Cortical bone material / compositional properties in growing children and young adults aged 1.5-23 years, as a function of gender, age, metabolic activity, and growth spurt.

Bone material / compositional properties are significant determinants of bone quality, thus strength. Raman spectroscopic analysis provides information on the quantity and quality of all three bone tissue components (mineral, organic matrix, and tissue water). The overwhelming majority of the published reports on the subject concern adults. We have previously reported on these properties in growing children and young adults, in the cancellous compartment. The purpose of the present study was to create normative reference data of bone material / compositional properties for children and young adults, in the cortical compartment. We performed Raman (Senterra (Bruker Optik GmbH), 50× objective, with an excitation of 785 nm (100 mW) and a lateral resolution of ~0.6 µm) microspectroscopic analysis of transiliac bone samples from 54 individuals between 1.5 and 23 years of age, with no known metabolic bone disease, and which have been previously used to establish histomorphometric, bone mineralization density distribution, and cancellous bone quality reference values. The bone quality indices that were determined were: mineral/matrix ratio (MM) from the integrated areas of the v2PO4 (410-460 cm-1) and the amide III (1215-1300 cm-1) bands, tissue water in nanopores approximated by the ratio of the integrated spectral area ~ 494-509 cm-1 to Amide III band, the glycosaminoglycan (GAG) content (ratio of integrated area 1365-1390 cm-1 to the Amide III band, the sulfated proteoglycan (sPG) content as the ratio of the integrated peaks ~1062 cm-1 and 1365-1390 cm-1, the pyridinoline (Pyd) content estimated from the ratio of the absorbance height at 1660 cm-1 / area of the amide I (1620-1700 cm-1) band, and the mineral maturity / crystallinity (MMC) estimated from the inverse of the full width at half height of the v1PO4 (930-980 cm-1) band. Analyses were performed at the three distinct cortical surfaces (endosteal, osteonal, periosteal) at specific anatomical microlocations, namely the osteoid, and the three precisely known tissue ages based on the presence of fluorescence double labels. Measurements were also taken in interstitial bone, a much older tissue that has undergone extensive secondary mineralization. Overall, significant dependencies of the measured parameters on tissue age were observed, while at any given tissue age, sex and subject age were minimal confounders. The established Raman database in the cortical compartments complements the previously published one in cancellous bone, and provides healthy baseline bone quality indices that may serve as a valuable tool to identify alterations due to pediatric disease.

Bone quality in an ovariectomized monkey animal model treated with two doses of teriparatide for either 18 months, or 12 months followed by withdrawal for 6 months.

Previous studies of ovariectomized (OVX) monkeys, treated with recombinant human parathyroid hormone (PTH) (1-34) at 1 or 5 µg/kg/day for 18 months or for 12 months followed by 6 months withdrawal from treatment, displayed significant changes in geometry, histomorphometry, and bone quality, but without strict tissue age criteria, of the midshaft humerus. Since bone quality significantly depends on tissue age among other factors, the aim of the present study was to establish the bone-turnover independent effects of two doses of PTH, as well as the effects of treatment withdrawal on bone quality by measuring bone material composition at precisely known tissue ages ranging from osteoid, to mineralized tissue older than 373 days. Raman microspectroscopic analysis of bone tissue from the mid-shaft humerus of OVX monkeys demonstrated that the clinically relevant dose of PTH administered for 18 months reverses the effects of ovariectomy on bone quality when compared against SHAM. Both doses investigated in this study restore the mineralization regulation mechanisms to SHAM levels. The study also showed that the beneficial effects induced by 12 months of clinically relevant PTH therapy were sustained after six months of therapy withdrawal.

Effect of hormone replacement therapy on bone formation quality and mineralization regulation mechanisms in early postmenopausal women.

Post-menopausal osteoporosis is characterized by a negative imbalance between bone formation and bone resorption resulting in a net bone loss, increasing the risk of fracture. One of the earliest interventions to protect against this was hormonal replacement therapy (HRT). Bone strength depends on both the amount and quality of bone, the latter including compositional / material and structural properties. Bone compositional / material properties are greatly dependent on both patient-, and tissue-age. Raman spectroscopy is an analytical tool ideally suited for the determination of bone compositional / material properties as a function of tissue age as it is capable of analyzing areas ~1 × 1 µm2 in tetracycline labeled bone forming areas. Using such analysis of humeri from an ovariectomized primate animal model, we reported that loss of estrogen results in alteration in the mineralization regulation mechanisms by osteoid organic matrix attributes at actively forming bone surfaces. In the present work, we used Raman microspectroscopic techniques to compare osteoid and youngest mineralized tissue composition, as well as relationships between osteoid organic matrix quality and quality attributes of the earliest mineralized tissue in paired iliac crest biopsies obtained from early postmenopausal women before and after two years of HRT therapy. Significant correlations between osteoid proteoglycans, sulfated proteoglycans, pyridinoline, and earliest mineralized tissue mineral content were observed, suggesting that in addition to changes in bone turnover rates, HRT affects the osteoid composition, mineralization regulation mechanisms, and potentially fibrillogenesis.

Interplay between mineral crystallinity and mineral accumulation in health and postmenopausal osteoporosis.

Osteoporosis is characterized by an imbalance between bone formation and resorption rates, resulting in bone loss. For ethical reasons, effects of antiosteoporosis drugs are compared against patients receiving vitamin D and calcium supplementation which is a mild antiresorptive regimen. Bone formation may be resolved into two phases: the initial formation of mineral crystals (primary nucleation) and the subsequent mineral accumulation (secondary nucleation and mineral growth) on them. In this study, we used Raman microspectroscopic analysis of iliac crest biopsies from healthy females (N = 108), postmenopausal osteoporosis patients receiving vitamin D and calcium supplementation (PMOP-S; N = 66), and treatment-naïve postmenopausal osteoporosis patients (PMOP-TN; N = 12) to test the hypothesis that at forming trabecular surfaces, mineral maturity / crystallinity of the youngest crystallites associates with the amount of subsequent mineral accumulation. The surfaces of analysis were chosen based on the presence of fluorescent double labels, defining three distinct tissue ages. The results indicated that when adjusted for age and tissue age, there were no differences in amount of mineral formed between healthy females and either PMOP-S or PMOP-TN, while both PMOP-S and PMOP-TN had larger crystallites compared to healthy females. Moreover, significant differences existed between PMOP-S and PMOP-TN in size of initial crystals formed as well as rate of mineral accumulation and maturation. These findings suggest an additional mechanism that may contribute to the decreased mineral content evident in PMOP, and provide a potential target for the development of new interventions. STATEMENT OF SIGNIFICANCE: We used Raman microspectroscopic analysis of iliac crest biopsies from healthy females and postmenopausal osteoporosis patients (PMOP) receiving placebo to test the hypothesis that at forming trabecular surfaces, mineral maturity / crystallinity (MMC) of the youngest crystallites associates with the amount of subsequent mineral accumulation. This can affect bone mechanical properties as larger crystallites have been shown to result in compromised mechanical attributes; and larger crystallites grow slower compared to smaller ones. The results of the present analysis indicate that increased MMC of the youngest formed mineral may contribute to the bone mineral loss evident in PMOP and the accompanying increased fracture risk independently of bone turnover rate.

Organic matrix quality discriminates between age- and BMD-matched fracturing versus non-fracturing post-menopausal women: A pilot study.

Women with similar areal Bone Mineral Densities (BMD) may show divergent fracture incidence due to differences in bone quality. The hypothesis tested in the present pilot study is that postmenopausal (PM) women who have sustained osteoporotic fractures have altered organic matrix quality compared to those who have not. We used Raman microspectroscopy to analyze transiliac biopsies collected from fracturing (n = 6, mean age 62.5 ±â€¯7.4 yrs; Cases) and non-fracturing PM women (n = 6, age- and BMD-matched; mean age 62.2 ±â€¯7.3 yrs; Controls). Previous results show differences in intrinsic material properties by nanoindentation that are more homogenously distributed and could facilitate microcrack propagation in Cases, along with lower mineral carbonate/phosphate ratio by Fourier transform infrared spectroscopic imaging, and no differences in bone tissue mineralization by digitized microradiography. No differences between groups were seen by conventional histomorphometry. Spectra were acquired 2 µm away from previously performed nanoindents, in cortical and cancellous compartments. The determined parameters were: mineral to matrix ratio (MM), and nanoporosity (a surrogate for tissue water (TW)), glycosaminoglycan (GAG), pyridinoline (Pyd; trivalent enzymatic collagen cross-link), N(6)-carboxymethyllysine (CML; advanced glycation endproduct), and pentosidine (PEN; advanced glycation endproduct) content. ANCOVA indicated no differences in any of the spectroscopic outcomes between cancellous and cortical compartments. On the other hand, Cases had lower nanoporosity (TW) and GAG, and elevated Pyd, and CML content compared to Controls. In conclusion, the results of the present study indicate significant differences in organic matrix quality in PM women that sustain fragility fractures versus age- and BMD-matched controls, highlighting its importance as a potential independent determinant of fracture incidence.

Estrogen depletion alters mineralization regulation mechanisms in an ovariectomized monkey animal model.

Ovariectomized animal models have been extensively used in osteoporosis research due to the resulting loss of bone mass. The purpose of the present study was to test the hypothesis that estrogen depletion alters mineralization regulation mechanisms in an ovariectomized monkey animal model. To achieve this we used Raman microspectroscopy to analyze humeri from monkeys that were either SHAM-operated or ovariectomized (N = 10 for each group). Measurements were made as a function of tissue age and cortical surface (periosteal, osteonal, endosteal) based on the presence of calcein fluorescent double labels. In the present work we focused on osteoid seams (defined as a surface with evident calcein labels, 1 µm distance away from the mineralizing front, and for which the Raman spectra showed the presence of organic matrix but not mineral), as well as the youngest mineralized tissue between the second fluorescent label and the mineralizing front, 1 µm inwards from the front with the phosphate mineral peak evident in the Raman spectra (TA1). The spectroscopically determined parameters of interest were the relative glycosaminoglycan (GAG) and pyridinoline (Pyd) contents in the osteoid, and the mineral content in TA1. At all three cortical surfaces, significant correlations were evident in the SHAM-operated animals between osteoid GAG (negative) and Pyd content, and mineral content, unlike the OVX animals. These results suggest that in addition to the well-established effects on turnover rates and bone mass, estrogen depletion alters the regulation of mineralization by GAGs and Pyd.

Baseline mineralizing surface determines the magnitude of the bisphosphonate effect on cortical bone mineralization in postmenopausal osteoporotic patients.

PURPOSE: To determine the effect of short- or long-term bisphosphonate treatment on cortical bone mineralization density distribution (BMDD). METHODS: BMDD was assessed by quantitative backscatter electron imaging in postmenopausal osteoporosis: in paired transiliac biopsy samples (n=36) at baseline and after 3 years risedronate treatment from a clinical study, in transiliac biopsy samples from patients who were treated with either risedronate (n=31) or alendronate (n=68) for 3 to 7 years from an observational study. Outcomes were related to premenopausal reference data (n=73) and to histomorphometric mineralizing surface per bone surface (MS/BS). RESULTS: In the clinical study, patients with lower (below cohort median) MS/BS had normal cortical CaMean at baseline. After 3 years risedronate, their CaMean was not different versus baseline but increased versus reference (+2.9%, p=0.003). Among the groups of the observational study, CaMean did not exceed reference level, was similar for alendronate versus risedronate and similar between 3 to 5 years versus longer than 5 years treatment duration. CONCLUSION: Baseline bone mineralizing surface appears to be important for the effect of bisphosphonate on cortical bone mineralization. In patients with lower baseline MS/BS, level of mineralization after treatment can exceed reference level. Whether this is beneficial in the long-term is unknown.

Vibrational spectroscopic techniques to assess bone quality.

Although musculoskeletal diseases such as osteoporosis are diagnosed and treatment outcome is evaluated based mainly on routine clinical outcomes of bone mineral density (BMD) by DXA and biochemical markers, it is recognized that these two indicators, as valuable as they have proven to be in the everyday clinical practice, do not fully account for manifested bone strength. Thus, the term bone quality was introduced, to complement considerations based on bone turnover rates and BMD. Bone quality is an "umbrella" term that incorporates the structural and material/compositional characteristics of bone tissue. Vibrational spectroscopic techniques such as Fourier transform infrared microspectroscopy (FTIRM) and imaging (FTIRI), and Raman spectroscopy, are suitable analytical tools for the determination of bone quality as they provide simultaneous, quantitative, and qualitative information on all main bone tissue components (mineral, organic matrix, tissue water), in a spatially resolved manner. Moreover, the results of such analyses may be readily combined with the outcomes of other techniques such as histology/histomorphometry, small angle X-ray scattering, quantitative backscattered electron imaging, and nanoindentation.

Ovarian hormone depletion affects cortical bone quality differently on different skeletal envelopes.

The physical properties of bone tissue are determined by the organic and mineral matrix, and are one aspect of bone quality. As such, the properties of mineral and matrix are a major contributor to bone strength, independent of bone mass. Cortical bone quality may differ regionally on the three skeletal envelopes that compose it. Each of these envelopes may be affected differently by ovarian hormone depletion. Identifying how these regions vary in their tissue adaptive response to ovarian hormones can inform our understanding of how tissue quality contributes to overall bone strength in postmenopausal women. We analyzed humeri from monkeys that were either SHAM-operated or ovariectomized. Raman microspectroscopic analysis was performed as a function of tissue age based on the presence of multiple fluorescent double labels, to determine whether bone compositional properties (mineral/matrix ratio, tissue water, glycosaminoglycan, lipid, and pyridinoline contents, and mineral maturity/crystallinity) are similar between periosteal, osteonal, and endosteal surfaces, as well as to determine the effects of ovarian hormone depletion on them. The results indicate that mineral and organic matrix characteristics, and kinetics of mineral and organic matrix modifications as a function of tissue age are different at periosteal vs. osteonal and endosteal surfaces. Ovarian hormone depletion affects the three cortical surfaces (periosteal, osteonal, endosteal) differently. While ovarian hormone depletion does not significantly affect the quality of either the osteoid or the most recently mineralized tissue, it significantly affects the rate of subsequent mineral accumulation, as well as the kinetics of organic matrix modifications, culminating in significant differences within interstitial bone. These results highlight the complexity of the cortical bone compartments, add to existing knowledge on the effects of ovarian hormone depletion on local cortical bone properties, and may contribute to a better understanding of the location specific action of drugs used in the management of postmenopausal osteoporosis.

Vitamin D and calcium supplementation for three years in postmenopausal osteoporosis significantly alters bone mineral and organic matrix quality.

Prospective, controlled clinical trials in postmenopausal osteoporosis typically compare effects of an active drug with placebo in addition to vitamin D and calcium supplementation in both treatment arms. While clinical benefits are documented, the effect of this supplementation in the placebo arm and in clinical practice on bone material composition properties is unknown. The purpose of the present study was to evaluate these bone quality indices (specifically mineral/matrix, nanoporosity, glycosaminoglycan content, mineral maturity/crystallinity, and pyridinoline content) in patients that either received long-term vitamin D (400-1200IU) and calcium (1.0-1.5g) supplementation, or did not. We have analyzed by Raman microspectroscopy the bone forming trabecular surfaces of iliac crest in pre-treatment samples of a teriparatide study and the endpoint biopsies of the control arm obtained from the HORIZON trial. In general, the mineral/matrix ratio and the glycosaminoglycan (GAG) content was higher while nanoporosity, (a surrogate for tissue water content), the mineral maturity/crystallinity (MMC) and the pyridinoline (Pyd) content was lower in patients without long-term supplementation. Moreover, all indices were significantly dependent on tissue age. In conclusion, vitamin D and calcium supplementation is associated with altered mineral and organic matrix properties.

Effects of long-term alendronate treatment on postmenopausal osteoporosis bone material properties.

UNLABELLED: Raman microspectroscopic analysis of iliac crest from patients that were treated with alendronate (ALN) for 10 years revealed minimal, transient alterations in bone material properties confined to actively forming bone surfaces compared to patients that were on ALN for 5 years. These changes were not encountered in the bulk tissue. INTRODUCTION: Alendronate (ALN) and other bisphosphonates (BPs) are the most widely prescribed therapy for postmenopausal osteoporosis. Despite their overall excellent safety record and efficacy in reducing fractures, questions have been raised regarding potential detrimental effects that may be related to prolonged bone turnover reduction, although no definite cause-effect relationship has been established to date. The purpose of the present study was to evaluate bone material properties in patients that were receiving ALN for 5 or 10 years. METHODS: Raman microspectroscopic analysis was used to analyze iliac crest biopsies from postmenopausal women with osteoporosis who had been treated with ALN for 5 years and were then re-randomized to placebo (PBO, N = 14), 5 mg/day ALN (N = 10), or 10 mg/day ALN (N = 6) for another 5 years. The parameters monitored and expressed as a function of tissue age were (i) the mineral/matrix ratio (MM), (ii) the relative proteoglycan content (PG), (iii) the relative lipid content (LPD), (iv) the mineral maturity/crystallinity (MMC), and (v) the relative pyridinoline content (PYD). RESULTS: The obtained data indicate that 10-year ALN use results in minimal, transient bone tissue composition changes compared to use for 5 years, confined to actively forming trabecular surfaces, implying potential differences in bone matrix maturation that nevertheless did not result in differences of these values in bulk tissue. CONCLUSIONS: The data suggest that prolonged reduction in bone turnover during 10 years of therapy with ALN by itself is unlikely to be associated with adverse effects on bone material properties.

Fourier transform Infrared spectroscopic characterization of mineralizing type I collagen enzymatic trivalent cross-links.

The most abundant protein of bone's organic matrix is collagen. One of its most important properties is its cross-linking pattern, which is responsible for the fibrillar matrices' mechanical properties such as tensile strength and viscoelasticity. We have previously described a spectroscopic method based on the resolution of the Amide I and II Fourier transform Infrared (FTIR) bands to their underlying constituent peaks, which allows the determination of divalent and pyridinoline (PYD) collagen cross-links in mineralized thin bone tissue sections with a spatial resolution of ~6.3 µm. In the present study, we used FTIR analysis of a series of biochemically characterized collagen peptides, as well as skin, dentin, and predentin, to examine the potential reasons underlying discrepancies between two different analytical methodologies specifically related to spectral processing. The results identified a novel distinct FTIR underlying peak at ~1,680 cm(-1), correlated with deoxypyridinoline (DPD) content. Furthermore, the two different methods of spectral resolution result in widely different results, while only the method employing well-established spectroscopic routines for spectral resolution provided biologically relevant results, confirming our earlier studies relating the area of the underlying 1,660 cm(-1) with PYD content. The results of the present study describe a new peak that may be used to determine DPD content, confirm our earlier report relating spectroscopic parameters to PYD content, and highlight the importance of the selected spectral resolution methodology.

Pediatric reference Raman data for material characteristics of iliac trabecular bone.

Bone material characteristics are important contributors in the determination of bone strength. Raman spectroscopic analysis provides information on mineral/matrix ratio, mineral maturity/crystallinity, relative pyridinoline (Pyd) collagen cross-link content, relative proteoglycan content and relative lipid content. However, published reference data are available only for adults. The purpose of the present study was to establish reference data of Raman outcomes pertaining to bone quality in trabecular bone for children and young adults. To this end, tissue age defined Raman microspectroscopic analysis was performed on bone samples from 54 individuals between 1.5 and 23 years with no metabolic bone disease, which have been previously used to establish histomorphometric and bone mineralization density distribution reference values. Four distinct tissue ages, three well defined by the fluorescent double labels representing early stages of bone formation and tissue maturation (days 3, 12, 20 of tissue mineralization) and a fourth representing old mature tissue at the geometrical center of the trabeculae, were analyzed. In general, significant dependencies of the measured parameters on tissue age were found, while at any given tissue age, sex and subject age were not confounders. Specifically, mineral/matrix ratio, mineral maturity/crystallinity index and relative pyridinoline collagen cross-link content index increased by 485%, 20% and 14%, respectively between days 3 and 20. The relative proteoglycan content index was unchanged between days 3 and 20 but was elevated in the old tissue compared to young tissue by 121%. The relative lipid content decreased within days 3 to 20 by -22%. Thus, the method allows not only the monitoring of material characteristics at a specific tissue age but also the kinetics of tissue maturation as well. The established reference Raman database will serve as sensitive tool to diagnose disturbances in material characteristics of pediatric bone biopsy samples.

Vibrational spectroscopic imaging for the evaluation of matrix and mineral chemistry.

Metabolic bone diseases manifesting fragility fractures (such as osteoporosis) are routinely diagnosed based on bone mineral density (BMD) measurements, and the effect of various therapies also evaluated based on the same outcome. Although useful, it is well recognized that this metric does not fully account for either fracture incidence or the effect of various therapies on fracture incidence, thus, the emergence of bone quality as a contributing factor in the determination of bone strength. Infrared and Raman vibrational spectroscopic techniques are particularly well-suited for the determination of bone quality as they provide quantitative and qualitative information of the mineral and organic matrix bone components, simultaneously. Through the use of microspectroscopic techniques, this information is available in a spatially resolved manner, thus, the outcomes may be easily correlated with outcomes from techniques such as histology, histomorphometry, and nanoindentation, linking metabolic status with material properties.

Bone quality of the newest bone formed after two years of teriparatide therapy in patients who were previously treatment-naïve or on long-term alendronate therapy.

UNLABELLED: The results of the present study, involving analysis of biopsies from patients who received teriparatide for 2 years and were previously either treatment-naïve or on long-term alendronate therapy, suggest that prior alendronate use does not blunt the favorable effects of teriparatide on bone quality. INTRODUCTION: Examine the effect of 2 years of teriparatide (TPTD) treatment on mineral and organic matrix properties of the newest formed bone in patients who were previously treatment-naïve (TN) or on long-term alendronate (ALN) therapy. METHODS: Raman and Fourier transform infrared microspectroscopic analyses were used to determine the mineral/matrix (M/M) ratio, the relative proteoglycan (PG) content, and the mineral maturity/crystallinity (MMC; determined by three methods: carbonate content, full width at half height of the v 1 PO4 band [FWHH], and wavelength at maxima of the v 1 PO4 band), as well as collagen maturity (ratio of pyridinoline/divalent cross-links), in paired iliac crest biopsies at trabecular, endosteal, and osteonal surfaces of newly formed bone in postmenopausal osteoporotic women who were previously either TN (n = 16) or receiving long-term ALN treatment (n = 24). RESULTS: Trabecular M/M ratio increased and matrix content decreased significantly in the ALN pretreated group. Collagen maturity decreased in both patient groups. Endosteal M/M ratio increased significantly in the TN group. Trabecular M/M ratio was higher at endpoint in the ALN pretreated group than in the TN group. Overall, no changes from baseline were observed in PG content, except that PG content was higher in the ALN pretreated group than in the TN group at endosteal surfaces at endpoint. The ability of TPTD treatment to reduce MMC in both patient groups and at the different bone surfaces depended on the measurement tool (relative carbonate content or wavelength at maxima of the v 1 PO4 band). None of the changes in MMC were different between the two patient groups. CONCLUSIONS: The results suggest some favorable impact of TPTD on bone mineral and organic matrix properties of in situ forming bone in terms of increased initial mineralization and decreased MMC and collagen maturity. Moreover, prior long-term ALN administration may have only limited influence on these properties in bone newly formed after 2 years of TPTD treatment.

Effects of 3 years treatment with once-yearly zoledronic acid on the kinetics of bone matrix maturation in osteoporotic patients.

UNLABELLED: Once-yearly administration of intravenous zoledronic acid for 3 years in humans affects the kinetics of matrix filling in by mineral, independent of bone turnover. INTRODUCTION: Yearly 5-mg infusions of zoledronic acid (ZOL) for 3 years have shown pronounced antifracture efficacy. The purpose of the present study was to test whether ZOL affects the kinetics of forming bone material properties maturation. METHODS: Iliac crest biopsies from the HORIZON-PFT clinical trial were analyzed by Raman microspectroscopy in actively bone-forming surfaces as a function of tissue age in trabecular and osteonal bone, to determine ZOL's effect on bone material quality indices maturation kinetics. RESULTS: Mineral/matrix ratio increased in both groups as a function of tissue age, at both osteonal- and trabecular-forming surfaces; ZOL exhibiting the greatest increase in the trabecular surfaces only. The proteoglycan content showed a dependency on tissue age in both trabecular and osteonal surfaces, with ZOL exhibiting lower values in the tissue age 8-22 days in the trabecular surfaces. Mineral crystallinity (crystallite length and thickness) showed a dependence on tissue age, with ZOL exhibiting lower crystallite length compared with placebo only in the 8- to 22-day-old tissue at trabecular surfaces, while crystal thickness was lower in the 1- to 5-day-old tissue at both osteonal and trabecular surfaces. CONCLUSIONS: The results of the present study suggest that once-yearly administration of intravenous ZOL for 3 years in humans does not exert any adverse effects on the evolution of bone material properties at actively forming osteonal and trabecular surfaces, while it may have a beneficial effect on the progression of the mineral-to-matrix ratio and mineral maturity bone quality indices.

Lathyrism-induced alterations in collagen cross-links influence the mechanical properties of bone material without affecting the mineral.

In the present study a rat animal model of lathyrism was employed to decipher whether anatomically confined alterations in collagen cross-links are sufficient to influence the mechanical properties of whole bone. Animal experiments were performed under an ethics committee approved protocol. Sixty-four female (47 day old) rats of equivalent weights were divided into four groups (16 per group): Controls were fed a semi-synthetic diet containing 0.6% calcium and 0.6% phosphorus for 2 or 4 weeks and ß-APN treated animals were fed additionally with ß-aminopropionitrile (0.1% dry weight). At the end of this period the rats in the four groups were sacrificed, and L2-L6 vertebra were collected. Collagen cross-links were determined by both biochemical and spectroscopic (Fourier transform infrared imaging (FTIRI)) analyses. Mineral content and distribution (BMDD) were determined by quantitative backscattered electron imaging (qBEI), and mineral maturity/crystallinity by FTIRI techniques. Micro-CT was used to describe the architectural properties. Mechanical performance of whole bone as well as of bone matrix material was tested by vertebral compression tests and by nano-indentation, respectively. The data of the present study indicate that ß-APN treatment changed whole vertebra properties compared to non-treated rats, including collagen cross-links pattern, trabecular bone volume to tissue ratio and trabecular thickness, which were all decreased (p<0.05). Further, compression tests revealed a significant negative impact of ß-APN treatment on maximal force to failure and energy to failure, while stiffness was not influenced. Bone mineral density distribution (BMDD) was not altered either. At the material level, ß-APN treated rats exhibited increased Pyd/Divalent cross-link ratios in areas confined to a newly formed bone. Moreover, nano-indentation experiments showed that the E-modulus and hardness were reduced only in newly formed bone areas under the influence of ß-APN, despite a similar mineral content. In conclusion the results emphasize the pivotal role of collagen cross-links in the determination of bone quality and mechanical integrity. However, in this rat animal model of lathyrism, the coupled alterations of tissue structural properties make it difficult to weigh the contribution of the anatomically confined material changes to the overall mechanical performance of whole bone. Interestingly, the collagen cross-link ratio in bone forming areas had the same profile as seen in actively bone forming trabecular surfaces in human iliac crest biopsies of osteoporotic patients.

T3 affects expression of collagen I and collagen cross-linking in bone cell cultures.

Thyroid hormones (T3,T4) have a broad range of effects on bone, however, its role in determining the quality of bone matrix is poorly understood. In-vitro, the immortalized mouse osteoblast-like cell line MC3T3-E1 forms a tissue like structure, consisting of several cell layers, whose formation is affected by T3 significantly. In this culture system, we investigated the effects of T3 on cell multiplication, collagen synthesis, expression of genes related to the collagen cross-linking process and on the formation of cross-links. T3 compared to controls modulated cell multiplication, up-regulated collagen synthesis time and dose dependently, and stimulated protein synthesis. T3 increased mRNA expressions of procollagen-lysine-1,2-oxoglutarate 5-dioxygenase 2 (Plod2) and of lysyloxidase (Lox), both genes involved in post-translational modification of collagen. Moreover, it stimulated mRNA expression of bone morphogenetic protein 1 (Bmp1), the processing enzyme of the lysyloxidase-precursor and of procollagen. An increase in the collagen cross-link-ratio Pyr/deDHLNL indicates, that T3 modulated cross-link maturation in the MC3T3-E1 culture system. These results demonstrate that T3 directly regulates collagen synthesis and collagen cross-linking by up-regulating gene expression of the specific cross-link related enzymes, and underlines the importance of a well-balanced concentration of thyroid hormones for maintenance of bone quality.

Cortical bone composition and orientation as a function of animal and tissue age in mice by Raman spectroscopy.

Important aspects of bone tissue quality include the physicochemical properties of its main constituents, the organic matrix and the mineral crystals. One of the most commonly reported measurements of Raman analysis of bone is the mineral to matrix ratio, obtained from the ratio of the integrated areas of any of the phosphate and amide peaks which depend on both tissue organization and composition. Cube-like samples of normal mouse cortical bone taken from the diaphysis and metaphysis of the femur were investigated within different age groups (2, 4, 8 and 12 weeks) by Raman microspectroscopy. Anatomically identical bone in both longitudinal and transverse directions was analyzed, enabling the discrimination between orientation and composition changes both as a function of animal age, and tissue age within the same animal. The results of the present study indicate that there is a parallel evolution of both orientation and chemical composition as a function of animal age, as well as tissue age within the same specimen. Our tissue age modified ratio of the carbonate to phosphate Raman peaks suggests that the bone mineral crystallite maturity remains relatively constant with animal age. Comparisons of polarized and depolarized experiments in the transversal plane of the diaphysis show a lack of orientation effects as a function of tissue age within the same animal, but exhibit differences as a function of animal age. In the metaphysis, the orientation effect is evident too, albeit less pronounced. This is most likely due to either the age difference between the two tissues within the same specimen in the long bone axis, as metaphyseal bone is generally younger than diaphyseal, or the more random orientation of the tissue collagen itself.