Age-related changes in cationic compositions of human cranial base bone apatite measured by X-ray energy dispersive spectroscopy (EDS) coupled with scanning electron microscope (SEM)

Bonicelli A., Kranioti E.F., Xhemali B., Arnold E., Zioupos P.

Understanding what maturity entails for bone, when it arrives, and its pre- and post-maturity traits and properties are very important for understanding its evolution and physiology. There is a clear but fine distinction between the chronological age of bone (the age of its donor) and the tissue age of the bone packets it comprises at the microscopic level. Whole bone fragility changes with age due to mass and architecture effects, but so do the properties of bone at the tissue level. Tissue age and tissue-level properties are therefore increasingly attracting a great deal of attention recently. The present study investigated compositional and material changes in the hydroxyapatite crystals, the collagenous phase, changes in bone matrix composition and its nanoindentation properties and their decline with chronological age in later life. The aim was to track the age threshold at which cortical bone arrives at maturity and what happens following that threshold. To do so FTIR, DSC/TGA, XRD, nanoindentation and microindentation were used to investigate rib cortical bone material across a cohort of 86 individuals from one ethnic group with age spanning between 17 and 82 years. Results of this cross-sectional study showed a clear increase in mineral content relative to the organic and water contents across all ages. Furthermore, an increase in crystal size and consequent decrease in strain (coherence length) was detected associated with secondary mineralisation and an increase in carbonate substitution. Overall, we observe a number of modifications which contribute to a typical functional behaviour of bone showing an increase in both indentation modulus and hardness until the age of about 35 after which both of these properties decline gradually and concomitantly to other physicochemical changes and seemingly until the end of one's life.

Murgoci A., Duer M.

Solid-state NMR spectroscopy has played an important role in multidisciplinary studies of the extracellular matrix. Here we review how solid-state NMR has been used to probe collagen molecular conformations, dynamics, post-translational modifications and non-enzymatic chemical changes, and in calcified tissues, the molecular structure of bone mineral and its interface with collagen. We conclude that NMR spectroscopy can deliver vital information that in combination with data from structural imaging techniques, can result in significant new insight into how the extracellular matrix plays its multiple roles.

Rondanelli M., Faliva M.A., Tartara A., Gasparri C., Perna S., Infantino V., Riva A., Petrangolini G., Peroni G.

In 2009 EFSA Panel concludes that a cause and effect relationship has been established between the dietary intake of magnesium (Mg) and maintenance of normal bone. After 2009, numerous studies have been published, but no reviews have made an update on this topic. So, the aim of this narrative review was to consider the state of the art since 2009 on relationship between Mg blood levels, Mg dietary intake and Mg dietary supplementation (alone or with other micronutrients; this last topic has been considered since 1990, because it is not included in the EFSA claims) and bone health in humans. This review included 28 eligible studies: nine studies concern Mg blood, 12 studies concern Mg intake and seven studies concern Mg supplementation, alone or in combination with other nutrients. From the various studies carried out on the serum concentration of Mg and its relationship with the bone, it has been shown that lower values are related to the presence of osteoporosis, and that about 30–40% of the subjects analyzed (mainly menopausal women) have hypomagnesaemia. Various dietetic investigations have shown that many people (about 20%) constantly consume lower quantities of Mg than recommended; moreover, in this category, a lower bone mineral density and a higher fracturing risk have been found. Considering the intervention studies published to date on supplementation with Mg, most have used this mineral in the form of citrate, carbonate or oxide, with a dosage varying between 250 and 1800 mg. In all studies there was a benefit both in terms of bone mineral density and fracture risk.

Shah F.A.

Whitlockite is a calcium phosphate that was first identified in minerals collected from the Palermo Quarry, New Hampshire. The terms magnesium whitlockite [Mg-whitlockite; Ca 18 Mg 2 (HPO 4 ) 2 (PO 4 ) 12 ] and beta-tricalcium phosphate [ β -TCP; β -Ca 3 (PO 4 ) 2 ] are often used interchangeably since Mg-whitlockite is not easily distinguished from β -Ca 3 (PO 4 ) 2 by powder X-ray diffraction although their crystalline structures differ significantly. Being both osteoconductive and bioresorbable, Mg-whitlockite is pursued as a synthetic bone graft substitute. In recent years, advances in development of synthetic Mg-whitlockite have been accompanied by claims that Mg-whitlockite is the second most abundant inorganic constituent of bone, occupying as much as 20–35 wt% of the inorganic fraction. To find evidence in support of this notion, this review presents an exhaustive summary of Mg-whitlockite identification in biological tissues. Mg-whitlockite is mainly found in association with pathological mineralisation of various soft tissues and dental calculus, and occasionally with enamel and dentine. With the exception of high-temperature treated tumoural calcified deposits around interphalangeal and metacarpal joints and rhomboidal Mg-whitlockite crystals in post-apoptotic osteocyte lacunae in human alveolar bone, this unusual mineral has never been detected in the extracellular matrix of mammalian bone. Characterisation techniques capable of unequivocally distinguishing between different calcium phosphate phases, such as high-resolution imaging, crystallography, and/or spectroscopy have exclusively identified bone mineral as poorly crystalline, ion-substituted, carbonated apatite. The idea that Mg-whitlockite is a significant constituent of bone mineral remains unsubstantiated. Contrary to claims that such biomaterials represent a bioinspired/biomimetic approach to bone repair, Mg-whitlockite remains, exclusively, a pathological biomineral. Magnesium whitlockite (Mg-whitlockite) is a unique calcium phosphate that typically features in pathological calcification of soft tissues; however, an alarming trend emerging in the synthetic bioceramics community claims that Mg-whitlockite occupies 20–35 wt% of bone mineral and therefore synthetic Mg-whitlockite represents a biomimetic approach towards bone regeneration. By providing an overview of Mg-whitlockite detection in biological tissues and scrutinising a diverse cross-section of literature relevant to bone composition analysis, this review concludes that Mg-whitlockite is exclusively a pathological biomineral, and having never been reported in bone extracellular matrix, Mg-whitlockite does not constitute a biomimetic strategy for bone repair.

Kranioti E.F., Bonicelli A., García-Donas J.G.

Yamanaka R., Shindo Y., Oka K.

Magnesium (Mg) is the second most abundant cation in mammalian cells, and it is essential for numerous cellular processes including enzymatic reactions, ion channel functions, metabolic cycles, cellular signaling, and DNA/RNA stabilities. Because of the versatile and universal nature of Mg2+, the homeostasis of intracellular Mg2+ is physiologically linked to growth, proliferation, differentiation, energy metabolism, and death of cells. On the cellular and tissue levels, maintaining Mg2+ within optimal levels according to the biological context, such as cell types, developmental stages, extracellular environments, and pathophysiological conditions, is crucial for development, normal functions, and diseases. Hence, Mg2+ is pathologically involved in cancers, diabetes, and neurodegenerative diseases, such as Parkinson’s disease, Alzheimer’s disease, and demyelination. In the research field regarding the roles and mechanisms of Mg2+ regulation, numerous controversies caused by its versatility and complexity still exist. As Mg2+, at least, plays critical roles in neuronal development, healthy normal functions, and diseases, appropriate Mg2+ supplementation exhibits neurotrophic effects in a majority of cases. Hence, the control of Mg2+ homeostasis can be a candidate for therapeutic targets in neuronal diseases. In this review, recent results regarding the roles of intracellular Mg2+ and its regulatory system in determining the cell phenotype, fate, and diseases in the nervous system are summarized, and an overview of the comprehensive roles of Mg2+ is provided.

Duman E., Şahin Kehribar E., Ahan R.E., Yuca E., Şeker U.Ö.

Hydroxyapatite (HAP) is the major biomineral of bone. Despite the large number of studies addressing HAP formation, a fundamental understanding of the critical roles of HAP-forming proteins in vitro is needed. Effects of two HAP-interacting proteins, osteocalcin (OCN) and osteopontin (OPN), on HAP formation was investigated via in vitro biomineralization experiments, and their outcomes on the crystal structure of calcium phosphate (CaP) was revealed. Our data suggest that OCN concentration is negatively correlated with crystal formation rate and crystal size, yet the presence of OCN leads to a more ordered HAP crystal formation. On the other hand, OPN protein promotes faster formation of CaP crystals potentially working as a growth site for mineral formation, and it decreases the Ca:P ratio. This effect results in a shift from HAP-type minerals to less ordered crystals. The crystal size, shape, and Ca:P ratio can be tuned to design improved mammalian hard tissue environment-mimicking matrices by taking advantage of the OCN and OPN proteins on crystal formation. We believe our current findings will lead to innovative approaches for bone biomineralization in regenerative medicine.

Von Euw S., Wang Y., Laurent G., Drouet C., Babonneau F., Nassif N., Azaïs T.

Some compositional and structural features of mature bone mineral particles remain unclear. They have been described as calcium-deficient and hydroxyl-deficient carbonated hydroxyapatite particles in which a fraction of the PO43− lattice sites are occupied by HPO42− ions. The time has come to revise this description since it has now been proven that the surface of mature bone mineral particles is not in the form of hydroxyapatite but rather in the form of hydrated amorphous calcium phosphate. Using a combination of dedicated solid-state nuclear magnetic resonance techniques, the hydrogen-bearing species present in bone mineral and especially the HPO42− ions were closely scrutinized. We show that these HPO42− ions are concentrated at the surface of bone mineral particles in the so-called amorphous surface layer whose thickness was estimated here to be about 0.8 nm for a 4-nm thick particle. We also show that their molar proportion is much higher than previously estimated since they stand for about half of the overall amount of inorganic phosphate ions that compose bone mineral. As such, the mineral-mineral and mineral-biomolecule interfaces in bone tissue must be driven by metastable hydrated amorphous environments rich in HPO42− ions rather than by stable crystalline environments of hydroxyapatite structure.

Wilson J.W., Werness P.G., Smith L.H.

Pyrophosphate, citrate and magnesium, inhibitors of hydroxyapatite crystal growth, were studied using a seeded crystal growth system of constant composition at pH 5.80, 6.60 and 7.40. With this technique, crystal growth was studied at constant supersaturation at different pH values without the induction of other calcium phosphate phases. One inhibitor unit (that concentration of material that results in a reduction of 50 per cent in the growth rate from control) was calculated using the Langmuir adsorption isotherm. Pyrophosphate and citrate increased inhibitor activity with decreasing pH, whereas magnesium increased inhibitor activity with increasing pH. These data suggest that, at the urinary concentrations of these inhibitors, pyrophosphate is the most potent inhibitor, citrate less, and magnesium least. Pooled urine collections were studied using the same system and were found to have decreased inhibitor activity as pH decreased. This suggests that other modulators of hydroxyapatite, either promoters or inhibitors, are active in this system at the pH values studied.

Kunutsor S.K., Whitehouse M.R., Blom A.W., Laukkanen J.A.

Magnesium, which is an essential trace element that plays a key role in several cellular processes, is a major component of bone; however, its relationship with risk of major bone fractures is uncertain. We aimed to investigate the association of baseline serum magnesium concentrations with risk of incident fractures. We analyzed data on 2245 men aged 42–61 years in the Kuopio Ischemic Heart Disease prospective cohort study, with the assessment of serum magnesium measurements and dietary intakes made at baseline. Hazard ratios [95% confidence intervals (CI)] for incident total (femoral, humeral, and forearm) and femoral fractures were assessed. During a median follow-up of 25.6 years, 123 total fractures were recorded. Serum magnesium was non-linearly associated with risk of total fractures. In age-adjusted Cox regression analysis, the hazard ratio (HR) (95% CIs) for total fractures in a comparison of the bottom quartile versus top quartile of magnesium concentrations was 2.10 (1.30–3.41), which persisted on adjustment for several established risk factors 1.99 (1.23–3.24). The association remained consistent on further adjustment for renal function, socioeconomic status, total energy intake, and several trace elements 1.80 (1.10–2.94). The corresponding adjusted HRs for femoral fractures were 2.56 (1.38–4.76), 2.43 (1.30–4.53) and 2.13 (1.13–3.99) respectively. There was no evidence of an association of dietary magnesium intake with risk of any fractures. In middle-aged Caucasian men, low serum magnesium is strongly and independently associated with an increased risk of fractures. Further research is needed to assess the potential relevance of serum magnesium in the prevention of fractures.

Langelier B., Wang X., Grandfield K.

Human bone is a complex hierarchical material. Understanding bone structure and its corresponding composition at the nanometer scale is critical for elucidating mechanisms of biomineralization under healthy and pathological states. However, the three-dimensional structure and chemical nature of bone remains largely unexplored at the nanometer scale due to the challenges associated with characterizing both the structural and chemical integrity of bone simultaneously. Here, we use correlative transmission electron microscopy and atom probe tomography for the first time, to our knowledge, to reveal structures in human bone at the atomic level. This approach provides an overlaying chemical map of the organic and inorganic constituents of bone on its structure. This first use of atom probe tomography on human bone reveals local gradients, trace element detection of Mg, and the co-localization of Na with the inorganic-organic interface of bone mineral and collagen fibrils, suggesting the important role of Na-rich organics in the structural connection between mineral and collagen. Our findings provide the first insights into the hierarchical organization and chemical heterogeneity in human bone in three-dimensions at its smallest length scale – the atomic level. We demonstrate that atom probe tomography shows potential for new insights in biomineralization research on bone.

Birkedal H.

Calcium phosphates are widespread in geochemistry, in biomineralization, and as biomaterials, and thus they have solicited great interest among researchers. In this chapter, prenucleation phenomena, amorphous phases, and crystallization processes from amorphous precursors are discussed for the calcium phosphate system. The focus is almost exclusively placed on pathways resulting in apatite formation, which is the mineral most employed in biomineralization. Phosphate speciation in solution is strongly pH dependent, and this in turn calls for extreme care when designing experiments to study calcium phosphate crystallization. This notion is illustrated by comparing in situ crystallization data obtained at two different pH values, one where the major species in the initial solution is phosphate and one where hydrogen phosphate dominates. Drastically different behavior is seen and discussed. Prior to crystallization, an amorphous calcium phosphate is typically observed, and it is argued that our current understanding of the structure of these phases is far from complete. Finally, prenucleation phenomena occurring before the formation of the first condensed phase are reviewed, and it is highlighted that this area in particular merits further attention, especially in the undersaturated concentration domain.

Querido W., Rossi A.L., Farina M.

The interest in effects of strontium (Sr) on bone has greatly increased in the last decade due to the development of the promising drug strontium ranelate. This drug is used for treating osteoporosis, a major bone disease affecting hundreds of millions of people worldwide, especially postmenopausal women. The novelty of strontium ranelate compared to other treatments for osteoporosis is its unique effect on bone: it simultaneously promotes bone formation by osteoblasts and inhibits bone resorption by osteoclasts. Besides affecting bone cells, treatment with strontium ranelate also has a direct effect on the mineralized bone matrix. Due to the chemical similarities between Sr and Ca, a topic that has long been of particular interest is the incorporation of Sr into bones replacing Ca from the mineral phase, which is composed by carbonated hydroxyapatite nanocrystals. Several groups have analyzed the mineral produced during treatment; however, most analysis were done with relatively large samples containing numerous nanocrystals, resulting thus on data that represents an average of many crystalline domains. The nanoscale analysis of the bone apatite crystals containing Sr has only been described in a few studies. In this study, we review the current knowledge on the effects of Sr on bone mineral and discuss the methodological approaches that have been used in the field. In particular, we focus on the great potential that advanced microscopy and microanalytical techniques may have on the detailed analysis of the nanostructure and composition of bone apatite nanocrystals produced during treatment with strontium ranelate.

Poralan G.M., Gambe J.E., Alcantara E.M., Vequizo R.M.

Biological hydroxyapatite (BHAp) derived from thermally-treated fish bones was successfully produced. However, the obtained biological HAp was amorphous and thus making it unfavorable for medical application. Consequently, this research exploits and engineers the crystallinity of BHAp powders by addition of CaCO3 and investigates its degree of crystallinity using XRD and IR spectroscopy. On XRD, the HAp powders with [Ca]/[P] ratios 1.42, 1.46, 1.61 and 1.93 have degree of crystallinity equal to 58.08, 72.13, 85.79, 75.85% and crystal size equal to 0.67, 0.74, 0.75, 0.72 nm, respectively. The degree of crystallinity and crystal size of the obtained calcium deficient biological HAp powders increase as their [Ca]/[P] ratio approaches the stoichiometric ratio by addition of CaCO3 as source of Ca2+ ions. These results show the possibility of engineering the crystallinity and crystal size of biological HAp by addition of CaCO3. Moreover, the splitting factor of PO4 vibration matches the result with % crystallinity on XRD. Also, the area of phosphate-substitution site of PO4 vibration shows linear relationship (R2 = 0.994) with crystal size calculated from XRD. It is worth noting that the crystallinity of the biological HAp with [Ca]/[P] ratios 1.42 and 1.48 fall near the range 60-70% for highly resorbable HAp used in the medical application.

Pigolkin Y.I., Zolotenkova G.V.

A comprehensive analysis of research and development results of Sechenov University forensic medicine department for the last 5 years (from 2018 to 2022) was performed. The thematic structure and citation indices of scientific publications were presented. The most promising directions of the department's research activities were identified.Представлен комплексный анализ результатов научно-исследовательской работы кафедры судебной медицины Сеченовского Университета за последние 5 лет (с 2018 по 2022 г.). Приведены тематическая структура и показатели цитируемости научных работ. Определены наиболее перспективные направления кафедральной научно-исследовательской деятельности.

Nefedova S.M., Novikov I.A., Kravchik M.V., Zolotenkov D.D., Subbot A.M., Pigolkin Y.I.

Was to assess the possibility of using chemical analysis of bone mineral content by the means of an energy dispersive X-Ray spectroscopy to determine the age of unidentified corpses for forensic identification. A semi-quantative chemical microanalysis of bone fragments of 85 male and female corpses aged between 21 and 91 was done through the use of energy dispersive X-Ray spectrometer. The association of bone tissue apatite mineral composition with age is confirmed and a formula, connecting age and chemical composition change, is proposed. The possibility of using quantitive evaluation of chemical elements content in the normative mineral to determine the unidentified corpse's age in a standard laboratory, equipped with an energy dispersive X-Ray spectrometer, was proved.Оценить возможность использования химического анализа минерального вещества костной ткани с помощью энергодисперсионной рентгеновской спектроскопии для установления возраста при судебно-медицинской идентификации личности неопознанных трупов. С помощью энергодисперсионного рентгеновского спектрометра проведен полуколичественный химический микроанализ фрагментов костной ткани от 85 трупов лиц мужского и женского полов в возрасте от 21 до 91 года. Подтверждена связь минерального состава апатита костной ткани с возрастом и предложена формула, связывающая возраст и изменение химического состава. Доказана возможность применения количественной оценки содержания химических элементов в нормативном минерале для установления возраста неопознанного трупа в условиях стандартной лаборатории, оснащенной энергодисперсионным рентгеновским спектрометром.

Minetti E., Palermo A., Malcangi G., Inchingolo A.D., Mancini A., Dipalma G., Inchingolo F., Patano A., Inchingolo A.M.

Background: The use of the human dentin matrix could serve as an alternative to autologous, allogenic, and xenogeneic bone grafts. Since 1967, when the osteoinductive characteristics of autogenous demineralized dentin matrix were revealed, autologous tooth grafts have been advocated. The tooth is very similar to the bone and contains many growth factors. The purpose of the present study is to evaluate the similarities and differences between the three samples (dentin, demineralized dentin, and alveolar cortical bone) with the aim of demonstrating that the demineralized dentin can be considered in regenerative surgery as an alternative to the autologous bone. Methods: This in vitro study analyzed the biochemical characterizations of 11 dentin granules (Group A), 11 demineralized using the Tooth Transformer (Group B), and dentin granules and 11 cortical bone granules (Group C) using scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) to evaluate mineral content. Atomic percentages of C (carbon), O (oxygen), Ca (calcium), and P (phosphorus) were individually analyzed and compared by the statistical t-test. Results: The significant p-value (p < 0.05) between group A and group C indicated that these two groups were not significantly similar, while the non-significant result (p > 0.05) obtained between group B and group C indicated that these two groups are similar. Conclusions: The findings support that the hypothesis that the demineralization process can lead to the dentin being remarkably similar to the natural bone in terms of their surface chemical composition. The demineralized dentin can therefore be considered an alternative to the autologous bone in regenerative surgery.

Zhang J., Treyer V., Sun J., Zhang C., Gietl A., Hock C., Razansky D., Nitsch R.M., Ni R.

AbstractPersonalized neurostimulation has been a potential treatment for many brain diseases, which requires insights into brain/skull geometry. Here, we developed an open source efficient pipeline BrainCalculator for automatically computing the skull thickness map, scalp-to-cortex distance (SCD), and brain volume based on T1-weighted magnetic resonance imaging (MRI) data. We examined the influence of age and sex cross-sectionally in 407 cognitively normal older adults (71.9±8.0 years, 60.2% female) from the ADNI. We demonstrated the compatibility of our pipeline with commonly used preprocessing packages and found that BrainSuite Skullfinder was better suited for such automatic analysis compared to FSL Brain Extraction Tool 2 and SPM12- based unified segmentation using ground truth. We found that the sphenoid bone and temporal bone were thinnest among the skull regions in both females and males. There was no increase in regional minimum skull thickness with age except in the female sphenoid bone. No sex difference in minimum skull thickness or SCD was observed. Positive correlations between age and SCD were observed, faster in females (0.307%/y) than males (0.216%/y) in temporal SCD. A negative correlation was observed between age and whole brain volume computed based on brain surface (females -1.031%/y, males -0.998%/y). In conclusion, we developed an automatic pipeline for MR-based skull thickness map, SCD, and brain volume analysis and demonstrated the sex-dependent association between minimum regional skull thickness, SCD and brain volume with age. This pipeline might be useful for personalized neurostimulation planning.