Session I - Basic Science

A Model for Persistent Transgene Expression of BMP-2 in Different Cell Types after Adenoviral Gene Transfer in a Femur Fracture Model of the Rat

Christian W. Mueller, MD¹; Martijn van Griensven, PhD¹; Andreas Barke, PhD¹; Wilhelm Lindenmaier, PhD²; Christian Krettek, MD, FRACS¹; Torsten G. Gerich, MD¹;

¹Medizinische Hochschule Hannover, Department of Trauma Surgery, Hannover, Germany;

²Gesellschaft fur Biotechnologische Forschung, Braunschweig, Germany

Purpose: Adenoviral vectors can be used to directly convert cells of callus formation in fracture healing to express a transgene, the bone morphogenetic protein 2 (BMP-2) vector. However, currently it is not known which cell types are preferentially infected and the time of expression during fracture healing. The aim of this study was to determine the possible tropism of an adenoviral BMP-2 vector in a fracture model and show the possible time course of expression of BMP-2 in the transfected cell types of the fracture healing site.

Methods: The adenoviral type 5 vectors used in this study were replication incompetent viruses. Ad5CMVlacZE1/E3 encodes ß-galactosidase, while Ad5cos45gfp encodes green fluorescent protein (GFP). Femora of 35 Sprague Dawley rats were fractured, and fractures were stabilized with a 1.2-mm K-wire. Fifteen rats were used for the Lacz-vector;15 rats were used for the GFP-vector; and 5 rats served as controls and received an injection with wild type adenovirus type 2. Three days after stabilization with K-wire, 10¹² pfu of viral suspension were injected into the fracture zone. As a control, five animals received injections of adenovirus type 2. Animals were sacrificed after 3 days, 1, 2, and 4 weeks. The fracture zone was prepared for histological evaluation. Staining and counterstaining for the Lacz-vector was performed using X-gal and eosin. Specimens transfected by the GFP vector were examined under fluorescent light at a wave length of 580 nm.

Results: Fractures healed radiographically within 2 to 3 weeks. All specimens were examined for ß-galactosidase and GFP expression. Fibroblast and osteoblasts within callus tissue displayed a high transgene expression (week 1). Chondral progenitor cells expressed both transgenes also. During ossification we found incorporation of the transduced cells into the newly mineralized bone. A decrease of expression was observed during the observation period, but a good level of gene expression was even found at the time of solid healing of the fracture. Additionally,
expression of transgenes occurred in all phases of fracture healing, as granulation tissue, ossification, and remodeling of the original bone, proving a persistent transgene expression.

Discussion: Bone morphogenetic proteins, especially BMP-2, have been shown to induce bone formation. Recombinant BMP-2 combined with different matrices has been shown to be capable of healing bony defects in rat femur and rabbit ulna models. Still, the kinetics of BMPs after a single application remains unclear, but it is expected not to provide a sufficient concentration for a sufficient period of time. In this experimental study, we have demonstrated that all cells of the primary callus can be transfected with adenoviral vectors. Transgene expression was found in differentiated cells of the callus, such as chondrocytes, osteoblasts, and osteoclasts. Furthermore, expression was persistent throughout the whole course of fracture healing.

Fracture management often is complicated by posttraumatic defects of bone substance. In the United States alone, more than 250,000 procedures per year require bone grafts to reconstruct bony defects. Currently, autogenous cancellous bone graft is regarded as the best material available for this use. Autogenous cancellous bone graft features osteo inductive growth factors, osteogenic bone marrow stem cells, and an osteoconductive bone matrix, which are considered essential for optimal fracture healing. Nevertheless, the excision of autogenous cancellous bone graft is associated with disadvantages, such as surgical morbidity, pain, and infection of the donor site. The amount of suitable material available is limited, too. Synthetic composite grafts which are capable of inducing bone formation mimic the stated features of fracture healing and could be used as an alternative to bone graft. Osteoinduction bone morphogenetic proteins (BMPs) are among the most promising growth factors. BMPs can either be applied as purified proteins or as vectors. Among the viral vectors, recombinant adenovirus is widely used as a feasible gene delivery vehicle.

Conclusion: The model presented in this study provides a useful tool for further investigation of the adenoviral transfer of growth factors such as BMP-2. This should help to elucidate the possibilities for influencing formation of callus and the process of fracture healing. Thus, the use of synthetic composite grafts could be greatly enhanced.