Density gradient fractionation of dentine and bone powder
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In order to obtain enough material to analyse the organic matrix of mineralizing tissue a technique for preparative separation according to the degree of mineralization was developed. The method employs ultracentrifugation of powdered material in density gradients made from acetone and bromoform. The density range of the fractions is checked by refractive index measurements of the gradient medium. The amount of the material in the fractions is checked by weighing and their degree of mineralization is estimated by determining the Ca/N and P/N ratios. The homogeneity of the fractions is determined by soft X-ray microscopy. Isolated dissected microscopic bone structure (osteones and lamellar bone fragments) with different degrees of mineralization were fractionated in this way. Chromatography on Sepharose 2B of proteoglycans from costal cartilage exposed to an acetone-bromoform gradient revealed no effect of the gradient medium on the molecular size of the proteoglycans.
Key wordsDentine Bone Mineralization Density gradient fractionation Proteoglycans
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