Infrared imaging microscopy of bone: Illustrations from a mouse model of Fabry disease

Bone is a complex tissue whose composition and properties vary with age, sex, diet, tissue type, health and disease. In this review, we demonstrate how infrared spectroscopy and infrared spectroscopic imaging can be applied to the study of these variations. A specific example of mice with Fabry disease (a lipid storage disease) is presented in which it is demonstrated that the bones of these young animals, while showing typical spatial variation in mineral content, mineral crystal size, and collagen maturity, do not differ from the bones of age- and sex-matched wild type animals.     

Osteochondrosis and Arthrosis in Pigs

Eight feeding experiments, each comprising 48 boars or gilts, were carried out. The factors feed level, Ca, P and protein levels were varied within practical physiological limits. The animals were slaughtered at close to 100 kg live weight. Lesions in joints and bones were of a noninfectious, non-rachitic nature and of the osteochondrosis and arthrosis type. Ten to 12 g Ca and approx. 10 g P/kg ration gave histomorphologically seemingly more optimum structure of the spongious bone tissue and a higher ash percentage in bones than 7 g Ca and 6 g P, or unbalanced mineral levels in the ration. However, there was no significant difference in degree or incidence of joint and bone lesions between mineral groups. Neither were there such differences present between feed level groups or protein level groups. This may for the feed level groups be partly due to the low feed level pigs having a longer period of time during which they were highly susceptible to the development of lesions. The low feed level animals had longer long bones at 100 kg live weight than the high feed level animals. The length of the vertebral column was, however, approximately similar in the 2 feed level groups. The angles of the condyles and head of the femur according to the length axis were influenced by feed level or growth rate, most probably by an effect on the endochondral ossification.     

Physique types and the skeletal mineral saturation of young men and women

The term "massiveness of the skeleton" does not reflect the amount of the mineral component in bones. As demonstrates application of the standard linear x-ray densitometry, in a more "massive" skeleton amount of mineral salts per a volume unit of the osseous tissue is less than in a thin "gracile" skeleton of the thoracal type of constitution. The muscular type of constitution concerning the mineral saturation occupies an intermediate position between the thoracal and digestive constitutional types.     

Influence of Osteoprotegerin (OPG) on Resorption of Heterotopically Induced Ossicle

In this paper, the effect of osteoprotegerin (OPG) on slowing down the resorption process of heterotopically induced bone tissue is described. The induced ossicle is resorbed ex inactivitate. This system mimics osteoporosis in immobilised skeletal bones. Bone induction was achieved in BALB/c mice after the injection of the suspension of 3 x 10(6) HeLa cells into thigh muscle of animals immuno-suppressed by a single dose of hydrocortisone. To slow down the process of resorption we applied OPG and measured quantitatively the effect by weighing the mass of mineral deposited in the induced ossicle after hydrolysis of soft tissues surrounding the induced ossicles. As the effect of application of OPG more than 340-540% of bone mineral is found in the induced ossicles following nine applications of 0.05 mg OPG per mouse, every second day--in comparison to the control animals.     

Preservation of Bone Structure and Function by Lithothamnion sp. Derived Minerals

Progressive bone mineral loss and increasing bone fragility are hallmarks of osteoporosis. A combination of minerals isolated from the red marine algae, Lithothamnion sp. was examined for ability to inhibit bone mineral loss in female mice maintained on either a standard rodent chow (control) diet or a high-fat western diet (HFWD) for 5, 12, and 18 months. At each time point, femora were subjected to μ-CT analysis and biomechanical testing. A subset of caudal vertebrae was also analyzed. Following this, individual elements were assessed in bones. Serum levels of the 5b isoform of tartrate-resistant acid phosphatase (TRAP) and procollagen type I propeptide (P1NP) were also measured. Trabecular bone loss occurred in both diets (evident as early as 5 months). Cortical bone increased through month 5 and then declined. Cortical bone loss was primarily in mice on the HFWD. Inclusion of the minerals in the diet reduced bone mineral loss in both diets and improved bone strength. Bone mineral density was also enhanced by these minerals. Of several cationic minerals known to be important to bone health, only strontium was significantly increased in bone tissue from animals fed the mineral diets, but the increase was large (5–10 fold). Serum levels of TRAP were consistently higher in mice receiving the minerals, but levels of P1NP were not. These data suggest that trace minerals derived from marine red algae may be used to prevent progressive bone mineral loss in conjunction with calcium. Mineral supplementation could find use as part of an osteoporosis-prevention strategy.     

Genetic randomization reveals functional relationships among morphologic and tissue-quality traits that contribute to bone strength and fragility

We examined femora from adult AXB/BXA recombinant inbred (RI) mouse strains to identify skeletal traits that are functionally related and to determine how functional interactions among these traits contribute to genetic variability in whole-bone stiffness, strength, and toughness. Randomization of A/J and C57BL/6J genomic regions resulted in each adult male and female RI strain building mechanically functional femora by assembling unique sets of morphologic and tissue-quality traits. A correlation analysis was conducted using the mean trait values for each RI strain. A third of the 66 correlations examined were significant, indicating that many bone traits covaried or were functionally related. Path analysis revealed important functional interactions among bone slenderness, cortical thickness, and tissue mineral density. The path coefficients describing these functional relations were similar for both sexes. The causal relationship among these three traits suggested that cellular processes during growth simultaneously regulate bone slenderness, cortical thickness, and tissue mineral density so that the combination of traits is sufficiently stiff and strong to satisfy daily loading demands. A disadvantage of these functional interactions was that increases in tissue mineral density also deleteriously affected tissue ductility. Consequently, slender bones with high mineral density may be stiff and strong but they are also brittle. Thus, genetically randomized mouse strains revealed a basic biological paradigm that allows for flexibility in building bones that are functional for daily activities but that creates preferred sets of traits under extreme loading conditions. Genetic or environmental perturbations that alter these functional interactions during growth would be expected to lead to loss of function and suboptimal adult bone quality.     

Yeast-Incorporated Gallium Promotes Fracture Healing by Increasing Callus Bony Area and Improving Trabecular Microstructure on Ovariectomized Osteopenic Rats

The purpose of this study was to analyze the impact of yeast-incorporated gallium on fracture healing in ovariectomized osteopenic rats. Forty Wistar female rats used were divided into three groups: sham-operated rats (SHAM), ovariectomized (OVX) rats, and ovx rats treated with yeast-bound gallium (YG). A standardized fracture-healing model with stable plate fixation was established for rat femoral. After 4-week stable fixation, animals were killed to prepare bones for Micro-CT, biomechanical testing, and histomorphometry. In addition, bone samples were obtained to evaluate the content of mineral substances in bones. Quantitative analysis of the bones from animals in the organic gallium group revealed significantly increased mineral contents compared to bones from OVX and SHAM groups. Micro-CT showed that treatment with yeast-incorporated gallium increased BV/TV and trabecular thickness and decreased trabecular separation in ovx animals. Histomorphometric evaluation demonstrated that YG increased callus area and callus bone formation. Yeast-bound gallium also improved the biomechanical properties of bone healing. In conclusion, this study suggests that yeast-incorporated gallium could promote fracture healing in ovariectomized rats.     

Effect of bone mineral content on the tensile properties of cortical bone: experiments and theory

The effect of mineral volume fraction on the tensile mechanical properties of cortical bone tissue is investigated by theoretical and experimental means. The mineral content of plexiform, bovine bone was lowered by 18% and 29% by immersion in fluoride solutions for 3 days and 12 days, respectively. The elastic modulus, yield strength and ultimate strength of bone tissue decreased, while the ultimate strain increased with a decrease in mineral content. The mechanical behavior of bone tissue was modeled by using a micromechanical shear lag theory consisting of overlapped mineral platelets reinforcing the organic matrix. The decrease in yield stress, by the 0.002 offset method, of the fluoride treated bones were matched in the theoretical curves by lowering the shear yield stress of the organic matrix. The failure criterion used was based on failure stresses determined from a failure envelope (Mohr's circle), which was constructed using experimental data. It was found that the model predictions of elastic modulus got worse with a decrease in mineral content (being 7.9%, 17.2% and 33.0% higher for the control, 3-day and 12-day fluoride-treated bones). As a result, the developed theory could not fully predict the yield strain of bones with lowered mineral content, being 12.9% and 21.7% lower than the experimental values. The predicted ultimate stresses of the bone tissues with lower mineral contents were within +/- 10% of the experimental values while the ultimate strains were 12.7% and 26.3% lower than the experimental values. Although the model developed in this study did not take into account the presence of hierarchical structures, voids, orientation of collagen molecules and micro cracks, it still indicated that the mechanical properties of the organic matrix depend on bone mineral content.     

The Identification of Proteoglycans and Glycosaminoglycans in Archaeological Human Bones and Teeth

Bone tissue is mineralized dense connective tissue consisting mainly of a mineral component (hydroxyapatite) and an organic matrix comprised of collagens, non-collagenous proteins and proteoglycans (PGs). Extracellular matrix proteins and PGs bind tightly to hydroxyapatite which would protect these molecules from the destructive effects of temperature and chemical agents after death. DNA and proteins have been successfully extracted from archaeological skeletons from which valuable information has been obtained; however, to date neither PGs nor glycosaminoglycan (GAG) chains have been studied in archaeological skeletons. PGs and GAGs play a major role in bone morphogenesis, homeostasis and degenerative bone disease. The ability to isolate and characterize PG and GAG content from archaeological skeletons would unveil valuable paleontological information. We therefore optimized methods for the extraction of both PGs and GAGs from archaeological human skeletons. PGs and GAGs were successfully extracted from both archaeological human bones and teeth, and characterized by their electrophoretic mobility in agarose gel, degradation by specific enzymes and HPLC. The GAG populations isolated were chondroitin sulfate (CS) and hyaluronic acid (HA). In addition, a CSPG was detected. The localization of CS, HA, three small leucine rich PGs (biglycan, decorin and fibromodulin) and glypican was analyzed in archaeological human bone slices. Staining patterns were different for juvenile and adult bones, whilst adolescent bones had a similar staining pattern to adult bones. The finding that significant quantities of PGs and GAGs persist in archaeological bones and teeth opens novel venues for the field of Paleontology.     

Regional distribution of mineral and matrix in the femurs of rats flown on Cosmos 1887 biosatellite

We combined biochemical measurements with novel techniques for image analysis in the rat femur to characterize the location and nature of the defect in mineralization known to occur in growing animals after spaceflight. Concentrations of mineral and osteocalcin were low in the distal half of the diaphysis and concentrations of collagen were low with evidence of increased synthesis in the proximal half of the diaphysis of the flight bones. X-ray microtomography provided semiquantitative data in computer-generated sections of whole wet bone that indicated a longitudinal gradient of decreasing mineralization toward the distal diaphysis, similar to the chemistry results. Analysis of embedded sections by backscattered electrons in a scanning electron microscope revealed distinct patterns of mineral distribution in the proximal, central, and distal regions of the diaphysis and also showed a net reduction in mineral levels toward the distal shaft. Increases in mineral density to higher fractions in controls were less in the flight bones at all three levels, with the most distal cross-sectional area most affected. The combined results from these novel techniques identified the areas of femoral diaphysis most vulnerable to the mineralization defect associated with spaceflight and/or the stress of landing.     

The Wnt antagonist secreted frizzled-related protein-1 is a negative regulator of trabecular bone formation in adult mice

Previous studies have associated activation of canonical Wnt signaling in osteoblasts with elevated bone formation. Here we report that deletion of the murine Wnt antagonist, secreted frizzled-related protein (sFRP)-1, prolongs and enhances trabecular bone accrual in adult animals. sFRP-1 mRNA was expressed in bones and other tissues of +/+ mice but was not observed in -/- animals. Despite its broad tissue distribution, ablation of sFRP-1 did not affect blood and urine chemistries, most nonskeletal organs, or cortical bone. However, sFRP-1-/- mice exhibited increased trabecular bone mineral density, volume, and mineral apposition rate when compared with +/+ controls. The heightened trabecular bone mass of sFRP-1-/- mice was observed in adult animals between the ages of 13-52 wk, occurred in multiple skeletal sites, and was seen in both sexes. Mechanistically, loss of sFRP-1 reduced osteoblast and osteocyte apoptosis in vivo. In addition, deletion of sFRP-1 inhibited osteoblast lineage cell apoptosis while enhancing the proliferation and differentiation of these cells in vitro. Ablation of sFRP-1 also increased osteoclastogenesis in vitro, although changes in bone resorption were not observed in intact animals in vivo. Our findings demonstrate that deletion of sFRP-1 preferentially activates Wnt signaling in osteoblasts, leading to enhanced trabecular bone formation in adults.     

Bone mineralization proceeds through intracellular calcium phosphate loaded vesicles: a cryo-electron microscopy study

Bone is the most widespread mineralized tissue in vertebrates and its formation is orchestrated by specialized cells - the osteoblasts. Crystalline carbonated hydroxyapatite, an inorganic calcium phosphate mineral, constitutes a substantial fraction of mature bone tissue. Yet key aspects of the mineral formation mechanism, transport pathways and deposition in the extracellular matrix remain unidentified. Using cryo-electron microscopy on native frozen-hydrated tissues we show that during mineralization of developing mouse calvaria and long bones, bone-lining cells concentrate membrane-bound mineral granules within intracellular vesicles. Elemental analysis and electron diffraction show that the intracellular mineral granules consist of disordered calcium phosphate, a highly metastable phase and a potential precursor of carbonated hydroxyapatite. The intracellular mineral contains considerably less calcium than expected for synthetic amorphous calcium phosphate, suggesting the presence of a cellular mechanism by which phosphate entities are first formed and thereafter gradually sequester calcium within the vesicles. We thus demonstrate that in vivo osteoblasts actively produce disordered mineral packets within intracellular vesicles for mineralization of the extracellular developing bone tissue. The use of a highly disordered precursor mineral phase that later crystallizes within an extracellular matrix is a strategy employed in the formation of fish fin bones and by various invertebrate phyla. This therefore appears to be a widespread strategy used by many animal phyla, including vertebrates.     

Spectrométrie infrarouge (DRIFT) des os de rongeurs fossiles de Tighenif (Pléistocène,Algérie)

Chemical analyses of Pléistocène rodent bones from Tighenif (Algeria) have shown some alterations of their contents. The infrared spectrometry (FTIR) of powdered bones of the various stratigraphic levels of this site confirms the diagenesis of the fossil bones. Content and composition of the organic matrix, as well as composition and crystallinity of the mineral phase have been altered. With Drift method (Diffuse Reflectance Infrared Fourier Transform), denaturation of the organic matrix is avoided, and spectra are obtained in a few minutes. Moreover, the powdered samples can be used for other analyses.     

Compressive axial mechanical properties of rat bone as functions of bone volume fraction,apparent density and micro-ct based mineral density

Mechanical testing has been regarded as the gold standard to investigate the effects of pathologies on the structure–function properties of the skeleton. With recent advances in computing power of personal computers, virtual alternatives to mechanical testing are gaining acceptance and use. We have previously introduced such a technique called structural rigidity analysis to assess mechanical strength of skeletal tissue with defects. The application of this technique is predicated upon the use of relationships defining the strength of bone as a function of its density for a given loading mode. We are to apply this technique in rat models to assess their compressive skeletal response subjected to a host of biological and pharmaceutical stimulations. Therefore, the aim of this study is to derive a relationship expressing axial compressive mechanical properties of rat cortical and cancellous bone as a function of equivalent bone mineral density, bone volume fraction or apparent density over a range of normal and pathologic bones.We used bones from normal, ovariectomized and partially nephrectomized animals. All specimens underwent micro-computed tomographic imaging to assess bone morphometric and densitometric indices and uniaxial compression to failure.We obtained univariate relationships describing 71–78% of the mechanical properties of rat cortical and cancellous bone based on equivalent mineral density, bone volume fraction or apparent density over a wide range of density and common skeletal pathologies. The relationships reported in this study can be used in the structural rigidity analysis introduced by the authors to provide a non-invasive method to assess the compressive strength of bones affected by pathology and/or treatment options.     

Quality assessment for processed and sterilized bone using Raman spectroscopy

To eliminate the potential for infection, many tissue banks routinely process and terminally sterilize allografts prior to transplantation. A number of techniques, including the use of scanning electron microscopy, bone graft models, and mechanical property tests, are used to evaluate the properties of allograft bone. However, as these methods are time consuming and often destroy the bone sample, the quality assessment of allograft bones are not routinely performed after processing and sterilization procedures. Raman spectroscopy is a non-destructive, rapid analysis technique that requires only small sample volumes and has recently been used to evaluate the mineral content, mineral crystallinity, acid phosphate and carbonate contents, and collagen maturity in human and animal bones. Here, to establish a quality assessment method of allograft bones using Raman spectroscopy, the effect of several common sterilization and preservation procedures on rat femoral bones were investigated. We found that freeze-thawing had no detectable effects on the composition of bone minerals or matrix, although heat treatment and gamma irradiation resulted in altered Raman spectra. Our findings suggest Raman spectroscopy may facilitate the quality control of allograft bone after processing and sterilization procedures.     

Mineral saturation, ossification and synostosis of the hand bones in adolescents and youth]

The results on X-ray densitometric studies of the hand bones in adolescent and young people at the age of 10--17 years (369 persons in all) are presented in the work. Processes of mineralization, ossification and synostosis, as the investigation has demonstrated, are closely connected with each other. From the beginning of ossification in the pisiform and sesamoid bones of the first metacarpophalageal joint (11--12 years of age) up to the completion of synostosis in short tubular bones of the hand (15--16 years of age), a decrease of mineral salts is noted in osseous tissue. Hence, accumulation of mineral salts in skeleton of children and adolescent persons does not occur smoothly, but rather distinctly reflects those functional changes which take place in the adolescent organism when the genital glands begin their increased activity.     

The effects of estrogen deficiency and bisphosphonate treatment on tissue mineralisation and stiffness in an ovine model of osteoporosis

While much research has been dedicated to understanding osteoporosis, the nature of mineral distribution and the mechanical property variation in diseased bone is poorly understood. The current study aimed to determine the effect of estrogen deficiency and bisphosphonate therapy on bone tissue properties using an ovine model of osteoporosis. Skeletally mature animals (4+ years) were divided into an ovariectomy group (ovx, n=20) and a non treatment control group (control, n=20). A zoledronic acid treated group was also included in which animals were estrogen deficient for 20 months prior to receiving treatment (Zol, n=4). Half of the control and ovx groups were euthanized 12 or 31 months post-operatively and all Zol animals were euthanised at 31 months. Individual trabeculae were removed from the proximal femur and were analysed at specific locations across the width of the trabeculae. The mineral content was measured using quantitative backscatter electron imaging and the modulus was measured using nanoindentation. The spatial distribution of tissue modulus and mineral content in bone from ovariectomised animals was similar to control. However, ovariectomy significantly reduced the overall mineral content and tissue modulus relative to the control group after 12 months. Interestingly, significant differences were not maintained 31 months post-OVX. Treatment with zoledronic acid increased the mineral content and tissue modulus relative to both the ovariectomised and control groups. Zoledronic acid was also found to alter the mineral and modulus gradients normally associated with healthy bone tissue. The current study provides evidence that both estrogen deficiency and zoledronic acid therapy significantly alter mineral content and the mechanical properties of trabecular tissue.     

Composition chimique de quelques Mammifères fossiles d’un milieu continental phosphaté (Aubrelong I, Lot, France, Oligocène inférieur)

Fossil mammal long bones from Lower Oligocene from Aubrelong 1 (Quercy) were analysed for a range of elements using electron microprobe technique and infrared spectrometry. Fossil mammal teeth were analysed using electron microprobe technique. Comparison of the fossil samples is made with those of recent samples. Infrared spectra show the decrease and the alteration of the organic matrix content in fossil bones. Values for the elements P, Ca are significantly higher, and S lower in the fossil samples relative to the recent samples. The high concentrations of these elements in the fossil samples are clearly a result of diagenetic alteration. In most fossil sites, all the tissues are enriched in Ca, P contents being higher or lower than in the recent tissues. All the fossil tissues of the Aubrelong site are enriched in P. Thus one may tentatively conclude that there is a relationship between the phosphatic composition of sediment and the behavior of P. The chemical composition of all the fossil tissues seems to converge. The organic and the mineral phases are altered by diagenetic processes.     

An infrared study of the interaction of polymethyl methacrylate with the protein and mineral components of bone.

We used Fourier transform infrared (FT-IR) microscopic mapping techniques to investigate the infiltration of polymethyl methacrylate (PMMA), a widely used medium for embedding biological tissues, into rat femur sections. Monitoring of the infrared absorbances of the PMMA carbonyl stretch, the protein amide I, and the apatite mineral phosphate stretch over a 225 x 975-microns region of the epiphyseal growth plate region of the rat femur enabled comparison of the relative amount of each component in distinct regions of the tissue. It was found that PMMA penetrates less into regions of greater mineral density and that the frequency of the PMMA carbonyl absorbance from the embedded tissue, 1729 cm-1, is identical to the free PMMA carbonyl frequency. This is consistent with a diffusion mechanism of infiltration of the PMMA, with no specific chemical interaction between the PMMA and the tissue components.     

DEXA analysis on the bones of rats exposed in utero and neonatally to static and 50 Hz electric fields

Effects of the electromagnetic fields on living bodies, bones in particular, are among the relevant issues of contemporary life. In this study, we report the influences of 50 Hz and 0 Hz (static) electric fields (EF), on intact rat bones, as evaluated by dual energy X-ray absorbtion (DEXA) measurements on bone content and density when these animals (n = 27) are continuously exposed in utero and neonatally to EFs (10 kV/m) 14 days before and 14 days after their birth, for 28 days in total. Differences between 50 Hz EF and static EF groups are found to be significant (95% confidence level) for total bone mineral content (BMC), TBMC (P = .002). Differences between 50 Hz and control groups are found to be significant for total bone mineral density (BMD), TBMD (P = .002), lumbar BMC, LBMC (P = .023), and TBMC (P = .001). Differences between static EF and control groups are found to be significant for femoral BMD, FBMD (P = .009), TBMD (P = .002), LBMC (P = .001), and TBMC (P = .001). Note that TBMC parameters are jointly significant for all differences between the three groups of test animals. These results have shown that both static and 50 Hz EFs influence the early development of rat bones. However, the influence of static EFs is more pronounced than that of the 50 Hz field.