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

Abstract

Cartilage tissue engineering can provide a valuable tool for controlled studies of tissue development. As an example, analysis of the spatial distribution of glycosaminoglycans (GAG) in sections of cartilaginous tissues engineered under different culture conditions could be used to correlate the effects of environmental factors with the structure of the regenerated tissue. In this paper we describe a computer-based technique for quantitative analysis of safranin-O stained histological sections, using low magnification light microscopy images. We identified a parameter to quantify the intensity of red color in the sections, which in turn was proportional to the biochemically determined wet weight fraction of GAG in corresponding tissue samples, 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 determine the thickness of an external region, with lower GAG fractions, and the spatial rate of change in GAG content. The method was applied to the quantitatation of GAG distribution in samples of natural and engineered cartilage, cultured for 6 weeks in three different vessels: static flasks, mixed flasks, and rotating bioreactors.