Florence Nicolas

Denatured thiolated collagen. II. Cross-linking by oxidation.

We have recently described a new method for the thiolation of denatured collagen, which allows precise amounts of SH groups to be attached onto the protein backbone. The oxidation of denatured thiolated collagen produces disulphide cross-linking. The cross-linking of these products has been studied, optimized and compared to the cross-linking of native and denatured collagen with 0.5% aqueous glutaraldehyde. Films have been prepared and their tensile mechanical properties and biodegradation rates with trypsin and collagenase have been evaluated. Our results indicate that the cross-linking in oxidized thiolated collagen depends on the number of the disulphide bridges formed and on their intermolecular versus intramolecular repartition. Since the number of disulphide bridges can be controlled by the level of thiol in the denatured collagen and by the oxidation procedure, it is possible to control the mechanical properties and the biodegradation rates of these new materials. Under optimized conditions, oxidized denatured thiolated collagen films are more resistant and rigid than glutaraldehyde-cross-linked collagen films Cross-linked thiolated collagen materials are also more resistant to collagenase degradation. However, because of the loss of the triple-helical structure, they are more susceptible to trypsin degradation relative to glutaraldehyde-cross-linked triple-helical collagen. Denatured collagen cross-linked by physiological bridges such as disulphide bridges, with controllable mechanical properties and biodegradation rates, is of considerable interest in biomedical applications.

Denatured thiolated collagen: I. Synthesis and characterization

A new thiolating reagent is used to introduce sulphur groups into denatured atelocollagen. The procedure is easy to control and applicable on a large scale. The reagent is a reactive dicarboxylic acid compound containing sulphur in the form of a disulphide functionality. It is prepared by reacting N,N-disuccinoylcystamine with 1,1-carbonyldiimidazole. When this reagent is added to a solution of denatured atelocollagen in dimethylsulphoxide, amide bonds are formed between the carbonyl functions of the reagent and epsilon-NH2 of lysine and hydroxylysine residues from the protein. The disulphide groups introduced can then be reduced by reaction with 1,4-dithiothreitol to give the-SH form of the modified protein. Control of the stoichiometry between the reagent and the protein can lead to varying modification levels. A maximum level of 0.33 mmol SH per gram of protein can be attained, which corresponds to complete thiolation of the lysine and hydroxylysine residues. Thiolated denatured atelocollagen exhibits gelatin-like behaviour, by being highly soluble in water at all pH values and by forming heat-reversible gels.