LETTERS Sacrificial bonds and hidden length dissipate energy as mineralized fibrils separate during bone fracture GEORG E. FANTNER 1 *, TUE HASSENKAM 1 , JOHANNES H. KINDT 1 , JAMES C. WEAVER 2 , HENRIK BIRKEDAL 3 , LEONID PECHENIK 1 , JACQUELINE A. CUTRONI 1 , GERALDO A. G. CIDADE 4 , GALEN D. STUCKY 3 , DANIEL E. MORSE 2 AND PAUL K. HANSMA 1 1 University of California, Santa Barbara, Department of Physics, California, 93106, USA 2 University of California, Santa Barbara, Institute for Collaborative Biotechnologies, California, 93106, USA 3 University of California, Santa Barbara, Department of Chemistry and Biochemistry, California, 93106, USA 4 Federal University of Rio de Janeiro, Biophysics Institute Carlos Chagas Filho, Rio de Janeiro 21491-590, Brazil *e-mail: fantner@physics.ucsb.edu Published online: 17 July 2005; doi:10.1038/nmat1428 P roperties of the organic matrix of bone 1 as well as its function in the microstructure 2 could be the key to the remarkable mechanical properties of bone 3 . Previously, it was found that on the molecular level, calcium-mediated sacrificial bonds increased stiffness and enhanced energy dissipation in bone constituent molecules 4,5 . Here we present evidence for how this sacrificial bond and hidden length mechanism contributes to the mechanical properties of the bone composite, by investigating the nanoscale arrangement of the bone constituents 6–8 and their interactions. We find evidence that bone consists of mineralized collagen fibrils and a non-fibrillar organic matrix 2 , which acts as a ‘glue’ that holds the mineralized fibrils together. We believe that this glue may resist the separation of mineralized collagen fibrils. As in the case of the sacrificial bonds in single molecules 5 , the effectiveness of this mechanism increases with the presence of Ca 2+ ions. It has become increasingly clear that, in addition to the influence of bone mineral density 9 , the microscopic architecture 10 and microdamage 11,12 , the nanoscopic arrangement of the bone constituents is vital for understanding the functioning 13 and mechanistic fracture criteria 14 of bone. Here we present data that shed light on nanoscale interactions between the mineralized collagen fibrils and a non-fibrous organic matrix present in the bone. These nanoscale interactions have profound implications for the behaviour of bone at the macroscopic scale. Figure 1a shows scanning electron microscope (SEM) images of mineralized collagen fibrils on a fractured surface of human trabecular bone. Some fibrils are closely packed whereas others have spaces between them. These spaces are sometimes spanned by small filaments (see the arrows in Fig. 1b). The separation of mineralized collagen fibrils under loading is consistent with the findings of atomic force microscope (AFM) studies that crack formation and bone fracture occur between the mineralized collagen fibrils 6 . In the AFM image of Fig. 1c, such mineralized collagen fibrils can be seen, again with filaments spanning between the individual fibrils (arrows). Figure 1b and c indicates the presence of extrafibrillar organic material that was originally a thin layer between two fibrils (Fig. 1d) and is now stretched between the separated fibrils (Fig. 1e). Do these filaments apply significant forces to resist the separation or shear of the mineralized collagen fibrils? The AFM can shed light on this question directly in a model system. We implemented a system, based on single-molecule force spectroscopy 15,16 , composed of two pieces of bone in solution, one on the AFM cantilever and one as a sample. The pieces can be pressed together and then pulled apart (Fig. 2a). This process is repeated for several cycles. This system simulates the molecular interactions that occur during the separation of mineralized fibrils within bone. After the two bone pieces are put in contact, a force has to be applied to separate the pieces again, which indicates the existence of adhesion between the two bone pieces. The forces experienced during the separation were of the order of nanonewtons and persisted for distances of micrometres (Fig. 2b and d). The area under the curve of force versus distance (Fig. 2b) represents the energy required to separate the bone pieces. Most of the energy is dissipated, although some of this energy is stored elastically and recovered as the force is relaxed, if not all of the filaments have been broken (upper curve of Fig. 2b). Energy dissipation at the single-molecule level has been previously shown 5,17,18 for biological materials and biological composites. An ion dependence of the molecular energy dissipation was found for bone and commercial bovine tendon collagen (which may also have included proteoglycans and other collagen-associated polymers); there was more energy dissipation when Ca 2+ ions were present in the buffer in which the experiment was conducted 5 . Moreover, the energy dissipation increased if there was a delay of the order of 10 s between cycles of straining the molecules. Relative to this work 5 , it has been pointed out that it is important to see whether this mechanism is involved in preventing the spread of microcracks 4 . Here we report that, for our experimental model that simulates the separation of mineralized fibrils, the energy dissipation is also greater if calcium ions are present in the buffer (Fig. 2c). For consistency, we used the same buffers as for the previously reported molecular pulling experiments 5 on bone (‘calcium buffer’: 40 mM CaCl 2 , 110 mM NaCl, 10 mM HEPES, pH 7; ‘sodium buffer’: 150 mM NaCl, 10 mM HEPES, pH 7). The idea was to look for a perhaps subtle dependence on calcium ions by using one buffer that was much higher in calcium than physiological saline and one that was much lower. With Ca 2+ ions present, the energy dissipation nature materials ADVANCE ONLINE PUBLICATION www.nature.com/naturematerials 1 © 2005 Nature Publishing Group