A new technique for imaging Mineralized Fibrils on Bovine Trabecular Bone Fracture Surfaces by Atomic Force Microscopy
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A new technique for imaging Mineralized Fibrils on Bovine Trabecular Bone Fracture Surfaces by Atomic Force Microscopy
Johannes H. Kindt, Georg E. Fantner, Philipp J. Thurner, Georg Schitter, Paul K. Hansma University of California Santa Barbara, Santa Barbara, CA, USA
ABSTRACT High resolution atomic force microscopy (AFM) images of bovine trabecular bone fracture surfaces reveal individual fibrils coated with extrafibrillar mineral particles. The mineral particles are distinctly different in different regions. In some regions the particles have average dimensions of (70 ± 35) nm along the fibrils and about half that across the fibrils. In other regions they are smaller and rounder, of order (53 ± 14) nm both along and across the fibrils. In other regions they are smaller and rounder, of order (25 ± 15) nm both along and across the fibrils, with more rounded top surfaces. Significantly, we rarely observed bare collagen fibrils. If the observed particles can be verified to be native extrafibrillar mineral, this could imply that the fractures which created the observed areas propagated within the mineralized extrafibrillar matrix.
INTRODUCTION The ultra-structure of bone and other mineralized tissues has been studied extensively(18). Bone consists of collagen fibrils with diameters on the order of 100nm, a small percentage of non-collagenous proteins, and mineral platelets of carbonated hydroxyapatite (dahllite), also referred to as bone-apatite or bio-apatite (9), with typical dimensions of 27.3nmx11.2 nm for bovine bone with a thickness of 2 - 4nm, as measured by X-Ray scattering (1, 2) and Atomic Force Microscopy (AFM)(10, 11). The mineral platelets can occur both inside and outside collagen fibrils. Fibrils appearing to be covered by mineral have been observed earlier by SEM(12). Here we present a high-resolution imaging technique of bovine trabecular bone fracture surfaces using AFM.
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EXPERIMENTAL DETAIL Solutions used: Ca-Buffer: (110mM NaCl, 40mM CaCl2, 10mM Hepes), pH 7.0 Na-Buffer (150mM NaCl, 10mM Hepes), pH 7.0 Bone preparation: The trabecular parts of bovine vertebrae were stored frozen until they were diced into blocks of 4x4x5mm3 under constant irrigation using a bone bandsaw (Marmed, Inc. Cleveland, OH, USA), and the marrow was removed with a pressured water system for dental hygiene (Waterpik Technologies, Fort Collins, CO, USA). The outside surface of the trabeculae was then stained with Coomassie Blue R (Sigma-Aldrich, 0.2g/ml) in Nabuffer for 5 minutes, and the samples were rinsed in Ca-buffer to remove excess staining solution. The wet samples were then stored at -20° C in a sealed container until immediately before AFM sample preparation. The maximum handling time at room temperature before fracture and drying was limited to less than 30 minutes in order to minimize sample degradation. AFM sample preparation: A stained bone cube was soaked in Ca buffer for 5 minutes, then clamped in a vice, with the natural bone load axis parallel with the vice grip edge, and half of the bloc
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