Erna De Maeyer

Isolation, proliferation and differentiation of osteoblastic cells to study cell/biomaterial interactions: comparison of different isolation techniques and source

A sufficient amount of easily obtained and well-characterized osteoblastic cells is a useful tool to study biomaterial/cell interactions essential for bone tissue engineering. Osteoblastic cells were derived from adult and fetal rat via different isolation techniques. The isolation and in vitro proliferation of primary cultures were compared. The osteogenic potential of subcultures was studied by culturing them in osteogenic medium and compared with respect to alkaline phosphatase activity, nodule formation and mineralization potential. Calvaria cells were easier to obtain and the amount of cells released by enzymatic isolation was higher than for the long bone cells. The expansion of the cells in primary culture was highest for fetal calvaria cells compared to fetal and adult long bone cells. All cultures expressed high alkaline phosphatase activity except for calvaria cells obtained by spontaneous outgrowth. Enzymatic isolation of fetal calvaria and long bone cells favoured the osteogenic differentiation. Enzymatically isolated calvaria cells formed well-defined three-dimensional nodules which mineralized restricted to this area. On the contrary, cultures derived from fetal as well as adult long bones mineralized in ill-defined deposits throughout the culture and only formed occasionally nodular-like structures. The mineral phase of all osteoblastic cultures was identified as a carbonate-containing apatite. The present study demonstrates that considering the isolation method, proliferation capacity and the osteogenic potential, the enzymatically released fetal calvaria cells are most satisfactory to study cell/biomaterial interactions.

Fluoride release profiles of restorative glass ionomer formulations

OBJECTIVES The amounts of fluoride released by different glass ionomer formulations were compared on the basis of individual fluoride release profiles in order to derive the effect of the physical and chemical formulation on the fluoride release process. METHODS The fluoride release profiles of each of five specimens of ten glass ionomer cements setting by an acid-base reaction were investigated. The profiles were obtained by determining the amount of fluoride released [F] after equilibrating the samples at 37 degrees C in distilled water for 140 d. The [[F],t]-profiles were compared with a Multivariate Data Analysis on the basis of a Principal Component Analysis. RESULTS The Multivariate Data Analysis reveals that eight of the ten glass ionomers can be classified into four distinct groups. When the cumulative amount of fluoride released by each sample, [F]c, is calculated and fitted as a function of time, a regression analysis (r > 0.99) reveals that [F]c for all samples is most adequately represented by [F]c = ([F]l x t)/(t + t1/2) + beta x square root of t, indicating that two kinetic processes are responsible for the fluoride release profiles. SIGNIFICANCE A comparison of the parameters of this equation shows that the physicochemical rationale for the classification of the glass ionomers conforms to differences in the kinetics of these processes which are determined by the qualitative and quantitative chemical composition as well as by the presentation (hand-mixed vs. capsules) of the glass ionomer. From the classification, it becomes apparent that different formulations can result in the same fluoride release profiles.

Fluoride release process of (resin-modified) glass-ionomer cements versus (polyacid-modified) composite resins

The fluoride release of conventional and resin-modified glass-ionomers is reviewed and compared to that of fluoride-releasing (polyacid-modified) composite resins. Each formulation displays a typical fluoride release profile. The cumulative amount of fluoride released is described by [F]c = [F](I)t/(t + t1/2) + beta square root t for glass ionomers whether resin-modified or not, whereas for composite resins this quantity is given by [F]c = [F](I)t/(t + t1/2) + alpha t. Both equations indicate that two kinetic processes are responsible for the fluoride release profiles. The kinetic parameters [F](I), t1/2, beta and alpha depend on the formulation. On the basis of the exchange characteristics for fluoride, an attempt is made to explain the mechanisms responsible for these fluoride release processes.

Conversion of octacalcium phosphate in calcium phosphate cements

This study investigated the in vitro conversion reaction in calcium phosphate cements (CPCs) containing octacalcium phosphate (OCP) as one of the reagents. OCP is known to be a precursor for apatite formation in vivo. The reaction products were characterized using infrared spectroscopy and X-ray diffraction. Although the conversion of OCP into hydroxyapatite is thermodynamically favorable, OCP only yields apatite formation in CPC provided it is combined with a highly soluble Ca(2+) and OH(-) releasing reaction partner. In this respect, tetracalcium phosphate is a promising compound. Adding small amounts of monocalcium phosphate monohydrate can stimulate the setting through intermediate brushite formation. The preparation method of OCP might drastically affect the performance of the cement. The reaction path of the setting of these CPC probably does not conform to the singular point principle described in the literature, and an in situ hydrolysis of OCP to apatite is conceivable. Simulation of apatite formation using OCP as the precursor and/or seed in CPC might be beneficial for some biomedical applications.

Surface roughening of glass ionomer cements by neutral NaF solutions

The objective of this study was to investigate the effect of repeated applications of a neutral NaF solution on the surface roughness of four conventional glass ionomer cements (GIC) (ChemFil Superior encapsulated, Fuji Cap II, Ketac-Fil and Hi Dense), three resin-modified (RM-) GIC (Fuji II LC encapsulated, Photac-Fil and Vitremer) and one polyacid-modified composite resin (PAM-C) (Dyract). Matured specimens were four times alternately eluted in water and exposed to 2% neutral NaF aqueous solutions for 1h. Control specimens were only subjected to elution in water for the same time period. After the treatment the surface roughness R(a) was determined using non-contact surface profilometry and selected samples were examined with SEM. Except for the PAM-C, R(a) increased drastically for the fluoride-treated samples compared to water-stored samples, the effect being most pronounced for the GIC. Surface roughening apparently is caused by a progressive disintegration or chemical erosion of the polysalt matrix of (RM-)GIC.

Fluoride release profiles of mature restorative glass ionomer cements after fluoride application

This study investigates the fluoridation of four conventional glass ionomer cements (GIC) (ChemFil Superior encapsulated, Fuji Cap II, Ketac-Fil and Hi Dense) and three resin-modified GIC (RM-GIC) (Fuji II LC encapsulated, Photac-Fil and Vitremer). The fluoride release of matured restorative GIC was measured as a function of time, after four repeated fluoridations in a 2% NaF aqueous solution for 1 h. This release was corrected for the intrinsic release as determined with a control group. It was demonstrated that application of fluoride is capable of recharging GIC but the subsequent high fluoride release only lasts for one or a few days. Moreover, the fluoride release behaviour depends on the cement formulation. Comparable to the intrinsic release, the net fluoride release after fluoridation is composed of a short- and a long-term process, the former being predominant after fluoridation. The total amount of fluoride released according to the short-term process increases with consecutive fluoridations. This is especially pronounced for the RM-GIC, who exhibit a relatively slow release after fluoridation as compared to the conventional GIC. An explanation for these results is suggested on the basis of the physicochemistry of the setting reaction of the cements and of the fluoridation process.

Effect of a neutral citrate solution on the fluoride release of resin-modified glass ionomer and polyacid-modified composite resin cements.

The effect of 0.01 mol/l citrate solution at pH = 7 on the fluoride release is compared for the resin-modified glass ionomer cements (RM-GIC) GC Lining LC, PhotacBond, Vitremer and Vitrebond and for the polyacid-modified composite resins (PAM-C) Variglass and Dyract by means of the six-month fluoride release profiles at 37 degrees C. The fluoride release of both RM-GIC and PAM-C increases in the neutral citrate solution as compared to water, which can be explained by the ability of citrate to complex metal ions and hence to degrade the glass as well as the polysalt matrix of the cement. Although RM-GIC release more fluoride than PAM-C in water as well as in citrate solution, the relative increase in fluoride release upon immersion in citrate solution is most pronounced for PAM-C. Whereas for the latter citrate affects both the short-term and long-term fluoride release, for RM-GIC only the long-term fluoride release is affected. This suggests that the action of citrate increases with decreasing importance of the polysalt formation in the hardening of the material. This could be explained on the basis of the difference in the chemical properties of the cement matrix.

Optimalization of the preparation of Na+- and CO2-3 -containing hydroxyapatite by the hydrolysis of monetite

In this study, we investigated the preparation of Na+- and CO2-3 containing apatites by the hydrolysis of monetite (CaHPO4) at 95°C in solutions with Na2CO3 concentrations ranging from 0.001M to 0.250M. When the pH of the solution is relatively low (<9), unreacted CaHPO4 might be found in the precipitates, indicating that high pH is necessary for the complete conversion of monetite to apatite. Moreover, low Na2CO3 concentrations (<0.005M) favour, even at high pH (∼ 10), the incorporation of HPO2-4 in the apatite lattice and suppress the CO2-3 incorporation corroborating the competition between CO2-3 and HPO2- 4 for an incorporation in the lattice. It was also found that a coprecipitation of CaCO3 (calcite) occurs after short reaction times. However, after hydrolysis for at least 5 h, no calcite could be detected in the precipitates. It is concluded that uncontaminated, well crystalline and single phase apatites with varying Na- and CO3 content can be obtained in a highly reproducible way by the hydrolysis of CaHPO4 for at least 5 h in Na2CO3 solutions with a concentration between 0.010M and 0.250M. A suitable choice of the solid/solution ratio permits to restrict the changes of CO2-3 and Na+ concentration in the solution to acceptable values.

Influence of the solution composition on the stoichiometry of Na+- and of K+-containing carbonated apatites obtained by the hydrolysis of monetite

In this study, Na+- and CO2−3-containing hydroxyapatites (NCAps) and K+- and CO2−3-containing hydroxyapatites (KCAps) were prepared by the hydrolysis of monetite in solutions with independently varied CO2−3 and alkalimetal (M+) concentration. The chemical and physical analysis results show that both the CO2−3 and the M+ incorporation in apatite can affect the dimensions of the hexagonal lattice. From the composition of the unit cell of these apatites, the contributions of the substitution mechanisms for the incorporation of CO2−3 and M+ could be calculated. Generally, the contributions of the predominant mechanisms, (Ca2+ + PO3−4 + OH− ↔ VCa + CO2−3 + VOH) and (Ca2+ + PO3−4 ↔ M+ + CO2−3) with VX a vacancy on a regular X lattice site, vary as can be expected on the basis of the variation of the driving force in the hydrolysis solution. Their behavior seems to suppress the appearance of the less stable mechanisms: (Ca2+ + OH− ↔ M+ + VOH) for the NCAps and (Ca2+ + 2PO3−4 ↔ VCa + 2CO2−3) for the KCAps. This suggests that, although there is no intrinsic coupling between the fundamental substitution mechanisms, an indirect correlation can occur.

Effect of maturation on the fluoride release of resin-modified glass ionomer and polyacid-modified composite resin cements

The effect of an early water contact on the fluoride release is studied for the resin-modified glass ionomer cements (RM-GIC) GC Lining LC, PhotacBond, Vitremer and Vitrebond and for the polyacid-modified composite resins (PAM-C) Variglass and Dyract. Six months fluoride release profiles were determined in regularly renewed water (37 degrees C), for the products directly after light curing and after 24 h maturation in a humid atmosphere (85% RH). ANOVA shows that both the short-term and the long-term fluoride release of a RM-GIC are influenced by this maturation. This indicates that direct water contact for this material should be avoided. For the RM-GIC a correlation is found between the initial fluoride release process and the long-term process. For the PAM-C materials, no differences in the fluoride release are found as a function of maturation, indicating that early water contact has no effect. The amounts of fluoride released by PAM-C are low compared to RM-GIC, which can affect their caries preventive potential. The results are explained on the basis of the setting reaction of both types of materials.