The adult mammalian bone is mainly composed of layered onion-like cylindrical structures called osteons. Each osteon is surrounded by a very thin sheath. Osteons and their layered structure were first identified in the late 17th Century, but to this day much remains to be learned about the structure of this most important material.
About 10 years ago a powerful new technique for 3D structural analysis was applied to bone after the mineral was removed. Focused ion beam-scanning electron microscopy (FIB SEM) involves removing a very thin nanometers thick layer from a block of the material and then imaging the newly exposed surface at high resolution. The cycle is repeated hundreds of times and all the images are then digitally stacked to form a volume that is tens of microns along each dimension and the images are at a resolution of around 10 nanometers. At this resolution the basic building block of bone, the mineralized collagen fibril can be visualized. And from this the organization of the building blocks in the relatively large volume can be reconstructed to provide a detailed characterization of bone structure. Weiner says that when his former student, Natalie Reznikov obtained the first such structures of demineralized osteonal lamellar bone, this was a eureka moment in that it not only confirmed that the collagen fibrils of the major component are organized in a plywood-like structure, but that an additional material was present with a much more complicated structure that appeared rather disordered. So we showed that osteonal bone is composed of two materials.
Those FIB SEM studies suffered from the fact that the mineral was removed in order to reveal the collagenous matrix. This chemical treatment could introduce artifacts, and of course provided no information on the major component of bone, namely the mineral. This technical barrier was broken when Dr. Emeline Raguin, Dr. Katya Rechav and Professor Steve Weiner from the Weizmann Institute of Science and Professor Ron Shahar from The Hebrew University of Jerusalem developed the methodology to visualize the collagen fibrils using FIB SEM without removing the mineral. The study published in the journal, Acta Biomaterialia, reported the 3D structure of osteonal lamellar bone and the so-called cement sheath surrounding each osteon using pig bone.
The study showed that the lamellae that make up the osteonal structure are composed of layers of mineralized collagen fibrils aligned in one direction that are separated by layers of mineralized collagen fibrils oriented in mainly two different directions. In the demineralized bone studied previously this latter layer appeared more disordered probably as a result of the mineral removal. This latest study not only confirmed that the lamellar structure is composed of two different materials, but showed that the cement sheath structure is even more complicated in that it is composed of layers with mineralized collagen fibrils aligned in 3 or more directions. Furthermore, Raguin et al could characterize the mineral distribution by comparing the same surface imaged using secondary electron with the image obtained using back scattered electrons. The latter is sensitive to mineral composition and density. The back scattered electron images showed the presence of a surprisingly large proportion of less mineralized fibrils surrounding the bundles of more mineralized fibrils in the lamellar structure. These back scattered images also showed that the extent of mineralization of the cement sheath is similar to that of lamellar bone. Another innovation reported in this study, is the capability to automatically analyse each of the thousands of images that make up one stack by taking advantage of the 67nm repeating structure that characterizes the collagen fibril. In this way the computer provides quantitative information on the predominant directions of the fibrils and on how well they are aligned. This eliminates the subjectivity involved in visual image analysis and makes possible a rapid and reliable quantitative assessment of the structure.
Specimen preparation for analyzing mineralized bone in the FIB SEM is straightforward and takes just a few hours to prepare a polished surface for imaging, whereas specimen preparation of the demineralized bone matrix is complicated and requires several weeks. A consequence of this is that many more image stacks can be prepared and can be automatically analyzed. This in turn opens up the possibility of studying the structures of pathological mineralized tissues that because of their inherent variability require the study of many samples. Such studies could include osteoporotic bones and bones that are malformed as a result of genetic mutations (osteogenesis imperfecta). Weiner commented that this exciting possibility may shed light both on how bones are formed, and even more important provide an assessment of how pathological bones may or may not function mechanically; an important medical issue.
Raguin, E., Rechav, K., Shahar, R., & Weiner, S. (2021). Focused ion beam-SEM 3D analysis of mineralized osteonal bone: lamellae and cement sheath structures. Acta Biomaterialia, 121, 497-513.Go To Acta Biomaterialia