Microstnictural characterization and x-ray microanalysis of human bone by SEM and image analysis

Abstract

IntroductionThe purpose of this work was to investigate a possible correlation between mechanical properties, microstructure and mineral content of human bone tissue. This study will improve our knowledge of bone mechanics and bone remodelling. Meunier et al. developed a new technique to obtain local mechanical properties by scanning acoustic microscopy (SAM). A good correlation was obtained between the average acoustic impedance and the elastic stiffness patterns of bone. We will present results concerning the evaluation of the microstructure and the mineral content of human tibiae and femora.Materials and MethodsIn order to assess whether correlation exists or not between morphology, mineral content and the acoustic impedance, we worked on the same specimens as Meunier; 20 fresh right paired human tibiae and femora (6 females and 14 males) retrieved at necropsy; age ranged from 48 to 92 years and weight from 40 to 90 kg.Using a low-speed diamond saw, samples of 100-120 μm thickness were cut, polished and dehydrated stepwise in ethanol (60-100%). The specimens were coated with carbon to eliminate charging effects and examined with JEOL JSM 840 scanning electron microscope equipped with a LINK LZ4 for x-ray spectrometer and with backscattered (BS) electron detector. Micrographs with BS electrons were taken at 20KV. BS images are transferred to an IBAS2 image analyser to quantify the microstructure by difference of grey levels. X-ray analyses are made at 15KV to obtain mineral content, mainly Ca and P. We performed x-ray analyses on areas of .45mm2 (covering about 30 osteons) on different zones on each sample (as defined on figure 1) . Synthetic hydroxyapatite was used as a standard in order to quantify the mineral content for each analysis. We studied the variation of the mineral content at different levels expressed in % of cortical thickness from the perioste (0%) to the endoste (100%).Results and DiscussionThe main features investigated were Microstructural characteristics (porosity (%), osteon density, Haversian canal areas) and Mineral content (Ca and P amount expressed in % of HAp, Ca/P ratio computed from x-ray net intensity, global amount of mineral present in the bone matrix). We had investigated differences between tibiae and femora and the evolution between successive levels within the same bone category. Figures 2a,b and 3a,b show typical results of the two categories studied for Haversian canal and osteon density, mineral content and Ca/P ratio respectively. In term of microstructural parameters, femur and tibia exhibit significant differences mostly in the central and endosteal part. This founding has to be related with results found using acoustic measurements in which the longitudinal elastic stiffness (C33) was significantly different between tibia and femur. Regarding the mineral content, no significant difference has been found between tibia and femur. However, the Ca/P ratio is significantly different between tibia and femur.