Raman imaging of two orthogonal planes within cortical bone M. Kazanci a,c , H.D. Wagner b , N.I. Manjubala a , H.S. Gupta a , E. Paschalis c , P. Roschger c , P. Fratzl a, ⁎ a Max Planck Institute of Colloids and Interfaces, Department of Biomaterials, 14424 Potsdam, Germany b Department of Materials and Interfaces, Weizmann Institute of Science, Rehovot 76100, Israel c Ludwig Boltzmann Institute of Osteology at the Hanusch Hospital of WGKK, AUVA Trauma Centre Meidling, 4th Med Department, Hanusch Hospital, Vienna, Austria Received 31 October 2006; revised 23 February 2007; accepted 19 April 2007 Available online 2 June 2007 Abstract The lamellar bone's strength is mainly affected by the organization of its mineralized collagen fibers and material composition. In the present study, Raman microspectroscopic and imaging analyses were employed to study a normal human femoral midshaft bone cube-like specimen with a spatial resolution of ∼ 1–2 μm. Identical bone lamellae in both longitudinal and transverse directions were analyzed, which allowed us to separate out orientation and composition dependent Raman lines, depending on the polarization directions. This approach gives information about lamellar bone orientation and variation in bone composition. It is shown that the ν 1 PO 4 to amide I ratio mainly displays lamellar bone orientation; and ν 2 PO 4 to amide III and CO 3 to ν 2 PO 4 ratios display variation in bone composition. The ν 2 PO 4 to amide III ratio is higher in the interstitial bone region, whereas the CO 3 to ν 2 PO 4 ratio has lower values in the same region. The present study provides fresh insights into the organization of a lamellar bone tissue from two orthogonal orientations. © 2007 Elsevier Inc. All rights reserved. Keywords: Raman; Imaging; Cortical bone; Orientation; Composition; Spectroscopy Introduction The mechanical properties of bone are influenced by a variety of material and structural properties, such as the tissue organization, the amount of mineral, and the orientation and cross-linking of the collagen component. All these structural aspects contribute to the quality of bone tissue [7,25] and to the resulting biomechanical properties of the bone organ. The mineral is stiff and brittle while the protein is much softer but also much tougher (that is, more fracture resistant) than the mineral. Remarkably, such composite combines complementary mechanical properties of both components. This rather unusual combination of material properties provides rigidity and resistance against fracture. The structural origin of such design optimization is still debated. Therefore, new methodologies are clearly needed for the characterization of bone material quality, since the mechanical performance of the mineralized tissue is of utmost importance for the fragility of bone, particularly in old age and in case of diseases. One of the difficulties in assessing the bone material quality is its hierarchical structure. Lamellar cortical bone, for example, comprises mineralized collagen fibers with alternating fiber orientation in successive lamellae. It has been recently shown that the deformation pattern during loading is also hierarchical and distributes the strain in an intriguing way between the mineral and organic components [10]. Giraud-Guille [8] and Weiner et al. [24] described the multiple layers of aligned fiber orientation in different directions as “twisted plywood”. The mechanical design implications of such a structure were sub- sequently investigated [1,2,25]. The lamellar organization has a profound influence on the fracture properties of cortical bone, as the energy for a crack propagating parallel to the layers is almost a hundred times smaller than for cracks extending perpendicular to them [17]. Different orientations of collagen fibers are thought to be advantageous from a structural design viewpoint, within an environment of multidirectional mechanical constraints. Studies of the tibia and fibula [5] and femur [19,20] provided the first Bone 41 (2007) 456 – 461 www.elsevier.com/locate/bone ⁎ Corresponding author. Fax: +49 331 567 9402. E-mail address: Peter.Fratzl@mpikg.mpg.de (P. Fratzl). 8756-3282/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bone.2007.04.200