Session III - Polytrauma Fracture Healing
Feasibility of a Gene Therapy Approach for the Treatment of an Atrophic Non-union: An Experimental Study in Rabbits
Christian Lattermann, MD; Janey D. Whalen, PhD; Axel Baltzer, MD; Kurt R. Weiss; Christopher H. Evans, PhD, DSc; Paul D. Robbins, PhD; Gary S. Gruen, MD, University of Pittsburgh, Orthopaedic Department, Pittsburgh, PA
Introduction: It is estimated that about 5.6 million fractures occur annually in the United States of which approximately 5-10% result in a delayed- or a non-union leading to 250,000 cancellous bone grafts every year. Thus the evolving costs of a nonunion pose a substantial burden to the socio-economic system. The incidence of delayed or nonunions due to an impaired healing response rises with patient age. In light of a growing patient population over 60 years of age, the tolerance to extensive operative procedures is significantly reduced. In order to restore a healing response in an atrophic tissue the presence of growth factors and precursor cells is critical. The direct application of growth factors, however, suffers from their short biological half-life (minutes to hours). One way to overcome this problem is the use of gene therapy. This technique provides a high potential for sustained growth factor delivery and can easily be combined with a minimally invasive, percutaneous injection technique. In this study we aim to evaluate the feasibility of a gene therapy protocol in an atrophic nonunion model using a percutaneous, minimally invasive, injection technique.
Material / Methods: We used 29 female NZW rabbits for this study. The animals were housed according to the NIH guidelines for the use and care of laboratory animals.
Operative procedure: The rabbits were anaesthetized and draped in a sterile fashion. An antero-medial incision was performed on the right tibia. The tibia was exposed and the periosteum was removed. A mid-diaphyseal cut through the tibia was performed using a dental burr. The marrow cavity was reamed and a retrograde Steinmann-pin was inserted into the tibia. A silastic tubing was placed around the fracture site and the fracture was reduced, using the silastic tubing as a reduction tool. The silastic tubing was then securely fixed around the fracture site with two cerclage wires. The rabbits were kept at free cage activity for 28 days. Then the silastic tubing was removed through a percutaneous incision. 4 weeks after removal of the silastic tubing the rabbits presented an established atrophic nonunion.
Establishment of the atrophic nonunion: 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.
Feasibility of percutaneous gene transfer to an atrophic nonunion site: 9 rabbits were used for this experiment. The rabbits were injected with 1x108 pfu (plaque forming units) of an adenoviral vector carrying the LacZ marker gene (Ad/CMV-LacZ). The injection was done under fluoroscopic control using a Hamilton syringe. The rabbits were sacrificed at 1,2 and 4 weeks after injection. Both rabbit tibiae were harvested and the soft tissue was stripped. The fibrous capsule of the nonunion was left intact. The right tibia was cut 2cm proximal and distal to the nonunion and the Steinmann-pin was removed. The tissue was fixed in 4% paraformaldehyde for 7 days. The detection of ß-galactosidase transgene expression was done for 48 hours at 37oC with 5-bromo-4-chloro-3-indolyl-b-galactopyranoside solution (X-gal; Sigma). Then the decalcification was performed in 20% EDTA for 4 weeks. The tissue was embedded in paraffin and 5-7mm sections were cut. Eosin staining was performed in order to detect the blue-colored, ß-galactosidase positive cells.
Results: 28/29 rabbits developed a nonunion 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 nonunion site at any given time. The bone ends appeared atrophic after 4 weeks showing empty osteocytic lacunae. There was no spontaneous healing after 16 weeks. The nonunion was clinically unstable and radiologically manifested at the time of euthanasia. Histologically there was no callus formation in the surrounding of the nonunion site. There was a marked focal necrosis of the bone ends and an accompanying soft tissue ingrowth into the nonunion site. However,an inflammatory response could not be detected at any of the time points. The histological appearance of the nonunion site closely resembled the human atrophic nonunion.
The percutaneous gene delivery of the LacZ marker gene into the nonunion site was successful at 1 and 4 weeks. We could not observe a significant inflammatory reaction to the LacZ infected cells. Gene expression was mainly found in the surrounding soft tissue of the atrophic bone. The bone itself did not show any LacZ staining .
Conclusion: We developed a reliable animal model for an atrophic nonunion. We demonstrated on this model that a protein can be delivered into the site of an atrophic nonunion in a sustained manner by a percutaneous minimally invasive injection technique using gene therapy. Protein expression lasted at least for 4 weeks, which might be sufficient for the reestablishment of a healing response in an atrophic situation. This approach offers great therapeutic potential for the enhancement of bone healing in an atrophic nonunion when therapeutic genes coding for vascular or bone growth factors are used.