Scanning SAXS/WAXD of Connective Tissue with 20 µm spatial resolution.

by I. Zizak (1), O. Paris (1), P. Roschger (2), H. Amenitsch (3), S. Bernstorff (4), K. Klaushofer (2), P. Fratzl (1)
(1) Erich Schmid Institut für Materialwissenschaft der ÖAW, Leoben & Institut für Materialphysik der Montanuniversität Leoben, Austria
(2) Ludwig Boltzmann Institut für Osteologie, 4. Med. Hanusch-Krankenhaus, Vienna, Austria
(3) Institute of Biophysics and X-Ray Structure Research, Austrian Academy of Science, Graz, Austria
(4) Sincrotrone Trieste, SS14, Km163.5, Basovizza (Italy)

( contacts: zizak@unileoben.ac.at, paris@unileoben.ac.at, fratzl@unileoben.ac.at,amenitsch@elettra.trieste.it, bernstorff@elettra.trieste.it )

Many complex materials providing optimized mechanical properties are hierarchically structured on different length scales down to the atomic or molecular level. Typical examples are biological tissues, such as bone, cartilage or wood. Investigation of the structure of such materials requires new experimental techniques in order to understand the interplay between mechanical properties and the structure at all levels of organization. A very promising attempt is the scanning of the specimen with a very narrow X-ray beam [1], taking SAXS (Small-Angle X-ray Scattering) and/or WAXD (Wide-Angle X-ray Diffraction) patterns for every scanning step. Such scanning experiments provide in principle structural information on three different length-scales: at the micrometer scale (absorption-image with a spatial resolution defined by the beam size), at the nanometer scale (SAXS) and/or at the scale of interatomic distances (WAXD).
In the last two years, we developed an experimental set-up at the SAXS beamline at ELETTRA, which allows us to perform extensive scans over large specimen areas in reasonable time. Using a laboratory x-ray source with pinhole geometry, the limited photon flux does not allow a reduction of the illuminated sample area below 200 µm [1]. The high brilliance of synchrotron radiation sources, such as ELETTRA, opens the possibility to reduce the illuminated area to about 10 µm. Note, that in order to obtain a certain spatial resolution, not only the beam area but also the sample thickness has to be proportionally reduced. Figure 1 shows schematically the experimental setup we built for the SAXS beam-line at ELETTRA.

Fig. 1: Experimental set-up (Coloured parts belong to our microfocus set-up, grey parts to the standard beam-line set-up).

At the nanometer scale, bone and mineralised cartilage consist of small mineral particles embedded in an organic matrix (collagen). For bone, these particles are known to be needle- or plate-shaped with an average thickness of about 3 nm and a length of a few hundred nanometers [2]. The size, shape and orientation of these mineral crystals within the collagen matrix can be determined experimentally by the method of SAXS. Moreover, the mineral type and crystallographic orientation of the crystals may be investigated with WAXD. The main intention of our experiment was to study the structural changes of the mineral particles at the interface between bone and mineralised cartilage in human femoral head. It was therefore necessary to scan the specimen using a small X-ray beam of only a few micrometer width. Using the above described scanning equipment, information at an atomic (WAXD) and a nanometer scale (SAXS) was derived with a spatial resolution of 20 µm and hence, structural changes at the bone-cartilage interface could be detected.
The first step of the experiment consisted in acquiring a radiography of the whole sample by measuring the transmitted intensity with the X-ray sensitive diode (Fig. 2b). This radiography provides an exact mapping of the mineral density-distribution in the sample which can be compared with e.g. light micrographs (Fig. 2a) or scanning electron micrographs. In the second step, the SAXS-signal was recorded with the 2D-detector along lines normal to the bone-cartilage interface. Finally, WAXD patterns were taken (at the same positions) with the 2D-detector. The 2D-patterns were evaluated with methods described elsewhere [3] and the results (shown in Fig.2c-e for one of the linear scans) can be summarised as follows:
1. in bone, the orientation of the mineral crystals follows exactly the direction of the trabeculae (Fig. 2c);
2. the mineral crystals are thicker in cartilage than in bone (Fig 2e);
3. the size and/or shape distribution of the mineral particles (as measured by the h-parameter [2]) in cartilage differs from that in bone (Fig 2d);
4. the orientation of the mineral particles changes at the bone-cartilage interface (Fig 2c). The degree of alignment of mineral crystals in bone is larger than in cartilage (Fig 2c);
5. the crystallographic orientation (measured using WAXD) correlates strongly with the orientation of the particles, the type of the mineral is however the same in both tissues.
These interesting results are a first step towards a more detailed understanding of the complex processes leading to the biochemically optimized structure of bone.

Fig. 2: Comparison of different parameters derived from a scanning-SAXS measurement.
Fig. 2b shows the x-ray radiography as compared to a polarised light micrograph (fig.2a). One-dimensional SAXS and WAXD scans were performed along the horizontal line across the cartilage (left) and bone (right) interface.
Fig. 2c shows the average direction of the mineral crystals (direction of the lines) and the degree of the alignment (length of the lines). The shape parameter h and the mean crystal thickness T are visualised in 2d and 2e.

References
[1] P. Fratzl et al., J. Appl. Cryst. 30, 765-769 (1997)
[2] P. Fratzl et al., J. Bone Miner. Res. 7, 329-334(1992)
[3] P. Fratzl, S. Schreiber, K. Klaushofer, Connective Tissue Resarch 34, p247(1996)

The project is supported by the Fonds zur Förderung der wissenschaftlichen Forschung FWF (project P11762-PHY).