Matrix metalloproteinases that associate with and cleave bone morphogenetic protein-2 in vitro are elevated in hypertrophic fracture nonunion tissue.

Abstract

A delayed union or a nonunion of a fracture is a potentially adverse complication. Understanding the mechanisms of nonunion development may lead to improved treatment modalities. Proteases such as the matrix metalloproteinases play important roles in bone remodeling and repair, in which an imbalance or a nonfunctioning enzyme may lead to defects in bone healing (nonunion). The purpose of this pilot study was twofold: first to define an mRNA expression profile of all the matrix metalloproteinases (MMPs), a disintegrin and metalloproteinases with thrombospondin motif (ADAMTS) enzymes, and their inhibitors (TIMPs) within fracture nonunion tissue, and second to compare this profile with mineralized fracture callus. Using a systematic real-time polymerase chain reaction, we screened the gene expression profiles of all members of the MMPs, ADAMTS, and their inhibitor TIMPs on human fracture nonunion tissue and matched mineralized callus tissue. Significant results were further analyzed using Western immunoblotting, immunohistochemistry, and in vitro protein interaction assays with bone morphogenetic protein-2. This analysis confirmed MMP-7 and MMP-12 as two unidentified enzymes expressed in fracture nonunion tissue. Both MMP-7 and MMP-12 mRNAs were significantly elevated in nonunion tissue when compared with local mineralized callus from the same site (P < 0.001); the elevated protein levels of interest were visualized through immunoblotting and immunohistochemistry. In addition, these two MMPs were found to directly bind to and degrade bone morphogenetic protein-2 in vitro. Collectively, our findings indicate that tissue present at the site of hypertrophic nonunions commonly expresses significantly higher levels of MMP-7 and MMP-12 in relation to mineralized fracture callus. Both were found to directly inactivate bone morphogenetic protein-2 in vitro, the best established growth factor in bone formation and repair.