Norbert Hassler

Sclerostin Deficiency Is Linked to Altered Bone Composition

High bone mass in animals and humans with sclerostin deficiency is associated with increased bone strength, which is not the case for all disorders with high bone mineral density, some of which are even associated with fragility fractures owing to unfavorable bone composition. In the current study we investigated whether alterations in bone composition may contribute to the bone strength characteristics associated with lack of sclerostin. We examined cortical bone of Sost‐knockout (KO) mice (n = 9, 16 weeks old) and sclerosteosis patients (young [4 to 14 years], n = 4 and adults [24 and 43 years], n = 2) by quantitative backscattered electron imaging and Raman microspectroscopy and compared it to bone from wild‐type mice and healthy subjects, respectively. In Sost‐KO mice endocortical bone exhibited altered bone composition, whereas subperiosteal bone was unchanged. When comparing endocortical bone tissue of identical tissue age as defined by sequential dual fluorochrome labeling the average bone matrix mineralization was reduced −1.9% (p < 0.0001, younger tissue age) and −1.5% (p < 0.05, older tissue age), and the relative proteoglycan content was significantly increased. Similarly, bone matrix mineralization density distribution was also shifted toward lower matrix mineralization in surgical samples of compact bone of sclerosteosis patients. This was associated with an increase in mineralization heterogeneity in the young population. In addition, and consistently, the relative proteoglycan content was increased. In conclusion, we observed decreased matrix mineralization and increased relative proteoglycan content in bone subcompartments of Sost‐KO mice—a finding that translated into sclerosteosis patients. We hypothesize that the altered bone composition contributes to the increased bone strength of patients with sclerostin deficiency.

Aging Versus Postmenopausal Osteoporosis: Bone Composition and Maturation Kinetics at Actively-Forming Trabecular Surfaces of Female Subjects Aged 1 to 84 Years.

Bone strength depends on the amount of bone, typically expressed as bone mineral density (BMD), determined by dual-energy X-ray absorptiometry (DXA), and on bone quality. Bone quality is a multifactorial entity including bone structural and material compositional properties. The purpose of the present study was to examine whether bone material composition properties at actively-forming trabecular bone surfaces in health are dependent on subject age, and to contrast them with postmenopausal osteoporosis patients. To achieve this, we analyzed by Raman microspectroscopy iliac crest biopsy samples from healthy subjects aged 1.5 to 45.7 years, paired biopsy samples from females before and immediately after menopause aged 46.7 to 53.6 years, and biopsy samples from placebo-treated postmenopausal osteoporotic patients aged 66 to 84 years. The monitored parameters were as follows: the mineral/matrix ratio; the mineral maturity/crystallinity (MMC); nanoporosity; the glycosaminoglycan (GAG) content; the lipid content; and the pyridinoline (Pyd) content. The results indicate that these bone quality parameters in healthy, actively-forming trabecular bone surfaces are dependent on subject age at constant tissue age, suggesting that with advancing age the kinetics of maturation (either accumulation, or posttranslational modifications, or both) change. For most parameters, the extrapolation of models fitted to the individual age dependence of bone in healthy individuals was in rough agreement with their values in postmenopausal osteoporotic patients, except for MMC, lipid, and Pyd content. Among these three, Pyd content showed the greatest deviation between healthy aging and disease, highlighting its potential to be used as a discriminating factor.

Application of special FTIR ATR techniques for quantitative structural analysis of thin surface layers

FTIR ATR spectroscopy is increasingly used for in situ investigations of processes at or near a surface. Particularly when thin layers (biomembranes, monolayers, thin films) are investigated with respect to surface concentration and molecular structure, very sensitive techniques have to be applied in order to achieve an adequate signal-to-noise ratio. This may lead to long measuring times due to extended data accumulation and averaging. However, this can cause new problems with respect to the stability of relevant experimental parameters, such as the sample itself, the spectrometer, and the atmosphere in the spectrometer. In this article we report on two techniques which were developed or improved in our laboratory and successfully applied over past years. Both methods, the so-called single-beam sample reference (SBSR) spectroscopy and the modulation or modulated excitation (ME) spectroscopy, are well suited to compensate instabilities that occur in the course of an experimental series. The SBSR technique converts a single-beam FTIR spectrometer into a pseudo double-beam instrument. By this technique there is always a reference with the same age as the sample available. Moreover, by alternating sample and reference measurements within short time periods, varying environmental conditions such as water vapor concentration in the spectrometer are easily compensated. Moreover SBSR technique enables data evaluation in the conventional single-beam mode (SB) in both the sample (S) and reference (R) channel. This kind of evaluation is important to gain information on the history of S and R. As examples for SBSR and SB applications we report on studies of the interaction of an endotoxin with an immobilized lipid bilayer membrane, as well as on the interaction of TNFa with a TNFa antibody. ME spectroscopy can be applied to systems that show a (pseudo-) reversible response to a periodic excitation. The response of the system measured with time-resolved FTIR spectroscopy is then processed by phase-sensitive detection (PSD). ME spectroscopy is able to determine kinetic constants of a system, allows a hardware separation of overlapping absorption bands, and eliminates all disturbing signal components which do not have the same frequency as the excitation itself. This improves the signal-to-noise ratio dramatically and leads in principal to a stable baseline. The binding of sodium cholate to an adsorbed protein layer of human serum albumin (HSA) is shown as an example that the required sensitivity to study specific molecular interaction is in the μAU range and can be reached by FTIR ME spectroscopy. In a second example, the measurement of structural changes of PLL induced by temperature modulation shows the feasibility of band separation and indicates the possible determination of kinetic properties of a system.

Evidence for a Role for Nanoporosity and Pyridinoline Content in Human Mild Osteogenesis Imperfecta

Osteogenesis imperfecta (OI) is a clinically and genetically heterogeneous connective tissue disorder characterized by bone fragility that arises from decreased bone mass and abnormalities in bone material quality. OI type I represents the milder form of the disease and according to the original Sillence classification is characterized by minimal skeletal deformities and near‐normal stature. Raman microspectroscopy is a vibrational spectroscopic technique that allows the determination of bone material properties in bone biopsy blocks with a spatial resolution of ∼1 µm, as a function of tissue age. In the present study, we used Raman microspectroscopy to evaluate bone material quality in transiliac bone biopsies from children with a mild form of OI, either attributable to collagen haploinsufficiency OI type I (OI‐Quant; n = 11) or aberrant collagen structure (OI‐Qual; n = 5), as a function of tissue age, and compared it against the previously published values established in a cohort of biopsies from healthy children (n = 54, ages 1 to 23 years). The results indicated significant differences in bone material compositional characteristics between OI‐Quant patients and healthy controls, whereas fewer were evident in the OI‐Qual patients. Differences in both subgroups of OI compared with healthy children were evident for nanoporosity, mineral maturity/crystallinity as determined by maxima of the v1PO4 Raman band, and pyridinoline (albeit in different direction) content. These alterations in bone material compositional properties most likely contribute to the bone fragility characterizing this disease.

In situ ATR FTIR monitoring of the formation of functionalized mono- and multilayers on germanium substrate: from 7-octenyltrichlorosilane to 7-carboxylsilane.

The development and optimization of biomimetic surfaces required for biosensors and medical assays are made more efficient by quantitatively monitoring the surface chemical reactions in situ by means of attenuated total reflection (ATR) FTIR spectroscopy. single-beam-sample-reference (SBSR) ATR, as well as modulated excitation (ME), techniques have been applied to get physicochemical information on growth and structure of the surface layer. SBSR and ME methods result in optimum background compensation and signal-to-noise ratio. Surface modification was performed on a germanium multiple internal reflection element (Ge-MIRE). Activation of the surface resulted in free Ge-OH groups used for a spontaneous chemical reaction with 7-octenyltrichlorosilane (7-OTCS) in toluene. Formation of Ge-O-Si bonds was enabled by hydrolization of Si-Cl3 after partial elimination of a tightly bound thin water layer covering the MIRE. Unwanted side-reaction by hydrolization of Si-Cl3 in solution followed by polymerization paralleled this process. Steady growing of the silane layer to multilayer thickness with increasing time was observed in all experiments. Most unexpectedly, in some experiments the end-standing double bond of the silane layer was found to be partly oxidized even after being exposed only to toluene, probably because of catalysis by molecular sieve nanoparticles remaining in toluene after drying. Finally, theoretical means are presented enabling the calculation of the spectrum of dissolved 7-OTCS in toluene, a prerequisite for background compensation during in situ studies of the growing layer.

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.

Real-Time Spectroscopic Analysis of Extracellular Matrix Produced by MC3T3-E1 Preosteoblastic Cells Cultured under Dynamic Conditions

The deposition of extracellular matrix (ECM) produced by osteoblasts is one of the first steps in bone formation. Composition and structure of the ECM influence the development and strength of bone, as well as the onset of its mineralization. Since ECM is secreted onto the surface where the cells attach, Fourier transform infrared (FT-IR) attenuated total reflection (ATR), as a surface sensitive technique, provides a useful tool for its investigation, as ECM instead of the cells is predominantly detected by the IR beam. The purpose of the present study was to develop the FT-IR ATR technique so that real-time measurements of the ECM produced by MC3T3-E1 osteoblasts could be obtained in situ. Measurements were performed using polarized incident IR light to apply a procedure for solvent compensation which reduces the influence of culture medium on the evaluation of the amide I and II bands. The formation of ECM took place in a flow-through chamber under dynamic conditions by applying a constant flow of culture medium and was tracked over a time period of two weeks by evaluation of the integrated absorbance of amide I and II bands reflecting the amount and isotropic arrangement of amide bonds in the ECM. Cultures without ascorbic acid had a reduced protein concentration that enabled the analysis of cell-mediated matrix accumulation. Presence and proliferation of cells after two weeks of permanent flow-through of culture medium was shown by cell counting exhibiting a 67% increase in cell number as well as by crystal violet and live/dead staining. These results demonstrate the application of FT-IR ATR spectroscopy for monitoring matrix formation.