Volume 51 Number 4 December 2006

Australian Dental Jo u r n a l
The official journal of the Australian Dental Association

SPECIAL RESEARCH SUPPLEMENT

Australian Dental Research Foundation

Introduction
Its show time again. The list of cast members can be found on the next page, each of them eager to grab your attention. Some are established stars, others are promising newcomers. Major credits include ADJ Productions for putting the show together. Then, of course, there are the showbiz angels who came up with the cash that made it all possible. They are hopeful, quietly confident even, that the reviews will be favourable. The gathered ensemble has put in many hundreds of hours of disciplined preparation. Those who have made it this far deserve our applause. Dental research has always enjoyed a sort of cult following. It is intended that this current presentation, together with similar annual offerings that have preceded it and others still to appear in the years ahead, will have the effect of appealing to an ever enlarging mainstream audience. This is one of the aims of the Australian Dental Research Foundation Inc (ADRF), the organization where the angels referred to above can be found. It is not necessary to be a cast member in order to appreciate, enjoy and support the theatre of oral health research. Australian researchers are ranked with the finest in the world. They should be accorded comprehensive acclamation within their own country. Dental research enjoys very little widespread, no strings attached aid other than through the ADRF. It seems not to possess the same degree of money-magnetism that attaches to cardiovascular, cancer, diabetes, asthma and many other areas of medical and pharmacological research. It deserves better than it gets. Dental disease is near the top of lists depicting causes of distress, diminished productivity and economic loss within our community. There is already a crisis, and this will worsen if, as seems inevitable, the number of trained dental personnel does not keep up with population increase. Moreover, dental disease has become linked as a co-factor in an increasing range of systemic conditions, many of which have potentially more serious morbidity than dental disease alone. It makes sense to spend money on research to improve understanding of oral health (or lack thereof) and its holistic interrelationships; to devise an expanded range of cures and preventive measures, both for individuals and the masses; to gain the micro-level understanding necessary to overcome various oral diseases (for example, there is still much ground to cover with periodontal disease); to introduce new materials and techniques; to simplify existing procedures; to reduce costs and otherwise increase access for dental services; and to assist with the development of the next generation of dental researchers. A less self-evident attribute of dental research, particularly in areas like immunology and molecular biology, is that its findings translate readily. The techniques employed and the results achieved can be picked up by investigative researchers in other fields. The tag dental could in fact be dropped. Basic processes that are revealed in oral situations will almost certainly have parallels elsewhere. This present assemblage showcases recent works in a series of divertissements, otherwise known as Abstracts. Some of the work featured will give rise to fuller expositions in specialized journals. The Australian Dental Journal (deservedly one of the most highly respected the world) is always made available for the publication of articles likely to be of relevance and interest to its largely general practitioner readership. Several ADRF Research Grant Reports meeting the requisite ADJ criteria have appeared therein as full papers during the year. These are listed within this Special Research Supplement for quick reference. The Abstracts now making up this Supplement are weighted more towards the esoteric. Nevertheless, their portrayal holds considerable fascination. At the same time it enhances the Journals coverage and attracts some attention to the ADRFs benevolent presence. There is much variety in the topics presented, which is testimony to the richness of Australian dental research. Prominent amongst the offerings this year are several contributions by Ashman et al. All of these seek to elucidate immunological reactions involved in oral candidiasis. Aside from the excellence of the research itself these people are world leaders in the field two other points are worthy of note. First, it is apparent that the separate investigations (and several other related projects that have over the years received ADRF funding) collectively contribute pieces of a bigger mosaic. Sadly, the Foundation is not able to offer large single grants to any particular individual or group: but the approach used here has worked within that restriction. The researchers have organized their campaign well. Second, as a good example of the translation effect mentioned above, their findings potentially have expanded relevance. Candidiasis is not confined to the oral cavity. As usual, there is a designated portion of the production wherein undergraduate researchers take the stage. Their efforts are impressive, especially as this is their first entrance. The material they work with frequently reflects the interests of their mentors and thus is enhanced by the expertise and enthusiasm of those persons. Perhaps this introductory experience will for some ignite a spark, such that they will aspire to be among our dental researchers of the future. If so, they must expect a certain amount of drudgery and disappointment; but for those who stay the course, the excitement of actual or potential discovery is a powerful stimulant. Finally, as well as "On with the show", I must also say thank you on behalf of the ADRF to the Editor and staff of the ADJ, to all the researchers who have featured in this production and to those who are waiting in the wings for next years. Fred Widdop Chairman Australian Dental Research Foundation

ADRF Special Research Supplement

Vol 51 No 4 December 2006

Contents
ADRF Research Grant Abstracts
S3 S3 S3 S4 S4 S4 S6 S6 Role of T lymphocytes and monocytes/macrophages in oral candidiasis RB Ashman, CS Farah T helper cytokine profile in oral candidiasis RB Ashman, CS Farah Epitope specificity and protective effects of candida-specific antibody RB Ashman, CS Farah, Y Hu Host responses to oral and systemic infection with different isolates of Candida albicans RB Ashman, CS Farah, Y Hu Macrophage gene activation in candida infection RB Ashman, CA Wells Synthesis of multiphosphorylated analogues of the anticariogenic casein phosphopeptides TJ Attard, KJ Cross, NM OBrien-Simpson, NL Huq, EC Reynolds Subsurface degradation of resin-based composites R Bagheri, MJ Tyas, MF Burrow Evaluation of human periodontal ligament fibroblast and human gingival fibroblast attachment to PRPcoated guided tissue regeneration membranes PM Bartold, Q Liu, T Chang, V Marino Problems with bite mark analysis SA Blackwell, RV Taylor, I Gordon, CL Ogelby, T Tanijiri, M Yoshino, MR Donald, JG Clement Effect of water sorption on resin cements MF Burrow, A Koiwa, J Palamara Bonding of resin composite to teeth affected by molar hypomineralization MF Burrow, V William, J Palamara, L Brearley Messer Immunohistochemical identification of mesenchymal stem cells in human periodontal ligament S Chen, S Gronthos, V Marino, PM Bartold Fluoride exposure, dental fluorosis and caries among South Australian children LG Do, AJ Spencer, A Puzio, J Armfield Th1/Th2 cytokine immune responses to Porphyromonas gingivalis in a mouse model CS Farah, S Ivanovski, E Gemmell, GJ Seymour The role of IL-12 in innate and adaptive resistance against oropharyngeal candidiasis CS Farah, AM Lichanska, MJ Waters, RB Ashman Expression of hTERT in relation to programmed cell death in premalignant and malignant oral epithelial lesions CS Farah, NW Savage, M Raiyon Development of a molecular tool for epidemiological investigation of recurrent oral candidiasis infections in HIV-positive individuals ML Fraser, RH Andrews, AH Rogers Identification of the osteoclastogenic factor from Eikenella corrodens surface-associated material NJ Gully, DR Haynes, AH Rogers, PS Zilm An investigation into the role of VEGF in oral dysplasia and oral squamous cell carcinoma S Johnstone, RM Logan Immunohistochemical study of bone sialoprotein and osteopontin in healthy and diseased root surfaces M Lao, V Marino, PM Bartold Oral mucositis clinical presentation, histological features and pro-inflammatory cytokine expression RM Logan, RJ Gibson, ST Sonis, DMK Keefe Development of integrated clinical assessment in dentistry: comparison of perceptions by students and assessors T McLean, J Fairley, TM Gerzina

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S15 S16 S17 S18 S19

Contents continued S20 Examination of the efficiency in removing bacterial contamination from dental unit water lines using current treatment protocols K Mahajani, PS Zilm, NJ Gully Mechanism of surface and subsurface demineralization of dentine P Mishra, MJ Tyas, MF Burrow, J Palamara Effect of prostaglandin E2 on the gene expression of RANKL and OPG, and TGF- 1 release from cultured osteoblasts G Ramirez, AL Symons Fluoride and apoptosis in amelogenesis JR Smid, D Harbrow, BJ Joseph Frictional resistance to sliding, with repeated displacements, in a multi-bracket model A Srinivasa, IA Meyers, CTC Ho Early detection of dental caries using laser fluorescence LJ Walsh, S Diklich Longitudinal assessment of changes in enamel mineral in vivo using laser fluorescence LJ Walsh, G Groeneveld, V Hoppe, F Keles, W van Uum, H Clifford Trabecular patterns in the temporomandibular condyle: the characterization of a young and older normal sheep model in reference to human specimens D Wilson, O Wiebkin, J Gardner, N Fazzalari The expression of GroEL and enolase by Fusobacterium nucleatum grown in continuous culture PS Zilm, NJ Gully The investigation and characterization of the co-aggregation of Fusobacterium nucleatum grown in continuous culture PS Zilm, NJ Gully ADRF Research Grant Reports published as full papers in the Australian Dental Journal 2006

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S23 S24 S25 S26 S26

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ADRF Undergraduate Research Grant Abstracts

S31 Effect of local application of PGE2 and lipoxin A4 on the immunoexpression of OPG and RANKL during healing of a surgically placed defect in the Lewis rat mandible R Chou; S Varanasi, AL Symons (Supervisors) Temporal variation of the different clinical presentations of histopathologically-proven oral lichen planus: a retrospective study of 391 patients S Kaing; M McCullough (Supervisor) An analysis of dental trauma in a large rural centre (Bunbury, WA) from 20002005 R Lam; PV Abbott (Supervisor) Staining potential of APF foam on restorative materials in vitro D Lin; B Huang (Supervisor) Non-carious cervical lesions: a proposed new system of classification JA Michael; GC Townsend, J Kaidonis (Supervisors) A 3-D CT analysis of craniofacial asymmetry in Malaysian infants with cleft lip and palate N Tziavaras; G Townsend, D Netherway (Supervisors) Interactions between the periodontopathogenic bacteria Treponema denticola and Porphyromonas gingivalis S Yoon; R Orth, S Dashper (Supervisors)

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S32 S32 S33 S33 S34

Trebitsch Grant Abstract 20052006

S35 An immunohistological study of co-infection in a mouse model J Lin; PS Bird, A Chan (Supervisors)

ADRF Research Grant Abstracts

Role of T lymphocytes and monocytes/macrophages in oral candidiasis
RB Ashman, CS Farah*

This project demonstrated a crucial role for both neutrophils and monocytes/macrophages in the clearance of Candida albicans from the oral cavity of mice,
*School of Dentistry, The University of Queensland.

and established that cytokines such as IFN- , IL-4, IL-6 and IL-10 were produced by lymphocytes from the cervical and submaxillary lymph nodes. It also led to the postulate that TNF- was an important mediator of host recovery from oral infection.

T helper cytokine profile in oral candidiasis

RB Ashman, CS Farah*

Lymph nodes and oral tissues were harvested from cytokine knockout animals infected with Candida albicans yeast and samples analysed by RT-PCR and histopathology. The results demonstrated a significant role for TNF- and IL-12 in the clearance of the oral
*School of Dentistry, The University of Queensland.

yeast. TNF- knockout mice developed a higher fungal load than controls, but the duration of infection was unchanged. In IL-12 knockout mice both fungal load and duration of infection were significantly increased, and these mice developed a chronic infection resembling that seen in T cell deficient mice. In contrast, mice deficient in IL-4, IL-10 and IFNshowed no difference compared to control mice.

Epitope specificity and protective effects of candida-specific antibody

RB Ashman, CS Farah, Y Hu*

Antibody production by inbred mice that are genetically resistant or susceptible to tissue damage was studied after systemic or oral infection with three distinct isolates of Candida albicans. The severity of infection in various anatomical locations had previously been shown to differ between both yeasts and mouse strains. Tissue-susceptible CBA/CaH mice produced both IgG1 and IgG2a antibodies, whereas BALB/c mice produced antibodies of predominantly IgG1 subclass. Systemic infection protected against rechallenge with the homologous, but not the

heterologous yeast; however the protective effect was more evident in the susceptible CBA/CaH mice than in the resistant BALB/c strain. Oral infection protected against both homologous and heterologous oral challenge, although this was significant only in the CBA/CaH mice. Western blotting demonstrated a more diverse spectrum of antibody specificities in serum from CBA/CaH, as compared to BALB/c mice. The following paper included data from this project Hu Y, Farah CS, Ashman RB. Isolates of Candida albicans that differ in virulence for mice elicit strainspecific antibody-mediated protective responses. Microbes and Infection 2006;8:612-620.

*School of Dentistry, The University of Queensland.

Australian Dental Journal ADRF Special Research Supplement 2006;51:4.

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Host responses to oral and systemic infection with different isolates of Candida albicans
RB Ashman, CS Farah, Y Hu*
Three distinct isolates of Candida albicans were used to establish systemic and oral infections in inbred mice that are genetically resistant or susceptible to tissue damage. Mice infected by either route showed significant differences both between yeasts and between mouse strains in the levels of infection in various anatomical regions. Western blotting demonstrated different patterns of epitope recognition when developed with antibodies specific for either IgG1 or IgG2a. There was substantial cross-reactivity of antibodies raised against each yeast when tested against the others, although some strain-specificity was evident. We also measured the candidacidal activity of both neutrophils and macrophages generated by culture in vitro, and showed consistent differences in the killing of the various candida strains. Thus, distinct isolates of yeast show different patterns of infection in susceptible and resistant mice, and elicit both qualitatively and quantitatively different host responses. The following paper included data from this project Hu Y, Farah CS, Ashman RB. Isolates of Candida albicans that differ in virulence for mice elicit strainspecific antibody-mediated protective responses. Microbes and Infection 2006;8:612-620.

*School of Dentistry, The University of Queensland.

Macrophage gene activation in candida infection

RB Ashman,* CA Wells
Macrophages represent an important component of natural resistance against candida infection. We have studied patterns of gene activation in macrophages from BALB/c (resistant) and CBA/CaH (susceptible) mice after one hour and six hours exposure to heatkilled Candida albicans 3630 yeasts in vitro. There were significant differences in the transcriptional responses of the macrophages, consistent with the known patterns of resistance and susceptibility in these two mouse strains. About 300 genes were regulated in BALB/c macrophages, whereas more than 800 were regulated in macrophages from the susceptible CBA/CaH mice. However, yeast infections are cleared efficiently by mice of both strains, and pathways involved in production of reactive oxygen species, apoptosis, and expression of TNF receptors were
*School of Dentistry, The University of Queensland. School of Biomolecular and Biomedical Science, Griffith University.

prominent in both. Induction of TNF- signalling components (including TNF- itself) indicated that signalling through TLR2, a known receptor for yeast membrane components, was intact in both strains, and TLR2 message was itself significantly up-regulated after one hours exposure to C. albicans. We also observed a shared expression pattern between Tlr2 and a novel c-type lectin, Mincle (Macrophage inducible c-type lectin). Mincle and Tlr2 were highly inducible in resistant BALB/c, but were poorly regulated in susceptible CBA/CaH bone marrow macrophages. Mincle protein was induced in BALB/c macrophages in response to C. albicans infection whereas CBA macrophages demonstrated higher spontaneous levels of protein but a poorer response to the yeast. Mincle was shown to co-localize to the phagocytic cup of macrophages ingesting yeast, and demonstrated different degrees of responsiveness to different isolates of the yeast.

Synthesis of multiphosphorylated analogues of the anticariogenic casein phosphopeptides

TJ Attard, KJ Cross, NM OBrien-Simpson, NL Huq, EC Reynolds*
Dental caries is initiated via the demineralization of tooth hard tissue by organic acids from the fermentation of dietary sugar by dental plaque odontopathogenic bacteria.1 Tryptic phosphopeptides derived from milk caseins are known to associate with amorphous calcium phosphate (ACP), forming stable
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complexes that behave as calcium phosphate delivery vehicles and which are effective in the remineralization of early enamel lesions. We have recently developed a simple and efficient purification procedure involving microfiltration of calcium phosphate-induced complexes of the multiple phosphoseryl-containing peptides from
Australian Dental Journal ADRF Special Research Supplement 2006;51:4.

a tryptic digest of casein. The peptides produced by this procedure have been comprehensively characterized. The major peptides of the preparation are -CN(1-25) [2] and S1-CN(59-79) [1] and their deamidated forms with smaller amounts of S2-CN(1-21) [3] and S2CN(46-70) [4]. The sequences are shown below, using the three letter codes for the amino acyl residues, where Ser(P) denotes an O-phosphoseryl residue. [1] Gln59-Met-Glu-Ala-Glu-Ser(P)-Ile-Ser(P)-Ser(P)Ser(P)-Glu-Glu-Ile-Val-Pro-Asn-Ser(P)-Val-GluGln-Lys79 S1-CN(59-79) 1 [2] Arg -Glu-Leu-Glu-Glu-Leu-Asn-Val-Pro-GlyGlu-Ile-Val-Glu-Ser(P)-Leu-Ser(P)-Ser(P)-CN(1-25) Ser(P)-Glu-Glu-Ser-Ile-Thr-Arg25. [3] Lys1-Asn-Thr-Met-Glu-His-Val-Ser(P)-Ser(P)Ser(P)-Glu-Glu-Ser-Ile-Ile-Ser(P)-Gln-Glu-ThrTyr-Lys21. S2-CN(1-21) [4] Asn 46-Ala-Asn-Glu-Glu-Glu-Tyr-Ser-Ile-GlySer(P)-Ser(P)-Ser(P)-Glu-Glu-Ser(P)-Ala-GluVal-Ala-Thr-Glu-Glu-Val-Lys70. S2-CN(46-70) All peptides contain the sequence motif -Ser(P)Ser(P)-Ser(P)-Glu-Glu-. These peptides have been shown to be calcium phosphate delivery vehicles. The potential anticariogenicity of the CPP-ACP has been demonstrated in the rat caries model, in situ human caries models, in vitro remineralization/demineralization models and short-term mouthwash trials, as reviewed recently.2 We are investigating the development of improved calcium phosphate delivery vehicles with enhanced anticariogenic properties. To achieve this we are studying the structure-function relationship of the casein phosphopeptides-ACP complexes using analogues of the casein phosphopeptides. The goal is to investigate the anticariogenicity of the casein phosphopeptides using synthetic analogues in order to develop anticariogenic peptides with enhanced functionality. Peptides were designed based on the phosphorylated motif. Fmoc chemistry was used to synthesize peptides. The following peptides relating to the S1-CN(59-79) casein peptide were prepared: Ser- Ser-Ser-Glu-Glu, Glu-Glu-Glu-Glu-Glu, Xaa-Xaa-Xaa-Glu-Glu [X=Ser(P), Thr(P) or Tyr(P)], Ile-Ser(P)-Ser(P)-Ser(P)-Glu-Glu and Gln59-Met-Glu-Ala-Glu-Ser(P)-Ile-Ser(P)-Ser(P)-Ser(P)Glu-Glu-Ile-Val-Pro-Asn-Ser(P)-Val-Glu-Gln-Lys 79. Syntheses was carried out using an AB 431A peptide synthesizer. Standard deprotection/coupling cycles were employed with extended coupling times required for incorporation of the specialized derivatives, FmocSer(PO3,Bzl,H)-OH, Fmoc-Tyr(PO3,Bzl,H)-OH and Fmoc-Thr(PO3,Bzl,H)-OH. Upon acidolytic deprotection and cleavage from the resin, the crude peptides were purified via semi-preparative RP-HPLC using a Zorbax 300SB-C18 column. Mass spectrometry was used to confirm the mass of the synthetic peptide. 1D NMR spectroscopy was used to confirm that the peptides were random coil in solution in the absence of
*Cooperative Research Centre for Oral Health Science, The University of Melbourne.
Australian Dental Journal ADRF Special Research Supplement 2006;51:4.

calcium ions. The peptide Ile-Ser(P)-Ser(P)-Ser(P)-GluGlu was examined by 2D NMR spectroscopy in the presence of five equivalents of calcium ions. The spectra were recorded with spectral widths of 6000.6Hz in F1 and F2 with 1024 complex data points. Phase-sensitive spectra were collected using the StatesTPPI method.3 The WET4 pulse sequence was used for solvent suppression in the TOCSY5,6 spectra. A mixing time of 80 min was used for the TOCSY spectrum. A total of 100 t1 increments of 16 transients were collected for the TOCSY spectrum. TOCSY spectra were zero-filled to 2k complex points in t2 prior to Fourier transformation. Linear prediction was used to extrapolate the t1 data to 2k data points. A Hamming window function7 was applied to both the t1 and t2 data. All data processing was performed using Varians VnmrS program on an SGI Indigo2 workstation with 256MB of RAM. The TOCSY spectra revealed all spin systems and showed dispersion of the phosphoseryl amide resonances similar to that observed in the larger peptide 59 S1-CN(59-79), Gln -Met-Glu-Ala-Glu-Ser(P)-Ile-Ser(P)Ser(P)-Ser(P)-Glu-Glu-Ile-Val-Pro-Asn-Ser(P)-Val-GluGln-Lys79. The secondary H and NH proton chemical shifts were calculated using the random coil chemical shifts reported for the phosphoseryl residues8 and other residues9 with the sequence-dependent corrections.10 Similar secondary amide chemical shifts were observed for the larger peptide S1-CN(59-79). In conclusion, synthesis of multiphosphorylated analogues of the casein phosphopeptides is the appropriate strategy to further our understanding of the role of peptide sequence on the uptake and release of calcium, and phosphate ions from these anticariogenic complexes. References
1. Loesche W. Role of Streptococcus mutans in human dental decay. Microbiol Revs 1986:50:353-380. 2. Cross KN, Huq L, Reynolds EC. Anticariogenic peptides. Nutraceutical Proteins and Peptides in Health and Disease. Invited Book Chapter. CRC Press, 2005. 3. Marion D, Ikura M, Tschudin R, Bax A . Rapid recording of 2D NMR spectra without phase cycling. Application to the study of hydrogen exchange in proteins. J Magn Reson 1989;85:393-399. 4. Smallcombe SH, Patt SL, Keifer PA. WET solvent suppression and its applications to LC NMR and high-resolution NMR spectroscopy. J Magn Reson, Ser. A 1983;117:295-303. 5. Braunschweiler L, Ernst RR. Coherence transfer by isotropic mixing: Application to proton correlation spectroscopy. J Magn Reson 1983;53:521-528. 6. Bax A, Davis DG. MLEV-17-based two-dimensional homonuclear magnetization transfer spectroscopy. J Magn Reson 1985;65:355-360. 7. Ernst RR, Bodenhausen G, Wokaun A. Principles of Nuclear Magnetic Resonance in One and Two Dimensions. Oxford: Clarendon Press, 1990. 8. Bienkiewicz EA, Lumb KJ. Random-coil chemical shifts of phosphorylated amino acids. J Biomol NMR 1999;15:203-206. 9. Merutka G, Dyson HJ, Wright PE. 'Random coil' 1H chemical shifts obtained as a function of temperature and trifluoroethanol concentration for the peptide series GGXGG. J Biomol NMR 1995;5:14-24. 10. Schwarzinger S, Kroon GJ, Foss TR, Chung J, Wright PE, Dyson HJ. Sequence-dependent correction of random coil NMR chemical shifts. J Am Chem Soc 2001;123:2970-2978.

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Subsurface degradation of resin-based composites

R Bagheri, MJ Tyas, MF Burrow*

The objective of this study was to test the hypothesis that a degraded subsurface layer is produced in dental composites as a result of exposure to lactic acid and NaOH, by observing the penetration of AgNO 3 solutions. Fifty-four disc shaped specimens were prepared from four resin composites: Point 4 (Kerr), Premise (Kerr), Filtek Supreme (3M ESPE) and Ceram X (Dentsply), and two polyacid-modified resin composites: Dyract (Dentsply) and F2000 (3M ESPE). The moulds were filled with the materials and cured against plastics matrix strips. Specimens were immersed in distilled water for one week at 60C and then transferred to one of the three aqueous media: distilled water, lactic acid or NaOH at 60C for an additional two weeks. Specimens were washed and immersed in 50 w/w % aqueous silver nitrate for 10 days at 60C, washed and exposed to a photo developer solution under light for eight hours. After reduction of the silver, specimens were embedded in epoxy resin, sectioned
*School of Dental Science, The University of Melbourne.

and polished using diamond pastes down to 1m, coated with carbon and examined by backscatter electron microscopy. The depth of silver penetration into the degraded area was measured using SEM micrographs. Energy dispersive X-ray analysis (EDAX) was used to confirm the presence of silver in the specimens. Data analysis by ANOVA and Tukeys test showed that the depth of silver penetration was material and solution dependent, and the difference was significant between most of the materials (P<0.05). NaOH produced the greatest depth of degradation and lactic acid the least. Premise incurred the greatest depth of silver penetration when it was subjected to NaOH, and Filtek Supreme the second greatest with peeling and cracking of the surface, whereas F2000 and Point 4 showed the least silver penetration depth in NaOH and lactic acid. Published: Bagheri R, Tyas MJ, Burrow MF. Subsurface degradation of resin-based composites. Dent Mater (in press).

Evaluation of human periodontal ligament fibroblast and human gingival fibroblast attachment to PRP-coated guided tissue regeneration membranes
PM Bartold, Q Liu, T Chang, V Marino*
The purpose of this study was to evaluate, using scanning electron microscopy (SEM), the attachment of human periodontal ligament (HPDL) and human gingival (HG) fibroblasts onto commercially available guided tissue regeneration (GTR) membranes coated with platelet rich plasma (PRP). Three commercially available GTR membranes were tested: Gore-Tex Regenerative Membrane (GTN1), Inion GTR Biodegradable system (INION) and GoreResolut XT Regenerative membrane (GTRX). Small pieces of membrane were prepared as specified by the manufacturers and pre-wetted with phosphate buffered saline before exposure to PRP for two hours. These membranes were then seeded with either HPDL or gingival fibroblasts and incubated in Dulbeccos Modified Eagles culture medium for 24 hours. Post incubation samples were fixed and processed for examination using SEM microscopy. Under SEM microscopy, PRP treated membranes showed greater cell attachment compared to nontreated membranes. The Inion membrane showed the greatest attachment followed by the GTN1 and GTRX membranes for both populations of fibroblasts. There were two major morphological features seen for both the HPDL and HG fibroblasts in the PRP treated membranes. The cells showed a distinct increase in cytoplasmic extensions and they also appeared more intertwined and wrapped around individual fibres of the GTR membranes compared to untreated control membranes. HPDL and HG fibroblasts have a distinct reaction with different GTR membranes depending on both microstructure and composition of the membrane. It is clear that PRP does have a significant impact on the way HPDL and HG fibroblasts attach to certain GTR membranes by both increasing the amount and the quality of attachment. However, how this increase in attachment is achieved and how well these in vitro results are translated into in vivo and actual clinical benefits still needs further investigation.

*Dental School, Colgate Australian Clinical Dental Research Centre, The University of Adelaide.

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Australian Dental Journal ADRF Special Research Supplement 2006;51:4.

Problems with bite mark analysis

SA Blackwell,* RV Taylor,* I Gordon, CL Ogelby, T Tanijiri, M Yoshino, MR Donald, JG Clement*
Bite mark analysis is currently contentious. For a subject with such potentially serious outcomes for both suspect and victim, little research analysing methods and evaluating outcomes has reached peer reviewed journals.1 Although admissibility of bite mark evidence has been established and routinely accepted in the USA and other legal systems for a long time,2 some odontologists argue that bite mark methodology has never undergone critical evaluation and legitimately passed the Frye3 test for admissibility, a problem also relevant for other areas such as earprint identification.4 Other legal observers are concerned that forensic odontologists are giving insufficient critical attention to the quality of bite mark evidence presented to the courts.5,6 Central to the problem of analysis is the difficulty of comparing two-dimensional images of a bite mark with three-dimensional replicas of dentitions which may have caused them. A deficiency in quantitative bite mark research has, over time, resulted in uncertainty in bite mark evidence in legal systems worldwide, particularly in Australia. The natural tendency to see what one wants to see, thereby tempting examiners to over-interpret bite mark evidence, has led to serious difficulties when bringing such evidence before the courts.7 This area of forensic science requires standardization to ensure consistency of expert opinions. Two notorious Australian cases8,9 have seen bite mark evidence rejected as unsafe and convictions overturned on appeal. Perhaps for such reasons, bite mark analysis is currently undergoing review. Generally, courts now look for quantitative rather than simply descriptive analysis before accepting scientific evidence and it can be anticipated that future developments in the analysis of bite marks will need to follow this general trend if convictions are going to be made with confidence. Many studies have described and quantified bite patterns in two dimensions (photographs, overlays etc.) and yet, despite the fact that the dentition of the biter and the corresponding bite marks are both 3-D phenomena, there have been few 3-D analyses.10-12 This is surprising but may reflect the lack of access to methods of measuring in three dimensions that have recently become available. Legal problems involving bite mark evidence suggest that alternative methods of analysis may be required, and the logical first step is to analyse bite marks in 3-D. There are three factors of three-dimensionality involved when one person bites another the curvature of the skin, the shape of the biting dentition and the depth of the injury should the tooth/teeth puncture the skin to create a depression (although this is infrequent). The injury, as it is being inflicted, is a three-dimensional
Australian Dental Journal ADRF Special Research Supplement 2006;51:4.

event the skin deforms to accommodate the shape of the teeth. However, once the teeth are withdrawn, the skin is restored to near its original shape and the resultant mark is represented without depth information on the curved surface of the skin. If the force of the bite is great enough to leave an indentation in the skin, then the mark is also three-dimensional. Injuries range from a defined mark/s, a diffuse bruise, complete removal of tissue, and swelling due to inflammation. This study presents a technique developed for 3-D imaging and quantitative comparison of human dentitions and simulated bite marks. A sample of 42 study models and the corresponding bites, made by the same subjects in acrylic dental wax, were digitised by laser scanning. This technique allows image comparison of a 3-D dentition with a 3-D bite mark eliminating distortion due to perspective, as experienced in conventional photography. Cartesian co-ordinates of a series of landmarks were used to describe the dentitions and bite marks and, using crossvalidation techniques, a matrix was created to compare all possible combinations of matches and non-matches. An algorithm was developed which estimated the probability of a dentition matching its corresponding bite mark. A receiver operating characteristic graph illustrated the relationship between values for specificity and sensitivity and showed that, for this sample, 15 per cent of non-matches could not be distinguished from the true match, translating to a 15 per cent probability of falsely convicting an innocent person. In other words, six out the 42 people in this sample are at risk of being falsely convicted. This result is indicative of this particular sample and may be lower in actual casework, but this is not certain. Although the morphology of each dentition in this study may be unique, we hypothesised that very similar and indeed indistinguishable bite marks may be produced by a number of different dentitions, despite the uniqueness of these dentitions. Bite marks produced by different dentitions in a firm substrate cheese, for example may be more unique with respect to each other, and more similar to their corresponding dentitions, than bite marks inflicted by the same set of dentitions on skin, a highly deformable substrate. Dynamic, tissue and postural distortion, as explained by Sheasby and MacDonald,13 have a significant impact on the quality of a bite mark in skin. The ideas and methods developed in this study for 3-D imaging and quantitative comparison of human dentitions and their corresponding bite marks are by no means a final solution to the complex problems bite mark analysis presents. This research is intended to
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increase awareness about the possibility that the number of false positives resulting from bite mark evidence given in court may be higher than we realise, a problem which is not unique to bite mark analysis but affects many other areas of forensic science. We hope researchers will be inspired to continue to investigate the three-dimensionality of bite marks and to improve the science of bite mark analysis. References
1. Pretty IA, Sweet D. The scientific basis for human bitemark analyses - a critical review. Sci Justice 2001;41:85-92. 2. People v. Marx, in 54 Cal. App. 3d 100, 126 Cal Rptr. 350. 1975. 3. Frye v. United States, in 293 F.1013 (D.C. Circ). 1923. 4. Rutty GN, Abbas A, Crossling D. Could earprint identification be computerised? An illustrated proof of concept paper. Int J Legal Med 2005;119:335-343. Epub 2005 Feb 10. 5. Rothwell BR. Bite marks in forensic dentistry: a review of legal, scientific issues. J Am Dent Assoc 1995;126:223-232. *School of Dental Science, The University of Melbourne. Department of Mathematics and Statistics, The University of Melbourne. Department of Geomatics, The University of Melbourne. Medic Engineering, Kyoto, Japan. National Research Institute of Police Science, Chiba, Japan.

6. Gundelach A. Lawyers reasoning and scientific proof: a cautionary tale in forensic odontology. J Forensic Odontostomatol 1989;7:11-16. 7. Wells D. Bitemarks (Teaching resource material for Forensic Diploma of Clinical Forensic Medicine). Monash University, Victoria, Australia. 1998. 8. Raymond John Carroll, in Australian Criminal Reports. Court of Criminal Appeal, Queensland, p. 410. 1985. 9. Lewis v The Queen, in Federal Law Reports. Court of the Appeal of the Northern Territory p. 104. 1987. 10. Forrest A, Davies I. Bite marks on trial the Carroll case. Aust Soc Forensic Dent 2001;18:6-8. 11. Thali MJ, Braun M, Markwalder ThH, et al. Bite mark documentation and analysis: the forensic 3D/CAD supported photogrammetry approach. Forensic Sci Int 2003;135:115-121. 12. Martin-de las Heras S, Valenzuela A, Ogayar C, Valverde AJ, Torres JC. Computer-based production of comparison overlays from 3D-scanned dental casts for bite mark analysis. J Forensic Sci 2005;50:127-133. 13. Sheasby DR, MacDonald DG. A forensic classification of distortion in human bite marks. Forensic Sci Int 2001;122:75-78.

Published: Blackwell SA, Taylor RV, Gordon I, Ogelby CL, Tanijiri T, Yoshino M, Donald MR, Clement JG. 3-D imaging and quantitative comparison of human dentitions and simulated bite marks. Int J Legal Med 2006;4:1-9 [Epub ahead of print].

Effect of water sorption on resin cements

MF Burrow,* A Koiwa, J Palamara*

The use of ceramic materials in clinical restorative dentistry has increased in recent years and associated with this is the increase of resin-based cements available. One of the critical factors for ceramic restoration survival is that the underlying resin cement and base materials do not absorb water so as to stress the restoration in tension, which may lead to premature failure. In addition, there is a change in philosophy in the design of some restorations in severely broken down teeth that places a heavy reliance on the strength and adhesive potential of the resin-based cement. Previous research has demonstrated that when resinbased bonding resins are placed in water the effect is for the tensile strength to decrease and the resin to expand. There is little information available describing water sorption and plasticisation of resin cements and how this might affect the physical properties and influence the longevity of direct restorations. The aim of this project was to investigate the water sorption and tensile strength of four resin cements, a polyacid-modified resin cement and a resin-modified glass-ionomer luting cement stored in water or 50 vol% ethanol and water solution for up to six months. Four resin-based cements: PermaFlo DC (Ultradent, USA), Panavia F (Kuraray Medical, Japan), RelyX ARC (3M ESPE, USA), and LINKMAX (ColteneWhaledent, Switzerland), one resin-modified glassionomer cement (GIC): FujiCem (GC International,
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Japan) and one polyacid-modified resin composite: PermaCem (DMG, Germany) were used in the study. Cylinders (4mm diameter and 6mm long) of the cement were made. Light-cured and dual-cured materials were light-cured for 60 seconds at each end of the cylinders. Specimens were placed in either distilled water or 50 vol% ethanol and water and stored at 37C. One set of samples was used for determining water sorption by weighing, and measuring the diameter and length of the specimens to calculate volumetric change. Measurements were made approximately after one hour, 1, 2, 3 and 7 days, then weekly until variations in dimensions and water sorption had stabilized. A further set of samples was made to determine tensile strengths of the cements. Cylinders of the resins were made then shaped to an hourglass shape using fine round diamond burs in a high-speed handpiece to a diameter of approximately 1mm at the narrowest portion. Five specimens were made for each of the test times of 1, 3, and 7 days, and 1, 3 and 6 months. Specimens were stressed in tension at a rate of 1mm/min in a Universal testing machine. The load at failure was recorded and calculated to MPa. Mean tensile strengths were calculated then analysed statistically using ANOVA and Fishers test. All materials demonstrated a significant increase in weight due to water sorption and stabilised within five
Australian Dental Journal ADRF Special Research Supplement 2006;51:4.

weeks. The resin-modified GIC absorbed most water, however water uptake quickly stabilised by three days. The other resin-based materials all absorbed water, but to a much lesser extent. All materials showed minimal increase in water sorption after one week. When immersed in the 50% water:ethanol solution the degree of water sorption was greater for all materials, with the resin-modified GIC showing most water uptake. The materials least affected were PermaFlo and LinkMax. All materials increased in size slightly, with the greatest increase in volume being approximately four per cent. The most stable material in the water solution was LinkMax. Little variation was noted for the specimens stored in the 50% water:ethanol solution. The tensile strength showed an initial increase in the first week for the resin-based materials. The resin-

*School of Dental Science, The University of Melbourne. Department of Cariology and Operative Dentistry, Graduate School, Medical and Dental University, Tokyo, Japan.

modified GIC failed during specimen preparation so was excluded. PermaFlow showed a significant decrease in tensile strength during the six months of storage. Panavia F showed little change over six months, and RelyX stabilised in strength after one month. The remaining materials showed a decrease in strength after one month and continued to decline for up to six months. It was concluded that the resin-based cements were more stable compared with the polyacid-modified resin composite or resin-modified glass-ionomer cement. It would seem that although the tensile strength decreases over time for most materials the decrease is unlikely to significantly affect retention of a restoration. The observed volumetric changes were quite small for the resin-based materials. There was little difference whether the cement was stored in water or 50% ethanol:water. The 50% ethanol:water solution compared with 100 per cent water showed little variation in strength and volume changes but showed greater sorption for the materials tested.

Bonding of resin composite to teeth affected by molar hypomineralization

MF Burrow, V William, J Palamara, L Brearley Messer*

Hypomineralized first permanent molars are at risk of enamel breakdown after eruption into the oral cavity. Restorative management of affected teeth is often very difficult and is dependent on the severity of the hypomineralized defects and patient co-operation. Literature on bonding to hypomineralized enamel is limited. The advent of the microshear bond strength test method has allowed testing of small areas of tooth structure such as the defects in molars exhibiting hypomineralization. The bonding mechanisms of phosphoric acid etchbased resin adhesive systems rely on the etching of the enamel surface to enable a micromechanical bond. The advent of the self-etching priming bonding systems employs a higher pH acidic resin to condition the enamel surface for bonding. This bond of the selfetching priming adhesives is partly micromechanical and also seems to incorporate chemical bonding. The enamel surface of hypomineralized teeth has a reduced content of mineral and increased amounts of interprismatic proteins and water. Due to these differences compared with normal enamel, it is important to better understand the bond of these two types of resin bonding systems to hypomineralized enamel. The aim of the current project was to investigate the microshear bond strength of resin composite bonded to hypomineralized enamel using either Single Bond (3M ESPE, USA) or Clearfil SE Bond (Japan). The bonded
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interfaces were also examined using scanning electron microscopy (SEM). Extracted molars from patients exhibiting molarincisor hypomineralization were used. The molar crowns were sectioned into four pieces with the buccal or lingual surfaces ground with 600-grit SiC paper to create a flat bonding surface. The enamel pieces were then embedded in plaster. Bonding was performed according to each manufacturers instructions for either Single Bond or Clearfil SE Bond. The bonded surface area was demarcated by placing a 1mm high piece of 0.975mm diameter tube onto the bonded enamel surface, which was light-cured. The tube was subsequently filled with resin composite. Specimens were stored in 37C water for 12 hours prior to testing the microshear bond strength using a universal testing machine with a crosshead speed of 1mm/min. The load at fracture was recorded and converted to MPa. Mean bond strengths were calculated and statistically analysed using ANOVA and the Students t test. In addition, the etched enamel and bonded interfaces were examined using SEM. Twenty-two specimens were tested for the normal enamel for each adhesive and for hypomineralized enamel, 29 specimens for Single Bond and 27 specimens for SE Bond. The bond strengths for each material were: Single Bond 16.3(10.0)MPa for normal enamel and 7.1(4.9)MPa for hypomineralized enamel; SE Bond 19.6(7.4)MPa for normal versus 10.4(7.6)MPa for
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hypomineralized enamel. The results were statistically significantly lower for the hypomineralized enamel compared with normal enamel for both adhesives (p<0.01). There were no differences between the materials bonded to the same enamel substrate. The examination of etch patterns after phosphoric acid etching showed typical enamel etch patterns on normal enamel whereas hypomineralized enamel showed an irregular etch pattern that was potentially less conducive for bonding to. The enamel conditioned with the self-etching primer showed no clear etch pattern on the normal enamel whereas the hypomimeralized enamel showed a more distinct etch pattern. However, the depth of etching was significantly less than the phosphoric acid etched enamel. The bonded interfaces for Single Bond were quite different between the two enamel substrates. The hypomineralized enamel showed a bonded interface that was porous and exhibited numerous cracks. Similarly, for SE Bond, the hypomineralized enamel surfaces showed cracks in the enamel and also porosities not seen in the normal enamel. The cracking was partly due to the desiccation of the specimens for the SEM observations. This was an indication that the
*School of Dental Science, The University of Melbourne.

hypomineralized enamel contained a greater content of water. The microshear bond strengths of the phosphoric acid-based adhesive and the self-etching priming adhesive were significantly less for the hypomineralized enamel. There was no difference between the two adhesive systems examined, although the self-etching priming system showed a slight indication that it may perform better on hypomineralized enamel. The poorer bonding to the hypomineralized enamel of phosphoric acidbased adhesive was most likely caused by inadequate tag formation due to the reduced porosity among crystals of the enamel. The self-etching priming adhesive showed more effective etching of the hypomineralized enamel but the bond was affected by other factors within this enamel substrate that need to be clarified. Cohesive failure in the hypomineralized enamel was frequently observed after the bond test, indicating the intrinsic weakness of this substrate for bonding. Published: William B, Burrow MF, Palamara JE, Messer LB. Microshear bond strength of resin composite to teeth affected by molar hypomineralization using 2 adhesive systems. Pediatr Dent 2006;28:233-241.

Immunohistochemical identification of mesenchymal stem cells in human periodontal ligament

S Chen,* S Gronthos, V Marino,* PM Bartold*
The human periodontium is comprised of four structures gingiva, alveolar bone, periodontal ligament and cementum. The phenotypes of cells in each structure are unique and they are important for the homeostasis of these tissues. The precise origins of the cells in the mature periodontium, and the location of their progenitors, are still unknown. It is unclear whether they originate from multi-potential stem cells or from multiple different ancestral cells for each separate lineage. This ambiguity is due, in part, to the lack of clear phenotype markers. Therefore the aim of this study was to identify and localize stem cells in diseased and healthy human periodontal ligament using immunohistochemistry. Twenty-five healthy and 25 periodontitis-affected teeth were collected from individual patients. The teeth were fixed in formalin, decalcified and embedded in paraffin in preparation for immunohistochemistry staining. The antibodies used to identify stem cells included Stro-1, CC9 (anti-CD146) and CD44. Stained sections were examined by light microscopy and an assessment of the distribution of positively stained cells within defined compartments of the periodontium was made. Stem cells were identified in both healthy and diseased periodontal ligament. These stem cells were mainly located in the paravascular region and small clusters of stem cells were also found in the extravascular region. Wider distributions of stem cells were detected in diseased sections compared to healthy sections. Stem cells have been identified in human periodontal ligament of both healthy and diseased teeth. In the future, tissue engineering through biological induction of the stem cells in periodontal ligament and functional construction of the tissue architecture may lead to more predictable regeneration of periodontium. Published: Chen S, Marino V, Gronthos S, Bartold PM. Location of putative stem cells in human periodontal ligament. J Periodontal Res (in press).
Australian Dental Journal ADRF Special Research Supplement 2006;51:4.

*Dental School, Colgate Australian Clinical Dental Research Centre, The University of Adelaide. Mesenchymal Stem Cell Group, Division of Haematology, Institute of Medical and Veterinary Science, Adelaide.
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Fluoride exposure, dental fluorosis and caries among South Australian children
LG Do, AJ Spencer, A Puzio, J Armfield*

The use of fluoride involves a balance between the protective effect against caries and the risk of having fluorosis. Fluorosis in Australian children was highly prevalent in the early 1990s. Policy initiatives were introduced to control fluoride exposure so as to reduce the prevalence of fluorosis. This study aimed to describe the prevalence, severity and risk factors for fluorosis, and the trend of fluorosis among South Australian children. The study also aimed to explore the effect of the change in fluoride exposure on dental fluorosis and caries. This research project was nested in a larger population-based study, the Child Oral Health Study (COHS) in Australia 20022005. The parent studys sample was chosen using a multi-stage, stratified random selection with probability of selection proportional to population size. Fluoride exposure history was retrospectively collected by a parental questionnaire. This nested study sample (n=1401) was selected from the pool of South Australian COHS participants. Children were selected by year of birth to form three birth cohorts: those born in 19891990; 19911992; and 19931994. Children were approached in two further stages: a dental health perception questionnaire, and a clinical examination for fluorosis. Some 898 children took part in the first stage. Among those, one trained dentist examined 677 children for fluorosis under clinical conditions using two indices (the Fluorosis Risk Index (Pendrys, 1990) and the TF Index (Thylstrup and Fejerskov, 1978)). The Dental Aesthetic Index score was also recorded. Caries experience extracted from dental records of all previous visits to school dental clinics was used to enable calculation of dmfs/DMFS scores at different anchor ages. Data were re-weighted by age and sex to represent the South Australian child population. Per cent lifetime exposure to fluoride in water and patterns of
*Australian Research Centre for Population Oral Health, School of Dentistry, The University of Adelaide.

discretionary fluoride use were calculated. Fluorosis data were used to calculate the prevalence and severity of fluorosis. Caries dmfs/DMFS scores were calculated at anchor ages six and eight years to enable comparison between birth cohorts. A higher proportion of children in the later birth cohorts used low concentration fluoride toothpaste, and a smaller amount of toothpaste was used when they commenced toothbrushing. There was a significant decline in the prevalence of fluorosis across the three successive birth cohorts. The prevalence of fluorosis defined as having a TF score of 1+ on upper central incisors of the cohorts 19891990, 19911992 and 19931994 were 34.7 per cent, 25.4 per cent and 22.1 per cent respectively (chi-square, p<0.05). Risk factors for fluorosis, defined by the two indices, were use of standard concentration fluoride toothpaste, an eating and/or licking toothpaste habit, and exposure to fluoridated water. Means (SD) of the deciduous caries dmfs scores at ages six and eight years were 1.45 (3.11) and 2.46 (3.93) respectively. Evaluation of the tradeoff between fluorosis and caries with fluoride exposure indicated that the use of low concentration fluoride toothpaste and preventing an eating/licking of toothpaste habit when children commence their toothbrushing in childhood could reduce the prevalence of fluorosis without a significant increase in caries experience. Analysis of oral health-related quality of life data revealed that having caries and malocclusion had a negative impact on affected childrens quality of life. Children with fluorotic teeth generally had less caries and were more likely to perceive better oral health. There was a marked decline in the prevalence of fluorosis across the three successive birth cohorts. The decline was linked with the reduction in exposure to fluoride. Exposure to fluoridated water and several components of toothpaste use were risk factors for fluorosis. Establishing an appropriate use of fluoride toothpaste could be successful in reducing fluorosis without a significant increase in caries experience.

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Th1/Th2 cytokine immune responses to Porphyromonas gingivalis in a mouse model

CS Farah, S Ivanovski, E Gemmell, GJ Seymour*

Studies examining Th1 and Th2 cytokine profiles in have been controversial and periodontitis inconsistent.1,2 Regardless of the various models for the role of Th1 and Th2 cytokines in disease progression, T cell derived cytokines remain critical in the immunoregulation of periodontal disease.3 Various animal models have been used to study the pathogenesis of periodontal disease, and these have been useful in elucidating the bacteria-mediated response.4 In the current study we utilized a wellestablished murine abscess model in genetically modified cytokine-specific knockout mice to elaborate on the role of Th1/Th2 cytokines in the immune response to Porphyromonas gingivalis. Specific pathogen-free female IL-10, IL-12p40, IFN- , and TNF knockout (-/-) mice on the C57BL/6J background, and IL-4 knockout mice on the BALB/c background were used in this study. Mice were injected subcutaneously in two sites on the dorsal surface approximately 1cm on either side of the midline as described previously.5 Mice were injected with 100l/site of 1010/ml P. gingivalis W50, or the equivalent volume of sterile PBS. Lesion size was determined, and serum analysed for Th1/Th2 cytokine expression and P. gingivalis specific antibodies. Splenic CD4 and CD8 T cells were assayed by intracellular cytokine staining for the expression on IL-4, IL-10 and IFN- . IL-12p40-/- mice exhibited a large lesion diameter at day 1 and day 10 compared to C57BL/6J wildtype mice, and there was no significant reduction in lesion size from day 1 to day 10, in contrast to other knockout mice. There was a significant increase in the percentage of CD4 and CD8 T cells positive for IL-4 and IFN- in P. gingivalis challenged IL-12p40-/- mice compared with sham injected IL-12p40-/- mice. Porphyromonas gingivalis challenged IL-12p40-/- mice also displayed higher numbers of CD4 and CD8 T cells positive for IL-4, IL-10 and IFN- , compared with P. gingivalis challenged wildtype mice. A similar pattern was seen in TNF-/- mice, but not in other knockout mice. High levels of IL-12 were detected in the serum of IL-10-/- mice after P. gingivalis challenge. No IL-10 was detected in IL-12p40-/- mice, but moderate levels were detected in TNF-/- mice. High levels of IgG1 were detected in IL-12p40-/- and IL-10-/- mice, with lower levels seen in IFN- -/-, TNF-/-, and IL-4-/- mice. Virtually no or very low levels of IgG2a were found. The results of our study support the important role for innate mechanisms in response to a primary P. gingivalis challenge. Our results show that
*Oral Biology and Pathology Research Unit, School of Dentistry, The University of Queensland.
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IL-12p40-/- and TNF-/- mice suffer from an altered local and systemic response, rendering them more susceptible to subcutaneous P. gingivalis challenge. Both these cytokines are critical in either innate resistance and/or in immunoregulation between innate and adaptive systems.6,7 Except for IL-12p40-/- and TNF-/mice, the remaining knockout mice were characterized by a weak splenic T cell cytokine response. IL-12p40-/- mice exhibited a marked initial inflammatory response to subcutaneous infection of P. gingivalis. In the absence of IL-12, the inflammatory response did not resolve, suggesting that these mice suffer from a poor or disregulated adaptive immune response that renders the mice unable to clear P. gingivalis. This was confirmed by the continued presence of skin lesions at the local level, and at the systemic level, by the up-regulated splenic Th1 and Th2 cytokine profile in IL-12p40-/- mice. The humoral response in all knockout mice was polarized towards a Th2 phenotype as indicated by the elevated levels of IgG1, and this is in agreement with previous studies showing that LPS from P. gingivalis polarizes murine dendritic cell responses towards a Th2 phenotype.8 Moreover, the P. gingivalis-induced polarization seen in our study was not altered by the absence of IL-10, IL-12, IFN- , TNF or IL-4. In conclusion, the results of our study indicate an important role for innate mechanisms when responding to a primary P. gingivalis challenge. We found that IL12p40-/- and TNF-/- mice exhibited an altered local and systemic response after subcutaneous P. gingivalis infection. References
1. Teng YT. The role of acquired immunity and periodontal disease progression. Crit Rev Oral Biol Med 2003;14:237-252. 2. Gemmell E, Carter CL, Grieco DA, Sugerman PB, Seymour GJ. P. gingivalis-specific T-cell lines produce Th1 and Th2 cytokines. J Dent Res 2002;81:303-307. 3. Seymour GJ, Taylor JJ. Shouts and whispers: An introduction to immunoregulation in periodontal disease. Periodontal 2000 2004;35:9-13. 4. Gemmell E, Yamazaki K, Seymour GJ. Destructive periodontitis lesions are determined by the nature of the lymphocytic response. Crit Rev Oral Biol Med 2002;13:17-34. 5. Gemmell E, Winning TA, Bird PS, Seymour GJ. Cytokine profiles of lesional and splenic T cells in Porphyromonas gingivalis infection in a murine model. J Periodontol 1998;69:1131-1138. 6. Ma X. TNF-alpha and IL-12: a balancing act in macrophage functioning. Microbes Infect 2001;3:121-129. 7. Lin YY, Huang JH, Lai YY, Huang HC, Hu SW. Tissue destruction induced by Porphyromonas gingivalis infection in a mouse chamber model is associated with host tumor necrosis factor generation. Infect Immun 2005;73:7946-7952. 8. Pulendran B, Kumar P, Cutler CW, Mohamadzadeh M, Van Dyke T, Banchereau J. Lipopolysaccharides from distinct pathogens induce different classes of immune responses in vivo. J Immunol 2001;167:5067-5076.
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The role of IL-12 in innate and adaptive resistance against oropharyngeal candidiasis
CS Farah, AM Lichanska, MJ Waters, RB Ashman*
Our studies on oropharyngeal candidiasis have centred on understanding the cellular and molecular mechanisms involved in resistance against oral Candida albicans infections by making use of immunodeficient murine models.1,2 We have shown that clearance of an oral C. albicans infection is dependant on CD4+ T cells augmented by macrophages and neutrophils,3 in the presence of Th1 cytokines IL-12, IFN- and TNF.2 We have also confirmed the role of these cytokines in resistance against infection by using cytokine specific gene knockout (KO) mice, and have shown that mice lacking TNF showed an early acute increase in the severity of infection, while in the absence of IL-12, the animals developed a severe, chronic non-resolving oral infection that persisted for more than three months.4 Histopathological examination of oral tissues from infected IL-12 KO mice showed extensive hyphal penetration into the squamous epithelium, and microabscess formation, similar to that seen in T cell deficient nude mice.4 In an effort to clarify the pathways involved in the pathogenesis of the IL-12p40 KO mouse, the applicants undertook an analysis of the gene expression profile in candida-infected IL-12p40 KO mice and C57/BL6 wildtype controls by using cDNA microarrays. Oral tissues and submandibular and superficial cervical draining lymph node cells were isolated from IL-12p40 KO and C57/BL6 mice on day 0, and six days after inoculation with the yeast (height of infection). Tissues were analysed by GeneChip Mouse Expression Set 430A microarrays (Affymetrix). Software packages (Affymetrix Microarray Suite, Data Mining Tool, GeneSpring, and SpotfireDecision) were used to define differences in expression patterns between various gene clusters that have plausible functional implications in the process of antigen presentation, macrophage activation and function, T cell signalling, and cytokine pathways. Only immune function genes showing more than a twofold change in expression have been included for analysis. Data analysis has implicated CD4, CD8a, RAG1 (recombination activating gene 1), NFAT5 (nuclear of activated T cells 5), and TNFSF5 (tumor necrosis factor ligand superfamily member 5) in the lymph nodes, and TRAF6 (tumour necrosis factor receptor associated factor 6), TREM1 (triggering receptor expressed on myeloid cells), SPP1 (secreted phosphoprotein 1 osteopontin), BD4 (beta defensin 4), and TNFRSF5 (tumor necrosis factor receptor superfamily member 5) in the oral tissues. Confirmation of these findings with quantitative real time reverse transcription PCR and immunohistochemistry will be required before more indepth analysis of the role of each of these factors can be undertaken. References
1. Farah CS, Elahi S, Drysdale K, et al. Primary role for CD4(+) T lymphocytes in recovery from oropharyngeal candidiasis. Infect Immun 2002;70:724-731. 2. Farah CS, Gotjamanos T, Seymour GJ, Ashman RB. Cytokines in the oral mucosa of mice infected with Candida albicans. Oral Microbiol Immunol 2002;17:375-378. 3. Farah CS, Elahi S, Pang G, et al. T cells augment monocyte and neutrophil function in host resistance against oropharyngeal candidiasis. Infect Immun 2001;69:6110-6118. 4. Farah CS, Hu Y, Riminton S, Ashman RB. Distinct roles for interleukin-12p40 and tumour necrosis factor in resistance to oral candidiasis defined by gene-targeting. Oral Microbiol Immunol 2006;21:252-255.

*Oral Biology and Pathology Research Unit, School of Dentistry, The University of Queensland.

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Expression of hTERT in relation to programmed cell death in premalignant and malignant oral epithelial lesions
CS Farah, NW Savage, M Raiyon*
Oral squamous cell carcinoma (OSCC) is the most common cancer of the head and neck.1 The oral neoplastic process is a continuum beginning with normal epithelium progressing through hyperplasia to dysplasia to carcinoma in situ and invasive carcinoma. Morphological changes observable under the light microscope are the key features in the diagnosis of OSCC, although histopathological diagnosis based on morphological changes is not a confident predictor of malignant change in precancerous lesions.2 Recently it has been shown that corresponding genetic alterations are reflected in clinical and microscopic pathology from hyperplasia through invasiveness, thus molecular changes and genetic alterations may be essential methods to determining malignant progression. During development of OSCC, apoptotic cells are detected with increasing frequency from normal oral epithelium to dysplasia to oral carcinoma.3-6 The regulation of apoptosis is tightly controlled, and defects in apoptosis can lead to the development of cancer. Another characteristic feature of cancer cells is their ability to continuously proliferate. In carcinomas, the sequence that is found in normal epithelial cells from cell division through maturity, differentiation, senescence and death, is somehow altered and cell death is prevented. In the current study, we examined the association between telomerase activity as a marker of proliferation, and apoptosis in the progression pathway of oral epithelial dysplasia and squamous cell carcinoma. Bcl-2 and hTERT expression were determined by immunohistochemistry performed on formalin-fixed paraffin-embedded sections of epithelial hyperplasia, dysplasia and squamous cell carcinoma. Telomerase activity was measured by quantitative real time RT-PCR, while apoptosis was determined by TUNEL. Bcl-2 anti-apoptotic expression was not significantly different between hyperplasia, dysplasia and squamous cell carcinoma, although it was slightly decreased in the latter. There were however significantly more TUNEL positive cells in dysplasia and carcinoma compared to hyperplasia, indicating more apoptosis was occurring in these tissues and confirming previous findings.3 There was no significant difference in mean hTERT expression between hyperplasia, dysplasia and carcinoma, but there was a significant difference in its expression between basal and suprabasal compartments regardless of the histopathological diagnosis. Quantitative real time RT-PCR could not determine any difference in expression of hTERT between the three different epithelial groups. The findings of this study in relation to Bcl-2 expression and apoptosis support those of Loro et al.3 and suggest that Bcl-2 expression in oral epithelium is more closely correlated with apoptosis than proliferation. References
1. Epstein JB, Zhang L, Rosin M. Advances in the diagnosis of oral premalignant and malignant lesions. J Can Dent Assoc 2002;68:617-621. 2. Scully C, Sudbo J, Speight PM. Progress in determining the malignant potential of oral lesions. J Oral Pathol Med 2003;32:251-256. 3. Loro LL, Johannessen AC, Vintermyr OK. Decreased expression of bcl-2 in moderate and severe oral epithelia dysplasias. Oral Oncol 2002;38:691-698. 4. Loro LL, Vintermyr OK, Johannessen AC. Cell death regulation in oral squamous cell carcinoma: methodological considerations and clinical significance. J Oral Pathol Med 2003;32:125-138. 5. Loro LL, Vintermyr OK, Liavaag PG, Jonsson R, Johannessen AC. Oral squamous cell carcinoma is associated with decreased bcl2/bax expression ratio and increased apoptosis. Hum Pathol 1999;30:1097-1105. 6. Macluskey M, Chandrachud LM, Pazouki S, et al. Apoptosis, proliferation, and angiogenesis in oral tissues. Possible relevance to tumour progression. J Pathol 2000;191:368-375.

*Oral Biology and Pathology Research Unit, School of Dentistry, The University of Queensland.

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Development of a molecular tool for epidemiological investigation of recurrent oral candidiasis infections in HIV-positive individuals
ML Fraser, RH Andrews, AH Rogers*
Oral candidiasis occurs in over 80 per cent of HIVpositive individuals. About 40 per cent of the healthy HIV-negative population also harbours candida species, making such people potential sources of infection. The ability to fingerprint an individuals candida strain(s) is clinically significant when considering acquisition of the organism by noncarriers, the origins of recurrent infections and the emergence of strains resistant to antifungal treatment. Development of an accurate and rapid molecular method would aid in the identification of sources of candida infection and mechanisms of the acquisition of antifungal resistance. Using allozyme electrophoresis, previous studies in this laboratory showed that, among candida strains, the metabolic enzyme pyruvate kinase (PK) is particularly variable in its nett charge; a reflection of diversity at the gene level. Accordingly, PK was targeted for the development of a molecular method for strain discrimination. Based on the electrophoretic profile of 16 independent metabolic enzymes, 12 candida isolates were selected. These varied at between 075 per cent of the selected loci and the PK allelic profiles revealed six differently-charged enzyme structures; and, therefore,
*Microbiology Laboratory, Dental School, The University of Adelaide.

six different genetic codes for the same enzyme. Potential primers were designed to bind to the least variable region within the 5-end of the PK gene, identified from the alignment of the PK sequences available for yeasts on Genebank. The sequences should be about 550 base pairs (bp) covering the same region as the published sequences, being amplified using a single pair of primers. In order to ensure that the primers bind only to the gene of interest, they would be about 20bp long so that they are more selective. Consequently, the gene sequences for the 5-end of the PK enzyme would be obtained for a number of closely as well as distantly-related isolates; and sequence alignments would identify regions of high-level variation between strains. Such regions would form the basis for the design of a PCR-RFLPbased rapid diagnostic tool. After obtaining a number of sequences, it was found that there was insufficient variation to allow us to distinguish between Candida albicans strains. We conclude that the huge variation seen in PK, using allozyme electrophoresis, was not reflected in the gene sequence we obtained. It is possible that the variable region may fall outside of the region we sequenced or that post-translational modification altered the charge of the active enzyme. Nevertheless, we feel that the general concept is valid but further work is obviously needed.

Identification of the osteoclastogenic factor from Eikenella corrodens surfaceassociated material

NJ Gully,* DR Haynes, AH Rogers,* PS Zilm*
Previous studies in our laboratory, examining the effects of Eikenella corrodens surface-associated material on the cells and cytokines that induce osteoclastogenesis, have shown that cytokine induction occurs in human monocytes (precursors of osteoclastic cells), and macrophages in the presence of this material. Other studies show that E. corrodens surface-associated material is able to induce increased numbers of osteoclasts. More recent studies have shown that non-LPS surface-associated products from some periodontopathogenic bacterial species induce the release of the pro-inflammatory cytokines IL-1 , TNF- and IL-6. These mediators are thought to be major pathological mediators of inflammatory diseases. The stimuli inducing pro-inflammatory cytokine induction in periodontal diseases is believed to be the accumulation
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of bacteria in the subgingival region. As these bacteria do not invade the diseased tissue in large numbers, it is believed that their soluble components or products interact with host cells to induce cytokine synthesis and therefore play a role in the pathology of the periodontal diseases. In this study, Eikenella corrodens strain 35EK, an isolate from the periodontal pocket of an adult patient, was grown in a tryptone-based medium supplemented with yeast extract under continuous culture conditions. The organism was grown at 35C, a pH of 7.2 and at a growth rate that resulted in a doubling time of 10 hours. Cells were harvested daily by centrifugation (3500g for 30 min at 4C) and the resulting bacterial cell pellets stored at -20C. Stored cells were thawed, resuspended in 0.85M NaCl and surface-associated material removed by
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gentle stirring at 4C for one hour. The cell suspension was centrifuged (3500g for 30 min at 4C), the resulting supernatant collected and dialysed and concentrated using an Amicon ultrafiltration stirred cell (molecular weight cut-off 10kD). The resulting concentrate was lyophilised and stored desiccated at 4C until required for fractionation. The smooth and rough form of lipopolysaccharide of E. corrodens was extracted according to the method of Darveau-Handcock. The levels of the human cytokine, IL-1 , in cell culture supernatants were determined via ELISA assay, developed in our laboratories using commercially available antibodies. Human peripheral blood monocytes were isolated by differential centrifugation from human blood buffy coats. After washing (three times in Hanks balanced salt solution) the monocytes were counted and resuspended (2 x 106 cells/ml) in a medium containing RPMI-1640, 25mM Hepes buffer, 10 per cent foetal calf serum, penicillin (5g/ml), streptomycin (50U/ml) and 2-mercaptoethanol (50M). Equal numbers of cells were added and adhered to micro-titre tray wells and exposed to various purified fractions of (0.5g/ml to 5g/ml) E. corrodens surface-associated material, E. corrodens LPS and E. coli LPS, suspended in complete RPMI medium. Unstimulated monocytes were used as negative controls. Cells were incubated at 37C for 24 hours at which time supernatants were removed for analysis. A variety of protein purification techniques were employed to fractionate surface-associated material in an attempt to isolate the biologically active components. Ammonium sulphate precipitation, hydrophobic interaction chromatography and ionexchange chromatography all failed to satisfactorily
*School of Dentistry, The University of Adelaide. School of Medical Sciences, The University of Adelaide.

resolve activity. Cytokine induction was observed in all fractions following purification with each of these methods. A significant amount (approximately 40 per cent) of activity was retained following ATP affinity chromatography suggesting at least one of the active components possesses an ATP binding region. Although we were able extract proteins from the surface of the E. corrodens capable of inducing significant amounts of inflammatory cytokines from human monocytes, we were unsuccessful in purifying them sufficiently to allow identification of the cytokine inducing components. The necessity to retain biological activity during the purification process and the lipophilic nature of the extracted material made purification difficult. It is noteworthy that significant activity was retained on an ATP ligand and this is the preferred method of purification for HSP-60 (GroEL). Others have shown that surface-associated proteins from A. actinomycetemcomitans, an organism widely recognized for its involvement in the aetiology of localised juvenile periodontitis, have similar pro-inflammatory activity as that exhibited by E. corrodens and that much of the activity was associated with a 62kD protein. The protein demonstrated >95 per cent homology with the E. coli heat shock protein GroEL. HSP-60 is produced by prokaryotic and eukaryotic cells in response to a variety of stresses and can modify the function and destiny of other proteins and play important roles in immunity. Immune responses against HSPs can be highly cross-reactive among bacterial and mammalian species and are capable of recognizing both foreign and self-stress proteins. Healthy individuals may use this capacity to respond to self-stress protein determinants to help eliminate infected autologous cells. It is possible that defects in the ability to regulate this anti-self capability may lead to chronic inflammatory reactions such as periodontal disease.

An investigation into the role of VEGF in oral dysplasia and oral squamous cell carcinoma
S Johnstone, RM Logan*
A significant increase in vascularity occurs during the transition from normal oral mucosa, through differing degrees of dysplasia, to invasive squamous cell carcinoma. This increase in vascularity has been associated with tumour progression and lymph node metastasis. Previous research has shown that an adequate blood supply is essential for solid tumour growth and metastasis. Vascular development and factors that regulate it have therefore been extensively studied in various tumour types. Vascular endothelial growth factor (VEGF), also known as VEGF-A and vascular permeability factor, has been shown to be a
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critical angiogenic cytokine involved in the development of a blood supply in several different tumours, including those of the head and neck. VEGF is a highly potent angiogenic agent that acts to increase vessel permeability and enhance endothelial cell growth, proliferation, migration and differentiation. Because VEGF is a powerful promoter of angiogenesis in many tumour types, its role in oral cancer has been the subject of numerous studies. In particular, the role of VEGF in tumour angiogenesis, disease progression and its use as a prognostic indicator has been investigated. However, few studies have considered
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VEGF and its involvement in oral dysplasias or their progression to invasive carcinomas. The aim of this study was to test the hypothesis that the increase in vascularity associated with oral tumour progression is related to an up-regulation of VEGF expression. In this study, we investigated the expression of VEGF in normal oral mucosa (NOM), oral dysplasia and oral squamous cell carcinoma (OSCC). VEGF expression was also assessed among varying grades of oral dysplasia and differing degrees of differentiation of OSCCs. Specimens consisting of NOM, oral dysplastic lesions and OSCC were stained using standard immunohistochemistry methods to determine VEGF expression. Statistical analysis indicated an up-regulation of VEGF
*Dental School, The University of Adelaide.

during the transition from NOM, through dysplasia to SCC. There was also a significant difference in expression according to differentiation of SCC, but not grade of dysplasia. As VEGF is a potent mediator of vascular development, these results suggest that VEGF may play an important role in the maintenance of a blood supply for developing pre-cancerous and invasive oral lesions. Published: Johnstone S, Logan RM. The role of vascular endothelial growth factor (VEGF) in oral dysplasia and oral squamous cell carcinoma. Oral Oncol 2006;337-342. Johnstone S, Logan RM. Expression of vascular endothelial growth factor (VEGF) in normal oral mucosa, oral dysplasia and oral squamous cell carcinoma. Int J Oral Maxillofac Surg (in press).

Immunohistochemical study of bone sialoprotein and osteopontin in healthy and diseased root surfaces
M Lao, V Marino, PM Bartold*
Periodontal disease is marked by inflammation and subsequent loss and/or damage to tooth-supporting tissues including bone, cementum, and periodontal ligament (PDL). There has been an increasing interest in regeneration of cementum on the surfaces of denuded tooth root. Of particular interest are factors present in cementum, which are thought to have the ability to influence the regeneration of surrounding tissues. Bone sialoprotein (BSP) is a major noncollagenous protein in mineralized connective tissues. High expression of bone sialoprotein may be involved in pre-cementoblast chemo-attraction, adhesion to the root surface and cell differentiation. Osteopontin (OPN) may also serve similar functions. The purpose of this investigation was to determine whether the expression and distribution of BSP and OPN on root surfaces affected by periodontitis is altered when compared to healthy, non-diseased root surfaces. In this study, 30 healthy and 30 periodontitisaffected teeth were collected. Following fixation and demineralization the specimens were embedded in paraffin and sectioned. The sections were then exposed to antibodies against BSP and OPN and counter stained with haematoxylin. Stained sections were then assessed using light microscopy. Eighteen healthy teeth and 30 diseased teeth were used to stain for BSP and 15 healthy and 24 diseased teeth were used to stain for OPN. Nine healthy teeth (50 per cent) and 17 diseased teeth (57 per cent) were immunoreactive for BSP. For OPN, six healthy teeth (40 per cent) and 12 diseased teeth (50 per cent) were immunoreactive for this protein. BSP was not detected in the exposed cementum (absence of overlying PDL) of diseased teeth. In most areas where the PDL was intact, BSP was detected for both healthy and diseased teeth. For teeth reactive for BSP, the matrix of the cementum just below the PDL was moderately stained. Similar immunoreactivity pattern for OPN was observed in that moderate staining was seen in the extracellular matrix of cementum with some light to moderate staining within the PDL and cells. In conclusion, cementum is an integral component of the periodontium. Periodontal disease may alter the structure and composition of the cementum matrix. The absence of BSP and OPN staining along exposed cementum surfaces may be due to structural and compositional changes in matrix components. This may influence the ability for regeneration and new connective tissue attachment onto previously diseased root surfaces. Published: Lao M, Marino V, Bartold PM. Immunohistochemical study of bone sialoprotein and osteopontin in healthy and diseased root surfaces. J Periodontol (in press).

*Dental School, Colgate Australian Clinical Dental Research Centre, The University of Adelaide.

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Oral mucositis clinical presentation, histological features and pro-inflammatory cytokine expression
RM Logan,* RJ Gibson, ST Sonis, DMK Keefe
Oral mucositis is a debilitating side effect of cancer chemotherapy which is difficult to prevent and is currently only treated symptomatically. There are no data on the morphological changes in the human oral mucosa following chemotherapy and this lack of knowledge has made treatment options difficult. In the present study we have begun to characterize the ultrastructural and histological changes that occur in the human oral mucosa following cytotoxic chemotherapy. Twenty patients (17f: 3m) receiving chemotherapy for cancer and four controls (3f: 1m) who had not received any prior chemotherapy were enrolled in this study after providing informed consent. Each patient had one biopsy prior to chemotherapy and a second biopsy at varying intervals after chemotherapy. Clinically, apart from mild erythema, no patients demonstrated obvious mucosal ulceration. Mucosal biopsies were assessed histologically and ultrastructurally by transmission electron microscopy. Transcription factor (NF- B) and cytokine (Cox-2) expression in the buccal mucosa were detected using standard immunohistochemical techniques. We demonstrated that there was no change to the mucosa light microscopically following chemotherapy, other than a mild inflammatory change in some, but not all, patients. However, when the mucosa was examined ultrastructurally, consistent changes in the buccal mucosa early after chemotherapy were found. These changes persisted until at least day 11 following treatment and included apoptosis in the basal cells of the buccal mucosa. The detection of apoptosis, both ultrastructurally and with the TUNEL assay, was a key finding of this study. Apoptosis was observed in the basal cells of the buccal mucosa at all time points following chemotherapy by the TUNEL assay. Ultrastructurally, we observed early stages of apoptosis, including nuclear separation and pyknotic nuclei at both days 1 and 2 after chemotherapy. Overall apoptosis increased in patients compared to normal volunteers for the first three days following administration of cytotoxic chemotherapy, before decreasing but not returning to the low levels of normal volunteers. However, when patients had received prior chemotherapy, their individual apoptosis levels were lower on days 1 and 2 compared with their prechemotherapy biopsies (which were taken on day 21 of a previous chemotherapy cycle). The reasons behind this are unclear and as such we have refined our project to only take biopsies from patients undergoing their first cycle of chemotherapy and to only take one biopsy at one time point after the commencement of chemotherapy. This study also investigated the role that transcription factors and cytokines play in mucositis. We demonstrated that in chemotherapy nave specimens there was no significant staining for either NF- B or Cox-2. Pre-chemotherapy specimens showed evidence of staining for both. There was significantly increased staining for Cox-2 and NF- B in all specimens following chemotherapy (Wilcoxon paired rank test p<0.05). Although preliminary, these findings have confirmed the presence of the transcription factor NF- B and the cytokine Cox-2 in the oral mucosa following administration of cytotoxic chemotherapy. These findings have provided further evidence of their involvement in the pathobiology of oral mucositis, and have extended our previous findings. Data from this study has confirmed that the early changes seen in the oral mucosa are similar to those seen after radiation therapy. This is an important finding. Further work now needs to be performed to determine the effects of individual chemotherapeutic agents on the expression of these factors in patients undergoing chemotherapy and their subsequent role in the pathobiology of mucositis. Published: Gibson RJ, Cummins AG, Bowen JM, Logan RM, Healey T, Keefe DMK. Apoptosis occurs early in the basal layer of the oral mucosa following cancer chemotherapy. Asia-Pacific Journal of Clinical Oncology 2006;2:39-49. Logan RM, Gibson RJ, Sonis ST, Keefe DMK. Nuclear factor -KappaB (NF-KappaB) and cyclooxygenase-2 (Cox-2) expression in the oral mucosa following cancer chemotherapy. Oral Oncol (in press).

*Oral Pathology, School of Dentistry, The University of Adelaide. Division of Tissue Pathology, Institute of Medical and Veterinary Science, Adelaide. Department of Medical Oncology, Royal Adelaide Hospital. Brigham and Womens Hospital, Boston, Massachusetts, USA. Division of Medicine, Faculty of Health Sciences, The University of Adelaide.

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Australian Dental Journal ADRF Special Research Supplement 2006;51:4.

Development of integrated clinical assessment in dentistry: comparison of perceptions by students and assessors
T McLean, J Fairley, TM Gerzina*
The learning environment of the clinic or hospital is a challenging area for both teacher and student. Recent reports in dental education point to the value of early introduction of students to the clinical learning environment, largely because of the demonstrated value of contextual learning and the facilitation of integration of knowledge from basic to clinical sciences.1,2 The student/clinical teacher relationship has also been suggested to mirror the therapeutic alliance that exists between patient and physician, in being an educational alliance.3 Effective clinical teachers have empathy, provide support, are flexible and track well, in addition to being interpretive, focused and practical.4 Valuable supervision as a teaching style involves joint problemsolving, feedback, reassurance and theory-practice linking and can have lasting effect.5 Greater positive patient outcome of the supervision, for example, has been shown to occur when direct supervision, combined with focused feedback, is provided.6 This study presents the findings of an exploration of dental clinical teaching by investigating the perceptions held by the central partners in that environment, namely the students and the teachers, and hypothesizes that these two groups have different views of the value of teaching methods. Qualitative methodology employed two participatory focus group discussions with ensuing discussion transcripts analysed for emergent themes. Quantitative methodology involved the use of a specifically designed questionnaire to identify student perceptions of their clinical learning and the usefulness of various teaching styles used in preparing to be a dental clinician. Final year students and clinical teacher volunteer participants came from dental programmes at the University of Sydney. Scores from the questionnaire for the different groups of participants were analysed to provide comparisons using Students t test with Bonferroni correction setting P<0.005. A theme that emerged in the focus group discussions was the nature of the teacher/student relationship in terms of student role and the nature/level of interaction between clinical teacher and student. Questions pertinent to this theme were then designed and added to the questionnaire. The students considered the use of clinical demonstration as the best form of clinical teaching. This style enables students to see how things are done so that you can understand them and functioned to back up theory. In direct reference to a clinical demonstration by a clinical supervisor, a centrally illustrative comment was: It is very helpful to see theory work applied or demonstrated as this adds depth and reinforces the theory.
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Demographics were students (where n=45, 69 per cent female, 76 per cent response rate, average age 23.7Y, age range 2133Y) and teachers (where n=21, 52 per cent female, 69 per cent response rate, average age 37.8Y, age range 2478Y). The questionnaire examined three themes: (i) the teacher/student relationship; (ii) educational theory applied to dental clinical teaching; and (iii) skills important for dental clinical practice. None of the comparisons for theme (i) were statistically significant. The majority of both teacher and student groups, in excess of 70 per cent, showed agreement for the following: the importance of empathic guidance by the clinical teacher (greater than 70 per cent for both teacher and student groups), discussion of alternative treatment plans during clinical sessions (greater than 90 per cent), the value of clinical demonstrations by the clinical tutor (greater than 80 per cent) and provision of continuous feedback during a clinical session by the clinical tutor (greater than 80 per cent). In theme (ii), two of the comparisons between results for teachers and students in this theme were statistically significant. Students found a clear link between theory and clinical practice in the programme (P<0.0029) and that a clinical log book was valuable for the preparation for clinical practice (P<0.0038). The teacher group was clearly divided on the value of problem based learning (PBL) in supporting the ability to provide clinical care of patients with an almost equal number agreeing as disagreeing (27 per cent and 26 per cent respectively); a larger majority of students disagreed than agreed (39 per cent and 7 per cent respectively). In theme (iii), only one of the comparisons between results for teachers and students was statistically significant (P<0.0013). This aspect was the recognition that a critical appreciation of evidence-based practice was an important part of dental clinical practice; 66 per cent of teachers agreed (and no teachers disagreed) with this statement, whereas only 42 per cent of students agreed and 20 per cent disagreed. Teachers agreed that all other skills listed were important for dental clinical practice, with no disagreement recorded for any skill. Some students, however, did disagree with the importance of self-assessment and self-confidence, in addition to the item about evidence-based practice. Overall a high level of alignment between student and teacher views was seen in the data presented. This shared perspective probably reflects the very close and cohesive relationship between clinical teachers and students that is fostered in that setting and a strong influence of teachers on student view. The intensity of the clinical environment may strongly reinforce the
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tendency of teachers to continue to conform to teaching in a way that has been perceived successful in terms of patient outcome and service needs and also a tendency of teachers to recognise and adopt a style of teaching they themselves experienced as a student clinician. This has been described as a teacher-centred style.7 Student self-confidence and teacher interactivity represent strong tensions in the dental clinic and, in balance, are essential in the progression of clinical skill acquisition by the student clinician. The Dreyfus model of skill acquisition, as described by Eraut,8 defines progression from novice (reliance on rules, no discretionary judgement and little situational perception) to expert (no reliance on rules, analytical approach only in novel and problematic times, intuitive approach based on deep understanding). In this study students strongly valued clinical demonstrations as a definitive expression of clinical theory in practice. The utility of PBL as a support to clinical activities was not seen by teachers or students, which supports the findings of Colliver,9 although it should be noted that neither group reported extensive current or past experience in this modality. The link between theory and practice is a central one in education and whilst students acknowledged that the link existed in the programme, teachers did not agree as strongly. PBL is a teaching modality which, whilst reported to have only modest effects on development of clinical skills, is increasingly associated with student satisfaction with teaching and development of appropriate learning skills.10,11 A clearer understanding of these concepts is recommended in view of the direct relationship to

quality of patient care and professional attributes in dentistry. References

1. Mullins G, Wetherell J, Robbe I. Learning in the clinical environment. In: Sweet J, Huttly S, Taylor I, eds. Effective learning and teaching in medical, dental and veterinary education. London: Kogan Page, 2003. 2. Rumelhart DE, Norman DA. Accretion, tuning and restructuring: Three modes of learning. In: Klatzky R, Cotton JW, eds. Semantic factors in cognition. Hillsdale NJ: Lawrence Erlbaum Associates, 1978. 3. Tiberius RG, Sinai J, Flak EA. The role of teacher-learner relationships in medical education. In: Norman GR, van der Vleuten CPM, Newble DI, eds. International handbook of research in medical education. Dordrecht: Kluwer Academic Publishers, 2002:463-497. 4. Kilminister S, Jolly B, van der Vleuten CP. A framework for effective training for supervisors. Med Teach 2002;24:385-389. 5. Hirons A, Velleman R. Factors which might contribute to effective supervision. Clinical Psychology Forum 1993 (July):1113. 6. Fallon WF Jr, Wears RL, Tepas JJ 3rd. Resident supervision in the operating room: does this impact on outcome? J Trauma 1993;35:556-560. 7. Schaefer KM, Zygmont D. Analyzing the teaching style of nursing faculty. Does it promote a student-centred or a teachercentred learning environment? Nurs Educ Perspect 2003:24:238245. 8. Eraut M. Developing professional knowledge and competence. London: Falmer Press, 1994:75-87. 9. Colliver J. Effectiveness of problem based learning curricula: research and theory. Acad Med 2000;75:259-266. 10. Bligh J, Lloyd-Jones G, Smith G. Early effects of a new problembased clinically oriented curriculum on students perceptions of teaching. Med Educ 2000:34;487-489. 11. Albanese M. Problem-based learning: why curricula are likely to show little effect on knowledge and clinical skills. Med Educ 2000;34:729-738.

*Faculty of Dentistry, The University of Sydney.

Published: Gerzina TM, McLean T, Fairley J. Dental clinical teaching: perceptions of students and teachers. J Dent Educ 2005;69:1377-1384.

Examination of the efficiency in removing bacterial contamination from dental unit water lines using current treatment protocols
K Mahajani, PS Zilm, NJ Gully*
Bacteria organize themselves in aqueous environments by forming clusters produced by ordered and specific aggregation and co-adhesion interactions which lead to the development of biofilms. Biofilms facilitate further bacterial attachment, confer protection and are widespread in nature. Dental unit water lines (DUWLs) harbour biofilms which, if seeded intra-orally during dental procedures, could prove pathogenic. Pathogens including Pseudomonas spp., Klebsiella spp and nontuberculous Mycobacterium species have been found in DUWLs and are capable of causing serious infections in compromised patients. Reports that conclusively link biofilms to diseases are uncommon but the emergence of multi-resistant organisms and the increased numbers
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of immunocompromised individuals warrants greater awareness of biofilms. Eliminating biofilms in DUWLs also has the benefit of increasing the structural longevity of dental units and associated equipment. A number of protocols currently exist to reduce bacterial counts to <200 colony forming units/ml (CFU/ml) as recommended by the American Dental Association (AmDA). The aims of this project therefore were to provide an accurate microbial assessment of current treatment protocols used to clean DUWLs, and to determine the rate of biofilm formation in new DUWLs. Potential cost-effective ways of controlling biofilm growth in DUWLs were also examined.
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Quantitative assessment of bacterial biofilms in DUWLs was done using standard bacterial culturing techniques. The second part of this study examined the application of Listerine (an oral antiseptic) to water lines to determine its ability to eradicate biofilm growth. For each experiment listed below, duplicate, 0.1ml aliqouts of water collected from DUWLs were sterilely plated directly onto nutrient agar plates before incubation at 37C or 15C for 48 hours. Following incubation, individual colonies were counted and the total cfu/ml were calculated. Experiment 1: 50ml samples of water from randomly selected dental units were collected in sterile containers. Samples were collected between each patient and before the lines were flushed. Samples were taken from the high-speed handpiece line (without the handpiece) and triplex line (without the nozzle in place). Samples were taken randomly during working hours. Experiment 2: DUWL samples were taken immediately before and after purging from the highspeed and the triplex waterlines to determine the effectiveness of this procedure (purging). Purging water lines means flushing the water lines forcefully under pressure with water or a chemical disinfectant. Tap water was used as a control so that microbial content of the DUWL could be determined. Experiment 3: Samples were collected from the highspeed and triplex lines at 9:00am after purging, but before the first patient was treated for the day. Another sample was taken at 4:30pm after the last patient was seen, but before purging. Experiment 4: Water samples were collected from a new, recently installed DUWL four times a day. Experiment 5: After evaluating the results of experiments listed above, two dental units with low and high bacterial counts were treated with 300ml of undiluted Listerine for 20 days. Following the usual cleaning protocol, the high-speed handpiece was flushed with Listerine towards the end of the day for
*Dental School, The University of Adelaide.

two minutes. The purpose was to keep Listerine in contact with the water lines overnight. Experiments 1 and 2: All the dental units except those with an inbuilt sanitation system had bacterial numbers 10 times higher than that recommended by the AmDA of <200CFU/ ml. The disinfectant used in the sanitation systems was either Dentosept or AlproJet, used to the manufacturers instructions. Water samples collected before purging had high bacterial counts (2,320CFU/ml) but the count significantly reduced to 20CFU/ml after purging and remained low until the end of the day. This shows that purging is effective at lowering CFU to below recommended levels. Tap water had a significantly lower bacterial count compared to water residing in the DUWL. Experiment 3: Water samples collected in (no sanitation system) the morning (after purging) and at the end of the day showed a high bacterial count throughout the week (MondayFriday). This indicates that purging is ineffective if the biofilm is established in the water lines. Experiment 4: Water samples collected before purging had 2,320CFU/ml but purging reduced bacterial counts significantly to 3CFU/ml. Bacterial numbers increased by midday to 4,550CFU/ml, and remained high (4020CFU/ml) for the rest of the day. Experiment 5: After flushing the water lines with Listerine, bacterial numbers were either reduced significantly or remained the same. The incidence of iatrogenic infections as a result of biofilm contamination from DUWLs is unknown. However, the emergence of antibiotic resistant micro-organisms and the increasing numbers of immunocompromised individuals suggests that contaminated water lines should not be used during dental procedures. It is clear that various modalities are effective in reducing bacterial biofilms to acceptable levels. This study concludes that in pre-contaminated water lines, simply purging the line does not remove bacterial biofilms. Treatments aimed at removing the biofilm are required and once cleared, purging regularly is effective in maintaining the water line.

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Mechanism of surface and subsurface demineralization of dentine

P Mishra, MJ Tyas, MF Burrow, J Palamara*

This study was designed to investigate the surface loss and subsurface demineralization depth of dentine as a function of loading and pH to try to model how tooth structure may be lost during the development of non-carious cervical lesions (NCCL). A laboratory model was developed to create surface loss and subsurface demineralization when the dentine beams were placed under tension and compression at pH levels of 4.5, 7 and 10. The aims of the study were: to investigate the loss of dentine as a function of loading and pH; to investigate surface and subsurface material loss as a function of magnitude of stress; and to develop a laboratory model which simulated NCCL found in human teeth. Freshly extracted bovine incisors were obtained and stored for no longer than one month in 1% chloramineT at 4C prior to use. Each tooth was sectioned using a slow speed diamond saw with water coolant to form rectangular dentine slabs, 7.5mm wide and 1.5mm thick, still attached to the root of the tooth. The slabs were polished on the labial side using wet silicon carbide papers up to 4000-grit, and sectioned from the incisal edge to the CEJ into two small beams each 10mm long, 1.45mm thick and 3.75mm wide. Sixty teeth were prepared. Each approximal half of the beam was coated with clear nail varnish on the polished surface and its undersurface to protect the tooth from acid. Samples were immersed in 0.1M lactic acid, adjusted to either pH 4.5, 7 or 10 at 20C for five days under load. The samples were removed and embedded in epoxy resin. Digital images of the beams were recorded and surface losses from acid-exposed and unexposed portions of both beams were measured at millimetre intervals in pixels and converted to micrometres by prior calibration. Subsurface demineralization was measured every 1mm from the incisal edge to the cervical area.

*School of Dental Science, The University of Melbourne.

Data were analysed using Genstat statistical software comparing surface loss from the exposed and unexposed surfaces of both loaded and unloaded beams, and between loaded and unloaded beams at the three different pH levels. Two-way ANOVA was used to observe the effect of load-location interaction at the three different pH levels. At pH 4.5, the interaction between load-location was not statistically significant for surfaces under tension (p=0.72) or compression (p=0.402). At pH 7 a significant load-location interaction (p=0.001) was observed for the loaded surfaces under tension or compression. A significant amount (p<0.001) of surface loss at the fixed end compared with the free end on the loaded beams was observed. However, the surface loss from the unloaded beams did not vary along the beam length. Similarly, at pH 10 there was a significant loadlocation interaction for surface loss on both tension (p<0.001) and compression (p<0.001) surfaces. At this pH, surface loss decreased as a function of distance from the fixed end on surfaces subjected to tensile or compressive stresses, while surface loss on the unloaded beam did not vary. For the subsurface demineralization the results showed that beams under compression sustained more subsurface demineralization than under tension (p=0.006). However, when subsurface demineralization was compared from the fixed to the free end of the beam at each millimetre increment, there was no statistical difference (p=0.112). No subsurface demineralization was observed at pH 7 and 10. The results showed that the beams at pH 4.5 and end-point loading sustained increased demineralization depth, while the beams under load at pH levels 7 and 10 did not show any subsurface changes. The results showed that the surface under compression sustained a greater depth of subsurface demineralization than the surface under tension. It would seem that the formation of NCCL may, in part, be caused by compressive and tensile forces being applied to teeth during function.

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Effect of prostaglandin E2 on the gene expression of RANKL and OPG, and TGF- 1 release from cultured osteoblasts
G Ramirez, AL Symons*
Osteoblasts produce regulatory cytokines which modulate bone metabolism and this regulation may be influenced by the presence of an inflammatory agent. The aims of this study were to determine the effect of exogenous PGE2 on the expression of genes for nuclear factor B-ligand (RANKL) and osteoprotegerin (OPG) and the release of transforming growth factor beta-1 (TGF- 1) in cultured primary osteoblasts. Osteoblasts obtained from the long bones and jaws of three Wistar rats were cultured and stimulated with 10-3M, 10-5M or 10-7M PGE2, or cultured in Dulbecos modified media for 5, 10, 15 or 20 days. Relative quantitation PCR was used to determine gene expression and ELISA the presence of TGF- 1 in supernatants. RANKL gene expression was significantly greater (p<0.001) in osteoblasts treated with all PGE2 treatments after five days in culture. Higher RANKL gene expression was maintained in cells treated with 10-3M PGE2 over the experimental periods, but gene expression was reduced in cultured cells treated with 10-5M and 10-7M PGE2. Osteoblasts
*School of Dentistry, The University of Queensland.

cultured with the two lower doses of PGE2 had higher levels of OPG. After five days of culturing, the amount of TGF- 1 present in supernatants significantly decreased (p<0.05) in cells treated with PGE2 at a concentration of 10-5M and significantly increased in supernatants from the 10-3M and 10-7M PGE2-treated groups (p<0.001). At day 10 the level of TGF- 1 in the supernatant was significantly higher in osteoblasts treated with 10-3M PGE2 (p<0.001) and significantly reduced in supernatant from osteoblasts cultured with 10-5M and 10-7M PGE2 (p<0.05). No significant differences were observed in the supernatant TGF- 1 levels following 15 days of culture. At 20 days higher levels of TGF- 1 were present in supernatants collected from 10-3M and 10-7M PGE2-treated osteoblasts (p<0.05 and p<0.01 respectively). The effect of PGE2 on cultured osteoblasts appears to be dose dependent. The lower doses of PGE2 resulted in higher levels of OPG and lower levels of RANKL which may enhance mineralized tissue formation. PGE2 treatment significantly varied TGF- 1 release into the culture media and this was related to the dosage and culture period. It appears that PGE2 may influence the extracellular release of TGF- 1 by osteoblasts.

Fluoride and apoptosis in amelogenesis

JR Smid,* D Harbrow,* BJ Joseph

Enamel fluorosis is a defect in tooth enamel mineral resulting from toxic doses of fluoride ingested during amelogenesis. Although no definitive mechanism is currently available to explain the formation of this abnormal enamel, it is generally accepted that the fluoride influences enamel development via an impact on ameloblast function. A well-established cellular phenomenon that occurs in many ameloblasts during amelogenesis is programmed-cell death or apoptosis.1 In the continuously erupting rat incisor, in particular, apoptosis occurs in the transitional stage ameloblasts.2 Characteristic apoptotic bodies are clearly visible, with the light microscope, in sections of the transitional enamel organ.2 Cells grown in culture treated with high doses of fluoride show increases in the number of cells undergoing apoptosis.3,4 Laboratory animals given high doses of fluoride also show increases in apoptosis of enamel organ cells.4,5 Many of these studies indicate the protease caspase-3 has a role in fluoride-induced cell death.3,4

Caspase-3 activity has not been demonstrated in ameloblasts, however, caspase-3 activity has been detected in the enamel knot cells of molar tooth germs.6,7 The inactive procaspase-3 has also been detected in ameloblastoma cells.8 The aim of this study was: first, to immunohistochemically detect active caspase-3 in the enamel organ of the rat incisor; and secondly, to ascertain whether this distribution of active caspase-3 in the enamel organ is affected by fluoride treatment. Twelve female, three week old Wistar rats were randomly divided into three groups. The rats were then given two intraperitoneal injections per day over 4.5 days of either isotonic saline, NaF, 10mg per kg body weight, or NaF, 20mg per kg body weight. Two hours after the final injection the rats were killed by decapitation under anaesthesia. The heads were bisected in the sagittal plane and then immersed in buffered tissue fixation solution (4% paraformaldehyde in 0.1M phosphate buffered saline, pH 7) for 24
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Australian Dental Journal ADRF Special Research Supplement 2006;51:4.

hours at 4C. The tissue blocks were then processed for immunohistochemistry. This included demineralization in neutral EDTA solution (confirmed by radiography) at 4C for three weeks, with daily solution changes and embedment in paraffin wax using standard histological procedures. Sections were then cut (to provide 5m thick lingual longitudinal sections of the maxillary incisors) and mounted on polylysine-coated slides. The incisor mounted slides were autoclaved (120C for 15 minutes) in antigen-retrieval solution (1mM EDTA, pH 8) and stained with a rabbit anti-active caspase-3 polyclonal antibody (Promega Corporation, USA), using a biotinylated-goat anti-rabbit second antibody (Dako Australia), a streptavidin-horseradish peroxidase conjugate (Amersham International, Australia) and 3,3-diaminobenzidine tetrahydrochloride (DAB; Dako Australia) as the brown chromagen and haematoxylin for a blue counter-stain. Other similar incisor slides were also stained either with a rabbit control antibody (Dako Australia) or a rabbit anti-Bcl2 antibody (an anti-cell apoptosis marker). The stain distribution of the active caspase-3 was localized in many of the late maturation ameloblasts in all of the rat maxillary incisors, but was completely absent in the corresponding secretory ameloblasts. Also, no active caspase-3 was able to be detected in the transitional ameloblasts, where apoptotic bodies were present. Because some of the maturation ameloblasts also have cytoplasmic organelles with a faint brown colouration due to the presence of an iron pigment, the active caspase-3 distribution was confirmed with the use of an alternative, purple, chromagen (VIP, Vector Labs, USA) and green counter-stain (methyl green, Vector Labs, USA). In contrast, the distribution of Bcl2 was mainly localized in pre-ameloblasts and secretory ameloblasts. In a comparison of the three fluoride
*School of Dentistry, The University of Queensland. Faculty of Dentistry, Kuwait University, Kuwait.

treatments in relation to the incisor immunohistochemistry, it was not possible to detect any marked differences in the active caspase-3 stain distribution. Since the rat incisor presents a spatial-temporal view of the amelogenesis, the results suggest that virtually all caspase-3-related apoptosis occurs in the late maturation phase of amelogenesis. The apoptosis occurring in the transitional ameloblasts may result from a lysosomal pathway of apoptosis. This proposition is not incompatible with our observations of a rich lysosomal enzyme distribution in the transitional ameloblasts.9 References
1. Bronckers AL, Goei SW, Dumont E, et al. In situ detection of apoptosis in dental and periodontal tissues of the adult mouse using annexin-V-biotin. Histochem Cell Biol 2000;113:293-301. 2. Joseph BK, Harbrow DJ, Sugerman PB, Smid JR, Savage NW, Young WG. Ameloblast apoptosis and IGF-1 receptor expression in the continuously erupting rat incisor model. Apoptosis 1999;4:441-447. 3. Anuradha CD, Kanno S, Hirano S. Oxidative damage to mitochondria is a preliminary step to caspase-3 activation in fluoride-induced apoptosis in HL-60 cells. Free Radic Biol Med 2001;31:367-373. 4. Kubota K, Lee DH, Tsuchiya M, et al. Fluoride induces endoplasmic reticulum stress in ameloblasts responsible for dental enamel formation. J Biol Chem 2005;280:23194-23202. 5. Lyaruu DM, Bervoets TJ, Bronckers AL. Short exposure to high levels of fluoride induces stage-dependent structural changes in ameloblasts and enamel mineralization. Eur J Oral Sci 2006;114(Suppl 1):111-115. 6. Shigemura N, Kiyoshima T, Sakai T, et al. Localization of activated caspase-3-positive and apoptotic cells in the developing tooth germ of the mouse lower first molar. Histochem J 2001;33:253-258. 7. Matalov E, Kov u F, M ek I. Caspase 3 activation in the r s primary enamel knot of developing molar tooth. Physiol Res 2006;55:183-188. 8. Kumamoto H, Kimi K, Ooya K. Immunohistochemical analysis of apoptosis-related factors (Fas, Fas ligand, caspase-3 and singlestranded DNA) in ameloblastomas. J Oral Pathol Med 2001;30:596-602. 9. Smid JR, Monsour PA, Rousseau EM, Young WG. Cytochemical localization of dipeptidyl peptidase II activity in rat incisor tooth ameloblasts. Anat Rec 1992;233:493-503.

Frictional resistance to sliding, with repeated displacements, in a multi-bracket model

A Srinivasa, IA Meyers, CTC Ho*
This in vitro study investigated the effects of ligation and repeated vertical displacement forces on sliding resistance, and the magnitude of displacement required to overcome classical friction in a multi-bracket model involving various bracket/ligation combinations. Six different types of brackets two stainless steel (conventional twin and In-Ovation self-ligating brackets), three ceramic (Transcend, In-Vu and Inspire) and one ceramic with a metal insert (Clarity) all having a slot dimension of 0.018"x0.025", were used.
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Three methods of ligation (elastomeric modules, ligature ties and cobalt chromium clips of self-ligating brackets) and one stainless steel archwire (0.016"x0.022") were used in this study. Three brackets were mounted on an acrylic block at distances of 14mm and 7mm, the former to simulate an extraction space and the latter to simulate a typical interbracket distance. The part of the acrylic in the simulated extraction space was removed to allow the displacement beam to act on it. The minimum amount of force required for continuous free sliding (f-m) for
Australian Dental Journal ADRF Special Research Supplement 2006;51:4.

each set of bracket/archwire/ligation combination was determined. The f-m was then reduced to 80 per cent and applied as the sliding force. Vertical displacement forces of 50g, 100g, 150g and 200g were applied to the archwire at the rate of 91 cycles/min to simulate intraoral mastication. Any wire movement was attributed to a decrease in resistance to sliding caused by vertical displacement forces. F-m values showed statistically significant differences (p<0.01) between the combinations of bracket, archwire and ligation. Self-ligating brackets offered the least resistance to sliding, followed by conventional twin brackets with ligature ties. In the ceramic category Transcend and Clarity demonstrated similar f-m values and were the lowest in that group, followed by In-Vu.

*School of Dentistry, The University of Queensland.

Inspire demonstrated the highest f-m value. Repeated vertical displacements have a significant effect (p<0.001) on classical friction at the archwire/bracket interface. The reduction in sliding resistance depended on the displacement loads applied. The 50g load demonstrated the least resistance to sliding. Greater loads to 100g, 150g and 200g showed an increase in resistance to movement by 3442 per cent, 4856 per cent and 6471 per cent respectively, in each set of bracket/archwire/ligation combinations. This study has demonstrated that: vertical displacement forces at the bracket archwire interface have a significant effect in decreasing resistance to sliding; displacement loads as low as 50g had a significant effect in decreasing resistance to sliding; and the type of ligation played an important role in increasing or decreasing resistance to sliding, even in the presence of vertical displacing forces.

Early detection of dental caries using laser fluorescence

LJ Walsh, S Diklich*

The advent of quantitative laser-induced fluorescence as a clinical adjunct to caries diagnosis raises issues as to the range of user, equipment and optical factors which can potentially affect the reliability and performance of this technique over time. In addition there is the possibility that extrinsic or intrinsic stains can alter the signal to noise ratio. This study examined the factors which could potentially influence the performance of the DIAGNOdent device. With regard to instrument factors, measurements of laser wavelength and laser power output over time showed no significant variation. Degradation with autoclaving of the A tip used for occlusal caries diagnosis was minimal with no decline evident after 50 cycles at 134C. Up to 150 cycles, the performance changes were well within the calibration capabilities of the instrument. The presence of plaque gave an intense signal, a funding which stresses the need for plaque removal before using laser fluorescence. A strong signal
*School of Dentistry, The University of Queensland.

was also found for dental calculus, even when overlying deposits of plaque were removed. For occlusal and smooth surface lesions, there was a spatial association with greater scores in the direction of the greatest depth of the lesion. Measurements made by the one operator were reproducible to within three units (on a 099 scale) from day to day for measurements of sound tooth structure or caries, while sound dentine showed the lowest variation with a consistent score of five or less. Moisture on the occlusal surface tended to reduce readings, whereas areas of mineral conversion (enamel fusion) for high fluence laser treatment with CO2 and ER:YAG lasers gave a false positive signal. Because of the range of bacterial substrates which can elicit fluorescence, clinical users must be stringent in plaque removal to gain the maximum performance from the DIAGNOdent system. The same point could also be exploited to extend the capabilities of the instrument for detecting subgingival calculus or cavitated lesions provided appropriate optical tips or fibres were employed.

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Longitudinal assessment of changes in enamel mineral in vivo using laser fluorescence

LJ Walsh,* G Groeneveld, V Hoppe, F Keles, W van Uum, H Clifford
The DIAGNOdent device exploits the fluorescence properties of bacterial porphyrins for detecting dental caries. It is used commonly in dental practice as an adjunct when assessing occlusal for incipient or occult caries, however it can also be used on smooth surfaces. Most previous studies of this device have employed permanent teeth. The aim of the present study was to determine the distribution of DIAGNOdent laser fluorescence readings for the various clinical grades of smooth surface caries for the buccal and lingual tooth surfaces of primary canines and molars. As part of the Toothbrushing in Primary Schools (TIPS) project, 2136 children aged between five and seven years at baseline were examined by a calibrated examiner each year for three consecutive years. DIAGNOdent readings scores were obtained after removal of plaque and clinical inspection. A total of 17,088 primary molars and canines were available for analysis (33,029 readings).
*School of Dentistry, The University of Queensland. Academisch Centrum Tandheelkunde Amsterdam. Queensland Health.

Taking both buccal and lingual tooth surfaces together, the mean values were: sound enamel 3; demineralized (white spot decalcification) 8; decay with cavitation into dentine 27; and gross decay with an open cavitation 62. There were small differences between lingual and buccal sites, and between canine and molar teeth. Over the three-year period, where the clinical score remained the same, the DIAGNOdent score did likewise. These data show that sound deciduous enamel, despite its physical and chemical differences from permanent enamel, has a low fluorescence reading when sound, and that there is a progression in laser fluorescence scores with increasing severity of the lesion, as bacterial activity increases. This study is the first to observe clear differences in scores between sound enamel and white spot caries. The DIAGNOdent has considerable value as an adjunctive method for monitoring the progress of incipient caries lesions on smooth surfaces in primary teeth, however visual inspection is still recommended as the primary diagnostic method.

Trabecular patterns in the temporomandibular condyle: the characterization of a young and older normal sheep model in reference to human specimens
D Wilson,* O Wiebkin,* J Gardner, N Fazzalari
Much of the literature regarding arthritic changes in the temporomandibular joint (TMJ) is based on the assumption, rather than the demonstration, that joint degeneration is pathologically and biochemically similar to that which has been described for other arthrodial joints. Understanding such changes is axiomatic of an understanding of the specific histomorphometric structure of the normal TMJ, in particular the condyle. Unfortunately, very little has been established about the trabecular bone patterns in the mandibular condyle as it develops. As a consequence of the obvious practical difficulties in investigations of the human TMJ, the sheep has been variously used as an animal model. In order to augment a fuller characterization of this animal model, this study focused on the quantitative histomorphometry of the trabeculae in the mandibular condyles of young and mature sheep. Quantitative histomorphometric analyses of condylar trabeculae were performed on histological sections prepared from mature and young sheep condyles. Lateral, central and medial sagittal sections, and anterior and posterior coronal sections of the condyle were analysed using a Quantimet 500MC image analysis system that had been programmed to provide structural index values of trabecular bone volume, trabecular surface, trabecular thickness, trabecular separation and trabecular number. Analysis of histoquantitation data revealed a significant concordance in trabecular structural index values in lateral, central and medial regions of both young and mature sheep as well as in anterior and posterior regions in young sheep. With the exception of a degree of variation in trabecular thickness values in lateral sections, there was no significant variation in quantitative trabecular values for any of the condylar regions when the two age groups were compared. The findings of this study reinforce the appropriateness of the sheep as an experimental animal in deriving quantitative data concerning TMJ condyle trabecular patterns and structure.
Australian Dental Journal ADRF Special Research Supplement 2006;51:4.

*Department of Oral Pathology, The University of Adelaide. Australian Jaw Joint Project, The University of Adelaide.
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The expression of GroEL and enolase by Fusobacterium nucleatum grown in continuous culture
PS Zilm, NJ Gully*
Adult periodontitis is a destructive inflammatory disease of the connective tissues and bone surrounding teeth, and is the major cause of tooth loss in adults. The growth of a number of pathogenic oral bacteria and the increase in bacterial products/toxins is associated with the production of host cell immune mediators such as pro-inflammatory cytokines. Cytokines have been shown to be produced by various cell types including fibroblasts, monocytes, lymphocytes and epithelial cells. The Eschericia coli, GroEL homologue, is included in the family of heat shock proteins (hsp) with a molecular weight of 60kDa, (hsp60) and is a dominant bacterial antigen that plays an important role in the pathogenesis of infection by stimulating the host immune response. The hsp60 family participates in energy-requiring processes like folding, assembly and translocation of proteins across membranes. The production of stress proteins such as GroEL by specific Gram-negative bacteria are also reported to be linked to autoimmunity and one of the several factors related to the progression of human periodontal disease. Heat shock proteins can also be highly cross-reactive among bacterial and mammalian species. It is believed that chronic inflammatory reactions, such as periodontal diseases, may be due to defects in the ability to regulate the antiself capability. The effect of hsp60 is unclear, but is thought to be associated with an increase in TNFrelease, causing activation of p38 MAPK, and eventually apoptotic cell death. Enolase (hsp48) is one of several enzymes in the glycolytic pathway that are thought to be up-regulated upon heat stress in yeast. The up-regulation of the glycolytic pathway may be vital in maintaining intracellular ATP levels. Fusobacterium nucleatum is a Gram-negative anaerobe which has been implicated in the pathogenesis of oral diseases, including pulpal infection, alveolar bone abscesses and periodontal disease. The bacterium is particularly important for its ability to form aggregates with other oral bacteria and aid in the establishment of hypoxic environments, allowing the late anaerobic colonizers to establish in dental plaque. During the onset of periodontitis, the sub-gingival temperature is slightly higher in diseased sites
*Dental School, The University of Adelaide.

compared to healthy sites, and the pH of the periodontal pocket increases with its depth and also with the severity of the inflammatory host response. To ensure their survival, these and other environmental insults such as nutrient availability and oxygen stress, could be expected to promote the up-regulation of stress proteins such as GroEL by pathogenic bacteria such as F. nucleatum. The main aims of this project were to compare the expression of GroEL (a chaperonin which requires ATP) with that of the stress indicator enolase (hsp48) in F. nucleatum. Fusobacterium nucleatum ATCC 10953 was grown in continuous culture using a chemostat and a chemically defined medium. Culture conditions were maintained anaerobically at a dilution rate of 0.08h-1 and pH 7.2 at 36C. After steady state was achieved, the culture was stressed by raising (7.8) or lowering (6.4) the pH. Aliquots of cell culture harvested over five consecutive days were pooled, lysed and centrifuged to remove cell membranes. Cytosolic proteins were separated by 2D-PAGE using a minimum of three replicate gels for each growth condition. PD Quest image analysis software was used to determine the levels of GroEL and enolase expression following R250 Coomassie blue staining. GroEL and enolase were identified by tandem mass spectrometry following trypsin digestion. GroEL expression was significantly (p<0.05) upregulated 1.6-fold at a growth pH of 7.8 and was down-regulated 1.6-fold at pH 6.4. Mass spectrometry analysis indicated 2D-PAGE had resolved several isozymes of GroEL. Data analysis of the isozymes was unable to determine the nature of possible posttranslational modifications. Enolase expression remained unaffected by a change in external growth pH although further investigation of other glycolytic enzymes revealed the fructose-bi-phosphate aldolase was up-regulated sixfold at pH 7.8. The up-regulation of GroEL by F. nucleatum has been demonstrated in response to an increase in external growth pH. The onset of periodontal disease is associated with an increase in the pathogenic red and orange complex bacteria, of which F. nucleatum is a member. The results support in vivo observations that the pH of the periodontal pocket increases with pocket depth and is associated with the increased transcription of hsp genes.

Australian Dental Journal ADRF Special Research Supplement 2006;51:4.

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The investigation and characterization of the co-aggregation of Fusobacterium nucleatum grown in continuous culture
PS Zilm, NJ Gully*
The mouth is a unique environment in that the teeth provide a non-shedding surface that can result in the accumulation of large masses of bacteria and their products at sites between teeth (approximal surfaces), in the pits and fissures on the occlusal surfaces and around the gums (gingival crevice). The build up of plaque beyond those compatible with health can lead to problems such as dental caries and periodontal diseases. Oral bacteria possess very specific multiple adhesins that serve to attach to surfaces and to other bacteria (co-aggregation). The co-aggregation between pairs of bacteria have been found to be highly specific and are typically mediated by a protein adhesin on one cell type and a complementary saccharide receptor on the other. Cell surface proteins can have several roles in biofilm development. S. gordonii, for example, can interact with salivary proteins in the pellicle as well as with receptor proteins on the surface of A. naeslundii. Fusobacteria have been widely reported in the literature as being able to co-aggregate with a wide range of bacterial genera. In particular, Fusobacterium nucleatum appears to be unique in that it can coaggregate with the early and late colonizers of dental plaque. Fusobacterium nucleatum is a Gram-negative anaerobic rod and in its absence many other secondary colonizers cannot become part of the dental plaque community. The multiplicity of its co-aggregation interactions makes F. nucleatum an essential organism in the development of dental plaque. A review of the current literature supports the view that F. nucleatum can adhere to a wide range of organisms and yet they are unable to co-aggregate with each other. Preliminary studies in our laboratory have shown that when grown in continuous culture, F. nucleatum will form coaggregates in the planktonic phase and this is associated with the later establishment of a homogenous biofilm. To our knowledge this has not been previously reported. The mechanism that produces co-aggregation has not yet been determined but an important physiological event in the early development of a biofilm is the increased production of polymeric substances. Further studies are required to characterize the process of co-aggregation, therefore the main aim of this project was to determine the physiological events that trigger the co-aggregation of F. nucleatum, and to characterize the nature of the interactions between cells. Fusobacterium nucleatum ATCC 10953 was grown in continuous culture using a chemostat and a chemically defined medium (CDM). Culture conditions were maintained anaerobically at a dilution rate of 0.08h-1 and pH 7.4 at 36C. After steady state was achieved, the culture was stressed by raising (8.1) or lowering (6.4) the pH. Co-aggregation was also monitored after the addition of chlorhexidine (15g/ml) to the culture. If co-aggregation occurred, cells were harvested and co-aggregation was quantified by measuring the decrease in optical density (560nm) in the planktonic phase over five minutes. Cellular hydrophobicity was also quantified inter alia by measuring the change in optical density after adding n-hexadecane to an aliquot of harvested cells. Cellular yield and intracellular polymer synthesis (IPS) were also measured following cell harvesting. Growth of F. nucleatum as a biofilm was assessed by scanning electron microscopy (SEM) following removal of glass slides from the chemostat; slides had been placed in the culture for a minimum of 30 generations. Growth of F. nucleatum at elevated pH (8.1) was the only test condition that produced homogenous biofilms and co-aggregation (0.150.003 OD/min) of F. nucleatum. Growth at pH 7.4 in the presence of glucose (20mM) in CDM only produced planktonic growth (0.01.007 OD/min) and cells contained significant IPS levels (250mol glucose/g protein). After raising the pH to 8.1 the IPS level fell to almost undetectable levels (5mol glucose/g protein) and co-aggregation/biofilm formation was observed. The growth of F. nucleatum in CDM (no glucose) at pH 8.1 also produced biofilms so it was deduced that IPS was not required for biofilm formation. This was supported by the inability to resolve a capsule following staining with Manevals stain. SEM analysis of glass slides revealed dense biofilms comprising elongated cells that appeared to be embedded in an extracellular matrix. Glass slides of planktonic phase growth (pH 7.8) displayed minimal cell attachment. Cellular hydrophobicity also increased following co-aggregation (34%7) compared to cells grown at pH 7.4 (155) and preliminary observations showed that co-aggregation could be disrupted with heamin while glucosamine, trypsin and lactose were ineffective. The addition of the antimicrobial chlorhexidine to the culture did not produce coaggregation or biofilm growth. This study examined the growth conditions that produce co-aggregation and biofilm formation from homogeneous planktonic cultures of F. nucleatum. It has been reported that the onset of periodontal diseases are associated with elevated pH in the periodontal
Australian Dental Journal ADRF Special Research Supplement 2006;51:4.

*Dental School, The University of Adelaide.

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pocket, and that the alkalinity increases with pocket depth and the severity of the host inflammatory response. We report a correlation between growth pH and the formation of biofilms. Biofilms are recognized as a cellular defense mechanism and may reflect an attempt by the organism to evade host defenses. Growth at pH 8.1 was the only growth condition tested

that produced biofilm growth and co-aggregation. Coaggregation appears associated with an increase in cell surface hydrophobicity. The disruption by heamin indicates that co-aggregation may be linked to a loss of charge on the cell surface resulting in greater hydrophobic interactions between cells.

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ADRF Research Grant Reports published as full papers in the Australian Dental Journal 2006
Aust Dent J 2006;51:6-10 Dental therapy in Western Australia: profile and perceptions of the workforce E Kruger, K Smith, M Tennant Aust Dent J 2006;51:11-15 Guided self diagnosis: an innovative approach to triage for emergency dental care K Smith, A Clark, K Dyson, E Kruger, L Lejmanoski, A Russell, M Tennant Aust Dent J 2006;51:16-22 Epidemiological analysis of tongue cancer in South Australia for the 24-year period, 19772001 L Lam, RM Logan, C Luke Aust Dent J 2006;51:117-123 Longitudinal comparison of factors influencing choice of dental treatment by private general practitioners DS Brennan, AJ Spencer Aust Dent J 2006;51:219-224 Prevalence and side preference for tooth grinding in twins KV Dooland, GC Townsend, JA Kaidonis Aust Dent J 2006;51:290-296 An analysis of complaints against Victorian dental care providers 20002004 M Hopcraft, D Sanduja

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Australian Dental Journal ADRF Special Research Supplement 2006;51:4.

ADRF Undergraduate Research Grant Abstracts

Effect of local application of PGE2 and lipoxin A4 on the immunoexpression of OPG and RANKL during healing of a surgically placed defect in the Lewis rat mandible
R Chou; S Varanasi, AL Symons (Supervisors)*
Lipoxin A4 (LXA4) and prostaglandin E2 (PGE2) modulate inflammation and may affect bone healing. Under physiological conditions, the local application of PGE2 to the rat mandible promotes bone formation but the presence of inflammation may modify this activity. LXA4 is known to suppress inflammation and may inhibit bone resorption. Receptor activator of nuclear factor B-ligand (RANKL) is released by osteoblasts and fibroblasts to stimulate the maturation, differentiation and survival of osteoclasts. The effect of RANKL is inhibited by osteoprotegerin (OPG) also released by osteoblasts. The aim of this study was to determine the effect of PGE2 and LXA4 on the expression of OPG and RANKL during bone healing of a surgical defect in the rat mandible. A surgical defect was placed in the left side of the mandible of 60 female rats. A 20-day, controlled-release PGE2 (0.05mg/kg/day) pellet was implanted adjacent to the bone defect in the PGE2treated groups. In the control groups, the defect was
*School of Dentistry, The University of Queensland.

left to heal and animals sacrificed after 24-hours, 5-, 10- and 20-days. For the LXA4-treated groups, a local dose of 500ngm of lipoxin was injected on days 3, 6 and 9 post-operatively for the 10-day group and, additionally on days 12, 15 and 18 post-operatively for the 20-day group. Transverse serial sections of the mandibles were immunohistochemically stained for OPG and RANKL expression. Compared with the 24hour controls the expression of OPG and RANKL was significantly greater in sections from the 5-, 10- and 20day post-healing groups (p<0.001). Immunoexpression of OPG was significantly reduced in the 10-day PGE2treated mandibles (p<0.05) compared with 10-day controls. No differences in OPG expression were observed in the 20-day animals. Application of LXA4 increased the number of cells expressing both OPG and RANKL in the 20-day group. PGE2 treatment did not alter the immunoexpression of RANKL but reduced the expression of OPG in the 10-day group. There was no evidence PGE2 promoted mineralized tissue formation, nor did LXA4 promote early bone healing in this model.

Temporal variation of the different clinical presentations of histopathologicallyproven oral lichen planus: a retrospective study of 391 patients
S Kaing; M McCullough (Supervisor)*
Oral lichen planus (OLP) is a chronic inflammatory disease that has various clinical presentations including reticular, erythematous, plaque-like, ulcerative and bullous. These clinical forms behave differently over time. The ulcerative form of OLP has been associated with a higher rate of malignant transformation. Patients with a clinical and biopsy-confirmed diagnosis of OLP at Royal Melbourne Dental Hospital between the years 19892000 were included in this study. Clinical photographs were analysed and classified into reticular, erythematous, plaque-like or ulcerative. Appearance changes were noted as improved, worsened, unchanged or variable. Over the 12-year period, 266 females and 125 males were diagnosed with OLP. On average, the patients were followed up for 6.3 years and it was found that
*School of Dental Science, The University of Melbourne.
Australian Dental Journal ADRF Special Research Supplement 2006;51:4.

most lesions remained unchanged (71 per cent). No statistically significant correlations could be found between appearance change and age or gender. Statistically significantly more patients with ulcerative lesions were more likely to improve while patients with reticular lesions were less likely to improve. The clinical appearance of OLP did not change or improve in the vast majority (84 per cent) of patients during this 12-year period. Further, this study supports the notion that ulcerative OLP lesions, non-responsive to treatment, warrant a high degree of suspicion. Published: Kaing S, McCullough M. Temporal variation of the different clinical presentations of histopathologically-proven oral lichen planus: a retrospective study of 391 patients. Oral Disease (in press).
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An analysis of dental trauma in a large rural centre (Bunbury, WA) from 20002005
R Lam; PV Abbott (Supervisor)*

There is little epidemiological research regarding dental trauma in Australia. Most of the previous research has focused on specific sub-populations and their data are not necessarily applicable to a general rural Australian population. Studies from other countries report great variability and the applicability of their findings to the Australian situation is also questionable. The aim of this study was to investigate the factors influencing dental trauma in a large rural centre in Australia. This study was a retrospective analysis of the dental records of 323 consecutive patients who had attended a private general dental practice in Bunbury, Western Australia following an injury to their teeth and/or mouths over a six-year period from 20002005 (inclusive). Injuries were classified using the Andreasen system (1994). Data analysis was carried out using SPSS software and chi-square tests were performed with the level of significance set at five per cent. There were 527 teeth injured and eight patients only had soft tissue injuries. Males (68 per cent) significantly
*School of Dentistry, The University of Western Australia.

outnumbered females (31.9 per cent) and the ages ranged from 10 months to 78 years. The highest number of injuries occurred in children and adolescents, specifically the 04 year old age group followed by the 59 and 1014 year old age groups. Trauma was most frequently the result of falls or accidents while playing and participating in sport. The maxillary central incisors were the most commonly injured teeth in both the primary and permanent dentitions. Uncomplicated crown fractures were the most common injury followed by luxations and subluxations. No significant differences in frequency were reported for the different days of the week, the different months or seasons of the year. Only one-third of the patients presented for dental treatment within 24 hours of the injury whilst the remainder delayed seeking treatment for varying times up to one year. This study has provided an insight into the types, causes and incidence of dental and oral injuries in a large Australian rural centre. Such knowledge should help the local dental profession to plan emergency services and provide advice regarding preventive measures.

Staining potential of APF foam on restorative materials in vitro

D Lin; B Huang (Supervisor)*

This study aimed to identify the staining potential of Acidulated Phosphate Fluoride (APF) foam on restorative materials in vitro. A random sample of 200 permanent molars from 10 deceased sheep was selected. Each received one of the five standard clinical procedures, including no preparation, preparation but no restoration, glassionomer cement (GIC) restoration, resin-modified glass-ionomer cement (RMGIC) restoration as well as composite resin restoration (CR). This was followed by topical APF application at a daily interval and a predetermined frequency ranging from zero to three times. Staining formation on teeth and restorations was evaluated and determined by three trained examiners. Fluoride staining on the teeth and/or the restorations varied, appearing with a distinguishable darker shade, an orange-coloured surface or a deep brown margin.
*School of Dentistry, The University of Western Australia.

The fluoride staining rates of GIC, RMGIC and CR were 50 per cent, 27.5 per cent and 17.5 per cent respectively. GIC had a higher staining potential than RMGIC ( 2=4.266, df=1, p=0.039) and CR ( 2=9.448, df=1, p=0.002), while difference of staining potential between RMGIC and CR was indiscernible ( 2=1.147, df=1, p=0.284). The occurrence of staining increased with the frequency of topical APF application on RMGIC ( 2=8.436 df=1, p=0.004) and CR ( 2=6.873, df=1, p=0.009) but not GIC ( 2=0, df=1, p=1). Staining was not associated with frequency of APF application on teeth without preparation and/or restoration ( 2=4.051, df=3, p=0.256). The staining of APF foam reported in the literature was confirmed in vitro. GIC was more susceptible to fluoride staining than RMGIC and CR. This study suggested aesthetic implications when topically applying fluorides to restored teeth. Further investigation on human teeth is indicated.

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Australian Dental Journal ADRF Special Research Supplement 2006;51:4.

Non-carious cervical lesions: a proposed new system of classification

JA Michael; GC Townsend, J Kaidonis (Supervisors)*

Non-carious cervical lesions (NCCLs) involve loss of dental hard tissue at the cervical third of the crown and subjacent root surface, through processes unrelated to caries. The aetiology of NCCLs is commonly multifactorial, with combinations of distinct processes, including abrasion, corrosion (erosion) and possibly abfraction, operating to varying degrees. The crosssectional form of NCCLs has been described but no formal morphological classification system has been developed. Lack of agreement regarding the most appropriate term to use when describing a given morphological form of NCCL adds to the confusion in this controversial area of dentistry. The primary aim of this study was to describe the spectrum of common morphological forms of NCCLs observed within a large sample of extracted teeth and to develop a simple and logical descriptive classification system for NCCLs. A large sample of extracted permanent anterior teeth, stored in the Murray Barrett Laboratory in the Adelaide Dental Hospital, was examined macro-

*Dental School, The University of Adelaide.

scopically under illumination at 2x magnification. Well-defined, descriptive categories were formed, based on dental and NCCL features and terminology currently used in the literature. Teeth were then sorted into these categories according to the cross-sectional form of the NCCLs. In total, 11,434 teeth were studied and 510 NCCLs observed. The NCCL categories developed were shallow (subdivided into flat-shallow and curvedshallow), concave, wedge-shaped, notched NCCLs, and irregular (subdivided into angular-irregular, curved-irregular and angular- and curved-irregular). This appears to be the first study to report the broad morphological spectrum of NCCLs that may be encountered. The new classification system should help to improve communication between oral health professionals regarding morphological forms of NCCLs described in the literature and between oral health professionals when examining and comparing NCCLs. It presents an alternative to the causally-based NCCL classification system used widely in the literature that may be difficult to apply in many cases due to the multifactorial aetiology of these lesions.

A 3-D CT analysis of craniofacial asymmetry in Malaysian infants with cleft lip and palate
N Tziavaras; G Townsend, D Netherway (Supervisors)*
The aim of this study was to compare craniofacial morphology, including asymmetry, in a sample of unoperated Malaysian infants with cleft lip and palate (CLP) with an unaffected group of infants matched for age, by referring to a midline plane. The cleft sample comprised 29 individuals including 10 with unilateral cleft lip and palate (UCLP), five with bilateral cleft lip and palate, seven with cleft lip and primary palate and seven with isolated cleft palate. The control sample consisted of 13 infants with no craniofacial abnormalities. The ages of the subjects ranged from 0.412.2 months. The software programme Persona was used to locate six landmarks on images of nasal bones derived from high resolution CT scans. A reference plane was created using the landmarks basion, sella and nasion, and 13 landmarks were
*Dental School, The University of Adelaide.

compared to that plane by calculating distances, degrees and ratios. Comparisons of mean values and variances between groups were made using unpaired t tests and F-tests with significance set at p<0.05. Comparisons between ipsilateral and contralateral sides in the UCLP cleft group and in the non-cleft (NC) sample were made using paired t tests. Differences in craniofacial distances and angles were found between CLP and NC groups. There was also some evidence of left-right facial dominance in the combined cleft group. This group had flatter, wider superiorly and longer nasal bones compared to the NC group. The nasal bones tended to deviate to the contralateral side of the cleft. This study has demonstrated that CLP affects the size and orientation of the nasal bones and also is associated with alterations in the morphology of other facial bones further from the region of the cleft.

Australian Dental Journal ADRF Special Research Supplement 2006;51:4.

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Interactions between the periodontopathogenic bacteria Treponema denticola and Porphyromonas gingivalis
S Yoon; R Orth, S Dashper (Supervisors)*
Periodontal diseases are bacteria-associated inflammatory diseases of the supporting tissues of the teeth, with advanced disease leading to loss of periodontal tissue attachment and destruction of alveolar bone. Chronic periodontal disease has a polymicrobial aetiology with Treponema denticola and Porphyromonas gingivalis strongly implicated in disease progression. The aim of this research was to determine interactions between T. denticola and P. gingivalis that may contribute to our understanding of their ability to cause disease. Treponema denticola ATCC 35405 was grown in continuous culture in a chemostat on a modified NOS medium under strictly anaerobic conditions at 37C. Unless otherwise stated, P. gingivalis ATCC 53978 was grown in batch culture in a modified NOS medium under strictly anaerobic conditions at 37C to a cell density of 0.62 (OD650 nm). Whole cell protease assays were performed to determine cell-surface-associated proline-specific and arginine-specific activities of both T. denticola and P. gingivalis using the synthetic substrates N-Succinyl-Ala-Ala-Pro-Phe p-Nitroanilide (SAAPNA) and N -Benzoyl-L-Arginine p-Nitroanilide (L-BAPNA). Porphyromonas gingivalis was found to have 29.801.97 U/1011 cells (Units of activity/1011 cells) of Arg-specific (BAPNA) activity, more than 10 times the activity of T. denticola, which had an initial rate of 2.480.23 U/1011 cells. The initial rate of
*School of Dental Science, The University of Melbourne.

proline-specific (SAAPNA) activity of T. denticola whole cells was found to be 2.740.35 U/1011 cells, approximately four times that of P. gingivalis which had an initial rate of 0.670.52 U/1011 cells. Treponema denticola and P. gingivalis were grown together in continuous culture. To achieve this, P. gingivalis grown in batch culture was added to a steady state T. denticola continuous culture with a generation time of 15.75 hours. After two days of growth the cell density of the culture increased from 0.24 (OD650 nm) to 1.36 (OD650 nm). Porphyromonas gingivalis established itself rapidly in this culture and a ratio of 2:15 T. denticola:P. gingivalis cells was observed. Microscopic analysis of the co-culture revealed physical interactions between the two species, with P. gingivalis binding to T. denticola in a string of pearls formation. This close interaction may aid the colonization of the periodontal pocket by T. denticola. The pH of the culture remained stable, implying that the environmental conditions were amenable to both bacterial species. These results suggest that it is possible to cultivate T. denticola and P. gingivalis together in continuous culture without any obvious detrimental effects to either species, enabling the future examination of protease activities under polymicrobial conditions. Further research is required on the long-term effects of co-culture of T. denticola and P. gingivalis to determine the effects of this close association on metabolic activities and proteolytic activities of the individual species.

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Australian Dental Journal ADRF Special Research Supplement 2006;51:4.

Trebitsch Grant Abstract 20052006

An immunohistological study of co-infection in a mouse model
J Lin; PS Bird, A Chan (Supervisors)*

The aim of this project was to investigate the pathogenicity of co-infection of Tannerella forsythensis, Porphyromonas gingivalis and Fusobacterium nucleatum using a mouse abscess model. This was determined by counting the infiltrating CD4+ T cells, CD8+ T cells, macrophages, and B cells in developing lesions resulting from exposure to a combination of pairs of each bacterium compared to exposure from a single bacterium. Non-immunized BALB/c (68 weeks old) mice were challenged with F. nucleatum ATCC 25586, T. forsythensis ATCC 43037 and P. gingivalis ATCC 33277. The mice were divided into seven groups of 1216 mice per group and injected with different combinations of bacteria. Group 1 mice received P. gingivalis and F. nucleatum; Group 2 mice, T. forsythensis and F. nucleatum; Group 3 mice, P. gingivalis and T. forsythensis; Group 4 mice,

*School of Dentistry, The University of Queensland.

P. gingivalis only; Group 5 mice, F. nucleatum only; Group 6 mice, T. forsythensis only; and Group 7 mice received injections of saline. Lesions were excised from 34 mice per group at days 4, 7 and 14. The lesions were fixed and embedded for cryostat sectioning. The sections were stained by hematoxylin and eosin to evaluate the lesions. The types and numbers of infiltrated cells were determined using an immunohistological method. The overall results showed that mice infected with combinations of P. gingivalis and F. nucleatum, P. gingivalis and T. forsythensis or F. nucleatum and T. forsythensis had elevated levels of infiltrating CD4+ positive T cells, and macrophages into the lesions at day 7 when compared to mice injected with P. gingivalis, F. nucleatum or T. forsythensis as single injections. Mice infected with only P. gingivalis had higher levels of infiltrating cells when compared with T. forsythensis or F. nucleatum. These results show there is a synergistic increase in pathogenicity resulting from the combination of bacteria.

Australian Dental Journal ADRF Special Research Supplement 2006;51:4.

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Notes