Quantitative observation on teeth during calcium deprivation in rats.

Adult female rats were subjected to severe calcium deprivation by feeding them a calcium-deficient diet containing oxalate (Group A). Furthermore, pregnant and lactating animals were subjected to the same diet (Group B). The mandibular teeth were collected for ash determination and for linear measurements. The ash content of the growing incisors was only slightly reduced in Group A, while the reduction in Group B was 27%. Linear measurements demonstrated that the outer dimensions of the incisors were not affected by the calcium-depriving regimen in either group, while the inner dimensions (pulp cavity) were considerably increased in Group B due to reduced thickness of the dentin mantle. The daily dentin apposition in Group A did not deviate much from the control values, while in Group B it was severely reduced. The thickness of incisor enamel was unaffected by the calcium-depriving regimen. The ash content of the molars was normal in Group A, but slightly reduced in Group B.     

Quantitative observation on teeth during calcium deprivation in rats

abstract – Adult female rats were subjected to severe calcium deprivation by Feeding them a calcium-deficient diet containing oxalate (Group A). Furthermore, pregnant and lactating animals were subjected to the same diet (Group B). The mandibular teeth were collected for ash determination and for linear measurements. The ash content of the growing incisors was only slightly reduced in Group A, while the reduction in Group B was 27%. Linear measurements demonstrated that the outer dimensions of the incisors were not affected by the calcium-depriving regimen in either group, while the inner dimensions (pulp cavity) were considerably increased in Group B due to reduced thickness of the dentin mantle. The daily dentin apposition in Group A did not deviate much from the control values, while in Group B it was severely reduced. The thickness of incisor enamel was unaffected by the calcium-depriving regimen. The ash content of the molars was normal in Group A, but slightly reduced in Group B.     

A morphometric and quantitative microradiographic study of dental tissues in the hypopituitary dwarf mouse

In transverse sections the areas of enamel, dentine and pulp were determined using image analysis morphometry, and the mineralization levels of enamel, dentine and alveolar bone using quantitative microradiography and a microphotometric-microdensitometric technique. There were significant differences in the ratios of dentine to pulp area, and in the enamel to total area in the incisors from dwarf and normal animals. The mean dentine to pulp ratio for dwarf incisors was more than six times greater than in normal incisors. There was also a significantly greater variation in the dentine to pulp ratio in dwarfs, which was attributed to their greater variation in pulp size. Values for mineral density of enamel, dentine and bone were similar to those found by other investigators. For example, for normal specimens, the mean mineral density of outer enamel was 2.84 g/cm3 (SD = 0.38), percentage mineralization 92%; outer labial dentine was 1.64 g/cm3 (SD = 0.15), percentage mineralization 52%; and alveolar bone was 1.53 g/cm3 (SD = 0.13), percentage mineralization 49%. The variation in mineralization within each tissue confirmed previous work in rodents. There was a consistent trend for mineralization levels of all hard tissues in the dwarf sample to be less than in the normal sample but the difference was not statistically significant for any one tissue.     

Effect of fluoride and gonadal steroid deficiency on enamel and dentin mineralization of female rats

The aim of this study was to verify the influence of estrogen deficiency on enamel and dentin microhardness of female rats' incisors, as well as the influence of chronic administration of fluoride on that response. Sham or OVX rats drank distilled water or distilled water with 10 ppm fluoride as NaF, for 90 days. The upper incisors were removed and the specimens submitted to microhardness analysis of enamel and dentin. The data were compared by variance analysis and Tukey's test (p<0.05). There was a significant reduction in the mineralization of crown (7.9% and 8.1%) and root (20.4% and 25.0%) in both groups treated with fluoride or distilled water, respectively and an increase (14.2%) in the mineralization of coronal dentin after ovariectomy. Fluoride treatment did not cause reduction in enamel mineralization; however, it reduced dentin mineralization following ovariectomy. It was possible to conclude that estrogens directly or indirectly influence the process of enamel and dentin mineralization of female rats' incisors. The results also indicate that enamel and dentin exhibit different responses to fluoride administration.     

Different enamel and dentin mineralization observed in VDR deficient mouse model

Vitamin D plays an important role in the bone mineralization process. Enamel and dentin are two mineralized tissues of different origins that combine to form teeth, but the mechanism by which vitamin D regulates these tissues remains unclear. We hypothesized that vitamin D affects enamel and dentin mineralization through different mechanisms.

Objective

To examine enamel and dentin mineralization in a vitamin D receptor (VDR) deficient mouse model by micro-computerized tomography (micro-CT) and scanning electronic microscopy (SEM).

Methods

VDR wild type mice (VDR+/+) and VDR deficient (VDR−/−) littermates were sacrificed at 70.5 days old, and their mandibles were dissected. Micro-CT was used to compare mineral density (MD) of enamel and dentin of the two groups at different levels along the axis of mandibular incisors. SEM was employed to examine the ultrastructure of incisors at the levels corresponding to the levels used for the micro-CT studies. Furthermore, an accelerated eruption procedure was performed to exclude the effect of delayed eruption on enamel and dentin mineralization.

Results

Different distribution patterns of enamel and dentin MD were observed between VDR+/+ and VDR−/− groups. Early enamel maturation, mineralization, and hypomineralization in dentin were observed in the VDR deficient mice.

Conclusion

Vitamin D may affect the mineralization of dentin systemically, and enamel mineralization may be regulated locally.     

Formation of the dentino–enamel interface in enamelysin (MMP-20)-deficient mouse incisors

An anomalous dentino–enamel junction (DEJ), manifested by delamination of the enamel layer, was reported in enamelysin [matrix metalloproteinase-20 (MMP-20)] knockout (KO) mice. To better understand the possible role of MMP-20 in the formation of the DEJ, we performed transmission electron microscopy (TEM) studies of the DEJ at early stages of tooth morphogenesis in KO mice. Our TEM analysis revealed that in the incisors from KO mice the mantle dentin is hypomineralized at the onset of enamel mineralization. At this early stage, TEM revealed no apparent differences in nascent aprismatic enamel between the KO mice and the controls. Hypomineralized mantle dentin was also observed in the incisors from KO mice, as assessed by back-scattered SEM at the secretory and early maturation stages, but not in the late-maturation stage, suggesting that the mineralization of mantle dentin is not completely arrested, but rather postponed. Histological studies indicate that the organic content in the initial enamel layer remains very high throughout amelogenesis. These results imply that MMP-20 is involved in the regulation of mineralization in mantle dentin and demonstrate the complex nature of DEJ formation. They also suggest that the structural and functional properties of the DEJ are determined during the initial mineralization stages.     

Matrix Gla protein inhibition of tooth mineralization

Extracellular matrix (ECM) mineralization is regulated by mineral ion availability, proteins, and other molecular determinants. To investigate protein regulation of mineralization in tooth dentin and cementum, and in alveolar bone, we expressed matrix Gla protein (MGP) ectopically in bones and teeth in mice, using an osteoblast/odontoblast-specific 2.3-kb Col1a1 promoter. Mandibles were analyzed by radiography, micro-computed tomography, light microscopy, histomorphometry, and transmission electron microscopy. While bone and tooth ECMs were established in the Col1a1-Mgp mice, extensive hypomineralization was observed, with values of unmineralized ECM from four- to eight-fold higher in dentin and alveolar bone when compared with that in wild-type tissues. Mineralization was virtually absent in tooth root dentin and cellular cementum, while crown dentin showed "breakthrough" areas of mineralization. Acellular cementum was lacking in Col1a1-Mgp teeth, and unmineralized osteodentin formed within the pulp. These results strengthen the view that bone and tooth mineralization is critically regulated by mineralization inhibitors.     

Enamel mineralization in the absence of maturation stage ameloblasts

The role of maturation stage ameloblasts is not clear yet. The aim of this study was to verify to which extent enamel mineralizes in the absence of these cells. Maturation stage ameloblasts and adjacent dental follicle cells from rat lower incisors were surgically removed and the limits of this removal were marked by notches made in the enamel. Histological analysis confirmed that the ameloblasts had been removed within the limits of the notches. The teeth erupted and when the notches appeared in the mouth, the enamel in the experimental teeth was hard but whitish compared to the yellowish colour of the contralateral incisors used as control. SEM images revealed similar enamel rod arrangement in both groups. Decreased mineral content was observed in some specimens by polarized light microscopy, and microhardness values were much lower in the experimental teeth. FTIR analysis showed that higher amounts of protein were found in most experimental teeth, compared with the control teeth. Enamel proteins could not be resolved on 15% SDS-PAGE gels, suggesting that most of them were below 5 kDa. These results suggest that the enamel matured in the absence of ameloblasts has increased protein content and a much lower mineral content, suggesting that maturation stage ameloblasts are essential for proper enamel mineralization.     

Effect of polymers used in saliva substitutes on demineralized bovine enamel and dentin

PURPOSE: To evaluate the effects of porcine mucin- and carboxymethylcellulose-based (CMC) solutions on the inhibition of demineralization of pre-demineralized bovine enamel and dentin in vitro. Additionally the sugar substitutes sorbit or xylit were added to the various solutions. METHODS: 54 enamel and dentin samples were prepared from 14 freshly extracted permanent bovine central incisors. The samples were demineralized (pH 5.0 for enamel and pH 5.5 for dentin; 14 days) and subsequently exposed to either mucin- or CMC-based solutions combined with xylit and sorbit at pH 5.5. After in vitro exposure, the specimens were cut perpendicular to the enamel/dentin surface and the sections were ground (4000 grit) to a uniform thickness (100 microm). Mineral loss and lesion depths were evaluated from microradiographs with a dedicated software package (TMR 2.0.27.2). RESULTS: For both dental hard tissues, storage in the mucin-containing solutions resulted in significantly higher lesion depths (deltaLD), but lower mineral loss values (deltadeltaZ) compared to the CMC-containing solutions (P< 0.05; 2-way ANOVA). For the dentin specimens, significant differences in deltadeltaZ were observed between sorbit and xylit (P< 0.05; 2-way ANOVA). The mineral loss and lesion depths of the enamel specimens did not differ significantly after storage in the various solutions (P> 0.05; Bonferroni post hoc test). For the dentin specimens stored in the solution containing mucin/xylit the mineral loss was significantly decreased compared to the other mucin groups as well as to the CMC/xylit solution (P< 0.05; Bonferroni post hoc test).     

Root development in mice lacking functional tissue non-specific alkaline phosphatase gene: inhibition of acellular cementum formation.

Tissue non-specific alkaline phosphatase (TNAP) is richly present in developing teeth including the cells of the periodontal ligament. Here, we investigated tooth and root development in mice lacking the TNAP gene. Heterozygous mutants were obtained from The Jackson Laboratory, Animal Resources (Bar Harbor, ME, USA) and bred. TNAP-deficient mice and their littermates were killed from 6 to 25 days after birth and their molar blocks processed for light and electron microscopy. It was observed that the eruption of the incisors into the oral cavity was delayed for 2 to 3 days. Also, the onset of mineralization of the mantle dentin in the roots of the developing molars was delayed for 2 to 3 days. Yet, dentin and enamel formation in the homozygous mutants showed a more or less normal pattern, with the exception of localized enamel hypoplasias. The most conspicuous finding was the defective formation of acellular cementum along the molar roots. Instead of a continuous layer, the cementum was deposited as very thin and irregularly shaped patches around the bases of the periodontal ligament fibers. Sharpey's fibers were short and poorly developed. In contrast, the development of the alveolar bone, the periodontal ligament, and the cellular cementum was seemingly unaffected. It is concluded that TNAP represents an essential factor in mantle dentin mineralization and in the formation of acellular cementum.     

Clinical, histologic and microradiographic study of natal, neonatal and pre-erupted teeth

abstract — A clinical, histologic and microradiographic investigation of 62 natal, neonatal and otherwise pre-erupted teeth from 51 children is presented. Such a comprehensive material has not previously been reviewed. Of the 62 teeth, 59 were mandibular incisors. A hereditary factor could be demonstrated in 12 cases. Fifty-three teeth showed more or less pronounced enamel hypoplasia. The enamel had a normal prism structure and mineralization with the exception of 22 cases where the prism structure was absent in the cervical part of the enamel. The cervical and apical dentin was atubular and in developing teeth the dentin in these regions changed to an irregularly formed hard tissue of osteodentin character, in which enclosed cells could be observed. Developing teeth often had no cementum, and in those cases where acellular cementum could be observed it was thinner than normal. In four patients atypically located carious lesions occurred.     

Clinical, histologic and micro-radiographic study of natal, neonatal and pre-erupted teeth.

A clinical, histologic and microradiographic investigation of 62 natal, neonatal and otherwise pre-erupted teeth from 51 children is presented. Such a comprehensive material has not previously been reviewed. Of the 62 teeth, 59 were mandibular incisors. A hereditary factor could be demonstrated in 12 cases. Fifty-three teeth showed more or less pronounced enamel hypoplasia. The enamel had a normal prism structure and mineralization with the exception of 22 cases where the prism structure was absent in the cervical part of the enamel. The cervical and apical dentin was atubular and in developing teeth the dentin in these regions changed to an irregularly formed hard tissue of osteodentin character, in which enclosed cells could be observed. Developing teeth often had no cementum, and in those cases where acellular cementum could be observed it was thinner than normal. In four patients atypically located carious lesions occurred.     

Influence of post placement in the fracture resistance of endodontically treated incisors veneered with direct composite

STATEMENT OF PROBLEM: Veneer preparations are considered to weaken endodontically treated maxillary incisors. Prefabricated posts have been controversially indicated to reinforce endodontically treated teeth before final restoration. PURPOSE: This in vitro study evaluated whether (1) veneer preparation in enamel or in enamel/dentin weakens endodontically treated maxillary incisors, (2) bonding of direct composite veneer restores the original strength of the unprepared teeth, and (3) use of prefabricated metal posts increases fracture resistance of prepared and restored teeth. MATERIAL AND METHODS: Ninety extracted human maxillary central incisors were submitted to conventional root canal treatment. Specimens were randomly divided into 8 experimental groups (veneer preparation in enamel or dentin with/without post and with/without direct composite veneer restoration) and a control group (n = 10). Specimens were loaded to fracture, and the data were analyzed statistically. RESULTS: Statistical analysis revealed that a conservative veneer preparation does not significantly reduce maxillary incisors' fracture resistance. For prepared incisors, bonding of direct composite veneer restored their original strength, and the use of posts did not increase their fracture resistance. CONCLUSION: Conservative veneer preparations involving enamel and enamel/dentin did not significantly reduce the fracture resistance of endodontically treated maxillary incisors. In addition, restoration of the intraenamel preparations with direct composite resulted in teeth more resistant to fracture than teeth having restorations in dentin. The use of posts did not improve fracture resistance of endodontically treated maxillary incisors reduced and veneered with direct composite.     

Maternal diabetes and changes in the hard tissues of primary teeth. III. A histologic and microradiographic study.

According to clinical studies, infants of diabetic mothers (IDM) seem to run a higher risk of mineralization disturbances in the enamel than normal healthy infants. In order to evaluate this observation at the histologic level, exfoliated primary incisors were collected from 74 IDM, representing two types of care during gestation and the perinatal period, and from 52 healthy children. The width of the neonatal line was evaluated in the enamel and the dentin by study of bucco-lingual undemineralized sections. Pre- and postnatal enamel was classified according to discolorations, deviations of prisms and hypoplasia. Microradiographic studies included measurements of the neonatal lines and classification of the degree of mineralization in the pre- and postnatal dental hard tissues. Irrespective of treatment, the IDM showed a significantly higher incidence of widened neonatal lines and also an increased frequency of postnatal disturbances compared to the controls. The observations are discussed against the background of the neonatal hypocalcemia reported in IDM.     

Relationships between dentin and enamel mineral at the dentino-enamel boundary: electron tomography and high-resolution transmission electron microscopy study

To better understand the nature of the relationships between mineral phases at the dentino-enamel boundary (DEB), we performed electron tomography (ET) and high-resolution transmission electron microscopy (HR-TEM) of the apical portions of rat incisors. The ET studies of the DEB at the secretory stage of amelogenesis revealed that nascent enamel crystals are co-aligned and closely associated with dentin crystallites in the mineralized von Korff fibers, with the distances between dentin and enamel crystals in the nanometer range. We have further studied the relationships between dentin and enamel crystals using HR-TEM lattice imaging of the DEB. Among dozens of high-resolution micrographs taken from the DEB we were able to identify only one case of lattice continuity between dentin and enamel crystals, indicating direct epitaxy. In other cases, although there was no direct continuity between the crystalline lattices, power spectra analysis of lattice images revealed a very high level of co-alignment between dentin and enamel crystals. Hence, we propose here that the high degree of alignment and integration between dentin and enamel mineral can be established either by epitaxy or without direct interactions between crystalline lattices, probably via regulation of mineral formation and organization by integrated organic matrices of dentin and enamel at the DEB.     

Relationships between protein and mineral during enamel development in normal and genetically altered mice

The purpose of this study was to quantify and compare the amounts of volatiles (mostly protein) and mineral present in developing incisor enamel in normal mice and in those genetically engineered for absence of intact enamelin, ameloblastin, matrix metalloproteinase 20 (MMP20) or kallikrein-related peptidase 4 (KLK4). Data indicated that all mice showed peaks in the gross weight of volatiles and a similar weight of mineral at locations on incisors normally associated with early maturation. Thereafter, the content of volatiles on normal incisors declined rapidly by as much as 62%, but not by 100%, over 2 mm, accompanied by increases of ≈ threefold in mineral weights. Enamelin heterozygous mice (lower incisors) showed a decrease in volatile content across the maturation stage, yet mineral failed to increase significantly. Mmp20 null mice showed no significant loss of volatiles from maturing enamel, yet the amount of mineral increased. Klk4 null mice showed normal mineral acquisition up to early maturation, but the input of new volatiles in mid to late maturation caused the final mineralization to slow below normal levels. These results suggest that it is not only the amount of protein but also the nature or type of protein or fragments present in the local crystallite environment that affects their volumetric expansion as they mature.     

The masticatory system under varying functional load. Part 2: Effect of reduced masticatory load on the degree and distribution of mineralization in the rabbit mandible

A reduction in mechanical loading of the mandible brought about by mastication of soft food is assumed to decrease the remodelling rate of bone, which, in turn, might increase the degree of bone mineralization. The effect of a reduction in masticatory functional load on the degree and distribution of mineralization of mandibular bone was investigated in male juvenile New Zealand White rabbits. The experimental animals (n=8) had been raised on a diet of soft pellets from 8 to 20 weeks of age, while the controls (n=8) had been fed pellets of normal hardness. The degree of mineralization of bone (DMB) was assessed at the attachment sites of various jaw muscles, the condylar head, and the alveolar process. Differences between groups and among sites were tested for statistical significance using a Student's t-test and one-way analysis of variance, respectively. The DMB did not differ significantly between the experimental and control animals at any of the sites assessed. However, in the rabbits that had been fed soft pellets, both cortical bone at the attachment sites of the temporalis and digastric muscles and cortical bone in the alveolar process had a significantly higher DMB than cortical bone at the attachment site of the masseter muscle, while there were no significant differences among these sites in the control animals. The results suggest that a moderate reduction in masticatory functional load does not significantly affect the remodelling rate and the DMB in areas of the mandible that are loaded during mastication but might induce a more heterogeneous mineral distribution.     

Effect of hypocalcemic state on enamel formation in rat maxillary incisors

Several authors have proposed that hypocalcemia can interfere with amelogenesis, resulting in enamel aberrations. Therefore, the purpose of this investigation was to study the effects of a diet-induced hypocalcemic state in young rats on enamel formation of the maxillary incisors. The experimental rats were fed a special diet, free from vitamin D and very low in calcium. The control rats were fed a normal diet. The experimental period was 3 wk. After termination, the blood analysis showed that the experimental rats had developed hypocalcemia with very low values of both total and ionized blood calcium. The experimental rats were smaller than their controls after 3 wk, with smaller skulls and teeth. At the light microscope level, the enamel and the ameloblasts did not seem to be affected, except in one rat, the smallest, which showed enamel hypoplasias in both maxillary incisors and a delayed increase of the mineral content during the maturation stage process. It is concluded that the hypocalcemic state induced did not greatly affect enamel formation. However, occasional enamel aberrations may occur.     

Effect of single-dose amoxicillin on rat incisor odontogenesis: a morphological study

The effect of exposure to amoxicillin on tooth development remains to be elucidated. The purpose of this study was to investigate the effect of amoxicillin on rat incisor odontogenesis. Male Wistar rats weighing approximately 100?g were given a single intraperitoneal injection of 3.0?g/kg body weight amoxicillin. One week after injection, the rats were fixed, and the lower incisors were demineralized and prepared into paraffin sections for light microscopy (LM) and immunohistochemistry. Undemineralized samples were embedded in resin and ground for processing for contact microradiography (CMR) and scanning electron microscopy (SEM). Serum calcium, phosphate, and magnesium concentrations were measured. At 1?week after amoxicillin administration, LM, CMR, and SEM revealed a clear increase in the area of interglobular dentin, representing disruption of mineralization by odontoblasts. Immunohistochemistry demonstrated moderate levels of the small integrin-binding ligand N-linked glycoprotein family dentin matrix protein 1 in large areas of interglobular dentin. On the other hand, no morphological alteration or hypomineralization was observed in the enamel. Serum calcium values showed no significant differences between the control and experimental rats during the experimental period although both serum phosphate and magnesium levels increased at day?1 after amoxicillin injection. The results suggest that a single dose of amoxicillin specifically affects normal tooth dentin mineralization, but not enamel mineralization in rat incisor odontogenesis. The present results further our understanding of the clinical association between dentin abnormality and amoxicillin exposure during tooth development.     

Alveolar ridge augmentation in rats by combined hydroxylapatite and osteoinductive material.

The purpose of this study was to examine if increased bony ingrowth developed when hydroxylapatite was implanted together with an osteoinductive substrate. Dense hydroxylapatite granules (HA) (Calcitite, Calcitek, San Diego, CA, USA) were mixed with equal volumes of allogenic, demineralized, lyophilized dentin or bone and implanted subperiosteally for alveolar ridge augmentation purposes and heterotopically in the abdominal muscles in rats. Light microscopic evaluation revealed that HA was surrounded by fibrous connective tissue containing foreign body giant cells and it had neither an osteoinductive nor an osteoconductive effect. The newly formed bone induced from the implanted allogenic, demineralized, lyophilized dentin or bone was never found in close contact with the HA and did not incorporate the implant.