Europäisches Patentamt *EP000693523B1* (19) European Patent Office Office européen des brevets (11) EP 0 693 523 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.7: C08H 1/06, A61L 27/00, of the grant of the patent: A61L 31/00 20.11.2002 Bulletin 2002/47 (21) Application number: 95111260.6 (22) Date of filing: 18.07.1995 (54) Collagen-based matrix Matrix auf der Basis von Kollagen Matrice à base de collagène (84) Designated Contracting States: • Noff, Matityahu AT BE CH DE DK ES FR GB GR IE IT LI LU MC NL Rehovot 76228 (IL) PT SE (74) Representative: Grünecker, Kinkeldey, (30) Priority: 19.07.1994 IL 11036794 Stockmair & Schwanhäusser Anwaltssozietät Maximilianstrasse 58 (43) Date of publication of application: 80538 München (DE) 24.01.1996 Bulletin 1996/04 (56) References cited: (73) Proprietor: COL-BAR R & D LTD. DE-A- 4 302 708 FR-A- 2 679 778 Ramat-Gan 52290 (IL) US-A- 4 971 954 (72) Inventors: Remarks: • Pitaru, Sandu The file contains technical information submitted Tel-Aviv 62300 (IL) after the application was filed and not included in this specification Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 0 693 523 B1 Printed by Jouve, 75001 PARIS (FR) EP 0 693 523 B1 Description [0001] The present invention concerns a collagen-based matrix and devices comprising this matrix. A particular example of such device is a collagen-based sheet useful in a guided tissue regeneration (GTR), which will be referred 5 to herein as "GTR membrane". [0002] A particular application of the GTR membrane of the invention is in dentistry, for guided tissue regeneration of periodontal tissue. [0003] The present invention also concerns a process for the preparation of the matrix. [0004] Guided tissue regeneration is a surgical procedure intended to restore or regenerate the morphology and 10 function of tissues or organs that were destroyed by disease or trauma. In tissue regeneration, the regenerating tissues have to repopulate the same site and space previously occupied by the healthy tissues that were destroyed. Further- more, to restore the morphological and functional relationships between the different regeneration tissues at the re- generation site, the repopulation of the affected site and the subsequent differentiation of the repopulating cells should be an orderly and concerted process. 15 [0005] The technique of GTR aims to allow orderly and concerted repopulation of an affected site by regenerating tissues. To this end, a barrier is interposed between the regenerating tissues and the tissue that might intervene with the regenerative process. The barrier is maintained in place until the affected site is repopulated by the proper tissues and the regenerating tissues reach maturity. [0006] Membrane barriers are currently used mainly in dentistry, for GTR of regenerating periodontal tissues that 20 were destroyed by periodontal disease or trauma. Generally, two types of membranes are in use, membranes made of non-degradable material and membranes made of degradable materials. [0007] Collagen are a family of proteins with a well determined triple helical configuration. Among these proteins, collagen Type I is most prevalent, constituting approximately 25% of the body's proteins and 80% of the connective tissues' proteins. Collagen Type I polymerizes to form aggregates of fibers and bundles. Collagen is continuously 25 remodeled in the body by degradation and synthesis. Collagen Type I is degraded only by a specific enzyme - colla- genase, and is resistant to any non-specific proteolytic degradation. [0008] Collagen is a weak antigen and most of its antigenicity resides in the non-helical terminals of the molecule. These terminals may be removed by enzymes such as pepsin. Its weak antigenicity and its relative resistance to degradation make collagen a good candidate as a building material of implantable devices. 30 [0009] A molecular solution of type I collagen can be prepared from a connective tissue rich in this protein and the molecular collagen can then be reassembled into fibrils which can then combine to form a collagen matrix. Collagen matrices can be molded in vitro into numerous implantable devices such as, for example collagen sheets or collagen tubes. [0010] When used to form implantable devices, collagen matrices should maintain their integrity for long periods of 35 time. The resistance towards degradation of the collagen fibrils can be increased by increasing the number of inter- molecular cross-links. Several agents, such as aldehyde fixatives and imides, and treatments such as radiations have been used to achieve this purpose. The main drawbacks of such treatments are toxicity and inability to accurately control the degree of cross-linking. [0011] US-A-4,971,954 is related to the provision of glycated collagen based matrices useful as an implant in clinical 40 and veterinary applications which support the growth of connective tissue cells and particularly allow fibroblast in- growth. [0012] It is the object of the present invention to provide a collagen matrix suitable for use in implantable devices such as membranes or tubes for guided tissue regeneration. [0013] It is furthermore the object of the present invention to provide a process for the preparation of such a matrix. 45 [0014] It is still further the object of the present invention to provide a kit comprising ingredients useful in guided tissue regeneration procedures, e.g. comprising space maintainers for use in guided tissue regeneration (GTR) pro- cedures. [0015] According to the first aspect of the present invention, said object is attained by a process for the preparation of a collagen matrix from collagen comprising: 50 (i) dissolving said collagen in a solvent; (ii) transferring said dissolved collagen to a mold and incubating it to obtain a membrane; (iii) compressing said membrane until a desired membrane thickness is obtained; (iv) reacting said compressed membrane with a reducing sugar or a reducing sugar derivative which is capable of 55 existing in an aldehyde or ketone state in an aqueous solution, to crosslink fibrils of the collagen to one another, thus forming a matrix; and (v) subjecting said matrix to critical point drying. 2 EP 0 693 523 B1 [0016] The process may further comprise after step (iv) the step of dehydrating said matrix prior to critical point drying. [0017] The present invention further provides a collagen matrix obtainable by the above process and an implantable device comprising said collagen matrix. [0018] The present invention is also directed to the use of said implantable device as a membrane barrier for guided 5 tissue regeneration. [0019] Furthermore, the present invention provides a kit for use in guided tissue regeneration, comprising said im- plantable device as a tissue regeneration guidance membrane and a substance for use as a space maintainer, said space maintainer being hyaluronic acid. [0020] The collagen matrix of the present invention comprises collagen fibrils, the molecules or microfibrils of which 10 are being cross-linked to one another by a cross-linking agent, the cross-linking agent comprising a reducing sugar, or a reducing sugar derivative. [0021] The process of the present invention for preparing a collagen matrix comprises reacting collagen with a re- ducing agent whereby fibrils of the collagen become cross-linked to one another. Preferably, following preparation, the collagen matrix is dehydrated, e.g. in alcohol solution, and then subjected to critical point drying. 15 [0022] Said cross-linking agent may be an aldehyde mono sugar or a mono sugar derivative wherein the α-carbon exists in an aldehyde or ketone state in an aqueous solution. [0023] Said cross-linking agent may be a compound represented by one of the following formulae I or II: 20 25 30 wherein: 35 R1 is H or lower alkyl or alkylene, an amino acid, a peptide, a saccharide, purine or pyrimidine base, a phospho- rylated purine or pyrimidine base; n is an integer between 2-9, and p and q are each independently an integer between 0 to 8, provided that p and q together are at least 2 and not 40 more than 8. [0024] A reducing sugar can form a Schiff base with an α or ε amino groups of amino acids of the collagen molecule. The Schiff base undergoes an Amadori Rearrangement to form a ketoamine product by the following reaction scheme: 45 50 55 3 EP 0 693 523 B1 a. Aldehyde sugar [0025] 5 10 15 20 25 30 b. Ketone sugar [0026] 35 40 45 50 55 4 EP 0 693 523 B1 5 10 [0027] Two adjacent ketoamine groups can then condense to form a stable intermolecular or intramolecular crosslink. 15 [0028] When the cross-linking agent is ribose, a stable product cross-linked via a pentosidine group may be formed by the following reaction scheme (in the following scheme "A" denotes a first collagen molecule and "B" a second collagen molecule): 20 25 30 35 40 45 50 55 5 EP 0 693 523 B1 5 10 15 20 [0029] Examples of said reducing agent are glycerose, threose, erythrose, lyxose, xylose, arabinose, ribose, allose, altrose, glucose, mannose, gulose, idose, galactose, talose, or any other diose, triose, tetrose, pentose, hexose, sep- tose, octose, nanose or decose. [0030] The degradation rate of the collagen matrix when in situ can be controlled by the extent of cross-linking between the collagen molecules in the matrix. This may in turn be controlled by the concentration of the sugar during 25 the preparation of the matrix, the temperature, and the extent of time during which the collagen is exposed to the sugar.