Dynamic Alterations in Acetylation and Modulation of Histone Deacetylase Expression Evident in the Dentine-Pulp Complex during Dentinogenesis.

Epigenetic modulation, including histone modification, alters gene expression and controls cell fate. Histone deacetylases (HDACs) are identified as important regulators of dental pulp cell (DPC) mineralisation processes. Currently, there is a paucity of information regarding the nature of histone modification and HDAC expression in the dentine-pulp complex during dentinogenesis. The aim of this study was to investigate post-translational histone modulation and HDAC expression during DPC mineralisation and the expression of Class I/II HDACs during tooth development and in adult teeth. HDAC expression (isoforms -1 to -6) was analysed in mineralising primary rat DPCs using qRT-PCR and Western blot with mass spectrometry being used to analyse post-translational histone modifications. Maxillary molar teeth from postnatal and adult rats were analysed using immunohistochemical (IHC) staining for HDACs (1-6). HDAC-1, -2, and -4 protein expression increased until days 7 and 11, but decreased at days 14 and 21, while other HDAC expression increased continuously for 21 days. The Class II mineralisation-associated HDAC-4 was strongly expressed in postnatal sample odontoblasts and DPCs, but weakly in adult teeth, while other Class II HDACs (-5, -6) were relatively strongly expressed in postnatal DPCs and adult odontoblasts. Among Class I HDACs, HDAC-1 showed high expression in postnatal teeth, notably in ameloblasts and odontoblasts. HDAC-2 and -3 had extremely low expression in the rat dentine-pulp complex. Significant increases in acetylation were noted during DPC mineralisation processes, while trimethylation H3K9 and H3K27 marks decreased, and the HDAC-inhibitor suberoylanilide hydroxamic acid (SAHA) enhanced H3K27me3. These results highlight a dynamic alteration in histone acetylation during mineralisation and indicate the relevance of Class II HDAC expression in tooth development and regenerative processes.

Role of alpha smooth muscle actin in odontogenic differentiation of dental pulp stem cells.

Pulpotomy is an effective treatment for retaining vital pulp after pulp exposure caused by caries removal and/or trauma. The expression of alpha smooth muscle actin (α-SMA) is increased during the wound-healing process, and α-SMA-positive fibroblasts accelerate tissue repair. However, it remains largely unknown whether α-SMA-positive fibroblasts influence pulpal repair. In this study, we established an experimental rat pulpotomy model and found that the expression of α-SMA was increased in dental pulp after pulpotomy relative to that in normal dental pulp. In vitro results showed that the expression of α-SMA was increased during the induction of odontogenic differentiation in dental pulp stem cells (DPSCs) compared with untreated DPSCs. Moreover, α-SMA overexpression promoted the odontogenic differentiation of DPSCs via increasing mitochondrial function. Mechanistically, α-SMA overexpression activated the mammalian target of rapamycin (mTOR) signaling pathway. Inhibition of the mTOR signaling pathway by rapamycin decreased the mitochondrial function in α-SMA-overexpressing DPSCs and suppressed the odontogenic differentiation of DPSCs. Furthermore, we found that α-SMA overexpression increased the secretion of transforming growth factor beta-1 (TGF-ß1). In sum, our present study demonstrates a novel mechanism by which α-SMA promotes odontogenic differentiation of DPSCs by increasing mitochondrial respiratory activity via the mTOR signaling pathway.

Differential expression of lncRNA/miRNA/mRNA and their related functional networks during the osteogenic/odontogenic differentiation of dental pulp stem cells.

Dentin-pulp regeneration requires dental pulp stem cells (DPSCs), but the role of long noncoding RNAs (lncRNAs) during this process remains unclear. Here, we cultured human DPSCs in osteogenic/odontogenic medium for 14 days and analyzed cells via RNA-sequencing. The data were validated by quantitative reverse transcription-polymerase chain reaction and lncRNA-microRNA (miRNA)-messenger RNA (mRNA) networks were constructed to reveal the potential competing endogenous RNA regulatory role of lncRNAs. Kyoto Encyclopedia of Genes and Genomes and Gene Ontology enrichment analysis were performed. One lncRNA, SNHG7, was identified and validated by genetic shRNA silencing. A total of 89 lncRNAs, 1,636 mRNAs, and 113 miRNAs were differentially expressed after differentiation. Bioinformatics identified an array of affected signaling pathways including phosphoinositide-3-kinase-protein kinase B, transforming growth factor-ß, and Wnt. mRNAs were enriched in cell migration, cell differentiation, stem cell development, ossification, and skeletal development. One lncRNA, SNHG7, was indentified to inhibit the odonto/osteogenic differentiation of DPSCs when silenced. In summary, we reveal several lncRNAs that significantly change during DPSC differentiation, including SNHG7. This reveals new targets for dentin-pulp complex regeneration and tissue engineering.

Gestational diabetes mellitus affects odontoblastic differentiation of dental papilla cells via Toll-like receptor 4 signaling in offspring.

Gestational diabetes mellitus (GDM) is an important factor involved in the pathogenesis of organ development in the offspring. Here, we analyzed the effects of GDM on odontoblastic differentiation of dental papilla cells (DPCs) and dentin formation in offspring and investigated their underlying mechanisms. A GDM rat model was induced by intraperitoneal injection of streptozotocin and offspring were collected. The results showed that GDM significantly affected odontoblast differentiation and dentin formation in offspring tooth. GDM activated the toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-ĸB) signaling pathway and inhibited SMAD1/5/9 signaling to modulate the odontoblastic differentiation of DPCs in offspring. Inhibition of TLR4 signaling by treated with TAK-242 significantly reverses the suppression of odonto-differentiation of DPCs in diabetic offspring. Taken together, these data indicate GDM activated the offspring DPCs TLR4/NF-ĸB signaling, which suppressed the SMAD1/5/9 phosphorylation and then inhibited odontoblasts differentiation and dentin formation.

Mouse Wnt1-CRE-RosaTomato Dental Pulp Stem Cells Directly Contribute to the Calvarial Bone Regeneration Process.

Stem cells endowed with skeletogenic potentials seeded in specific scaffolds are considered attractive tissue engineering strategies for treating large bone defects. In the context of craniofacial bone, mesenchymal stromal/stem cells derived from the dental pulp (DPSCs) have demonstrated significant osteogenic properties. Their neural crest embryonic origin further makes them a potential accessible therapeutic tool to repair craniofacial bone. The stem cells' direct involvement in the repair process versus a paracrine effect is however still discussed. To clarify this question, we have followed the fate of fluorescent murine DPSCs derived from PN3 Wnt1-CRE- RosaTomato mouse molar (T-mDPSCs) during the repair process of calvaria bone defects. Two symmetrical critical defects created on each parietal region were filled with (a) dense collagen scaffolds seeded with T-mDPSCs, (b) noncellularized scaffolds, or (c) no scaffold. Mice were imaged over a 3-month period by microcomputed tomography to evaluate the extent of repair and by biphotonic microscopy to track T-mDPSCs. Histological and immunocytochemical analyses were performed in parallel to characterize the nature of the repaired tissue. We show that T-mDPSCs are present up to 3 months postimplantation in the healing defect and that they rapidly differentiate in chondrocyte-like cells expressing all the expected characteristic markers. T-mDPSCs further maturate into hypertrophic chondrocytes and likely signal to host progenitors that form new bone tissue. This demonstrates that implanted T-mDPSCs are able to survive in the defect microenvironment and to participate directly in repair via an endochondral bone ossification-like process. Stem Cells 2019;37:701-711.

Examination of the radiographic visibility of the root pulp of the mandibular second molars as an age marker.

Forensic odontologists often confront with conceptually simple medicolegal question of whether an individual is a juvenile or an adult. The demand for additional research into extending dental age estimation methods in late adolescence is never the less, especially in those who have passed 15 years and are suspected older than 18 years. The present research investigated the regressive dental characteristic, i.e. radiographic visibility of the root pulp in mandibular second molars for the purpose of age assessment, especially for determining the age over 18 years. Nine hundred thirty-six orthopantomograms comprised of 436 males and 500 females aged from 14 to 22 years were examined. Descriptive statistics were performed for each stage in both sexes. The correlation statistics revealed that there is a strong, positive correlation between the chronological age and root pulp visibility stages. The analysis revealed that stage 0 occurred first at the age of 14 years in both sexes. Stage 1 first occurred at the age of 14.26 years in females and 14.74 years in males. Stage 2 was first achieved at the age of 18.6 and 15.2 years in males and females, respectively. The suitability of the studied characteristics in mandibular second molars for age estimation could be confirmed but of limited value. The presence of this stage 2 root pulp visibility in male subjects represents a potential criterion for indicating the age over 18 years. In future studies, the pattern of secondary dentin formation in other tooth types should be investigated.

Usability of dental pulp visibility and tooth coronal index in digital panoramic radiography in age estimation in the forensic medicine.

Age estimation has a great importance due to legal requirements. The aim of our study was to determine the applicability of two different methods in age estimation; one of them based on the calculation of the visibility of the root pulp of mandibular third molar teeth and the other based on the calculation of the tooth coronal index (TCI) in the mandibular first and second molar teeth in the 9059 digital orthopantomogram of people aged between 15 and 40 in Bursa. In the first method in which the visibility of the fully mineralized root pulp of the mandibular third molar teeth was evaluated in 4 stages; the stages 0, 1, 2, and 3 were observed regardless of sex at the earliest 17.2; 19.1; 20.1, and 25.1 years, respectively. In the second method in which TCI was regressed on chronological age using measuring crown and coronal pulp cavity heights, the most accurate age estimation model based on simple linear regression for all cases without any sex difference was found to be with right first molar tooth (SEE ±7304 years) and the most accurate age model based on multiple regression model was found to be with bilateral first molar teeth (SEE ±7413 years). In conclusion, we believe that the root pulp visibility of the third molar teeth can be applied safely for stages 1, 2, and 3 at 16, 17, and 21 years of ages, respectively. A correlation was also found between TCI and age, and our findings have shown that both methods are available for forensic purposes.

Root Pulp Visibility as a mandibular maturity marker at the 18-year threshold in the Maltese population.

INTRODUCTION: The determination of age of majority (the 18-year-old threshold) using the popular Demirjian tooth staging method is unreliable, so other maturity markers are required. This study examines whether the Root Pulp Visibility (RPV) of the mandibular third molar is a useful indicator of age. METHODS: One thousand six dental panoramic radiographs (DPTs) were examined and the left mandibular third molar assessed according to the RPV stages using the method of Olze et al. (Int J Legal Med 124:183-186, 2010) as modified by Lucas et al. (Forensic Sci Int 270:98-102, 2017). Six hundred sixty-two DPTs, 288 males and 374 females, satisfied the inclusion criteria. RESULTS: Individuals who had reached stages RPV-A and RPV-B were represented in a wide range of ages spanning the 18-year-old threshold. Individuals who had reached stages RPV-C and RPV-D were all above the 18-year-old threshold. It was not possible to analyse a large number of DPTs for various reasons. DISCUSSION: Individuals whose mandibular third molar exhibited stages RPV-C and RPV-D may be deemed to have reached the age of majority. These results are consistent with other published studies. Variation in the rate of development of the third molar limits the applicability of this method. CONCLUSIONS: RPV staging is an accurate method of determining the 18-year-old threshold. Substitute methods are required in a large number of cases.

Sonic Hedgehog Signaling and Tooth Development.

Sonic hedgehog (Shh) is a secreted protein with important roles in mammalian embryogenesis. During tooth development, Shh is primarily expressed in the dental epithelium, from initiation to the root formation stages. A number of studies have analyzed the function of Shh signaling at different stages of tooth development and have revealed that Shh signaling regulates the formation of various tooth components, including enamel, dentin, cementum, and other soft tissues. In addition, dental mesenchymal cells positive for Gli1, a downstream transcription factor of Shh signaling, have been found to have stem cell properties, including multipotency and the ability to self-renew. Indeed, Gli1-positive cells in mature teeth appear to contribute to the regeneration of dental pulp and periodontal tissues. In this review, we provide an overview of recent advances related to the role of Shh signaling in tooth development, as well as the contribution of this pathway to tooth homeostasis and regeneration.

N-Acetyl Cysteine Modulates the Inflammatory and Oxidative Stress Responses of Rescued Growth-Arrested Dental Pulp Microtissues Exposed to TEGDMA in ECM.

Dental pulp is exposed to resin monomers leaching from capping materials. Toxic doses of the monomer, triethyleneglycol dimethacrylate (TEGDMA), impact cell growth, enhance inflammatory and oxidative stress responses, and lead to tissue necrosis. A therapeutic agent is required to rescue growth-arrested tissues by continuing their development and modulating the exacerbated responses. The functionality of N-Acetyl Cysteine (NAC) as a treatment was assessed by employing a 3D dental pulp microtissue platform. Immortalized and primary microtissues developed and matured in the extracellular matrix (ECM). TEGDMA was introduced at various concentrations. NAC was administered simultaneously with TEGDMA, before or after monomer addition during the development and after the maturation stages of the microtissue. Spatial growth was validated by confocal microscopy and image processing. Levels of inflammatory (COX2, NLRP3, IL-8) and oxidative stress (GSH, Nrf2) markers were quantified by immunoassays. NAC treatments, in parallel with TEGDMA challenge or post-challenge, resumed the growth of the underdeveloped microtissues and protected mature microtissues from deterioration. Growth recovery correlated with the alleviation of both responses by decreasing significantly the intracellular and extracellular levels of the markers. Our 3D/ECM-based dental pulp platform is an efficient tool for drug rescue screening. NAC supports compromised microtissues development, and immunomodulates and maintains the oxidative balance.

Age estimation using canine pulp volumes in adults: a CBCT image analysis.

Secondary dentine deposition is responsible for the decrease in the volume of the pulp cavity with age. Therefore, the volume of the pulp cavity can be considered as a predictor for estimating age. The aims of this study were to investigate the relationship strength between canine pulp volumes and chronological age from homogenous (approximately equal numbers of individuals in each age range) age distribution and to assess the effect of sex as predictor in age estimation. This study was performed on 719 subjects of Pakistani origin. Cone beam computed tomography images of 521 left maxillary and 681 left mandibular canines were collected from 368 females and 349 males aged from 15 to 65 years. Planmeca Romexis® software was used to trace the outline of the pulp cavity and to calculate pulp volumes. Regression analysis was performed to assess the correlation between pulp volumes considering with and without sex as a predictor with chronological age. The obtained results showed that mandibular canine pulp volume and sex have the highest predictive power (R2 = 0.33). The relationship between mandibular canine pulp volume and sex with chronological age demonstrates an odd S-shaped non-linear relationship. A statistically significant difference in volumes of pulp was found (p = 0.000) between males and females. The conclusion was that predictions using the pulp volume of the mandibular canine and sex produced the best estimates of chronological age.

Age estimation using pulp/enamel volume ratio of impacted mandibular third molars measured on CBCT images in a northern Chinese population.

The present study aims to evaluate the relation between chronological age and the ratio of pulp volume (PV) to enamel volume (EV) of impacted mandibular third molars (IMTMs) by using cone-beam computed tomography (CBCT) images and an improved 3D image segmentation technique. A sample of CBCT images of IMTM was collected from 414 northern Chinese subjects (214 male and 200 female clinical patients) ranging in age from 20 to 65 years. The GrowCut effect image segmentation (GCEIS) module algorithm was used to calculate the PV and EV from CBCT images. The total sample was divided into a training group and validation group in a ratio of 7 to 3. The PV/EV ratio (PEr) in the training sample was used to develop a mathematical formula for age estimation as follows: age = - 5.817-21.726 × Ln PEr (p < 0.0001) (Ln, natural logarithm). The mean absolute error (MAE) and root mean square error (RMSE) were used to determine the precision and accuracy of the mathematical formula in the validation group and all samples. The MAEs in the male, female, and pooled gender samples were 9.223, 7.722, and 8.41, respectively, and the RMSEs in the male, female, and pooled gender samples were 10.76, 9.58, and 9.986, respectively. The precise and accurate results indicate that the PEr of IMTM in CBCT images is a potential index for dental age estimation and is possible to be used in forensic medicine.

Evidence for changing nerve growth factor signalling mechanisms during development, maturation and ageing in the rat molar pulp.

AIM: To examine rat molar pulp innervation and identify complex cellular signalling systems involving nerve growth factor (NGF) and its p75 receptors (NGFR) at different stages of development, maturation and ageing. METHODOLOGY: Decalcified mandibular first molar mesial cusps from Wistar rats of ages 0 day; 1, 2, 3, 4, 6, 9, 12 and 24 weeks (n = 5 per group) were sectioned (10 µm) and incubated with antibodies for NGF, NGFR, calcitonin gene-related peptide (CGRP) and neurofilament. Nerve densities in worn and intact regions of 3- to 24-week-old rats were compared by anova, Bonferroni and t-tests. RESULTS: During odontogenesis, differences in NGF and NGFR expression were observed, with no evidence of nerve fibres, suggesting a signalling mechanism controlling cellular differentiation and dentine formation. Tooth wear in 4-week rats was associated with reduced NGF expression and significantly decreased CGRP axons within affected odontoblast regions. The underlying subodontoblasts started expressing NGF which continued until 9 weeks. This may promote a significant increase in CGRP nerve density in affected regions. Nerve density in intact odontoblast regions increased gradually and reached significant levels in 12-week rats. Reduction in nerve densities within worn and intact regions of cusps was observed at 24 weeks. CONCLUSIONS: Age-related changes and responses to tooth wear may be controlled by the NGF signalling mechanism, with roles in odontoblast/subodontoblast communication and control of sensory innervation at different stages of tooth development, maturation and ageing. Greater understanding of cellular and nerve regulation in the injured pulp may promote therapeutic strategies for pulp survival.

Use of 0.4-Tesla static magnetic field to promote reparative dentine formation of dental pulp stem cells through activation of p38 MAPK signalling pathway.

AIM: To investigate whether static magnetic fields (SMFs) have a positive effect on the migration and dentinogenesis of dental pulp stem cells (DPSCs) to promote reparative dentine formation. METHODOLOGY: In vitro scratch assays and a traumatic pulp exposure model were performed to evaluate the effect of 0.4-Tesla (T) SMF on DPSC migration. The cytoskeletons of the DPSCs were identified by fluorescence immunostaining and compared with those of a sham-exposed group. Dentinogenic evaluation was performed by analysing the expressions of DMP-1 and DSPP marker genes using a quantitative real-time polymerase chain reaction (qRT-PCR) process. Furthermore, the formation of calcified deposits was examined by staining the dentinogenic DPSCs with Alizarin Red S dye. Finally, the role played by the p38 MAPK signalling pathway in the migration and dentinogenesis of DPSCs under 0.4-T SMF was investigated by incorporating p38 inhibitor (SB203580) into the in vitro DPSC experiments. The Student's t-test and the Kruskal-Wallis test followed by Dunn's post hoc test with a significance level of P < 0.05 were used for statistical analysis. RESULTS: The scratch assay results revealed that the application of 0.4-T SMF enhanced DPSCs migration towards the scratch wound (P < 0.05). The cytoskeletons of the SMF-treated DPSCs were found to be aligned perpendicular to the scratch wound. After 20 days of culture, the SMF-treated group had a greater number of out-grown cells than the sham-exposed group (nonmagnetized control). For the SMF-treated group, the DMP-1 (P < 0.05) and DSPP genes (P < 0.05), analysed by qRT-PCR, exhibited a higher expression. The distribution of calcified nodules was also found to be denser in the SMF-treated group when stained with Alizarin Red S dye (P < 0.05). Given the incorporation of p38 inhibitor SB203580 into the DPSCs, cell migration and dentinogenesis were suppressed. No difference was found between the SMF-treated and sham-exposed cells (P > 0.05). CONCLUSION: 0.4-T SMF enhanced DPSC migration and dentinogenesis through the activation of the p38 MAPK-related pathway.

Pentraxin-3 Modulates Osteogenic/Odontogenic Differentiation and Migration of Human Dental Pulp Stem Cells.

Pentraxin-3 (PTX3) is recognized as a modulator of inflammation and a mediator of tissue repair. In this study, we characterized the role of PTX3 on some biological functions of human dental pulp stem cells (HDPSCs). The expression level of PTX3 significantly increased during osteogenic/odontogenic differentiation of HDPSCs, whereas the knockdown of PTX3 decreased this differentiation. Silencing of PTX3 in HDPSCs inhibited their migration and C-X-C chemokine receptor type 4 (CXCR4) expression. Our present study indicates that PTX3 is involved in osteogenic/odontogenic differentiation and migration of HDPSCs, and may contribute to the therapeutic potential of HDPSCs for regeneration and repair.

Ion Channels Involved in Tooth Pain.

The tooth has an unusual sensory system that converts external stimuli predominantly into pain, yet its sensory afferents in teeth demonstrate cytochemical properties of non-nociceptive neurons. This review summarizes the recent knowledge underlying this paradoxical nociception, with a focus on the ion channels involved in tooth pain. The expression of temperature-sensitive ion channels has been extensively investigated because thermal stimulation often evokes tooth pain. However, temperature-sensitive ion channels cannot explain the sudden intense tooth pain evoked by innocuous temperatures or light air puffs, leading to the hydrodynamic theory emphasizing the microfluidic movement within the dentinal tubules for detection by mechanosensitive ion channels. Several mechanosensitive ion channels expressed in dental sensory systems have been suggested as key players in the hydrodynamic theory, and TRPM7, which is abundant in the odontoblasts, and recently discovered PIEZO receptors are promising candidates. Several ligand-gated ion channels and voltage-gated ion channels expressed in dental primary afferent neurons have been discussed in relation to their potential contribution to tooth pain. In addition, in recent years, there has been growing interest in the potential sensory role of odontoblasts; thus, the expression of ion channels in odontoblasts and their potential relation to tooth pain is also reviewed.

Senescence and odontoblastic differentiation of dental pulp cells.

Cellular senescence has been suggested to be involved in physiological changes of cytokine production. Previous studies showed that the concentration of tumor necrosis factor-α (TNF-α) is higher in the blood of aged people compared with that of young people. So far, the precise effects of TNF-α on the odontoblastic differentiation of pulp cells have been controversial. Therefore, we aimed to clarify how this cytokine affected pulp cells during aging. Human dental pulp cells (HDPCs) were cultured until reaching the plateau of their growth, and the cells were isolated at actively (young HDPCs; yHDPCs) or inactively (senescent HDPCs; sHDPCs) proliferating stages. sHDPCs expressed senescence-related molecules while yHDPCs did not. When these HDPCs were cultured in an odontoblast-inductive medium, both young and senescent cells showed mineralization, but mineralization in sHDPCs was lower compared with yHDPCs. However, the administration of TNF-α to this culture medium altered these responses: yHDPCs showed downregulated mineralization, while sHDPCs exhibited significantly increased mineralization. Furthermore, the expression of tumor necrosis factor receptor 1 (TNFR1), a receptor of TNF-α, was significantly upregulated in sHDPCs compared with yHDPCs. Downregulation of TNFR1 expression led to decreased mineralization of TNF-α-treated sHDPCs, whereas restored the reduction in TNF-α-treated yHDPCs. These results suggested that sHDPCs preserved the odontoblastic differentiation capacity and TNF-α promoted odontoblastic differentiation of HDPCs with the progress of their population doublings through increased expression of TNFR1. Thus, TNF-α might exert a different effect on the odontoblastic differentiation of HDPCs depending on their proliferating activity. In addition, the calcification of pulp chamber with age may be related with increased reactivity of pulp cells to TNF-α.

Application of age assessment based on the radiographic visibility of the root pulp of lower third molars in a northern Chinese population.

The purpose of the present study was to explore the potential application of radiographic visibility of the root pulp in lower third molars in a northern Chinese population, in order to determine if this methodology can be used to prove whether a person has surpassed the thresholds of 18 and 21 years of age. A total of 1300 orthopantomograms comprising equal numbers of females and males evenly distributed between the ages of 15 and 40 years were analyzed. The radiographic visibility of the root pulp of the lower third molars was assessed using the stages described by Olze et al. (2010). Stage 1 first appeared at 19.25 years in males and at 20.73 years in females. The earliest appearance of stage 2 happened at 22.33 years in males and at 22.41 years in females. Stage 3 was achieved first at 26.45 years in males and at 27.66 years in females. It was concluded that stages 1, 2, and 3 can be used to show that a person is over 18 years of age. If stages 2 or 3 are determined, it is possible to prove that an individual has already attained the age of 21 years.

Lysyl oxidase-mediated VEGF-induced differentiation and angiogenesis in human dental pulp cells.

AIM: To investigate the effects of recombinant human vascular endothelial growth factor (rhVEGF) on odontoblastic differentiation, in vitro angiogenesis, and expression and activity of lysyl oxidase (LOX) in human dental pulp cells (HDPCs), compared with rhFGF-2. To identify the underlying molecular mechanisms, the study focused on whether LOX was responsible for the actions of rhVEGF. METHODOLOGY: Recombinant human vascular endothelial growth factor (rhVEGF) was constructed using the pBAD-HisA plasmid in Escherichia coli. HDPCs were treated with 1-50 µg mL-1 rhVEGF for 14 days. Alkaline phosphatase (ALP) activity was measured, and the formation of calcified nodules was assessed using alizarin red staining after the induction of odontogenic differentiation of HDPCs. The expression level of the odontogenic differentiation markers was detected by reverse transcription polymerase chain reaction. Signal pathways were assessed by Western blot and immunocytochemistry. The data were analysed by anova with Bonferroni's test (α = 0.05). RESULTS: Recombinant human vascular endothelial growth factor significantly increased cell growth (P < 0.05), ALP activity (P < 0.05) and mineralization nodule formation and upregulated the mRNA expression levels of the osteogenic/odontogenic markers that were lower with rhFGF-2. rhVEGF significantly increased amine oxidase activity (P < 0.05) and upregulated LOX and LOXL mRNA expression in HDPCs. Additionally, rhVEGF dose-dependently upregulated angiogenic gene mRNAs and capillary tube formation to a greater degree than rhFGF-2. Inhibition of LOX using ß-aminopropionitrile (BAPN) and LOX or LOXL gene silencing by RNA interference attenuated rhVEGF-induced growth, ALP activity, mineralization, the expression of marker mRNAs and in vitro angiogenesis. Furthermore, treatment with rhVEGF resulted in phosphorylation of Akt, ERK, JNK and p38, and activation of NF-κB, which was inhibited by LOX or LOXL silencing and BAPN. CONCLUSION: Recombinant human vascular endothelial growth factor promoted cell growth, odontogenic potential and in vitro angiogenesis via modulation of LOX expression. These results support the concept that rhVEGF may offer therapeutic benefits in regenerative endodontics.

Forensic age assessment of asylum seekers in Finland.

In Finland, forensic age assessment is strictly regulated by legislation. According to the Aliens Act (301/2004) and the amendment of the Act (549/2010), the police authorities, the frontier guard authorities, and the immigration authorities have the right to refer asylum seekers to the University of Helsinki, Department of Forensic Medicine, for age assessment. These assessments are especially performed to solve if the person is of major age, the cutoff being 18 completed years. The forensic age assessment is largely based on dental development, since the special permit of the Radiation and Nuclear Safety Authority (STUK) to the Department of Forensic Medicine of the University of Helsinki, allowing the use of ionizing radiation for non-medical purposes, includes dental and hand X-rays. Forensic age assessment is always performed by two forensic odontologists. In 2015, the total number of forensic age assessment examinations was 149, and the countries of origin of the asylum seekers were most commonly Iraq, Afghanistan, and Somalia. The current legislation on forensic age assessment has been well received and approved. Radiological and other examinations can be performed in different parts of Finland, but the forensic odontologist at the University of Helsinki is always involved in the process and ensures joint quality standards for the forensic age assessment.