BIOMATERIAL IMPLANT WITH A NET POSITIVELY CHARGED SURFACE

Abstract: A biomaterial implant, preferably derived from glutaraldehyde-treated animal tissue, is covalently bonded to a compound which imparts a net positive charge to the implant. Preferably, the implant is treated with a polyamino acid which has free amino groups so as to impart the net positive charge to the implant's surface.

Description: BIOMATERIAL IMPLANT WITH A NET POSITIVELY CHARGED SURFACE Background of the Invention This invention relates to preventing calcification of biαmaterial implants. Biomaterial implants, e.g., bioprosthetic heart 5 halves for use in human patients, are often derived from human or animal tissue. These implants generally are pre-treated with glutaraldehyde to prevent biodegradation and immunogenic responses following implantation. One problem with glutaraldehyde-treated 10 implantc is pathologic calcification, defined as the deposition of calcium phosphate mineral salts in association with a disease process. Pathologic calcification represents the main source of bioprosthetic heart valve failure. 15 Summary of the Invention In general, the iny.ention features a biomaterial implant covalently bonded-to a compound that imparts a positive charge to the implant sufficient to substantially repel calcium ions in the environment of 20 use, i.e., sufficient to prevent calcium ions from adhering to the implant to the extent that the functioning of the implant is impaired. In preferred embodiments, the compound, after bonding to said implant, has at least one free basic 25 functional group (e.g., an amino group) that imparts the positive charge to the implant; the compound has at least six amino groups; the compound has a molecular weight of greater than 600; and the compound is a polypeptide such as protamine, polylysine, or 30 polyargininβ; and the implant is made of natural tissue or collagen and is stabilized with a stabilizing reagent (e.g., glutaraldehyde) that forms a covalent bond with an amino group of the implant. As used herein, the term "natural tissue" refers to tissue derived from humans or animals. According to the invention, calcification is prevented by the addition of positive charges to the implant. The positive charge repels positively charged calcium ions which could otherwise attach to the implant and act as nucleation sites for calcification. Covalently bonding the compound to the implant minimizes long-term calcification because the compound remains attached to the implant. Particular advantage is gained where the compound is used with implants that have been treated with gluteraldehyde, which consumes the naturally-present basic functionality (e.g., amino groups) of the implant; it is hypothesized that such consumption normally is a cause of calcification. The compounds are easy to use and non-toxic. Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. Description of the Preferred Embodiments We now describe the structure, preparation, and use of preferred embodiments of the invention. Structure Preferred biomaterial implants are produced from collagen or derived from natural tissue, e.g., porcine aortic valves or bovine pericardium, and are stabilized with glutaraldehyde, which reacts with some of the amino groups of the implants. Examples of biomaterial implants include heart valves, vascular grafts, breast implants, hip and tendon prostheses, intracardiac patches, and artificial heart devices having biolized surfaces. The biomaterial implants preferably are covalently bonded, prior to implantation, to a compound that has a plurality of amino groups. When such a compound is reacted with the implant in accordance with the method described below, at least one of the amino groups covalently bonds to the implant. If the compound has more then two amino groups, some of the amino groups do not bond to the implant and thus remain free. If the compound has two amino groups,- generally only one of the amino groups bonds to the implant, thus leaving one amino group per compound free. The free, unreacted amino groups are basic, i.e., are positively charged at physiological pH. The positive charge imparted to the implant by the charged amino groups inhibits calcificaion of the implant. Preferably, the compound is a nontoxic, water soluble, organic polymer that has a molecular weight of greater than 600, more preferably greater than 2000, and has at least 6 amino groups. Such polymers molecules are efficient at spreading through the implant the positive charges associated with each molecule. Examples of such polymers include protamine (which is comprised of approximately 80% arginine residues, each of which has a free amino group), polylysine (each lysine residue having a free amino group), polyarginine, polymyxin, and polyornithine, or their pharmaceutically acceptable salts, e.g., sulfates, chlorides, or nitrates. Preparation In general, the biomaterial implants of the invention are prepared by contacting a conventionally fabricated biomaterial implant with the compound in a suitable aqueous buffer solution, e.g., HEPES, to allow the compound to penetrate into the tissue. The pH of the buffer solution is between 7.0 and 8.0. The implants are then contacted with a buffered solution of glutaraldehyde. To stabilize the covalent bonding between the compound and the biomaterial implant, the implant can be reduced with sodium borohydride following glutaraldehyde treatment. It is believed that the compound covalently bonds to the implant through the glutaraldehyde, which reacts with both an amino group of the compound and an amino group of the implant. The glutaraldehyde thus both stabilizes the tissue and serves as the crosslinking reagent that attaches the compound to the implant. A collagen sponge treated according to the invention was prepared as follows. Type I collagen " sponges were cut into small pieces (1 5 cm) and immersed for 24 hrs. in 10 ml of a solution containing 10% protamine sulfate (available from Sigma Chemical Co., St. Louis, MO, catalog no. P 4380) and 5 ml HEPES buffer (pH = 7.5) (available from Sigma Chemical Co., catalog no. H 3375). Analytical grade glutaraldehyde was then added to a concentration of 0.2%, and the resulting solution incubated for 96 hrs, after which the sponges were rinsed free of buffer solution and reduced with sodium borohydride. The sponges were then rinsed and dried. Use The biomaterial implants of the invention can be surgically implanted using conventional surgical techniques. It is believed that calcification is prevented as follows. Biomaterial implants in general are substantially composed of collagen Type I. Collagen Type I contains 30-35 lysine and hydroxylysine residues per 1000 amino acid residues; these residues have basic, positively charged amino groups. Collagen Type I also contains many acidic, negatively charged σarboxylate groups. When a biomaterial implant is treated with gluteraldehyde to stabilize the implant, the gluteraldehyde reacts primarily with the amino group-, of the lysine and hydroxylysine residues of collagen. As a result, following the reaction, the implant has fewer basic amino groups. Because the number of carboxylate groups is unchanged, the implant will have more of a negative charge associated with it following glutaraldehyde treatment. The increased negative charge will tend to attract Ca +2 to the implant; the most likely affinity sites for the ions are the carboxylate groups. Bonding the compound to the implant compensates for the amino groups that are consumed during glutaraldehyde treatment. The amino groups effectively neutralize the acidic groups of the tissue, thus making the carboxylate groups less attractive to Ca +2. In addition, the positively charged groups repel calcium ions, thus preventing the ions from interacting with the tissue. Other Embodiments Other embodiments are within the following claims. For example, the compounds can be bound to synthetic polymers used to fabricate biomaterial implants, e.g., silicone rubbers, polyurethane elastomers, and polymer hydrogels. The polymers must contain suitable functional groups which are capable of covalently bonding to the compound. Compounds can also be contacted with, the implant after or during the period in which the implant is treated with gluteraldehyde. Compounds can be covalently bonded to the implant through standard crosslinking reagents such as 1,2 cyclohexanedione, formaldehyde, DMA, and CNBr (all of which are available from PIERCE).

Claims: _ _ _ Claims 1. A biomaterial implant covalently bonded to a compound that imparts a positive charge to said implant sufficient to substantially repel calcium ions in the environment of use. 2. The biomaterial implant of claim 1 wherein said compound, when bonded to said implant, has at least one free basic functional group that imparts said positive charge to said implant. 3. The biomaterial implant of claim 2 wherein said functional group is an amino group. 4. The biomaterial implant of .claim 1 wherein said compound has at least 6 amino groups. 5. The biomaterial implant of claim 1 wherein said compound has a molecular weight greater than about 600. 6. The biomaterial implant of claim 1 wherein said compound comprises protamine, polylysine, or polyarginine, or a pharmaceutically acceptable salt thereof. 7. The biomaterial implant of claim 1 wherein said compound comprises a polypeptide. 8. The biomaterial implant of claim 1 wherein said implant is stabilized with a stabilizing reagent that forms a covalent bond with an amino group of said implant. 9. The biomaterial implant of claim 8 wherein said stabilizing reagent is glutaraldehyde. 10. The biomaterial implant of claim 8 wherein said implant comprises natural tissue or collagen. 11. A method of inhibiting calcification of a biomaterial implant comprising the steps of providing a basic compound; and covalently bonding a sufficient amount of said compound to said implant to impart a positive charge to said implant. 12. The method of claim 11 further comprising the step of treating said implant with glutaraldehyde. 13. The method of claim 11 wherein said compoumd comprises protamine, polylysine, or polyarginine, or a -pharmaceutically acceptable salt thereof. 14. The method of claim 11 wherein said compound comprises a polypeptide. 15. The method of claim 11 wherein said compound has a molecular weight greater than about 600. 16. The method of claim 11 wherein said implant comprises natural tissue or collagen. 17. The method of claim 11 wherein said compound has at least two amino groups. 18. The method of claim 10 wherein said compound has at least six amino groups.

Inventors: GOLOMB GERSHON, LEVY ROBERT J, GOLOMB, GERSHON, LEVY, ROBERT, J.
Applicants: CHILDRENS MEDICAL CENTER, THE CHILDREN'S MEDICAL CENTER CORPORATION
Application year: 1987
Main classification: A61F2
IPC codes: A61L26, A61L27