X-ray diffraction studies of a partially demineralized oriented cortical bone with the controlled depth of analysis.

The in vitro demineralization of bone tissue is used for simulating the osteoporosis related bone loss. This way would be helpful in observations of bone apatite dissolution in microstructural level and may give significant input for understanding crystal-chemistry of bone resorption. In the case of cortical bone, demineralization occurs inhomogeneously, with the formation of a superficial demineralized layer and a transition zone with a gradient of concentration and structural characteristics perpendicular to the reaction advance front. Changes in the microstructural parameters of the bone mineral in this interface zone are of great interest for understanding the resorptive processes in the bone associated with osteoporosis. In this work, the SEM-EDX method was used to estimate the sizes of the demineralized and interface layers in the cortical bone during stepwise demineralization in HCl water solution; the general patterns of changes in the concentrations of Ca, P, and Cl in these layers were established. The calculations of the effective penetration depth of X-rays in diffraction mode for the intact and partially demineralized cortical bone were performed. It is shown that the use of CoKα radiation (instead of the usual CuKα) ensures the depth of probing within the interface zone, which allows to adequately assess the microstructural parameters (crystallite sizes and lattice microdeformations) of altered bioapatite in the zone of its interaction with an acid agent. A nonmonotonic change in the average size of crystallites and microdeformations of the apatite lattice was revealed during acid demineralization of the bone. Using asymmetric XRD geometry, the evidence was obtained that the affected mineral of the transition zone does not contain other crystalline phases except for weakly crystallized apatite. For the first time, the depth-controlled XRD analysis was applied to such a complex (surface-gradient) object as partially demineralized cortical bone. Additionally, we propose a rapid, averaging, and non-destructive method for estimating the depth of the reaction front dividing the demineralized and non-demineralized portions of the bone by XRD. The consistency of XRD and SEM-EDX data on the thickness values of the demineralized layer is shown.

A Simple Method to Determine the Fractions of Labile and Mineral-Bound Microelements in Bone Tissue by Atomic Absorption Spectrometry.

In this work a simple and inexpensive method to assess the concentration ratio of the labile and mineral-bound microelements of the bone tissue was developed. The approach is based on the separation of the components of bone tissue by their selective solubility with the subsequent determination of microelements with atomic absorption spectrometry. The total concentrations of Mg, Zn, Fe, Sr, Al, Cu, and Mn and the concentrations of these elements in aqueous solutions with pH 6.5, 10, and 12 after their ultrasonically activated interaction with the powder of dried bone were determined. Two quite different bone samples were analyzed: a cortical fragment of the femur of a mature healthy cow and the spongy part of a human femoral head affected by osteoporosis. Some common and individual features of the both type of bones in regard to the total concentrations and fractional distribution of microelements are discussed. The obtained concentrations of the "soluble" fractions of microelements were critically analyzed taking into account the possible reactions leading to new insoluble phases' formation in alkaline solutions. Based on the data obtained, the ability of elements to form labile fractions in the bone tissue could be arranged in the following descending series: Mg ≥ Zn > Al > Fe > Mn > Cu > Sr.

Formation of a Bacteriostatic Surface on ZrNb Alloy via Anodization in a Solution Containing Cu Nanoparticles.

High strength, excellent corrosion resistance, high biocompatibility, osseointegration ability, and low bacteria adhesion are critical properties of metal implants. Additionally, the implant surface plays a critical role as the cell and bacteria host, and the development of a simultaneously antibacterial and biocompatible implant is still a crucial challenge. Copper nanoparticles (CuNPs) could be a promising alternative to silver in antibacterial surface engineering due to low cell toxicity. In our study, we assessed the biocompatibility and antibacterial properties of a PEO (plasma electrolytic oxidation) coating incorporated with CuNPs (Cu nanoparticles). The structural and chemical parameters of the CuNP and PEO coating were studied with TEM/SEM (Transmission Electron Microscopy/Scanning Electron Microscopy), EDX (Energy-Dispersive X-ray Dpectroscopy), and XRD (X-ray Diffraction) methods. Cell toxicity and bacteria adhesion tests were used to prove the surface safety and antibacterial properties. We can conclude that PEO on a ZrNb alloy in Ca-P solution with CuNPs formed a stable ceramic layer incorporated with Cu nanoparticles. The new surface provided better osteoblast adhesion in all time-points compared with the nontreated metal and showed medium grade antibacterial activities. PEO at 450 V provided better antibacterial properties that are recommended for further investigation.

Anisotropic aspects of solubility behavior in the demineralization of cortical bone revealed by XRD analysis.

Dissolution of cortical bone mineral under demineralization in 0.1 M HCl and 0.1 M EDTA solutions is studied by X-ray diffraction (XRD). The bone specimens (in the form of planar oriented pieces) were cut from a diaphysial fragment of a mature mammal bone so that a cross-section surface and a longitudinal section surface could be analyzed individually. This permitted to compare the dissolution behavior of bone apatite of different morphologies: crystals having the c-axis of the hexagonal unit-cell generally parallel to the long axis of the bone (major morphology) and those having the c-axis almost perpendicular to the bone axis (minor morphology). For these two types of morphology, the crystallite sizes in two mutually perpendicular directions (namely, [002] and [310]) were estimated by Scherrer formula in the initial and the stepwise-demineralized specimens. The data obtained reveal that the crystals belonging to the minor morphology dissolve faster than the crystals of the major morphological type, despite the fact that the crystallites of the minor morphology seem to be only a little smaller than those of the major morphology; the apatite crystallites irrespective of the morphology type are elongated in the c-axis direction. We hypothesize that the revealed difference in solubility may be caused by diverse chemical modifications of apatite of these two morphological types, since the solubility of apatite is strictly regulated by anionic and cationic substitutions in the lattice. The anisotropy effect in solubility of bone mineral seems to be functionally predetermined and this should be a crucial factor in the resorption and remodeling behavior of a bone. Some challenges arising at XRD examination of partially decalcified cortical bone blocks are discussed, as well as the limitations of estimation of bone crystallite size by XRD line-broadening analysis.

Structural and crystal-chemical characteristics of the apatite deposits from human aortic walls.

Thermal behavior of biological apatite is the object of several studies. Crystal size, carbonate content, phase composition, and other parameters change during annealing up to 900 °C in biological minerals with apatite structure. The way these parameters change reflects the specific properties of the initial bioapatite. This work presents data on thermal transformations of pathological bioapatite from the human cardiovascular system, namely aortic wall deposits. Some minor elements, foreign to calcium hydroxyapatite (e.g., Na and Mg), can be both incorporated in the apatite structure and localized in the surface layers of crystals, modifying functions of the mineral. A new approach was proposed to determine the predominant location of minor elements, such as Mg, Na, and K, in the mineral of pathological deposits. Mg and Na in pathological apatite can be in both structurally bound (substituting calcium in lattice) and labile (localized on the crystal surface) states, while K is not able to join the apatite structure in significant amount or be chemically bound to it. This approach, based on atomic spectrometry, can be used effectively in combination with a set of traditional techniques, such as like EDS, IRS, and XRD.

Icariin prevents bone loss by inhibiting bone resorption and stabilizing bone biological apatite in a hindlimb suspension rodent model.

Bone loss induced by microgravity is a substantial barrier to humans in long-term spaceflight. Recent studies have revealed that icariin (ICA) can attenuate osteoporosis in postmenopausal women and ovariectomized rats. However, whether ICA can protect against microgravity-induced bone loss remains unknown. In this study, the effects of ICA on a hindlimb suspension rodent model were investigated. Two-month-old female Wistar rats were hindlimb suspended and treated with ICA (25 mg·kg-1·d-1, i.g.) or a vehicle for 4 weeks (n = 6). The bone mass density of the hindlimbs was analyzed using dual-energy X-ray absorptiometry and micro-CT. mRNA expression of osteogenic genes in the tibia and the content of bone metabolism markers in serum were measured using qRT-PCR and ELISA, respectively. The bone mineral phase was analyzed using X-ray diffraction and atomic spectrometry. The results showed that ICA treatment significantly rescued the hindlimb suspension-induced reduction in bone mineral density, trabecular number and thickness, as well as the increases in trabecular separation and the structure model index. In addition, ICA treatment recovered the decreased bone-related gene expression, including alkaline phosphatase (ALP), bone glaprotein (BGP), and osteoprotegerin/receptor activator of the NF-κB ligand ratio (OPG/RANKL), in the tibia and the decreased bone resorption marker TRACP-5b levels in serum caused by simulated microgravity. Notably, ICA treatment restored the instability of bone biological apatite and the metabolic disorder of bone mineral elicited by simulated microgravity. These results demonstrate that ICA treatment plays osteoprotective roles in bone loss induced by simulated microgravity by inhibiting bone resorption and stabilizing bone biological apatite.

Dielectric and electric properties of new chitosan-hydroxyapatite materials for biomedical application: Dielectric spectroscopy and corona treatment.

Chitosan-hydroxyapatite composite materials were synthesized and the possibility to make their surface charged by corona discharge treatment has been evaluated. Dielectric and electric properties of the materials were studied by dielectric spectroscopy, including application of equivalent circuits method and computer simulations. Dielectric spectroscopy shows behavior of the materials quite different from that of both chitosan and HA alone. The obtained dielectric permittivity data are of particular interest in predicting the materials' behavior in electrostimulation after implantation. The ε values observed at physiological temperature in the frequency ranges applied are similar to ε data available for bone tissues.

Characterization and in vivo evaluation of chitosan-hydroxyapatite bone scaffolds made by one step coprecipitation method.

Chitosan/hydroxyapatite scaffolds could be used for bone regeneration in case the application of auto- or allografts is impossible. The objective of the present work was to characterize and study in vivo biodegradation of simple chitosan/hydroxyapatite scaffolds. For this purpose, a series of chitosan/hydroxyapatite composites has been synthesized in aqueous medium from chitosan solution and soluble precursor salts by a one step coprecipitation method. A study of in vivo behavior of the materials was then performed using model linear rats. Cylindrical-shaped rods made of the chitosan/hydroxyapatite composite material were implanted into tibial bones of the rats. After 5, 10, 15, and 24 days of implantation, histological and histo-morphometric analyses of decalcified specimens were performed to evaluate the stages of biodegradation processes. Calcified specimens were examined by scanning electron microscopy with X-ray microanalysis to compare elemental composition and morphological characteristics of the implant and the bone during integration. Porous chitosan/hydroxyapatite scaffolds have shown osteoconductive properties and have been replaced in the in vivo experiments by newly formed bone tissue.