Mineralization of calcium phosphate crystals in starch template inducing a brushite kidney stone biomimetic composite

Dicalcium phosphate dihydrate (brushite) and octacalcium phosphate (OCP) crystals are precursors of hydroxyapatite (HAp) for tooth enamel, dentine, and bones formation in living organisms. Here, we introduce a new method for biomimicking brushite and OCP in starch using single and double diffusion techniques. Brushite and OCP crystals were grown by precipitation in starch after gelation. The obtained materials were analyzed by infrared spectroscopy (IR), scanning electron microscopy (SEM), X-ray diffraction (XRD), and confocal laser scanning microscopy (CLSM). IR spectra demonstrate starch inclusion by peak shifts in the 2900–3500 cm–1 region. SEM showed two different morphologies: plate-shaped and needle-like crystals. Calcium phosphate/starch aggregates bear strong resemblance to prismatic brushite kidney stones. This may open up a clue to understand the mechanism of kidney stone formation.

Preparation and optimization of calcium fluoride particles for dental applications.

Fluorides are used in dental care due to their beneficial effect in tooth enamel de-/remineralization cycles. To achieve a desired constant supply of soluble fluorides in the oral cavity, different approaches have been followed. Here we present results on the preparation of CaF2 particles and their characterization with respect to a potential application as enamel associated fluoride releasing reservoirs. CaF2 particles were synthesized by precipitation from soluble NaF and CaCl2 salt solutions of defined concentrations and their morphology analyzed by scanning electron microscopy. CaF2 particles with defined sizes and shapes could be synthesized by adjusting the concentrations of the precursor salt solutions. Such particles interacted with enamel surfaces when applied at fluoride concentrations correlating to typical dental care products. Fluoride release from the synthesized CaF2 particles was observed to be largely influenced by the concentration of phosphate in the solution. Physiological solutions with phosphate concentration similar to saliva (3.5 mM) reduced the fluoride release from pure CaF2 particles by a factor of 10-20 × as compared to phosphate free buffer solutions. Fluoride release was even lower in human saliva. The fluoride release could be increased by the addition of phosphate in substoichiometric amounts during CaF2 particle synthesis. The presented results demonstrate that the morphology and fluoride release characteristics of CaF2 particles can be tuned and provide evidence of the suitability of synthetic CaF2 particles as enamel associated fluoride reservoirs.

Impact of Acquired Pellicle Modification on Adhesion of Early Colonizers

New preventive approaches against dental erosion caused by acidic drinks and beverages include fortification of beverages with natural polymers. We have shown that the mixture of casein and mucin significantly improved the erosion-inhibiting properties of the human pellicle layer. This study aimed to investigate the effect of pellicle modification by casein, mucin and a casein-mucin mixture on the adhesion of early bacterial colonizers. Test specimens of human tooth enamel were prepared, covered with saliva and coated with 0.5% aqueous (aq.) casein, 0.27% aq. mucin or with 0.5% aq. casein-0.27% aq. mucin, after which the adhesion of Streptococcus gordonii, Streptococcus oralis, and Actinomyces odontolyticus was measured after incubation for 30 min and 2 h. log10 colony-forming units were compared by nonparametric tests. All three bacterial strains adhered in higher number to pellicle-coated enamel than to native enamel. The protein modifications of pellicle all decreased the counts of adhering bacteria up to 0.34 log10/mm2, the most efficient being the casein-mucin mixture. In addition to the recently shown erosion-reducing effect by casein-mucin, modification of the pellicle may inhibit bacterial adherence compared to untreated human pellicle.

Development and Characterization of an in vitro Four-species Anaerobic Dental Biofilm Model

Dental caries is the most common chronic disease worldwide. It is characterized by the demineralization of tooth enamel caused by acid produced by cariogenic dental bacteria growing on tooth surfaces, termed bacterial biofilms. Cariogenesis is a complex biological process that is influence by multiple factors and is not attributed to a sole causative agent. Instead, caries is associated with multispecies microbial biofilm communities composed of some bacterial species that directly influence the development of a caries lesion and other species that are seemingly benign but must contribute to the community in an uncharacterized way. Clinical analysis of dental caries and its microbial populations is challenging due to many factors including low sensitivity of clinical measurement tools, variability in saliva chemistry, and variation in the microbiota. Our laboratory has developed an in vitro anaerobic biofilm model for dental carries to facilitate both clinical and basic research-based analyses of the multispecies dynamics and individual factors that contribute to cariogenicity. The rational for development of this system was to improve upon the current models that lack key elements. This model places an emphasis on physiological relevance and ease of maintenance and reproducibility. The uniqueness of the model is based on integrating four critical elements: 1) a biofilm community composed of four distinct and representative species typically associated with dental caries, 2) a semi-defined synthetic growth medium designed to mimic saliva, 3) physiologically relevant biofilm growth substrates, and 4) a novel biofilm reactor device designed to facilitate the maintenance and analysis. Specifically, human tooth sections or hydroxyapatite discs embedded into poly(methyl methacrylate) (PMMA) discs are incubated for an initial 24 hr in a static inverted removable substrate (SIRS) biofilm reactor at 37°C under anaerobic conditions in artificial saliva (CAMM) without sucrose in the presence of 1 X 106 cells/ml of each Actinomyces odontolyticus, Fusobacterium nucleatum, Streptococcus mutans, and Veillonella dispar. During days 2 and 3 the samples are maintained continually in CAMM with various exposures to 0.2% sucrose; all of the discs are transferred into fresh medium every 24 hr. To validate that this model is an appropriate in vitro representation of a caries-associated multispecies biofilm, research aims were designed to test the following overarching hypothesis: an in vitro anaerobic biofilm composed of four species (S. mutans, V. dispar, A. odontolyticus, and F. nucleatum) will form a stable biofilm with a community profile that changes in response to environmental conditions and exhibits a cariogenic potential. For these experiments the biofilms as described above were exposed on days 2 and 3 to either CAMM lacking sucrose (no sucrose), CAMM with 0.2% sucrose (constant sucrose), or were transferred twice a day for 1 hr each time into 0.2% sucrose (intermittent sucrose). Four types of analysis were performed: 1) fluorescence microscopy of biofilms stained with Syto 9 and hexidium idodine to determine the biofilm architecture, 2) quantitative PCR (qPCR) to determine the cell number of each species per cm2, 3) vertical scanning interferometry (VSI) to determine the cariogenic potential of the biofilms, and 4) tomographic pH imaging using radiometric fluorescence microscopy after exposure to pH sensitive nanoparticles to measure the micro-environmental pH. The qualitative and quantitative results reveal the expected dynamics of the community profile when exposed to different sucrose conditions and the cariogenic potential of this in vitro four-species anaerobic biofilm model, thus confirming its usefulness for future analysis of primary and secondary dental caries.

Paleoclimatate of the Middle Burdigalian-Langhian period in Turkey

The Miocene is the last warm episode in Earth history, and this episode was well recorded in Turkey as shown by plant distribution and inferred numerical temperature values. In this study, Ören-Kultak, Hüssamlar and Karacaagac palynofloras from western Turkey, which are characterized by the thermophilous plants (Engelhardia, Sapotaceae, Cyrillaceae, Avicennia, Arecaceae, Palmae), are described. Age determinations of these palynofloras (middle Burdigalian-Langhian) are strengthened by the mammalian fossil record (MN4-5) and strontium isotope results. Palaeoclimate is humid and warm subtropical during the middle Burdigalian-Langhian time interval in Europe and Turkey. However, temperature difference has been observed between Europe and Turkey during this time interval and it could be explained by the palaeogeographic position of countries. Despite some discrepancies in the climatic values and palaeovegetation groups, warm climatic conditions are recorded, based on the palynofloras, in Turkey (Cayyrhan, Havza, Can, Etili, Gönen, Bigadic, Emet, Kirka and Kestelek, Sabuncubeli, Soma, Tire, Kulogullary, Bascayyr, Hüssamlar and Karacaagac), Greece and elsewhere in Europe throughout the middle Burdigalian-Langhian period. This warming is related to the Middle Miocene Climatic Optimum period. Carbon and oxygen isotope values obtained from tooth enamel of Gomphotherium sp. from Kultak and Hüssamlar indicate similar ecological condition during the Burdigalian-Langhian time. This isotopic result and high MAPDRY value from the Kultak locality are in agreement with ecological interpretation of mammalian fossils. Besides, according to the precipitation values, central and northwestern Anatolian sites provide more rainfall during the Burdigalian-Langhian time interval than the western Anatolian sites.

A new Eocene archaeocete (Mammalia, Cetacea) from India and the time of origin of whales

Himalayacetus subathuensis is a new pakicetid archaeocete from the Subathu Formation of northern India. The type dentary has a small mandibular canal indicating a lack of auditory specializations seen in more advanced cetaceans, and it has Pakicetus-like molar teeth suggesting that it fed on fish. Himalayacetus is significant because it is the oldest archaeocete known and because it was found in marine strata associated with a marine fauna. Himalayacetus extends the fossil record of whales about 3.5 million years back in geological time, to the middle part of the early Eocene [≈53.5 million years ago (Ma)]. Oxygen in the tooth-enamel phosphate has an isotopic composition intermediate between values reported for freshwater and marine archaeocetes, indicating that Himalayacetus probably spent some time in both environments. When the temporal range of Archaeoceti is calibrated radiometrically, comparison of likelihoods constrains the time of origin of Archaeoceti and hence Cetacea to about 54–55 Ma (beginning of the Eocene), whereas their divergence from extant Artiodactyla may have been as early as 64–65 Ma (beginning of the Cenozoic).

Dated co-occurrence of Homo erectus and Gigantopithecus from Tham Khuyen Cave, Vietnam.

Tham Khuyen Cave (Lang Son Province, northern Vietnam) is one of the more significant sites to yield fossil vertebrates in east Asia. During the mid-1960s, excavation in a suite of deposits produced important hominoid dental remains of middle Pleistocene age. We undertake more rigorous analyses of these sediments to understand the fluvial dynamics of Pleistocene cave infilling as they determine how skeletal elements accumulate within Tham Khuyen and other east Asian sites. Uranium/thorium series analysis of speleothems brackets the Pleistocene chronology for breaching, infilling, and exhuming the regional paleokarst. Clast analysis indicates sedimentary constituents, including hominoid teeth and cranial fragments accumulated from very short distances and under low fluvial energy. Electron spin resonance analysis of vertebrate tooth enamel and sediments shows that the main fossil-bearing suite (S1-S3) was deposited about 475 thousand years ago. Among the hominoid teeth excavated from S1-S3, some represent Homo erectus and Gigantopithecus blacki. Criteria are defined to differentiate these teeth from more numerous Pongo pygmaeus elements. The dated co-occurrence of Homo erectus and Gigantopithecus blacki at Tham Khuyen helps to establish the long co-existence of these two species throughout east Asia during the Early and Middle Pleistocene.

Desenvolvimento de microssensores eletroquímicos e aplicações no estudo de processos dinâmicos interfaciais utilizando microscopia eletroquímica de varredura

O trabalho descrito nesta tese mostra de forma detalhada a fabricação e caracterização de diferentes microssensores eletroquímicos os quais têm sido recentemente utilizados como sondas em grupo de técnicas conhecida como Scanning Electrochemical Probe Microscopy (SEPM). Desta forma, a caracterização de superfícies pode ser feita explorando diferentes fenômenos interfaciais relevantes à Ciência. Neste sentido, as interfaces de materiais cristalinos como hidroxiapatita (materiais dentários) e calcita foram o foco de estudo neste trabalho. Assim, diferentes técnicas SEPM foram exploradas no sentido de se obter informações relevantes relacionadas aos processos dentários, como a erosão ácida e hipersensibilidade. Inicialmente, microeletrodos de platina foram desenvolvidos empregando uma metodologia convencional na qual são utilizados microfibras encapsuladas em capilares de vidro. Scanning Electrochemical Microscopy (SECM) no modo amperométrico foi utilizada para obtenção de informações com relação às alterações de topografia do esmalte dentário causadas pelo contato com substâncias ácidas. Adicionalmente, SECM foi empregada no estudo do transporte de espécies eletroativas em amostras de dentina e investigações relacionadas ao bloqueio dos túbulos empregando-se cremes dentais comerciais foram realizadas. A permeação de peróxido de hidrogênio pela dentina também foi estudada. Os resultados de SECM foram comparados com imagens SEM obtidas nas mesmas condições experimentais. Microeletrodos de membrana ionófora íon-seletiva (Ion Selective Microelectrodes-ISMEs) sensíveis a íons cálcio também foram desenvolvidos e caracterizados, com subsequente aplicação em SECM no modo potenciométrico. A dissolução ácida de esmalte bovino (erosão dentária) foi investigada em diferentes valores de pH (2,5; 4,5; 6,8). Além disso, o transporte de íons cálcio através de membranas porosas sintéticas foi avaliado a uma distância tip/substrato de 300µm. Alterações no fluxo de íons cálcio foram correlacionadas em experimentos realizados na presença e ausência de campos magnéticos gerados por nanopartículas de magnetita incorporadas à membrana porosa. Microcristais de calcita facilmente sintetizados pelo método de precipitação foram utilizados como superfície modelo para investigações interfaciais, cujos resultados podem ser correlacionados aos materiais dentários. Desta forma, nanopipetas de vidro preenchidas com eletrólito suporte foram fabricadas e utilizadas como sonda em Scanning Ion Conductance Microscopy (SICM). O mapeamento topográfico de alta resolução espacial da superfície de um microcristal de calcita foi obtido utilizando o modo de varredura hopping mode. Adicionalmente, sondas multifuncionais ISME-SICM também foram desenvolvidas e caracterizadas para investigações simultâneas com relação às alterações topográficas e quantificação de íons cálcio sobre a superfície de um microcristal de calcita. A adição de reagentes ácidos no canal SICM promoveu a dissolução da superfície do microcristal, sendo obtidos dados cinéticos de dissolução. Investigações em meio neutro também foram realizadas utilizando a sonda ISME-SICM. Os resultados experimentais obtidos também foram comparados com aqueles oriundos de simulação computacional.

Identifying mobility in populations with mixed marine/terrestrial diets : strontium isotope analysis of skeletal material from a passage grave in Resmo, Öland, Sweden

Strontium isotope analysis of skeletal material as a means to reconstruct prehistoric residential patterns has previously mainly been applied to populations with terrestrial diets. Here we present a model for populations with mixed marine/terrestrial diets, which is based on two-component mixing of strontium isotopes. Applying this model, we can estimate the original strontium isotope value of the terrestrial component of the diet. Accordingly it is possible to identify non-local individuals even if they had a mixed marine/terrestrial diet. The model is applied to tooth enamel samples representing nine individuals recovered from a passage grave in Resmo, on the island of Öland in the Baltic Sea, where at least five non-local individuals, representing at least two different geographical regions of origin, were identified. Non-local individuals were more frequent during the Bronze Age than during previous phases.

Table 2: Water stable isotope results and sampling site information of samples taken between 2003 and 2009 on the island of Corsica (western Mediterranean)

The Mediterranean is regarded as a region of intense climate change. To better understand future climate change, this area has been the target of several palaeoclimate studies which also studied stable isotope proxies that are directly linked to the stable isotope composition of water, such as tree rings, tooth enamel or speleothems. For such work, it is also essential to establish an isotope hydrology framework of the region of interest. Surface waters from streams and lakes as well as groundwater from springs on the island of Corsica were sampled between 2003 and 2009 for their oxygen and hydrogen isotope compositions. Isotope values from lake waters were enriched in heavier isotopes and define a local evaporation line (LEL). On the other hand, stream and spring waters reflect the isotope composition of local precipitation in the catchment. The intersection of the LEL and the linear fit of the spring and stream waters reflect the mean isotope composition of the annual precipitation (dP) with values of -8.6(±0.2) per mil for d18O and -58(±2) per mil for d2H. This value is also a good indicator of the average isotope composition of the local groundwater in the island. Surface water samples reflect the altitude isotope effect with a value of -0.17(±0.02) per mil per 100 m elevation for oxygen isotopes. At Vizzavona Pass in central Corsica, water samples from two catchments within a lateral distance of only a few hundred metres showed unexpected but systematic differences in their stable isotope composition. At this specific location, the direction of exposure seems to be an important factor. The differences were likely caused by isotopic enrichment during recharge in warm weather conditions in south-exposed valley flanks compared to the opposite, north-exposed valley flanks.

Estudio in vitro de la resistencia adhesiva en la interfaz de resina entre la base del pad y el resinoso utilizado en cementación indirecta de la técnica lingual

Con el fin de aminorar retrasos por descementación de cualquier accesorio durante el tratamiento de Ortodoncia Lingual, se ha considerado que la fuerza de adhesión es muy importante, especialmente cuando está ubicada en las diferentes interfaces presentes entre el bracket y la resina del PAD; entre la resina del PAD y el cemento resinoso fotopolimerizable y entre este cemento y el esmalte dental. Por lo que este estudio se ha enfocado en determinar la resistencia adhesiva en la interfaz localizada entre la resina de la base del PAD y el cemento resinoso fotopolimerizable utilizando ácido fluorhídrico y óxido de aluminio como tratamiento de superficie previo a la cementación indirecta de la técnica lingual. MATERIALES Y METODOS: El tipo de estudio fue experimental "in vitro", con una muestra de 30 cuerpos de prueba hechos con resina Transbond XT, utilizando para su confección un blíster de brackets, se siguieron tres protocolos diferentes; G1 o grupo control sin ninguna preparación, G2 con aplicación de óxido de aluminio, 50 micrones durante 10 segundos en la superficie del cuerpo de prueba, G3 con aplicación de ácido fluorhídrico al 9% en la superficie del cuerpo de prueba durante 10 minutos. Previo al test de resistencia adhesiva, se realizó los cortes de precisión en cada cuerpo de prueba, obteniendo así 45 tiras de prueba, cada una de las muestras fue adherida a un porta muestra para la prueba de micro tracción, la misma que fue realizada con la máquina universal Mini-Instron modelo 5942, a una velocidad de deformación constante de 0.5 mm/min. Los datos fueron sometidos al test de Normalidad de residuos de Shapiro Wilk (p>0,05) y de LEVENE para el análisis de homogeneidad de las varianzas. La resistencia adhesiva fue comparada entre los grupos por medio del Análisis de Varianza (ANOVA) como factor único para el procesamiento de los datos. Para todos los análisis el nivel de significancia fue del 5% (p< 0,05) con un nivel de confianza del 95% (IC95%). Se consideró estadísticamente significativo valores por debajo de 0,05. RESULTADOS Y CONCLUSIONES: El resultado del test de ANOVA, reveló que el factor de tratamiento de superficie F(2,12)=2,52;p=0,12 no es significante, por lo tanto los diferentes tratamientos de superficie (óxido de aluminio y ácido fluorhídrico) utilizados son equivalentes al grupo control, indicando que no ejercen influencia de manera significativa en los valores de Resistencia Adhesiva (RA) en la preparación de la interfaz localizada entre la resina de la base del PAD y el cemento resinoso fotopolimerizable; concluyendo que se puede utilizar cualquier protocolo de tratamiento de superficie indicado en la presente investigación.

COMMUNITY KNOWLEDGE, ATTITUDE AND PRACTICES (KAP) ON FLUOROSIS AND ITS MITIGATION IN ENDEMIC AREAS OF ETHIOPIA

Optimum fluoride intake plays an essential role in the prevention of dental caries while fluoride consumption above recommended level interferes with the normal formation of tooth enamel and bones and may increase risk of dental and skeletal fluorosis. The knowledge and practices of endemic communities on etiology of fluorosis will help in its mitigation and prevention. The objective of this study was to investigate the knowledge, attitude and practices of endemic community on fluoride contamination, fluorosis and prevention practices in order to devise coordinated and targeted prevention mechanisms. Focus group discussions (FGD) and key-informant interview were conducted in three dietary areas to collect knowledge, attitude and practices (KAP) of the endemic community in July 2013.The results indicated that health consequences of fluoride contaminated water are fairly understood. None of the discussants mentioned the word “fluoride”. The knowledge and perception of the community on fluoride ingestion is poor. Health extension workers (HEWs) did not teach about fluoride and related health consequences. Dental fluorosis was reported to start at early ages and not commonly perceived as a major problem. However, adolescents worried and felt that they might be singled out when going to other areas. Older people have a skeletal fluorosis, which interferes with their day to day activities. In severely affected people, the teeth were weak and fragile and thus create difficulty in chewing hard foods like unfermented dry flat bread, sugar cane and toasted grains. People prefer rain water rather than water from borehole because of the inconvenient taste of the latter. The endemic communities have no sufficient knowledge and skills on potential sources of fluoride intake, the debilitating effect of high fluoride ingestion, and preventive and mitigatory measures to reduce fluoride intake. The effect of fluoride contamination and mitigatory methods should get sufficient attention by the community, health workers and concerned governmental bodies. The trend of harvesting and using rain water should be encouraged as it reduces fluoride intake. Future studies should focus on information communication on possible fluoride risks, intervention and evaluation studies on defluoridation, rain water harvesting and mitigatory techniques.

Influence of enamel matrix derivative (Emdogain((R))) and sodium fluoride on the healing process in delayed tooth replantation: histologic and histometric analysis in rats

Although it has already been shown that enamel matrix derivative (Emdogain((R))) promotes periodontal regeneration in the treatment of intrabony periodontal defects, there is little information concerning its regenerative capacity in cases of delayed tooth replantation. To evaluate the alterations in the periodontal healing of replanted teeth after use of Emdogain((R)), the central incisors of 24 Wistar rats (Rattus norvegicus albinus) were extracted and left on the bench for 6 h. Thereafter, the dental papilla and the enamel organ of each tooth were sectioned for pulp removal by a retrograde way and the canal was irrigated with 1% sodium hypochlorite. The teeth were assigned to two groups:in group I, root surface was treated with 1% sodium hypochlorite for 10 min (changing the solution every 5 min), rinsed with saline for 10 min and immersed in 2% acidulated-phosphate sodium fluoride for 10 min; in group II, root surfaces were treated in the same way as described above, except for the application of Emdogain((R)) instead of sodium fluoride. The teeth were filled with calcium hydroxide (in group II right before Emdogain((R)) was applied) and replanted. All animals received antibiotic therapy. The rats were killed by anesthetic overdose 10 and 60 days after replantation. The pieces containing the replanted teeth were removed, fixated, decalcified and paraffin-embedded. Semi-serial 6-mu m-thick sections were obtained and stained with hematoxylin and eosin for histologic and histometric analyses. The use of 2% acidulated-phosphate sodium fluoride provided more areas of replacement resorption. The use of Emdogain((R)) resulted in more areas of ankylosis and was therefore not able to avoid dentoalveolar ankylosis. It may be concluded that neither 2% acidulated-phosphate sodium fluoride nor Emdogain((R)) were able to prevent root resorption in delayed tooth replantation in rats.

Light microscopy and computer three-dimensional reconstruction of the blood capillaries of the enamel organ of rat molar tooth germs

We performed a light microscope and a computer three-dimensional reconstruction study of serial sections of the molar enamel organ of 3- and 5-day-old rats perfused with Indian ink through the arterial system. The tooth germs were fixed in Bouin's solution, embedded in paraffin, sectioned and stained with haematoxylin and eosin. For the three-dimensional reconstruction, light micrographs of the serial sections were digitized, and aligned using the serial EM Align software downloaded from http://synapses.bu.edu/tools/. After alignment, the boundaries of the India-ink-filled blood vessels were manually traced with a mouse using the software IGL trace (version 1.26b), also downloaded from the above website. After tracing, a three-dimensional representation of the blood vessel contours was generated in a VRML format and visualized with the help of the software Cortona Web3D viewer (version 4.0) downloaded from http://www.parallelgraphics.com/products/cortona. Our results showed that in regions where ameloblasts are polarized the capillaries are arranged in three distinct levels: (1) penetrating and leaving capillaries in relation to the outer enamel epithelium; (2) capillaries crossing and branching inside the stellate reticulum; and (3) capillaries branching and anastomosing profusely within the stratum intermedium, thereby forming an extensive capillary plexus intimately associated with the cells of the stratum intermedium. The existence of a conspicuous capillary plexus intermingled with cells of the stratum intermedium, as shown in our results, suggests that some molecules produced by cells of the stratum intermedium could be released into the capillary plexus and thereafter carried to the dental follicle.