Expression of EDA+ and EDB+ fibronectin splice variants in bone $ O. Kilian a, * , R. Dahse b , V. Alt a , L. Zardi c , J. Rosenhahn d , U. Exner e , A. Battmann f , R. Schnettler a , H. Kosmehl b a Department of Trauma Surgery, University of Giessen, Germany b Department of Pathology, HELIOS Medical Center GmbH Erfurt, Germany c Dipartimento di Immunologia Clinica e sperimentale Genova, Italy d Institute of Human Genetics and Anthropology, University of Jena, Germany e Department of Orthopedic Surgery, University Balgrist of Zurich, Switzerland f Department of Pathology, University of Giessen, Germany Received 14 April 2004; revised 9 August 2004; accepted 10 August 2004 Available online 19 October 2004 Abstract The extracellular matrix component fibronectin (fn) has fundamental functions in cell attachment, differentiation, proliferation, and migration. Isoforms of cellular fibronectin, named EDA+ fibronectin or embryonal EDB+ fibronectin, are generated by alternative splicing of its mRNA precursors. Little is known about the expression of EDA+ and EDB+ fibronectin splice variants in human bone. The aim of this study was to investigate the expression pattern of fibronectin splice variants in bone cell lines and in different human bone tissue samples (mature bone, early stages of fracture healing, hypotrophic nonunion, osteosarcoma). Analysis was done by immunostaining with recombinant and monoclonal antibodies, qualitative RT–PCR and LightCycler-based real-time quantitative RT–PCR assay. In osteoblast and osteosarcoma cell lines, abundant expression of EDA+ and EDB+ fibronectin was found in immunocytochemistry. High transcription levels of both splice variants mRNA were seen in quantitative RT–PCR in osteosarcoma cell lines. In mature bone, EDA+ and EDB+ were not detectable in immunohistochemistry. Transcription of mRNA in both splice variants was absent in these samples. Early stages of fracture healing and osteosarcoma cell samples exhibited extensive staining for EDA+ and EDB+ fibronectin, and high mRNA levels were found. Both osteosarcoma and bone fracture healing tissue expressed high mRNA levels of the fibronectin splice variants independent of benign or malignant behavior. Low level of EDA+ fibronectin mRNA transcription and focal immunohistochemical staining of EDA+ fibronectin was found in hypotrophic nonunions, whereas EDB+ fibronectin was not detected by immunohistochemistry and qualitative or quantitative PCR. EDA+ fibronectin was found in granulation tissue-forming processes in bone independent from bone-forming activity. EDB+ fibronectin was seen only in high-turnover new osteoid-forming processes like early stages of fracture healing and osteosarcoma and was absent in low- turnover processes like mature bone and hypotrophic nonunion. Both EDA+ and EDB+ fibronectin mark active processes in bone without differentiation between malignant or benign activity. In conclusion, EDA+ and EDB+ fibronectin splice variants are strong markers for active fibrogenetic and osteoid-forming processes in human bones. D 2004 Elsevier Inc. All rights reserved. Keywords: Extracellular matrix protein; Fibronectin; Fibronectin splice variants; EDA+ fibronectin; EDB+ fibronectin Introduction Fibronectin (fn) is a high molecular mass glycoprotein that can be found in soluble form and in high concentration in blood plasma (plasma fibronectin) [31,32,46]. In low concentration, cellular fibronectin is distributed throughout 8756-3282/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bone.2004.08.008 $ This publication reflects only the authors’ view. The European Commission is not liable for any use that may be made of the information contained. * Corresponding author. Department of Trauma Surgery, University of Giessen, Rudolf-Buchheim-Str. 7, 35292 Giessen, Germany. Fax: +49 641 9944609. E-mail address: olaf.kilian@chiru.med.uni-giessen.de (O. Kilian). Bone 35 (2004) 1334 – 1345 www.elsevier.com/locate/bone