Enzymatic and Non-enzymatic Collagen Cross-Links and Fracture Occurrence in Type 1 Diabetes Patients.

Increased fracture risk in type 1 diabetes (T1D) patients is not fully captured by bone mineral density (BMD) by DXA. Advanced glycation end-products (AGEs) have been implicated in the increased fracture risk in T1D, yet recent publications question this. To test the hypothesis that enzymatic collagen cross-links rather than AGEs correlate with fracture incidence in T1D, we analyzed iliac crest biopsies from sex-matched, fracturing T1D patients (N = 5; T1DFx), 6 non-fracturing T1D patients (T1DNoFx), and 6 healthy subjects, by Raman microspectroscopy as a function of tissue age (based on double fluorescent labels), in intracortical and trabecular bone, to determine pyridinoline (Pyd), ε-N-Carboxymethyl-L-lysine, and pentosidine (PEN)). There were no differences in the clinical characteristics between the T1DFx and T1DNoFx groups. At trabecular forming surfaces, T1DFx patients had higher PEN and Pyd content compared to T1DNoFx ones. Previous studies have shown that elevated PEN does not necessarily correlate with fracture incidence in postmenopausal, long-term T1D patients. On the other hand, the elevated Pyd content in the T1DFx patients would be consistent with published studies showing a significant correlation between elevated trivalent enzymatic collagen cross-links and fracture occurrence independent of BMD. Collagen fibers with high Pyd content are more brittle. Thus, a plausible suggestion is that it is the enzymatic collagen cross-links that either by themselves or in combination with the adverse effects of increased AGE accumulation that result in fragility fracture in T1D.

Nothobranchius furzeri, the Turquoise Killifish: A Model of Age-Related Osteoporosis?

INTRODUCTION: Osteoporosis is a frequent age-related disease, which affects millions of people worldwide. Despite significant progress in the treatment of the disease, a high number of patients still are underdiagnosed and undertreated. Therefore, novel animal models for the investigation of the disease are necessary. Nothobranchius furzeri is the shortest-lived vertebrate (with a lifespan of 3-7 months) that can be kept in captivity. Although it is an established model for aging research, studies on bone are lacking. The aim of this study was therefore to characterize N. furzeri as a potential model for age-related osteoporosis. MATERIALS AND METHODS: Bone properties of aging N. furzeri were investigated in male and female fish of the Gona Re Zhou strain, which were between 8 and 20 weeks old. Micro-computed tomography (Scanco Medical µCT35) was performed to determine the bone properties of the vertebral bodies. Bone structure and remodeling were investigated by different histological staining techniques and histomorphometry. The chemical composition of fish vertebrae and intervertebral discs was analyzed by Raman microspectroscopy. RESULTS: Osteoblasts, mono- and multinucleated osteoclasts but no osteocytes could be observed in the vertebral area of N. furzeri. Histomorphometric evaluations revealed a significant decrease of the number of osteoblasts/bone perimeter and for osteoid volume/bone volume (BV) a trend toward a decrease in old male N. furzeri. Comparing male and female fish, males showed higher BV densities and cortical thickness. The relative values of the bone volume density of 20-week-old male N. furzeri were significantly lower than 10-week-old ones. The mineral to matrix ratio increased with age in male and female fish. In the intervertebral discs, proteoglycans in relation to the organic matrix were significantly lower in older female fish. CONCLUSION: Our finding of a lack of osteocytes is in agreement with the fact that N. furzeri belongs to the evolutionarily advanced teleost fish. Furthermore, not only age-specific but also sex-specific differences were visible in the bone properties of N. furzeri, which can be taken into consideration for the study of gender aspects of age-related musculoskeletal diseases.

Bone properties in osteogenesis imperfecta: what can we learn from a bone biopsy beyond histology?

Transiliac bone biopsy samples are used to evaluate histology and bone cell activity in unclear pathological conditions. However, much additional information can be obtained from such bone samples. Using the example of osteogenesis imperfecta (OI), the current article describes how biopsy samples can be further used to study bone material characteristics including the degree of matrix mineralization, organic matrix properties, mineral particle size and bone nanoporosity. OI is a heritable collagen-related disorder that is phenotypically and genetically extremely heterogeneous. One essential finding was that OI bone is hypermineralized independently of clinical severity. Moreover, mineral particles in OI bone are of normal size or even smaller, but more densely packed than normally. Another recent finding was that in some forms of OI, collagen orientation is highly disorganized, indicating that the collagen-mineral particle network is profoundly altered in OI. These findings have contributed to the understanding of impaired bone strength in OI.

Alterations of bone material properties in adult patients with X-linked hypophosphatemia (XLH).

X-linked hypophosphatemia (XLH) caused by PHEX mutations results in elevated serum FGF23 levels, renal phosphate wasting and low 1,25-dihydroxyvitamin D. The glycophosphoprotein osteopontin, a potent inhibitor of mineralization normally degraded by PHEX, accumulates within the bone matrix. Conventional therapy consisting of supplementation with phosphate and vitamin D analogs is burdensome and the effects on bone material poorly characterized. We analyzed transiliac bone biopsies from four adult patients, two of them severely affected due to no diagnosis and no treatment until adulthood. We used light microscopy, qBEI and FTIRI to study histology, histomorphometry, bone mineralization density distribution, properties of the organic matrix and size of hypomineralized periosteocytic lesions. Non-treatment resulted in severe osteomalacia, twice the amount of mineralized trabecular volume, multiple osteon-like perforations, continuity of lamellae from mineralized to unmineralized areas and distinctive patches of woven bone. Periosteocytic lesions were larger than in treated patients. The latter had nearly normal osteoid thicknesses, although surface was still elevated. The median calcium content of the matrix was always within normal range, although the percentage of lowly mineralized bone areas was highly increased in non-treated patients, resulting in a marked heterogeneity in mineralization. Divalent collagen cross-links were evident independently of the mineral content of the matrix. Broad osteoid seams lacked measurable pyridinoline, a mature trivalent cross-link and exhibited considerable acidic lipid content, typically found in matrix vesicles. Based on our results, we propose a model that possibly integrates the relationship between the observed mineralization disturbances, FGF23 secretion and the known osteopontin accumulation in XLH.

Identification of Pyridinoline Trivalent Collagen Cross-Links by Raman Microspectroscopy.

Intermolecular cross-linking of bone collagen is intimately related to the way collagen molecules are arranged in a fibril, imparts certain mechanical properties to the fibril, and may be involved in the initiation of mineralization. Raman microspectroscopy allows the analysis of minimally processed bone blocks and provides simultaneous information on both the mineral and organic matrix (mainly type I collagen) components, with a spatial resolution of ~1 µm. The aim of the present study was to validate Raman spectroscopic parameters describing one of the major mineralizing type I trivalent cross-links, namely pyridinoline (PYD). To achieve this, a series of collagen cross-linked peptides with known PYD content (as determined by HPLC analysis), human bone, porcine skin, predentin and dentin animal model tissues were analyzed by Raman microspectroscopy. The results of the present study confirm that it is feasible to monitor PYD trivalent collagen cross-links by Raman spectroscopic analysis in mineralized tissues, exclusively through a Raman band ~1660 wavenumbers. This allows determination of the relative PYD content in undecalcified bone tissues with a spatial resolution of ~1 µm, thus enabling correlations with histologic and histomorphometric parameters.

Homocysteine induces serum amyloid A3 in osteoblasts via unlocking RGD-motifs in collagen.

Hyperhomocysteinemia is a risk factor for osteoporotic fractures. Homocysteine (Hcys) inhibits collagen cross-linking and consequently decreases bone extracellular matrix (ECM) quality. Serum amyloid A (A-SAA), an acute-phase protein family, plays an important role in chronic and inflammatory diseases and up-regulates MMP13, which plays an important role in bone development and remodeling. Here, we investigate the effect of Hcys on expression of SAA3, a member of the A-SAA gene family, in osteoblasts characterizing underlying mechanisms and possible consequences on bone metabolism. MC3T3-E1 osteoblast-like cells were cultured up to 21 d with Hcys (low millimolar range) or reseeded onto ECM resulting from untreated or Hcys-treated MC3T3-E1 cells. Fourier-transformed infrared spectroscopy and a discriminative antibody were used to characterize the resulting ECM. Gene expression and signaling pathways were analyzed by gene chip, quantitative RT-PCR, and immunoblotting. Transcriptional regulation of Saa3 was studied by promoter transfection assays, chromatin immunoprecipitation, and immunofluorescence microscopy. Hcys treatment resulted in reduced collagen cross-linking, uncovering of RGD-motifs, and activation of the PTK2-PXN-CTNNB1 pathway followed by RELA activation. These signaling events led to increased SAA3 expression followed by the production of MMP13 and several chemokines, including Ccl5, Ccl2, Cxcl10, and Il6. Our data suggest Saa3 as link between hyperhomocysteinemia and development of osteoporosis.

Bone intrinsic material and compositional properties in postmenopausal women diagnosed with long-term Type-1 diabetes.

The incidence of diabetes mellitus and the associated complications are growing worldwide, affecting the patients' quality of life and exerting a considerable burden on health systems. Yet, the increase in fracture risk in type 1 diabetes (T1D) patients is not fully captured by bone mineral density (BMD), leading to the hypothesis that alterations in bone quality are responsible for the increased risk. Material/compositional properties are important aspects of bone quality, yet information on human bone material/compositional properties in T1D is rather sparse. The purpose of the present study is to measure both the intrinsic material behaviour by nanoindentation, and material compositional properties by Raman spectroscopy as a function of tissue age and microanatomical location (cement lines) in bone tissue from iliac crest biopsies from postmenopausal women diagnosed with long-term T1D (N = 8), and appropriate sex-, age-, BMD- and clinically-matched controls (postmenopausal women; N = 5). The results suggest elevation of advanced glycation endproducts (AGE) content in the T1D and show significant differences in mineral maturity / crystallinity (MMC) and glycosaminoglycan (GAG) content between the T1D and control groups. Furthermore, both hardness and modulus by nanoindentation are greater in T1D. These data suggest a significant deterioration of material strength properties (toughness) and compositional properties in T1D compared with controls.

Homocysteine suppresses the expression of the collagen cross-linker lysyl oxidase involving IL-6, Fli1, and epigenetic DNA methylation.

Elevated homocysteine (Hcys) serum levels represent a risk factor for several chronic pathologies, including cardiovascular disease, atherosclerosis, and chronic renal failure, and affect bone development, quality, and homeostasis. Hcys influences the formation of a stable bone matrix directly through the inhibition of the collagen cross-linking enzyme lysyl oxidase (Lox) and, as we have shown recently, by repressing its mRNA expression. The aim of this study was to investigate the mechanisms involved in this process. Through evaluation of gene arrays, quantitative RT-PCR, immunoblots, and ELISA, we identified a Hcys-dependent stimulation of interleukin 6 (IL-6) and genes involved in IL-6/Janus kinase 2 (JAK2)-dependent signal transduction pathways in pre-osteoblastic MC3T3-E1 cells. Moreover, up-regulation of genes essential for epigenetic DNA methylation (DNA (cytosine-5)-methyltransferases and helicase lymphoid-specific (Hells) was observed. Further investigations demonstrated that Hcys increased via IL-6/JAK2 the expression of Fli1 (Friend leukemia virus integration 1), a transcription factor, which we found essential for IL-6-dependent Dnmt1 stimulation. CpG methylation analysis of CpG-rich Lox proximal promoter revealed an increased CpG methylation status after treatment of the cells with Hcys indicating an epigenetic origin for Hcys-dependent Lox repression. Inhibition of the IL-6/JAK2 pathway or of CpG methylation reversed the repressive effect of Hcys on Lox expression. In conclusion, we demonstrate that Hcys stimulates IL-6 synthesis in osteoblasts, which is known to affect bone metabolism via osteoclasts. Furthermore, IL-6 stimulation results via JAK2, Fli1, and Dnmt1 in down-regulation of Lox expression by epigenetic CpG methylation revealing a new mechanism negatively affecting bone matrix formation.

Bone Matrix Mineralization and Response to Burosumab in Adult Patients With X-Linked Hypophosphatemia: Results From the Phase 3, Single-Arm International Trial.

X-linked hypophosphatemia (XLH) is characterized by excess fibroblast growth factor 23 (FGF23) secretion, renal phosphate wasting, and low 1,25(OH)2 D3 . Adult patients present with osteomalacia, hypomineralized periosteocytic lesions, bone fragility, and pain. Burosumab is a fully human monoclonal FGF23 antibody approved for XLH treatment. UX023-CL304 was an open-label, phase 3 study investigating the effects of burosumab on osteomalacia in adults with XLH, who remained untreated at least 2 years prior enrollment. Here, we present the effect of burosumab on bone material properties. We analyzed transiliac bone biopsy samples from 11 individuals before and after 48 weeks of subcutaneous burosumab treatment (1.0 mg/kg administered every 4 weeks). We used quantitative backscattered electron imaging (qBEI) and Fourier transform infrared imaging (FTIRI) to assess bone mineralization density distribution (BMDD), mineralized bone volume, properties of the organic matrix, and size of periosteocytic lesions. The outcomes were compared with reference values from healthy adults and with four XLH patients either untreated or treated by conventional therapy. Prior to burosumab, the average mineralization in cancellous bone was lower than in healthy reference. CaLow, the fraction of lowly mineralized matrix, and CaHigh, the fraction of highly mineralized matrix, were both elevated resulting in a broad heterogeneity in mineralization (CaWidth). Burosumab resulted in a decrease of CaHigh toward normal range, whereas CaLow and CaWidth remained elevated. The mineralized bone volume was notably increased (+35.9%). The size of the periosteocytic lesions was variable but lower than in untreated XLH patients. FTIRI indicated decreased enzymatic collagen crosslink ratio heterogeneity. In summary, matrix mineralization in XLH is very heterogeneous. Highly mineralized regions represent old bone packets, probably protected from osteoclastic resorption by osteoid seams. The concomitant decrease of highly mineralized matrix, persistence of lowly mineralized matrix, and increase in mineralized bone volume after burosumab suggest a boost in mineralization of preexisting unmineralized or very lowly mineralized matrix, providing a potential explanation for previously observed improved osteomalacia. © 2022 American Society for Bone and Mineral Research (ASBMR).

Treatment of postmenopausal osteoporosis patients with teriparatide for 24 months reverts forming bone quality indices to premenopausal healthy control values.

Postmenopausal osteoporosis (PMOP) therapies are frequently evaluated by bone mineral density (BMD) gains against patients receiving placebo (calcium and vitamin D supplementation, a mild bone turnover-suppressing intervention), which is not equivalent to either healthy or treatment-naive PMOP. The aim of the present observational study was to assess the effects of TPTD treatment in PMOP (20 µg, once daily) at 6 (TPTD 6m; n = 28, age 65 ± 7.3 years), and 24 (TPTD 24m; n = 32, age 67.4 ± 6.15 years) months on bone quality indices at actively forming trabecular surfaces (with fluorescent double labels). Data from the TPTD-treated PMOP patients were compared with those in healthy adult premenopausal women (HC; n = 62, age 40.5 ± 10.6 years), and PMOP receiving placebo (PMOP-PLC; n = 94, age 70.6 ± 4.5 years). Iliac crest biopsies were analyzed by Raman microspectroscopy at three distinct tissue ages: mid-distance between the second label and the bone surface, mid-distance between the two labels, and 1 µm behind the first label. Mineral to matrix ratio (MM), mineral maturity/crystallinity (MMC), tissue water (TW), glycosaminoglycan (GAGs), and pyridinoline (Pyd) content were determined. Outcomes were compared by ANCOVA with subject age and tissue age as covariates, and health status as a fixed factor, followed by Sidak's post-hoc testing (significance assigned to p < 0.05). Both TPTD groups increased MM compared to PMOP-PLC. While TPTD 6m had values similar to HC, TPTD 24m had higher values compared to either HC or TPTD 6m. Both TPTD groups had lower MMC values compared to PMOP-PLC and similar to HC. TPTD 6m patients had higher TW content compared to HC, while TPTD 24m had values similar to HC and lower than either PMOP-PLC or TPTD 6m. Both TPTD groups had lower GAG content compared to HC group, while TPTD 6m had higher values compared to PMOP-PLC. Finally, TPTD 6m patients had higher Pyd content compared to HC and lower compared to PMOP-PLC, while TPTD 24m had lower values compared to PMOP-PLC and TPTD 6m, and similar to HC group. The results of the present study indicate that effects of TPTD on forming trabecular bone quality indices depend on treatment duration. At the recommended length of 24 m, TPTD restores bone mineral and organic matrix quality indices (MMC, TW, Pyd content) to premenopausal healthy (HC) levels.

Vitamin C epigenetically controls osteogenesis and bone mineralization.

Vitamin C deficiency disrupts the integrity of connective tissues including bone. For decades this function has been primarily attributed to Vitamin C as a cofactor for collagen maturation. Here, we demonstrate that Vitamin C epigenetically orchestrates osteogenic differentiation and function by modulating chromatin accessibility and priming transcriptional activity. Vitamin C regulates histone demethylation (H3K9me3 and H3K27me3) and promotes TET-mediated 5hmC DNA hydroxymethylation at promoters, enhancers and super-enhancers near bone-specific genes. This epigenetic circuit licenses osteoblastogenesis by permitting the expression of all major pro-osteogenic genes. Osteogenic cell differentiation is strictly and continuously dependent on Vitamin C, whereas Vitamin C is dispensable for adipogenesis. Importantly, deletion of 5hmC-writers, Tet1 and Tet2, in Vitamin C-sufficient murine bone causes severe skeletal defects which mimic bone phenotypes of Vitamin C-insufficient Gulo knockout mice, a model of Vitamin C deficiency and scurvy. Thus, Vitamin C's epigenetic functions are central to osteoblastogenesis and bone formation and may be leveraged to prevent common bone-degenerating conditions.

Increased strontium uptake in trabecular bone of ovariectomized calcium-deficient rats treated with strontium ranelate or strontium chloride.

Based on clinical trials showing the efficacy to reduce vertebral and non-vertebral fractures, strontium ranelate (SrR) has been approved in several countries for the treatment of postmenopausal osteoporosis. Hence, it is of special clinical interest to elucidate how the Sr uptake is influenced by dietary Ca deficiency as well as by the formula of Sr administration, SrR versus strontium chloride (SrCl(2)). Three-month-old ovariectomized rats were treated for 90 days with doses of 25 mg kg(-1) d(-1) and 150 mg kg(-1) d(-1) of SrR or SrCl(2) at low (0.1% Ca) or normal (1.19% Ca) Ca diet. Vertebral bone tissue was analysed by confocal synchrotron-radiation-induced micro X-ray fluorescence and by backscattered electron imaging. Principal component analysis and k-means clustering of the acquired elemental maps of Ca and Sr revealed that the newly formed bone exhibited the highest Sr fractions and that low Ca diet increased the Sr uptake by a factor of three to four. Furthermore, Sr uptake in bone of the SrCl(2)-treated animals was generally lower compared with SrR. The study clearly shows that inadequate nutritional calcium intake significantly increases uptake of Sr in serum as well as in trabecular bone matrix. This indicates that nutritional calcium intake as well as serum Ca levels are important regulators of any Sr treatment.

Effects of risedronate in Runx2 overexpressing mice, an animal model for evaluation of treatment effects on bone quality and fractures.

Young mice overexpressing Runx2 specifically in cells of the osteoblastic lineage failed to gain bone mass and exhibited a dramatic increase in bone resorption, leading to severe osteopenia and spontaneous vertebral fractures. The objective of the current study was to determine whether treatment with a bisphosphonate (risedronate, Ris), which reduces fractures in postmenopausal as well as in juvenile osteoporosis, was able to improve bone quality and reduce vertebral fractures in mice overexpressing Runx2. Four-week-old female Runx2 mice received Ris at 2 and 10 µg/kg subcutaneously twice a week for 12 weeks. Runx2 and wild-type mice received vehicle (Veh) as control. We measured the number of new fractures by X-ray and bone mineral density (BMD) by DEXA. We evaluated bone quality by histomorphometry, micro-CT, and Fourier transform infrared imaging (FTIRI). Ris at 20 µg/kg weekly significantly reduced the average number of new vertebral fractures compared to controls. This was accompanied by significantly increased BMD, increased trabecular bone volume, and reduced bone remodeling (seen in indices of bone resorption and formation) in the vertebrae and femoral metaphysis compared to Runx2 Veh. At the femur, Ris also increased cortical thickness. Changes in collagen cross-linking seen on FTIRI confirmed that Runx2 mice have accelerated bone turnover and showed that Ris affects the collagen cross-link ratio at both forming and resorbing sites. In conclusion, young mice overexpressing Runx2 have high bone turnover-induced osteopenia and spontaneous fractures. Ris at 20 µg/kg weekly induced an increase in bone mass, changes in bone microarchitecture, and decreased vertebral fractures.

Infrared assessment of bone quality: a review.

BACKGROUND: Bone strength depends on both bone quantity and quality. The former is routinely estimated in clinical settings through bone mineral density measurements but not the latter. Bone quality encompasses the structural and material properties of bone. Although its importance is appreciated, its contribution in determining bone strength has been difficult to precisely quantify partly because it is multifactorial and requires investigation of all bone hierarchical levels. Fourier transform infrared spectroscopy provides one way to explore these levels. QUESTIONS/PURPOSES: The purposes of our review were to (1) provide a brief overview of Fourier transform infrared spectroscopy as a way to establish bone quality, (2) review the major bone material parameters determined from Fourier transform infrared spectroscopy, and (3) review the role of Fourier transform infrared microspectroscopic analysis in establishing bone quality. METHODS: We used the ISI Web of Knowledge database initially to identify articles containing the Boolean term "infrared" AND "bone." We then focused on articles on infrared spectroscopy in bone-related journals. RESULTS: Infrared spectroscopy provides information on bone material properties. Their microspectroscopic versions allow one to establish these properties as a function of anatomic location, mineralization extent, and bone metabolic activity. It provides answers pertaining to the contribution of mineral to matrix ratio, mineral maturity, mineral carbonate substitution, and collagen crosslinks to bone strength. Alterations of bone material properties have been identified in disease (especially osteoporosis) not attainable by other techniques. CONCLUSIONS: Infrared spectroscopic analysis is a powerful tool for establishing the important material properties contributing to bone strength and thus has helped better understand changes in fragile bone.

Mineral and organic matrix composition at bone forming surfaces in postmenopausal women with osteoporosis treated with either teriparatide or zoledronic acid.

The ability of bone to resist fracture is dependent on the composite nature of its mineral and organic matrix content. Teriparatide (TPTD) and zoledronic acid (ZOL) are approved anabolic and antiresorptive therapies, respectively, to reduce fracture risk in women with postmenopausal osteoporosis. In the SHOTZ study, postmenopausal women with osteoporosis were treated with TPTD (20 µg daily, subcutaneous) or ZOL (5 mg/year, intravenous infusion) for 24 months. Iliac crest biopsies were obtained at 6 months and again at 24 months from approximately one third of the original study cohort. To investigate the early effects of these two drugs on the quality of newly formed bone, we used vibrational spectroscopic techniques to analyze tetracycline-labelled transiliac biopsies obtained from participants at the 6-month time point. Raman spectra were acquired at forming trabecular and intra-cortical surfaces (identified by fluorescent double labels), to determine mineral, organic matrix, glycosaminoglycan, and tissue water content, as well as mineral maturity/crystallinity at three specific tissue ages (1-5, 15, and ≥25 days). Fourier transformed infrared microspectroscopy was used to determine pyridinoline/divalent collagen cross-link ratios. At 6 months, treatment with TPTD versus ZOL resulted in lower mineral and higher organic matrix content, increased tissue water content, and lower mineral/matrix, mineral maturity/crystallinity, glycosaminoglycan content, and pyridinoline/divalent enzymatic collagen cross-link ratio. Our results suggest that TPTD and ZOL have differential effects on material properties of newly formed bone at individual remodeling sites, highlighting their different mechanisms of action.

Bone tissue material composition is compromised in premenopausal women with Type 2 diabetes.

Type 2 diabetes mellitus (T2DM) patients are at an increased risk of fracture despite normal to high bone mineral density (BMD) values. In this cross-sectional study we establish bone compositional properties in tetracycline labeled iliac crest biopsies from premenopausal women diagnosed with T2DM (N = 26). Within group comparisons were made as a function of tissue age (TA), presence of chronic complications (CC), glycosylated haemoglobin (HbA1c) levels, and morphometric fracture (MFx). We also compared these data at actively trabecular bone forming surfaces against sex- and age-matched healthy controls (N = 32). The bone quality indices determined by Raman microspectroscopic analysis were: mineral/matrix (MM), tissue water content (nanoporosity; NanoP), mineral maturity/crystallinity (MMC), and glycosaminoglycan (GAG), pyridinoline (Pyd), N-(carboxymethyl)lysine (CML), and pentosidine (PEN) content. Within the T2DM group, at the oldest tissue, CML and PEN contents were significantly elevated in the cancellous compared to cortical compartment. The outcomes were not dependent on MFx. On the other hand, both were significantly elevated in patients with CC, as well as those with HbA1c levels > 7%. At actively forming surfaces, the cortical compartment had higher NanoP compared to cancellous. Still within the T2DM group, patients with MFx had significantly elevated MM and GAGs compared to the ones that did not. At actively forming trabecular surfaces, compared to healthy women, T2DM patients had elevated GAGs content and MMC. The results of this study indicate increased AGEs in those with poor glycation control and chronic complications. Additionally, T2DM patients had elevated MMC and decreased GAGs content compared to healthy controls. These alterations may be contributing to the T2DM inherent elevated fracture risk and suggest a role for hyperglycemia on bone quality.

Newly formed and remodeled human bone exhibits differences in the mineralization process.

During human skeletal growth, bone is formed via different processes. Two of them are: new bone formation by depositing bone at the periosteal (outer) surface and bone remodeling corresponding to a local renewal of tissue. Since in remodeling formation is preceded by resorption, we hypothesize that modeling and remodeling could require radically different transport paths for ionic precursors of mineralization. While remodeling may recycle locally resorbed mineral, modeling implies the transport over large distances to the site of bone apposition. Therefore, we searched for potential differences of size, arrangement and chemical composition of mineral particles just below surfaces of modeling and remodeling sites in femur midshaft cross-sections from healthy children. These bone sites were mapped using scanning synchrotron X-ray scattering, Raman microspectroscopy, energy dispersive X-ray analysis and quantitative backscattered electron microscopy. The results show clear differences in mineral particle size and composition between the sites, which cannot be explained by a change in the rate of mineral apposition or accumulation. At periosteal modeling sites, mineral crystals are distinctly larger, display higher crystallinity and exhibit a lower calcium to phosphorus ratio and elevated Na and Mg content. The latter may originate from Mg used for phase stabilization of mineral precursors and therefore indicate different time periods for mineral transport. We conclude that the mineralization process is distinctively different between modeling and remodeling sites due to varying requirements for the transport distance and, therefore, the stability of non-crystalline ionic precursors, resulting in distinct compositions of the deposited mineral phase. STATEMENT OF SIGNIFICANCE: In growing children new bone is formed either due to apposition of bone tissue e.g. at the outer ridge of long bones to allow growth in thickness (bone modeling), or in cavities inside the mineralized matrix when replacing tissue (bone remodeling). We demonstrate that mineral crystal shape and composition are not the same between these two sites, which is indicative of differences in mineralization precursors. We suggest that this may be due to a longer mineral transport distance to sites of new bone formation as compared to remodeling where mineral can be locally recycled.

Fourier Transform Infrared Imaging of Bone.

Fourier transform infrared imaging (FTIRI) is a technique that can be used to analyze the material properties of bone using tissue sections. This chapter describes the basic principles of FTIR and the methods for capturing and analyzing FTIR images in bone sections.

Material properties and osteoporosis.

The main clinical tool for the diagnosis and treatment of skeletal diseases such as osteoporosis is the determination of bone mineral density by dual x-ray absorptiometry. Although this outcome contributes to the determination of bone strength, the clinical evidence to date suggests that it does not correlate strongly with fracture incidence. The main reason for this discrepancy is the fact that several other bone properties, such as material properties, are not taken into account. This short review summarizes the reasons why material properties are important in the determination of bone strength and briefly discusses some of them as well as their influence on bone's mechanical performance.

Biomechanical and Bone Material Properties of Schnurri-3 Null Mice.

Schnurri-3 (Shn3) is an essential regulator of postnatal skeletal remodeling. Shn3-deficient mice (Shn3-/-) have high bone mass; however, their bone mechanical and material properties have not been investigated to date. We performed three-point bending of femora, compression tests of L3 vertebrae. We also measured intrinsic material properties, including bone mineralization density distribution (BMDD) and osteocyte lacunae section (OLS) characteristics by quantitative backscatter electron imaging, as well as collagen cross-linking by Fourier transform infrared microspectroscopy of femora from Shn3-/- and WT mice at different ages (6 weeks, 4 months, and 18 months). Moreover, computer modeling was performed for the interpretation of the BMDD outcomes. Femora and L3 vertebrae from Shn3-/- aged 6 weeks revealed increased ultimate force (2.2- and 3.2-fold, p < .01, respectively). Mineralized bone volume at the distal femoral metaphysis was about twofold (at 6 weeks) to eightfold (at 4 and 18 months of age) in Shn3-/- (p < .001). Compared with WT, the average degree of trabecular bone mineralization was similar at 6 weeks, but increased at 4 and 18 months of age (+12.6% and +7.7%, p < .01, respectively) in Shn3-/-. The analysis of OLS characteristics revealed a higher OLS area for Shn3-/- versus WT at all ages (+16%, +23%, +21%, respectively, p < .01). The collagen cross-link ratio was similar between groups. We conclude that femora and vertebrae from Shn3-/- had higher ultimate force in mechanical testing. Computer modeling demonstrated that in cases of highly increased bone volume, the average degree of bone matrix mineralization can be higher than in WT bone, which was actually measured in the older Shn3-/- groups. The area of 2D osteocyte lacunae sections was also increased in Shn3-deficiency, which could only partly be explained by larger remnant areas of primary cortical bone. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.