Effect of hydrazine deproteination on bone mineral phase: a critical view

Over the last 30 years several techniques have been developed to separate bone matrix and bone mineral, in order to allow for a study of each component independently of the other. Preservation of original characteristics of the phase studied after isolation has always been a great challenge for all such techniques. The hydrazine deproteination procedure, first proposed by Termine, has been one of the processes most widely used for studying bone mineral. It is found to be one of the most effective, notwithstanding controversy over its efficiency in bone deproteination and criticism regarding possible changes it could make to the characteristics of bone mineral. In this work, we have studied the possible chemical and physical alterations caused by the hydrazine deproteination process to bone mineral from rats and to other materials of biological interest. Materials were characterized by scanning electron microscopy (SEM), thermogravimetric analysis (TGA), X-ray diffractometry (XRD), inductive coupled plasma-optical emission spectroscopy (ICP-OES), C-H-N analysis and infrared spectroscopy (FTIR), before and after hydrazine deproteination. Finally, here we present a comprehensive discussion on the criticism of hydrazine deproteination. The experimental results obtained in this work, even when compared to the results in the literature, show that most widespread criticism to the hydrazine deproteination process is not completely justified.     

The Mineral Phase of Calcified Cartilage: Its Molecular Structure and Interface with the Organic Matrix

We have studied the atomic level structure of mineralized articular cartilage with heteronuclear solid-state NMR, our aims being to identify the inorganic species present at the surfaces of the mineral crystals which may interact with the surrounding organic matrix and to determine which components of the organic matrix are most closely involved with the mineral crystals. One-dimensional ¹H and ³¹P and two-dimensional ¹H-³¹P heteronuclear correlation NMR experiments show that the mineral component is very similar to that in bone with regard to its surface structure. ¹³C{³¹P} rotational echo double resonance experiments identify the organic molecules at the mineral surface as glycosaminoglycans, which concurs with our recent finding in bone. There is also evidence of γ-carboxyglutamic acid residues interacting with the mineral. However, other matrix components appear more distant from the mineral compared with bone. This may be due to a larger hydration layer on the mineral crystal surfaces in calcified cartilage.     

Evidence of low molecular weight components in the organic matrix of the reef building coral, Stylophora pistillata

Biominerals contain both inorganic and organic components. Organic components are collectively termed the organic matrix, and this matrix has been reported to play a crucial role in mineralization. Several matrix proteins have been characterized in vertebrates, but only a few in invertebrates, primarily in Molluscs and Echinoderms. Methods classically used to extract organic matrix proteins eliminate potential low molecular weight matrix components, since cut-offs ranging from 3.5 to 10 kDa are used to desalt matrix extracts. Consequently, the presence of such components remains unknown and these are never subjected to further analyses. In the present study, we have used microcolonies from the Scleractinian coral Stylophora pistillata to study newly synthesized matrix components by labelling them with 14C-labelled amino acids. Radioactive matrix components were investigated by a method in which both total organic matrix and fractions of matrix below and above 5 kDa were analyzed. Using this method and SDS-PAGE analyses, we were able to detect the presence of low molecular mass matrix components (<3.5 kDa), but no free amino acids in the skeletal organic matrix. Since more than 98% of the 14C-labelled amino acids were incorporated into low molecular weight molecules, these probably form the bulk of newly synthesized organic matrix components. Our results suggest that these low molecular weight components may be peptides, which can be involved in the regulation of coral skeleton mineralization.     

Do stress-whitening and optical clearing of collagenous tissue occur by the same mechanism?

Bone is biphasic with an organic matrix and an inorganic mineral component. As we age bone's susceptibility to fracture increases. It has been shown that there is no change in mean mineralization with aging, but bone nevertheless becomes less tough. This aging effect is therefore likely related to the organic phase.     

Enhanced cell attachment and osteoblastic activity by P-15 peptide-coated matrix in hydrogels

The cells in bone grow on a composite matrix made up of mineral and organic (mainly type-I collagen) components. In this study, anorganic bone mineral (ABM) particles were coated with a cell-binding domain of type-I collagen (P-15 peptide) to mimic the bone matrix components and suspended in injectable hyaluronate (Hy) hydrogels. The ABM/P-15/Hy was compared to ABM/Hy-the same matrix without P-15 peptide. Osteoblast-like HOS cells migrated through the hydrogels around ABM/P-15 or ABM particles; however, more cells adhered to ABM/P-15/Hy particles, and the cells formed better surface coverage and had more stress fibers on ABM/P-15/Hy. HOS cells cultured on ABM/P-15/Hy had increased osteogenic gene expression for alkaline phosphatase and bone morphogenetic proteins, and deposited more mineralized matrix. Studies with two different hydrogels (carboxymethylcellulose and sodium alginate) showed similar enhanced cell attachment and mineralization. The studies suggest that the ABM/P-15 in hydrogels can be used as an injectable biomimetic matrix to facilitate bone repair.     

Tensile behavior of cortical bone: dependence of organic matrix material properties on bone mineral content

A porous composite model is developed to analyze the tensile mechanical properties of cortical bone. The effects of microporosity (volksman's canals, osteocyte lacunae) on the mechanical properties of bone tissue are taken into account. A simple shear lag theory, wherein tensile loads are transferred between overlapped mineral platelets by shearing of the organic matrix, is used to model the reinforcement provided by mineral platelets. It is assumed that the organic matrix is elastic in tension and elastic-perfectly plastic in shear until it fails. When organic matrix shear stresses at the ends of mineral platelets reach their yield values, the stress-strain curve of bone tissue starts to deviate from linear behavior. This is referred as the microscopic yield point. At the point where the stress-strain behavior of bone shows a sharp curvature, the organic phase reaches its shear yield stress value over the entire platelet. This is referred as the macroscopic yield point. It is assumed that after macroscopic yield, mineral platelets cannot contribute to the load bearing capacity of bone and that the mechanical behavior of cortical bone tissue is determined by the organic phase only. Bone fails when the principal stress of the organic matrix is reached. By assuming that mechanical properties of the organic matrix are dependent on bone mineral content below the macroscopic yield point, the model is used to predict the entire tensile mechanical behavior of cortical bone for different mineral contents. It is found that decreased shear yield stresses and organic matrix elastic moduli are required to explain the mechanical behavior of bones with lowered mineral contents. Under these conditions, the predicted values (elastic modulus, 0.002 yield stress and strain, and ultimate stress and strain) are within 15% of experimental data.     

Biomineralization of calcium carbonate in the cell wall of Lithothamnion crispatum (Hapalidiales,Rhodophyta): correlation between the organic matrix and the mineral phase

Over the past few decades, progress has been made toward understanding the mechanisms of coralline algae mineralization. However, the relationship between the mineral phase and the organic matrix in coralline algae has not yet been thoroughly examined. The aim of this study was to describe the cell wall ultrastructure of Lithothamnion crispatum, a cosmopolitan rhodolith‐forming coralline algal species collected near Salvador (Brazil), and examine the relationship between the organic matrix and the nucleation and growth/shape modulation of calcium carbonate crystals. A nanostructured pattern was observed in L. crispatum along the cell walls. At the nanoscale, the crystals from L. crispatum consisted of several single crystallites assembled and associated with organic material. The crystallites in the bulk of the cell wall had a high level of spatial organization. However, the crystals displayed cleavages in the (104) faces after ultrathin sectioning with a microtome. This organism is an important model for biomineralization studies as the crystallographic data do not fit in any of the general biomineralization processes described for other organisms. Biomineralization in L. crispatum is dependent on both the soluble and the insoluble organic matrix, which are involved in the control of mineral formation and organizational patterns through an organic matrix‐mediated process. This knowledge concerning the mineral composition and organizational patterns of crystals within the cell walls should be taken into account in future studies of changing ocean conditions as they represent important factors influencing the physico‐chemical interactions between rhodoliths and the environment in coralline reefs.     

Microstructure and crystallographic-texture of giant barnacle (Austromegabalanus psittacus) shell

Barnacle shell is a very complex and strong composite bioceramic composed of different structural units which consist of calcite 15 microcrystals of very uniform size. In the study reported herein, the microstructural organization of these units has been examinated in detail with optical and scanning electron microscopy, and X-ray diffraction techniques. These analyses showed that the external part of the shell has a massive microstructure consisting of randomly oriented crystals. Toward the interior, the shell became organized in mineral layers separated by thin organic sheets. Each of these mineral layers has a massive microstructure constituted by highly oriented calcite microcrystals with their c-axes aligned [(001) fibre texture] perpendicular to the organic sheets and the shell surface. Interestingly, in another structural unit, the shell shield, the orientation of the c-axis calcite crystals shifts from being perpendicular to being parallel to the shell surface across its thickness. This study provides evidence that the organic matrix is responsible for the organization of the shell mineral and exterts strong a strict control on the polymorphic type, size and orientation of shell-forming crystals.     

Atomic force microscopy of the morphology of the matrix and mineral components of the otolith of Hyperoglyphe antarctica

The sagittal otolith of Hyperoglyphe antarctica (Centrolophidae: Teleostei) has a prismatic structure in which the anti-sulcal growth axes of each prism consist of a series of nested cones each composed of a mineral layer followed by an organic matrix layer. Broken sections show the mineral layers to be composed of stacks of crystals. Otolith matrix that has been decalcified and air-dried, or critical-pont-dried, retains a periodic structure of repeating high and low matrix density. At high magnifications, both broken whole crystal surfaces and decalcified matrix surfaces have a granular structure. Chloroxbleached whole otoliths also show a granular crystalline structure. At higher magnifications, the air-dried matrix showed a parallel fiber structure with similar dimensions to keratin fibers. © 1995 Wiley-Liss, Inc.     

Recent studies of the mineral phase in bone and its possible linkage to the organic matrix by protein-bound phosphate bonds

The most widely accepted hypothesis to account for maturational changes in the X-ray diffraction characteristics of bone mineral has been the 'amorphous calcium phosphate theory', which postulates that an initial amorphous calcium phosphate solid phase is deposited that gradually converts to poorly crystalline hydroxyapatite. Our studies of bone mineral of different ages by X-ray radial distribution function analysis and 31P n.m.r. have conclusively demonstrated that a solid phase of amorphous calcium phosphate does not exist in bone in any significant amount. 31P n.m.r. studies have detected the presence of acid phosphate groups in a brushite-like configuration. Phosphoproteins containing O-phosphoserine and O-phosphothreonine have been isolated from bone matrix and characterized. Tissue and cell culture have established that they are synthesized in bone, most likely by the osteoblasts. Physiochemical and pathophysiological studies support the thesis that the mineral and organic phases of bone and other vertebrate mineralized tissues are linked by the phosphomonester bonds of O-phosphoserine and O-phosphothreonine, which are constituents of both the structural organic matrix and the inorganic calcium phosphate crystals.     

The density and protein content of calcium oxalate crystals precipitated from human urine: a tool to investigate ultrastructure and the fractional volume occupied by organic matrix

One of the key debates in biomineralisation studies is the extent to which components of the organic matrix become occluded into the crystal lattice during growth. Here, the relationship between protein content and density of calcium oxalate crystals grown in human urine has been investigated in order to determine which fraction of crystal volume is non-mineral. The density of crystals varied from 1.84 to 2.08 g/cm3 while the protein content ranged from 0.1 to 2.1% (w/w). There was an inverse relationship between measured density and protein content which was qualitatively and quantitatively consistent with predictions based on reasonable densities for the mineral and non-mineral components. The coefficients of the fitted equation suggest that, at 2% protein (w/w), the volume of non-mineral would be 5.0% (v/v). The density values we observed are incompatible with fractional volumes of 20%. The results confirm that the occlusion of a small but possibly significant amount of protein into a crystal lattice is possible, but cast doubt on the hypothesis that protein acts as a major intracrystalline ultrastructural element. Moreover, the methodology developed for this study offers a simple and robust method for interrogating organic/inorganic associations in a range of biological and medical systems.     

Comparaison de l'état de conservation des phases minérales et organiques d'os fossiles. Implications pour les reconstitutions paléoenvironnementales et phylétiques

Bones have been collected from different geological sites, the sediment and the age of which being different. Comparizon between the mineral and organic phases shows the very fast alteration of the organic matrix in recent bones. Fossil bones show a loss in the organic matrices. Moreover, insoluble and soluble organic matrix alterations differ in a single bone. Implications of the diagenesis in palaeoenvironmental reconstruction and phylogeny are evoked.     

Modeling the tensile mechanical behavior of bone along the longitudinal direction

The tensile stress-strain behavior of bone along its longitudinal axis is modeled by using a simple shear-lag theory, wherein, stresses and strains in a unit cell consisting of an organic matrix reinforced by overlapped mineral platelets are computed. It is assumed that loads are transferred between overlapped mineral-platelets by shear in the organic matrix. The mechanical behavior of bone in which the matrix partially or completely debonds from the sides of the overlapped mineral platelets (after an ultimate interfacial shear stress value is exceeded) is modeled. It is shown that the tensile mechanical behavior of bone can be modeled only by assuming little or no debonding of the organic from the mineral. A physical phenomenon that explains the tensile behavior of bone is, after the interfacial shear stress has reached a constant value over the length of the mineral platelets, the collagen molecules/microfibrils (with the associated mineral platelets) move relative to one another. The tensile stress-strain curve of bovine bone is modeled using this model. The theory predicts the mechanical behavior of the tissue in the elastic, yield and post-yield region. The ultimate strain and strengths are not predicted in the present model.     

The composition and structure of bovine peritubular dentin: mapping by time of flight secondary ion mass spectroscopy

The dentin layer of the tooth is a complex mineralized tissue traversed by a closely packed system of tubules. Each tubule is surrounded by highly mineralized tissue referred to as peritubular dentin (PTD). The remaining mineralized collagen network between the tubules is the intertubular dentin (ITD). A TOF-SIMS analysis of the PTD constituents has been used to compare the PTD to the ITD. The PTD differs from the ITD not only in the degree of mineralization but also in the amount and nature of the mineral elements and amino acids. The organic matrix of the PTD consists of a unique collagen free assembly of proteins rich in glutamic acid, where the ITD organic matrix is collagen-rich and Asp-rich. The apparent concentration of organic fragment ions observed in the PTD in the TOF-SIMS negative ion mode was much higher than expected. The PTD was found to be rich in Ca2+, Na+, Mg2+, and K+. The amount of Mg2+ and K+ in the PTD was significantly reduced after deproteination, while Ca2+ and Na+ were still accumulated in the PTD. This implies that Mg2+ and K+ are mainly associated with the organic matrix rather than with the mineral of the PTD.     

On the ultrastructure and the supposed function of the mineralizing matrix coat of sea urchins (Echinodermata,Echinoida)

Summary Echinoderm ossicles are part of the mesenchyme. Their formation and growth, with respect to the underlying tissues, is studied using echinoid spines and teeth and applying different methods of fixation. The calcification process in echinoderms is strictly intracellular and needs (1) syncytial sclerocytes which completely enclose (2) a vacuolar cavity which in turn contains (3) an organic matrix coat. Strictly speaking, each ossicle is nothing but the calcified vacuolar space of a single syncytium of sclerocytes. In fully grown parts, however, the continuous sheath may split open and the matrix-coated mineral may come into contact with the extracellular space. According to biochemical analyses the matrix consists of insoluble components, but most (95%) of its constituents are soluble in EDTA or weak acids. If routine transmission electron microscope methods are used the soluble components are lost and the matrix at best looks electron light. If tannic acid is added to the fixative the soluble matrix components are preserved and reveal further ultrastructural details of the biomineralization process in echinoderms. The matrix coat looks extremely electron dense. Further soluble material is to be found within the vacuolar space or attached to the vacuolar surface of the cytoplasmic sheath. The results lead to the opinion that the matrix coat consists of a hydrophobic framework of insoluble components that contains soluble components which guide the Ca through pores in the hydrophobic layers into the interior of the matrix-coated space. It is only within this space that the mineral is deposited.     

Microstructure of matrix and mineral components of eggshells from White Leghorn chickens (Gallus gallus)

The avian eggshell is a composite structure of organic matrix and mineral (calcium carbonate) that is rapidly and sequentially fabricated in the oviduct in <24 hr. The eggshell is an excellent vehicle for the study of biomineralization processes and the role of the organic matrix in the mineral-matrix composite. The organic matrix components of eggshells from White Leghorn chickens (Gallus gallus) were examined by transmission electron microscopy (TEM) and optical microscopy. The mineral phase was analyzed by TEM, scanning electron microscopy (SEM), X-ray compositional microanalysis, and electron diffraction. Ultrastructural examination of the matrices within the calcified eggshell reveals a complex architecture that differs within each of the major zones of the eggshell: the shell membranes, the mammillary zone, the palisade region, and the cuticle. The mammillary layer consists of the calcium reserve assembly (CRA) and crown region, each with a unique substructure. TEM images show that the matrix of the CRA consists of a dense, flocculent material partially embedded within the outer shell membrane (a mostly noncalcified region of the shell). The mantle of the collagen fibers of the shell membranes is rich in polyanions (cuprolinic blue-positive), as is the CRA matrix. The CRA is capped by a centrally located calcium reserve body sac (CRB sac) that contains numerous 300–400 nm, electron-dense, spherical vesicles. Directly above the CRB sac is a zone of matrix consisting of stacks of interconnected vesicles (similar in morphology to CRA vesicles) that are interspersed with a granular material. The palisade region, the largest of the mineralized zones, contains hollow vesicles ∼450 nm (s.d. = 75 nm) in diameter, with a crescent-shaped, electron-dense fringe. An interconnecting matrix material is also found between the vesicles in the palisades region. The cuticle is composed of two layers, a mineralized inner layer and an outer layer consisting of only organic matrix. The bulk of the mineral within the eggshell is calcite, with small amounts of needlelike hydroxyapatite in the inner cuticle and occasionally, vaterite micro crystals found at the base of the palisade (cone) region. The well-crystallized calcite crystals within the palisade are columnar, typically ∼20 μm wide by 100–200 μm long; aside from numerous entrapped vesicles and occasional dislocations, they are relatively defect-free. The bulk of the matrix found in the palisade and crown regions are thought to be residual components of the rapid mineralization process. The unique matrix structure within the CRB corresponds to the region of preferentially solubilized calcite used by the developing embryo and the hydroxyapatite found in the inner cuticle may play a role in the cessation of mineral growth. © 1996 Wiley-Liss, Inc.     

Mineral phase in linguloid brachiopod shell: Lingula adamsi

The linguloid brachiopod shell family has been the focus of several studies because of the similarity in the composition of the mineral phase of these shells to that of human bone. However, ultrastructural features of Lingula shells have not yet been fully demonstrated at high magnification using Transmission Electron Microscopy (TEM) and Electron Diffraction. Ultrastructural characterization of the mineral phase in Lingula shells will improve our understanding of the biomineralization processes and mineral/organic interaction in more complex systems such as in bone or in other human mineralized tissues. In this study, the mineral phase of Lingula adamsi was characterized using a combination of ultrastructural and crystallographic techniques. The results showed that L. adamsi shells consist of apatite crystals of varying size, shape, and orientation in different areas of the shell. The c-axis of apatite was parallel to the shell surface and crystals were organized in different laminae. Compared to trabecular bovine bone, L. adamsi shells demonstrated a higher crystallinity and a lower amount of carbonate and organic compounds. This study therefore demonstrated how dissimilar organic matrix between L. adamsi shell and trabecular bone can modify the ultrastructural characteristics of apatite crystals in these two biomineralized tissues.     

Localization of calcium and phosphorus in early predentin-matrix components by electron spectroscopic imaging (ESI)-analysis in rat molars

Summary The subcellular distribution of the inorganic elements calcium (Ca) and phosphorus (P) was studied in the first-formed dentin matrix during initial mineralization in neonatal rat molars. This most peripheral matrix region is comprised of a proteoglycan-rich ground substance, interwoven by a collagenous network, matrix vesicles, aperiodic fibrils derived from the dental basal lamina, and apical odontoblastic cell processes. All matrix components may possibly serve as templets for mineral deposition during initial calcification of first-formed mantle dentin and predentin. By means of the very sensitive ESI-analysis we studied the subcellular localization of Ca and P and their possible association with distinct organic extracellular matrix components and odontoblasts. Ca-signals were found in the ground substance, at striated collagen fibrils and plasma membranes of odontoblasts in the cuspal early matrix region, but occurred only sparsely in the ground substance of the more distal matrix region where odontoblast processes attach to aperiodic fibrils of the dental basal lamina. Ca was generally absent in matrix vesicles. In contrast, P-signals were found in matrix vesicles, at aperiodic fibrils and at the plasma membranes of odontoblasts. Ca and P co-localized at striated collagen fibrils (type I or II). These results suggest that striated collagen fibrils might serve as primary deposition sites for calcium phosphate during early biological calcification of organic extracellular macromolecules.     

Pyrolysis-field ionization mass spectrometry of agricultural soils and humic substances: Effect of cropping systems and influence of the mineral matrix

Whole soil samples, extracted humic substances, the corresponding fulvic (FA) and humic acids (HA) and the extraction residues (humins) from long-term, agricultural test plots were investigated by in-source pyrolysis-field ionization mass spectrometry (Py-FIMS). For the soils distinct differences in the chemical composition of the organic matter in differently managed fields were observed. The FI mass spectra of the extracted humic substances gave complementary chemical information, as they cover a larger mass range compared to the whole soil spectra. The chemical, structural information of the conventional alkaline extraction residues was demonstrated by Py-FIMS spectra to be similar to that of the related soil samples. Influences of mineral matrix to organic matter ratios were studied on mixtures of extracted humic substances with defined mineral components such as quartz, basalt, iron oxide (Fe₂O₃), Ca-montmorillonite, kaolinite and illite. It was shown that in these mixtures the number of mass signals detected and the covered mass range decreased, when organic carbon concentrations (C_org) in this synthetic mineral matrix dropped below 2% (w/w). Limitations in the direct application of Py-FIMS might arise in the case of natural soil samples with C_org concentrations below 0.5% (w/w), high contents of swelling clay minerals and iron oxides. ei]{gnR}{fnMerckx}     

稻作系统有机肥替代部分化肥的土壤氮循环特征及增产机制

采用有机肥替代部分化肥是实现化肥使用零增长和作物稳产增产的重要途径。基于近年来的研究进展,探讨了稻作系统有机肥替代部分化肥对水稻产量、氮素利用效率、土壤氮库组分和微生物固氮、氨化、硝化和反硝化等氮循环关键过程的影响。同时,就单施化肥与有机肥替代部分化肥的氮素循环特征进行了比较。有机肥替代部分化肥通过改变稻田土壤氮素循环多个环节(增强氨化过程、协调硝化和反硝化过程、降低氨挥发和减少氮素损失等),改善土壤氮素供给状态(提高小分子有机氮供给、协调无机氮组分与比例、提高土壤微生物量氮和总氮固持),进而促进水稻氮素吸收并协调植株氮素分配过程,最终实现水稻稳产增产。