Szilvia Arany

Radiographic Survey of Third Molar Development in Relation to Chronological Age Among Japanese Juveniles

The aim of the present study was to establish Japanese reference material on the third molar development of Japanese juveniles for forensic application. Observations were performed on the orthopantomograms of 1282 Japanese patients between the ages of 14.0 and 24.0 years. Demirjian formation stages of the maxillary and mandibular third molars were recorded for chronological evaluation of wisdom teeth and applied for further statistical analysis. Statistically significant differences were noted between the upper and lower jaws and genders. Accordingly, males achieved root developmental grades earlier than females. We assessed the mean ages for all formation grades and predicted the probability that a Japanese juvenile would be older than the relevant ages of 14, 16, and 20 as defined by Japanese Juvenile Law. We determined the likelihood that a Japanese youth is older than the relevant age of 18 as defined by legislation in the United States.

Nerve growth factor promotes differentiation of odontoblast‐like cells

Contemporary strategies in tooth repair markedly rely on the newest findings on the cellular and biological components of dental development. Among several identified bioactive molecules, neurotrophins were recently proposed to affect tooth germ cell proliferation, differentiation, and cell–extracellular matrix interactions. The present study attempted to explore the effect of nerve growth factor (NGF) on a spontaneously immortalized dental papilla mesenchymal cell line. NGF induced differentiation of odontoblast‐lineage cells with subsequent biomineralization in vitro. Here we showed that normalized transcript levels of tissue‐specific markers such as DSPP and DMP1 were elevated significantly, indicating cell differentiation and maturation processes. We performed innovative gene expression analysis of TM14, a matricellular protein and novel member of the fibulin family. TM14 expression followed a pattern similar to that of DMP1, which suggests its important role in cell–matrix and intercellular interactions during dentin calcification. Alkaline phosphatase enzyme assay confirmed the extracellular matrix calcifications in NGF‐supplemented groups. Thus, NGF was characterized as a potent promoter of mineralization during dentin formation. For the first time, we included TM14 in odontoblast genotype analysis and proved that NGF also promotes in vitro odontoblast differentiation. Collectively, these results highlight the importance of NGF during tooth morphogenesis, as well as urge the elaboration of complex epithelial–mesenchymal tissue cultures, where further elucidation of the signaling factor network could be completed. J. Cell. Biochem. 106: 539–545, 2009.

Ascl3 knockout and cell ablation models reveal complexity of salivary gland maintenance and regeneration.

Expression of the transcription factor, Ascl3, marks a population of adult progenitor cells, which can give rise to both acinar and duct cell types in the murine salivary glands. Using a previously reported Ascl3(EGFP-Cre/+) knock-in strain, we demonstrate that Ascl3-expressing cells represent a molecularly distinct, and proliferating population of progenitor cells located in salivary gland ducts. To investigate both the role of the Ascl3 transcription factor, and the role of the cells in which it is expressed, we generated knockout and cell-specific ablation models. Ascl3 knockout mice develop smaller salivary glands than wild type littermates, but secrete saliva normally. They display a lower level of cell proliferation, consistent with their smaller size. In the absence of Ascl3, the cells maintain their progenitor function and continue to generate both acinar and duct cells. To directly test the role of the progenitor cells, themselves, in salivary gland development and regeneration, we used Cre-activated expression of diphtheria toxin (DTA) in the Ascl3-expressing (Ascl3+) cell population, resulting in specific cell ablation of Ascl3+ cells. In the absence of the Ascl3+ progenitor cells, the mice developed morphologically normal, albeit smaller, salivary glands able to secrete saliva. Furthermore, in a ductal ligation model of salivary gland injury, the glands of these mice were able to regenerate acinar cells. Our results indicate that Ascl3+ cells are active proliferating progenitors, but they are not the only precursors for salivary gland development or regeneration. We conclude that maintenance of tissue homeostasis in the salivary gland must involve more than one progenitor cell population.

Acute effects on the lung and the liver of oral administration of cerium chloride on adult, neonatal and fetal mice

We evaluated tissue changes associated with cerium chloride administration via gavage to adult mice, via milk to neonatal mice and transplacentally to fetal mice. Change in adults consisted of extensive pulmonary hemorrhage, pulmonary venous congestion, thickened alveolar septae, hepatic necrosis and neutrophil infiltrations. Those in fetal mice consisted of pulmonary and hepatic congestion. These results indicate that gavage cerium administration elicited subtle tissue changes, though oral toxicity is rather low. These changes were less severe in neonatal and fetal mice. When cerium was injected into adult mice through the tail vein, cerium was distributed mainly to the liver, spleen and lung dose-dependently with the cerium concentration gradually decreasing after 3 days. A study of cerium anticoagulation in mouse plasma showed that clotting time was significantly prolonged when cerium was added to plasma. These results suggest that cerium may disturb blood coagulation and cause pulmonary and hepatic vascular congestion.

Racemization in enamel among different types of teeth from the same individual

We measured the quantity of D-aspartic acid (degree of racemization of aspartic acid) in the enamel of different types of teeth from the same individual. We studied the correlation between the degree of racemization and the time of formation of each particular tooth, as well as the applicability of the degree of racemization to estimation of chronological age. If the environmental condition of the teeth is the same, the degree of racemization is expected to be highest in teeth that completed formation in the earliest period of time. Different degrees of racemization in enamel were found among different types of teeth, even in the same individual. The degree of racemization in enamel was found to be higher in molars than in incisors, and showed a tendency that did not necessarily coincide with the time of formation. This seemed to be due to the fact that the environmental temperature was higher in the molar region located deeper in the oral cavity than the front region, and that enamel was more affected by breathing air than dentin because the D/L ratios in enamel were lower than those in dentin. Using enamel, a better estimation of chronological age was obtained from calculations based on the degree of racemization of each type of tooth than from all the different teeth together. However, these estimated ages were not better than those from dentin.

Comparison of Age Estimated from Degree of Racemization of Aspartic Acid, Glutamic Acid and Alanine in the Femur

Aspartic acid (Asp) is generally used for estimation of age by measuring the degree of racemization. For other amino acids, however, there are few reports regarding the usefulness of the degree of racemization for the estimation of age. Accordingly, in this study using the femur (obtained from 21 cadavers) as the specimen, we measured the degree of racemization of glutamic acid (Glu) and alanine (Ala) along with Asp in the total amino acid (TAA) fraction as well as in acid-insoluble collagen-rich (IC) and acid-soluble peptide (SP) sub-fractions. We compared the degrees of racemization of each amino acid and the accuracy of the ages estimated from them. The degree of racemization and the reaction rate of racemization were ranked in the order of Asp > Glu > Ala in the TAA and IC fractions, but Asp > Ala > Glu in the SP fraction. It is noteworthy that the degrees of racemization differed between the three amino acids depending on the fraction tested. The correlation coefficient (r) between the degree of racemization and the chronological age was higher in the SP than in the TAA or IC fraction. Among three amino acids, Asp showed the highest correlation coefficient as predicted. The present study confirmed that Asp from the SP fraction is the best indicator for age estimation using racemization rates.

Application of spontaneously immortalized odontoblast cells in tooth regeneration

Here, we report on the first attempt to bioengineer tooth using a spontaneously immortalized mesenchymal cell line. To assess the odontogenic potential of this cell line, odontoblast-lineage cells (OLC) were re-associated with competent dental epithelium isolated from E14.5 mice. A novel three-dimensional organ germ culture method was applied to nurture the constructs in vitro. Additionally, recombinants were transplanted under the kidney capsule in host animals for 2 weeks. Transplants developed into tooth tissues in one-third of the cases. OLC-derived GFP-positive cells could be identified in mineralizing tooth germs by immunohistochemistry. OLCs were capable of intercellular and cell-matrix communication, thus they eventually differentiated into functional odontoblasts. In summary, we managed to utilize OLCs for dental mesenchyme substitution in tooth regeneration experiments. Therefore, our spontaneously transformed cell line proved its potential for future complex, tooth developmental and bioengineering studies.

Pro-apoptotic gene knockdown mediated by nanocomplexed siRNA reduces radiation damage in primary salivary gland cultures.

A critical issue in the management of head and neck tumors is radioprotection of the salivary glands. We have investigated whether siRNA‐mediated gene knock down of pro‐apoptotic mediators can reduce radiation‐induced cellular apoptosis in salivary gland cells in vitro. We used novel, pH‐responsive nanoparticles to deliver functionally active siRNAs into cultures of salivary gland cells. The nanoparticle molecules are comprised of cationic micelles that electrostatically interact with the siRNA, protecting it from nuclease attack, and also include pH‐responsive endosomolytic constituents that promote release of the siRNA into the target cell cytoplasm. Transfection controls with Cy3‐tagged siRNA/nanoparticle complexes showed efficiently internalized siRNAs in more than 70% of the submandibular gland cells. We found that introduction of siRNAs specifically targeting the Pkcδ or Bax genes significantly blocked the induction of these pro‐apoptotic proteins that normally occurs after radiation in cultured salivary gland cells. Furthermore, the level of cell death from subsequent radiation, as measured by caspase‐3, TUNEL, and mitochondrial disruption assays, was significantly decreased. Thus, we have successfully demonstrated that the siRNA/nanoparticle‐mediated knock down of pro‐apoptotic genes can prevent radiation‐induced damage in submandibular gland primary cell cultures. J. Cell. Biochem. 113: 1955–1965, 2012.

Age estimation of bloodstains: a preliminary report based on aspartic acid racemization rate.

This study describes an innovative application of a well-established method of age determination. The conventional method of aspartic acid racemization (AAR) is based on estimation of the d-l-aspartic acid ratio in slow turnover tissues, such as tooth tissue, to reflect the age of an individual. This method has been recently applied to age estimation in forensic investigations, and is also widely used for archeological dating of fossils. We suggest that the aspartic acid racemization method could be applied to a significant, although unresolved, forensic issue: that of bloodstain dating. Standard kinetic experiments were used to describe the characteristics of the racemization reaction in bloodstains, which were then employed to estimate the age of various samples. The soluble protein fraction of a bloodstain produced a stronger correlation between elapsed time and d-aspartic acid content than total amino acid fractions. According to our preliminary results, the time lapse after the creation of a bloodstain can be determined ex vivo by measuring the extent of aspartic acid racemization. Our analysis highlights the need for further study into the preservation and composition of bloodstains to assist in further development of this pioneering application.

Salivary Glands Stem Cells, Self-duplication, or Both?

Understanding the intrinsic potential for renewal and regeneration within a tissue is critical for the rational design of reparative strategies. Maintenance of the salivary glands is widely thought to depend on the differentiation of stem cells. However, there is also new evidence that homeostasis of the salivary glands, like that of the liver and pancreas, relies on self-renewal of differentiated cells rather than a stem cell pool. Here, we review the evidence for both modes of turnover and consider the implications for the process of regeneration. We propose that the view of salivary glands as postmitotic and dependent on stem cells for renewal be revised to reflect the proliferative activity of acinar cells and their role in salivary gland homeostasis.