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.
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.
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
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.
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).