Human identification through DNA analysis of restored postmortem teeth.

The identification of human remains using DNA analysis can be extremely challenging and its success is certainly influenced by the time elapsed since death. In that context, intact teeth have been shown to be highly successful in DNA analysis. However, restored teeth are usually available and, surprisingly, these specimens have been poorly studied. In fact, there are no reports regarding forensic DNA analysis of those types of samples in real cases. Therefore, the aim of this study was to perform DNA typing on healthy and restored teeth from exhumed human remains, which had been buried for 46 years. A powder-free DNA extraction protocol specifically designed for teeth was followed and human DNA quantitation and degradation assessment was performed using an in-house qPCR assay. Samples were amplified with commercial human identification kits for autosomal and Y chromosome markers. The obtained DNA profiles were compared to those of a previously processed femur sample as well as a buccal swab from a putative son. One healthy and one restored tooth yielded complete, concordant and compatible DNA profiles with previously typed samples from the femur and the putative son. Biostatistical calculations supported the paternity relationship with a likelihood ratio greater than 11 million. The present study highlights the use of restored teeth in a real exhumation case and the powder-free approach specifically designed for the extraction of DNA from teeth is discussed.

Factors affecting dental DNA in various real post-mortem conditions.

Post-mortem DNA degradation is still the real challenge of DNA-based identification in forensic practise. It is a complicated multifactorial process occurring as a result of the combination of several different environmental effects along with the crucial effect of the elapsed post-mortem interval (PMI). The main purpose of the present study is to evaluate the effect of ante- and post-mortem factors on dental DNA in real forensic cases. Ninety-five teeth extracted from 39 corpses, whose bodies were subject to 6 different post-mortem conditions, were used to evaluate dental DNA amount. In total, 179 DNA extracts isolated from the root of the teeth were examined after removing the crown and sectioning each root into apical and cervical portions. DNA concentration was measured using real-time polymerase chain reaction DNA quantitation kit (PowerQuant™ System/Promega). Our results indicate that the post-mortem interval (PMI) is the most important influential factor on dental DNA quantification (p < 0.001). However, in the actual data set, it was confounded with several ante- and post-mortem factors, rendering its actual net effect difficult. The time period of the first 10 days after death yielded the best DNA results from all analysed dental samples. Afterwards, a dramatic decrease in dental DNA was observed in the following time period. Teeth extracted from burnt and fresh corpses yielded the highest amount of DNA, while skeletonized exhumed corpses resulted in the lowest DNA amount. Indeed, dry and indoor conditions demonstrated better results than those in water, outdoors, or buried in the ground. On the other hand, ante-mortem factors including sex, age, tooth type, and tooth root portions did not reveal significant effect on dental DNA yield. We suggest that ante-mortem factors are considerably more subjected to individual variations. Post-mortem factors including PMI, post-mortem conditions, and the relevant surrounding environments have substantial influence on the dental DNA amount yielded.

Debris extrusion and foraminal deformation produced by reciprocating instruments made of thermally treated NiTi wires

Abstract Objective To evaluate the amount of apically extruded debris, percentage of foraminal enlargement and apical foramen (AF) deformation that occurred during root canal preparation with different reciprocation systems: Reciproc, WaveOne (M-Wire), and ProDesign R (Shape Memory Technology Wire) at two different working lengths (WLs): 0.0 and 1.0 mm beyond the AF. Material and methods The AF of 120 root canals in 60 mesial roots of mandibular molars were photographed with stereomicroscope and randomly assigned into four groups: manual, Reciproc (REC), WaveOne (WO), and ProDesign R (PDR); subsequently, they were further subdivided according to the WL (n=15). Teeth were instrumented, coupled to a dual collecting chamber, and then another photograph of each AF was captured. Extrusion was analysed by determining the weight of extruded debris. Each AF diameter was measured in pre- and post-instrumentation images to determine deformation, which was analysed, and afterwards the final format of AFs was classified (circular/oval/deformed). Results We found no significant differences when analysing each system at different WLs. When considering each WL, REC and WO showed highest extrusion values (P<.05); for AF enlargement, differences were observed only for WO, when it was used beyond the AF; differences were observed among M-Wire groups beyond the AF (P<.05). AF deformation was observed in all groups; PDR showed the lowest AF deformation values at both WLs; M-Wire groups showed 50% strain beyond the AF. Conclusion Authors concluded that beyond the apical limit, the alloy and taper are important aspects when considering extrusion and deformation.

Forensic DNA typing from teeth using demineralized root tips.

Teeth are widely used samples in forensic human genetic identification due to their persistence and practical sampling and processing. Their processing, however, has changed very little in the last 20 years, usually including powdering or pulverization of the tooth. The objective of this study was to present demineralized root tips as DNA sources while, at the same time, not involving powdering the samples or expensive equipment for teeth processing. One to five teeth from each of 20 unidentified human bodies recovered from midwest Brazil were analyzed. Whole teeth were demineralized in EDTA solution with daily solution change. After a maximum of approximately seven days, the final millimeters of the root tip was excised. This portion of the sample was used for DNA extraction through a conventional organic protocol. DNA quantification and STR amplification were performed using commercial kits followed by capillary electrophoresis on 3130 or 3500 genetic analyzers. For 60% of the unidentified bodies (12 of 20), a full genetic profile was obtained from the extraction of the first root tip. By the end of the analyses, full genetic profiles were obtained for 85% of the individuals studied, of which 80% were positively identified. This alternative low-tech approach for postmortem teeth processing is capable of extracting DNA in sufficient quantity and quality for forensic casework, showing that root tips are viable nuclear DNA sources even after demineralization.

Removal of Radioactively Marked Calcium Hydroxide from the Root Canal: Influence of Volume of Irrigation and Activation.

INTRODUCTION: The purpose of this study was to evaluate the amount of calcium hydroxide (Ca[OH]2) removed by irrigation with different volumes and activation methods. METHODS: One hundred thirty extracted straight, single-rooted human teeth were instrumented to size 45/.04. One hundred twenty teeth were filled with radioactively marked Ca(OH)2 and a gutta-percha point; 10 teeth with only gutta-percha served as a negative control. All specimens were stored in saline solution (7 days at 35°C). After storage, teeth were randomly divided into 12 groups (n = 10). The gutta-percha was taken out, and Ca(OH)2 was removed either by irrigation with different volumes (0 mL, 0.5 mL, 1 mL, 2 mL, 4 mL, or 8 mL) or mechanical activation with a 2- or 4-mL volume using a file (Instr) (FlexMaster size 45/.04; VDW, Munich, Germany), a brush (CanalBrush [CB]; Coltène/Whaledent, Langenau, Germany), or passive ultrasonic irrigation (PUI, smooth wire). Irrigation was performed by alternating 40% citric acid and 3% sodium hypochlorite. Residual Ca(OH)2 was measured by scintillation and expressed as a percentage of the original Ca(OH)2. RESULTS: Increasing the irrigation volume led to a significant decrease (P < .05) of residual Ca(OH)2 (0 mL [98.5%], 0.5 mL [21.7%], 1 mL [16.5%], 2 mL [12.9%], 4 mL [8.7%], 8 mL [5.0%], and negative control [0.0%]). Activation led to less residual Ca(OH)2 (2 mL Instr [12.0%], 2 mL CB [11.7%], 2 mL PUI [9.1%], 4 mL Instr [8.5%], 4 mL CB [7.4%], and 4 mL PUI [6.2%]), with significant differences according to the PUI (P < .05). CONCLUSIONS: No irrigation procedure was able to remove Ca(OH)2 completely. PUI was the most effective activation method. However, irrigation with an 8-mL volume was the most effective.

The effects of ageing on the biomechanical properties of root dentine and fracture.

OBJECTIVES: Knowledge of the mechanical behaviour of root dentine can facilitate better understanding of spontaneous vertical root fracture (VRF), an age-related disease initiated mainly at the root apex. We tested the hypothesis that the biomechanical properties of root dentine change with ageing. METHODS: Sixteen human premolars were divided into "old" (17-30 years) and "young" (50-80 years) groups. The elastic modulus, nano-hardness, micro-hardness, elemental contents, tubular density/area of root dentine in cervical, middle and apical root regions were evaluated using atomic force microscopy-based nano-indentation, Knoop indentation, scanning electron microscopy and energy dispersive X-ray spectroscopy, respectively. RESULTS: The apical dentine showed a lower nano-hardness, a lower elastic modulus, a lower calcium content, a lower calcium-to-phosphorus ratio and a smaller tubular density/area than the cervical dentine in both age groups, whereas spatial differences in micro-hardness were observed only in old roots. Compared with young dentine, old dentine showed a greater hardness, a higher elastic modulus, a greater mineral content and a smaller tubular size in the cervical portion, whereas the age-induced changes in tubular density were insignificant. Finite element analysis revealed that due to its higher elastic modulus, old apical dentine has a higher stress level than young dentine. CONCLUSIONS: The intrinsic material properties of root dentine have spatial variations, and they are altered by ageing. The higher stress level in old apical dentine may be one reason, if not the most important one, why spontaneous VRFs are more likely to occur in the elderly population.

Efeito do tratamento térmico nas propriedades físicas e mecânicas de fios e instrumentos endodônticos de NiTi / Effect of heat treatment on mechanical and physical properties of NiTi wires and endodontic instruments

O objetivo deste trabalho foi comparar as propriedades químicas, físicas e mecânicas de fios de níquel-titânio convencionais (CW) e termomecanicamente tratados (MW), utilizados na fabricação de instrumentos endodônticos por dois fornecedores diferentes (Dentsply Tulsa Dental Specialities, Tulsa - OK, USA e dentsply maillefer - Ballaigues, Switzerland). Foram ainda avaliados o torque e a força apical requerida pelos novos instrumentos endodônticos ProTaper NextÕ. A composição química foi determinada por espectroscopia de energia dispersiva de raios-X, a constituição de fase por difratometria de raio-X e as temperaturas de transformação por calorimetria exploratória diferencial. Ensaios de tração em carga e descarga e até ruptura, flexão em três pontos, flexão rotativa/fadiga e medidas de microdureza Vickers foram realizados para avaliar o comportamento mecânico dos fios. As superfícies de fratura foram observadas por microscopia eletrônica de varredura e as microestruturas não deformadas por microscopia eletrônica de transmissão...

Qualitative SEM/EDS analysis of microleakage and apical gap formation of adhesive root-filling materials.

OBJECTIVE: The aim of this study was to compare the correspondence between gap formation and apical microleakage in root canals filled with epoxy resin-based (AH Plus) combined or not with resinous primer or with a dimethacrylate-based root canal sealer (Epiphany). MATERIAL AND METHODS: Thirty-nine lower single-rooted human premolars were filled by the lateral condensation technique (LC) and immersed in a 50-wt% aqueous silver nitrate solution at 37ºC (24 h). After longitudinal sectioning, epoxy resin replicas were made from the tooth specimens. Both the replicas and the specimens were prepared for scanning electron microscopy (SEM). The gaps were observed in the replicas. Apical microleakage was detected in the specimens by SEM/energy dispersive spectroscopy (SEM/EDS). The data were analyzed statistically using an Ordinal Logistic Regression model and Analysis of Correspondence (α=0.05). RESULTS: Epiphany presented more regions containing gaps between dentin and sealer (p<0.05). There was correspondence between the presence of gaps and microleakage (p<0.05). Microleakage was similar among the root-filling materials (p>0.05). CONCLUSIONS: The resinous primer did not improve the sealing ability of AH Plus sealer and the presence of gaps had an effect on apical microleakage for all materials.

Sealability of MTA and calcium hydroxidecontaining sealers.

OBJECTIVES: The aim of this study was to evaluate the apical sealability of Fillapex®, endo-CPM-Sealer® and Sealapex®. MATERIAL AND METHODS: Ninety-four freshly extracted single-rooted teeth were selected and decoronated. All teeth were radiographed to confirm the existence of a single and straight root canal, which was prepared using Protaper Universal and 2.5% sodium hypochlorite. The teeth were randomly divided in groups of 10 specimens each according to the sealer, and the canals were filled using the single cone technique and one of the sealers. Four additional teeth were used as controls. The teeth were submitted to dye leakage with Rhodamine B for 24 h but using vacuum on the initial 15 min. Thereafter, they were cut longitudinally and the leakage was measured in a linear fashion from apex to crown. Data were analyzed by ANOVA and Tukey's tests at 5% significance level. RESULTS: Fillapex® and Sealapex® showed significantly less dye leakage than endo-CPM-Sealer® (p<0.05). CONCLUSIONS: It was concluded that Fillapex® and Sealapex® were able to prevent apical dye leakage differently from endo-CPM-Sealer®.

Qualitative SEM/EDS analysis of microleakage and apical gap formation of adhesive root-filling materials

OBJECTIVE: The aim of this study was to compare the correspondence between gap formation and apical microleakage in root canals filled with epoxy resin-based (AH Plus) combined or not with resinous primer or with a dimethacrylate-based root canal sealer (Epiphany). MATERIAL AND METHODS: Thirty-nine lower single-rooted human premolars were filled by the lateral condensation technique (LC) and immersed in a 50-wt% aqueous silver nitrate solution at 37ºC (24 h). After longitudinal sectioning, epoxy resin replicas were made from the tooth specimens. Both the replicas and the specimens were prepared for scanning electron microscopy (SEM). The gaps were observed in the replicas. Apical microleakage was detected in the specimens by SEM/energy dispersive spectroscopy (SEM/EDS). The data were analyzed statistically using an Ordinal Logistic Regression model and Analysis of Correspondence (α=0.05). RESULTS: Epiphany presented more regions containing gaps between dentin and sealer (p<0.05). There was correspondence between the presence of gaps and microleakage (p<0.05). Microleakage was similar among the root-filling materials (p>0.05). CONCLUSIONS: The resinous primer did not improve the sealing ability of AH Plus sealer and the presence of gaps had an effect on apical microleakage for all materials.

Sealability of MTA and calcium hydroxidecontaining sealers

OBJECTIVES: The aim of this study was to evaluate the apical sealability of Fillapex®, endo-CPM-Sealer® and Sealapex®. Material and Methods: Ninety-four freshly extracted single-rooted teeth were selected and decoronated. All teeth were radiographed to confirm the existence of a single and straight root canal, which was prepared using Protaper Universal and 2.5% sodium hypochlorite. The teeth were randomly divided in groups of 10 specimens each according to the sealer, and the canals were filled using the single cone technique and one of the sealers. Four additional teeth were used as controls. The teeth were submitted to dye leakage with Rhodamine B for 24 h but using vacuum on the initial 15 min. Thereafter, they were cut longitudinally and the leakage was measured in a linear fashion from apex to crown. Data were analyzed by ANOVA and Tukey's tests at 5% significance level. Results: Fillapex® and Sealapex® showed significantly less dye leakage than endo-CPM-Sealer® (p<0.05). Conclusions: It was concluded that Fillapex® and Sealapex® were able to prevent apical dye leakage differently from endo-CPM-Sealer®.

Fluorosis in rats exposed to oscillating chronic fluoride doses.

Considering that blood fluoride concentration varies according to fluoride exposure and that dental fluorosis is related to the amount of enamel formed under a given fluoride dose, the present study investigated whether the fluorosis produced by an oscillating chronic fluoride dose would be similar to that caused by exposure to a constant dose, representing the mean of the oscillation during a given time. Rats received during 78 days water with fluoride concentrations of 0, 12.5, 25 or 37.5 microg F/mL, or oscillating concentrations of 12.5 and 37.5 microg F/mL every 72 h (mean exposure=25 microg F/mL). The concentrations of fluoride in the plasma, femur and incisors of the rats were determined at the end of the experimental period. Also, the enamel dental fluorosis index was determined in the incisors using a quantitative method developed by our research group named Dental Fluorosis by Image Analysis (DFIA). Fluoride concentrations in plasma, femur and teeth, and DFIA increased linearly for constant fluoride concentrations in water (p<0.0001, r values=0.87-0.98). The results of the oscillating group and the groups receiving 25 microg F/mL did not differ significantly (p>0.05). The findings of this study suggest that in animals chronically exposed to symmetrically oscillating fluoride doses, the resulting dental fluorosis reflects the metabolic effect of the mean of the oscillating doses.

Fluorosis in rats exposed to oscillating chronic fluoride doses

Considering that blood fluoride concentration varies according to fluoride exposure and that dental fluorosis is related to the amount of enamel formed under a given fluoride dose, the present study investigated whether the fluorosis produced by an oscillating chronic fluoride dose would be similar to that caused by exposure to a constant dose, representing the mean of the oscillation during a given time. Rats received during 78 days water with fluoride concentrations of 0, 12.5, 25 or 37.5 µg F/mL, or oscillating concentrations of 12.5 and 37.5 µg F/mL every 72 h (mean exposure=25 µg F/mL). The concentrations of fluoride in the plasma, femur and incisors of the rats were determined at the end of the experimental period. Also, the enamel dental fluorosis index was determined in the incisors using a quantitative method developed by our research group named Dental Fluorosis by Image Analysis (DFIA). Fluoride concentrations in plasma, femur and teeth, and DFIA increased linearly for constant fluoride concentrations in water (p<0.0001, r values=0.87-0.98). The results of the oscillating group and the groups receiving 25 µg F/mL did not differ significantly (p>0.05). The findings of this study suggest that in animals chronically exposed to symmetrically oscillating fluoride doses, the resulting dental fluorosis reflects the metabolic effect of the mean of the oscillating doses.

Considerando que a concentração de fluoreto no sangue varia de acordo com a exposição ao fluoreto, e que a fluorose dental está relacionada com a quantidade de esmalte formado sob determinada dose de fluoreto, este estudo avaliou se a fluorose resultante da exposição a doses oscilantes de fluoreto seria semelhante àquela causada pela exposição a uma dose constante, representativa da média das oscilações durante um determinado tempo. Durante 78 dias, ratos receberam água com concentrações constantes de fluoreto de 0; 12,5; 25 ou 37,5 µg F/mL, ou concentrações oscilantes de 12,5 e 37,5 µg F/mL alternados a cada 72 h (média de exposição = 25 µg F/mL). Concentrações de fluoreto no plasma, fêmur e dentes incisivos dos ratos foram determinadas após o período experimental. O índice de fluorose, observado nos incisivos dos ratos, foi quantificado usando um método de análise de imagem desenvolvido para essa pesquisa, denominado de índice de fluorose por análise de imagem (DFIA, em Inglês). A concentração de fluoreto no plasma, fêmur e incisivo dos ratos, assim como o DFIA, aumentaram de forma linear para as concentrações constantes de fluoreto na água (p<0,0001, r=0,87-0,98). Não houve diferença significativa entre o grupo que recebeu doses oscilantes e o grupo que recebeu 25 µg F/mL (p>0,05). Os resultados sugerem a fluorose dental decorrente de exposição crônica de animais a doses de fluoreto oscilantes e simétricas reflete o efeito metabólico da média da oscilação.

A Raman spectroscopic investigation of dentin and enamel structures modified by lactic acid.

Confocal Raman microspectrometry allows a thorough molecular analysis of mineralised dental tissues. The output information is provided in the form of curves representing the intensity of the signal according to the frequency, and its mathematical exploitation permits all sorts of comparative and quantitative analyses. By this process, we investigated the in vitro action of lactic acid on enamel and dentin from human permanent teeth. Modifications due to the acidic attack essentially concern phosphate grouping PO(4)(3-), which represents the mineral phase in enamel and dentin (hydroxyapatite): on Raman spectra, changes in intensity of the PO(4)(3-) band are linked to the type of dentin, to its anatomical location, and to the age of the subject. The variability of the dentinal chemical structure was confirmed by a quantitative statistical analysis, revealing a significant spectral difference between coronal and root dentin.

Structure and chemical composition of an experimentally formed apical barrier after the application of calcium-glycerophosphate.

Transformation in the structure and composition of calcium-glycerophosphate (Ca-GP) was investigated using a scanning electron microscope fitted with an energy dispersive X-ray microanalysis (EDX) system. Ca-GP was packed inside roots that were subsequently implanted into the mandibles of rats to stimulate the formation of an apical barrier. Scanning electron microscopic observations of packed Ca-GP revealed three types of structures: globular aggregates, plate or flake-like aggregates, and granular aggregates. The average Ca/P molar ratios of EDX by area mode--postoperatively obtained at days 1, 3, 5, and 7--were 1.300, 1.533, 1.472, and 1.495 (analytic area: 0.01 to 0.03 mm2) and those of EDX by point mode were 1.220, 1.451, 1.487, and 1.467 (analytic point: 0.05 micron 2). The magnesium (Mg) weight percentage was found to be approximately 1.9 at days 3, 5, and 7, similar to Mg-substituted whitlockite. These findings suggest that Ca-GP are transformed, over a period of 3 days into Mg-substituted whitlockite as a result of being hydrolyzed by tissue fluid.

Phosphoprotein analysis of sequential extracts of human dentin and the determination of the subsequent remineralization potential of these dentin matrices.

Phosphoprotein appears to play an important role in the mineralization of dentin during tooth development and remineralization after demineralization by dental caries. To better understand this role, we describe the extraction and characterization of phosphoprotein from immature, human root apex dentin during and after EDTA demineralization. The extraction procedure included dissociation of the demineralized dentin matrix by guanidine hydrochloride (Gdn.HCl) followed by subsequent digestion with cyanogen bromide (CNBr) and collagenase. Characterization of these extracts included 'Stains-All' staining of SDS polyacrylamide gels (SDS-PAGE) and amino acid, protein and phosphorus analyses. The ability of these matrices to remineralize was determined by TEM and measuring calcium levels in the remineralized tissue by atomic absorption spectroscopy. The staining of SDS-PAGE gels and amino acid analysis showed that an intact phosphophoryn was extracted from the dentin of the immature apices during EDTA demineralization and that it had an apparent Mr approximately 140,000. In the subsequent extracts and digests, the phosphoprotein has a range of molecular weights, some of which may have been degraded products of the intact phosphoprotein. A greater quantity of phosphoprotein was found in the EDTA-demineralized dentin matrices than in dentin after Gdn.HCl, CNBr and collagenase digests. These EDTA-demineralized matrices also remineralized to a greater extent than those dissociated with Gdn.HCl. The differences in both the quantity and the quality, as defined by the amino acid residue profile, of the phosphoprotein in the sequential extracts of the root apex dentin may be important in affecting the ability of this tissue to remineralize.

Apexificaçäo em dentes permanentes jovens com ápice incompleto e necrose pulpar indicaçöes, técnica e caso clínico / Apexification in nonvital young permanent teeth with incommplete root formation treatment procedures and report of case

Säo tecidas consideraçöes em torno das indicaçöes e técnicas utilizadas no tratamento de dentes permanentes jovens com ápice radicular incompleto e necrose pulpar, enfatizando o uso do hidróxido de cálcio associado ao iodofórmio nos procedimentos de apexificaçäo