Survival of chondrocytes in rabbit septal cartilage after electromechanical reshaping

Abstract

Electromechanical reshaping (EMR) has been recently described as an alternative method for reshaping facial cartilage without the need for incisions or sutures. This study focuses on determining the short- and long-term viability of chondrocytes following EMR in cartilage grafts maintained in tissue culture. Flat rabbit nasal septal cartilage specimens were bent into semi-cylindrical shapes by an aluminum jig while a constant electric voltage was applied across the concave and convex surfaces. After EMR, specimens were maintained in culture media for 64 days. Over this time period, specimens were serially biopsied and then stained with a fluorescent live-dead assay system and imaged using laser scanning confocal microscopy. In addition, the fraction of viable chondrocytes was measured, correlated with voltage, voltage application time, electric field configuration, and examined serially. The fraction of viable chondrocytes decreased with voltage and application time. High local electric field intensity and proximity to the positive electrode also focally reduced chondrocyte viability. The density of viable chondrocytes decreased over time and reached a steady state after 2-4 weeks. Viable cells were concentrated within the central region of the specimen. Approximately 20% of original chondrocytes remained viable after reshaping with optimal voltage and application time parameters and compared favorably with conventional surgical shape change techniques such as morselization.

FIGURE 1

Experimental procedure to determine viability of chondrocytes after electroforming. A rectangular cartilage specimen is excised from a rabbit septum and placed into the electroforming jig (a). After EMR, cartilage specimen is removed from the jig (specimens reshaped at 5 V for 2 min (i) and 0 V for 2 min (ii) are shown) and thin (>200 μm) section of tissue is dissected from it (b). The thin section is stained with a “live–dead assay” and then imaged using a confocal fluorescent microscope (c). The bulk of the specimen is returned to cell culture (d). After 48–72 h, the specimen is again removed from the culture, and another thin section is excised (e), stained, and imaged for viable cells (f). The process is repeated, and the specimen is returned to culture (g)

FIGURE 2

(a) Magnified image of cartilage section acquired from the green (live) channel of confocal microscope. Live cells are visible as bright round dots in the middle and right-hand side of grayscale image. (b) The same image thresholded for an automated cell count of green (live) cells. (c) Correlation between automated and manual count of green (live) cells

FIGURE 3

Distribution of (a) non-viable (red) and (b) viable (green) cells in septal cartilage observed using, respectively, red and green channels of confocal fluorescent microscope. Non-viable (red) and viable (green) cells are visible as bright round dots on the corresponding grayscale images: (i) control, (ii) immediately after electroforming at 5 V for 2 min, (iii) after electroforming at 5 V for 2 min with electric insulation tape (EIT) inserted between cartilage and electrode. Indicated position of positive and negative electrodes is the same on all images

FIGURE 4

The normalized density (number of cells per unit area) of the green (viable) cells in cartilage after electroforming for (a) 1, (b) 2, and (c) 3 min as a function of time in culture. The content is shown as a percentage of the average number of viable cells observed in samples from control group II before placement in culture. Standard deviation is indicated

FIGURE 5

Digital montage of a thin section of a septal cartilage electroformed at 5 V for 2 min after 62 days in culture media showing distributions of (a) non-viable and (b) viable cells obtained using, respectively, red and green channels of confocal fluorescent microscope. Regular streak pattern on the red-channel image is due to interference of laser light in the cover glass. Positive and negative electrodes were at the concave and convex sides of the sample, respectively

FIGURE 6

Average percentage of live (green) cells in the half of the sample closest to the cathode after treatment for 1 (a) and 2 min (b) and anode after treatment for 1 (c) and 2 min (d). Standard deviation is indicated