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(12) Patent Application Publication (10) Pub. No.: US 2008/0305517 A1 Griffin Et Al US 2008O305517A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2008/0305517 A1 Griffin et al. (43) Pub. Date: Dec. 11, 2008 (54) TRANSGLUTAMINASE CROSSLINKED (30) Foreign Application Priority Data COLLAGEN BOMATERAL FOR MEDICAL MPLANT MATERALS Sep. 10, 2004 (GB) ................................... O42O091.1 (75) Inventors: Martin Griffin, Nottingham (GB); Publication Classification Russell Collighan, Birmingham (51) Int. Cl. (GB); David Chau, Birmingham CI2N 5/06 (2006.01) (GB); Elisabetta Verderio CI2P 2L/02 (2006.01) Edwards, Nottingham (GB) A6IF 2/28 (2006.01) A6F I3/00 (2006.01) Correspondence Address: FSH & RICHARDSON PC (52) U.S. Cl. ..................... 435/68.1; 435/395; 623/16.11; 602/48 P.O. BOX 1022 MINNEAPOLIS, MN 55440-1022 (US) (57) ABSTRACT (73) Assignee: ASTON UNIVERSITY, The present invention provides a method for producing an Birmingham, West Midlands (GB) improved biomaterial comprising treating a collagen bioma terial with a transglutaminase under conditions which permit (21) Appl. No.: 111574,918 the formation of cross-links within the collagen. Preferably, the transglutaminase is a tissue transglutaminase, a plasma (22) PCT Filed: Sep. 12, 2005 transglutaminase or a microbial transglutaminase. In a pre ferred embodiment, the collagen biomaterial further com (86). PCT No.: PCT/GBOS/O352O prises a cell adhesion factor, Such as fibronectin. The inven tion further provides biomaterials obtainable by the methods S371 (c)(1), of the invention, and medical implants and wound dressings (2), (4) Date: Aug. 11, 2008 comprising the same. -- Collagen (3 mg/ml) -O- Coll-mTG (50ug/ml) -v- Co-mTG (250ug/ml) -v- Coll-mTG-R281 -- Coll-R281 O 5 10 15 20 25 Time (min) Patent Application Publication Dec. 11, 2008 Sheet 1 of 32 US 2008/0305517 A1 FIGURE 1(A) (7: - f I -O- Collagen (3mg/ml) -O- Coll-mTG (50ug/ml) -v- Col-mTG (250ug/ml) -v- Coll-mi G-R281 -- Co-R281 O 5 10 15 20 25 Time (min) Patent Application Publication Dec. 11, 2008 Sheet 2 of 32 US 2008/03055.17 A1 FIGURE (B) -O- Collagen (3 mg/ml) -O- Coll-tTG (50ug/ml) -v- Coll-tTG (25Oug/ml) -v- Coll-tTG-R281 -- Coll-R281 O 5 10 15 20 25 Time (min) Patent Application Publication Dec. 11, 2008 Sheet 3 of 32 US 2008/0305517 A1 FIGURE 2CA) 0.7 -o- Collagen ill (3mg/ml) -O- Coll-mTG (50ug/ml) -v- Coll-mTG (250ug/ml) -v- Coll-mTG-R28 -- Coll-R281 O 5 10 15 20 25 Time (min) Patent Application Publication Dec. 11, 2008 Sheet 4 of 32 US 2008/03055.17 A1 FIGURE 2(B) -1) 1.1 . -- Collagen ill (3mg/ml) -O- Coll-tTG (50ug/ml) -v- Coll-tTG (250ug/ml) -v- Colli-tTG-R281 -- Coll-R281 O 5 10 15 20 25 Time (min) Patent Application Publication Dec. 11, 2008 Sheet 5 of 32 US 2008/0305517 A1 FGURE 3 Coliagen (6mg/ml) Collagen-tTG (50 g/ml) Collagen-mTG (50 g/ml) Patent Application Publication Dec. 11, 2008 Sheet 6 of 32 US 2008/0305517 A1 FIGURE 4 EE Collagen Coll-tTG E Coll-mTG Patent Application Publication Dec. 11, 2008 Sheet 7 of 32 US 2008/0305517 A1 FGURES Collagen (6mg/ml) Collagen-ti G (50pg/ml) Collagen-mTG (50pug/ml) 0 hours Patent Application Publication Dec. 11, 2008 Sheet 8 of 32 US 2008/03055.17 A1 FGURE 6 Collagen Coll-tTG Coll-mTG Patent Application Publication Dec. 11, 2008 Sheet 9 of 32 US 2008/0305517 A1 FGURE 7 200 kDa 6 kDa 97 KDa 66 koa 45kDa 3 kDa Patent Application Publication Dec. 11, 2008 Sheet 10 of 32 US 2008/03055.17 A1 FIGURE 8(A) 2 is . Col-G 3. Coll-tTG-FN (5ug/ml) Coll-tTG-FN (50ug/ml) Patent Application Publication Dec. 11, 2008 Sheet 11 of 32 US 2008/03055.17 A1 FIGURE 8(B) 2 - 1 ess. Co-mTG Coll-mTG-FN (5uglml) Coll-mTG-FN (5Oug/ml) Patent Application Publication Dec. 11, 2008 Sheet 12 of 32 US 2008/0305517 A1 FIGURE 8(C) 2 Coll-tTG Coll-tTG-FN (5uglml) Coll-tTG-FN (5Oug/ml) Patent Application Publication Dec. 11, 2008 Sheet 13 of 32 US 2008/03055.17 A1 FIGURE 8(D) 2 2 O)E - s C) 2 1 - O C 9 9 D- 3. a Coll-mTG Cofi-mTG-FN (5ug/ml) E. Coll-mTG-FN (5Ouglml) Patent Application Publication Dec. 11, 2008 Sheet 14 of 32 US 2008/0305517 A1 FEGURE 9(A) -- Collagen -O- Co-tG -V - Co-mC O 50 OO 150 2OO Time (h) Patent Application Publication Dec. 11, 2008 Sheet 15 of 32 US 2008/0305517 A1 FIGURE 9(B) -- Collagen -O- Co-tG -V- Co-mTG O 5O 1 OO 150 2OO Time (h) Patent Application Publication Dec. 11, 2008 Sheet 16 of 32 US 2008/03055.17 A1 FIGURE 9(C) -o- Collagen -O- Co-tTG -v- Coll-mTG O 50 1 OO 150 200 Time (h) Patent Application Publication Dec. 11, 2008 Sheet 17 of 32 US 2008/0305517 A1 FIGURE 9(D) -O- Collagen -O- Co-tTG -- Co-mTG O 50 100 150 200 Time (h) Patent Application Publication Dec. 11, 2008 Sheet 18 of 32 US 2008/0305517 A1 FIGURE 9(E) -0- Col-tTG -O- Coll-tTG-FN (5ug/ml) -v- Coll-tTG-FN (5Oug/ml) -V- Co-mTG -- Coll-mTG-FN (5ug/ml) -O- Coll-mTG-FN (5Ougiml) Time (h) Patent Application Publication Dec. 11, 2008 Sheet 19 of 32 US 2008/0305517 A1 FIGURE 10(A) 120 100 80 60 40 -e- Collagen -O- Co-tTG 20 -v- Co-mTG Patent Application Publication Dec. 11, 2008 Sheet 20 of 32 US 2008/0305517 A1 FIGURE 10(B) 120 CD i t d 80 CD CD s s 60 g 9, 40 -- Collagen 3. -O- Co-tTG -V- Coll-mTOG & 20 Patent Application Publication Dec. 11, 2008 Sheet 21 of 32 US 2008/03055.17 A1 FIGURE 10(C) 120 100 8 O r 6 O e -O- Collagen -O-- Coll-tTG 20 -v- Co-mTC Patent Application Publication Dec. 11, 2008 Sheet 22 of 32 US 2008/0305517 A1 FIGURE 10(ED) 120 1 OO 8 O 6 O 4.O -O- Collagen -O- Co-tTG 2 O - V - Co-mTG Patent Application Publication Dec. 11, 2008 Sheet 23 of 32 US 2008/03055.17 A1 FIGURE 10(E) 120 1 O O 8 O 60 Coll-tTG 4 O Coll-tTG-FN (5ug/ml) Coll-tTG-FN (50ug/ml) Co-mTG 20 A. Coll-mTG-FN (5ug/ml) Coll-miG-FN (50ug/ml) Patent Application Publication Dec. 11, 2008 Sheet 24 of 32 US 2008/0305517 A1 FIGURE 10(F) 120 1 OO 8O 60 -(- Co-tTG 40 -O- Col-tTG-FN (5ugml) -v- Coll-tTG-FN (50tug/ml) -V- Co-mTG 2O -H - Coll-mTG-FN (5ug/ml) -O- Coll-mTG-FN (50ugml) Patent Application Publication Dec. 11, 2008 Sheet 25 of 32 US 2008/0305517 A1 FIGURE 11(A) 30 Collagen O Coll-tTG 9 iii. COll-mTG l d d O "C9 r C. OO Gy O) s 9 10 < S Patent Application Publication Dec. 11, 2008 Sheet 26 of 32 US 2008/0305517 A1 FIGURE 11(B) 3 O Collagen Co-tTG E. Co-mTOG 2 O Patent Application Publication Dec. 11, 2008 Sheet 27 of 32 US 2008/03055.17 A1 FIGURE 11(C) a Collagen Coll-tTG E.g. Co-TG Patent Application Publication Dec. 11, 2008 Sheet 28 of 32 US 2008/0305517 A1 FIGURE 11(D) 30 | Collagen Co-tTG E. Co-mTG 20 - 10 - Patent Application Publication Dec. 11, 2008 Sheet 29 of 32 US 2008/0305517 A1 FIGURE 11 (E) 50 — . Col-G Coll-tTG-FN (5ug/ml) 4. O SS Coll-tTG-FN (5Oug/ml) Co-mTG EE Coll-mTG-FN (5ug/ml) SS Coll-mTG-FN (5Oug/ml) 3. O Time (h) Patent Application Publication Dec. 11, 2008 Sheet 30 of 32 US 2008/0305517 A1 FIGURE 11(F) 70 - ... Co-tTG Coll-tTG-FN (5ug/ml) 60 - E. Coli-tTG-FN (50ug/ml) Co-mTG 50 - Coll-mTO-FN (5ug/ml) Coll-mTG-FN (5Oug/ml) 40 - 30 - 2O - 10 - Time (h) Patent Application Publication Dec. 11, 2008 Sheet 31 of 32 US 2008/0305517 A1 FGURE 2 g S HOB w E s s s E C c. es t : HOB S w 2 - ''': HFDF d . o o s m Patent Application Publication Dec. 11, 2008 Sheet 32 of 32 US 2008/0305517 A1 FIGURE 13 0.20 sta Collagen Coll-tTG (50ug/ml) 0.18 - E3 Coll-tTG (100ug/ml) Coll-tTG (25Oug/ml) asa 2s. Coll-mTG (50ug/ml) s E. Coll-mTG (OOug/ml) S 0.16 - E Coll-mTG (25Oug/ml) > - C o 4 O. 12 - 0.10 -2 2 4. 6 8 10 Time (days) US 2008/03055.17 A1 Dec. 11, 2008 TRANSGLUTAMINASE CROSSLINKED for the usefulness of collagen in biomedical application is that COLLAGEN BOMATERAL FOR MEDICAL collagen can form fibres with extra strength and Stability MPLANT MATERALS through its self-aggregation and cross-linking (Lee et al., 2001). Unfortunately, collagen, like many natural polymers once extracted from its original source and then reprocessed, 0001. The present invention relates to materials for use in suffers from weak mechanical properties, thermal instability medicine, in particular medical implant materials. The inven and ease of proteolytic breakdown. To overcome these prob tion further provides a method of improving the biocompat lems, collagen has been cross-linked by a variety of agents ibility of a medical implant material. and is the subject of much recent research to find methods of preventing rapid absorption by the body.
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