Quantitative assessment of chondrocyte viability after laser mediated reshaping: a novel application of flow cytometry

BACKGROUND AND OBJECTIVES: Lasers can be used to reshape cartilage by accelerating mechanical stress relaxation. In this study, fluorescent differential cell viability staining and flow cytometry were used to determine chondrocyte viability following laser heating. STUDY DESIGN/MATERIALS AND METHODS: Porcine septal cartilages were irradiated with an Nd:YAG laser (lambda = 1.32 microm, 25 W/cm(2)) 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 seconds). Irradiated samples were then divided into two groups analyzed immediately and at 5 days following laser exposure. Chondrocytes were isolated following serial enzymatic digestion, and stained using SYTO/DEAD Red (Molecular Probes, Eugene, OR). A flow cytometer 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. RESULTS: Nearly 60% of chondrocytes from reshaped cartilage samples isolated shortly after one irradiation, were viable while non-irradiated controls were 100% viable. Specimens irradiated two or three times demonstrated increasing amounts of cellular debris along with a reduction in chondrocyte viability: 31 and 16% after two and three exposures, respectively. In those samples maintained in culture medium and assayed 5 days after irradiation, viability was reduced by 28-88%, with the least amount of deterioration in untreated and singly irradiated samples. CONCLUSIONS: Functional fluorescent dyes combined with flow cytometric analysis successfully determines the effect of laser irradiation on the viability of reshaped cartilage.