Ultrastructure of early amelogenesis in wildâ type, Amelxâ /â , and Enamâ /â mice: enamel ribbon initiation on dentin mineral and ribbon orientation by ameloblasts

IntroductionDental enamel is comprised of highly organized, oriented apatite crystals, but how they form is unclear.MethodsWe used focused ion beam (FIB) scanning electron microscopy (SEM) to investigate early enamel formation in 7â weekâ old incisors from wildâ type, Amelxâ /â , and Enamâ /â 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.ResultsWe demonstrate that wildâ 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 fingerâ 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 Enamâ /â 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. Amelxâ /â mice produce oriented enamel ribbons, but the ribbons fuse into fanâ like structures. The matrix does not expand sufficiently to support formation of the Tomes process or establish rod and interrod organization.ConclusionAmelogenin 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.This is the first report using focused ion beam microscopy to visualize enamel ribbons at high resolution as they form, which are shown to initiate on the underlying dentin crystals. This continuity between dentin and enamel mineral has been difficult to establish with conventional thin sectioning techniques and has been debated for many years. We converted serial images into movies that allowed us to better appreciate the complex infolding of cell membranes and intercellular compartmentalization that are integral to the complex mechanism of enamel biomineralization.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/135052/1/mgg3253-sup-0002-FigS13-21.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/135052/2/mgg3253-sup-0005-FigS41-51.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/135052/3/mgg3253_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/135052/4/mgg3253-sup-0003-FigS24-26.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/135052/5/mgg3253-sup-0004-FigS29-40.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/135052/6/mgg3253.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/135052/7/mgg3253-sup-0001-FigS1-12.pd

Porcine dentin sialoprotein glycosylation and glycosaminoglycan attachments

<p>Abstract</p> <p>Background</p> <p>Dentin sialophosphoprotein (Dspp) is a multidomain, secreted protein that is critical for the formation of tooth dentin. Mutations in <it>DSPP </it>cause inherited dentin defects categorized as dentin dysplasia type II and dentinogenesis imperfecta type II and type III. Dentin sialoprotein (Dsp), the N-terminal domain of dentin sialophosphoprotein (Dspp), is a highly glycosylated proteoglycan, but little is known about the number, character, and attachment sites of its carbohydrate moieties.</p> <p>Results</p> <p>To identify its carbohydrate attachment sites we isolated Dsp from developing porcine molars and digested it with endoproteinase Glu-C or pronase, fractionated the digestion products, identified fractions containing glycosylated peptides using a phenol sulfuric acid assay, and characterized the glycopeptides by N-terminal sequencing, amino acid analyses, or LC/MSMS. To determine the average number of sialic acid attachments per N-glycosylation, we digested Dsp with glycopeptidase A, labeled the released N-glycosylations with 2-aminobenzoic acid, and quantified the moles of released glycosylations by comparison to labeled standards of known concentration. Sialic acid was released by sialidase digestion and quantified by measuring β-NADH reduction of pyruvic acid, which was generated stoichiometrically from sialic acid by aldolase. To determine its forms, sialic acid released by sialidase digestion was labeled with 1,2-diamino-4,5-methyleneoxybenzene (DMB) and compared to a DMB-labeled sialic acid reference panel by RP-HPLC. To determine the composition of Dsp glycosaminoglycan (GAG) attachments, we digested Dsp with chondroitinase ABC and compared the chromotagraphic profiles of the released disaccharides to commercial standards. N-glycosylations were identified at Asn³⁷, Asn⁷⁷, Asn¹³⁶, Asn¹⁵⁵, Asn¹⁶¹, and Asn¹⁷⁶. Dsp averages one sialic acid per N-glycosylation, which is always in the form of N-acetylneuraminic acid. O-glycosylations were tentatively assigned at Thr²⁰⁰, Thr²¹⁶and Thr³¹⁶. Porcine Dsp GAG attachments were found at Ser²³⁸and Ser²⁵⁰and were comprised of chondroitin 6-sulfate and chondroitin 4-sulfate in a ratio of 7 to 3, respectively.</p> <p>Conclusions</p> <p>The distribution of porcine Dsp posttranslational modifications indicate that porcine Dsp has an N-terminal domain with at least six N-glycosylations and a C-terminal domain with two GAG attachments and at least two O-glycosylations.</p

Quantitative analysis of the core 2D arrangement and distribution of enamel rods in crossâ sections of mandibular mouse incisors

Considerable descriptive information about the overall organization of mouse mandibular incisor enamel is available but almost nothing is known about the quantitative characteristics of enamel rod arrangement and distribution in these teeth. This has important implications concerning cell movement during the secretory stage because each ameloblast makes one enamel rod. Knowing how many enamel rods are cut open in a crossâ section of the enamel layer could provide insights into understanding the dynamics of how groups of ameloblasts form the enamel layer. In this study, crossâ sections of fully mineralized enamel were cut on 24 mandibular mouse incisors, polished and etched, and imaged by scanning electron microscopy in backscatter mode. Montaged maps of the entire enamel layer were made at high magnification and the enamel rod profiles in each map were colorâ coded based upon rod category. Quantitative analyses of each color layer in the maps were then performed using standard routines available in imagej. The data indicated that that there were on average 7233 ± 575 enamel rod profiles per crossâ section in mandibular incisors of 7â weekâ old mice, with 70% located in the inner enamel layer, 27% located in the outer enamel layer, and 3% positioned near the mesial and lateral cementoenamel junctions. All enamel rod profiles showed progressive increases in tilt angles, some very large in magnitude, from the lateral to mesial sides of the enamel layer, whereas only minor variations in tilt angle were found relative to enamel thickness at given locations across the enamel layer. The decussation angle between alternating rows of rod profiles within the inner enamel layer was fairly constant from the lateral to central labial sides of the enamel layer, but it increased dramatically in the mesial region of the enamel layer. The packing density of all rod profiles decreased from lateral to central labial regions of the enamel layer and then in progressing mesially, decreased slightly (inner enamel, mesial tilt), increased slightly (outer enamel layer) or almost doubled in magnitude (inner enamel, lateral tilt). It was concluded that these variations in rod tilt angle and packing densities are adaptations that allow the tooth to maintain a sharp incisal edge and shovelâ shape as renewing segments formed by around 7200 ameloblasts are brought onto the occluding surface of the tooth by continuous renewal.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/147012/1/joa12912_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/147012/2/joa12912.pd

Characterization of Periodontal Structures of Enamelinâ Null Mice

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141513/1/jper0195.pd

Mutational analysis of candidate genes in 24 amelogenesis imperfecta families

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72969/1/j.1600-0722.2006.00278.x.pd

Characterization of porcine dentin sialoprotein (DSP) and dentin sialophosphoprotein (DSPP) cDNA clones

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/74726/1/j.1600-0722.2003.00009.x.pd

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

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/90093/1/EOS_871_sm_SupportingInformation.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/90093/2/j.1600-0722.2011.00871.x.pd

Enamel ribbons, surface nodules, and octacalcium phosphate in C57BL/6 Amelxâ /â mice and Amelx+/â lyonization

BackgroundAmelogenin is required for normal enamel formation and is the most abundant protein in developing enamel.MethodsAmelx+/+, Amelx+/â , and Amelxâ /â molars and incisors from C57BL/6 mice were characterized using RTâ PCR, Western blotting, dissecting and light microscopy, immunohistochemistry (IHC), transmission electron microscopy (TEM), scanning electron microscopy (SEM), backscattered SEM (bSEM), nanohardness testing, and Xâ ray diffraction.ResultsNo amelogenin protein was detected by Western blot analyses of enamel extracts from Amelxâ /â mice. Amelxâ /â incisor enamel averaged 20.3 ± 3.3 μm in thickness, or only 1/6th that of the wild type (122.3 ± 7.9 μm). Amelxâ /â incisor enamel nanohardness was 1.6 Gpa, less than half that of wildâ type enamel (3.6 Gpa). Amelx+/â incisors and molars showed vertical banding patterns unique to each tooth. IHC detected no amelogenin in Amelxâ /â enamel and varied levels of amelogenin in Amelx+/â 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 Amelxâ /â enamel extending from mineralized dentin collagen to the ameloblast. The Amelxâ /â enamel ribbons were not well separated by matrix and appeared to fuse together, forming plates. Xâ ray diffraction determined that the predominant mineral in Amelxâ /â enamel is octacalcium phosphate (not calcium hydroxyapatite). Amelxâ /â ameloblasts were similar to wildâ type ameloblasts except no Tomesâ processes extended into the thin enamel. Amelxâ /â and Amelx+/â molars both showed calcified nodules on their occlusal surfaces. Histology of D5 and D11 developing molars showed nodules forming during the maturation stage.ConclusionAmelogenin forms a resorbable matrix that separates and supports, but does not shape early secretoryâ 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 Tomesâ process.We thoroughly characterized enamel formation in amelogenin null mice and determined that the mineral covering dentin in these animals is octacalcium phosphate. The initial enamel mineral has a ribbon shape, similar to the wild type. Thus, amelogenin is not required to shape the ribbons, as is currently thought, but is required to ensure that the final mineral phase is calcium hydroxyapatite.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/134766/1/mgg3252_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/134766/2/mgg3252-sup-0001-AppendixS1-21.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/134766/3/mgg3252.pd