Full-Field Calcium K-Edge X-ray Absorption Near-Edge Structure Spectroscopy on Cortical Bone at the Micron-Scale: Polarization Effects Reveal Mineral Orientation.

Here, we show results on X-ray absorption near edge structure spectroscopy in both transmission and X-ray fluorescence full-field mode (FF-XANES) at the calcium K-edge on human bone tissue in healthy and diseased conditions and for different tissue maturation stages. We observe that the dominating spectral differences originating from different tissue regions, which are well pronounced in the white line and postedge structures are associated with polarization effects. These polarization effects dominate the spectral variance and must be well understood and modeled before analyzing the very subtle spectral variations related to the bone tissue variations itself. However, these modulations in the fine structure of the spectra can potentially be of high interest to quantify orientations of the apatite crystals in highly structured tissue matrices such as bone. Due to the extremely short wavelengths of X-rays, FF-XANES overcomes the limited spatial resolution of other optical and spectroscopic techniques exploiting visible light. Since the field of view in FF-XANES is rather large the acquisition times for analyzing the same region are short compared to, for example, X-ray diffraction techniques. Our results on the angular absorption dependence were verified by both site-matched polarized Raman spectroscopy, which has been shown to be sensitive to the orientation of bone building blocks and by mathematical simulations of the angular absorbance dependence. As an outlook we further demonstrate the polarization based assessment of calcium-containing crystal orientation and specification of calcium in a beta-tricalcium phosphate (ß-Ca3(PO4)2 scaffold implanted into ovine bone. Regarding the use of XANES to assess chemical properties of Ca in human bone tissue our data suggest that neither the anatomical site (tibia vs jaw) nor pathology (healthy vs necrotic jaw bone tissue) affected the averaged spectral shape of the XANES spectra.

Alterations of mass density and 3D osteocyte lacunar properties in bisphosphonate-related osteonecrotic human jaw bone, a synchrotron µCT study.

Osteonecrosis of the jaw, in association with bisphosphonates (BRONJ) used for treating osteoporosis or cancer, is a severe and most often irreversible side effect whose underlying pathophysiological mechanisms remain largely unknown. Osteocytes are involved in bone remodeling and mineralization where they orchestrate the delicate equilibrium between osteoclast and osteoblast activity and through the active process called osteocytic osteolysis. Here, we hypothesized that (i) changes of the mineralized tissue matrix play a substantial role in the pathogenesis of BRONJ, and (ii) the osteocyte lacunar morphology is altered in BRONJ. Synchrotron µCT with phase contrast is an appropriate tool for assessing both the 3D morphology of the osteocyte lacunae and the bone matrix mass density. Here, we used this technique to investigate the mass density distribution and 3D osteocyte lacunar properties at the sub-micrometer scale in human bone samples from the jaw, femur and tibia. First, we compared healthy human jaw bone to human tibia and femur in order to assess the specific differences and address potential explanations of why the jaw bone is exclusively targeted by the necrosis as a side effect of BP treatment. Second, we investigated the differences between BRONJ and control jaw bone samples to detect potential differences which could aid an improved understanding of the course of BRONJ. We found that the apparent mass density of jaw bone was significantly smaller compared to that of tibia, consistent with a higher bone turnover in the jaw bone. The variance of the lacunar volume distribution was significantly different depending on the anatomical site. The comparison between BRONJ and control jaw specimens revealed no significant increase in mineralization after BP. We found a significant decrease in osteocyte-lacunar density in the BRONJ group compared to the control jaw. Interestingly, the osteocyte-lacunar volume distribution was not altered after BP treatment.