Are OPG and RANKL Involved in Human Fracture Healing? Julia Ko ¨ ttstorfer, 1 Anita Thomas, 2 Markus Gregori, 1 Mathias Kecht, 1 Georg Kaiser, 1 Stefan Eipeldauer, 1 Kambiz Sarahrudi 1 1 Medical University Vienna, University Clinic for Trauma Surgery, Wa ¨hringer Gu ¨rtel 18-20 1090, Vienna, Austria, 2 Department of Internal Medicine III, Division of Endocrinology and Metabolism, Medical University Vienna, Gender Medicine Unit, Wa ¨hringer Gu ¨ rtel 18-20 1090, Vienna, Austria Received 5 April 2014; accepted 24 July 2014 Published online 12 September 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jor.22723 ABSTRACT: Human fracture healing is a complex interaction of several cytokines that regulate osteoblast and osteoclast activity. By monitoring OPG (osteoprotegerin) and sRANKL we aimed to possibly predict normal or impaired fracture healing. In 64 patients with a fracture of a long bone serum level of sRANKL and OPG were evaluated with respect to bony union (n ¼ 57) or pseudarthrosis (n ¼ 7). Measurements were carried out at admission and at 1, 2, 4, 6, 8, 12, 24, and 48 weeks after the injury. Patients’ serum levels were compared to 33 healthy controls. Fracture hematoma contained significantly higher sRANKL and OPG concentrations compared to patients serum (p ¼ 0.005, p ¼ 0.028). OPG level in fracture hematoma was higher compared to the unions serum level (p ¼ 0.028). sRANKL was decreased in unions during the observation period. In non-unions sRANKL and OPG levels showed a variable course, with no statistical significance. This is the first study to document the course of OPG and sRANKL in normal and delayed human fracture healing emphasizing its local and systemic involvement. We provide evidence of strongly enhanced OPG levels in patients with a long bone fracture compared to healthy controls. Further, levels of free sRANKL were decreased during regular fracture repair. ß 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1557–1561, 2014. Keywords: fracture healing; RANKL; OPG BACKGROUND Fracture healing in humans is a cascade of intra- membranous bone formation and enchondral ossifica- tion. This physiologic process is regulated by complex interactions of hormones, growth factors, and extracel- lular matrix proteins. 1 Within the last decade the bone remodeling process has been intensively investigated. Numerous cytokines, angiogenic factors, proteases, and morphogens with significant roles in fracture healing were identified. 2,3 Among these, cytokines that interact with the TNFa (tumor necrosis factor a) receptor family seem to be crucial in osteoclastogene- sis. The cellular interactions of osteoblasts and osteo- clasts are critical and mainly regulated upon the RANKL/OPG/RANK complex. 4,5 RANKL (receptor ac- tivator of N-kB ligand) is a potent physiological induc- er of osteoclast activity. RANKL, by interacting with the RANK (receptor activator of N-kB), stimulates the preosteoclast differentiation and increases osteoclast activity. Membrane RANKL is expressed by many cell types especially osteoblasts. The cell-bound form of RANKL is the most common and has been suggested to be somewhat more potent than soluble RANKL in stimulating osteoclastogenesis in vitro. But the soluble form evolves by splitting a truncated ectodomain by a TNF-converting enzyme-like protease and is measur- able in circulation. 5,6 OPG (osteoprotegerin), a glycoprotein mainly syn- thesized by osteoblasts is the corresponding decoy receptor. OPG, by binding to RANKL is a competitive inhibitor of RANKL action and causes its anti-resorp- tive effect. Thereby, OPG suppresses bone resorption and increases bone mass. 7 Thus, the OPG/RANKL ratio reflects the rate of bone resorption in general and its role is well recognized in osteoporosis. 8 Interesting- ly, it has been shown, that an increased RANKL/OPG ratio in iliac bone samples is accompanied by increased fracture susceptibility. 8 Comparable findings were reported by Mizuno et al. who identified a relative OPG deficiency causing bone loss in a murine model. 9 On the other hand, OPG administration has been shown to be effective in preventing bone resorbtion. 10 Quantification of serum RANKL and OPG concen- tration during the course of human fracture healing after long bone fracture is still lacking in current literature. We therefore observed the expression of these cytokines in patients with physiologic and delayed fracture healing. METHODS This study was approved by the Ethic Committee of the Medical University of Vienna and conducted in accordance with the declaration of Helsinki. Patients gave informed written consent to be enrolled in the study. The recruitment parameters, sample collection schedule, matching process, patient demographics as well as exclusion criteria of this study have been previously published in detail. 11 In brief, between April 2006 and 2008 a consecutive series of 113 patients with meta-/diaphyseal fractures of long bone (humer- us, femur, lower leg, and forearm) and surgical treatment were included. In order to have a homogenous study group and due to the strict selection criteria 49 patients with incomplete data were excluded from further investigation. Finally the data of 64 patients were analyzed (33 males and 31 females). Mean age of the patients was 59.3 years (range, 17–90 years). Patients’ serum was collected following a stand- ardized time schedule. The follow up examination was based on clinical and radiological examination at 1, 2, 4, 6, 8, 12, 24, and 48 weeks after trauma. All patients were followed up for at least six months after the operation. The diagnosis of bony consolidation or delayed union was based on exercise-induced Conflict of interest: None. Grant sponsor: Medical Scientific Fund of the Mayor of the City of Vienna; Grant number: 12069. Correspondence to: Kambiz Sarahrudi (T: þ43 1 40400 5901; F: þ43 1 40400 5949; E-mail: kambiz.sarahrudi@meduniwien.ac.at) # 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. JOURNAL OF ORTHOPAEDIC RESEARCH DECEMBER 2014 1557