Biomedical subjects

H C Margolis

Publications and source records attributed to H C Margolis.

At least 19 recordsLinked to original sources

PubMed2006

Role of macromolecular assembly of enamel matrix proteins in enamel formation.

Unlike other mineralized tissues, mature dental enamel is primarily (> 95% by weight) composed of apatitic crystals and has a unique hierarchical structure. Due to its high mineral content and organized structure, enamel has exceptional functional properties and is the hardest substance in the human body. Enamel formation (amelogenesis) is the result of highly orchestrated extracellular processes that regulate the nucleation, growth, and organization of forming mineral crystals. However, major aspects of the mechanism of enamel formation are not well-understood, although substantial evidence suggests that protein-protein and protein-mineral interactions play crucial roles in this process. The purpose of this review is a critical evaluation of the present state of knowledge regarding the potential role of the assembly of enamel matrix proteins in the regulation of crystal growth and the structural organization of the resulting enamel tissue. This review primarily focuses on the structure and function of amelogenin, the predominant enamel matrix protein. This review also provides a brief description of novel in vitro approaches that have used synthetic macromolecules (i.e., surfactants and polymers) to regulate the formation of hierarchical inorganic (composite) structures in a fashion analogous to that believed to take place in biological systems, such as enamel. Accordingly, this review illustrates the potential for developing bio-inspired approaches to mineralized tissue repair and regeneration. In conclusion, the authors present a hypothesis, based on the evidence presented, that the full-length amelogenin uniquely regulates proper enamel formation through a process of cooperative mineralization, and not as a pre-formed matrix.

Amelogenesis↗

PubMed2005

Multi-component adsorption model for pellicle formation: the influence of salivary proteins and non-salivary phospho proteins on the binding of histatin 5 onto hydroxyapatite.

The acquired enamel pellicle formed by selective adsorption of proteins in whole saliva is a protective integument on the tooth surface. The purpose of the present study was to investigate the formation of human acquired enamel pellicle using an in vitro hydroxyapatite (HA) model and 3H-histatin 5 to allow accurate measurement of histatin 5 binding in a multi-component experimental system. A binary system was employed by mixing 3H-histatin 5 with one unlabeled protein prior to incubation with HA or by first incubating 3H-histatin 5 with the HA which had been pre-coated with one of a panel of unlabeled proteins (human albumin, salivary amylase, lysozyme, acidic PIFs, statherin, the N-terminal fragment of statherin, and egg yolk phosvitin). A ternary system was employed by mixing 3H-histatin 5 with HA sequentially pre-coated with two different unlabeled proteins, including recombinant histatin 1. The results showed that only salivary statherin and egg yolk phosvitin promote histatin 5 adsorption significantly. The amount of histatin 5 adsorbed was also found to increase as a function of the amount of phosvitin and statherin used to pre-coat HA up to a maximum level that was two- to four-fold greater than that observed on untreated HA. These data suggest that specific protein-protein interactions may play important roles in pellicle formation in vivo.

Adsorption↗

PubMed2005

The onset of amelogenin nanosphere aggregation studied by small-angle X-ray scattering and dynamic light scattering.

Proteins with predominantly hydrophobic character called amelogenins play a key role in the formation of the highly organized enamel tissue by forming nanospheres that interact with hydroxyapatite crystals. In the present investigation, we have studied the temperature and pH-dependent self-assembly of two recombinant mouse amelogenins, rM179 and rM166, the latter being an engineered version of the protein that lacks a 13 amino acid hydrophilic C-terminus. It has been postulated that this hydrophilic domain plays an important role in controlling the self-assembly behavior of rM179. By small-angle X-ray and neutron scattering, as well as by dynamic light scattering, we observed the onset of an aggregation of the rM179 protein nanospheres at pH 8. This behavior of the full-length recombinant protein is best explained by a core-shell model for the nanospheres, where hydrophilic and negatively charged side chains prevent the agglomeration of hydrophobic cores of the protein nanospheres at lower temperatures, while clusters consisting of several nanospheres start to form at elevated temperatures. In contrast, while capable of forming nanospheres, rM166 shows a very different aggregation behavior resulting in the formation of larger precipitates just above room temperature. These results, together with recent observations that rM179, unlike rM166, can regulate mineral organization in vitro, suggest that the aggregation of nanospheres of the full-length amelogenin rM179 is an important step in the self-assembly of the enamel matrix.

Amelogenin↗

PubMed2003

Physical parameters of hydroxyapatite adsorption and effect on candidacidal activity of histatins.

Histatins 1, 3 and 5 are the major members of a histidine-rich protein family present in human salivary secretions. These proteins are distinct from many salivary proteins in their high positive charge density at neutral pH, and their antibacterial and antifungal properties. In this study, the hydroxyapatite adsorption characteristics of histatin 1, containing a single phosphoserine residue, recombinantly expressed histatin 1, native histatin 3, synthetic histatin 5 and an internal 12-residue sequence of histatin 5 were investigated. A Langmuir-type model was used to analyse the adsorption. A comparison of the affinities and binding sites of phosphorylated and recombinant histatin 1 provided an estimate of the positive influence of the single phosphoseryl group on mineral adsorption. Furthermore, an apparent correlation was shown to exist between peptide chain length and the number of binding sites. The influence of histatin 5 adsorption on its anticandidal activity was also investigated by performing Candida albicans killing assays with histatin 5 and histatin 5/hydroxyapatite suspensions. A decrease in killing activity was observed with the increase of hydroxyapatite present. The results suggest that the anticandidal properties of histatin 5 could be impaired by the conformations resulting from mineral adsorption, or that putative cellular receptors necessary for candidacidal activity are inaccessible when histatin 5 is adsorbed on hydroxyapatite.

Adsorption↗

PubMed2002

PLGA bone plates reinforced with crosslinked PPF.

In this study a matrix of poly(propylene fumarate) (PPF) was crosslinked with N-vinylpyrrolidone (NVP), 2-hydroxyethylmethacrylate (HEMA), or a mixture of NVP and ethyleneglycol dimethacrylate (EGDMA) in the presence of poly(lactide-co-glycolide) (PLGA) to reinforce and preserve the form of PLGA bone plates. The degree of crosslinkage varied depending on the crosslinker as shown by the rapid and almost complete leaching of NVP upon incubation in phosphate buffered saline at 37 degrees C in 900 h and retention of 92% of HEMA. With the reinforced bone plates extracted for 72 h at room temperature methylene chloride, the extracted PLGA from NVP/PPF, NVP-EGDMA/PPF, and HEMA/PPF were 75.42% (w/w), 59.52% (w/w), and 30.86% (w/w), respectively. The flexural modulus and compressive strength of the crosslinked PPF reinforced bone plates were higher than that of the unreinforced bone plate. Atomic force microscopy showed that NVP/PPF reinforced PLGA bone plates eroded substantially (a mean surface roughness of 19.319 nm) whereas NVP-EGDMA-PPF reinforced bone plate showed a distinct crystalline organization (and a higher roughness, 43.525 nm). In conclusion, we propose the consideration of NVP-EGDMA/PPF reinforced PLGA as a biodegradable orthopedic implant material that has a lower likelihood of warping or failing catastrophically than the currently available materials.

Journal Article↗

PubMed2001

Association of caries activity with the composition of dental plaque fluid.

This study tests the hypothesis that caries activity is associated with lower degrees of saturation with respect to enamel mineral in dental plaque fluid following sucrose exposure. Plaque fluids were obtained from caries-free, caries-positive, and caries-active subjects. Samples were collected before and at 3 and 7 min after a sucrose rinse on consecutive weeks and analyzed for organic acids, inorganic ions, pH, calcium activity, and, in selected samples, total protein. After sucrose, pH values were significantly lower in the caries-active group in comparison with the caries-free and caries-positive groups. Total and free calcium concentrations increased with decreasing pH, with free calcium being about one-third of total calcium. The caries-active group exhibited significantly lower degrees of saturation with respect to enamel mineral, after sucrose, and had significantly higher mutans streptococci levels in plaque than did the caries-free samples. Thus, saturation levels in post-sucrose plaque fluids reflect the cariogenic potential of dental plaque.

Adolescent↗

PubMed2000

Enamel demineralization under driving forces found in dental plaque fluid.

The present study was carried out to examine the kinetics of enamel demineralization in vitro under driving forces for demineralization (i.e., the degree of saturation with respect to enamel, DS(En)) similar to those found in dental plaque fluid. Thin sections of human enamel were exposed at 25 degrees C to lactic acid solutions with DS(En) values (DS(En) = [(Ca2+)5(OH-)(PO4(3-))3/K(En)]1/9; K(En) = 5.5 x 10(-55)) ranging from 0.28 to 0.79. Lesion development was monitored by quantitative microradiography. Enamel mineral loss in solutions with DS(En) values of 0.28, 0.32 and 0.36 was first detected after 3, 3, and 7 wk of continuous exposure, respectively. Consistent with previous findings, subsurface demineralization was observed and rates of mineral loss increased significantly with decreasing DS(En) values. However, no mineral loss was observed in sections of enamel exposed to solutions with DS(En) values of 0.41 and 0.79, even after 11 months. These results suggest that (outer) enamel mineral behaves as a mineral phase that is less soluble than that dictated by the solubility product constant (K(En)) used in this study. Furthermore, these results indicate that the kinetics and general features of the demineralization process are maintained over a wide range of DS(En) values, including conditions that better reflect those found in the oral cavity. These findings are particularly relevant to the assessment of the cariogenic potential of dental plaque fluids.

Cariogenic Agents↗

PubMed1999

Release of mineral ions in dental plaque following acid production.

The release of appreciable amounts of calcium, phosphate and fluoride found in whole plaque into the plaque-fluid phase, following bacterial acid production, can potentially reduce the driving force for tooth demineralization. However, limited information is available on this topic, particularly on the release of fluoride. This study sought to determine the change in calcium, phosphate and fluoride concentrations in plaque fluid after sucrose exposure. 48 h overnight-fasted supragingival plaque samples were collected from all tooth surfaces (with the exception of the lower lingual anterior teeth) of one half of an individual mouth, following a 1 min water rinse. Plaque samples were then collected from the other half of the same mouth, following a 292 mM sucrose rinse. Plaque fluid was isolated by centrifugation and analysed for total calcium and phosphate (ion chromatography) and for free fluoride (ion-specific electrode). Samples were collected from seven individuals. Following sucrose exposure, plaque-fluid pH decreased significantly from 6.5+/- 0.3 to 5.4+/-0.2; calcium concentrations (mmol/l) also increased significantly (p < 0.01) from 1.9+/-0.5 to 5.0+/-2.1. Fluoride and phosphate concentrations in plaque fluid, however, did not increase significantly after sucrose exposure: mean concentrations (mmol/l) of fluoride after the water and sucrose rinses were 0.006+/-0.003 and 0.005+/-0.002, respectively, and mean phosphate concentrations (mmol/l) were 11.0+/-2.0 and 12.0+/-3.0, respectively. When results were expressed per wet plaque weight, phosphate concentrations were also found to increase significantly. The same trends were observed when additional plaque samples were treated in vitro with sucrose: fluoride-ion activity did not increase in plaque under in vivo-like conditions.

Adolescent↗

PubMed1999

Effect of in situ plaque mineral supplementation on the state of saturation of plaque fluid during sugar-induced acidogenesis.

Dental plaque fluid is normally supersaturated with respect to enamel mineral but this may change to a state of undersaturation when plaque pH falls following sugar exposure, placing the adjacent enamel at risk of caries. We have determined the saturation status of the fluid in both resting and fermenting plaque following mineral supplementation. Eleven subjects abstained from oral hygiene and rinsed their mouth 3 times/d for 3 d with a placebo solution or with test solutions designed to enrich plaque with hydroxyapatite or fluorhydroxyapatite. On the morning of day 4, plaque samples were collected before and after exposure to 10% sucrose. Compared to the placebo, use of the test rinses resulted in significantly higher concentrations of Ca, P and F in plaque residue. In plaque fluid, higher post-sucrose Ca2+ free concentrations and saturation levels with respect to enamel mineral and fluorapatite were found after use of the hydroxyapatite rinse compared to the placebo, effects that probably resulted from the release of cell-bound Ca2+ as well as from the dissolution of apatite. Thus, some evidence was obtained that the test mouthrinses can counteract the fall in saturation level found when plaque is exposed briefly to sucrose. Potential long-term benefits of the test mouthrinses deserve further study.

Acetates↗

PubMed1999

Kinetics of enamel demineralization in vitro.

Previously, we reported that the rate (R) of hydroxyapatite dissolution in acetic, lactic, and phosphoric acid solutions is a function of the degree of saturation with respect to the dissolving mineral, DS (defined as the ratio of the mean ionic activity product for hydroxyapatite [Ca5OH(PO4)3] in solution to its solubility product constant), and the sum of the acid activities (sumBiH) in solution: R = K(1-DS)m(sumBiH)n. The present study was undertaken to explore the general validity of this model in describing the kinetics of enamel demineralization. Thin sections of human enamel were exposed to partially saturated 0.1 mol/L lactic acid solutions, at two different DS levels, and at pH values of 4.3 to 6.0. Thin sections of human enamel were also exposed to solutions with four different concentrations of acetic and lactic acids (pH 4.3) with three different DS values and, at one DS value, to solutions of propionic acid. Mineral loss was monitored by quantitative microradiography. In solutions with pH values of 4.3 and 5.0, "lesions" were formed with well-defined surface layers, whereas, in solutions with pH 6.0, "lesions" were produced with no apparent surface layers. The formation of relatively intact surface layers was consistent with predicted phase transformations. Rates of mineral loss were found to be inversely proportional to both the degree of saturation with respect to enamel mineral, DS(En), and the pH of the solution and increased with increased activities of each organic acid, consistent with the proposed model. However, at the same DS(En) and acid activity, rates of demineralization were the same in the acetic and propionic acid solutions, whereas rates of demineralization in lactic acid were greater. It is suggested that specific interactions of acid species with enamel mineral may modify the rate of enamel demineralization. These in vitro findings suggest that relatively small differences in DS(En) values found in plaque fluid may result in very significant differences in the rate of enamel demineralization in vivo.

Acetates↗

PubMed1998

Purification, characterization, and cloning of enamel matrix serine proteinase 1.

The maturation of dental enamel succeeds the degradation of organic matrix. Inhibition studies have shown that this degradation is accomplished by a serine-type proteinase. To isolate and characterize cDNA clones encoding this proteinase, we used two degenerate primer approaches to amplify part of the coding region using polymerase chain-reaction (PCR). First, we purified the proteinase from porcine transition-stage enamel matrix and characterized it by partial protein sequencing. The enzyme was isolated from the neutral soluble enamel extract by successive ammonium sulfate precipitations, hydroxyapatite HPLC, reverse-phase HPLC, DEAE ion exchange, and affinity chromatography with a Benzamidine Sepharose 6B column. The intact protein and lysylendopeptidase-generated cleavage products were characterized by amino acid sequence analyses. Degenerate oligonucleotide primers encoding two of the polypeptide sequences were synthesized. In a complementary strategy, degenerate oligonucleotide primers were designed against highly conserved active-site regions of chymotrypsin-like proteinases. Both approaches yielded PCR amplification products that served as probes for screening a porcine enamel organ epithelia-specific cDNA library. The longest full-length clone is 1133 nucleotides and encodes a preproprotein of 254 amino acids. We designate this protein enamel matrix serine proteinase 1 or EMSP1. The active protein has 224 amino acids, an isotope-averaged molecular mass of 24.1 kDa, and an isoelectric point of 6.0. Multiple-tissue Northern analysis indicates that EMSP1 is a tooth-specific protein. Gelatin enzymography shows a dramatic increase in EMSP1 activity in the transition-stage enamel matrix. EMSP1 is most homologous to kallikriens and trypsins.

Amelogenesis↗

PubMed1998

Edgard C. Moreno, PhD: a tribute to his major contributions to oral biology research.

The seminal contributions of Dr. Edgard C. Moreno to physical chemical aspects of oral biology research are reviewed, with personal insights provided by the author. Dr. Moreno has made major contributions to our understanding of fundamental processes which occur within the oral cavity and which control the formation of mineralized tissues. These contributions include extensive research in the areas of: the chemistry of synthetic calcium phosphates and biominerals, the interaction of salivary proteins and other molecules of biological interest with calcium phosphate surfaces, the mechanism of dental caries formation and the effects of fluoride on this process, the mechanism of dental calculus formation and prevention, and the mechanism of dental enamel formation.

Chemistry, Physical↗

PubMed1998

Enamelysin mRNA displays a developmentally defined pattern of expression and encodes a protein which degrades amelogenin.

Previously, a cDNA encoding a novel matrix metalloproteinase (enamelysin) was isolated from a porcine enamel organ-specific cDNA library. The cloned mRNA is tooth-specific and contains an open reading frame encoding a protein composed of 483 amino acids (Gene, 183:(1-2), p123-128, 1996). Here, we show that: 1) The expression of enamelysin mRNA is not limited to the enamel organ as previously reported. The enamelysin message is also expressed at very low levels in the pulp organ. 2) Northern analysis reveals that the enamelysin mRNA displays a developmentally defined pattern of expression in the enamel organ. The message is expressed at relatively high levels during the presecretory and early transition stages of development. However, during late maturation, the quantity of enamelysin mRNA is greatly reduced. Conversely, the low message levels in the pulp organ remain relatively constant throughout these developmental stages. 3) The enamelysin cDNA was ligated into a prokaryotic expression vector and recombinant enamelysin containing a His tag was purified from E. coli. Zymographic analysis utilizing recombinant murine amelogenin as the substrate, reveals that the purified enamelysin degrades amelogenin. Since enamelysin is developmentally regulated and is capable of degrading amelogenin, it is likely to play a significant role during enamel biomineralization.

Amelogenin↗

PubMed1997

In vivo detection of calcium from dicalcium phosphate dihydrate dentifrices in demineralized human enamel and plaque.

A series of studies was conducted to clarify the role of DCPD in improving the effects of fluoride in the mouth. We performed plaque fluid studies to see if the use of DCPD dentifrices increases the levels of free Ca2+ ions and the DSenamel in plaque fluid. The results of these studies showed that plaque fluid was undersaturated with respect to DCPD; therefore, DCPD would dissolve in the mouth. The Ca2+ activity in plaque fluid measured 12 hours post-treatment was significantly elevated with the use of DCPD dentifrices in comparison with silica dentifrices. The use of DCPD dentifrices also increased the DSenamel of plaque fluid in comparison with a NaF/silica dentifrice. To determine whether Ca from DCPD could be detected in demineralized enamel and whole plaque after in vivo usage of a DCPD/ fluoride dentifrice, we conducted a 44Ca labeling study. Secondary Ion Mass Spectrometry (SIMS) was used to detect and measure changes in 44Ca levels in enamel before and after 6 days of in vivo treatment with a 44DCPD/fluoride dentifrice. The results showed that 44Ca from DCPD was incorporated into enamel and detected in plaque 18 hours post-treatment. The findings from these studies show that brushing with a DCPD dentifrice introduces additional, exogenous Ca into the oral environment, which fosters improved remineralization of teeth in combination with fluoride.

Calcium↗

PubMed1997

[Association of the degree of saturation with respect to enamel in plaque fluid with caries activity].

The aim of this study was to investigate changes of the degree of saturation with respect to enamel in plaque fluid by sucrose rinse, and their association with caries activity. Three groups of subjects were selected by their caries status: caries free, caries positive and caries active. Plaque samples were collected before, 3 and 7 minutes after a one-minute 5% sucrose rinse. Plaque fluids were isolated and analyzed for organic acids (capillary electrophoresis), inorganic ions (ion exchange chromatography), calcium activity and pH (selective electrodes). The degrees of saturation (DS) were calculated based on the total composition or calcium activity. The results showed that there were significantly more pH drops, and particularly more DS drops within caries active group after a sucrose rinse, compared with caries free and positive groups. This suggests that the degree of saturation is a much sensitive index to describe the cariogenetic status of individuals.

Adult↗

PubMed1996

Molecular cloning and mRNA tissue distribution of a novel matrix metalloproteinase isolated from porcine enamel organ.

A cDNA encoding a novel matrix metalloproteinase (MMP) was isolated from a porcine enamel organ-specific cDNA library. Multiple tissue northern blot analysis revealed the presence of two mRNA transcripts which were expressed only in the enamel organ. The transcripts were 1968 bp or 3420 bp in length and resulted from the utilization of alternative polyadenylation sites. The open reading frame of the cloned mRNA encodes a protein composed of 483 amino acids. The MMP has a predicted molecular mass of 54.1 kDa, which is similar to that of the stromelysins or collagenases, although it is not a member of either of these two classes of MMPs. A motif analysis revealed that the cloned MMP does not contain a consensus hemopexin-like domain because it lacks a critical tryptophan and proline residue at the appropriate positions. Since the cloned MMP is a new member of the MMP gene family and its expression appears limited to the enamel organ, we have named it enamelysin.

Amino Acid Sequence↗

PubMed1996

Developmental changes in the pH of enamel fluid and its effects on matrix-resident proteinases.

The objectives of this study were to measure pH in developing enamel at progressively older (more mature) stages of amelogenesis in vivo, and then to formulate synthetic enamel fluid mixtures that approximated these pH values for in vitro studies. The ultimate goal was to characterize the molecular weights of proteinases visualized by enzymograms incubated in synthetic enamel fluid using gelatin and casein as substrates. For most experiments, the proteinases were extracted en masse from small freeze-dried enamel strips directly into a non-reducing sample preparation buffer. In some experiments, we pre-treated the enamel strips with acetic acid to determine if this common method for demineralization and protein extraction caused any changes in the activity levels of the enamel proteinases. In other experiments, we first soaked enamel strips in synthetic enamel fluid to determine solubility of the proteinases within an aqueous phase. The results indicated that the pH of developing enamel remained fairly constant near pH 7.23 across the secretory stage, but it was generally more acidic (6.93) and fluctuated in focal areas between mildly acidic (6.2-6.8) and near-neutral (7.2) conditions across the maturation stage. The pH then slowly rose to near 7.35 when the enamel was almost mature (hard). The acidic conditions were generally inhibitory to most enamel proteinases, but there were some caseinase activities in mid-maturation-stage enamel near 23-30 kDa which appeared to be activated by weakly acidic conditions (pH 6.28). Pre-treatment of enamel samples with 0.5 M acetic acid markedly altered the overall profile of enamel proteinases, causing activation of some latent proteinase activities and permanent inhibition of other activities. Most proteinases in whole homogenates were insoluble in synthetic enamel fluid. This suggests that they may be tightly bound, directly or indirectly, to matrix proteins or mineral components in situ.

Acetic Acid↗

PubMed1994

Composition and cariogenic potential of dental plaque fluid.

Our understanding of the chemical events that take place at the tooth-plaque interface has improved greatly through studies of the chemical composition and properties of dental plaque fluid. In the absence of fermentable carbohydrate, plaque fluid has been found to be supersaturated with respect to tooth mineral and other calcium phosphate phases, thus exhibiting the potential to support calculus formation and the remineralization of incipient carious lesions. Following the exposure to fermentable carbohydrate, the degree of saturation of plaque fluid decreases rapidly, primarily due to lactic acid production and the lowering of plaque fluid pH. The extent of these chemical changes has been shown to be associated with differences in caries history. Such studies have been facilitated by the recent development of microanalytical techniques. Unfortunately, little is known about the relationship between the observed chemical changes in plaque fluid and the microbial composition of plaque. Limited information is also available on the association of immune factors in plaque fluid with dental disease.

Calcium Phosphates↗