piR-368 promotes odontoblastic differentiation of dental papilla cells via the Smad1/5 signaling pathway by targeting Smurf1.

PURPOSE: The important role of non-coding RNAs in odontoblastic differentiation of dental tissue-derived stem cells has been widely demonstrated; however, whether piRNA (a subclass of non-coding RNA) involved in the course of odontoblastic differentiation is not yet available. This study aimed to investigate the expression profile of piRNA during odontogenic differentiation of mDPCs and the potential molecular mechanism in vitro. MATERIALS AND METHODS: The primary mouse dental papilla cells (mDPCs) were isolated from the first molars of 1-day postnatal Kunming mice. Then, they were cultured in odontogenic medium for 9 days. The expression profile of piRNA was detected by Small RNA sequencing. RT-qPCR was used to verify the elevation of piR-368. The mRNA and protein levels of mineralization markers were examined by qRT-PCR and Western blot analysis. Alkaline phosphatase (ALP) activity and alizarin red S staining were conducted to assess the odontoblastic differentiation ability. RESULTS: We validated piR-368 was significantly upregulated and interference with piR-368 markedly inhibited the odontogenic differentiation of mDPCs. In addition, the relationship between Smad1/5 signaling pathway and piR-368-induced odontoblastic differentiation has been discovered. Finally, we demonstrated Smurf1 as a target gene of piR-368 using dual-luciferase assays. CONCLUSION: This study was the first to illustrate the participation of piRNA in odontoblastic differentiation. We proved that piR-368 promoted odontoblastic differentiation of mouse dental papilla cells via the Smad1/5 signaling pathway by targeting Smurf1.

Identification of ancient viruses from metagenomic data of the Jomon people.

Ancient DNA studies provide genomic information about the origins, population structures, and physical characteristics of ancient humans that cannot be solely examined by archeological studies. The DNAs extracted from ancient human bones, teeth, or tissues are often contaminated with coexisting bacterial and viral genomes that contain DNA from ancient microbes infecting those of ancient humans. Information on ancient viral genomes is useful in making inferences about the viral evolution. Here, we have utilized metagenomic sequencing data from the dental pulp of five Jomon individuals, who lived on the Japanese archipelago more than 3000 years ago; this is to detect ancient viral genomes. We conducted de novo assembly of the non-human reads where we have obtained 277,387 contigs that were longer than 1000 bp. These contigs were subjected to homology searches against a collection of modern viral genome sequences. We were able to detect eleven putative ancient viral genomes. Among them, we reconstructed the complete sequence of the Siphovirus contig89 (CT89) viral genome. The Jomon CT89-like sequence was determined to contain 59 open reading frames, among which five genes known to encode phage proteins were under strong purifying selection. The host of CT89 was predicted to be Schaalia meyeri, a bacterium residing in the human oral cavity. Finally, the CT89 phylogenetic tree showed two clusters, from both of which the Jomon sequence was separated. Our results suggest that metagenomic information from the dental pulp of the Jomon people is essential in retrieving ancient viral genomes used to examine their evolution.

An outbreak of relapsing fever unmasked by microbial paleoserology, 16th century, France.

OBJECTIVES: Depicting past epidemics currently relies on DNA-based detection of pathogens, an approach limited to pathogens with well-preserved DNA sequences. We used paleoserology as a complementary approach detecting specific antibodies under a mini line-blot format including positive and negative control antigens. METHODS: Mini line blot assay incorporated skim milk as negative control, Staphylococcus aureus as positive control, and antigens prepared from lice-borne pathogens Rickettsia prowazekii, Borrelia recurrentis, Bartonella quintana, and Yersinia pestis. Paleoserums were extracted from rehydrated dental pulp recovered from buried individuals. Mini line blots observed with the naked eye, were quantified using a scanner and appropriate software. Paleoserology was applied to the indirect detection of lice-borne pathogens in seven skeletons exhumed from a 16th-17th century suspected military burial site (Auxi-le-Château); and 14 civils exhumed from a 5th-13th century burial site (Saint-Mont). Direct detection of pathogens was performed using quantitative real-time PCR. RESULTS: In Auxi-le-Château, paleoserology yielded 7/7 interpretable paleoserums including 7/7 positives for B. recurrentis including one also positive for B. quintana. In Saint-Mont, paleoserology yielded 8/14 interpretable paleoserums and none reacted against any of the four pathogens. Antibodies against R. prowazekii and Y. pestis were not detected. The seroprevalence was significantly higher in the military burial site of Auxi-le-Château than in the civil burial site of Saint-Mont. Real-time PCR detection of B. quintana yielded 5/21 positive (3 at Saint-Mont and 2 at Auxi-le-Château) whereas B. recurrentis was not detected. CONCLUSIONS: Paleoserology unmasked an outbreak of relapsing B. recurrentis fever in one 16th - 17th century military garrison, missed by real-time PCR. Paleoserology offers a new tool for investigating past epidemics, in complement to DNA sequence-based approaches.

Optimization of DNA extraction from dental remains.

Efficient DNA extraction procedures is a critical step involved in the process of successful DNA analysis of such samples. Various protocols have been devised for the genomic DNA extraction from human tissues and forensic stains, such as dental tissue that is the skeletal part that better preserves DNA over time. However DNA recovery is low and protocols require labor-intensive and time-consuming step prior to isolating genetic material. Herein, we describe an extremely fast procedure of DNA extraction from teeth compared to classical method. Sixteen teeth of 100-year-old human remains were divided into two groups of 8 teeth and we compared DNA yield, in term of quantity and quality, starting from two different sample preparation steps. Specifically, teeth of group 1 were treated with a classic technique based on several steps of pulverization and decalcification, while teeth of group 2 were processed following a new procedure to withdraw dental pulp. In the next phase, the samples of both group underwent the same procedure of extraction, quantification and DNA profile analysis. Our findings provide an alternative protocol to obtain a higher amount of good quality DNA in a fast time procedure, helpful for forensic and anthropological studies.

Dental pulp as a source of low-contaminated DNA.

The in-laboratory contamination of the ancient samples hinders the result interpretation of the investigations in the field of paleomicrobiology. We had promoted the dental pulp as a sample that limits the risks of in-laboratory contamination of the ancient material. In this work, we measured the contamination of the dental pulp manipulated according to paleomicrobiology protocol, used as a source of a total DNA for metagenomics. First, total DNA extracted from two dog canines was sequenced using next generation sequencing. This yielded a total of 487,828 trimmed reads with a length of 227 ± 35 bp. Sequence analysis of the final dataset using Blast algorithm search and stringent thresholds for sequence identity and coverage against a database including both Canis lupus familiaris and Homo sapiens complete genomes showed that 95% of reads were assigned to C. familiaris whereas 0.03% was assigned to H. sapiens. In a second step, two teeth collected from two 12th century mammals were manipulated following the same protocol. A total of 13,890 trimmed reads with a 157 ± 67 bp length yielded 0-0.35% reads assigned to H. sapiens. This study indicates that the dental pulp is a useful for detecting the significant nucleic sequences in both modern and ancient samples.

High proportions of Staphylococcus epidermidis in dental caries harbor multiple classes of antibiotics resistance, significantly increase inflammatory interleukins in dental pulps.

Staphylococcus epidermidis is one of most prevalent in dental caries or dental pulp which has the capability of horizontal genetic transfer between different bacterial species in the oropharynx, suggesting that it may evolve with the dissemination of resistant determinants, This study was performed to molecularly characterize and differentiate S. epidermidis isolated from dental caries and healthy individual. Also, two important cytokines in inflammation were assayed caused due to S. epidermidis of health and dental caries sources. Dental caries strains were more resistant with high MIC 50 and MIC 90 value. These isolates also showed the presence of mecA gene and another virulence gene i. e sea and seb comparatively more than healthy individual isolates. SCCmec types, III and IV was more prevalent in dental caries isolates where an as healthy individual was more non-typable. Additionally, the quantity of IL-1ß and IL-8 caused due to dental caries isolates was seen more which indicate dental caries isolates are able to induce. This study showed that S. epidermidis a normal flora of oropharyngeal are more diverse to those strains which cause dental caries. S. epidermidis owns a prodigious genetic plasticity that permits to obtain, lose or regulate genetic elements that provide compensations to improve its colonization in the host.

The rate of RNA degradation in human dental pulp reveals post-mortem interval.

Post-mortem interval (PMI) is the amount of time elapsed since the time of death. Over the years, many methods were developed to assess PMI, but their precision and time frame of applicability are often limited. Our present pilot study aimed to prove if RNA degradation of human dental pulp can be used for PMI estimation. RNA was isolated from the pulps of healthy wisdom teeth and premolars. RNA degradation was determined as RNA integrity number (RIN) with Agilent Bioanalyzer and subsequently by amplification of different length products by PCR after reverse transcription. The RNA integrity analysis allowed us to determine the time of post-mortem interval with high confidence level in the first 21 days. With the PCR-based method, we were able to perform a crude estimation of incubation time of teeth between 20 and 42 days post extraction. These results show that this method might be a promising new tool for PMI estimation despite the limitations.

Dental pulp in mature replanted human teeth: morphological alterations and metalloproteineses-2 and -9, Annexin-5, BCL-2 and iNOS modulation.

Tooth replantation, as a treatment concept, has been subject to controversies regarding the mechanism as well as the various parameters underlying this process. This work aimed to study time-related changes in the pulp of replanted mature human premolars through the changes in the levels of certain factors involved in the underlying mechanisms of pulpal tissue healing after replantation. Eleven experimental mature teeth were extracted, immediately replanted in the original socket and left without any other intervention for 1, 2, 3 and 12 weeks before re-extraction. Three premolars served as control. All specimens were subject to histological analysis and the levels of MMP-2, MMP-9, Annexin V, iNOS and BCL-2 (anti-apoptotic family) were analyzed employing immunohistochemistry. The results showed degradation of the extracellular matrix (ECM), inflammatory cell infiltrate, loss in pulpo-dentine interface and loss of odontoblasts in the dental pulp tissue. This was accompanied by increase over time of MMP-9, Annexin V, iNOS and a decrease of BCL-2 and MMP-2, suggesting that apoptosis increased throughout the experimental period.

Biomarkers in the dentin-pulp complex: role in health and disease.

Biomarkers are functional elements at the cellular or molecular level, playing important roles in health and disease. The dentin-pulp complex of the tooth houses several biomarkers at different stages of development, and a lack of these biomarkers results in developmental disorders. Furthermore, biomarkers play a very important role in the pathogenesis of dental caries, pulpal and periapical pathoses in two ways - they are essential elements in the pathological process and their detection helps in accurate diagnosis of the pathological condition. The aim of this paper is to review the literature regarding the important biomarkers involved in the development of the dentin-pulp complex and in the pathological conditions involving the dentin-pulp complex.

A pilot study on post-mortem determination of drug abuse on dental tissues.

BACKGROUND: Post-mortem toxicology constantly deals with the research of reliable alternative matrices to be applied in case of highly damaged corpses (such us carbonized, skeletonized, human remains, etc.). Teeth represent a promising alternative matrix since dental tissues are endowed by different features, resistance and stability after death. SCOPE: Since scant literature reported on the pharmacokinetics and mechanism of incorporation of xenobiotics into dental tissues, this pilot research aims to investigate whether in the pulp can be detected the same substances found in blood in drug related death cases. Secondly, the study is addressed to disclose the possible deposit of drugs in dental hard tissues (dentine and/or enamel), thus contributing to reconstruct the drug abuse history (timing, e.g.). MATERIALS AND METHODS: The study experimented with a novel method to separately analyse dental enamel, dentin, and pulp, applied to 10 teeth collected during autopsies of drug-related deaths along with blood and hair samples for classic toxicological analyses. Each tooth was prepared by "pulverization technique" and then analysed by gas chromatography paired with mass spectrometry (GC-MS) and ultra high performance liquid chromatography coupled to high resolution mass spectrometry (UHPLC/HR-MS) for searching cocaine, opiates, and metabolites. The results were then compared with those obtained from blood and hair samples. RESULTS: Preliminary results demonstrated that teeth differ from any other classic matrix (blood and hairs) since the qualitative correspondence of the detected substances between pulp and blood as well as dental hard tissues and hair suggests that they can be useful in post-mortem evaluation as a unique matrix for both acute and chronic assumptions of drugs. The mechanism of accumulation of substances in mineralized dental tissues emerged the most significant result, being influenced by the type of molecule and the method of assumption. The main limitation of this study is the limited availability of the sample and the absence of anamnestic information of the time, rates and method of drug assumption during life. Further research is necessary to systematically investigate the distribution of different substances within the different tissues of the tooth.

Orthodontic stress Bcl-2 modulation and human odontoblast survival.

This study assessed the effect of orthodontic traction on Bcl-2 expression and apoptosis in human dental pulp. It also explored, in absence of noxious stimuli the regeneration of odontoblasts during the entire life of the tooth. Twenty young patients, with Class II malocclusion and severe to moderate crowding, were referred for orthodontic assessment. Whole pulps were removed. Half the pulps were fixed, paraffin-embedded and processed for histology and immunohistochemistry using anti Bcl-2, Caspase 9 cleaved and Caspase 9 not cleaved antibodies. The rest of the samples, both orthodontically treated and not treated dental pulps, were immediately frozen at -80ºC after the extraction and quantitative PCR was performed. Histology showed alterations in pulp microanatomy after 8 months of treatment. Immunohistochemistry depicted a decreasing expression of Bcl-2 in dental pulp over time in the non-treated while a very weak to absent Bcl-2 expression was detected in the orthodontically treated tissues. Active and non-active forms of Caspases, were expressed in both groups of dental pulp, however staining for the non active form was stronger than the corresponding cleaved form in all samples. The increased expression was detected mainly at nuclear level. Real time qPCR results correlated with those of immunohistochemistry and exhibited a decreasing expression of Bcl-2 in the treated samples. Orthodontic traction may inhibit the expression of Bcl-2, favoring the onset of apoptosis and leading us to conclude that the physical stress in the absence of noxious stimuli might make odontoblasts regeneration less likely.

Teeth as a source of DNA for forensic identification of human remains: a review.

Teeth and bones are frequently the only sources of DNA available for identification of degraded or fragmented human remains. The unique composition of teeth and their location in the jawbone provide additional protection to DNA compared to bones making them a preferred source of DNA in many cases. Despite this, post-mortem changes in the structure and composition of teeth, and the location and diagenesis of DNA within them are poorly understood. This review summarises current knowledge of tooth morphology with respect to DNA content and preservation, and discusses the way in which post-mortem changes will affect the recovery of DNA from teeth under a range of commonly used extraction protocols. We highlight the benefits and pitfalls of using specific tooth tissues for DNA extraction and make recommendations for tooth selection and sampling that will maximise DNA typing success. A comprehensive understanding of tooth structure and an appreciation of the relationship between DNA and mineralized tissues in post-mortem teeth are critical for optimal sample selection. More informed sampling methods that target specific tooth tissues will increase the likelihood of successful genetic analysis and allow for efficient and timely missing persons case work and disaster victim identification response.

Utilization of dental pulp DNA as diagnostic molecular marker for fertility detection in men.

UNLABELLED: Recent advances in DNA technology have revolutionized forensic identification procedures. Teeth dentin and pulp are rich sources of DNA material, which can be successfully extracted and it provides us with valuable information on individuals, systemic health including fertility status. AIM OF STUDY: The aim of this study was to use DNA material extracted from human teeth pulp for detection of fertility status of men. MATERIALS AND METHODS: Twenty extracted premolar teeth of systemic disease free male Saudi individuals (45 years average age) were collected; eight of them were infertile while others were fertile and were used as control group. This information was concealed until the PCR analysis was performed. The results of recorded patient information was matched with the results of the DNA analysis. RESULTS: Results showed that the gene (sY83) an important gene of AZFa region in Y chromosome is important for male fertility. It was later evident that the infertile patients suffered from azoospermia, and that information is completely matched with our results. CONCLUSION: Using DNA extracted from dental pulp can be used successfully in determining fertility status of human which may help in an accurate personal identification specially in extreme circumstances.

Prostaglandin E2 to diagnose between reversible and irreversible pulpitis.

The aim of this work is to verify a correlation between the grade of inflammation and the concentration of PGE2 in human dental pulp. A total of 25 human dental pulps were examined by histological analysis and radioimmunologic dosage of PGE2. The pulps used in this experiment were from healthy and symptomatic teeth; the first ones were collected from teeth destined to be extracted for orthodontic reasons. An increase was observed of PGE2 in reversible pulpitis compared with healthy pulps and with the irreversible pulpitis and the clear decrease of these when NSAIDs are taken. This study demonstrates that PGE2 level is correlated to histological analysis thus allowing to distinguish symptomatic teeth in reversible and irreversible pulpitis.

Human dental pulp cell apoptosis: immunohistochemical study after applying orthodontic traction.

The aim of this study was to compare human dental pulp stress and programmed cell death after 3 and 6 months of orthodontic treatments by assessing the degree of apoptosis and related proteins. Human dental pulps were collected from twenty young patients orthodontically treated by Straight Wire technique. Samples were fixed, paraffin-embedded and processed for histology and immunohistochemistry using anti-heat shock protein 60 kDa (Hsp60), -caspase 3, -caspase 9, and -PCNA antibodies, as well as TUNEL reactions. Moreover, we performed immunoprecipitation for Hsp60 and caspase 3, and for Hsp60 and caspase 9, from paraffin extracted tissues. Increased levels of both caspases and Hsp60 occurred in 6-months treated samples; at the same time, we found increased levels of proliferating cell nuclear antigen and terminal deoxynucleotidyl transferase dUTP nick end labeling positive cells. Immunoprecipitation showed that Hsp60 forms a complex with both Pro-caspase 3 and Caspase 3, and this may accelerate Pro-caspase 3 activation, especially in the 6-months treated group. On the contrary, no complex between Hsp60 and Pro-caspase 9 was detected. The orthodontic tractions may be a cause of stress, apoptosis and proliferation in pulp tissue. These results suggest the need of further studies about the effects of long term orthodontic treatments on the dental pulp.

Antibacterial activity of dentine and pulp extracellular matrix extracts.

AIM: To determine whether extracellular matrix (ECM) preparations from pulp (pECM) and dentine (dECM) possess antimicrobial activity. METHODOLOGY: Dentine and pulp ECM preparations were isolated with 10% ethylenediaminetetraacetic acid (EDTA), pH 7.2 and sequential use of 0.5mol L(-1) NaCl, pH 11.7 and 0.1mol L(-1) tartaric acid, pH 2.0, respectively, with protease inhibitor inclusion throughout. Antimicrobial activity against Streptococcus mutans, Streptococcus oralis and Enterococcus faecalis was assessed using turbidity as a measure of bacteria growth. The cytotoxicity of the extracts on primary pulp cells was also determined by lactate dehydrogenase (LDH) release. Statistical analysis of data was performed using paired student's t-tests. RESULTS: Extracellular matrix extracts from the pulp and dentine showed antibacterial activity against three types of anaerobic bacteria associated with dental disease (P< 0.05). The ECM extracts demonstrated no significant cytotoxic effect on pulpal cells at the concentrations used for antibacterial activity. CONCLUSIONS: The bacteriostatic antibacterial activity of pECM and dECM indicates that the release of these matrix molecules from pulp and dentine may contribute to defence responses during dental disease, treatment and repair.

Immunolocalization of RANK and RANKL along the root surface and in the periodontal membrane of human primary and permanent teeth.

OBJECTIVE: Root resorption, impaired tooth eruption and early tooth loss have been described in relation to diseases that involve defects in the RANK-RANKL-OPG-expression. The aim of the present immunhistochemical study was to localize and compare the reactions for RANK and membrane-bound RANKL along root surfaces and in the periodontal membrane in close proximity to the root surface of human primary and permanent teeth. MATERIALS AND METHODS: The material comprised extracted human teeth (11 primary teeth and six permanent teeth) from 10 different patients. Paraffin sections were prepared of each tooth and sections of each tooth were immunohistochemically stained with antibodies specific for membrane-bound RANKL and RANK. RESULTS: The root surface and the periodontal membrane in close proximity to the root surface did not show immunoreactivity for RANKL. RANKL was only located in odontoblasts and in cells along denticles in one primary tooth. RANK was located in mononuclear cells in the pulp and in multinucleated odontoclasts along resorbed root surfaces and along resorbed dentin surfaces in the pulp in primary teeth and one permanent tooth. CONCLUSIONS: This study demonstrated RANK positivity in resorption areas in primary and permanent teeth. RANKL was positive in the pulp of one primary tooth. RANK expression in odontoclasts and RANKL expression in the pulp may indicate that RANK/RANKL play a role during resorption.

Comparative evaluation of different human dental tissues and alveolar bone for DNA quantity and quality for forensic investigation.

In this study, the efficacy of dental tissues (cementum, dentine and pulp) and alveolar bone as a potential source of DNA was tested in terms of the quality and quantity using nuclear and mitochondrial markers for forensic investigation.This study found dentine as the best source of DNA with only 5.36% imbalanced (PHR<0.7) heterozygous loci. Pulp showed the highest quantity of DNA but exhibited 22.3% imbalanced (PHR<0.7) heterozygous loci. Cementum with highest (46.67%) heterozygote imbalance proved to be the last choice as a source of DNA. Alveolar bone exhibited the second-highest total yield of DNA/mg of tissue. All Global Filer™ STR loci were amplified in 70% samples of fresh alveolar bone whereas for 30% samples, only partial profile was generated along with successful sex determination. All the dental tissues and alveolar bone samples amplified non STR markers (D-loop, Cytochrome Oxidase I, SRY, AMEL). Of the alveolar bones from archival samples, one sample exhibited full STR profile whereas other alveolar bone samples gave partial profiles. This study substantiates alveolar bone as an alternate source of nuclear and mitochondrial DNA.

Chemical aspects on dental hard tissues in primary teeth from preterm infants.

Preterm children with very low birth weight suffer from several neonatal and postnatal complications that may affect the mineralization of teeth. Clinical and morphological studies have shown enamel aberrations in teeth from preterm children. In this study, the chemical composition in enamel and dentin was compared in primary teeth from preterm children and full-term children, and the relationship between the chemical composition and the morphological appearance was investigated. Enamel and dentin in 17 exfoliated primary teeth, from 14 children with a gestational age below 29 wk, were investigated and compared with 36 exfoliated primary teeth from full-term children, using X-ray microanalyses (XRMA). In comparison with the teeth from the controls, the teeth from preterm children had a higher relative value of carbon (C), a lower relative value of calcium (Ca), a lower ratio of calcium/phosphorus (Ca/P) and a lower ratio of Ca/C throughout the outer part of the enamel. In dentin, the relative values for P were higher, and Ca/P ratio was lower, at the dentin-pulp junction. The Ca/P ratio indicated normal hydroxyapatite in the crystals in enamel and dentin. The lower ratio of Ca/C in the bulk and outer part of the enamel indicated more porous enamel.