Development of preparation methods for and insights obtained from atomic force microscopy of fluid spaces in cortical bone.

Several preparation methods were developed to investigate the dimensions and surface structure of fluid spaces within cortical bone, using atomic force microscopy (AFM). Of special interest was the morphology of the lacunocanalicular system, which serves as a conduit between osteocytes encased in bone tissue, the intramedullary cavity, blood vessels running through the bone, and the periosteal surface of bone. Fracture and the removal of either the mineral or the organic component is a method by which each component can be investigated at a very high resolution in situ. Although fractured bone was too rough to image details of the lacunocanalicular system, post-treatment with ethylene diamine tetraacetic acid (EDTA) or papain allowed for investigation of the collagen matrix or the mineral crystals of bone, respectively. Cut and polished bone was smooth enough for identifying the lacunae of bone using AFM, but unambiguous differentiation between the canaliculi and cracks in the bone surface was not possible. However, when the lacunocanalicular system was filled with polymethylmethacrylate (PMMA), it was possible to image casts of the lacunocanalicular system by selectively etching away the surrounding bone matrix. Using this method, we identified individual canaliculi and measured their dimensions. Furthermore, by carefully etching away the bone matrix in successive etches, it was shown that the wall structure of the canaliculus is dominated by collagen fibrils. These observations have important implications for fluid flow in bone.