perfumed cosmetics on the sampling day and substantial false dentition. On the evening prior to the test day volunteers were instructed to continue their normal oral hygiene habit but on the morning of their assessments they were asked to avoid oral hygiene (brushing their teeth) and food intake.

All participants were provided with their individual protocol, a diary and appointment dates/times for attending the laboratory. An adverse reaction form was available on request from the principal investigator. With the exception of the treatment mornings, subjects were not asked to alter their normal oral hygiene regime throughout the 6-week study.

Oral test rinses

Five oral rinses, all of which are commercially available, were compared along with water as the negative control; SB12 ® , Listerine ® , BreathRX ® , Smarth Mouth ® and Lacer Fresh ® . Table 1 lists the mouthrinses, the manufacturers and a summary of their ingredients (and amounts) as far as this information is available.

Ethics and study conduct

The protocol and informed consent form were approved by the local Ethics Committee. The study was conducted in a manner consistent with the ethics encompassed within the 'Declaration of Helsinki'.

Organoleptic assessment

One trained odour judge scored breath odour levels using the 0 to 5 organoleptic scale as outlined by [START_REF] Rosenberg | Reproducibility and sensivity of oral malodour measurements with a portable sulphide monitor[END_REF] and modified in term of odour descriptives by [START_REF] Greenman | Study on the organoleptic intensity scale for measuring oral malodor[END_REF], 0 = no odour, 1 = barely noticeable, 2 = slight odour, 3 = moderate odour, 4 = strong odour, 5 = very strong odour (saturation).

Instrumental analysis:

Measurements with Halimeter and OralChroma were taken according to the manufacturer's instructions. Two halimeter readings were taken and the calculated average was recorded as ppb. OralChroma readings were taken using a 1ml gas sample from a "2-minute" closed mouth via plastic syringe.

H 2 S was obtained by measurements of area-under-curve (AUC) of the separated chromatographic peaks from the output trace. However, due to the 10 minute time period required for running samples, only one sample per person per time point was taken.

Trial procedures

On the test day, volunteers reported to the breath odour judge who carried out a baseline breath assessment. Two assessments were taken within 5 minutes and an average value calculated for each time point. Following organoleptic assessment, the laboratory technician undertook baseline halimeter and OralChroma readings. The volunteers where then given 15 ml of one of six test mouthrinses, in a randomised and double-blind manner and instructed to rinse the mouth for two-minutes. The breath assessments and instrumental readings were repeated at 30, 60, 90 and 180 minutes following test or control "treatment". The volunteers were not allowed to eat or drink between sampling. Excel Microsoft and goodness of fit expressed as the coefficient of determination (r 2 ).

Statistical analysis

Results

Table 2 shows the range and overall average readings for the pretreatment (baseline) conditions for the three measured parameters. Since this involved readings from 14 individual trialists on 6 different occasions (5 treatments and control) the total data points are n = 84. The mean and range are suitable for a designed study to show reductions in malodour parameters. Efficacy in terms of reduction in VSC compared to control substance F (water) as measured by the Halimeter (figure 1 and table 3) varied amongst the mouthrinse formulations, ranging from no significant effect with product D (LacerFresh ® ), moderate effect for products B (BreathRX ® ) and C (SmartMouth ® ) p≤0.01, to good marked with products E (SB12 ® ) and A (Listerine ® ) p≤0.001.

Comparing the Halimeter TM readings between products, SB12 ® (p≤0.01) and Listerine ® (p≤0.05) both showed statistical separation from LacerFresh at all time points using ANOVA plus Bonferroni's Multiple Comparison post test. The separation for SB12 was larger and was maintained throughout the 3 hour observation period.

Efficacy as measured by reduction in breath odour using the organoleptic scale showed water to have a very slight breath reduction at 30 minutes, but then odour levels increase to above the initial, time zero, pre-treatment level.

All products separated statistically from water at all time points (range p ≤ 0.05 -0.001). As can be seen in figure 2, product D (LacerFresh ® ) had the least benefit, products A (Listerine ® ), B (BreathRX ® ) and C (SmartMouth ® ) show more marked reductions whilst product E (SB12 ® ) reduced breath odour levels to a measurably greater extent than all other products, and maintained the reduction up to 180 minutes.

SB12 ® , (figure 2, table 3), showed statistical separation from LacerFresh ® at all time points (p<0.001), from SmartMouth ® at 90 minutes (p≤0.05) and at 180 minutes (p<0.001), and from Listerine ® and BreathRX ® by 180 minutes (p<0.01). Listerine ® showed statistical separation from LacerFresh ® at all time points (p≤0.05). Smart Mouth ® and BreathRX ® separated from LacerFresh ® from 60 minutes (p<0.001).

The organoleptic data support the Halimeter TM results with all products maintaining their positions of efficacy F<D<C<B<A<E. Figure 3 shows the results obtained for H 2 S using the OralChroma TM . These data followed a similar profile of reduction and recovery over time as halimetry or organoleptic scores. Relationships between organoleptic scores, Halimeter TM , and OralChroma were between R 2 = 0.795 to 0.926 as seen in figures 4-6.

Discussion

Oral Five oral rinses, SB12 ® (containing a low concentration of Zn and CHX), Listerine ® , BreathRX ® , SmartMouth ® and Lacer Fresh ® , all of which are commercially available, were compared along with water as the negative control. The odour-inhibiting capacity of the mouthwash formulations was determined using the organoleptic scale, the Halimeter and the OralChroma. Malodour levels of 14 orally healthy volunteers were assessed at baseline and at the same time periods during the day. The organoleptic assessment of individuals prior to and after treatment was performed by one trained odour judge in a completely double-blind manner. It is well accepted that humans have the capacity to determine the strength (i.e. concentration) of odour molecules. Models relating the organoleptic score to the occupancy of odour binding sites (degree of receptor saturation) have been proposed [START_REF] Greenman | Study on the organoleptic intensity scale for measuring oral malodor[END_REF]2005). Judges can be trained to score the strength of odour (0 to 5) and it is clear that to have a useful meaning, a zero score must relate to no detectable odour and a 5 must be as strong as it gets. When subjected to pure odour compounds of known concentrations, judges are able to discriminate and respond in a dose dependant manner even when the order of concentrations is randomised. Moreover, the judges can repeat their measurements at a later date and be shown to give similar (reproducible) responses. Another useful method to validate the organoleptic judge is to see how their scores compare with other objective measurements of the same or similar samples, using an instrumental gas sensor (e.g. Halimeter) or GC.

In this study, it was important to see whether any correlations between sensory and instrumental measurements existed so that one type of measure could be used to validate the other. Although some reports have shown a relationship between organoleptic score and either halimeter or GC [START_REF] Rosenberg | Reproducibility and sensivity of oral malodour measurements with a portable sulphide monitor[END_REF][START_REF] Doran | A clinical study on the antimicrobial and breath-freshening effect of zinc-containing lozenge formulations[END_REF][START_REF] Winkel | Clinical association of volatile sulfur compounds, Halimeter values, organoleptic score and tongue coating in oral malodour[END_REF]Van del Velde et al., 2009), no relationships between all three methods have been reported. In the current work, it was noticed that whether an inhibitory effect from an active mouthwash was calculated as a change in malodour value from time zero or as an absolute measurement at each time point, the correlations between the three methods of breath measurement were high. This finding implies that all methods are equally capable of assessing oral malodour and that any method on its own might also be sufficient.

The inhibitory effects on H 2 S and oral malodour can be descrbied as follows: slight effect (Lacer Fresh ® ), a moderate effect (BreathRX ® , SmartMouth ® ) and a marked effect (Listerine ® , SB12 ® ). However, in comparison with a clinically proven mouthwash such as Listerine [START_REF] Pitts | Mechanism of action of an antiseptic, anti-odor mouth-wash[END_REF], SB12 was shown to be numerically and at some time points, statistically, superior.

Chlorhexidine, a cationic bis-biguanide with low mammalian toxicity and broad spectrum activity against Gram-negative and Gram-positive bacteria [START_REF] Denton | Chlorhexidine Disinfection, Sterilization and Preservation[END_REF] has been used for in vitro and in vivo studies (Kinniment et al., 1996[START_REF] Jones | Chlorhexidine is it still the gold standard[END_REF]. The cationic properties of CHX explain how its electrostatic attraction by the anionic bacterial surfaces may lead to membrane disruption, increased permeability and cell death and as a result, to a reduction in bacterial load [START_REF] Jones | Chlorhexidine is it still the gold standard[END_REF]Kuyyakanond, 1992, Quirynen and[START_REF] Quirynen | Review of the stategies for oral malodour[END_REF] Oral and malodour. CHX is also known for its high substantivity to buccal surfaces and has been shown to reduce gingival inflammation and dental plaque [START_REF] Cummins | Delivery of antiplaque agents from dentifrice, gels and mouthwashes[END_REF]Addy and Moran, 1997;[START_REF] Bollen | Microbiological response to mechanical treatment in combination with adjunctive therapy: a review of the literature[END_REF]. The strong antimicrobial action and increased substantivity in the mouth of CHX justify its use for malodour reduction [START_REF] Bosy | Relationship of oral malodor to periodontitis :evidence of independence in discrete subpopulations[END_REF]De Bover et al., 1996). More recently, CHX has been used in association with other anti-malodour agents such as CPC and Zn and the efficacy of this combination was shown to be more effective than CHX alone [START_REF] Quirynen | Review of the stategies for oral malodour[END_REF][START_REF] Roldan | Clinical and microbiological efficacy of an antimicrobial mouthrinse on oral halitosis[END_REF] suggesting a more synergistic effect by CHX when present with other active compounds.

The efficacy of CHX against microbes has been shown to be both dose and time-dependant [START_REF] Quirynen | Review of the stategies for oral malodour[END_REF] and different product formulations may use CHX at different concentrations which might explain the variability of side effects such as discolouration of the oral mucosa and teeth as well as an alteration of taste (Bossy et al., 1994;[START_REF] Flötra | Side effects of Chlorhexidine mouth washes[END_REF][START_REF] Quirynen | Review of the stategies for oral malodour[END_REF].

From the 1970's onwards, zinc has been extensively studied either on its own or in association with other compounds used to control oral malodour, [START_REF] Tonzetich | Production and origin of oral malodour: a review of mechanisms and methods of analysis[END_REF][START_REF] Schmidt | The effect of oral rinses on organoleptic mouth odor ratings and levels of volatile sulphur compounds[END_REF][START_REF] Wåler | The effect of some metal ions on volatile sulphur-containing compounds originating from the oral cavity[END_REF][START_REF] Young | Effects of metal salts on the oral production of volatile sulphur-containing compounds (VSC)[END_REF]. In addition to its antimicrobial properties, zinc is relatively non-toxic, non-cumulative and gives no visible coloration [START_REF] Quirynen | Review of the stategies for oral malodour[END_REF]. It is believed that zinc binds to the membrane of microorganisms, interfering with, and reducing cell growth rate (Sugman, 1983;[START_REF] Radke | Effect of abcess fluid supernatant on kinetics of Candida albicans growth[END_REF]. It has also been suggested that zinc reacts with VSC by forming an insoluble complex (ZnS) which is non-volatile and thus non-odiferous [START_REF] Boulware | Sanguinaria extract, a new agent for the control of volatile sulphur compounds in the oral cavity[END_REF].

In previous clinical trials using mouthwashes containing zinc, volunteers have reported an unpleasant metallic taste [START_REF] Young | Combined effects of zinc ions and cationic antibacterial agents on intraoral volatile sulphur compounds (VSC)[END_REF]. It has also been shown that low concentrations of zinc alone do not produce an unpleasant taste but are not very effective against oral malodour. Likewise, CHX at high concentrations produces taste effects as well as staining. Young et al showed that low concentrations of CHX still maintained an effect over time. It could be that a low concentration of CHX may reduce the staining of the teeth without loosing all of its anti malodour properties. A synergistic effect between low zinc and low CHX, previously observed by others (

Figure 2 :Figure 3 :Figure 4 :Figure 5 :

 2345 Figure 2: Organoleptic changes for 5 products plus control ( SB12, Listerine, 3BreathRX, ▲SmartMouth, LacerFresh, •Control)

Figure 4 :

 4 Figure 4: Correlation between organoleptic score and Log10 halimeter readings 177x103mm (96 x 96 DPI)

Figure 5 :

 5 Figure 5: Correlation between organoleptic score and Log10 H2S readings from Oral ChromaTM 177x102mm (96 x 96 DPI)

Figure 6 :

 6 Figure 6: Correlation between Log10 Halimeter readings and Log10 H2S from Oral ChromaTM

  Comparative effects of various commercially availablemouth-rinse formulations on oral malodour.

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	Introduction
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Organoleptic scores, VSC concentrations (by halimetry) and H 2 S (by OralChroma) were taken at baseline, 30, 60, 90 and 3 hours per person, per treatment. GraphPad Prism (GraphPad Software, San Diego, Ca, USA) was used to log transform, plot (as change in readings from time zero) and statistically analyse the data using ANOVA plus Bonferroni's multiple comparison post test. Correlation tests were performed using Oral Diseases -Manuscript Copy Oral Diseases -Manuscript Copy

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Thrane et al., 2007; Young et al., 2003) may reduce oral malodour and decrease the above mentioned side-effects. It is likely that zinc and CHX have different high- affinity binding sites within the cell, and that occupation of one type of site makes the cell more sensitive to the inhibitory or cidal effects of the other type of ligand.

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	In conclusion, a combination of CHX and Zn in low concentration, such as SB12, was shown to reduce significantly (for up to 3 hours) H 2 S in the oral cavity which is considered to be the main contributor to oral malodour. o p y o p y o p y o p y o p y
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Table 1 :

 1 Mouthrinse, ingredients and code

	Code Mouthrinse			Ingredients
	A	Listerine		Aqua, Alcohol, Sorbitol, Aroma, Poloxamer 407,
		antibacterial	Benzoic Acid, Eucalyptol, Methyl salicylate, Thymol,
		Mouthwash-Total Care ®	Menthol, Sodium Fluoride, Zinc Chloride, Sucralose, Sodium saccharin, Sodium benzoate, Benzyl alcohol,
		Pfizer		Cl 16035, Cl 42090. Contains Sodium fluoride
		Consumer		(0.022% w/v 100ppm F).
		Healthcare
	B	BreathRX ®	Aqua,	Sorbitol,	Propylene	glycol,	PEG-40,
		Discus	Dent	hydrogenated Castor oil, Polaxamer 407, Xylitol,
		Inc USA		Aroma (Mint, thymol and Eucalyptus oil), Zinc
				gluconate, Cocamidopropyl Betaine, Cetylpyridinium
				Chloride, Sodium Saccharin, Citric Acid, Cl 42090.
	C	SmartMouth Wash ®	Solution 1: Purified water, Sodium Benzoate, Benzoic Acid, and Sodium Chlorite.
		Triumph		Solution 2: Purified water, glycerine, Polaxamer 407,
		Pharmaceutical	Propylene Glycol, Benzoic acid, Flavour, Polaxamer
		Inc		124, Sodium Benzoate, Sodium Chloride, Sodium
		US		Saccharin, Zinc Chloride, D&C Yellow No 10, and
				FD&C Blue No 1.
	D	LacerFresh Mouthwash ®	Triclosan 0.15%, Zinc Chloride 0.05%, Sodium Fluoride 0.05% (225ppm), Xylitol 1%
		Lacer, S.A
		Sardenya
		Barcelona
	E	SB12 ®		Zinc acetate (0.3%), chlorhexidine diacetate
		Antula		(0.025%), sodium fluoride (0.05%), mint/menthol
		Healthcare	flavour (in alcohol)
		Stockhlom
	F	Control		Water

Table 2 :

 2 Average, minimum and maximum values of baseline readings (pre-treatment measurements) for organoleptic scores, VSC and H 2 S (from OralChroma) recorded for 14 trialists

	Measurements	Average (± SD; n=84) Minimum (± SD) Maximum (± SD)
	Organoleptic score 3.55 (0.23)	2.50 (0.00)	4.33 (0.25)
	Halimeter readings 156 (49.21)	36.16 (3.65)	379.50 (86.00)
	H 2 S OralChroma	416.40 (213)	50.75 (27.80)	544 (61.09)

Table 3 :

 3 Summary of ANOVA plus Bonferroni statistical data

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Acknowledgements

This study was supported by Healthcare Brands International Ltd, 2nd Floor, The Clock Tower, Talbot Street, Nottingham, NG1 5GG, UK.


Dr Samira ; Osailan
email: samira.osailan@kcl.ac.uk

Rashida Pramanik

Soha Shirodaria

Stephen J Challacombe

Burgess Gordon

Proctor

King'

Prof Gordon B Proctor
email: gordon.proctor@kcl.ac.uk

Investigating the relationship

Keywords: salivary gland disease, Diagnostics, Pathology xerostomia, mucosal wetness, salivary secretion

published or not. The documents may come

 (Fox et al., 1985)and objective (hyposalivation) feelings of dry mouth [START_REF] Bergdahl | Low unstimulated flow and subjective oral dryness: association with medication, anxiety, depression and stress[END_REF]. It is known that drugs are the most common cause of the dry mouth condition and complaints of xerostomia are a frequent side effect of many drugs (reviewed by Scully, 2003). Hyposalivation is especially known in those drugs used to treat anxiety, depression, and stress but is usually reversible. Salivary gland diseases associated with hyposalivation include primary or secondary Sjögren's syndrome, (Sjögren, 1933;Navazesh et al., 1996;Price & Venables, 2002;Kassan & Moutsopoulos, 2004;[START_REF] Atkinson | Salivary hypofunction and xerostomia: diagnosis and treatment[END_REF], and Sialadenitis, Nodal

Osteoarthiritis, Xerostomia syndrome (SNOX; Kassimos et al., 1995). Sjögren's syndrome affects approximately 0.4% of the population and has a male: female ratio of 1: 10 (Fox 2005). Other conditions and systemic diseases where dry mouth could be a relevant complaint include diabetes, thyroid disorders, connective tissue diseases and graft versus host disease [START_REF] Atkinson | Salivary gland dysfunction: causes, symptoms, treatment[END_REF]Scully, 2003). The prevalence of xerostomia in the general population ranges between 10-20% in different published studies (Fox et al., 1985;Pujol, 1998).

Prevalence is greater in females and increases with increased medication (Nederfors, 1997;Schein et al., 1999). In the elderly (60+ years) population prevalence is approximately 20% [START_REF] Ben-Aryeh | xerostomia in elderly: prevalence, diagnosis, complications and treatment[END_REF]Nederfors et al., 1997;Nayak et al., 2004).

As with most symptoms, it has been difficult to quantify dry mouth complaints precisely and reproducibly. Investigators have used a variety of methods to assess oral dryness including: questionnaires, Visual Analog-Scales (VAS), simple functional measures such as observing if a tongue blade adheres to the buccal mucosa or if a patient can chew and swallow dried biscuits without water (Fox, 2005). Dry mouth can also be assessed by measuring the volume of residual saliva on mucosal surfaces using filter paper and micro-moisture meter -Mordarski & Kleinberg, 1996;Won et al., 2001;Lee et al. , 2002;and Eliasson et al. , 2005) and more recently mucosal wetness devices have been used (Kakinoki et al., 2004;Takahashi et al., 2005).

Collins & Dawes (1987) calculated the average surface area of the mouth to be 214.7 cm 2 and calculated the thickness of the salivary film in the mouth to be 44 Fifty healthy age matched subjects who did not complain of dry mouth were selected as controls and had a mean age of 60 ± 15 years (range 22-83). They were recruited from members of staff and from a residential home for the elderly.

All patients and participants were given an explanation and information sheet of the study and all gave their informed consent prior to the procedure. The study was performed under ethical approval of Guy's & St Thomas' Hospitals (Local)

Research Committee. Ten healthy volunteers with mean age ± SD is 35 ± 9.5 years (n=10) from the 50 controls were used to validate the reproducibility of mucosal wetness measurements.

Assessment of patients and collection of samples

a. Clinical oral dryness score

The signs of dryness in the mouth were examined using a scoring system (the Clinical Oral Dryness Score, CODS) which is composed of ten features: 1) Mirror sticks to buccal mucosa, 2) Mirror sticks to tongue, 3) Saliva frothy, 4) No saliva pooling in floor of mouth, 5) Tongue shows loss of papillae, 6) Altered gingival architecture/ smooth (especially anterior), 7) Glassy appearance to oral mucosa (especially palate), 8) Tongue lobulated / deeply fissured, 9) Cervical caries (more than two teeth), 10) Mucosal debris on palate (excluding under dentures).

This technique was validated and the data presented elsewhere [START_REF] Challacombe | A Clinical Score of oral Dryness: relation to salivary flow rates[END_REF].

c. Mucosal wetness (MW) measurements

The thickness of residual saliva (oral mucosal wetness) was measured in dry mouth patients (n =100) and aged matched healthy subjects (n = 50) using a filter paper strip (Oraflow Inc, USA) and micro-moisture meter (Periotron® 8000;

Oraflow Inc, USA). A filter paper strip with a diameter 7.5 mm covering an area of 44 mm 2 was placed immediately on the mucosa after swallowing and was gently pressed flat with a finger of a gloved hand. After 10 sec the paper strip was transferred to the sensors of the micro-moisture meter. Four mucosal sites were measured; Anterior Hard palate (AHP), Buccal (BUC), Anterior Tongue (AT), and

Lower Lip (LL) (Figure 1). A calibration curve previously constructed using volumes of UWS saliva was used to calculate the volumes (µl) of residual saliva collected from mucosal surfaces and then mucosal thickness (µm) was calculated. For the validation of mucosal wetness measurements 10 volunteer subjects were assessed over 10 visits, 5 morning (9-12 am) and 5 afternoon (2-5 pm) visits. UWS was also measured on each occasion.

Statistics

Statistical analysis was carried out using SPSS computer software version 15.

An analysis of variance (ANOVA) and intra-class correlation coefficient (ICC)

were used in order to validate the mucosal wetness measurement between and within subjects. For the purpose of analysis, dry mouth patients' data was grouped according to either diagnostic or to UWS flow rate groups. All groups were compared with age matched controls. Correlations between MW and UWS salivary flow rate were determined using Pearson (parametric) correlation analysis.

Validation of mucosal wetness measurement

Measurement of MW using filter paper strips and the micro-moisture meter showed good reproducibility. Intra-class correlation coefficients (ICC) for MW measurements from the same subjects (n =10) at different visits for AHP, BUC, AT and LL were 0.49, 0.48, 0.58, 0.53 respectively (P < 0.02 for all surfaces). No significant difference was found between morning and afternoon for oral mucosal wetness values for AHP, BUC, AT, and LL (Figure 2) and UWS salivary flow rate.

Mucosal wetness of dry mouth patients and controls

In dry mouth patients, the means ± SD MW of all four surfaces were significantly reduced by approximately 50% compared with age-matched controls (Figure 3) but the trend was the same. That is AHP had the thinnest and AT tongue had the thickest MW amongst both patients and controls (Figure 3).

Correlation between UWS salivary flow and mucosal wetness

Overall UWS was significantly (p < 0.05) directly correlated with MW at all four sites. Pearson correlation coefficients for each site were r = 0.22 (AHP), r = 0.18 (BUC), r = 0.4 (AT), r= 0.3 (LL) respectively.

Subjects were grouped according to UWS salivary flow rate as follows: three patient groups with low flow (0-0.1ml/min, n = 57), moderate flow (0.1-0.2 ml/min, n=25), high flow (>0.2 ml/min, n=18) and a fourth group of controls (mean flow = 0.45 ml/min, range 0.2-1.0 ml/min, n = 50). The group with lowest flow (< 0.1ml/min) showed a significant (p < 0.05) reduction in MW at all four sites (AHP, BUC, AT and LL) compared with controls (Figure 4a). The AHP, BUC and LL mucosal surfaces showed no significant differences between the low and high UWS patient groups whilst AT showed a significant (p < 0.05) reduction in MW between all UWS flow rate patient groups.

In addition, when a patient group (n = 14) with UWS flow rate > 0.2-0.3 ml/min with a mean ± SD (0.24 ± 0.01 ml/min) was compared with a similar UWS flow 4b). The AHP showed no difference in wetness whilst the LL showed a significant difference only by a one-tailed t-test (p<0.05). The different flow rate groups of control subjects did not show any statistically significant differences in MW of different oral surfaces.

The relationship between CODS and UWS salivary flow rate

There was inverse correlation between CODS and UWS salivary flow rate of dry mouth patients and healthy subjects (aged matched controls). Even in dry mouth patients with low CODS (1-3) has a significant (p < 0.01) reduction in there UWS compared with the controls (Figure 5). For example, the surface vestibular to the upper right molars appears to have a 50-60% contribution from parotid saliva whilst the surfaces vestibular or lingual to the lower incisors have only a 5-7% contribution. These differences in composition along with differing densities of minor salivary glands in the submucosae of oral surfaces will also presumably contribute in determining the wetness of the different surfaces measured in the present study.

Measurement of MW by filter paper sampling and micro-moisture meter measurement showed good reproducibility and consistency at all four sites. AT and BUC surfaces were very consistent sites while anterior hard palate showed variations between individuals. Previously it has been reported that unstimulated whole salivary flow rates show a circadian rhythm (Dawes, 1972). However, in the present study, neither mucosal wetness nor UWS flow rate showed a significant difference between samples taken in the morning (9-12) or in the afternoon (2-5). This suggests that mucosal wetness and UWS flow rate can be measured during the hours of normal clinics.

In 1998;Won et al., 2001;Lee et al., 2002;Eliasson et al., 2005). In addition, the distribution pattern of the MW on the four mucosal surfaces was the same in patients and controls. i.e., the AT had the thickest and anterior AHP had the thinnest layers of MW which is in agreement with Lee et al, (2002). Whilst Wolff and Kleinberg (1998) found that the posterior tongue had the thickest layer of mucosal wetness. Although there appeared to be a decrease in wetness of the AHP in the dry mouth patients compared to controls there was more variation between individuals with means of 7 ± 7.2 µm and 11 ± 11.3 µm for patients and controls respectively. In the present study all of the patient groups showed a mean MW of <10 µm but the normal control group showed a mean thickness of only 11 µm. Others have shown that there was no significant difference in palatal saliva secretion between Sjögren's syndrome patients and healthy control (Marton et al, 2004).

Oral dryness assessed using CODS was significantly increased in all patients complaining of dry mouth. Subjects with lower UWS salivary flow rates showed the highest CODS values. Since mucosal wetness of all surfaces (AHP, AT, BUC, LL) showed a significant decrease with a reduction in UWS salivary flow rate it can be inferred that CODS and mucosal wetness also show an inverse relationship. Thus reduced MW is linked with increased CODS and clinical features of oral dryness. A positive correlation between MW and UWS salivary flow rate has also been previously reported (Wolff & Kleinberg 1998).The anterior tongue showed a different pattern to the other surfaces with a 'stepped' decrease in wetness that mirrored the decrease in UWS flow rate. An explanation for this is that the tongue is the mobile part in the mouth and its fluid coating is derived from all contributions to the whole mouth saliva volume.

Patients with higher UWS flow rates (>0.2-0.3 ml/min) still showed a significant reduction in MW of the BUC and AT surfaces compared to controls with similar UWS flow rates. Others have observed a decrease in labial mucosal wetness in (Dawes, 1987;Wolff and Kleinberg, 1999). It may also be that the patients with higher UWS flows but reduced mucosal wetness have saliva with altered mucosal coating properties due to changes in composition.

There is evidence of reduced mucin sulphation in Sjögren's syndrome and this may impact on surface coating properties or water retention (Allende et al., 2008). Changed composition could result from a relatively greater reduction in submandibular secretion which might reduce mucin levels in whole mouth saliva, although results from a previous study do not support this idea (van den Berg et al., 2007). It would be interesting to examine the rheological and wetting properties and mucin content of salivas from such patients. When control subjects were divided into UWS flow rate groupings it is evident that there was little difference in wetness of the oral surfaces with increase UWS flow rate. It can be suggested that above a UWS flow rate of 0.2ml/ min there is no further significant retention of residual fluid on oral surfaces.

In conclusion, mucosal wetness can potentially be used as an index of oral dryness. It is a reliable, simple method which can be used at the chair side to measure oral dryness. There is a positive correlation between oral mucosal wetness and unstimulated salivary flow rate. -Marton K, Boros I, Fejerdy P, Madlena M. Evaluation of Unstimulated flow rates of whole and palatal saliva in healthy patients wearing complete denture and in patients with Sjögren's syndrome. J. Prosthet. Dent. 2004;91 (6):577-81.

-Navazesh M., Brightman V.J.,Pogoda J.M. Relation of medical status, medication, and salivary flow rates in adults of different age. Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod. 1996;81:172-6.

-Nayak L. Wolff A., Fedele S., Martin-Granizo R., Reichart P., Russo L., -Shern R.J., Fox P.C., Cain J.L., and Li S-H. A method for measuring the flow of saliva from the minor salivary glands. J. Dent. Res. 1990,69(5):1146-1149.

-Sjögren H. Zur Kenntnis der Keratoconjunctivitis sicca (Keratitis filiformis bei

  Mignogna M., Strietzel F. The Burden of Xerostomia in Independent Communitydwelling Older Adults: Results from the Saliwell Project. Oral Biosci. Med. 2004; 1(4): 283-89. -Niedermeier, W., Hüber, M. Quantitative studies on the secretory output of the palatine salivary glands. Dtsch Zahnärztl Zschr 1989; 44: 37-40. Isaksson R, Mornstad H, Dahlof C. Prevalence of perceived symptoms of dry mouth in an adult Swedish population--relation to age, sex and pharmacotherapy. Community Dent Oral Epidemiol. 1997; 25(3):211-6. -Pujol T, Coma M, Pujol M, Postigo P. Prevalence of xerostomia in the general population. Aten. Primaria. 1998; 21(4):225-8. -Price E.J., and Venables P.J.W. Dry eyes and mouth syndrome-a subgroup of patients presenting with sicca symptoms. Rheumatology 2002; 41: 416-422 -Sas R., Dawes C. The intra-oral distribution of unstimulated and chewing gum stimulated parotid saliva. Arch oral. Biol. 1997; 42: 469-474 -Scully C. Drug effects on salivary glands: dry mouth oral Dis. 2003; 9(4):165-76. -Schein O.D., Hochberg M.C., Munoz B, Tielsch J. M., Bandeen-Roche K., Provost T, Anhalt G. J., West S. Dry Eye and Dry Mouth in the Elderly: A Population-Based Assessment. Arch.Intern. Med. 1999; 159:1359-1363.

  strip™ and four mucosal wetness surfaces inside the mouth, (a) Anterior hard palate (AHP), (b) Buccal (BUC), (c) Anterior Tongue (AT), (d) Lower Lip (LL) surfaces on a healthy subjects.

Figure 2 .

 2 Figure 2. Mean values of mucosal wetness in µm from four oral surfaces (AHP, BUC, AT, LL) were measured from 10 subjects in the morning (am, light bars)) and the afternoon (pm, dark bars) and 5 visits for each time point of the same subjects. It showed there are no significant (P < 0.05) differences in wetness of all the four surfaces between morning and afternoon. Keys: AHP= Anterior Hard Palate, BUC= Buccal, AT= Anterior Tongue, LL= Lower Lip. Error bars represent SEM.

Figure 3 .Figure 4 .

 34 Figure 3. Mean values of mucosal wetness at four surfaces (AHP, BUC, AT, LL) from dry mouth patients (n=100, dark bars) and healthy subjects as controls (n=50, light bars). There is a significant (p < 0.05) reduction in wetness on all mucosal sites from dry mouth patients compared with controls. Keys: AHP= Anterior Hard Palate, BUC= Buccal, AT= Anterior Tongue, LL= Lower lip. *= statistically significant (P < 0.05). Error bars represent SEM

Figure 5 .

 5 Figure 5. The relationship between Clinical Oral Dryness Score (CODS) and Unstimulated Whole salivary flow rate (UWS) of dry mouth patients and healthy subjects (aged matched controls). Patients in the lowest UWS flow rate group have the highest CODS. The CODS was significantly (p < 0.01) increased in all three patients groups compared with the controls. Keys: 1 = significantly different in all three patients groups compared with the controls. 2 = statistically significant between patients group 0-0.1and >0.1-0.2 ml/min. 3 = statistically significant between 0-0.1 and > 0.2-0.3 ml/min. Error bars represent SEM.

  µm, by dividing the mean residual saliva in the mouth by surface area. The thickness of the salivary film is governed it part by the rheological properties of saliva. It is apparent that the thickness and composition of the salivary film will vary in different parts of the mouth depending upon the position in relation to salivary glands.

	Oral Diseases -Manuscript Copy
	2. MATERIAL & METHOD
	2.1. Study subjects
	Samples were collected from a total of 100 patients with a mean age of 62 ± 11 O years (range 22-82 years) attending Oral Medicine clinics at Guy's Hospital. They r all complained of dry mouth and were divided into five groups according to their a l diagnosis: primary and secondary Sjögren's syndrome (SS1 & SS2); Drug
	induced Hyposalivation (DIH); non-Sjögren's but presence of sialadenitis, nodular D i s e s a e s osteoarthritis, xerostomia (SNOX); none of the above (NOS).
	-
	M a n
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	Oral Diseases -Manuscript Copy	6

The current study aims are to determine the normal variation of Mucosal Wetness (MW) at different oral mucosa sites and secondly to determine the relationship between mucosal wetness, unstimulated whole mouth (UWS) salivary flow rate, Clinical Oral Dryness Score (CODS).

Page 6 of 26 Oral Diseases -Manuscript Copy Oral Diseases -Manuscript Copy

	b.
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Unstimulated whole mouth (UWS) salivary flow UWS was

  measured for 10 minutes and the subject was asked to spit into a pre-

	weighed vessel and not to swallow any saliva. UWS flow rate was calculated and
	expressed ml/min, taking 1g saliva =1ml.

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	3. RESULTS
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  The findings show that the mucosal wetness differ on oral surfaces; AHP, BUC, AT and LL. The trend of MW follows the same trend in healthy subjects as well as dry mouth patients. In both groups the wettest surface was AT, followed by BUC, LL then AHP. Our findings are similar to those in previous studies(DiSabato-Mordarski & Kleinberg, 1996; Won et al., 2001; Lee et al., 2002). For AHP the values of at least 10 µm on the palate appeared necessary to avoid complaints of dry mouth(Wolff and Kleinberg, 1998). Previous studies suggest that the percentage contribution of different glandular salivas to the total saliva on different oral surfaces varies. Thus the percentage contribution of parotid saliva to the total saliva on different oral surfaces is not the same(Sas & Dawes, 1997).

	Oral Diseases -Manuscript Copy
	4. DISCUSSION
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	Oral Diseases -Manuscript Copy	10


Christian Walter
email: walter@mkg.klinik.uni-mainz.de

Andreas Pabst

Thomas Ziebart

Marcus Oliver Klein

Klein Mo

Pabst A Contributed Equally

Dr W Wagner

Bisphosphonates affect migration

Keywords: bisphosphonate, bisphosphonate associated osteonecrosis of the jaws, HUVEC, fibroblast, osteoblast, osteogenic cells Oral Diseases - bisphosphonate, bisphosphonate-associated-osteonecrosis, HUVEC, fibroblast, osteoblast, osteogenic cells

HAL is

Objective: Bisphosphonate-associated-osteonecrosis of the jaw (BP-ONJ) is a side effect in patients being treated with bisphosphonates. The bisphosphonates most often associated with BP-ONJ are the highly potent nitrogen-containing bisphosphonates e.g. pamidronate or zoledronate. In terms of BP-ONJ aetiology, several theories are being discussed: inhibition of bone remodelling, effect on soft tissues, and antiangiogenic effect of bisphosphonates. The aim of this in-vitro study was to investigate the effect of different potent bisphosphonates on osteoblasts, fibroblasts and human umbilicord vein endothelial cells (HUVEC).

Materials and Methods: Three nitrogen-containing bisphosphonates (ibandronate, pamidronate and zoledronate) and one non-nitrogen-containing bisphosphonate (clodronate)

were compared concerning their potency on apoptosis induction (tunel), cell viability (calcein assay) and migration potency (boyden chamber) on osteoblasts, fibroblasts and HUVEC.

Results: The nitrogen-containing bisphosphonates, particularly pamidronate and zoledronate, affect cell viability, cell migration and the induction of apoptosis of osteoblasts, fibroblasts and HUVEC.

Conclusions: These results support the theory that BP-ONJ is a multifactorially caused disease because several cell lines of the oral cavity which are responsible for integrity and wound healing are negatively affected by nitrogen-containing bisphosphonates. Perioperative interruption of bisphosphonate application during dental surgical procedures -if possiblemight be feasible to promote better wound healing. For over 30 years, bisphosphonates have been used in medicine [START_REF] Bassett | Diphosphonates in the treatment of myositis ossificans[END_REF]. They are separated into highly potent nitrogen-containing and low potent non-nitrogen-containing bisphosphonates. Through inhibition of osteoclasts, bisphosphonates prevent skeletal related events such as pathological fractures or hypercalcaemic episodes. Their positive effects are without controversy and increase the quality of life in these patients [START_REF] Ruggiero | American Association of Oral and Maxillofacial Surgeons position paper on bisphosphonate-related osteonecrosis of the jaws--2009 update[END_REF].

The negative side effects can be separated into four major groups: acute-phase reactions, gastrointestinal effects, renal side effects and bisphosphonate associated osteonecrosis of the jaw (BP-ONJ) [START_REF] Diel | Adverse effects of bisphosphonates: current issues[END_REF]. The American Association of Oral and Maxillofacial Surgeons defines BP-ONJ as exposed bone in the maxillofacial region that has persisted for more than eight weeks in combination with a current or previous bisphosphonate therapy, and a lack of head and neck radiation in patient's history [START_REF] Ruggiero | American Association of Oral and Maxillofacial Surgeons position paper on bisphosphonate-related osteonecrosis of the jaws--2009 update[END_REF]. The frequency of BP-PNJ is higher in patients with malignancies and intravenously delivered, highly potent bisphosphonates such as pamidronate and zoledronate, and lower in patients receiving oral bisphosphonates due to osteoporosis [START_REF] Ruggiero | American Association of Oral and Maxillofacial Surgeons position paper on bisphosphonate-related osteonecrosis of the jaws--2009 update[END_REF]. Only a few cases of BP-ONJ have been reported in connection with the administration of non-nitrogen containing bisphosphonates such as clodroante [START_REF] Montazeri | Oral sodium clodronate induced osteonecrosis of the jaw in a patient with myeloma[END_REF].

Several theories on BP-ONJ development are being discussed in the literature [START_REF] Allen | The pathogenesis of bisphosphonate-related osteonecrosis of the jaw: so many hypotheses, so few data[END_REF]. The most common theory concentrates on the impact of bisphosphonates on bone, especially on osteoclasts. Osteoclasts are the main cellular target of bisphosphonates, inhibiting enzymes of the mevalonate pathways (nitrogen containing bisphosphonates) or synthesizing ATP derivates (non nitrogen containing bisphosphonates) [START_REF] Frith | Clodronate and liposome-encapsulated clodronate are metabolized to a toxic ATP analog, adenosine 5'-(beta, gamma-dichloromethylene) triphosphate, by mammalian cells in vitro[END_REF][START_REF] Rogers | Cellular and molecular mechanisms of action of bisphosphonates[END_REF]. In addition to osteoclasts, osteocytes ( (Walter et al., 2009). The reduced potency of the latter is desired in terms of suppressed angiogenesis tumor therapy [START_REF] Green | Mechanisms of bisphosphonate effects on osteoclasts, tumor cell growth, and metastasis[END_REF]. For some bisphosphonates, a proapoptotic effect on cancer cells is reported [START_REF] Santini | The antineoplastic role of bisphosphonates: from basic research to clinical evidence[END_REF].

Therefore bisphosphonates might inhibit several cell types necessary for wound healing, such as osteoblasts, fibroblasts and vessel cells. In summary, a broad range of cells and their functions is affected by bisphosphonates, and not just osteoclasts. Since BP-ONJ frequencies differ among the several bisphosphonates, the impact on cell viability, cell migration and apoptosis of osteoblasts, fibroblasts and endothelial cells was analyzed. with 1% Penicillin-Streptomycin-Neomycin antibiotic mixture (PSN), 10% FCS, and 500ng basic fibroblast growth factor per 500ml medium.

All cell cultures were incubated and cultured in an incubator with 5% CO 2 and 95% air at 37 o C till the third passage of cell generation. Cells were passaged at regular intervals depending on their growth characteristics using 25% trypsin (Seromed Biochrom, Berlin, Germany).

Calcein viability assay: To analyze the effect of bisphosphonates on cell viability, we evaluated a Calcein Viability Assay. HUVEC, fibroblasts and human osteoblasts were cultivated in standard cell culture as described. All cells were incubated in standard 24-well culture plates with different bisphosphonates (clodronat, ibandronate, pamidronate, zoledronate) in gradient concentrations (0 µmol, 5 µmol, 50 µmol, 100 µmol, 200 µmol, 500 µmol) for 72 hours. This range of concentrations was selected because in-vivo concentrations in plasma shortly after zoledronate infusion are about 5 µmol, and concentrations in bone shortly after infusion can be 100 fold higher [START_REF] Chen | Pharmacokinetics and pharmacodynamics of zoledronic acid in cancer patients with bone metastases[END_REF]. After removing the used Osteoblasts: At lower concentrations (5 -50 µmol), the impact of zoledronate on osteoblasts was the strongest compared to the other bisphosphonates, and was significant compared to all bisphosphonates except for clodronate, which depended on concentration. At 5µmol, only zoledronate had a significant reduction on cell viability compared to the control (p=0.006).

Osteoblasts were affected by clodronate, which turned out to be significant compared to the control at 100µmol (p=0.036). There were no further significant differences between pamidronate and zoledronate, and there were no differences at any concentration between ibandronate and clodronate.

Fibroblasts: There was no significant difference at 5µmol between the groups. At 50µmol, the differences between the control and all the other bisphosphonates (p=0.002 up to 0.037) were significant. From this concentration level and higher, the difference between clodronate and all the other bisphosphonates were significant. The differences between the nitrogen containing bisphosphonates were significant as of 200µmol, with a significant difference between pamidronate and ibandronate (p<0.001), and pamidronate and zoledronate (p=0.03).

At 500µmol there is no longer a difference between pamidronate and zoledronate, but a significant difference between ibandronate and clodronate (p<0.001), and ibandronate and pamidronate (p=0.008) was found.

HUVEC: There were no significant differences at 5µmol. At 200µmol, clodronate has a significant impact on the cells (p=0.047). All other bisphosphonates turned out to have a significant impact at 50µmol (p<0.001). The differences between zoledronate and all the other bisphosphonates was significant as well (p<0.001). At a concentration of 100µmol, the difference of the impact of zoledronate and pamidronate was no longer significant.

TUNEL-Assay (figure 2 Osteoblasts: Zoledronate and pamidronate had the greatest impact but there was no significant difference between these two bisphosphonates. The effect of ibandronate was significantly weaker (p<0.001 compared to zoledronate, and p=0.006 compared to pamidronate). There was no significant difference of clodronate compared to the control, and no significant difference compared to ibandronate.

Fibroblasts: All differences between the respective bisphosphonates were statistically significant (p between p<0.001 and 0.007). Zoledronate had the strongest impact, followed by pamidronate, ibandronate and clodronate. The level of apoptosis was nearly 77% for zoledronate and 61% for pamidronate.

HUVEC: Pamidronate and zoledronate had the strongest effect (p<0.001) but there was no significant difference between these two bisphosphonates (p=0.522). Ibandronate and clodronate had a considerably smaller but nevertheless significant effect on the apoptosis rate (p<0.001 for ibandronate and p=0.005 for clodronate). The impact difference between ibandronate and clodronate was not significant (p=0.351).

Boyden Migration Assay (figure 3):

For each cell type, the ANOVAs conducted showed significant differences among the experimental groups and control group. Each ANOVA yielded a p-value <0.001.

Osteoblasts: Only the nitrogen containing bisphosphonates inhibited the migration ability of osteoblasts. There was no significant difference between zoledronate, pamidronate and ibandronate.

Fibroblasts: All bisphosphonates inhibited the migration ability, save clodronate. The strongest effect was measured for pamidronate (p<0.001 compared to all other bisphosphonates). There was no significant difference between zoledronate and ibandronate.

HUVEC: All bisphosphonates inhibited the migration ability of HUVEC. Zoledronate and pamidronate had the strongest effect, but there was no significant difference between these Clodronate, representing the non-nitrogen containing bisphosphonates, has nearly no impact on the apoptosis rate of the investigated cell lines. The effect of clodronate on cell viability of HUVEC fibroblasts and osteoblasts was not as large compared to the other bisphosphonates, yet still yielded significant results in higher concentrations. As well, the effect on cell migration was marginal and statistically significant only for HUVEC, so that the over all effect of clodronate is minimal, which could explain the rarity of BP-ONJ in patients receiving clodronate. Among the nitrogen containing bisphosphonates represented by ibandronate, pamidronate and zoledronate, pamidronate and zoledronate had the greatest effect on all cell types and functions. One exception is the effect of zoledronate and Pamidronat on the migration rate of fibroblasts with a way higher effect of pamidronat. The apoptosis rate in cells incubated with ibandronate is either as high as the cells incubated with clodronate (osteoblasts, HUVEC) or is little higher (fibroblasts). A similar results was obtained for the cell viability, with an effect just as high as clodronate for osteoblasts. The effect on fibroblasts and HUVEC was stronger, but not as strong as the effect of zoledronate and pamidronate. These last two bisphosphonates had the greatest impact on the apoptosis rate, the cell viability and the migration ability of all cell lines with only a few exceptions.

Similar results regarding migration ability and cell viability have previously been described (Walter et al., 2009). A migration assay was performed with a scratch wound assay, and results showed that clodronte had had the weakest effect on all cell types, which is similar to the present study. effect on all tested cell types as well. Among the nitrogen containing bisphosphonates, pamidronate and zoledronate had a greater effect on the cell viability compared to ibandronate. The cell viability measured with the calcein assay seems to be higher compared to the ones evaluated with the MTT-test. This is well in accordance with the underlying test methods, as MTT is a metabolism test and therefore might be more sensitive than calcein staining alone. It should however be noted, that in these in vitro experiments for all cell types and functions tested, the influence of the different bisphosphonates is reproducible. This is even of more significance as the effect ranking of the bisphosphonates is also nearly the same in these experiements.

This in vitro study verified an impact on osteoblasts, fibroblasts and HUVEC thus supporting the idea of a multifactor pathology of BP-ONJ. It supports the clinical theory of damage to osteogenic and blood vessel cells and soft tissue repair.

These results should be verified in an animal model and might be verified in specimens from patients with BP-ONJ.

  Bisphosphonates affect migration ability & cell viability of HUVEC, fibroblasts and osteoblasts in-vitro

  the wells, they were filled with 500µl fresh culture medium and 7µl Calcein-AM (invitrogen TM , Eugene, Oregon, USA) per well. The plates were incubated in a dark cell incubator for 1 hour. Subsequently, the Calcein-AM medium was also removed and cells were detached with 500 µl Trypsin-EDTA solution per well. 200 µl of the cell-Trypsin solution was carried in standard black 24-well cell culture plates and the extent of cell viability was measured by Microplate Fluorescence Reader (BioTek FLx800, bad Friedrichshall, Germany) with an extinction wave length of 480nm and an emission wave length of 520 nm.Tunel assay: For the apoptosis TUNEL-Assay, the three cell lines (HUVEC, HOB, NHDF, 5x104 ) in EBM, DMEM and SCGM medium were seeded to 6 well plates. After 24 hours, all cell lines were incubated with 50µmol bisphosphonates (clodronate, ibandronate, pamidronate and zoledronate) including a negative control group for 72 hours. Cells were solved by Trypsin-EDTA and centrifuged onto cover slips by 2000 rpm for 5 minutes. Then cells were fixed with 3% paraformaldehyd for 10 minutes at room temperature (RT), permeabilized with 0.1% Triton X-100, 0.1% sodium citrate for 2 minutes on ice, washed twice with PBS, pH 7.4, and incubated for one hour at 37 o C in the dark with a TUNEL reaction mixture (Boehringer, Ingelheim, Germany), for in situ detection of cell death. After washing twice with PBS, pH 7.4, cells were incubated at RT with the Hoechst solution for 5 minutes. All Hoechst-positive nuclei as well as TUNEL-positive nuclei were visualized using a Zeiss Axioplan fluorescence microscope. Apoptosis was expressed as a percent of fragmented Hoechst-positive nuclei versus total Hoechst-positive nuclei, and as a percent of TUNELpositive nuclei versus total Hoechst-positive nuclei and are shown as fold increase versus control.Migration Assay (Boyden-Chamber): To examine the effects of bisphosphonates on the migration ability of HUVEC, osteoblasts and fibroblasts, a 24-well Boyden Chamber Assay system (ThinCert TM , pore size 8µm translucent, Greiner BioOne, Essen, Germany) with a 8µm was used according to the manual (Greiner BioOne, Essen, Germany). All cell lines were incubated for 72 hours with different bisphosphonates and 50 µmol. Cells were harvested, washed twice in PBS and re-suspended in cell culture medium at a final concentration of 10 6 ml -1 . After 24 hours, the cells were stained with Calcein-AM fluorescent dye (invitrogen TM , Eugene, Oregon, USA). Thereafter, the culture medium was removed from the inserts, and the inserts were transferred to the wells of a new 24-well plate containing 500µl Trypsin-EDTA per well. This plate was incubated for 10 minutes in a cell culture incubator at 37 o C and 5% CO 2 . The inserts were removed, and 200 µl of the Trypsin-EDTA solution, now containing the detached migratory cells, was transferred from each well of the 24 well-plate to a well of a flat-bottom black 24-well plate. For quantification of cell migration, we used a Microplate Fluorescence Reader (BioTek FLx800, Bad Friedrichshall, Germany) with an extinction of 480 nm and emission of 520 nm. For each cell line and each bisphosphonate the experiments were conducted 12 times.Statistical analysis: The hypothesis was that incubation of bisphosphonates with varying potencies has a distinct effect on cell viability, cell migration ability and apoptosis of osteoblasts, fibroblasts and endothelial cells. As a control, non-incubated cells were used.Continuous variables are expressed as mean +/-standard deviation. Comparisons between groups were analysed by ANOVA (post hoc test: Tukey) for experiments with more than two subgroups (SPSS version 17.0). P values <0.05 were considered statistically significant.

Figure 1 :

 1 Figure 1: Cell viability test for osteogenic cells (a), fibroblasts (b) and human umbilicord vein endothelial cells (c) influenced by clodronate, ibandronate, pamidronate and zoledronate at different concentrations compared to a control set to 100%. Asterixs indicate concentrations that are significantly different from control.

Figure 2 :

 2 Figure 2: Tunel assay for osteogenic cells, fibroblasts and human umbilicord vein endothelial cells influenced by 50µmol clodronate, ibandronate, pamidronate or zoledronate compared to a control without bisphosphonates. The black bar in the middle of each box represents the median. The box includes all values between the 25 th and 75 th percentile. Whiskers indicate values still within 1.5 interquartile range (IQR). Outliers within 3 IQR are represented as circles, outliers over 3 IQR are represented as asterix.

Figure 3 :

 3 Figure 3: Boyden chamber migration assay for osteogenic cells, fibroblasts and human umbilicord vein endothelial cells influenced by 50µmol clodronate, ibandronate, pamidronate or zoledronate compared to a control without bisphosphonates. The black bar in the middle of each box represents the median. The box includes all values between the 25 th and 75 th percentile. Whiskers indicate values still within 1.5 interquartile range (IQR). Outliers within 3 IQR are represented as circles.

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	Materials and Methods
	Cell culture: Cell cultures were prepared and maintained according to standard cell culture
	procedures. Commercially available human umbilicord vein endothelial cells (HUVEC;
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Lonza, Basel, Switzerland) were cultured in an endothelial basal medium (EBM) supplemented with 1µg/ml hydrocortisone, 12 µg/ml bovine brain extract, 50 µg/ml gentamicin, 50 ng/ml amphotericin-B, 10 ng/ml epidermal growth factor and 10% fetal calf serum (FCS).

Human osteogenic cells (HOB; Promocell, Heidelberg, Germany), were cultivated in a solution composed of Dulbecco's Modified Eagle's Medium (DMEM) with 1% PSN, 1% Lglutamin and 10% FCS. Before the experiments, the osteogenic cells were positively characterized by immunhistochemical expression of alkaline phosphatase and osteocalcin (labelled streptavidin-botin/horseradish peroxidase).

Fibroblasts (Lonza, Basel, Switzerland) were grown in Stroma Cell Growth Medium (SCGM)

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  There were similar results for the significantly less potent clodronate and ibandronate with no significant difference between them.

	Page 9 of 19 two bisphosphonates. Page 10 of 19 Oral Diseases -Manuscript Copy Oral Diseases -Manuscript Copy Discussion
	In addition to reduced bone remodelling several theories regarding the aetiology of
	bisphosphonate osteonecrosis of the jaw are being discussed in literature (Allen and Burr,
	O r a 2009). The range of aetiological possibilities has been broadened with the realization that not O only osteoclasts, but rather several cell types are affected by bisphosphonates, as shown in r a this study.
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  The cell migration was most strongly inhibited by zoledronate and the exception of fibroblast cell migration of where ibandronat had a greater effect than zoledronate. A similar effect is seen in the present study with the greatest effect of pamidronate on fibroblast cell migration. The cell viability test in the previously published study(Walter et al., 2009) conducted with the MTT-test clodronate evaluated the weakest

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	pamidronate, with
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  The distribution of bisphosphonates in patients with BP-ONJ is reflected by this study's results. Within the natural limitations of in vitro studies, these data support the theory that bisphosphonates influence soft tissue cells such as vessel cells and fibroblasts, in addition to osteoblasts. Although the half-life of bisphosphonates can be more than ten years (Wang et al., 2007), the finding that soft tissue cells are affected by bisphosphonates could indicate that interruption of bisphosphonate treatment to promote better wound healing in the oral cavity may be reasonable.There was no funding for this study.Christian Walter and Bilal Al-Nawas got speecher's fees from Roche, Christian Walter received funding from Novartis for a different study. Klein MO, Pabst A, Al-Nawas B, Duschner H and Ziebart T (2009). Influence of bisphosphonates on endothelial cells, fibroblasts, and osteogenic cells. Clin Oral Investig 19. Wang EP, Kaban LB, Strewler GJ, Raje N and Troulis MJ (2007). Incidence of osteonecrosis of the jaw in patients with multiple myeloma and breast or prostate cancer on intravenous bisphosphonate therapy. J Oral Maxillofac Surg 65: 1328-1331

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	O r a Acknowledgement: O r a O r a 18. Walter C, O r a
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		M a M a M a M a n u s n u s n u s n u s	Deleted: This study verified an impact on osteoblasts, fibroblasts and HUVEC thus supporting the idea of a multifactor pathology of BP-ONJ.
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Conflict of interest: no funding was received for this study