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.

The contribution of the pericanalicular matrix to mineral content in human osteonal bone.

The osteocyte lacunar-canalicular network (LCN) penetrates bone and houses the osteocytes and their processes. Despite its rather low volume fraction, the LCN represents an outstanding large surface that is possibly used by the osteocytes to interact with the surrounding mineralized bone matrix thereby contributing to mineral homeostasis. The aim of this study was to quantitatively describe such contributions by spatially correlating the local density of the LCN with the mineral content at the same location in micrometer-sized volume elements in human osteons. For this purpose, 65 osteons from the femur midshaft from healthy adults (n = 4) and children (n = 2) were structurally characterized with two different techniques. The 3D structure of the LCN in the osteons was imaged with confocal laser scanning microscopy after staining the bone samples with rhodamine. Subsequent image analysis provided the canalicular length density, i.e. the total length of the canaliculi per unit volume (µm/µm3). Quantitative information on the mineral content (wt%Ca) from the identical regions was obtained using quantitative backscattered electron imaging. As the LCN-porosity lowers the mineral content, a negative correlation between Ca content and network density was expected. Calculations predict a reduction of around -0.97 fmol Ca per µm of network. However, the experiment revealed for 62 out of 65 osteons a positive correlation resulting in an average additional Ca loading of +1.15 fmol per µm of canalicular network, i.e. an accumulation of mineral has occurred at dense network regions. We hypothesize that this accumulation happens in the close vicinity of canaliculi forming mineral reservoirs that can be utilized by osteocytes. Significant differences found between individuals indicate that the extent of mineral loading of the reservoir zone reflects an important parameter for mineral homeostasis.

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.

Cortical bone properties in the Brtl/+ mouse model of Osteogenesis imperfecta as evidenced by acoustic transmission microscopy.

Higher skeletal fragility has been established for the Brtl/+ mouse model of osteogenesis imperfecta at the whole bone level, but previous investigations of mechanical properties at the bone material level were inconclusive. Bone material was analyzed separately at endosteal (ER) and periosteal regions (PR) on transverse femoral midshaft sections for 2-month old mice (wild-type n = 6; Brtl/+ n = 6). Quantitative backscattered electron imaging revealed that the mass density computed from mineral density maps was higher in PR than in ER for both wild-type (+2.1%, p < 0.05) and Brtl/+ mice (+1.8%, p < 0.05). Electron induced X-ray fluorescence analysis indicated significantly lower atomic Ca/P ratios and higher Na/Ca, Mg/Ca and K/Ca ratios in PR bone compared to ER independently of genotype. Second harmonic generation microscopy indicated that the occurrence of periodically alternating collagen orientation in ER of Brtl/+ mice was strongly reduced compared to wild-type mice. Scanning acoustic microscopy in time of flight mode revealed that the sound velocity and Young's modulus (estimated based on sound velocity and mass density maps) were significantly greater in PR (respectively +6% and +15%) compared to ER in wild-type mice but not in Brtl/+ mice. ER sound velocity and Young's modulus were significantly increased in Brtl/+ mice (+9.4% and +22%, respectively) compared to wild-type mice. These data demonstrate that the Col1a1 G349C mutation in Brtl/+ mice affects the mechanical behavior of bone material predominantly in the endosteal region by altering the collagen orientation.

Novel familial mutation of LRP5 causing high bone mass: Genetic analysis, clinical presentation, and characterization of bone matrix mineralization.

The Wnt signalling pathway is a critical regulator of bone mass and quality. Several heterozygous mutations in the LRP5 gene, a Wnt co-receptor, causing high bone mass (LRP5-HBM) have been described to date. The pathogenic mechanism is thought to be a gain-of-function caused by impaired inhibition of the canonical Wnt signalling pathway, thereby leading to increased bone formation. We report the cases of two affected family members, a 53-year-old mother and her 23-year-old daughter, with high bone mass (T-scores mother: lumbar spine 11.4, femoral neck 10.5; T-scores daughter: lumbar spine 5.4, femoral neck 8.7), increased calvarial thickness, and thickened cortices of the long bones but no history of fractures. Whereas the mother did not show any indications of the mutation, the daughter suffered from congenital hearing impairment resulting in cochlear implantation, recurrent facial palsy, and migraine. In addition, she had stenosis of the foramen magnum. In both individuals, we detected a novel heterozygous duplication of six basepairs in the LRP5 gene, resulting in an insertion of two amino acids, very likely associated with a gain-of-function. When the daughter had part of the occipital bone surgically removed, the bone sample was used for the visualization of bone lamellar structure and bone cells as well as the measurement of bone mineralization density distribution (BMDD). The bone sample revealed two distinctly different regions: an intra-cortical region with osteonal remodeling, typical osteonal lamellar orientation, associated with relatively higher heterogeneity of bone matrix mineralization, and another periosteal region devoid of bone remodeling, with parallel bone lamellae and lower heterogeneity of mineralization. In conclusion, we present data on bone tissue and material level from an LRP5-HBM patient with a novel mutation in the LRP5 gene. Our findings indicate normal morphology of osteoclasts and osteoblasts as well as normal mineralization in skull bone in LRP5-HBM.

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.

Use of proton pump inhibitors and mortality after hip fracture in a nationwide study.

We analyzed the association of proton pump inhibitors (PPIs) with mortality after osteoporosis-related hip fracture in Austria. PPIs were associated with reduced 90-day mortality but elevated mortality after half a year when initiated pre-fracture. Inpatients and discharged patients on PPIs showed lowered in-hospital and 90-day mortality, respectively. INTRODUCTION: We herein investigated use of proton pump inhibitors (PPIs) and mortality among hip fracture patients in a nationwide study in Austria. METHODS: In this retrospective cohort study, data on use of PPIs were obtained from 31,668 Austrian patients ≥50 years with a hip fracture between July 2008 and December 2010. All-cause mortality in patients without anti-osteoporotic drug treatment who had received their first recorded PPI prescription in the study period either before or after fracture was compared with hip fracture patients on neither PPIs nor anti-osteoporotic medication using logistic and Cox regression analysis. RESULTS: With PPI use, 90-day mortality was significantly reduced, both at initiation before (OR 0.66; p < 0.0001) and after hip fracture (OR 0.23; p < 0.0001). 90-day mortality was also reduced when PPIs were prescribed not until after discharge from the last recorded hip fracture-related hospital stay (OR 0.49; p < 0.0001) except for patients aged <70 years. In a sub-cohort of patients beginning PPIs during hospital stay, in-hospital mortality (0.2%) was substantially reduced relative to matched control patients (3.5%) (p < 0.0001). Longer-term mortality significantly increased after half a year post-fracture only among those who started PPI prescription before fracture. CONCLUSIONS: PPI use during and after hospital stay due to hip fracture is associated with a considerable decrease in mortality. These findings could have implications for hip fracture treatment.

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.

Synchrotron radiation micro X-ray fluorescence spectroscopy of thin structures in bone samples: comparison of confocal and color X-ray camera setups.

In the quest for finding the ideal synchrotron-radiation-induced imaging method for the investigation of trace element distributions in human bone samples, experiments were performed using both a scanning confocal synchrotron radiation micro X-ray fluorescence (SR-µXRF) (FLUO beamline at ANKA) setup and a full-field color X-ray camera (BAMline at BESSY-II) setup. As zinc is a trace element of special interest in bone, the setups were optimized for its detection. The setups were compared with respect to count rate, required measurement time and spatial resolution. It was demonstrated that the ideal method depends on the element of interest. Although for Ca (a major constituent of the bone with a low energy of 3.69 keV for its Kα XRF line) the color X-ray camera provided a higher resolution in the plane, for Zn (a trace element in bone) only the confocal SR-µXRF setup was able to sufficiently image the distribution.

Antiresorptive therapy and risk of mortality and refracture in osteoporosis-related hip fracture: a nationwide study.

UNLABELLED: We analyzed the association of bisphosphonate therapy with mortality and hip refracture incidence among osteoporosis-related hip fracture patients in Austria. Mortality was lower in primarily female bisphosphonate users, while hip refracture incidence was generally elevated relative to controls, indicating beneficial effects of bisphosphonates other than on bone. INTRODUCTION: The purpose of this study was to analyze mortality and hip refracture risk in osteoporotic hip fracture patients with and without antiosteoporotic medication. METHODS: We retrospectively analyzed data on 31,668 Austrian patients ≥50 years with a hip fracture between July 2008 and December 2010 for antiosteoporotic drug treatment with respect to outcome parameters all-cause mortality, hip refracture incidence, and hip refracture-free days. Outcomes when bisphosphonate (BP) treatment was begun before or after fracture were compared with an age- and sex-matched hip fracture control without antiosteoporotic medication. RESULTS: 27.69 % of patients (33.01 % of women, 13.13 % of men) were prescribed antiosteoporotic medication, primarily BPs. Females having initiated BP treatment before first fracture had lower odds for mortality 1 and 3 year(s) post-fracture, whereas hip refracture incidence under pre-fracture BP initiation was generally higher. Treatment that was started after fracture, however, entailed significantly lower mortality hazards for both genders (HR 0.43, 95 %CI 0.36-0.52, p < 0.0001 after 1 year) but significantly higher hip refracture incidence except for patients aged 50-69 years and more hip refracture free days for females. Hip refractures overall amounted to 29.22/1000 patient years differing significantly between women and men (31.03 vs. 23.89, respectively, p < 0.0001), and longer hip refracture free survival was observed for women than for men (499 vs. 466 median days, respectively, p < 0.0001). CONCLUSIONS: Although BP use is associated with reduced mortality after hip fracture, notably among women, hip refracture incidences are likewise elevated, which is most likely accounted for by a high probability of BP prescription to more comorbid patients suffering from more severe osteoporosis. Concomitantly, through possible effects other than on bone, BPs might be able to curtail mortality. Male hip fracture patients' low treatment frequency in particular reflects underdiagnosis and undertreatment of osteoporosis in Austria.

Bone matrix mineralization is preserved during early perimenopausal stage in healthy women: a paired biopsy study.

UNLABELLED: Bone matrix mineralization based on quantitative backscatter electron imaging remained unchanged during the first year of menopause in paired transiliac biopsy samples from healthy women. This suggests that the reported early perimenopausal reductions in bone mineral density are caused by factors other than decreases in the degree of mineralization. INTRODUCTION: It is unknown whether perimenopausal loss of bone mass is associated with a drop in bone matrix mineralization. METHODS: For this purpose, we measured the bone mineralization density distribution (BMDD) by quantitative backscatter electron imaging (qBEI) in n = 17 paired transiliac bone biopsy samples at premenopausal baseline and 12 months after last menses (obtained at average ages of 49 ± 2 and 55 ± 2 years, respectively) in healthy women. For interpretation of BMDD outcomes, previously measured bone mineral density (BMD) and biochemical and histomorphometric markers of bone turnover were revisited for the present biopsy cohort. RESULTS: Menopause significantly decreased BMD at the lumbar spine (-4.5 %) and femoral neck (-3.8 %), increased the fasting urinary hydroxyproline/creatinine ratio (+60 %, all p < 0.01) and histomorphometric bone formation rate (+25 %, p < 0.05), but affected neither cancellous nor cortical BMDD variables (paired comparison p > 0.05). Mean calcium concentrations of cancellous (Cn.CaMean) and cortical bone (Ct.CaMean) were within normal range (p > 0.05 compared to established reference data). Ct.CaMean was significantly correlated with Cn.CaMean before (R = 0.81, p < 0.001) and after menopause (R = 0.80, p < 0.001) and to cortical porosity of mineralized tissue (Ct.Po.) after menopause (R = -0.57, p = 0.02). CONCLUSIONS: Surprisingly, the BMDD was found not affected by the changes in bone turnover rates in this cohort. This suggests that the substantial increase in bone formation rates took place shortly before the second biopsy, and the bone mineralization changes lag behind. We conclude that during the first year after the last menses, the degree of bone matrix mineralization is preserved and does not contribute to the observed reductions in BMD.

Subtle changes in bone mineralization density distribution in most severely affected patients with chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is associated with low aBMD as measured by DXA and altered microstructure as assessed by bone histomorphometry and microcomputed tomography. Knowledge of bone matrix mineralization is lacking in COPD. Using quantitative backscatter electron imaging (qBEI), we assessed cancellous (Cn.) and cortical (Ct.) bone mineralization density distribution (BMDD) in 19 postmenopausal women (62.1 ± 7.3 years of age) with COPD. Eight had sustained fragility fractures, and 13 had received treatment with inhaled glucocorticoids. The BMDD outcomes from the patients were compared with healthy reference data and were correlated with previous clinical and histomorphometric findings. In general, the BMDD outcomes for the patients were not significantly different from the reference data. Neither the subgroups of with or without fragility fractures or of who did or did not receive inhaled glucocorticoid treatment, showed differences in BMDD. However, subgroup comparison according to severity revealed 10% decreased cancellous mineralization heterogeneity (Cn.CaWidth) for the most severely affected compared with less affected patients (p=0.042) and compared with healthy premenopausal controls (p=0.021). BMDD parameters were highly correlated with histomorphometric cancellous bone volume (BV/TV) and formation indices: mean degree of mineralization (Cn.CaMean) versus BV/TV (r=0.58, p=0.009), and Cn.CaMean and Ct.CaMean versus bone formation rate (BFR/BS) (r=-0.71, p<0.001). In particular, those with lower BV/TV (<50th percentile) had significantly lower Cn.CaMean (p=0.037) and higher Cn.CaLow (p=0.020) compared with those with higher (>50th percentile) BV/TV. The normality in most of the BMDD parameters and bone formation rates as well as the significant correlations between them suggests unaffected mineralization processes in COPD. Our findings also indicate no significant negative effect of treatment with inhaled glucocorticoids on the bone mineralization pattern. However, the observed concomitant occurrence of relatively lower bone volumes with lower bone matrix mineralization will both contribute to the reduced aBMD in some patients with COPD.

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.

Relationship of bone mineralization density distribution (BMDD) in cortical and cancellous bone within the iliac crest of healthy premenopausal women.

Bone mineralization density distribution (BMDD) is an important determinant of bone mechanical properties. The most available skeletal site for access to the BMDD is the iliac crest. Compared to cancellous bone much less information on BMDD is available for cortical bone. Hence, we analyzed complete transiliac crest bone biopsy samples from premenopausal women (n = 73) aged 25-48 years, clinically classified as healthy, by quantitative backscattered electron imaging for cortical (Ct.) and cancellous (Cn.) BMDD. The Ct.BMDD was characterized by the arithmetic mean of the BMDD of the cortical plates. We found correlations between Ct. and Cn. BMDD variables with correlation coefficients r between 0.42 and 0.73 (all p < 0.001). Additionally to this synchronous behavior of cortical and cancellous compartments, we found that the heterogeneity of mineralization densities (Ct.Ca(Width)), as well as the cortical porosity (Ct.Po) was larger for a lower average degree of mineralization (Ct.Ca(Mean)). Moreover, Ct.Po correlated negatively with the percentage of highly mineralized bone areas (Ct.Ca(High)) and positively with the percentage of lowly mineralized bone areas (Ct.Ca(Low)). In conclusion, the correlation of cortical with cancellous BMDD in the iliac crest of the study cohort suggests coordinated regulation of bone turnover between both bone compartments. Only in a few cases, there was a difference in the degree of mineralization of >1wt % between both cortices suggesting a possible modeling situation. This normative dataset of healthy premenopausal women will provide a reference standard by which disease- and treatment-specific effects can be assessed at the level of cortical bone BMDD.

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.