Biomedical subjects

A Veis

Publications and source records attributed to A Veis.

At least 19 recordsLinked to original sources

PubMed2006

Amelogenin gene splice products A+4 and A-4 implanted in soft tissue determine the reorientation of CD45-positive cells to an osteo-chondrogenic lineage.

Several molecules such as bone morphogenetic protein-7, bone sialoprotein (BSP), or amelogenin gene splice products (A+4 or A-4) have been shown to induce reparative dentin formation in a rat model. However, at the moment, the origin and the mechanism of differentiation of the pulp cells stimulated by the bioactive molecules remain poorly understood. The present investigation was undertaken to validate an ectopic oral mucosal mouse model to evaluate the effects of amelogenin gene splice product implantation in a non-mineralizing tissue. Agarose beads, alone or coated with amelogenin gene splice products, were implanted in the mucosa of the cheeks in mouse. An immunohistochemical characterization of the recruited cells was undertaken for 3 days, 8 days, and 30 days after the implantation. The results showed that the implantation of agarose beads in mucosa induced the recruitment of inflammatory CD45 positive cells. When the beads were coated with amelogenin gene splice products (A+4 or A-4), the expression of osteo-chondrogenic markers (RP59, Sox9, or BSP) was also observed. However, no mineralization nodule was observed, even after 30 days of implantation. The present investigation suggests that amelognin gene splice products have the capacity of recruiting among inflammatory cell mesenchymal progenitors that eventually differentiate into osteo-chondrogenic cells. Altogether, the results obtained in the pulp model and the present data suggest the existence of different pathways of cell recruitment and differentiation in different cellular environments.

Absorbable Implants↗

PubMed2006

Mapping of magnesium and of different protein fragments in sea urchin teeth via secondary ion mass spectroscopy.

Mature portions of sea urchin are comprised of a complex array of reinforcing elements yet are single crystals of high and very high Mg calcite. How a relatively poor structural material (calcite) can produce mechanically competent structures is of great interest. In teeth of the sea urchin Lytechinus variegatus, we recorded high-resolution secondary ion mass spectrometry (SIMS) maps of Mg, Ca ,and specific amino acid fragments of mineral-related proteins including aspartic acid (Asp). SIMS revealed strong colocalization of Asp residues with very high Mg. Demineralized specimens showed serine localization on membranes between crystal elements and reduced Mg and aspartic acid signals, further emphasizing colocalization of very high Mg with ready soluble Asp-rich protein(s). The association of Asp with nonequilibrium, very high magnesium calcite provides insight to the makeup of the macromolecules involved in the growth of two different composition calcites and the fundamental process of biomineralization.

Animals↗

PubMed2005

Transmission electron microscopy characterization of macromolecular domain cavities and microstructure of single-crystal calcite tooth plates of the sea urchin Lytechinus variegatus.

The calcite plates and prisms in Lytechinus variegatus teeth form a complex biocomposite and employ a myriad of strengthening and toughening strategies. These crystal elements have macromolecule-containing internal cavities that may act to prevent cleavage. Transmission electron microscopy employing a small objective aperture was used to quantify several characteristics of these cavities. Cavity diameters ranged from 10 to 225 nm, the mean cavity diameter was between 50 and 60 nm, and cavities comprised approximately 20% of the volume of the crystal. Some cavities exhibited faceting and trace analysis identified these planes as being predominately of {1014} type. Through focus series of micrographs show the cavities were homogeneously distributed throughout the foil. The electron beam decomposed a substance within cavities and this suggests that these cavities are filled with a hydrated organic phase.

Animals↗

PubMed2003

Multiple microscopy modalities applied to a sea urchin tooth fragment.

Two synchrotron X-ray microscopy methods, phase-contrast microradiography (the propagation method) and absorption microCT (high-resolution computed tomography or microtomography), and laser-scanning confocal microscopy (visible wavelength) were used to study a fragment of the keel of a tooth of the sea urchin Lytechinus variegatus. Stripes observed in the phase-contrast images of the fragment were also seen in confocal micrographs. MicroCT showed that the stripes were due to two parallel planar arrays of low-absorption channels within the bulk of the keel. In the phase microradiographs, maximum contrast stripes appear when a channel image from one row coincides with a channel image from the second row; otherwise, contrast is minimal. Long channels do not appear to have been observed previously in keels of sea urchin teeth.

Animals↗

PubMed2003

Synchrotron X-ray studies of the keel of the short-spined sea urchin Lytechinus variegatus: absorption microtomography (microCT) and small beam diffraction mapping.

In sea urchin teeth, the keel plays an important structural role, and this paper reports results of microstructural characterization of the keel of Lytechinus variegatus using two noninvasive synchrotron x-ray techniques: x-ray absorption microtomography (microCT) and x-ray diffraction mapping. MicroCT with 14 keV x-rays mapped the spatial distribution of mineral at the 1.3 microm level in a millimeter-sized fragment of a mature portion of the keel. Two rows of low absorption channels (i.e., primary channels) slightly less than 10 microm in diameter were found running linearly from the flange to the base of the keel and parallel to its sides. The primary channels paralleled the oral edge of the keel, and the microCT slices revealed a planar secondary channel leading from each primary channel to the side of the keel. The primary and secondary channels were more or less coplanar and may correspond to the soft tissue between plates of the carinar process. Transmission x-ray diffraction with 80.8 keV x-rays and a 0.1 mm beam mapped the distribution of calcite crystal orientations and the composition Ca(1-x)Mg(x)CO(3) of the calcite. Unlike the variable Mg concentration and highly curved prisms found in the keel of Paracentrotus lividus, a constant Mg content (x = 0.13) and relatively little prism curvature was found in the keel of Lytechinus variegatus.

Animals↗

PubMed2003

Amelogenin gene splice products: potential signaling molecules.

The amelogenins, the major proteins of the developing tooth enamel matrix, are highly conserved throughout most species studied. The gene structure is similar, with a set of seven exons and intervening introns, and remarkable conservation of particular exon sizes over divergent species. Studies of exon skipping and consequent alternative gene splicing suggest that, in vertebrates, exon definition is crucial. In this mechanism, exon size is important. If too small, an exon can be readily skipped, if too large, internal cryptic splice sites may be utilized. Other factors, such as intron length and specific nucleotide sequences at the splice boundaries also modulate splicing efficiency, but amelogenin gene splicing conforms well to the generalized exon length model. Exons 1, 2 and 7 are not subject to splicing that affects the secreted protein product, but exons 3, 4 and 5 are at the lower boundary of exon size, rendering them, 4 and 5 especially, subject to skipping. On the other hand, exon 6 is very long and has cryptic splicing sites that can be used. In the mouse, nine distinct splice product proteins have been detected. The question now is the functions of these products. The larger forms, those that contain the intact proline-rich, hydrophobic exon 6 domains, are important for enamel mineralization. Recent work suggests that the small proteins resulting from deletion of a major part of amelogenin gene exon 6 via utilization of a cryptic site may have signal transduction functions during tooth development. Furthermore, new work also suggests that odontoblasts transiently express the small amelogenins during the period that epithelial-mesenchymal signaling between preodontoblasts and preameloblasts determines the course of tooth development. The same peptides have been demonstrated to act on non-odontogenic cells and effect their phenotypic expression patterns in vitro, and to induce bone formation in implants in vivo.

Alternative Splicing↗

PubMed2003

Three-dimensional microarchitecture of the plates (primary, secondary, and carinar process) in the developing tooth of Lytechinus variegatus revealed by synchrotron X-ray absorption microtomography (microCT).

This paper reports the first noninvasive, volumetric study of entire cross-sections of a sea urchin tooth in which the individual calcite structural elements could be resolved. Two cross-sectionally intact fragments of a Lytechinus variegatus tooth were studied with synchrotron microCT (microcomputed tomography) with 1.66 microm voxels (volume elements). These fragments were from the plumula, that is the tooth zone with rapidly increasing levels of mineral; one fragment was from a position aboral of where the keel developed and the second was from the zone where the keel was developing. The primary plates, secondary plates, carinar process plates, prisms, and elements of the lamellar-needle complex were resolved. Comparison of the microCT data with optical micrographs of stained thin sections confirmed the identifications and measured dimensions of the characteristic microarchitectural features. The interplay of reinforcing structures (plates and prisms) was more clearly revealed in the volumetric numerical data sets than in single or sequential slices. While it is well known that the primary plates and prisms in camarodont teeth are situated to improve resistance to bending (which can be termed primary bending), the data presented provide a new understanding of the mechanical role of the carinar process plates, that is, a geometry consistent with that required in the keel to resist lateral or transverse bending of the tooth about a second axis. The increase in robustness of teeth incorporating lateral keel reinforcement suggests that the relative development of carinar processes (toward a geometry similar to that of L. variegatus) is a character which can be used to infer which sea urchins among the stirodonts are most primitive and among the camarodonts which are more primitive.

Animals↗

PubMed2003

X-ray microCT study of pyramids of the sea urchin Lytechinus variegatus.

This paper reports results of a novel approach, X-ray microCT, for quantifying stereom structures applied to ossicles of the sea urchin Lytechinus variegatus. MicroCT, a high resolution variant of medical CT (computed tomography), allows noninvasive mapping of microstructure in 3-D with spatial resolution approaching that of optical microscopy. An intact pyramid (two demipyramids, tooth epiphyses, and one tooth) was reconstructed with 17 microm isotropic voxels (volume elements); two individual demipyramids and a pair of epiphyses were studied with 9-13 microm isotropic voxels. The cross-sectional maps of a linear attenuation coefficient produced by the reconstruction algorithm showed that the structure of the ossicles was quite heterogeneous on the scale of tens to hundreds of micrometers. Variations in magnesium content and in minor elemental constitutents could not account for the observed heterogeneities. Spatial resolution was insufficient to resolve the individual elements of the stereom, but the observed values of the linear attenuation coefficient (for the 26 keV effective X-ray energy, a maximum of 7.4 cm(-1) and a minimum of approximately 2 cm(-1) away from obvious voids) could be interpreted in terms of fractions of voxels occupied by mineral (high magnesium calcite). The average volume fraction of mineral determined for a transverse slice of the demipyramid near where it joins an epiphysis was 0.46; for a slice 3.3 mm adoral it was 0.70. Local volume fractions of mineral approached 1, and, away from resolvable voids, considerable portions of the demipyramids had volume fractions of calcite at or below approximately 0.33. MicroCT imaging of a demipyramid before and after infiltration with a high absorptivity fluid (sodium polytungstate) confirmed the determination of the volume fractions of minerals.

Animals↗

PubMed2002

[Mineralization of the dental pulp: contributions of tissue engineering to tomorrow's therapeutics in odontology].

When bioactive molecules such as bone sialoprotein (BSP), bone morphogenetic protein-7 (BMP-7, also termed OP-1) and chondrogenic Inducing Agents (CIA, A+4 and A-4) were implanted in the pulp of the first upper molars, mineralizations were induced. They were either limited to the formation of a reparative dentinal bridge closing the pulpal wound (CIA A+4), or filled the mesial part of the coronal pulp (BSP), or filled totally the pulp located in the root canal (BMP-7 and CIA A-4). Consequently, these molecules may change in the next future the every day practice in dentistry.

Animals↗

PubMed2002

X-ray absorption microtomography (microCT) and small beam diffraction mapping of sea urchin teeth.

Two noninvasive X-ray techniques, laboratory X-ray absorption microtomography (microCT) and X-ray diffraction mapping, were used to study teeth of the sea urchin Lytechinus variegatus. MicroCT revealed low attenuation regions at near the tooth's stone part and along the carinar process-central prism boundary; this latter observation appears to be novel. The expected variation of Mg fraction x in the mineral phase (calcite, Ca(1-x)Mg(x)CO(3)) cannot account for all of the linear attenuation coefficient decrease in the two zones: this suggested that soft tissue is localized there. Transmission diffraction mapping (synchrotron X-radiation, 80.8 keV, 0.1 x 0.1mm(2) beam area, 0.1mm translation grid, image plate area detector) simultaneously probed variations in 3-D and showed that the crystal elements of the "T"-shaped tooth were very highly aligned. Diffraction patterns from the keel (adaxial web) and from the abaxial flange (containing primary plates and the stone part) differed markedly. The flange contained two populations of identically oriented crystal elements with lattice parameters corresponding to x=0.13 and x=0.32. The keel produced one set of diffraction spots corresponding to the lower x. The compositions were more or less equivalent to those determined by others for camarodont teeth, and the high Mg phase is expected to be disks of secondary mineral epitaxially related to the underlying primary mineral element. Lattice parameter gradients were not noted in the keel or flange. Taken together, the microCT and diffraction results indicated that there was a band of relatively high protein content, of up to approximately 0.25 volume fraction, in the central part of the flange and paralleling its adaxial and abaxial faces. X-ray microCT and microdiffraction data used in conjunction with protein distribution data will be crucial for understanding the properties of various biocomposites and their mechanical functions.

Animals↗

PubMed2001

Dentin regeneration in vital pulp therapy: design principles.

The nature and specificity of the mechanisms by which the amputated dentin-pulp interface is therapeutically healed determine the properties of the barrier at this site and play a critical role in the outcome of vital pulp therapy. Healing of the dentin-pulp complex proceeds either by natural repair-which results in defensive hard-tissue formation, or therapeutically regulated dentin regeneration, which aims to reconstitute the normal tissue architecture at the pulp periphery. Progress in biomedical research opens new directions for the design of biologically effective pulp therapies. Application of biocompatible and biodegradable carrier vehicles for local delivery of signaling molecules in pulp-capping situations showed induction of fibrodentin/reparative dentin formation, but often at the expense of underlying pulp tissue. An alternative pre-clinical model aiming to reconstitute normal tissue architecture directly at the dentin-pulp interface should be designed on the basis of the direct induction of odontoblast-like cell differentiation and reparative dentin formation at the pulp-capping material interface. Experimental data clearly showed that pulpal cells can differentiate directly into odontoblast-like cells in association with specific extracellular matrices (dentinal or fibrodentinal matrix) or TGF beta 1-containing artificial substrates. Dentin-induced dentinogenesis can be used as a master plan for the achievement of new therapeutic opportunities. In the present study, several short-term experimental studies on dog teeth for potential direct induction of odontoblast-like cell differentiation at the surface of rhTGF beta 1-containing artificial substrates (Millipore filters, hydroxyapatite granules, calcium hydroxide, pure titanium) failed to induce any specific reparative dentinogenic effects.

Animals↗

PubMed2000

Specific amelogenin gene splice products have signaling effects on cells in culture and in implants in vivo.

Low molecular mass amelogenin-related polypeptides extracted from mineralized dentin have the ability to affect the differentiation pathway of embryonic muscle fibroblasts in culture and lead to the formation of mineralized matrix in in vivo implants. The objective of the present study was to determine whether the bioactive peptides could have been amelogenin protein degradation products or specific amelogenin gene splice products. Thus, the splice products were prepared, and their activities were determined in vitro and in vivo. A rat incisor tooth odontoblast pulp cDNA library was screened using probes based on the peptide amino acid sequencing data. Two specific cDNAs comprised from amelogenin gene exons 2,3,4,5,6d,7 and 2,3,5,6d, 7 were identified. The corresponding recombinant proteins, designated r[A+4] (8.1 kDa) and r[A-4] (6.9 kDa), were produced. Both peptides enhanced in vitro sulfate incorporation into proteoglycan, the induction of type II collagen, and Sox9 or Cbfa1 mRNA expression. In vivo implant assays demonstrated implant mineralization accompanied by vascularization and the presence of the bone matrix proteins, BSP and BAG-75. We postulate that during tooth development these specific amelogenin gene splice products, [A+4] and [A-4], may have a role in preodontoblast maturation. The [A+4] and [A-4] may thus be tissue-specific epithelial mesenchymal signaling molecules.

Amelogenin↗

PubMed2000

A transmission electron microscope study using vitrified ice sections of predentin: structural changes in the dentin collagenous matrix prior to mineralization.

The assembly of the collagenous organic matrix prior to mineralization is a key step in the formation of bones and teeth. This process was studied in the predentin of continuously forming rat incisors, using unstained vitrified ice sections examined in the transmission electron microscope. Progressing from the odontoblast surface to the mineralization front, the collagen fibrils thicken to ultimately form a dense network, and their repeat D-spacings and banding patterns vary. Using immunolocalization, the most abundant noncollagenous protein in dentin, phosphophoryn, was mapped to the boundaries between the gap and overlap zones along the fibrils nearest the mineralization front. It thus appears that the premineralized collagen matrix undergoes dynamic changes in its structure. These may be mediated by the addition and interaction with the highly anionic noncollagenous proteins associated with collagen. These changes presumably create a collagenous framework that is able to mineralize.

Animals↗

PubMed2000

Effect of alendronate on immature human dental root explants.

This study investigated the use of alendronate in the formation of new dentin in vitro. Extracted human premolar and molar teeth with immature apices were grown in tissue culture medium for 60 days. Six control specimens were grown without alendronate in the medium, and 22 experimental specimens were grown with alendronate at [10(-9) M] in the medium. Newly formed dentin was stained with tetracycline and procion brilliant red at days 1 to 3, 30 to 33, and 60. Specimens were decalcified and 5-micron sections were prepared for examination using fluorescent microscopy. New dentin formation was measured in microns at the most apical region, at 125-micron from the apical measurement and at 250-micron from the apical measurement. The alendronate group had 57.15% more growth than the control group at the most apical region, and this difference was significant (p = 0.0001). The results indicate that alendronate at [10(-9) M] is effective in accelerating dentin formation in vitro.

Alendronate↗

PubMed2000

A case report of a compound odontoma causing delayed eruption of a central maxillary incisor: clinical and microscopic evaluation.

A case of a compound odontoma caused delayed eruption of a central incisor in the maxilla is presented with clinical, radiographic, and microscopic findings. The odontoma was surgically removed and microscopic examination showed a lot of crown-like structures in a very irregular form, some of which were fused to each other at their apical parts. Enamel and pre-enamel were totally abnormal, whereas the inside of the pulp chamber tissue did not present any histological sign of functional tissue. The most homogeneous tissue was dentin. The removal of the odontoma was followed by a rapid eruption of the impacted central incisor.

Child↗

PubMed2000

The non-collagenous dentin matrix proteins are involved in dentinogenesis imperfecta type II (DGI-II).

Dentinogenesis Imperfecta type II (DGI-II) is a localized form of mesodermal dysplasia of the dentin affecting both the primary and permanent dentitions. This is an autosomal-dominant disease in which there is a disorder in dentin mineralization. Several studies have localized DGI-II to human chromosome 4 in the region 4q 12-21. Many ECM genes-such as OPN, DMP1, DMP2, DMP3 (DSPP), and BSP-have been mapped to the same locus. Biochemical studies indicated that dentin phosphophoryn (DMP2) might be a candidate gene in DGI-II. In this study, we have used histological and RFLP analyses of tissues from a DGI-II-affected patient, as compared with two normal controls, to determine if DMP1, 2, or 3 was linked to DGI-II. The histology of the affected tooth was very different in the DGI-II patient as compared with the normals. In particular, the dentinal tubules in the DGI-II patient were very irregular, which could be the result of perturbations in the process of dentin formation. Patient and control DNA samples were digested with EcoRI or PstI and Southern-hybridized with the DMP1, DMP2, and DMP3 cDNAs. Few differences in the restriction pattern were observed between affected and normal samples for DMP1 and DMP3-3' region (phosphophoryn-like sequences) probes. On the other hand, DMP2 showed a dramatic shift in the restriction pattern in DGI-II. This study suggests that the different restriction enzyme digestion profiles of the DNA from the DGI-II patient, as probed by DMP2, might be related to the defective mineralization of dentin in DGI-II.

Chromosomes, Human, Pair 4↗

PubMed1999

Peritubular dentin formation: crystal organization and the macromolecular constituents in human teeth.

Peritubular dentin (PTD) is a relatively dense mineralized tissue that surrounds the tubules of coronal tooth dentin. It is composed mainly of crystals of carbonated apatite together with a small amount of collagen. Its mode of formation has been investigated by studying the relatively dense particles isolated from a powdered preparation. Electron microscopic examination of the PTD particles, including 3-dimensional image reconstruction and electron diffraction, shows that the organization of the crystals of PTD is very similar to that of the adjacent intertubular dentin (ITD). The latter contains relatively large amounts of collagen and the carbonated apatite crystals are closely associated with the collagen matrix. The proteins present in the PTD particles are soluble after decalcification and stain with Stains All. The principal protein has higher molecular weight and a quite different amino acid composition than the phosphophoryns of the intertubular dentin. The interface between the PTD and the ITD shows structural continuity. These data show how two distinct carbonated apatite-based mineralized tissues can be organized and formed contiguously within the same organ by utilizing different sets of matrix proteins.

Adult↗

PubMed1999

Assembly of the type 1 procollagen molecule: selectivity of the interactions between the alpha 1(I)- and alpha 2(I)-carboxyl propeptides.

Assembly of the heterotrimeric procollagen I molecule is initiated by interactions between the carboxyl propeptide domains of the completed nascent pro alpha chains. The [pro alpha 1(I)]2[pro alpha 2(I)] heterotrimer is the predominant molecule, with much smaller amounts of stable [pro alpha 1(I)]3 homotrimer also being formed. However, the [pro alpha 2(1)]3 homotrimer has not been detected, raising questions as to the mechanism of chain assembly and why [pro alpha2(1)]3 homotrimers are not formed. These questions have been examined here by expressing the intact and amino- or carboxyl-terminal truncated C-propeptides of the pro alpha chains recombinantly in bacteria and in a coupled transcription/translation reticulocyte lysate system. Their interactions were studied in vitro by binding analyses and in vivo by using the yeast two-hybrid system. The C-pro alpha 1(I) interacted with itself, and with C-pro alpha 2(I), as expected. Surprisingly, the C-pro alpha 2(I) also interacted with itself, both in vitro and in vivo. While the interaction of C-pro alpha 2(I) with itself and C-pro alpha 1(I) in vitro was strong, these interactions were weaker in vivo. Deletion of 36 amino acids from the C-terminal domain of C-pro alpha 1 had no effect on its binding to intact self or intact C-pro alpha 2, but the same deletion in C-pro alpha 2 completely abolished its binding to intact C-pro alpha 2 and to C-pro alpha 1. Comparable N-terminal deletions in C-pro alpha 1 or C-pro alpha 2 diminished, but did not abolish, their binding to intact C-pro alpha 1 and C-pro alpha 2. In the yeast two-hybrid system, C-pro alpha 2 interacted with itself more weakly than with C-pro alpha 1. Molecular modeling and circular dichroism analyses showed that C-pro alpha 1 and C-pro alpha 2 have different folded structures and stability. Studies with antibodies specific to the C-pro alpha1 and alpha2 peptides showed them to precipitate different, specific, and distinct cell proteins from fibroblast lysates. The C-pro alpha 2(I) antibody complexed with more cell proteins. We hypothesize that the lack of pro alpha 2(I) homotrimers is not due to the inability of the C-pro alpha 2(I) to interact with itself, but rather to the competing presence of other cell proteins. The specificity of these interactions may reside in conformational differences in N- and C-terminal sequences of the two propeptides or in their different folding patterns.

Amino Acid Sequence↗