Naomi Eidelman

Graded microstructure and mechanical properties of human crown dentin.

The mineralized tissue of intertubular dentin is a collagen-mineral composite with considerable local variations of mechanical properties. Area scans of human coronal dentin were made by complementary methods to investigate correlations between local mechanical properties and the density, size, and crystallinity of the mineral particles. Scanning images from the same specimen were collected with Fourier-transform infrared microspectroscopy in reflectance mode (FTIR-RM), small angle X-ray scattering (SAXS), quantitative backscattered electron imaging (qBEI), and Nanoindentation in an atomic force microscope. The mineral content of dentin was found to decrease and the thickness of mineral crystals to increase towards the dentin-enamel junction (DEJ). Hardness and elastic modulus both decreased towards the DEJ. In a correlation analysis, the mineral content and, even more, the thickness of mineral crystals were found as the best predictors of hardness. The dentin layer close to the DEJ corresponds to a local minimum in hardness and elastic modulus, a configuration known to be an effective obstacle for crack propagation. Hence, the observed variations of mechanical and structural properties in an area between 0 and 1.5 mm below the DEJ define crown dentin as a gradient material optimized for its mechanical function.

3D mapping of polymerization shrinkage using X-ray micro-computed tomography to predict microleakage.

OBJECTIVES The objectives of this study were to (1) demonstrate X-ray micro-computed tomography (microCT) as a viable method for determining the polymerization shrinkage and microleakage on the same sample accurately and non-destructively, and (2) investigate the effect of sample geometry (e.g., C-factor and volume) on polymerization shrinkage and microleakage. METHODS Composites placed in a series of model cavities of controlled C-factors and volumes were imaged using microCT to determine their precise location and volume before and after photopolymerization. Shrinkage was calculated by comparing the volume of composites before and after polymerization and leakage was predicted based on gap formation between composites and cavity walls as a function of position. Dye penetration experiments were used to validate microCT results. RESULTS The degree of conversion (DC) of composites measured using FTIR microspectroscopy in reflectance mode was nearly identical for composites filled in all model cavity geometries. The shrinkage of composites calculated based on microCT results was statistically identical regardless of sample geometry. Microleakage, on the other hand, was highly dependent on the C-factor as well as the composite volume, with higher C-factors and larger volumes leading to a greater probability of microleakage. Spatial distribution of microleakage determined by microCT agreed well with results determined by dye penetration. SIGNIFICANCE microCT has proven to be a powerful technique in quantifying polymerization shrinkage and corresponding microleakage for clinically relevant cavity geometries.

Triglycidylamine Crosslinking of Porcine Aortic Valve Cusps or Bovine Pericardium Results in Improved Biocompatibility, Biomechanics, and Calcification Resistance: Chemical and Biological Mechanisms

We investigated a novel polyepoxide crosslinker that was hypothesized to confer both material stabilization and calcification resistance when used to prepare bioprosthetic heart valves. Triglycidylamine (TGA) was synthesized via reacting epichlorhydrin and NH(3). TGA was used to crosslink porcine aortic cusps, bovine pericardium, and type I collagen. Control materials were crosslinked with glutaraldehyde (Glut). TGA-pretreated materials had shrink temperatures comparable to Glut fixation. However, TGA crosslinking conferred significantly greater collagenase resistance than Glut pretreatment, and significantly improved biomechanical compliance. Sheep aortic valve interstitial cells grown on TGA-pretreated collagen did not calcify, whereas sheep aortic valve interstitial cells grown on control substrates calcified extensively. Rat subdermal implants (porcine aortic cusps/bovine pericardium) pretreated with TGA demonstrated significantly less calcification than Glut pretreated implants. Investigations of extracellular matrix proteins associated with calcification, matrix metalloproteinases (MMPs) 2 and 9, tenascin-C, and osteopontin, revealed that MMP-9 and tenascin-C demonstrated reduced expression both in vitro and in vivo with TGA crosslinking compared to controls, whereas osteopontin and MMP-2 expression were not affected. TGA pretreatment of heterograft biomaterials results in improved stability compared to Glut, confers biomechanical properties superior to Glut crosslinking, and demonstrates significant calcification resistance.

Calcium phosphate saturation levels in ultrafiltered serum.

SummaryCalcifications occurring in arteriosclerotic plaque and other pathological deposits are important health concerns, and the nature of these deposits and their mechanisms of formation warrant investigation. Crystals of the relevant calcium phosphates were equilibrated with the undiluted ultrafiltered human serum (u.f.s.) at 37°C by constant stirring and periodically removing samples for calcium and phosphate analysis and for pH measurement. The solubility measurements were carried out both with and without a 5.5% CO2 atmosphere, the physiological partial pressure of CO2. The apparent ion activity products of well-crystallized dicalcium phosphate dihydrate (DCPD), octacalcium phosphate (OCP), and hydroxyapatite (OHAp) equilibrated, in u.f.s. were calculated from the calcium and phosphate concentrations and pH in each case for comparison with their known, solubility products. In this way the well-crystallized calcium phosphates serve as fiducial solubility standards, thereby minimizing errors due to complexing of calcium and phosphate ions by u.f.s. constituents. Under 5.5%, CO2 native u.f.s. was found to be substantially undersaturated with respect to DCPD, slightly supersaturated with respect to OCP, and highly supersaturated with respect to OHAp. The ion activity product of DCPD in DCPD-saturated u.f.s. was 2.4×10−7, and the ion activity product of OCP in OCP-saturated u.f.s. was 4×10−49, slightly above their solubility products (Ksp(DCPD)=2.3×10−7, Ksp(OCP)=2.5×10−49). The ion activity products of DCPD and OCP in u.f.s. under CO2 indicate that the concentrations of calcium and phosphate complexing agents (except bicarbonate) are quite low. The u.f.s. remained supersaturated with respect to OHAp even after 2 months of equilibration. This is attributed to the presence of crystal growth inhibitors in u.f.s.

Characterization of Combinatorial Polymer Blend Composition Gradients by FTIR Microspectroscopy

A new FTIR technique was developed for characterizing thin polymer films used in combinatorial materials science. Fourier transform infrared microspectroscopy mapping technique was used to determine the composition of polymer blend gradients. Composition gradients were made from poly(L-lactic acid) (PLLA) and poly(D,L-lactic acid) (PDLLA) in the form of thin films (6 cm × 2 cm) deposited on IR reflective substrates. Three composition gradient films were prepared and characterized. The results demonstrate the reproducibility and feasibility of a new, high-throughput approach for preparing and characterizing polymer composition gradients. The combination of composition gradient film technology and automated nondestructive FTIR microspectroscopy makes it possible to rapidly and quantitatively characterize polymer composition gradients for use in combinatorial materials science.

Calcium phosphate phase transformations in serum

SummaryA better knowledge of the pathological calcification mechanisms should provide a rational basis for their control. In the present study, dicalcium phosphate dihydrate (DCPD, CaHPO4·2H2O) was used as a source of calcium and phosphate ions to investigate the mechanism of formation of more basic and more insoluble calcium phosphates in ultrafiltered serum (u.f.s.). DCPD crystals were suspended in u.f.s. at 37°C by constant stirring; samples were removed periodically for calcium and phosphate analysis and pH measurement. Occasionally, samples of solids were removed for X-ray diffraction. The experiments were carried out both with and without a 5.5% CO2 atmosphere. After initially becoming saturated with DCPD, the u.f.s. composition changed and became saturated with respect to octacalcium phosphate (OCP, Ca8H2 (PO4)6·5H2O). At this point OCP crystals were detected in the solid phase by X-ray diffraction. Further stirring changed the composition so that it became undersaturated with both DCPD and OCP and shifted toward, but did not reach, a value so low as to be saturated with hydroxyapatite (OHAp, (Ca5(PO4)3OH). The presence of CO2 in the atmosphere slowed down, but did not prevent, the above sequence of events. The above results strongly suggest that calcifications, beneficial and pathological, that take place in serum may involve OCP as a precursor, which hydrolyzesin situ to a more basic apatitic product. The results also indicate that direct formation of OHAp in u.f.s. is a very slow process and may occur only rarely. The process appears to be similar in whole serum.

Magnetic resonance microscopy of collagen mineralization.

A model mineralizing system was subjected to magnetic resonance microscopy to investigate how water proton transverse (T(2)) relaxation times and magnetization transfer ratios can be applied to monitor collagen mineralization. In our model system, a collagen sponge was mineralized with polymer-stabilized amorphous calcium carbonate. The lower hydration and water proton T(2) values of collagen sponges during the initial mineralization phase were attributed to the replacement of the water within the collagen fibrils by amorphous calcium carbonate. The significant reduction in T(2) values by day 6 (p < 0.001) was attributed to the appearance of mineral crystallites, which were also detected by x-ray diffraction and scanning electron microscopy. In the second phase, between days 6 and 13, magnetic resonance microscopy properties appear to plateau as amorphous calcium carbonate droplets began to coalesce within the intrafibrillar space of collagen. In the third phase, after day 15, the amorphous mineral phase crystallized, resulting in a reduction in the absolute intensity of the collagen diffraction pattern. We speculate that magnetization transfer ratio values for collagen sponges, with similar collagen contents, increased from 0.25 +/- 0.02 for control strips to a maximum value of 0.31 +/- 0.04 at day 15 (p = 0.03) because mineral crystals greatly reduce the mobility of the collagen fibrils.

Microstructure and mineral composition of dystrophic calcification associated with the idiopathic inflammatory myopathies.

IntroductionCalcified deposits (CDs) in skin and muscles are common in juvenile dermatomyositis (DM), and less frequent in adult DM. Limited information exists about the microstructure and composition of these deposits, and no information is available on their elemental composition and contents, mineral density (MD) and stiffness. We determined the microstructure, chemical composition, MD and stiffness of CDs obtained from DM patients.MethodsSurgically-removed calcinosis specimens were analyzed with fourier transform infrared microspectroscopy in reflectance mode (FTIR-RM) to map their spatial distribution and composition, and with scanning electron microscopy/silicon drift detector energy dispersive X-ray spectrometry (SEM/SDD-EDS) to obtain elemental maps. X-ray diffraction (XRD) identified their mineral structure, X-ray micro-computed tomography (μCT) mapped their internal structure and 3D distribution, quantitative backscattered electron (qBSE) imaging assessed their morphology and MD, nanoindentation measured their stiffness, and polarized light microscopy (PLM) evaluated the organic matrix composition.ResultsSome specimens were composed of continuous carbonate apatite containing small amounts of proteins with a mineral to protein ratio much higher than in bone, and other specimens contained scattered agglomerates of various sizes with similar composition (FTIR-RM). Continuous or fragmented mineralization was present across the entire specimens (μCT). The apatite was much more crystallized than bone and dentin, and closer to enamel (XRD) and its calcium/phophorous ratios were close to stoichiometric hydroxyapatite (SEM/SDD-EDS). The deposits also contained magnesium and sodium (SEM/SDD-EDS). The MD (qBSE) was closer to enamel than bone and dentin, as was the stiffness (nanoindentation) in the larger dense patches. Large mineralized areas were typically devoid of collagen; however, collagen was noted in some regions within the mineral or margins (PLM). qBSE, FTIR-RM and SEM/SDD-EDS maps suggest that the mineral is deposited first in a fragmented pattern followed by a wave of mineralization that incorporates these particles. Calcinosis masses with shorter duration appeared to have islands of mineralization, whereas longstanding deposits were solidly mineralized.ConclusionsThe properties of the mineral present in the calcinosis masses are closest to that of enamel, while clearly differing from bone. Calcium and phosphate, normally present in affected tissues, may have precipitated as carbonate apatite due to local loss of mineralization inhibitors.

Nondestructive quantification of leakage at the tooth-composite interface and its correlation with material performance parameters.

Current methods to determine debonding/leakage at the tooth-composite interface are qualitative or semi-quantitative. Our previous work introduced a 3D imaging technique to determine and visualize leakage and its distribution at the interface of cavity wall and composite restoration in model cavities. In this study, an automated program was developed to quantify leakage in terms of area and volume. 3D leakage distribution obtained via the image analysis program was shown to have excellent agreement with leakage visualized by dye penetration. The relationship between leakage and various material performance parameters including processability, shrinkage, stress, and shrinkage strain-rate was determined using a series of experimental composites containing different filler contents. Results indicate that the magnitude of leakage correlated well with polymerization stress, confirming the validity of the common approach utilizing polymerization stress to predict bonding durability. 3D imaging and image analysis provide insight to help understand the relations between leakage and material properties.

In vitro inhibition of membrane-mediated calcification by novel phosphonates

The effects of a series of novel phosphonates on the kinetics of mineral development in an ionophore-primed 7:2:1 phosphatidylcholine (PC): dicetylphosphate (DCP): cholesterol (Chol) liposomal model system are reported. When present at 2.5 μmol/liter or 25 μmol/liter concentrations in the solution surrounding the liposomes, the investigated phosphonates did not significantly delay the initial formation of hydroxyapatite-like calcium phosphate salts (HAP) within the liposomes or the penetration of HAP crystals through the enclosing membranes. However, the phosphonates variably retarded the subsequent growth and proliferation of the HAP crystals once they became directly exposed to the phosphonate-containing solution. The effectiveness of phosphonates in inhibiting extraliposomal precipitation strongly depended on their structure. The inhibitory action on active surface growth sites of released intraliposomal crystals was found to be the most effective if the phosphonate molecule, contained two phosphonic groups linked to the same C atom. At a phosphonate concentration of 25 μmol/liter, the, following general order of effectiveness was established: geminal bisphosphonate≥geminal tetrakisphosphonate > bisacylphosphonates > monoacyl-phosphonate > bisalkylphosphonate. Within the bisacylphosphonate family, the highest inhibitory action was observed when four of five-CH2-groups separated the ketophosphonic groups.