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Biomedical subjects

P Fratzl

Publications and source records attributed to P Fratzl.

At least 37 records · Page 2Linked to original sources
PubMed2001

Alendronate increases degree and uniformity of mineralization in cancellous bone and decreases the porosity in cortical bone of osteoporotic women.

The strength of bone is correlated with bone mass but is also influenced significantly by other factors such as structural properties of the matrix (e.g., collagen mutations) and the mineral. Changes at all levels of this organization could contribute to fracture risk. We investigated the effects of alendronate (Aln) treatment on the density of mineralization and the ultrastructure of the mineral/collagen composite, size and habitus of mineral particles in iliac cancellous bone, as well as on the porosity of iliac cortical bone from postmenopausal osteoporotic women. Twenty-four transiliac bone biopsies from Phase III Aln (10 mg/day) trials (placebo and Aln after 2 and 3 years of treatment, n = 6 per group) were studied. The mineral structure was investigated by quantitative backscattered electron imaging (qBEI) and by scanning small-angle X-ray scattering (scanning-SAXS). qBEI histograms reflect the bone mineralization density distribution (BMDD), whereas SAXS patterns characterize the size and arrangement of the mineral particles in bone. We found that: (i) the relative calcium content of osteoporotic bone was significantly lower than that of data-base controls; (ii) mineralization was significantly higher and more uniform after Aln treatment; (iii) size and habitus of the mineral particles was not different between placebo and Aln-treated groups; and (iv) the porosity of cortical bone was reduced significantly by Aln treatment. We conclude that Aln treatment increases the degree and uniformity of bone matrix mineralization without affecting the size and habitus of the mineral crystals. It also decreases the porosity of the corticalis. Together these effects may contribute to the observed reduction in fractures.

Alendronate↗
PubMed2001

Age- and genotype-dependence of bone material properties in the osteogenesis imperfecta murine model (oim).

Cortical mineralization of long bones was studied in collagen alpha2(I)-deficient mice (oim) used as a model for human osteogenesis imperfecta. Aspects of the age development of the mice were characterized by combining nanometer- to micrometer-scale structural analysis with microhardness measurements. Bone structure was determined from homozygous (oim/oim) and heterozygous (oim/+) mice and their normal (+/+) littermates as a function of animal age by small-angle X-ray scattering (SAXS) and quantitative backscattered electron imaging (qBEI) measurements. SAXS studies found anomalies in the size and arrangement of bone mineral crystals in both homozygous and heterozygous mice aged 1-14 months. Generally, the crystals were smaller in thickness and less well aligned in these mice compared with control animals. An increase in the mean crystal thickness of the bone was found within all three genotypes up to an age of 3 months. Vicker's hardness measurements were significantly enhanced for oim bone (homozygotes and heterozygotes) compared with controls. The microhardness values were correlated directly with increased mineral content of homozygous and heterozygous compared with control bone, as determined by qBEI analysis. There was also a significant increase of mineral content with age. Two possibilities for collagen-mineral association are discussed for explaining the increased hardness and mineral content of oim/oim bone, together with its decreased toughness and thinner mineral crystals. As a consequence of the present measurements, one model for oim bone could incorporate small and densely packed mineral crystals. A second model for possible collagen-mineral association in oim material would consist of two families of mineral crystals, one being smaller and the other being much larger than the crystals found in normal mouse long bones.

Age Factors↗
PubMed2000

Mineralized collagen fibrils: a mechanical model with a staggered arrangement of mineral particles.

Both elastic modulus and fracture stress are known to increase with the amount of mineral deposited within collagen fibrils. Current mechanical models of mineralized fibrils, where mineral platelets are arranged in parallel arrays, reproduce the first effect but fail to predict an increase in fracture stress. Here, we propose a model with a staggered array of platelets that is in better agreement with results on molecular packing in collagen fibrils and that accounts for an increase of both elastic modulus and fracture stress with the amount of mineral in the fibril. Finally, we explore the dependence of the mechanical properties within the model, when the degree of mineralization and the thickness of the platelets as well as their distance varies.

Animals↗
PubMed2000

Analysis of the hierarchical structure of biological tissues by scanning X-ray scattering using a micro-beam.

The outstanding mechanical properties of biological tissues such as wood or bone are mainly due to their hierarchical structure and to their optimization at all levels of hierarchy. It is therefore essential to characterize the structure at all levels to understand the complex behavior of such tissues. Structures down to the micrometer level are accessible to light or scanning electron microscopic observation. In the case of bone this includes, for example, morphometry of the trabecular architecture or the bone mineral density distribution in cortical and trabecular bone. To characterize the sub-micrometer structure of, e.g., the collagen-mineral composite in the case of bone or the cellulose microfibrils in the case of wood, other methods, such as transmission electron microscopy or X-ray scattering are necessary. The recent availability of extremely brilliant synchrotron X-ray sources has led to the development of the new techniques of scanning small-angle X-ray scattering and scanning X-ray microdiffraction, which are capable of providing structural information on the micrometer and the nanometer level, simultaneously. As a basic principle of the method the specimen is scanned across an X-ray beam which has a diameter of few micrometers. Measuring the X-ray absorption at each position provides an image of the specimen (microradiography) with resolution similar to light microscopy, in the micrometer range. Moreover, the X-ray scattering pattern is analyzed at each specimen position to provide parameters characterizing the structure in the nanometer range. The present paper reviews the principles of the techniques and demonstrates their application to biological materials, such as wood or bone.

Biomechanical Phenomena↗
PubMed1999

Variation of cellulose microfibril angles in softwoods and hardwoods-a possible strategy of mechanical optimization.

Position-resolved small-angle X-ray scattering was used to investigate the nanostructure of the wood cell wall in two softwood species (Norwegian spruce and Scots pine) and two hardwood species (pedunculate oak and copper beech). The tilt angle of the cellulose fibrils in the wood cell wall versus the longitudinal cell axis (microfibril angle) was systematically studied over a wide range of annual rings in each tree. The measured angles were correlated with the distance from the pith and the results were compared. The microfibril angle was found to decrease from pith to bark in all four trees, but was generally higher in the softwood than in the hardwood. In Norwegian spruce, the microfibril angles were higher in late wood than in early wood; in Scots pine the opposite was observed. In pedunculate oak and copper beech, low angles were found in the major part of the stem, except for the very first annual rings in pedunculate oak. The results are interpreted in terms of mechanical optimization. An attempt was made to give a quantitative estimation for the mechanical constraints imposed on a tree of given dimensions and to establish a model that could explain the general decrease of microfibril angles from pith to bark.

Cell Wall↗
PubMed1999

Scanning small angle X-ray scattering analysis of human bone sections.

Scanning small-angle X-ray scattering (scanning SAXS) was applied for the first time on bone to compare results from SAXS directly with those from other position-sensitive methods, such as light and polarized light microscopy, back-scattered electron imaging, and radiographic imaging. Since scanning SAXS is a nondestructive method of investigation, images from all these techniques could be obtained from the same bone sections. Thus, it could be shown that both the collagen and the mineral crystals were predominantly aligned parallel to the trabeculae and, therefore, to principle stress directions. Moreover, the mean crystal thickness as determined by scanning SAXS was found to be different at various positions inside the trabecular and cortical structure. Finally, it could be shown that scanning SAXS is suitable for detecting local changes in bone material, e.g., due to fluoride treatment.

Adolescent↗
PubMed1999

Complementary information on bone ultrastructure from scanning small angle X-ray scattering and Fourier-transform infrared microspectroscopy.

Scanning small angle X-ray scattering (scanning SAXS) and Fourier-transform infrared microspectroscopy (FT-IRM) have previously been utilized independently to characterize the structural properties of bone in an anatomical position-resolved fashion. Whereas SAXS provides a direct measure of the physical characteristics of apatitic crystals, FT-IRM assesses structure of both mineral and organic matrix at the molecular level. In the present study both methods were applied to examine the same developing bone tissue from the L-4 vertebra of a 14-month-old (accidental death). A 200-microm-thick section was processed for examination by scanning electron microscopy and SAXS. Spectra were collected at 200 microm spatial resolution at specific locations in cortical and cancellous bone. Parameters determined included total SAXS intensity, crystal thickness (T), and degree and direction of predominant crystal orientation. For FT-IRM analysis, a section 4 microm thick was cut longitudinally from the top of the sample. Spectra of regions 100 x 100 microm2 were acquired from the same locations as the SAXS spectra. Integrated areas of the phosphate nu(1,3) collagen amide I, and carbonate nu2 absorbances, were calculated to obtain mineral: matrix and carbonate:mineral ratios. The relative quantities of types A, B, and labile carbonate (substituted for apatite hydroxyl, phosphate, and surface positions, respectively) were also evaluated. Polarized FT-IRM data were collected to determine molecular orientation of the apatite and collagen components. The results of this study show that the information obtained from the two techniques is complementary. Both SAXS and FT-IRM data revealed that the crystals were significantly larger in the cancellous region compared with the cortical region, that mineralization was greater in the cortex, and that the crystals were oriented to a larger degree in the cancellous compared with the cortical bone. The scanning SAXS measure of crystal thickness was significantly correlated to the FT-IRM measures of crystallinity, type A carbonate substitution, and crystal orientation. In conclusion, it was found that the combined use of SAXS and FT-IRM provides valuable, unique information on structural changes in bone at both the microstructural and ultrastructural level. Although each method can be used individually, the combination of techniques provides additional insights into the mechanism of bone crystal maturation.

Apatites↗
PubMed1998

Fibrillar structure and mechanical properties of collagen.

Collagen type I is among the most important stress-carrying protein structures in mammals. Despite their importance for the outstanding mechanical properties of this tissue, there is still a lack of understanding of the processes that lead to the specific shape of the stress-strain curve of collagen. Recent in situ synchrotron X-ray scattering experiments suggest that several different processes could dominate depending on the amount of strain. While at small strains there is a straightening of kinks in the collagen structure, first at the fibrillar then at the molecular level, higher strains lead to molecular gliding within the fibrils and ultimately to a disruption of the fibril structure. Moreover, it was observed that the strain within collagen fibrils is always considerably smaller than in the whole tendon. This phenomenon is still very poorly understood but points toward the existence of additional gliding processes occurring at the interfibrillar level.

Animals↗
PubMed1998

Validation of quantitative backscattered electron imaging for the measurement of mineral density distribution in human bone biopsies.

The measurement of bone mineral density (BMD) using X-rays is usually employed to monitor the mineral content in a given portion of bone. However, this method cannot differentiate between changes in bone volume or in degree of mineralization of the bone matrix. In contrast to BMD, bone mineral density distribution (BMDD), as measured on bone sections by quantitative backscattered electron imaging (qBEI), is able to distinguish differences in the degree of mineralization. For routine clinical research, we have validated the method of calibration and standardization of the backscattered electron (BE) signal. Carbon and aluminum were used as reference materials for BE gray levels and osteoid and apatite for calcium concentration. Experiments were performed to get knowledge about precision (intraassay variance-instrumental stability and interassay variance-reproducibility) and accuracy (standardization) of this method as well as the biological variance (intraindividual and interindividual) in human bone. On transiliac biopsies or necropsies from 20 individuals having had accidental death (13 females, 7 males, age 30-85 years) BMDD measurements were conducted. The patients' medical history as well as the histomorphology of these bones showed no evidence of metabolic bone disease. For instance, the standard deviations of the weighted mean calcium concentrations were <0.3%, <0.4%, <0.9%, and <2.6% of the mean for the intraassay, interassay, intraindividual, and interindividual variations, respectively. In addition, a mean BMDD histogram for transiliac bone specimens was calculated from the 20 aforementioned individuals. The method used allows detection of the degree of mineralization independently from the actual bone volume, a result that seems to be of special interest in the assessment of the effect of treatments for osteoporosis. The power of this technique is demonstrated by using bone from a patient with a metabolic bone disease. In this case of osteomalacia due to celiac disease, the mean calcium concentration in the bone matrix was reduced by 19.3% as compared with normal.

Adult↗
PubMed1998

Matrix mineralization in MC3T3-E1 cell cultures initiated by beta-glycerophosphate pulse.

MC3T3-E1 cells, grown in the presence of serum and ascorbate, express alkaline phosphatase and produce an extensive collagenous extracellular matrix that can be mineralized by the addition of beta-glycerophosphate (beta-GP). In the present work, we study the influence of concentration and duration of beta-GP treatment on the mineralization pattern in 4-week-old cell cultures. Amount and structure of mineral deposition were monitored by von Kossa staining, light, and electron microscopy, as well as small-angle X-ray scattering (SAXS) of unstained specimens. SAXS measures the total surface of the mineral phase and is therefore preferentially sensitive to very small crystals (typically <50 nm). It was used to determine the ratio (M) of small crystals to collagen matrix. A variety of mineralization patterns was observed to occur simultaneously, some associated with collagen within nodules or in deeper layers of the cultures and some independent of it. At a beta-GP concentration of 10 mmol, mineralization was initiated after about 24 h and continued to increase, irrespective of whether the high level of beta-GP was maintained or reduced to 2 mmol. With shorter pulses (<24 h), no significant mineralization was observed in the week following beta-GP pulse. With continuous treatment at 5 mmol beta-GP, the first signs of mineralization were detected 14 days after the beginning of treatment in the 4-week-old cultures, but no mineralization at all occurred at lower beta-GP concentrations. When cells were grown without ascorbic acid for 4 weeks, only two cell layers without collagen matrix were found. In these cultures, no mineralization detectable by SAXS could be induced with beta-GP. These data indicate that, in viable cells, high doses of beta-GP are essential for the nucleation of mineral crystals, but not for the progression of mineralization once crystals had been nucleated. In contrast, when 4-week-old cell cultures were devitalized, M was found to increase immediately, even at 2 mmol beta-GP. These results suggest that, in MC3T3-E1 cell cultures, cell viability is essential for prevention of spontaneous mineralization of the extracellular matrix.

3T3 Cells↗
PubMed1998

Collagen fibrils in the human corneal stroma: structure and aging.

PURPOSE: Transparency and biomechanical properties of the cornea depend on the structure and organization of collagen fibrils. The authors determined diameter, axial period, and lateral molecular spacing of collagen fibrils in human corneal stroma as a function of age. METHODS: Seventeen normal human corneas were investigated in their native state by means of small-angle and wide-angle x-ray scattering. RESULTS: The mean radius of collagen fibrils, the axial period of collagen fibrils, and the lateral intermolecular Bragg spacing were found to be age dependent. The authors determined fibril radii of 16.1 +/- 0.5 nm in persons older than 65 years of age (n = 10) and 15.4 +/- 0.5 nm (mean +/- SD) in persons younger than 65 years (n = 7) (P < 0.022). The related age-dependent values were 66.4 +/- 0.7 nm (> 65 years) and 65.2 +/- 0.8 nm (< 65 years) for the axial period (P < 0.006) and 1.515 +/- 0.010 nm (> 65 years) and 1.499 +/- 0.013 nm (< 65 years) for the intermolecular Bragg spacing (P < 0.022). CONCLUSIONS: Aging is related to a three-dimensional growth of collagen fibrils in the human corneal stroma. The age-related growth of the fibril diameter was mostly a result of an increased number of collagen molecules and, in addition, to some expansion of the intermolecular Bragg spacing probably resulting from glycation-induced cross-linking. The observed expansion of the fibrils in an axial direction may result from reduction of the molecular tilting angle within collagen fibrils. The observed alterations of the collagen framework may have implications for refractive surgery and ocular tonometry achieved through related changes in the biomechanical properties of the cornea.

Aged↗
PubMed1997

Collagen from the osteogenesis imperfecta mouse model (oim) shows reduced resistance against tensile stress.

Osteogenesis imperfecta (OI) is a disease attributable to any of a large number of possible mutations of type I collagen. The disease is clinically characterized in part by highly brittle bone, the cause of this feature being unknown. Recently a mouse model of OI, designated as osteogenesis imperfecta murine (oim), and having a well defined genetic mutation, has been studied and found to contain mineral crystals different in their alignment with respect to collagen and in their size. These observations are consistent with those reported in human OI and the unusual crystal alignment and size undoubtedly contribute to the reduced mechanical properties of OI bone. While the mineral has been investigated, no information is available on the tensile properties of oim collagen. In this study, the mechanical properties of tendon collagen under tension have been examined for homozygous (oim/oim), heterozygous (+/oim), and control (+/+) mice under native wet conditions. The ultimate stress and strain found for oim/oim collagen were only about half the values for control mice. Assuming that prestrained collagen molecules carry most of the tensile load in normal bone while the mineral confers rigidity and compression stability, the reported results suggest that the brittleness of OI bone in the mouse model may be related to a dramatic reduction of the ultimate tensile strain of the collagen.

Analysis of Variance↗
PubMed1997

A new molecular model for collagen elasticity based on synchrotron X-ray scattering evidence.

Collagen is the most abundant structural protein in vertebrates. The specific shape of its stress-strain curve is crucial for the function of a number of organs. Although the macroscopic mechanical behavior of collagen is well known, there is still no explanation of the elastic process at the supramolecular level. We have performed in situ synchrotron x-ray scattering experiments, which show that the amount of lateral molecular order increases upon stretching of collagen fibers. In strain cycling experiments the relation between strain and diffuse equatorial scattering was found to be linear in the "heel" region of the stress-strain curve. A new molecular model for collagen elasticity is proposed, which, based on the existence of thermally activated molecular kinks, reproduces this linearity and gives a simple explanation for the form of the stress-strain curve of collagen.

Animals↗
PubMed1997

Mineralization of cancellous bone after alendronate and sodium fluoride treatment: a quantitative backscattered electron imaging study on minipig ribs.

Fluoride stimulates bone formation, whereas bisphosphonates reduce bone resorption. In clinical trials, both treatments increase bone density, although sodium fluoride (NaF) increases and alendronate (bisphosphonate, ALN) decreases bone turnover. In a comparative study using minipigs an inverse correlation has been reported between bone turnover and elastic modulus. Small-angle X-ray scattering (SAXS) measurements of these bones revealed no structural deterioration of the collagen/mineral composite at the nanometer range for ALN-treated vertebra, whereas a slight increase of the average thickness of the mineral crystals as well as changes of the structure of the collagen/mineral composite were found in the bones of NaF-treated animals. In this study we used quantitative backscattered electron imaging (qBSE) to investigate the cancellous bones from ribs of minipigs treated with vehicle, NaF, or ALN. This method provides information on the local mineral concentration in the micrometer range. Mineralization spectra were obtained from each treatment group, and statistically significant differences between ALN and controls were found for the peak position, the peak height, the peak width, and the average calcium (Ca) concentration of the mineral distribution. The results reveal that the cancellous bone matrix was more uniformly mineralized after ALN treatment. The reduced bone turnover induced by ALN, documented histomorphometrically could be at the origin of this phenomenon. No significant differences were detected between NaF and control. Together with the earlier SAXS data these results may explain in part the increase in bone density and the improvement of biomechanical properties observed after ALN treatment in animals and in osteoporotic patients.

Alendronate↗
PubMed1997

Collagen fibril orientation in the human corneal stroma and its implication in keratoconus.

PURPOSE: The kind and the degree of preferred collagen fibril orientation in normal human corneal stroma were investigated as important qualities of the cornea with respect to its mechanical properties and, hence, to refractive surgery. To determine whether this information is relevant to corneal disease, the authors investigated collagen fibril orientation in several corneas with keratoconus. METHODS: By means of low-angle x-ray scattering, 17 normal human corneas and four corneas of eyes with keratoconus were investigated. RESULTS: Collagen fibrils in the normal human corneal stroma showed two preferred orientations orthogonal to each other. These were the horizontal and the vertical directions. The authors defined a degree of orientation gamma, determined to be gamma = 0.49 +/- 0.10 (mean +/- SD). This means that the excess of the preferentially oriented fibrils in relation to the total number of fibrils was approximately 49%. It follows from this value that approximately two thirds of the fibrils (66%) were within in a 45 degrees sector (+/-22.5 degrees) around the horizontal and vertical meridians, whereas approximately one third (34%) is oriented in the oblique sectors in between. No statistically significant variation of gamma within a central 7 mm zone could be detected in normal corneas. The orthogonal arrangement of the collagen fibrils was, however, profoundly altered in keratoconus, in which nonorthogonal orientations were found inside the apical scar. CONCLUSIONS: The normal human corneal stroma shows a considerable degree of structural anisotropy. It is characterized by two preferred collagen fibril orientations orthogonal to each other. Alteration of the regular orthogonal arrangement of the fibrils in keratoconus may be related to the biomechanical instability of the tissue.

Adult↗
PubMed1996

Bone mineralization in an osteogenesis imperfecta mouse model studied by small-angle x-ray scattering.

We have studied the size and orientation of mineral crystals in cortical bone of oim/oim mice, which are known to produce only alpha 1(I) collagen homotrimers and which may serve as a model for human osteogenesis imperfecta. Long bones (femur and tibia) from young (5 wk old) oim/oim mice and from unaffected heterozygous counterparts were investigated by small-angle x-ray scattering (SAXS), which is sensitive to structures smaller than 50 nm. Mineral crystals were compared in terms of their thickness and their alignment with respect to the long bone axis. While electron microscopic tomography has recently shown the existence of large mineral blocks (with all dimensions typically exceeding 50 nm) in mineralized tendons of oim/oim mice, SAXS revealed a family of thin, possibly needle-like, crystals in cortical bone. These crystals were similar in shape to those observed previously in normal mice, but they were thinner and less well aligned in oim/oim mice relative to heterozygotes. Moreover, the crystal thickness and their alignment with the bone axis were more variable in oim/oim bone, with a close correlation (r = 0.94, P < 0.001) between the two parameters. The presence of smaller crystals with more variable alignment in corticalis of oim/oim mice may contribute to the brittleness of their bone, similar to that of human osteogenesis imperfecta.

Animals↗