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During embryological limb formation mesenchymal cells condense and differentiate into chondrocytes, in a process known as chondrogenesis. These chondrocytes synthesize glycosaminoglycans (GAGs), thus playing an important role in this process. A simplified system in vitro chondrogenesis, using adult mesenchymal stromal cells (MSCs) has been demonstrated. This differentiation potential is usually assessed by histological staining. Objective. Establishment of a semi-automatic grading system for histochemistry stains and immunohistochemistry assays. Materials and methods. For chondrogenesis cells were cultured for three weeks in aggregates with inducing media. Total GAGs were measured using dimethylmethylene blue (DMB) method. For histological analyses aggregates were stained with Alcian blue for total GAGs detection and immunohistochemistry (IHC) for aggrecan was performed. Semi-automatic grading for all slides was obtained after ImageJ analysis. Results. MSCs cultured as aggregates in chondrogenic differentiation media had similar protein concentrations for all time points, suggesting cellularity remained homogenous during culture. Total GAGs was higher for aggregates cultured in chondrogenic compared to complete media. The same trend was observed for Alcian blue stain grades by blinded observer and analysis using ImageJ software. Aggrecan’s IHC analysis had a decreasing tendency with time for aggregates in chondrogenic media for blinded observer and ImageJ evaluation. Conclusion. We developed a functional system for semi-automatic slide grading. We corroborated these results by biochemical analysis with comparable results. To our knowledge, for in vitro chondrogenesis, this is the first report to evaluate stains using this methodology. This procedure might be useful for other applications in the field of Biology and Medical Sciences.

Semi-automatic grading system in histologic and immunohistochemistry analysis to evaluate in vitro chondrogenesis