%0 Journal Article
%T Semi-automatic grading system in histologic and immunohistochemistry analysis to evaluate in vitro chondrogenesis
%A Mar¨ªa Luc¨ªa Guti¨¦rrez
%A Johana Guevara Morales
%A Luis Alejandro Barrera
%J Universitas Scientiarum
%D 2012
%I Pontificia Universidad Javeriana
%X During embryological limb formation mesenchymal cells condense and differentiate into chondrocytes, in a process known aschondrogenesis. 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. Thisdifferentiation potential is usually assessed by histological staining. Objective. Establishment of a semi-automatic grading systemfor histochemistry stains and immunohistochemistry assays. Materials and methods. For chondrogenesis cells were culturedfor 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 culturedas aggregates in chondrogenic differentiation media had similar protein concentrations for all time points, suggesting cellularityremained 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 IHCanalysis 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 biochemicalanalysis with comparable results. To our knowledge, for in vitro chondrogenesis, this is the first report to evaluate stains usingthis methodology. This procedure might be useful for other applications in the field of Biology and Medical Sciences.
%K mesenchymal stromal cells
%K in vitro chondrogenesis
%K glycosaminoglycans
%K ImageJ
%U http://revistas.javeriana.edu.co/index.php/scientarium/article/view/3691/2761