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In the serum-free culture medium of bovine odontoblasts we detected active gelatinolytic metalloproteinases, matrix metalloproteinase (MMP)-2 and MMP-9 (gelatinases A and B). The activity of MMP-2, in particular, appeared suddenly around day 21 in the culture, coinciding with the development of odontoblastic cell processes and the loss of alkaline phosphatase. Reverse transcriptase-polymerase chain reaction analysis of these odontoblasts demonstrated that messages of MMP-2 but not MMP-9 increased significantly between day 15 and day 21. The in vitro observation indicates that medium conditioned by these odontoblasts and containing significant amounts of MMP-2 degrades not only the collagenous substrates but also purified dentin phosphophoryn as well. We have also observed that dephosphorylated dentin phosphoprotein becomes a better substrate for casein kinase II after limited proteolysis with MMP-2. These results support our working hypothesis that MMP-2-mediated proteolytic processing is an important step in accelerating the process of dentin matrix maturation, which includes phosphorylation and subsequent mineralization. As has been suggested previously, extracellular phosphorylation of matrix proteins is an important step in biomineralization both in bone and in dentin (Mikuni-Takagaki et al., J Bone Miner Res 1995;10:231-42; Zhu et al., Biochem J 1997; 323:637-43). Our present histochemical analysis in MMP-2 knockout mice confirms the concept with the delayed formation of mineralized tissues, dentin, and bone.

Integrin adhesion and signaling events may contribute to the progressive differentiation of the osteoblast and to the initiation of a mineralized matrix. The purpose of our study was to begin to analyze the role of integrin receptors, in particular alpha2beta1, alpha5beta1, and alphaVbeta3, regarding mediation of the initiation of a mineralized matrix. Integrin-perturbation assays were conducted in microdot cultures of UMR-106-01 Bone Sialoprotein (BSP) osteoblast cells. For phenotypic analysis, we performed bright-field microscopy and Aliziran Red S staining to analyze effects on mineralization initiation. Mineralization was reduced significantly (P < 0.001) following the addition of alpha5- or beta1-integrin subunit antibody by approximately 20% and 45%, respectively--alphaVbeta3 integrin by nearly 65%, and alpha2beta1 integrin by nearly 95%. This effect was reversible following the removal of the antiintegrin antibody. These results suggest that integrin adhesion and signaling events may contribute to the ability of this cell line to mediate the initiation of the mineralization phenotype biologically.

Considerable knowledge has accumulated over the years on the structure and function of the dental pulp and dentin. Some of this knowledge has important clinical implications. This review, which is the first of seven articles, will be limited to those parts of the normal structure and physiology of the pulp and dentin that have been shown to result in, or are likely lead to, tissue reactions associated with the clinical treatment of these tissues. Although certain normal structures will be highlighted in some detail, a basic knowledge of pulpal and dentinal development and structure is a prerequisite for an understanding of this text.

In a study from Finland in 1986 it was shown that celiac disease was often associated with tooth enamel defects of permanent teeth. This study also showed a strong association between the time of gluten challenge in the diagnostic procedure and enamel defects. In the current study, dental examinations were carried out for a group of 40 children and adolescents suffering from celiac disease diagnosed according to the criteria of The European Society of Paediatric Gastroenterology and Nutrition (ESPGAN) at the Department of Paediatrics, Hospital of Uddevalla, Sweden. A control group made of 40 healthy children of the same age, sex and living area was examined in the same way. The results failed to show disturbances of the same type, degree of severity or frequency as was reported in Finland and no statistically significant differences concerning enamel defects were found between the patients with celiac disease and the controls.

Purpose: The purpose of this study was to observe developing teeth in a lengthened mandible after distraction., Material: Ten mongrel dogs with deciduous dentitions were used., Methods: A corticotomy was carefully made around a tooth bud and the external distractor (Orthofix M-100) was connected. After a 5-day latent period, distraction was started at a rate of 0.75 mm per day for 10 consecutive days. Then, the lower jaw was stabilized by an external fixation to allow ossification. While the operation was performed on the left side (Distraction group), the contralateral side was studied for comparison (Control). In addition, a corticotomy, artificial fracture and external fixation were carried out to confirm the influence of the operation (Fracture group). Then macroscopic, radiographic and histological evaluations were carried out., Results: In the Distraction group, the space between the wall of the dental follicle and the crown expanded as distraction began. The end of the calcified root became wider and irregular during the distraction period, and finally, the apex closed. In the Fracture group, the teeth erupted although slight alterations of the root shape were observed in association with the operation period., Conclusion: The root became irregular, but the teeth erupted within the distraction area.

Fourier transform infrared microspectroscopy (FTIRM) was used to investigate the organic and mineral phases of a calcified tissue (dentin) as a function of its location from predentin toward enamel. Thin dentin slices (decalcified or not) were fixed in formaldehyde and embedded in glycolmethylmethacrylate (GMA). Fixation did not denature collagen, and GMA did not interact with organic or mineral constituents of dentin. The v1v3 PO4 domain was studied in particular in order to estimate mineral maturity and amide I, II, A, and B to obtain data on protein conformation. The results showed that dentin apatite became increasingly mature (stoichiometric) from the mineralization front toward the enamel, especially through loss of HPO4(2-) groups and vacancies. Moreover, collagen fibrils became less and less hydrated, suggesting that intrafibrillar mineralization partially dehydrated the collagen. Combined study of the organic and mineral fractions of calcified tissues may help clarify their relationships in physiological and pathological tissues.

The predominant proteoglycans present in predentin and dentin are the chondroitin-sulphate-rich decorin and biglycan and the keratan-sulphate-rich lumican and fibromodulin. These are small, interstitial, leucine-rich proteoglycans which have recently been shown to exist in gradients across the predentin. Antibodies recognizing chondroitin sulphate show a decreasing gradient from the pulpal aspect toward the mineralizing front, the converse being true for keratan sulphate. Antidecorin shows an increase toward the mineralization front. Evidence from biochemical, autoradiographic, and immunohistochemical studies implies that such changes may be brought about by gradients of metalloproteinases. This offers the possibility that the proteoglycans organize the collagen network for receipt of phosphoproteins and phospholipids, the former being evident only at the onset of dentin formation. The suggestion is raised that glycosaminoglycan-depleted leucine-rich protein cores act as sequester points for receipt of phosphoproteins in particular. The rigid, spatially oriented glycosaminoglycan chains on decorin and biglycan are known to bind calcium and may feature directly in mineral initiation.

In vitro studies on interactions between amelogenins and calcium phosphate crystals are critical for elucidating biomineralization mechanisms of tooth enamel. This work was aimed at investigating the effects of native porcine amelogenins on octacalcium phosphate (OCP) crystal growth in a gelatin gel. We prepared OCP mineral discs by circulating calcium and phosphate solutions on the opposite ends of the gels loaded with 0-2% amelogenin for one week. A dose-dependent modulation of OCP crystal habit by amelogenins was observed by scanning electron microscopy. While the incorporation of 0.125, 0.25, or 0.5% amelogenins showed no significant effect on the crystal morphology, in the presence of 1 and 2% amelogenins, the crystals were remarkably longer, having an average aspect ratio 3-5 times greater than that of those formed in the control gels. Transmission electron microscopy and atomic force microscopy suggested that amelogenin assemblies selectively blocked b-axial development, resulting in the c-axial elongation of OCP crystals.

This early development was studied from a serial histological sections of eight elephant embryos with masses varying between 1 and 240 g. The tush and the tusk develop from one tooth germ in a deciduous to permanent tooth relation. In order to study the mineralization of the dental organ of the tush and cap and bell stage of the tusk, embryos older than 3-months' gestation (weighing more than 250 g) would be required.

Although type X collagen is one of the key molecules in endochondral ossification, no data are available on whether it is present in dental structures when mineralization is proceeding. We therefore monitored the appearance of type X collagen in tooth germs of human samples ranging in gestational age from 17-week-old fetuses to 9-week-old newborn. Using immunohistochemistry, ELISA techniques, and Western blotting, we show that type X collagen is present in human tooth germ during enamel maturation. Intense immunohistochemical staining for collagen type X was observed in the enamel and in the apical parts of secretory ameloblast at the bell stage when the dentine and enamel matrix were already under formation. The odontoblasts, the dentine, and the pulp were not stained. In the early (9-week) postnatal stage, the staining for collagen type X in the enamel matrix was diminished, and only a very weak signal could be detected in the secretory ameloblasts. A positive reaction for collagen type X was also observed in ELISA assay of extracts obtained from human embryonic enamel and hypertrophic cartilage samples. The Western blot analysis of the enamel demonstrated that size of the molecule detected by MoAb X53 is characteristic of the type X collagen. This correlates well with our immunohistochemical findings. Based on these data, we propose that type X collagen is one of the candidate molecules present in the enamel matrix that might be involved in mineralization of the enamel.

We tested the capacity of cementum attachment protein (CAP) to recruit putative cementoblastic populations to root surfaces in vitro by determining the phenotypic expression of periodontal ligament cloned cell populations. The clones were derived from cells that attached to either CAP-coated (experimental) or uncoated (control) root slices. Root slices were co-cultured with primary human periodontal ligament cells. Cloned and parent populations were analyzed for their capacity to express alkaline phosphatase (AP), osteopontin, bone sialoprotein (BSP), and CAP and to form mineralized tissue in vitro. The percentage of CAP- and BSP-positive clones was significantly higher in the experimental clones than in the controls. The percentage of cells positive for AP, BSP, and CAP was higher in the experimental clones than in their control counterparts. Mineralized tissue formation was observed only in the cell populations derived from the CAP-coated root slices. These results indicate that CAP is capable of recruiting putative cementoblastic populations on root slices in vitro and therefore might play an important role in cementogenesis during periodontal homeostasis and wound healing.

An etiologic connection between palatally ectopic canines and small and missing teeth is well established in the literature. Additionally, it has been observed that patients with palatally ectopic canines have a delayed dental development. The present study was designed to examine the validity of this latter observation. We radiographically assessed the subjects' dental ages using criteria of tooth calcification, rather than tooth eruption pattern. A similar determination was made in relation to subjects in whom buccally ectopic canines were present. The experimental group consisted of panoramic radiographs of 55 consecutively treated patients with palatally displaced maxillary canines and of 47 consecutively treated patients with buccally displaced canines. They were compared with a control group of 57 consecutively treated patients with normally placed canines. Approximately half the subjects with palatal displacement exhibited a late-developing dentition, whereas the timing of dentition in the remaining subjects appeared to be normal. Buccal displacement was not associated with a retarded dental development, and the ranges of the dental age values were similar to those seen in the control group. The results support the idea that there are different etiologies for the occurrence of buccal versus palatal ectopia in maxillary canines. They also suggest that dentitions with a palatal canine appear to be of 2 distinct varieties, with different dental characteristics and, perhaps, different etiologies.

Intrusion injuries of the primary dentition are highly associated with developmental disturbances of their successor teeth. The age of the child at the time of injury, the direction and severity of the intrusion, and the presence of alveolar bone fracture are important variables influencing the effect of primary tooth intrusion on the developing permanent germ. The developmental defects of the permanent successor tooth range from mild alteration in enamel mineralization to severe sequestration of the developing germ. This article will review the developmental anomalies of the permanent incisors induced by intrusion injuries of their predecessors.

The subjects of the investigation comprised 95 girls and 73 boys with juvenile rheumatoid arthritis (JRA), and 102 girls and 66 boys representing healthy controls, all with a chronological age from 6.3 to 14.4 yr. The dental development was assessed from panoramic radiographs using a seven-tooth model. The radiographs were evaluated on three separate occasions with a minimum interval of one month in a randomized order, and blind with respect to absence or presence of JRA. In both JRA patients and healthy controls, dental maturity was ahead of chronological age. In addition, dental maturity was significantly advanced in JRA patients with 0.26 yr in girls and 0.28 yr in boys. It is tentatively suggested that the advanced dental development in JRA patients compared with healthy children was partly an effect of treatment with cortisone, while the influence of the disorder per se remains to be elucidated.

Seven daily subcutaneous injections of 1-hydroxyethylidene-1, 1-bisphosphonate (HEBP) can induce enamel hypoplasia. Several enamel-free zones were observed along the crown-analogue side of rat incisors during the secretory stage of amelogenesis. Ameloblasts related to the enamel-free zones lay directly on the abnormally non-mineralized mantle dentine, whereas the adjacent ameloblasts, which were forming the enamel matrix layer, were associated with the region where mineralization of dentine was proceeding. The further purpose of this study was to investigate the synthetic and secretory activity of these two groups of ameloblasts and to trace the fate of the radioactively labelled proteins. [(3)H]-proline was administered to Wistar rats 12 h after the last injection of HEBP. Light-microscopic autoradiography was performed. Quantitative analysis indicated that the ameloblasts of the enamel-forming zones in the drug-treated group showed a distribution pattern of silver grains similar to that of the controls. The ameloblasts of the enamel-free zones also demonstrated incorporation of [(3)H]-proline at the same level. There was some labelling over the non-mineralized mantle dentine, which was supposed to indicate the penetration of ameloblast products. From these results, it is concluded that HEBP does not affect the ameloblast activity in protein synthesis. The complete failure of enamel-layer formation in some specific regions is probably due to the failure in protein secretion and protein deposition. This study provides additional evidence that the mineralization of dentine is an essential factor in successful enamel matrix secretion and deposition.

It has been reported that multiple injections of 1-hydroxyethylidene- 1,1-bisphosphonate (HEBP) to rats prevent mineralization of incisor dentin, thereby revealing high concentrations of calcium in the non-mineralized matrix of circumpulpal dentin. To identify the molecules responsible for calcium accumulation in circumpulpal dentin matrix, rats were injected daily with HEBP (8 mg P/kg) for 7 d, and the incisors processed for various histochemical and immunohistochemical staining of non-collagenous matrices of dentin. Cuprolinic blue reactions for proteoglycans (PGs) were equally distributed in non-mineralized matrix of mantle and circumpulpal dentin layers. Dentin sialoprotein (DSP) and osteopontin (OPN) immunoreactions were found in non-mineralized circumpulpal dentin matrix, but not in mantle dentin. In normal incisors, however, predentin matrix showing significant DSP immunoreactivity was negative for Ca-GBHA reactions. HEBP-affected, non-mineralized OPN immunopositive bone matrix was also non-reactive for calcium. From these observations, neither PGs, OPN nor DSP appear to be responsible for calcium accumulation in HEBP-affected circumpulpal dentin. Stains-all reactive component, possibly dentin phosphoprotein (DPP), only showed the same distribution as that of Ca-GBHA in both HEBP-affected and normal dentin matrix, implicating a possible contribution of DPP to calcium accumulation in circumpulpal dentin and, hence, to appositional mineralization of dentin.

The aim of the present study was to find an accurate estimation of chronological age using a small number of selected teeth. For this purpose, the method devised by Nolla [C. Nolla, The development of the permanent teeth, J. Dent. Child. 27 (1960) 254-266.] was used: the development of each of the teeth was determined according to this method on 374 radiographs, 195 of boys (mean age 8.59) and 179 of girls (mean age 8.75). The 28 variables representing the calcification stages were analyzed using cluster analysis followed by multivariate analysis (multiple linear regression model). Patient age was considered to be a dependent variable. Our study showed that antimere teeth are the most homogeneous as regards stages of development. The prediction was more accurate for boys and girls below 10 years of age, using teeth 21, 43 and 46 from boys and teeth 21, 46 and 47 from girls. These teeth accounted for 80% total variance of chronological age for dental calcification. Standard error was +/-1.4 years for boys and +/-1.2 years for girls. When the age of the children remained completely unknown, the best estimates were provided by teeth 43, 47, 46 and 44 from boys and teeth 44, 47 and 43 from girls.

The aim of this study was to assess the influence of age at start, duration, and completion of enamel formation, as well as of tooth-eruption age and enamel thickness on the severity of dental fluorosis within the permanent dentition. The material comprised Ugandan children (n = 219), aged 10-14 years, with 28 teeth and at least 1 tooth with fluorosis. The children were permanent residents in districts with either 0.5 mg or 2.5 mg fluoride/l in the drinking water. Fluorosis was assessed on the vestibular surfaces of all teeth using the modified Thylstrup and Fejerskov (TF) index. In order to relate fluorosis to the dental variables, the material was divided into a test group (n = 103), with fluorosis on all teeth, and a reference group (n = 116), with fluorosis on up to 27 teeth. The reference group was used to confirm or refute the findings in the test group. Paired comparisons showed significantly higher median TF scores for the late than for the early mineralizing and erupting teeth. In multiple regression analyses, the age at start, duration, and completion of enamel formation as well as tooth eruption was significantly related to the severity of fluorosis after controlling for enamel thickness (P < 0.05, n = 14). The effect (R2change) of the dental variables on the variation in severity of fluorosis within the dentition was in decreasing order: the duration of enamel formation, age at completion of enamel formation, tooth-eruption age, and the start of enamel formation.

The aim of this investigation was to evaluate the crypt formation and calcification of the maxillary and mandibular third molars according to age and gender on panoramic radiographs in a group of Turkish children. Four hundred panoramic radiographs of 188 female and 212 male dental patients, ages six to thirteen years, were examined by two observers. The interrater reliability was tested by Kappa statistics. The earliest age for maxillary third molar crypt formation was eight years; mandibular third molars could be seen radiographically as early as seven years.

The calcification degree of extracted human teeth was observed by using high resolution microfocus X-ray CT. As samples, upper and lower first premolars extracted from a 21-year-old female were used. The computed tomograms were produced by high resolution microfocus X-ray CT with a open vacuum X-ray source, rotating sample stage, and image sensor. The distinction between enamel and dentin was very clear, and the shape of the pulp cavity was also clearly identified. The secondary dentin was visible in the circumpulpal dentin. The color map displays showed the heterogeneity of the calcification degree not only in the dentin but also in the enamel. The enamel was divided into three layers according to the calcification degree. High resolution microfocus X-ray CT was very useful for the observation of the internal structure of human teeth without destroying the samples.

Dental pulps contain sulfated glycosaminoglycans (GAGs), such as chondroitin 4-sulfate (CSA/4CS), dermatan sulfate (CSB/DS), and chondroitin 6-sulfate (CSC/6CS). Sulfated GAGs play important roles in mineralization and collagen fibrillogenesis during primary, secondary, and reparative dentin formations. Transforming growth factor-beta (TGF-beta) is a potent regulator for several extracellular matrix (ECM) components and modulates the proliferation and differentiation. Using rat clonal dental pulp cells (RPC-C2A), we investigated the constituents of GAGs synthesized by the cells and the effect of TGF-beta on their synthesis by measuring the radioactivity of [35S]sulfate incorporated into GAG fractions. Cellulose acetate electrophoresis analysis revealed that RPC-C2A cells synthesized CSA and CSB but not CSC and that 10 ng/ml of TGF-beta increased the production of CSA and CSB in the cell/ECM fraction. Measurement of [35S]sulfate incorporation showed a significant increase in the amount of GAGs by TGF-beta, 1.3-fold CSA, and 1.2-fold CSB in the cell/ECM fraction. In the medium fraction the most secreted GAG was CSA, whereas CSB was stored in the cell/ECM fraction. Secreted CSA in the medium was markedly increased by 10 ng/ml of TGF-beta (1.7-fold). These findings indicate that CSA and CSB are major sulfated GAGs synthesized by RPC-C2A cells and that TGF-beta acts as a stimulator of sulfated GAG synthesis in dental pulp cells.

Osteopontin (OPN) is one of the major non-collagenous proteins in root cementum and other mineralized tissues. Although most of this mineral-seeking protein is thought to be produced by local tissue cells, some of it might enter the mineralizing matrix from the blood. To test this hypothesis, we followed the distribution of a single dose of purified porcine or rat 125I-labeled OPN injected i.v. in rats, in mineralizing and non-mineralizing tissues and in subcutaneously implanted collagenous implants. The animals were killed 30 or 48 hrs after injection. Tissues (calvaria, tibia, lower and upper jaws) were harvested and processed for radioautography and biochemical analysis. Tissues as well as calcifying collagenous implants proved to have taken up radiolabel. In EDTA extracts of long bones, the majority of the radiolabel was demonstrated to be associated with intact OPN. The iodinated protein was also found in the acellular extrinsic fiber cementum (acellular cementum) layer investing the continuously growing incisors, in laminae limitantes, cement lines, and in forming bone near the mineralization front. Further, the label was present in the circumpulpal dentin of the incisors, and some of it appeared to have been incorporated into developing enamel. It is concluded that OPN in acellular cementum and other mineralizing tissues may-at least partially-originate from sources outside the direct environment following its transportation via serum.

Enamel is secreted as a protein matrix by the ameloblasts. These same cells then control the maturation of the enamel matrix, secreting proteinases that hydrolyze proteins as mineralization progresses, until mature enamel containing less than 1% protein by weight remains. Further understanding of the factors that control ameloblast function and differentiation requires an in vitro cell culture system. In this study, we report immortalization of enamel organ epithelial cells and the selection of a cell line with characteristics of ameloblasts. Porcine enamel organ cells were dissected from unerupted porcine molars, cultured in serum-free medium, and passaged twice. These cells were transfected with an origin-of-replication defective SV40 plasmid by calcium phosphate precipitation, and a cell line with mRNA expression characteristic of ameloblasts was cloned. This cell line (PABSo-E) expressed mRNA for amelogenin, matrix metalloproteinase-20 (enamelysin), and enamel matrix serine proteinase 1 (EMSP1), but not ameloblastin. PABSo-E cells have been passaged more than 55 times, while continuing to maintain characteristics of ameloblasts. These cells will be useful for future studies of ameloblast function.

The purpose was to examine the formation and mineralization of molar roots in rats with hypocalcaemia, induced by a low-calcium diet and the changes after returning to a normal diet. Two-week-old male Wistar rats were fed a low-calcium diet (0.03% calcium) during the period when the first molar roots form and then a diet containing normal amounts of calcium (1.83% calcium) was restored. The blood calcium, the length and mineralization of the first upper molar roots, and the density of surrounding alveolar bone were measured, and the morphological and histological features of the roots examined. While the rats were fed the low-calcium diet, they had a significantly lower blood calcium than normal controls. Morphologically, the upper first molar roots were shorter and the predentine layer was thicker. The mineralization of dentine and the surrounding alveolar bone was significantly less than in the controls and no mineralization was detected in the thickened predentine. After a normal calcium diet had been restored, the blood calcium, thickness of dentine, alveolar bone density, and length of the roots caught up with the normal. In addition, hypomineralized dentine and interglobular dentine were observed. Subsequently, the mineralization of the dentine increased and the amount of interglobular dentine gradually decreased. These results suggest that in rats a low-calcium diet induces hypocalcaemia, causing the formation of interglobular dentine and hypomineralization of the dentine of the roots. Most physiological variables recovered completely with the return to a normal-calcium diet, although some hypomineralization of the dentine remained.

The present study reveals the inhibitory effect of iron intoxication on the process of dentine mineralization. Wistar rats were injected intraperitoneally with iron dextran at 0.88 g/kg body weight per day for 10 days during the period of odontogenesis. An age-matched group was injected intraperitoneally with bisodium etidronate (EHBP) at 20 mg/kg body weight per day for 10 days. Another age-matched group was treated with similar amounts of saline intraperitoneally and considered as control. At the end of the experimental period the animals intoxicated with iron exhibited non-mineralized dentine and mineralized bone. The animals treated with EHBP showed non-mineralized dentine and bone. These findings would suggest the existence of different mineralization mechanisms for bone and dentine.

Objective: To study the relationship between mandibular canine calcification and skeletal maturity., Methods: Hand wrist and mandibular canine radiographs of 106 boys and 152 girls in Beijing area were assessed., Results: Mandibular canine calcification coincides with the initiation of puberty in male (93.8%) and with the deceleration of puberty in female(89.7%). However the coincidence with the indicators of other pubertal stages was less than 50%., Conclusion: Mandibular canine calcification just can give an initial assessment for estimating the timing of puberty.

The objective of this investigation was to determine if there is a difference in dental age of maturation between adolescents treated in the 1970s and those treated in the 1990s. Records of 150 Caucasian patients, 8.5 to 14.5 years old and treated in a private orthodontic office between 1972 and 1974, were randomly selected; records of another 150 patients of the same race and age range but treated between 1992 and 1994 were also collected. The percentage of calcification of the mandibular canines was rated according to methods used by Demirjian, who divided tooth development into eight segments, A to H. Using stage G to compare the 1970 and 1990 patient samples, we demonstrated dental age reductions of 1.21 years for males and 1.52 years for females, and a combined reduction of 1.40 years.

Biological apatite-crystal formation is a complex process starting with heterogeneous nucleation of inorganic calcium phosphate on an organic extracellular matrix [Cuisinier et al. (1995), J. Cryst. Growth, 156, 443-453]. Further stages of crystal growth are also controlled by the organic matrix and both nucleation and growth processes are under cellular control [Mann (1993), Nature (London), 367, 499-505]. The final mineral in calcified tissue is constituted by poorly crystalline hydroxyapatite (HA) with a low Ca:P ratio, containing foreign ions such as carbonate and fluoride. This study reports the first observation of octacalcium phosphate (OCP) [Brown (1962), Nature (London), 196, 1048-1055] in a biological tissue; OCP was found in the central part and HA at the extremities of the same crystal of calcifying dentine. This observation is of key importance in understanding the first nucleation steps of biological mineralization. The presence of OCP in a forming human dentine crystal and the observation in the same tissue of nanometer-sized particles with a HA structure [Houllé et al. (1997), J. Dent. Res. 76, 895-904] clearly proves that two mechanisms, direct nucleation of non-stoichiometric HA crystals and nucleation of OCP, occur simultaneously in same area of mineralization. OCP is found to be a transient phase during the growth of biological crystals. In small crystals, OCP is completely transformed into HA by a hydrolysis reaction (Brown, 1962) and can only be detected in larger crystals because of its slow kinetics of transformation.

The biogenetic formation of mineral crystals, one aspect of biomineralization, is a multistep process of apatite formation throughout the growth of dentin tissue. An important step is the transformation of the non-mineralized predentin matrix to mineralizing dentin matrix and its biological control. In this study, the high capacity of elemental mapping is combined with single x-ray point measurements to elucidate whether special elements are involved in initiation or regulation of mineral nucleation. Directly at the mineralization front, micro-areas with a strong co-enrichment of phosphorus (e.g., as phosphate) and potassium are found. During the beginning of the calcium enrichment and the subsequent apatite mineral formation in the characteristic micro-areas, the content of potassium decreases significantly. These findings indicate that potassium is involved in the process of dentin mineralization.

In this communication, we summarized our recent experimental approaches to an unsettled issue, i.e., the nature and role of an acidic precursor in enamel mineralization. The objectives we specially focused our attention on are: the composition, structure and high resolution images of enamel crystals at various developmental stages, thermodynamic and kinetic consideration of octacalcium phosphate (OCP) vs hydroxyapatite (HA) precipitation in physiological media simulating the enamel fluid, reversible changes in the composition and structure of OCP, effects of fluoride at low concentrations and enamel proteins on OCP hydrolysis, and adsorption of enamel proteins onto OCP and fluoridated hydrolysates at neutral pH and room temperature. On the basis of all experimental evidence, we propose that enamel crystal growth comprises two events: the two-dimensional growth of an OCP-like precursor in a narrow outermost zone adjacent to the ameloblasts and the subsequent overgrowth of apatite units on the template under discrete fluid environment in the underlying region distant from the cell layer. The experimental data also support the concept that the whole process of enamel mineralization is modulated substantially through interaction between enamel proteins and crystals including the acidic precursor.

One of the difficulties in simulating in vivo calcification by in vitro experiments is how to prepare and apply a suitable calcifying solution. We have previously developed an entirely new model system consisting of 40% acrylamide gel blocks that contains matrix proteins and is immersed in fetal calf serum at 37 degrees C. (40% gel system) for 18 hr (Connect. Tissue Res., 33, 185, 1995). The gels were analyzed for immobilized calcium. In this system bovine enamel proteins (0.1% in the gel) showed the highest calcifiability among the tested matrices, followed by insoluble bovine dentin, bone and skin collagens. The 40% gel system provides a barrier for high molecular weight inhibitor molecules in the body fluid. The new calcifying system developed in this study consists of the matrix protein sealed in dialysis tubing within a glass chromatography column that was eluted with a calcifying solution. In this system (dialysis tubing system), again the enamel protein showed higher calcifiability than dentin, bone and skin collagens. It was also shown that enamel proteins became not only a reversible opaque gel, but also a relatively-irreversible coagulant, if the solution contained calcium and phosphate ions at concentration below saturation (1 mM calcium and 1 mM phosphate). With both systems combined, deposition and crystal growth of minerals in enamel proteins will be better understood than with previous methods.

Phosphoprotein appears to play an important role in the mineralization of dentin during tooth development and remineralization after demineralization by dental caries. To better understand this role, we describe the extraction and characterization of phosphoprotein from immature, human root apex dentin during and after EDTA demineralization. The extraction procedure included dissociation of the demineralized dentin matrix by guanidine hydrochloride (Gdn.HCl) followed by subsequent digestion with cyanogen bromide (CNBr) and collagenase. Characterization of these extracts included 'Stains-All' staining of SDS polyacrylamide gels (SDS-PAGE) and amino acid, protein and phosphorus analyses. The ability of these matrices to remineralize was determined by TEM and measuring calcium levels in the remineralized tissue by atomic absorption spectroscopy. The staining of SDS-PAGE gels and amino acid analysis showed that an intact phosphophoryn was extracted from the dentin of the immature apices during EDTA demineralization and that it had an apparent Mr approximately 140,000. In the subsequent extracts and digests, the phosphoprotein has a range of molecular weights, some of which may have been degraded products of the intact phosphoprotein. A greater quantity of phosphoprotein was found in the EDTA-demineralized dentin matrices than in dentin after Gdn.HCl, CNBr and collagenase digests. These EDTA-demineralized matrices also remineralized to a greater extent than those dissociated with Gdn.HCl. The differences in both the quantity and the quality, as defined by the amino acid residue profile, of the phosphoprotein in the sequential extracts of the root apex dentin may be important in affecting the ability of this tissue to remineralize.

This review focuses on the process of enamel maturation, a series of events associated with slow, progressive growth in the width and thickness of apatitic crystals. This developmental step causes gradual physical hardening and transformation of soft, newly formed enamel into one of the most durable mineralized tissues produced biologically. Enamel is the secretory product of specialized epithelial cells, the ameloblasts, which make this covering on the crowns of teeth in two steps. First, they roughly "map out" the location and limits (overall thickness) of the entire extracellular layer as a protein-rich, acellular, and avascular matrix filled with thin, ribbon-like crystals of carbonated hydroxyapatite. These initial crystals are organized spatially into rod and interrod territories as they form, and rod crystals are lengthened by Tomes' processes in tandem with appositional movement of ameloblasts away from the dentin surface. Once the full thickness of enamel has been formed, ameloblasts initiate a series of repetitive morphological changes at the enamel surface in which tight junctions and deep membrane infoldings periodically appear (ruffle-ended), then disappear for short intervals (smooth-ended), from the apical ends of the cells. As this happens, the enamel covered by these cells changes rhythmically in net pH from mildly acidic (ruffle-ended) to near-physiologic (smooth-ended) as mineral crystals slowly expand into the "spaces" (volume) formerly occupied by matrix proteins and water. Matrix proteins are processed and degraded by proteinases throughout amelogenesis, but they undergo more rapid destruction once ameloblast modulation begins. Ruffle-ended ameloblasts appear to function primarily as a regulatory and transport epithelium for controlling the movement of calcium and other ions such as bicarbonate into enamel to maintain buffering capacity and driving forces optimized for surface crystal growth. The reason ruffle-ended ameloblasts become smooth-ended periodically is unknown, although this event seems to be crucial for sustaining long-term crystal growth.

We have found, at high EM magnification, on ultrathin sections of shock-frozen, freeze-dried, embedded pieces of the developing hard tissues, that the primary crystallites consist of strands composed of nanometer-sized apatitic islands, which rapidly coalesce to needles and afterward to platelets. By small-area electron diffraction, with energy-filtered electrons, it was clarified that these strands are already crystallographically oriented along the bipolar c-axis so that the center-to-center distances between the islands would reflect the distances between crystal-nucleating sites along the matrix. The EM analysis of the cross-cut stained unmineralized and of the unstained mineralized collagen fibers of turkey tibia tendon shows that the staining "nuclei" and the early crystallites, appearing as dark dots, surround "light" round structures, which we interpret as the collagen microfibrils, surrounded by the apatitic crystallites.

The purpose of this study was to examine the differences in the times of eruption and calcification of the permanent dentition between skeletal class III and class II groups. (And also to examine the relationship between the time of eruption and the type of malocclusion) Fifty-three children, ages seven to ten years, were selected. Of these, twenty-six children (twelve boys and fourteen girls) were Angle class III with minus ANB and twenty-seven children (eleven boys and sixteen girls) were Angle class II with five or more ANB. Panoramic radiographs and cephalometric radiographs were used. The panoramic radiographs showed that the calcification of the maxillary second molars in class II were earlier than in class III using Nolla's classification. There was no statistically significant difference, however, for mean values of calcification stages, using Nolla's classification, between boys and girls. The cephalometric and panoramic radiographs showed that the times of eruption and calcification were earlier in the maxillary second molars than in the mandibular second molars for class II. In contrast, the times of eruption and calcification were earlier in the mandibular second molars than in the maxillary second molars for class III. The times of eruption and calcification of the maxillary molars were significantly related to the length of the ANS-PNS. The longer the ANS-PNS, the earlier were the times of eruption and calcification. There was a significant relationship between the ANB angle and the time of eruption, as well as the ANB angle and calcification. The larger ANB had earlier calcification and chronology of maxillary second molars.

The interfacial free-energy is an important factor in the regulation of mineralization and dissolution at the surfaces of dental hard tissues. However, few thermodynamic studies have been aimed at the elucidation of the interfacial terms. Contact angle measurements (sessile drop and thin layer wicking) and kinetic dissolution and growth techniques have been used to study the interfacial properties of root dentin (D), human enamel (E), and hydroxyapatite (HAP). The interfacial tensions between water (w) and each of these phases were calculated from contact angle data according to surface tension components theory. The values gamma wD = 4.5 x 10(-3) J m-2, gamma wE = 8.8 x 10(-3) J m-2, and gamma w,HAP = 10.4 x 10(-3) J m-2 were of the same order of magnitude as those obtained from dissolution kinetic data (pH = 4.5): gamma wD = 1.4 x 10(-3) J m-2, gamma wE = 3.2 x 10(-3) J m-2, and gamma wHAP = 9.3 x 10(-3) J m-2. Kinetics studies of the crystallization of HAP on HAP, dentin, and enamel yielded the interfacial free-energy values, gamma wHAP = 17.1 x 10(-3) J m-2, 17.7 x 10(-3) J m-2, and 9.4 x 10(-3) J m-2, respectively, probably reflecting the interfacial energies of the deposited phases rather than those of the dental hard-tissue substrata. The lower interfacial tension values are consistent with the higher solubilities of these solid phases: logKSO = -52.0, -55 approximately 57, and -58 approximately 59, for root dentin, enamel, and HAP, respectively, expressed as an equivalent HAP ionic product. The higher interfacial free-energy is also consistent with the slower mineralization of HAP on dentin and enamel surfaces.

The effect of early weaning on caries progression, dentin formation, and dentin mineralization was examined in four groups of rats. Two groups received a normal diet and were weaned on day 18 or 21, and another two received a sucrose-rich diet and were weaned on day 18 or 21. At age 35 days the lower molars were sectioned sagittally, and the areas of dentin formation and of the dentinal caries were quantified. The width of the predentin zone was measured from histologically stained sections of maxillary molars. Early weaning reduced dentin formation in the group on the high-sucrose diet only the first days; later this effect was partially caught up with. A high-sucrose diet significantly increased caries frequency and extension of caries lesions compared with a normal diet in both early weaned and normally weaned groups. The effect of early weaning on caries frequency and extension in the high-sucrose group was insignificant compared with the normally weaned group on a high-sucrose diet. The predentin zone was wider in the sucrose groups than in the control groups at the end of the experiment. These results indicate that the effect of sucrose on dentin formation was dependent on the stage of physiologic dentin formation, but early weaning as such did not affect this.

Human dental papilla cells were enzymatically separated from deciduous tooth germs of an 8-month-old embryo legally aborted. The second passage cells were cultured up to 35 days in 3 groups. The beta-GP group was cultured in the Dulbecco MEM containing ascorbic acid and beta-glycerophosphate supplemented with 15% fetal bovine serum. The Dex group was in the same medium, in addition containing dexamethasone. The control group contained none of the 3 chemicals. Mineralized nodules were formed after 15 days in the beta-GP and Dex groups. Only in the presence of ascorbic acid and organic phosphate did they mineralize. The addition of dexamethasone caused a significant increase in the number of nodules. By electron microscopy, the nodules contained needle-shaped crystals associated with a network of collagen fibrils. Calcium and phosphorus were detected by energy-dispersive X-ray microanalysis in the nodules. Furthermore, the crystalline material exhibited a pattern consistent with hydroxyapatite and dentin when examined by X-ray diffractometry. Cells showed high levels of alkaline phosphatase activity, which was increased 2-3 times in the presence of the 3 chemicals. These results indicated that human dental papilla cells have the ability to form dentin in culture. The formation of mineralized nodules by human dental papilla in vitro provides a useful model for studying the morphogenesis and differentiation of dental papilla ectomesenchyme.

Morphological features of the cap enameloid and dental epithelial cells were investigated by light and transmission electron microscopy during the various stages of enameloid mineralisation and early maturation in the tilapia. The pattern of mineralisation along collagen fibrils in the enameloid differed from that in the dentine. Many matrix vesicles were found in the predentine and in the enameloid, suggesting that they may be involved in the initial mineralisation in both regions. Most of the organic matrix disappeared from the cap enameloid during mineralisation and maturation. The disappearance of the organic matrix could be divided into 2 stages. Initially a fine network-like matrix, which probably consisted of glycosaminoglycans and extended between collagen fibrils, began to disappear. At the same time, fine crystallites and electron-dense, fine granular material covered the collagen fibrils as mineralisation of the enameloid began. In the second stage, the maturation of the enameloid, the collagen fibrils degenerated completely and disappeared from the cap enameloid, being replaced by large numbers of large crystals. At the mineralisation stage, the numbers of lysosomal bodies tended to increase in the inner dental epithelial (IDE) cells, which contained a well developed Golgi apparatus and rough endoplasmic reticulum (rER). At the early stage of maturation, a ruffled border was noted at the distal ends of the IDE cells, which contained many mitochondria and lysosomal bodies, but less rER. These features suggest that the cells actively absorb the organic matrix, which includes collagen fibrils, in the cap enameloid. The outer dental epithelial (ODE) cells were translucent cells that contained well developed labyrinthine canalicular spaces from the onset of the mineralisation stage to the middle stage of maturation. The IDE and ODE cells were clearly involved in the mineralisation of the cap enameloid at the mineralisation and maturation stages.

Enamel maturation is characterized by massive crystal growth in both width and thickness, resulting in the most highly mineralized of all mammalian skeletal tissues. The control of this process is mediated via a carefully orchestrated series of events that are temporally and spatially regulated, and it requires the co-ordinated degradation and removal of the endogenous enamel matrix. This is affected by both neutral metalloproteases and serine proteases, which are developmentally restricted and may be further modulated by changes in the chemistry of the enamel crystals themselves. Failure of these mechanisms, or the adventitious entry of mineral-binding proteins during the later stages of maturation, may result in the incomplete maturation of the enamel crystals and the eruption of dysplastic tissue.

Tuftelin is a novel acidic enamel protein thought to play a major role in enamel mineralization. Its identity and localization has been confirmed by amino acid composition, enzyme-linked immunosorbant assay, Western blots, indirect immunohistochemistry and high resolution protein-A gold immunocytochemistry. The deduced tuftelin protein (pI 5.2) contains 389 amino acids and has a calculated peptide molecular mass of 43,814 Da. Immunological studies suggest conservation of tuftelin structure between species throughout vertebrate evolution. The cDNA sequence encodes for several putative post-translation sites including one N-glycosylation consensus site, seven O-glycosylation sites and seven phosphorylation sites, as well as an EF-hand calcium-binding domain (with mismatch), localized towards the N-terminal region. At the C-terminal region (residues 252-345) tuftelin contains structurally relevant determinants for self assembly. We recently cloned and partially sequenced the human tuftelin gene (four exons have now been sequenced). These sequences include exon 1 and over 1000 bases of the putative promoter region. Employing fluorescent in situ hybridization, we mapped the human tuftelin gene to chromosome 1q 21-31. Localization of the human tuftelin gene to a well-defined cytogenetic region may be important in understanding the aetiology of autosomally inherited amelogenesis imperfecta, the most common enamel hereditary disease.

Secretory ameloblasts have multiple functions including the synthesis and resorption of enamel matrix proteins and calcium transport during enamel formation. We have examined these functions by means of cytochemistry and immunocytochemistry. Enamel proteins, amelogenins and enamelins are localized in the biosynthetic pathways of ameloblasts and in the forming enamel. Sulfated glycoconjugates are present in secretory ameloblasts. The distal junctional complex of ameloblasts may act as a permeability barrier to enamel proteins, thereby confining the secreted proteins to the growing enamel front. Secretory ameloblasts contain lysosomal enzymes in the Golgi lysosome endoplasmic reticulum system and also exhibit absorptive capacity, which might be associated with an early decrease in extracellularly degraded enamel proteins. Active calcium transport through the ameloblasts towards the growing enamel is indicated by the demonstration of Ca-ATPase activity along the plasma membranes. A calcium-dependent modulator protein, calmodulin, is localized in ameloblasts, suggesting that early enamel mineralization is dependent upon calmodulin-regulated Ca-ATPase in ameloblasts. These results suggest that the secretory ameloblast is a highly specialized multifunctional cell in the production, resorption and degradation of enamel matrix and in the active calcium transport essential for matrix mineralization during enamel formation.

The formation of enamel takes place through a sequence of processes that can be mimicked in inorganic materials chemistry. This chapter describes the generic features of enamel biomineralization in terms of a house-building analogy. Four stages are identified: supramolecular preorganization and spatial patterning; interfacial molecular recognition in inorganic nucleation; vectorial crystallization; and pattern evolution and hierarchy. Each of these concepts can be translated into synthetic approaches to the formation of inorganic materials with organized architectures. An example of applying this biomimetic paradigm is described. Supersaturated water-in-oil microemulsions have been used to synthesize microskeletal calcium phosphates by controlled nucleation and vectorial growth in constrained reaction environments. The results of these preliminary studies suggest that biomimetic concepts could be useful in the fabrication of biomaterial implants with controlled porosity and microstructure.

Hertwig's epithelial root sheath (HERS) cells were isolated and recombined with ectomesenchymal cells in vitro utilizing extracellular matrix components as substrate. After 14 days in culture, HERS cells were differentiated and exhibited a stratified organization. These features resembled those observed in vivo as epithelial rests of Malassez. A mineralization process was also present in HERS cells, in which calcium salts were deposited. This mineralization was correlated with the strong immunoexpression of osteopontin by HERS. The results obtained add support to the possible role of HERS in the secretion of hypocalcified material on the root during early cementogenesis.

The purpose of this study was to establish transgenic mouse lines as a tool to investigate the function of amelogenin during mineralization by causing ectopic production of amelogenin and studying its effect. The mouse amelogenin (mAme) was cloned from a 16-day-old whole mouse embryo cDNA library and was determined to be "full-length" mouse amelogenin (with a complete coding region) by comparison with the mouse amelogenin reported previously by Snead et al. (1985) and Lau et al. (1992). The overexpression construct contained: (1) the rat osteocalcin (OC) promoter (1.8 kb); (2) the adenovirus splicing casettes, including introgenic (Int) sequence (0.3 kb); (3) the full-length mAme cDNA (0.8 kb); and (4) the polyadenylation signal sequence from the pSG5 mammalian expression vector. Both Southern blotting and polymerase chain-reaction (PCR) analyses were performed, by means of a specific probe and a pair of oligodeoxynucleotides to OcIntmAme(A)+, respectively. The animals which showed transgene-positive in both analyses were further used to establish F1 animals. Heterozygocity was confirmed with F1 animals by PCR analysis of DNA from the F0 x FVB/N pups. Three independent transgenic F1 heterozygous lines (640t, 706t, and 708t) have now been established. The generation of F2 homozygous lines is under way. The heterozygous transgenic animals are currently being analyzed for alterations in the morphology and structure of various bone tissues.

Our previous studies revealed intense membrane-associated labeling for Ca(2+)-Mg2+ ATPase (Ca(2+)-pump) in secretory and maturation ameloblasts in the rat incisor, both by enzyme cytochemistry and by immunohistochemical techniques. The purpose of the present study was to map the distribution of Ca(2+)-pump protein at the cellular and subcellular levels by means of a Ca(2+)-pump-specific monoclonal antibody and electron microscopic immunogold cytochemistry. Tissue specimens were dissected from secretory, early, and late enamel maturation zones. We quantified results by comparing gold particle densities over ameloblast lateral and distal plasma membrane regions, supranuclear cytoplasm, regions of the ruffled borders, and nuclei. The highest concentration of gold particles was seen over the distal membranes of early-maturation ameloblasts relative to those in late-maturation and secretory stages. Cytoplasmic labeling was less than that of the distal and lateral membranes, and gold particles located over nuclei were considered to be due to non-specific binding. These results are consistent with our earlier findings and suggest a role for the plasma membrane Ca(2+)-pump in the regulation of calcium availability to mineralizing enamel.

The effect of accelerated eruption of the rat lower incisor on enamel was studied in a series of segments obtained when the incisor was cut repeatedly out of occlusion over a five-week period. The segments were ground, cleaned, acid-etched, observed with SEM, and analyzed with EDX. Pigmentation was lost within 11 days. Pigmented superficial enamel was more acid-resistant than the rest of the enamel, but this quality decreased with decreasing iron content. Hypomineralized enamel first appeared in a restricted area at the mesio-labial angle of the tooth in the 6th-7th segment obtained after 11-14 days. Later, hypomineralization became more generalized. All enamel zones were retained throughout the experiment. The geometry of the prism pattern was affected. The angle between prism rows and the enamel-dentin junction increased from 44 degrees to 48 degrees, while the angle of decussation increased from 60 degrees to 70 degrees. The angle between the enamel surface and prisms in the outer enamel was more difficult to assess, but tended to increase from about 25 degrees to 29 degrees. However, the prisms retained their incisal direction. The connection between enamel and dentin was partly disrupted from about the 9th segment onward. The depth of the mesial concavity of the enamel-dentin junction decreased from about the 10th segment onward. Accelerated eruption affects all stages of enamel formation and is a suitable and predictable model for studying regulatory mechanisms in amelogenesis.