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Methods

  • Cellular and molecular biology
  • Preparation of bone-samples
  • Histology and histomorphometry
  • Scanning electrone microscopy
  • Small angle x-ray scattering
  • Nano Indentation
  • Confocal laser scanning microscopy
  • FTIR
  • Osteodensitometry
  • Bone biopsy

METHODS for the characterization
of the micro- and nano
structure of the bone material

Quantitative Backscattered Electron Imaging (qBEI)

Quantitative backscattered electron imaging (qBEI) at the scanning electron microscope (SEM) was developed for the investigation of the mineralization pattern of bone samples and other mineralized tissues. The information on the local variation of the calcium concentration within the bone sample is essential since the amount of mineral and its distribution within the specimen are important contributors to the mechanical quality of the bone.

1For the investigation by qBEI, the unmineralized sample has to be embedded into polymethylmethacrylate (PMMA). The block samples are then polished and carbon coated. In the SEM, the surface of the sample is scanned by the electron beam and the intensity of the backscattered electron (BE)-signal from each measured site is recorded.

 


This BE-signal is mainly determined by the local calcium concentration of the specimen, as reflected by different gray-scales in the image. High calcium concentration can be identified by its bright gray-level in the image, low calcium concentration by its dark gray-level. The embedding material PMMA appears black in this image.


From the BE-gray-level images, histograms can be obtained which are displaying the percentage of the sample occupied by a specific gray-level. If the gray-levels are transferred to Ca-values, the bone samples can be analyzed quantitatively for their calcium concentrations by evaluating the bone mineral density distribution (BMDD). The accuracy of this technique (the minimum difference in calcium concentration that can be resolved by the method) is 0.17wt% Ca (weight-percent calcium). It was shown that the BMDD of trabecular bone from healthy adult individuals is similar independent of age, gender, ethnic origin and skeletal site (vertebra, patella, femoral neck, iliac crest), which is a strong indication for the optimization of the mineral pattern of bone. In the case of diseases, deviations from the “normal” BMDD could be observed and therefore the BMDD has become a powerful tool for the diagnosis of specific bone diseases and can be used as an indicator for the effectiveness of treatments.


For the comparison of a measured BMDD with the “normal” BMDD from healthy individuals, several parameters are obtained from the distribution and compared to the reference-values, such as the peak position CaPeak (which displays the most frequently occurring calcium concentration within the studied bone area) and CaMean (which is the weighted mean calcium concentration).


Additionally, CaWidth, the width of the distribution (full width at half maximum) which is reflecting the homogeneity of mineralization (the lower this parameter, the more homogeneous is the mineralization pattern of the sample) and CaLow (a parameter which displays the percentage of calcium concentrations below 17.68 wt% Ca referring to the amount of newly formed bone matrix) can be deduced from the BMDD and compared to the normal values of these parameters.

Detailed description of qBEI can be found in: Roschger et al., Scan Microsc 9:75-88;1995; Roschger et al, Bone 23:319-326;1998;Roschger et al, Bone 32:316-323,2003.

 

Morphometrical Analysis

Morphometrical parameters are obtained from digital BE-images, light microscopy images of specifically stained or flourescence labelled bone sections. One can distinguish between static and dynamic histomorphometrical parameters. Static morphometric parameters are reflecting the structure and the cellular activity at the time-point of the biopsy while the dynamic parameters give information on amount of bone formation between two given time points.


Typically, structural parameters such as bone volume per tissue volume (BV/TV), trabecular number (Tb.Nb.), trabecular thickness (Tb. Th.) etc. are used to characterize the trabecular features, while cortical width (Ct.Wi.) and porosity are providing information on cortical bone. These parameters can be obtained easily from the digital BE-images.

 


Specific staining (Giemsa and trichrome Goldner's) of 3 micrometer thick undecalcified microtom sections of bone tissue allows the determination of static parameters of bone formation and resorption.

 

 


By computer assisted light microscopy  parameters such as osteoid volume (OV/BV), osteoid surface (OS/BS), osteoid thickness (O.Th), osteoblasts surface per bone surface (Ob.S./BS), osteoclast number per bone surface (Oc.N/BS) and eroded surface per bone surface (ES/BS) etc. are measured.


For the determination of the dynamic parameters, the biopsies are usually fluorescence double labelled by intake of tetracycline for 3 days followed by 12 days free followed again by 3 days tetracycline (3/12/3). The bone biopsy (Bx) is then taken 5 days after. Tetracycline is incorporated to the mineralizing areas of the bone and is therefore labelling the newly mineralizing bone sites. Subsequently, the newly formed bone matrix can be identified in the fluorescence light microscope by fluorescent bands. Parameters describing the dynamics of bone formation can be obtained such as mineral apposition rate (MAR) and bone formation rate per bone surface (BFR/BS).

 

Fourier Transform Infrared Imaging (FTIRI)

The FTIRI method is mainly used to get information on the maturity of the mineral and on the organic component of the mineralized tissue. Both of these outcomes are greatly dependent on tissue age and are very sensitive to pharmaceutical interventions.


Several absorbance bands are determined for bone tissue for:
i) the estimation of the relative amount of mineral to organic matter; (ii) the stoichiometry of the apatite mineral for the characterization of the maturity of the mineral; (iii) the relative carbonate content of the hydroxyapatite – a second indicator for the maturity of the mineral; (iv) and the ratio of the non-reducible/reducible collagen cross-links in bone and thus the maturity of the collagen.

For details see  Paschalis et al, Calcified Tissue International, 59:480-487, 1996;  Paschalis et al, J of Bone and Mineral Research, 16(10), 1821-8, 2001.

 

Scanning Small Angle X-Ray Scattering (SAXS)

In cooperation with the Max Planck Institute of Colloids and Interfaces in Potsdam, Germany (www.mpikg.mpg.de), small angle X-ray scattering has been used extensively to characterize the bone material at nanometer-scale by geometrical properties of the mineral particles, such as thickness (T-parameter) as well as the degree of orientation (rho which represents the fraction of non-isotropically aligned mineral particles) and the function G(x), which characterizes mineral particle shape, size, and arrangement. Recently, a method was established to obtain additional parameters from this G(x)-function giving information on the typical platelet distance and the order/disorder in the arrangement of the mineral platelets.


The SAXS experiments on bone are done by scanning the sample by the X-ray beam (with a spatial resolution of 5-15mm at the synchrotron and 100-150mm at the laboratory). Subsequently, images are obtained by mapping of the determined parameter and mostly these maps are directly combined with qBEI images of the same bone region. This combination of techniques allows the correlation of several parameters characterizing the nanocomposite material of bone and other mineralized tissues.

For further information see: Fratzl et al., Progr. Colloid Polym Sci 130;2005; Fratzl et al.,  J Appl Cryst 36, 2003; Rinnerthaler et al., Calcif Tissue Int, 64, 1999

 

Raman microspectroscopy


In Raman microspectroscopy the information of the mineral component and the organic matrix is obtained simultaneously by creating a complete picture of bone composition. The collection of Raman spectra from both, the organic and mineral constituent is crucial for better understanding the bone physiology.

 


Bone is a composite material, comprising mineral, organic, and water phases at multiple levels of hierarchy. The mineral fraction of bone is a highly impure carbonated apatite situated between collagen fibril cross-links and fibril ends. In Raman spectra of bone tissues the phosphate ν1 band (960 cm-1) and the bands associated with collagen (amide III at 1250 cm-1 and amide I at 1665 cm-1) are of particular interest for bone compositional studies.


Bone has a heterogeneous nature and therefore simple point Raman microspectroscopy cannot adequately describe the chemical microstructure of bone. For this reason Raman spectroscopic imaging is increasingly popular for the analysis of complex organized systems. By using the Raman imaging it is possible to collect spectra point by point across a defined bone sample area.

 

In cooperation with the Max Planck Institute of Colloids and Interfaces in Potsdam, Germany (www.mpikg.mpg.de) bone osteonal tissues have been measured by Raman imaging to demonstrate the versatility of the analytical technique, and provide insights into the organization of bone tissue at the ultra structural level.For further information see: Kazanci et al. J Struct Biol 156, 2006; Kazanci et al. Calcif Tissue Int 79, 2006; Kazanci et al. Bone 41, 2007.

 

 

 

 

 

 

METHODS for the characterization of the mechanical properties of the bone material

 

Nano-Indentation

For the mechanical characterization of the collagen-mineral composite of bone and other mineralized tissues, a combination of an atomic force microscope (AFM) and an add-on nanoindentation device consisting of a three-plate capacitor is used in cooperation with the Max Planck Institute of Colloids and Interfaces (www.mpikg.mpg.de), Potsdam, Germany.


When voltage is applied to the outer plates of the AFM, the electrostatic force on the center plate drives the indenter into the sample and the changes of the electrostatic capacity due to the displacement of the center plate is a measure for the penetration of the indenter (a three side pyramidal tip) into the sample. The indentations can then be viewed by using the same tip in the AFM allowing high spatial accuracy. The loading-unloading cycles consist of five linear segments and the elastic modulus and hardness are obtained from the first linear region of unloading.

 

 

For further information see Tesch et al., Calcif Tissue Int 69;2001, Gupta et al., J Struct Biol 194;2005.

 

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