Langenbecks Arch Surg (2003) 388:356–365 DOI 10.1007/s00423-003-0403-5 Received: 1 April 2003 Accepted: 17 June 2003 Published online: 20 September 2003 © Springer-Verlag 2003 Abstract Background: Ex vivo gene therapy can induce bone formation when delivery cells carrying the bone morphogenetic protein (BMP) gene are used. The hypothesis for this study was that the cell-mediated gene therapy could improve the heal- ing of bony lesions with severe soft tissue damage. Method: An animal model with a femoral osteotomy lesion associated with soft tissue damage was developed in rats. Muscle-derived cells, genetically engineered to express BMP4, were inserted within the osteotomy gap. Cells genetically engineered to ex- press LacZ were used for the control group. The groups were subdivided with regard to the fixation method: stable and unstable fixation. The rats were killed for histological and ra- diographic evaluation 3 and 6 weeks post-surgery. Results: No callus for- mation was found in the control group at any time point, whereas sufficient callus formation appeared in the treatment group after 6 weeks. A bridging callus with woven bone and hypertrophic chondrocytes was achieved in the treatment group when a stable fixation was used, but failed to appear in unstable fixation. Conclusion: The combination of muscle-derived cells expressing BMP4 and a stable fixation were able to bridge the bone defect within 6 weeks, but with prolonged osteo- chondral ossification. Therefore, the ex vivo gene therapy could be an efficient biological approach to im- prove the treatment of bone lesions with severe soft tissue damage. Keywords Soft tissue damage · Bone healing · Ex vivo gene therapy · Bone morphogenetic proteins MUSCULOSKELETAL SOFT TISSUE CONDITIONING Tim Rose Hairong Peng Arvydas Usas Ryosuke Kuroda Helmut Lill Freddie H. Fu Johnny Huard Gene therapy to improve osteogenesis in bone lesions with severe soft tissue damage Introduction Open fractures in humans, mostly caused by high-veloci- ty trauma, are often associated with a high incidence of non-unions [21, 40]. Animal studies have also shown that the effect on the periosteum and the soft tissue leads to non-union [8, 10, 17], whereas the missing mineralisation of the connective tissue is the result of the missing initial differentiation to cartilage, which is usually calcified to a bony union [9], called endochondral bone formation. This process can be improved by the bone morphogenetic pro- teins (BMPs), which are part of the TGF-b superfamily, first described in 1965 by Urist [42]. The BMPs initiate a multi-step cascade of events, i.e. migration of progenitor cells, proliferation of mesenchymal cells, differentiation to chondrogenic or osteogenic cells, vascular invasion and remodelling of bone [36]. The up-regulated expres- sion of BMPs following a fracture, particularly the BMP 2/4 and the osteoprogenitor protein 1 (OP-1), is caused by the osteogenic cells of the thickened periosteum near the fracture gap [9, 31]. Additionally, an intense expression of the receptors for the BMPs was shown in the osteo- blasts and in the periostal cells [7, 31]. The osteo-precur- sor cells from the surrounding muscle tissue of the frac- ture can also participate in endochondral bone formation, due to their positive response to BMPs and their differen- tiation to chondrogenic and osteogenic cells [5, 19, 20, 24, 27, 30, 42, 43]. T. Rose ( ✉ ) · H. Lill Department of Trauma and Reconstructive Surgery, University of Leipzig, Liebigstrasse 20a, 04103 Leipzig, Germany e-mail: rost@medizin.uni-leipzig.de Tel.: +49-341-9717300 Fax: +49-341-9717319 T. Rose · H. Peng · A. Usas · R. Kuroda J. Huard Growth and Development Laboratory, Department of Orthopedic Surgery, Children’s Hospital of Pittsburgh and University of Pittsburgh, Pittsburgh, Pennsylvania, USA T. Rose · R. Kuroda · F. H. Fu Department of Orthopedics, University of Pittsburgh, Pittsburgh, Pennsylvania, USA