Spatio-temporal immunolocalization of VEGF-A, Runx2, and osterix during the early steps of intramembranous ossification of the alveolar process in rat embryos.

Vascular endothelial growth factor A (VEGF-A) is expressed by several cell types and is a crucial factor for angiogenic-osteogenic coupling. However, the immunolocalization of VEGF-A during the early stages of the alveolar process formation remains underexplored. Thus, we analyzed the spatio-temporal immunolocalization of VEGF-A and its relationship with Runt-related transcription factor 2 (Runx2) and osterix (Osx) during the early steps of intramembranous ossification of the alveolar process in rat embryos. Embryo heads (E) of 16, 18 and 20-day-old rats were processed for paraffin embedding. Histomorphometry and immunohistochemistry to detect VEGF-A, Runx2, and Osx (osteoblast differentiation markers) were performed. The volume density of bone tissue including bone cells and blood vessels increased significantly in E18 and E20. Cells showing high VEGF-A immunoreactivity were initially observed within a perivascular niche in the ectomesenchyme; afterwards, these cells were diffusely located near bone formation sites. Runx2-and Osx-immunopositive cells were observed in corresponded regions of cells showing strong VEGF-A immunoreactivity. Although these immunostained cells were observed in all specimens, this immunolocalization pattern was more evident in E16 specimens and gradually decreased in E18 and E20 specimens. Double immunofluorescence labelling showed intracellular co-localization of Osx and VEGF-A in cells surrounding the developing alveolar process, indicating a crucial role of VEGF-A in osteoblast differentiation. Our results showed VEGF-A immunoexpression in osteoblasts and its precursors during the maxillary alveolar process formation of rat embryos. Moreover, the VEGF-A-positive cells located within a perivascular niche at the early stages of the alveolar process development suggest a crosstalk between endothelium and ectomesenchymal cells, reinforcing the angiogenic-osteogenic coupling in this process.

Antagonistic interaction between Ezh2 and Arid1a coordinates root patterning and development via Cdkn2a in mouse molars.

Patterning is a critical step during organogenesis and is closely associated with the physiological function of organs. Tooth root shapes are finely tuned to provide precise occlusal support to facilitate the function of each tooth type. However, the mechanism regulating tooth root patterning and development is largely unknown. In this study, we provide the first in vivo evidence demonstrating that Ezh2 in the dental mesenchyme determines patterning and furcation formation during dental root development in mouse molars. Mechanistically, an antagonistic interaction between epigenetic regulators Ezh2 and Arid1a controls Cdkn2a expression in the dental mesenchyme to regulate dental root patterning and development. These findings indicate the importance of balanced epigenetic regulation in determining the tooth root pattern and the integration of roots with the jaw bones to achieve physiological function. Collectively, our study provides important clues about the regulation of organogenesis and has general implications for tooth regeneration in the future.

Subphenotyping and Classification of Cleft Lip and Alveolus in Adult Unoperated Patients: A New Embryological Approach.

OBJECTIVE: Previously, a new embryological classification was introduced subdividing oral clefts into fusion and/or differentiation defects. This subdivision was used to classify all subphenotypes of cleft lip with or without alveolus (CL±A). Subsequently, it was investigated whether further morphological grading of incomplete CLs is clinically relevant, and which alveolar part is deficient in fusion/differentiation defects. DESIGN: Observational cohort study. SETTING: Three hundred fifty adult unoperated Indonesian cleft patients presented themselves for operation. Cephalograms, dental casts, and intraoral and extraoral photographs-eligible for the present study-were used to determine morphological severity of CL±A. PATIENTS: Patients with unilateral or bilateral clefts of the primary palate only were included. MAIN OUTCOME MEASURES: Clefts were classified-according to developmental mechanisms and timing in embryogenesis-as fusion and/or differentiation defects. Grades of incomplete CLs were related to the severity of alveolar clefts (CAs) and hypoplasia, and permanent dentition was used to investigate which alveolar part is deficient in fusion/differentiation defects. RESULTS: One hundred eight adult patients were included. All subphenotypes-96 unilateral and 12 bilateral clefts-could be classified into differentiation (79%), fusion (17%), fusion-differentiation (2%), or fusion and differentiation (2%) defects. The various grades of incomplete CLs were related to associated CAs and hypoplasia, and all alveolar deformities were located in the premaxillae. CONCLUSIONS: This study showed that all CL±A including the Simonart bands can be classified, that further morphological grading of incomplete CLs is clinically relevant, and that the premaxilla forms the deficient part in alveolar deformities.

Fluoxetine effects on periodontogenesis: histomorphometrical and immunohistochemical analyses in rats.

OBJECTIVE: This study aimed to assess the effects of fluoxetine, a selective serotonin reuptake inhibitor, on the formation of the periodontal ligament during pregnancy and lactation in rat pups. MATERIAL AND METHODS: Twelve pregnant rats of Wistar lineage were divided into four study groups. In the control group, 0.9% sodium chloride solution was administered orally, throughout the entire period of the 21 days of pregnancy (CG group) and in the CGL group, it was administrated during pregnancy and lactation (from day 1 of pregnancy to the 21st day after birth). Fluoxetine was administered orally at the dose of 20 mg/kg in a group treated during pregnancy only (FG group), and during pregnancy and lactation (FGL group). Histometrical, histochemical and immunohistochemical analysis of the maxillary first molar periodontium region of the 24 rat pups was made under light microscopy, and periodontal ligament collagen was qualitatively evaluated under a polarizing light microscope. RESULTS: The quantity of fibroblasts (p=0.006), osteoblasts (p=0.027) and cementoblasts (p=0.001) was reduced in pups from the rats that received fluoxetine during pregnancy and lactation. No alterations were seen in the collagen fibers. CONCLUSION: These findings suggest that periodontal tissue may be sensitive to fluoxetine, and its interference in reducing periodontal cells depends on exposure time during lactation.

Fluoxetine effects on periodontogenesis: histomorphometrical and immunohistochemical analyses in rats

Abstract Reports have indicated that serotonin plays an important role in cell migration and differentiation during the organogenesis of several tissues, including the oral types. Administration of selective serotonin reuptake inhibitor (SSRI) drugs during pregnancy could affect the delivery of serotonin to embryonic tissues altering its development. Objective This study aimed to assess the effects of fluoxetine, a selective serotonin reuptake inhibitor, on the formation of the periodontal ligament during pregnancy and lactation in rat pups. Material and Methods Twelve pregnant rats of Wistar lineage were divided into four study groups. In the control group, 0.9% sodium chloride solution was administered orally, throughout the entire period of the 21 days of pregnancy (CG group) and in the CGL group, it was administrated during pregnancy and lactation (from day 1 of pregnancy to the 21st day after birth). Fluoxetine was administered orally at the dose of 20 mg/kg in a group treated during pregnancy only (FG group), and during pregnancy and lactation (FGL group). Histometrical, histochemical and immunohistochemical analysis of the maxillary first molar periodontium region of the 24 rat pups was made under light microscopy, and periodontal ligament collagen was qualitatively evaluated under a polarizing light microscope. Results The quantity of fibroblasts (p=0.006), osteoblasts (p=0.027) and cementoblasts (p=0.001) was reduced in pups from the rats that received fluoxetine during pregnancy and lactation. No alterations were seen in the collagen fibers. Conclusion These findings suggest that periodontal tissue may be sensitive to fluoxetine, and its interference in reducing periodontal cells depends on exposure time during lactation.

Dual embryonic origin of maxillary lateral incisors: clinical implications in patients with cleft lip and palate.

INTRODUCTION: Cleft lip and palate are craniofacial anomalies highly prevalent in the overall population. In oral clefts involving the alveolar ridge, variations of number, shape, size and position are observed in maxillary lateral incisors. The objective of this manuscript is to elucidate the embryonic origin of maxillary lateral incisors in order to understand the etiology of these variations.Contextualization: The hypothesis that orofacial clefts would split maxillary lateral incisor buds has been previously reported. However, recent studies showed that maxillary lateral incisors have dual embryonic origin, being partially formed by both the medial nasal process and the maxillary process. In other words, the mesial half of the lateral incisor seems to come from the medial nasal process while the distal half of the lateral incisor originates from the maxillary process. In cleft patients, these processes do not fuse, which results in different numerical and positional patterns for lateral incisors relating to the alveolar cleft. In addition to these considerations, this study proposes a nomenclature for maxillary lateral incisors in patients with cleft lip and palate, based on embryology and lateral incisors position in relation to the alveolar cleft. CONCLUSION: Embryological knowledge on the dual origin of maxillary lateral incisors and the use of a proper nomenclature for their numerical and positional variations renders appropriate communication among professionals and treatment planning easier, in addition to standardizing research analysis.

Dual embryonic origin of maxillary lateral incisors: clinical implications in patients with cleft lip and palate

Introduction:Cleft lip and palate are craniofacial anomalies highly prevalent in the overall population. In oral clefts involving the alveolar ridge, variations of number, shape, size and position are observed in maxillary lateral incisors. The objective of this manuscript is to elucidate the embryonic origin of maxillary lateral incisors in order to understand the etiology of these variations.Contextualization: The hypothesis that orofacial clefts would split maxillary lateral incisor buds has been previously reported. However, recent studies showed that maxillary lateral incisors have dual embryonic origin, being partially formed by both the medial nasal process and the maxillary process. In other words, the mesial half of the lateral incisor seems to come from the medial nasal process while the distal half of the lateral incisor originates from the maxillary process. In cleft patients, these processes do not fuse, which results in different numerical and positional patterns for lateral incisors relating to the alveolar cleft. In addition to these considerations, this study proposes a nomenclature for maxillary lateral incisors in patients with cleft lip and palate, based on embryology and lateral incisors position in relation to the alveolar cleft.Conclusion:Embryological knowledge on the dual origin of maxillary lateral incisors and the use of a proper nomenclature for their numerical and positional variations renders appropriate communication among professionals and treatment planning easier, in addition to standardizing research analysis.

Introdução:as fissuras de lábio e palato são malformações de alta prevalência na população. Nas fissuras que envolvem o rebordo alveolar, o incisivo lateral superior mostra variações de número, forma, tamanho e posição, o que o torna objeto de estudo, na tentativa de elucidar sua origem embrionária para compreender a etiologia dessas alterações.Contextualização:existia a hipótese de que a fissura orofacial seria capaz de segmentar o botão embrionário do incisivo lateral. No entanto, estudos recentes evidenciaram que o incisivo lateral superior possui dupla origem embrionária, sendo formado parcialmente pelo processo nasal medial e pelo processo maxilar. Em outras palavras, a metade mesial do incisivo lateral provém do processo nasal medial, enquanto a metade distal do incisivo lateral origina-se do processo maxilar. No paciente com fissura, não há fusão desses processos, o que resulta nos diferentes padrões numéricos e posicionais do incisivo lateral em relação à fissura. Além dessas considerações, propõe-se também uma nomenclatura para o incisivo lateral em pacientes com fissura labiopalatina, com embasamento na Embriologia, considerando-se sua posição em relação à fissura alveolar.Conclusão:o conhecimento embriológico da dupla origem do incisivo lateral superior e o emprego de uma nomenclatura adequada para as suas variações numéricas e posicionais facilita a comunicação entre profissionais, o planejamento dos casos e possibilita a realização de estudos clínicos comparativos.

Histometric evaluation of dental alveolar repair in malnourished rats in the intrauterine or postnatal phase.

OBJECTIVE: Nutritional aggravations during pregnancy or during the early stages of postnatal development can impair bone development; thus, we aimed to assess the effects of food restriction on the dental alveolar bone repair process using histometric analysis. DESIGN: Thirty-six Wistar rats were divided into three groups: (C) 12 pups were obtained from control mothers with food intake at ease; (GR) 12 pups from mothers subjected to 70% food restriction during pregnancy; (PNR) 50% of maternal food restriction during lactation and 50% of restriction for the 12 pups after weaning. At three months of age, the upper right incisor was extracted from the pups. After 14 or 28 days, the pups were sacrificed for evaluation of newly formed bone area (NB) and total bone area (TA) in the medial and apical thirds of the alveolus. RESULTS: In the apical third of the alveolus, the ratio of NB/TA was greater at 28 days for all groups and there was no damage to any of the groups. In the medial third, the ratio was higher at 28 days for the C and GR groups. The PNR group did not show an evolution of alveolar dental repair. Compared between the thirds, all groups exhibited a higher percentage of newly formed bone in the medial third area, at any time point after surgery. CONCLUSIONS: The percentage of the total alveolar area covered by newly formed bone (NB/TA) revealed a late preference in the process of alveolar repair in the medial third, although only in the PNR group.

Facial cleft detected: is the palate normal?

Despite advances in ultrasound technology, the sensitivity for detection of facial clefts at the routine mid-trimester details scan remains relatively poor. This can be improved by the use of a three-point ultrasound screening protocol, although this is not routine in many countries. When a facial cleft is suspected at the routine scan, further imaging is usually required to detail the extent of the cleft and presence or absence of any other abnormalities. Involvement of the fetal palate is an important finding that will determine the requirement for surgery, audiology, and orthodontic services well into teenage years. There remains little uniformity in how a facial cleft is described antenatally, with involvement of the alveolar ridge frequently and incorrectly taken to mean involvement of the palate. Further, midline clefts of the hard and soft palates, where the fetal lips and alveolar ridge are intact, are a feature of many genetic conditions, but are almost never diagnosed by prenatal ultrasound. In this chapter, we detail issues surrounding the nomenclature of facial clefts in relation to the palate, and describe some of the more commonly used two-dimensional and three-dimensional methodologies for imaging the fetal palate.

Interactions of the tooth and bone during development.

The tooth works as a functional unit with its surrounding bony socket, the alveolar bone. The growth of the tooth and alveolar bone is co-ordinated so that a studied distance always separates the 2, known as the tooth-bone interface (TBI). Lack of mineralization, a crucial feature of the TBI, creates the space for the developing tooth to grow and the soft tissues of the periodontium to develop. We have investigated the interactions between the tooth and its surrounding bone during development, focusing on the impact of the developing alveolar bone on the development of the mouse first molar (M1). During development, TRAP-positive osteoclasts are found to line the TBI as bone starts to be deposited around the tooth, removing the bone as the tooth expands. An enhancement of osteoclastogenesis through RANK-RANKL signaling results in an expansion of the TBI, showing that osteoclasts are essential for defining the size of this region. Isolation of the M1 from the surrounding mesenchyme and alveolar bone leads to an expansion of the tooth germ, driven by increased proliferation, indicating that, during normal development, the growth of the tooth germ is constrained by the surrounding tissues.

Expression of clock proteins in developing tooth.

Morphological and functional changes during ameloblast and odontoblast differentiation suggest that enamel and dentin formation is under circadian control. Circadian rhythms are endogenous self-sustained oscillations with periods of 24h that control diverse physiological and metabolic processes. Mammalian clock genes play a key role in synchronizing circadian functions in many organs. However, close to nothing is known on clock genes expression during tooth development. In this work, we investigated the expression of four clock genes during tooth development. Our results showed that circadian clock genes Bmal1, clock, per1, and per2 mRNAs were detected in teeth by RT-PCR. Immunohistochemistry showed that clock protein expression was first detected in teeth at the bell stage (E17), being expressed in EOE and dental papilla cells. At post-natal day four (PN4), all four clock proteins continued to be expressed in teeth but with different intensities, being strongly expressed within the nucleus of ameloblasts and odontoblasts and down-regulated in dental pulp cells. Interestingly, at PN21 incisor, expression of clock proteins was down-regulated in odontoblasts of the crown-analogue side but expression was persisting in root-analogue side odontoblasts. In contrast, both crown and root odontoblasts were strongly stained for all four clock proteins in first molars at PN21. Within the periodontal ligament (PDL) space, epithelial rests of Malassez (ERM) showed the strongest expression among other PDL cells. Our data suggests that clock genes might be involved in the regulation of ameloblast and odontoblast functions, such as enamel and dentin protein secretion and matrix mineralization.

Prenatal assessment of the antero-posterior jaw relationship in human fetuses: from anatomical to ultrasound cephalometric analysis.

OBJECTIVES: We wished to develop an ultrasound cephalometric analysis, particularly of the antero-posterior jaw relationship, to increase the accuracy of prenatal diagnosis of retrognathism during the routine midterm test. METHODS: Anatomical cephalometric analysis was performed in 18 formalin-fixed human fetuses (between 16 and 39 gestational weeks), and ultrasound cephalometry was prospectively carried out in 52 pregnant women (21 to 25 gestational weeks). The same landmarks were used in the anatomical and ultrasound median sagittal planes for comparison. Four cephalometric angles were measured relative to the anterior cranial base: alveolar projection of the maxilla and the mandible, chin projection, and facial angle. The antero-posterior jaw discrepancy was calculated. RESULTS: The projection of the maxilla was similar in the two cephalometric analyses (IC [-3.39, 0.23]), whereas the values of the projection of the mandible were lower in the ultrasound sample. The slope of the regression line of the antero-posterior jaw discrepancy on fetuses' age did not show significant differences (IC [-0.05, 1.54]) between anatomical and ultrasound cephalometry, although a difference of 3.23° ± 0.78° (IC [1.69, 4.77]) was observed. Despite this variability, the projections of mandible and chin were well determined by the projection of the maxilla both in the anatomical and ultrasound sample. CONCLUSIONS: Cephalometric analysis by prenatal sonography can be performed to study the antero-posterior jaw relationship. We think that this procedure could be useful to improve prenatal diagnosis of retrognathism in high-risk pregnancies. Further studies should address the reproducibility and accuracy of such analysis.

Genetic and transcriptional control of bone formation.

An exquisite interplay of developmental cues, transcription factors, and coregulatory and signaling proteins support formation of skeletal elements of the jaw during embryogenesis and dynamic remodeling of alveolar bone in postnatal life. These molecules promote initial condensation of the mesenchyme, commitment of the mesenchymal progenitor to osteogenic lineage cells, and differentiation of committed osteoblasts to mature osteocytes within mineralized bone. Parallel regulatory networks promote formation of the functional osteoclast from mononuclear cells to support continuous bone remodeling within the alveolar bone. With an ever expanding list of new regulatory factors, the complexities of the molecular mechanisms that control gene expression in skeletal cells are being further appreciated. This article examines the multifunctional roles of prominent nuclear proteins, cytokines, hormones, and paracrine factors that control osteogenesis.

Formation of the tooth-bone interface.

Not only are teeth essential for mastication, but also missing teeth are considered a social handicap due to speech and aesthetic problems, with a resulting high impact on emotional well-being. Several treatment procedures are currently available for tooth replacement with mostly inert prosthetic materials and implants. Natural tooth substitution based on copying the developmental process of tooth formation is particularly challenging and creates a rapidly developing area of molecular dentistry. In any approach, functional interactions among the tooth, the surrounding bone, and the periodontium must be established. Therefore, recent research in craniofacial genetics searches for mechanisms responsible for correct cell and tissue interactions, not only within a specific structure, but also in the context of supporting structures. A tooth crown that is not functionally anchored to roots and bone is useless. This review aims to summarize the developmental and tissue homeostatic aspects of the tooth-bone interface, from the initial patterning toward tooth eruption and lifelong interactions between the tooth and its surrounding alveolar bone.

Expression patterns of the Fam83h gene during murine tooth development.

AIM: Recently a novel gene, FAM83H, was identified by a genetic linkage study in the hypocalcified form of the amelogenesis imperfecta family with an autosomal dominant hereditary pattern. Little is known about this novel gene, and so we investigated the expression pattern of Fam83h in murine tooth development using serial sectional in situ hybridisation. METHODS AND MATERIALS: Using mandibles of ICR mouse at specific developmental stages, in situ hybridisation was performed by DIG-labeled RNA probe. RESULTS: Faint expression was detected in limited cells at embryonic day 14 (E14) in the molar. At the bell stage, E16, Fam83h was localised in the outer and inner enamel epithelium, as well as dental papilla. Fam83h expression begins on E15 in the developing incisor. At E18, Fam83h was expressed in the inner enamel epithelium of the apical bud, ameloblasts and odontoblasts. The expression was stronger in the presecretory stages than the secretory stages. CONCLUSION: Fam83h was detected in the ameloblasts from the presecretory to the secretory stage, and also the odontoblasts layer and surrounding alveolar bone.

Immunohistochemical PGP 9.5 positivity in human osteoblasts may indicate that compensatory and dysplastic craniofacial growth are under control by peripheral nerves.

OBJECTIVES: The purpose was to examine human osteoblasts immunohistochemically in order to clarify the significance of the innervation for alveolar bone growth. SETTING AND SAMPLE POPULATION: Unstained sections available from 21 normal human mandibles (foetal gestational ages: 14-22 weeks). MATERIAL AND METHODS: Before sectioning in 1980-1990, the mandibular tissue had been fixed in 4% neutral-buffered formaldehyde for 5 days. Tissue blocks were then decalcified in equal parts of 2% citric acid and 20% sodium citrate for 7-15 days, dehydrated, embedded in paraffin, and sagittally cut into 4-mum-thick serial sections and mounted on Superfrost Plus microscope slides. Sections were dried overnight at 40 degrees C. In the present study, paraffin sections were deparaffinized and treated with Tris-EDTA (Merck, Germany), pH 9.0, and immunohistochemically tested with polyclonal rabbit anti-PGP 9.5, and the EnVision +/HRP dual link (K4065; DAKO Denmark A/S, Denmark) method. RESULTS: A pronounced protein gene product (PGP) 9.5 activity was registered in osteoblasts from alveolar bone in all specimens. In all cases, the activity was intense at the top of and labially to the alveolar bone, while less or no activity was observed on the inner lingual aspects of the alveolar processes. Osteoclasts and osteocytes reacted vaguely or negatively. CONCLUSION: As the present study has demonstrated that human osteoblast activity in the alveolar bone seemingly responds to innervation, it is suggested that the peripheral nervous system via the trigeminal ganglion regulates compensatory and dysplastic alveolar bone formation.

Abnormal maxillary trapezoid pattern in human fetal cleft lip and palate.

OBJECTIVE: To elucidate abnormal growth patterns of human fetal maxillae with cleft lip and palate (CLP). SUBJECT: A total of 71 fetal maxillae with CLP were obtained from aborted human fetuses. METHOD: Dimensions of the maxillary trapezoid (MT), formed by the maxillary primary growth centers (MxPGC), were taken from radiographic images. The CLP dimensions were compared with maxillary trapezoid dimensions of normal fetuses from a previous study (Lee et al., 1992). MAIN OUTCOME MEASURES: Cleft lip subjects without a cleft palate, unilateral cleft lip-alveolar cleft or cleft palate (UCL+A/UCLP), and bilateral cleft lip-alveolar cleft or cleft palate (BCL+A/BCLP) displayed abnormal MT patterns. MT abnormalities were most marked in the BCL+A/BCLP cohort. RESULTS: The MT growth of prenatal CLP maxillae was severely arrested, resulting in abnormal MT shape on palatal radiograms. BCL+A/BCLP subjects had a more protruded nasal septum than subjects with other types of CLPs, while UCL+A/UCLP subjects showed severe deviation of the protruded nasal septum toward the noncleft side. Cleft lip-only subjects also exhibited abnormal MT growth. CONCLUSION: MT is primarily involved in CLPs, so that the MT shape could be utilized as a sensitive indicator for the analysis of maxillary malformation in different types of CLPs.

Effect of cyclic 3', 5' adenosin-monophosphate on osteogenesis embryonic bone cultivated in tissue culture: an autoradiographic study.

UNLABELLED: The aim of this study was to evaluate the effect of cyclic 3', 5' adenosin-monophosphate (cAMP) on DNA synthesis of embryonic alveolar bone in tissue culture. MATERIAL AND METHODS: Bone fragments were cultured in the medium composed of 80% medium 199, 15% horse serum, 4 mg/ml glucose, 100 microg/ml penicillin using the grid method. The explants were cultured up to 12 days. In the second series, the effect of cAMP in a concentration of 10(-6)M on bone during 12 days was studied. DNA synthesis was determined by calculating mitotic labelling indices for (3)H-thymidine incorporation into cells within cultured explants at 3 to 12 days. The medium was supplemented with 1 microCurie/ml (3)H-thymidine for 4 hours and processed for autoradiography. The mitotic labelling index was determined in the histological sections. All values wer presented as mean+/-standard deviation. Statistical significance was defined by p-values of 0.05 or less. RESULTS: Morphological and statistical analysis showed that there were differences in mitotic incidence (MI) and silver grain densities over osteoblasts in control cultures and with cAMP. The mean value of MI was 4,627+/-1,001 in control and 7,706+/-1,188 in the cultures where cAMP was added (p<0.05). CONCLUSION: Thus cAMP inhibited bone resorption and stimulated new bone formation in tissue culture. This study provides a novel concept that may help to identify new therapeutic targets.