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.     

The lateral enamel lamina--component of tooth primordia in selected mammalian species

The lateral enamel lamina (LEL) is a part of the enamel organ, which is probably not involved in tooth formation. It represents, besides the "stalk" of the tooth primordium, a second interconnection between enamel organ and oral epithelium or vestibular lamina. We detected the LEL in the sheep (Ovis aries), the dolphin (Stenella attenuata), and the vole (Microtus agrestis) by light microscopy and computer-aided three-dimensional reconstruction. The LEL could be found in cap to bell stage tooth primordia, most clearly in slowly developing tooth germs. LEL-like structures have been furthermore described or depicted in tooth germs of the mouse, the elk (Alces alces), the dugong (Dugong dugong), the elephant (Loxodonta africana), and the human. Probably it is a part of all mammalian tooth primordia that undergoes regression during morphogenesis of the enamel organ. As a reducing structure, it should be considered in studies of tooth development.     

Predicting negligence in female sterilization failure using time interval to sterilization failure: analysis of 131 cases

BACKGROUND: Sterilization failure due to 'tubal non-occlusion' or 'wrong structure sterilization' is considered negligent, whereas 'spontaneous tubal recanalization' or 'fistula formation' is considered non-negligent. We examined whether interval to pregnancy failure was predictive of a negligent rather non-negligent failure mechanism. We aim to test this hypothesis in a selected population series of known mechanisms of sterilization failure and their time interval to failure. METHODS: Analyses of 131 failed sterilizations pooled from UK (NHS Litigation Authority, Medical Protection Society and our hospital), Australia and a qualitative systematic review. RESULTS: We identified 88 negligent and 43 non-negligent sterilization failures. Filshie and ring methods failed earlier than diathermy and Pomeroy methods. Sterilization failure occurred significantly earlier in negligent than non-negligent failure mechanisms [median failure intervals 7.0 versus 12.0 months; Hazard ratio (2.35 95% CI 1.31-4.21)]. Knowing that sterilization failure occurred early, increased the probability that the failure mechanism was likely to be negligent rather than non-negligent. CONCLUSIONS: A short interval to failure is suggestive of a negligent failure mechanism. There is less certainty in the predictive value of longer time intervals on the mechanism of failure due to a paucity of cases. A national register of failed sterilizations that have been systematically investigated is needed to improve our understanding of negligent and non-negligent failure mechanisms.     

Predicting the outcome of renal replacement therapy in severe acute renal failure

Continuous renal replacement therapy (CRRT), such as continuous venovenous hemofiltration, has theoretical advantages over intermittent hemodialysis (IHD) that are related to cardiorespiratory stability, metabolic control, and fluid balance allowing nutritional supplementation. However, retrospective and controlled studies fail to show these advantages because of comorbidity associated with triage to CRRT. To compare outcomes using IHD versus CRRT, we applied published risk stratification models (Cleveland Clinic Foundation, Lohr index, and APACHE II) to the 349 patients with acute renal failure requiring renal replacement therapy at University of Michigan over the 2 year period including 1995 and 1996. The Cleveland Clinic Foundation model best predicted overall mortality, but our CRRT patients had excess, unpredicted mortality that was particularly prominent in the lower risk categories. The Lohr clinical score predicted mortality less accurately but also was associated with higher, unpredicted mortality at lower risk scores among the CRRT patients. APACHE II scores did not predict mortality very well among IHD, CRRT, or the combined group of patients. We conclude that the need for CRRT itself predicts mortality over and above that included in published risk models. Either CRRT is associated with some unidentified morbidity (e.g., treatment associated infection) or, more likely, triage to CRRT is associated with as yet unspecified comorbidity not detected in existing risk stratification schemes. It will be important to address these issues in any future studies evaluating outcome or comparing renal replacement therapy modalities among patients with severe acute renal failure.     

Degradation of enamel matrix proteins in porcine secretory enamel

To elucidate the progressive disappearance of 25 kDa amelogenin occurring in a narrow space near the surface of enamel, the alkaline soluble fraction which contained 80% of the total proteins was extracted from a newly formed porcine enamel. When this fraction was incubated with the addition of Ca ions in an in vitro system, the degradation of the coexisting amelogenin and enamelin occurred without activation during the incubation period. Although the fraction contained mainly two kinds of metalloproteinases, 56 kDa and 61 kDa gelatinolytic, and 41 kDa and 46 kDa caseinolytic activities, it was demonstrated on amelogenin enzymography that the caseinolytic one was concerned with the conversion of the 25 kDa amelogenin into the 20 kDa amelogenin. The protein distribution of the newly formed enamel indicated that the metalloproteinases degraded the coexisting enamelin and amelogenin imperfectly. Nevertheless, during the next developing stage they demonstrated their full activities. It is suspected that these activities are regulated by Ca ions, which may be increased by a cascade system.     

Origin of enamel prisms and Hunter-Schreger bands in reptilian enamel

Enamel prisms in mammalian enamel structure including human are considered to have originated in the reptilian enamel. The author has reported the original pattern of enamel prisms and of Hunter-Schreger bands in fossil reptiles of Mosasaurus sp. and Phytosaurus sp. The origin of the patterns of crystallite groups appear to arise in the fossil reptiles. Mosasaurus sp. had an island pattern and Phytosaurus sp. had a slender pattern. These two types are of various sizes, and are formed by variable numbers of ameloblasts. The number of ameloblasts involved in the formation of the crystallite group may change during amelogenesis. The present study observed the initial crystallite groups as enamel islands, in the surface of the early secretory stage of amelogenesis of living Alligator mississippiensis. These crystallite groups become bigger and more uniform in size during amelogenesis, being formed by several ameloblasts in the maturation stage. The border of crystallite groups observed appear as the lamella in human enamel. These crystallite groups may develop into the enamel prisms and Hunter-Schreger bands.     

Predicting in vivo failure of pseudoelastic NiTi devices under low cycle, high amplitude fatigue

Due to the large reversible strains achievable through the stress-induced austenite-martensite phase transformation in NiTi alloys, NiTi has replaced stainless steel in the majority of large-strain biomedical applications such as root canal enlargement. However, the pseudoelasticity of NiTi is currently overshadowed by the short fatigue life of NiTi wires used in this low cycle (200-2000 rpm), high amplitude (epsilon(a) > 2.5%) application, resulting in in vivo fracture or premature retirement of otherwise reusable NiTi-based wire devices. In this study, the failure of pseudoelastic 55.8 wt % Ni-Ti wire is investigated experimentally, as a function of experimental parameters that include the clinically relevant regime. The effects of radius of curvature, angle of curvature, wire diameter, strain amplitude, cyclic frequency, volume under strain, and specific heat of the surrounding environmental fluid are considered systematically. These data indicate that the lifetime or cycles to failure N(f) of a rotating NiTi wire can be predicted via a modified Coffin-Manson relation that is a strong function of both strain amplitude and volume under strain, and a weaker function of frequency and fluid specific heat. The resulting quantitative relation can be used to predict useful device lifetime under clinically relevant conditions and thereby reduce incidences of in vivo failure.     

Organization of crystals in enamel

It has been variously suggested that the organic matrix associated with the mineral phase of enamel is present as either calcified fibrils, central dark lines, peripheral sheaths around hexagonal crystals, or organic ghosts apparently contained within crystal profiles. The most consistent findings confirm the crystal ghost conception. Grid decalcification of nearly mature sectioned enamel and staining revealed hollow, noncrystalline structures whose external measurements were statistically identical to those of the dissolved crystallites, but with internal measurements too small to accommodate the crystallites. To explain these apparent ghosts in view of the incompatibility of ghosts with crystal structure, it has been proposed that the crystallites are not hexagonal in cross-sections and the hexagonal appearance is due to projections of parallelepiped-shaped crystallite segments with cut surfaces that are rhomboidal in shape. Material on the surface of such profiles would project as if it were contained within the profile. Hexagonal forms could not be demonstrated in isolated crystallites examined by transmission electron microscopy, high-resolution scanning electron microscopy, and replicas made of the isolated crystallite preparations examined by transmission electron microscopy. Existing evidence does not rule out the possibility that the noncrystalline profiles represent stain drawn into the holes left by the dissolved crystallites as a result of high capillarity forces.     

The orientation of the enamel prisms at the enamel surface

The angulation that prisms obtain with the surface in human permanent teeth was analysed on broken enamel by means of the scanning electron microscope. In the cervical region the prisms end nearly perpendicular towards the surface, while the angle becomes more and more acute towards the occlusal region of the crown. In the cuspal area the deviation from the perpendicular direction of the prisms approaches 70 degrees. The size discrepancy between the inner and the outer surface of the enamel mantle can be explained by the more or less angulated position of the prisms.     

The ultrastructure of the enamel organ related to enamel formation

The ultrastructure of the cells of the enamel organ related to enamel formation was studied using the lower incisors of adult male rats. In the region of enamel deposition, stratum intermedium cells are stabilized by a system of intercellular bridges and intracellular fibrils. The mitochondria in these cells are positioned toward the extracellular channels through which any direct intercellular exchange between the capillaries and ameloblasts must occur. Tentatively, the mitochondrial arrangement is considered to be related to the movements of electrolytes and water across the capillary-ameloblast interval. In the region of transition, enamel deposition ceases and the ergastoplasm of the ameloblasts is removed, apparently by cytosegresomes, with an accompanying reduction in the height of the ameloblasts. Here, vesicles containing stippled material are infrequent compared to their occurrence in ameloblasts concerned with enamel deposition. Other vesicles, characteristically found in ameloblasts related to maturing enamel, first appear in the transition region and seem to originate from the cell membrane abutting on the enamel. In the region of maturation, cytosomes are common in the Golgi region whereas vesicles and mitochondria predominate in the distal ends of the ameloblasts. The papillary cells contain an unusually large number of mitochondria, elaborate microvilli and vesicles, which suggests that these cells are extremely active, presumably in the movement of materials related to enamel maturation. The changes in structure of the papillary cells, which occur concomitantly with those of the ameloblasts during enamel formation, are indicative of interrelated functional changes and strongly support the concept of ameloblasts and papillary cells acting together as a functional unit.     

Hereditary enamel hypoplasia

An extensive family with a total of forty–one individuals who are either affected or reputed to be affected with hereditary hypoplasia of the enamel is presented. The distribution of patients, which spans four generations in this pedigree, appears to be consistent with X–linked dominant inheritance of the disorder. Considerable variability in the severity of affection was observed among both male and female patients. Whereas in the case of the latter, random X–chromosome inactivation may provide an explanation, in the case of the former another mechanism may have to be sought.     

Overexpression of TRAP in the enamel matrix does not alter the enamel structural hierarchy

The secreted, full-length amelogenin is the dominant protein of the forming enamel organ. As enamel mineralization progresses, amelogenin is quickly subjected to proteolytic activity, and eliminated from the enamel environment. Mature enamel contains only traces of structural proteins, including enamelin and the sheath protein ameloblastin. In addition, a proteolytic fragment of amelogenin, known as the tyrosine-rich amelogenin peptide or TRAP, is present in low but isolatable quantities. By overexpressing TRAP during enamel development we sought to determine if such overexpression would result in structural alterations to the mature enamel. We reasoned that overexpressing a protein associated with enamel maturation, at an inappropriate developmental stage, would result in alterations to the enamel protein assembly and hence, alterations in enamel structure and morphology. As judged by transmission and scanning electron microscopy, the enamel formed by overexpressing TRAP showed little morphological differences when compared to the enamel of normal nontransgenic animals. Based on scanning electron-microscopic images, there was modest hypomineralization evident in the interrod enamel of the TRAP-overexpressing animals. However, this finding was inconsistent and inconsequential from a structural and functional perspective. From these results it appears that additional amounts of TRAP protein in the immature enamel matrix are not sufficient to alter the properties of the enamel extracellular matrix to an extent that the hierarchical structure of mature enamel is altered.     

Pitted enamel hypoplasia in tuberous sclerosis

Thirty patients with tuberous sclerosis (from 29 different families) were examined for evidence of macroscopically visible pitted enamel hypoplasia. Of 23 patients with permanent teeth, 11 (48%) showed multiple enamel pits (mean 4.6 pits, range 3-9), but none were seen in six patients with deciduous teeth. Five of 563 controls (0.88%) had similar pitted enamel hypoplasia. Simple dental examination may be a useful adjunct in the assessment of patients with permanent teeth when a diagnosis of tuberous sclerosis is being considered, but is less likely to be helpful in the pre-school child.