A specific quantitative assay for collagen synthesis by cells seeded in collagen-based biomaterials using sirius red F3B precipitation.
The measurement of collagen synthesis by seeded cells in vitro is a prerequisite for the assessment of biocompatibility of many biomaterials. Existing methods are either complicated or not applicable to systems utilizing collagen-based materials, and the development of a rapid and simple technique would be an advantage. In the current paper, a method is described which relies on the radiolabelling of newly synthesized protein with [3H]-proline followed by specific precipitation of collagen using 1% sirius red dissolved in water. The results indicate that collagen binding to sirius red is unaffected by using water rather than picric acid as a solvent and the dye binds in a similar fashion to collagen type I, II and III. Cycloheximide treatment of the gels indicated that precipitated [3H]-proline was restricted to macromolecular protein. Collagenase treatment eradicated labelled precipitation formation when using 1% sirius red in water, indicating a high degree of specificity for collagen whilst specificity was poor when using 1% sirius red in picric acid. The method described is both simple and rapid and shows a high degree of specificity and sensitivity. For these reasons it is highly suited for the assessment of collagen synthesis by cells in collagen-based materials.
Lee
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IRC in Biomedical Materials, Institute of Orthopaedics, University College London Medical School, Brockley Hill, Stanmore, Middlesex HA7 4LP UK.
Assoku
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Doyle
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