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MAS-NMR spectroscopy studies in the setting reaction of glass ionomer cements.

OBJECTIVES: The main objective is the characterisation of the setting reaction in glass ionomer cements based on experimental ionomer glasses with different fluorine content and a commercial glass ionomer cement liquid by using 13C CP/MAS-NMR, 29Si, 27Al and 31P MAS-NMR spectroscopy in order to receive information specifically about the cross-linking process. METHODS: Different fluorine containing glass compositions based on 4.5SiO2-3Al2O3-1.5P2O5-(5-z)CaO-zCaF(2) where z=0-3, were mixed with a commercially available polymer liquid to form glass ionomer cements. The cements were subjected to 27Al, 13C CP/MAS, 29Si, and 31P MAS-NMR analysis. RESULTS: The 27Al spectra showed clearly the formation of six-fold coordinate Al(VI), that may crosslink the carboxyl groups in the poly-acid molecules. A shift towards to more positive values of the carboxyl peak in the 13C CP/MAS-NMR spectra showed clearly the proton dissociation of the carboxyl groups. A shift towards more negative values was observed in the 29Si MAS-NMR spectra, suggesting formation of hydrated silica gel and consequently formation of additional Si-O-Si bonds. 31P MAS-NMR spectra also reflected changes in the coordination state around a PO4(3-) tetrahedron. Increasing the fluorine content of the glasses resulted generally in increased reactivity during setting, due to promoting cross-linking and repolymerisation of the silicate phase, followed by clear changes in the MAS-NMR spectra. CONCLUSIONS: The cross-linking process during the setting reaction of glass ionomer cements can be followed by MAS-NMR spectroscopy observing the conversion of Al(IV) to Al(VI). The acid base setting reaction is completed in 1 day and no further significant changes in the MAS-NMR spectra can be observed. Further study is required in order to understand the role of phosphorus.

Aluminum↗

Altered vitronectin receptor (alphav integrin) function in fibroblasts adhering on hydrophobic glass.

Function of integrins is crucial for adhesion, movement, proliferation, and survival of cells. In a recent study we found impaired fibronectin receptor function on hydrophobic substrata (G. Altankov et al. J Biomater Sci Polym Edn 1997;8:712-740). Here, we have studied the distribution and function of the vitronectin receptor (alphav integrin) in fibroblasts adhering on hydrophilic glass and hydrophobic octadecyl glass (ODS). The morphology of fibroblasts and the organization of actin cytoskeleton were studied and found to be altered on ODS, where the cells did not spread and possessed condensed actin. Pretreatment of the surfaces with serum or pure vitronectin improved cell morphology on both substrata, resulting in the development of longitudinal actin stress fibers. It was found with biotinylated vitronectin that comparable quantities of vitronectin were adsorbed from single vitronectin solutions or serum on glass and on hydrophobic ODS. The organization of the vitronectin receptors on the ventral cell surface was investigated in permeabilized cells showing normal focal adhesions in fibroblasts plated on glass but none of these structures on ODS. The distribution of alphav integrin on the dorsal cell surface was studied on nonpermeabilized living cells after antibody tagging. While fibroblasts adhering on plain or serum-treated glass developed a linear organization of alphav integrin, cells on plain and serum-treated ODS were not able to reorganize the vitronectin receptor. Studies on signal transduction with antiphosphotyrosine antibodies revealed co-localization of alphav integrin and phosphotyrosine in focal adhesions on glass and serum-treated glass. However, signaling was almost absent on plain ODS and weak on serum-treated ODS. It was concluded that alterations in vitronectin receptor function on the ventral cell surface caused by the hydrophobic material surface inhibit signal transfer and subsequent intracellular events that are important for the organization and function of integrins.

Actins↗

A rejuvenation method for poly(N,N-dimethylacrylamide)-coated glass microfluidic chips.

As microfluidic chips come to integrate the higher levels of functionality required for the implementation of advanced bioanalytical protocols, a crucial factor is that of cost. Although glass chips provide advantages in multilayer integrations, their cost is far higher than that of polymer chips. However, a simple and effective rejuvenation protocol for glass microchips may enable higher levels of integration and functionality on glass microchips. Here we present a method to rejuvenate glass microchips that had been used for capillary electrophoresis to the extent that their performance was degraded. This degradation was due to one of the two mechanisms: (i) a deterioration of the polymer coating on the inner surface of the microchannel or (ii) an aging of the glass substrate. Using the method presented here, we have rejuvenated more than 50 such "aged" microchips. The performance of these microchips was fully restored after the rejuvenation and lasted for hundreds of DNA separation runs. Our experiments indicate that the loss of resolution in microchip separations was not associated with glass aging, but was due to the degradation of the polymer coating on the inner surface of microchannels. This suggests that it is possible to extend the microchip lifetime "forever" using the rejuvenation protocol and that the exploration of higher levels of integration and functionality on glass microchips (or of hybrid structures involving materials capable of withstanding the reagents and elevated temperatures used) is feasible.

Acrylamides↗

Nitrocellulose and polyvinyl coatings prevent sperm adhesion to glass without affecting the motility of intact and demembranated human spermatozoa.

The effectiveness of several glass coating agents in preventing sperm adherence to glass surfaces for both intact and demembranated human spermatozoa was investigated. These agents included bovine serum albumin (BSA), Sigmacote, poly glu-lys, Collodion and Formvar. The presence of at least 5% of seminal plasma in sperm suspensions prevented sperm adhesion to glass surfaces. On the other hand, 56% of washed spermatozoa resuspended in an isoosmotic buffer containing 1 mg/ml of BSA attached to glass. Collodion, Formvar and Sigmacote reduced sperm attachment to glass to 2, 5 and 7%, respectively. BSA was partially effective, with 20% sperm adherence to glass, and poly glu-lys was totally ineffective. Whereas Sigmacote and BSA coatings lacked transparency, Collodion always achieved the best light transmission. Demembranated reactivated spermatozoa were all attached to glass within 90 seconds of contact. This adhesion was prevented by Collodion and Formvar. Other agents were less effective or interfered with motility. In contrast to intact spermatozoa, demembranated spermatozoa have a very low progressiveness ratio (vector speed/track speed), a wide beating amplitude and because of their whiplash flagellar movement, their motility resembles that of hamster capacitated spermatozoa.

Cell Adhesion↗

Mechanical properties and osteoconductivity of new bioactive composites consisting of partially crystallized glass beads and poly(methyl methacrylate).

New bioactive composites consisting of partially crystallized glass beads as inorganic fillers and poly(methyl methacrylate) (PMMA) as an organic matrix were developed. Two kinds of partially crystallized glass beads, designated Cry820 and Cry850, were newly prepared by the heating of MgO-CaO-SiO(2)-P(2)O(5) glass at 820 and 850 degrees C, respectively. The glass beads were mixed with PMMA to form two new composites designated Cry820C and Cry850C, respectively. The goal of this study was to produce a highly osteoconductive and mechanically strong composite cement with these new fillers. A previously reported composite cement designated AWC, which was composed of apatite- and wollastonite-containing glass ceramic (AW-GC) as a powder filler and the same PMMA polymer used in the new composites, was used as a reference material. The quantity of filler added to each composite was 70 wt %. The bending strength of Cry820C was significantly higher than that of Cry850C. Composites were packed into intramedullary canals of rat tibiae to evaluate their osteoconductivity, as determined by an affinity index. The affinity index, which equaled the length of bone in direct contact with the composite surface expressed as a percentage of the total length of the composite surface, was calculated for each composite. The rats were euthanized at 4, 8, and 25 weeks after implantation. At each time interval studied, Cry820C showed a significantly higher affinity index than AWC up to 25 weeks after implantation. Cry850C showed a significantly higher affinity index than AWC up to 8 weeks and a higher affinity index than AWC at 25 weeks, although the difference was not significant. The values for each composite increased significantly with time up to 25 weeks. Our study revealed that the higher osteoconductivity of the new composites was due to the larger quantity of the glassy phase of the crystallized glass beads at the composite surface and the lower solubility of the PMMA powder to methyl methacrylate monomer. In addition, the spherical shape of the crystallized glass beads gave the new composites strong enough mechanical properties to be useful under weight-bearing conditions. The new composites show promise as alternatives, with improved properties, to conventional PMMA bone cement.

Animals↗

Effect of additives on the microstructure and thermal properties of a mica-based glass-ceramic.

Previous work has shown that lithium mica glass-ceramics were excellent potential candidates as dental ceramics. The purpose of this study was to evaluate the effect of various additives on the microstructure and thermal properties of a mica glass-ceramic in the system Li(2)O-K(2)O-SiO(2)-MgO-F. Five glass compositions were prepared: a base composition and four compositions with various additives. The compositions were melted at 1400 C for 2 h and cast into 50 x 8 mm ingots. Differential thermal analyses were performed on the glasses up to 1400 C. Bars (4 x 8 x 25 mm) were cut from the ingots and heat treated at various temperatures. The crystalline phases were analyzed by x-ray diffraction. The microstructure was investigated by scanning electron microscopy. The percent crystallinity, crystal density, and average particle size were calculated from stereology measurements. X-ray diffraction revealed that the major crystalline phase was taeniolite for all glass-ceramics. Differential thermal analyses showed that the crystallization exotherm occurred in the temperature range 600--700 C. The addition of calcium fluoride was most efficient in promoting the growth of mica crystals. Larger mica plate diameter could be beneficial to the crack-propagation resistance of the glass-ceramic. The addition of aluminum phosphate promoted nucleation rather than crystal growth and led to a significantly lower glass transition temperature and crystallization exotherm temperature.

Aluminum Compounds↗

Interface reactions between machinable bioactive glass-ceramics and bone.

A new biomaterial for bone substitution, a "machinable bioactive glass-ceramic" has been developed. The material contains two main crystal phases, mica and apatite, and is therefore machinable and bioactive. It has the advantage to be workable by the surgeon, if necessary, during operation. The preparation method of this glass-ceramic is described. Different types of the material can be produced in dependence of the composition, nucleation, and crystallization of the basic glass. In vivo and in vitro investigations showed a characteristic solubility of the material. A Ca-phosphate-rich interface layer with apatite crystals (from the basic glass-ceramic) and a thickness of about 5-10 micrometers grows as solid-state reaction between glass-ceramic and bone. This interface reaction is interpreted as a chemical process which includes a slight solubility of the glass-ceramic and a solid state reaction between the stable apatite crystals in the glass-ceramic and the bone.

Aluminum Silicates↗

Calcium phosphate formation at the surface of bioactive glass in vitro.

The calcium phosphate formation at the surface of bioactive glass was studied in vitro. Glass rods and grains were immersed in different aqueous solutions and studied by means of scanning electron microscopy and energy dispersive x-ray analysis. Surface morphological changes and weight loss of corroded grains were monitored. In-depth compositional profiles were determined for rods immersed in the different solutions. The solutions used were tris-buffer (tris-hydroxymethylaminomethane + HCl), tris-buffer prepared using citric acid (tris-hydroxymethylaminomethane + C6H8O7.H2O), and a simulated body fluid, SBF, containing inorganic ions close in concentration to those in human blood plasma. It was found that the calcium phosphate formation at the surface of bioactive glass in vitro proceeds in two stages. When immersing the glass in tris or in SBF a Ca,P-rich surface layer forms. This accumulation takes place within the silica structure. Later, apatite crystals forming spherulites appear on the surface. The Ca/P-ratio of initially formed calcium phosphate was found to be about unity. It is proposed that this is due to bonding of phosphate to a silica gel. The surface is stabilized, i.e., leaching is retarded, by the rapid Ca,P-accumulation within the silica structure before apatite crystals are observed on the surface. It is proposed that the initially formed calcium phosphate is initiated within the silica gel. The crystallizing surface provides nucleation sites for extensive apatite formation on the glass surface. In the presence of citrate no Ca,P-accumulation occur at the glass surface, but soluble Ca-citrate complexes form. By comparing the weight loss during corrosion in tris with that in the calcium and phosphate containing SBF, it is possible to establish whether the glass can induce apatite formation at its surface or not.

Biocompatible Materials↗

Preparation of dense hydroxylapatite or rhenanite containing bioactive glass composites.

The effect of time at 600 degrees C and of small additions of Al2O3 and B2O3 on the sintering of two composite materials of (1) hydroxylapatite (Ca10(PO4)6(OH)2) and bioactive glass (SiO2-CaO-P2O5-Na2O) or (2) rhenanite (CaNaPO4) and bioactive glass were studied. Scanning microscopy, quantitative EDX, x-ray diffraction, helium gas density measurements, and diametral measurements were performed on the resulting composites. No reactions were observed with the SEM or XRD between the hydroxylapatite particles and the glass matrix within sufficient sintering times to achieve maximum density. The rheunanite-containing composites were observed to form Na2O2CaO3SiO2 crystals by x-ray diffraction, probably as a result of dissolution of the rhenanite particle surfaces into the glass phase, the crystals formed in the glass or at the interface of the glass, and the ceramic particles. However, within the short sintering times needed to achieve maximum density the rhenanite particles remained mostly intact. The rhenanite-containing materials gave better results than the hydroxylapatite-containing materials. The glass composition had a great effect on the densification process.

Biocompatible Materials↗

Bioactive glass promoted formation of nodules in periodontal-ligament fibroblasts in vitro.

The effects of bioactive glass and vitamin D3 on cultured fibroblasts derived from periodontal-ligament, with respect to their proliferation and alkaline-phosphatase activity were studied. The cells were cultured with or without the bioactive glass and/or vitamin D3, then the number and alkaline-phosphatase activity of the cells were measured periodically until the 33rd day. Formation of mineralized deposits was assessed by staining with alizarin red and von Kossa staining techniques. Fewer fibroblasts grew when they were cultured in the presence of bioactive glass and/or vitamin D3 as compared to those cultures without them. Alkaline-phosphatase activity was greater in the fibroblasts cultured with bioactive glass and vitamin D3 than in the cells grown without them. Mineralized deposits assessed by alizarin red and von Kossa staining techniques were observed microscopically around the fibroblasts cultured with bioactive glass and/or vitamin D3. A nodule visible after drying was evident only when both bioactive glass and vitamin D3 were present in culture. The results showed that although the bioactive glass and vitamin D3 decreased cell proliferation, they increased the alkaline-phosphatase activity of the fibroblasts which formed a nodule, suggesting an effect which might be useful for implant materials.

Alkaline Phosphatase↗

Dissolution and scanning electron microscopic studies of Ca,P particle-containing bioactive glasses.

Calcium phosphate (Ca,P) precipitation behavior on the surface of two bioactive glasses and four bioactive glass composites--two with hydroxylapatite (Ca10(PO4)6 (OH)2) and two with rhenanite (CaNaPO4)--were studied in simulated body fluid (SBF) and in Tris-Buffer at 5, 8, 16, 24, 48, 72, and 144 h. The weight loss of the materials was measured and the amount of precipitation was estimated using scanning electron microscopy with electrochemical detection (SEM-EDX) analysis. The test was repeated for one glass and its respective rhenanite composite every 3 h until 60 h and thereafter every 10 h until 150 h in SBF. Atomic absorption spectroscopy, spectrophotometry, SEM-EDX analysis, and pH measurements were performed on these samples. It is shown that in vitro the composite materials have a higher capacity for Ca,P precipitation than the glasses. Weight losses of the materials correlate well with their composition. Both the glass and Ca,P phases influence the precipitation mechanism and rate. Precipitation begins preferably from the glass phase. Ca,P particles clearly influence the time of onset and rate of precipitation. Cross-sectional EDX analysis of the samples revealed an absence of a clear Si-rich layer in glass A0B0 (SiO2 53.9 mol %, Na2O 27.5, CaO 12.4, P2O5 6.2, Al2O3 0.0 and B2O3 0.0) composites. This was attributed to the presence of extra calcium and phosphate ions on the surface of the material. The ion-concentration and pH change curves offered insight into the mechanism of precipitation. A connection was established between SEM-EDX results and the release curves. Formation of an Si,Ca,Na film was observed that seemed to initiate the Ca,P precipitation.(ABSTRACT TRUNCATED AT 250 WORDS)

Biocompatible Materials↗

Cellular response to calcium phosphate glasses with controlled solubility.

In the last decades, the research on materials for bone regeneration has focused on materials that are degradable and capable of stimulating tissue regeneration. In this context, phosphate glasses offer an interesting alternative, given the wide range of solubility they present and their similarity with respect to the chemical composition of the bone mineral phase. In the current work, two different formulations of phosphate glasses in the system P(2)O(5)[bond]CaO[bond]Na(2)O[bond]TiO(2) are developed. The incorporation of TiO(2) into the glass network allows for better control of the glass dissolution rate. Although these glasses have been studied extensively from the physicochemical point of view, little is known about their biocompatibility. To evaluate the biological response to these materials, we have used a human skin fibroblast model. The cells were incubated in vitro following two different methods. The first was incubated in direct contact with the glasses and the second one, in the presence of their extracts. The effects of the materials on cell growth were determined by means of toxicity (WST assay), adhesion, and proliferation tests. The results showed that the in vitro behavior of soluble phosphate glasses is strongly affected by their solubility. On the other hand, the results showed that the cellular response is highly affected by the testing procedure.

Calcium Phosphates↗

The influence of novel bioactive glasses on in vitro osteoblast behavior.

Implant success requires a direct bond between bone and implant surface. Bioinert implants, such as titanium alloys, are commonly plasma-spray-coated with a bone-bonding, bioactive material such as hydroxyapatite. Such coatings tend to be chemically and topographically inhomogeneous without reproducible properties. A family of bioactive glasses that can be enameled and reliably adheres to titanium alloy has been developed. In this study the cytocompatibility of two of these glass compositions was tested in the as-cast condition. The effects of these glasses on the early and late events of osseous tissue formation in vitro were determined with MC3T3-E1.4 mouse osteoblast-like cells. MC3T3-E1.4 cells were cultured on glasses containing 55 and 50 wt % SiO(2), with titanium alloy (Ti6Al4V) and tissue culture polystyrene as controls. Cellular adhesion and proliferation, and alkaline phosphatase activity were studied over 5 to 15 days in culture. Qualitative and quantitative assays of mineralization were conducted. The osteoblast-like cells showed increased proliferation when grown on a bioactive glass containing 50 wt % silica. However, the adhesion, differentiation and mineralization behavior were similar on both glass compositions used in this study. These bioactive glasses proved to be cytocompatible substrata for osteoblast-like cell culture, and yielded higher cellular proliferation than titanium alloy.

Alkaline Phosphatase↗

Effect of heat pressing on the mechanical properties of a mica-based glass-ceramic.

Previous work has shown that heat pressing of mica-based glass-ceramics can lead to crystal alignment along the direction of pressing. The purpose of this study was to evaluate the effect of heat pressing on the fracture toughness of mica-based glass-ceramics. Glass rods (12 x 60 mm) were prepared by melting the glass composition at 1400 degrees C for 2 h. Ingots (12 x 12 mm; n = 5) and discs (12 x 1.5 mm; n = 10) were cut from the rods. The discs were heat treated to simulate heat pressing and served as controls. A machinable mica-based glass- ceramic (Dicor MGC) also served as control. Bar-shaped wax patterns (2 x 4 x 22 mm) were invested and heat pressed at 875 degrees C. The elastic constants were determined with the use of the pulsed ultrasonic velocity method. The fracture toughness was measured by the indentation strength technique. The degree of texture was assessed qualitatively by X-ray diffraction and quantitatively on digital SEM micrographs. The results showed that the mean fracture toughness of the heat-pressed specimens (1.96 +/- 0.19 MPa. m(0.5)) was significantly higher than that of the heat-treated controls (1.51 +/- 0.21 MPa. m(0.5)) or the proprietary mica glass-ceramic (Dicor MGC; 1.66 +/- 0.04 MPa. m(0.5)) (p <.001). It was concluded that heat pressing led to a significant increase in fracture toughness in mica glass-ceramics because of crystal alignment along the direction of pressing.

Aluminum Silicates↗

The effect of production regime and crucible materials on the thermal properties of sodium phosphate glasses produced from salts.

Changes in the thermal properties of sodium phosphate glasses during melt production have been investigated using Pt/Au and fused alumina crucibles. Glasses were produced from NaH(2)PO(4) as a starting material, providing an intrinsic Na(2)O:P(2)O(5) ratio of 1:1 and giving an O/P = 3, that is, a metaphosphate. In Pt/Au crucibles, glass transition temperatures rose to a plateau value of 295 degrees C at a rate determined by melt temperature. No contamination of the glass by platinum or gold was detected or indicated in the results. E(a) for the reaction was found to be 66.4 kJ mol(-1). In fused alumina crucibles, glass transition temperatures rose to over 450 degrees C, with these values showing some convergence at higher furnace temperatures. Extensive erosion of the alumina crucibles was observed. The amount of alumina incorporation within the glasses correlated well with the rise in glass transition temperature up to a maximum of 15.5 mol % Al(2)O(3) content. Al(2)O(3) incorporation above this value caused a reduction in the value of the T(g).

Aluminum Oxide↗

Effect of niobium content on the microstructure and thermal properties of fluorapatite glass-ceramics.

Niobium oxide has been shown to improve biocompatibility and promote bioactivity. The purpose of this study was to evaluate the effect of niobium oxide additions on the microstructure and thermal properties of fluorapatite glass-ceramics for biomedical applications. Four glass-ceramic compositions with increasing amounts of niobium oxide from 0 to 5 wt % were prepared. The glass compositions were melted at 1,525 degrees C for 3 h, quenched, ground, melted again at 1,525 degrees C for 3 h and furnace cooled. The coefficient of thermal expansion was measured by dilatometry. The crystallization behavior was evaluated by differential thermal analysis. The nature of the crystalline phases was investigated by X-ray diffraction. The microstructure was studied by SEM. In addition, the cytotoxicity of the ceramics was evaluated according to the ASTM standard F895--84. The results from X-ray diffraction analyses showed that fluorapatite was the major crystalline phase in all glass-ceramics. Differential thermal analyses revealed that fluorapatite crystallization occurred between 800 and 934 degrees C depending on the composition. The coefficient of thermal expansion varied from 7.6 to 9.4 x 10(-6)/ degrees C. The microstructure after heat treatment at 975 degrees C for 30 min consisted of submicroscopic fluorapatite crystals (200--300 nm) for all niobium-containing glass-ceramics, whereas the niobium-free glass-ceramic contained needle-shaped fluorapatite crystals, 2 microm in length. None of the glass-ceramics tested exhibited any cytotoxic activity as tested by ASTM standard F895--84.

Apatites↗

Studies on the origin of the tip potential of glass microelectrode.

1. Tip potential (TP) of glass microelectrodes filled with 3 M KCl increased remarkably with the increase in the storage period in 3 M KCl solution at 37 degrees C, while the electrode resistances decreased gradually. 2. The electrical conductivity through the thin glass wall near the tip was found to increase in parallel with the TP increase. 3. The e.m.f. across the thin glass wall in the tip region was directly measured. This seems to contribute to the TP generation of the microelectrode when the conductivity of the glass wall is significantly high in the tip region. 4. Effects of the acid treatment of glass employed and the acidification of fillant electrolyte solution suggested that fixed negative charges on the glass wall play a fundamental role in the TP formation. 5. Based on these experimental results, it was concluded that not only the diffusion potential through the tip pore but also the interfacial potential through the thin glass wall near the tip contributes to the TP generation, and the contribution of the latter increases with a long exposure period of the electrodes to electrolyte solution. 6. In this connection, technical problems related to reduction of the tip potential were also discussed.

Chlorides↗

Macroporous calcium phosphate glass-ceramic prepared by two-step pressing technique and using sucrose as a pore former.

Macroporous calcium phosphate glass-ceramic with an initial glass composition of 60CaO.30P2O5.3TiO2.7Na2O in mol% was successfully prepared by sintering the mixture compact consisting of calcium phosphate glass and sucrose powders, which was formed using a two-step pressing technique. After burning off the sucrose phase, a 3D interconnected macroporous structure was formed in the sintered body, in which the skeleton consisting of the calcium phosphate glass-ceramic (including beta-calcium pyrophosphate and beta-tricalcium phosphate as the crystalline phases) was transformed from the initial glass during the sintering. The macropores with several hundred microns in diameter and the large interconnection size (approximately 100 microm), which result from the controllably large-sized sucrose particles and the hot-pressing at a little higher temperature than the sucrose's melting point, are believed to meet the requirements for cell adhesion and bone tissue regeneration well. Moreover, in vitro dissolution behavior study indicates that the calcium phosphate glass-ceramic is soluble to an acetic acid solution of pH 5-7. These, together with the simplicity and feasibility of the innovative fabrication method itself, show that the formed porous glass-ceramic has a promising potential for application to a scaffold for bone tissue engineering.

Biocompatible Materials↗