TISSUE ENGINEERING OF TENDONS AND LIGAMENTS

Participants: P.V. Patil, K.M. Kozloff, J.E. Carpenter, S.A. Goldstein

Keywords: gene therapy, healing response, soft tissue

Introduction

Ruptures or tears of tendons and ligaments pose substantial problems to patients. These injuries often result in significant loss of joint function and long term problems such as degenerative joint disease. With current techniques, the time to effective repair is long and substantially delays the return to normal activity.

Our global objective is to model injuries or defects of tendons or ligaments in an animal model and apply tissue engineering principles to test whether the injury or defect can be healed more effectively than in clinical practice. The specific goals of this project are to evaluate the potential for a localized gene therapy to aid in the healing or replacement of tendons and ligaments.

Materials and Methods

In order to evaluate the possibility of delivering a therapeutic gene to the wound site, we have chosen a reporter gene to test for transfection into local cells. In this model we have chosen to use plasmid DNA that encodes for b -galactosidase. b -galactosidase has been used in many different applications as a reporter gene and histologic analysis is well documented.

Middle-third patellar tendon defects were created in 4-week-old Sprague-Dawley rats through a lateral approach using fine scalpel resection of the middle-third. Collagen sponges with or without plasmid DNA encoding for b -galactosidase were placed within the created defect. Soft tissue was closed in layers in order hold sponge material within the defect. Rats resumed normal cage activity and were sacrificed at 4 weeks. Upon sacrifice, the knee and tibia was dissected free of the femur, keeping the patellar tendon and patella intact for mechanical testing and histologic analysis.

The patellar tendon was prepared for b -galactosidase staining according to standard staining protocols previously used in this laboratory forb b -galactosidase. The tissue was fixed in 4% paraformaldehyde and embedded in JB-4. The tissue was then sectioned at 7µm and analyzed for marker gene expression.

Tendons indicated for mechanical testing were prepared by embedding the tibia in PMMA and gripping the intact patella by a custom-made adjustable grip apparatus. The tendon-bone complex will be tested mechanically in tension to failure to determine the mechanical integrity of the wound site when compared to controls.

Progress

To date, four rats have been operated on. One of these served as a surgical unilateral control, implanting a collagen sponge without gene into one patellar tendon defect and leaving the other leg unoperated. The remaining three rats had sponges implanted bilaterally. An experimental and control sponge were randomly assigned to each limb and implanted into the site. To date, all rat tendon-patella-tibia complexes have been dissected free of soft tissue. The cohort of three animals have been embedded in JB-4 currently await analysis. The fourth collagen only and unoperated contralateral limb will be used to establish a mechanical testing protocol. The custom adjustable grip apparatus for tensile testing is currently being developed and manufactured.