This article discusses the visualization of proteoglycans through a method involving mesenchymal stem cells (MSCs) and collagen-based scaffolds. The process includes chondrogenic differentiation of MSCs into mature chondrocytes, which secrete proteoglycans that interact with collagen.
Proteoglycans are composed of a core protein covalently attached to sulfated glycosaminoglycan chains.
To visualize proteoglycans, begin with collagen-based scaffolds - the porous polymeric substrate - and seed the mesenchymal stem cell, or MSC, suspension onto them. This provides structural support to the MSCs and allows them to grow on the surface as well as inside the cavities of the scaffolds.
Treat the constructs in a chondrogenic differentiation medium containing transforming growth factors that drive the transformation of MSCs into mature chondrocytes - cartilaginous cells. Mature chondrocytes secrete proteoglycans that form a network with the collagen in the scaffold.
Take a section prepared from the chondrocyte-laden construct. Add an acidic solution of alcian blue stain supplemented with guanidium hydrochloride and incubate. During incubation, the guanidine hydrochloride in the dye solution dissociates any proteoglycan aggregates for better staining.
Subsequently, the negatively charged sulfated glycosaminoglycans from the proteoglycans bind to the copper-containing positively charged alcian blue dye molecules, resulting in blue deposits.
Next, immerse the slide in a destaining solution containing magnesium chloride. The magnesium ions compete with the positively charged dye molecules for binding sites on the glycosaminoglycans, thus, preventing overstaining.
Image the section. Blue coloration confirms the presence of proteoglycans in the sample
To carry out chondrogenic differentiation from a sponge-shaped medical device constituted from lyophilized collagen type 1, cut cubes 3 millimeters per side. Seed MSCs on top of the cubes at a concentration of 4 x 106 cells/ml.
After allowing the cells to adhere to the cubes for 30 minutes at room temperature, maintain MSC-collagen constructs in chondrogenic medium, as listed in the text protocol. Add 0.4% Alcian blue solution to the sections, and incubate overnight at 4 degrees Celsius.
The next day, use 40% DMSO and 0.05 molar magnesium chloride to wash the cells for 30 minutes before using nuclear fast red to counterstain the cells.
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