Method for quantitative analysis of glycosaminoglycan distribution in cultured natural and engineered cartilage

Cartilage tissue engineering can provide a valuable tool for controlled stu dies of tissue development. As an example, analysis of the spatial distribu tion of glycosaminoglycans (GAG) in sections of cartilaginous tissues engin eered under different culture conditions could be used to correlate the eff ects of environmental factors with the structure of the regenerated tissue. In this paper we describe a computer-based technique for quantitative anal ysis of safranin-O stained histological sections, using low magnification l ight microscopy images. We identified a parameter to quantify the intensity of red color in the sections, which in turn was proportional to the bioche mically determined wet weight fraction of GAG in corresponding tissue sampl es, and to describe the spatial distribution of GAG as a function of depth from. the section edge. A broken line regression model was then used to det ermine the thickness of an external region, with lower GAG fractions, and t he spatial rate of change in GAG content. The method was applied to the qua ntitatation of GAG distribution in samples of natural and engineered cartil age, cultured for 6 weeks in three different vessels: static flasks, mixed flasks, and rotating bioreactors. (C) 1999 Biomedical Engineering Society. [S0090-6964(99)00505-6].