Human septal cartilage was obtained from 10 patients undergoing septal surgery. The average age was 23.8 ± 4.0 years (range, 18 to 30 years) with 7 men and 3 women. Harvested cartilage was carved to 1-2 mm thick with a shape of rectangle, and stored in normal saline solution at room temperature until use. Total 56 specimens were irradiated by 1,460 nm diode laser (CyT-ML1450, CyTroniQ, Cheonan, Korea) within 1 hour after harvest of the cartilage. Ethics approval was obtained from the Internal Review Board of the Dankook University Hospital.

Laser was delivered via an optic fiber (2 mm in spot diameter) in two different patterns: spot and linear. In spot pattern, the specimens were irradiated with laser powers of 0.5 W, 1.0 W, and 2.0 W for exposure time of 5, 10, and 20 seconds. In linear pattern, specimens were irradiated with laser powers of 0.5 W, 1.0 W, and 2.0 W in a to-and-fro manner for 10 and 20 seconds each.

Two separate 3×4 cm-sized segments of the auricular cartilage was harvested from six New Zealand white rabbits after elevating submucoperichondrial flap in each ear. The harvested cartilage was immediately divided into 5×10 mm sized rectangular slabs with a razor blade. More than 16 specimens were irradiated by 1,460 nm diode laser for 5 seconds at laser powers of 0.3 W, 0.5 W, and 1 W, respectively. The schematic diagram of experimental protocol was described in Fig. 1. These settings were decided after a preliminary study to obtain a range of laser parameters required to change the shape of the cartilage without significant thermal injury.

The average depth and width of the thermally damaged area were measured by a single observer who was unaware of the experimental conditions using embedded software in the Olympus microscope system.

The control and reshaped cartilage were reimplanted into a subperichondrial pocket of the rabbit ear. The quilting sutures were performed to provide fixation between perichondrium and reimplanted cartilages. Ceftizoxime was administered intramuscularly to prevent wound infection.

Cartilage specimens were reharvested at 1, 2, and 4 weeks after reimplantation. Subperichondrial planes were dissected carefully with iris scissors, and cartilages were harvested from the subperichondrial pockets and prepared for viability assessment and histologic examination (Fig. 1).

Cell viability assay were performed on the human and rabbit cartilage immediately after laser irradiation. The cartilages were sliced into 400 µm-thick-cross sections. They were stained with calcein acetoxymethylester (calcein AM) and ethidium homodimer-1 (EthD-1; Molecular Probes, Eugene, OR, USA). Calcein AM is an indicator of living cells that have intact cell membranes and esterase activity and Eth-D-1 is an indicator of dead cell. It can only permeate cells with compromised membranes. Stained specimens were inspected with a confocal microscope at 10× magnification (LSM 510 META, Carl Zeiss, Jena, Germany).

Harvested rabbit cartilages were examined at 1, 2, and 4 weeks after reimplantation by the same method to evaluate regeneration of damaged cartilage.

Hoechst and propidium iodide (PI) staining was used to evaluate the mechanism of thermal injury in rabbit cartilage after laser irradiation. Hoechst 33342 (Sigma-Aldrich Co., St Louis, MO, USA), a blue-fluorescence dye (excitation/emission maxima ≈350/461 nm when bound to DNA), stains the condensed chromatin in apoptotic cells more brightly than normal chromatin. PI, a red-fluorescence dye (excitation/emission maxima ≈535/617 nm when bound to DNA), is only permeant to dead cells. The staining pattern resulting from the simultaneous use of these 2 dyes makes it possible to distinguish normal, apoptotic, and necrotic dead cell populations by fluorescence microscopy.

The first stain (Hoechst solution, 4 µL) was added to 2 mL of medium. After incubating the sample in Hoechst solution overnight, the samples were rinsed with Dulbecco's phosphate-buffered saline for a few seconds. Then, the second stain (PI, 4 µL) was added to 2 mL of medium. Sample slides were finally examined by confocal microscopy (405 nm and 543 nm).

The remaining portions of the specimens were fixed in formalin and serially dehydrated using graded ethanol solutions and subsequently embedded in paraffin. Then, 6-µm sections were obtained, and stained with hematoxylin-eosin (H&E), and finally examined with a light microscope at 200× magnification.

Laser irradiation with given power and exposure time produced shape changes in all human septal cartilages without definite tissue ablation (Fig. 2). Cell viability test using live/dead assay showed a clear demarcation between live cells (green) and dead cells (red) in histology and the extent of the damaged area increased accordingly as the laser power and the exposure time increased (Fig. 3). The average depth and width of the thermally damaged area showed a linear correlation according to the laser power and the exposure time, irrespective of the irradiation method (spot or linear) (Fig. 3).

Laser exposure to rabbit auricular cartilage created a similar shape changes like in human septal cartilages. The extent of the damaged area in the rabbit auricular cartilage increased with the increase of the laser power (0.3-1.0 W) and the extent of dead chondrocytes in the 0.3 W and 0.5 W treatment groups was less extensive than in the 1 W treatment group (Fig. 4A). The depth of thermal damage showed no significant difference among treatment groups due to thin thickness of the auricular cartilage. However, the width of thermal damage increased from 736 µm in 0.3 W treatment group to 1,377 µm in 1 W treatment group (Table 1).

Hoechst and PI staining showed a dominance of red staining without blue fragmented nucleus-like an apoptotic body after laser irradiation, suggesting that cell death by laser irradiation resulted from mainly necrosis, rather than apoptosis (Fig. 4B).

In terms of regeneration capacity, we implanted irradiated cartilages in rabbit ear until 4 weeks. The angle of curvature had a tendency to recover to its original position over time and it reduced slightly at the time of harvest as compared with that immediately after irradiation. Chondrocytes in 0.3 W treatment group regenerated completely at 2 weeks and those in 0.5 W treatment group regenerated at 4 weeks, showing relationship between radiation power and recovery time. In 1 W treatment group, chondrocytes could not recover until 4 weeks (Fig. 5A). At 1 week after implantation, 75% of damaged chondrocytes in the 0.3 W treatment group regenerated. In 0.5 W treatment group, 33%, 73%, and 99% of damaged chondrocytes regenerated at 1, 2, and 4 weeks, respectively. In contrast, chondrocyte could not regenerate in 1 W treatment group until 4 weeks (Fig. 5B). Table 2 shows changes in the average depth and width of thermally damaged tissue. The width of the damaged area in the 0.3 W and 0.5 W laser groups decreased with time, while the width of the damaged area in 1 W laser group increased slightly.

H&E staining revealed loss of chondrocytes and condensation of nuclei in laser irradiated area and the degree of changes was different according to the laser power and reimplantation time (Fig. 5C). However, H&E staining could not precisely identify the thermally damaged area and regeneration of chondrocytes like cell viability test. Cell viability test showed much superior result in determining cellular change by thermal damage. The extent of thermal damage demonstrated by the cell viability test was found to be greater than that demonstrated by conventional histologic examination.