Genetic basis for tooth malformations: from mice to men and back again.

Teeth arise from sequential and reciprocal interactions between the oral epithelium and the cranial neural crest-derived mesenchyme. Their formation involves a precisely orchestrated series of molecular and morphogenetic events. Numerous regulatory genes that have been primarily found in organisms such as Drosophila, zebrafish, xenopus and mouse are associated with all stages of tooth formation (patterning, morphogenesis, cytodifferentiation and mineralization). Most of these genes belong to evolutionary conserved signaling pathways that regulate communication between epithelium and mesenchyme during embryonic development. These signaling molecules together with specific transcription factors constitute a unique molecular imprint for odontogenesis and contribute to the generation of teeth with various and function-specific shapes. Mutations in several genes involved in tooth formation cause developmental absence and/or defects of teeth in mice. In humans, the odontogenic molecular program is not as well known as that of mice. However, some insight can be obtained from the study of mutations in regulatory genes, which lead to tooth agenesis and/or the formation of defective dental tissues.

Biodegradation, soft and hard tissue integration of various polyethylene glycol hydrogels: a histomorphometric study in rabbits.

OBJECTIVES: (i) To evaluate biodegradation, hard and soft tissue integration using various polyethylene glycol (PEG) hydrogels; (ii) to evaluate the influence of arginine-glycine-aspartic acid (RGD) on two types of PEG hydrogels. MATERIAL AND METHODS: In seven rabbits, six treatment modalities were randomly applied subperiosteally on the skull: (1) a dense network PEG hydrogel (PEG1), (2) PEG1 modified with RGD (PEG1-RGD), (3) a looser network PEG hydrogel (PEG2), (4) PEG2 modified with RGD (PEG2-RGD), (5) a collagen membrane, and (6) a polylactide/polyglycolide/trimethylene carbonate membrane. The animals were sacrificed at 14 days. Histomorphometric analyses were performed on undecalcified Epon sections using a standardized region of interest. For statistical analysis, paired t-test and signed rank test were applied. RESULTS: PEG1 and PEG1-RGD remained intact and maintained the shape. PEG2 and PEG2-RGD completely degraded and were replaced by connective tissue and bone. The largest amount of mineralized tissue was found for PEG2-RGD (21.4%), followed by PEG 2 (9.5%). The highest percentage of residual hydrogel/membrane was observed for PEG1-RGD (55.6%), followed by PEG1 (26.7%). CONCLUSIONS: Modifications of the physico-chemical properties of PEG hydrogels and the addition of RGD influenced soft and hard tissue integration and biodegradation. PEG1 showed an increased degradation time and maintained the shape. The soft tissue integration was enhanced by adding an RGD sequence. A high turn-over rate and extensive bone regeneration was observed using PEG2. The addition of RGD further improved bone formation and soft tissue integration.

Stem cells for tooth engineering.

Tooth development results from sequential and reciprocal interactions between the oral epithelium and the underlying neural crest-derived mesenchyme. The generation of dental structures and/or entire teeth in the laboratory depends upon the manipulation of stem cells and requires a synergy of all cellular and molecular events that finally lead to the formation of tooth-specific hard tissues, dentin and enamel. Although mesenchymal stem cells from different origins have been extensively studied in their capacity to form dentin in vitro, information is not yet available concerning the use of epithelial stem cells. The odontogenic potential resides in the oral epithelium and thus epithelial stem cells are necessary for both the initiation of tooth formation and enamel matrix production. This review focuses on the different sources of stem cells that have been used for making teeth in vitro and their relative efficiency. Embryonic, post-natal or even adult stem cells were assessed and proved to possess an enormous regenerative potential, but their application in dental practice is still problematic and limited due to various parameters that are not yet under control such as the high risk of rejection, cell behaviour, long tooth eruption period, appropriate crown morphology and suitable colour. Nevertheless, the development of biological approaches for dental reconstruction using stem cells is promising and remains one of the greatest challenges in the dental field for the years to come.

Bone Morphogenetic Protein 2 Coordinates Early Tooth Mineralization.

Formation of highly organized dental hard tissues is a complex process involving sequential and ordered deposition of an extracellular scaffold, followed by its mineralization. Odontoblast and ameloblast differentiation involves reciprocal and sequential epithelial-mesenchymal interactions. Similar to early tooth development, various Bmps are expressed during this process, although their functions have not been explored in detail. Here, we investigated the role of odontoblast-derived Bmp2 for tooth mineralization using Bmp2 conditional knockout mice. In developing molars, Bmp2LacZ reporter mice revealed restricted expression of Bmp2 in early polarized and functional odontoblasts while it was not expressed in mature odontoblasts. Loss of Bmp2 in neural crest cells, which includes all dental mesenchyme, caused a delay in dentin and enamel deposition. Immunohistochemistry for nestin and dentin sialoprotein (Dsp) revealed polarization defects in odontoblasts, indicative of a role for Bmp2 in odontoblast organization. Surprisingly, pSmad1/5/8, an indicator of Bmp signaling, was predominantly reduced in ameloblasts, with reduced expression of amelogenin ( Amlx), ameloblastin ( Ambn), and matrix metalloproteinase ( Mmp20). Quantitative real-time polymerase chain reaction (RT-qPCR) analysis and immunohistochemistry showed that loss of Bmp2 resulted in increased expression of the Wnt antagonists dickkopf 1 ( Dkk1) in the epithelium and sclerostin ( Sost) in mesenchyme and epithelium. Odontoblasts showed reduced Wnt signaling, which is important for odontoblast differentiation, and a strong reduction in dentin sialophosphoprotein ( Dspp) but not collagen 1 a1 ( Col1a1) expression. Mature Bmp2-deficient teeth, which were obtained by transplanting tooth germs from Bmp2-deficient embryos under a kidney capsule, showed a dentinogenesis imperfecta type II-like appearance. Micro-computed tomography and scanning electron microscopy revealed reduced dentin and enamel thickness, indistinguishable primary and secondary dentin, and deposition of ectopic osteodentin. This establishes that Bmp2 provides an early temporal, nonredundant signal for directed and organized tooth mineralization.

Onset of human aging estimated from hazard functions associated with various causes of death.

In an attempt to estimate the onset of aging-associated mortality in humans, Swiss national survival and mortality data from 1978 to 1983 were analyzed. The nonparametric kernel method served to estimate gender-specific survival and hazard functions related to five major as well as all causes of death. On the basis of graphical models, it was hypothesized that the onset of aging conceivably was associated with prominent acceleration in mortality rates. The earliest maximal accelerations in hazard functions for most causes of death occurred during the second and again during the fourth age decade. Overall mortality rates of males and females exhibited prominent humps between the two periods of acceleration. These humps were accounted for largely by a high incidence of deaths from violence (accidents, suicide, and crime), which have to be attributed to environmental factors rather than to senescence. On the other hand, no plausible argument could be found against the assumption that maximal acceleration in death rates from ischemic heart and other circulatory diseases around 20 years of age was related to aging. Therefore, these data were interpreted to indicate that in the population examined, senescent mortality sets in around 20 years of age, about 5 years earlier in males than in females. However, when considering overall hazard rates, aging is hidden from view by mortality associated with environmental factors, which predominates up to ages of 30-35 years in both genders.

Light and electron microscopic morphology of the temporomandibular joint in growing and mature crab-eating monkeys (Macaca fascicularis): the condylar calcified cartilage.

In an attempt to show maturational alterations in the calcified cartilage, mandibular condyles of four growing and four adult male monkeys (Macaca fascicularis) were studied using light microscopy as well as transmission and scanning electron microscopy. All specimens were initially fixed by perfusion in the presence of ruthenium red. For examination of the hard tissue surfaces in the scanning electron microscope, uncalcified tissues were removed with sodium hypochlorite. In growing animals, almost the entire hard tissue surface in the joint region of the condyle was formed by calcified cartilage, while in adult animals, calcified cartilage was confined to load-bearing regions. In growing animals, the appearance of the calcified cartilage surface suggested a continuously advancing mineralizing front similar to that seen in the epiphyseal plate. Chondrocytes mostly exhibited a terminal stage of hypertrophy, and seemed to die and get lost through vascular invasion and subsequent endochondral ossification. In adult animals, most of the calcified cartilage surface appeared comparatively stable, and resembled the tidemark of articular cartilage. Chondrocytes were usually small and appeared viable. However, on the adult condyles, there were always circumscribed islands where chondrocytes and the pattern of mineralization resembled those seen in growing animals. In these regions, prominent chondroclastic activity indicated extensive articular remodelling. These observations suggest that at the end of somatic growth, condylar calcified cartilage undergoes considerable maturation from a type reminiscent of hyaline growth cartilage to a type resembling articular cartilage. Concomitantly, chondrocytes appear to change their developmental program, in that they stop enlarging and lose their commitment to death. However, they may be able to retain, or switch back to, a more immature stage, in case there is need for extensive articular remodelling.

Light and electron microscopic morphology of the temporomandibular joint in growing and mature crab-eating monkeys (Macaca fascicularis): the condylar articular layer.

In an attempt to establish maturational alterations in the morphology of the articular tissue layer, mandibular condyles of four immature and four mature male monkeys (Macaca fascicularis) were studied using light microscopy as well as scanning and transmission electron microscopy. Specimens were fixed in situ by perfusion in the presence of ruthenium red to stabilize proteoglycans. Preparations intended for observation in the scanning electron microscope were first dehydrated and sputtered for the examination of articular surfaces, and afterwards treated with trypsin to expose the spatial arrangement of collagen fibrils. Gross anatomical relations between joint components indicated that the anterior and central, but not the posterior region of the condylar articular surface can be subject to compressional load. Load-bearing and non-load-bearing regions differed with respect to the morphology of the articular layer. Load-bearing surfaces were covered by a prominent articular surface lamina similar to that observed on articular cartilage. This lamina seemed to constitute an integral part of the articular layer, distinct from the lining of synovial fluid, and to be composed largely of proteoglycans. It was unaffected by maturation. The subjacent, load-bearing articular layer differed markedly in structure, both from articular cartilage, and between immature and mature animals. Articular cells of immature animals were classified as fibroblastlike, but unlike typical fibroblasts, were surrounded by a thin, often incomplete halo of fibril-free pericellular matrix, presumably consisting of proteoglycans. In mature animals, articular cells closely resembled chondrocytes, but exhibited prominent nuclear fibrous laminae, which usually are found only in fibroblasts. Thus, the load-bearing part of the articular layer seems to undergo a maturation-dependent metaplastic conversion, from a dense connective tissue with some features of fibrocartilage, to a fibrocartilage-like tissue containing chondrocyte-like cells with some features of fibroblasts. This conversion might reflect an adaptation to a maturation-associated increase in articular stress.

Number and size-spectra of myelinated nerve fibers of human premolars.

The primary objective of this study was to determine the number and size of myelinated nerve fibers at the subcervical, midroot and juxta-apical levels of human premolars. Sixty-seven healthy premolars extracted from adolescents were utilized. Root-discs were prepared from the three sites and processed for light and electron microscopy. The myelinated nerve fibers were counted from semithin sections using a sampling microscope. The measurements were taken from composite electron micrographs using an electronic image processing unit. A total of 1883 myelinated axons from seven mandibular second premolars was gauged. The 67 teeth had an average of 312 +/- 149 myelinated nerve fibers at the juxta-apical level (range 18 to 728). The contra- and ipsilateral differences in means among the four groups of premolars were not significant (P > 0.05). The number of nerves increased significantly (P < 0.05) toward midroot and subcervical (P < 0.001) levels in all groups. The average neural diameter was 3.5 + 1.0 microns at the juxta-apical level, and the between-teeth difference in mean was found to be significant (P < 0.01). There was no decline (P > 0.05) in the diameter of myelinated nerve fibers toward midroot and subcervical levels.

Nature and attachment of cementum formed under guided conditions in human teeth. An electron microscopic study.

In an attempt at characterizing the nature and attachment of cementum formed under conditions of guided tissue regeneration (GTR) in humans, front teeth from 4 patients aged 42 to 72 years were examined at the electron microscopic level. All teeth were affected by complex periodontitis associated with advanced loss of periodontal support. Roots were surgically planed and notched, but not chemically conditioned. Either the mesial or distal surface of each tooth represented the experimental site and was covered with a biodegradable polyglactin 910 barrier, while the opposite approximal surface served as control. Following 3 months of healing, teeth were removed together with surrounding periodontal tissues including some alveolar bone. These blocks were fixed histologically, decalcified, embedded in epoxy, and sectioned for examination in the scanning (backscatter mode) and transmission electron microscope. Both experimental and control sites disclosed 2 types of regenerative cementum that seemed to be formed by cells resembling cementoblasts. The first type was characterized by a thin fringe of collagen fibrils which were arranged perpendicular to the root surface and appeared mineralized in a zone extending about 1 to 3 microm from the dentin. The second type occurred as thick patches which revealed scattered cementocytes and sheets of collagen fibrils oriented mainly parallel to the root surface, running both circularly and axially. In both situations, a continuous, thin, electrondense layer was interposed between newly formed cementum and preexisting radicular hard tissues. Interdigitation of collagen fibrils from cementum and dentin, such as observed along the natural cemento-dentinal junction, did not occur. Thus, regenerative cementum laid down in humans under guided conditions on previously diseased and planed, but not otherwise treated root surfaces shares some morphologic features with cementum formed during spontaneous repair of root resorptions. However, unlike in the course of such repair, a fibrous attachment of new cementum resembling the natural cemento-dentinal junction does not seem to be regenerated under guided conditions.

Growth direction in the mandibular condyle of pre-pubertal and pubertal monkeys (Macaca fascicularis) studied by morphometry and radioautography.

Eight male monkeys with prepubertal or pubertal skeletal maturity were used to assess age-related changes in anterior-posterior gradients of parameters which characterize growth velocity. The animals received single intravenous doses of 1 mCi/kg body weight of [3H]-proline at 24 h, and of 0.5 mCi/kg body weight [3H]-thymidine, 3 h before death. Four perichondral and chondral layers, and the subchondral zone of erosion of each condyle, were analysed morphometrically and radioautographically at 12 sampling sites distributed systematically over the joint surface. Values for cell density, cell proliferation, extracellular cartilaginous matrix production, cartilage resorption and subchondral bone formation produced characteristic curves which generally peaked in the central joint region. These peaks did not shift, either in relation to age or growth velocity, suggesting a uniform posterior-superior growth direction. Individual variability in both shape and prominence of the curves usually prevailed over the group effects attributable to skeletal maturity or growth velocity. Thus the gradual age-dependent shift in human condylar growth direction may be revealed only in longitudinal studies and would be masked by pronounced individual variation in cross-sectional studies of small samples.

Articular degeneration and remodeling in human temporomandibular joints with normal and abnormal disc position.

Temporomandibular joints with normal and abnormal disc positions, obtained from 15 females and 38 males (most between 15 and 50 years of age), were examined under a light microscope. Histologic changes attributed to degeneration and remodeling of the articular tissues were graded according to their prominence and extension along the articular surfaces. These changes seemed little affected by internal derangement in the condyle and the temporal component. In contrast, degeneration and regressive remodeling of the disc and, although to a much lesser degree, its attachments were more severe and increased more steeply with age in internally deranged as compared to normal joints. The present findings suggest that the disc indeed suffers from an abnormal position. However, this conclusion seems tenable only for adults, as rather prominent progressive remodeling changes have been observed in joints from adolescents, but no significant degenerative variations were noted.

Histometric study of synovial cavity dimensions of human temporomandibular joints with normal and anterior disc position.

To evaluate effects of internal derangement, the sagittal lengths of the condylar, temporal, and disc articular surfaces, as well as those of the disc attachments, were measured in histologic sections of human temporomandibular joints obtained at autopsy, mainly from adolescent, young adult, and middle-aged subjects. While the upper joint compartment appeared little affected, anterior disc position was significantly associated with comparatively long inferior disc attachments and a short condylar articular surface, indicative of possibly aberrant insertions of the attachments. Such discrepancies in size or alignment between condyle and disc complex could primarily reflect a constitutional deviation or result secondarily from remodeling.

Facial pattern and anterior apical base.

A cephalometric study finding significant association between variations in facial pattern and three measurements commonly used to evaluate apical base relationships (the horizontal distance between points A and B, and angles A-N-B and A-B/Occlusal plane).

Wall architecture and disc attachment of the human temporomandibular joint.

In an attempt to evaluate the potential behaviour of the temporomandibular joint wall during mandibular movements, joint specimens of 18 individuals, ranging in age from 15-45 years, were examined macroscopically and in the light microscope. The inner part of the joint wall was consistently formed by the superior and inferior lamina of the disc attachment. A distinct fibrous capsule existed in the posterior and lateral joint region, while anteriorly and medially, it could hardly be identified. The posterior attachment to the temporal bone of both the disc and capsule was clearly anterior to the squamotympanic and petrosquamosal fissure. A collagenous sheet inserting in and around these fissure was considered the deep lamina of the parotid-masseteric fascia. With reference to collagen arrangement, content in elastic fibres, and type of synovial lining, the postero-superior disc attachment appeared to be highly extensible, although not elastic enough to effectively pull the disc back upon posterior movements of the condyle. On the other hand, the latero-inferior disc attachment gave the impression of a rigid band well suited to drag the disc along with the condyle and still allowing condylar rotation. In addition, however, the postero-inferior disc attachment in combination with condylar rotation might also contribute effectively to the control of disc position on the moving condyle.

Morphological and labeling evidence supporting and extending a modern theory of tooth eruption.

As the interest in biological mechanisms of tooth eruption has recently been revived by a new eruption theory, the present study was an attempt to contribute new data to this problem. Four male Macaca fascicularis monkeys, two infant (about 13 months old) and two juvenile (about 44 months old), were labeled either by sequential fluorochrome or by single 3H-proline injections and then served for studying the bone apposition patterns around erupting premolars and molars. About 100 microns thick ground sections cut either in the mesiodistal or bucco-oral direction and the corresponding micrographs, microradiographs and autoradiographs, as well as fluorescence micrographs were used. In the multirooted teeth studied, bone apposition was most prominent and fast in the inter-radicular region, while at the fundus of the alveoli, bone apposition was slight or negligible. Around maxillary premolars and molars, bone apposition pointed in the mesial as well as in the axial direction. This was true for the intraosseous and the supraosseous phase of tooth eruption. Using these observations in addition to preliminary data calculated for the rates of bone apposition in the inter-radicular, apical and crestal regions, and for the rate of root elongation, the new eruption hypothesis could be extended. It is suggested that eruption of multirooted teeth, in the presence of corresponding coronal resorption, is entirely explained by forces generated through inter-radicular bone apposition and that their dental follicle is in a stimulating mode inter-radicularly but neutral apically at the bottom of the alveolar fundus.

Age estimation in an Indian population using pulp/tooth volume ratio of mandibular canines obtained from cone beam computed tomography.

The present study assessed the suitability of pulp/tooth volume ratio of mandibular canines for age prediction in an Indian population. Volumetric reconstruction of scanned images of mandibular canines from 140 individuals (aged ten - 70 years), using computed tomography was used to measure pulp and tooth volumes. Age calculated using a formula reported earlier for a Belgian sample, resulted in errors > ten years in almost 86% of the study population. The regression equation obtained for the Indian population: Age = 57.18 + (- 413.41 x pulp/tooth volume ratio), was applied to an independent control group (n = 48), and this resulted in mean absolute errors of 8.54 years which was significantly (p < 0.05) lower than those derived with the Belgian formula. The pulp/tooth volume ratio is a useful indicator of age, although correlations may vary in different populations and hence, specific formulae should be applied for the estimates.

Histological, ultrastructural and quantitative investigations on the response of healthy human pulps to experimental capping with mineral trioxide aggregate: a randomized controlled trial.

AIM: To investigate the pulpal response to direct pulp capping in healthy human teeth with mineral trioxide aggregate (MTA) as against calcium hydroxide cement (Dycal) as control. METHODOLOGY: Twenty healthy human third molars had iatrogenic pulpotomy and direct pulp capping with MTA. Another 13 teeth were capped with Dycal as controls. The teeth were restored, with IRM, clinically reviewed and extracted after a number of pre-determined intervals (1 week, 1 month and 3 months). The specimens were fixed, decalcified, subdivided axially into two halves in the oro-buccal (lingual-buccal) plane, embedded in plastic, serial sectioned and evaluated qualitatively and quantitatively by correlative light and transmission electron microscopy with appropriate statistical evaluation of the quantitative data. RESULTS: Iatrogenic pulpal wounds treated with MTA were mostly free from inflammation after 1 week and became covered with a compact, hard tissue barrier of steadily increasing length and thickness within 3 months following capping. Control teeth treated with Dycal revealed distinctly less consistent formation of a hard tissue barrier that had numerous tunnel defects. The presence of pulpal inflammation up to the longest observation period (3 months) after capping, was a common feature in Dycal specimens. CONCLUSIONS: The MTA was clinically easier to use as a direct pulp-capping agent and resulted in less pulpal inflammation and more predictable hard tissue barrier formation than Dycal. Therefore, MTA or equivalent products should be the material of choice for direct pulp capping procedures instead of hard setting calcium hydroxide cements.

Effect of sodium hypochlorite on human root dentine--mechanical, chemical and structural evaluation.

AIM: To investigate the mechanical, chemical and structural alterations of human root dentine following exposure to ascending sodium hypochlorite concentrations. METHODOLOGY: Three-point bending tests were carried out on standardized root dentine bars (n = 8 per group, sectioned from sound extracted human third molar teeth) to evaluate their flexural strength and modulus of elasticity after immersion in 5 mL of water (control), 1% NaOCl, 5% NaOCl or 9% NaOCl at 37 degrees C for 1 h. Additional dentine specimens were studied using microelemental analysis, light microscopy following bulk staining with basic fuchsin, and scanning electron microscopy (SEM). Numerical data were compared using one-way ANOVA. Bonferroni's correction was applied for multiple testing. RESULTS: Immersion in 1% NaOCl did not cause a significant drop in elastic modulus or flexural strength values in comparison to water, whilst immersion in 5% and 9% hypochlorite reduced these values by half (P < 0.05). Both, carbon and nitrogen contents of the specimens were significantly (P < 0.05) reduced by 5% and 9% NaOCl, whilst 1% NaOCl had no such effect. Exposure to 5% NaOCl rendered the superficial 80-100 mum of the intertubular dentine permeable to basic fuchsin. Three-dimensional SEM reconstructions of partly demineralized specimens showed NaOCl concentration-dependent matrix deterioration. Backscattered electron micrographs revealed that hypochlorite at any of the tested concentrations left the inorganic dentine components intact. CONCLUSIONS: The current data link the concentration-dependent hypochlorite effect on the mechanical dentine properties with the dissolution of organic dentine components.

Tubular sclerosis rather than the smear layer impedes dye penetration into the dentine of endodontically instrumented root canals.

AIM: To evaluate the effect of different root canal irrigating regimes on dentine penetration of Patent Blue dye. METHODOLOGY: Eighty extracted single-rooted human mandibular premolar teeth with narrow root canals were prepared using ProFile instruments. After each instrument, canals were irrigated with 1% sodium hypochlorite. Subsequently, teeth were randomly assigned to receive a 10 mL rinse of aqueous 17% (w/v) ethylenediaminetetraacetic acid or tap water for 2 or 10 min, followed by a final rinse with a 2% Patent Blue dye solution for 2 or 10 min (eight groups, n = 10 teeth per group). Teeth were then horizontally sectioned 3, 6 and 9 mm from the apex. Sections were digitally photographed and dye penetration was calculated as percentage of total dentine area using NIH Image J. Values were compared using one-way anova and Bonferroni correction with the alpha-type error set at <0.05. Representative tooth sections from all groups were further analysed using scanning electron microscopy. RESULTS: No significant impact of irrigating protocols on dye penetration was found. Dye penetration was significantly (P < 0.001) greater in the coronal than middle, and in middle than in apical root thirds. When observed microscopically, irrigant penetration was independent of the presence of a smear layer, but was rather a function of tubular sclerosis. CONCLUSIONS: Tubular sclerosis, a physiological phenomenon that starts in the third decade of life in the apical root region and advances coronally with age, was the main factor influencing penetrability of root dentine.