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QUANTITATIVE IMPEDANCE ANALYSIS OF CARTILAGE GRAFT TISSUE FOLLOWING IMPLANTATION INTO CANINE DEFECTS

INTRODUCTION: A minimally invasive, hand-held impedance diagnostic device was used to quantify changes in articular cartilage after surgical treatment of chondral defects created in the canine knee joint model. Effects of the procedure on the impedance of both treated and uninvolved tissue were examined. Previous studies have shown electrical impedance to be a sensitive indicator of biochemical composition and tissue integrity. The objectives in this study were (1) to validate the ability of the device to gauge graft repair by comparing impedance measurements with biomechanical measurements and histology, and (2) to quantify the effects of the harvest and graft procedure on the impedance of untreated articulating tissue with comparison to tissue hydration and GAG content. METHODS: Probe manufacture: A 4.5-mm diameter, 10-cm long arthroscopic probe was designed and developed [1,2] to measure the electrical and electromechanical integrity of cartilage at articulating joint surfaces. The four 1.59 mm Ag-AgCl electrodes are placed on the cartilage (Fig. 1) and deliver 10 μA/mm of sinusoidal current at the tissue surface. Surgical treatment: Autologous articular cartilage was harvested from the trochlear ridge of the left knees of six mature hound dogs. A strip of approximately 2025mm by 3-5 mm tissue was removed down to the tidemark. The chondrocytes were extracted and expanded in monolayer up to third passage and then seeded at 1x10 cells/disk into EDAC cross-linked, 95% porous, porcine type-II collagen matrices. (Geistlich Biomaterials, Wulhusen, Switzerland). After four weeks the matrices were implanted in partialthickness 4-mm diameter surgically constructed defects in the proximal and distal patellar groove of the right knees of each dog. The animals were sacrificed sixteen weeks after implantation. This animal experiment was approved by the Brockton/West Roxbury VA Animal Care Committee. Impedance Measurements were made in situ at the two defect sites in the patellar groove and then on adjacent visually normal tissue, as well as at three sites on the medial and lateral halves of the excised right and left patellae (Fig. 2). The feedback-controlled constant current density was applied at frequencies of 1000, 300, 100 Hz with a penetration depth of 0.7mm[2]. Tissue impedance was calculated as the measured electrode voltage divided by the driving current, and was normalized to the averaged electrodeelectrolyte interfacial impedance. This interfacial impedance was measured in 0.15M phosphate buffer solution at intervals before, during, and after each series of tissue measurements. Mechanical Testing: In situ indentation tests were performed on repair tissue (on bone) from the distal graft and on articular cartilage from the corresponding site in the contralateral joint. Three sequential step displacements to 10, 15, and 20% strain were applied, and the relaxed equilibrium loads recorded. Sinusoidal displacements at 1-% strain amplitude were applied in the 1.0 to 0.005 Hz frequency range, superimposed on the 15% static strain, and the resulting loads recorded. The thickness of the cartilage was measured using a needle-ramp method. Histology: Proximal grafts were placed in 10% formalin immediately after specimen procurement. Distal grafts were fixed in formalin immediately after mechanical testing. After fixation, specimens were decalcified in 15% EDTA, dehydrated, and embedded in paraffin. 5 μm sections were stained with Safranin O; semiquantitative assessment of the degradative changes was made using the Mankin scale. Biochemical Analysis: The articular cartilage was scraped from the surface of the patellae. The samples were weighed wet, lyophilized (48-72 hours), weighed dry, and then digested in 1mg/mL proteinase K and analyzed for total sulfated glycosaminoglycan (GAG) using the dimethylmethylene blue (DMMB) dye-binding assay with shark chondroitan-6-sulfate as the standard. Tissue hydration was calculated from the wet and dry weights. GAG content was normalized to the wet weight of the undigested tissue. Statistics: Repair tissue in the grafts was compared to adjacent visually normal tissue on the femoral patellar groove. Tissue on the right patellae, articulating against the graft sites, was compared with that on the left patellae, articulating against the harvest sites. The Student’s t-test and the Fisher Exact test were used, with the criterion of p<0.05 for significance.