Analysing the bioactive makeup of demineralised dentine matrix on bone marrow mesenchymal stem cells for enhanced bone repair.

Dentine matrix has proposed roles for directing mineralised tissue repair in dentine and bone; however, the range of bioactive components in dentine and specific biological effects on bone-derived mesenchymal stem cells (MSCs) in humans are less well understood. The aims of this study were to further elucidate the biological response of MSCs to demineralised dentine matrix (DDM) in enhancing wound repair responses and ascertain key contributing components. Dentine was obtained from human teeth and DDM proteins solubilised with ethylenediaminetetraacetic acid (EDTA). Bone marrow derived MSCs were commercially obtained. Cells with a more immature phenotype were then selected by preferential fibronectin adhesion (FN-BMMSCs) for use in subsequent in vitro assays. DDM at 10 µg/mL reduced cell expansion, attenuated apoptosis and was the minimal concentration capable of inducing osteoblastic differentiation. Enzyme-linked immunosorbent assay (ELISA) quantification of growth factors indicated physiological levels produced the above responses; transforming growth factor ß (TGF-ß1) was predominant (15.6 ng/mg DDM), with relatively lower concentrations of BMP-2, FGF, VEGF and PDGF (6.2-4.7 ng/mg DDM). Fractionation of growth factors from other DDM components by heparin affinity chromatography diminished osteogenic responses. Depletion of biglycan from DDM also attenuated osteogenic potency, which was partially rescued by the isolated biglycan. Decorin depletion from DDM had no influence on osteogenic potency. Collectively, these results demonstrate the potential of DDM for the delivery of physiological levels of growth factors for bone repair processes, and substantiate a role for biglycan as an additional adjuvant for driving osteogenic pathways.

Growth Factor Liberation and DPSC Response Following Dentine Conditioning.

Liberation of the sequestrated bioactive molecules from dentine by the action of applied dental materials has been proposed as an important mechanism in inducing a dentinogenic response in teeth with viable pulps. Although adhesive restorations and dentine-bonding procedures are routinely practiced, clinical protocols to improve pulp protection and dentine regeneration are not currently driven by biological knowledge. This study investigated the effect of dentine (powder and slice) conditioning by etchants/conditioners relevant to adhesive restorative systems on growth factor solubilization and odontoblast-like cell differentiation of human dental pulp progenitor cells (DPSCs). The agents included ethylenediaminetetraacetic acid (EDTA; 10%, pH 7.2), phosphoric acid (37%, pH <1), citric acid (10%, pH 1.5), and polyacrylic acid (25%, pH 3.9). Growth factors were detected in dentine matrix extracts drawn by EDTA, phosphoric acid, and citric acid from powdered dentine. The dentine matrix extracts were shown to be bioactive, capable of stimulating odontogenic/osteogenic differentiation as observed by gene expression and phenotypic changes in DPSCs cultured in monolayer on plastic. Polyacrylic acid failed to solubilize proteins from powdered dentine and was therefore considered ineffective in triggering a growth factor-mediated response in cells. The study went on to investigate the effect of conditioning dentine slices on growth factor liberation and DPSC behavior. Conditioning by EDTA, phosphoric acid, and citric acid exposed growth factors on dentine and triggered an upregulation in genes associated with mineralized differentiation, osteopontin, and alkaline phosphatase in DPSCs cultured on dentine. The cells demonstrated odontoblast-like appearances with elongated bodies and long extracellular processes extending on dentine surface. However, phosphoric acid-treated dentine appeared strikingly less populated with cells, suggesting a detrimental impact on cell attachment and growth when conditioning by this agent. These findings take crucial steps in informing clinical practice on dentine-conditioning protocols as far as treatment of operatively exposed dentine in teeth with vital pulps is concerned.

A novel ex vivo culture model for inflammatory bone destruction.

Pathological alterations in the balance of bone metabolism are central to the progression of inflammatory bone diseases such as periodontal disease. We have developed and characterized a novel ex vivo murine mandible model of inflammatory bone destruction. Slices of mandible were cultured for 14 days in the presence or absence of P. gingivalis lipopolysaccharide (LPS) or pro-inflammatory cytokines. Following culture, cell viability and tissue histomorphometry were assessed with quantification of matrix proteins, resident osteoclasts, ligament cells, monocytes, macrophages, and neutrophils. In the absence of inflammatory factors, culture viability, osteoclasts, and matrix components were maintained. LPS or TNFα stimulation demonstrated an increase in cellular proliferation, monocyte cells, osteoclast differentiation, and matrix degradation. Pathophysiological bone metabolism can be induced via exposure to LPS and direct influence of TNFα within the model despite the absence of systemic circulation, providing a model for inflammatory bone destruction and investigation of the effects of novel therapeutics.

An ex vivo culture model for orthodontically induced root resorption.

OBJECTIVES: Root resorption is a ubiquitous although undesirable sequela to orthodontic treatment. Current methods to investigate the pathophysiology have certain limitations. In pursuit to understand and develop treatment modalities for orthodontically induced root resorption, the ability to manipulate cells within their natural extracellular matrix in a three dimensional organotypic model is invaluable. The study aimed to develop a laboratory-based organotypic model to investigate the effect of orthodontic forces on the periodontium. METHODS: Mandibular slices of male Wistar rats were maintained in Trowel-typed cultures at 37°C in 5% carbon dioxide in air for 7 days with test specimens subjected to compressive forces at 50 g and 100g by stainless steel springs. Tissue architecture and cell viability were maintained under culture conditions. RESULTS: Osteoclast numbers increased significantly in both test groups whilst odontoclasts increased in the 50 g group. Immunohistochemistry demonstrated increased dentine sialoprotein expression in both test groups, suggesting changes in mineralization-related activity due to mechanical strain. CONCLUSION: The study showed initial cellular and molecular changes of key markers that relate to root resorption in response to mechanical loading. CLINICAL SIGNIFICANCE: Severe root resorption may occur when forces applied are heavy or transmitted over an extended period and could lead to mobility and tooth loss. This ex vivo model can be used to investigate cellular and molecular processes during orthodontic tooth movement which may advance the clinical management of root resorption.

TGF-beta1 exposure from bone surfaces by chemical treatment modalities.

Growth factors are known to be sequestered to the mineralised matrix of bone. The aim of this study was to investigate the ability of citric acid, EDTA, calcium hydroxide and sodium hydroxide to release active growth factors from bone surfaces, able to promote osteoblast differentiation. All chemical treatments increased surface levels of TGF-beta1 (used as a biomarker of growth factor release), compared to control bone surfaces treated with PBS. Differences were observed in the kinetics of TGF-beta1 exposure at the surface and its subsequent release into the aqueous environment for the different chemical treatments. Surface levels of growth factor following chemical treatment were low, but of sufficient concentration to stimulate cell expansion and osteoblast differentiation of bone marrow stromal cells grown on EDTA and calcium hydroxide treated surfaces compared to PBS treated surfaces. The increased osteogenic potential on these surfaces may relate to an increase in growth factor availability and changes to the surface chemistry and topography.

TGF-beta/extracellular matrix interactions in dentin matrix: a role in regulating sequestration and protection of bioactivity.

TGF-beta isoforms sequestrated in dentin matrix potentially provide a reservoir of bioactive molecules that may influence cell behavior in the dentin-pulp complex following tissue injury. The association of these growth factors with dentin matrix and the influence of such associations on the bioactivity of growth factors are still unclear. We used surface plasmon resonance technology in the BIAcore 3000 system to investigate the binding of TGF-beta isoforms 1 and 3 to purified decorin, biglycan, and EDTA soluble dentin matrix components. TGF-beta isoforms 1 and 3 were immobilized on sensorchips CM4 through amine coupling. For kinetic studies of protein binding, purified decorin and biglycan, isolated EDTA soluble dentin matrix, and dentin matrix immunodepleted of decorin and/or biglycan were injected over TGF-beta isoforms and allowed to interact. Programmed kinetic analysis software provided sensorgrams for each concentration of proteoglycan or dentin matrix extract injected. Purified decorin and biglycan and dentin matrix extract bound to the TGF-beta isoforms. However, the association with TGF-beta3 was much weaker than that with TGF-beta1. After immunoaffinity depletion of the dentin matrix extract, the level of interaction between the dentin matrix extract and TGF-beta was significantly reduced. These results suggest isoform-specific interactions between decorin/biglycan and TGF-beta isoforms 1 and 3, which may explain why TGF-beta3 is not detected in the dentin matrix despite being expressed at higher levels than TGF-beta1 in odontoblasts. These proteoglycans appear to play a significant role in TGF-beta/extracellular matrix interactions and may be important in the sequestration of these growth factors in the dentin matrix.

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.

Extracellular matrix metabolites as potential biomarkers of disease activity in wound fluid: lessons learned from other inflammatory diseases?

The new era of pharmacogenetics has identified a potential for individuals to receive customized treatments for a variety of disease states. For such individualized treatments to fulfil their potential, it will be essential for clinicians to be able to monitor disease activity, ideally in a rapid, noninvasive fashion. The accessibility of the skin offers much potential to develop noninvasive tests of metabolic and disease activity for clinical use. Impaired human wound healing in the skin is a chronic inflammatory disorder in which the development of such tests has considerable potential, aiding clinical decision making and monitoring responses to treatment. This review article discusses how studies in other human diseases have highlighted potential biochemical markers (biomarkers) of disease activity in secreted biofluids, as aids to determining disease and metabolic activity within tissues. Using, as examples, lessons learned in the study of disease activity and prognosis of other chronic inflammatory conditions, such as osteoarthritis and periodontal disease, this review highlights the potential of dermal extracellular matrix (ECM) components (collagens, proteoglycans, hyaluronan and glycoproteins) for such uses. The limitations of currently utilized techniques and the concept that analysis of ECM components in wound fluid may represent useful biomarkers of disease activity are also discussed.

Differential roles for small leucine-rich proteoglycans in bone formation.

This paper reviews our current state of knowledge of the roles the small leucine-rich proteoglycans (SLRPs) play in the formation of connective tissue and mineralised tissue matrices. Both, the SLRPs biglycan and decorin are highly expressed in extracellular bone matrix and there is now substantial evidence to support an increasing role for biglycan and decorin in influencing bone cell differentiation and proliferative activity. In addition decorin and biglycan have been implicated in regulating mineral deposition and crystal morphology, whilst decorin has also roles in organic matrix assembly. In order to further assess the role of these SLRPs during bone formation we have initiated studies investigating primary bone cell culture models from rats (bone marrow stromal cells, and bone cells from alveolar bone explants), and identified periods relating to cell proliferation, organic matrix deposition, remodeling of the osteoid, and mineral deposition. Analysis of mRNA levels and the nature of the proteoglycan demonstrated that dermatan sulphate substituted biglycan was expressed during phases relating to cell proliferation, ceased at early matrix deposition, and then biglycan was re-expressed at the onset of mineralisation, but was conjugated to chondroitin sulphate. Decorin was expressed later than biglycan, was associated with early matrix deposition, but then continued to the mineralisation stages. Again, dermatan sulphate-decorin prevailed earlier within osteoid matrix, whilst chondroitin sulphate-decorin predominated later within the mineralizing matrix. The nature of the GAG chain conjugated to SLRP and the timing of its expression would seem to dictate the functions biglycan and decorin play in bone formation.

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.

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.

Altered expression of matrix metalloproteinases within mineralizing bone cells in vitro in the presence of fluoride.

Fluoride is known to alter mineralization within bone, although the mechanism for its action is unclear. An important stage in the formation of mineralized tissues is the remodeling of the osteoid, facilitating mineral deposition. Using a bone mineralizing culture system derived from rat femur washes, this study investigated the influence of fluoride on MMP expression at a developmental stage relating to the onset of mineralization. Bone cells cultured in the absence of fluoride synthesized an active form of a 45-kD MMP, which was immunoreactive with an antibody to human MMP-1 (although full characterization of this MMP was not achieved), trace levels of an MMP immunoreactive with anti-MMP-3, and a 66-kD proteolytic species. Incubation in 10(-7) and 10(-5) M fluoride resulted in a decrease in expression of the 45-kD MMP, sharp increases in the expression of MMP-3, and the appearance of a band at 110 kD, which showed immunoreactivity for MMP-9. The influence of fluoride on MMP expression is likely to influence the composition of the remodeling matrix and subsequent mineralization and offers a potential mechanism by which fluoride alters mineralization.

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 wound dressing materials--carboxymethylcellulose, hyaluronan benzyl ester and hyaluronan, towards polymorphonuclear leukocyte-derived reactive oxygen species.

In chronic wounds, factors are released which perpetuate inflammatory processes, including polymorphonuclear leukocyte (PMN)-derived reactive oxygen species (ROS), such as superoxide radical (O(2)*-) and hydroxyl radical (*OH) species. The glycosaminoglycan, hyaluronan, has established antioxidant properties towards ROS, although the antioxidant potential of wound dressing biomaterials, such as 75% benzyl esterified hyaluronan (BEHA) and carboxymethylcellulose (CMCH), are less characterised. This study compared the antioxidant properties of high and low molecular weight hyaluronan (HMWT HA and LMWT HA), BEHA and CMCH towards ROS, generated by stimulated PMN in vitro. The antioxidant capacities of each biomaterial were assessed by their inhibition of O(2)*- -induced cytochrome C reduction, generated via PMN stimulation by phorbol myristyl acetate (PMA); and their inhibition of *OH-induced 2-deoxy-D-ribose degradation, generated by PMA stimulated PMN in the presence of a ferric chloride-EDTA chelate. All biomaterials, except LMWT HA, possessed dose-dependent antioxidant properties against O(2)*-, BEHA having greatest antioxidant potential, followed by HMWT HA and CMCH. HMWT HA exhibited the highest dose-dependent antioxidant properties towards *OH, followed by BEHA and CMCH. LMWT HA demonstrated no antioxidant properties towards *OH. These antioxidant activities, particularly towards O(2)*-, may be beneficial in removing the initial source of ROS necessary for the secondary formation of *OH, implicated as a causal factor for the extensive metabolic alterations observed in chronic wounds.

TGF-beta latency-associated peptides (LAPs) in human dentin matrix and pulp.

Transforming growth factor (TGF)-beta s in dentin matrix provide a pool of bioactive molecules, but association with latency-associated peptides (LAPs) may influence their activity. We investigated TGF-beta 1, -beta 2, and -beta 3 LAP expression in sound and carious human teeth. Teeth were fixed and processed immediately following extraction prior to staining with rabbit polyclonal antibodies to the TGF-beta LAPs. A soluble dentin matrix fraction was prepared from dissected human dentin and sequential extraction of pulpal ECM was performed prior to purification. Fractions were Western blotted and probed with the LAP antibodies. All three LAPs were present in odontoblasts, cells of the pulp, and predentin; however, no staining of mineralized dentin matrix was seen. Similar patterns of expression were seen in carious tissue. Expression of TGF-beta LAPs in cells and pulpal matrix of healthy and carious teeth will be important in regulation of TGF-beta activity and may modulate the tissue response to injury.

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.