Validation of a viral and bacterial inactivation step during the extraction and purification process of porcine collagen.

In the last few years, regulations for biomolecule production, and especially for extraction and purification of animal molecules such as collagen, have been reinforced to ensure the sanitary safety of the materials. To be authorized to market biomaterials based on collagen, manufacturers now have to prove that at least one step of their process is described in guidelines to inactivate prion, viruses, and bacteria. The present study focuses on the inactivation step performed during the extraction and purification of porcine type I atelocollagen. We chose to determine the reduction factor of a 1 M NaOH step on porcine parvovirus and four bacterial strains inactivation. During the extraction step, we deliberately inoculated the collagen suspension with the different microorganisms tested. Then, 1 M NaOH was added to the suspension for 1 hour at 20 degrees C. We demonstrated that this treatment totally inactivated S. aureus, P. aeruginosa, C. albicans and A. niger which are bacterial strains responsible of severe human pathology. The reduction factors reached more than 4 logs for B. cereus spores and 4 logs for the porcine parvovirus. are encouraging as those two microorganisms are known to be very resistant to inactivation.

Influence of gradual introduction of hydrophobic groups (stearic acid) in denatured atelocollagen on fibroblasts behavior in vitro.

To prepare new biocompatible hydrophobic collagen films for medical devices, innovative collagen derivatives were synthesized by reaction of the lysyl amino groups of the alpha-chains with activated stearic acid. Different collagens having different substitution degrees were obtained and used to prepare films crosslinked with oxidized glycogen. Their physicochemical surface properties were evaluated, and in vitro assays were performed to analyze the behavior of fibroblasts in contact with the materials. The assays were performed with cells in adhesion and growth phases. The hydrophobic properties increased with the number of stearic acid introduced in the collagen but only in the range of 1-12 stearic acids per molecule. For higher modifications a decrease of hydrophoby was observed. All the films induced a decrease of cells growth and adhesion but without cytotoxicity. These effects were more pronounced for the collagen containing about eight stearic acid residues. Cells behavior on modified collagens films seems to be related to the chemical groups exposed on the surface of the films. Indeed, the surface chemistry directly influences the adsorption of adhesion proteins and modulates their conformation therefore modifying the cell adhesion.

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.

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.