Australian Dental Journal Research Supplement 2011

Volume 56 Number 4 December 2011
Australian Dental Journal

The ofcial journal of the Australian Dental Association
SPECIAL RESEARCH SUPPLEMENT

Australian Dental Research Foundation
ISSN 00450421
The ofcial journal of the Australian Dental Association
Australian Dental Journal
Introduction
The Australian Dental Research Foundation is moving into its fth decade of supporting dental research in Australia and the evidence of the Foundations contribution to dental research can be seen in the range and quality of abstracts presented in this supplement. The dental profession is dependent on research and scientic evidence to move clinical practice forwards and to ensure best practice, evidence based treatment for patients. In this respect, the profession owes a great debt to the dedicated scientists and researchers who continually contribute to our knowledge and expertise. I congratulate all those who have committed their time and expertise to undertaking the research, and openly presenting their results and information in this supplement. I also congratulate those who have managed to complete their research despite some very difcult nancial times and limitations in funding. There is no doubt this has required a higher level of commitment by all researchers. Obtaining research funding in dentistry is difcult, and recruiting and inspiring undergraduates and graduates into undertaking dental research is challenging. Dental disease continues to be a major concern within our society and it is unfortunate that this is not fully recognized by larger government research funding agencies and thereby does not receive the attention and funding that it deserves. As the number of dental schools in Australia increases and the demand for high quality dental education increases there is an essential need to ensure there is a balance of clinical education and research. Dental education and training is at risk of becoming focused purely on the technical skills required and it is vital that research underpinning dental education is enhanced and promoted. Universities throughout Australia must continue to be supported by research funding, particularly from ADRF, which without ongoing support from the profession, is becoming increasingly difcult in these tough economic times. I encourage you to read and enjoy this research supplement and realize the benets of research in maintaining the development of the profession. I am sure you will nd information relevant to your clinical practice and in doing so I encourage every reader to support the Foundation and keep innovation alive and assist with moving the profession forward. Ian Meyers Chairman Australian Dental Research Foundation
CONTENTS
ADRF Special Research Supplement

Volume 56 No 4 December 2011
ADRF Research Grant Abstracts

S5 S6 S6 S7 The role of phenytoin induced fetal hypoxia in cleft lip and maxillary hypoplasia D Abela, H Ritchie, D Ababneh, A Howe, D Oakes, W Webster Dental issues in Australian newspapers: a pilot study H Baros, LM Slack-Smith Effect of PDGF on bone formation around dental implants PM Bartold, V Marino, T Choo A vibrational spectroscopic analysis of the composition, microstructural arrangement and nanomechanical characteristics of hypomineralized enamel S Brent, M Swain Regulation of reactionary dentine formation by odontoblasts in response to polymicrobial invasion of dentine matrix N Charadram, RM Farahani, D Harty, C Rathsam, MV Swain, N Hunter Immunolocalization of CPP-ACP in remineralized enamel subsurface lesions and high resolution imaging of the enamel crystallites NJ Cochrane, S Rubanov, F Cai, Y Yuan, JEA Palamara, EC Reynolds Observational study of changes in lifestyle habits and behaviours of naval recruits and their caries risk N Cusack, G Mahoney, L Walsh Identication of molecular markers of oral epithelial stem cells A Dalley, J Saunus, R Dawson, Z Upton, CS Farah Inuence of implant abutment angulations on the fracture resistance of overlaying bre reinforced composite single crowns A Ellakwa Correlation of serum and gingival crevicular uid cytokines in obese subjects RA Fell, KY Zee, M Arora Structural and functional changes underlying orofacial pain SM Gustin, CC Peck, S Wilcox, GM Murray, LA Henderson The effect of titanium surface topography and chemistry on macrophage and monocyte gene expression SM Hamlet, S Ivanovski The effect of growth factors PDGF and IGF on cementum-associated gene expression by periodontal cells H Hannawi, SM Hamlet, S Ivanovski Establishing a mouse model to investigate the relationship between periodontal disease and inammatory arthritis DR Haynes, PM Bartold, V Marino, MD Cantley Vibrational spectroscopic investigation of the compositional, microstructural and nanomechanical characteristics of enamel LH He, MV Swain An evaluation of alternate splinting adhesives in the management of dental trauma J-Y Hu, GS Heithersay Neurophysiological changes underlying orofacial pain Z Karami, T Salameh, C Peck Association of strong premature contacts and tooth mobility using T-Scan S Knoepel, K Nguyen, M Bickel, J Rincon Porphyromonas gingivalis GroEL and atherosclerosis in a mouse model SJ Leishman, HL Do, JE Palmer, GJ Seymour, KW Beagley, PJ Ford Mucosal inoculation of Porphyromonas gingivalis and atherosclerosis SJ Leishman, GJ Seymour, PJ Ford
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S9 S10 S10
S11 S12 S13 S14 S14 S15
S16 S17 S18 S18 S19
Contents continued
S19 S20 S20 S21 S22 S22 S23 Predicting unscheduled dental visits for members of the military G Mahoney, G Slade, S Kitchener, A Mamun Inuence of Thl cytokine deciency on osteogenic and angiogenic markers involved in bone healing MA Matias, CS Farah Localization of MMP-9 in oral soft tissue wounds in the p40 knockout mice MA Matias, CS Farah The effects of acidic beverages on the demineralization rate of incipient enamel lesions: a pilot study JM McIntyre, I White, J Kibble The effect of remineralization treatments on demineralized dentine: an in vitro study E Moloney, S Varanasi, IA Meyers, L Rintoul, AL Symons GABA in human dental pulp DAS Parker, K Nassery, V Marino In vitro analysis of cytotoxicity and genotoxicity in human oral epithelial cells after treatment with alcohol-containing mouthwashes J Saunus, S Zhao, CS Farah Process analysis of the Monitor Dental Practice programme implementation A Sbaraini, SM Carter, W Evans, A Blinkhorn The effectiveness of sonic activation, laser activation or syringe irrigation of 4% sodium hypochlorite in eradicating an Enterococcus faecalis biofilm A Seet, P Cathro, P Zilm, T Fitzsimmons, N Gully Characterization of the response to Porphyromonas gingivalis using a co-culture method A Shah, HL Do, SJ Leishman, PJ Ford The effect of adding calcium and phosphate to acidic beverages on dental enamel erosion in vitro P Shen, NJ Cochrane, Y Yuan, GD Walker, C Reynolds, EC Reynolds Ultramicroscopy of bone at oral implant site: a comparison of ectodermal dysplasia patients and regular patients P Silthampitag, AS Jones, T Whittle, S Scholz, C Johansson, T Albrektsson, I Klineberg Cancer comorbidity and oral cancer survival: a cancer registry pilot study LM Slack-Smith, AM Frydrych, R Parsons, T Threlfall, SE Hall Survival of rat dental pulp cells in vascularized tissue engineering chambers T Srisuwan, D Tilkorn, A Vashi, A Penington, H Messer, K Abberton, E Thompson Reproducibility of ICDAS-II and DIAGNOdent for detection of occlusal caries by undergraduate dental students M Stacey Factors inuencing learning of psychomotor skills by dental students N Suksudaj, TA Winning, G Townsend, D Lekkas, J Kaidonis A controlled study of the oral health of twin children SS Taji, GC Townsend, WK Seow Dental insurance, level of cover and dental service use DN Teusner, DS Brennan, AJ Spencer The effect of the local application of different dosages of lipoxin A4 on bone healing following the placement of a surgical defect in the mandible S Varanasi, AL Symons, JR Smid The effect of low dose prostaglandin E2 on bone healing of surgical defects over time S Varanasi, AL Symons, JR Smid, GO Ramirez The effect of lipoxin A4 on the expression of osteoblasts and broblasts cells markers in vitro S Varanasi, AL Symons, JR Smid, GJ Seymour
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S30 S31 S32 S33
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Contents continued
S36 S37 Regulation of BMP-2 and BMP-6 expression by titanium surface topography and chemistry J Vlacic-Zischke, SM Hamlet, S Ivanovski Identication and characterization of novel cell surface markers that are essential for periodontal ligament stem cell phenotype N Wada, K Mrozik, S Gronthos, PM Bartold Students understandings, approaches and outcomes of PBL contexts in two PBL dental curricula T Winning, V Skinner, A Kinnell, G Townsend, G Svensa ter, M Rohlin, J Davies The effect of exercise training on jaw muscle recruitment strategies A Wirianski, C Peck, G Murray Dentists perceptions of a new local anaesthetic drug the uptake of new technology in dentistry KE Yapp, MS Hopcraft, P Parashos c-Src kinase mediates enhanced oral epithelial barrier function induced by ligation of CD24 P Ye, H Yu, N Hunter Magnitude of bacteraemia induced by ossing and by scaling and root planing in patients with chronic periodontitis W Zhang, C Daly, D Mitchell, B Curtis The role of S-layer protein in biolm formation by Fusobacterium nucleatum in response to high growth pH P Zilm, J Chew, N Gully, A Rogers Proteomic analysis of human mesenchymal stem cell surface proteins using uorescent Cye dye labelling P Zilm, K Mrozik, S Gronthos, J Xiong, PM Bartold
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S42 S43 S44
ADRF Research Grant Reports published as full papers in the Australian Dental Journal in 2011
ADRF Undergraduate Research Grant Abstracts

S45 S45 S46 S46 Risk assessment for children at high risk of early childhood caries a pilot qualitative study MK Cable; WK Seow, L Hobbs (Supervisors) Erosive potential of powdered sports drinks CH Chang; Y Yuan, GD Walker, P Shen, C Reynolds, EC Reynolds, NJ Cochrane (Supervisors) Expression of calmodulin-like skin protein in oral epithelial dysplasia DE Fan; PJ Ford, CS Farah (Supervisors) Awareness and understanding of osteonecrosis of the jaw by general practitioners and their referral pattern to dentists prior to commencing bisphosphonate treatment of osteoporosis J Hsieh; P Reher (Supervisor) Instrumental, Detrimental and TranscenDental: experiences of dental emergency and micro-rationing practices in a Victorian public dental clinic E Kahan; E Willis (Supervisor) A SEM evaluation of the effect of bicarbonate soda in root canal irrigation N Leow; P Abbott, J Castro Salgado (Supervisors) Perceptions of medical practitioners regarding oral and dental health in Western Australian aged care JG Martins; LM Slack-Smith (Supervisor) Quantifying the selection of maxillary anterior teeth using intraoral and extraoral anatomical landmarks K McNamara, J Sandhu, K James; A El-Sheikh, A Ellakwa, I Klineberg (Supervisors) An investigation into dental trauma prevalence and understanding of its management in New South Wales rugby union clubs K Metcalfe, EA Ilia, E Tsolakis; M Heffernan (Supervisor) Exploring the dimensions of oral health literacy in Chinese mothers M Nga-Man Liu; A Arora (Supervisor) Immunohistochemical analysis of alveolar bone at oral implant sites A Samakeh, H Badreddine; I Klineberg (Supervisor)
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S48 S49 S49 S50
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Contents continued
S51 S52 A three-year retrospective study of emergency visits at a community based oral health clinic for children N Wong; C Tran, WK Seow (Supervisors) The incidence of chronic pain following successful endodontic treatment GH Yoo, ML Lawless, A Forte; CC Peck (Supervisor)
RW Hession Award 2008

S53 Remineralization of natural white spot lesions and their characterization by X-ray microtomography and synchrotron X-ray microtomography analysis NJ Cochrane, P Anderson, GR Davis, DJ Vine, S Rubanov, CDL Thomas, EC Reynolds
Colin Cormie Grant 2010

S55 Analysis of remineralization produced by various products using transverse microradiography and surface microhardness TK Park; NJ Cochrane, EC Reynolds (Supervisors)
ADRF Research Grant Abstracts

The role of phenytoin induced fetal hypoxia in cleft lip and maxillary hypoplasia
D Abela,* H Ritchie, D Ababneh,* A Howe,* D Oakes, W Webster*
The anticonvulsant drug phenytoin is a known human teratogen that causes an increase risk of facial clefting as well as other birth defects if taken during pregnancy. An animal model developed by the authors show that if phenytoin is administered to pregnant rats on gestation day 11 (E11), about half way through rat pregnancy many of the fetuses develop similar facial defects to the phenytoin embryopathy. It has been hypothesized that the mechanism of the teratogenesis of phenytoin is blockade of hERG potassium channels in the embryonic heart resulting in embryonic bradycardia and arrhythmia. This would cause hypoxia in the embryo and tissue destruction leading to malformations. Similarly hyperoxia should reduce the effect of phenytoin on the embryo. The drug dofetilide is a known hERG channel blocker and when administered on E11 causes severe fetal arrhythmia and facial clefting was used as a positive control with phenytoin. In these present studies aspects of this hypoxia hypothesis for phenytoin facial clefting were tested; dofetilide was used to compare its hypoxic effects with the proposed hypoxic effects of phenytoin. Three separate experiments were used to test hypoxic effects of phenytoin on the rat fetus. The rst study uses the hypoxia marker pimonidazole and immunostaining to examine the location and extent of fetal hypoxia following phenytoin and dofetilide administration; fetuses were euthanased at intervals from E11 for 24 hours. In the second study pregnant rats were kept in a hyperoxiac environment (100% oxygen) following a teratogenic dose of phenytoin for 48 hours in an attempt to see if increased oxygen levels would reverse the affects of phenytoin and reduce the prevalence of cleft lip and maxillary hypoplasia in the offspring. The third study involves high denition ultrasound to examine the heart rate in rat embryos in vivo after teratogenic doses of dofetilide or phenytoin. Study one showed that dofetilide caused the exposed embryos to become extremely hypoxic (as indicated by pimonidazole staining) for 12 hours or more following a teratogenic dose. In contrast no increase in hypoxia was recorded in rat embryos tested that were exposed to a teratogenic dose of phenytoin. Preliminary results for study two indicated a protective effect of hyperoxia against cleft lip and maxillary hypoplasia in rat embryos exposed to a teratogenic dose of phenytoin; this study is continuing. Preliminary results for study three indicated severe bradycardia and arrhythmia was detectable in the dofetilide embryos showing that the technique is viable in measuring fetal heart rate. Phenytoin treated embryos are yet to be studied at the time of publication. In conclusion, although the rst study appears to preclude hypoxia as the cause of phenytoin teratogenicity, the second incomplete study supports the hypothesis that the teratogenic effect of phenytoin (facial clefting and maxillary hypoplasia) is caused by hypoxia. Dofetilide was shown to induce severe bradycardia, cardiac arrhythmia, facial clefting and hypoxia in the rat embryo and that with in vivo ultrasound is possible of detecting fetal heart rate. Studies in the teratogenicity of phenytoin and facial clefting are continuing.
*Discipline of Anatomy and Histology, School of Medical Science, Faculty of Medicine, The University of Sydney. Discipline of Biomedical Sciences, School of Medical Science, Faculty of Medicine, The University of Sydney. Email: andrew@anatomy.usyd.edu.au.
2011 Australian Dental Association
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Abstracts
Dental issues in Australian newspapers: a pilot study

H Baros, LM Slack-Smith*
Mass media is an important source of health information for the general public. Newspapers have the advantage of providing a variety of information to a broad range of age groups, with wide regional coverage of both urban and rural areas and they can be an important source of dental information. There is limited research literature regarding portrayal of oral health dentistry in the newspaper media. A study carried out in Japan looked at coverage by Japanese newspapers of oral health messages on the prevention of dental caries and found that most articles appeared frequently in health columns and most frequently described dietary control as a preventive method. Another study reviewed oral malodour information in Japanese newspapers and investigated peoples knowledge on the causes of oral malodour. The study found that less than half of those surveyed knew that oral problems were associated with malodour. Dental professionals should be aware of the importance of media advocacy in order to provide appropriate oral health information to the public. This pilot study investigated dentistry oral health articles in the newspaper media. The overall aim of this pilot study was to investigate dental articles in Australian major newspapers. The specic objectives were to: (1) determine whether Factiva (http://www.factiva.com) searches could assist identication of Australian newspaper articles with a dental oral health theme and which keywords should be used; and (2) examine newspaper articles relevant to dentistry and oral health using content analysis. Holsti dened content analysis as the application of scientic methods to documentary information. The current study was a simple content analysis to determine the value of investigating newspaper articles relevant to dentistry in Australian major newspapers using the Factiva database. A manual search of The West Australian, The Age, The Sydney Morning Herald and The Australian over three months (January March 2008) was compared to searching using dental related terms in Factiva to refine our approach and determine important terms. We investigated dental terms to determine those found frequently when searching the newspaper article in full or when searching articles which had a focus (using Factiva search features) on the term. We included all Australian newspaper articles for 2007 to investigate frequency of dental terms using Factiva. We initially compared a Factiva search for January March 2008 with a manual search of 4 newspapers. Factiva search was much quicker and was useful for searching for articles. This comparison allowed us to determine which terms to search. We reviewed all Australian newspaper articles for 2007. When we looked at any mention of a dental term, we noted that dentistry (in 306 articles) and dental (1170) were much more frequently used than oral health (145) and most commonly found other relevant terms were toothpaste (262), gum disease (44), dental decay (32), dental hygiene (26), mouth cancer (12) and periodontal disease (11). When we counted articles with a focus on the term (as dened by Factiva), dental (332) and dentistry (57) were much more frequent than oral health (42) and other terms of relevance included toothpaste (60), dental hygiene (8) and gum disease (6). Factiva was an effective way to undertake broad searches of newspaper article content. Factiva searching provided a viable approach to investigate the newspaper media.
*School of Dentistry, The University of Western Australia. Email: linda.slack-smith@uwa.edu.au.
Effect of PDGF on bone formation around dental implants

PM Bartold, V Marino, T Choo*
Immediate placement of implants into fresh extraction sockets has the potential of shortening the total treatment time for patients. However, when implants are placed at the time of tooth extraction, there is often a gap between the walls of the extraction socket and the implant surface. This gap is usually widest in the coronal aspect of the socket. Hence, management of this bony defect around dental implants may play an important role in improving implant success in terms of aesthetic outcomes. The use of growth or differentiation factors is an emerging approach with signicant
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potential to improve bone regeneration. One such agent which has been clinically tested and shown to be safe and effective for human intraoral use in the treatment of periodontal osseous defects is platelet-derived growth factor-BB in combination with an alloplastic material, b-TCP. However, to date, there have been no studies evaluating this combination for bone regeneration in defects surrounding implants. The aim of this investigation was to examine the effect of a combination of puried recombinant human platelet-derived growth factor (rhPDGF-BB) mixed
Abstracts with a synthetic betatricalcium phosphate (b-TCP) on bone healing around dental implants with critical size circumferential defects. Three critical-size circumferential defects (10 3 mm) were prepared in the ilium of six sheep. Three dental implants were placed into the centre of each defect and the 3.25 mm circumferential gap was lled with either: (1) rhPDGF-BB (0.3 mg ml) with b-TCP; (2) b-TCP; or (3) blood clot alone. The sheep were sacriced at 2 and 4 weeks and histologic and histomorphometric analysis was performed to determine the percentage of new mineralized bone formation and residual b-TCP graft particles in the defects. Defects lled with (1) showed the highest rate of bone formation after 2 and 4 weeks with limited degradation of the b-TCP particles over 4 weeks. Defects lled with (2) showed the least bone ll after 2 and 4 weeks, and faster degradation of the b-TCP particles over 4 weeks compared with defects lled with (1). Percentage of new mineralized bone was comparable for (3) and (2) after 4 weeks of healing, but the control defects (3) showed a collapse in the defect area, whereas the space was maintained in defects lled with (1) and (2) at 4 weeks. The combination of rhPDGF-BB with b-TCP enhanced bone regeneration in contained periimplant bone defects during the early stages of bone healing. The findings of this research were presented at the University of Adelaide School of Dentistry Research Day 2010.
*Colgate Australian Clinical Dental Research Centre, School of Dentistry, The University of Adelaide, South Australia. Email: mark.bartold@adelaide.edu.au.
A vibrational spectroscopic analysis of the composition, microstructural arrangement and nanomechanical characteristics of hypomineralized enamel
S Brent, M Swain*
The aim of this study was to utilize Fourier-Transform Raman spectroscopy to investigate the enamel of 3 rst permanent molars affected by enamel hypomineralization and one rst permanent molar with clinically normal enamel. Three rst permanent molars with clinically discernable enamel hypomineralization and one clinically sound rst permanent molar were obtained following orthodontic extractions. Post-extraction teeth were cleaned and placed in a collection vessel containing deionized water and a small amount of thymol. The affected teeth were designated test teeth 13 and classied according to severity of the enamel hypomineralization exhibited. All four teeth were embedded in epoxy resin, sectioned bucco-lingually using a diamond saw and polished to 0.6 microns with aluminium polishing paste and stored in Hanks Solution at room temperature. Raman spectra were then obtained utilizing a Fourier-Transform Raman Spectrometer. The incident light was 1024 nm and scans were collected at a spectral resolution of 4 cm)1, laser power 900 mW and 2000 scans. The Raman spectra obtained were reconstructed and manipulated utilizing Grams software. The Raman spectra for the sound molar tooth showed no difference whether taken from buccal, lingual or occlussal surfaces. The characteristic features of a Raman spectrum for enamel were identied. Phosphate (PO43)) v1 peak at 960 cm)1; v2 phosphate peak at 430 cm)1; v4 phosphate at 590 cm)1; v3 phosphate at 1045 cm)1; a v1 carbonate band at 1072 cm)1 and a small peak at 3200 cm)1 within the region of -CH bond. Affected teeth show reversal of the prominence of the v1 carbonate band and v3 phosphate bands, with the carbonate band becoming larger and more dominant in the spectra obtained from the regions of hypomineralization, whilst the v3 phosphate bands become smaller and less distinct. All phosphate peak heights in spectra obtained from the hypomineralized enamel were smaller than those seen in spectra obtained from normal enamel regions of test teeth and the development of an additional peak within the -CH stretching region (at 2932 cm)1) was seen for test tooth 2 and 3. The extent of the changes seen in the spectra reected the degree of hypomineralization seen clinically. Utilizing Fourier-Transform Raman spectroscopy, it is possible to successfully analyse hypomineralized enamel in rst permanent molars. Hypomineralized enamel shows a higher degree of B type carbonate substitution and retains more organic matrix than enamel in sound teeth. Increased carbonate substitution and protein content within the visually normal cervical enamel of affected teeth was also demonstrated.
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*Biomaterials Department, Faculty of Dentistry, The University of Sydney, New South Wales. Email: suzannebrent@yahoo.com.au.
Abstracts
Regulation of reactionary dentine formation by odontoblasts in response to polymicrobial invasion of dentine matrix
N Charadram,* RM Farahani,* D Harty,* C Rathsam,* MV Swain, N Hunter*
Odontoblast synthesis of dentine proceeds through discrete but overlapping phases characterized by formation of a patterned organic matrix followed by remodelling and active mineralization. Microbial invasion of dentine in caries triggers an adaptive response by odontoblasts, culminating in formation of a structurally altered reactionary dentine, marked by biochemical and architectonic modications including diminished tubularity. The aim of this study was to dissect the pathophysiological cascade culminating in the formation of reactionary dentine with particular focus on the following: (1) analysis of the micro-structure of reactionary dentine with reference to the collagenous framework and associated NCPs; (2) the enzymatic machinery and associated regulatory proteins involved in reactionary dentineogenesis; and (3) molecular basis for the functional shift of odontoblasts in cariesinduced reactionary dentineogenesis. The pattern of the collagenous scaffold of reactionary dentine was analysed and compared to physiologic dentine. Further, the anchorage pattern of dentine sialoprotein (DSP) overlayed on collagen was assessed. Together with investigation of the associated enzymatic machinery responsible for synthesis of altered reactionary dentine, the data provide the rst evidence for radical adaptive reprogramming of odontoblasts to synthesize a modied mineralized matrix in a non-stochastic context. Statistical signicance was determined by comparison between carious (physiological and reactionary dentine) and healthy as a
*Institute of Dental Research, Westmead Millennium Institute and Westmead Centre for Oral Health, Westmead, New South Wales. Faculty of Dentistry, The University of Sydney, New South Wales. Email: ncha1431@uni.sydney.edu.au.
control using a two-tailed Students t test assuming equal variance. In the present study, a p-value < 0.05 was considered as statistically significant. Scanning electron microscopy of the collagen framework in reactionary dentine revealed a radically modied yet highly organized meshwork. Immuno-gold labelling demonstrated increased density of DSP in reactionary dentine. DSP contributes putative hydroxyapatite nucleation sites on the collagen scaffold. Analysis of extracted dentine protein indicated increased activity of matrix metalloproteinase-2 (MMP-2) in the reactionary zone referenced to physiologic dentine. Likewise, gene expression analysis of micro-dissected odontoblast layer revealed upregulation of mmp-2. Parallel upregulation of tissue inhibitor of MMP-2 (TIMP-2) and membrane type I metalloproteinase (MT1-MMP) was observed in response to caries. Next, modulation of odontoblastic dentinogenic enzyme repertoire was addressed. In the odontoblast layer expression of toll-like receptors was markedly altered in response to bacterial invasion. In carious teeth genes encoding TLR-4 and adaptor proteins TRAM and MAL TIRAP were upregulated. TLR-4 signalling mediated by binding of bacterial products has been linked to upregulation of MMP-2. Further, increased expression of genes encoding components of the TGF-b signalling pathway, namely SMAD2 and SMAD4, may explain the increased synthesis of collagen by odontoblasts in caries. These ndings indicate a radical reprogramming of odontoblasts in response to microbial invasion of dentine with resultant synthesis of a relatively atubular reactionary dentine. The findings of this research were presented at the IADR (ANZ Division) meeting, Kiama, Australia, 2010 and the ORCA meeting, Kaunas, Lithuania, 2011.
Immunolocalization of CPP-ACP in remineralized enamel subsurface lesions and high resolution imaging of the enamel crystallites
NJ Cochrane,* S Rubanov, F Cai,* Y Yuan,* JEA Palamara,* EC Reynolds*
Casein phosphopeptides-amorphous calcium (uoride) phosphate complexes (CPP-ACP (CASRN691364-495) and CPP-ACFP) have been shown to remineralize enamel subsurface lesions. Although much is known about these complexes, it is not known whether the CPP enter the lesion and what crystallographic changes are produced by these treatments.
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The aim of this study was to examine CPP-ACP CPP-ACFP remineralized enamel subsurface lesions to determine whether CPP penetrates into the interior and to visualize the treated enamel crystallites. Enamel subsurface lesions were produced in human enamel using a Carbopol demineralization buffer and divided into test and control halves. The test halves
Abstracts were remineralized in 1% (w v) CPP-ACP or CPPACFP solutions for 10 days. Immunolocalization of CPP within the lesion was conducted using a confocal microscope, primary anti-CPP antibody and a secondary FITC-conjugated goat anti-Rabbit IgG antibody. The enamel lesion was broken through the lesion to produce a cross-sectional surface of the lesion interior and imaged using an Axiovert 200 M inverted microscope (Carl Zeiss, Germany) tted with a Zeiss LSM 510 META Confocal scan head using the 458 477 488 nm Argon laser and a 10X plan apochromatic objective. Samples for transmission electron microscopy were prepared using a FEI Nova dual beam focused ion beam system and analysed using a FEI Tecnai F20 transmission electron microscope. The confocal images of the lesion interior showed intense red staining showing that the CPP had penetrated
*Oral Health Cooperative Research Centre, Melbourne Dental School, Bio21 Institute, The University of Melbourne, Victoria. Electron Microscopy Unit, Bio21 Institute, The University of Melbourne, Victoria. Email: n.cochrane@unimelb.edu.au.
into the interior of the remineralized subsurface lesion. The bright eld transmission electron micrographs showed that the demineralized enamel crystals contained numerous central defects whereas following remineralization, the central defects showed substantial occlusion. The diffraction patterns of this newly formed mineral were consistent with apatite. These results further the understanding of CPP-ACP CPP-ACFP remineralization as it has been shown that CPP can diffuse into the lesion and control crystal growth. Further, they show that the crystals are repaired by growth of existing crystallites. CPP was found to penetrate into enamel subsurface lesions and the demineralized enamel crystals were shown to be repaired by crystal growth of apatite. Published: Cochrane NJ, Cai F, Huq NL, Burrow MF, Reynolds EC. New approaches to enhanced remineralization of tooth enamel. J Dent Res 2010;89:11871197. The findings of this research were presented at the European Organisation for Caries Research meeting in Hungary, 2009.
Observational study of changes in lifestyle habits and behaviours of naval recruits and their caries risk
N Cusack,* G Mahoney,* L Walsh
It is hypothesized that as anecdotal changes in lifestyle have been observed in the recruit population, these have the potential to adversely affect the saliva and increase the risk of caries. The aim of the study was to conduct a prospective observational cohort study into the changes in caries risk assessment and lifestyle habits and behaviours of naval recruits over a six-month period. A prospective cohort study was conducted using 105 new recruits to the Royal Australian Navy. Salivary proling and plaque testing were used for risk assessment for dental caries and dental erosion. Additionally, participants were asked to complete a questionnaire relating to their lifestyle and behaviours in order to gather baseline data. Individual saliva and plaque parameters were tabulated to facilitate the comparison
*Centre of Military and Veterans Health, School of Population Health, The University of Queensland. School of Dentistry, The University of Queensland. Email: g.mahoney@uq.edu.au.
with the prospective parameter data using symmetry analysis to determine improvements or deteriorations in parameter scores. Analysis of the changes in the saliva and plaque parameters found that there were signicant changes in the saliva resting ow (p < 0.01) and consistency (p = 0.03) and in the plaque age (p < 0.01). There were no signicant changes to participants oral hygiene, alcohol consumption, medications, carbonated drinks and healthy food choices. There were few changes to measured putative risk factors over the six months and these changes could not account for the changes in the saliva and plaque parameters alone. The changes in caries risk assessment appear to be due to the improved hydration and efcacy of their oral hygiene. The findings of this research were presented at the FDI Section of Defence Forces Dental Services meeting, Singapore, September 2009.
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Abstracts
Identication of molecular markers of oral epithelial stem cells

A Dalley, J Saunus, R Dawson, Z Upton, CS Farah*
The objectives of this project were to isolate oral epithelial cells from oral biopsies and expand these on organotypic mucosal constructs, and characterize them by ow cytometry in terms of stem cell markers. Primary epithelial cell growth was undertaken using Keratinocyte-Serum Free Media (Invitrogen), PCT Oral 24 (Cell n Tech), or FAD media. Sterilized acellular human dermis was seeded with primary oral epithelial cells or normal oral epithelial cell lines (OKF6) and cultured at the air liquid interface for 22 and 44 days. Flow cytometry of both primary cells and cell lines was undertaken, with markers for CD44, p75 and ALDH1 activity (as determined by the Aldeuor assay). Primary epithelial cell growth was supported by both KSFM and PCT Oral 24, with KSFM preferentially selecting for holoclone-like colony growth (a trait indicative of stem cell isolation). Growth was not supported by FAD media. A 50% net success rate was achieved despite the primary cell yield being low from 2 mm biopsies. A notable improvement to primary epithelial cell yield was the adoption of a tissue explant outgrowth methodology as opposed to enzymatic digestion. Immunouorescent staining of the human oral mucosa constructs revealed an appropriate level of differentiation displaying cytokeratin 1, cytokeratin 13 and cytokeratin 19. Flow cytometric analysis of primary epithelial cells was hampered by very low cell numbers irrespective of whether the primary tissue was used or whether expansion occurred on the dermal substrate. Flow cytometric analysis of OKF6 cells demonstrated ALDH1 functional activity with co-expression of CD44 and p75NGF antigens. OKF6 cells exhibited 8.5% co-staining of CD44 and p75NGF in the ALDH1 positive fraction. Putative oral stem cells can be isolated and grown in culture, and express CD44, p75NGF and ALDH1. Further work is being undertaken to explore other novel stem cells markers and combinations utilizing the methodology demonstrated here. The findings of this research were presented at the 7th Annual Stem Cell Research and Therapeutics Conference, Boston, USA, May 2011.
*The University of Queensland School of Dentistry, UQ Centre for Clinical Research and Queensland University of Technology. Email: c.farah@uq.edu.au.
Inuence of implant abutment angulations on the fracture resistance of overlaying bre reinforced composite single crowns
A Ellakwa*
An in vitro study was performed to assess the effect of three implant abutment angulations and two types of fibres on the fracture resistance of overlaying fibre reinforced composite (FRC) Cermage single crowns. Three groups, coded A to C, with different implant abutment angulations (group A 0, group B 15 and group C 30 angulation) were used to construct 45 overlay composite restorations 15 Ceramage crowns for each angulation. For each group, another three subdivided groups coded 1 = 5 Ceramage crowns without bre reinforcement, 2 = 5 crowns with glass reinforcement and 3 = 5 crowns with UHMWPE reinforcement. All unreinforced reinforced crowns were constructed by one technician using Ceramage System. The nal restorations were prepared and stored in distilled water at mouth temperature (37 C) for 24 hours prior to testing. The restorations were cemented
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using Temp Bond. The load required to break each crown and the mode of failure were recorded. All the results obtained were statistically analysed by the ANOVA test (level of signicance p < 0.05). Tested crowns were examined using a stereo microscope at X40 and selected crowns (ve randomly selected from each group were further examined by scanning electron microscopy SEM) to reveal the composite-bre interface and to determine the fracture origin. Implant abutment angulations not signicantly (p > 0.05) reduced the fracture resistance of overlaying Ceramage single-crowns. The fracture loads of Ceramage crowns cemented onto abutment preparations with a 0 angulation were the lowest of the groups tested. The bre type did not signicantly (p > 0.05) affect the fracture resistance of the Ceramage singlecrowns. SEM showed that the origin of the fracture appeared to be located at the occlusal surfaces of the
Abstracts crowns and the crack propagation tended to radiate from the occlusal surface towards the gingival margin.
*Faculty of Dentistry, The University of Sydney. Email: ayman.ellakwa@sydney.edu.au.
The implant angulations not signicantly (p > 0.05) reduced the fracture resistance of overlaying Ceramage single-crowns. Reinforcing the crowns with two types of bres did not affect (p > 0.05) the fracture resistance of overlaying Ceramage single-crowns.
Correlation of serum and gingival crevicular uid cytokines in obese subjects

RA Fell,* KY Zee,* M Arora
Inammatory cytokines (IL-6, TNF-a) released by adipose tissue in obese subjects may enhance the local immune response in the periodontal tissues. It is unclear whether the cytokines released by adipose tissue into serum are correlated to the amount found in the gingival crevicular uid (GCF). The aim of this study was to investigate the correlation, in obese patients, between the GCF levels of IL-6 and TNF-a with the levels in serum. Twenty-six obese patients were recruited from obesity clinics at Westmead Hospital. Obesity was dened using a body mass index (BMI) 30 kg m2. Patients with conditions known to affect periodontal tissues, such as uncontrolled diabetes (HbA1c >8%) and smoking, were excluded. Serum and GCF samples were collected and analysed for IL-6 and TNF-a using commercially available ELISA kits. Within each subject GCF was collected from two healthy sites (n = 26 subjects) and two gingivitis sites, dened by bleeding on probing (n = 22 subjects). The GCF samples were collected using standardized periopaper strips (Proow). The levels of IL-6 and TNF-a in the GCF were compared and correlated with the levels found in serum. A BlandAltman analysis was used to determine the level of agreement between serum and GCF samples. TNF-a was detectable in 64% of serum samples but in only 52% and 36% of the GCF samples from healthy and gingivitis sites, respectively. In contrast, IL-6 was found in all serum but only 73% and 95% of samples taken from healthy and gingivitis sites, respectively. There was no signicant correlation between the TNF-a in serum and GCF samples from healthy (r = 0.27, p = 0.22) and gingivitis (r = )0.19, p = 0.40) sites. In contrast, positive correlations were found for IL-6 when the serum levels were compared with GCF samples from healthy (r = 0.48, p = 0.03) and gingivitis (r = 0.79, p = 0.0001) sites. The correlation and agreement was strongest for IL-6 between serum and gingivitis GCF samples. These results suggested a lack of correlation between the level of TNF-a in serum and GCF. This might be explained by the number of samples below the detection threshold. IL-6 from healthy GCF samples showed a low correlation and poor agreement. The high correlation between serum and GCF samples from gingivitis sites might indicate that higher serum IL-6 increases the GCF IL-6 in response to plaque accumulation. Alternatively, the presence of gingival inammation might lead to higher serum levels of IL-6. Overall, the results suggested that future studies examining the relationship between BMI and gingival cytokine levels should also examine the serum samples. The findings of this research were presented at ANZAP 2009 (Adelaide) and RACDS 2010 (Perth).
*Discipline of Periodontics, Faculty of Dentistry, The University of Sydney, New South Wales. Periodontics Unit, Westmead Centre for Oral Health, Westmead Hospital, Westmead, New South Wales. Evidence-Based Practice and Research Methods, Faculty of Dentistry, The University of Sydney, New South Wales. Institute of Dental Research, Westmead Centre for Oral Health, Westmead Hospital, New South Wales. Email: kyzee@usyd.edu.au.
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Abstracts
Structural and functional changes underlying orofacial pain

SM Gustin,* CC Peck, S Wilcox,* GM Murray, LA Henderson*
Trigeminal neuropathic pain (TNP) and temporomandibular disorders (TMD) are two chronic orofacial pain conditions that are thought to have fundamentally different aetiologies. While TMD appears to result from or appears to be initiated by, at least in part, the activation of peripheral nociceptors, TNP arises through damage to somatosensory afferents in the trigeminal nerve. The pain symptoms and management strategies also differ between these two chronic orofacial pain conditions. The hypotheses are that: (a) neuropathic orofacial pain but not painful TMD are associated with reductions in grey matter thalamic volume and changes in thalamic biochemistry; and (b) the changes in thalamic structure and biochemistry in neuropathic orofacial pain subjects are correlated with the intensity of ongoing pain. A better understanding of any differences in brain anatomy associated with different forms of chronic pain may provide insights as to the underlying mechanisms responsible for these changes in brain structure and ultimately lead to a better understanding of how to treat more effectively patients with various forms of chronic pain. The aim of this investigation was to use voxel based morphometry (VBM) and magnetic resonance spectroscopy (MRS) to determine if changes in regional brain anatomy and biochemistry occur in both conditions. Twenty-one subjects with TNP, 20 subjects with TMD, and 47 healthy controls without facial pain were recruited. VBM analysis was performed on T1-weighted anatomical images, functional MRI (fMRI) image volumes were acquired with blood oxygen level dependent contrast during brushing of the lower lip, and MRS was performed on the thalamus. Analysis employing voxel-based morphometry in the TMD patients did not identify any signicant changes in regional grey matter volume compared to the healthy control subjects. However, the grey matter volume of the TNP patients was reduced, in comparison with controls, in the primary somatosensory cortex, anterior insular, putamen, nucleus accumbens and the thalamus, whereas grey matter volume was increased in the posterior insula. Furthermore, the volume decrease within the thalamus was only seen in the TNP patients who were classied as having trigeminal neuropathy but not in those TNP patients who were classied with trigeminal neuralgia. The N-Acetylaspartate:Creatine ratio was also assessed and this is a biochemical marker of neural viability. There was a signicant reduction in N-Acetylaspartate:Creatine ratio in the region of thalamic volume loss in the trigeminal neuropathy patients but not in the TMD patient group. The results of this investigation demonstrate that neuropathic orofacial pain was associated with significant changes in grey matter volume but that these changes did not occur in patients with non-neuropathic orofacial pain. Furthermore, within the thalamus, the volume decrease was specically associated with one subgroup of neuropathic pain patients, i.e. the thalamic volume was decreased in those patients with trigeminal neuropathy but not trigeminal neuralgia. Additionally, in the region of thalamic volume loss of the trigeminal neuropathy patients, a signicant reduction in neural viability was observed. In direct contrast, there was no signicant change in brain grey matter volume or in thalamic neural viability in the patients with chronic non-neuropathic orofacial pain (i.e. TMD). The data suggest that the pathogenesis underlying neuropathic and non-neuropathic pain conditions are fundamentally different and that neuropathic pain conditions that result from peripheral injuries may be generated and or maintained by structural changes in regions such as the thalamus. Published: Gustin SM, Peck CC, Wilcox SL, Nash PG, Murray GM, Henderson LA. Different pain, different brain: thalamic anatomy in neuropathic and nonneuropathic chronic pain syndromes. J Neurosci 2011;31: 59565964. The findings of this research were presented at the International Association for the Study of Pain Congress, August 2010.
*Department of Anatomy and Histology, The University of Sydney, New South Wales. Faculty of Dentistry, Jaw Function and Orofacial Pain Research Unit, The University of Sydney, Westmead Hospital, Westmead, New South Wales. Email: greg.murray@sydney.edu.au.
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Abstracts
The effect of titanium surface topography and chemistry on macrophage and monocyte gene expression
SM Hamlet, S Ivanovski*
It is a well established clinical nding that osseointegration occurs at different rates and degrees around implants with different surface texture, with rough surfaces having superior properties compared to smooth or machined surface implants. More recently, nanoscale surface modication of titanium dental implants with calcium phosphate (CaP) has been shown to achieve superior bone wound healing and osseointegration compared with smooth or microrough surfaces. Wound healing and osseointegration are complex processes involving the temporal cooperation of many factors. Macrophages play an important role in the healing response as their actions occur prior to the arrival of undifferentiated mesenchymal cells to the wound site and therefore may be responsible for driving the response of these progenitor cells, which ultimately differentiate into osteoblasts. As the recruitment of osseoinductive precursors to the wound site is facilitated by the action of cytokines released from macrophages, this study examined whether changes in macrophage cytokine gene expression from cells cultured on conventional microrough or nanoscale crystalline CaP-modied microrough titanium surfaces, may inuence downstream events in bone wound healing and osseointegration. Titanium discs with microrough (Osseotite Biomet 3i, Palm Beach Gardens, FL, USA) or nanoscale CaPmodied (NanoTite, Biomet 3i, Palm Beach Gardens, FL, USA) microrough surfaces were used in the study. The surface topography and composition of each surface was conrmed by scanning electron microscopy and X-ray photoelectron spectroscopy. The proliferation and attachment of murine macrophage-like RAW 264.7 cells was assessed following 24 and 72 hours culture on the titanium surfaces. Quantitative analysis of mRNA expression of inammatory cytokines and chemokines was also determined at these times using a PCR array. A commercially available multi-analyte ELISA array was subsequently used to determine whether observed changes in cytokine gene expression were translated into detectable differences in the level of secreted cytokine by the RAW 264.7 cells. Whilst no signicant difference in the attachment or proliferation of RAW 264.7 cells on the microrough or CaP-modied titanium surfaces was observed, activation of RAW 264.7 cells by the nanoscale CaP modied surface resulted in a marked down-regulation of the pro-inammatory macrophage genotype compared to the conventional microrough surface. Gene expression of the pro-inammatory cytokines TNFa, IL-1b, IL-1f8, IFNc, chemokines CCL2, CCL3, CCL5, CCL8, CCL11, CCL12 and chemokine receptors CCr1 and XCr1 were all signicantly down-regulated. This downregulation was subsequently conrmed at the protein level for TNFa and CCL5. This study demonstrated that surface topography and chemical composition of non-phagocytosable titanium surfaces can affect macrophage function. This in vitro observational study suggests a novel mechanism whereby down-regulation of pro-inflammatory cytokine gene expression may facilitate the enhanced bone wound healing and osseointegration observed clinically with nanoscale CaP-modified implant surfaces. Published: Hamlet S, Ivanovski S. Inflammatory cytokine response to titanium chemical composition and nanoscale calcium phosphate surface modification. Acta Biomater 2011;7:23452353. The findings of this research were presented at the IADR meeting, Barcelona, Spain, 2010.
*School of Dentistry and Oral Health, Grifth University, Gold Coast Campus, Queensland. Email: s.ivanovski@grifth.edu.au.
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Abstracts
The effect of growth factors PDGF and IGF on cementum-associated gene expression by periodontal cells
H Hannawi, SM Hamlet, S Ivanovski*
Regenerative periodontal therapy, which aims to restore the lost or injured periodontal apparatus, is a complex process requiring the formation of both hard and soft structures, such as cementum, alveolar bone and periodontal ligament. A critical step in this process is the formation of new cementum which subsequently facilitates the attachment of the tooth to the bone via the bres of the periodontal ligament. The periodontal ligament has been demonstrated to contain progenitor cells capable of differentiating into the required cell types for regeneration. Polypeptide growth factors may promote the wound healing process, by promoting the proliferation and or differentiation of progenitor cells capable of facilitating periodontal regeneration. We thus sought to determine the ability of two growth factors, PDGF and IGF, to promote the expression of novel cementum-associated genes; cementum attachment protein (CAP), cementum protein-1 (CEMP-1) and F-spondin (SPON) in cells derived from the periodontium. Primary periodontal ligament broblasts (PLF), osteoblasts (OB) and gingival broblasts (GF) were each explanted from three patients and cultured in vitro in the presence of the growth factors IGF and PDGF either alone or in combination for six weeks. Real-time PCR was subsequently utilized to quantify the expression of the cementum-associated genes; CAP, CEMP-1 and F-SPON. Some heterogeneity in the relative expression of the cementum-associated genes within each cell type in response to the growth factors was observed. Despite this, PLFs were clearly shown to be the most responsive cell type with increased expression of both CEMP-1 and F-SPON in response to IGF and PDGF, both alone and in combination. PLFs were also the only cell type to demonstrate any upregulation of CAP expression with IGF + PDGF. OB and GF expression proles were similar with upregulation of CEMP-1 and F-SPON expression in response to PDGF alone and in combination with IGF. Overall, F-SPON expression was generally upregulated with all growth factor treatments while CEMP-1 expression was only upregulated by PDGF or combined IGF + PDGF treatment. Temporally, the observed upregulation of any of the cementum-associated genes in any cell type was only seen after 6 weeks of cell culture. The growth factors used in this study were shown to have the potential to inuence the differentiation of cells derived from the periodontium by upregulating the expression of cementum-associated genes although the differences observed were not statistically signicant due to the small numbers of subjects in the study and the heterogeneity found within the same cell types. Combined IGF + PDGF treatment had the greatest potential for upregulating PLF differentiation along a cementum-producing cell phenotype. Interestingly, the expression of cementum-associated genes by GFs after prolonged treatment suggests these cells may also contain undifferentiated progenitor cells. The findings of this research were presented at the IADR meeting, Barcelona, Spain, 2010.
Establishing a mouse model to investigate the relationship between periodontal disease and inammatory arthritis
DR Haynes, PM Bartold, V Marino, MD Cantley*
Previous studies have suggested a possible relationship between periodontal disease (PD) and rheumatoid arthritis (RA) with the two having many remarkable similarities. Higher incidences of alveolar bone loss have been observed in patients with RA and patients with PD have been reported to be at a greater risk of having RA. The aim of this study was to investigate the relationship between these chronic inammatory
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diseases by developing a novel mouse model in which PD is initially induced using the known periodontal pathogen Porphyromonas gingivalis (P. gingivalis) followed by induction of inflammatory arthritis using a collagen antibody induced arthritis (CAIA) model. The method of inducing arthritis in mice using CAIA was initially established, in which female balb c mice received a 1.5 mg tail vein injection of a monoclonal
Abstracts antibody against type II collagen followed three days later by an intraperiotoneal injection of E. Coli LPS (5 lg). Periodontitis was induced in mice by oral gavage with P. gingivalis bacteria. Both experimental arthritis (EA) and PD were induced in one group of mice, whilst PD and EA were induced alone for comparison. Visual changes in paw swelling were assessed to determine clinical development of EA. Alveolar bone and joint changes were assessed using micro-CT, histological analyses and immunohistochemistry. Serum levels of C-reactive protein were used to monitor systemic inflammation. It was found that mice with pre-existing periodontitis developed more severe arthritis as it developed at a faster rate. This was evidenced by signicantly higher paw scores on days 6, 8, 9 and 10 through macroscopic assessment and also microscopically by histological assessment of H&E stained sections. There were also signicantly higher CRP levels in the PD and EA group. Micro CT analysis demonstrated no signicant differences in bone destruction of the radio carpal joints with a 14% decrease in bone volume during the arthritis stage compared to a 18% normal increase in control mice indicating that although there are signicant differences in inammation during this time the extent of bone destruction is similar. There was however more
*Discipline of Anatomy and Pathology, School of Medical Sciences, The University of Adelaide, South Australia. Email: david.haynes@adelaide.edu.au.
intense staining for RANKL in the EA and PD group compared to EA alone. Mice with periodontitis only also showed evidence of bone effects within the radio carpal joint as evidenced by CT analysis and by more intense RANKL staining in comparison to controls. There was also evidence of alveolar bone loss in mice with EA alone as assessed by CT analysis and also by mild RANKL staining the gingival tissues of EA only group which was more than control. We have effectively established a mouse model in which we can induce both EA and PD in the same animal to investigate the potential relationship between the two. Results indicate that there is a bidirectional relationship between inammatory arthritis and PD which needs further investigation. This model could now be effectively used to investigate the mechanisms of this relationship and to test the effects of potential treatments. Published: Cantley MD, Haynes DR, Marino V, Bartold PM. Pre-existing periodontitis exacerbates experimental arthritis in a mouse model. J Clin Periodontol 2011;38:532541. The findings of this research were presented at the University of Adelaide School of Dentistry Research Day 2010 and at the American Society for Bone and Mineral Research (ASBMR) Annual Meeting 2011 by Melissa Cantley where it received a Presidents Poster Competition Award.
Vibrational spectroscopic investigation of the compositional, microstructural and nanomechanical characteristics of enamel
LH He, MV Swain*
As the outer cover of the teeth, human enamel has superb mechanical behaviours against fatigue and crack propagation. To understand the compositional, microstructural and nanomechanical characteristics of the natural material will help the development of new composite materials. The aim of the project was to investigate the compositional characteristics of human enamel in respect to its hierarchial microstructure by vibrational spectroscopes and thus provide further evidence to explain the superb mechanical behaviour of enamel as load bearing hard tissue. Healthy premolar teeth extracted for orthodontic reasons were embedded with a cold curing epoxy resin (Epox, Struers, Copenhagen, Denmark) and cut in differentd directions, cross-sectionally or tangentially, using a low speed saw (Isomet, Buehler Ltd., Lake
Bluff, USA) under water irrigation, in which the enamel rod units were either parallel or perpendicular to the testing surface. All specimens were polished (RotoPol22, Struers, Copenhagen, Denmark) to 0.4 mm alumina polishing paste. Nanoindentations (UMIS 2000, CSIRO, Australia) were made on the surface of prepared specimens. Micro-Raman spectroscopy (Renishaw Raman InVia Reex Microscope (Renishaw plc., Wotton-under-Edge, UK) and scanning electron microscope (SEM) (XL-30, Phillips. Netherland) were also used to examine the specimens. Cross-sectionally, inner enamel (close to enameldentine junction) has lower elastic modulus and hardness but higher creep and stress redistribution abilities than the outer counterpart, which is related to the gradual compositional change through the enamel and is reected by Raman-spectroscopy. Inner enamel has
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Abstracts higher C-H stretch peak at the Raman shin of 2800 3000 cm)1 than the outer counterpart, which is an indication of the organic content of the specimen. Tangentially, the residual micro stresses within and surrounding the indentation impressions were monitored by mapping the position of the v1 (PO4) band at the Raman shift of 960 cm)1 of (crystalline) hydroxyapatite. The Raman maps coincide well with the optical micrographs and SEMs of the specimens. Repeated measurement illustrate that residual stress within enamel had time-dependent reduction. Moreover, in comparison with sintered hydroxyapatite disk, enamel has better ability to redistribute stress and thus protect itself from excessive plastic deformation and fracture. Micro-Raman spectroscopy provides a novel high resolution and non-destructive method to explore the role of microstructure on the residual stress distribution within natural biocomposites. Small amounts of protein remnants in enamel play an important role in regulating the mechanical responses of the natural material. Enamel can be regarded as a functionally graded natural biocomposite, which will require special attention using numerical analysis to fully appreciate the consequences of such a structure for the mechanical behaviour of teeth and restorations placed therein. In short, natures smart design provides us with interesting indications for new composite development and graded materials coatings design. Published: He LH, Carter EA, Swain MV. Characterization of nanoindentation induced residual stresses in human enamel by micro-Raman spectroscopy. Anal Bioanal Chem 2007;389:11851192. He LH, Carter EA, Swain MV. Enamel a functionally graded natural coating. J Dent 2009;37:596603. The findings of this study were presented at the International Association for Dental Research meeting, Toronto, Canada, July 2008.
*Biomaterials Science Research Unit, Faculty of Dentistry, The University of Sydney, Sydney Dental Hospital, Surry Hills, New South Wales. Email: mswain@mail.usyd.edu.au.
An evaluation of alternate splinting adhesives in the management of dental trauma

J-Y Hu, GS Heithersay*
A exible splint which is generally recommended in the management of luxated, root fractured or replanted teeth usually consists of ne stainless steel wire, nylon line or other exible materials attached with an adhesive traditionally composite resin which has been easy to apply, but time-consuming and difcult to remove from the etched enamel surface, with an inherent danger of enamel damage or retained residual composite material. The objective of this study was to investigate alternative adhesive materials which could be not only easy to apply, but more signicantly, easy to remove without causing any damage to the enamel surfaces of the splinted teeth. Freshly extracted premolars, selected with intact buccal enamel, no caries, restorations, defects or visible evidence of abnormal cracking were stored in a solution of 0.1% thymol at room temperature prior to a series of tests designed to develop a standardized method of testing conventional and alternative adhesives and to make selections for the nal phase of the study. The test materials chosen for the nal assessments were composite resin (Filtek Supreme XT), Luxatemp Solar (DMG), Fiber Splint with Adper Bond 2, Triad Provisional Material VLC, Fiji-Ortho 11LC and Maxcem Elite. A total of 180 teeth were divided randomly into six groups of 10 test specimens, each containing three extracted teeth. The teeth were mounted in acrylic resin
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after their roots had been covered with a 0.10 mm thick layer of autopolymerizing silicone (Aquasil, Dentsply) to simulate a periodontal ligament. A template was then constructed using laboratory putty (Coltene Whaledent) to allow the application of the test materials to attach either nylon line or Fiber Splint to a standardized and dened area on the enamel surface of the test teeth. Prior to the application of the test adhesive, a stereomicroscopic analysis of a dened area of the enamel surface of each test specimen was made to form a baseline for post-removal analyses. The adhesives were then applied to the test widows, noting the time and ease of application for each material. The mounted teeth were then subjected to a 24-hour thermocycling and a functional wear test which was followed immediately by a shear-bond test using a Hounseld universal testing machine. The dened enamel surfaces were then examined stereomicroscopically to assess surface roughness, enamel damage (Enamel Damage Index) and the presence of residual adhesive material (Adhesive Remnant Index). The gathered data were then subjected to power study calculations and pairwise test for signicance. Triad, Fiber-splint and Fuji Ortho had shear bond strength values that were in the low to intermediate range, indicating that these materials were suitable for short 2011 Australian Dental Association
Abstracts term splinting periods (i.e. 710 days), while composite resin, Luxatemp and Maxcem which displayed the greatest bond strength would appear to be more suitable for long-term splinting. Triad produced the smoothest enamel surface upon splint removal, followed by Fuji Ortho and Luxatemp. Composite resin and Maxcem were intermediate with moderate enamel roughness while Fiber-splint with Apner bond 2 produced signicantly the roughest enamel surface of all materials. In respect to the Adhesive Remnant Index (ARI), Triad was statistically superior to the other adhesive materials. However, Fuji Ortho also showed minimal to no adhesive on the tooth surfaces after debonding. Luxatemp, Maxcem Elite and Composite resin were all relatively similar in their ARI scores where remnants of residual resin were found on the enamel surface. When assessing enamel damage, the largest frequency of enamel fracture occurred within the adhesive systems that displayed the greatest bond strengths; Maxcem Elite, Luxatemp, Composite resin, Fiber-splint with Apner Bond 2, in descending order. Triad and Fuji-Ortho were
*School of Dentistry, The University of Adelaide, South Australia. Email: geoffrey.heithersay@adelaide.edu.au.
the only adhesive systems that produced insignicant or no damage to the enamel surface upon debonding. Based on this research into alternative adhesives for splinting, Triad showed excellent properties for temporary splinting purposes because it provided adequate bond strength and a clear separation on debonding from the enamel surface. However, there are currently difculties in its delivery and ease of application. FujiOrtho11LC also showed desirable properties as an adhesive splinting material, particularly with its comparative ease of delivery and application, and no pretreatment enamel etching. Both these materials are considered to be clinically advantageous because the enamel is restored as closely as possible to its pretreatment condition, and minimal or no polishing of the tooth surface is required. This contrasts with the other adhesive materials tested where the presence of residual resin on the tooth surface can result in severe adverse effects on enamel surfaces, both when debonding and polishing the enamel surface. The findings of this research were presented at the Australian and New Zealand Academy of Endodontists meeting, Brisbane, 2011 and the American Association of Endodontists meeting, San Antonio, 2011.
Neurophysiological changes underlying orofacial pain

Z Karami, T Salameh, C Peck*
Chronic pain in the oral and facial region occurs in 20% of Australians and can be extremely debilitating. The mechanisms for this pain are unknown. This study used nerve testing equipment to explore possible contributing factors to orofacial pain. The aim of this study was to investigate if orofacial pain is associated with changes in thermal sensitivity thresholds. Twenty-ve adult subjects (age range: 30 to 75 years, mean age: 58 years) participated in this study. The subjects were diagnosed (from established criteria of the RDC TMD and the Neuropathic Pain Liverpool criteria) with temporomandibular disorders, trigeminal neuralgia or post-traumatic neuropathic pain. Thermal sensitivity was assessed between the painful and the non-painful sides of the face in each subject with a neuro-sensory analyser system (Medoc Advanced Medical Systems, Ramat Yishay, Israel). A mini-thermode was used to determine the thermal thresholds for sensitivity and pain. Statistical differences between the painful side and the non-painful side
*Faculty of Dentistry, Jaw Function and Orofacial Pain Research Unit, The University of Sydney, Westmead Hospital, Westmead, New South Wales. Email: greg.murray@sydney.edu.au.
of a subject were tested by paired t-tests with SPSS version 17. The mean threshold temperature for thermal sensitivity and pain were lower on the painful sides of the mouth compared to the non-painful sides. There were statistically signicant differences in thermal pain thresholds between the painful and non-painful sides of the face. Heat sensitivity was signicantly lower on the painful side (mean difference of 1.7 C; p = 0.006) and cold sensitivity was signicantly increased in the painful side (mean difference of 2.5 C; p = 0.000). There were no differences in thermal-induced sensitivity between both sides of the face. Our ndings show that orofacial pain is associated with thermal sensitivity changes and we propose that these changes are induced by the orofacial pain. Central and peripheral neurophysiological changes (sensitization and neuroplasticity) have been demonstrated in patients with chronic pain. Future work will focus on characterizing differences in quantitative sensory testing between sub-groups of orofacial pain subjects. The findings of this research were presented at the International Association for Dental Research meeting 2011, San Diego, USA.
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Abstracts
Association of strong premature contacts and tooth mobility using T-Scan

S Knoepel,* K Nguyen, M Bickel, J Rincon*
This study aimed to use the Periotest and the T-Scan as instruments to determine the association of strong premature contacts comparing mobile and non-mobile teeth at initial examination, after occlusal adjustment of the mobile tooth, and 4 and 8 weeks later. A second aim was to determine if there was a reduction in tooth mobility after the alleviation of the strong contact. Patients diagnosed with teeth with traumatic occlusion were examined from the periodontal clinics. The Periotest was used to measure the mobility of the teeth and the T-Scan was used to identify the contacts and the associated occlusal load. A relative force contact index (RFCI) was created. The association between RFCI and tooth mobility was assessed at the baseline examination, after the experimental teeth were selectively adjusted and at reviews after 4 and 8 weeks.
*Department of Periodontics, School of Dentistry, The University of Western Australia. Department of Prosthodontics, School of Dentistry, The University of Queensland. Email: julio.rincon@uwa.edu.au.
The results showed that the T-Scan device was useful in identifying and alleviating strong occlusal contacts in teeth with increased mobility. Tooth mobility measured by the Periotest (PTV) was signicantly higher for the experimental teeth than the control teeth at baseline. Some patients showed a decrease in tooth mobility using the Periotest after contact alleviation. The RFCI from the T-Scan showed a signicant difference in the premature contact force comparing mobile and non-mobile teeth at initial assessment. After adjustment the mean RFCI decreased for mobile teeth and some non-mobile teeth had an increase. At 4 and 8 weeks there was a rebound trend for mobile teeth. There was no correlation found between rebound of mobile teeth and rebound in RFCI. The calculated RFCI showed an association between strong occlusal contact and increased tooth mobility. The electronic device T-Scan was a useful clinical tool to identify and assisted to alleviate premature occlusal contacts. After 4 weeks a signicant reduction in tooth mobility (PTV) was demonstrated but some teeth experienced a rebound in tooth mobility at week 8.
Porphyromonas gingivalis GroEL and atherosclerosis in a mouse model

SJ Leishman,* HL Do,* JE Palmer,* GJ Seymour, KW Beagley, PJ Ford*
Cross-reactivity between Porphyromonas gingivalis GroEL and endogenous heat-shock-protein-60 (HSP60) has been associated with atherogenesis. The objective of this study was to examine the effect of the immune response to P. gingivalis GroEL on atherosclerotic lesion development in a mouse model. Ten-week-old male apolipoprotein E-decient () )) mice received weekly intraperitoneal immunizations of recombinant P. gingivalis GroEL or incomplete Freunds Adjuvant (IFA; vehicle) over a period of 11 or 27 weeks. Atherosclerotic lesion size in the proximal aorta was quantified by histomorphometric analysis. Serum levels of antibodies to P. gingivalis GroEL and mouse HSP60 (IgG, IgG1 and IgG2a) as well as levels of IL-17 were quantified using ELISA. Over the time course of the study mice immunized with P. gingivalis GroEL tended to have an acceler*The University of Queensland, Brisbane, Queensland, Australia. The University of Otago, Dunedin, New Zealand. Queensland University of Technology, Brisbane, Queensland, Australia. Email: s.leishman@uq.edu.au.
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ated rate of atherosclerotic lesion development compared with control mice. All mice immunized with P. gingivalis GroEL demonstrated elevated IgG titres of anti-P. gingivalis GroEL and anti-mouse HSP60 antibodies compared with control mice. IgG1 and IgG2a subclasses of anti-P. gingivalis GroEL and anti-mouse HSP60 antibodies were induced in immunized mice, however IgG1 responses were dominant indicating a dominant Th2 immune response. No differences were seen in IL-17 levels among the groups suggesting that Th17 immune responses were not induced by immunization with P. gingivalis GroEL. The results show that immunization with P. gingivalis GroEL resulted in accelerated atherosclerosis in this mouse model which, in this case, was associated with a dominant Th2 immune response. This supports the hypothesis that anti-GroEL immune responses are involved in atherogenesis. The findings of this research were presented at the 88th General Session of the International Association for Dental Research, Barcelona, Spain, July 2010.
Abstracts
Mucosal inoculation of Porphyromonas gingivalis and atherosclerosis

SJ Leishman,* GJ Seymour, PJ Ford*
Epidemiological studies and mouse models have provided evidence to support the role of infections, including those caused by periodontal pathogens, in cardiovascular disease. This study used the apolipoprotein E-decient () )) mouse model to examine the immune response and atherosclerosis development following oral inoculation with Porphyromonas gingivalis. It was hypothesized that repeated oral mucosal exposure to this bacterium would lead to immune recognition and consequently a systemic immune response. We had previously demonstrated that intraperitoneal injections of this organism in apoE deficient mice resulted in a systemic immune response and enhanced atherosclerosis. The current experimental design was set up to more closely mimic the route of exposure of P. gingivalis in periodontal disease in humans. Six-week-old male apoE () )) mice received daily oral inoculations of P. gingivalis ATCC33277 (3 1010 organisms 100 ll) in 2% carboxymethylcellulose PBS or 2% carboxymethylcellulose PBS over a period of three weeks. Mice were subsequently sacriced at 24 weeks of age. Atherosclerotic lesion size in the proximal aorta was quantied by histomorphometric analysis. Serum levels of antibodies to P. gingivalis were quantified using an enzyme linked immunosorbent assay (ELISA) technique. There was no signicant difference in atherosclerotic lesion development between mice inoculated with P. gingivalis and those inoculated with 2% carboxymethylcellulose PBS. Similarly, there was no significant difference in antibody response to P. gingivalis between control and treatment mice. Whilst other studies have demonstrated that oral inoculation with P. gingivalis enhances atherosclerosis, the protocol used by the current study did not result in a systemic immune response or changes in atherosclerosis development. A number of variables may explain the observed inconsistencies between studies including sample size, bacterial strain, and inoculation dose and regime. Further studies are now required to elucidate the events that occur as a result of repeated mucosal exposure to this organism and to combinations of organisms in the form of biofilms. While this route of exposure poses complex questions and experimental challenges, including the role of mucosal tolerance, it has much higher physiologic importance than studies utilizing intravenous or intraperitoneal routes of exposure.
*The University of Queensland, Brisbane, Queensland, Australia. The University of Otago, Dunedin, New Zealand. Email: s.leishman@uq.edu.au.
Predicting unscheduled dental visits for members of the military

G Mahoney,* G Slade, S Kitchener,* A Mamun
Whether an Australian serviceman is dentally t for deployment is determined by the dental tness standard. Recent operations have found that this standard is not predictive of whether or not a member is likely to have a dental problem. The aim of this study was to investigate a set of variables that can more accurately predict a servicemans odds of becoming an unscheduled dental visit (UDV) than the present system. A prospective cohort study was conducted on 875 Australian service personnel. At enrolment clinical details were recorded and a questionnaire completed
*Centre for Military and Veterans Health, School of Population Health, The University of Queensland, Australia. Department of Dental Ecology, The University of North Carolina, USA. School of Population Health, The University of Queensland, Australia. Email: g.mahoney@uq.edu.au.
on demographic and lifestyles. The data were rst analysed for associations between each risk factor and whether or not subjects had a UDV in the following 12 months. Modelling was then carried out using the risk factors which yielded moderate associations. Prediction models were most accurate when created for two cohorts; the dentally t and dentally unt. The dentally t model has a sensitivity = 82.61% and a specicity = 70.23%, while the dentally unt model has a sensitivity = 62.86% and specicity = 77.71% and both models are better than the present system. Our proposed model can more accurately predict an ADF members likelihood of becoming a UDV than the present Denclas system. The findings of this research were presented at the IADR meeting in San Diego, USA, March 2011.
S19
Abstracts
Inuence of Thl cytokine deciency on osteogenic and angiogenic markers involved in bone healing
MA Matias, CS Farah*
Calvarial wounds in IL-12 23p40 knockout (KO) mice were used to investigate the potential role of IL12 23p40, a component of IL-l2 and IL-23, in bone regeneration. Two millimetre calvarial wounds were created in IL-12 23p40 KO mice and their respective C57BL6 J controls. The degree of bone regeneration was examined using several parameters including: morphometrics, densitometry, histology and chromogenic (bone stain). Wounds were harvested on day 0, 2, 8 and 21 weeks. Bone regeneration occurred unimpeded in both test and control groups. Morphometric analysis demonstrated bone defects healed in an irregular pattern and bony-ll was not homogenous. The rate of regeneration in IL-12 23p40 KO mice and C57BL6 J controls were similar. However, the trend for early healing and smaller defect size was seen in the KO mice. Villanueva osteochrome bone stain conrmed that at 21 weeks, all defects in test and control groups were covered with osteoid material. Further investigations are required to conrm the early bone healing trends seen in IL-12 23p40 KO mice. Potentially, the effects of IL-12 and IL-23 should be further dened to explore the regulatory roles of these cytokines in bone regeneration and elucidate their role in bony pathological conditions such as periodontal disease.
*The University of Queensland School of Dentistry and UQ Centre for Clinical Research (UQCCR). Email: c.farah@uq.edu.au.
Localization of MMP-9 in oral soft tissue wounds in the p40 knockout mice
MA Matias, CS Farah*
To further elucidate mechanisms underlying the accelerated healing phenotype of oral mucosal wounds in the IL-12 IL-23p40) ) mice, rstly we investigated the role of MMP-9 by localizing MMP-9 and TIMP-1 in wounds of IL-12 IL-23p40) ) and wildtype mice. Secondly, we investigated differences in broblast population in IL-12 IL-23p40) ) and wildtype control using an in vitro scratch wound assay. IL-12 IL-23p40) ) and C57BL 6J control mice were wounded on the dorsal surface of the tongue using a 2 mm biopsy punch. Harvested wounds (n = 3) were prepared for immunostaining at time points 0 (unwounded), 1 and 4 days after wounding. Serial sections from the central wound area were used to stain for MMP-9 and TIMP-l. Primary broblast cell cultures were grown from IL-12 IL-23p40) ) and C57BL 6J mice. Cells were seeded to conuence and single scratch wounds generated. Images were acquired from time 0 hours and every 2 to 3 hours for a minimum of 48 hours to assess the degree of wound closure. The IncuCyteTM scratch wound software was used to assess wound closure. Immunohistochemistry conrmed the presence of MMP-9 in healing wounds in both strains of mice. A more prominent presence of MMP-9 at day 1 in the knockout mice compared to wildtype counterparts was observed. MMP-9 appeared to be markedly associated with neutrophils, blood vessels and interstitially at the base of the wound bed. A similar pattern of TIMP-l staining was observed but the intensity of staining was less pronounced. The in vitro wound assay demonstrated innate differences in the behaviour of primary fibroblast cultures between the strains of mice. The primary fibroblast cultures from the knockout mice achieved wound confluence significantly earlier than the C57BL 6J fibroblasts (n = 8). The results from Logrank (Mantel-Cox) Test showed a significant difference (p value < 0.0002) between the two groups. The localization and spatial distribution of MMP-9 in the healing wounds of IL-12 IL-23p40) ) mice potentially inuence several aspects of wound repair and may account for the accelerated healing seen in these mutant mice. Differences in the broblast population may also account for the faster healing seen in the knockout mice. More experiments are required to elucidate the exact mechanism underlying this accelerated healing phenotype.
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Abstracts
The effects of acidic beverages on the demineralization rate of incipient enamel lesions: a pilot study
JM McIntyre, I White, J Kibble*
One possible contributing factor to the current increased caries incidence in young adult Australians could be the very large increase in acidic beverages consumed by this age group over the last three decades. It is possible that this rapidly progressing caries can result from a super-imposition of an erosive demineralization often over an already advancing chronic subsurface caries lesion. The aim of this project was to determine whether the rate of erosive mineral loss was greater in already carious enamel compared with that in intact enamel following exposure to two common acidic beverages. Diet Solo and Diet Coco Cola [Diet Coke], representing citric acid and phosphoric acid containing drinks, were the test beverages. Articial caries demineralizing solution was also used to generate articial caries and as a negative control. The crowns of 12 intact extracted human molar teeth, collected with appropriate ethical approval, were sectioned vertically mesio-distally to provide labial (test) and lingual (control) surfaces of enamel. These were painted with nail varnish, leaving a window of exposed enamel 10 2 mm in size. The 12 buccal sections were placed in 40 mL of an articial caries demineralizing solution at 37 C, static, to generate four each 7-, 14- and 21-day carious lesions. One specimen each of 7-, 14- and 21-day caries was sectioned to ensure a constant depth of caries was present across the 10 mm length of the carious lesions. Prior to experimentation, a quarter (2.5 mm) of the window of three 7-, 14- and 21-day specimens, and their intact pairs, were painted with nail varnish to provide a baseline control, the margin marked with a scalpel blade score. The samples were then placed in 40 mL of each acidic beverage separately and subjected to slow rotation in a rotary incubator at 37 C for 14 hours, removed and a further 2.5 mm painted and scored. This was repeated for a further 14 and then 28 hours, resulting in 14-, 28- and 56-hour exposure times. Each test (pre-carious) and control (intact) section was rinsed in acetone to remove nail varnish and vertical sectioned through the lesion centre. The cut sections were then viewed using a Zeiss Stereoscopic microscope at x320 and the images directed to a computer using a Pro-MicroScan digital camera (ProSciTech, Kirwan, Australia). As caries had also developed under the eroded surfaces in the intact
*School of Dentistry, The University of Adelaide, South Australia. Email: john.mcintyre@adelaide.edu.au.
enamel, all carious lesion depths were measured as well as erosion depths, using Scope Photo software (ProSciTech, Kirwan, Australia). Electron Probe Micro-Analysis (EPMA) was also carried out on all carious lesions using a Cameca SX51 (Cameca, Corbevoir, France) microanalyser. Exposure to Diet Solo in most precarious specimens resulted in a doubling of the depths of erosive demineralization compared with those on intact enamel (14 > 22 > 53 lm compared with 69 > 84 > 108 lm for 21-day caries). The rate of erosion progressed more slowly with Diet Coke, though by 56 hours, depth of erosion on precarious enamel was substantially greater than on intact enamel (123 lm compared with 75 lm). However, the depth of erosion of intact enamel exposed to each acidic beverages did not differ greatly by 56 hours (mean of 66.6 for Diet Solo and 70.4 for Diet Coke). No erosion resulted from a similar exposure period to articial caries buffers. The depth of caries in intact enamel, and its increase in pre-carious enamel from up to 56 hours of exposure to Diet Solo were relatively similar (mean of 171 lm for intact enamel and 190 lm for all precarious). The increases were somewhat less for Diet Coke, particularly for precarious enamel (mean of 120 lm for intact enamel and 48 lm for precarious). Caries progression following continued challenge to ACD solution remained relatively normal for articial caries generation (a mean of 94 lm by 56 hours for intact and 44, 90 and 130 lm respectively for 7-, 14- and 21-day precarious lesions.) EPMA showed that the calcium and phosphorus proles in weight percent for carious lesions did not vary greatly, whether generated initially by the acidic beverages or from an increase in depth following erosive challenge. As this was a pilot study, it is possible to draw only tentative conclusions from the data. However, the results strongly indicate that incipient enamel lesions experience a greater depth of erosion than intact enamel, particularly when exposed to citric acid containing beverages. The nding that these beverages also generate caries beneath the erosive lesions and deepen that already present strongly suggests that these types of drinks could greatly increase the rate and depth of caries progression in vivo. These data, if substantiated by a comprehensive statistical study, would clearly implicate acidic beverages in the development of advanced caries for a substantial segment of the young adult population.
S21
Abstracts
The effect of remineralization treatments on demineralized dentine: an in vitro study

E Moloney, S Varanasi, IA Meyers, L Rintoul, AL Symons*
Dentine is a vital, hydrated and complex tissue that forms the largest component of the human tooth. Once exposed to the oral cavity dentine is susceptible to demineralization and remineralization. Enhancing dentine remineralization is important for the treatment of dentinal hypersensitivity, cervical dentine wear and root caries. Commercially available bioactive agents effective in remineralizing dentine include casein phosphopeptide-amorphous calcium phosphate (CPPACP) and uoride containing products. The aim of this in vitro study was to determine the effect of the commercially available products, Tooth Mousse, Tooth Mousse Plus, Gel-Kam and NeutraFlour 5000, on the demineralized dentine surface. Dentine slices were prepared from 40 extracted third molars. Slices were divided into three sections to provide a specimen for each experimental group. Group 1 specimens acted as controls and were stored in distilled water. The remaining specimens were demineralized and stored in saliva (Group 2) or treated with Tooth Mousse, Tooth Mousse Plus, Gel-Kam or NeutraFluor 5000, for 7 days (Group 3). Dentine specimens were assessed using microhardness testing, scanning electron microscopy (SEM) and energy dispersive and Raman spectroscopy. The demineralization process decreased the microhardness of dentine and this was not restored following storage in saliva. Only the Tooth Mousse and NeutraFluor 5000 groups demonstrated a signicant increase (p < 0.05) in microhardness following treatment. SEM analyses showed that following demineralization the dentine tubule openings were more clearly dened and treatment with saliva did not alter this surface appearance. The dentine tubules appeared to be occluded following treatment with Tooth Mousse Plus and GelKam, and partially occluded following treatment with Tooth Mousse and NeutraFluor 5000. With the exception of uoride, few signicant differences in elemental content between Tooth Mousse, Tooth Mousse Plus and NeutraFluor 5000 were observed. Gel-Kam treated dentine demonstrated signicantly (p < 0.05) higher levels of magnesium and lower levels of oxygen, calcium and phosphate. All products signicantly increased (p < 0.01) uoride levels compared with control, demineralized and saliva stored specimens. The increase in surface uoride levels was signicantly higher for Gel-Kam and NeutraFluor 5000 (p < 0.001). Raman spectrometry indicated that uoride was incorporated into apatite crystals within the dentine following treatment with Tooth Mousse Plus and NeutraFluor 5000. The findings of this research were presented at the University of Queensland School of Dentistry Research Day 2008.
*School of Dentistry, The University of Queensland. Email: emmamoloney@optusnet.com.au.
GABA in human dental pulp

DAS Parker, K Nassery, V Marino*
The aim of this study was to examine the inuence of c-aminobutyric acid, GABA, on the release of the neurotransmitter, noradrenaline (NA), in sympathetic nerves in human dental pulp and the nature of the presynaptic GABA heteroceptors involved. Pulp removed from extracted teeth was incubated in gassed (95%O25%CO2) Krebs solution containing 3 HNA (0.6 lM) for 30 minutes, placed in a small chamber and superfused with Krebs solution containing ascorbic acid (0.3 mM), desipramine (DMI; 0.3 lM), rauwolscine (Ra; 0.1 lM) and for the GABA experiments aminooxyacetic acid (0.05 mmol L), DL-c-amino-b-hydroxybutyric acid (100 lM). Sympathetic nerves were stimulated after 70 (S1) and 105 (S2) minutes of
S22
superfusion by square wave pulses (5 or 20 Hz, 1 msec, 40 V). GABA, a GABA receptor agonist and GABA receptor antagonists were added to the superfusion medium at least 10 minutes prior to the second period of stimulation (S2). Superfusate was collected and assayed throughout each experiment and residual 3H in the tissues at the end of each experiment was extracted and assayed. From these data the fractional overow of 3 H in each collection period was calculated. The effect of an agonist antagonist was determined by comparing the overow of 3H at S2 relative to that at the rst period of stimulation in the presence of the compound with that in its absence. Statistical analysis was conducted using oneway analysis of variance followed by Tukeys Multiple
Abstracts Comparison Tests (GraphPad Prism 5.01). Signicance levels were set at p < 0.05. Initially, tissues were superfused in Krebs solution containing DMI, stimulated at 5 Hz for 500 pulses and the effects of the GABAB receptor agonist, baclofen (1 lM300 lM), tested. Baclofen had little effect on the release of 3HNA. In subsequent experiments pulp was stimulated at 20 Hz for 500 pulses in the presence of DMI and Ra. Baclofen (3 lM) inhibited the release of 3HNA by 16.6% (IC50 = 1.8 lM); however this effect was not reversed by the GABAB receptor antagonist (+)-(S)-5,5-dimethylmorpholinyl-2-acetic acid (Sch 50911; 10 lM).
*School of Dentistry, The University of Adelaide, South Australia. Email: david.parker@adelaide.edu.au.
GABA (100 lM), a non-specic GABA receptor agonist, inhibited the release of 3HNA by 25.5% (IC50 = 80 lM). This effect was not reversed by Sch 50911 (10 or 50 lM) or the specic GABAA receptor antagonist, bicuculline (10 or 50 lM). Picrotoxin (100 lM) which blocks the CI) channels associated with GABAA receptors prevented the inhibitory action of GABA. Neither the antagonists nor picrotoxin alone affected the release of NA. The results of this study imply that CI) channels associated with GABAA receptors, rather than the receptors per se, mediate the release of NA from sympathetic nerves in human dental pulp under the conditions used in these experiments. It appears from this study that GABAB heteroceptors do not modulate the release of NA from these nerves.
In vitro analysis of cytotoxicity and genotoxicity in human oral epithelial cells after treatment with alcohol-containing mouthwashes
J Saunus, S Zhao, CS Farah*
The aim of this study was to determine whether alcohol-containing mouthwashes can induce cellular toxicity and or genotoxicity in human cells in vitro with the single-cell gel (comet) assay and morphological assessment of apoptosis and mitosis, in order to clarify suggestive but inconclusive epidemiological data concerning a potential oral cancer risk associated with regular use of alcoholic mouthwash. Normal and dysplastic human oral epithelial cells were treated for 30 seconds with a 1:5 alcoholcontaining mouthwash dilution and equivalent concentration of ethanol (5.4%) for comparison. The negative control group was treated with phosphate buffered saline solution and the positive control group was treated with 650 lM H2O2. Recovery time points of 5, 10 and 20 minutes were allowed before trypsinization and layering onto slides with low-melting point agarose for electrophoresis. DNA damage was scored using the visual method established by Speit and Hartmann. Parameters from the comet assays and cytotoxicity testing were analysed by one-way ANOVA and Tukeys test. Alcohol-containing mouthwash and ethanol treatment groups consistently demonstrated signicantly greater DNA damage (p < 0.001) in the comet assay when compared to negative controls. No signicant difference was noted between recovery time points. No signicant differences in apoptosis and mitosis were noted between treatments and controls in the cytotoxicity assay, however OKF-6 cells (normal) were shown to have increased apoptosis and decreased mitosis when compared to DOK cells (dysplastic). Alcohol-containing mouthwashes do have a genotoxic effect in oral epithelial cells, however they do not induce cellular toxicity.
S23
Abstracts
Process analysis of the Monitor Dental Practice programme implementation

A Sbaraini,* SM Carter,* W Evans, A Blinkhorn*
This study was built on a previous Australian randomized controlled trial (RCT). Intervention practices in the RCT were provided with evidence-based preventive protocols to guide their treatment of dental caries. During that trial, the numbers of decayed, missing and lled teeth (DMFT) were monitored. Outcomes in the intervention practices varied widely; this qualitative study was designed to explain how dentists, their teams and patients adopted evidence-based preventive care in practice. We interviewed 40 participants (10 dentists, 2 hygienists, 9 dental assistants, 2 practice managers and 17 patients) about their experience and work processes. Analysis involved transcript coding, detailed memo writing and data interpretation. Dentists, their teams and patients talked about a process of slowly adapting their practices towards preventive care. Dentists spoke spontaneously about two assumptions or rules underpinning continued restorative treatment. They said that these assumptions were deeply held, and acted as a barrier to provide preventive care. Dentists believed that: (1) some patients were too unreliable to benet from prevention; and (2) patients thought that only tangible restorative treatment offered value for money. Dentists also described other factors that could hinder prevention, in particular, having an historical restorative background and being focused on cutting cavities fast and well. On the positive side, successful adaptation was possible if: (1) the dentist-in-charge brought the whole dental team together including other dentists and got everyone interested and actively participating during preventive activities; (2) the physical environment of the practice was reorganized around preventive activities; (3) the dental team was able to devise new and efcient routines to accommodate preventive activities; and (4) the fee schedule was amended to cover the delivery of preventive services, which hitherto was considered as unproductive time. Despite being able to adapt or not, all dentists trusted the concrete clinical evidence that they have produced, that is, seeing results in their patients mouths made them believe in a specic treatment approach. Patients talked about having to adapt to: (1) toothbrushing and ossing routines; and (2) dental appointments frequency, while dealing with children, home duties and work. Being offered preventive care was an amazing revelation for these patients they realized that dentists could actually practice dentistry without having to drill and ll their teeth. Patients made clear that they need to be made aware of preventive options, to be educated, and supported and reassured by the dental team during this process. Translating evidence into dental practice entailed a slow and complex adaptation process, requiring more than the removal of barriers. We suggest that dentists should be encouraged to look at preventive care as a central part of their practices, to try to adapt to preventive care and to experience results that are selfreinforcing and offer benets to all involved. Published: Sbaraini A, Carter SM, Evans RW, Blinkhorn A. Evidence of prevention: what do dental professionals perceive as evidence? J Dent Res Issue 89 (Spec Issue B):868. July 2010 (http://www.dentalresearch.org). Sbaraini A, Carter SM, Evans RW, Blinkhorn A. Adapting to evidence-based nonsurgical preventive protocols: barriers and facilitators. J Dent Res Issue 89 (Spec Issue C):l l. September 2010. Sbaraini A, Carter SM, Evans RW, Blinkhorn A. How to do a grounded theory study: a worked example of a study of dental practices. BMC Med Res Methodol 2011;11:128. Sbaraini A, Carter SM, Evans RW. How do dentists understand evidence and adopt it in practice? Health Educ J (in press). The findings of this research were presented at: 1. Sbaraini A, Carter SM, Evans RW, Blinkhorn A. Theorising the process of preventive dental care while combining two theories in practice. ACSPRI Conference 2010, North America, September 2010. Available at: http://conference.acspri.org.au/index. php/conf/2010/paper/view/35. Accessed 16 June 2011. 2. Sbaraini A. The process of implementing noninvasive caries management in private practice. Minimal Intervention Dentistry Day at Bio21 Institute, The University of Melbourne, 26 March 2010. 3. Sbaraini A. The process of adapting to non-surgical caries management in private practice. IADR Dental Forum hosted at the 2010 IADR ANZ Division Golden Jubilee Meeting, 29 September 2010.
*Centre for Values, Ethics and the Law in Medicine, The University of Sydney, New South Wales. Population Oral Health, Faculty of Dentistry, The University of Sydney, New South Wales. Email: alexandra.sbaraini@sydney.edu.au.
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Abstracts
The effectiveness of sonic activation, laser activation or syringe irrigation of 4% sodium hypochlorite in eradicating an Enterococcus faecalis biofilm
A Seet, P Cathro, P Zilm, T Fitzsimmons, N Gully*
It is well established that the causative agent of endodontic disease is the presence and growth of bacteria. Therefore, eradication of bacteria such as Enterococcus faecalis is essential to prevent or eliminate apical periodontitis. Studies have shown that elimination of bacteria prior to obturation has resulted in a more favourable outcome for endodontic therapy. Endodontic therapy is founded upon three principles: mechanical instrumentation, irrigation with antimicrobial agents and placement of an intracanal medicament. However, the complex anatomy of the root canal system often prevents the penetration of irrigants and medicaments into recesses that cannot be accessed by mechanical instrumentation. The advent of sonic, ultrasonic and laser instruments has led to many investigations looking at their potential for the activation of irrigants. However, most of these studies have concentrated on the removal of dentinal debris and smear layer, while only a few have investigated the effects of activated irrigation against bacteria that has been cultivated as a biofilm. The aim of this study was to evaluate and compare the effectiveness of three modes of irrigation: syringe irrigation, sonic activation and laser activation of the irrigant in eradicating E. faecalis that had been cultivated within the root canals of extracted single rooted teeth. A ow cell was designed and constructed. The extracted teeth were decoronated, and prepared with rotary instruments to #40 to 1 mm beyond the apex of the tooth. This was to allow nutrient media to ow through the root canals. The ow cell was connected to a nutrient reservoir containing Todd Hewitt Broth, which was pumped into the ow cell via a peristaltic pump. The ow cell was inoculated with E. faecalis (ATCC 700802) and cultivated for a period of 4 weeks. The flow cell was then dismantled and the teeth were assigned to 6 treatment groups: (1) syringe irrigation with saline; (2) syringe irrigation with 4% sodium hypochlorite; (3) sonic activation of saline (EndoActivator supplied by Dentsply); (4) sonic activation of 4% sodium hypochlorite; (5) laser activation of saline (Er,Cr:YSGG Waterlase laser supplied by Biolase Technology); and (6) laser activation of 4% sodium hypochlorite.
*School of Dentistry, The University of Adelaide, South Australia. Email: peter.cathro@adelaide.edu.au.
The teeth were irrigated with 5 ml of either saline or 4% sodium hypochlorite for 1 minute. The 4% sodium hypochlorite solution was inactivated with 5% sodium thiosulphate. Teeth that received sonic activation were irrigated by hand for 5 seconds, followed by 10 seconds of sonic activation, and this was repeated four times over 1 minute. Laser activation of the irrigants was also performed. Irrigant was introduced into the canal for 10 seconds, followed by 5 seconds of laser activation (0.25 W, 20 Hz). This cycle was repeated four times. The teeth were then crushed and serial dilution was performed to determine the number of viable bacteria (cfu ml) remaining in the root canals. Protein assays were conducted to quantitate the amount of biolm obtained. Samples were also taken from each treatment group and the radicular dentinal surfaces of the root canals were viewed under scanning electron microscopy (SEM). The results showed little difference when teeth were irrigated with saline or 4% sodium hypochlorite. In both groups, the numbers were in the order of 106 cfu ml. When sonic activation was used on the saline irrigant, there was an insignicant reduction in viable cells. However, sonic activation of 4% sodium hypochlorite resulted in a 10 fold reduction in viable cells, whereas a dramatic decrease was seen with laser activation of 4% sodium hypochlorite (10 000 fold). SEM analysis showed that sonic activation with saline only caused minimal disruption to the biolm. Teeth irrigated with sodium hypochlorite showed fewer bacterial cells on the radicular dentine but was not effective in eliminating E. faecalis that had invaded the dentinal tubules. Laser activation of sodium hypochlorite resulted in clean dentine walls and minimal bacteria within the dentinal tubules. Sonic or laser activation of an antimicrobial irrigant resulted in more effective bacterial elimination compared to hand irrigaton. Compared to syringe irrigation and sonic activation of sodium hypochlorite, laser activation of sodium hypochlorite was able to effectively disinfect the root canal. The findings of this research were presented at the University of Adelaide School of Dentistry Research Day 2009.
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Abstracts
Characterization of the response to Porphyromonas gingivalis using a co-culture method

A Shah, HL Do, SJ Leishman, PJ Ford*
Porphyromonas gingivalis stimulates a host immune response in the gingival tissues which involves epithelial cells and monocytes macrophages. This in vitro investigation aimed to characterize the inflammatory response to P. gingivalis in a co-culture of epithelial cells and peripheral blood mononuclear cells. Peripheral blood mononuclear cells were exposed to viable and non-viable P. gingivalis ATCC 33277 (type I fimA) and P. gingivalis ATCC 53977 (type II fimA). A co-culture model of epithelial-like transformed cells (HeLa cell line) and peripheral blood mononuclear cells was also established and exposed to viable and nonviable P. gingivalis ATCC 53977 (type II fimA). Flow cytometry was used to determine the intracellular production of IL-6 and IL-1b within monocytes and epithelial cells. There was an inhibition of IL-6 production by monocytes when exposed to P. gingivalis ATCC 33277 (type I fimA) or non-viable bacteria of either fimA type. However, viable P. gingivalis ATCC 53977 (type II fimA) stimulated a greater than 6 fold increase
*The University of Queensland. Email: p.ford1@uq.edu.au.
in IL-6 production. There was an inhibition of IL-1b production by monocytes when exposed to P. gingivalis ATCC 33277 (type I fimA) or non-viable bacteria of either fimA type. Viable P. gingivalis ATCC 53977 (type II fimA), however, stimulated a greater than 12 fold increase in IL-1b production at 6 hours. When cocultures were exposed to P. gingivalis ATCC 53977 (type II fimA) at 50MOI there was an increase in HeLa intracellular IL-6 levels for viable and non-viable bacteria at 30 minutes, and for non-viable bacteria at 6 hours. This study used a modication of an established coculture method. Modications included the use of peripheral blood mononuclear cells rather than monocyte cells lines and of ow cytometry instead of supernatant analysis. The effect of mA type on inammatory cytokine production following exposure to P. gingivalis was also examined and type II fimA was associated with a stimulation of cytokine production. This study emphasizes the importance of differentiating which cell types are responsible for production of specic inammatory mediators in co-cultures. If expanded, this model could further examine the role of host cell interactions in the response to P. gingivalis.
The effect of adding calcium and phosphate to acidic beverages on dental enamel erosion in vitro
P Shen, NJ Cochrane, Y Yuan, GD Walker, C Reynolds, EC Reynolds*
The prevalence of dental erosion is increasing in recent years due to increased consumption of acidic drinks. Casein phosphopeptide stabilized amorphous calcium phosphate (CPP-ACP) has been proposed to reduce erosion by localizing calcium and phosphate to the enamel surface. The study aimed to determine the effect of addition of calcium and phosphate as CPP-ACP on the erosive potential of acidic drinks. The pH and titratable acidity of two commercially available acidic beverages (A and B), Coca Cola and Mount Franklin, were determined. CPP-ACP was added at 0.1% and 0.2% (w v) to Beverage A and B. Enamel blocks with 1 2 mm windows were prepared from extracted human molars and suspended in 50 mL of beverage for 30 minutes. Enamel loss was determined using prolometry. Enamel surface hardness was determined using a microhardness tester.
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The pH was lowest for Coca-Cola (2.45 0.07) and highest for Mount Franklin (7.56 0.10). Addition of 0.1% and 0.2% CPP-ACP to Beverage A and B caused an increase in pH and a decrease in titratable acidities. Coca-Cola produced the highest amount of surface loss (3.22 0.52 lm) and Mount Franklin, Beverage A + 0.1% and 0.2% CPP-ACP, and Beverage B + 0.2% CPP-ACP did not produce detectable surface loss (all < 0.1 lm). Beverage A and B produced 0.35 0.13 lm and 2.33 0.52 lm surface loss, respectively. Surface hardness was reduced by approximately 50%, 36% and 43% when exposed to Coca-Cola, Beverage A and Beverage B, respectively. Addition of 0.1% and 0.2% CPP-ACP to Beverage A and 0.2% CPP-ACP to Beverage B signicantly reduced enamel surface softening. The addition of 0.1% CPP-ACP signicantly reduced surface loss yet did not reduce surface softening. The
Abstracts addition of 0.2% CPP-ACP eliminated measurable surface loss. These results suggest that a minimum 0.2% CPP-ACP should be added to Beverage B to reduce its erosive potential. The addition of 0.1% and 0.2% CPP-ACP to Beverage A reduced the surface loss to an undetectable level and greatly reduced surface softening.
*Oral Health Cooperative Research Centre, Melbourne Dental School, The University of Melbourne, Victoria. Email: peiyan@unimelb.edu.au.
The ability of CPP-ACP to reduce the erosive potential of acidic beverages may be attributed to its ability to localize calcium and phosphate and bind to enamel surface, slight increase in the pH, and a reduction in the titratable acidity. The addition of calcium and phosphate as CPP-ACP to acidic beverages resulted in signicant reductions in surface loss and softening in vitro. This study indicates that the calcium and phosphate as CPP-ACP could be added to beverages to reduce their erosive potential.
Ultramicroscopy of bone at oral implant site: a comparison of ectodermal dysplasia patients and regular patients
P Silthampitag,* AS Jones,* T Whittle,* S Scholz,* C Johansson, T Albrektsson, I Klineberg*
Osseointegrated dental implants are accepted as clinically desirable and have predictable outcomes for the management of partially and fully edentulous patients. However, implant failures continue to be reported and the reason for osseointegration failure is often not known. Overloading and poor bone quality at the implant site and the need for primary stability are considered likely factors. Bone quantity and quality are important factors for osseointegration and implant survival and should be considered during treatment planning. A consideration of bone structure as a correlate to implant failure may provide further insight into the failure mechanism. Data on bone structure and outcomes of implant-supported restorations is needed. In addition, genetic conditions such as ectodermal dysplasia (ED), which affect the jaws and associated tissues, may further inuence implant osseointegration. Bone microarchitecture in the maxilla and mandible needs to be considered to determine whether the local bone characteristics inuence implant survival. The aim of this study was to analyse the microarchitecture (ultrastructure and histology) of mandibular and maxillary bone in association with implant placement. The hypothesis is that the structural and morphological features of bone microarchitecture are different in different regions of the jaw bone and between regular and ED patients. This project seeks to develop a protocol for sampling bone at implant sites to allow details of structural analysis to be undertaken. The study is not additionally invasive, as the harvesting of bone was made at the time and site of implant placement. Bone samples have been allocated into 2 groups, ED and regular patients and specied by the site of bone harvesting. MicroCT analysis at 5 micron resolution is conducted on each bone sample. Computer analysis applying specialized CT analysis and VGStudiomax software allowed analysis of the three 2011 Australian Dental Association
dimensional microstructure of the bone samples for comparison of structural parameters. Four bone samples (2 ED, 2 regular) have been sent to The University of Gothenberg for histological analysis. Thirty-ve bone samples, 22 ED and 13 regular patients have been harvested. These data conrmed a distinct variation of bone samples harvested from the 2 groups. The ED group has signicantly greater bone volume (BV), percent bone volume (BV TV), bone surface (BS), bone surface density (BS TV), and trabecular number (Tb.N) with lower trabecular pattern factor (Tb.Pf), and structure model index (SMI) than specimens from the regular group. The histological analysis indicated that regular bone samples were smaller and contained more cancellous bone. The ED bone samples were larger and consisted of more compact bone with circumferential lamella observed, especially in one region. In the sample analysed, no bone forming or bone resorption sites have been observed and thus no osteoid rims with osteoblasts or regions with osteoclasts have been identied. Microstructural and histological analyses revealed that bone samples from ED patients consisted of more compact bone and better trabecular connectedness than regular bone samples. It is hypothesized that this structural feature has greater load bearing capacity through masticatory than bone from regular patients. Age-and gender-related changes are considered to be important in trabecular bone structural changes and this needs to be considered in further data analysis. The data have been derived from bone harvested from both maxilla and mandible of regular patients, whilst bone sampled from ED patients have been harvested mostly from the anterior mandible. Further bone samples will provide more data on whether broader areas of bone harvesting affect the quality and quantity of the bone that may inuence implant treatment outcome.
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Abstracts Published: Silthampitag P, Klineberg I, Jones AS, et al. Ultramicroscopy of bone at oral implant sites: a comparison of ED and regular patients. Part 1defining the protocol. Int J Prosthodont 2010;24:147154.
*The University of Sydney, Australia. The University of Gothenberg, Sweden. Email: terry.whittle@sydney.edu.au.
Silthampitag P, Klineberg I, Jones AS, et al. Bone microarchitecture at oral implant site in ectodermal dysplasia (ED) patients: microstructural and histological analysis. Clin Oral Implants Res (in press). The results of this study were presented at the International Association for Dental Research General Session in Barcelona, Spain, 1417 July 2010.
Cancer comorbidity and oral cancer survival: a cancer registry pilot study
LM Slack-Smith,* AM Frydrych,* R Parsons, T Threlfall, SE Hall
Analysis of the effect of comorbidity on the trajectory for oral squamous cell carcinomas (SCC) is rare, yet an important component of disease specic survival. This pilot study examined the effect of cancer comorbidity by determining survival among those with oral squamous cell carcinoma (oral SCC) with a prior cancer only and those who developed a later cancer only. This study arose from a study of oral SCC, biopsy type and survival. Total population data from the Western Australian Cancer Registry (WACR), comprising all primary oral SCC cases (ICD10 codes C01-C06) diagnosed between 1 January 1990 and 31 December 1999, were examined. Simple analyses presented here provide an overview of comorbidity and survival only. For this study year of biopsy, details of cancer comorbidity and date of death. Records were excluded if the diagnosis was ne needle aspiration based, or was not that of oral SCC. Univariate analyses were performed to look at the proportion of people who died each year (any cause) after their diagnosis of oral SCC and those who survived more than ve years. This was repeated for those cases: (i) with a cancer diagnosed prior to their oral SCC and no other cancer diagnosed (prior only); and (ii) with a second cancer diagnosed after their oral
*School of Dentistry, The University of Western Australia. School of Occupational Therapy and Social Work, Curtin University, Perth, Western Australia. Western Australian Cancer Registry, Department of Health (WA), Perth, Western Australia. School of Population Health, The University of Western Australia. Email: linda.slack-smith@uwa.edu.au.
SCC and no other cancer diagnosed (later only). The data were analysed using Stata 11. Approvals for the study were obtained from the Human Research Ethics Committees of the University of Western Australia and the Department of Health (WA). During the decade, 683 cases of oral SCC were identied. One in three patients (31.9%) had a cancer diagnosed before or after their oral SCC. A prior only cancer was diagnosed in 88 cases, and a later cancer in 130 cases. Of the 683 cases, 595 had no prior cancer and 553 were not diagnosed with a later cancer. One in three people (35.6%) diagnosed with oral SCC died within two years; the proportion of those with a prior cancer who died within two years, was four times higher than the proportion of those with a subsequent cancer who died within two years (51.1% vs. 13%). People with a prior cancer were less likely to survive more than ve years; only 19.3% of people with a prior cancer were alive compared with 56.9% of those diagnosed with a later cancer. Overall, only 39.8% of oral SCC patients were alive beyond ve years. This initial research demonstrated that comorbidity is associated with survival and further research is required. Published: Frydrych AM, Parsons R, Threlfall T, Austin N, Davies GR, Booth D, Slack-Smith LM. Oral cavity squamous cell carcinoma survival by biopsy type: a cancer registry study. Aust Dent J 2010;55:378384.
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Abstracts
Survival of rat dental pulp cells in vascularized tissue engineering chambers

T Srisuwan,* D Tilkorn,* A Vashi,* A Penington,* H Messer, K Abberton,* E Thompson*
Regenerative endodontics is a new eld in clinical dentistry which aims to preserve, repair and or regenerate the dental pulp tissue in diseased teeth. More recently, tissue engineering concepts have been introduced to dentistry, offering promise for a cell-based treatment approach in regenerative endodontics. Based on this concept, three main components are required: a source of cells, a scaffold, and inductive signalling molecules. Currently, several types of stem cells have been discovered in dental tissues including dental pulp and the surrounding support cells, which may have a potential use in dental pulp tissue generation. However, the specic requirements to drive the dental pulp tissue generation, especially odontoblast differentiation, are still undetermined and need to be claried. The objective of this study was to establish a model for studying the isolated dental pulp cells (DPC) and their potential to generate dental pulp tissue. This model combines rat vascularized tissue engineering chamber model with selected factors for dental tissue generation. Single cell suspensions of DiI labelled rat DPC were suspended within a collagen hydrogel scaffold, with or without gelatin microspheres containing bone morphogenic protein-4 (BMP-4) and broblast growth factors2 (FGF2). BMP-4 and FGF2 were chosen because of their roles in dental pulp repair. This mixture was then added into silicone and or dentine tissue engineering
*The OBrien Institute, Fitzroy, Victoria. The University of Melbourne Department of Surgery, St Vincents Hospital Campus, Victoria. Melbourne Dental School, The University of Melbourne, Victoria. Department of Restorative Dentistry and Periodontology, Faculty of Dentistry, Chiang Mai University, Chiang Mai, Thailand. Email: kerena@obi.edu.au.
chambers implanted in the rat groin. Tooth dentine was selected as a chamber component (either as a shell or as ground fragments) as it should provide the most suitable environmental cues for odontoblast differentiation in vivo. The chambers were harvested after 2, 4, 6, and 8 week timepoints and processed for analysis, including morphology, histology, and immunohistochemistry. After 2 weeks newly formed, highly vascularized tissues were observed inside the chamber. Labelled DPC were found around the dentine, particularly when located near the vascular pedicle and adjacent to the gelatin microspheres. Cell survival, as conrmed by double uorescent staining with DiI and DAPI displaying normal cell and nuclear morphology, was observed up to 8 weeks after transplantation. Dentin Sialophosphoprotein (DSPP) positive matrix production was observed in the chamber, indicating functionality of dental pulp progenitor cells. However, dentine production was not clearly observed. The system provides a useful model for the in vivo study of dental pulp progenitor cells and their differentiation requirements. Using a tissue engineering model, rat DPC mixed with collagen scaffold, dentine fragments and growth factors can survive and are able to secret DSPP in the chamber for up to 8 weeks. Whether this survival is related to improved angiogenesis, enhanced cell proliferation or other beneficial effects such as cell protection is unclear. The results of this study were presented at the International Association for Dental Research, Thessaloniki, Greece, 2007.
Reproducibility of ICDAS-II and DIAGNOdent for detection of occlusal caries by undergraduate dental students
M Stacey*
The aim of this study was to compare the reproducibility of the ICDAS-II visual-based system for occlusal caries detection with the reproducibility of the DIAGNOdent laser uorescence-based system for occlusal caries detection when used by undergraduate dental students. Six undergraduate dental students were trained in the use of ICDAS-II using a commercially available computer programme, and in the use of the DIAGNOdent by
means of the manufacturers instructions. The students then assessed the occlusal surfaces of 120 permanent molars twice, with a 2-week interval between the rst and second assessments, using the ICDAS-II, and later repeated the cycle using the DIAGNOdent. DIAGNOdent readings were classied as described by Lussi and Hellwig (J Dent 2006;34:467471). Intra-examiner Cohens kappa values for the students ranged from 0.56 to 0.66 for ICDAS-II and 0.49 to 0.58
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Abstracts for the DIAGNOdent. Each student scored a higher intra-examiner Cohens kappa score for ICDAS-II than for DIAGNOdent. Inter-examiner Cohens kappa values ranged from 0.37 to 0.62 for ICDAS-II, and from 0.29 to 0.71 for DIAGNOdent. Agreement between students was variable, with some achieving higher agreement with ICDAS-II and others with DIAGNOdent. Intra- and inter-examiner reproducibility was good for ICDAS-II codes 0, 5 and 6, and most problematic for ICDAS-II codes 1 and 2. With DIAGNOdent, intra- and inter-examiner reproducibility was good for sound tooth surface and deep dentine caries, but only poor to fair for caries in enamel and caries extending into outer dentine. DIAGNOdent offered no improvement in intra- or inter-examiner reproducibility over ICDAS-II for early occlusal lesions. Despite the fact there was no formal calibration process for the ICDAS-II, the reproducibility of occlusal caries detection was greater for ICDAS-II than DIAGNOdent for this group of students. The findings of this research were presented at the European Organisation for Caries Research Congress, Montpellier, France, 2010.
*Melbourne Dental School, The University of Melbourne, Victoria. Email: mastacey@unimelb.edu.au.
Factors inuencing learning of psychomotor skills by dental students

N Suksudaj, TA Winning, G Townsend, D Lekkas, J Kaidonis*
The acquisition of psychomotor skills is a key competence in dentistry. Several factors have been identied that relate to performance of this skill: (a) motivation, e.g. effort, value and importance; and (b) abilities for skill acquisition, e.g. cognitive, perceptual speed and psychomotor. Research has also shown that students perceptions of their learning context, e.g. what is required of them in the learning tasks assessment, is a critical element in the approaches they adopt for learning and the resultant quality of their learning outcomes. Recently, the use of skill acquisition and motor learning theories have informed the understanding of skill learning in surgical training. However, our understanding of key factors associated with the development of psychomotor skills in dental education is varied. This study aims to explore individual differences in factors that inuence the acquisition of psychomotor skills in dental students and to clarify what learning activities students perceive are effective or ineffective in supporting their learning of skills in an operative technique course. Consenting second-year students enrolled in the Dental Clinical Practice 2 course in 2008 (n = 52, 68%) at the University of Adelaide, were assessed for their cognitive, perceptual speed and psychomotor abilities using standard psychometric tests. After administration of the tests, a written exercise was completed that assessed theoretical knowledge about an MOD cavity preparation. Immediately following the written exercise, students completed a related cavity preparation exercise to assess motor performance. Students motivation related to this exercise was then assessed using the Intrinsic Motivation Inventory Survey. The teeth were graded into four categories (Unsatisfactory, Unsatisfactory Redeemable, Satisfactory and Good) based on clinical acceptability.
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During the acquisition phase of psychomotor skills, the actions of two sub-groups of students (n = 11 from each end of the performance range) were observed as part of their usual participation in class. The time spent by students from these sub-groups on a range of activities carried out during cavity preparation exercises was recorded. This component of the study was conducted with the same group of students over semester 1 and involved approximately 40 minutes observation per student in four consecutive laboratory sessions. Feedback was obtained on what students perceived as being effective and ineffective learning experiences in four laboratory sessions (2 semester), using a written critical incident approach and follow-up interview. Students involved in the interview component were chosen based on their performance and or issues in their critical incident report that specically linked to key learning constructs derived from motor learning theory. Ordinal regression models were used to analyse the associations of scores on the psychometric tests and the motivation survey with the grades. To determine whether the relationship between scores on the psychometric tests and the grades obtained for cavity preparation depended on time, the grades obtained for cavity preparations produced in the pre-learning stage, end of semester 1 and 2, were analysed using Generalized Estimating Equation (GEE) models. To compare the differences in average time spent on each operation between the two sub-groups of students, the MannWhitney U test was used. A signicant positive association was found between cognitive (visuo-spatial) and psychomotor ability and motivation scores related to the value students placed on the activity and their grades obtained for the cavity
Abstracts preparation exercise (p < 0.05). However, there was no signicant association between other ability scores and grades. There was evidence of a general improvement in grades over time for those with higher scores on a visuo-spatial ability test compared with those with lower scores. In addition, higher scores on tracing (psychomotor ability) were associated with higher grades overall (p < 0.05). Results showed that in preparing an occlusal cavity (week 6 of the course), students in the lower performance group changed their activity to observe their cavity twice as often as students in the higher performance group (p < 0.05). However, there were no differences in time spent in each operation or frequency of changes in operations at later weeks in the course. In relation to students perceptions of aspects of the practice environment that supported learning, they noted the importance of the role of laboratory tutors. Key aspects included: (1) arranging group discussion to clarify processes and outcomes; (2) providing specic and positive feedback about their performance; and (3) providing demonstrations, i.e. tell-show-do approaches. Feedback that focused on mistakes but not on how to improve performance was reported to result in ineffective learning experiences. Cognitive and psychomotor ability and also motivation of students were positively associated with cavity preparation outcomes. From the students perspectives, tutor interaction plays a key role in promoting an effective practice environment. When designing activities to support learning in operative dentistry courses, educators should address students motivation, and also aim to facilitate effective demonstrations, discussion and feedback with tutors to create a positive learning environment. Published: Suksudaj N, Townsend GC, Kaidonis J, Lekkas D, Winning TA. Acquiring psychomotor skills in operative dentistry: do innate ability and motivation matter? Eur J Dent Educ (in press). The findings of this research were presented at the International Association for Dental Research (IADR) Pan Asian Pacific Federation (PAPF) and combined meeting of the IADR Asia Pacific Region (APR) in Wuhan, China, 2024 September 2009.
*School of Dentistry, Faculty of Health Sciences, The University of Adelaide, South Australia. Email: tracey.winning@adelaide.edu.au.
A controlled study of the oral health of twin children

SS Taji,* GC Townsend, WK Seow*
There have been no previous clinical studies which examined the distribution of enamel hypoplasia in the primary dentition of monozygotic and dizygotic twins compared with singleton children. Similarly, the prevalence of dental erosion in twin children has not been investigated. We hypothesize that genetic factors inuence the presentation of enamel hypoplasia but not dental erosion. Therefore, the aim of the research project was to investigate the oral health of twin children compared to singleton controls to gain an insight into the contribution of hereditary and environmental factors in the aetiology of enamel hypoplasia and dental erosion. The twin subjects were part of a large cohort of Australian twins being followed up from birth to investigate various aspects of dental development in children. A total of 88 twin children (31 monozygotic, 50 dizygotic and 7 unknown zygosity) and 40 singleton controls aged 24 years and residing in the state of Queensland were studied. Medical, dental and dietary histories were obtained and the children examined for various oral and dental abnormalities. Enamel defects were charted using a modied Developmental Defects
of Enamel (DDE) Index, and tooth wear was assessed using a modied Tooth Wear Index. The results for developmental defects of enamel showed that enamel hypoplasia was observed in 21% of the teeth in monozygotic twins (MZ), 22% in dizygotic twins (DZ), and 15% in singleton control children (p < 0.05). The main risk indicators for enamel hypoplasia in twins were neonatal complications including low birthweight and endotracheal intubation. There were no signicant differences in site concordance of enamel hypoplasia within the MZ twin pairs compared with DZ twin pairs when only the presence of enamel hypoplasia was considered. On the other hand, when both the presence and absence of enamel hypoplasia were considered, a greater concordance was observed between MZ twin pairs compared with the DZ twin pairs. The results for dental erosion showed that the subject prevalence in the primary dentition was 77% in monozygotic twins (MZ), 74% in dizygotic twins (DZ), and 75% in singleton controls (results not statistically signicant). Of the erosion lesions, 12% were scored as mild, 10% moderate, and 1% severe. In addition, there was an association of more severe erosion with increasing age of the children (p < 0.05).
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Abstracts Concordance rates for erosion lesions in MZ and DZ co-twins were not statistically signicant. The results suggest that environmental factors probably exert a greater inuence on enamel hypoplasia in the primary dentition although both genetic and environmental factors contribute to the observed variation of enamel hypoplasia. In the case of dental erosion the contribution of genetic factors is negligible.
*Centre for Paediatric Dentistry, School of Dentistry, The University of Queensland. School of Dentistry, The University of Adelaide, South Australia. Email: k.seow@uq.edu.au.
Published: Taji S, Seow WK. A literature review of dental erosion in children. Aust Dent J 2010;55:358367. Taji S, Seow WK, Townsend GC, Holcombe T. A controlled study of dental erosion in 24 year old twins. Int J Paediatr Dent 2010;20:400409. Taji S, Seow WK, Townsend GC, Holcombe T. Enamel hypoplasia in monozygotic and dizygotic twins compared with singleton controls. Int J Paediatr Dent 2010;21:175184.
Dental insurance, level of cover and dental service use

DN Teusner, DS Brennan, AJ Spencer*
In cross sectional surveys, approximately 70% of middle-aged dentate adults (45 to 64 years) report a perceived need for dental treatment, while a third report that they have avoided or delayed dental care because of cost, indicating that there may be substantial unmet demand for dental care in this age group. Understanding the associations between insurance, the level of cover and use of dental services may indicate the impact on demand for dental care if policies aiming to expand insurance coverage or subsidize insurance were implemented. The aim of this study was to explore associations between dental insurance status, level of cover and use of dental services by dentate adults aged 45 to 54 years. Participants (South Australians aged 45 to 54 years) of an existing two-year prospective study (20052007) investigating dental service use were sent a follow-up survey collecting their insurance status and plan details. Dental visits and service types comprised the main outcome variables in the analysis and were collected from the study baseline questionnaire. Service types included exams, scale and cleans, llings and extractions. Insurance status and level of cover were the main explanatory variables. Insurance status comprised the categories of insured and uninsured. Percentage rebate for a periodic exam was used to categorize insurance plans by level of cover as this plan benet was positively correlated with other benets. Level of cover was classied as LOW if the rebate for an exam was less than 60% and HIGH if it was 60% or more. Response bias was investigated by comparing the age, gender, household income group and education level of the respondents to follow-up survey versus nonrespondents. Bivariate associations between receipt of services, insurance, level of cover and other known covariates (age, gender, household income and education) were explored. Poisson regression models estiS32
mated prevalence ratios (PR) of service use controlling for covariates (PROC GENMOD SAS V9.1). Response rate for the baseline questionnaire (adjusted for return to sender) was 43.8% and 87.2% for the follow-up insurance survey. Respondent characteristics: 46.9% were male, 61.2% were 50 years or older, 29.4% were in the highest income group ($80 000), 43.3% had a tertiary degree or diploma and 75.2% were insured for dental services. Analysis of dental visiting was limited to dentate persons who had known insurance status (n = 529) and analysis by level of cover was limited to the uninsured and those who provided sufcient plan details to enable categorization by level of cover (n = 354). Of the insured providing sufcient details (n = 223, 56%), three-quarters (77.6%) were categorized as LOW and had a mean rebate of 47% for an exam, 22.4% were classied as HIGH and had a mean rebate of 71%. Those responding to the follow-up survey, compared to the non-respondents, were more likely to be in the highest income group and were more likely to have a tertiary diploma or degree (chi-square, p < 0.01). The majority of adults had made a dental visit in the previous 12 months (69.1%) and of those that had visited, 92.3% received one or more exams, 76.3% received one or more scale and cleans, 52.2% received one or more llings and 12.4% received one or more extractions. Making a dental visit, receiving an exam and receiving a scale and clean were positively associated with insurance (chi-square, p < 0.01) while receiving a lling was not associated with insurance. The uninsured were signicantly more likely to receive an extraction (27.8%) compared to the insured (8.5%) (chi-square, p = 0.000). There was no variation in visiting by level of cover (chisquare, p > 0.05). Amongst service types, only receiving an extraction was associated with level of cover, counter intuitively, those with LOW cover were less likely to
Abstracts receive an extraction (4.0%) than those with HIGH cover (13.2%) (chi-square, p < 0.05). After controlling for covariates there was no association between insurance status and making a visit. The insured had a 17% higher prevalence of making a visit, but this difference was not signicant (PR = 1.17, 95% CI = 0.961.43). However, the signicant associations between having insurance and services types persisted. Compared to the uninsured, the insured were more likely to receive an exam (PR = 1.14, 95% CI = 1.02 1.28) and a scale and clean (PR = 1.52, 95% CI = 1.201.92) and less likely to receive an extraction (PR = 0.52, 95% CI = 0.330.83). The association between level of cover and receiving an extraction did not persist after controlling for covariates. The nding that insurance status and level of cover was not associated with dental visits may be related to response bias, which increased the homogeneity
*Australian Research Centre for Population Oral Health, School of Dentistry, The University of Adelaide, South Australia. Email: dana.teusner@adelaide.edu.au.
of the analysis sample, and potentially diminished variation in visiting behaviours. Alternatively, it may be a reection of the low level of rebate provided by the insurance cover held by the majority of the insured. While insurance status was reported by nearly all respondents, there was poor response to the plan details question; this excluded just under half of the insured respondents from the level of cover analysis. Although there was no observable relationship between level of cover and visiting, it is possible that a larger sample may have detected slight differences by service type. Furthermore, it is possible that visiting and service use may be associated with higher levels of cover not examined in this study. For middle-aged Australian adults dental visiting was not signicantly associated with insurance status or level of cover. Types of services received were associated with insurance status but did not appear to be associated with level of cover. Policies that result in increasing dental insurance coverage may not result in an increase of middle-aged adults accessing care but may result in a shift in pattern of services received.
The effect of the local application of different dosages of lipoxin A4 on bone healing following the placement of a surgical defect in the mandible
S Varanasi, AL Symons, JR Smid*
Bone healing involves an intricate series of events in which progenitor cells from the periosteum and the endosteum differentiate into osteoblasts. These differentiating osteoblasts which subsequently assist in new bone formation are mediated by cytokines present within the extracellular matrix. These cytokines can also promote, inhibit or delay deposition of type 1 collagen, matrix formation or mineralization, in response to growth factors and hormones. Lipoxin A4 (LXA4), a primary eicosanoid in humans, is produced by leukocytes via biosynthetic pathways and acts as an anti-inammatory agent both in vivo and in vitro. Local administration of LXA4 in experimentally induced periodontitis decreased the levels of COX2 and PGE2, and the intensity of neutrophil mediated tissue injury and inflammation, thereby minimizing gram-negative bacteria induced alveolar bone loss. The aims of this study were to determine the effect of a local application of various dosages of LXA4 on the healing of a surgical defect in the rat mandible and the distribution of extracellular bone matrix proteins, osteopontin (OPN), osteocalcin (OCN), alkaline phosphatase (ALP), osteopretegerin (OPG) and receptor activator of nuclear factor-kappa B ligand (RANKL). The undecalcied mandibles from the healing, LXA4treated and PBS-treated groups did not show signs of resorption on the buccal surface of alveolar bone. In the healing and LXA4 treated groups, the depth of the
surgical defect signicantly decreased compared to the depth of the defect at day 1. At day 20 the depth of the surgical cavity in the LXA4 treated groups were signicantly less than the 20-day sham treated group. At day 10, the serial sections of the decalcied tissues stained with Massons trichrome and haematoxylin and eosin staining showed no evidence of inammation or bone loss along the internal surface of the surgical defect. At day 10, in the LXA4 treated groups, the percentage area of new bone formation along the base of the surgical defect was signicantly more than the control and sham treated groups. By day 20, the percentage area of new bone formation in the LXA4 treated groups were not signicantly increased compared to the control group. The number of positive osteoclasts that had signicantly reduced in the LXA4 treated groups were signicantly reduced in 10 days 500 ng LX4 treated groups compared with the control, 1.0 lg LXA4 treated and 2.0 lg LXA4 treated groups. However, by day 20, TRAP-positive osteoclast-like cells were signicantly increased in the 1.0 lgm LXA4 treated groups compared to the control, 500 ng LXA treated and 2.0 lgm LXA4 treated groups. OPN, OCN, ALP, OPG and RANKL immune-expression was upregulated in 10- and 20-day control and treatment groups on the left treated side of the mandible compared to the day 1 control group. By day 20, the number of OCN immunopositive cells were signicantly increased in the 1.0 lgm treated
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Abstracts group compared to the control (p < 0.01) and 2.0 lgm LXA4 treated groups. By day 20, the number of ALP immunopositive cells were signicantly increased in the 1.0 lgm treated group compared to the control (p < 0.01) and sham treated (p < 0.05) groups. Throughout the experimental procedure, there was no signicant increase in TRAP-positive osteoclast-like cells, OPN, OCN, ALP, OPG and RANKL cell numbers in the untreated right side compared with the treated left side of the mandible at days 10 and 20. Compared with the healing group, LXA4 treatment enhanced new bone formation. In LXA4 treated animals, the depth of the defect was reduced at day 10 and the number of positive osteoclasts was significantly less than that observed for the 10-day healing group. By day 20, the depth of the defect was reduced and new bone formation continued to increase. However, new bone formation was signicantly less in the 20-day LXA4 treated animals compared with the 20day healing group, although immunopositive OPN and OCN cell numbers in the 20-day LXA4 treated group were not different from the 20-day healing group. The osteoclast cells numbers had increased by 20-day compared with 10-day in the LXA4 treated group. In the PBS treated group, the 10-day group showed early mineralized tissue formation although osteoclast cell numbers were greater than for the 10-day LXA4 treated group. However, by 20-day PBS treated group, the rate of new bone formation had reduced than normal healing of the surgical cavity. The findings of this research were presented at the International IADR meeting 2006; the University of Queensland School of Dentistry Research Day 2007 and 2008; and the IADR ANZ Division meeting, Kiama, 2010.
*School of Dentistry, The University of Queensland. Email: srinivas.varanasi@uq.edu.au.
The effect of low dose prostaglandin E2 on bone healing of surgical defects over time
S Varanasi, AL Symons, JR Smid, GO Ramirez*
Bone healing and new bone formation involves a complex series of events which begins with the interaction of cellular elements that may be delayed or inhibited by residual inammation and associated growth factors. Prostaglandin E2 (PGE2) is a naturally occurring substance that is known to have an anabolic effect on load bearing bones. Low doses of PGE acting over a long period of time are reported to have an anabolic effect on bone leading to bone formation both in vivo and in vitro. The aims of this study were to determine the effect of a local application of slow releasing low dosage of PGE2 on the healing of a surgical defect in the rat mandible and the distribution of extracellular bone matrix proteins, osteopontin (OPN), osteocalcin (OCN) and alkaline phosphatase (ALP). Following placement of the surgical defect, a slow releasing 20-day pellet of 1 mg (0.05 mg day) prostaglandin E2 was placed adjacent to the defect. The incision was sutured and left to heal. For the sham treated group, a sham pellet containing no prostaglandin E2 was placed adjacent to the defect. The incision was sutured and left to heal. Healing of the defect was monitored postoperatively at day 15 and day 30 for control and treatment groups. Eight animals were examined per experimental group, four providing undecalcified mandibles for confocal microscopy and the remaining four decalcified mandibles for light microscopy examination. Transverse serial sections of decalcified mandibles were stained with haematoxylin
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and eosin, for tartrate-resistant acid phosphatase and osteocalcin, osteopontin and alkaline phosphatase. Morphometrical and histological measurements were analysed using one-way ANOVA to compare the different treatment groups. The undecalcied mandibles from the PGE2 treated group showed some signs of inammation and bone destructions along the margins of the surgical defect on the buccal surface of the mandible. By day 30, there were no signs of inammation and bone destruction. In the sham treated group and healing group, there were no signs of inammation or bone resorption on the buccal surface of alveolar bone and the defect gradually occluded with time. At day 15, the depth of the surgical defect was signicantly less in the control and treatment groups compared to the mean depth of the cavity at day 1 (995.5 43.4 lm). By day 30 the depth of the surgical defect in the control group was signicantly less compared to the depth of the surgical cavity in PGE2 and the sham treatment groups. In the treatment and control groups, there was a signicant increase (p < 0.001) in the expression of TRAP positive osteoclast-like cells and in the expressions of OPN, OCN and ALP on the left treated side of the mandible compared to the non-treated right side of the mandible. By day 30, a signicant increase in the TRAP positive osteoclast-like cell numbers were observed in PGE2 treated group compared with the sham treated group. There was no signicant difference in the quantitative
Abstracts expression of OPN, OCN, OPG and RANKL between control and treatment groups at day 15 and day 30. At day 30, the immunopositive ALP cell numbers were signicantly higher in the control group compared with the sham treated group. There were minor signs of inammation and destruction to the buccal surface of the mandible in the PGE 2 treated animal groups at day 15. As healing progressed, the destruction on the buccal surface of the mandible and the depth of the surgical defect decreased over time by occluding new bone. PGE2 initially appeared to decrease healing of the defect and new bone formation and increase osteoclast cell numbers. However, this was not evident with increased expression of extracellular bone matrix proteins. By day 30, the surgical defect appeared to have completely obturated with new bone formation in the normal healing group. In the PGE2 and sham treatment groups, healing of the surgical defect appeared to be delayed over time compared to the control groups. The findings of this research were presented at the University of Queensland School of Dentistry Research Day 2007 and 2008.
*School of Dentistry, The University of Queensland. Email: srinivas.varanasi@uq.edu.au.
The effect of lipoxin A4 on the expression of osteoblasts and broblasts cells markers in vitro
S Varanasi,* AL Symons,* JR Smid,* GJ Seymour
Fibroblasts and osteoblasts are the most common cells involved in an intricate series of events during bone repair and are regulated by various growth factors and hormones. Lipoxin A4 is an endogenous anti-inammatory lipid mediator derived from arachidonic acid. Lipoxin A4 (LXA4) has shown to promote resolution of inammation and minimize localized alveolar bone loss in experimentally induced periodontitis in rabbits. The aims of this in vitro study were to determine the effect of various dose levels of LXA4 on primary cultured rat periodontal fibroblasts with respect to the proliferation and viability of fibroblasts and immunoexpression of fibroblast-markers basic Fibroblast Growth Factor-2 (bFGF-2) and Vimentin (VMT). Additionally, to determine the effect of various dose levels of LXA4 on primary cultured rat calvaria osteoblasts cells, with respect to the immunoexpression of osteocalcin (OCN), osteopontin (OPN), alkaline phosphatase (ALP) and enzyme alkaline phosphatase activity (eALP). Four Lewis female rats sourced from the Animal Resource Centre, Perth, were used to establish primary cell lines of fibroblasts and osteoblasts. Primary fibroblasts and osteoblasts were established in culture medium Dulbeccos Modified Eagle Medium (DMEM, pH 7.4) supplemented with 10% FCS, 1% penicillin (10 000 units ml) and streptomycin, 10 000 lg ml (PNS) and 0.1% Fungizone (1 lg ml). Various doses of LXA4 (500 ng, 1 lg and 2 lg dissolved in 10 lL of PBS) was delivered to the cultured cells, 1 104 (x3), per treatment group every 48 hours starting from zero hours, 48 hours and 96 hours. At days 1, 3 and 5, 24 hours post-dosage administration, cell proliferation and viability of broblasts and enzyme cytotoxic assays, osteocalcin and alkaline phosphatase activity for osteoblasts was determined.
The primary broblasts showed a signicant increase in the proliferation in day 1 control, LXA4 treated and sham treated groups. By day 3 and day 5, there was a signicant increase in the proliferation of primary broblasts in 2.0 lg LXA4 treated groups compared to control, 500 ng LXA4 treated and 1.0 lg LXA treated groups. Over days 1, 3 and 5, the number of viable live broblasts had signicantly increased (p < 0.001) compared to the numbers of cells initially used for the control and treatment groups. At day 3, the viability of primary broblasts signicantly increased in sham and 2.0 lg LXA4 treated groups compared to control, 500 ng LXA4 treated and 1.0 lg LXA treated groups. By day 5, the viability of primary broblasts was signicantly higher in 2.0 lg LXA4 treated groups compared to control, 500 ng and 1.0 lg LXA4 treated groups and sham treated groups. At day 1, there was a signicant increase (p < 0.001) in bFGF-2 and VMT immunopositive cell numbers in control, sham and LXA4 treated groups compared to the cells initially used for the control and treatment groups. By day 5, a significant increase (p < 0.001) in the bFGF-2 immunopositive cell numbers in control, 500 ng LXA treated and 1.0 lg LXA4 treated groups was observed compared to their respective groups at day 3. At day 3, a significant increase (p < 0.001) in VMT immunopositive cell numbers in 1.0 lg LXA4 treated group compared to 1.0 lg LXA4 treated group at day 1. By day 5, VMT immunopositive cell numbers in 500 ng LXA treated and 2.0 lg LXA4 treated groups had significantly increase (p < 0.001) compared to day 3 LXA4 treated groups. For osteoblasts, there was a gradual and signicant increase (p < 0.001) in OCN, ALP and OPN immunopositive cell numbers in control, sham and LXA4
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Abstracts treated groups compared to the numbers of cells initially used for the control and treatment groups. By day 5, no signicant difference in the OCN immunopositive cell numbers was observed between control, sham and LXA4 treated groups. For ALP, at day 1, there was a signicant increase in the number of ALP immunopositive osteoblasts cell numbers in sham treated and 2.0 lg LXA4 treated groups compared to control and 500 ng LXA4 treated groups. However, at days 3 and 5, no signicant difference in ALP immunpositive cell numbers was observed between control, sham and LXA4 treated groups. For OPN, at day 3, a relative increase in the OPN immunopositive cell numbers in control, sham 1.0 lg LXA4 treated and 2.0 lg LXA4 treated groups was not signicantly different compared to their respective groups at day 1. However, by day 5, OPN immunopositive cell numbers in control, and LXA4 treated groups were signicantly increased compared to control and LXA4 treatment groups at day 1. Additional tests for enzyme immuno assay for eALP, at day 1, showed a significant increase (p < 0.001) in the eALP levels of the 500 ng LXA4 treated group compared with control, 1.0 lg LXA4 treated and 2.0 lg LXA4 treated groups. By day 5, eALP levels in the control group was significantly less (p < 0.001) compared to the day 1 control group. However, by day 5, no significant difference in the eALP levels were detected between control, 500 ng LXA4 treated, 1.0 lg LXA4 treated and 2.0 lg LXA4 treated groups. This study was to determine the local effects of LXA4 on the proliferation, viability and cell metabolism of primary rat broblasts and rat osteoblasts that forms into new bone during bone regeneration and bone repair. LXA4 did not appear to inhibit proliferation or viability of broblasts cells. Over time, high dose levels of LXA4 enhanced the proliferation and viability of the primary rat periodontal broblast cell numbers. An increase in the quantitative immunoexpressions of broblast and osteoblast cell markers conrms the potential application of LXA4 as a therapeutic agent in the management of traumatic or inammatory conditions. Additionally, LXA4 did not decrease the enzyme alkaline phosphatase levels in osteoblast cell culture media. The findings of this research were presented at the University of Queensland School of Dentistry Research Day 2009 and 2010; the IADR meeting in Barcelona, July 2010; and the IADR ANZ Division meeting in Kiama, September 2010.
*School of Dentistry, The University of Queensland, Australia. School of Dentistry, The University of Otago, New Zealand. Email: srinivas.varanasi@uq.edu.au.
Regulation of BMP-2 and BMP-6 expression by titanium surface topography and chemistry
J Vlacic-Zischke, SM Hamlet, S Ivanovski*
Surface topography has been identied as an important factor in governing the reaction of tissues to surgically placed implants and the degree of osseointegration achieved. The microrough SLA surface has been shown to promote osteoblast differentiation, increased production of osteogenic factors, cytokines and growth factors and increase bone to implant contact. Chemical modication of the SLA surface resulting in increased hydrophilicity (modSLA) has been shown to induce even further bone to implant contact, osteoblast differentiation, growth factor production and osteogenic gene expression. We have previously demonstrated the expression of genes critically associated with osteoblast differentiation, bone morphogenetic protein (BMP)-2 and BMP6, were differentially regulated in response to topographically and chemically modied implant surfaces. This study aimed to conrm this expression by quantitating BMP-2 and BMP-6 protein secreted by
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osteoblasts cultured on both SLA and modSLA titanium surfaces. SLA and modSLA titanium surfaces were used for cell culture. Glass cover slips served as the control surface. Primary human osteoblasts were established from redundant tissues obtained following third molar extraction surgery. The level of BMP-2 and BMP-6 secreted into the culture supernatants by osteoblasts was determined by ELISA after 1, 3 and 7 days culture. The attachment and proliferation of osteoblasts to the titanium surfaces at 1, 3, 5 and 7 days was also determined. Osteoblast attachment to both titanium surfaces was signicantly higher compared to the control surface; however there was no signicant difference in attachment between the SLA and modSLA surfaces. Over a 7-day period, there was a progressive and signicant (p < 0.001) increase in osteoblast cell numbers on all three surfaces with the highest numbers seen on the control surface. Compared to the control surface, the
Abstracts proliferation of osteoblasts on both titanium surfaces was also signicantly lower at each time point (p < 0.001). By day 7, a signicant (p < 0.05) difference in proliferation was seen between the SLA and modSLA surfaces, with the modSLA surface eliciting a lower level of proliferation. Compared to both the control surface and SLA surface, a signicant (p < 0.05) increase in BMP-2 secretion was observed from osteoblasts cultured on the modSLA surface on both day 3 and day 7. No signicant difference in secretion of BMP-2 was detected between the control and SLA surfaces at either time point. BMP-6 was undetectable in all samples.
Decreased osteoblast proliferation on the modSLA surface coincides with greater differentiation as demonstrated by secretion of signicantly higher levels of BMP-2 protein. The secretion of another potent inducer of hMSC differentiation into osteoblasts (BMP-6) could not be detected in the culture media via the method used in this study. These results suggest that chemical (hydrophilic) modication of the SLA surface may result in more successful osseointegration through BMP-2 signalling. Published: Vlacic-Zischke J, Hamlet SM, Friis T, Tonetti MS, Ivanovski S. The influence of surface microroughness and hydrophilicity of titanium on the up-regulation of TGFb BMP signalling in osteoblasts. Biomaterials 2011;32:665671.
Identication and characterization of novel cell surface markers that are essential for periodontal ligament stem cell phenotype
N Wada, K Mrozik, S Gronthos, PM Bartold*
Periodontal ligament stem cells (PDLSCs) have recently been proposed for the development of new periodontal regenerative therapies. To isolate the stem cell population of high purity from PDL heterogeneous cell population, cell surface markers expressed on stem cell population are used. Using specic antibodies for antigens of cell surface marker protein, positive cells are able to be isolated by immunomagnetical isolation system or uorescence activated cell sorter. The aims of this study were to identify signicant differentially expressed genes specic to PDLSCs in comparison to PDL cells at different differentiation stages and to identify novel cell surface markers of PDLSCs for isolating stem cells of high purity from heterogeneous cell population of periodontal ligament. To identify the differences of genes expression present in cell clones of varying proliferation and differentiation potentials, comparative DNA microarray analysis was performed on three different types of PDL cell clones exhibiting high proliferation bi-differentiation potential (both of mineralization and adipogenic potential: bi-potential clone), high proliferation uni-potential (only mineralization potential: uni-potential clone) and low proliferation and
*Colgate Australian Clinical Dental Research Centre, School of Dentistry, The University of Adelaide, South Australia. Email: mark.bartold@adelaide.edu.au.
differentiation potential (low potential clone). To verify these results, real-time PCR was performed to analyse the expression of transmembrane protein genes picked up from microarray analysis data. The expression of these gene proteins in heterogeneous PDL cell population were examined by immunocytochemical staining, ow cytometric analysis and western blotting. The transmembrane protein genes such as epithelial membrane protein-1 (EMP1), interleukin-1 receptor 1 (IL1R1), CD13, transforming growth factor-beta receptor 3 (TGFBR3) and platelet derived growth factoralpha (PDGFRA) were expressed higher in bi-potential clone than in low-potential clone by both of DNA microarray analysis and real-time PCR. In these genes, EMP1 was expressed in some PDL cells by immunocytochemical staining and western blotting. PDL cells were also strongly positive for CD13 and TGFBR3 by ow cytometric analysis. Novel cell surface markers identied from DNA microarray analysis data might be specic to PDLSC. Although further study is required, these identied cell surface markers would provide new knowledge for the PDL and stem cell study and could be novel useful antigens for isolating mesenchymal stem cell population from heterogeneous PDL cell population. If successful, these studies will signicantly advance the development of the treatment of periodontal disease with bone destruction by PDLSC implantation.
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Abstracts
Students understandings, approaches and outcomes of PBL contexts in two PBL dental curricula
T Winning,* V Skinner,* A Kinnell,* G Townsend,* G Svensa ter, M Rohlin, J Davies
In contemporary health professions education contexts, the rationale for PBL curricula is to prepare students to learn discipline-specic ways of thinking and acting. Various studies of PBL curricular outcomes have demonstrated variations in the intended student experiences and outcomes. A theoretical approach to understanding student learning outcomes, Student Approaches to Learning (SAL) theory, explains how students understanding of the learning context inuences how they approach their learning and consequently, inuences the quality of their learning outcomes. The aim of this study was to use SAL theory to investigate rst-year students understandings of PBL and to examine the learning approaches and outcomes of these students at the beginning and end of the rst semester of rst year in two different curricular contexts. The curricula at the Faculty of Odontology, Malmo University, Sweden (MU) and School of Dentistry, The University of Adelaide, Australia (UA) were well established at the time data were collected (MU: implemented in 1990; UA: implemented in 1993). Examination of key features of PBL curricula indicated there were many common aspects across the two curricula, e.g. induction of students to PBL, problem selection, purpose, presentation, format and processes, as well as scope and format of course outcomes, resources students used, assessment format and early clinical exposure. The major differences related to the level of integration (MU: fully integrated, consecutive courses; UA: hybrid curriculum, four parallel courses), length of PBL cases (MU: longer sessions and cases), reduced contact time with timetabling of noncontact time for research (MU: 16h week contact; UA: 25h week contact) and the number of assessment tasks across the programme (UA: more tasks across the four courses). In the rst and last week of rst semester in rst year, students were asked to indicate what they would say to a friend regarding what PBL is about. Responses were analysed using an inductive thematic approach. Students also completed the revised two-factor study processes questionnaire (R-SPQ-2F; Biggs et al., Br J Educ Psych 2001;71:133). Student responses to the R-SPQ-2F were summarized into deep (DA) and surface approach (SA) scores. Differences in scores between the beginning and end of semester 1 were analysed using paired t-tests (p < 0.05). Examination grades were categorized as high or low pass, and associations between learning approaches and performance were analysed using v2 tests. There were no signicant differences in age, gender and performance on examinations between participants
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and non-participants. However, signicantly more MU participants were female. Initially, the majority of students described PBL as being about looking for information to solve problems. Some students also noted a high level of students responsibility for learning. In both Schools, some students indicated learning- and team-skills were outcomes of their PBL experiences. Only a few Malmo students noted the professional relevance of PBL and its intention for learning skills and process in contrast to several Adelaide students who noted the relevance of the practice context. After one semester of PBL, Malmo and Adelaide students descriptions of PBL were more congruent with curricular intentions, particularly related to using a systematic process for investigating a patient case. Responses continued to recognize the responsibility of the individual student in their learning. However, the major focus remained on fact-nding or information collection with only a few students noting the collaborative role of the group in supporting their learning. Learning of key skills and the role of the tutor in PBL were not consistently included in the key features identied by students. The relevance of the practice context to their learning remained unchanged for students at each site. In both institutions, the majority of students had high DA (>30) and low SA scores on both testing occasions (MU: 6668%; UA: 5473%). A smaller group of students had both low DA and SA scores (MU: 2728; UA: 2138%). Only a few students had high DA and SA scores or high SA and low DA scores (approx 2 4%). At the end of rst semester, the number of Malmo students with high DA scores remained constant (66%). This contrasted with Adelaide, where a signicant decrease in mean DA and SA scores was found (p < 0.05). Associations between the students understandings of PBL, DA SA scores and performance were not evident. First-year students understandings of PBL in both Malmo and Adelaide developed over one semester to more closely align with planned intentions. However, several key characteristics of PBL were not noted by students in their rst semester. Curricular experiences of students at Malmo were consistent with the adoption of deep approaches while Adelaide students PBL experiences were associated with decreased deep approaches. This was despite Adelaide students acknowledging the professional relevance of their learning in PBL. Consistent with other reports, these ndings demonstrate that students, learning approaches and associated outcomes are not solely inuenced by theoretically informed curricular designs. Reviewing
Abstracts students understandings, approaches and outcomes of their curriculum are critical to monitor the impact of our curricula. Clarication of key curricular components that have been reported to support or hinder our desired outcomes is needed. These ndings will inform how our curricula can be designed to support the planned understandings of the learning context with associated quality learning outcomes. Published: Winning TA, Skinner VJ, Kinnell A, Townsend GC, Svensa ter G, Rohlin M, Davies JR. The influence of two PBL curricular contexts on firstyear students understandings of PBL, approaches to learning and outcomes. In: Researching Problembased Learning in Clinical Education: The Next Generation. S Bridges, C McGrath, T Whitehill, eds, In Innovations and Change in Professional Education (ICPE) Series (Series editor: Wim Gijselaars), 2011:77101. The results of this study were presented at the 5th and 6th International Conferences on PBL in Dentistry, Rhode Island, USA, 2008 and Hong Kong, 2009.
*School of Dentistry, The University of Adelaide, South Australia, Australia. Faculty of Odontology, Malmo University, Sweden. Email: tracey.winning@adelaide.edu.au.
The effect of exercise training on jaw muscle recruitment strategies

A Wirianski, C Peck, G Murray*
Temporomandibular disorders (TMD) are primarily musculoskeletal conditions that affect around 10% to 26% of the adult population. Resistance exercise can relieve some symptoms of TMD. One possible mechanism whereby exercise may relieve the symptoms of TMD is by altering muscle activity. Changes in electromyographic (EMG) activity have been described in patients suffering low back pain and pattellofemoral pain. Prescription of specic exercise programmes for these patients can result in resolution of these abnormal EMG activities with concomitant improvements in pain and function. Therefore, the prescription of specic exercise programmes to patients suffering TMD could result in changes in muscle activity patterns and help to resolve their symptoms. The aim of this study was to determine whether resistance exercise alters the patterns of EMG activity of the jaw muscles during standardized jaw movements. Electromyographic activity was acquired from the masseter, temporalis and digastric muscles in 12 healthy adults during a standardized lateral jaw movement task. Subjects were randomly allocated into an exercise or control group. The exercise group performed isotonic resistance exercises, 3 times a day for 4 weeks, by applying nger pressure to the mandible against right lateral (ipsilateral) jaw movement. The standardized movements were repeated after this and again 4 weeks later. Raw EMG signals were rectied, Butterworth ltered and normalized to maximum voluntary contraction. Statistical analyses of total EMG activity, maximum EMG activity, rate of EMG activity increase, duration of EMG activity and timing of EMG activity onset and offset of this processed EMG signal were undertaken, between the control and
exercise groups, and before and after exercise sessions (ANOVA, Mann-Whitney and Friedman tests). All subjects performed the same standardized right lateral movements during and between testing sessions. The total EMG activity of all the tested muscles showed no statistically signicant (p > 0.05) changes between the exercise or control groups over the testing period. The left (contralateral) anterior temporalis, left (contralateral) masseter and right (ipsilateral) anterior digastric, showed signicant (p < 0.05) changes in 3 out of the 4 remaining tested variables. Initial exercise training resulted in a signicant (p < 0.05) reduction in the duration of EMG activity in the ipsilateral anterior temporalis, with a concomitant signicant (p < 0.05) increase in the duration of EMG activity in the ipsilateral anterior digastric. This study demonstrated that the application of isotonic resistance exercise can produce changes in some EMG parameters in the muscles of mastication of asymptomatic individuals. A reduction of the duration of EMG activity, as seen in the ipsilateral temporalis, may imply that other muscles of mastication may have increased their activity in order to complete the standardized jaw movement task. Along with the increased duration of EMG activity in the ipsilateral digastric, there may have also been changes in the lateral and or medial pterygoids which are the primary agonists of right lateral jaw movement but were not tested in this study. Such results would be in agreement with biomechanical modelling studies which suggest that different muscle activation strategies are capable of producing the same jaw movement. Further investigation into the effects of lateral resistance exercise on the pterygoid muscles
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Abstracts would help determine the mechanism of the changes in EMG duration as seen in this study. Resistance exercise can alter the patterns of EMG activity of the jaw muscles during standardized jaw movements. Changes in the duration of EMG activity may imply that other jaw muscles altered their activity in order to complete the standardized jaw movement
*Faculty of Dentistry, Jaw Function and Orofacial Pain Research Unit, The University of Sydney, Westmead Hospital, Westmead, New South Wales. Email: greg.murray@sydney.edu.au.
task. Future studies should focus on determining the central and peripheral mechanisms by which these changes occur. This will help facilitate the development and appropriate application of rehabilitation programmes in order to reduce the impact of TMD and return symptomatic individuals to normal jaw function. The findings of this research were presented at the 2nd Meeting of the IADR Pan Asian Pacific Federation (PAPF) and the 1st Meeting of the IADR Asia/Pacific Region (APR), 2224 September 2009, Wuhan, China.
Dentists perceptions of a new local anaesthetic drug the uptake of new technology in dentistry
KE Yapp, MS Hopcraft, P Parashos*
The aim of this study was to elicit information regarding the use of articaine in Australia, and factors that inuence attitudes toward adoption of new technology. A self-administered postal questionnaire was sent to a stratied systematic sample of dentists who were members of the Australian Dental Association, with questions regarding details about articaine use and how inuences such as education and scientic literature affect their adoption of new technology. Of the sample, 53% responded and over 70% of dentists surveyed used articaine, with 95 and 97% of respondents identifying scientic literature and continuing professional development courses respectively as inuencing factors in their adoption of new technology. The most common reason for not using articaine was no perceived advantage and the most inuential factor that would encourage non-users to start using articaine was a scientically proven advantage. Senior clinicians, specialists and public sector dentists were less likely to use articaine. A majority of Australian dentists were using articaine and cited CPD courses, scientic literature and anecdotal peer reports as being inuential in their adoption of new technology. Published: Yapp KE, Hopcraft MS, Parashos P. Dentists perceptions of a new local anaesthetic drug articaine. Aust Dent J (in press). The findings of this research were presented at the 2011 International Association for Dental Research meeting, San Diego, California, USA.
*Melbourne Dental School, The University of Melbourne, Victoria. Email: keyapp@hotmail.com.
c-Src kinase mediates enhanced oral epithelial barrier function induced by ligation of CD24
P Ye,* H Yu, N Hunter*
In stratied epithelium, the role of point contacts with morphological characteristics of tight junctions remains incompletely understood. In a previous study we showed that the phosphoinositol anchored glycoprotein CD24 is strongly expressed by human junctional epithelium that mediates attachment of the gingiva to teeth and by cultured oral epithelial cells with characteristics of the junctional epithelium. We reported that ligation of CD24 in model epithelial
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monolayers induced assembly of the tight junction proteins occludin and zona occludens-1 (ZO-1) and 2 (ZO-2) at the cell periphery in association with enhancement of barrier function. Silencing of mRNA for CD24 increased permeability in oral epithelial monolayers. The Src family of tyrosine kinases mediates transduction and processing of many extra- and intracellular signals including regulation of tight junction assembly.
Abstracts In the present study we investigated the role of Src kinases in the signal transduction capacity of CD24. The oral epithelial culture model was used to establish that inhibitors of c-Src kinase ablate the effect of anti-CD24 peptide antibody in inducing reduction of paracellular diffusion of Mr 10 000 uorochromelabelled dextran across the monolayer by permeability assays and live cell imaging analysis using an adaptation of laser scanning microscopy. The role of c-Src kinase was conrmed by demonstrating increase in phosphorylation typical of activated c-Src kinase by Western blot. All data were analysed by paired t-test (mean SD, two-tailed, 95% CI range) from at least three consecutive experiments for permeability assays. Findings conrmed that inhibitors of Src kinases ablate the effect of anti-CD24 peptide antibody on barrier function of epithelial monolayers. The broad spectrum protein kinase inhibitors herbimycin A,
*Institute of Dental Research, Westmead Millennium Institute and Westmead Centre for Oral Health, Westmead Hospital, Westmead, New South Wales. Microscopy Laboratory, Westmead Millennium Institute, Westmead Hospital, Westmead, New South Wales. Email: p.ye@sydney.edu.au.
genistein and PP2 were effective in suppressing the barrier promoting effect of anti-CD24 antibody whereas PP3 as a negative control for the Src family of protein tyrosine kinase inhibitor PP2, had no detectable effect. Further indication of the essential role for c-Src kinase was obtained by demonstrating that the Src kinase inhibitor saracatinib blocked antiCD24 enhancement of barrier function. Live cell imaging indicated these effects were related to paracellular diffusion of uorochrome-dextran. Western blot showing strong staining for afnity-isolated c-Src phospho tyr418 indicated the kinase had been activated. The combined data implicate activation of c-Src kinase as a mediator of the effect of ligand binding to CD24 on the assembly and function of effective barrier function in this epithelial model. It is concluded that enhanced barrier function induced by ligation of CD24 is mediated by activation of c-Src kinase. Published: Ye P, Yu H, Simonian M, Hunter N. Ligation of CD24 expressed by oral epithelial cells induces kinase dependent decrease in paracellular permeability mediated by tight junction proteins. Biochem Biophys Res Commun 2011;412:165169.
Magnitude of bacteraemia induced by ossing and by scaling and root planing in patients with chronic periodontitis
W Zhang, C Daly, D Mitchell, B Curtis*
Recent changes to antibiotic prophylaxis guidelines for invasive dental treatment in patients at risk of infective endocarditis have been based, in part, on the concept that everyday oral hygiene procedures may represent a greater risk of infective endocarditis by exposing individuals to repetitive bacteraemia. However, no reliable data exist for the magnitude of bacteraemia caused by ossing and no studies have investigated either the incidence or magnitude of bacteraemia caused by ossing and by scaling and root planing (SRP) in the same individual. The aims of this study were to investigate: (1) the incidence and magnitude of total oral bacteraemia (TOB) and viridans streptococcal bacteraemia (VSB) due to ossing as compared with SRP in the same individual with chronic periodontitis; (2) any possible association between patient and clinical parameters and the incidence and magnitude of bacteraemia; and (3) micro-organisms associated with bacteraemia. Full-mouth ossing and single quadrant SRP were performed for 30 patients with moderate severe chronic periodontitis at separate visits. Twenty millilitres of blood were collected as a baseline prior to each procedure and further samples were obtained at 30 seconds and 10 minutes after completion of ossing,
at 5 minutes after initiation of SRP and 30 seconds and 10 minutes after completion of SRP. Magnitude of bacteraemia was investigated with lysis centrifugation and expressed as CFU ml. Bacteria were identied to species level. The incidence of TOB was 30% (9 30) for ossing and 43.3% (13 30) for SRP with no statistically signicant difference (p = 0.21). Flossing and SRP caused the same incidence of VSB, both at 26.7% (8 30). Flossing caused a higher mean magnitude of TOB than that caused by SRP (7.4 16.2 CFU ml vs. 2 3.4 CFU ml) but the difference was not statistically signicant (p = 0.2). Flossing caused a higher mean magnitude of VSB than that caused by SRP (1.2 1.6 CFU ml vs. 0.4 0.2 CFU ml) but the difference was not statistically signicant (p = 0.09). A wide variety of oral micro-organisms was identied in the isolates including a number of periodontal pathogens. Viridans streptococci accounted for 11.4% (61 535) of the microbial isolates from ossing and 7.6% (17 225) of the microbial isolates from SRP. No signicant correlations were found between any of the patient or clinical parameters and the incidence or magnitude of bacteraemia following ossing. Gingival index was found to be signicantly associated with the
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Abstracts incidence of TOB (p = 0.01) and VSB (p = 0.001) following SRP, but no other statistically signicant predictors of outcome were identied. No signicant correlations were found for any of the patient or clinical parameters and the magnitude of TOB or VSB following SRP. The similar incidences and magnitudes of bacteraemia, especially VSB, caused by ossing as compared with SRP in the same individual provide further evidence that an everyday oral hygiene activity such as ossing should be considered a risk procedure for infective endocarditis. It may therefore be inconsistent to recommend antibiotic prophylaxis for individuals at risk of infective endocarditis for invasive dental procedures such as SRP, but not for ossing. The preliminary results of this study were presented at RACDS Convocation, Perth, 1114 March 2010.
*School of Dentistry, The University of Sydney, New South Wales. Email: christopher.daly@sydney.edu.au.
The role of S-layer protein in biolm formation by Fusobacterium nucleatum in response to high growth pH
P Zilm, J Chew, N Gully, A Rogers*
Approximately one-fth of the Australian population suffers moderate or acute periodontal disease. The adverse quality of life and the overall cost to the patient and public health programs is signicant. Basic research investigating the complex genotypic changes that occur in bacteria associated with periodontal diseases is the rst step in developing treatment protocols. The bacterium Fusobacterium nucleatum (Fn) is a Gram negative anaerobe considered to play a crucial role in the progression of periodontal disease and is commonly found in clinical infections of other body sites. Its cell surface properties enable it to attach to gingival epithelia, collagen and other oral bacterial species. The development of periodontitis is associated with a rise in pH in the gingival sulcus and this is thought to occur by the catabolism of proteins supplied by gingival crevicular fluid. Fn is commonly isolated from periodontally diseased sites and is also able to survive in root canal systems at pH 9.0 after calcium hydroxide treatment. In order to survive hostile environmental conditions, such as nutrient deprivation and fluctuating temperature and pH, bacteria form biofilms that are usually made up of multi-species co-aggregates. We have previously grown Fn in a chemostat at a growth rate consistent with that of oral bacteria in vivo and have shown that, at an environmental pH of 8.2, Fn co-adheres and forms homogeneous biofilms. Our studies have shown that biofilm formation is associated with an increase in cell surface hydrophobicity and a decrease in the levels of intracellular polyglucose. Genomic analysis of Fn has revealed the existence of a gene (butB) that encodes for a predicted surface (S-layer) protein. S-layers are usually composed of a single protein or glycoprotein and although they may have many functions, have been found to act as adhesins. The expression of an S-layer by Fn has not been reported previously. Preliminary qRT-PCR results suggest that Fn does express the S-layer protein at both pH 7.2 and pH 8.2. The level and or change of expression is yet to be determined. Therefore, the aim of our investigation was to determine whether Fn produces an S-layer in response to growth pH or other disease-associated environmental changes (e.g. redox potential, temperature). Fusobacterium nucleatum ATCC 25586 was grown via continuous culture and after steady state growth had been achieved, cells were harvested at either pH 7.2 (planktonic) or pH 8.2 (biofilm). Cells were harvested and cell surface material and extracellular proteins were prepared using published protocols. Proteins were separated by SDSPAGE and regions corresponding to the predicted molecular weight of the S-layer protein (68 kDa and 71.5 kDa) bands were excised and identied by mass spectrometry. Unfortunately, the reduced budget for the project meant that a glycoprotein specic dye was not used. Thirty-ve proteins were identied in the cell associated fraction, however none were identied as an S-layer protein. The cell surface samples are awaiting analysis at the Adelaide Proteomics Centre following repair to the mass spectrometer. The results indicated that the S-layer was not detectable in relation to the cellular components of Fn. It remains undetermined at this time whether or not the S-layer is associated with the surface layer of Fn.
*School of Dentistry, The University of Adelaide, South Australia. Email: peter.zilm@adelaide.edu.au.
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Abstracts
Proteomic analysis of human mesenchymal stem cell surface proteins using uorescent Cye dye labelling
P Zilm,* K Mrozik, S Gronthos, J Xiong, PM Bartold
Approximately one-fth of the Australian population suffers moderate or acute periodontal disease. The adverse quality of life and the overall cost to the patient and public health programmes is signicant. The use of stem cells derived from dental pulp or periodontal ligament to regenerate periodontal tissues represents a new and innovative treatment modality. In a broader context, understanding the signalling processes and the interactions with the environment which guide the differentiation of multi-potential stem cells is of major importance for medical science and regenerative therapy. Previous studies have demonstrated the tissue regeneration capacity of mesenchymal stem cell-like populations derived from periodontal ligament and dental pulp. Ongoing studies are aimed at regenerating bone and dental tissues by utilizing the different unique properties of these cell types. Cell surface proteins are involved in many cellular processes such as attachment, solute transport and cell signalling and their resolution in whole cell lysates can be difcult against a background of highly abundant cytosolic proteins. Typically, in the human proteome, cell surface proteins represent only 510% of all membrane coding genes. In the present study, we selectively stained cell surface proteins with an impermeable uorescent dye so that the expression can be compared between cell types. The aim of the present project was to use a proteomic approach to investigate the expression of cell surface
*Oral Microbiology Proteomics Laboratory, School of Dentistry, The University of Adelaide, South Australia. Colgate Australian Clinical Dental Research Centre, School of Dentistry, The University of Adelaide, South Australia. SA Pathology Hanson Institute, Division of Haematology, Adelaide, South Australia. Email: peter.zilm@adelaide.edu.au.
proteins in periodontal ligament stem cells, dental pulp stem cells and gingival broblasts which are unique to each cell type and which may serve as stem cell markers to identify multipotential stem cells in heterogeneous populations of stromal cells. Human periodontal ligament broblast stem cells (PDLSC) and human gingival broblasts (HGF) were cultured in a-minimum essential media supplemented with 10% foetal calf serum and 100 lM l-ascorbate-2phosphate, 1 mM sodium pyruvate, 50 lg mL streptomycin 50 U mL penicillin, and 2 mM Lglutamine. Cells were harvested following detachment using either collagenase or EDTA. Cell surface labelling was done by addition of uorescent Cye dye and cells were then lysed and cell membrane proteins were partially puried. Membrane proteins were separated by two dimensional gel electrophoresis (2DE) and identied by mass spectrometry. Detachment of tissue culture cells using EDTA produced unacceptable levels of cell morbidity (20%) and produced a high background of uorescently stained cytosolic proteins. Collagenase detachment reduced the level of cell death to acceptable levels. From these, a total of 72 proteins were excised from the gel and identied by mass spectrometry. Of these, 27 were cell membrane associated. Differences seen between HGF and PDLSC populations included 5-nucleotidase (CD73), Thy-1 membrane glycoprotein (CD90), heat shock protein 70 (HSP70). Fluorescent Cye dye labelling of cell surface exposed membrane proteins proved to be technically difcult and was dependent upon the protocol used for cell detachment. However, from the study, 27 cell membrane proteins were identied that may prove important in the recognition of periodontal ligament stem cell populations.
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Abstracts
ADRF Research Grant Reports published as full papers in the Australian Dental Journal in 2011
Aust Dent J 2011;56:1622 Australian population norms for the Index of Dental Anxiety and Fear (IDAF-4C) JM Armeld Aust Dent J 2011;56:2332 Job satisfaction of the oral health labour force in Australia L Luzzi, AJ Spencer Aust Dent J 2011;56:112121 Applied anatomy of the pterygomandibular space: improving the success of inferior alveolar nerve blocks JN Khoury, S Mihailidis, M Ghabriel, G Townsend Aust Dent J 2011;56:132140 Influence of implant abutment angulations on the fracture resistance of overlaying CAM-milled zirconia single crowns A Ellakwa, T Raj, S Deeb, G Ronaghi, FE Martin, I Klineberg Aust Dent J 2011;56:141147 Augmentation of linear facial anthropometrics through modern morphometrics: a facial convexity example R Wei, P Claes, M Walters, C Wholley, JG Clement Aust Dent J 2011;56:272277 Self-reported oral health of a metropolitan homeless population in Australia: comparisons with population-level data EJ Parker, LM Jamieson, MA Steffens, P Cathro, RM Logan Aust Dent J 2011;56:352357 Improved periodontal health and cardiovascular risk S Rose-Hill, PJ Ford, SJ Leishman, HL Do, JE Palmer, NCK Heng, MJ West, GJ Seymour, MP Cullinan
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Abstracts
ADRF Undergraduate Research Grant Abstracts

Risk assessment for children at high risk of early childhood caries a pilot qualitative study
MK Cable; WK Seow, L Hobbs (Supervisors)*
Despite the fact that early childhood caries is a preventable disease, many young children are affected and dental general anaesthesia is a regular management option. It was hypothesized that preventive programmes targeting specic risk factors may be a successful strategy in reducing the levels of cariogenic bacteria and prevalence of early childhood caries in high risk children. This study aimed to assess whether children who underwent aggressive treatment for early childhood caries under dental general anaesthesia and followed up for preventive care show changes in mutans streptococci (MS) levels. This pilot study sample included ve children in the Metro South District of Brisbane, aged between ve and seven years who required dental general anaesthesia for treatment of early childhood caries. A questionnaire obtained information on the childs oral hygiene regime, dietary habits and medical history. A baseline test for MS was performed on each participant immediately before dental treatment. In-depth interviews were conducted with mothers relating to lifestyle routines and parenting behaviours. Preventive advice was also provided. A four-week recall appointment was provided to determine changes in MS levels after dental treatment. At the review appointment, positive short-term changes were noted since the dental general anaesthesia. Common attributes discovered in the interview included families needing to move house due to unemployment or separation of the parents, difculty in disciplining the child and mothers nding that parenting was either difcult or challenging. One mother had taken on sole responsibility of her childs oral hygiene regime. Child cooperation with toothbrushing had improved with parents reporting compliance in most cases. Interproximal cleaning had been adopted by two mothers who had been using dental oss three to four times per week with the participant. Both mutans streptococci and Lactobacilli numbers either decreased or no bacteria were found at the review appointment compared to the initial dental general anaesthesia appointment. The reduction in microbial levels suggests that the oral hygiene and dietary counselling that was given at the dental general anaesthesia appointment was effective and these changes in habits were practised in the home. Prevention strategies such as dietary and oral hygiene education may enable parents and children reduce the occurrence of future dental caries. This pilot study may be extended to determine whether the initial short-term changes can be maintained in the longer term.
*School of Dentistry, The University of Queensland. Email: megan.cable@uqconnect.edu.au.
Erosive potential of powdered sports drinks

CH Chang; Y Yuan, GD Walker, P Shen, C Reynolds, EC Reynolds, NJ Cochrane (Supervisors)*
A 2007 survey of Australian school children found that 68% showed signs of dental erosion. It has been suggested that this high prevalence may be related to the consumption of acidic beverages such as carbonated soft drinks and fruit juices. Another product category that is regularly consumed in Australia are sports drinks. The Australasia region, representing only 0.4% of the global population, accounts for 1.1% of the global sports drinks market which is disproportionally high. Consumption of sports drinks have also been shown to be related to dental erosion due to their acidic nature. Recently, an Australian company has developed a sports drink that they claim has a low erosive potential.
The objective of this study was to evaluate the erosive potential of different sports drinks, including those claiming to have a reduced erosive potential, and compare them with their chemical composition. Freshly extracted human molars and premolars were sectioned to obtain 70 enamel blocks of either buccal or palatal surfaces. The enamel blocks were then embedded in epoxy resin, lapped and polished to expose the enamel and covered in acid resistant nail polish to create a window. The blocks were then randomly divided into 10 groups of 7. Ten drinks were purchased for analysis: (1) Mount Franklin water ()ve control); (2) Coca-cola (+ve control); (3) Gatorade powder;
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Abstracts (4) Gatorade liquid; (5) Powerade powder; (6) Powerade liquid; (7) Staminade; (8) Sukkie Lemon; (9) Sukkie Berry; and (10) Endura. Sports drink powders were diluted in distilled deionized water according to manufacturer instructions. The pH and titratable acidity of each drink was determined using a pH probe and autoburette. Each enamel block was exposed to 50 mL of their respective drink for 30 minutes at 25 C with agitation at 120 rpm. The surface loss produced by each drink was determined by non-contact white light prolometry and surface softening determined by change in Knoop surface microhardness.
*Oral Health Cooperative Research Centre, Melbourne Dental School, The University of Melbourne, Victoria. Email: n.cochrane@unimelb.edu.au.
The pH of drinks 37 ranged from 2.81 to 3.55 whereas drinks 810 ranged from 4.85 to 4.93. The positive and negative control had the greatest and least surface loss and change in surface microhardness respectively. Surface loss produced by the sports drinks ranged from 2.55 lm (7) to undetectable levels (<0.1 lm, 810). Surface softening produced by the sports drinks ranged from )188 (4) to )23 (9). Drinks 810 had a similar softening effect to 1. The pH of the beverages was found to correlate strongly with their erosive potential (p < 0.01). Sukkie and Endura had a low erosive potential, while all other sports drinks produced signicant amounts of surface softening and surface loss. The in vitro erosive potential of sports drinks correlated strongly with their pH.
Expression of calmodulin-like skin protein in oral epithelial dysplasia

DE Fan; PJ Ford, CS Farah (Supervisors)*
It is often difcult to diagnose, classify and prognosticate oral epithelial dysplasia. Traditional histopathogical diagnosis using biopsy specimens is subjective with a high degree of intra- and inter-observer variability. Molecular biomarkers have much potential to enhance current diagnostic methods. We have chosen to study the expression of calmodulin-like skin protein (CLSP) in oral tissues. This novel protein is involved in keratinocyte differentiation and has been implicated by a number of studies as a potential biomarker for dysplasia. We hypothesized that expression of CLSP is downregulated in lesions with dysplasia or oral squamous cell carcinoma (OSCC). Our rst aim was to compare
*The University of Queensland School of Dentistry and UQ Centre for Clinical Research. Email: david.fan@uqconnect.edu.au.
the expression pattern between oral dysplasia, OSCC and a control group. Secondly, we aimed to determine if there was any relationship between the level of expression of the protein and the degree of dysplasia. Parafn sections were obtained from a clinical archive of biopsies from patients with potentially malignant or malignant oral lesions. The lesions were diagnosed histopathologically by an oral pathologist. The sections were stained with polyclonal antibodies against CLSP. The level of expression in each section was calculated by determining the average of the percent positive cells in 10 views under 40x magnication. Expression of CLSP was upregulated in mild dysplasia only. There was no signicant difference in CLSP expression in the other diagnostic groups. CLSP expression may be upregulated in mild dysplasia because these lesions are usually leukoplakias and involve increased keratinization.
Awareness and understanding of osteonecrosis of the jaw by general practitioners and their referral pattern to dentists prior to commencing bisphosphonate treatment of osteoporosis
J Hsieh; P Reher (Supervisor)*
Osteonecrosis of the jaw (ONJ) is a rare but serious complication associated with bisphosphonate therapy. About three-quarters of ONJ occur after dental extractions. The risk is higher with intravenous bisphosphonate use than its oral formulation, commonly used to treat osteoporosis. There is consensus internationally to recommend dental review prior to intravenous
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bisphosphonate administration if the patients systemic condition permits. However, recommendations for oral bisphosphonate are less consistent. In Australia, dental health assessment is recommended prior to initiation of oral bisphosphonate therapy and such recommendations have been published in a number of journals commonly encountered by general practitioners, the
Abstracts largest group of oral bisphosphonate prescribers and initiators. The study aimed to investigate awareness and understanding of ONJ among general practitioners and their referral pattern for oral health assessment prior to commencing patients on bisphosphonate treatment for osteoporosis. The study also aimed to identify possible factors that would limit general practitioners from making dental referrals. A two-page survey was sent out to all general practitioners practising in the Gold Coast Division of General Practice as dened by the Australian Government Department of Health and Ageing. Follow-up telephone calls were made to general practitioners who
*School of Dentistry and Oral Health, Grifth University, Queensland. Email: rachel.hsieh@grifthuni.edu.au.
had not yet responded at four weeks. Data collected were subjected to statistical analysis. A total of 497 surveys were sent with a response rate of 13.5%. The majority of GPs never or only occasionally (29% and 25% respectively) referred patients for dental health assessment and only 12% of GPs always referred. Dental extraction was identied by 76.1% of GPs as a risk factor for BRONJ while only 31.3% of GPs identied poorly-tting dentures as a risk factor. The results conrm that the majority of GPs on the Gold Coast have not adopted Australian guidelines which recommend dental referral for patients prior to commencement of oral bisphosphonate therapy into their clinical practice. The findings of this research were presented at the ADA Dentsply Student Clinician Program, 34th Australian Dental Congress, Brisbane, 30 March 3 April 2011.
Instrumental, Detrimental and TranscenDental: experiences of dental emergency and micro-rationing practices in a Victorian public dental clinic
E Kahan; E Willis (Supervisor)*
Since the abolition of federal funding of public dental services in 1996, the Australian public dental health system has been under pressure. In Victoria this has been evident by long waiting periods and an increased demand for emergency services, with many socially disadvantaged individuals experiencing delay or denial of treatment that will relieve their dental pain. While there have been several studies that have identied these inequalities and described the dental emergency cycle, where eligible individuals attend public services for repeated emergency service while languishing in the waiting lists, little is known of what it means to rely on public clinics for the relief of dental pain, or how is the implementation of current policies played out in daily interactions between service seekers and providers. The principal focus of this work provides a sociological analysis of the denition and experience of the phenomenon of dental emergency in public clinics in Victoria. From historical and theoretical perspectives, this analysis problematizes the denition of emergency in the public policy discourse as a service rationing device and its impact on the interaction between service users and providers. From an empirical perspective, this work applies concepts of Narrative Theory to explain the experience of dental emergency treatment of individuals attending an outer suburban public dental clinic. Another focus of this work applies the concept of Micro-rationing to explore the impact of dental emergency on service providers in different locations through three case studies in inner city, outer suburban and rural settings.
Ethics approval was sought and granted by the La Trobe University Human Ethics Committee and the Peninsula Health Care Network Ethics Committee. Participants in the study were selected through a voluntary recruitment process that was informed by the conceptual framework, opportunity and convenience. This data collection process was consistent with conventional qualitative research practice. In-depth interviews of 40 service users and nine service providers were recorded verbatim and transcripts were coded for recurring themes. Other sources of data included the researchers observations and eld notes as well as elements of material culture, such as signs on walls, policy documents and software manuals. Data were consequently analysed using two conceptual methods. Firstly, the interview tapes and transcripts were reviewed by the researcher using the principles of Grounded Theory, aiming at generating theory through a thematic analysis, identifying and ordering concepts into a systematic account of the phenomena found in the eld. Secondly, the service attenders interview transcripts were explored using narrative analysis techniques. The work gives voice to service user stories absent from ofcial documents that relate to what it means to receive emergency dental treatment for disadvantaged individuals. The stories describe instances when the service fulls their expectations (Instrumental) but also when it exacerbates their feelings of dejection in their condition of welfare payment recipients (Detrimental). For some, however, their experience offers meaning
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Abstracts that transcends conventional user-provider interaction outcomes (TranscenDental). In these stories, the narrator endures an experience that provides insight into their own identity and circumstances that is valued beyond the immediate benet of a resolved dental problem. A theme of interest in the participants narratives is the description of their perception of the public dentist. The case study component disclosed how staff view and categorize their clients depending on the degree to which they conform to their expectations of compliance with their own perception of professional role and service regulations. Hence clients are portrayed as nice, aggressive, smart or naive. In turn, staff ideology in regards to service regulations allowed for provider proles and practices, namely the marshalling, conforming and maverick. Marshalling practices resulted in the enforcement of regulations and punished clients. Conforming practices usually created confusion amongst clients. Maverick practices saw staff collude to subvert regulations in favour of the interests of clients.
*School of Sociology, Faculty of Humanities and Social Sciences, La Trobe University, Bundoora, Victoria. Email: enrique@gatewaydental.com.au.
This work provides insight into the experience of receiving and providing dental emergency services in a public setting in Victoria. The participants stories reect on the meaning of the health and illness of their mouth. They also describe the context in which they are affected by dental treatment needs and how current policies and practices impact on their experience, with increasingly fragmented and impersonal interactions. Likewise, service providers describe the impact of increased demand for emergency treatment on their interactions with service users and fellow staff, in a political environment that imposes ideologies of efciency and economic accountability over those of professional discretion and care. This investigation aims to assist policy development in the area of public dental health care that takes into consideration the broader social context of oral health and illness of service users and professional interaction dynamics in public dental clinics. This in turn will hopefully address issues of disconnection between dental health policy makers, service providers and users that is manifest in victim blaming rhetoric and service irrelevance. Parts of this research were presented at TASA Conferences 2003 and 2004, and BSA Conference 2005.
A SEM evaluation of the effect of bicarbonate soda in root canal irrigation

N Leow; P Abbott, J Castro Salgado (Supervisors)*
The aim of this study was to investigate bicarbonate soda as a potential root canal irrigant during endodontic treatment and to compare it to commonly used irrigating solutions (EDTAC and NaOCl). As a secondary objective, a new and simple model was evaluated to determine whether it could be used to predictably create smear layer on the root canal walls and to test irrigating solutions for smear layer removal. An experimental model was developed using extracted human single-canal teeth sectioned into two halves mounted in a device that allowed root canal preparation and SEM evaluation. A uniform method of hand ling with Hedstro m les and distilled water was applied to the root canal walls to create a smear layer on each sample. This was evaluated by SEM examination and was found to be able to predictably create a smear layer. In the main part of the study, samples were irrigated with test solutions, including distilled water as a control, 1% NaOCl, 17% EDTAC and bicarbonate soda, at concentrations of 1%, 5%, 10% and 15%. Some samples were treated with multiple solutions in different sequences EDTAC NaOCl EDTAC and NaOCl EDTAC NaOCl. Each sample was then examined under a scanning electron microscope and representative images were captured. The images were de-identied and scored according to the amount of smear layer present and the number of dentinal tubules visible. The results were statistically analysed with the SAS system, using the GENMOD procedure. Complete smear layer coverage of the root canal walls was found in samples of all test solutions except when EDTAC was used alone. There were no signicant differences between the two irrigation sequences, EDTAC NaOCl EDTAC and NaOCl EDTAC NaOCl (p-value = 0.8903). There were also no significant differences between the groups with and without the use of bicarbonate soda (p-value = 0.9415). The model predictably produced an endodontic smear layer. Bicarbonate soda did not remove this smear layer and it did not provide any additional cleaning effects on the walls of root canals irrigated with EDTAC and NaOCl. EDTAC was the only irrigant tested that removed the smear layer.
*School of Dentistry, The University of Western Australia. Email: paul.v.abbott@uwa.edu.au.
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Abstracts
Perceptions of medical practitioners regarding oral and dental health in Western Australian aged care
JG Martins; LM Slack-Smith (Supervisor)*
The Australian population is following the international trend of an ageing society. Due to our ageing society, there are an increasing number of people living in residential care. As a result, the medical well-being of these individuals depends signicantly upon the care provided by the staff of the nursing home. Despite the overall improvement in the medical care of nursing home patients worldwide, there are studies indicating there is often suboptimal oral and dental care of patients. Though there have been previous studies on oral health care for nursing home residents, there is limited information available regarding the perspectives of general medical practitioners.
*Oral Epidemiology, School of Dentistry, The University of Western Australia. Email: linda.slack-smith@uwa.edu.au.
Through qualitative data collection, we consulted with general (medical) practitioners to determine their perceptions regarding oral care in nursing homes and views on improving the current system. Six general (medical) practitioners were interviewed about various aspects of nursing home oral health and access to dental care. Based on the results of this study, it was evident that oral care was perceived to be suboptimal in nursing homes; however, this was complicated by the residents poor general health, lack of access to dental care and the limited involvement of general medical practitioners in oral health issues. It would be benecial for patients to receive an initial dental review on admission to the nursing home and then receive regular dental care to improve their overall oral health.
Quantifying the selection of maxillary anterior teeth using intraoral and extraoral anatomical landmarks
K McNamara, J Sandhu, K James; A El-Sheikh, A Ellakwa, I Klineberg (Supervisors)*
The aim of this study was to determine if a relationship exists between intraoral and extraoral facial measurements that could assist dental practitioners in selecting aesthetically appropriate prosthetics in the anterior maxilla in the absence of pre-extraction records. A cross sectional design was used with a sample size of 120 participants. A questionnaire was used to identify selection criteria and a digital photograph was taken for facial measurements using digitally calibrated software. Ninety-eight participants met the selection criteria and were included in the study. Measurements of intraoral landmarks were taken from stone casts of maxillary impressions using calibrated digital calipers. Each measurement was completed by two assessors to obtain average values. The data were statistically analysed using SPSS version 10 software. The average values for intraoral maxillary landmarks were: central incisor width = 8.39 mm, circumferential canine tip to canine tip distance = 34.89 mm, arch width = 48.24 mm, arch length left = 45.24 mm. arch
*Jaw Function and Orofacial Pain Research Unit, School of Dentistry, The University of Sydney. Email: iven.klineberg@sydney.edu.au.
width right = 45.56 mm. The average values for extraoral measures were: intercanthal = 33.24 mm, interpupillary = 60.68 mm, interalar = 38.27 mm, commisure of lips = 50.61 mm. Differences existed within subgroups for all intraoral and extraoral measures. A weak positive correlation existed between intraoral (r < 0.4) and extraoral measurements (r < 0.38) that remained consistent when examined by gender. This study showed that the average length and width of the maxillary arch and interalar width were the anatomical landmarks that provided the strongest predictive relationship with anterior maxillary teeth (r = 0.380.4). Using these dimensions an average multiplying factor can be used to calculate maxillary incisor width or canine tip to canine tip distance. As the predictive strength is not strong, the authors recommend its use only as a preliminary guide for determining the width of the maxillary anterior teeth during the initial selection of articial teeth in the absence of preextraction records. The findings of this research were presented at the University of Sydney Faculty of Dentistry Research Day, September 2010.
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Abstracts
An investigation into dental trauma prevalence and understanding of its management in New South Wales rugby union clubs
K Metcalfe, EA Ilia, E Tsolakis; M Heffernan (Supervisor)*
The purpose of this study was to investigate the prevalence of oral trauma in adult, amateur rugby union players in New South Wales, Australia and to assess player understanding of injury prevention and rst aid management of dental injuries. There is a high prevalence of orofacial trauma in rugby union players. Mouthguards prevent and reduce the severity of dental injuries. Accurate and immediate rst aid management can prevent complications and improve the prognosis of traumatic dental injuries. It is important that players and support staff understand how orofacial trauma can be prevented and the most appropriate rst aid management for oral and dental injuries. Questionnaires were distributed to players in New South Wales, suburban and country rugby union clubs. The questionnaire asked players about their orofacial
*Faculty of Dentistry, Department of Endodontics, The University of Sydney, New South Wales. Email: m.heffernan@usyd.edu.au.
trauma experience, rst aid training and mouthguard use. Players were given dental trauma scenarios and were asked what rst aid management would be most appropriate for each scenario. The risk of orofacial trauma as perceived by the players was 6.67 ( 2.27) out of 10, with 10 representing the highest risk of injury. Results showed a high prevalence of orofacial trauma in rugby union players at 64.90%. The most common injury reported was laceration to the intraoral and extraoral soft tissues at 44.57% of all orofacial injuries. Participant knowledge of appropriate rst aid for dental injuries was poor, with only 24.44% recommending replantation of an avulsed tooth. Despite 97.30% of respondents knowing mouthguards prevent dental injury, only 76.89% wore mouthguards regularly. This study has highlighted the high prevalence of orofacial injuries in amateur rugby union players and the need for education about the prevention and rst aid management of orofacial injuries amongst players of contact sports and laypeople.
Exploring the dimensions of oral health literacy in Chinese mothers

M Nga-Man Liu; A Arora (Supervisor)*
Health education leaets are available to give dental advice to parents with preschool children. The aim of this study was to record the views of Chinese mothers living in south-western Sydney on the value of commonly used dental health education materials which gave advice on looking after the oral health of young children. This qualitative study was nested within a large cohort study in south-western Sydney. Chinese speaking mothers (n = 27) with young children were approached for a face-to-face, semi-structured interview at their home. Three dental leaets in English which gave advice on looking after young childrens oral health were sent to each mother prior to the interview. On the day of the interview, mothers were also given translated versions of the leaets for comparison. Interviews were recorded and subsequently transcribed verbatim. Transcripts
*Population Oral Health, Faculty of Dentistry, The University of Sydney, Westmead, New South Wales. Email: amit.arora@sydney.edu.au.
were analysed by interview debrieng and a thematic coding. Mothers reported that the leaets were not tailored to match the different levels of English literacy of the Chinese community and participants favoured health information material written in their mother language. However, translations had to take account of the Chinese culture as they did not reect Chinese family values. Mothers also felt that the information should be specic to gain a better understanding of the rationale for advice. Producers of dental health information literature for the Chinese population should not translate leaets directly from those intended for an English speaking audience. The text should reect Chinese culture and also offer specic advice. The findings of this research were presented at the IADR (ANZ Division) meeting, Kiama, New South Wales, September 2010.
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Abstracts
Immunohistochemical analysis of alveolar bone at oral implant sites

A Samakeh, H Badreddine; I Klineberg (Supervisor)*
Osseointegrated implants are used to restore function and improve aesthetics in patients with missing teeth. Osseointegration, a measure of implant success, is the direct structural and functional connection between living bone and the surface of a loaded-bearing implant. Osseointegration of implants varies with bone quality (mass and architecture) and implant material properties; which are important for osseointegration. Alveolar bone matrix protein expression levels over different age groups and implant sites have not been well documented. The aim of this pilot study was to analyse bone quality by measuring the differences between matrix protein levels in alveolar bone at different oral implant sites of patients of varying ages using immunohistochemical analysis. Our aim was to develop an immunohistochemical method to quantitatively measure collagen-I and osteopontin expression levels in alveolar bone at varying implant sites and to investigate any differences in expression levels over different age groups and implant sites. It is hypothesized that there are quantitative differences in matrix protein constituents of alveolar bone in different age groups and in different implant sites. Bone was harvested from alveolar bone implant sites of patients (n = 9) with a 2 mm trephine bur and xed in 10% neutral buffered formalin. The bone was decalcied with 0.5M EDTA pH 7.4 for six
*Jaw Function and Orofacial Pain Research Unit, Faculty of Dentistry, The University of Sydney and the Westmead Centre for Oral Health, New South Wales. Email: ivenk@mail.usyd.edu.au.
weeks. Following decalcication (n = 4), samples were immunohistostained for human collagen-I and osteopontin monoclonal antibodies and identied with a HRP-DAB chromagen system (brown) and a haematoxylin counterstain (blue). Percentage of sample stained for collagen-I and osteopontin per unit area was measured using the Aperio positive pixel algorithm. Pearsons correlation test was used to identify any trends. Four of the nine bone samples harvested decalcied using EDTA solutions. These four patients were 46, 59, 61 and 89 years of age and implant sites were 12, 26, 22 and 43 respectively. The percentage per mm2 of each sample expressing collagen-I was 7.72, 33.6, 4.84 and 23.3 respectively. The percentage per mm2 of each sample expressing osteopontin was 0.696, 8.30, 1.24 and 2.95 respectively. A very weak positive but nonsignicant correlation was calculated between patient age and collagen-I (r = 0.391, p = 0.609) and osteopontin (r = 0.139, p = 0.861). An immunohistochemical quantitative analytical method was developed to measure bone protein matrix quality; however a more efcient and reliable decalcication procedure needs to be identied. Our results show that anterior mandible and posterior maxilla have similar and better bone matrix quantity than anterior maxilla. Also, a very weak positive but non-signicant correlation was calculated between patient age and bone collagen-I and osteopontin expression. However, the sample size was too small to eliminate confounding factors (such as gender, dental histories and particularly the medications Raloxifene and Lipitor) and generate a meaningful correlation.
A three-year retrospective study of emergency visits at a community based oral health clinic for children
N Wong; C Tran, WK Seow (Supervisors)*
Although emergency visits are commonly encountered in dental practice, there is little information currently available regarding paediatric dental emergencies in Australia. This study investigated the number and nature of emergencies managed at a community based oral health clinic for children. From a register of emergency cases kept at a community based oral health clinic in a low socio 2011 Australian Dental Association
economic district of Queensland, we analysed the monthly number of emergency visits for children over a three-year period (January 2008 to August 2010). The types of emergency and treatments provided were categorized as caries-, trauma-, or orthodontic-related, and other types of emergencies. There was a total of 10 537 patient appointments seen at this facility, and 3038 were for emergency care
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Abstracts for children aged between 2 to 18 years of age. The mean age of a child presenting for an emergency visit was 9.0 4.0 years old. From 2008 to 2010, a mean of 196 86 cases reported for emergency care each month. Approximately 74% presented for caries*School of Dentistry, The University of Queensland. Email: nicholas.wong@uqconnect.edu.au.
related problems, 8% for trauma and 3% each fororthodontic and other reasons. Another 7% cases reported pain for reasons unrelated to caries or trauma, and 8% of cases presented for other reasons. Trends in the past three years showed that the number of emergency cases is increasing in this district, and dental caries constitute nearly three-quarters of all paediatric emergency appointments.
The incidence of chronic pain following successful endodontic treatment

GH Yoo, ML Lawless, A Forte; CC Peck (Supervisor)*
There are occurrences in which chronic persistent tooth pain following endodontic treatment does not respond to dental treatment and such cases may be attributed to non-odontogenic sources including neuropathic pain disorders induced by nerve injuries sustained during endodontic procedures. Epidemiological research estimates an incidence of chronic neuropathic pain between 3% to 12%. However, the dental profession remains largely in the dark about the risk and incidence of pain persisting longer than 6 months among dened populations due to a lack of accepted diagnostic and treatment criteria. Without the capability to condently identify risk factors for the occurrence and set criteria for diagnosis of neuropathic tooth pain, treatment remains empirical at best. Through a prospective study our aim was to determine the incidence of chronic pain following successful endodontic treatment in a private and public health population within Australia. We also attempted to identify predictors for the development of nonodontogenic chronic pain following clinically successful root canal therapy. Ninety-seven patients teeth requiring endodontic treatment were examined clinically and radiographically and a detailed pain history and questionnaire was
*Faculty of Dentistry, The University of Sydney, New South Wales. Email: chris.peck@sydney.edu.au.
obtained pre-treatment. The inclusion criteria for the participants were those requiring endodontic treatment. Clinical and radiographic status was checked throughout treatment and patients were followed up at 3- and 6-months post-obturation via phone interview to determine the presence of pain associated with the treated tooth. Patients with persistent pain at 6-months post-treatment associated with the treated tooth were invited for a clinical and radiographic exam, and were asked to complete a descriptive pain survey. Of the 97 patients who were treated for endodontic treatment, 7 (6.7%) presented with persistent pain at 6 months after successful endodontic treatment. Successful endodontic treatment was determined by the absence of clinical and radiographic signs of pathology. The only statistically signicant variable contributing to the presence of chronic pain was a history of frequent headaches (p = 0.029). These results suggest that the incidence of chronic neuropathic tooth pain after successful endodontic treatment may be less common than what the current literature shows. The single signicant predictor for chronic pain at 6 months was shown to be a history of frequent headaches. The results also suggest that the burden of post-endodontic pain is not uncommon but the cause of which may not be directly due to the integrity of the root canal treatment itself.
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Abstracts
RW Hession Award 2008

Remineralization of natural white spot lesions and their characterization by X-ray microtomography and synchrotron X-ray microtomography analysis
NJ Cochrane,* P Anderson, GR Davis, DJ Vine, S Rubanov, CDL Thomas,* EC Reynolds*
White spot enamel lesions are a stage of the carious process where mineral has been lost from the subsurface with an intact surface layer overlying the mineral poor region. These lesions can be clinically assessed as either active or inactive and are ideally managed by non-invasive treatments such as remineralization. The repair of damaged crystals requires ions to diffuse into the lesion body. This has renewed interest in the structure of white spot lesions, in particular their surface layers and how they can be modied to promote remineralization. One suggested modication technique is the use of acid etchants to remove or increase the porosity of the surface layer. The visualization of the changes produced by remineralization treatments at a nanometer resolution in three dimensions would improve our understanding of this process. High-resolution coherent diffractive X-ray imaging can be combined with computed tomography to reconstruct three-dimensional volumes spanning micrometres with nanometre resolution. However, this technique requires small samples in the micrometre range suspended in the eld of view by a minimally diffractive material. The aims of this study were to: (1) characterize 12 natural white spot lesions using X-ray microtomography; (2) determine the effect of acid etching on the remineralization of articial and natural white spot lesions; and (3) develop a sample preparation method to conduct Synchrotron high-resolution coherent diffraction tomography on enamel and dentine samples. Twelve molars with long buccal white spot enamel lesions were collected, photographed and sectioned to produce three 500 lm thick sections from each. The sections were mounted and imaged using quantitative X-ray microtomography at a 15 lm voxel size. Line proles through the middle of each 3D image were taken using Image J and analysed to measure the mineral densities at various points across, and deep to the lesion. The depth from the surface to various points in the lesion were measured. A further six pathology-free molars and three molars with long buccal lesions were collected, photographed and sectioned to produce four and two adjacent 500 lm thick sections from each respective tooth type. Articial lesions were prepared by exposing two sections from each sound tooth to a Carbopol demineralization buffer
for 12 or 30 days. One of each of the paired sections of articial or natural lesion was etched with a 15% HCl etchant for 2 or 48 minutes respectively. The longer etching time for the natural lesions was due to their variable surface layer thickness which was determined via X-ray microtomography. The sections were mounted in a scanning microradiography system and exposed daily to a remineralization solution (2.5 mL) for 10 days. The rate and amount of remineralization was determined by change in X-ray absorption with time. Samples of enamel and dentine for synchrotron analysis were prepared from sections of tooth using a FEI Nova dual beam focussed ion beam system. A large deep trough was prepared around a 10 lm2 region of interest. A 1 lm2 small hollow was prepared in the surface of the block and a bre optic needle with a 70 nm diametre tip inserted using a micromanipulator. Platinum was deposited into the 1 lm2 hollow to connect the block to the bre optic needle. The stage was tilted and the <10 lm3 block freed from the section. The block of enamel or dentine on the needle was then transported to the SPring 8 Synchrotron in Japan for high-resolution coherent diffraction tomography. The surface layer thickness of the natural lesions ranged between 35 and 130 lm with the maximum mineral density in this layer being between 74% and 100% of that of sound enamel. The average mineral density across the lesions varied widely from 1.73 to 2.48 g cm3. No signicant differences could be found between the active and inactive lesions. However, for a depth matched active and inactive lesion the active lesion had a more porous surface layer than the inactive lesion. The rate of remineralization followed a linear pattern for both articial and natural lesions. After remineralization 15 7 and 12 4 lg hydroxyapatite was returned to the control and etched 12 day articial lesions respectively. There was signicantly less mineral returned to the etched 30-day articial lesions (13 6 lg) compared with the control (21 4 lg). However, acid etching did improve the remineralization of the natural lesions (Control: 9, 5, 6 lg Etched: 11, 9, 20 lg respectively). The FEI Nova dual beam focused ion beam system was able to prepare blocks of enamel and dentine in a micrometre range that were connected to a minimally diffractive bre optic needle mount. These small
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Abstracts samples were suitable for analysis with high-resolution coherent diffraction tomography at the SPring 8 Synchrotron. The white spot enamel lesions studied were highly variable with surface layers of considerable thickness. Acid etching did not improve the response of articial lesions to remineralization. The remineralization of natural lesions could be improved by etching. However, a large amount of mineral was lost in the etching process. The FEI Nova dual beam focused ion beam system could be used to prepare samples suitable for Synchrotron high-resolution coherent diffraction tomography. Published: Cochrane NJ, Anderson P, Davis GR, Adams GG, Stacey MA, Reynolds EC. An X-ray microtomographic study of natural white spot enamel lesions. J Dent Res 2011 (in press). The findings of this research were presented at the European Organisation for Caries Research meeting, Montepellier, France, 2010 and the European Organisation for Caries Research meeting, Kaunas, Lithuania, 2011.
*Oral Health Cooperative Research Centre, Melbourne Dental School, Bio21 Institute, The University of Melbourne, Melbourne, Australia. Centre for Oral Growth and Development, Barts and The London, Queen Marys School of Medicine and Dentistry, London, United Kingdom. Centre of Excellence for Coherent X-ray Science, School of Physics, The University of Melbourne, Melbourne, Australia. Electron Microscopy Unit, Bio21 Institute, The University of Melbourne, Melbourne, Australia. Email: n.cochrane@unimelb.edu.au.
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Abstracts
Colin Cormie Grant 2010

Analysis of remineralization produced by various products using transverse microradiography and surface microhardness
TK Park; NJ Cochrane, EC Reynolds (Supervisors)*
A goal of modern dentistry is to treat the early stages of dental caries through non-invasive remineralization treatments. Recently, a number of remineralization technologies (Recaldent CPP-ACP, Novamin, functionalised tricalcium phosphate, amorphous calcium phosphate, hydroxyapatite) have become commercially available, purporting to supply the ions required for remineralization. The aim of this in vitro study was to explore the pattern and extent of remineralization produced by these products using surface microhardness and transverse microradiography. Seventy-seven extracted human molar teeth were prepared and allocated to 11 groups, consisting of three control groups and eight test groups. The control groups included an artificial saliva solution (no product control), standard fluoride toothpaste (fluoride control) and a CPP-ACFP solution (positive control). The test groups consisted of various products diluted in artificial saliva, with the fluoride concentration of the solutions (if contained in the product) standardized at 100 ppm. The total, soluble and free ion concentrations of each solution were measured using ion
*Oral Health Cooperative Research Centre, Melbourne Dental School, Bio21 Institute, The University of Melbourne, Victoria. Email: n.cochrane@unimelb.edu.au.
chromatography. Demineralized enamel subsurface lesions were exposed to the various solutions for 10 days and analysed for mineral changes. All calcium phosphate containing products produced greater remineralization than the artificial saliva alone. Those that contained Recaldent had the greatest remineralization efficacy with mineral deposition observed throughout the lesion body. Amorphous calcium phosphate and functionalised tricalcium phosphate technologies produced greater remineralization compared to the standard fluoride toothpaste but less than the Recaldent containing products. There was no significant difference in the level of remineralization produced by the Novamin and hydroxyapatite containing products compared to the standard fluoride toothpaste. The amount of remineralization achieved was significantly correlated with the apparent activity of free calcium ions in the solution. Surface microhardness was found to correlate poorly with transverse microradiography. Additionally, surface microhardness did not provide information on the pattern of subsurface remineralization and was found to be negatively affected by surface precipitation. The product containing Recaldent plus fluoride was found to be the best product for returning mineral to subsurface lesions in this in vitro model.
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Australian Dental Journal Research Supplement 2011

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Volume 56 Number 4 December 2011

Australian Dental Journal

SPECIAL RESEARCH SUPPLEMENT

The ofcial journal of the Australian Dental Association

Australian Dental Journal

ADRF Special Research Supplement

ADRF Research Grant Abstracts

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S16 S17 S18 S18 S19

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S38 S39 S40 S40 S41

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ADRF Undergraduate Research Grant Abstracts

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RW Hession Award 2008

Colin Cormie Grant 2010

ADRF Research Grant Abstracts

2011 Australian Dental Association

Dental issues in Australian newspapers: a pilot study

*School of Dentistry, The University of Western Australia. Email: linda.slack-smith@uwa.edu.au.

Effect of PDGF on bone formation around dental implants

2011 Australian Dental Association

Identication of molecular markers of oral epithelial stem cells

Correlation of serum and gingival crevicular uid cytokines in obese subjects

2011 Australian Dental Association

Structural and functional changes underlying orofacial pain

2011 Australian Dental Association

2011 Australian Dental Association

An evaluation of alternate splinting adhesives in the management of dental trauma

Neurophysiological changes underlying orofacial pain

Association of strong premature contacts and tooth mobility using T-Scan

Porphyromonas gingivalis GroEL and atherosclerosis in a mouse model

Mucosal inoculation of Porphyromonas gingivalis and atherosclerosis

Predicting unscheduled dental visits for members of the military

2011 Australian Dental Association

2011 Australian Dental Association

The effect of remineralization treatments on demineralized dentine: an in vitro study

*School of Dentistry, The University of Queensland. Email: emmamoloney@optusnet.com.au.

GABA in human dental pulp

2011 Australian Dental Association

Process analysis of the Monitor Dental Practice programme implementation

2011 Australian Dental Association

Characterization of the response to Porphyromonas gingivalis using a co-culture method

2011 Australian Dental Association

Survival of rat dental pulp cells in vascularized tissue engineering chambers

*Melbourne Dental School, The University of Melbourne, Victoria. Email: mastacey@unimelb.edu.au.

Factors inuencing learning of psychomotor skills by dental students

A controlled study of the oral health of twin children

Dental insurance, level of cover and dental service use

*School of Dentistry, The University of Queensland. Email: srinivas.varanasi@uq.edu.au.

*School of Dentistry, The University of Queensland. Email: srinivas.varanasi@uq.edu.au.

2011 Australian Dental Association

The effect of exercise training on jaw muscle recruitment strategies

*Melbourne Dental School, The University of Melbourne, Victoria. Email: keyapp@hotmail.com.

*School of Dentistry, The University of Adelaide, South Australia. Email: peter.zilm@adelaide.edu.au.

2011 Australian Dental Association

2011 Australian Dental Association

2011 Australian Dental Association

ADRF Undergraduate Research Grant Abstracts

*School of Dentistry, The University of Queensland. Email: megan.cable@uqconnect.edu.au.