The Nanoscale Structure of Fully Dense Human Cortical Bone

Abstract

The nanoscale structure of fully dense human cortical bone is explored using advanced transmission electron microscopy (TEM) techniques. Studies of fully dense cortical bone are rare because of the sample preparation challenges. In this work, cryogenic ion milling is compared favourably with traditional ultramicrotoming methods because of the clearer imaging results and better preservation of biological structures in the ion milled samples. Ion milled samples were prepared parallel, perpendicular and at a 45 degree angle to the long axis of a human femur. The samples are cooled with liquid nitrogen while being milled to prevent heating damage to the bone structure. Bright-field and dark-field imaging show that mineral mainly occurs as 65 nm wide, 5 nm thick mineral structures, external to the collagen fibrils, and with the long axis of the mineral running parallel to the fibrils. In samples cut parallel to the long axis of the bone, the mineral structures have their c-axes aligned with the collagen fibril long axis. In these sections the mineral structures extend up to 200 nm and are grouped into stripe-like bunches, 53 nm apart. Samples cut perpendicular to the long axis of the bone show open areas roughly 45 nm in diameter. These open areas are assumed to be the location of collagen fibrils within the structure and are tangentially surrounded by 65 nm wide, 5 nm thick mineral structures. On average, there are 22 nm of mineral structures between adjoining collagen fibrils. Samples cut at 45 degrees to the long axis of the bone confirm that the open structure seen in the perpendicular section is not an artefact of sample preparation. By tilting the sample, the 45 degree sample shows the structure of both the parallel and perpendicular sections. The parallel structure strongly resembles images of embryonic bone and other mineralized tissues seen in the literature, so the perpendicular open structure is not caused by sample preparation. An examination of ultramicrotoming’s effect on mineral structure size compared with that of ion milling shows that the mineral structures in ion milled samples are twice as long as in ultramicrotomed samples, indicating that bone mineral may be damaged by the forces applied to the complex composite structure existing in fully dense cortical bone. Using energy dispersive X-ray spectroscopy (EDXS) results and a simplified model of the locations of mineral within the collagen/mineral framework, a calculation of the percentage of external mineral was performed. The result showed that 80+_ 6 % of the mineral in fully dense cortical bone must be external to the collagen fibrils to obtain the EDXS results. Finally, Z-contrast tomography, based on the use of high angle annular darkfield (HAADF) imaging, was used to prepared tomographic reconstructions of the external mineral in fully dense cortical bone. Unlike bright-field tomography, the Z-contrast technique allows examination of crystalline materials as the contrast in HAADF images is mass-thickness dependent instead of diffraction based. These reconstructions again showed the mineral tangentially surrounding 50 nm diameter cylindrical holes, assumed to be the location of collagen fibrils in all directions. This work shows the importance of mineral that is external to the collagen fibrils to the nanoscale structure of fully dense cortical bone.