Adsorption and interactions of dentine phosphoprotein with hydroxyapatite and collagen.

Dentine phosphoprotein (DPP) has been proposed to both promote and inhibit mineral deposition during dentinogenesis. The present study aimed to investigate the molecular interactions of DPP and dephosphorylated DPP (DPP-p) with hydroxyapatite (HAP). Bovine DPP was purified and dephosphorylated by alkaline phosphatase to obtain DPP-p. DPP and DPP-p adsorption to HAP was determined along with their ability, when free in solution or bound to collagen, to influence HAP-induced crystal growth. Absorption isotherms suggested that lower DPP concentrations (1.5-6.25 microg ml(-1)) demonstrated a reduced affinity for HAP compared with higher protein concentrations (12.5-50.0 microg ml(-1)). Dephosphorylated DPP had a much reduced affinity for HAP compared with DPP. Dentine phosphoprotein inhibited seeded HAP crystal growth, in a dose-dependent manner, whilst removal of the phosphate groups reduced this inhibition. When bound to collagen fibrils, DPP significantly promoted the rate of HAP crystal growth over 0-8 min. Conversely, DPP-p and collagen significantly decreased the rate of crystal growth over 0-18 min. These results indicate a major role for the phosphate groups present on DPP in HAP crystal growth. In addition, concentration-dependent conformational changes to DPP, and the interaction with other matrix components, such as collagen, are important in predicting its dual role in the mineralization of dentine.

Modulation of collagen fibrillogenesis by dentinal proteoglycans.

Studies have identified different pools of proteoglycan (PG) species present within the unmineralized matrix of the predentine, the transitional phase at the predentine-dentine interface and the mineralized dentine. These PGs alter with respect to the chemical nature of their glycosaminoglycan (GAG) chains and as a result of extracellular processing of the macromolecule in the matrix. The present study has examined the influence of the PGs isolated from these phases and the influence of the attached GAG chains, upon their ability to control collagen fibrillogenesis. PGs isolated from the three phases were characterized and determined to contain a mixture of decorin and biglycan. Results have indicated that predentine PGs, which are substituted with a higher proportion of dermatan sulfate, significantly delayed fibril formation while ultimately promoting the formation of thicker fibrils. Removal of the GAG chains further delayed fibrillogenesis, leading to the formation of thinner fibrils, compared with the collagen-only control. PGs isolated from predentine-dentine, which contained a higher proportion of chondroitin sulfate, also significantly delayed fibrillogenesis, resulting in thicker collagen fibrils. GAG chains attached to the predentine-dentine interface PGs played a role in the timing of fibrillogenesis with fibril formation initiated at the same time as the collagen control, but yielding thicker fibrils. Dentine PGs significantly inhibited fibrillogenesis and fibril thickness over concentrations of 50-25 microg/mL protein. In conclusion, the PGs isolated from the distinct phases have indicated differing roles in the orchestrated organization of the extracellular matrix during dentinogenesis, with roles for both the core protein and attached GAG chains indicated.

Effect of serum albumin on glycosaminoglycan inhibition of hydroxyapatite formation.

The interaction of proteins with hydroxyapatite (HAP), specifically proteoglycans and their spatial arms, glycosaminoglycans (GAGs) in addition to serum proteins, play an important role in regulating biological mineralisation. In this study, seeded HAP growth experiments revealed that inhibition of HAP formation by bovine serum albumin (BSA) was significantly elevated in combination with chondroitin 4-sulphate (C4S) versus BSA with heparin. Data suggest that C4S and BSA adsorb to different calcium sites on the HAP surface. In contrast, complexes of heparin and BSA may adsorb to HAP, thus reducing the rate of inhibition due to steric effects. In addition, the amount of protein bound to HAP growth seed was significantly higher in the presence of heparin versus C4S, confirming this suggestion. Furthermore, the data indicate that a GAG-induced conformational change in BSA occurs, which affects inhibition. This work provides novel information concerning binary molecule modulation of HAP growth.

Identification of proteinaceous material in the bone of the dinosaur Iguanodon.

This study has directed attention at the search for bone-related proteins in an extract of demineralized rib bone of the 120 mya Iguanodon. The inner compact bone was demineralized and the GuCl extract resolved into 11 fractions using anion exchange chromatography, which all contained silver-reactive proteins with various amino acid profiles. Two specific fractions, iv and xi, revealed characteristics typical of contemporary phosphoproteins and proteoglycans, respectively. Fraction iv, 43-57 kDa, contained a high ratio of aspartate and serine, although no phosphate was discernable. Fraction xi contained a band of 41-47 kDa and was rich in chondroitin sulphate and hyaluronan. In addition an early eluting fraction was immunoreactive with an antibody against osteocalcin. A cancellous bone fraction from the same bone sample was also analyzed using N-terminal sequencing and revealed potential similarities with cystatin. While we do not claim to have identified the presence of intact proteins, this study has value in demonstrating that extruded extracellular matrix is protected by its capacity to induce mineralization, which subsequently is important in conserving detectable protein products in ancient skeletal tissues.

The interaction of recombinant decorin with alpha2HS-glycoprotein-implications for structural and functional investigations.

Isolated protein preparations of the small leucine-rich proteoglycans (SLRPs) associated with mineralized tissues have provided important information in understanding their structural and functional interactions within extracellular matrices and their potential roles in mineralization. Two important SLRPs, decorin and biglycan, copurify following extraction and purification from mineralized tissues using standard procedures, and to overcome this problem decorin was synthesized within a mammalian expression system to obtain pure preparations. The expressed protein was purified from the culture medium using anion-exchange chromatography, and characterization confirmed the presence of a decorin-rich fraction. However, N-terminal sequencing revealed the additional presence of alpha2HS-glycoprotein (alpha2HSG), representing approximately 35% of the total purified fraction. The decorin-rich fraction was subjected to selected further purification techniques to separate decorin from alpha2HSG. Application of the sample at a low concentration (1 mg/ml) to a second anion-exchange procedure and elution over an expanded sodium chloride gradient resulted in a high degree of purity (98%), with a single protein isolate demonstrable by SDS-PAGE. Electroelution achieved partial purification ( approximately 89%), but immunoprecipitation with antibodies against the glycosaminoglycan chain and the polyhistidine tag failed to separate the two proteins. This study suggests there is a strong interaction between recombinantly produced decorin and alpha2 HSG and highlights the importance of the purification technique to the application of recombinantly produced proteins or those that have been extracted from mineralized tissues for use in structural and functional interactions.

Hyaluronan and its potential role in periodontal healing.

Hyaluronan is a natural tissue component, which plays a vital role in the functioning of extracellular matrices, including those of the periodontium. This molecule is also important in relation to the mechanisms associated with inflammation and wound healing. The application of exogenous hyaluronan and hyaluronan-based biomaterials has been successful in manipulating and accelerating the wound healing process in a number of medical disciplines, as evident in ophthalmology, dermatology and rheumatology. It is conceivable that hyaluronan administration to periodontal sites could achieve comparable beneficial effects in periodontal healing and surgery, hence aiding treatment of periodontal disease.

Cell-associated collagenolytic activity by Candida albicans.

Cell associated collagenolytic activity of Candida albicans was quantified by measuring the degradation of synthetic peptide 2-furanacryloyl-Leu-Gly-Pro-Ala (FALGPA), which is a specific substrate for collagenase, by the freeze-thaw procedure method. This collagenolytic activity was enhanced by cells cultured in the presence of bovine serum albumin (BSA) in culture medium. However, this activity was inhibited in the presence of ethylenediaminetetraacetic acid disodium salt (EDTA-2Na), but not by the serine proteinase inhibitor p-amidinophenyl methanesulfonyl fluoride (APMSF), nor the aspartyl proteinase inhibitor pepstatin A. These results suggested the presence of a metalloenzyme on pericellular C. albicans.

Inhibition of hydroxyapatite crystal growth by bone proteoglycans and proteoglycan components.

The small leucine-rich proteoglycans (SLRPs) interact with hydroxyapatite (HAP) and have been demonstrated to be important modulators of mineralisation. In the present study we have examined the effect of bone SLRPs, purified bone glycosaminoglycan (GAG) chains and core proteins as well as commercial chondroitin 4-sulphate, chondroitin 6-sulphate and desulphated chondroitin on HAP crystal growth. Seeded HAP growth experiments revealed that addition of bone GAG chains resulted in almost complete inhibition of crystal growth (93%), with addition of core proteins and intact PGs resulting in 55 and 37% inhibition, respectively. In contrast, commercial chondroitin 4-sulphate was significantly less inhibitory compared with the bone SLRPs and components, yielding only a 6% reduction in HAP-induced crystal growth at the same concentration. Significantly, chondroitin 6-sulphate was found to be noninhibitory, whilst desulphated chondroitin was inhibitory to seeded HAP growth. The data indicate that direct adsorption of SLRPs to growth sites and their ability to bind calcium are significant determinants in the inhibitory process. In addition, PG/GAG chemistry and the conformation of the macromolecules in solution have also been shown to be important. This work provides new information regarding the role of bone SLRPs and their components in the regulation of the mineralisation process.

Interaction of glucuronic acid and iduronic acid-rich glycosaminoglycans and their modified forms with hydroxyapatite.

Proteoglycans and their spatial arms, glycosaminoglycans (GAGs), are known to interact with hydroxyapatite (HAP) and have been implicated as important modulators of mineralisation. In the present study isotherm data (0.02 M sodium acetate, pH 6.8) revealed that the iduronic-rich GAGs heparan sulphate, heparin and dermatan sulphate showed greater binding onto HAP with higher adsorption maxima compared with the glucuronic acid-rich GAGs chondroitin-4-sulphate, chondroitin-6-sulphate and hyaluronan. Chemically desulphated chondroitin showed no adsorption onto HAP. With the exception of hyaluronan, the GAGs studied showed no desorbability in sodium acetate buffer only, whereas in di-sodium orthophosphate, desorption occurred much more readily. The data indicates that GAG chemistry and conformation in solution greatly influence the interaction of these molecules with HAP. The conformational flexibility of iduronic acid residues may be an important determinant in the strong binding of iduronic acid-rich GAGs to HAP, increasing the possibility of the appended anionic groups matching calcium sites on the HAP surface, compared with more rigid glucuronic acid residues. This work provides important information concerning interfacial adsorption phenomena between the organic-inorganic phases of mineralised systems.