Principles of demineralization: modern strategies for the isolation of organic frameworks. Part II. Decalcification

This is the second paper on principles of demineralization. The initial paper is dedicated to the common definitions and the history of demineralization. In present work we review the principles and mechanisms of decalcification, i.e., removing the mineral Ca-containing compounds (phosphates and carbonates) from the organic matrix in its two main aspects: natural and artificial. Natural chemical erosion of biominerals (cavitation of biogenic calcareous substrata by bacteria, fungi, algae, foraminifera, sponges, polychaetes, and mollusks) is driven by production of mineral and organic acids, acidic polysaccharides, and enzymes (cabonic anhydrase, alkaline and phosphoprotein phosphataes, and H(+)-ATPase). Examples of artifical decalcification includes demineralization of bone, dentin and enamel, and skeletal formations of corals and crustacean. The mechanism and kinetics of Ca-containing biomineral dissolution is analyzed within the framework of (i) diffusion-reaction theory; (ii) surface-reaction controlled, morphology-based theories, and (iii) phenomenological surface coordination models. The application of surface complexation model for describing and predicting the effect of organic ligands on calcium and magnesium dissolution kinetics is also described. Use of the electron microscopy-based methods for observation and visualization of the decalcification phenomenon is discussed.     

Mechanical heterogeneity of dentin at different length scales as determined by AFM phase contrast

In this study we sought to gain insights of the structural and mechanical heterogeneity of dentin at different length scales. We compared four distinct demineralization protocols with respect to their ability to expose the periodic pattern of dentin collagen. Additionally, we analyzed the phase contrast resulting from AFM images obtained in tapping mode to interrogate the viscoelastic behavior and surface adhesion properties of peritubular and intertubular dentin, and partially demineralized dentin collagen fibrils, particularly with respect to their gap and overlap regions. Results demonstrated that all demineralization protocols exposed the gap and overlap zones of dentin collagen fibrils. Phase contrast analyses suggested that the intertubular dentin, where the organic matrix is concentrated, generated a higher phase contrast due a higher contribution of energy dissipation (damping) than the highly mineralized peritubular region. At increasing amplitudes, viscoelasticity appeared to play a more significant contribution to the phase contrast of the images of collagen fibrils. The overlap region yielded a greater phase contrast than the more elastic gap zones. In summary, our results contribute to the perspective that, at different length scales, dentin is constituted of structural features that retain heterogeneous mechanical properties contributing to overall mechanical performance of the tissue. Furthermore, the interpretation of phase contrast from images generated with AFM tapping mode appears to be an effective tool to gain an improved understanding of the structure and property relationship of biological tissues and biomaterials at the micro- and nano-scale.     

Three-Dimensional Porous Network Structure Developed in Hydroxyapatite-Based Nanocomposites Containing Enzyme Pretreated Silk Fibroin

Chemically modified silk fibroin (SF) with an enzyme, Proteinase K, has been incorporated into hydroxyapatite (HAp)-based nanocomposite attempting to strengthen the interfacial bonding between the mineral phase and the organic matrix. Particular emphasis is laid on the microstructure and microhardness of the composite along with the crystallographic properties of HAp. The whisker-like HAp crystallites of nanometer size show the preferential self-assembly and anisotropic crystal growth along c-axis. There appears porous microstructure with 70% of open porosity and pore size distribution of 10–115 um in the composite. Attributed to the enzyme modification, the crosslinkage between HAp clusters and SF matrix is improved to form an enhanced three-dimensional network extending throughout the composites and an increase of 35% in microhardness of the composite is achieved as well.     

Meretrix lusoria--a natural biocomposite material: in situ analysis of hierarchical fabrication and micro-hardness

The ultrastructure of clam (Meretrix lusoria) was investigated by means of scanning electron microscope (SEM), transmission electron microscope (TEM) and X-ray diffraction analyzer (XRD) combining with in situ texture decalcified technique and the micro-hardness of clam was determined, in order to understand the spatial relationship between the mineral phase and organic matrix and further explain the correlation between the property and structure. The results showed that hierarchical fabrication is the major structure character of this mollusc shell. There is specific braided structure forming from domains composed of needle-like structure made up of the single crystal of aragonite. High magnification TEM image of clam indicates the intracrystal region of the aragonite single crystal is made up of subgrain phase and some amorphous substance. There are various crystal grain growth preferential orientations in the different growth direction of the shell. An amount of organic microtubule distribute evenly in the base of calcium carbonate as reinforcement phase. The mechanical property of this natural biological composite is better than other aragonite layer of mollusc shells and pearls according to the data of micro-hardness testing. The braided structure and organic microtubule reinforcement phase are responsible for its high mechanical performance. The stereo hierarchical fabrication of clam was elucidated for the first time.     

Fourier transform infrared spectroscopy (FTIR) application chemical characterization of enamel,dentin and bone

Fourier transform infrared spectroscopy (FTIR) has been used extensively for chemical characterization of mineralized tissues in the past few decades. FTIR is an ideal technique to analyze chemical structural properties of natural materials, since the frequencies of several vibrational modes of organic and inorganic molecules are active in the infrared. This review discusses the use of FTIR methodology, highlighting the attenuated total reflection (ATR) sampling mode, particularly for characterization of enamel, dentin and bone tissues. Enamel, dentin and bone, are composed of an organic and a mineral phase. The mineral phase is characterized essentially as nonstoichiometric substituted apatite, being the carbonate and phosphate spectral peaks the main representative of these phase. Organic matrix of the post-eruptive enamel is small (～1% weight (wt)). The dentin and bone organic phases are mainly composed of type I collagen that appears as spectral bands of amide I, amide II, amide III bands. Furthermore, synthetic apatite materials are being designed for total or partial replacement, restoration or augmentation of these biological tissues with FTIR assistance.     

Evaluation of enamel crystallites in subsurface lesion by microbeam X‐ray diffraction

Early caries lesion is a demineralization process that takes place in the top 0.1 mm layer of tooth enamel. In this study, X‐ray microbeam diffraction was used to evaluate the hydroxyapatite crystallites in the subsurface lesion of a bovine enamel section and the results are compared with those obtained by transversal microradiography, a method commonly used for evaluation of tooth mineral. Synchrotron radiation from SPring‐8 was used to obtain a microbeam with a diameter of 6 µm. Wide‐angle X‐ray diffraction reports the amount of hydroxyapatite crystals, and small‐angle X‐ray scattering reports that of voids in crystallites. All three methods showed a marked decrease in the enamel density in the subsurface region after demineralization. As these diffraction methods provide structural information in the nanometre range, they are useful for investigating the mechanism of the mineral loss in early caries lesion at a nanometre level.     

Molecular structural analysis of noncarious cervical sclerotic dentin using Raman spectroscopy

Molecular structure of the sclerotic dentin in noncarious cervical lesions (NCCLs) including both the inorganic phase and organic phase was investigated using Raman spectroscopy. It was found that NCCL sclerotic dentin was hypermineralized with the mineral/matrix ratios 2–3 times higher than those of normal dentin, which was caused by both the increase of mineral content and decrease of organic matrix (collagen) content in the sclerotic dentin. For the inorganic phase, the phosphate band (PO₄³⁻, ν₁, symmetric stretching vibrational mode) in NCCL sclerotic dentin was shifted from 960 to 963 cm⁻¹, and the width of this band was decreased from 16.4 to 10.4 cm⁻¹, indicating that the degree of mineral crystallinity in NCCL sclerotic dentin was higher than that of normal dentin. In addition, the carbonate content in the mineral of NCCL sclerotic dentin was less than that of normal dentin. As compared to the inorganic phase, the changes within the organic phase were not dramatic. However, the changes in collagen cross‐link density along with other spectral changes were still detectable. There was a noteworthy reduction in the ratio of nonreducible to reducible cross‐links in the NCCL sclerotic dentin, indicating that cross‐link breaks occurred in the collagen matrix of the lesions. Copyright © 2009 John Wiley & Sons, Ltd.     

High-affinity integration of hydroxyapatite nanoparticles with chemically modified silk fibroin

Hydroxyapatite (HA)-based nanocomposites were prepared by a co-precipitation method with silk fibroin (SF) serving as organic matrix. Silk fibroin was chemically modified with an alkali solution or an enzyme attempting to improve the interface between the mineral and the organic matrix. The influences of the alkali and enzyme pretreatments on microstructure and physicochemical properties of HA–SF composite were examined and compared. The results reveal that both the two kinds of pretreatments facilitate the formation of highly ordered three-dimensional porous network throughout the composites, increase the microhardness of the composite, and promote the preferential growth of HA crystallites along c-axis. Among all the as-prepared samples, the composite containing the enzyme pretreated SF shows desirable hierarchical microstructure with higher degree of organization and more uniform pore size distribution. Due to the enzyme pretreatment, HA crystallites undergo obvious changes in morphology from rod-like to␣whisker-like and in crystal growth towards more apparent epitaxy along c-axis. The alkali pretreatment induces the stronger chemical interactions between HA and SF and thus to strengthen the inorganic–organic interfacial adhesion. The newly developed HA–SF composites are expected to be attractive biomedical materials for bone repair and remodeling.     

Chemical-mechanical stability of the hierarchical structure of shell nacre

The hierarchical structure and mechanical property of shell nacre are experimentally investigated from the new aspects of chemical stability and chemistry-mechanics coupling. Through chemical deproteinization or demineralization methods together with characterization techniques at micro/nano scales,it is found that the nacre of abalone,haliotis discus hannai,contains a hierarchical structure stacked with irregular aragonite platelets and interplatelet organic matrix thin layers. Yet the aragonite platelet i...     

Alterations in enamel remineralization in vitro induced by blue light

Blue light, especially from LED devices, is a very frequently used tool in dental procedures. However, the investigations of its effects on dental enamel are focused primarily on enamel demineralization and fluoride retention. Despite the fact that this spectral region can inhibit enamel demineralization, the effects of the irradiation on demineralized enamel are not known. For this reason, we evaluated the effects of blue LED on remineralization of dental enamel. Artificial lesions were formed in bovine dental enamel blocks by immersing the samples in undersaturated acetate buffer. The lesions were irradiated with blue LED (455 nm, 1.38 W/cm², 13.75 J/cm², and 10 s) and remineralization was induced by pH-cycling process. Cross-sectional hardness was used to asses mineral changes after remineralization. Non-irradiated enamel lesions presented higher mineral content than irradiated ones. Furthermore, the mineral content of irradiated group was not significantly different from the lesion samples that were not submitted to the remineralization process. Results obtained in the present study show that the blue light is not innocuous for the dental enamel and inhibition of its remineralization can occur.     

Nanostructures of the aragonitic otolith of cod (Gadus morhua)

Topographical relationships between the mineral and organic components are important to understand the process involved in biomineralisation. We examined nanostructural features of cod (Gadus morhua) otoliths using atomic force microscope (AFM). Acicular aragonitic fibres that form primary increments observed in scanning electron or light microscopy were composed of lamellae, themselves being composite arrangements of round granules (diameter<50 nm) surrounded by an organic (?) cortex. The lamellae presented a banding pattern, which might correspond to a growth period of about 15 min. Cod otolith granules were similar in size, shape and arrangement to that previously reported for another fish species. The existence of smallest otolith organization units is suggested since granules are themselves composite. Finally, the intra-daily growth rhythm makes it possible to consider very high-resolution biological and environmental reconstructions in the future.     

Surface and interface properties of carbon fiber composites under cyclical aging

Carbon fiber-reinforced BMI composites have been subjected to combination accelerated aging comprising a hygrothermal process, a thermal-oxidative process, and a freezing process in order to simulate their responses under complicated service environments. This cyclical condition, including the freezing process, has not been investigated by other researchers so far. The effects of this combination accelerated aging on the mechanical properties have been characterized by FTIR, SEM/EDXA, XRD, and moisture-uptake determination. The results indicated that combination accelerated aging had great effects on the mechanical properties of the composite, the network structure of the BMI matrix, and the moisture uptake by the composite. After a third cycle of accelerated aging, moisture reached the center layer of the composite and as a result led to an obvious decrease in ILSS due to deterioration of the carbon fiber-BMI interface. Sufficient moisture absorption on the composite surface made the network structure of the BMI matrix more open, which facilitated stress relaxation and the creation of micro-cracks, with a consequent obvious decrease in flexural strength. With increasing number of combined-action accelerated aging cycles, ever more moisture was absorbed during each hygrothermal process due to the plasticizing effect of water, and micro-cracks propagated as a result of internal stresses caused by the hygrothermal process, the thermal-oxidative process, and the freezing process of each cycle. XRD analysis indicated that moisture penetrated through the amorphous region of the BMI matrix.     

Effects of structure and composition of the CaP composite coatings on apatite formation and bioactivity in simulated body fluid

The surface properties of biomaterials determine the interactions between biomedical devices and the surrounding biological environment. The surface modification of biomaterials is extensively recognized as a key strategy in the design of the next generation of bone implants and tissue engineering. In this study, the highly ordered octacalcium phosphate (OCP) coating and OCP/protein coating with hierarchically porous structure in nano-micro scale were constructed on titanium substrate by electrochemically-induced deposition (ED). The formation behavior of apatite on OCP and OCP/protein coatings immersed in simulated body fluid (SBF) was investigated in physicochemical aspects. It is indicated that soaked in SBF, the OCP and OCP/protein coatings are possible to induce relevant apatite formation on their surface, and the apatite-forming behavior in body environment is depended on the chemical composition and structure of the coatings. The apatite formed on OCP/protein composite coating possesses carbonated structure, needle-like crystals in nano scale, lower Ca/P ratio and higher degree of the preferred c-axis orientation, which are similar to the mineral composition and structure in natural bone, and hence called as bone-like apatite.     

Polypropylene-natural fibre composites. Analysis of fibre structure modification during compounding and its influence on the final properties

The final properties of composite materials are highly dependent on the residual geometrical parameters (length, diameter, aspect ratio), orientation and distribution of the fibres in the matrix, which in turn are related to the processing conditions. This study analysed the fibre structure variation during the processing of a polypropylene matrix reinforced with cellulose flax pulp for different reinforcement concentrations. The fibre's geometrical parameters, length, diameter and aspect ratio have been measured and their statistical distributions have been assessed for each concentration. Furthermore, the effect of the microstructure variation on the final mechanical properties was analysed. In particular, changes in the interfacial area were evaluated based on the hypothesis that the fibres were cylindrical in shape and considering the average values of the diameters and the lengths calculated using a statistical distribution approach. The fibre interfacial area after the process decreases as the fibre concentration increases and this evaluation explains how the adhesion methods that are used for fibre surface modification fail because of the decrement in the modifier interfacial density. The Halpin–Tsai approach was used to model the experimental data obtained from tensile tests for different composites, so as to confirm the effect of fibre parameters, such as aspect ratio and interfacial area values, in the PP/cellulose blends final properties.     

Effect of interfacial debonding and matrix cracking on mechanical properties of multidirectional composites

In composites, debonding at the fiber–matrix interface and matrix cracking due to loading or residual stresses can effect the mechanical properties. Here three different architectures — 3-directional orthogonal, 3-directional 8-harness satin weave and 4-directional in-plane multidirectional composites — are investigated and their effective properties are determined for different volume fractions using unit cell modeling with appropriate periodic boundary conditions. A cohesive zone model (CZM) has been used to simulate the interfacial debonding, and an octahedral shear stress failure criterion is used for the matrix cracking. The debonding and matrix cracking have significant effect on the mechanical properties of the composite. As strain increases, debonding increases, which produces a significant reduction in all the moduli of the composite. In the presence of residual stresses, debonding and resulting deterioration in properties occurs at much lower strains. Debonding accompanied with matrix cracking leads to further deterioration in the properties. The interfacial strength has a significant effect on debonding initiation and mechanical properties in the absence of residual stresses, whereas, in the presence of residual stresses, there is no effect on mechanical properties. A comparison of predicted results with experimental results shows that, while the tensile moduli E ₁₁, E ₃₃and shear modulus G ₁₂ match well, the predicted shear modulus G ₁₃ is much lower.     

Spray Freeze-drying – The Process of Choice for Low Water Soluble Drugs?

Most of the novel highly potent drugs, developed on the basis of modern molecular medicine, taking into account cell surface recognition techniques, show poor water solubility. A chemical modification of the drug substance enhancing the solubility often decreases the pharmacological activity. Thus, as an alternative an increase of the solubility can be obtained by the reduction of the size of the drug particles. Unfortunately, it is often difficult to obtain micro or nanosized drug particles by classical or more advanced crystallization using supercritical gases or by milling techniques. In addition, nanosized particles are often not physically stable and need to be stabilized in an appropriate matrix. Thus, it may be of interest to manufacture directly nanosized drug particles stabilized in an inert hydrophilic matrix, i.e. nanostructured and nanocomposite systems. Solid solutions and solid dispersions represent nanostructured and nanocomposite systems. In this context, the use of the vacuum-fluidized-bed technique for the spray-drying of a low water soluble drug cosolubilized with a hydrophilic excipient in a polar organic solvent is discussed. In order to avoid the use of organic solvents, a special spray-freeze-drying technique working at atmospheric pressure is presented. This process is very suitable for temperature and otherwise sensitive drugs such as pharmaproteins.     

Immobilisation and characterisation of the demineralised, fully hydrated organic matrix of nacre - An atomic force microscopy study

Mimicry of the tough natural composite nacre in future bioengineering requires knowledge of the biomineralisation process. The insoluble organic matrix isolated from the shell of the gastropod Haliotis laevigata was characterised by protein chemistry, topographical and mechanical measurements. Demineralisation of nacre in dilute acetic acid or ethylenediaminetetraacetic acid revealed a set of soluble proteins and the insoluble matrix. The insoluble matrix contains a chitin core and firmly attached proteins, which could be removed by sodium dodecyl sulfate and glycerol indicating a hydrophobic interaction. Atomic force microscopy images of the native insoluble matrix showed a filamentous network with pores or holes, where the filaments showed globular attachments of different sizes, possibly the attached protein molecules. During direct observation of protein degradation imaged by atomic force microscopy the insoluble matrix gets smooth and flat indicating the removal of the attached proteins by proteases. We propose a model of protein coated chitin filaments for the insoluble matrix of nacre. Mechanical measurements by force mapping revealed a Young's modulus depending on the hydration state of the organic layers. The fully hydrated organic matrix has an elastic modulus below 1MPa comparable to some hydrogels.     

Search for retrogressive reactions accompanying demineralization in native and air-oxidized coals

This solid state 13C NMR study confirms, for coals, and extends, for air-oxidized coals, the claim that the Bishop and Ward strong aqueous-acid coal demineralization procedure [M. Bishop, D.L. Ward, Fuel 37 (1958) 191.] does not lead to the formation of detectable levels of cross-links in the organic matrix of these materials. The study was prompted by the demonstration that polymerization accompanies strong acid demineralization in lignin, and the recognition that the chemical environment created in air-oxidized coals contains introduced reactive functionality similar to that in the lignin. In particular, both contain oxidized benzylic carbon functionality that can undergo acid-catalyzed polymerization reactions. For two coals of differing rank, no acid-induced cross-link formation has been observed in the pristine or air-oxidized coals, within the 5 parts per thousand (ppt) sensitivity of the NMR technique used in this study.     

东升庙硫多金属矿床氧化微粒的XRD,FTIR和XPS分析

利用XRD,FTIR和XPS现代分析测试技术对东升庙硫多金属矿床氧化微粒的物质组成、相结构和表面元素状态进行了表征以及转化机理研究。结果表明,样品主要由石膏、方解石、石英、白云母、针铁矿、有机质等组成,深部氧化带原生矿石主要发生了氧化、水解、脱水和碳酸化作用,且氧化过程、氧化条件不及地表氧化带复杂,未发现新的矿物,少见西北干旱和极端干旱气候条件下地表氧化带极其发育的硫酸盐类矿物。研究结果不仅可以用于该类型矿床氧化微粒矿物鉴定方面,而且对于这部分矿体的找矿勘探、采矿、选矿等方面具有重要意义。     

改性钢渣-矿粉复合橡胶填料补强-阻燃机理的光谱学分析

钢渣作为炼钢过程中产生的固体废弃物,矿渣作为高炉炼铁过程中的副产品,其存在难以利用与附加值的问题。面对上述问题,利用钢渣与矿渣开发一种价格低廉的复合橡胶填料用于橡胶领域。采用磁选热闷渣、未磁选热闷渣、矿粉和助磨-改性复合剂制备改性钢渣-矿粉复合橡胶填料,并且用于复合橡胶体系。研究磁选热闷渣用量、未磁选热闷渣用量、矿粉用量和助磨-改性复合剂用量对改性钢渣-矿粉基橡胶复合材料性能的影响,并且分析其影响机理。结果表明,以磁选热闷渣用量150 g、未磁选热闷渣用量150 g、矿粉用量150 g和助磨-改性复合剂用量9 g制备的改性钢渣-矿粉复合橡胶填料补强-阻燃性能最优。按改性钢渣-矿粉复合橡胶填料∶炭黑质量比20∶30制备的改性钢渣-矿粉基橡胶复合材料,其拉伸强度为21.83 MPa、撕裂强度为46.23 kN·m-1、邵尔A硬度为62、磨耗量为159 mm3、极限氧指数为19.8%与燃尽时间为187 s。助磨-改性复合剂不仅降低粒径尺寸、提高粒径均匀性,而且改善钢渣-矿粉复合橡胶填料的表面结构与性质,有利于改性钢渣-矿粉复合橡胶填料在复合橡胶体系中均匀分散,提高相容性。钢渣与矿粉在助磨-改性复合剂的作用下发生化学反应,改变了钢渣与矿粉的物相组成,提高补强性能与阻燃性能。