Fri., 10/17/08 Basic Science, Paper #18, 9:42 am OTA-2008
Analysis of Long-Bone Fracture Nonunion by an Expression Profile of the Matrix Metalloproteinases and ADAMTS Enzyme Families
Marc R. Fajardo, MD (n); Chuanju Liu, PhD (n); Kenneth A. Egol, MD (a-Smith + Nephew,
Stryker, Synthes; a,b-Biomet; b-Exactech, Inc; d-Johnson & Johnson);
New York University-Hospital for Joint Diseases, New York, New York, USA
Purpose: The remodeling process in bone is attributable to the matrix metalloproteinases (MMPs) and a disintegrin-like metalloproteinase with thrombospondin type I motif (ADAMTS) enzymes. Delays in bone healing or even development of a nonunion could be related to the concentrations and functions of these enzymes. The purpose of this study was threefold: (1) to define an RNA expression profile of most MMPs/ADAMTS enzymes and their inhibitors (tissue inhibitors of metalloproteinases [TIMP]) in fracture nonunion tissue, (1) to correlate their expression in normal healing fracture callus, and (3) to correlate the expression on a protein level using molecular biology techniques.
Methods: 15 consecutive patients with established fracture nonunions of at least 6 months duration (who provided informed consent) were enrolled for this OTA-funded study. Tissue samples taken from the nonunion site were prospectively collected utilizing a standard protocol. At the time of nonunion repair surgery, multiple tissue samples were collected from two distinct sites in each patient: fibrous tissue from the nonunion site and bony callus from the surrounding region. The samples were stored and the RNA extracted. The RNA expression patterns of 45 different MMPs, ADAMTS, and TIMP enzymes were subsequently determined in the nonunion and callus tissue using quantitative real-time polymerase chain reaction. The significant results were further characterized on a protein level using Western immunoblotting and immunohistochemistry techniques.
Results: The mean age of the patient cohort was 46 years (range, 32-80). The anatomic sites involved are: 2 femoral shafts, 2 subtrochanteric femurs, 2 tibial shafts, 1 tibial plateau, 1 distal tibia, 2 fibular shafts, 3 midshaft clavicles, 1 proximal humerus, and 1 ulnar shaft. Five patients were smokers. Mean length of established nonunion was determined to be 16 months. Comparison between both groups of tissue samples revealed significantly elevated concentrations of MMP-7 and MMP-12 in nonunion tissue when compared to normal callus (P = 0.01). Western immunoblotting and immunohistochemistry verified these findings on a protein level. No correlation was found between the expression of these enzymes and other clinical parameters (ie, patient smoking, use of a bone stimulator, number of previous surgeries).
Conclusion and Significance: Our findings indicate that tissue present at the nonunion site commonly expresses statistically significant levels of MMP-7 and MMP-12. These data suggest that these enzymes may play an integral role in the development of a fracture nonunion. These enzymes may likewise be used as biomolecular markers for failed fracture healing in patients. Further studies are needed to characterize these enzymes.
If noted, the author indicates something of value received. The codes are identified as a-research or institutional support; b-miscellaneous funding; c-royalties; d-stock options; e-consultant or employee; n-no conflicts disclosed, and *disclosure not available at time of printing.
• The FDA has not cleared this drug and/or medical device for the use described in this presentation (i.e., the drug or medical device is being discussed for an “off label” use). ◆FDA information not available at time of printing. Δ OTA Grant.