Dentinal proteoglycans demonstrate an increasing order of affinity for hydroxyapatite crystals during the transition of predentine to dentine.

The transition from an unmineralized predentine to a mineralized dentine involves a variety of molecular extracellular matrix interactions and protein degradation events. Previous studies have identified that different pools of proteoglycan (PG) species are present within the matrix of the predentine, the transitional phase at the predentine-dentine border, and the mineralized dentine. These PGs alter with respect to the chemical nature of the glycosaminoglycan (GAG) chain and as a result of extracellular processing of the macromolecule in the matrix. This study has examined the hydroxyapatite (HAP) binding affinity of the PGs isolated from these phases and the influence of the attached GAG chains upon their binding characteristics. PGs isolated from the three phases were characterized to contain a mixture of decorin and biglycan, substituted with chondroitin sulfate GAG chain(s). Maximal binding for dentine PGs onto HAP was achieved at 15.60 microg/ml protein and for predentine-dentine interface PGs at 0.125 mg/ml. A significantly increasing gradient of affinity was observed moving toward dentine, with dentine PGs exhibiting 19 times greater binding affinity for HAP than predentine PGs and 7.5 times greater affinity than predentine-dentine interface PGs. Removal of the GAG chains from dentine PGs significantly reduced binding affinity for HAP but did not influence the number of binding sites. The difference in binding ability observed for the different PG pools gives further support for the involvement of these macromolecules in regulating the transition from predentine to dentine and suggests key roles for the GAG chains in the mineralisation process.

Fluoride-induced changes to proteoglycan structure synthesised within the dentine-pulp complex in vitro.

Fluoride is known to influence mineralisation patterns within dentine, where alterations in the post-translational modification of proteoglycans (PG) have been proposed as an implicating factor. In light of recent studies elucidating changing PG profiles in the transition of predentine to mineralised dentine, this study investigates the influence of fluoride on the major PG populations (decorin, biglycan and versican) within the pulp, predentine and dentine. Tooth sections from rat incisors were cultured for 14 days in the presence 0, 1 and 6 mM sodium fluoride and the PG extracted from the pulp, predentine and dentine matrices. PG species and corresponding metabolites were identified by their immuno-reactivity to antibodies against decorin, biglycan and versican. Component glycosaminoglycan chains were characterised with respect to their nature, chain length and disaccharide composition. Levels of PG extracted from pulp and predentine were reduced, particularly for biglycan. Fluoride did not influence levels of decorin or versican within predentine or dentine, although the processing of these macromolecules within pulp and predentine was affected, particularly at higher fluoride concentrations. Levels of dermatan sulfate were reduced within pulp and predentine, although the effect was less pronounced for predentine. Fluoride reduced sulfation of glycosaminoglycan chains within pulp and predentine tissues, with a notable reduction in Deltadi6S evident. In all three tissues, glycosaminoglycan chain length was reduced. Considering the various roles for PG in the dentine-pulp complex, either directly or indirectly in the mineralisation process, changes in the synthesis, structure and processing of the different PG species within the pulp, predentine and dentine matrices provides a further molecular explanation for the altered mineralisation patterns witnessed during fluorosis.

The influence of fluoride exposure on dentin mineralization using an in vitro organ culture model.

This study aimed to characterize fluoride-induced alterations in dentin mineralization within a dentin-pulp organ culture system. Tooth sections derived from male Wistar rat incisors were cultured in Trowel-type culture for 14 days, in the presence of 0 mM, 1 mM, 3 mM and 6 mM sodium fluoride. Tooth sections were processed and analyzed for uptake of fluoride, its subsequent effect on dentin mineralization by tetracycline hydrochloride incorporation and mineral composition, expressed as calcium/phosphorous (Ca/P) ratios. Tetracycline hydrochloride incorporation was demonstrated to decrease with increased fluoride exposure, accompanied by significant increases in both Ca/P ratios and fluoride incorporation. These findings provide further evidence that the established alterations in dentin formation during fluorosis are a consequence of disruption to the mineralization process, and provide a model system with which to investigate further the potential role the extracellular matrix plays in inducing the apparent changes in mineral composition.

Proteoglycans in predentin: the last 15 micrometers before mineralization.

Small leucine-rich proteoglycans (SLRPs) regulate extracellular matrix organization. In order to investigate the distribution and potential functions of decorin, biglycan (BGN), and fibromodulin (3 SLRPs, potentially related to dentinogenesis), we performed light and electron immunochemistry on teeth from rats, and on wild-type and biglycan knockout mice (BGN KO). Immunohistochemical data demonstrate that chondroitin sulfate/dermatan sulfate (CS/DS) and keratan sulfate (KS) distributions displayed reverse gradients in predentin. The decrease of CS/DS labeling from the proximal to the distal predentin contrasted with the sharp decorin increase observed in the distal predentin near the predentin/dentin transition, an effect possibly attributable to the deglycosylation action of stromelysin-1. In contrast, BGN concentration was apparently constant throughout the whole predentin. Additional immunolabelings showed, for the first time, the presence of fibromodulin in predentin. Compared with the wild-type mouse, the mean diameter of collagen fibrils in the BGN KO was smaller in the proximal predentin but larger in the central and distal predentin, the metadentin was broader, and the dentin mineralization appeared altered and heterogeneous. Altogether, our data suggest an important role for BGN in dentin formation and mineralization.

Molecular interaction of recombinant decorin and biglycan with type I collagen influences crystal growth.

This study has investigated the interaction of recombinant chondroitin sulphate (CS)-substituted decorin and biglycan on collagen fibrillogenesis, their interaction with hydroxyapatite (HAP), and HAP-induced crystal growth. The core proteins of the recombinant decorin and biglycan were obtained following chondroitinase ABC digestion and their influence on the above physical mechanisms were investigated in parallel. CS-decorin promoted collagen fibrillogenesis, with the interaction mediated principally through the core protein. Both decorin and biglycan demonstrated a strong association for HAP, predominately facilitated through the glycosaminoglycan chains. HAP-induced crystal growth was inhibited by decorin and biglycan, although the degree of inhibition was reduced when these proteoglycans were complexed with type I collagen. Thus, this study has highlighted potentially differing roles for decorin and biglycan, as both promoters and inhibitors in the regulation of the mineralization process.

Changing profiles of proteoglycans in the transition of predentine to dentine.

Proteoglycans and their constituent glycosaminoglycans have been proposed to play important roles in matrix mediated formation of mineralised tissues, such as dentine. This study has examined the changing profile of proteoglycan species during the transition of unmineralised predentine to mineralised dentine. Three-week-old calves teeth were collected and proteoglycans purified from the predentine, the predentine/dentine interface and dentine. Decorin and biglycan, together with related degradation products, were identified in the predentine fraction, alongside degradation products of versican, indicating metabolism of the proteoglycan components within this tissue. Decorin and biglycan were also identified as major proteoglycan species within extracts from the predentine/dentine interface and dentine. Analysis of the glycosaminoglycan constituents within each fraction demonstrated significant changes in their composition. Predentine contained a high proportion of dermatan sulfate (DS) (51.5%), with chondroitin sulfate (CS) (17.8%) and hyaluronan (HA) (30.7%) additionally identified. Within the predentine/dentine interface the proportion of CS increased greatly (62.5%), with corresponding decrease in the proportion of DS (21.4%) and HA (16.1%) also evident. CS only was identifiable within the dentine matrix. A four-fold increase in the level of sulfation was identified for glycosaminoglycans extracted from the predentine/dentine interface compared with the predentine and dentine fraction. The ratio of DeltaDi4S:DeltaDi6S was higher for glycosaminoglycans isolated from the predentine fraction. Glycosaminoglycans extracted from the dentine fraction possessed longer chain lengths than those present in the predentine and predentine/dentine fractions. The results indicate that the proteoglycans within each fraction undergo subtle structural modification, particularly at the onset of mineralisation, indicating an active involvement of these macromolecules in the overall mineralisation process.

Odontoblast transport of sulphate--the in vitro influence of fluoride.

The present study reports the development of a culture system for the analysis of 35S-sulphate release from odontoblasts in vitro. Pulpless longitudinally split rat incisors were cultured in supplemented minimum essential medium (alphaMEM) with 20 microCi 35S-sulphate per ml, 20 microCi 3H-mannitol per ml for 1h. Teeth were then transferred to fresh unlabelled media and aliquots of media were removed and the level of 35S-sulphate 3H-mannitol determined. Results indicated a two phase release of 35S-sulphate into the media, and comparison with pulp tissue indicated a specific release pattern. Transport of sulphate is essential for correct synthesis and glycosylation of macromolecules such as proteoglycans (PG). Previous studies have shown that post-translational modifications of these proteins can be influenced by excess fluoride, resulting in decreased sulphation and elongation of glycosaminoglycan (GAG) chains. Therefore the influence of fluoride on sulphate transport, using the optimised culture system was also investigated. Inclusion of 6mM fluoride during pulse labelling caused a significant decrease of 35S-sulphate (P<0.0001) during the initial release phase. Inclusion of 3 and 6mM fluoride only in the post-labelling incubation media resulted in a significant decrease in the release of 35S-sulphate (P<0.0001), during the total time course. The influence of fluoride was not dose dependent. Inclusion of a specific chloride channel blocker SITS, into the culture system indicated that 35S-sulphate transport may in part be via this route. Fluoride would therefore appear to influence the transport of 35S-sulphate across the odontoblast membrane, potentially via a chloride channel.

The influence of fluoride on the cellular morphology and synthetic activity of the rat dentine-pulp complex in vitro.

Exposure to high fluoride concentrations in the immediate environment of the tissue is recognized to result in the post-translational modification of non-collagenous dentine extracellular matrix (ECM) components, potentially altering dentine mineralization. However, less is known about the effects of fluoride exposure on the morphology or metabolism of the cells associated with the dentine-pulp complex. This study examined the effects of fluoride exposure at defined concentrations on the cellular morphology and ECM synthetic activities of odontoblasts and pulpal fibroblasts by the culture of tooth sections from male Wistar rat incisors in Trowel-type cultures for up to 14 days, in the presence and absence of 6mM sodium fluoride. Histomorphometric analysis of the dentine-pulp complex of sodium fluoride-exposed tooth sections demonstrated no obvious gross morphological differences with respect to the odontoblasts and pulpal fibroblasts throughout the 14-day culture period, in comparison with unexposed tooth sections. No significant differences in odontoblast and pulpal fibroblast cell numbers were determined in the absence and presence of fluoride. Image analysis examination of odontoblast cytoplasmic:nuclear (C/N) ratios also showed no significant differences in fluoride-exposed and unexposed tooth sections, although reductions in the C/N ratios of pulpal fibroblasts were evident in fluoride-exposed sections at days 10 and 14. No significant differences in predentine width were observed in fluoride-exposed and unexposed tooth sections over the 14-day culture period. Autoradiography following [3H]proline incorporation into the dentine-pulp complex demonstrated inhibition of collagen synthesis, particularly by the odontoblasts in tooth sections exposed to 6mM sodium fluoride. These findings, in association with those from previous studies, imply that dentine ECM alterations may contribute to the altered mineralization of dentine during fluorosis, rather than secretory-related changes in odontoblast morphology.

Comparison of the antioxidant properties of HYAFF-11p75, AQUACEL and hyaluronan towards reactive oxygen species in vitro.

In chronic wounds, a number of host factors are released which perpetuate the inflammatory process, including polymorphonuclear leukocyte (PMN)-derived reactive oxygen species (ROS), such as superoxide radical (O2*-) and hydroxyl radical (*OH) species. The glycosaminoglycan. hyaluronan, has been shown to act as an antioxidant towards ROS, although the potential for biomaterials, such as HYAFF -11p75 (the 75% benzyl ester of hyaluronan) and AQUACEL (carboxymethylcellulose), to act in this manner has yet to be elucidated. This study compared the antioxidant properties of high and low molecular weight hyaluronan (HMWT HA and LMWT HA), HYAFF -11p75, AQUACEL and an AQUACEL /hyaluronan composite (AQUACEL /HA) against O2*- and *OH. The antioxidant capacities of each material were assessed by their ability to inhibit cytochrome C reduction by O2*- fluxes, generated via the oxidation of hypoxanthine by xanthine oxidase, and their inhibition of 2-deoxy-D-ribose degradation by *OH fluxes, generated by the reaction of hydrogen peroxide (H2O2) and iron (Fe2+). All materials studied possessed dose dependent antioxidant properties towards O2*-, with HYAFF 11p75 having the greatest antioxidant potential towards these species, followed by AQUACEL, HMWT HA, AQUACEL /HA and LMWT HA. Only HMWT HA exhibited dose dependent antioxidant properties towards *OH at the fluxes examined. Gas chromatography/mass spectrometry analysis implied that ester bonds between the hyaluronan backbone and benzyl groups of HYAFF -11p75 are highly susceptible to O2*- hydrolysis, with the de-esterified benzyl alcohol being rapidly degraded in the presence of *OH. This data supports the hypothesis that HYAFF -11p75 has greater antioxidant capacity towards O2*-, due to the esterified benzyl groups providing alternative sites for O2*- attack other than the hyaluronan backbone of HYAFF -11p75 itself and explains the inability of HYAFF -11p75 to scavenge *OH, due to benzyl alcohol degradation by *OH. The antioxidant activities of these biomaterials, particularly HYAFF -11p75 and AQUACEL, towards O2*- could be beneficial, as the scavenging of PMN-derived O2*- may remove initial sources of O2*- and further prevent the secondary formation of *OH. These ROS are thought to be a primary causal factor for the extensive degradation and metabolic alterations observed in chronic wounds.

Interaction of bone proteoglycans and proteoglycan components with hydroxyapatite.

The small leucine-rich proteoglycans (SLRPs) of bone interact with hydroxyapatite (HAP) and are proposed to play an important role in the regulation of the mineralisation process. The present study has examined the interaction of bone SLRPs, purified, liberated bone glycosaminoglycan (GAG) chains and core proteins, as well as commercial chondroitin 4-sulphate (C4S) with HAP. Isotherm data (0.02 M sodium acetate) revealed that the intact proteoglycans (PGs) and bone GAGs showed greater binding onto HAP with higher adsorption maxima than the constituent core proteins and commercial C4S. Adsorption was dependent on pH and ionic strength, increasing with decreasing pH and in the presence of calcium whilst decreasing in the presence of phosphate, suggesting that electrostatic effects are important. The data indicates that PG/GAG chemistry and conformation in solution are significant determinants in the adsorption process and provides important information concerning interfacial adsorption phenomena between the organic-inorganic phases of mineralised systems.

Proteoglycans and orthodontic tooth movement.

Proteoglycans represent an important and diverse family of extracellular matrix components within the connective tissues of the periodontium. This review focuses on the function and metabolism of the various proteoglycans in periodontal tissues, such as alveolar bone and periodontal ligament, and considers their potential fate in response to an orthodontic force. Such considerations provide an important background in evaluating the potential for proteoglycan metabolites, alongside other connective tissue metabolites, as biomarkers for assessing the deep-seated metabolic changes and as a diagnostic tool in monitoring orthodontic tooth movement.

Immunoelectron microscopic visualization of pro- and secreted forms of decorin and biglycan in the predentin and during dentin formation in the rat incisor.

Using antibodies raised against the proform and fully processed (secreted) forms of the proteoglycans decorin and biglycan, combined with gold electron immunohistochemistry, we observed in the incisors of five Sprague-Dawley rats that the proforms were mostly located in the cell bodies of odontoblasts, with a presence reduced to one-third or one-fourth in the processes. Proforms, also present in the extracellular matrix, were uniformly distributed throughout predentin, with higher labeling for probiglycan than pro-decorin. Both were present in lesser amounts in metadentin and dentin. With respect to the secreted form, grain density was at a constant level for biglycan in predentin and dentin, whereas a gradient was detected for decorin, the grain density being increased three times in the distal predentin. Although decorin labeling was diminished in metadentin, staining in circumpulpal dentin was similar to that found in distal predentin. We have previously reported a reverse gradient for chondroitin sulfate/dermatan sulfate distribution. To reconcile these diverging data, our hypothesis is that enzymatic proteolytic cleavage may remove the glycosylated N-terminal-containing region, resulting in a non- proteoglycan form of the molecule. Although substantial differences in distribution were apparent between the two proteoglycans, increasing interactions between proteoglycans and collagen, facilitated by the cleavage and loss of the N-terminal glycosaminoglycan chain region in the distal predentin, may be a prerequisite for dentin mineralization.

Proteoglycans in dentinogenesis.

The predominant proteoglycans present in predentin and dentin are the chondroitin-sulphate-rich decorin and biglycan and the keratan-sulphate-rich lumican and fibromodulin. These are small, interstitial, leucine-rich proteoglycans which have recently been shown to exist in gradients across the predentin. Antibodies recognizing chondroitin sulphate show a decreasing gradient from the pulpal aspect toward the mineralizing front, the converse being true for keratan sulphate. Antidecorin shows an increase toward the mineralization front. Evidence from biochemical, autoradiographic, and immunohistochemical studies implies that such changes may be brought about by gradients of metalloproteinases. This offers the possibility that the proteoglycans organize the collagen network for receipt of phosphoproteins and phospholipids, the former being evident only at the onset of dentin formation. The suggestion is raised that glycosaminoglycan-depleted leucine-rich protein cores act as sequester points for receipt of phosphoproteins in particular. The rigid, spatially oriented glycosaminoglycan chains on decorin and biglycan are known to bind calcium and may feature directly in mineral initiation.

Glycosaminoglycans in peri-implant sulcus fluid from implants placed in sinus-inlay bone grafts.

Glycosaminoglycans (GAG) present in peri-implant sulcus fluid (PISF) were used as an indicator of the metabolic activity in the supporting tissues of implants placed in maxillary bone or maxillary bone and bone grafts together. The study included 16 patients who received implants (Brånemark system(R)) and sinus-inlay bone grafts. In 12 of these patients, the implants were placed in either maxillary bone alone or maxillary bone and sinus-inlay bone grafts in combination. Altogether the patients received a total of 102 implants, of which 73 implants were placed in bone grafts and 29 implants in maxillary bone alone. Samples of PISF were tested at 2-8 days and at 6 months after abutment connection. Levels of the GAG's chondroitin-4-sulphate (C4S) and hyaluronan (HA) were assessed using cellulose acetate electrophoresis and densitometric scanning of Alcian blue-stained strips against known GAG standards. The C4S was used as a bone metabolic marker, and HA was used to reflect the progress of soft tissue healing. Comparing grafted and non-grafted regions, there was no significant difference in either C4S levels or HA levels during the first 8 days or at the 6 months period. The levels of HA from the first week collection did not differ significantly from the HA level after 6 months in either type of bone. However, the level of C4S was significantly lower after 6 months than during the first week, in both maxillary and grafted bone but consistent with a normal metabolic turnover. C4S can therefore be used as an indicator of the progressive healing of bone adjacent to implants.

Fluoride alters casein kinase II and alkaline phosphatase activity in vitro with potential implications for dentine mineralization.

Dentine phosphoprotein (DPP), a major non-collagenous acidic protein of dentine, undergoes altered phosphorylation in vivo in the presence of high fluoride concentrations. This has major implications for the altered mineralization patterns found during fluorosis. In dentine, casein kinase II is involved in phosphorylating DPP, and alkaline phosphatase (ALP) is ascribed roles in the dephosphorylation of DPP, increasing the inorganic phosphate at the mineralization front and the removal of pyrophosphate. Here the influence of fluoride in vitro on the activity of purified casein kinase II and ALP and its relation to altered patterns of mineralization were examined. Kinetic analysis showed that casein kinase II activity was completely inhibited at 0.04 M NaF. Vmax when compared to the control assay was significantly decreased (P < 0.0001) between concentrations 4 x 10(-4)-4 x 10(-8) M NaF. Significant changes to the Km (P < 0.0001) were also observed. ALP activity was inhibited by NaF (0.09-9 x 10(-8) M), with Vmax significantly decreased (P < 0.0001) at 0.09 M NaF. Alterations in the activity of these enzymes in the presence of fluoride may in part explain the decreased phosphorylation observed in DPP isolated from fluorotic dentine and may aid understanding of the altered matrix mediated mineralization patterns found during fluorosis.

Reactive oxygen species: a potential role in the pathogenesis of periodontal diseases.

The pathological events leading to the destruction of the periodontium during inflammatory periodontal diseases are likely to represent complex interactions involving an imbalance in enzymic and non-enzymic degradative mechanisms. This paper aims to review the increasing body of evidence implicating reactive oxygen species (ROS), derived from many metabolic sources, in the pathogenesis of periodontal tissue destruction. ROS are generated predominantly by polymorphonuclear leukocytes (PMN) during an inflammatory response and are regarded as being highly destructive in nature. The detection of ROS oxidation products, the elevation of iron and copper ions, which catalyse the production of the most reactive radical species, and the identification of an imbalance in the oxidant/antioxidant activity within periodontal pockets, suggests a significant role for ROS in periodontal tissue destruction. In vitro studies have shown that ROS are capable of degrading a number of extracellular matrix components including proteoglycans, resulting in the modification of amino acid functional groups, leading to fragmentation of the core protein, whilst the constituent glycosaminoglycan chains undergo limited depolymerisation. The identification and characterisation of connective tissue metabolites in gingival crevicular fluid (GCF) resulting from the degradation of periodontal tissues, notably alveolar bone, provides further evidence for a role for ROS in tissue destruction associated with inflammatory periodontal diseases.

The isolation and detection of non-collagenous proteins from the compact bone of the dinosaur Iguanodon.

This report describes the isolation of guanidinium chloride extractable protein from demineralised bone extracts obtained from the 125-130 mya dinosaur Iguanodon. Protein products were isolated in the Mr. range 5,000-66,000 using SDS-PAGE and represent the first electrophoretically defined proteins isolated from dinosaur tissues. The levels of glycine, aspartate and serine tentatively suggest the presence of phosphoproteins. Hydroxylysine and hydroxyproline were not detected, confirming the presence of non-collagenous material. In addition the absence of ornithine confirmed lack of bacterial contamination. The relatively high level of leucine in the 2MNaCl NaCl fractions together with the abolition of alcian blue reactivity following protease-free chondroitinase digestion suggests the presence of proteoglycans. The study is of interest in describing the early proteins laid down in mineralised tissues for epitactic crystal growth and may provide evidence on evolutionary aspects of bone proteins.

Effect of environmental haemin upon the physiology and biochemistry of Prevotella intermedia R78.

The effect of environmental haemin on the physiology and biochemistry of Prevotella intermedia R78 grown in batch culture was assessed. Extent and rate of growth increased as the environmental haemin concentration was raised. In addition, cell morphology was predominantly cocco-bacillary when cultured in high haemin environments, while bacillary forms were prevalent in low haemin conditions (< 2.5 mumol l-1). Cells harvested from low haemin environments produced greater numbers of extracellular vesicles and greater amounts of peptidolytic activity, haemagglutinating potential and haemin binding activity when compared with cells harvested from high haemin conditions. The results of the present study indicate that aspects of the biochemistry and physiology of P. intermedia are influenced by changes in environmental haemin levels.