Assessment of physiological disturbances during pre- and early postnatal development based on microscopic analysis of human deciduous teeth from the Late Epipaleolithic site of Shubayqa 1 (Jordan).

OBJECTIVES: To study pre- and early postnatal tooth formation and to analyze the effects of physiological disturbances on enamel and dentin formation in deciduous teeth of infants from the Late Epipaleolithic (Natufian) site Shubayqa 1. MATERIALS AND METHODS: Ten deciduous teeth from six infants (ages at death between 21 and 239 days) were analyzed by light and scanning electron microscopy. RESULTS: Marked prism cross-striations and an abnormal wavy course of the prisms were recorded in pre- and postnatal enamel of all analyzed teeth. Single or multiple accentuated incremental lines were observed in prenatal enamel of nine teeth and in postnatal enamel of eight teeth. Accentuated Andresen lines and broader zones exhibiting an enhanced calcospheritic pattern were recorded in the pre- and postnatally formed dentin of nine teeth. DISCUSSION: The structural abnormalities in the pre- and postnatally formed enamel of the infants are considered indicative of chronic stress that negatively affected the activity of secretory ameloblasts. The structural aberrations in pre- and postnatal dentin denote that odontoblasts were also affected by this stress. The presence of single or multiple accentuated incremental lines in pre- and postnatal enamel is interpreted as reflecting (short-term) impacts of higher intensity superimposed on the chronic stress. Our findings suggest compromised maternal health affecting the late fetus and compromised health in newborns. Although limited by the small number of analyzed individuals, the present study contributes to the knowledge of maternal and early infant health conditions in Late Epipaleolithic populations.

Biomineralization of Collagen-Based Materials for Hard Tissue Repair.

Hydroxyapatite (HA) reinforced collagen fibrils serve as the basic building blocks of natural bone and dentin. Mineralization of collagen fibrils play an essential role in ensuring the structural and mechanical functionalities of hard tissues such as bone and dentin. Biomineralization of collagen can be divided into intrafibrillar and extrafibrillar mineralization in terms of HA distribution relative to collagen fibrils. Intrafibrillar mineralization is termed when HA minerals are incorporated within the gap zone of collagen fibrils, while extrafibrillar mineralization refers to the minerals that are formed on the surface of collagen fibrils. However, the mechanisms resulting in these two types of mineralization still remain debatable. In this review, the evolution of both classical and non-classical biomineralization theories is summarized. Different intrafibrillar mineralization mechanisms, including polymer induced liquid precursor (PILP), capillary action, electrostatic attraction, size exclusion, Gibbs-Donnan equilibrium, and interfacial energy guided theories, are discussed. Exemplary strategies to induce biomimetic intrafibrillar mineralization using non-collagenous proteins (NCPs), polymer analogs, small molecules, and fluidic shear stress are discussed, and recent applications of mineralized collagen fibers for bone regeneration and dentin repair are included. Finally, conclusions are drawn on these proposed mechanisms, and the future trend of collagen-based materials for bone regeneration and tooth repair is speculated.

Biological clocks and incremental growth line formation in dentine.

Dentine- and enamel-forming cells secrete matrix in consistent rhythmic phases, resulting in the formation of successive microscopic growth lines inside tooth crowns and roots. Experimental studies of various mammals have proven that these lines are laid down in subdaily, daily (circadian), and multidaily rhythms, but it is less clear how these rhythms are initiated and maintained. In 2001, researchers reported that lesioning the so-called master biological clock, the suprachiasmatic nucleus (SCN), halted daily line formation in rat dentine, whereas subdaily lines persisted. More recently, a key clock gene (Bmal1) expressed in the SCN in a circadian manner was also found to be active in dentine- and enamel- secretory cells. To probe these potential neurological and local mechanisms for the production of rhythmic lines in teeth, we reexamined the role of the SCN in growth line formation in Wistar rats and investigated the presence of daily lines in Bmal1 knockout mice (Bmal1-/- ). In contrast to the results of the 2001 study, we found that both daily and subdaily growth lines persisted in rat dentine after complete or partial SCN lesion in the majority of individuals. In mice, after transfer into constant darkness, daily rhythms continued to manifest as incremental lines in the dentine of each Bmal1 genotype (wild-type, Bmal+/- , and Bmal1-/- ). These results affirm that the manifestation of biological rhythms in teeth is a robust phenomenon, imply a more autonomous role of local biological clocks in tooth growth than previously suggested, and underscore the need further to elucidate tissue-specific circadian biology and its role in incremental line formation. Investigations of this nature will strengthen an invaluable system for determining growth rates and calendar ages from mammalian hard tissues, as well as documenting the early lives of fossil hominins and other primates.

Blurred time resolution of tooth dentin serial sections.

OBJECTIVES: The growth of tooth dentin is incremental, so its formation represents a dietary record in early life. With archeological skeletons, applying sequential stable isotope analysis to the horizontal sections of tooth dentin has revealed weaning patterns and dietary changes that took place during childhood. However, the assignment of ages to dentin serial sections (DSSs) is problematic due to the changing extension rate and oblique growth layers of dentin, and these effects have not been quantified. This study presents a mathematical model for investigating the corresponding age range of the horizontal DSSs of human permanent incisors, canines, and molars. METHODS: Parameters describing the tooth dentin microstructure were taken from previous studies, and dentin growth patterns were modeled. The model was implemented as the R package MDSS. RESULTS: The developed model shows that the true corresponding age of the sections differed by a few years on average from the estimated age with equal temporal divisions, that the model gave values extending across a wide range, and that these differences become large for sections formed at older ages. The stable isotope ratio of the sections presented an aggregate representation of possibly complex dietary changes across a few years, and dietary changes over short times, such as several months, could not be accurately reconstructed even when using a finer horizontal sectioning method. CONCLUSIONS: These results demonstrate that DSSs correspond to an unexpectedly wider (i.e., three to four times) and different (i.e., -2 to 0.5 years on average) age range than previously assumed and that complicated patterns of dietary change blur in the isotopic trajectory of the sections. Alternative experimental methods, such as imaging-assisted oblique sampling, should be used to retrieve an accurate and precise sequential dietary record from tooth dentin.

Growth response of dental tissues to developmental stress in the domestic pig (Sus scrofa).

OBJECTIVES: To compare relative response of enamel, dentin and bone to developmental stressors between attritional and catastrophic mortality assemblages of pigs. MATERIALS AND METHODS: Heads from 70 Sus scrofa of known sex, weight and age comprising an attritional sample of 50 sick pen (SP) pigs that died prematurely versus 20 control pigs slaughtered at 6 months (Catastrophic assemblage). Hard tissue changes (alveolar bone thinning), abnormal bone formation (Harris lines) and re-modeling (auditory bullae) were recorded. Areas and volumes of coronal enamel and dentin were recorded from microCT scans with Avizo 6.3 and Geomagic Wrap. RESULTS: Attritional and catastrophic assemblages are metrically indistinguishable. Ages at death and tissue measures in the SP pigs are differentially distributed, necessitating partition into developmental outcome cohorts. SP "late death" pigs are of lesser physiological maturity than expected, free of disease, with large dental tissue dimensions, comparable to "Controls". SP "early death" pigs have 5% less dentin and enamel and chronic bone infection. Older cohorts of the SP "early deaths" mortality assemblage show progressively reduced enamel. SP pigs show dental evidence of reduced bone mass in the maxilla. DISCUSSION: Bone, dentin and enamel tissues, each, respond distinctively to developmental stressors. Bone mass evinces malnutrition not disease. Both dental tissue reduction and abnormal bone formation link to chronic infection. Paradoxically, reduced dentin mass signals lower survivorship while reduced enamel signals enhanced survivorship. Meaningful comparison of Attritional and Catastrophic assemblages necessitates recognition of developmental outcome cohorts, stratified by age at death and physiological maturity, to reveal heterogeneity of survivorship, tissue measures and lesions.

Activation of αSMA expressing perivascular cells during reactionary dentinogenesis.

AIM: To examine the contribution of perivascular cells expressing αSMA to reactionary dentinogenesis. METHODOLOGY: An inducible, Cre-loxP in vivo fate-mapping approach was used to examine the contribution of the descendants of cells expressing the αSMA-CreERT2 transgene to reactionary dentinogenesis in mice molars. Reactionary dentinogenesis was induced by experimental mild injury to dentine without pulp exposure. The Student's t test was used to determine statistical significance at *P ≤ 0.05. RESULTS: The lineage tracing experiments revealed that mild injury to dentine first led to activation of αSMA-tdTomato+ cells in the entire pulp chamber. The percentage of areas occupied by αSMA-tdTomato+ in injured (7.5 ± 0.7%) teeth were significantly higher than in teeth without injury (2 ± 0.5%). After their activation, αSMA-tdTomato+ cells migrated towards the site of injury, gave rise to pulp cells and a few odontoblasts that became integrated into the existing odontoblast layer expressing Col2.3-GFP and Dspp. CONCLUSION: Mild insult to dentine activated perivascular αSMA-tdTomato+ cells giving rise to pulp cells as well as a few odontoblasts that were integrated into the pre-existing odontoblast layer.

Uncovering neurodevelopmental windows of susceptibility to manganese exposure using dentine microspatial analyses.

BACKGROUND: Associations between manganese (Mn) and neurodevelopment may depend on dose and exposure timing, but most studies cannot measure exposure variability over time well. OBJECTIVE: We apply temporally informative tooth-matrix biomarkers to uncover windows of susceptibility in early life when Mn is associated with visual motor ability in childhood. We also explore effect modification by lead (Pb) and child sex. METHODS: Participants were drawn from the ELEMENT (Early Life Exposures in MExico and NeuroToxicology) longitudinal birth cohort studies. We reconstructed dose and timing of prenatal and early postnatal Mn and Pb exposures for 138 children by analyzing deciduous teeth using laser ablation-inductively coupled plasma-mass spectrometry. Neurodevelopment was assessed between 6 and 16 years of age using the Wide Range Assessment of Visual Motor Abilities (WRAVMA). Mn associations with total WRAVMA scores and subscales were estimated with multivariable generalized additive mixed models. We examined Mn interactions with Pb and child sex in stratified models. RESULTS: Levels of dentine Mn were highest in the second trimester and declined steeply over the prenatal period, with a slower rate of decline after birth. Mn was positively associated with visual spatial and total WRAVMA scores in the second trimester, among children with lower (< median) tooth Pb levels: one standard deviation (SD) increase in ln-transformed dentine Mn at 150 days before birth was associated with a 0.15 [95% CI: 0.04, 0.26] SD increase in total score. This positive association was not observed at high Pb levels. In contrast to the prenatal period, significant negative associations were found in the postnatal period from ~ 6 to 12 months of age, among boys only: one SD increase in ln-transformed dentine Mn was associated with a 0.11 [95% CI: - 0.001, - 0.22] to 0.16 [95% CI: - 0.04, - 0.28] SD decrease in visual spatial score. CONCLUSIONS: Using tooth-matrix biomarkers with fine scale temporal profiles of exposure, we found discrete developmental windows in which Mn was associated with visual-spatial abilities. Our results suggest that Mn associations are driven in large part by exposure timing, with beneficial effects found for prenatal levels and toxic effects found for postnatal levels.

A histologic assessment of a HYBENX® oral tissue decontaminant in vital pulp therapy in dogs.

The aim of this study was to assess HYBENX® Oral Tissue Decontaminant (HOTD) in treating vital pulp exposure in a canine model. The use of HOTD solution was compared to an accepted and standard regimen for vital pulp exposure, an application of a commercial calcium hydroxide product (Ca(OH)2). Both control and experimental treatments were followed by restoration with a commercial zinc oxide and eugenol obtundant intermediate restorative material and thermal insulator (ZOE). At 7 days there was 100% pulp vitality with HOTD and 50% with Ca(OH)2. New dentin formation was seen in 62.5% of the HOTD treated pulps and none of the Ca(OH)2 treatment group. The vital pulp exposures at day 21 post treatment with HOTD also showed significant improvement over Ca(OH)2 in the presence of odontoblasts, new dentin formation and pulp survivability. The presence of odontoblasts and new dentin was noted in 71% of the HOTD cases versus 50% of the survivable Ca(OH)2 cases. Furthermore, 100% of HOTD cases had vital pulps versus 62.5% of Ca(OH)2 cases. The 60-day specimens of both experimental and control techniques exhibited histologically similar appearances and were similar in outcomes. HOTD treatment at day 7 showed a significant positive difference, both in the formation of new dentin and tooth vitality. HOTD proved better for the post 21-day specimens and equivalent for the 60-day pulp specimens with no evidence of untoward tissue reactions or results.

Dual role of the Trps1 transcription factor in dentin mineralization.

TRPS1 (tricho-rhino-phalangeal syndrome) is a unique GATA-type transcription factor that acts as a transcriptional repressor. TRPS1 deficiency and dysregulated TRPS1 expression result in skeletal and dental abnormalities implicating TRPS1 in endochondral bone formation and tooth development. Moreover, patients with tricho-rhino-phalangeal syndrome frequently present with low bone mass indicating TRPS1 involvement in bone homeostasis. In addition, our previous data demonstrated accelerated mineralization of the perichondrium in Trps1 mutant mice and impaired dentin mineralization in Col1a1-Trps1 transgenic mice, implicating Trps1 in the mineralization process. To understand the role of Trps1 in the differentiation and function of cells producing mineralized matrix, we used a preodontoblastic cell line as a model of dentin mineralization. We generated both Trps1-deficient and Trps1-overexpressing stable cell lines and analyzed the progression of mineralization by alkaline phosphatase and alizarin red staining. As predicted, based on our previous in vivo data, delayed and decreased mineralization of Trps1-overexpressing odontoblastic cells was observed when compared with control cells. This was associated with down-regulation of genes regulating phosphate homeostasis. Interestingly, Trps1-deficient cells lost the ability to mineralize and demonstrated decreased expression of several genes critical for initiating the mineralization process, including Alpl and Phospho1. Based on these data, we have concluded that Trps1 serves two critical and context-dependent functions in odontoblast-regulated mineralization as follows: 1) Trps1 is required for odontoblast maturation by supporting expression of genes crucial for initiating the mineralization process, and 2) Trps1 represses the function of mature cells and, consequently, restricts the extent of extracellular matrix mineralization.

Nuclear factor I-C (NFIC) regulates dentin sialophosphoprotein (DSPP) and E-cadherin via control of Krüppel-like factor 4 (KLF4) during dentinogenesis.

Odontoblasts are a type of terminally differentiated matrix-secreting cells. A number of molecular mechanisms are involved in the differentiation of odontoblasts. Several studies demonstrated that Krüppel-like factor 4 (KLF4) promotes odontoblast differentiation via control of dentin sialophosphoprotein (DSPP). Because nuclear factor I-C (NFIC) is also known to control DSPP, we investigated the relationship between NFIC and KLF4 during odontoblast differentiation. Klf4 mRNA expression was significantly decreased in Nfic(-/-) pulp cells compared with wild type cells. In immunohistochemistry assays, dentin matrix protein 1 (Dmp1), and DSP protein expression was barely observed in Nfic(-/-) odontoblasts and dentin matrix. Nfic bound directly to the Klf4 promoter and stimulated Klf4 transcriptional activity, thereby regulating Dmp1 and DSPP expression during odontoblast differentiation. Nfic or Klf4 overexpression promoted mineralized nodule formation in MDPC-23 cells. In addition, Nfic overexpression also decreased Slug luciferase activity but augmented E-cadherin promoter activity via up-regulation of Klf4 in odontoblasts. Our study reveals important signaling pathways during dentinogenesis: the Nfic-Klf4-Dmp1-Dspp and the Nfic-Klf4-E-cadherin pathways in odontoblasts. Our results indicate the important role of NFIC in regulating KLF4 during dentinogenesis.

Temporo-spatial requirement of Smad4 in dentin formation.

The TGF-ß/BMP family plays an important role in multiple stages of tooth development. TGF-ß/BMP signaling is required for odontoblast differentiation and dentin formation; however, the precise molecular mechanisms underlying dentin formation remain unclear. To address the role of TGF-ß/BMP signaling in dentin formation, we analyzed mice in which Smad4, a key intracellular mediator of TGF-ß/BMP signaling, was subjected to tissue-specific ablation under the control of Dspp, OC, or Col1a1 promoters. Three independent Smad4 conditional knockout mice exhibited various dentin defects in the crowns and roots of their molars depending on the transactivator. In all mutant molars, crown dentin thickness was thinner than that of the control. In addition, impaired dentin was found in the cervical region and root furcation area. Although the initial differentiation of odontoblasts was normal, odontoblast polarity abruptly decreased and the expression of Col1a1, OC, and Dspp was reduced in the odontoblasts of mutant molars. In Dspp-Cre-mediated Smad4 disruption mice, primary dentin formation was slightly delayed, while secondary dentin formation was severely affected in the cervical region of the molars. These results indicate that TGF-ß/BMP signaling is required for odontoblast maturation and dentin formation in a stage- and site-dependent manner.

Oral histories: a simple method of assigning chronological age to isotopic values from human dentine collagen.

BACKGROUND: Stable isotope ratios of carbon (δ(13)C) and nitrogen (δ(15)N) in bone and dentine collagen have been used for over 30 years to estimate palaeodiet, subsistence strategy, breastfeeding duration and migration within burial populations. Recent developments in dentine microsampling allow improved temporal resolution for dietary patterns. AIM: A simple method is proposed which could be applied to human teeth to estimate chronological age represented by dentine microsamples in the direction of tooth growth, allowing comparison of dietary patterns between individuals and populations. The method is tested using profiles from permanent and deciduous teeth of two individuals. SUBJECTS AND METHODS: Using a diagrammatic representation of dentine development by approximate age for each human tooth (based on the Queen Mary University of London Atlas), this study estimated the age represented by each dentine section. Two case studies are shown: comparison of M1 and M2 from a 19th century individual from London, England, and identification of an unknown tooth from an Iron Age female adult from Scotland. RESULTS AND CONCLUSIONS: The isotopic profiles demonstrate that variations in consecutively-forming teeth can be aligned using this method to extend the dietary history of an individual or identify an unknown tooth by matching the profiles.

Human dental pulp stem cells cultured onto dentin derived scaffold can regenerate dentin-like tissue in vivo.

Regeneration of dentin tissues in the pulp space of teeth serves the ultimate goal of preserving teeth via endodontic approaches. In recent times, many studies suggested that human dentin scaffolds combined with dental stem cells was a potential strategy for the complete dentin tissue regeneration. In this study, human dental pulp stem cells (DPSCs) were isolated and cultured. Dentin specimens were prepared from human third molars and treated with ethylene diamine tetra-acetic acid and citric acid to remove the smear layer. Then, DPSCs were cultured onto human treated dentin (hTD) and implanted in mouse model for 4, 6 and 8 weeks. The resulting grafts were assessed by hematoxylin and eosin stain and immunohistochemical stains. As a result, DPSCs were supported and induced to regenerate of dentin-like tissues which expressed specific dentin markers such as dentin sialophosphoprotein and dentin matrix protein 1 by combination with hTD in vivo. Furthermore, cells existed in the newly-formed dentin-like tissues also expressed typical human mitochondria antibodies, demonstrated that new tissues originated from human. In conclusion, the obtain results extend hopefully newly-established therapy to apply in endodontics and traumatic dental hard tissues.

Ring1a/b polycomb proteins regulate the mesenchymal stem cell niche in continuously growing incisors.

Rodent incisors are capable of growing continuously and the renewal of dental epithelium giving rise to enamel-forming ameloblasts and dental mesenchyme giving rise to dentin-forming odontoblasts and pulp cells is achieved by stem cells residing at their proximal ends. Although the dental epithelial stem cell niche (cervical loop) is well characterized, little is known about the dental mesenchymal stem cell niche. Ring1a/b are the core Polycomb repressive complex1 (PRC1) components that have recently also been found in a protein complex with BcoR (Bcl-6 interacting corepressor) and Fbxl10. During mouse incisor development, we found that genes encoding members of the PRC1 complex are strongly expressed in the incisor apical mesenchyme in an area that contains the cells with the highest proliferation rate in the tooth pulp, consistent with a location for transit amplifying cells. Analysis of Ring1a(-/-);Ring1b(cko/cko) mice showed that loss of Ring1a/b postnatally results in defective cervical loops and disturbances of enamel and dentin formation in continuously growing incisors. To further characterize the defect found in Ring1a(-/-);Ring1b(cko/cko) mice, we demonstrated that cell proliferation is dramatically reduced in the apical mesenchyme and cervical loop epithelium of Ring1a(-/-);Ring1b(cko/cko) incisors in comparison to Ring1a(-/-);Ring1b(fl/fl)cre- incisors. Fgf signaling and downstream targets that have been previously shown to be important in the maintenance of the dental epithelial stem cell compartment in the cervical loop are downregulated in Ring1a(-/-);Ring1b(cko/cko) incisors. In addition, expression of other genes of the PRC1 complex is also altered. We also identified an essential postnatal requirement for Ring1 proteins in molar root formation. These results show that the PRC1 complex regulates the transit amplifying cell compartment of the dental mesenchymal stem cell niche and cell differentiation in developing mouse incisors and is required for molar root formation.

Roles of heparan sulfate sulfation in dentinogenesis.

Cell surface heparan sulfate (HS) is an essential regulator of cell signaling and development. HS traps signaling molecules, like Wnt in the glycosaminoglycan side chains of HS proteoglycans (HSPGs), and regulates their functions. Endosulfatases Sulf1 and Sulf2 are secreted at the cell surface to selectively remove 6-O-sulfate groups from HSPGs, thereby modifying the affinity of cell surface HSPGs for its ligands. This study provides molecular evidence for the functional roles of HSPG sulfation and desulfation in dentinogenesis. We show that odontogenic cells are highly sulfated on the cell surface and become desulfated during their differentiation to odontoblasts, which produce tooth dentin. Sulf1/Sulf2 double null mutant mice exhibit a thin dentin matrix and short roots combined with reduced expression of dentin sialophosphoprotein (Dspp) mRNA, encoding a dentin-specific extracellular matrix precursor protein, whereas single Sulf mutants do not show such defective phenotypes. In odontoblast cell lines, Dspp mRNA expression is potentiated by the activation of the Wnt canonical signaling pathway. In addition, pharmacological interference with HS sulfation promotes Dspp mRNA expression through activation of Wnt signaling. On the contrary, the silencing of Sulf suppresses the Wnt signaling pathway and subsequently Dspp mRNA expression. We also show that Wnt10a protein binds to cell surface HSPGs in odontoblasts, and interference with HS sulfation decreases the binding affinity of Wnt10a for HSPGs, which facilitates the binding of Wnt10a to its receptor and potentiates the Wnt signaling pathway, thereby up-regulating Dspp mRNA expression. These results demonstrate that Sulf-mediated desulfation of cellular HSPGs is an important modification that is critical for the activation of the Wnt signaling in odontoblasts and for production of the dentin matrix.

Neural crest deletion of Dlx3 leads to major dentin defects through down-regulation of Dspp.

During development, Dlx3 is expressed in ectodermal appendages such as hair and teeth. Thus far, the evidence that Dlx3 plays a crucial role in tooth development comes from reports showing that autosomal dominant mutations in DLX3 result in severe enamel and dentin defects leading to abscesses and infections. However, the normal function of DLX3 in odontogenesis remains unknown. Here, we use a mouse model to demonstrate that the absence of Dlx3 in the neural crest results in major impairment of odontoblast differentiation and dentin production. Mutant mice develop brittle teeth with hypoplastic dentin and molars with an enlarged pulp chamber and underdeveloped roots. Using this mouse model, we found that dentin sialophosphoprotein (Dspp), a major component of the dentin matrix, is strongly down-regulated in odontoblasts lacking Dlx3. Using ChIP-seq, we further demonstrate the direct binding of Dlx3 to the Dspp promoter in vivo. Luciferase reporter assays determined that Dlx3 positively regulates Dspp expression. This establishes a regulatory pathway where the transcription factor Dlx3 is essential in dentin formation by directly regulating a crucial matrix protein.

Localization of SUMOylation factors and Osterix in odontoblast lineage cells during dentin formation and regeneration.

Small ubiquitin-related modifier (SUMO) conjugation (SUMOylation) is a post-translational modification involved in various cellular processes including the regulation of transcription factors. In this study, to analyze the involvement of SUMOylation in odontoblast differentiation, we examined the immunohistochemical localization of SUMO-1, SUMO-2/3, and Osterix during rat tooth development. At the bud and cap stages, localization of SUMOs and Osterix was hardly detected in the dental mesenchyme. At the bell stage, odontoblasts just beginning dentin matrix secretion and preodontoblasts near these odontoblasts showed intense immunoreactivity for these molecules. However, after the root-formation stage, these immunoreactivities in the odontoblasts decreased in intensity. Next, to examine whether the SUMOylation participates in dentin regeneration, we evaluated the distribution of SUMOs and Osterix in the dental pulp after cavity preparation. In the coronal pulp chamber of an untreated rat molar, odontoblasts and pulp cells showed no immunoreactivity. At 4 days after cavity preparation, positive cells for SUMOs and Osterix appeared on the surface of the dentin beneath the cavity. Odontoblast-like cells forming reparative dentin were immunopositive for SUMOs and Osterix at 1 week, whereas these immunoreactivities disappeared after 8 weeks. Additionally, we further analyzed the capacity of SUMO-1 to bind Osterix by performing an immunoprecipitation assay using C2C12 cells, and showed that Osterix could undergo SUMOylation. These results suggest that SUMOylation might regulate the transcriptional activity of Osterix in odontoblast lineage cells, and thus play important roles in odontoblast differentiation and regeneration.

Evaluation of blood cell attachment on Er: YAG laser applied root surface using scanning electron microscopy.

BACKGROUND: Periodontal regeneration is dependent on the uninterrupted adhesion, maturation and absorption of fibrin clots to a periodontally compromised root surface. The modification of the root surface with different agents has been used for better fibrin clot formation and blood cell attachment. It is known that Er:YAG laser application on dentin removes the smear layer succesfully. AIM: The aim of this study is to observe blood cell attachment and fibrin network formation following ER:YAG laser irradiation on periodontally compromised root surfaces in comparison to chemical root conditioning techniques in vitro. MATERIALS AND METHODS: 40 dentin blocks prepared from freshly extracted periodontally compromised hopeless teeth. Specimens were divided in 5 groups; those applied with PBS, EDTA, Citric acid and Er:YAG. They were further divided into two groups: those which had received these applications, and the control group. The specimens were evaluated with scanning electron microscope and micrographs were taken. Smear layer and blood cell attachment scoring was performed. RESULTS: In the Er:YAG laser applied group, smear layer were totally removed. In the blood applied specimens, better fibrin clot formation and blood cell attachment were observed in the Er:YAG group. In the group that had been applied with citric acid, the smear layer was also removed. The smear layer could not be fully removed in the EDTA group. CONCLUSION: Er:YAG laser application on the root dentin seems to form a suitable surface for fibrin clot formation and blood cell attachment. Further clinical studies to support these results are necessitated.

New bone formation around xenogenic dentin grafts to rabbit tibia marrow.

PURPOSE: From traumatology, it is well known that dentoalveolar ankylosis results in osseous replacement and formation of new bone. This principle is used after decoronation for preservation of the height and width of the alveolar bone crest after trauma. Dentin possesses bone-forming properties and may possibly also be used as a bone augmentation material prior to implant placement. The aim of this study was to investigate whether xenogenic dentin particles inserted into the marrow space of rabbit tibia, a space where there is no solid bone tissue initially, would contribute to new bone formation. MATERIALS AND METHODS: Dentin chips from human teeth were inserted into tibias of ten New Zealand rabbits. The tibial bones were processed for histology after 6 months, and new bone formation was quantified. RESULTS: Bone formation was ranging from 0 to 86% on the dentin fragments, and there was minor inflammation. Bone formation was seen to a larger extent on dentin grafts located close to the native tibial bone wall. There was a significant correlation (r = -0.579, P < 0.001) between the amount of bone formation around the dentin graft and distance to the tibial cortical wall. CONCLUSION: Dentin promotes new bone formation when located close to native cortical bone and may have a potential as a bone augmentation material.

The role of runt-related transcription factor 2 (Runx2) in the late stage of odontoblast differentiation and dentin formation.

Runx2, of the Runx family, is an essential transcription factor that controls bone and tooth development by regulating osteoblast and odontoblast differentiation. However, the function of Runx2 in late stage odontoblast differentiation is not clear. We studied the function of Runx2 in dentinogenesis by generating transgenic mice expressing Runx2 specifically in odontoblasts. We observed dentin formation in postnatal day 3 (P3), P7 and P28 mice and measured the expression levels of Runx2 and matrix proteins in dentin. The odontoblasts in transgenic mice (Tg) lost their tall columnar shape and polarization and dentinal tubules were absent. The dental pulp chamber was dramatically enlarged and the dentin in Tg mice was thinner. Osteoblast-like cells were seen instead of normal odontoblasts and were embedded in a bone-like matrix, indicating that dentin formation was replaced with bone. Predentin was disorganized possessing lacunae that contained odontoblasts. The mandibular molars of Tg mice showed noticeable defects by Micro-CT. Using quantitative real-time PCR, the expression of dentin matrix proteins, particularly dentin sialophosphoprotein (DSPP), was found to be upregulated in 3-day-old Tg mice and downregulated at 1 month of age. These findings indicate that Runx2 inhibited odontoblast terminal differentiation and induced transdifferentiation of odontoblasts to osteoblasts at the late cell differentiation stage. Therefore, Runx2 should be inhibited in odontoblasts to encourage normal cell maturation, differentiation and dentinogenesis.