Melbourne Dental School - Theses
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ItemInvestigations into the effects of head and neck radiotherapy and the potential anticariogenicity of casein phosphopeptide-amorphous calcium phosphate and stannous fluoride( 2017)Radiotherapy is the primary treatment modality for most head and neck cancers, in particular, nasopharyngeal carcinoma (NPC). The use of advanced radiotherapy techniques such as intensity modulated radiotherapy (IMRT) improves tumour targeting whilst minimising radiation dose to normal non-target tissues. However, salivary gland dysfunction still persists as the salivary glands remain in the treatment portal. The major post-irradiation oral complication is dry mouth resulting from hyposalivation with subsequent development of radiation caries, a highly destructive form of dental caries which has a rapid onset and progression. The use of high strength fluoride products, 5,000 ppm fluoride as 1.1% sodium fluoride (NaF) has been recommended in the United States and Europe as the standard of care for the control of radiation caries. These products are not available in Singapore and the recommended radiation caries control is via the use of 1,000 ppm fluoride as 0.4% stannous fluoride (SnF2). Fluoride-driven remineralisation is limited by the bioavailability of calcium and phosphate and the use of the saliva biomimetic, casein phosphopeptide amorphous calcium phosphate (CPP-ACP), has been reported to be effective as an adjunctive treatment to fluoride therapy in the management of early carious lesions in healthy individuals. The use of CPP-ACP to augment fluoride remineralisation in patients with hyposalivation has not yet been explored, in particular, as an adjunct to SnF2 therapy. In an in vitro six species polymicrobial biofilm model representative of supragingival plaque cultured on sound human enamel substrata and subjected to a high acid challenge, the rate of demineralisation produced by the individual CPP-ACP and SnF2 treatments was similar at 112.1 vol%min.µm/day, a significant 50.2% reduction compared with the control. For the same period, the combined CPP-ACP/SnF2 treatment resulted in a significant 72% reduction in demineralisation rate to 64.1 vol%min.µm/day. Significant biofilm compositional changes were produced by the CPP-ACP-based treatments, indicating a CPP-ACP prebiotic effect on the biofilm development favouring the growth of commensal species. The combined CPP-ACP/SnF2 treatment also produced the highest net remineralisation (30.6%) of enamel subsurface lesions in an in situ study. This in situ study is the first to show that the extent of remineralisation of CPP-ACP/F is dependent on the form of fluoride added; fluoride added as CPP-ACP/SnF2 exhibited significantly greater net remineralisation of enamel subsurface lesions compared with fluoride added as CPP-ACP/NaF at the same fluoride concentration. The CPP-ACP/SnF2 treatment produced the highest mean weight percent fluoride deposition throughout the depths of the lesions with the mineral deposited consistent with the formation of fluorapatite. The CPP-binding kinetics showed that the presence of stannous ions in the CPP-ACFP nanocomplexes enhanced the stability of the complexes even at low pH, delivering greater amounts of calcium, phosphate and fluoride ions to the tooth surface and promoting greater remineralisation of enamel subsurface lesions. This study is the first to show that the delivery of stable nanocomplexes to the tooth surface is critical to maximise remineralisation, providing better subsurface remineralisation in vivo. The adjunctive use of CPP-ACP with a SnF2/NaF regime was efficacious in a placebo-controlled randomised clinical study in NPC patients undergoing IMRT. The CPP-ACP group exhibited a significant reduction in coronal caries progression for total surfaces (47%); smooth surfaces (38%) and occlusal surfaces (79%) compared with the placebo group three months post-radiotherapy. This is of great benefit as these patients were at risk of caries even at the early stage of the cancer treatment as reflected by the significantly low salivary flow rates, pH and buffering capacity. Access to critical calcium, phosphate and fluoride ions required for remineralisation was restricted as evidenced by the significant decline in ion velocity of these ions and the mineral saturation indices. In summary, the superior pre-clinical in vitro and in situ results of CPP-ACP/SnF2 treatment in significantly reducing the rate of enamel demineralisation and promoting remineralisation were confirmed in vivo by the first placebo-controlled randomised clinical study in NPC patients undergoing IMRT. This novel study suggests that CPP-ACP/SnF2 has beneficial application for caries control in high risk individuals.
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ItemEnhancing remineralisation using casein phosphopeptide complexes( 2017)Casein phosphopeptide (CPP) complexes have been shown to promote remineralisation of dental tissues affected by dental caries. However, remineralisation takes time and can be limited by the delivery and composition of the remineralisation agent. The aim of this thesis was to enhance remineralisation by CPP complexes through clinical and laboratory studies assessing various chemical changes to the remineralisation process. Using an in vitro enamel remineralisation model, it was determined that intra-lesion serum albumin did not interfere with remineralisation by casein phosphopeptide stabilised amorphous calcium fluoride phosphate (CPP-ACFP). A high pH pre-treatment significantly increased remineralisation by CPP-ACFP. To expand on this finding, a cyclic in vitro remineralisation model tested intra-lesion pH modulation whereby enamel subsurface lesions were periodically exposed to CPP-ACFP (pH 5.5) and either sodium hypochlorite (pH 12.9), sodium hydroxide (pH 12.9) or distilled deionised water. Enamel subsurface lesions that had cyclic treatment with CPP-ACFP and sodium hydroxide were observed to have significantly higher remineralisation, displaying intra-lesion pH modulation enhanced remineralisation. Cyclic treatment with CPP-ACFP and sodium hypochlorite was observed to further demineralise and cause a surface precipitation due to a disadvantageous interaction of the treatment solutions. A second short-term cyclic in vitro remineralisation experiment revealed intra-lesion pH modulation with CPP-ACFP and sodium hydroxide was more effective than an equivalent exposure to CPP-ACFP alone. The use of x-ray microtomography (XMT) to measure remineralisation by CPP-ACFP in vitro was assessed using conventional polychromatic and monochromatic synchrotron x-ray sources. These methods of analysis were compared with transverse microradiography (TMR) analysis to investigate the accuracy and practicality of each method. XMT analysis from both x-ray sources detected remineralisation in enamel lesions however the amount of remineralisation detected was significantly less than that detected by TMR. Due to a range of artefacts unique to the x-ray source and the devices used, it was determined that XMT analysis of remineralisation under the conditions used was less sensitive compared with TMR. The remineralisation potential of a combined casein phosphopeptide stabilised amorphous calcium phosphate (CPP-ACP) and stannous fluoride (SnF2) solution was tested in vitro and in situ. The combined CPP-ACP and SnF2 solution showed significantly higher enamel remineralisation than all other treatments due to an increase in CPP complex stability and ion delivery. The interaction of a combined CPP-ACP and SnF2 solution with surface dentine in vitro displayed an organic ‘nanocoating’ suggesting stannous ions mediated CPP cross-linking and ion release at the dentine surface. A crossover clinical study was conducted on low caries-risk individuals to assess changes in the abundance of Streptococcus sanguinis in supragingival plaque following a two week intervention period chewing either CPP-ACP sugar-free gum, sugar-free gum or no gum. It was determined that chewing the CPP-ACP gum significantly increased the abundance of S. sanguinis, as well as other commensal, alkaline-producing microorganisms. This demonstrated chewing CPP-ACP gum exerted a prebiotic effect in supragingival plaque. The promising results expounded in this thesis indicate modifications to the composition and delivery of CPP complexes have the potential to improve the rate and amount of remineralisation.
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ItemMolar-incisor hypomineralisation: perception, clinical characteristics and associated risk factors in an Iraqi community( 2013)A growing awareness of the epidemiology of molar-incisor hypomineralisation (MIH) highlights the need to investigate the distribution and causes of this defect across a range of communities. Together with establishing the extent of the disorder comes a need to identify the cause and understand how to prevent MIH from occurring in the future. So that appropriate care can be provided to those with existing lesions, a clear picture of clinicians’ current level of knowledge about MIH is fundamental. Little research investigating the distribution of MIH-like opacities in the second primary molar (HSPM), exists. The dynamic properties of enamel hypomineralisation may predispose the affected tooth to deteriorate post-eruptively by developing dental caries in the presence of an unfavourable oral environment and absence of treatment intervention. The influence of salivary characteristics and existing caries experience on the clinical presentation of MIH is unknown. While investigations into the impact of the defect on the individual are lacking, the sequalae of untreated MIH include pain and high treatment costs which add to the public health burden of disease in the community. The present research project had two major aims: (1) to assess the perception of 146 Iraqi dental academics regarding the prevalence, severity and possible aetiological factors of MIH and HSPM (2) to investigate the distribution, risk factors and the impact of these defects on the oral health of 823 schoolchildren. The results indicated that the majority of the Iraqi dental academics (81.2%) encountered MIH in their clinical activities. Fewer than half of the respondents observed MIH affected teeth on a monthly basis. HSPM was less commonly seen than MIH. A variation in views about MIH specific aetiological factor/s was evident. Respondents advocated the need for clinical training regarding MIH-aetiological and therapeutic fields. Analysis of the prevalence and severity of the defect revealed 153 (18.6%) of the children examined had at least one affected first permanent molar (FPM) or FPMs and incisors and were considered as MIH-affected, while 53 (6.6%) had hypomineralisation defects in at least one SPM and were considered as HSPM-affected. Maxillary index teeth had the highest defect prevalence (61.5%). Demarcated creamy white opacities were the most frequent lesion type (48.7%). The severity of defects increased with age and the prevalence was greater in boys compared to girls. The more severe the lesion the greater was the involved tooth surface area. Of those with HSPM, 39.6% were diagnosed with affected FPMs simultaneously, whereas only 7.5% of them reported HSPM with affected incisor but not with affected FPMs. Analysis of the medical history revealed approximately 94% of the children with demarcated lesions had experienced one or more medical conditions compared with 70% of the defect free children. Post-natal health conditions were the most commonly associated health events in the MIH-affected group (33.3%), whereas peri-natal health conditions were the most frequently reported health events for HSPM-affected group (45.3%). When the data were split into the possible risk-effect groups, risk factors contributing to MIH were also identified as risk factors for HSPM. Analysis of the relationship between the defects with caries severity and saliva characteristics illustrated that hypomineralised-affected children have significantly higher mean caries scores compared to the non-affected group. Dentinal carious lesions were ten-times more frequent in teeth with post-eruptive enamel breakdown (PEB) than with teeth with opacities only. Low salivary flow rates (LSFR), moderately viscous saliva and low pH were significantly more common in the affected group. LSFR, moderate-and highly-acidic saliva were more likely to be associated with PEB. Analysis of the potential MIH-impact on oral health burden indicated that MIH-affected children had significantly higher frequency of seeking dental care than their non-affected counterparts (82.4%, 68.2%; respectively). They were over three times (OR 3.2) more likely to visit the dentist complaining of pain and were over six times (OR 6.4) more likely to seek dental care due to tooth sensitivity than their non-affected peers. Affected molars required more than twice the amount of restorative care than unaffected molars. No significant difference was found between the study groups in terms of tooth-brushing and toothpaste-use history, with brushing frequency “once-a-day” commonly reported in both groups (75.5%). Early exposure to fluoridated water appeared to have a protective effect for MIH (OR 0.4).
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ItemCharacterization of casein phosphopeptides( 1995)Casein phosphopeptides (CPP), containing the cluster sequence Ser(P)-Ser(P)-Ser(P)-Glu-Glu-, have been shown to stabilize amorphous calcium phosphate at neutral and alkaline pH and to be anticariogenic in various in vitro, animal and human experiments. Anticariogenic casein phosphopeptides (ACPP) therefore have commercial potential as toothpaste, mouthwash and food additives for the prevention of dental caries. The aim of this project was to comprehensively characterize the CPP produced under industrially-relevant conditions using the commercially available enzymes Novo trypsin PTN 3.0 S, Novo Alcalase® 2.4L and Pancreatin 4NF. To facilitate studies on the identification and characterization of CPP a simple and efficient purification procedure involving selective precipitation of Ca2+/ethanol-induced aggregates of the phosphopeptides from enzymic digests was developed. The individual peptides of the precipitates were purified using anion exchange FPLC and reversed-phase HPLC and then identified by solid-phase sequence analysis and amino acid composition analysis after vapour-phase hydrolysis. Prior to sequence analysis the phosphopeptides were covalently coupled to arylamine membranes and the phosphoseryl residues converted to S-ethylcysteinyl residues by calcium-ion-catalysed β-elimination in the presence of ethanethiol. Alternatively, in the event of low coupling efficiency to arylamine membranes, phosphoseryl residues were converted to S-ethylcysteinyl or S-propylcysteinyl residues and sequenced from polybrene-treated glass fibre discs. A method for the separation and identification of CPP using high performance capillary electrophoresis (HPCE) was also developed. The ability of HPCE to rapidly resolve phosphopeptides exhibiting varying degrees of phosphorylation, truncation and deamidation make it ideal for the product quality control of CPP. HPCE was also used to develop relationships between absolute electrophoretic mobility (µ) and peptide charge and size for CPP containing 2-5 phosphoseryl residues, with µ found to be proportional to q/M2/3 where q is the net negative charge and M is molecular mass of the peptide. The results showed that CPP can be produced using Novo trypsin and pancreatin with only minor modifications, relative to CPP produced using analytical-grade trypsin, such as slight truncation, deamidation and methionine oxidation. Deamidation and methionine oxidation most likely resulted from an elevated hydrolysis temperature and/or conditions employed during commercial casein (CN) production. Studies on the hydrolysis of casein at different enzyme:substrate (E/S) ratios by these enzymes suggest that for CPP production using Novo trypsin, a minimum degree of hydrolysis (DH) of 17.3% is required for maximal release of ACPP whilst for pancreatin, a DH of 19.0% is required. For pancreatin, it is recommended that this value is not exceeded so as to minimize the release of the truncated β-CN-4P(f7-24). CPP produced using alcalase were severely truncated relative to those prepared using Novo trypsin and pancreatin, resulting in the loss of residues suggested to be necessary for full anticariogenic activity. Decreasing E/S ratio slightly lowered the degree of truncation caused by alcalase, however, low E/S ratios also resulted in a reduction in CPP yield. It is likely that ACPP produced using alcalase would have much lower specific anticariogenic activity than those produced using Novo trypsin or pancreatin. In conclusion, it has been demonstrated that Novo trypsin PTN 3.0 S and Pancreatin 4NF are suitable enzymes for the production of CPP on a commercial scale and that Alcalase® 2.4L is unsuitable. For the production of CPP using Novo trypsin, a minimum DH value of 17.3% is required for maximal ACPP release whilst for pancreatin, a DH value of 19.0% is required. HPCE has been demonstrated to be an ideal method to monitor CPP quality.