Effects of deproteinization and ashing on site-specific properties of cortical bone

Buffered sodium hypochlorite (NaOCl) solution was used to remove selectively the collagen phase from bovine cortical bone. Changes in the mechanical behaviour and material properties were studied over a wide range of resolution (from 5 m to 3 mm) using an integrated combination of experimental techniques. Optical microscopy indicated that timed immersion in NaOCl results in cortical bone specimens that consist of a mineralized tissue core surrounded by a layer of deproteinized or anorganic bone. With increased NaOCl treatment, the mechanical behaviour in three-point flexure of the intact specimens became increasingly characteristic of a brittle ceramic material. Localized material properties were evaluated using histology, scanning electron microscopy and microhardness testing. The site-specific properties and the mineralization of the cores were not significantly affected by the treatment; however, the interactions and structural framework of the hydroxyapatite crystallites within the anorganic material were compromised. This destruction of crystallite interlocking was not observed in samples in which the organic phase was removed by ashing at 800°C. The ashed samples maintained microhardness values three times those of the bleached samples. Because of its damaging effects on cortical bone structural integrity, the NaOCl treatment did not provide a reasonable means of studying, as a function of the phasic mass fraction, incremental changes in bone mechanical behaviour or the relative roles of collagen and mineral within the structural hierarchy.