H.B. Wen

Elongated Growth of Octacalcium Phosphate Crystals in Recombinant Amelogenin Gels under Controlled Ionic Flow

Amelogenin proteins constitute the primary structural entity of the extracellular protein framework of the developing enamel matrix. Recent data on the interactions of amelogenin with calcium phosphate crystals support the hypothesis that amelogenins control the oriented and elongated growth of enamel carbonate apatite crystals. To exploit further the molecular mechanisms involved in amelogenin-calcium phosphate mineral interactions, we conducted in vitro experiments to examine the effect of amelogenin on synthetic octacalcium phosphate (OCP) crystals. A 10% (wt/vol) recombinant murine amelogenin (rM179, rM166) gel was constructed with nanospheres of about 10- to 20-nm diameter, as observed by atomic force microscopy. The growth of OCP was modulated uniquely in 10% rM179 and rM166 amelogenin gels, regardless of the presence of the hydrophilic C-terminal residues. Fibrous crystals grew with large length-to-width ratio and small width-to-thickness ratio. Both rM179 and rM166 enhanced the growth of elongated OCP crystals, suggesting a relationship to the initial elongated growth of enamel crystals.

Dose-dependent Modulation of Octacalcium Phosphate Crystal Habit by Amelogenins:

In vitro studies on interactions between amelogenins and calcium phosphate crystals are critical for elucidating biomineralization mechanisms of tooth enamel. This work was aimed at investigating the effects of native porcine amelogenins on octacalcium phosphate (OCP) crystal growth in a gelatin gel. We prepared OCP mineral discs by circulating calcium and phosphate solutions on the opposite ends of the gels loaded with 0-2% amelogenin for one week. A dose-dependent modulation of OCP crystal habit by amelogenins was observed by scanning electron microscopy. While the incorporation of 0.125, 0.25, or 0.5% amelogenins showed no significant effect on the crystal morphology, in the presence of I and 2% amelogenins, the crystals were remarkably longer, having an average aspect ratio 3-5 times greater than that of those formed in the control gels. Transmission electron microscopy and atomic force microscopy suggested that amelogenin assemblies selectively blocked b-axial development, resulting in the c-axial elongation of OCP crystals.

Progressive accretion of amelogenin molecules during nanospheres assembly revealed by atomic force microscopy.

Amelogenin proteins, the principal components of the developing dental enamel matrix, self-assemble to form nanosphere structures that are believed to function as structural components directly involved in the matrix mediated enamel biomineralization. The self-assembly behavior of a recombinant murine amelogenin (rM179) was investigated by atomic force microscopy (AFM) for further understanding the roles of amelogenin proteins in dental enamel biomineralization. Recombinant rM179 amelogenin was dissolved in a pH 7.4 Tris-HCl buffer at concentrations ranging from 12.5 to 300 microg/ml. The solutions were adsorbed on mica, fixed with Karnovsky fixative and rinsed thoroughly with water for atomic force microscopy (AFM). At low concentrations (12.5-50 microg/ml), nanospheres with diameters varying from 7 to 53 nm were identified while at concentrations ranging between 100-300 microg/ml the size distribution was significantly narrowed to be steadily between 10 and 25 nm in diameter. These nanospheres were observed to be the basic building blocks of both engineered rM179 gels and of the developing enamel extracellular matrix. The stable 15-20-nm nanosphere structures generated in the presence of high concentrations of amelogenins were postulated to be of great importance in facilitating the highly organized ultrastructural microenvironment required for the formation of initial enamel apatite crystallites.

Modulation of apatite crystal growth on Bioglass by recombinant amelogenin

The effects of a recombinant mouse amelogenin (rM179) on the growth of apatite crystals nucleated on a bioactive glass (45S5 type Bioglass) surface were investigated with a view to gaining a better understanding of the role of amelogenin protein in tooth enamel formation and of its potential application in the design of novel enamel-like biomaterials. Bioglass discs were incubated in phosphate-buffered saline (PBS) to preform a calcium phosphate surface layer and subsequently immersed in blank, bovine serum albumin (BSA)- and rM179-containing supersaturated calcification solutions (SCS(B), SCS(BSA) and SCSrM179), respectively. Calcium phosphate layers formed on all the treated samples and were characterized to be apatite by X-ray diffraction and Fourier transmission infrared spectrophotometry. Under scanning electron microscopy, plate-shaped crystals (approximately 50 nm thick and 300-600 nm across) were observed on the samples after PBS incubation. The crystals grown from SCS(B) were of the typical plate shape except for an increased thickness, while needle-shaped crystals (200-300 nm long and 50-70 nm thick) were precipitated on the SCS(BSA)-immersed samples. Interestingly, it was found that the crystals deposited on the SCSrM179-immersed samples adopted an elongated, curved shape (approximately 500 nm long and approximately 120 nm thick). Further TEM observations showed that the crystals generated by the SCSrM179 immersion appeared to be composed of bundles of lengthwise crystals (15-20 nm thick) orientated parallel to one another, much alike the long and thin crystals observed in the very early stage of enamel formation. The significant modulation by the rM179 protein of apatite crystal growth is quite different from the overall inhibition observed by BSA and most likely is relevant to the specific function of the amelogenin matrix in controlling enamel crystal growth in vivo.

Effects of amelogenin on the transforming surface microstructures of Bioglass in a calcifying solution

Topographies of a bioactive glass (45S5 type Bioglass(R)) during 0-4 h of immersion in a supersaturated calcifying solution (SCS) and the SCS containing recombinant porcine amelogenin rP172 (SCS(rP172)) were observed by atomic force microscopy. Other techniques including X-ray diffraction, scanning electron microscopy coupled with energy dispersive X-ray spectroscopy, and transmission electron microscopy were used for some complementary microstructural investigations. The smooth Bioglass surface changed to be very rough after 0.5 h of SCS immersion because of glass network dissolution. Spherical silica-gel particles with diameters of 150-300 nm consisting of substructures of 20-60 nm across had formed on the sample surfaces after 1 h of SCS immersion. The chemisorption of amorphous calcium phosphate and crystallization of nanophase apatite were seen to occur epitaxially on the silica-gel structures during 1-4 h of SCS immersion. During the first 0.5 h of SCS(rP172) immersion, more than 95% of rP172 protein in solution was adsorbed onto the sample surfaces and generated spherical assemblies of 10-60 nm diameters. During 0.5-4 h of SCS(rP172) immersion, the protein assemblies of rP172 remarkably induced the formation of orientated silica-gel plates (approximately 100-nm wide and 50-nm thick) and subsequently of long and thin apatite needle crystals. The recombinant amelogenin rP172-modulated apatite crystals resembled those formed in the early stage of tooth enamel biomineralization, suggesting the functional roles of amelogenins during the oriented growth of enamel crystallites and a great potential for amelogenins in applications designed to fabricate enamel-like calcium phosphate biomaterials.

Assembly of amelogenin proteolytic products and control of octacalcium phosphate crystal morphology.

The formation of enamel apatite crystals involves extracellular molecular events among which matrix assembly, interactions with growing crystals, and protein processing and removal are the subject of numerous investigations. Following the description of amelogenin nanospheres and the evidence for their presence in vivo as the principal structural component of developing dental enamel, we have focused our studies on investigating at the molecular level the process of nanosphere assembly and evaluating the effects of amelogenin on crystal growth and morphology. This paper is a short review of our recent studies with a focus on the assembly of amelogenin proteolytic products and their modulating effect on octacalcium phosphate (OCP) crystal morphology. In addition, we report that incorporation of amelogenins into 10% gelatin gel does not affect diffusion of calcium. This remarkable finding indicates that the observed modulation effect by amelogenin on OCP crystal morphology is not due to alteration of calcium diffusion into the gels but is the result of direct amelogenin-mineral interactions.

Effects of Ionic Flow and Amelogenins on the Lengthwise Growth of Octacalcium Phosphate Crystals in a Model System of Tooth Enamel Formation

Abstract : This paper briefly reviews our recent studies which aimed to investigate the effects of 1) the Ca(exp 2+) and PO4(exp 3-) ions flow and 2) amelogenins on the lengthwise growth of octacalcium phosphate (OCP), which is a potent precursor of enamel apatite crystal. OCP crystals were groan at 37 degrees C in a dual membrane system under various amount of ionic inflow into a reaction space using 1) 5-30mM Ca and PO4 solutions as ionic sources arid 2) extracted bovine amelogenin arid recombinant murine amelogenins (rM179, rM166). With an increase in the amount of Ca(exp 2+) and/or PO4(3-) ions flow, the length of OCP crystal increased, while the width decreased. As a result, the length to width (L/W) ratio of crystal changed from 3 to 95, while the width to thickness (W/T) ratio from 32 to 9. The effect of amelogenins was unique, regardless of the type of amelogenins Rod-like arid prism-like OCP crystals with large L/W (61-107) and small W/T(1.3-2.2) ratios were formed in 10% amelogenin gels. In contrast, characteristic ribbon-like OCP crystals grew without protein arid with gelatin, albumin, polyacrylamide gel and agarose gel. Specific interaction of amelogenins with OCP crystal was ascribed to the self-assembly property of amelogenin molecules and their hydrophobic nature. It was suggested that ionic flow and amelogenins play some critical roles in the elongated growth of enamel crystals.

Amelogenin Nanospheres Modulate Crystal Habit of Octacalcium Phosphate and Hydroxyapatite Crystals in In Vitro Model Systems

This paper is a short review of recent studies, which were undertaken to investigate interactions of amelogenin with octacalcium phosphate (OCP), and apatite. OCP crystals were grown using two independent experimental systems; (a) in a 10% gelatin gel, containing 0-2% amelogenin, where the crystals were formed in a double-diffusion chamber, and (b) in a 10% pure amelogenin gel, where crystal growth took place in between a cation-selective and a dialysis membrane. Apatite crystals were grown from a supersaturated calcifying solution on a bioactive glass in the absence (SCS B ) and the presence of amelogenin (SCSrM179). It was found that OCP crystals formed in 10% gelatin gel containing 1-2% amelogenin were longer (3-5 times larger in aspect ratio) than the OCP crystals formed in 10% gelatin without amelogenin. A profound effect was that found in the cation selective membrane system when 10% amelogenin inhibited the growth morphology in a specific manner. Affected crystals had a length to width ratio twice larger than that of control crystals while the width to thickness ratio was about 1/12 of that of the control crystals. Amelogenin promoted the formation of bundles of lengthwise apatite crystals, which were all oriented parallel to their c axes when grown on SCSrM179. It was found that individual apatite crystals within those bundles adopted an elongated, curved shape. The data presented here suggest that amelogenin nanospheres modulate the growth morphology of apatite and OCP crystals and indicate significant functional roles for amelogenin proteins during the in vivo oriented growth of enamel crystallites.