Use of flow cytometry to assess chondrocyte viability after laser reshaping of cartilage

Abstract

Lasers have been shown to cause permanent shape change in cartilage via photothermally induced mechanical stress relaxation. While the biophysical properties of cartilage during laser irradiation have been studied, tissue viability following laser irradiation has not been fully characterized. In this study, cell viability staining and flow cytometry were used to determine chondrocyte viability following photothermal stress relaxation. Porcine septal cartilage slabs (10 X 25 X 1.5 mm) were irradiated with light from a Nd:YAG laser ((lambda) equals 1.32 micrometer, 25 W/cm<SUP>2</SUP>) while surface temperature, stress relaxation, and diffuse reflectance were recorded. Each slab received one, two, or three laser exposures (respective exposure times of 6.7, 7.2, 10 s), determined from measurements of diffuse reflectance, which correlate with mechanical stress relaxation. Irradiated samples were then divided into two groups analyzed immediately and at five days following laser exposure (the latter group was maintained in culture). Chondrocytes were isolated following serial enzymatic digestion with hyaluronidase, protease, and collagenase II for a total of 17 hours. Chondrocytes were then stained using SYTO<SUP>R</SUP>/DEAD Red<SUP>TM</SUP> (Molecular Probes; Eugene, OR) wherein live cells stained green (530 nm) and dead cells stained red (630 nm) when excited at 488 nm. A flow cytometer (FACScan, Becton Dickinson, Franklin Lakes, NJ) was then used to detect differential cell fluorescence; size; granularity; and the number of live cells, dead cells, and post irradiation debris in each treatment population. Nearly 60% of chondrocytes from reshaped cartilage samples isolated shortly after irradiation, were viable as determined using flow cytometry while non- irradiated controls were 100 percent viable. Specimens irradiated two or three times with the laser demonstrated increasing amounts of cellular debris along with a reduction in chondrocyte viability: 31 percent following two laser exposures, and 16 percent after three laser exposures. In those samples maintained in culture medium and assayed 5 days after irradiation, viability was reduced by 28 to 88 percent, with the least amount of deterioration in untreated and singly irradiated samples. Functional fluorescent dyes combined with flow cytometric analysis successfully determines the effect of laser irradiation on the viability of reshaped cartilage. The flow cytometric approach to viability is accurate, fast, and can handle large sample numbers and sizes. Most importantly, since the method reveals that a single laser exposure of 6.7 s (sufficient for sustained shape change) causes less than 40 percent acute reduction in viability, photothermal reshaping of cartilage may be further researched as a clinical alternative to conventional techniques.