Demonstration and semi-quantification of mtDNA from human dentine and its relation to age

In order to study if mitochondrial DNA (mtDNA) could be retrieved from isolated human dentine, small pieces of dentine were cut out from the central part of the apical half of wisdom teeth from 21 individuals aged 15 to 85 years. The dental pulp was used as a control. After extraction, amplification and agarose gel electrophoresis the amount of mtDNA was semi-quantified from the intensity of the stained bands in the gel. Mitochondrial DNA was retrieved from all samples and the sequences were identical in pulp and dentine from each individual. There was a clear age-dependent decrease in the amount of amplified mtDNA. Since the odontoblastic processes in the apical dentine undergo degeneration with age and the dentinal tubules subsequently become occluded with calcium phosphate crystals, the conclusion was drawn that even after dissolution of the odontoblastic processes, at least remains of the mtDNA are trapped in the dentine. This well protected mtDNA could thus be regarded a good source of DNA in identification cases with severe degradation. Received: 17 March 1998 / Received in revised form: 21 May 1998     

Visibility of dental pulp spaces in dental ultrasound

The purpose of this study was to assess the feasibility of dental ultrasound with conventional sonographic equipment. The teeth of three adult volunteers who had cone beam CT examinations performed previously with clinical indications and one extracted tooth were examined using linear and compact (hockey stick) sonographic probes. The sonographic images were compared with cone beam CT images reconstructed accordingly. Dental pulp spaces were demonstrated in all teeth not covered with prosthetic crowns. The dentin and pulp were best visualized at the level of the neck of the teeth. The dentin was hypoechoic, and the superficial layer comprising the cementum and the pulp spaces were hyperechoic. Dental ultrasound is feasible with general purpose sonographic machines. The buccal surfaces of all teeth are accessible with a compact (hockey stick) probe. Visualization and differentiation of dental pulp spaces, dentin and the superficial layer comprising cementum is possible in the portions of teeth not covered by the alveolar bone or prosthetic crowns. The dental pulp spaces are best seen at the level of the tooth neck. Pulp and endodontic fillings can be distinguished on ultrasound.     

Effect of sodium hypochlorite decontamination on the DNA recovery from human teeth

Genetic identification of skeletal human remains is often realized by short tandem repeat (STR) genotyping of nuclear DNA. Dental DNA is preferred to DNA from bone for the better protection of the endogenous DNA. Especially if whole tooth grinding is intended to access the DNA, contaminations with exogenous DNA have to be avoided. The immersion of the tooth in sodium hypochlorite (NaOCl, known as bleach) is one common procedure to clean the outer surface from extraneous DNA and PCR inhibitors. To investigate the impact of bleaching on endogenous DNA and the decontamination success, 71 recently extracted teeth were differently treated with sodium hypochlorite (2.5 or 5.0% NaOCl for 30 or 60 s, 5.0% NaOCl for 10 min, and control group) in the beginning of the extraction process, whereas equally handled afterwards. Quantitative and qualitative evaluation of the extracted DNA was performed. There was a great variation for the DNA concentration of the extracts even within a group of the same NaOCl treatment. Complete DNA profiles from single persons with alleles for the 16 ESS (European Standard Set) STR loci were obtained for all regarded teeth. A statistically significant difference between the DNA yields of the treatment groups was not determined. Moreover, a negative effect of NaOCl (2.5% and 5.0%) on the DNA recovery could not be observed. Significant larger amounts of DNA were extracted from anterior teeth in contrast to posterior teeth.     

Multivariate comparison of elemental concentrations in human teeth

R-mode factor analysis was applied to characterize the chemical composition of human teeth investigated by particle induced X-ray emission (PIXE), Rutherford backscattering spectrometry (RBS) and X-ray fluorescence (XRF) techniques. The approach developed in this study enabled the separation between essential mineral teeth components and the pollutants deposited in teeth tissues during the human life. The three independent sources of metals incorporated in human teeth were found. The first source, representing about 43% of the variance of the concentration data, was characterized by pollutant elements of power industry emissions. The second factor was loaded with toxic elements of general urban pollution. The third factor represented the tooth source as it contained mainly large fractions of the mineral components of the tooth tissue as Ca and K.     

Coronal displacement of cementum in impacted teeth: As age marker

Dental cementum is a vital tissue that demonstrates continuous apposition throughout the life of a tooth. This study was conducted on 130 impacted mandibular third molars extracted from healthy patients, aged between 18 and 38 years. The buccolingual ground sections were prepared and the distance between the edges of enamel and cementum were measured with micrometer attached to a light microscope. A significant correlation (r = 0.56) between age and coronal displacement of cementum in impacted teeth was calculated and a regression equation was derived for age determination.     

A mathematical model for calculation of 90Sr absorbed dose in dental tissues: elaboration and comparison to EPR measurements.

A mathematical model for calculation of the 90Sr absorbed doses in dental tissues is presented. The results of the Monte-Carlo calculations are compared to the data obtained by EPR measurements of dental tissues. Radiometric measurements of the 90Sr concentrations. TLD and EPR dosimetry investigations were performed in animal (dog) study. The importance of the irregular 90Sr distribution in the dentine for absorbed dose formation has been shown. The dominant dose formation factors (main source-tissues) were identified for the crown dentine and enamel. The model has shown agreement with experimental data which allows to determine further directions of the human tooth model development.     

EPR dose reconstruction for bone-seeking 90Sr.

The results of the EPR dose reconstruction in calcified tissues of dog injected with 90Sr are presented. It has been established that there is no essential difference in the values of doses absorbed in tooth tissues of teeth in symmetric positions in the mouth, whereas a significant difference occurs in the values of absorbed doses in teeth in non-symmetric positions. In the case of 90Sr internal exposure the dose reconstruction in crown dentine plays an important role. It has been found that its quantity is close to the dose in diaphyseal cortical bone of the femur, dose at the endosteal bone surface and in femural fatty marrow. The fact that these values exceed doses absorbed in tooth enamel points out the predominant contribution of internal exposure. The highest absorbed doses have been observed in metaphyseal trabecular femur bones, tooth alveolar bone walls, and cortical and trabecular vertebra that can be considered as suitable candidates for biomarkers of internal 90Sr exposure for post-mortal autopsy. The satisfactory correlation has been found between the doses reconstructed in calcified dog tissues and the doses measured by EPR in alanine dosimeters fixed in (or nearby) the sites of autopsy of bones/teeth. The experiments provide support for the view that EPR retrospective dosimetry with calcified tissues for internal exposure is unique in providing useful information on the doses obtained.     

Ultrasound crack detection in a simulated human tooth

OBJECTIVE: Currently, diagnosis of cracked teeth generally depends upon the overall clinical assessment, or on exclusion of other clinical possibilities, not primarily on the direct identification of cracks themselves. Owing to its short wavelength in hard tissues and associated high resolution, ultrasound has the potential to allow detection of cracks within tooth structure. However, ultrasound detection of dental cracks has not previously been achieved. The purpose was to determine if an ultrasound imaging system was capable of imaging cracks in simulated tooth structure. METHODS: A complete ultrasound system including a novel transducer made of PLZT-98, a novel gallium-indium alloy coupling agent, and customized electronic and digital signal processing (DSP) algorithms was developed for the specific application of optimizing crack detection within teeth. A simulated tooth with a known and uniform internal structure and acoustic properties similar to those of natural enamel and dentin was designed to model a human tooth with a crack located in dentin deep to the dentino-enamel junction (DEJ). The distance between the DEJ and a crack of the simulated tooth were calculated. RESULTS: The system unequivocally distinguished between areas with and without a simulated crack. CONCLUSION: A unique ultrasound dental crack detection system using a novel transducer; a novel coupling agent; and customized electronic and digital signal processing (DSP) algorithms has been validated in a simulated tooth.     

Soft tissue removal by maceration and feeding of <Emphasis Type="Italic">Dermestes</Emphasis> sp.: impact on morphological and biomolecular analyses of dental tissues in forensic medicine

Maceration techniques remove soft tissue by the destruction of biomolecules, but the applied techniques may also affect the morphology and the molecular integrity of the hard tissue itself. The impact of seven different techniques for soft tissue removal on morphological and biomolecular parameters of teeth and dental tissues was systematically examined. All methods tested showed significant changes in dental morphology and in the molecular integrity of DNA and the dental proteins, as revealed by aspartic acid racemisation (AAR). In forensic casework this may have severe impacts on the results of morphological methods (e.g. age estimation based on root translucency) and of biomolecular analyses (e.g. age estimation based on AAR and DNA analysis). Therefore, age estimation based on AAR should not be applied to tissue treated in such a manner, and it is recommended that teeth for analysis should be extracted before soft tissue removal. DNA in the hard tissue seems to be less susceptible to soft tissue removal than proteins, and several of the tested maceration techniques appear not to have a damaging effect on DNA. Generally, the indication for soft tissue removal demands a careful case management to avoid methodological collisions.     

Does luminosity and smear layer influence 810 nm laser energy transmission through human dentine?

PurposeThe aim of this study was to evaluate the influence of luminosity values and smear layer on transmission characteristics of 810 nm near infrared laser energy through dentine, using extracted human teeth.MethodsA total of 200 teeth treated with endodontic medicaments to cause discolouration (Ledermix, Doxypaste or Odontopaste) were selected, to give a range of values for tooth shade (luminosity values). Sectioned teeth had smear layer created or removed. Near-infrared laser energy transmission was assessed using an 810 nm diode laser (90 mW, CW mode) with the tip placed at 2 mm and 4 mm from the external root surface, and a laser power metre.ResultsBoth tooth shade and smear layer had independent attenuating effects. Transmission of laser energy was significantly lower in teeth with darker shades. There was a strong positive correlation between relative luminosity (lighter tooth colour) and transmitted laser power (r = 0.9271 for the Spearman rank correlation, P = 0.0022). When a smear layer was present, this significantly reduced the transmission of laser energy through the sample, compared to when no smear layer was present.ConclusionThe results of this laboratory study highlight the attenuating influence of a darker tooth shade and the presence of smear layer on the transmission of 810 nm light when directed onto the surface of human dentine. Such effects may be relevant to photo biomodulation, Photodynamic Therapy and other dental laser treatments.     

Die Zuordungswahrscheinlichkeit eines numerisch-topographischen Zahnstatus bei der Identifikation unbekannter Leichen

The numerical topographical dental findings of 29,862 adult patients from several German dental practices were evaluated. Taking into account the age-dependent loos of teeth the various decades were investigated separately for increased incidences of identical findings. The total number of teeth comprising the dental findings in the form of tooth number classes was a first-line criterion aiding classification. In accordance with an underlying binominal distribution, a raised incidence of identical dental findings was found both in patients edentulous jaws or few residual teeth and in patients with largly complete dentition. The raised incidence was confined to a few standard findings in each of these high or low tooth number classes. However, more than 50% of all cases were either in the intermediate tooth number classes, in which raised incidences of ≥ 0.2% generally do not occur, or showed correspondingly rare constellations of findings in the high or low tooth number classes. In more than half of all cases, there was therefore a numerical topographical dental status which can be used in identification with a matching probability of ≥ 99.8%. Dental findings which occur less frequently can be arranged clearly according to tooth number classes.     

Femur, rib, and tooth sample collection for DNA analysis in disaster victim identification (DVI)

Although much literature is available on DNA extraction from tissue samples to obtain the best possible genotyping results, to the best of our knowledge no written recommendations exist on how to excise or extract bone and tooth samples from a victim to facilitate this. Because the possibility of cross-contamination is high, especially when excising numerous samples under disaster conditions, it is important to minimize this risk and to keep samples in optimum condition. In this paper a standard operating procedure is proposed for collection of femur, rib, and tooth samples to aid victim identification both after mass disasters and in (single) forensic investigations.     

Nails as a primary sample type for molecular genetic identification of highly decomposed human remains

International Journal of Legal Medicine - For identification of badly preserved cadavers, only a few tissues can be used as a source of DNA, mostly bones and teeth, from which sampling and DNA...     

Application of DNA techniques for identification using human dental pulp as a source of DNA

Summary Dental pulp tissue could be obtained in most cases from materials obtained under experimental conditions and from forensic casework (air accidents, burned and putrefied bodies). Teeth extracted during dental treatment (n = 30) were stored for 6 weeks and 4 years at room temperature. In addition teeth (n = 10) extracted from jaw fragments that had been stored for 15 years at room temperature, and teeth extracted post mortem from actual identification cases (n = 8) were investigated. Following extraction from dental pulp tissue the DNA concentration was measured by fluorometry. The amount of DNA obtained from the dental pulp tissue of a single tooth varied from 6 g to 50 g DNA. In most cases high molecular weight DNA was still present although the major portion consisted of degraded DNA. Genomic dot blot hybridization for sex determination using the biotinylated repetitive DNA probe pHY 2.1 was performed and sex was correctly classified in all cases using 50–100 ng target DNA. PCR typing of the HLA-DQ and ApoB 3 VNTR systems from dental pulp tissue DNA was in agreement with the results obtained from blood, bloodstains, or lung tissue. In addition, Southern blot analysis of selected samples using the single locus VNTR probe pYNH24 was successfully performed. In all cases the DNA recovered from dental pulp was unsuitable for multilocus probe analysis.

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Abbreviations BCIP 5-bromo-4-chloro-3-indolylphosphate - RFLP Restriction fragment length polymorphism - VNTR Variable number of tandem repeats - AMP-FLP Amplified fragment length polymorphism Presented in part as poster demonstration at the International Symposium on Mass Disasters, Lausanne 1991     

Evaluation of carrier RNA and low volume demineralization for recovery of nuclear DNA from human teeth

Purpose

Teeth and bones are frequently used in the genetic analysis of degraded and ancient human and animal remains. Standard extraction methods, including most commercially available systems, may not yield sufficient DNA to enable successful genetic analysis. Addition of a carrier molecule and demineralization (via EDTA) can increase yields from samples containing limited amounts of DNA. However the benefits of carrier molecules have not been demonstrated for bones and teeth and demineralization introduces large reagent volumes that are difficult to integrate into commercial DNA extraction systems.

Methods

We compared nuclear DNA yields recovered from small samples of partially decomposed human teeth using a commercial silica-based DNA extraction system with and without the addition of carrier RNA and/or a low-volume demineralization step.

Results

DNA yield was significantly improved with demineralization, but there was no significant effect of carrier RNA. The DNA content of a sample did not influence the significance of the effect of demineralization.

Conclusion

Using a simple low-volume (1 mL) demineralization step, prior to DNA extraction with the QIAmp DNA Investigator kit (Qiagen), as little as 50 mg of tooth powder can yield more than 500 ng of nuclear DNA.     

Neue Erkenntnisse zur Ätiologie der sogenannten Strahlenkaries

Aim

In spite of a great number of relevant studies the etiology of radiation induced caries still is discussed in controversy: The assumption of direct radiation induced lesions in the hard tissue is in contrast to an indirect pathogenesis mediated via radio-xerostomia.

Methods

A systematic study is presented, comparing teeth with a manifest radiation caries (group 1, about 60 Gy, long interval to the extraction) and clinically caries free teeth (group 2, about 30 Gy, short interval) with tooth specimens after an experimental enoral (in situ) irradiation (60 Gy, group 3) and after in vitro irradiation (500 to 2.500 Gy, group 4).⁶⁰Co was the irradiation source. Sound teeth were used as a standard (group 5). For non destructive visualisation of subsurface histotomograms by confocal laser scanning microscopy (CLSM) teeth were either used as fresh sections or as Technovit embedded thin slices (sawing grinding technique).

Results

Tooth samples from radiotherapy patients (cancer therapeutic doses, long interval before extraction; group 1) showed three characteristic changes: 1. rarefication of the branching (ramification) of odontoblast processes near the junction, 2. dentine tubules end infront of the interface to the hard tissue and 3. in dentine the interface is characterised by an zone (about 10 μm wide) of low intensity of the remitted light.

Conclusions

The obliteration of the dentine tubules, preceded by a degeneration of the odontoblast processes, is obviously the result of a direct radiogenic cell damage with hampered vascularisation and metabolism particularly in the area of the terminations of the odontoblast processes. The deficit in metabolism combined with a latent damage of the parenchyma (hypo-remitting zone) is evidence for the functional symptoms (subsurface caries). The prerequisite for the micromorphological manifestation of this direct irradiation damage is a vital tooth and in consequence cannot be simulated in situ or in vivo.     

Odontological identification of human remains from mass graves in Croatia

This paper reports the results and methods of dental identification of 1000 human remains exhumed from mass graves in Croatia up to July 1998. Personal identification of the victims was performed at the Department of Forensic Medicine and Criminology at the School of Medicine in Zagreb. A forensic odontologist participated in the identification process by carrying out the dental identification. A total of 824 victims were positively identified, while 176 victims remained unidentified. Dental identification based on available dental antemortem data was achieved in 25% of the cases. Dental identification based on dental charts was achieved in 35%, on x-rays in 15%, on photographs of teeth in 22%, on interviews in 18%, and on confirmation by odontologists in 10% of the cases. Teeth, in combination with anthropological parameters, age, sex and height, as well as other specific characteristics such as tattoos, personal identification cards, clothes, jewellery and DNA, were helpful for identification of 64% of the victims, but the significance for the identification was not dominant. Only in 11% of the cases was identification achieved by other relevant means and teeth not used at all. Identification procedures in Croatia will continue until another 1700 people who are still missing or kept as prisoners of war since the aggression on Croatia in 1991 are found and/or identified. Received: 6 August 1999 / Received in revised form: 9 November 1999     

Reliable genetic identification of burnt human remains

The identification of severely burnt human remains by genetic fingerprinting is a common task in forensic routine work. In cases of extreme fire impact, only hard tissues (bones, teeth) may be left for DNA analysis. DNA extracted from burnt bone fragments may be highly degraded, making an amplification of genetic markers difficult or even impossible. Furthermore, heavily burnt bones are very prone to contamination with external DNA.We investigated whether authentic DNA profiles can be generated from human bones showing different stages of fire induced destruction (well preserved, semi-burnt, black burnt, blue–grey burnt, blue–grey–white burnt). DNA was extracted from 71 bone fragments derived from 13 individuals. Obtained genetic patterns (STRs and mtDNA sequences) were compared to the genetic pattern of the respective bodies.Our results show that the identification via DNA analysis is reliably and reproducibly possible from well preserved and semi-burnt bones. In black burnt bones the DNA was highly degraded and in some cases no nuclear DNA was left, leaving mitochondrial DNA analysis as an option. Blue–grey burnt bones lead only sporadically to authentic profiles. The investigation of blue–grey–white burnt bones barely led to reliable results.     

Dental caries risk assessment

The United Kingdom Defence Dental Agency assesses United Kingdom servicemen as being at low, moderate, or high risk of developing morbidity during the next year, based on the examining dental officer's assessment of their dental status. The most frequent reason for assessing a patient as being at high risk of experiencing a dental emergency is the presence of extensive caries; however, dental officers' perceptions of which caries render a patient at high risk vary considerably. An investigation was carried out with records for Royal Air Force recruits who had been assessed as needing restorative work at their initial dental inspections but for whom this treatment had not been provided for > or = 1 year. Intraoral radiographs taken at the initial dental inspection were examined, and the width of apparently sound dentine remaining between the edge of the carious lesion and the pulp was measured. The results indicate that, when there is > or = 2 mm of apparently sound dentine remaining, there is very little risk that the patient will experience symptoms during the next year. Personnel with a carious tooth with < 2 mm of sound dentine remaining should be classified as high risk.     

Inkrementelle Strukturen im Schmelz der Milchzähne

The neonatal line (NNL) is a distinct incremental marking in the dental enamel, especially in deciduous teeth. In the course of tooth development the NNL represents the position of the enamel-forming front at the time of birth in all the teeth already forming hard tissues at this time. If birth is survived for a certain period of time, deposition of new enamel can be detected outside the NNL. The material properties of teeth, the lack of remodeling, and the known chronology of incremental structures in enamel make teeth an important source of information of vital data in retrospective analyses. Counting enamel incremental markings with circadian periodicity in postnatal enamel enables the determination of the duration of the postnatal lifespan. The lack of an NNL indicates death in utero. In this respect an important contribution can be made by structural analyses of teeth to the investigation of decomposed remains of fetuses and young infants.