Chemistry: molecular biology and microbiology – Differentiated tissue or organ other than blood – per se – or...
Reexamination Certificate
1998-02-13
2001-06-26
Stucker, Jeffrey (Department: 1648)
Chemistry: molecular biology and microbiology
Differentiated tissue or organ other than blood, per se, or...
C424S423000, C623S002220, C204S157630
Reexamination Certificate
active
06251579
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates, in general, to a process for cross-linking and stabilizing proteinaceous material, and in particular, to a process for oxidizing collagenous material in the absence of a photo-catalyst but in the presence of chemicals that promote an oxidation reaction. The invention also relates to the resulting cross-linked product.
2. Description of the Related Art
It has been stated that degeneration of collagen and elastin are major factors in the malfunction of bioprosthestic heart valves (A. Carpentier, Biological Tissue in Heart Valve Replacement, M.I. Ionescue et al. (Eds.), Butterworth, London, 1972). Carpentier developed a method for treating the tissue to inhibit inflammatory reactions by host cells while enhancing strength and flexibility, and to prevent the degeneration of collagen and elastin. This method involved washing the tissue in an acid, i.e., in Hanks solution, then oxidizing mucopolysaccharide and glycoprotein with metaperiodate to form aldehyde groups, and finally binding and cross-linking the aldehyde groups with amines. The cross-linkages were then stabilized with sodium borohydride. This method suffered because the tissue had a tendency to calcify.
Reagents and processes currently used for protein cross-linking generally depend upon the incorporation of the cross-linking reagent into the protein matrix to cross-link the &egr;-amino groups of lysine hydroxylysine, and/or other groups in the protein. Common cross-linking reagents in such processes include formaldehyde and glutaraldehyde; other processes include the introduction of a phthaloyl or adipoyl moiety into the protein via phthaloyl dichloride or adipoyl dichloride, respectively, and/or the introduction of a mereaptan for oxidization to a disulfide bond. The cross-linking processes, reactions and reagents of the prior art vary, but most involve incorporating the reagent into or around the protein. For example, when collagen fibrils are cross-linked with the reagent glutaraldehyde, a polymeric-like coating forms around the fibrils, resulting in stiffer collagen matrix (Cheung and Nimni, Connec. Tissue Res. 10:201, 1982 and Connec. Tissue Res. 13:109, 1984). the incorporation of glutaraldehyde in the collagen and in the coating lead to problems such as an increased tendency of the collagen-containing tissue to calcify and a slow bleeding of the fixating agent from the tissue after it has been implanted into a host animal.
Acid has the well-known effect of denaturing the protein comprising the collagen fibril. It is, of course, the three-dimensional structure of the proteins comprising the collagen fibril which imparts to the fibril the unique properties of collagen. If there are changes to the structure of the collagen fibril, the protein cannot interact in the manner needed to give rise to those unique properties. Collagen molecules extracted by acid and neutral salt procedures differ in many respects from the natural product, including the extent to which they are covalently cross-linked, the size, shape, interaction properties and rate of fiber formation. (P. H. von Hipple, “Structural and Stabilization of the Collagen Molecule in Solution” in Treatise on Collagen, Vol. 1: Chemistry of Collagen, G. N. Ramachandran (Ed.), London: Academic Press Inc. (London) Ltd. (1967), pp. 253-338 at 262). Acid extraction does not provide collagen in a form useful for many medical applications.
A dye catalyzed process said to be useful for preparing thermostable, irreversibly cross-linked collagenous polymers is described in U.S. Pat. No. 3,152,976. This product is more susceptible to enzymatic degradation than uncross-linked collagen. Another type of dye catalyzed oxidative stabilization is described in U.S. Pat. No. 5,147,514. The tissue treated in this manner was resistant to calcification and provided material that was resistant to chemical and enzymatic degradation. However, these dyes are large and complex compounds, and there is concern about dyes remaining in the collagen bundles or tissue after treatment.
What is needed is a process that will provide cross-linked, stabilized proteinaceous products. in particular, there is a need for cross-linked, stabilized collagenous products that are suitable materials for use in the replacement and/or repair of diseased or damaged body tissues. Moreover, it is desirable that these products do not contain any foreign compounds which may later react with compounds in the host environment or leach out of the material into the host. The present invention is directed to overcoming or at least reducing the effects of one or more of the problems set forth above.
SUMMARY OF THE INVENTION
This invention relates to a process for cross-linking and stabilizing proteinaceous material, and in particular, to a process for oxidizing collagenous material. The invention also relates to the resulting cross-linked product. The process comprises immersing the proteinaceous material in a solution. The solution comprises an oxidizing agent. The oxidizing agents are those molecules that, in combination with other molecules in the solution, provide the energy to form localized reduction-oxidation reactions via a transfer of electrons. No external energy source is required. Typical oxidizing agents include (a) a mixture of copper chloride and hydrogen peroxide, (b) a mixture of ascorbate and ferrous chloride or (c) ferric sulfate. The solution usually comprises a pH buffer. At acidic pH values, below about 6.5, the acid interacts with the proteins. At lower pH values the acid will denature the protein.
The tissue remains immersed in the solution for a specified period of time at a specified temperature. While the temperature is not important, at higher temperatures the reaction proceeds at a faster rate, until heat begins denaturing the protein.
REFERENCES:
patent: 5412076 (1995-05-01), Gagnieu
Moore Mark A.
Phillips Richard E.
Robinson Melanie D.
Barrow Kenneth S.
Lyren Philip S.
Scott Timothy L.
Stucker Jeffrey
Sulzer Carbomedics Inc.
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