Gene therapy for treatment of infected non-unions and novel methods for early diagnosis of impaired fracture healing

Abstract

Non-union and infected non-union are devastating complications of long-bone fracture repair of both human and veterinary patients. Novel methods for treatment of impaired fracture healing, particularly infected non-union, are required. While studies have demonstrated the efficacy of adenoviral transfer of bone morphogenetic protein-2 (Ad-BMP-2) for enhancing healing in non-union models, there have been no studies evaluating the use of Ad-BMP-2 for enhancing healing of infected non-unions.

Early diagnosis of non-union and infected non-union is essential for a favorable outcome. Currently available techniques have limitations for use, particularly when metallic implants are used to stabilize the fracture. Therefore new techniques for early diagnosis of non-union and infected non-union are needed.

The purpose of this study was to evaluate the use of gene therapy for enhancing fracture healing in an infected non-union model and to evaluate the use of nuclear scintigraphy and serum bone marker concentration for early diagnosis of non-union and infected non-union.

The objective of the first part of this study (Chapter 2) was to develop and evaluate an infected non-union model in rabbits. Rabbits were used for economical reasons. A 10-mm middiaphyseal femoral defect, stabilized with bone plates and cortical screws was the basic model. A sclerosing agent was used on the end of the bone fragments adjacent to the defect to prevent healing and to facilitate the development of osteomyelitis. Rabbits were inoculated with 107 colony-forming units of Staphylococcus aureus 48 hours after surgery. While there were some complications associated with this model, overall the methods used appeared to result in an infected non-union model that was adequate for evaluating methods to enhance fracture healing.

The objective of the second part of this study (Chapter 3) was to evaluate the use of Ad-BMP-2 for enhancing fracture healing in the infected non-union rabbit model. Radiography, dual energy x-ray absorptiometry, and histomorphometry were used to evaluate healing. Radiographically. rabbits treated with Ad-BMP-2 had earlier initial- and bridging-callus formation, and there were more rabbits with bridging-callus formation compared to control rabbits. Rabbits in the Ad-BMP-2 group also had a higher grade for callus formation. However, there was no difference in bone formation, measured histologically, at 16 weeks between the control and treated rabbits; but treated rabbits that were euthanized prior to 16 weeks did have more endochondral ossification and bone formation compared to control rabbits.

The objective of the final part of this study (Chapters 4 and 5) was to evaluate the use of nuclear scintigraphy and serum bone markers for early diagnosis of non-union and infected non-union. Nuclear scintigraphy using both technetium-labeled diphosphonate and ciprofloxacin was useful for differentiating infected from non-infected fractures in the late phase of healing; however, the accuracy was lower during the early phase. Technetium-labeled diphosphonate may be useful for evaluating callus formation and defect ossification.

Serum markers of bone resorption (deoxypyridinoline crosslinks) peaked at 4 weeks, and markers of bone formation (osteocalcin and bone-specific alkaline phosphatase) peaked at 8 weeks after surgery. Markers of bone formation were lower in infected rabbits at 4 weeks, and osteocalcin was higher in infected rabbits later in healing. The deoxypyridinoline crosslinks were higher in infected rabbits at all time periods. There were only weak associations between serum bone marker concentration and callus formation.

Overall, the results of this study suggest that Ad-BMP-2 may be useful for enhancing healing of infected non-unions: however, in cases with severe cell and tissue damage, ex vivo gene transfer may be better. Nuclear scintigraphy has many limitations for diagnosing osteomyelitis associated with long-bone fractures early in the course of fracture healing: however, serum bone markers may be more useful and require further investigation.