Abstract
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.
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References
M. E. Nimni, in “Encyclopaedic handbook of biomaterials and bioengineering”, edited by D. L. Wise, D. Trantola, D. E. Altobelli, M. J. Yaszemski, J. D. Gresser and E. R. Schwartz, (Marcel Dekker, New York, 1995) pp. 1229-43.
M. E. Nimni, D. Cheung, B. Strates, M. Kodama and K. Sheikh, J. Biomed. Mater. Res. 21 (1987) 741.
E. Bell, B Ivarsson and C. Merrill, Proc. Nat. Acad. Sci. 76 (1979) 1274.
I. V. Yannas, in “Collagen”, Vol. 3, edited by M. E. Nimni, (CRC Press, Boca Raton, FL, 1988) pp. 88-115.
C. A. Edwards and W. D. O'Brien, Clin. Chim. Acta. 104 (1980) 161.
S. I. Rennard, G. R. Martin and R. G. Crystal, in “Immunochemistry of the Extracellular Matrix”, Vol. 2, edited by H. Furthmayr, (CRC Press, Elkins Park, PA, 1982) pp. 237-51.
J. Melkko, S. Niemi, L. Risteli and J. Risteli, Clin. Chem. 36 (1990) 1328.
S. D. Breul, K. H. Bradley, A. J. Hance, M. P. Schafer, R. A. Berg and R. G. Crystal, J. Biol. Chem. 255 (1980) 5250.
J. G. Clark and J. N. Hildebran, Analyt. Biochem. 140 (1984) 478.
J. N. Hildbran, J. Airhart, W. S. Stirewalt and R. B. Low, Biochem. J. 198 (1981) 249.
F. Sweat, H. Puchtler and S. I. Rosenthal, Archiv. Pathol. 78 (1964) 69.
G. S. Montes and L. C. U. Junqueira, in “Collagen”, Vol. 2, edited by M. E. Nimni, (CRC Press, Boca Raton, FL, 1988) 41-72.
B. J. Walsh, S. C. Thornton, R. Penny and S. N. Breit, Analyt. Biochem. 203 (1992) 187.
L. C. U. Junqueira, G. Bignola and R. R. Brentani, Histochem. J. 11 (1979) 447.
J. Rao and W. R. Otto, Analyt. Biochem. 207 (1992) 186.
L. Feldskov-Nielson, D. Moe, S. Kirkeby and C. Garbarsch, in “Proceedings of the XVth Federation of Europeans Connective Tissue Societies, FECTS” (1996) u5.
C. A. Lambert, E. P. Soudant, B. V. Nusgens and C. M. Lapriere, Lab. Invest. 66 (1992) 444. ??
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LEE, D.A., ASSOKU, E. & DOYLE, V. A specific quantitative assay for collagen synthesis by cells seeded in collagen-based biomaterials using sirius red F3B precipitation. Journal of Materials Science: Materials in Medicine 9, 47–51 (1998). https://doi.org/10.1023/A:1008882628142
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DOI: https://doi.org/10.1023/A:1008882628142