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Focal stacks are an alternative spatial arrangement of enamel rods within the inner enamel of mandibular mouse incisors where short rows comprised of 2–45 enamel rods are nestled at the side of much longer rows, both sharing the same rod tilt directed mesially or laterally. The significance of focal stacks to enamel function is unknown, but their high frequency in transverse sections (30% of all rows) suggests that they serve some purpose beyond representing an oddity of enamel development. In this study, we characterized the spatial distribution of focal stacks in random transverse sections relative to different regions of the inner enamel and to different locations across enamel thickness. The curving dentinoenamel junction (DEJ) in transverse sections complicated spatial distribution analyses, and a technique was developed to “unbend” the curving DEJ allowing for more linear quantitative analyses to be carried out. The data indicated that on average there were 36 ± 7 focal stacks located variably within the inner enamel in any given transverse section. Consistent with area distributions, focal stacks were four times more frequent in the lateral region (53%) and twice as frequent in the mesial region (33%) compared to the central region (14%). Focal stacks were equally split by tilt (52% mesial vs. 48% lateral, not significant), but those having a mesial tilt were more frequently encountered in the lateral and central regions (2:1) and those having a lateral tilt were more numerous in the mesial region (1:3). Focal stacks having a mesial tilt were longer on average compared to those having a lateral tilt (7.5 ± 5.6 vs. 5.9 ± 4.0 rods per row, p, The objective of this study is to ascertain if current techniques in 2D spatial point pattern analyses can be applied to mouse incisor enamel to establish if the packing of rows across the thickness of the inner enamel is spatially random or shows clustering or some type of repeating pattern. Knowledge of the spatial distribution characteristics of rows of enamel rods could provide new insights into how rows of ameloblasts initially form at the start of enamel formation.

The hepatitis C virus (HCV) epidemic was forecasted through 2030 for 17 countries in Africa, Asia, Europe, Latin America and the Middle East, and interventions for achieving the Global Health Sector Strategy on viral hepatitis targets-"WHO Targets" (65% reduction in HCV-related deaths, 90% reduction in new infections and 90% of infections diagnosed by 2030) were considered. Scaling up treatment and diagnosis rates over time would be required to achieve these targets in all but one country, even with the introduction of high SVR therapies. The scenarios developed to achieve the WHO Targets in all countries studied assumed the implementation of national policies to prevent new infections and to diagnose current infections through screening.

Background ENAM mutations cause autosomal dominant or recessive amelogenesis imperfecta (AI) and show a dose effect: enamel malformations are more severe or only penetrant when both ENAM alleles are defective. Methods Whole exome sequences of recruited AI probands were initially screened for mutations in known AI candidate genes. Sanger sequencing was used to confirm sequence variations and their segregation with the disease phenotype. The co-occurrence of ENAM and LAMA3 mutations in one family raised the possibility of digenic inheritance. Enamel formed in Enam+/+ Ambn+/+ , Enam+/- , Ambn+/- , and Enam+/- Ambn+/- mice was characterized by dissection and backscattered scanning electron microscopy (bSEM). Results ENAM mutations segregating with AI in five families were identified. Two novel ENAM frameshift mutations were identified. A single-nucleotide duplication (c.395dupA/p.Pro133Alafs*13) replaced amino acids 133-1142 with a 12 amino acid (ATTKAAFEAAIT*) sequence, and a single-nucleotide deletion (c.2763delT/p.Asp921Glufs*32) replaced amino acids 921-1142 with 31 amino acids (ESSPQQASYQAKETAQRRGKAKTLLEMMCPR*). Three families were heterozygous for a previously reported single-nucleotide ENAM deletion (c.588+1delG/p.Asn197Ilefs*81). One of these families also harbored a heterozygous LAMA3 mutation (c.1559G>A/p.Cys520Tyr) that cosegregated with both the AI phenotype and the ENAM mutation. In mice, Ambn+/- maxillary incisors were normal. Ambn+/- molars were also normal, except for minor surface roughness. Ambn+/- mandibular incisors were sometimes chalky and showed minor chipping. Enam+/- incisor enamel was thinner than normal with ectopic mineral deposited laterally. Enam+/- molars were sometimes chalky and rough surfaced. Enam+/- Ambn+/- enamel was thin and rough, in part due to ectopic mineralization, but also underwent accelerated attrition. Conclusion Novel ENAM mutations causing AI were identified, raising to 22 the number of ENAM variations known to cause AI. The severity of the enamel phenotype in Enam+/- Ambn+/- double heterozygous mice is caused by composite digenic effects. Digenic inheritance should be explored as a cause of AI in humans.

Background Ameloblastin (AMBN) is a secreted matrix protein that is critical for the formation of dental enamel and is enamel‐specific with respect to its essential functions. Biallelic AMBN defects cause non‐syndromic autosomal recessive amelogenesis imperfecta. Homozygous Ambn mutant mice expressing an internally truncated AMBN protein deposit only a soft mineral crust on the surface of dentin. Methods We characterized a family with hypoplastic amelogenesis imperfecta caused by AMBN compound heterozygous mutations (c.1061T>C; p.Leu354Pro/ c.1340C>T; p.Pro447Leu). We generated and characterized Ambn knockout/NLS‐lacZ (Ambn lacZ/lacZ) knockin mice. Results No AMBN protein was detected using immunohistochemistry in null mice. ß‐galactosidase activity was specific for ameloblasts in incisors and molars, and islands of cells along developing molar roots. Ambn lacZ/lacZ 7‐week incisors and unerupted (D14) first molars showed extreme enamel surface roughness. No abnormalities were observed in dentin mineralization or in nondental tissues. Ameloblasts in the Ambn lacZ/lacZ mice were unable to initiate appositional growth and started to degenerate and deposit ectopic mineral. No layer of initial enamel ribbons formed in the Ambn lacZ/lacZ mice, but pockets of amelogenin accumulated on the dentin surface along the ameloblast distal membrane and within the enamel organ epithelia (EOE). NLS‐lacZ signal was positive in the epididymis and nasal epithelium, but negative in ovary, oviduct, uterus, prostate, seminal vesicles, testis, submandibular salivary gland, kidney, liver, bladder, and bone, even after 15 hr of incubation with X‐gal. Conclusions Ameloblastin is critical for the initiation of enamel ribbon formation, and its absence results in pathological mineralization within the enamel organ epithelia.

WOS: 000458956100004 PubMed ID: 30506946 Amelogenesis imperfecta (AI) is a collection of isolated (non-syndromic) inherited diseases affecting dental enamel formation or a clinical phenotype in syndromic conditions. We characterized three consanguineous AI families with generalized irregular hypoplastic enamel with rapid attrition that perfectly segregated with homozygous defects in a novel gene: RELT that is a member of the tumor necrosis factor receptor superfamily (TNFRSF). RNAscope in situ hybridization of wild-type mouse molars and incisors showed specific Relt mRNA expression by secretory stage ameloblasts and by odontoblasts. Relt(-/-) mice generated by CRISPR/Cas9 exhibited incisor and molar enamel malformations. Relt(-/-) enamel had a rough surface and underwent rapid attrition. Normally unmineralized spaces in the deep enamel near the dentino-enamel junction (DEJ) were as highly mineralized as the adjacent enamel, which likely altered the mechanical properties of the DEJ. Phylogenetic analyses showed the existence of selective pressure on RELT gene outside of tooth development, indicating that the human condition may be syndromic, which possibly explains the history of small stature and severe childhood infections in two of the probands. Knowing a TNFRSF member is critical during the secretory stage of enamel formation advances our understanding of amelogenesis and improves our ability to diagnose human conditions featuring enamel malformations. National Institute of Dental and Craniofacial Research [DE015846DE016276]; National Research Foundation of Korea [NRF-2017R1A2A2A05069281NRF-2018R1A5A2024418] National Institute of Dental and Craniofacial Research, Grant/Award Number: DE015846DE016276; National Research Foundation of Korea, Grant/Award Number: NRF-2017R1A2A2A05069281NRF-2018R1A5A2024418

Introduction Dental enamel is comprised of highly organized, oriented apatite crystals, but how they form is unclear. Methods We used focused ion beam (FIB) scanning electron microscopy (SEM) to investigate early enamel formation in 7-week-old incisors from wild-type, Amelx-/-, and Enam-/- C56BL/6 mice. FIB surface imaging scans thicker samples so that the thin enamel ribbons do not pass as readily out of the plane of section, and generates serial images by a mill and view approach for computerized tomography. Results We demonstrate that wild-type enamel ribbons initiate on dentin mineral on the sides and tips of mineralized collagen fibers, and extend in clusters from dentin to the ameloblast membrane. The clustering suggested that groups of enamel ribbons were initiated and then extended by finger-like membrane processes as they retracted back into the ameloblast distal membrane. These findings support the conclusions that no organic nucleator is necessary for enamel ribbon initiation (although no ribbons form in the Enam-/- mice), and that enamel ribbons elongate along the ameloblast membrane and orient in the direction of its retrograde movement. Tomographic reconstruction videos revealed a complex of ameloblast membrane processes and invaginations associated with intercellular junctions proximal to the mineralization front and also highlighted interproximal extracellular enamel matrix accumulations proximal to the interrod growth sites, which we propose are important for expanding the interrod matrix and extending interrod enamel ribbons. Amelx-/- mice produce oriented enamel ribbons, but the ribbons fuse into fan-like structures. The matrix does not expand sufficiently to support formation of the Tomes process or establish rod and interrod organization. Conclusion Amelogenin does not directly nucleate, shape, or orient enamel ribbons, but separates and supports the enamel ribbons, and expands the enamel matrix to accommodate continued ribbon elongation, retrograde ameloblast movement, and rod/interrod organization.

Background Amelogenin is required for normal enamel formation and is the most abundant protein in developing enamel. Methods Amelx+/+, Amelx+/−, and Amelx−/− molars and incisors from C57BL/6 mice were characterized using RT-PCR, Western blotting, dissecting and light microscopy, immunohistochemistry (IHC), transmission electron microscopy (TEM), scanning electron microscopy (SEM), backscattered SEM (bSEM), nanohardness testing, and X-ray diffraction. Results No amelogenin protein was detected by Western blot analyses of enamel extracts from Amelx−/− mice. Amelx−/− incisor enamel averaged 20.3 ± 3.3 μm in thickness, or only 1/6th that of the wild type (122.3 ± 7.9 μm). Amelx−/− incisor enamel nanohardness was 1.6 Gpa, less than half that of wild-type enamel (3.6 Gpa). Amelx+/− incisors and molars showed vertical banding patterns unique to each tooth. IHC detected no amelogenin in Amelx−/− enamel and varied levels of amelogenin in Amelx+/− incisors, which correlated positively with enamel thickness, strongly supporting lyonization as the cause of the variations in enamel thickness. TEM analyses showed characteristic mineral ribbons in Amelx+/+ and Amelx−/− enamel extending from mineralized dentin collagen to the ameloblast. The Amelx−/− enamel ribbons were not well separated by matrix and appeared to fuse together, forming plates. X-ray diffraction determined that the predominant mineral in Amelx−/− enamel is octacalcium phosphate (not calcium hydroxyapatite). Amelx−/− ameloblasts were similar to wild-type ameloblasts except no Tomes’ processes extended into the thin enamel. Amelx−/− and Amelx+/− molars both showed calcified nodules on their occlusal surfaces. Histology of D5 and D11 developing molars showed nodules forming during the maturation stage. Conclusion Amelogenin forms a resorbable matrix that separates and supports, but does not shape early secretory-stage enamel ribbons. Amelogenin may facilitate the conversion of enamel ribbons into hydroxyapatite by inhibiting the formation of octacalcium phosphate. Amelogenin is necessary for thickening the enamel layer, which helps maintain ribbon organization and development and maintenance of the Tomes’ process.

Matrix metalloproteinase 20 (MMP20) and kallikrein-related peptidase 4 (KLK4) are secreted proteinases that are essential for proper dental enamel formation. We characterized and compared enamel formed in wild-type, Mmp20 (-/-), Klk4 (-/-), Mmp20 (+/-) Klk4 (+/-), and Mmp20 (-/-) Klk4 (-/-) mice using dissecting and light microscopy, backscattered scanning electron microscopy (bSEM), SEM, microcomputed tomography (μCT), and energy-dispersive X-ray analysis (EDX). Following eruption, fractures were observed on Mmp20 (-/-), Klk4 (-/-), Mmp20 (+/-) Klk4 (+/-), and Mmp20 (-/-) Klk4 (-/-) molars. Failure of the enamel in the Mmp20 (+/-) Klk4 (+/-) molars was unexpected and suggested that digenic effects could contribute to the etiology of amelogenesis imperfecta in humans. Micro-CT analyses of hemimandibles demonstrated significantly reduced high-density enamel volume in the Mmp20 (-/-) and Klk4 (-/-) mice relative to the wild-type, which was further reduced in Mmp20 (-/-) Klk4 (-/-) mice. bSEM images of 7-week Mmp20 (-/-) and Mmp20 (-/-) Klk4 (-/-) mandibular incisors showed rough, pitted enamel surfaces with numerous indentations and protruding nodules. The Mmp20 (+/-) and Mmp20 (+/-) Klk4 (+/-) incisors showed prominent, evenly spaced, horizontal ridges that were more distinct in Mmp20 (+/-) Klk4 (+/-) incisors relative to Mmp20 (+/-) incisors due to the darkening of the valleys between the ridges. In cross sections, the Mmp20 (-/-) and Mmp20 (-/-) Klk4 (-/-) exhibited three distinct layers. The outer layer exhibited a disturbed elemental composition and an irregular enamel surface covered with nodules. The Mmp20 null enamel was apparently unable to withstand the sheer forces associated with eruption and separated from dentin during development. Cells invaded the cracks and interposed between the dentin and enamel layers. MMP20 and KLK4 serve overlapping and complementary functions to harden enamel by removing protein, but MMP20 potentially serves multiple additional functions necessary for the adherence of enamel to dentin, the release of intercellular protein stores into the enamel matrix, the retreat of ameloblasts to facilitate thickening of the enamel layer, and the timely transition of ameloblasts to maturation.

Truncation mutations in FAM83H (family with sequence similarity 83, member H) cause autosomal dominant hypocalcified amelogenesis imperfecta (ADHCAI), but little is known about FAM83H function and the pathogenesis of ADHCAI. We recruited three ADHCAI families and identified two novel (p.Gln457*; p.Lys639*) and one previously documented (p.Q452*) disease-causing FAM83H mutations. We generated and characterized Fam83h-knockout/lacZ-knockin mice. Surprisingly, enamel thickness, density, Knoop hardness, morphology, and prism patterns were similar in Fam83h (+/+), Fam83h (+/-), and Fam83h (-/-) mice. The histology of ameloblasts in all stages of development, in both molars and incisors, was virtually identical in all three genotypes and showed no signs of pathology, although the Fam83h (-/-) mice usually died after 2 weeks and rarely survived to 7 weeks. LacZ expression in the knockin mice was used to report Fam83h expression in the epithelial tissues of many organs, notably in skin and hair follicles, which manifested a disease phenotype. Pull-down studies determined that FAM83H dimerizes through its N-terminal phospholipase D-like (PLD-like) domain and identified potential FAM83H interacting proteins. Casein kinase 1 (CK1) interacts with the FAM83H PLD-like domain via an F(270)-X-X-X-F(274)-X-X-X-F(278) motif. CK1 can phosphorylate FAM83H in vitro, and many phosphorylation sites were identified in the FAM83H C-terminus. Truncation of FAM83H alters its subcellular localization and that of CK1. Our results support the conclusion that FAM83H is not necessary for proper dental enamel formation in mice, but may act as a scaffold protein that localizes CK1. ADHCAI is likely caused by gain-of-function effects mediated by truncated FAM83H, which potentially mislocalizes CK1 as part of its pathological mechanism.

Nonsyndromic dentin defects classified as type II dentin dysplasia and types II and III dentinogenesis imperfecta are caused by mutations in DSPP (dentin sialophosphoprotein). Most reported disease‐causing DSPP mutations occur within the repetitive DPP (dentin phosphoprotein) coding sequence. We characterized the DPP sequences of five probands with inherited dentin defects using single molecule real‐time (SMRT) DNA sequencing. Eight of the 10 sequences matched previously reported DPP length haplotypes and two were novel. Alignment with known DPP sequences showed 32 indels arranged in 36 different patterns. Sixteen of the 32 indels were not represented in more than one haplotype. The 25 haplotypes with confirmed indels were aligned to generate a tree that describes how the length variations might have evolved. Some indels were independently generated in multiple lines. A previously reported disease‐causing DSPP mutation in Family 1 was confirmed and its position clarified (c.3135delC; p.Ser1045Argfs*269). A novel frameshift mutation (c.3504_3508dup; p.Asp1170Alafs*146) caused the dentin defects in Family 2. A COL1A2 (c.2027G>A or p.Gly676Asp) missense mutation, discovered by whole‐exome sequencing, caused the dentin defects in Family 3. We conclude that SMRT sequencing characterizes the DPP repeat region without cloning and can improve our understanding of normal and pathological length variations in DSPP alleles.

Defects in WDR72 (WD repeat-containing protein 72) cause autosomal recessive hypomaturation amelogenesis imperfecta. We generated and characterized Wdr72-knockout/lacZ-knockin mice to investigate the role of WDR72 in enamel formation. In all analyses, enamel formed by Wdr72 heterozygous mice was indistinguishable from wild-type enamel. Without WDR72, enamel mineral density increased early during the maturation stage but soon arrested. The null enamel layer was only a tenth as hard as wild-type enamel and underwent rapid attrition following eruption. Despite the failure to further mineralize enamel deposited during the secretory stage, ectopic mineral formed on the enamel surface and penetrated into the overlying soft tissue. While the proteins in the enamel matrix were successfully degraded, the digestion products remained inside the enamel. Interactome analysis of WDR72 protein revealed potential interactions with clathrin-associated proteins and involvement in ameloblastic endocytosis. The maturation stage mandibular incisor enamel did not stain with methyl red, indicating that the enamel did not acidify beneath ruffle-ended ameloblasts. Attachment of maturation ameloblasts to the enamel layer was weakened, and SLC24A4, a critical ameloblast calcium transporter, did not localize appropriately along the ameloblast distal membrane. Fewer blood vessels were observed in the papillary layer supporting ameloblasts. Specific WDR72 expression by maturation stage ameloblasts explained the observation that enamel thickness and rod decussation (established during the secretory stage) are normal in the Wdr72 null mice. We conclude that WDR72 serves critical functions specifically during the maturation stage of amelogenesis and is required for both protein removal and enamel mineralization.

FAM20C, also known as Golgi Casein Kinase (G-CK), is proposed to be the archetype for a family of secreted kinases that phosphorylate target proteins in the Golgi and in extracellular matrices, but FAM20C serving an extracellular function is controversial. FAM20C phosphorylates secretory calcium-binding phosphoproteins (SCPPs), which are associated with the evolution of biomineralization in vertebrates. Current models of biomineralization assume SCPP proteins are secreted as phosphoproteins and their phosphates are essential for protein conformation and function. It would be a radical departure from current theories if proteins in mineralizing matrices were dephosphorylated as part of the mineralization mechanism and rephosphorylated in the extracellular milieu by FAM20C using ATP. To see if such mechanisms are possible in the formation of dental enamel, we tested the hypothesis that FAM20C is secreted by ameloblasts and accumulates in the enamel extracellular matrix during tooth development. FAM20C localization was determined by immunohistochemistry in Day 5 mouse incisors and molars and by Western blot analyses of proteins extracted from pig enamel organ epithelia (EOE) and enamel shavings. FAM20C localized intracellularly within ameloblasts and odontoblasts in a pattern consistent with Golgi localization. Western blots detected FAM20C in the EOE extracts but not in the enamel matrix. We conclude that FAM20C is not a constituent of the enamel extracellular matrix and functions intracellularly within ameloblasts.

One factor that seems to exert major control over enamel development is the matrix proteins that help to promote and stabilize initial deposits of mineral as the enamel layer thickens by appositional growth (1). However, these same proteins and/or their fragments unless removed are believed capable of ‘poisoning’ the secondary mineralization process that is associated with volumetric growth of apatite crystallites later during the maturation stage of amelogenesis (2–4). This notion is based largely on findings that (i) the degradation and seemingly total removal of enamel matrix proteins (amelogenin, enamelin, and ameloblastin) normally occurs before the crystallites start undergoing their rapid volumetric expansion (2); and (ii) excess amounts of residual enamel matrix proteins/fragments, in some cases supplemented with serum proteins such as albumin, are usually detected at sites where hypomineralization defects occur in the enamel (2, 5). Undermineralized enamel typically appears chalky and white, and the affected areas abrade easily when these teeth erupt into occlusion. This can have disastrous consequences in cases of genetic mutations where the entire crowns of teeth are covered with poor-quality enamel. This substantially reduces the functional longevity of the affected dentition. The exact method(s)/pathway(s) by which matrix proteins are removed from maturing enamel in vivo are presently only partially defined (4). This is understandable considering the unique nature of such an event within the context of the way in which mineralization normally occurs in all other mammalian hard tissues. In bone and dentin, for example, the formative cells (osteoblasts and odontoblasts) intentionally create a delay between the time when matrix proteins are secreted and the time when mineral is deposited in association with them. This delay, as osteoid and predentin, is necessary to allow tropocollagen molecules, secreted by the cells, to aggregate into fibrils and cross-link themselves for stability (6). It is only once the fibrils have reached an optimal size and become spatially organized, in some cases into very complex lamellar patterns, that specific non-collagenous components and mineral are added onto the fibrils (7). The numerous, small, plate-like crystallites of hydroxyapatite appearing along the mineralizing front in these hard tissues do not change much in size thereafter, and the supporting scaffold of collagen fibrils does not change unless resorbed by osteoclasts/odontoclasts. In contrast, the main matrix protein in developing enamel, amelogenin, is globular in nature and has a propensity to self-aggregate/assemble at the submicroscopic/nanoscale level into spheres, ovals, and/or ribbons (1, 8, 9) rather than to form rigid cross-linked fibrils as occurs in the collagen-based hard tissues. Forming and mature enamel show considerable structural complexity in terms of rod and inter-rod organizations, as viewed by both light and electron microscopy (4), but this complexity results from the complicated three-dimensional (3D) arrangements of constituent and highly elongated hydroxyapatite crystallites, rather than from the organic matrix itself where constituent proteins start degrading incrementally within minutes or hours from when they are secreted (10). These may be among the reasons why enamel does not linger for any length of time as an unmineralized pre-enamel, but instead has mineral deposited simultaneously with exocytosis of matrix proteins from the ameloblasts (11). The lack of structural complexity and rigidity within the organic framework of enamel is also probably an attribute that makes enamel matrix proteins accessible and easier to break down and remove from the enamel layer during development. Current evidence suggests that at least two pathways are utilized to remove proteins from the enamel layer during development. One involves the sequential secretion of two proteinases possessing vastly different properties and cleavage-site specificities (12, 13). The first proteinase to appear in enamel development is matrix metalloproteinase 20 (MMP20) which is released extracellularly along with amelogenin, enamelin, and ameloblastin as the enamel layer thickens by appositional growth during the secretory stage (14, 15). Matrix metalloproteinase 20 serves an important function to initiate certain key hydrolytic cleavages on the enamel matrix proteins within minutes or hours of their secretion. This results in the formation of variably sized protein fragments that gradually become the primary organic component in the extracellular milieu as development proceeds (4). There is increasing evidence that these fragments have physical properties which are different from their original secretory forms, and that the fragments, which in many cases show distinct distribution patterns within the enamel layer, may serve functions that are different from their intact parental forms (2, 4, 16–18). Ameloblasts switch their emphasis from producing MMP20 to expressing and secreting the second, more active, proteinase called kallikrein-related peptidase 4 (KLK4) as they undergo postsecretory transition into maturation (19–21). This hydrolase is active throughout the maturation stage and appears to further degrade fragments of amelogenin, enamelin, and ameloblastin so they can be removed and thus provide the space needed for the volumetric expansion of crystallites seeded and lengthened in the secretory stage (22). The second pathway that is suspected to be important for the removal of proteins/fragments from the enamel layer is based on a currently ill-defined biological process called ‘resorptive activity of ameloblasts’ (4, 23–26). In this process, organic material within the enamel layer flows in a direction towards the enamel organ and comes into contact with the apical surfaces of ameloblasts that lie against and cover the enamel surface (4). From here, the residual organic material is taken up into endocytotic vesicles and transferred to the lysosomal system of ameloblasts for final degradation (4, 27). There has never been much direct evidence for such a garbage-disposal mechanism operating during the maturation stage, but it is clear that modulating maturation-stage ameloblasts contain numerous vacuoles and dense bodies suggestive of high endocytotic activity (28– 31). These cells are also capable of moving peroxidase and other fluid-phase tracers from the cell surface into lysosomes (29, 30, 32). Additionally, enamel will remain undermineralized and infused with higher-than-normal amounts of residual protein if the enamel organ cells are damaged or removed shortly after the maturation stage begins (33). The purpose of this study was to gain insight into the relationships between the quantity of mineral and protein that co-exist as the enamel forms and matures. In particular, we were most interested in defining the time in amelogenesis when the maximum amounts of matrix proteins are present within the enamel layer and the quantity of mineral associated with the proteins at this time. We also wanted to determine the amount of proteins lost up to the point where the enamel accumulates enough mineral to render it physically hardened. Another goal was to look at what happens to mineral and bulk protein content when there is loss of function of either one of the two non-amelogenins (enamelin and ameloblastin), believed to be important for mineral induction and cell adhesion, or of either one of the two secreted enamel proteinases (MMP20 and KLK4) that are known to be responsible for postsecretory processing and degradation of enamel matrix proteins.

Kallikrein-related peptidase 4 (KLK4) is critical for proper dental enamel formation. Klk4 null mice, and humans with two defective KLK4 alleles have obvious enamel defects, with no other apparent phenotype. KLK4 mRNA or protein is reported to be present in tissues besides teeth, including prostate, ovary, kidney, liver, and salivary gland. In this study we used the Klk4 knockout/NLS-lacZ knockin mouse to assay Klk4 expression using β-galactosidase histochemistry. Incubations for 5 h were used to detect KLK4 expression with minimal endogenous background, while overnight incubations susceptible to false positives were used to look for trace KLK4 expression. Developing maxillary molars at postnatal days 5, 6, 7, 8, and 14, developing mandibular incisors at postnatal day 14, and selected non-dental tissues from adult wild-type and Klk4(lacZ/lacZ) mice were examined by X-gal histochemistry. After 5 h of incubation, X-gal staining was observed specifically in the nuclei of maturation-stage ameloblasts in molars and incisors from Klk4(lacZ/lacZ) mice and was detected weakly in the nuclei of salivary gland ducts and in patches of prostate epithelia. We conclude that KLK4 is predominantly a tooth-specific protease with low expression in submandibular salivary gland and prostate, and with no detectable expression in liver, kidney, testis, ovary, oviduct, epididymis, and vas deferens.

The crowns of matrix metalloproteinase 20 (Mmp20) null mice fracture at the dentino-enamel junction (DEJ), whereas the crowns of kallikrein-related peptidase 4 (Klk4) null mice fracture in the deep enamel just above the DEJ. We used backscatter scanning electron microscopy to assess enamel mineralization in incisors from 9-wk-old wild-type, Klk4 null, and Mmp20 null mice, and in developing pig molars. We observed a line of hypermineralization along the DEJ in developing wild-type mouse and pig teeth. This line was discernible from the early secretory stage until the enamel in the maturation stage reached a similar density. The line was apparent in Klk4 null mice, but absent in Mmp20 null mice. Enamel in the Klk4 null mice matured normally at the surface, but was progressively less mineralized with depth. Enamel in the Mmp20 null mice formed as a mineral bilayer, with neither layer looking like true enamel. The most superficial mineral layer expanded during the maturation stage and formed irregular surface nodules. A surprising finding was the observation of electron backscatter from mid-maturation wild-type ameloblasts, which we attributed to the accumulation and release of iron. We conclude that enamel breaks in the deep enamel of Klk4 null mice because of decreasing enamel maturation with depth, and at the DEJ in Mmp20 null mice because of hypomineralization at the DEJ.

The three major proteins in the enamel matrix of developing teeth are amelogenin, enamelin, and ameloblastin (1). These proteins are expressed from related genes, all belonging to the secretory calcium-binding phosphoprotein (SCPP) family (2). Intact enamelin is a large, proline-rich, phosphorylated glycoprotein. Secreted human enamelin has 1,099 amino acids. Unlike amelogenin and ameloblastin, the enamelin primary RNA transcript does not undergo alternative splicing, so there is only a single isoform of enamelin. In pigs, secreted enamelin is a 186-kDa protein that localizes to the mineralization front along the secretory surface of ameloblasts (3). Matrix metalloproteinase 20 (MMP20) rapidly degrades it into many fragments, one of which is a stable 32-kDa domain that accumulates within the matrix (4, 5). Expression of the enamelin gene (ENAM, 4q13) is largely restricted to developing teeth. The human enamelin expressed sequence tag (EST) profile ({"type":"entrez-nucleotide","attrs":{"text":"Hs667018","term_id":"317654759","term_text":"HS667018"}}Hs667018) from normal tissues (which does not include developing teeth) lists only 10 enamelin cDNAs out of 3,360,307 characterized. No tissue has more than two enamelin ESTs, which is a pattern that is characteristic of trace expression. Defects in ENAM contribute to the aetiology of non-syndromic forms of amelogenesis imperfecta (AI), a collection of inherited diseases featuring enamel malformations as the only phenotype (6–8). ENAM mutations cause AI that generally follows an **autosomal-dominant pattern of inheritance (9), with variations in both penetrance and expressivity. When a single ENAM allele is defective, the phenotype can be non-penetrant (10), or be manifested as enamel pits (11), horizontal grooves (12), or generalized thin enamel (13). There is also a dose effect: when both ENAM alleles are defective the enamel is extremely thin or non-existent (11). Enamelin gene and deduced amino acid sequences are available from species representing the main groups of tetrapods (14). There are 25 unchanging amino acid positions when the 36 known mammalian enamelin protein sequences are aligned with the few non-mammalian sequences that go back to amphibians (frog). Sites of enamelin post-translational modifications are highly conserved, including several potential and known phosphorylation and N-linked glycosylation sites, as well as six cysteines believed to form disulphide bridges (14). Evolutionary analyses have not only identified functionally critical positions in the enamelin protein, but they have also demonstrated that all of the major secretory-stage enamel proteins serve necessary functions only in tooth formation and specifically in dental enamel formation. Genes encoding enamel matrix proteins have degenerated into pseudogenes in multiple toothless or enamel-less species that descended from ancestors with teeth covered by enamel. The genes encoding amelogenin, ameloblastin, and enamelin have degenerated in birds (14, 15) and in toothless (baleen) whales (16). A functional enamelin gene is absent in enamel-less (Kogia) whales (16) and in four different orders of placental mammals with toothless and/or enamel-less taxa (17). Genetic studies in humans strongly corroborate the evolutionary findings. ENAM defects in 10 different kindreds with non-syndromic AI have been reported. One of the cases is caused by the substitution of leucine with a highly conserved phosphoserine (p.S216L) (9). Enamelin knockout mice provide still more support for the tooth-specificity of enamelin function, as the phenotype of these mice is restricted exclusively to defects in the enamel layer (18–20). The genetically engineered Enam knockout/lacZ knockin mouse model has also provided strong support for ameloblast specificity of enamelin expression through X-gal histostaining for nuclear-localized β-galactosidase activity to mark tissues that normally express enamelin. To date, nuclear staining has been detected only in ameloblasts in developing teeth (18). Enam knockout mice also provide a means to investigate normal and pathological enamel formation. Enamelin knockouts demonstrate that enamelin is not only required for the deposition of tooth enamel, but it is also necessary to maintain the ameloblast phenotype, as is the case for ameloblastin (21). Degeneration of the ameloblast layer and the subsequent appearance of cyst-like enamel organ-based structures suggested that enamelin and ameloblastin are cell-adhesion proteins. However, as no enamel layer forms in the Enam and ameloblastin (Ambn) knockout mice, ameloblasts could fail to adhere to the unnatural underlying surface even if their attachment apparatus was intact and did not normally contain enamelin or ameloblastin. RGD sequences are found in enamel proteins from some species, but they are not a conserved feature, and mouse enamelin and ameloblastin both lack these potential integrin-binding motifs. Recombinant ameloblastin shows some affinity for heparin (22) and fibronectin (23) in vitro, but these molecular partners have not been localized in ameloblasts or shown to be important for ameloblast adhesion in vivo. The observations that enamelin is critical for dental enamel formation and for maintenance of the ameloblast phenotype emphasize the dynamic roles played by ameloblasts besides simply creating an extracellular space and environment conducive for mineral deposition. In this study we characterized ameloblasts in Enam heterozygous and null mice by documenting the extent of apoptosis in maxillary first molars starting in the secretory stage at day 5 and continuing until tooth eruption at day 17. We demonstrated that ameloblasts in restricted areas of developing teeth undergo apoptosis when enamelin is completely missing, and that reducing enamelin production by one-half, as occurs in heterozygous mice, may result in more apoptosis later in crown development, despite the fact that normal enamel eventually forms on these teeth.

Matrix metalloproteinase 20 (MMP20) and kallikrein-related peptidase 4 (KLK4) are thought to be necessary to clear proteins from the enamel matrix of developing teeth. We characterized Mmp20 and Klk4 null mice to better understand their roles in matrix degradation and removal. Histological examination showed retained organic matrix in Mmp20, Klk4, and Mmp20/Klk4 double-null mouse enamel matrix, but not in the wild-type. X-gal histostaining of Mmp20 null mice heterozygous for the Klk4 knockout/lacZ knockin showed that Klk4 is expressed normally in the Mmp20 null background. This finding was corroborated by zymogram and western blotting, which discovered a 40-kDa protease induced in the maturation stage of Mmp20 null mice. Proteins were extracted from secretory-stage or maturation-stage maxillary first molars from wild-type, Mmp20 null, Klk4 null, and Mmp20/Klk4 double-null mice and were analyzed by SDS-PAGE and western blotting. Only intact amelogenins and ameloblastin were observed in secretory-stage enamel of Mmp20 null mice, whereas the secretory-stage matrix from Klk4 null mice was identical to the matrix from wild-type mice. More residual matrix was observed in the double-null mice compared with either of the single-null mice. These results support the importance of MMP20 during the secretory stage and of KLK4 during the maturation stage and show there is only limited functional redundancy for these enzymes.

Kallikrein-related peptidase 4 (KLK4) is a glycosylated serine protease that functions in the maturation (hardening) of dental enamel. Pig and mouse KLK4 contain three potential N-glycosylation sites. We isolated KLK4 from developing pig and mouse molars and characterized their N-glycosylations. N-glycans were enzymatically released by digestion with N-glycosidase F and fluorescently labeled with 2-aminobenzoic acid. Normal-phase high-performance liquid chromatography (NP-HPLC) revealed N-glycans with no, or with one, two, or three sialic acid attachments in pig KLK4 and with no, or with one or two sialic acid attachments in mouse KLK4. The labeled N-glycans were digested with sialidase to generate the asialo N-glycan cores that were fractionated by reverse-phase HPLC, and their retention times were compared with similarly labeled glycan standards. The purified cores were characterized by mass spectrometric and monosaccharide composition analyses. We determined that pig and mouse KLK4 have NA2 and NA2F biantennary N-glycan cores. The pig triantennary core is NA3. The mouse triantennary core is NA3 with a fucose connected by an α1–6 linkage, indicating that it is attached to the first N-acetyglucosamine (NA3F). We conclude that pig KLK4 has NA2, NA2F, and NA3 N-glycan cores with no, or with one, two, or three sialic acids. Mouse KLK4 has NA2, NA2F, and NA3F N-glycan cores with no, or with one or two sialic acids.

Dentin sialophosphoprotein (DSPP) mutations cause dentin dysplasia type II (DD-II) and dentinogenesis imperfecta types II and III (DGI-II and DGI-III, respectively). We identified two kindreds with DGI-II who exhibited vertical bands of hypoplastic enamel. Both families had a previously reported DSPP mutation that segregated with the disease phenotype. Oral photographs and dental radiographs of four affected and one unaffected participant in one family and of the proband in the second family were used to document the dental phenotypes. We aligned the 33 unique allelic DSPP sequences showing variable patterns of insertions and deletions (indels), generated a merged dentin phosphoprotein (DPP) sequence that includes sequences from all DSPP length haplotypes, and mapped the known DSPP mutations in this context. Analyses of the DSPP sequence changes and their probable effects on protein expression, as well as published findings of the dental phenotype in Dspp null mice, support the hypothesis that all DSPP mutations cause pathology through dominant-negative effects. Noting that Dspp is transiently expressed by mouse pre-ameloblasts during formation of the dentino-enamel junction, we hypothesize that DSPP dominant-negative effects potentially cause cellular pathology in pre-ameloblasts that, in turn, causes enamel defects. We conclude that enamel defects can be part of the dental phenotype caused by DSPP mutations, although DSPP is not critical for dental enamel formation.

Previously, mutational analyses identified six disease-causing mutations in 24 amelogenesis imperfecta (AI) kindreds. We have since expanded the number of AI kindreds to 39, and performed mutation analyses covering the coding exons and adjoining intron sequences for the six proven AI candidate genes [amelogenin (AMELX), enamelin (ENAM), family with sequence similarity 83, member H (FAM83H), WD repeat containing domain 72 (WDR72), enamelysin (MMP20), and kallikrein-related peptidase 4 (KLK4)] and for ameloblastin (AMBN) (a suspected candidate gene). All four of the X-linked AI families (100%) had disease-causing mutations in AMELX, suggesting that AMELX is the only gene involved in the aetiology of X-linked AI. Eighteen families showed an autosomal-dominant pattern of inheritance. Disease-causing mutations were identified in 12 (67%): eight in FAM83H, and four in ENAM. No FAM83H coding-region or splice-junction mutations were identified in three probands with autosomal-dominant hypocalcification AI (ADHCAI), suggesting that a second gene may contribute to the aetiology of ADHCAI. Six families showed an autosomal-recessive pattern of inheritance, and disease-causing mutations were identified in three (50%): two in MMP20, and one in WDR72. No disease-causing mutations were found in 11 families with only one affected member. We conclude that mutation analyses of the current candidate genes for AI have about a 50% chance of identifying the disease-causing mutation in a given kindred.

Myocardial infarction (MI) is a leading cause of death and disabilities. Inflammatory cells play a vital role in the process of postinfarction remodeling and repair. Inflammatory cell infiltration into the infarct site can be monitored using T-weighted MRI following an intravenous administration of iron oxide particles. In this study, various doses of micrometer-sized iron oxide particles (1.1–14.5 μg Fe/g body weight) were injected into the mouse blood stream before a surgical induction of MI. Cardiac MRIs were performed at 3, 7, 14, and 21 days postinfarction to monitor the signal attenuation at the infarct site. A dose-dependent phenomenon of signal attenuation was observed at the infarct site, with a higher dose leading to a darker signal. The study suggests an optimal temporal window for monitoring iron oxide particles-labeled inflammatory cell infiltration to the infarct site using MRI. The dose-dependent signal attenuation also indicates an optimal iron oxide dose of approximately 9.1–14.5 μg Fe/g body weight. A lower dose cannot differentiate the signal attenuation, whereas a higher dose would cause significant artifacts. This iron oxide-enhanced MRI technique can potentially be used to monitor cell migration and infiltration at the pathological site or to confirm any cellular response following some specific treatment strategies. Magn Reson Med, 2011. © 2011 Wiley Periodicals, Inc.

How stem cells promote myocardial repair in myocardial infarction (MI) is not well understood. The purpose of this study was to noninvasively monitor and quantify mesenchymal stem cells (MSC) from bone marrow to MI sites using magnetic resonance imaging (MRI). MSC were dual-labeled with an enhanced green fluorescent protein and micrometer-sized iron oxide particles prior to intra-bone marrow transplantation into the tibial medullary space of C57Bl/6 mice. Micrometer-sized iron oxide particles labeling caused signal attenuation in T(2)*-weighted MRI and thus allowed noninvasive cell tracking. Longitudinal MRI demonstrated MSC infiltration into MI sites over time. Fluorescence from both micrometer-sized iron oxide particles and enhanced green fluorescent protein in histology validated the presence of dual-labeled cells at MI sites. This study demonstrated that MSC traffic to MI sites can be noninvasively monitored in MRI by labeling cells with micrometer-sized iron oxide particles. The dual-labeled MSC at MI sites maintained their capability of proliferation and differentiation. The dual-labeling, intra-bone marrow transplantation, and MRI cell tracking provided a unique approach for investigating stem cells' roles in the post-MI healing process. This technique can potentially be applied to monitor possible effects on stem cell mobilization caused by given treatment strategies.

Manganese ions (Mn(2+) ) enter viable myocardial cells via voltage-gated calcium channels. Because of its shortening of T(1) and its relatively long half-life in cells, Mn(2+) can serve as an intracellular molecular contrast agent to study indirect calcium influx into the myocardium. One major concern in using Mn(2+) is its sensitivity over a limited range of concentrations employing T(1)-weighted images for visualization, which limits its potential in quantitative techniques. Therefore, this study assessed the implementation of a T(1) mapping method for cardiac manganese-enhanced MRI to enable a quantitative estimate of the influx of Mn(2+) over a wide range of concentrations in male Sprague-Dawley rats. This MRI method was used to compare the relationship between T(1) changes in the heart as a function of myocardium and blood Mn(2+) levels. Results showed a biphasic relationship between ΔR(1) and the total Mn(2+) infusion dose. Nonlinear relationships were observed between the total Mn(2+) infusion dose versus blood levels and left ventricular free wall ΔR(1) . At low blood levels of Mn(2+) , there was proportionally less cardiac enhancement seen than at higher levels of blood Mn(2+) . We hypothesize that Mn(2+) blood levels increase as a result of rate-limiting excretion by the liver and kidneys at these higher Mn(2+) doses.

Prolonged ischemia causes cellular necrosis and myocardial infarction (MI) via intracellular calcium (Ca2+) overload. Manganese-enhanced MRI indirectly assesses Ca2+ influx movement in vivo as manganese (Mn2+) is a Ca2+ analog. To characterize myocardial Mn2+ efflux properties, T1-mapping manganese-enhanced MRI studies were performed on adult male C57Bl/6 mice in which Ca2+ efflux was altered using pharmacological intervention agents or MI-inducing surgery. Results showed that (1) Mn2+ efflux rate increased exponentially with increasing Mn2+ doses; (2) SEA0400 (a sodium–calcium exchanger inhibitor) decreased the rate of Mn2+ efflux; and (3) dobutamine (a positive inotropic agent) increased the Mn2+ efflux rate. A novel analysis technique also delineated regional features in the MI mice, which showed an increased Mn2+ efflux rate in the necrosed and peri-infarcted tissue zones. The T1-mapping manganese-enhanced MRI technique characterized alterations in myocardial Mn2+ efflux rates following both pharmacologic intervention and an acute MI. The Mn2+ efflux results were consistent with those in ex vivo studies showing an increased Ca2+ concentration under similar conditions. Thus, T1-mapping manganese-enhanced MRI has the potential to indirectly identify and quantify intracellular Ca2+ handling in the peri-infarcted tissue zones, which may reveal salvageable tissue in the post-MI myocardium. Magn Reson Med, 2010. © 2010 Wiley-Liss, Inc.

Dentin sialophosphoprotein (Dspp) is critical for proper dentin biomineralization because genetic defects in DSPP cause dentin dysplasia type II and dentinogenesis imperfecta types II and III. Dspp is processed by proteases into smaller subunits; the initial cleavage releases dentin phosphoprotein (Dpp). We incubated fluorescence resonance energy transfer (FRET) peptides containing the amino acid context of the Dpp cleavage site (YEFDGKSMQGDDPN, designated Dspp-FRET) or a mutant version of that context (YEFDGKSIEGDDPN, designated mutDspp-FRET) with BMP-1, MEP1A, MEP1B, MMP-2, MMP-8, MMP-9, MT1-MMP, MT3-MMP, Klk4, MMP-20, plasmin, or porcine Dpp and characterized the peptide cleavage products. Only BMP-1, MEP1A, and MEP1B cleaved Dspp-FRET at the G–D peptide bond that releases Dpp from Dspp in vivo. We isolated Dspp proteoglycan from dentin power and incubated it with the three enzymes that cleaved Dspp-FRET at the G–D bond. In each case, the released Dpp domain was isolated, and its N-terminus was characterized by Edman degradation. BMP-1 and MEP1A both cleaved native Dspp at the correct site to generate Dpp, making both these enzymes prime candidates for the protease that cleaves Dspp in vivo. MEP1B was able to degrade Dpp when the Dpp was at sufficiently high concentration to deplete free calcium ion concentration. Immunohistochemistry of developing porcine molars demonstrated that astacins are expressed by odontoblasts, a result that is consistent with RT-PCR analyses. We conclude that during odontogenesis, astacins in the predentin matrix cleave Dspp before the DDPN sequence at the N-terminus of Dpp to release Dpp from the parent Dspp protein. © 2011 American Society for Bone and Mineral Research.

Lee T-H, Hu C-C, Lee S-S, Chou M-Y, Chang Y-C. Cytotoxicity of chlorhexidine on human osteoblastic cells is related to intracellular glutathione levels. International Endodontic Journal, 43, 430–435, 2010. Abstract Aim To evaluate the mechanisms of cytotoxicity of chlorhexidine (CHX) in human osteoblastic cells in vitro. Methodology Cytotoxicity, cell proliferation and collagen synthesis assays were performed to elucidate the toxic effects of CHX on the human osteoblastic cell line U2OS. To determine whether glutathione (GSH) levels were important in the cytotoxicity of CHX, cells were pre-treated with 2-oxothiazolidine-4-carboxylic acid (OTZ) to boost GSH levels or buthionine sulfoximine (BSO) to deplete GSH. Results CHX demonstrated a cytotoxic effect to U2OS cells in a dose-dependent manner (P

Micrometer-sized iron oxide particles (MPIO) are a more sensitive MRI contrast agent for tracking cell migration compared to ultrasmall iron oxide particles. This study investigated the temporal relationship between inflammation and tissue remodeling due to myocardial infarction (MI) using MPIO-enhanced MRI. C57Bl/6 mice received an intravenous MPIO injection for cell labeling, followed by a surgically induced MI seven days later (n=7). For controls, two groups underwent either sham-operated surgery without inducing an MI post-MPIO injection (n=7) or MI surgery without MPIO injection (n=6). The MRIs performed post-MI showed significant signal attenuation around the MI site for the mice that received an intravenous MPIO injection for cell labeling, followed by a surgically induced MI seven days later, compared to the two control groups (P

Although the nonamelogenin proteins, ameloblastin and enamelin, are both low-abundance and rapidly degrading components of forming enamel, they seem to serve essential developmental functions, as suggested by findings that an enamel layer fails to appear on teeth of mice genetically engineered to produce either a truncated form of ameloblastin (exons 5 and 6 deleted) or no enamelin at all (null). The purpose of this study was to characterize, by direct micro weighing, changes in enamel mineralization occurring on maxillary and mandibular incisors of mice bred for these alterations in nonamelogenin function (Ambn(+/+, +/-5,6, -5,6/-5,6), Enam(+/+, +/- ,-/-)). The results indicated similar changes to enamel-mineralization patterns within the altered genotypes, including significant decreases by as much as 50% in the mineral content of maturing enamel from heterozygous mice and the formation of a thin, crusty, and disorganized mineralized layer, rather than true enamel, on the labial (occlusal) surfaces of incisors and molars along with ectopic calcifications within enamel organ cells in Ambn(-5,6/-5,6) and Enam(-/-) homozygous mice. These findings confirm that both ameloblastin and enamelin are required by ameloblasts to create an enamel layer by appositional growth as well as to assist in achieving its unique high level of mineralization.

The sodium-calcium exchanger (NCX) is one of the transporters contributing to the control of intracellular calcium (Ca(2+)) concentration by normally mediating net Ca(2+) efflux. However, the reverse mode of the NCX can cause intracellular Ca(2+) concentration overload, which exacerbates the myocardial tissue injury resulting from ischemia. Although the NCX inhibitor SEA0400 has been shown to therapeutically reduce myocardial injury, no in vivo technique exists to monitor intracellular Ca(2+) fluctuations produced by this drug. Cardiac manganese-enhanced MRI (MEMRI) may indirectly assess Ca(2+) efflux by estimating changes in manganese (Mn(2+)) content in vivo, since Mn(2+) has been suggested as a surrogate marker for Ca(2+). This study used the MEMRI technique to examine the temporal features of cardiac Mn(2+) efflux by implementing a T(1)-mapping method and inhibiting the NCX with SEA0400. The change in (1)H(2)O longitudinal relaxation rate, Delta R(1), in the left ventricular free wall, was calculated at different time points following infusion of 190 nmol/g manganese chloride (MnCl(2)) in healthy adult male mice. The results showed 50% MEMRI signal attenuation at 3.4 +/- 0.6 h post-MnCl(2) infusion without drug intervention. Furthermore, treatment with 50 +/- 0.2 mg/kg of SEA0400 significantly reduced the rate of decrease in Delta R(1). At 4.9-5.9 h post-MnCl(2) infusion, the average Delta R(1) values for the two groups treated with SEA0400 were 2.46 +/- 0.29 and 1.72 +/- 0.24 s(-1) for 50 and 20 mg/kg doses, respectively, as compared to the value of 1.27 +/- 0.28 s(-1) for the control group. When this in vivo data were compared to ex vivo absolute manganese content data, the MEMRI T(1)-mapping technique was shown to effectively quantify Mn(2+) efflux rates in the myocardium. Therefore, combining an NCX inhibitor with MEMRI may be a useful technique for assessing Mn(2+) transport mechanisms and rates in vivo, which may reflect changes in Ca(2+) transport.

The activities of two proteases - enamelysin (MMP-20) and kallikrein 4 (KLK4) - are necessary for dental enamel to achieve its high degree of mineralization. We hypothesize that the selected enamel protein cleavage products which accumulate in the secretory-stage enamel matrix do so because they are resistant to further cleavage by MMP-20. Later, they are degraded by KLK4. The 32-kDa enamelin is the only domain of the parent protein that accumulates in the deeper enamel. Our objective was to identify the cleavage sites of 32-kDa enamelin that are generated by proteolysis with MMP-20 and KLK4. Enamelysin, KLK4, the major amelogenin isoform (P173), and the 32-kDa enamelin were isolated from developing porcine enamel. P173 and the 32-kDa enamelin were incubated with MMP-20 or KLK4 for up to 48 h. Then, the 32-kDa enamelin digestion products were fractionated by reverse-phase high-performance liquid chromatography (RP-HPLC) and characterized by Edman sequencing, amino acid analysis, and mass spectrometry. Enamelysin cleaved the 32-kDa enamelin only after it was deglycosylated. Kallikrein 4 digestion of the 32-kDa enamelin generated nine major cleavage products, six of which were successfully characterized. After 12 h of digestion with KLK4, all of the 32-kDa enamelin had been cleaved, but some cleavage products persisted after 48 h of digestion.

The ability of MnCl2 to enhance canine myocardium and to delineate ischemic areas is demonstrated. A dose–response curve was measured using T1 weighted images in 11 dogs. MnCl2 (36, 113, 360, and 3600 mol) was infused over a period of 3 min. Signal intensity increased linearly with MnCl2 dose. At 113 mol (10 mol/kg) the steady-state increase in intensity averaged 212 34%. No significant physiologic effects due to the infused MnCl2 were detected except at the highest dose where there was a cardiac depressive effect. Ischemia was induced by occluding the left anterior descending coronary artery in 5 dogs. At an infused dose of 113 mol, MnCl2 clearly demarcated the ischemic zone during coronary occlusion. Contrast enhancement in the ischemic zone was less than 30% compared with normal tissue (P < 0.03). In conclusion, the intracellular contrast agent MnCl2 enhances the canine heart and shows promise in detecting ischemia at doses that do not cause adverse cardiac effects. Magn Reson Med 54:196 –200, 2005. Published 2005 Wiley-Liss, Inc. †

Purpose To compare atherosclerotic plaque uptake of a first (ferumoxtran-10) and second generation (ferumoxytol) ultrasmall superparamagnetic iron oxide (USPIO) contrast agent with different pharmacokinetic/pharmacodynamic properties. Materials and Methods New Zealand White rabbits maintained on a high cholesterol/fat diet were subjected to balloon injury to the abdominal aorta. Ferumoxtran-10 or ferumoxytol (500 μmol/kg) was administered at 2, 4, and 8 weeks following injury. In vivo magnetic resonance imaging (MRI) was performed immediately prior to, immediately after, and 6 days post-contrast administration. Ex vivo MRI, histologic, and inductively coupled plasma-mass spectrometry (ICP-MS) iron analyses were performed on the excised vessels. Results The blood pool clearance of ferumoxytol (t½ ≤ 6 hours) was more rapid than that of ferumoxtran-10 (t½ ≤ 48 hours). Decreased in vivo MRI signal intensity in the abdominal aorta was observed at 2, 4, and 8 weeks following injury with ferumoxtran-10, but not with ferumoxytol. Consistent with these observations, ex vivo MRI signal intensity was decreased in the ferumoxtran-10 vessels, and to a lesser degree in the ferumoxytol vs. control vessels (− contrast agent). In contrast, in vitro macrophage phagocytosis of USPIO was four to six fold greater with ferumoxytol than with ferumoxtran-10. Additionally, the absolute iron content correlated with ex vivo MRI signal intensity in all vessels (r = −0.86, P < 0.0001). Conclusions These data suggest that the exposure period of atherosclerotic plaque to USPIO rather than the kinetics of the USPIO uptake by plaque alone is a critical criterion for experimental design of in vivo studies. J. Magn. Reson. Imaging 2005;21:432–442. © 2005 Wiley-Liss, Inc.

To the Editor: Amelogenesis imperfecta (AI) is a collection of non-syndromic inherited diseases featuring a variety of abnormal enamel phenotypes, patterns of inheritance, and causative genes. The term is also used to indicate the presence of an enamel phenotype in syndromes. Dental enamel is the most highly mineralized tissue in the body, lacks collagen, and is the product of specialized epithelial cells (ameloblasts). During the formation of enamel, ameloblasts rely upon many genes that function in other tissues, like laminin-332 and type XVII collagen. Mutations in such genes typically cause syndromes that include enamel malformations as a phenotype. Non-syndromic AI is caused by defects in genes that are more functionally specialized for dental enamel formation. Mutations in AMELX, ENAM, FAM83H, WDR72, KLK4 and MMP20 cause non-syndromic AI, and account for about half of all AI cases (1). The other causative genes are unknown. To date, no non-dental phenotype or history of an associated non-dental phenotype has been reported in any person with an AI-causing defect in any of these genes. Increasingly, AI is classified according to the pattern of inheritance and genetic etiology. Most cases of autosomal dominant amelogenesis imperfecta are caused by mutations in FAM83H (family with sequence similarity, member H; 8q24.3) or ENAM (enamelin; 4q21), the rest are of unknown etiology. FAM83H mutations cause autosomal dominant hypocalcified AI (OMIM: #130900), which has a distinctive enamel phenotype. At eruption, the enamel has a pale-cream color and is poorly mineralized. It is soft and undergoes rapid attrition and staining so that only the cervical enamel near the gingiva may remain. In contrast, ENAM mutations are dose dependent and cause local hypoplastic AI (OMIM: #104500), featuring regions of abnormally thin enamel or a nearly complete lack of enamel when both alleles are defective. Recently, we recruited a Caucasian family with autosomal dominant hypoplastic AI that falls within the range of phenotypes characteristic of ENAM mutations. The proband was a 6.5 years old Caucasian male. His mixed dentition displayed thin enamel that was only slightly more radiopaque than dentin and was secondarily affected with dental caries (Fig. 1a–c). The incisal edges of the primary anterior teeth were chipped. The permanent mandibular central incisors had recently erupted and exhibited particularly thin enamel over the incisal two thirds of the crown. This was not due to attrition, but was how the teeth formed. The proband's father had similar enamel defects (Fig. 1d–f) and reported that his teeth came in small, just like his baby teeth. His dentition was characterized as having small, well-separated teeth with extensive carious lesions and attrition. The remaining enamel surfaces were rough and pitted. Fig. 1 Oral photos and dental radiographs. (a–c) Frontal and maxillary and mandibular occlusal photos of the proband at age 6.5 years. Note the chips on the incisal edges of primary anterior teeth and that the enamel is particularly thin on the incisal ... The pedigree indicated an autosomal dominant pattern of inheritance (Fig. 2a). Mutational analyses identified a novel heterozygous frameshift mutation in exon 4 of ENAM (c.107delA; g.2979delA; p.Asn36Ilefs56) in both the proband and his father. The frameshift occurs in the coding region for the signal peptide and would have precluded synthesis of the entire secreted protein (Fig. 2c). This sequence variation is not listed among the known polymorphisms in the current single nucleotide polymorphism database (dbSNP, National Center for Biotechnology Information). Fig. 2 Identifying the AI-causing ENAM mutation. (a) Pedigree showing the autosomal dominant pattern of inheritance. A dot indicates individuals who were examined and contributed DNA. (b) DNA sequencing chromatograms showing the single nucleotide deletion (c.107delA; ... This AI-causing ENAM mutation is the 12th to be reported (Fig. S1). To help clinicians identify autosomal dominant AI patients with probable ENAM mutations based upon their dental phenotypes, previously published oral photographs of patients with defined mutations in a single ENAM allele are provided in Figs S2 and S3. When only one ENAM allele is defective, the enamel malformations vary in severity, but rarely show a complete lack of penetrance. In its mildest form, the enamel defects are well-circumscribed enamel pits, often arranged in horizontal lines. With increasing severity, the enamel defects appear as horizontal grooves, usually in the cervical third of the crown, or as slightly thin enamel that transitions abruptly to very thin enamel with a border that runs horizontally around the crown. When both ENAM alleles are defective, the enamel layer is either completely absent or appears as a very thin mineral layer that only partially covers the crown (Fig. S4). The International Classification of Diseases lists AI under hereditary disturbances in tooth structure, not elsewhere classified (K00.5) (2). As AI is a diverse collection of diseases caused by more than six genes that can be transmitted in an autosomal dominant, autosomal recessive, or X-linked pattern and can be mistaken for enamel malformations associated with non-dental manifestations, a more definitive diagnosis is warranted when practicable. When the pattern of inheritance and genotype–phenotype correlations implicate a single gene as being the probable cause, mutational analyses should be considered to establish a specific diagnosis.

Kallikrein-4 (KLK4) is a serine proteinase believed to be important in the normal development of dental enamel. We isolated native KLK4 from developing pig enamel and expressed four recombinant forms. Pig KLK4 was expressed in bacteria with and without the propeptide, and in two eukaryotic systems. Recombinant pig KLK4 was secreted as a zymogen by '293' cells and purified. The proKLK4 was activated in vitro by thermolysin and recombinant pig enamelysin, but not by native KLK4. These results were confirmed using a fluorescent peptide analog of the KLK4 propeptide-enzyme junction. Native KLK4 appears as a doublet at 37 kDa and 34 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Removal of N-linked oligosaccharides by digestion with deglycosidase-F reduced the doublet to a single band at approximately 28 kDa, demonstrating that the active enzyme is glycosylated, and that the 37 kDa and 34 kDa forms differ only in their number of glycosylations. Deglycosylation was also associated with a loss of proteolytic activity. We digested recombinant pig amelogenin with native KLK4 and characterized the cleavage products by N-terminal sequencing and mass spectrometry. Eleven cleavage sites in the amelogenin protein were identified, demonstrating that KLK4 degrades amelogenin and is likely to participate in the degradation of enamel proteins in vivo.

Proteolytic processing and degradation of enamel matrix proteins appears to be an essential feature of dental enamel formation. The source and character of proteolytic activity in the enamel matrix of developing teeth changes as enamel formation progresses. Two proteinases have been isolated from the extracellular enamel matrix of developing teeth: enamelysin (MMP-20), a matrix metalloproteinase, and kallikrein-4 (KLK4), a serine proteinase. Here, we ask if the expression of MMP-20 and KLK4 correlate with the stage-associated changes in the digestion of enamel proteins. Using in situ hybridization, we localized MMP-20 and KLK4 mRNA in mouse maxillary first molars on postnatal days 1, 2, 3, 5, 6, 7, 9, 11, and 14. Enamelysin signal was first detected in preameloblasts, ameloblasts, and odontoblasts on day 2, but not in ameloblasts covering the enamel-free zone. Enamelysin signal declined in ameloblasts on day 6 but persisted in the dental pulp. In contrast, KLK4 transcripts were first observed on day 3 in pulp and on day 6 in ameloblasts covering the enamel-free zone. the KLK4 signal was present in maturation-stage ameloblasts on days 9, 11, and 14. The expression patterns of MMP-20 and KLK4 by ameloblasts in mouse molars are stage-specific and complementary.

The nature of tooth enamel is of inherent interest to dental professionals. The current-day clinical practice of dentistry involves the prevention of enamel demineralization, the promotion of enamel remineralization, the restoration of cavitated enamel where demineralization has become irreversible, the vital bleaching of dental enamel that has become discolored, and the diagnosis and treatment of developmental enamel malformations, which can be caused by environmental or genetic factors. On a daily basis, dental health providers make diagnostic and treatment decisions that are influenced by their understanding of tooth formation. A systemic condition during tooth development, such as high fever, can produce a pattern of enamel defects in the dentition. Knowing the timing of tooth development permits estimates about the timing of the disturbance. The process of enamel maturation continues following tooth eruption, so that erupted teeth can become less susceptible to decay over time. Mutations in the genes encoding enamel proteins lead to amelogenesis imperfecta, a collection of inherited diseases having enamel malformations as the predominant phenotype. Defects in the amelogenin gene cause X-linked amelogenesis imperfecta, and genes encoding other enamel proteins are candidates for autosomal forms. Here we review our current understanding of dental enamel formation, and relate this information to clinical circumstances where this understanding may be particularly relevant.

Amelogenin and enamelin are structural proteins in the enamel matrix of developing teeth. The temporal and spatial patterns of enamelin expression in developing mouse molars have not been characterized, while controversy remains with respect to amelogenin expression by odontoblasts and cementoblasts. Here we report the results of in situ hybridization analyses of amelogenin and enamelin expression in mouse molars from postnatal days 1, 2, 3, 7, 9, 14, and 21. Amelogenin and enamelin mRNA in maxillary first molars was first observed in pre-ameloblasts on the cusp slopes at day 2. The onsets of amelogenin and enamelin expression were approximately synchronous with the initial accumulation of predentin matrix. Both proteins were expressed by ameloblasts throughout the secretory, transition, and early maturation stages. Enamelin expression terminated in maturation stage ameloblasts on day 9, while amelogenin expression is still detected in maturation stage ameloblasts on day 14. No amelogenin expression was observed in day 21 mouse molars. Amelogenin and enamelin RNA messages were restricted to ameloblasts. No expression was observed in pulp, bone, or along the developing root. We conclude that amelogenin and enamelin are enamel-specific and do not directly participate in the formation of dentin or cementum in developing mouse molars.

Recently the dual properties of manganese ion (Mn(2+)) as an MRI contrast agent and a calcium analogue to enter excitable cells has been used to mark specific cells in brain and as a potential intracellular cardiac contrast agent. Here the hypothesis that in vivo manganese-enhanced MRI (MEMRI) can detect changes in inotropy in the mouse heart has been tested. T(1)-weighted images were acquired every minute during an experimental time course of 75 min. Varying doses of Mn(2+) (3.3-14.0 nmoles/min/g BW) were infused during control and altered inotropy with dobutamine (positive inotropy due to increased calcium influx) and the calcium channel blocker diltiazem (negative inotropy). Infusion of MnCl(2) led to a significant increase in signal enhancement in mouse heart that saturated above 3.3 +/- 0.1 nmoles/min/g BW Mn(2+) infusion. At the highest Mn(2+) dose infused there was a 41-47% increase in signal intensity with no alteration in cardiac function as measured by MRI-determined ejection fractions. Dobutamine increased both the steady-state level of enhancement and the rate of MRI signal enhancement. Diltiazem decreased both the steady-state level of enhancement and the rate of MRI signal enhancement. These results are consistent with the model that Mn(2+)-induced enhancement of cardiac signal is indicative of the rate of calcium influx into the heart. Thus, the simultaneous measurement of global function and calcium influx using MEMRI may provide a useful method of evaluating in vivo responses to inotropic therapy.

A kindred with dominant hypophosphatasia resulting from an alanine to threonine substitution at position 99 of the alkaline phosphatase protein is described. The clinical findings of individual members of the kindred were assessed by oral and physical examinations, or from the descriptions of multiple family members. The proband displayed enamel hypoplasia and premature loss of fully rooted primary anterior teeth, which were shown by histological examination to lack cementum. Serum alkaline phosphatase (ALP) and a vitamin B6 panel, and urine phosphoethanolamine (PEA) were measured on 21 family members. Based upon the clinical and laboratory tests, affected and unaffected status was assigned. Parametric linkage analysis of the kindred using different dominant models and frequency distributions for the disease allele and the mutation gave lodscores > 4.2 and confirmed the strong linkage between the disease and the mutation. Assuming the defined mutation causes the disease, the reliability of clinical and laboratory tests is assessed.

Amelogenins are the main component of the developing enamel matrix. In placental mammals, amelogenins are rapidly cleaved following their secretion. HPLC fractionation of tooth extracts produces a complex chromatographic profile. The fractions are rich in amelogenin cleavage products that generally retain the amino-terminus of the parent protein but have varying lengths of peptide removed from the original carboxyl-terminus. In contrast, HPLC fractionation of opossum tooth extracts produces a simple profile with a single major chromatographic peak. SDS-and Western blot analyses demonstrated that most of the amelogenin consisted of a prominent protein band that migrated at 28 kDa. Mass spectroscopy confirmed the presence of two uncleaved, alternatively spliced forms of opossum amelogenin, Op202 and Op57, but did not detect major amelogenin cleavage products evident in tooth extracts from placental mammals. Amino acid composition analysis supported the conclusion that uncleaved amelogenin is the major component in the developing enamel matrix. Enzymogram analyses using gelatin, casein and recombinant amelogenin as substrates, comparing porcine, rat and opossum tooth extracts, suggested that fewer proteinases are present in opossum. These results identify potentially significant differences in the proteolytic processing of amelogenins between metatherian and eutherian mammals.

Hamster enamel protein extracts were analyzed by RP-HPLC and the isolated fractions by SDS-and Western blotting using polyclonal antibodies against recombinant mouse amelogenin and anti-peptide antibodies against the mouse exon 4-encoded sequence. Total RNA was extracted from enamel organ epithelia and, using a 3' rapid amplification of cDNA ends (3' RACE) technique, the coding regions for three different amelogenin isoforms were cloned along with the 3' non-coding region. DNA sequencing revealed that the hamster amelogenin isoforms are 180, 73 and 59 amino acids in length, respectively. The 59-residue amelogenin corresponds to the leucine-rich amelogenin protein (LRAP), the 73-residue amelogenin corresponds to LRAP with the inclusion of the exon 4-encoded sequence, while the 180-residue amelogenin is the most abundant amelogenin isoform. Edman degradation was performed on purified hamster amelogenin, which provided the amino acid sequence in the region encoded by the 5' PCR amplification primer used in cloning. Therefore, the entire derived amino acid sequence of hamster amelogenin was revealed. The hamster amelogenin amino acid sequence was aligned with all its known homologues. Hamster differs from rat and mouse amelogenin at only three amino acid positions. Southern blot analysis using a panel of restriction enzymes gave the same pattern for hamster DNA obtained from males and females, suggesting that in hamster, as in mouse, amelogenin is expressed from a single gene located on the X chromosome.

Purpose: To quantify the cellular response in myocardial infarction by using iron oxide particles cell labeling and MR imaging techniques. Method and Materials:Iron oxide particles can produce signal attenuation in MRI and were used to label either macrophages or mesenchymal stem cells. Macrophages were labeled via an intravenous administration of iron oxide particles whereas mesenchymal stem cells were labeled in vitro and then transplanted into the animal bone marrow. In the macrophage study varied doses (1.1–14.5 μg Fe/g body weight) were applied. After surgically inducing a myocardial infarction in the C57 mouse the labeled cells would mobilize to the infarction site attempting to repair the damaged tissue. To monitor the cellular response T2*‐weighted MRI was used to acquire short‐axis cardiac images at 3 7 14 and 21 days post‐infarction. Signal intensity normalized to the maximum signal from the ventricular blood was used to quantify labeled cells at infarction sites. Results: Cellular response during myocardial infarction was temporally and noninvasively monitored. In the macrophage study linear regression of normalized signal intensity at each time point revealed a linear relationship between the normalized signal intensity and iron oxide dose at 7 days (r2=0.92) and 14 days (r2=0.98) post‐infarction. An optimum dose as well as imaging time window for monitoring inflammatory cell response was also obtained. Either labeled macrophages or mesenchymal stem cells were evidenced at the infarction site in histology. Conclusions: A linear signal‐dose relationship at 7 and 14 days post‐infarction suggests that the number of labeled cells at the infarction site can be interpreted from the MR signal within this time window. This linear relationship may further apply to quantify labeled mesenchymal stem cells. Therefore the cell labeling and MR imaging technique have a potential to quantify cellular response in pathological processes as well as during cell therapy.

Purpose: To monitor the MPIO and enhanced green fluorescenceprotein (eGFP) labeled mesenchymal stem cells (MSCs) infiltration into the myocardial infarction (MI) site using T 2 * ‐weighted MRI; To monitor the MRI contrast around the MI site post‐GCSF modulation. Methods: C57Bl/6 male mice (6–8 weeks old) were irradiated with an 8‐Gy dose. The labeled MSCs (3–7×105) were transplanted into the tibial medullary space 2 days post‐irradiation. The mice were divided into: 1) a sham‐operated group (Sham, n=7); 2) a MI group without GCSF injection (MI‐GCSF, n=7); and 3) a MI group with GCSF treatment (MI+GCSF, n=3). At 14 days post‐labeled MSCs transplantation, the two MI groups underwent surgery via permanent ligation of the left anterior descending coronary artery while the Sham group underwent open‐chest operation without perturbing the heart. The MI+GCSF group received subcutaneous GCSF injection 1 day post‐MI to enhance MSC mobilization. T 2 * ‐weighted short‐axis cardiac MRI was performed at baseline, 3, 7 and 14 days (D14) post‐surgery. Results: The MRI signal at the MI site was temporally attenuated for both MI groups, with more attenuated for MI+GCSF group (SNR 18.17±6.06 vs 11.37±1.01 at D14, p

Purpose: Ca 2+ is an important regulator of contractile function in the heart. Efflux mechanisms of the intracellular Ca 2+ concentration are regulated by the Na + / Ca 2+ exchanger (NCX) and plasma membrane Ca 2+ ‐ATPase (PMCA). During myocardial ischemic‐reperfusion intracellular Ca 2+ overloads via the reverse mode of the NCX, exacerbating myocardial injuries. Protocols that selectively inhibit this exchanger have shown potential therapeutic effects. Cardiac manganese‐enhanced MRI (MEMRI) can be implemented to quantify Mn 2+ concentration in vivo, where Mn 2+ has be sugested as a surrogate marker for Ca 2+ . This study introduces a potential technique to study cardiac Mn 2+ efflux by inhibiting the NCX using SEA0400. Method and Materials: Male C57Bl/6 mice (6–13 weeks) were separated into two groups to study the rate of Mn 2+ efflux; a control group and a group treated with SEA0400. Both groups were infused with a single dose of 190±2 nmoles/g BW Mn 2+ . The SEA0400 group were injected with 50 mg/kg SEA0400 one hour post‐ Mn 2+ infusion. Images were acquired on a horizontal 7.0 T Bruker BioSpec MRI spectrometer equipped with a micro imaging gradient. T1‐maps were acquired pre‐ Mn 2+ infusion and at various time points post‐ Mn 2+ infusion using an ECG‐gated, flow‐compensated Look‐Locker MRI pulse sequence. The change in relaxivity, ΔR1, in the left ventricular free wall (LV Wall), was calculated at different time points post‐infusion. Results: In the LV Wall 50% of the signal enhancement is attenuated within ∼3–4 hours post‐ Mn 2+ infusion. SEA0400 demonstrates the effectiveness of reducing the rate of Mn 2+ efflux. At a SEA0400 dose of 50 mg/kg the Mn 2+ efflux half‐life was approximately two times longer than the control group. Conclusion: This T1‐mapping technique can be used to quantify Mn 2+ efflux rates from the myocardium. By using a NCX inhibiting agent this technique can potentially be employed to interrogate individual Mn 2+ efflux mechanisms and rates in vivo.