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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., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2021. Published by Elsevier Inc.)

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., Competing Interests: JJ, JF, JL, XH, JH, TH, JD, XZ, MU, YC No competing interests declared, (© 2019, Jing et al.)

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.

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.

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.

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., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

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.

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.

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., (Copyright © 2010 Elsevier B.V. All rights reserved.)

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., (Copyright 2010 Elsevier Inc. All rights reserved.)

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.

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.

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.

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.

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.

In the developing mammalian tooth, the cranial neural crest derived dental mesenchyme consists of the dental papilla and dental follicle. The dental papilla gives rise to odontoblasts and dental pulp and the dental follicle gives rise to the periodontium, including the osteoblasts that contribute to the alveolar process. The alveolar process is a specialized intramembranous bone that forms the primary support structure for the dentition. The Msx1 gene controls many aspects of craniofacial development, as evidenced by craniofacial abnormalities seen in Msx1(-/-) mice, including the arrest of tooth development and the absence of the alveolar bone. Previous studies demonstrated that ectopic expression of Bmp4, a downstream target of Msx1, in the Msx1(-/-) dental mesenchyme rescued alveolar bone formation. Here we confirm an early requirement of BMP activity for alveolar bone formation. We show that the expression of Cbfa1 and Dlx5, two genes encode transcription factors that are critical for bone differentiation, overlaps with that of Msx1 and Bmp4 in the developing tooth and alveolar process. We have demonstrated that Dlx5 and Cbfa1 expression is down-regulated in Msx1(-/-) dental mesenchyme and that Msx1 and Bmp4 expression are unaltered in Cbfa1(-/-) mice. These data place Dlx5 and Cbfa1 downstream from the Msx1/Bmp4 in the genetic pathway that regulates tooth development. Ectopic expression of Bmp4 in Msx1 mutants restores the expression of Dlx5, but not Cbfa1, in the dental mesenchyme, and rescues the expression of both Dlx5 and Cbfa1 in the developing alveolar bone. Therefore, the early expression of Cfba1 in the dental mesenchyme appears dispensable for the development of the alveolar bone. Taken together with in vitro gene induction studies, our results demonstrate that BMP4 controls Dlx5 expression in dental mesenchyme, and functions upstream to both Dlx5 and Cbfa1 to regulate alveolar bone formation during tooth development.

The management of alveolar clefts has changed through the years as medical knowledge has improved. An alveolar cleft is the result of abnormal primary palate formation during weeks 4 to 12 of gestation. The rationale for its closure includes 1) stabilizing the maxillary arch, 2) permitting support for tooth eruption, 3) eliminating oronasal fistulae, and 4) providing improved esthetic results. Methods for closure of the alveolar cleft have been solidified during the last century with the use of bone grafting. Secondary bone grafting is now the preferred method of treatment, because early grafting has proven detrimental to midfacial growth. Various materials for bone grafting have been proposed, including iliac crest, cranium, tibia, rib, and mandibular symphysis. Regardless of the timing and materials used, the main principles in approaching alveolar clefts have been well described. They include 1) appropriate flap design, 2) wide exposure, 3) nasal floor reconstruction, 4) closure of oronasal fistula, 5) packing bony defect with cancellous bone, and 6) coverage of bone graft with gingival mucoperiosteal flaps. Certain alveolar clefts are difficult to manage by grafting alone, and orthodontic preparation may be required. Complications of alveolar bone grafts include donor site morbidity as well as graft exposure and loss.

Alveolar bone resorption by osteoclasts is essential for tooth eruption. Osteoclast-deficient Csfm(op) homozygous (op/op) mice, which lack functional macrophage colony-stimulating factor (M-CSF), suffer from osteopetrosis and completely lack tooth eruption. Although osteoclasts appear, and osteopetrosis is cured with age in op/op mice, tooth eruption is never seen. This fact suggests that there is a critical period when osteoclasts are required for tooth eruption. In this study, to detect the critical period, we administered an antagonistic antibody directed against c-Fms, a receptor for M-CSF, to inbred C57BL/6 mice for various periods. Administration of this antibody decreased tartrate-resistant acid phosphatase-positive (TRAP) osteoclasts, and incisor eruption was completely inhibited by continual administration of this antibody from embryonic day 15.5 (E15.5) until postnatal day 12.5 (D12.5). A 1-day delay of this administration abolished the inhibition of incisor eruption. The number of TRAP-positive osteoclasts was significantly reduced between E16.5 and E18.5 in the mice treated with antibody from E15.5 compared with those treated from E16.5. These results indicate that this period, during which the number of osteoclasts decreases significantly, is critical for inhibiting incisor eruption in C57BL/6 mice.

In a previous study, it was shown that tooth germs of neonatal homozygous parathyroid hormone-related protein (PTHrP)-knockout mice are penetrated or compressed by the surrounding alveolar bone, suggesting an important role for PTHrP in the formation and activation of osteoclasts around growing tooth germs. In order to elucidate the role of PTHrP during the development of the tooth germ and related structures, mandibular explants containing cap stage tooth germs of embryonic day 14, homozygous mice were here cultured with or without surrounding alveolar bone. There was no difference in the number of tartrate-resistant acid phosphatase-positive multinucleated osteoclastic cells around the first molars of homozygous and wild-type mice. After 10 days of culture, osteoclastic cells were rarely present in explants from homozygous mice and penetration of alveolar bone into the dental papilla was observed. The decline in osteoclast number was partly restored by the addition of PTHrP to the culture. Tooth germs of both wild-type and homozygous mice cultured without alveolar bone developed well, with no apparent structural abnormality; dentine formation was evident after 10 days. These data suggest that PTHrP is not required for the development of the tooth germ proper but is indispensable in promoting the osteoclast formation required to accommodate that development.

Tissue recombination experiments and cell lineage analyses of the developing neural crest have documented the role and central pathways of migratory cells during early craniofacial development. In the present study, regional pathways of cells during late peripheral morphogenesis were investigated using the crown stage tooth organ as a model. Homing targets during tooth integument formation were analyzed to understand the fate of migratory cells involved in late tooth organogenesis and the developmental origin of periodontal tissues. After surgical removal of the oral mucosa, the oral aspect of the dental follicle of lower first mouse molar teeth was labeled using a fluorescent contact dye. Following sacrifice after 0, 2, 4, and 6 days, labeled cells were detected in the dental follicle, in the alveolar bone, and in the periodontal ligament adjacent to the molar root. The distribution of labeled tissues was reconstructed three-dimensionally via confocal microscopy. Using a tooth molar organ culture system, labeled cells within the dental follicle were documented traveling in the apical direction. Our results indicated that cell migration during tooth organogenesis was following specific pathways and that cells within the circumference of the dental follicle were migrating in the apical direction. We speculate that migratory cells passing through the dental follicle connective tissue may contribute to the formation of the periodontium. The present documentation visualizes pathways, role, and dynamics of extensive cell movements during late tooth organogenesis.

Objective: Cleft lip/palate (CLP) is a common human congenital defect in which the maxillary lateral incisors are often absent, malformed, and malpositioned. The present study was designed to examine the origin of the upper primary lateral incisor relative to the medial nasal process (MNP) and maxillary process (MP) fusion area and to the premaxillary/maxillary (incisive) suture in monkeys., Method: Scanning electron microscopy, histology, skeletal staining, and drying techniques were used to study facial development in embryo and fetal monkey specimens. A teratogenic dose of cyclophosphamide was administered to pregnant monkeys prior to fusion of the MNP and MP and fetuses were examined for CLP., Results: Formation of the anterior maxilla involved fusion of the MNP and MP at stages 14-18. At stages 18-20, the palatal portion of the MNP had formed the medial and lateral incisive mounds. By stage 22, the upper primary lateral incisor has formed within the MP, lateral to the MNP/MP fusion area and to the ossifying premaxilla. Ossification of the premaxilla begins in the MNP and subsequently spreads laterally across the MNP/MP fusion area into the MP. Accordingly, the lateral incisor undergoes a complex positional shift (mainly medial) relative to the incisive suture both prenatally and postnatally and is finally located medial to the suture. Examination of the cyclophosphamide-induced CLP fetuses showed that the lateral incisor is located lateral to the alveolar cleft and does not shift medial to the incisive suture., Conclusion: Understanding the origin of the lateral incisor (the tooth closest to the cleft) and the shift after its formation provides clues to high incidence of malformations and ectopia of this incisor in cleft patients.

Objective: The objective of this study was to obtain a nomogram of the fetal alveolar ridge development, as a basis for the diagnosis of primary cleft palate., Design: A cross-sectional study of 323 normal pregnant women of 14-32 weeks' gestation. Several biometric measurements were obtained throughout pregnancy, including the width of the fetal alveolar ridge., Results: A nomogram of the width of the fetal alveolar ridge during 14-32 weeks' gestation is presented. A linear growth function was observed between alveolar ridge width and gestational age, biparietal diameter, head circumference, femoral length and humeral length. The alveolar ridge width in all eight cases of cleft palate was above two standard deviations and the 90th centile of our nomogram., Conclusion: We provide a nomogram of the growth of the fetal alveolar ridge between 14 and 32 weeks' gestation. This will aid the detection of primary cleft palate during routine prenatal sonography.

The formation of the human alveolar process around the incisors and canines is a hitherto unreported aspect of fetal oral development. The question is how, where, and when the alveolar process is formed. The purpose of this study was to elucidate the questions where and when and hence to analyze the pattern of formation of the alveolar bone around developing human deciduous canine and incisor teeth. The study was conducted with material selected from a sample of 61 normal human fetuses, legally or spontaneously aborted. Fertilization ages ranged from 9 to 22 weeks, crown-rump Length from 25 to 205 mm, and foot length from 4 to 34 mm. From this material, 7 maxillae and 10 mandibles were selected for the study according to developmental stages of the canines and incisors and according to the orientation of the histological sections. The skeletal investigation consisted of macroscopic and microscopic analysis of the mandibles and maxillae. The investigations revealed the absence of bone between the central and lateral incisors in the mandibles and maxillae of fetuses below the ages of 21 and 22 weeks, respectively. In three specimens, alveolar bone between the central and lateral incisors was recorded (21, 21, and 22 weeks of fertilization). Absence of bone labial to the canine was recorded in all fetuses investigated. In all other regions, alveolar bone occurred as a regular finding. The present study has shown that alveolar bone formation is strictly coordinated with tooth formation. Alveolar bone occurs later in the area labial to the canine and in the region between the central and lateral incisors than it does in other areas associated with the incisor/canine regions. Knowledge of this pattern in the formation of the alveolar process seems to be of importance for a future elucidation of how the alveolar process is initially formed.

Objective: The purpose of this study was to investigate, in a normal human fetal sample, the development of the anterior area of the palate. Emphasis has been placed on the bilateral epithelial fusion lines between the soft-tissue palatal shelves and the premaxillary mucosa, and on the relationship between these fusion lines and the locations of the inclusive fissures and of the tooth buds. The goal was to evaluate how the location of clefts in cleft lip and palate (CLP) patients interferes with tooth buds that are often affected in clefting., Design: Nineteen maxillas of human fetuses underwent histologic examination after serial horizontal sectioning. Superimpositions of tracings from photographs of the histologic sections yielded information on the maxillary morphology at different levels., Results: After elevation and fusion of the soft-tissue palatal shelves, the palatal fusions were Y-shaped. During subsequent osseous palate development, including formation of the incisive fissure and intermaxillary suture, the Y-shaped epithelial fusions disintegrated. The locations of the anterior arms of the epithelial Y and of the incisive fissure did not coincide., Conclusions: The incisive fissure is likely not the location of clefts in CLP. The location of anterior palatal clefting in CLP patients follows the original course of the epithelial fusion between the palatal shelves and the premaxillary mucosa found in the region of the lateral incisors.

Osteoblasts and bone tissue of the mandibular and maxillary alveolar processes substantially differ from osteoblasts and bone in other parts of the skeleton. These differences are apparent during embryonic development, maturation, and aging of these bones. The cellular and molecular basis for these differences is still not clear, but it is unfolding at record speed.

Recent studies on prenatal innervation of the jaws have shown that three separate main innervation paths, constituting three bilateral neural developmental fields (incisor field, canine/premolar field, molar field) exist in each jaw. In this communication the sequences in which the fields are innervated are indicated. These correspond to the sequences of formation of teeth and jawbone. The normal pattern of tooth agenesis is closely related to the neural fields, as the region within a single field were innervation occurs last is always the area most often affected by tooth agenesis. The initial manifestations of juvenile periodontitis also appear at the sites within the different fields where innervation occurs last. It is suggested that the pubertal growth of the alveolar process does not occur in these regions due to deficient innervation, and that the infection in juvenile periodontitis might be secondary to this regional lack of bone apposition.

There are numerous problems encountered among individuals with a cleft lip and/or cleft palate. Addressing these problems necessitates a multidisciplinary approach, requiring a team of experts to facilitate care for these individuals. Dentists play a vital role on the team. An orthodontist, pediatric dentist, oral maxillofacial surgeon, and prosthodontist usually represent the dental members of the team. The success of this team depends on expertise within each discipline but also depends on each member having a broad base of knowledge in general about oral clefts. An understanding of other disciplines and how they approach the treatment of oral clefts is important for each team member. Also, each team representative should appreciate the need to understand the causes of oral clefts, how clefts develop in utero, how various populations are affected, and how to recognize and classify an oral cleft. It is difficult to communicate effectively within the team if we fail to recognize the importance of expanding our knowledge to include other aspects of oral clefts beyond treatment perspectives. This article provides the orthodontist with basic information as it relates to the etiology of oral clefts (ie, genetics, teratogens, and medical conditions), the demographics and incidence of oral clefts, the embryology and classification of clefts.

The A/J inbred strain of mice spontaneously develops cleft lip and/or palate (CL/P). Ovarian transplantation and several test-matings with C57BL/6 strain mice (0% CL/P) were performed to investigate a relationship between mother and fetus in regard to this order. The fetuses obtained showed that regardless of whether the genetic background of the mother was A/J or F1, CL/P was produced only in cases where the fetuses were genetically pure A/J. CL/P frequency was 7.46% in A/J fetuses developed in A/J mothers; it was 5.88% in F1 mothers with transplanted A/J ovaries. Isolated cleft palate (CP) and cleft lip with cleft palate (CLP) were found in A/J mothers. However, the frequency of CP was lower in F1 mothers than in A/J mothers. These results suggest that the occurrence of CLP depends more on the genetic constitution of the fetus than on maternal environmental factors, and the occurrence of CP was influenced by the mother's condition.

Mandibular first molar tooth germs were dissected from Mus musculus (CDI) and Mus caroli (age range: 14-day embryo to 1-day postnatal). Most of the tooth germs were separated enzymically into epithelial and mesenchymal components. Interspecific tissue recombinations and intact M. caroli tooth germs were grown in the anterior chamber of the eye of adult CDI mice for 24 weeks. Recombinations of M. caroli enamel-organ epithelium with M. musculus, dental papilla and follicle mesenchyme developed into normal teeth with advanced root, periodontal ligament and bone formation, thereby confirming extensive epithelial-mesenchymal interactions across the species barrier. Labelling sections by in situ hybridization with a M. musculus-specific DNA probe (pMSat5) showed that almost all cells in the pulp, periodontal ligament and bone were M. musculus, including cementoblasts. Reduced enamel epithelium and epithelial cell rests derived from donor M. caroli enamel organ were unlabelled. This indicates that any cementogenic role of Hertwig's epithelial root sheath must be short-lived. The immunological privilege of the intraocular transplantation site in M. musculus CDI mice did not extend to grafts including xenogeneic M. caroli dental mesenchyme. Thus, intact M. caroli tooth germs and recombinations of M. musculus enamel organ with M. caroli dental papilla and follicle showed limited development, with no root formation, and were populated almost exclusively with labelled host M. musculus lymphocytes.

On the basis of examination of 50 fetal mandibles, development of the mandibular canal is described. The development of the foramen mandibulae, foramen mentale, the lingula, the alveolar process, and the morphological changes in the mandible due to the effect of the development of the mandibular canal are discussed. The anatomical conditions examined may also have clinical implications.

Mandibular first-molar tooth germs were dissected from 16-day-embryo and new-born CD1 mice. By incubation in collagenase they were separated into enamel organ, papilla and follicle. Dental pulp was obtained from mandibular first molars of 3-, 7- and 10-day-old mice. Various combinations of epithelial and mesenchymal tissues were grown for periods up to four weeks in the anterior chamber of the eye of homologous adult male mice. Recombinations of enamel organ and papilla formed teeth with regeneration of the investing layer of follicle and a root-related periodontal ligament, but no formation of alveolar bone. Bone only formed in those grafts which also included follicle. Recombinations of enamel organ and pulp produced dysplastic dentine with no enamel formation or proper tooth development. It was impossible, therefore, to assess whether the potential to regenerate an investing layer extends to the pulp later in development. At an earlier stage, however, the papillary mesenchyme has the ability to regenerate investing-layer cells which lack the capacity to form bone.

Microperoxidase (MP) was used to investigate the cytological and functional features of preosteoclasts and osteoclasts during rat alveolar bone development. We observed mononuclear cells as preosteoclasts and multinuclear cells with and without ruffled borders (RB). In the bone facing multinuclear cells with RB as active osteoclasts, MP was extensively deposited along the external bone matrix undergoing resorption, and was phagocytosed with bone components into the vacuoles of osteoclasts. Neither preosteoclasts nor multinuclear cells without RB took up MP and bone components. Only multinuclear cells with RB seemed to resorb bone. Monocytes/macrophages (MMP) phagocytosed MP through all regions of the plasma membrane, whereas osteoclasts took up MP only through the RB which was a part of the plasma membrane. Endogenous peroxidase was detected in the MMP but not in preosteoclasts and osteoclasts. Thus, osteoclasts were considerably different from the MMP. The numbers of MMP were extremely few close to the osteoclasts, whereas moderate numbers of preosteoclasts were located close to the osteoclasts. Except for the nucleus and RB, there were many morphological similarities between preosteoclasts and osteoclasts. We therefore suggest that preosteoclasts, rather than MMP, are the precursors of osteoclasts during alveolar bone development of neonatal rats.

Ingrowth of dental nerve fibres into the mesenchyme of the tooth-bearing areas of the mouth takes place at an relatively early stage, when the dental laminae are forming but the tooth buds are undeveloped. By the time a developing tooth has reached the cap and bell stages small nerve bundles have begun to enter the mesenchyme from which the dental papilla and sac arise.

The in utero and neonatal development of the molar teeth in osteopetrotic grey lethal mice and normal litter mates was observed in timed embryos which were obtained by caeserian section, and in neonatal litters which were sacrificed on the day of birth and each day thereafter until day 7. Serial coronal and sagittal sections through the heads were prepared. The earliest disturbances in the alveolar bone-tooth germ complex were seen at day 19 in utero. At birth, the grey lethal first molar germs are severely distorted by encroaching alveolar bone. The abnormalities become progressively more marked until day 7. The failure in alveolar bone remodeling is the factor which prevents tooth eruption in grey lethal mice.

Specimens of known age, weight, and crown-rump length were used to characterize the distribution of the dental lamina and deciduous tooth development in the mandible of the domestic pig.

The qualitative structural development of the inferior alveolar nerve was studied by electron microscopy in 56 pre- and postnatal kittens and 21 young and old adult cats. At 25 days post conception the nerve was composed of a bundle of small axons enclosed by primitive sheath cells. Three weeks later myelination had been initiated. Axons measuring 2-3 micrometers underwent local demyelination from 2 weeks before to 3 weeks after birth. This was accompanied and followed by nodalization of larger axons. A typical perineurium was first apparent in the newborn kitten. Six to eight weeks postnatally, the nerve appeared qualitatively mature, although axonal growth was far from completed. This coincides with achievement of a fully mature primary dentition shortly after the weanling period. Apart from a continued size growth, no changes were observed in the nerve during the transition from the primary to the permanent dentition. In the inferior alveolar nerve of old cats, axonal and perineurial changes co-existed with signs of dental attrition and pathology.

A study was made of the effects of cAMP, db-cGMP and db-cAMP on the mitotic activity of the cells of the tooth anlage and alveolar bone in tissue culture of mouse embryo aged 15 days. The data indicate that db-cGMP and db-cAMP at concentrations 10(-6) M and 10(-8) M do not have any essential effect on the mitotic activity of the cells of the tooth anlage whereas cAMP inhibits the mitotic activity in these cells as compared with control. A definite relationship was established between the character of differentiation of the osseous and dental tissues in tissue culture and the concentration of cyclic nucleotides. db-cAMP, db-cGMP and cAMP raise the mitotic activity of the osteogenic cells, inhibit resorption of the alveolar bone, and stimulate formation of a new osseous tissue.

In cases of advanced periodontal disease, it is likely that vertical osseous defects will be present and that selected teeth will require extraction. The healing extraction site provides a source of vital osseous grafting material for the treatment of these infrabony defects. The preparation and grafting of the recipient site, as well as the management of the donor area, are described in detail. Three cases are presented which illustrate the efficacy of this type of therapy.