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Material and Methods:Operative Technique: 20 New Zealand white rabbits were used. The operative procedure was performed according to the observations of Oni et al. (1). After anaesthesia the right tibia was exposed using an anteromedial approach. The tibia was cut in the diaphysis using a dental burr. The periosteum was stripped proximal and distal from the created cut. The bone marrow was reamed. Then a Steinman-pin was introduced into the marrow cavity in a retrograde manner. A silastic tubing was placed over the fracture site and fixed with two cerclage wires. The rabbits were housed at free cage activity postoperatively. After 4 weeks the silastic tubing is removed percutaneously.
Validation of the Model: The rabbits were followed up radiologically after 1, 2, 4, 6, 8, and 16 weeks. Histology was examined after 8 and 16 weeks using H&E staining techniques. 2 rabbits were used for the assessment of new bone formation after 4 and 12 weeks using Tetracyclin labelling and plastic sectioning.
Validation of a Percutaneous Gene Therapy Approach: Using an in vivo gene therapy procedure an adenoviral vector carrying a marker gene (Ad/CMV-LacZ coding for bacterial β-galactosidase) was injected percutaneously into the non-union site. Expression of the marker gene was assessed after 2, 7, 14, 21 and 28 days. The samples were fixed and stained for bacterial β-galactosidase using the X-Gal staining protocol. Histological sections were cut after decalcification in 20% EDTA.
Results: 19/20 rabbits developed a non-union after 8 weeks. 1 rabbit had a low grade infection after the primary procedure and had to be euthanised. There was no callus formation around the non-union site at any given time. The bone ends appeared atrophic after 4 weeks. There was no spontaneous healing after 16 weeks. The non-union was clinically unstable and radiologically manifested at the time of euthanasia. Histologically there was no callus formation in the surrounding of the non-union site. There was a marked focal necrosis of the bone ends and an accompanying soft tissue ingrowth into the non-union site. However, an inflammatory response could not be detected at any of the time points. The tetracyclin labeling did not show any apositional bone growth after the sheath removal. The histological appearance of the non-union site closely resembled the human atrophic non-union. The feasibility study for the percutaneous gene delivery using an adenoviral marker vector showed gene expression near the non-union site within the fibrous scar tissue. The gene expression causing blue staining of the infected cells could be detected up to 28 days.
Conclusion: The described atrophic non-union model is reliable and shows a true atrophic non-union. The local percutaneous gene delivery into the non-union site using an adenoviral vector is feasible. This approach offers great therapeutic potential for the enhancement of bone healing in an atrophic non-union when therapeutic genes coding for growth factors are used.
