Collagen is a ubiquitous protein with remarkable mechanical properties. It is highly elastic, shows large fracture strength and enables substantial energy dissipation during deformation. Most of the connective tissue in humans consists of collagen fibrils composed of a staggered array of tropocollagen molecules, which are connected by intermolecular cross-links. In this study, we report a three-dimensional coarse-grained model of collagen and analyze the influence of enzymatic cross-links on the mechanics of collagen fibrils. Two representatives immature and mature cross-links are implemented in the mesoscale model using a bottom-up approach. By varying the number, type and mechanical properties of cross-links in the fibrils and performing tensile test on the models, we systematically investigate the deformation mechanisms of cross-linked collagen fibrils. We find that cross-linked fibrils exhibit a three phase behavior, which agrees closer with experimental results than what was obtained using previous models. The fibril mechanical response is characterized by: (i) an initial elastic deformation corresponding to the collagen molecule uncoiling, (ii) a linear regime dominated by molecule sliding and (iii) the second stiffer elastic regime related to the stretching of the backbone of the tropocollagen molecules until the fibril ruptures. Our results suggest that both cross-link density and type dictate the stiffness of large deformation regime by increasing the number of interconnected molecules while cross-links mechanical properties determine the failure strain and strength of the fibril. These findings reveal that cross-links play an essential role in creating an interconnected fibrillar material of tunable toughness and strength.

Mechanical injury to connective tissue causes changes in collagen structure and material behaviour, but the role and mechanisms of molecular damage have not been established. In the case of mechanical subfailure damage, no apparent macroscale damage can be detected, yet this damage initiates and potentiates in pathological processes. Here, we utilize collagen hybridizing peptide (CHP), which binds unfolded collagen by triple helix formation, to detect molecular level subfailure damage to collagen in mechanically stretched rat tail tendon fascicle. Our results directly reveal that collagen triple helix unfolding occurs during tensile loading of collagenous tissues and thus is an important damage mechanism. Steered molecular dynamics simulations suggest that a likely mechanism for triple helix unfolding is intermolecular shearing of collagen α-chains. Our results elucidate a probable molecular failure mechanism associated with subfailure injuries, and demonstrate the potential of CHP targeting for diagnosis, treatment and monitoring of tissue disease and injury.

Osteopontin (OPN) is a non-collagenous protein involved in biomineralization of bone tissue. Beyond its role in biomineralization, we show that osteopontin is essential to the quality of collagen fibrils in bone. Transmission electron microscopy revealed that, in Opn-/- tissue, the organization of the collagen fibrils was highly heterogeneous, more disorganized than WT bone and comprised of regions of both organized and disorganized matrix with a reduced density. The Opn-/- bone tissue also exhibited regions in which the collagen had lost its characteristic fibrillar structure, and the crystals were disorganized. Using nanobeam electron diffraction, we show that damage to structural integrity of collagen fibrils in Opn-/- bone tissue and their organization causes mineral disorganization, which could ultimately affect its mechanical integrity. STATEMENT OF SIGNIFICANCE: This study presents new evidence about the role of osteopontin (OPN) - a non-collagenous protein - on the structure and organization of the organic and mineral matrix in bone. In previous work, osteopontin has been suggested to regulate the nucleation and growth of bone mineral crystals and to form sacrificial bonds between mineralized collagen fibrils to enhance bone's toughness. Our findings show that OPN plays a crucial role before mineralization, during the formation of the collagen fibrils. OPN-deficient bones present a lower collagen content compared to wild type bone and, at the tissue level, collagen fibrils organization can be significantly altered in the absence of OPN. Our results suggest that OPN is critical for the formation and/or remodeling of bone collagen matrix. Our findings could lead to the development of new therapeutic strategies of bone diseases affecting collagen formation and remodeling.

Bone metastasis is a complication occurring in up to 70% of advanced breast cancer patients. The estrogen receptor-related receptor alpha (ERRα) has been implicated in breast cancer and bone development, prompting us to examine whether ERRα may function in promoting the osteolytic growth of breast cancer cells in bone. In a mouse xenograft model of metastatic human breast cancer, overexpression of wild-type ERRα reduced metastasis, whereas overexpression of a dominant negative mutant promoted metastasis. Osteoclasts were directly affected and ERRα upregulated the osteoclastogenesis inhibitor, osteoprotegerin (OPG), providing a direct mechanistic basis for understanding how ERRα reduced breast cancer cell growth in bone. In contrast, ERRα overexpression increased breast cancer cell growth in the mammary gland. ERRα-overexpressing primary tumors were highly vascularized, consistent with an observed upregulation of angiogenic growth factor, the VEGF. In support of these findings, we documented that elevated expression of ERRα mRNA in breast carcinomas was associated with high expression of OPG and VEGF and with disease progression. In conclusion, our results show that ERRα plays a dual role in breast cancer progression in promoting the local growth of tumor cells, but decreasing metastatic growth of osteolytic lesions in bone. Cancer Res; 71(17); 5728–38. ©2011 AACR.