BMP-7–Induced Ectopic Bone Formation and Fracture Healing Is Impaired by Systemic NSAID Application in C57BL/6-Mice Alexander S. Spiro, 1,2 F. Timo Beil, 1,2 Anke Baranowsky, 1,2 Florian Barvencik, 1,2 Arndt F. Schilling, 1,2 Khoa Nguyen, 1,2 Shahram Khadem, 1,2 Sebastian Seitz, 1,2 Johannes M. Rueger, 1,2 Thorsten Schinke, 1,2 Michael Amling 1,2 1 Department of Trauma-, Hand-, and Reconstructive Surgery, University Medical Center Hamburg–Eppendorf, Martinistr. 52, 20246 Hamburg, Germany, 2 Center for Biomechanics and Skeletal Biology, Experimental Trauma Surgery, University Medical Center Hamburg– Eppendorf, Hamburg, Germany Received 2 December 2008; accepted 24 September 2009 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jor.21044 ABSTRACT: Nonsteroidal antiinflammatory drugs (NSAIDs) are known to potentially impair the fracture healing process. The aim of the present study was to determine if the impairment of bone healing by systemic NSAID application is, at least in part, due to an interaction of NSAIDs with the bone anabolic BMP-7 pathway. Therefore, we first analyzed fracture healing in control and diclofenac-treated mice, where we not only found a significant impairment of fracture healing due to diclofenac treatment as assessed by biomechanical testing and mCT imaging, but also found high coexpression of bone morphogenetic protein-7 (BMP-7) and cyclooxygenase-2 (COX-2) within the fracture callus of both groups. To experimentally address the possible interaction between BMP-7 and COX-2, we then induced ectopic bone formation in control (n ¼ 10) and diclofenac-treated mice (n ¼ 10) by application of BMP-7 (recombinant human OP-1, rhOP-1) into the hamstring muscles. After 20 days of treatment, each ectopic bone nodule was analyzed by contact-radiography, mCT, histology, and histomorphometry. Diclofenac application decreased the trabecular number and bone mass in the ectopic bone nodules significantly due to reduced osteoblast number and activity. These data demonstrate that the bone anabolic effect of BMP-7 and fracture healing is impaired by diclofenac application, and suggest that the potential negative impact of NSAIDs on fracture healing is, at least in part, due to interference with BMP-7 signaling. ß 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res Keywords: BMP-7 (OP-1); NSAIDs; fracture healing; ectopic bone formation; histology Nonsteroidal antiinflammatory drugs (NSAIDs) are often used to treat pain and inflammation in patients with chronic musculoskeletal diseases and acute injuries, such as fractures. 1 The action of NSAIDs is based on the inhibition of cyclooxygenase (COX) leading to a reduction in prostaglandin production and its effects on inflammation and pain. 2 Two genes, Cox1 and Cox2, encode cyclooxygenases. The first generation of drugs developed, the nonsteroidal antiinflammatory drugs (e.g., diclofenac), inhibit the activities of both COX enzymes. Newer generations of NSAIDs (coxibs) that selectively inhibit COX-2 were developed. 3 These drugs were shown to have less adverse effects, like gastric bleeding, but still have antiinflammatory and analgetic effects. 4 In recent years, many studies reported an inhibitory effect of NSAIDs on fracture healing in animals due to impaired bone formation. 5–8 There is also a large number of studies that demonstrated an interaction of COX and bone morphogenetic proteins (BMPs) in bone and the role of NSAIDs. 9–13 Bone morphogenetic protein-7 (BMP-7), clinically known and used as OP-1, plays an essential role in bone formation and fracture healing by inducing mesenchymal cell differentiation, proliferation, and maturation into osteoblasts. 14–20 BMP-7 is a member of the transforming growth factor-beta superfamily, and acts through receptors on local connective tissue stem and progenitor cells, either as a soluble factor or a matrix-bound protein. 21 The biological activity of recombinant human OP-1 (rhOP-1) to improve the healing of bone defects has been shown in a variety of animal models. 22–24 Also, clinical studies provided supportive evidence for the use of OP-1 in the treatment of nonunions. 25–27 Furthermore, rhOP-1 has been shown to induce ectopic bone formation in a rat model of osteoinductivity. 16,28 The aim of the present study was to determine if the impairment of bone healing by systemic NSAID applica- tion is, at least in part, due to an interaction of NSAIDs with the bone anabolic BMP-7 pathway. Therefore, we analyzed fracture healing and BMP-7–induced ectopic bone formation in control and diclofenac-treated mice by contact-radiography, mCT, histomorphometry, immuno- histochemistry, and biomechanical testing. METHODS Mice Twelve-week-old female C57BL/6-mice were purchased from the Charles River Laboratories GmbH (Sulzfeld, Germany). All animal experiments were approved by the Institutional Committee for Animal Care and Experiments of Hamburg University. Fracture Healing (Fracturing, mCT, Biomechanical Testing) Fracturing of the right femora was performed on 50 mice. After intraperitoneal anesthesia with a mixture of ketamine (50 mg/ml; 12 mg/10 g body weight; BayerVital, Leverkusen, Germany) and rompun (20 mg/ml; 1.6 mg/10 g body weight; Parke-Davis, Freiburg, Germany), the right femora were fractured in a closed manner with a guillotine-like fracture device under standardized conditions. 29 After closed reduction, the femora were stabilized with an intramedullary Kirschner wire (0.8 mm steel K-wire). Postoperative X-ray examinations were performed to document the fracture configuration, and to verify the correct fracture reduction and stabilization. After JOURNAL OF ORTHOPAEDIC RESEARCH 1 Alexander S. Spiro and F. Timo Beil contributed equally to this work and therefore share first authorship. Correspondence to: Michael Amling (T: þþ49-40-74105-6083; F: þþ49-40-74105-8010; E-mail: amling@uke.uni-hamburg.de) ß 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.