Biomedical subjects

R C Shore

Publications and source records attributed to R C Shore.

At least 19 recordsLinked to original sources

PubMed2001

Amelin extracellular processing and aggregation during rat incisor amelogenesis.

Amelin (also known as ameloblastin and sheathlin) is a recently described protein that is secreted by ameloblasts during enamel formation. Here, the extracellular distribution and processing of amelin during rat incisor amelogenesis were investigated by Western blot probing using anti-recombinant rat amelin antibodies. In addition, the solubility behaviour and aggregative properties of rat amelin were investigated using a sequential extraction procedure involving (1) extraction with simulated enamel fluid to extract proteins most likely to be soluble in vivo; (2) extraction with phosphate buffer to desorb proteins bound to enamel crystal surfaces; (3) extraction with sodium dodecyl sulphate (SDS) to extract proteins present as insoluble aggregates; followed by (4) a final acid demineralization step to release any remaining proteins. Proteins immunoreactive to the anti-amelin antibodies were detectable in secretory- and transition-stage enamel. Maturation-stage enamel appeared devoid of amelin. The largest immunoreactive protein detected migrated at 68 kDa on SDS gels, corresponding to the M(r) of nascent amelin. Other immunoreactive bands at 52, 40, 37, 19, 17, 16, 15, 14 and 13 kDa were presumably amelin processing products. The sequential extraction procedure revealed that the 68-, 52-, 40-, 37- and 13-kDa amelins were completely extracted under solution conditions similar to those reported to exist in vivo. In contrast, the 19-, 17- and 16-kDa amelins were only partially extracted, whilst the 15- and 14-kDa amelins could not be extracted with simulated enamel fluid. A proportion of the remaining 17- and 16-kDa amelins was desorbed from the enamel crystals with phosphate buffer and appeared to have been mineral-bound. The 15- and 14-kDa amelins and the remainder of the 17- and 16-kDa amelins were extracted with SDS only, suggesting that these species were present in vivo as an insoluble aggregate. The results provide additional information on amelin processing and degradation, and on how such processing influences the solubility and aggregative properties of amelin-derived proteins.

Ameloblasts↗

PubMed2000

The chemical composition of tooth enamel in junctional epidermolysis bullosa.

The junctionalis form of epidermolysis bullosa (EBJ) is associated with a number of clinical problems involving tooth enamel, including increased susceptibility to caries. The aim here was to carry out a chemical characterization of the enamel of teeth from EBJ patients compared with that of unaffected controls. The results showed that while protein concentration, amino acid composition and carbonate content were similar in both groups, EBJ enamel contained a significantly reduced mineral per volume content, resulting in enamel hypoplasia. In addition, Western blotting revealed the presence of serum albumin (a known inhibitor of enamel crystal growth) in EBJ enamel. This was not detected in control enamel or in enamel of teeth from patients with the dystrophic form of the disease. It is concluded that EBJ enamel is developmentally compromised and that the enamel defects are commensurate with the reported genetic lesions.

Amelogenesis↗

PubMed2000

Spatially related amelogenin interactions in developing rat enamel as revealed by molecular cross-linking studies.

A cleavable cross-linker (dithiobis[succinimidyl propionate], DTSP) was used to investigate the subunit structure of the developing enamel matrix. Intact matrix was cross-linked under conditions chosen to simulate those found in vivo. The cross-linked complexes were isolated by preparative sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and their subunit composition determined by analytical SDS-PAGE following reductive cleavage of the cross-links. Western blotting using antiamelogenin antibodies was used to confirm the identity of the proteins involved. The results showed that nascent amelogenins tended to be cross-linked to other nascent amelogenins while amelogenin-processing products tended to be cross-linked to other processed molecules at the same stage of processing. The results suggest that nascent amelogenins are in close association after secretion and during extracellular processing, and that processed products are not free to associate with nascent molecules, presumably due to diffusion constraints in the tissue. This conclusion implies that individual amelogenin molecules within supramolecular aggregates (nanospheres) are processed in situ and remain in the same nanosphere while all the individual component amelogenins undergo processing. The biological function of amelogenin processing remains unclear but the fact that amelogenin-amelogenin associations are maintained during processing indicates that matrix stability is an important factor while the enamel layer is being deposited.

Amelogenesis↗

PubMed2000

Trabecular architecture in women and men of similar bone mass with and without vertebral fracture: II. Three-dimensional histology.

We recently developed a simple and inexpensive method that complements established bone histomorphometry procedures by enabling the two-dimensional imaging of cancellous bone to be viewed within its three-dimensional context with the marrow tissue in place and without detriment to the material for other histological purposes. The method, based on the preparation and superficial staining of slices 300 microm thick, enables "real" (i.e., unstained) trabecular termini to be separated from "artifactual" (i.e., stained) termini, providing a direct measure of cancellous connectivity in osteopenic bone. The technique was applied to osteopenic age-matched, white, postmenopausal women (31 with and 22 without vertebral compression fractures) with a similar bone status, as measured at the spine by absorptiometry and at the iliac crest by histology (see part I of this study). Despite the similarity in the mass of trabecular bone at either site, the results showed a significant difference (p < 0. 05) in the number of "real" trabecular termini between the groups, such that the fracture group had almost four times as many termini (mean +/- SE: 1.98 +/- 0.51/30 mm(2)) at the iliac crest as the nonfracture group (mean +/- SE: 0.53 +/- 0.31/30 mm(2)). Previous histomorphometry of the same material failed to detect a structural distinction between the two groups using established variables. It was concluded that a mass-independent trabecular discontinuity contributes to skeletal failure and that determination of the number of "real" disconnections (i.e., unstained termini) by the direct method proposed may provide a more sensitive discriminant of fracture than the present indirect procedures. A group of fracture and nonfracture men (see part I) suggested a similar distinction (fracture: 0.69 +/- 0.30/30 mm(2); nonfracture: 0.18 +/- 0.18/30 mm(2)), although the difference was not significant.

Aged↗

PubMed2000

Architecture of intact natural human plaque biofilms studied by confocal laser scanning microscopy.

Determination of the structure of human plaque will be of great benefit in the prediction of its formation and also the effects of treatment. However, a problem lies in the harvesting of undisturbed intact plaque samples from human volunteers and the viewing of the biofilms in their natural state. In this study, we used an in situ device for the in vivo generation of intact dental plaque biofilms on natural tooth surfaces in human subjects. Two devices were placed in the mouths of each of eight healthy volunteers and left to generate biofilm for 4 days. Immediately upon removal from the mouth, the intact, undisturbed biofilms were imaged by the non-invasive technique of confocal microscopy in both reflected light and fluorescence mode. Depth measurements indicated that the plaque formed in the devices was thicker round the edges at the enamel/nylon junction (range = 75-220 microm) than in the center of the devices (range = 35-215 microm). The reflected-light confocal images showed a heterogeneous structure in all of the plaque biofilms examined; channels and voids were clearly visible. This is in contrast to images generated previously by electron microscopy, suggesting a more compact structure. Staining of the biofilms with fluorescein in conjunction with fluorescence imaging suggested that the voids were fluid-filled. This more open architecture is consistent with recent models of biofilm structure from other habitats and has important implications for the delivery of therapeutics to desired targets within the plaque.

Biofilms↗

PubMed2000

Evidence for charge domains on developing enamel crystal surfaces.

The control of hydroxyapatite crystal initiation and growth during enamel development is thought to be mediated via the proteins of the extracellular matrix. However, the precise nature of these matrix-mineral interactions remains obscure. The aim of the present study was to use a combination of atomic and chemical force microscopy to characterize developing enamel crystal surfaces and to determine their relationship with endogenous enamel matrix protein (amelogenin). The results show regular and discrete domains of various charges or charge densities on the surfaces of hydroxyapatite crystals derived from the maturation stage of enamel development. Binding of amelogenin to individual crystals at physiological pH was seen to be coincident with positively charged surface domains. These domains may therefore provide an instructional template for matrix-mineral interactions. Alternatively, the alternating array of charge on the crystal surfaces may reflect the original relationship with, and influence of, matrix interaction with the crystal surfaces during crystal growth.

Amelogenesis↗

PubMed2000

The chemistry of enamel caries.

The chemical changes which occur during the process of carious destruction of enamel are complex due to a number of factors. First, substituted hydroxyapatite, the main component of dental enamel, can behave in a very complex manner during dissolution. This is due not only to its ability to accept substituent ions but also to the wide range of calcium phosphate species which can form following dissolution. In addition, the composition, i.e., the extent of substitution, changes throughout enamel in the direction of carious attack, i.e., from surface to interior. Both surface and positively birefringent zones of the lesion clearly illustrate that carious destruction is not simple dissolution. Selective dissolution of soluble minerals occurs, and there is the probability of reprecipitation. The role of fluoride here is crucial in that not only does it protect enamel per se but also its presence in solution means that rather insoluble fluoridated species can form very easily, encouraging redeposition. The role of organic material clearly needs further investigation, but there is the real possibility of both inhibition of repair and facilitation of redeposition. For the future, delivering fluoride deep into the lesion would appear to offer the prospect of improved repair. This would entail a delivery vehicle which solved the problem of fluoride uptake by apatite at the tooth surface. Elucidation of the role of organic material may also reveal putative mechanisms for encouraging repair and/or protecting the enamel mineral.

Animals↗

PubMed2000

Morphology and elemental composition of subgingival calculus in two ethnic groups.

BACKGROUND: The aim of the present study was to compare the morphology and elemental composition of subgingival calculus between Indo-Pakistani and Caucasian patient groups. METHODS: Extracted teeth from 14 Indo-Pakistani and 19 Caucasian subjects were collected. Of these, 12 Indo-Pakistani and 10 Caucasian teeth had sufficient subgingival calculus for analysis. Subgingival calculus present on the 22 teeth was classified into six morphological types: 1) crusty/spiny/nodular; 2) ledge/ ring; 3) thin, smooth veneers; 4) finger/fern-like; 5) individual islands/spots; or 6) supramarginal on submarginal. Subgingival calculus was zoned: coronal, mid, and apical. A sample obtained from each zone was subdivided to allow 3 separate analyses: transmission electron microscopic (TEM) x-ray microanalysis for elemental composition, fluoride analysis, and carbonate analysis. RESULTS: Crusty/spiny/nodular, ledge/ring, and thin, smooth veneers were more commonly found in the Indo-Pakistani group; individual islands were more prevalent in the Caucasian subjects. Supramarginal on submarginal calculus was found only in the Indo-Pakistani group. No finger/fern-like deposits were found. No differences within or between the two ethnic groups were found with regard to calcium:phosphate ratios, fluoride, or carbonate content. However, the Indo-Pakistani group showed significantly lower levels of sodium in apical samples than in coronal samples (ANOVA, F1,16 = 5.98, P= 0.03), and significantly lower levels of sodium (ANOVA, F1,12 = 4.75, P= 0.05) and magnesium (ANOVA, F1,12 = 5.16, P= 0.04) in apical samples than in those from Caucasians. After adjusting for smoking, the magnesium results remained significant (ANOVA, F2,11 = 4.64, P= 0.05). CONCLUSIONS: Subgingival calculus demonstrated differences in morphology between these two ethnic groups and differences in elemental composition, which may influence its overall solubility and contribute to its greater accretion in the Indo-Pakistani subjects.

Adult↗

PubMed2000

A three-dimensional histological method for direct determination of the number of trabecular termini in cancellous bone.

Osteoporotic fractures occur frequently in aging populations. Established methods for analyzing microarchitecture indicate that cancellous bone loss in the elderly is associated with progressive reduction in the connectivity of the trabecular network. This disconnection may explain the increased skeletal fragility that is sometimes out of proportion to the amount of bone lost. Connectivity, however, is difficult to measure and usually requires indirect methods. We describe development of a simple, inexpensive and direct procedure for counting sites of trabecular disconnection. The method is based upon preparation of 300-500 microm thick slices of methylmethacrylate embedded material rather than the more usual thin 8 microm histological sections. The marrow tissue is retained within the thick slice; this is essential for conservation of any detached bone fragments. In such preparations differential superficial staining of the upper and lower surfaces with alizarin red and light green, respectively, allows the two-dimensional image to be viewed at the same time as its three-dimensional counterpart. In this way, "real" (i. e., unstained) trabecular termini can be distinguished from "apparent" (i. e., stained red or green) termini that are artifacts of the plane of section. Partly polarized light enhances the microscope image. The method does not destroy the material for subsequent bone histomorphometry and, therefore, may be a useful adjunct to iliac bone biopsy analysis in studies of metabolic bone disease.

Adult↗

PubMed1998

The developing enamel matrix: nature and function.

The hydroxyapatite crystals of mature enamel are unusually large, uniform and regularly disposed within the tissue, implying that their development is a highly controlled process. The organic matrix of developing enamel is presumed to play an important role in the modulation of mineral deposition and growth during tooth morphogenesis but the precise functions of individual matrix proteins remain unclear. The aim of this review was to survey the current knowledge of enamel matrix proteins with a view to suggesting possible functions. The organic matrix is highly heterogeneous, comprising proteins derived from a number of different genes, including amelogenin, enamelin, ameloblastin (amelin/sheathlin), tuftelin, dentine sialophosphoprotein, enzymes and serum proteins such as albumin. Each of these classes appears to undergo post-secretory sequential degradation which contributes further towards matrix heterogeneity. Possible functions of these proteins include de novo mineral nucleation/initiation (dentine sialophosphoprotein, tuftelin), mineral ion binding as crystal precursors (amelogenin, enamelin), control of crystal growth (amelogenin, enamelin, ameloblastin), support of growing crystals (amelogenin, enamelin), determination of prismatic structure (ameloblastin), cell signalling (tuftelin, ameloblastin), control of secretion (breakdown products) and protection of the mineral phase (amelogenin, enamelin). Failure of these mechanisms could lead to incomplete maturation of the enamel and the eruption of dysplastic tissue.

Ameloblasts↗

PubMed1998

Identification of human serum albumin in human caries lesions of enamel: the role of putative inhibitors of remineralisation.

Carious attack on enamel is not a unidirectional process but involves both demineralisation and remineralisation. The chemistry of carious attack on enamel has, to a large extent, now been clarified as far as mineral components are concerned but little attention, however, has been paid to the identity of organic material in carious lesions and its possible role in the caries process. The only clear information available is that organic material accumulates with time within enamel lesions. The present study was aimed at identifying a specific protein component known to bind to hydroxyapatite (albumin) in carious lesions with a view to investigating its role in the disease process. The distribution of albumin within both white spot and fissure lesions and adjacent sound enamel of extracted human teeth was investigated using SEM immunohistochemistry on undermineralised sections of human enamel and employing a polyclonal antibody to human serum albumin. The nature of the protein, i.e. whether it was in the form of intact molecules or degraded fragments, was investigated by Western blotting, employing the same antibody. The immunohistochemistry revealed the presence of albumin within both interproximal white spot and fissure lesions with little if any present in sound enamel. The Western blotting indicated that the albumin was in the intact form with no evidence of degradation products. The ability of albumin to bind and to inhibit growth of calcium phosphate crystals raises the question as to the possible role of such a molecule in the development of carious lesions.

Antibodies↗

PubMed1998

Atomic force microscopy studies of crystal surface topology during enamel development.

During the secretory stage of enamel development, the hydroxyapatite crystals appear as thin ribbons which grow substantially in width and thickness during the later maturation stage. In this study, the atomic force microscope (AFM) was used to investigate developmentally-related changes in deproteinized enamel crystal surface topography in normal animals and in those receiving daily doses of fluoride. The AFM revealed previously undescribed surfaces features, some of which may represent growth sites or different crystalline phases. Secretory stage crystals had greater surface rugosity and were more irregular, with spherical sub-structures of 20-30 nm diameter arranged along the "c"-axis. Maturation stage crystals were smoother and larger but revealed both subnanometer steps and lateral grooves running parallel to the "c"-axis. Crystals from fluorotic tissue showed similar features but were more irregular with a higher degree of surface roughness, suggesting abnormal growth. The AFM may prove an important adjunct in determination of the mechanisms controlling crystal size and morphology in skeletal tissues.

Animals↗

PubMed1998

Identification of rat enamel organ RNA transcripts using differential-display.

Enamel formation is a complex process which involves the expression of a number of genes, the most obvious being those related to the mineralized extracellular matrix. In this study the differential-display technique, first described by Liang and Pardee, has been used to identify genes specifically expressed in enamel organ cells. By comparing results obtained from RNA derived from rat enamel organ with RNA derived from other cellular sources, a number of differentially expressed transcripts have been identified. The nucleotide sequences of two of these have been analyzed and shown to have no homology with any previously published sequences. Further analysis will provide information on the type of protein that they may encode, their tissue distribution and their potential role in enamel formation.

Amino Acid Sequence↗

PubMed1998

Enzyme compartmentalization during biphasic enamel matrix processing.

Processing of enamel matrix proteins is essentially biphasic. Secretory stage metalloprotease activity generates a discrete, presumably functional, spectrum of molecules which may also undergo dephosphorylation. Maturation stage serine proteases almost completely destroy the matrix. The present aim was to examine the tissue compartmentalization of these enzyme activities in relation to their possible function. A sequential extraction using synthetic enamel fluid, phosphate buffer and SDS was used to identify enzymes free in the enamel fluid, crystal bound or aggregated with the bulk matrix respectively. Results indicated that the metallo-proteases and alkaline phosphatase were free in the secretory stage enamel fluid while the serine proteases appeared to be largely bound to the maturation stage crystals. The mobility of the metallo-proteases and alkaline phosphatase would ensure efficient initial processing of secretory matrix, while the largely mineral bound serine proteases would ensure retention of protease activity despite massive destruction and protein removal.

Animals↗

PubMed1997

Enamel maturation.

Enamel maturation is characterized by massive crystal growth in both width and thickness, resulting in the most highly mineralized of all mammalian skeletal tissues. The control of this process is mediated via a carefully orchestrated series of events that are temporally and spatially regulated, and it requires the co-ordinated degradation and removal of the endogenous enamel matrix. This is affected by both neutral metalloproteases and serine proteases, which are developmentally restricted and may be further modulated by changes in the chemistry of the enamel crystals themselves. Failure of these mechanisms, or the adventitious entry of mineral-binding proteins during the later stages of maturation, may result in the incomplete maturation of the enamel crystals and the eruption of dysplastic tissue.

Albumins↗

PubMed1997

A method for the quantitative site-specific study of the biochemistry within dental plaque biofilms formed in vivo.

The study of plaque biofilms in the oral cavity is difficult as plaque removal inevitably disrupts biofilm integrity precluding kinetic studies involving the penetration of components and metabolism of substrates in situ. A method is described here in which plaque is formed in vivo under normal (or experimental) conditions using a collection device which can be removed from the mouth after a specified time without physical disturbance to the plaque biofilm, permitting site-specific analysis or exposure of the undisturbed plaque to experimental conditions in vitro. Microbiological analysis revealed plaque flora which was similar to that reported from many natural sources. Analytical data can be related to plaque volume rather than weight. Using this device, plaque fluoride concentrations have been shown to vary with plaque depth and in vitro short-term exposure to radiolabelled components may be carried out, permitting important conclusions to be drawn regarding the site-specific composition and dynamics of dental plaque.

Bacterial Adhesion↗

PubMed1996

Preparation of guide planes using a reciprocating handpiece.

A reciprocating handpiece and attachments have been found effective in preparing guide planes prior to the construction of removable partial dentures. A comparison was made of the enamel surface prepared using the reciprocating handpiece and a conventional rotary handpiece. Two-stage and three-stage techniques for each handpiece were compared. Scanning electron micrographs showed that a smoother surface was created by the reciprocating handpiece.

Bicuspid↗

PubMed1996

The chemical composition of tooth enamel in recessive dystrophic epidermolysis bullosa: significance with respect to dental caries.

Previous reports have linked the prevalence of tooth abnormalities with high caries experience in the different types of epidermolysis bullosa (EB). However, it is not known to what extent the apparent susceptibility to enamel caries is due to disease-related altered enamel chemistry in these cases. The aim of this study was to characterize the enamel of teeth from patients suffering from recessive epidermolysis bullosa dystrophica (rEBD) in terms of its mineral content, carbonate content, protein content, and amino acid composition. The results showed that dental enamel from these patients was essentially normal in terms of its chemistry. It is therefore concluded that the high caries experience in recessive dystrophic epidermolysis bullosa patients is probably related to other factors, such as compromised oral hygiene and prolonged oral clearance due to extensive oral soft tissue damage and a cariogenic diet.

Amino Acids↗