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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., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper, (© 2023 The Authors.)

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.

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.