The origin of tooth cusps--a hypothesis

An embryological concept is offered to explain the formation of tooth cusps. The epithelium is considered to play the active part in morphogenesis. The folding of the inner sheath of the enamel organ must be seen to be the origin of tooth cusps. This folding can be considered to be a consequence of the ability to span only a certain area without folding. The regularity of the folding--and thus the regularity of the molar occlusal pattern--might be a result of the unique material properties of the inner enamel epithelium.     

Analysis of fracture and deformation modes in teeth subjected to occlusal loading

An analysis of fracture and deformation modes in tooth enamel subjected to occlusal loading is presented. Several competing modes are identified: deformation by yield beneath the indenter; median cracking from the ensuing plastic zone and analogous radial cracking from the dentin–enamel junction along the load axis; and margin cracking from the cervical enamel–cement junction. The analysis, based on a simple model of tooth geometry, presents relations for the critical loads to initiate these damage modes within the enamel, and to drive ensuing cracks longitudinally around the tooth walls to failure. The relations are explicit in their dependence on characteristic tooth dimensions – enamel thickness and cuspal radius – and on material properties – modulus, hardness, toughness and strength. Provision is made to incorporate properties of the occlusal contact, whether from opposing dentition or intervening food particles. All these features are demonstrated on critical-load master diagrams. A characteristic feature of the damage evolution is the gradual evolution of each mode with increasing load, so that failure is generally a prolonged rather than abrupt event. This accounts for the remarkable damage tolerance of natural teeth. The equations may enable basic predictions of tooth responses for humans and animals under a variety of specified dietary and functional conditions.     

The induction of enamel and dentin complexes by subcutaneous implantation of reconstructed human and murine tooth germ elements

Tooth induction by xenogenic graft of reconstructed human tooth germ components has never been attempted. Here we report our first attempt at a transplantation of human tooth germ components, heterologously recombined with mouse dental epithelia, into immunocompromised animals. Human third molar tooth germs enucleated from young patients as prophylactic treatment for orthodontic reasons were collected. The whole or minced human dental papilla was reconstructed with human- or mouse molar enamel epithelium, and transplanted in the dorsal aspect of C.B-17/Icr-scid Jcl mice. The transplant of human dental papilla reconstructed with human enamel epithelium formed thin dentin and immature enamel layers by 3 to 4 weeks, but remained extremely small in quantity due to a shortage of epithelial components in the graft. The addition of E16 mouse molar enamel organs (n=10-12) to each graft augmented the formation of tooth germ-like structures, but the differentiation of mouse molar ameloblasts was suppressed. However, once a solid layer of mineralized dentin was established, mouse ameloblasts accelerated their differentiation, and completed the enamel matrix formation and maturation within the following 4 weeks, whereas human ameloblasts, which had interacted with human dental papilla, remained in the stage of matrix formation during the same period. These data imply that, in reconstructed transplants, the differentiation of mouse dental epithelia is restrained by putative suppressive factors derived from human dental papilla until they are separated by mineralized dentin layers that serve as a diffusion barrier. The mouse enamel organ nevertheless retains its own phenotypic characteristics and intrinsic timing of cell differentiation and function.     

Mechanics of longitudinal cracks in tooth enamel

A study is made of longitudinal "channel" cracking in tooth enamel from axial compressive loading. The cracks simulate those generated in the molar and premolar teeth of humans and animals by natural tooth function. Contact loading tests are made on extracted human molars with hard and soft indenting plates to determine the evolution of such cracks with increasing load. Fracture is largely stable, with initial slow growth followed by acceleration as the cracks approach completion around an enamel side wall. A simple power law relation expresses the critical load for full fracture in terms of characteristic tooth dimensions-base radius and enamel thickness-as well as enamel toughness. Extended three-dimensional finite element modeling with provision for growth of embedded cracks is used to validate this relation. The cracks leave "fingerprints" that offer valuable clues to dietary habits, and provide a basis for a priori prediction of bite forces for different animals from measured tooth dimensions.     

On the mechanical properties of tooth enamel under spherical indentation

The mechanical properties of tooth enamel generally exhibit large variations, which reflect its structural and material complexity. Some key properties were evaluated under localized contact, simulating actual functioning conditions. Prominent cusps of extracted human molar teeth were polished down ∼0.7 mm below the cusp tip and indented by tungsten carbide balls. The internal damage was assessed after unloading from longitudinal or transverse sections. The ultimate tensile stress (UTS) was determined using a novel bilayer specimen. The damage is characterized by penny-like radial cracks driven by hoop stresses and cylindrical cracks driven along protein-rich interrod materials by shear stresses. Shallow cone cracks typical of homogeneous materials which may cause rapid tooth wear under repeat contact are thus avoided. The mean stress vs. indentation strain curve is highly nonlinear, attributable to plastic shearing of protein between and within enamel rods. This curve is also affected by damage, especially radial cracks, the onset of which depends on ball radius. Several material properties were extracted from the tests, including shear strain at the onset of ring cracks γ_F (= 0.14), UTS (= 119 MPa), toughness K_C (= 0.94 MPa m^1/2), a crack propagation law and a constitutive response determined by trial and error with the aid of a finite-element analysis. These quantities, which are only slightly sensitive to anatomical location within the enamel region tested, facilitate a quantitative assessment of crown failure. Causes for variations in published UTS and K_C values are discussed.     

Predicting failure in mammalian enamel

Dentition is a vital element of human and animal function, yet there is little fundamental knowledge about how tooth enamel endures under stringent oral conditions. This paper describes a novel approach to the issue. Model glass dome specimens fabricated from glass and back-filled with polymer resin are used as representative of the basic enamel/dentine shell structure. Contact loading is used to deform the dome structures to failure, in simulation of occlusal loading with opposing dentition or food bolus. To investigate the role of enamel microstructure, additional contact tests are conducted on two-phase materials that capture the essence of the mineralized-rod/organic-sheath structure of dental enamel. These materials include dental glass-ceramics and biomimicked composites fabricated from glass fibers infiltrated with epoxy. The tests indicate how enamel is likely to deform and fracture along easy sliding and fracture paths within the binding phase between the rods. Analytical relations describing the critical loads for each damage mode are presented in terms of material properties (hardness, modulus, toughness) and tooth geometry variables (enamel thickness, cusp radius). Implications in dentistry and evolutionary biology are discussed.     

A simplified genetic design for mammalian enamel

A biomimetic replacement for tooth enamel is urgently needed because dental caries is the most prevalent infectious disease to affect man. Here, design specifications for an enamel replacement material inspired by Nature are deployed for testing in an animal model. Using genetic engineering we created a simplified enamel protein matrix precursor where only one, rather than dozens of amelogenin isoforms, contributed to enamel formation. Enamel function and architecture were unaltered, but the balance between the competing materials properties of hardness and toughness was modulated. While the other amelogenin isoforms make a modest contribution to optimal biomechanical design, the enamel made with only one amelogenin isoform served as a functional substitute. Where enamel has been lost to caries or trauma a suitable biomimetic replacement material could be fabricated using only one amelogenin isoform, thereby simplifying the protein matrix parameters by one order of magnitude.     

小鼠牙胚在肾包膜及口腔黏膜环境下成牙能力的比较研究

目的:通过对比分析肾包膜和口腔黏膜环境对小鼠牙胚成牙的适宜度,期望为牙齿再生技术探寻较为理想的培养环境。方法:构建小鼠牙胚肾包膜下及口腔黏膜下移植模型,将14.5 d胚龄(ED14.5)的胚鼠下颌第1磨牙牙胚分别移入肾包膜及口腔黏膜下,于植入后3、4周取材,进行大体标本观察、组织学切片分析、硬度、弹性模量测定及拉曼光谱分析等,以观察牙胚的成牙情况。结果:(1)牙胚在肾包膜下及口腔黏膜下均可发育成牙齿状结构,但口腔黏膜组在各时点牙冠体积均小于肾包膜组,且牙齿尖窝发育状态不如肾包膜组明显。(2)组织切片染色显示:在口腔黏膜下形成的牙釉质层和牙本质层比肾包膜组薄,成釉细胞及成牙本质细胞分化不明显。(3)在釉质的硬度比较上,仅口腔黏膜4周组的釉质硬度比正常鼠牙低(P0.05);在釉质的弹性模量比较上,肾包膜下3周后的移植物釉质弹性模量比成年鼠牙略低,但差别无统计学意义(P0.05),肾包膜下4周和口腔黏膜下4周后的移植物釉质弹性模量均低于正常鼠牙和肾包膜3周的釉质弹性模量(P0.01);各组间牙本质的硬度及弹性模量无显著差异(P0.05)。(4)拉曼光谱分析结果显示:2组的拉曼光谱趋势基本一致,在961 cm-1处有最大峰值,但是在2 947 cm-1处,口腔黏膜3周组有明显的峰值,远高于其它各组。结论:(1)对于ED14.5的牙胚来说,相对口腔黏膜环境而言,肾包膜下环境培育3~4周更能发育成相对完整的牙齿。(2)口腔黏膜环境所形成牙齿的诸多性状与肾包膜环境下发育的牙齿相比虽有差异,但其对牙胚的生长发育还是具有一定的影响。     

Megadontia, striae periodicity and patterns of enamel secretion in Plio-Pleistocene fossil hominins

Early hominins formed large and thick-enamelled cheek-teeth within relatively short growth periods as compared with modern humans. To understand better the developmental basis of this process, we measured daily enamel increments, or cross striations, in 17 molars of Plio-Pleistocene hominins representing seven different species, including specimens attributed to early Homo. Our results show considerable variation across species, although all specimens conformed to the known pattern characterised by greater values in outer than inner enamel, and greater cuspal than cervical values. We then compared our results with the megadontia index, which represents tooth size in relation to body mass, for each species to assess the effect of daily growth rates on tooth size. Our results indicate that larger toothed (megadont) taxa display higher rates or faster forming enamel than smaller toothed hominins. By forming enamel quickly, large tooth crowns were able to develop within the constraints of shorter growth periods. Besides daily increments, many animals express long-period markings (striae of Retzius) in their enamel. We report periodicity values (number of cross striations between adjacent striae) in 14 new specimens of Australopithecus afarensis, Paranthropus aethiopicus, Paranthropus boisei, Homo habilis, Homo rudolfensis and Homo erectus, and show that long-period striae express a strong association with male and average male–female body mass. Our results for Plio-Pleistocene hominins show that the biological rhythms that give rise to long-period striae are encompassed within the range of variation known for modern humans, but show a lower mean and modal value of 7 days in australopithecines. In our sample of early Homo, mean and modal periodicity values were 8 days, and therefore similar to modern humans. These new data on daily rates of enamel formation and periodicity provide a better framework to interpret surface manifestations of internal growth markings on fossil hominin tooth crowns. Importantly, our data on early hominin cross striation variation may now contribute towards solving difficult taxonomic diagnoses where much may depend on fragmentary molar remains and enamel structure.     

Short-term mineralization of dentin and enamel in the mouse embryonic molars cultured in serum-free, chemically-defined medium.

The present study was designed to demonstrate the short term mineralization of dentin and enamel, and to investigate the effects of sodium beta-glycerophosphate (Na-beta-GPO4) on calcification in a serum-free, chemically-defined medium. The first mandibular molars at the bell stage dissected from 18-day-old mouse embryos were used as explants, which were cultured by an improved flotation method. Calcification of enamel in the 18-day-old embryonic molars occurred within the 6th day of culture. In another experiment, the molar germs were cultured in a serum-free, chemically-defined medium supplemented with 1, 5 and 10 mM Na-beta-GPO4. Promotion of tooth mineralization was recognizable at very low concentrations, such as 1 mM Na-beta-GPO4, in 18-day-old embryonic tooth germs. The culturing system reported here shortens the time required for dentin and enamel calcification to one half or one third of that reported previously and therefore should prove useful for examining regulations for cytodifferentiation and morphogenesis in tooth germs and the mineralization of dentin and enamel.     

Expression of E- and P-cadherin during tooth morphogenesis and cytodifferentiation of ameloblasts

 Cell-cell adhesion is fundamental in morphogenesis and is known to be mediated by several groups of cell adhesion molecules. Cadherins are a group of such molecules involved in the Ca²⁺-dependent cell-cell adhesion mechanism and are found in most kinds of tissue. In this study using indirect immunofluorescence microscopy, we analyzed the distribution of two kinds of cadherins, E- and P-cadherin, in developing tooth germs. In the molar tooth germs at the early bud stage, marginal cells of the epithelial tooth bud expressed both E- and P-cadherin, whereas central cells expressed only E-cadherin. At the cap stage, in addition to the cells of the inner and outer enamel epithelium, which outline the enamal organ, cells of the enamel knot, which is thought to control tooth morphogenesis, strongly expressed P-cadherin. The expression of P-cadherin was prominent in the inner enamel epithelium during the early to mid bell stage, and was also evident in the non-dividing cell masses at future cusp tips, which are the so-called secondary enamel knots. In the tooth germ at the late bell stage when the cells of the inner enamel epithelium began to polarize to differentiate into ameloblasts, the polarizing ameloblasts lost P-cadherin and strongly expressed E-cadherin. However, E-cadherin was also lost from polarized ameloblasts at later stages. The stratum intermedium and the stellate reticulum were E-cadherin positive from the bell stage onward even at the stages when the ameloblasts became E-cadherin negative again. These results suggest that the differential expression of E- and P-cadherin during morphogenetic stages plays a role in the regulation of tooth morphogenesis, whereas alteration of E-cadherin expression during later stages of tooth development is related to differentiation and function of the ameloblasts and other cells supporting amelogenesis. Accepted: 29 January 1998     

A spontaneous mutation: amelogenesis imperfecta with cysts in rats

Amelogenesis imperfecta (AI) is an inherited dental disease of enamel formation in humans, and there are various phenotypes due to the combination of enamel quality and quantity. We encountered four female IGS rats with spontaneous AI including odontogenic cysts in the incisor teeth. Histopathologically, in the incisors of the rats, the enamel organ was disorganized with the remaining enamel matrix residing within the enamel space. The expanding cysts derived from the enamel organ were formed in the periosteal connective tissue on the labial side. At the bottom of the tooth germs, the precursor cells of the epithelial root sheath were arranged regularly and the enamel organs were preserved to the same degree as those of normal rats. In the molar teeth of the affected rats an enamel matrix remained on the neck and crown of the erupted teeth; however, no abnormality was observed at the tooth root. Although an animal model of AI has been developed from mutants of the SHR-SP rat strain, the present cases represent another potential model of the disease because of the differences in the way the enamel matured and the odontogenic cyst formation in the incisors.     

Production of monoclonal antibodies against tooth germs of hamster or rat by in vitro immunization

The in vitro immunization procedure for the production of monoclonal antibodies has several advantages over the in vivo procedure; e.g. it requires a much smaller amount of immunogen and only a few days are required for immunization. However, no in vitro immunization procedure for the production of monoclonal antibodies against tooth germs has been reported. By means of in vitro immunization, we tried to produce monoclonal antibodies against the components of tooth germs which would be useful for immunohistochemical investigation of the development of tooth germs. Spleen cells of mice were immunized with a homogenate of molar tooth germs from hamsters or rats. The hybridomas produced were screened by immunohistochemical examination of paraffin sections of tooth germs. We obtained five monoclonal antibodies reacting with the dental tissues; ameloblasts, odontoblasts, enamel, dentin, predentin, and dental sac. One of them reacted specifically with only ameloblasts and one with ameloblasts and enamel, but the others are not specific for individual dental tissues. The results demonstrate that in vitro immunization is a satisfactory method for producing monoclonal antibodies useful for immunohistochemical investigation of tooth germs.     

Abortive secretion of an enamel matrix in the inner enamel epithelial cells during an enameloid formation in the gar-pike, Lepisosteus oculatus (Holostei, Actinopterygii)

The tooth in the gar-pike, Lepisosteus oculatus, an actinopterygian fish, is characterized by the occurrence of both enamel and enameloid, the former covering the tooth shaft and the latter, the tooth cap. Our previous research demonstrated that the enamel in this species was, as in the lungfish, immunoreactive for amelogenin, indicating its homologous nature with the mammalian tooth enamel, whereas the enameloid was completely immunonegative. The present study demonstrates that, during the early maturation stage of the enameloid formation, the inner enamel epithelial cells (IEECs) synthesize through a well-developed Golgi apparatus a fine-granular substance which is intensely immunoreactive for amelogenin. This substance was accumulated in a large saccule extended in a suprabasal zone of the cell; we were unable to find any morphological sign indicating a connection of the substance with the enameloid matrix. The abortive secretion of the enamel matrix-like substance in the IEEC during an enameloid formation was considered to be an instance of rudimental enamel formation. In the gar-pike, the synthesis of amelogenin in the IEEC has been demonstrated to occur independently from that of the enameloid matrix. The present findings demonstrate a prominent difference between the tooth enamel and enameloid.     

Modulation of activin/bone morphogenetic protein signaling by follistatin is required for the morphogenesis of mouse molar teeth.

Teeth form as ectodermal appendages, and their morphogenesis is regulated by conserved signaling pathways. The shape of the tooth crown results from growth and folding of inner dental epithelium, and the cusp patterning is regulated by transient signaling centers, the enamel knots. Several signal proteins in the transforming growth factor-beta (TGF beta) superfamily are required for tooth development. Follistatin is an extracellular inhibitor of TGF beta signaling. To investigate the roles of follistatin during tooth development, we analyzed in detail the expression patterns of follistatin, activin beta A, as well as Bmp2, Bmp4, and Bmp7 during tooth morphogenesis. We also examined the tooth phenotypes of follistatin knockout mice and of transgenic mice overexpressing follistatin in the epithelium under the keratin 14 (K14) promoter. The folding of the dental epithelium was aberrant in the molars of follistatin knockout mice, and the cusps were shallow with reduced cell proliferation and lack of anteroposterior polarization. The functions of both primary and secondary enamel knots were apparently disturbed. In K14-follistatin transgenic mice, the molar cusp pattern was also seriously affected (although different from the follistatin knockouts) and the occlusal surfaces of the molars were whorled. Their enamel was prematurely worn. In addition, all of the third molars were missing. Our results indicate that follistatin regulates morphogenesis and shaping of the tooth crown. We propose that finely tuned antagonistic effects between follistatin and TGF beta superfamily signals are critical for enamel knot formation and function, as well as for patterning of tooth cusps.     

A simple model for enamel fracture from margin cracks

We present results of in situ fracture tests on extracted human molar teeth showing failure by margin cracking. The teeth are mounted into an epoxy base and loaded with a rod indenter capped with a Teflon insert, as representative of food modulus. In situ observations of cracks extending longitudinally upward from the cervical margins are recorded in real time with a video camera. The cracks appear above some threshold and grow steadily within the enamel coat toward the occlusal surface in a configuration reminiscent of channel-like cracks in brittle films. Substantially higher loading is required to delaminate the enamel from the dentin, attesting to the resilience of the tooth structure. A simplistic fracture mechanics analysis is applied to determine the critical load relation for traversal of the margin crack along the full length of the side wall. The capacity of any given tooth to resist failure by margin cracking is predicted to increase with greater enamel thickness and cuspal radius. Implications in relation to dentistry and evolutionary biology are briefly considered.     

Hunter-Schreger Band patterns in human tooth enamel

Using light microscopy, we examined Hunter‐Schreger Band (HSB) patterns on the axial and occlusal/incisal surfaces of 160 human teeth, sectioned in both the buccolingual and mesiodistal planes. We found regional variations in HSB packing densities (number of HSBs per mm of amelodentinal junction length) and patterns throughout the crown of each class of tooth (maxillary and mandibular: incisor, canine, premolar, and molar) examined. HSB packing densities were greatest in areas where functional and occlusal loads are greatest, such as the occlusal surfaces of posterior teeth and the incisal regions of incisors and canines. From this it is possible to infer that the behaviour of ameloblasts forming enamel prisms during amelogenesis is guided by genetic/evolutionary controls that act to increase the fracture and wear resistance of human tooth enamel. It is suggested that HSB packing densities and patterns are important in modern clinical dental treatments, such as the bonding of adhesive restorations to enamel, and in the development of conditions, such as abfraction and cracked tooth syndrome.     

2D mapping of texture and lattice parameters of dental enamel

We have used synchrotron X-ray diffraction to study the texture and the change in lattice parameter as a function of position in a cross section of human dental enamel. Our study is the first to map changes in preferred orientation and lattice parameter as a function of position within enamel across a whole tooth section with such high resolution. Synchrotron X-ray diffraction with a micro-focused beam spot was used to collect two-dimensional (2D) diffraction images at 150 microm spatial resolution over the entire tooth crown. Contour maps of the texture and lattice parameter distribution of the hydroxyapatite phase were produced from Rietveld refinement of diffraction patterns generated by azimuthally sectioning and integrating the 2D images. The 002 Debye ring showed the largest variation in intensity. This variation is indicative of preferred orientation. Areas of high crystallite alignment on the tooth cusps match the expected biting surfaces. Additionally we found a large variation in lattice parameter when travelling from the enamel surface to the enamel-dentine junction. We believe this to be due to a change in the chemical composition within the tooth. The results provide a new insight on the texture and lattice parameter profiles within enamel.     

The initial process of enamel prism arrangement and its relation to the Hunter-Schreger bands in dog teeth

The three-dimensional architecture of enamel prisms at early stages of enamel formation and its spatial relationship to the Hunter-Schreger bands were examined in canine tooth germs by light and electron microscopy. In serial semithin sections of demineralized tooth germs tangential to the enamel-dentin junction, a straight row of enamel prisms was depicted along the longitudinal tooth axis at the level of the enamel-dentin junction and then their three-dimensional arrangement was reconstructed using computer software. The spatial arrangement of the groups of enamel rods oriented in specific sideward directions was also reconstructed in deep layers of the enamel. Initially, all enamel prisms were parallel to perpendicular toward the enamel-dentin junction, but at 10μm from the enamel-dentin junction, some small specks, or groups of enamel prisms--tilting to the right or the left--emerged as small islands. In each speck of enamel prism, the inclined prisms were uniformly oriented in a sideward direction and gradually expanded their boundary until merging with the neighboring specks inclined in the same direction. Consequently, at 50μm from the enamel-dentin junction, the group of enamel prisms oriented either to the right or the left formed alternately arranged horizontal belt-like zones, corresponding to the parazone or the diazone of the Hunter-Schreger bands. Reversed images of scanning electron-micrographs of the exposed surfaces of the developing enamel revealed round and bulb-like profiles of Tomes' processes at early amelogenesis and its changes into a characteristic structure combined with flat secretory and enclosing nonsecretory faces that dictated the orientation of corresponding enamel prisms. The results suggest that the groups of enamel prisms oriented in sideward directions first appear as small island-like specks near the enamel-dentin junction, which later merge and form alternating horizontal belt-like zones as a consequence of morphological changes of the Tomes' processes. However, the mechanisms whereby the functional grouping of secretory ameloblasts with similarly oriented Tomes' processes is induced are yet to be determined.