A timeseries analysis of the fracture callus extracellular matrix proteome during bone fracture healing.

While most bones fully self-heal, certain diseases require bone allograft to assist with fracture healing. Bone allografts offer promise as treatments for such fractures due to their osteogenic properties. However, current bone allografts made of decellularized bone extracellular matrix (ECM) have high failure rates, and thus grafts which improve fracture healing outcomes are needed. Understanding specific changes to the ECM proteome during normal fracture healing would enable the identification of key proteins that could be used enhance osteogenicity of bone allograft. Here, we performed a timeseries analysis of the fracture callus in mice to investigate proteomic and mineralization changes to the ECM at key stages of fracture healing. We found that changes to the ECM proteome largely coincide with the distinct phases of fracture healing. Basement membrane proteins (AGRN, COL4, LAMA), cartilage proteins (COL2A1, ACAN), and collagen crosslinking enzymes (LOXL, PLOD, ITIH) were initially upregulated, followed by bone specific proteoglycans and collagens (IBSP, COL1A1). Various tissue proteases (MMP2, 9, 13, 14; CTSK, CTSG, ELANE) were expressed at different levels throughout fracture healing. These changes coordinated with mineralization of the fracture callus, which increased steeply during the initial stages of healing. Interestingly the later timepoint was characterized by a response to wound healing and high expression of clotting factors (F2, 7, 9, 10). We identified ELANE and ITIH2 as tissue remodeling enzymes having no prior known involvement with fracture healing. This data can be further mined to identify regenerative proteins for enhanced bone graft design.