The application of dental age estimation methods

Die Studie “The chronology of third molar mineralization in the Austrian population—a contribution to forensic age estimation” legt Referenzdaten zur Mineralisation des dritten Molaren in der österreichischen Population vor. Im Rahmen dieser Querschnittsuntersuchung wurden Panoramaröntgenaufnahmen von 610 ÖsterreicherInnen im Alter zwischen 12 und 24 Jahren gemäß dem 8-gradigen Entwicklungsschema nach Demirjian et al. (1973) beurteilt. Es wurde festgestellt, dass 78,2% der beurteilten Individuen beide mandibulären Molaren besaßen; bei 11,3% der Subjekte wurde ein unterer dritter Molar erfasst. Bisherige Erfahrungen über den zeitigeren Beginn der Weisheitszahnmineralisation bei Frauen wurden bestätigt. Ein statistisch signifikanter Sexualdimorphismus wurde in den Stadien E (p<0,01) und F (p<0,05) nach Demirjian beobachtet. Weiters wurde die Wahrscheinlichkeit eines österreichischen Individuums ermittelt wenigstens 18 Jahre alt zu sein. Waren beide mandibulären Molaren vorhanden, so betrug die Wahrscheinlichkeit wenigstens 18 Jahre alt zu sein 99,5 bzw. 99,3 % für Männer bzw. für Frauen, jeweils im Falle der vollendeten Zahnmineralisation (Stage H nach Demirjian). Dies legt nahe, dass Demirjians Stage H eine wertvolle Hilfe in der dentalen Altersschätzung darstellt. Die gewonnenen Daten sind ausschließlich für österreichische Individuen gültig und können nicht zur Altersschätzung in anderen Populationen herangezogen werden. Gemäß den Empfehlungen der interdisziplinären Arbeitsgemeinschaft für Forensische Altersdiagnostik inkludiert eine Altersbestimmung am Lebenden außerdem ein Handröntgen sowie eine körperliche Untersuchung. Die Autorin befürwortet eine kombinierte Technik die dazu beiträgt, inkorrekte Altersschätzungen aufgrund der biologischen Varianz eines einzelnen Merkmals zu vermeiden. Trotzdem fehlt noch immer eine große Studie, welche die Verwendung der Methodenkombination validiert, obwohl sich selbige bereits in Verwendung befindet. Die zweite Untersuchung “On the applicability of secondary dentin formation to radiological age estimation in young adults” untersuchte, ob bisher publizierte Regressionsgleichungen zur dentalen Altersschätzung, basierend auf radiographischen, zweidimensionalen Messungen der Pulpagröße, zu reproduzierbaren und korrekten Ergebnissen führen. Es wurden Panoramaröntgenaufnahmen von 44 österreichischen Individuen, im Alter zwischen 13 und 24 Jahren, ausgesucht um die Messungen nach Kvaal et al. (1995) vorzunehmen. Die Verwendung der Regressionsgleichung von Paewinsky et al. (2005) führte zu einer konsistenten Überschätzung, die Gleichungen nach Kvaal et al. (1995) resultierten in einer konstanten Unterschätzung des Alters. Aufgrund dieser Ergebnisse kann gefolgert werden, dass die Formeln The Application of Dental Age Estimation Methods Zusammenfassung auf Deutsch 93 nach Kvaal et al. (1995) und Paewinsky et al. (2005) für die dentale Altersschätzung junger Erwachsener nicht geeignet sind. Es wird empfohlen Sekundärdentinbildung anhand einer großen Stichprobe der vorliegenden Altersgruppe zu evaluieren um verlässliche Referenzdaten hierzu erhalten. Weiters könnte es hilfreich sein, die Verminderung der Pulpagröße anhand zusätzlicher Parameter, wie von Cameriere et al. (2007b) vorgeschlagen zu ermitteln, oder aber mittels Computertomographie (Yang et al. 2006). Die dritte Studie „Comparison of the validity of three dental methods for the estimation of age at death“ konzentrierte sich auf den Vergleich der Genauigkeit, des Bias sowie der Praktikabilität zweier makroskopischer und einer histologischen dentalen Methode zur Schätzung des Sterbealters. Die Stichprobe bestand aus 67 Zähnen von 37 Individuen im Alter zwischen 20 und 91 Jahren. Die Schätzung des chronologischen Alters erfolgte gemäß den Methoden nach Lamendin et al. (1992) (LAM), Bang and Ramm (1970) (BR), sowie der Quantifizierung von Zahnzementannulationen (TCA). Die höchste Genauigkeit des Schätzergebnisses zeigte die TCA- Methode bei Zähnen der jungen, mittleren und alten Altersgruppe. LAM zeigte die höchste Präzision in der jungen und alten Altersgruppe, wohingegen sich in der mittleren Altersgruppe TCA als präziser erwies. TCA war die präziseste Methode, sofern die Altersgruppen nicht getrennt analysiert wurden. Bezüglich des Bias hatten alle Methoden die Tendenz das Alter in jungen Proben zu überschätzen, sowie in alten Proben zu unterschätzen. Die Ausnahme war hier TCA, die junge Erwachsene ohne Bias schätzte. Aufgrund der höheren Genauigkeit und Präzision wird es empfohlen die TCA-Methode LAM und BR vorzuziehen, sofern entsprechendes Wissen und Ausstattung vorhanden sind. Weitere Studien in Bezug auf den genauen Mechanismus und die Chronologie der Zahnzementannulationen, der Sekundärdentinapposition, sowie der Dentintransparenz würden die Genauigkeit von Altersschätzungen verbessern. Weiters wäre eine allgemein anerkannte Prozedur zur Präparation und Evaluation von Zahnzementannulationen wünschenswert um einen gemeinsamen Standard zu etablieren.The study “The chronology of third molar mineralization in the Austrian population—a contribution to forensic age estimation” provides reference data on third molar mineralization in the Austrian population. Within the present cross-sectional evaluation panoramic radiographs of 610 Austrians between 12 and 24 years of age were assessed using the 8 developmental stages of Demirjian et al. (1973). It was found that 78.2% of the analyzed individuals possessed both mandibular third molars; 11.3% of the subjects were recorded to have one lower third molar. Previous records about an earlier occuring third molar mineralization in females were confirmed. A statistically significant sexual dimorphism was noted in Demirjian’s stages E (p<0.01) and F (p<0.05). A comparison with previous study data suggests ethnic differences in the timing of third molar mineralization. Moreover, the probability of whether an Austrian individual is at least 18 years old was determined. In the case where two mandibular third molars were present, the probability for an Austrian individual to be at least 18 years is 99.5% and 99.3% for males and females, respectively in the case were tooth mineralization is completed (Demirjian’s stage H). This indicates that Demirjian’s stage H constitutes a reliable aid in dental age estimation. The obtained data is exclusively valid in Austrian individuals and cannot be used for dental age estimation in other populations. According to the recommendations of the interdisciplinary Study Group on Forensic Age Diagnostics, an age analysis of living persons also includes an X-ray of the hand and a physical examination. The author supports a combined technique that helps to avoid incorrect age estimates due to the biological variance of a single trait. Nonetheless, a large study that validates the use of the combination of methods is still lacking although this procedure is already in use. The second investigation “On the applicability of secondary dentin formation to radiological age estimation in young adults” aimed to explore whether previously published regression formulae for dental age estimation based on radiographic two-dimensional measurements of the pulp size lead to reproducible and statistically sound results. Panoramic radiographs of 44 Austrian individuals, aged between 13 and 24 years, were selected to carry out the measurements proposed by Kvaal et al. (1995). The use of the regression formulae reported by Paewinsky et al. (2005) led to a consistent overestimation, the equations of Kvaal et al. (1995) resulted in a constant underestimation of age. From these results it can be concluded that the formulae of Kvaal et al. (1995) and Paewinsky et al. (2005) are not suitable for dental age The Application of Dental Age Estimation Methods Summary of the Results and Outlook 91 estimation in young adults. It is suggested to evaluate secondary dentin formation with a large sample size in the present age group to obtain reliable reference data. Furthermore, it could be helpful to assess the decrease of the pulp size also by additional parameters as proposed by Cameriere et al. (2007b) or via computerized tomography (Yang et al. 2006). The third study “Comparison of the validity of three dental methods for the estimation of age at death” focused on the comparison of accuracy, bias, and practicability of two macroscopic and one histological dental age at death estimation methods. The study sample comprised of 67 teeth, obtained from 37 individuals aged 20 to 91 years. Chronological age was estimated by means of the methods proposed by Lamendin et al. (1992) (LAM), Bang and Ramm (1970) (BR), and the quantification of tooth cementum annulations (TCA). TCA was found to be the most accurate age at death estimation method in young, middle, and old age group teeth. LAM displayed the highest precision in the young, and the old age group whereas TCA was more precise in the middle age group. TCA showed to be the most precise method when the precision was calculated for all ages. Considering the bias, all methods displayed a tendency to overestimate age in young and to underestimate it in old specimens. The exception to this rule was TCA, which provided unbiased estimates for young adults. The higher accuracy and precision recommends favouring TCA over LAM and BR, provided that the required knowhow and equipment are available. Further studies in order to assess the exact mechanism and timing of tooth cementum layering, secondary dentin apposition, and dentin sclerosis would be helpful to improve the accuracy of age estimates. Moreover, a generally accepted procedure for the preparation and evaluation of root cementum annulations would be desirable to establish common standards

Silk fibroin microparticles as carriers for delivery of human recombinant bone morphogenetic protein-2 : in vitro and in vivo bioactivity

The in vitro and in vivo efficiency of fibroin microparticles as a delivery carrier for bone morphogenetic protein-2 (BMP-2) was evaluated. BMP-2 was encapsulated in silk fibroin particles that were produced by a simple and very mild processing method. The dose-response of BMP-2-loaded fibroin particles was examined in C2C12 cells, after 5 days of culture. The BMP-2 retained most of its activity as observed by the increase in alkaline phosphatase activity, which was much higher when BMP-2 was encapsulated into the particles rather than just surface-adsorbed. After 2 weeks of culture, increased mineralization was observed with BMP-2-loaded particles in comparison to soluble added growth factor. No significant cytotoxicity was detected. When implanted in a rat ectopic model, bone formation was observed by in vivo micro-computed tomography after 2 and 4 weeks postimplantation, with particles loaded with 5 or 12.5 microg BMP-2. An increase in bone density was observed over time. Histology revealed further evidence of ectopic bone formation, observed by strong alizarin red staining and osteocalcin immunostaining. Our findings show that fibroin microparticles may present an interesting option for future clinical applications in the bone tissue engineering field, and therefore, further studies have been planned.The authors acknowledge Anna Hofmann and Anna Khadem for additional help with some experiments, and Karin Brenner for animal maintenance. This work was supported by Fundacao para a Ciencia e Tecnologia (Ph.D. grant SFRH/BD/17049/2004), project ElastM (POCI/CTM/57177/2004 funded by FEDER and the Fundacao para a Ciencia e Tecnologia), Marie Curie Alea Jacta EST short-term grant (MEST-CT-2004-8104), and European STREP Project HIP-POCRATES (NMP3-CT-2003-505758). This work was carried out under the scope of the European NoE EXPERTISSUES (NMP3-CT-2004-500283)

Non-invasive in vivo tracking of fibrin degradation by fluorescence imaging

Fibrin-based sealants consist of natural coagulation factors involved in the final phase of blood coagulation, during which fibrinogen is enzymatically converted by thrombin to form a solid-phase fibrin clot. For applications in tissue regeneration, a controlled process of matrix degradation within a certain period of time is essential for optimal wound healing. Hence, it is desirable to follow the kinetics of fibrinolysis at the application site. Non-invasive molecular imaging systems enable real-time tracking of processes in the living animal. In this study, a non-invasive fluorescence based imaging system was applied to follow and quantify site-specific degradation of fibrin sealant. To enable non-invasive tracking of fibrin in vivo, fibrin-matrix was labelled by incorporation of a fluorophore-conjugated fibrinogen component. Protein degradation and release of fluorescence were, in a first step, correlated in vitro. In vivo, fluorophore-labelled fibrin was subcutaneously implanted in mice and followed throughout the experiment using a multispectral imaging system. For the fluorescent fibrin, degradation correlated with the release of fluorescence from the clots in vitro. In vivo it was possible to follow and quantify implanted fibrin clots throughout the experiment, demonstrating degradation kinetics of approximately 16 days in the subcutaneous compartment, which was further confirmed by histological evaluation of the application site

Gelatin embedding for the preparation of thermoreversible or delicate scaffolds for histological analysis

Thermoreversible hydrogels for tissue engineering (TE) purposes have gained increased attention in recent years as they can be combined with cells and drugs and directly injected into the body. Following the fate of transplanted cells in situ is essential in characterizing their distribution and survival, as well as the expression of specific markers or cell-matrix interactions. Existing histological embedding methods, such as paraffin wax embedding, can mechanically damage some biomaterials during processing. In this study, we describe a broadly applicable preparation protocol that allows the handling of delicate, thermoreversible scaffolds for histological sectioning. The gelatin solution permits the embedding of samples at 37 °C, which suits the solid phase of most TE scaffolds. A thermoreversible scaffold of polycaprolactone microparticles, combined with poly(polyethylene glycol methacrylate ethyl ether) and containing human adipose-derived stem cells, was prepared for histology by an initial gelatin embedding step in addition to the standard cryosectioning and paraffin processing protocols. Sections were evaluated by hematoxylin eosin staining and immunostaining for human vimentin. The gelatin embedding retained the scaffold particles and permitted the complete transfer of the construct. After rapid cooling, the solid gelatin blocks could be cryosectioned and paraffin infiltrated. In contrast to direct cryosectioning or paraffin infiltration, the extended protocol preserved the scaffold structure as well as the relevant cell epitopes, which subsequently allowed for immunostaining of human cells within the material. The gelatin embedding method proposed is a generalizable alternative to standard preparations for histological examination of a variety of delicate samples

Sunlight exposure exerts immunomodulatory effects to reduce multiple sclerosis severity

Multiple sclerosis (MS) disease risk is associated with reduced sunexposure. This study assessed the relationship between measures of sun exposure (vitamin D [vitD], latitude) and MS severity in the setting of two multicenter cohort studies (n(NationMS) = 946, n(BIONAT) = 990). Additionally, effect-modification by medication and photosensitivity-associated MC1R variants was assessed. High serum vitD was associated with a reduced MS severity score (MSSS), reduced risk for relapses, and lower disability accumulation over time. Low latitude was associated with higher vitD, lower MSSS, fewer gadolinium-enhancing lesions, and lower disability accumulation. The association of latitude with disability was lacking in IFN-beta-treated patients. In carriers of MC1R:rs1805008(T), who reported increased sensitivity toward sunlight, lower latitude was associated with higher MRI activity, whereas for noncarriers there was less MRI activity at lower latitudes. In a further exploratory approach, the effect of ultraviolet (UV)-phototherapy on the transcriptome of immune cells of MS patients was assessed using samples from an earlier study. Phototherapy induced a vitD and type I IFN signature that was most apparent in monocytes but that could also be detected in B and T cells. In summary, our study suggests benefidal effects of sun exposure on established MS, as demonstrated by a correlative network between the three factors: Latitude, vitD, and disease severity. However, sun exposure might be detrimental for photosensitive patients. Furthermore, a direct induction of type I IFNs through sun exposure could be another mechanism of UV-mediated immune-modulation in MS