The main goal of bone repair is to regenerate pre-existing properties and restore tissue integrity and function. It has been reported that bone contains numerous growth factors which are proposed to be released from the matrix during injury and mediate the repair process. These molecules act in synergistic action causing recruitment of progenitor stem cells to sites of bone injury, which then proliferate and differentiate into mature bone synthesising cells capable to initiate repair processes. It has been demonstrated that combinations of growth factors, such as the combinations found in the bone matrix, may be more effective in promoting bone healing compared with single growth factor therapy. This project focuses on understanding bone repair processes by stimulating ex vivo fractured rat mandible model with either endogenous growth factors released by chemical treatment or by exogenous single growth factor therapy and investigating their effects on cellular behaviour. This project utilised the ex vivo mandible model as a promising alternative to current model and fractures were made within the ex vivo mandible slices to mimic bone fracture repair scenario. In summary, ex vivo experimental models were used successfully to investigate mechanism of bone repair. The results demonstrated that bioactive growth factors, particularly TGF-β1, BMP2 and VEGF successfully released from the bone matrix by EDTA, citric acid and calcium hydroxide. These growth factors found to affect cellular behaviour, by influencing proliferation and differentiation of osteoprogenitor cells. Calcium hydroxide derived endogenous growth factors mediated the repair process of mandibular bone greater than exogenously applied BMP2. Calcium hydroxide may provide a novel therapeutic approaches to utilise the synergistic effect of cocktail growth factors entrapped in bone matrix to stimulate optimal bone regeneration and avoid issues regard single growth factor therapy.

Despite the rich oral microflora, infections within the oral cavity are rare. Rapid wound healing within the oral mucosa occurs, potentially due to the presence of oral mucosa lamina propria-progenitor cells (OMLP-PCs). OMLP-PCs are a novel population of multipotent cells known to possess immunosuppressive properties,through contact-independent mediated mechanisms. Many immunomodulatory soluble factors are also documented to have dual functions as antimicrobials; leading to the hypothesis that OMLP-PCs possess antibacterial properties in addition to their published immunoregulatory actions. The aim of this study was to investigate the antibacterial properties of OMLP-PCs and to define the mechanisms of action. A further aim of this study was to determine whether the antibacterial potential of OMLP-PCs was affected during disease, specifically Graft Versus Host Disease (GVHD). The antibacterial properties of OMLP-PCs were compared between cells isolated from healthy donors and patients with oral chronic GVHD. During this study it was determined that OMLP-PCs possess constitutive antibacterial properties against Gram positive and Gram negative bacteria which are mediated through the release of soluble factors. LL37 and Indoleamine 2,3-Dioxygenase are known to mediated the antibacterial properties of bone marrow-mesenchymal stem cells, however this study determined that these factors did not play a role in the OMLP-PCs antibacterial effects. It was established that osteoprotegerin, haptoglobin and prostaglandin E2 in part mediate the antibacterial effects of OMLP-PCs. For the first time, direct antibacterial properties of osteoprotegerin were demonstrated against Gram positive bacteria. Furthermore, OMLP-PCs isolated from GVHD patients did not display antibacterial properties. It was further established that the secretion of innate cell chemoattractants was dysregulated in OMLP-PCs isolated from GVHD patients compared to healthy controls. This finding demonstrates that during GVHD, the oral mucosa is unable to regulate the oral microflora and sufficiently recruit innate immune cells during infection.

The role that dental team members can play in the smoking cessation field is largely unevaluated. The work of this PhD thesis encompasses two phases, the first was to develop a means of determining smoking status, using analysis of continine, a nicotine metabolite. The second phase looked at the efficacy of dental hygienist-delivered smoking cessation advice in a dental setting. The aim of the first study was to compare continue levels in different biological fluids collected from both smokers and non-smokers, and to relate the findings to self-reported smoking status. Patients recruited to the study were asked to provide samples of urine, blood and saliva (both stimulated and unstimulated). Data collected from patients by questionnaire included information on smoking behaviour, such as daily number of cigarettes smoked, and environmental exposure to smoke. Following sample collection, patients were asked to rate the acceptability of each sampling method. Samples were analysed using enzyme immunoassay (EIA) kits. In total, 80 patients participated, with 49 smokers and 31 non-smokers. There was clear differentiation between smokers and non-smokers (p<0.001) for all the different samples in terms of cotinine concentration. A significant relationship was seen between cotinine levels and daily number of cigarettes for both salivas and urine (all p < 0.001) but not for serum. Participants found serum and urine collection methodologies 'very acceptable' (67% and 66%, respectively) whereas 9% found collection of stimulated saliva 'not at all acceptable'. Thus, continine, as analysed by EIA kits, whatever the collection method, shows good differentiation between smokers and non-smokers. Salivary samples have the advantage of being non-invasive. However, collection methodology is important, as continine levels may vary.

Over the past few decades stem cells have been extensively investigated due to their potentially invaluable therapeutic use. Embryonic stem cells (ESCs) have wide-ranging therapeutic applications in tissue repair and regeneration due to their pluripotent properties and their ability to self-renew indefinitely. However, ethical concerns surround their use and hence alternatives are sought. Adult stem cells (ASCs) have been isolated from various adult tissues including the oral mucosa lamina propria (OMLP). This study aims to isolate ASCs from the OMLP, reprogram these cells to induced pluripotent stem cells (iPSCs) and determine the potential for both to differentiate into functional neurons due to the limited regeneration of neurons in the central nervous system. Such investigations into strategies for the treatment of neural damage are invaluable and timely due to current limitations in the availability of human-derived cells for potential autologous or allogeneic tissue repair. OMLP-PCs represent an ideal cell source for use in regenerative medicine given their ease of isolation, proliferative potential, multipotent properties and immunosuppressive activities. Work in this Thesis has now demonstrated that these oral progenitors expressed numerous pluripotency markers and for the first time, that they could be reprogrammed to iPSCs utilising safer, non-integrating plasmids, thus increasing their potential for use in clinical applications. OMLP-iPSCs were positive for a number of pluripotent stem cell markers including SSEA-4, SSEA-5, TRA-1-60, TRA-1-81, Oct-4 and Sox-2. Moreover, their expression of early stage germ layer markers indicated their potential to differentiate into cell types of the mesoderm, endoderm and ectoderm. OMLP-PCs were also demonstrated within this Thesis to differentiate down an early neural lineage as evidenced by the presence of typical neural markers (Nestin, βIII tubulin, MAP-2 and NF-M). The presence of both ligand-gated and voltage-sensitive calcium channels indicated some limited potential functional phenotype. Unfortunately, utilising the same neural differentiation methodology, OMLP-iPSCs were not able to be similarly driven down a neural pathway. None-the-less this data suggests that OMLP-PCs and OMLP-iPSCs may hold great promise for a wide range of regenerative medicine applications

Streptococcus pneumoniae, also known as the pneumococcus, is an important human pathogen, with high burdens of disease and mortality worldwide. There are over 90 serotypes of this pathogen, demonstrating the vast amounts of diversity present. Currently, there are two pneumococcal vaccines, both targeting the polysaccharide capsule. However, one vaccine is ineffective in the paediatric population, whilst the other only targets a minority of disease-causing serotypes, and has increased disease caused by serotypes not present in the vaccine. One solution is a new pneumococcal vaccine targeting a protein virulence factor possessed by all pneumococci, which would afford cross-serotype protection. As a result, it is important to assess the diversity of pneumococcal virulence factors in order to determine their potential as vaccine candidates, as excess diversity present may prevent full serotype-independent protection of a vaccine. Furthermore, diversity studies offer important insight on pneumococcal biology, epidemiology and pathogenesis. The diversity in the toxin pneumolysin (Ply) was greater than previously thought, with 14 protein alleles discovered. However, diversity remained significantly lower than surface-exposed virulence factors, indicating this toxin may be more suitable as a vaccine candidate. Furthermore, all 14 alleles were recognised by polyclonal antibodies, showing the potential cross-serotype protection of a vaccine targeting this toxin. A novel non-haemolytic Ply allele was associated with clones recently expanding in the pneumococcal population, as well as serotypes associated with outbreaks of pneumococcal disease. The non-haemolytic toxin may therefore play a role in driving clonal expansion in certain genetic backgrounds, or be involved in establishing outbreaks of pneumococcal disease. The diversity in the neuraminidase A (NanA) enzyme was significantly higher than in Ply, with many point mutations, mosaic blocks and insertions regions present in 18 divergent alleles. This level of diversity should not be prohibitive to the use of this protein as a vaccine candidate, as polyclonal antibodies recognised 4 NanA alleles of significant diversity, indicating the possibility of cross-serotype protection. The role of NanA in pathogenesis of pneumococcal haemolytic uraemic syndrome (p-HUS) was investigated, although there was no correlation between p-HUS and NanA allele or activity. The novel discovery that pneumococcal NanA was inhibited by viral neuraminidase inhibitors of influenza allowed insight into the synergistic relationship between these two deadly pathogens, and showed for the first time that treatment with these drugs acts on both the primary and secondary pathogen. One of these inhibitors, Oseltamivir, was found to have potential in treating secondary pneumococcal pneumonia, which may help decrease the significant burden of this disease, as well as reducing the over-reliance on antibiotics for treating pneumococcal diseases. Homologues of Ply and NanA were identified and characterised in the related species Streptococcus mitis and Streptococcus pseudopneumoniae, giving insight into the evolutionary relationships between these species. Furthermore, the presence of these homologues in related species gives rise to the possibility of pneumococci acquiring altered genes through horizontal gene transfer. The selective pressure of a vaccine targeting these proteins may give evolutionary advantage to these pneumococci, resulting in evasion of a vaccine. Microarray studies have been used to assess pneumococcal diversity at a genome-wide level. Gene expression studies identified candidate genes which may play a role in p-HUS pathogenesis. Further studies into this area will improve the diagnosis and treatment of this disease. Furthermore, a large number of established pneumococcal virulence factors, many of which are vaccine candidates, were found to have homologues in closely related commensal species. These results must be taken into consideration for future protein-based pneumococcal vaccine design.

The work was undertaken to examine the effects of nutritional deficiencies on cancer induction. Two of the most common and widely distributed nutrients, iron and folic acid, were examined to evaluate the effects of their deficiency on animals. The Syrian golden hamster was the animal model for all experimental work. In the first part of the study an attempt was made to induce iron deficiency in young adult male hamsters by feeding iron deficient diet coupled with repeated venesection of 1.5ml every two weeks. Following twelve weeks on this regime a superficial biopsy was taken, on week 13, from the medial wall of one pouch in each hamster in order to evaluate the effect of iron depletion on the epithelial compartment thicknesses. After allowing the biopsy sites to heal for two weeks, a solution of 0.25% DMBA in acetone was painted, three times per week, for eight weeks, on a defined one square centimetre area in each pouch of each hamster of the experimental and control groups. The hamsters were then maintained on the same dietary regimes for twelve weeks before being killed at the beginning of week 37 for analysis. Iron deficiency anaemia could not be induced in the experimental animals of this study. The effect of the iron deficient diet on epithelial compartment thicknesses at the stage of the biopsy was not clear. However, restriction of iron intake did cause animals to develop significantly fewer grossly seen tumours and histologically identified carcinomas than control animals. In the second part of this thesis an attempt was made to investigate alternative hamster dietary components that have less iron contamination than the diet given in the first part of this thesis. Casein and calcium lactate were the main contributers to iron in the hamster diet. Casein could not be substituted by another source of protein for hamsters. However, other sources for calcium with less iron contamination were available and therefore investigated in this part of the study. Three groups of young adult male and female hamsters were given the fully nourishing powdered diet used in previous studies. However, calcium lactate was substituted for by either calcium acetate, calcium chloride or calcium sulphate in each group. None of the three diets was accepted by the animals and many of them died of starvation. When calcium salts were replaced by calcium lactate the surviving animals accepted the diet and recovered quickly afterwards. This study proved that calcium lactate could not be substituted by any other calcium salt with less iron content and therefore iron contamination in the hamster diet could not be further reduced by this method. In the third part of this thesis the effect of nutritional folate deficiency on cancer induction was studied. A group of young adult female hamsters was given folate deficient diet for four weeks. On week 5, DMBA in acetone at a concentration of 0.25% was painted on a defined one square centimetre area of the medial wall of each pouch in each hamster in folate deficient and control groups. The carcinogen was applied three times per week for eight weeks following which animals were maintained on the same dietary regimes for a further 13 weeks before being killed at the beginning of week 27 for the final analysis of the study. It was found that nutritional folate deficiency had significantly reduced the number of animals developing grossly counted tumours and histologically identified carcinomas. The folate deficient animals also developed significantly less tumours and carcinomas compared to control groups. In the last part of this thesis, the effect of combined iron and folate deficiency was examined for its role in carcinogenesis of the hamster cheek pouch. Two groups of young adult male hamsters were fed powdered diet lacking iron and folic acid and a third group was fed diet lacking iron only. One of the combined deficiency groups and the iron deficiency group were bled 1.0-1.3ml every week. On week 6 of the study DMBA in acetone at a concentration of 0.25% was painted three times per week for eight weeks on the same area of the pouch used in the previous studies. The animals were then maintained on the same experimental regimes for a further eleven weeks before being sacrificed, on week 25, for the final analysis of the study. In this study, iron deficiency anaemia was induced in animals of the bleeding groups. Animals in the group with combined iron and folate deficiency without bleeding showed low normal folate levels and normal haemoglobin levels. The two groups that were bled repeatedly showed iron deficiency anaemia. In all groups, the numbers of tumours counted grossly and the numbers of carcinomas identified histologically were significantly reduced compared to control animals in the previous studies. The folate deficient diet did not appear to influence the induction of iron deficiency. The studies reported in this thesis proved that nutritional folate deficiency not only reduces the incidence, but it also reduces the numbers of tumours and carcinomas in the hamster cheek pouch. Iron deficiency anaemia was also found to significantly reduce the numbers of tumours and carcinomas of the hamster cheek pouch. It was not possible to produce combined iron and folate deficiency under the conditions of these studies. However, animals fed on a diet lacking iron and folic acid had significantly reduced numbers of grossly seen tumours and histologically identified carcinomas in the cheek pouch in response to DMBA applications. In each of the reported studies, the nutritional deficiency of iron and folic acid, whether individually or combined was found to significantly reduce the growth rate of affected animals.

Ultrasonic cutting has existed in surgery since the 1950s. However, it was not until the end of the 20th century that advances in ultrasonic tool design, transduction and control allowed commercially viable ultrasonic cutting devices to enter the market. Ultrasonic surgical devices, like those in other power ultrasonic applications such as drilling and welding, require devices to be driven at high power to ensure sufficient output motion is produced to fulfil the application it is designed to perform. With the advent of novel surgical techniques surgeons require tuned ultrasonic tools which can reduce invasiveness while giving access to increasingly difficult to reach surgical sites. To fulfil the requirements of novel surgical procedures new tuned tools need to be designed. Meanwhile, it is well documented that power ultrasonic devices, whilst driven at high power, are inherently nonlinear and, if no attempt is made to understand and subsequently control these behaviours, it is likely that these devices will suffer from poor performance or even failure. The behaviour of the commercial ultrasonic transducer used in bone surgery (Piezosurgery® Device) is dynamically characterised through finite element and experimental methods whilst operating in conjunction with a variety of tuned inserts. Finite element analysis was used to predict modal parameters as well as stress levels within the tuned devices whilst operating at elevated amplitudes of vibration, while experimental modal analysis validated predicted resonant frequencies and mode shapes between 0-80kHz. To investigate the behaviour of tuned devices at elevated vibrational amplitudes near resonance, responses were measured whilst the device was excited via the burst sine sweep method. In an attempt to provide an understanding of the effects that geometry, material selection and wavelength of tuned assemblies have on the behaviour of an ultrasonic device, tuned inserts consisting of a simple rod horn design were characterised alongside more complex cutting inserts which are used in maxillofacial and craniofacial surgery. From these results the aim will be to develop guidelines for design of tuned inserts. Meanwhile, Langevin transducers, commonly known as sandwich or stack transducers, in their most basic form generally consist of four parts; a front mass, a back mass, a piezoceramic stack and a stud or bolt holding the parts together under a compressive pre-load. It is traditionally proposed that the piezoceramic stack is positioned at or close to the vibrational nodal point of the longitudinal mode, however, this also corresponds with the position of highest dynamic stress. It is also well documented that piezoceramic materials possess a low linear stress threshold, therefore this research, in part, investigates whether locating the piezoceramic stack away from a position of intrinsic high stress will affect the behaviour of the device. Through experimental characterisation it has been observed that the tuned devices under investigation exhibited; resonant frequency shifts, jump amplitudes, hysteretic behaviour as well as autoparametric vibration. The source of these behaviours have been found to stem from device geometry, but also from heating within the piezoceramic elements as well as joints with different joining torques.

Feline chronic gingivostomatitis (FCGS) is an inflammatory disease of the oral cavity that causes severe pain and distress. No specific treatment methods are available and little is known about its aetiology. The aims of this study were:- 1) to identify the bacterial flora, including uncultivable and potentially novel species, in healthy cats and those with FCGS, using 16S rRNA gene sequencing in combination with conventional culture methods; 2) to investigate the viral status of cats with and without FCGS; 3) to assess the immune response by investigating the expression of cytokine and Toll-like receptor (TLR) genes in tissue biopsies from normal cats and those with FCGS; 4) to investigate the histopathological changes in tissue biopsies from normal cats and those with FCGS, 5) to assess putative risk factors for FCGS by the use of a questionnaire-based study. Oral swabs, mucosal biopsies and blood were collected and the location of the oral lesions was recorded. A total of 32 cats with FCGS and 16 normal cats were included in the study. Bacteria were identified from swabs by use of 16S rRNA gene sequencing and by conventional culture methods. Blood samples and swabs were used for diagnosis of infection with feline leukaemia virus (FeLV), feline immunodeficiency virus (FIV), feline herpes virus 1 (FHV-1), feline calicivirus (FCV) and for blood biochemistry and haematology. Gene expression levels for TLR2, TLR3, TLR4, TLR7 and TLR9, and cytokines IL-1β, IL-4, IL-6, IL-10, TNF-α and IFN-γ mRNA were determined using quantitative PCR in biopsy samples from healthy cats and cats with FCGS. Histopathological examination of the tissue biopsies was done using hematoxylin and eosin (H&E) staining. In the healthy group, 16S rRNA gene sequencing demonstrated that the most prevalent bacteria were part of the Proteobacteria and Bacteroidetes phyla, plus a group of uncultured bacteria. The most prevalent species in the healthy group were Xanthomonadaceae bacterium (6.2 % of clones analysed), Capnocytophaga canimorsus (5.4%), Capnocytophaga cynodegmi (4.8%), Bergeyella species (4.5%) and Pasteurella multocida subspecies septica (4.4%). Uncultured bacteria accounted for 29% of the clones analysed. In the FCGS group most of the identified species were part of the phylum Proteobacteria. The most prevalent species in the FCGS group were P. multocida subsp. multocida (14.1%) P. multocida subsp. septica (11.5%), Pseudomonas sp. (7.3%), Tannerella forsythia (6.6%) and Porphyromonas circumdentaria (5.6%). A variety of uncultured bacteria represented 7.7% of all analysed FCGS clones. The culture data showed the most prevalent bacteria in the healthy group were P. multocida subsp. septica (9.9%), and uncultured bacteria (30.5%). In the FCGS group the most prevalent isolates were P. multocida subsp septica and P. multocida subsp. multocida (both 9.9%). Uncultured bacteria accounted for 21.7% of all isolates. FCV was detected in 71% of cats with FCGS and in 13.3% of normal cats. FeLV antigen was detected in 33.3% of normal cats but not in any cats with FCGS. FIV antibodies were detected in 3.4% of cats with FCGS and in 33.3% of normal cats. FHV-1 was detected in 6.9% of cats with FCGS, but was not detected any of the normal cats. In the FCGS group a significant increase was seen in the expression of TLR2 and TLR7 genes as well as TNF-α, IFN-γ, IL-1β and IL-6 cytokine genes. The healthy cats and cats with FCGS in the study that were found to harbour T. forsythia and P. circumdentaria showed an increase in the expression of several TLR and cytokine genes when compared to the group of cats in which these bacterial species were absent. The most severely inflamed sites in the oral cavity of cats with FCGS included the tissue lateral to the palatoglossal folds and the maxillary attached gingiva. Histopathological analysis of the tissue from the palatoglossal folds showed two types of infiltrates:- 1) a combination of lymphocytes and plasma cells, most often seen in the milder inflamed tissue samples; 2) a predominantly plasmacytic infiltration, most often seen in the severely inflamed tissue samples. Preliminary data from a questionnaire-based epidemiological study showed that the presence of potential environmental stress factors such as no ability to roam outdoors and the presence of more than one cat in the household is significantly higher in cats with FCGS when compared to normal cats. This study highlights the possibility of a multifactorial aetiology for FCGS in which FCV, specific bacteria and stress factors may play an important role. Although species from the Bacteroidetes phylum appeared to be capable of eliciting an immune response, these were not the most prevalent species in the FCGS group. A shift could be seen in the composition of the bacterial flora when healthy cats and those with FCGS were compared.

This thesis examines the development of a novel in vitro biological caries model and its suitability in testing the efficacy of anti-caries approaches. Dental caries remains a public health concern worldwide; with extensive treatment costs and impacts on quality of life. Ineffective removal of all dental plaque from tooth surfaces after brushing, the insufficient delivery of anti-caries therapies; along with continuing shifts towards high frequency, sucrose-rich food consumption, expedites the caries disease process. It is, therefore, important to explore caries risk and development at these sites, particularly when representatively assessing the efficacy of a test agent in preventing caries. This caries model enabled the study of the anti-caries effects of fluoride to assess its efficacy in conditions simulating the modern diet. The current methodology employed the Constant Depth Film Fermenter (CDFF) to investigate the caries disease process in response to fluoride delivered continuously; twice and thrice daily; and at different concentrations. The approach is the first in CDFF research modelling caries inclusive of a biologically relevant microcosm biofilm in addition to enamel demineralisation. Specific members of multispecies biofilm were selectively enumerated using traditional microbiological culture techniques whilst caries was simultaneously quantified with Transverse Microradiography (TMR), Quantitative Light-Induced Fluorescence (QLF), and Non-Contact Surface Profilometry (NCSP). The fluorescence of biofilm illuminated by QLF was also investigated. Results indicated that quantities of total or specific members of the microbial community are not direct indicators of caries risk and turning focus towards the metabolism of oral biofilm bacteria, and how it may be affected, is vital in caries research. TMR and QLF agree when quantifying caries whilst NCSP shows promise in studying surface changes. At 0.05 ppm, fluoride was unable to exert a significant anti-caries effect despite being continuously present during and between sucrose exposures. Laminated lesions confirmed the importance of maintaining elevated levels of fluoride in the oral environment throughout the day. At higher concentrations (1,450 and 228 ppm fluoride) the anti-caries efficacy of fluoride when supplied in frequent applications throughout the day was confirmed. A third application of fluoride did not appear to additively benefit enamel, since all strategies were effective after 10 days regardless of frequency. Nonetheless the increased plaque fluoride reservoir and subtle antimicrobial effects than in twice daily pulsed biofilm, mean the benefit of a third application is likely more discernible in the long term. Scanning Electron Microscopy (SEM) and Energy-Dispersive X-ray Spectroscopy (EDX) elucidated significant calcium fluoride deposits of enamel surfaces beneath biofilm exposed to 1,450 ppm fluoride continuously. In conclusion, the CDFF can produce multispecies biofilm under conditions similar to those of the oral milieu and investigate its cariogenicity in response to diet and experimental anti-caries agents. The model could be examined using an array of techniques to obtain information about aspects of the biofilm, the substratum, and to validate upcoming methods in an orally representative environment. In this regard, the current study contributes not only to enamel caries research but to biofilm research in general by minimising variation.

In forensic investigations involving severely burned human remains, dental analysis stands alone as other means of identification are often destroyed. Therefore, the aim of the present work was to investigate the influence of duration of heat exposure and heating regimes regarding the macroscopic, compositional, structural and crystalline alterations of dental tissues. Experiments were carried out using 215 freshly extracted human teeth, exposed to temperatures of 400 to 1000°C. Shrinkage and shape preservation was analysed using micro-CT, whilst crystalline alterations were evaluated with synchrotron-based SAXS/WAXS experiments. The alterations of organic constituents were assessed using TGA and FTIR. Moreover, calibrated digital photographs were used to document and analyse colour alterations. Although dentinal shrinkage was found at 400°C, tooth morphology was well preserved even at 1000°C. Surface colour alterations were linked to the degradation of organic components, and were highly dependent upon the duration of heat exposure and the heating regime, whilst crystalline alterations were less influenced by these factors. The combination of novel analytical approaches enabled the documentation and quantification of heat-induced alterations of dental tissues, providing results that can be used in the forensic identification process and allow an improved estimation of the cremation temperature range based on human dental remains.