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 4O 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. This has been BACKGROUND accomplished by the use of cross-linking agents such as glu 0002 The shortage of organ or tissue donors has required taraldehyde (Barbani et al., 1995), formaldehyde (Ruderman the use of new biological Substitutes regenerated from tissue et al., 1973), chrome tanning (Bradley and Wilkes, 1977), cells or synthetic polymer matrices. From which, tissue epoxy compounds (Tu et al., 1993), acyl azide (Petite et al., replacement has become an important part of modern medical 1990), carbodiimides (Nimni et al., 1993) and hexamethyl treatments; whether artificial, such as joint replacements or enediisocyanate (Chvapil et al., 1993). The use of UV light, living, Such as skin and organ transplants. A new alternative gamma irradiation and dehyrothermal treatment has also for the medical industry is the use of artificial living tissues shown to be effective at introducing cross-links into collagen designed to mimic the native tissue and induce tissue forma (Harkness et al., 1966; Stenzel et al., 1969; Miyata et al., tion. Replacement of skin with artificial collagen-GAG matri 1971; Gorham et al., 1992). However, these methods suffer ces has been investigated since the early 1980s and is now in from the problem that the residual catalysts, initiators and clinical use (Bell et al., 1981: Burke et al., 1981). Tissue unreacted or partially reacted cross-linking agents used can engineering materials must satisfy several crucial factors: be toxic or cause inflammatory responses if not fully removed they must be resorbable, they must not elicit inflammation or or, simply, not cost-effective or practical at the large-scale a foreign body response, they must possess adequate (Matsuda et al., 1999; Ben-Slimane et al., 1988; Dunn et al., mechanical strength to perform its on-site function and they 1969). must encourage and promote cellular invasion, proliferation 0005 Hence, the present invention seeks to provide and differentiation. At its simplest characteristic, the material improved biomaterials which overcome the above problems serves as a bridge guiding cell-mediated remodelling to of existing biomaterials. reproduce the structure and organisation of the intended tis SUMMARY OF THE INVENTION SUS. 0003. Although many matrices currently exist and have 0006. A first aspect of the invention provides a method for been optimised for their individual applications; not many producing a biocompatible biomaterial comprising crosslink materials have general multi-application capabilities. Syn ing collagen using a transglutaminase. Thus, the method thetic biodegradable polymers, such as aliphatic polyester, comprises treating collagen with a transglutaminase under (e.g. polyglycolic acid, polylactic acid, polyesters and their conditions which permit the formation of crosslinks within copolymers, are the most commonly used for tissue engineer the collagen. ing applications. However, these synthetic polymers posses a 0007. By biomaterial we include any material compris Surface chemistry that does not promote general cell adhe ing collagen which is suitable for use within or on a mamma Sion. In addition, they can produce high local concentrations lian host body (and, in particular, a human host body). Pref of acidic by-products during degradation that may induce erably, the biomaterial is suitable for use as a medical implant adverse inflammatory responses or create local environments material and/or a wound dressing. that may not favour the biological activity of Surrounding 0008. By biocompatible we mean the biomaterial is able cells (Sachlos et al., 2003). Hydrogels have gained popularity to support its colonisation by host cells and their proliferation as potential materials for tissue engineering due to their high therein. Thus, biocompatibility is not intended to cover mere water content, good biocompatibility, and consistency similar adhesion of host cells to the biomaterial, but rather relates to to soft tissue. (Schmedlen et al., 2002). However, because of an interaction between the host cells and biomaterial which their complex, three-dimensional hydrophobic structure, they permits colonisation to occur. In particular, biocompatibility are capable of absorbing excess amounts of aqueous solution includes the ability of said material to support cell attach and undergoing degradation via erosion, hydrolysis, Solubili ment, cell spreading, cell proliferation and differentiation. sation and other biodegradation mechanisms. (Einerson et al., 0009. In a preferred embodiment of the first aspect of the 2002). Other bioactive materials. Such as glasses, ceramics or invention, the biocompatible biomaterial exhibits an gels, possess unsuitable physical and mechanical character enhanced ability to Support cell attachment, cell spreading, istics that prevent them from being used in many applications. cell proliferation and/or differentiation compared to non Additionally, many of these have not had their biological crosslinked collagen. activity assessed using in vitro cell culture systems. (Rhee et 0010 Advantageously, the biomaterial exhibits an al., 2003). enhanced ability to Support attachment, spreading, prolifera 0004 Collagen is the major component of skin bones and tion and/or differentiation of osteoblasts compared to non connective tissue. Collagen is a very popular biomaterial due crosslinked collagen. to its biocompatibility; the ability to support cell adhesion and 0011 Thus, the invention provides a method of improving proliferation. It is also biodegradable and only weakly anti the biocompatibility of collagen. Biocompatibility of a bio genic, and is thus able to persist in the body without devel material Such as collagen may be assessed using methods oping a foreign body response that could lead to its premature known in the art (see Examples). For example, increased rejection (Goo et al., 2003). Nevertheless, the primary reason biocompatibility of a biomaterial is associated with an US 2008/03055.17 A1 Dec. 11, 2008
increase in the ability of the material to facilitate cell attach 0022. A characterising feature of the methods of the ment, cell spreading, cell proliferation and differentiation. In present invention is that a transglutaminase enzyme is used as addition, the biomaterial should not induce any substantial a crosslinking agent in place of existing chemical and physi loss in cell viability, i.e. via the induction of cell death through cal crosslinking means. either apoptosis or necrosis. The differentiation of a cell type 0023 Transglutaminases (Enzyme Commission System is measured in different ways depending on the cell type in of Classification 2.3.2.13) are a group of multifunctional question. For example, for osteoblasts cells in culture, alka enzymes that cross-link and stabilise proteins in tissues and line phosphate together with extracellular matrix (ECM) body fluids (Aeschlimann & Paulsson, 1994& Greenberg et deposition, e.g. collagen 1, fibronectin, osteonectin and al., 1991). In mammals, they are calcium dependent and osteopontin, can be used as a marker. In addition, the ability catalyse the post-translational modification of proteins by of cells to proliferate and deposit ECM is important to any forming inter and intra-molecular e(Y-glutamyl)lysine cross links. The bonds that form are stable, covalent and resistant to material that is to be used as an implant, this includes endot proteolysis, thereby increasing the resistance of tissues to helial cells, chondroctes and epithelial cells etc. chemical, enzymatic and physical disruption. In contrast to 0012. In a further preferred embodiment the methods of transglutaminases of mammalian origin, microbial trans the first aspect of the invention, the biocompatible biomaterial glutaminases are generally not Ca"-dependent. exhibits enhanced resistance to cell-mediated degradation 0024. It will be appreciated that the term transglutami compared to non-crosslinked collagen. In particular, the bio nase is intended to include any polypeptide, or derivative compatible biomaterial preferably exhibits enhanced resis thereof, which is able to catalyse the formation of inter tance to one or more protease enzymes produced by mamma and/or intra-molecular e(Y-glutamyl)lysine crosslinks in col lian cells. lagen. Thus, the transglutaminase may be a naturally occur 0013. It will be appreciated by persons skilled in the art ring transglutaminase, or a variant, fragment of derivative that the methods of the first aspect of the invention may be thereof which retainstansglutaminase crosslinking activity. used to improve the biocompatibility of any collagen-based 0025. In a preferred embodiment of the first aspect of the starting material, provided that the collagen is present in invention the transglutaminase is a tissue transglutaminase. Sufficient concentration to enable Successful formation of a Alternatively, a plasma transglutaminase may be used. Solidgel matrix. Preferably, the collagen-based starting mate 0026. Preferably, the transglutaminase is derived or pre rial comprises collagen at a concentration of 1 to 10 mg/ml. pared from mammaliantissue or cells. For example, the trans 0014 Preferably, the collagen-containing starting mate glutaminase may be guinea pig liver tissue transglutaminase. rial consists of Substantially pure collagen. By substantially 0027 More preferably, the transglutaminase is prepared pure we mean that the starting material is at least 50% by from human tissue or cells. For example, the transglutami weight collagen, preferably at least 60%, 70%, 80%, 90%, or nase may be extracted from human tissue sources Such as 95% by weight collagen. More preferably, the starting mate lung, liver, spleen, kidney, heart muscle, skeletal muscle, eye rial is 100% by weight collagen. lens, endothelial cells, erythrocytes, Smooth muscle cells, 0015. Alternatively, the collagen-containing starting bone and macrophages. Advantageously, the transglutami material may comprise one or more additives. For example, in nase is a tissue transglutaminase derived from human red a preferred embodiment the starting material comprises a cell cells (erthrocytes), or a plasma transglutaminase derived adhesion factor. from either human placenta or human plasma. 0016. By cell adhesion factor we mean a component (e.g. 0028. Alternatively, the transglutaminase may be obtained polypeptide) that possesses specific binding sites for cell from a culture of human cells that express a mammalian Surface receptors, thus enabling cell attachment, cell spread transglutaminase, using cell culture methodology well ing and differentiation. known in the art. Preferred cell line sources of such trans glutaminases include human endothelial cell line ECV304 0017 Preferably, the cell adhesion factor is selected from (for tissue transglutaminase) and human osteosarcoma cell the group consisting of fibronectin, fibrin, fibrillin, glyco line MG63. Soaminoglycans, hyaluronic acid laminin, vitronectin and 0029. It will be appreciated by those skilled in the art that elastin. the Source of the transglutaminase may be selected according 0018 More preferably, the cell adhesion factor is to the particular use (e.g. site of implantation) of the bioma fibronectin. terial. For example, if the biomaterial is to be used as artificial 0019 Most preferably, the fibronectin is present at a con bone, it may be beneficial for the material to comprise a centration of 5 to 1000 ug/ml. bone-derived transglutaminase. 0020. In a further preferred embodiment, the additives is 0030. In an alternative embodiment of the first aspect of selected from the group consisting of polylactic acid, poly the invention, the transglutaminase is a microbial trans hydroxybutyrate, poly(e-caprolactone), polyglycolic acid, glutaminase. For example, the transglutaminase may be polysaccharides, chitosans and silicates. derived or prepared from Streptoverticillium mobaraenase, 0021. In a further preferred embodiment, the collagen Streptoverticillium ladakanum, Streptoverticillium cinnamo containing biomaterial could is coated on an inert medical neum, Bacillus subtilis or Phytophthora cactorum. implant, such as metals, bioceramics, glass or bio-stable poly 0031. It will be appreciated by skilled persons that the mers (for example polyethylene, polypropylene, polyure transglutaminase used in the methods of the invention may be thane, polytetrafluoroethylene, poly(vinyl chloride), polya a recombinant transglutaminase. mides, poly(methy-7-methacrylate), polyacetal, 0032) Nucleic acid molecules encoding a transglutami polycarbonate, poly(-ethylene terphthalate), polyetherether nase are known in the art. For example, the coding sequence letone, and polysulfone). The biomaterial may also be coated for human coagulation factor XII A1 polypeptide is disclosed or mixed with silk. in Grundmann et al., 1986 (accession no. NM 000129). The US 2008/03055.17 A1 Dec. 11, 2008
coding sequence for human tissue transglutaminase is dis time and under appropriate conditions known to those skilled closed in Gentile et al., 1991 (accession no. M55153). in the art in view of the teachings disclosed herein to permit 0033 Nucleic acid molecules encoding a transglutami the expression of the transglutaminase, which can then be nase may be used in accordance with known techniques, recovered. appropriately modified in view of the teachings contained 0038. The recombinant transglutaminase can be recovered herein, to construct an expression vector, which is then used and purified from recombinant cell cultures by well-known to transform an appropriate host cell for the expression and methods including ammonium Sulphate or ethanol precipita production of the polypeptide of the invention. Methods of tion, acid extraction, anion or cation exchange chromatogra expressing proteins in recombinant cells lines are widely phy, phosphocellulose chromatography, hydrophobic inter known in the art (for example, see Sambrook & Russell, 2001, action chromatography, affinity chromatography, Molecular Cloning, A Laboratory Manual. Third Edition, hydroxylapatite chromatography and lectin chromatography. Cold Spring Harbor, N.Y.). Exemplary techniques also Most preferably, high performance liquid chromatography include those disclosed in U.S. Pat. No. 4,440,859 issued 3 (“HPLC) is employed for purification. Apr. 1984 to Rutter et al., U.S. Pat. No. 4,530,901 issued 23 0039. Many expression systems are known, including sys Jul.1985 to Weissman, U.S. Pat. No. 4,582,800 issued 15 Apr. tems employing: bacteria (e.g. E. coli and Bacillus subtilis) 1986 to Crowl, U.S. Pat. No. 4,677,063 issued 30 Jun. 1987 to transformed with, for example, recombinant bacteriophage, Mark et al., U.S. Pat. No. 4,678,751 issued 7 Jul. 1987 to plasmid or cosmid DNA expression vectors; yeasts (e.g. Sac Goeddel, U.S. Pat. No. 4,704,362 issued 3 Nov. 1987 to charomyces cerevisiae) transformed with, for example, yeast Itakura et al., U.S. Pat. No. 4,710,463 issued 1 Dec. 1987 to expression vectors; insect cell Systems transformed with, for Murray, U.S. Pat. No. 4,757,006 issued 12 Jul.1988 to Toole, example, viral expression vectors (e.g. baculovirus); plant Jr. et al., U.S. Pat. No. 4,766,075 issued 23 Aug. 1988 to cell systems transfected with, for example viral or bacterial Goeddeletal and U.S. Pat. No. 4,810,648 issued 7 Mar. 1989 expression vectors; animal cell systems transfected with, for to Stalker, all of which are incorporated herein by reference. example, adenovirus expression vectors. 0034. The nucleic acid molecule, e.g. cDNA, encoding the transglutaminase may be joined to a wide variety of other 0040. The vectors include a prokaryotic replicon, such as DNA sequences for introduction into an appropriate host. The the Col E1 ori, for propagation in a prokaryote, even if the companion DNA will depend upon the nature of the host, the vector is to be used for expression in other, non-prokaryotic manner of the introduction of the DNA into the host, and cell types. The vectors can also include an appropriate pro whether episomal maintenance or integration is desired. moter Such as a prolaryotic promoter capable of directing the 0035 Generally, the DNA is inserted into an expression expression (transcription and translation) of the genes in a vector, Such as a plasmid, in proper orientation and correct bacterial host cell, such as E. coli, transformed therewith. reading frame for expression. If necessary, the DNA may be 0041 A promoter is an expression control element formed linked to the appropriate transcriptional and translational by a DNA sequence that permits binding of RNA polymerase regulatory control nucleotide sequences recognised by the and transcription to occur. Promoter sequences compatible desired host, although Such controls are generally available in with exemplary bacterial hosts are typically provided in plas the expression vector. Thus, the DNA insert may be opera mid vectors containing convenient restriction sites for inser tively linked to an appropriate promoter. Bacterial promoters tion of a DNA segment of the present invention. include the E. coli lacI and lacZ promoters, the T3 and T7 0042 Typical prokaryotic vector plasmids are: puC18, promoters, the gpt promoter, the phage W. PR and PL promot pUC19, pBR322 and pBR329 available from Biorad Labo ers, the phoA promoter and the trp promoter. Eukaryotic ratories (Richmond, Calif., USA); pTrc99A, pKK223-3, promoters include the CMV immediate early promoter, the pKK233-3, plDR540 and pRIT5 available from Pharmacia HSV thymidine kinase promoter, the early and late SV40 (Piscataway, N.J., USA); p3S vectors, Phagescript vectors, promoters and the promoters of retroviral LTRs. Other suit Bluescript vectors, pNH8A, pNH16A, pNH18A, pNH46A able promoters will be known to the skilled artisan. Alterna available from Stratagene Cloning Systems (La Jolla, Calif. tively, the Baculovirus expression system in insect cells may 92037, USA). be used (see Richardson et al., 1995). The expression con 0043 A typical mammalian cell vector plasmid is pSVL structs will desirably also contain sites for transcription ini available from Pharmacia (Piscataway, N.J., USA). This vec tiation and termination, and in the transcribed region, a ribo toruses the SV40 late promoter to drive expression of cloned some binding site for translation. (see WO98/16643) genes, the highest level of expression being found in T anti 0036. The vector is then introduced into the host through gen-producing cells. Such as COS-1 cells. Examples of an standard techniques. Generally, not all of the hosts will be inducible mammalian expression vectors include pMSG, also transformed by the vector and it will therefore be necessary to available from Pharmacia (Piscataway, N.J., USA), and the select for transformed host cells. One selection technique tetracycline (tet) regulatable system, available form Clon involves incorporating into the expression vector a DNA tech. The pMSG vector uses the glucocorticoid-inducible sequence marker, with any necessary control elements, that promoter of the mouse mammary tumour virus long terminal codes for a selectable trait in the transformed cell. These repeat to drive expression of the cloned gene. The tet regulat markers include dihydrofolate reductase, G418 or neomycin able system uses the presence or absence of tetracycline to resistance for eukaryotic cell culture, and tetracyclin, kana induce protein expression via the tet-controlled transcrip mycin or amplicillin resistance genes for culturing in E. coli tional activator. and other bacteria. Alternatively, the gene for such selectable 0044) Useful yeast plasmid vectors are pRS403-406 and trait can be on another vector, which is used to co-transform pRS413-416 and are generally available from Stratagene the desired host cell. Cloning Systems (La Jolla, Calif. 92037, USA). Plasmids 0037 Host cells that have been transformed by the recom pRS403, pRS404, pRS405 and pRS406 are Yeast Integrating binant DNA of the invention are then cultured for a sufficient plasmids (YIps) and incorporate the yeast selectable markers US 2008/03055.17 A1 Dec. 11, 2008
HIS3, TRP1, LEU2 and URA3. Plasmids pRS413-416 are increased ability of the variant enzyme to facilitate the adhe Yeast Centromere plasmids (YCps). sion and spreading of cells on medical implants may be mea 0.045 Methods well known to those skilled in the art can sured by the methods disclosed herein. be used to construct expression vectors containing the coding 0053 Variant transglutaminases may be made using meth sequence and, for example appropriate transcriptional or ods of protein engineering and site-directed mutagenesis translational controls. One such method involves ligation via commonly known in the art (for example, see Sambrook & homopolymer tails. Homopolymer polydA (or polydC) tails Russell, Supra.). are added to exposed 3'OH groups on the DNA fragment to be 0054 Advantageously, the variant transglutaminase is a cloned by terminal deoxynucleotidyl transferases. The frag fragment of a naturally occurring transglutaminase which ment is then capable of annealing to the polydT (or polydC) retains the ability of said naturally occurring transglutami tails added to the ends of a linearised plasmid vector. Gaps left nase to promote collagen crosslinking. following annealing can be filled by DNA polymerase and the 0055. It will be appreciated that in the methods of the first free ends joined by DNA ligase. aspect of the invention, the treatment of the collagen-contain 0046. Another method involves ligation via cohesive ends. ing starting material with a transglutaminase must be per Compatible cohesive ends can be generated on the DNA formed under conditions which allow the formation of e-(y- fragment and vector by the action of suitable restriction glutamyl) lysine crosslinks in the collagen. Such conditions enzymes. These ends will rapidly anneal through comple may readily be determined by persons skilled in the art. For mentary base pairing and remaining nicks can be closed by example, the formation of e-(Y-glutamyl) lysine crosslinks the action of DNA ligase. may be measured as described in the Examples below. 0047 A further method uses synthetic molecules called 0056 Preferably, the collagen starting material is neutra linkers and adaptors. DNA fragments with blunt ends are lised prior to treatment with the transglutaminase (in order to generated by bacteriophage T4 DNA polymerase or E. coli facilitate collagen fibril formation and to promote trans DNA polymerase I which remove protruding 3' termini and glutaminase activity). full in recessed 3' ends. Synthetic linkers, pieces of blunt 0057 Advantageously, the transglutaminase is used at a ended double-stranded DNA which contain recognition sequences for defined restriction enzymes, can be ligated to concentration of between 50 and 1000 ug per ml of reaction blunt-ended DNA fragments by T4 DNA ligase. They are mixture. Preferably, the collagen concentration within the Subsequently digested with appropriate restriction enzymes reaction mixture is 3 to 6 mg/ml. to create cohesive ends and ligated to an expression vector 0058. The crosslinking reaction mixture containing the with compatible termini. Adaptors are also chemically syn collagen and the transglutaminase may further comprise one thesised DNA fragments which contain one blunt end used or more of the following: for ligation but which also possess one pre-formed cohesive (i) a reducing agent (for example, DTT); end. (ii) calcium ions (for example, CaCl); and/or 0048 Synthetic linkers containing a variety of restriction (iii) a buffering agent which buffers the reaction mixture at endonuclease sites are commercially available from a number pH 7.4. of sources including International Biotechnologies Inc., New 0059 Preferably, treatment with the transglutaminase is Haven, Conn., USA. performed at 37° C. 0049. A desirable way to modify the nucleic acid molecule 0060 A second aspect of the invention provides a bioma encoding the transglutaminase is to use the polymerase chain terial comprising crosslinked collagen obtained or obtainable reaction as disclosed by Saiki et al. (1988). In this method the by a method according to the first aspect of the invention. nucleic acid molecule, e.g. DNA, to be enzymatically ampli fied is flanked by two specific oligonucleotide primers which 0061 Preferably, the biomaterial is substantially free of themselves become incorporated into the amplified DNA. catalysts, initiators and/or unreacted or partially reacted The said specific primers may contain restriction endonu crosslinking agents, wherein the unreacted or partially clease recognition sites which can be used for cloning into reacted crosslinking agent is not a transglutaminase. expression vectors using methods known in the art. 0062. A third aspect of the invention provides the use of a 0050 Conveniently, the transglutaminase is a variant biomaterial according to the second aspect of the invention in transglutaminase. the manufacture of a medical implant or wound dressing. 0051. By “a variant' we include a polypeptide comprising 0063 A fourth aspect of the invention provides a medical the amino acid sequence of a naturally occurring trans implant comprising a biomaterial according the first aspect of glutaminase wherein there have been amino acid insertions, the invention. Preferably, the medical implant material is deletions or Substitutions, either conservative or non-conser artificial bone. Vative, such that the changes do not Substantially reduce the 0064. It will be appreciated that the medical implant may activity of the variant compared to the activity of the activated consist solely of a biomaterial of the invention or, alterna naturally occurring transglutaminase. For example, the Vari tively, may comprise a biomaterial of the invention together ant may have increased crosslinking activity compared to the with one or more other biomaterials. For example, the medi crosslinking activity of the naturally occurring transglutami cal implant may comprise a biomaterial of the invention aSC. which is coated, impregnated, covalently linked or otherwise 0052. The enzyme activity of variant transglutaminases mixed with a known biomaterial. Such as metals, bioceram may be measured by the biotin-cadaverine assay, as described ics, glass or biostable polymers (for example polyethylene, in the Examples and as published in (Jones et al., 1997). For polypropylene, polyurethane, polytetrafluoroethylene, poly example, reduced expression of tissue transglutaminase in a (vinyl chloride), polyamides, poly(methylmethacrylate), human endothelial cell line leads to changes in cell spreading, polyacetal, polycarbonate, poly(-ethylene terphthalate), cell adhesion and reduced polymerisation of fibronectin. polyetheretherketone, and polysulfone). Alternatively, transglutaminase activity may be measured by 0065. A fifth aspect of the invention provides a wound the incorporation of "C-putrescine incorporation into dressing comprising a biomaterial according the first aspect N,N'-dimethylcasein, as outlined by Lorand et al., 1972. The of the invention. US 2008/03055.17 A1 Dec. 11, 2008
0066. The medical implants and wound dressings of the using the incorporation technique. HOB cells were then invention may take the form of a sponge or a freeze-dried seeded at 2000 cells/well onto the different substrates, using lattice after TGase crosslinking, or may easily be made in a complete media, in a humidified-atmosphere incubator at 37° variety of ways (see below). C. with 5% CO. At the relevant time points, the cells were removed and the substrates washed twice with PBS and dis tilled water. Samples were then stained using 0.1% Coo massie brilliant blue stain solution. Pictures were then taken FORM OF using an Olympus microscope and digital camera under x400 COLLAGEN APPLICATIONS magnification. Fibres Suture material, weaving blood vessels, valve prosthesis, (0075 FIG. 4. Residual protein concentration (after 72 haemostatic fleece, knitted or woven fabric as tissue hours) of native and TG-treated collagen gels following the Support culture of HOB cells. Collagen (6 mg/ml) was pre-treated Flour or Haemostatic agent powder with either 50 g/ml tTG or mTO (activities: tTG: 11500 Film, Corneal replacement, contact lens, haemodialysis, Units/mg. mTO: 16000 Units/mg). HOB cells were then membrane artificial kidneys, membrane oxygenators, wound seeded at 2000 cells/well onto the different substrates, using or tape dressing, patches (aneurism, bladder, hernia) complete media, in a humidified-atmosphere incubator at 37° Gel Vitreous body, cosmetics (creams) Solution Plasma expander, vehicle for drug delivery system, C. with 5% CO. After 72 hours, the cells were removed and injectable in skin and lip cosmetic defects the residual collagen-Substrates, if any, were washed twice Sponge or Wound dressing, bone-cartilage Substitute, Surgical with PBS and distilled water. Further treatment with micro felt tampons, laparotomy pads, contraceptives, vessel bial collagenase and trypsin for 24 hours was performed as prosthesis, reservoir for drug delivery described in the Materials and Methods section. The protein Tubing Reconstructive Surgery of hollow organs (oesophagus, concentrations of these were determined by the Lowry assay. trachea) Results are from three independent experiments, each with Taken from: Chvapil, 1979. In Fibrous Proteins: Scientific, Industrial and triplicate samples, and are expressed as mean values with Medical Aspects Vol. 1, 4th International Conference on Fibrous Proteins SD. (Massey University) (Editors: Parry D A D and Creamer L. K). London Aca (0076 FIG. 5. HFDF cell mediated collagen degradation demic Press. p 259 monitored using Coomassie blue staining. Collagen (6 0067. In a preferred embodiment, the medical implants mg/ml) was pre-treated with either 50 ug/ml tTG or mTO and wound dressings of the invention are provided in a sealed (activities: tTG: 11500 Units/mg. mTO: 16000 Units/mg) package. Preferably, the package is sterile. Methods of pro using the incorporation technique. HFDF cells were then ducing Such packages are well known in the art. seeded at 2000 cells/well onto the different substrates, using 0068 A sixth aspect of the invention provides a kit for complete media, in a humidified-atmosphere incubator at 37° producing a biomaterial according to the first aspect of the C. with 5% CO. At the relevant time points, the cells were invention comprising collagen, a transglutaminase and, removed and the substrates washed twice with PBS and dis optionally, a cell adhesion factor (Such as fibronectin). tilled water. Samples were then stained using 0.1% Coo 0069. In a preferred embodiment, the kit is provided in a massie brilliant blue stain solution. Pictures were then taken sealed package. Preferably, the package is sterile. using an Olympus microscope and digital camera under x400 0070 Advantageously, the kit further comprises instruc magnification. tions for performing a method according to the first aspect of (0077 FIG. 6. Residual protein concentration (after 72 the invention. hours) of native and TG-treated collagen gels following the (0071. The invention will now be described in detail with culture of HFDF cells. Collagen (6 mg/ml) was pre-treated reference to the following figures and examples: with either 50 g/ml tTG or mTO (activities: tTG: 11500 0072 FIG.1. Type I collagen fibrillogenesis after neutrali Units/mg. mTO: 16000 Units/mg). HFDF cells were then sation in the presence of transglutaminases. Collagen (3 seeded at 2000 cells/well onto the different substrates, using mg/ml) was neutralised as in the methods and was treated complete media, in a humidified-atmosphere incubator at 37° with 0, 50 or 250 g/ml of microbial TG (A) or tTG (B). 500 C. with 5% CO. After 72 hours, the cells were removed and uM of the TGase inhibitor N-Benzyloxycarbonyl-L-phenyla the residual collagen-Substrates, if any, were washed twice lanyl-6-dimethyl-sulfonium-5-oxo-L-norleucine (R281) with PBS and distilled water. Further treatment with micro was used to confirm that the effects were due to transglutami bial collagenase and trypsin for 24 hours was performed as nase activity. The absorbance at 325 nm was measured using described in the Materials and Methods section. The protein a PYE Unicam SP1800 UV spectrophotometer. The tempera concentrations of these were determined by the Lowry assay. ture was controlled at 25°C. using a Techne C-85A circulator. Results are from three independent experiments, each with Results are from the average of 3 independent experiments. triplicate samples, and are expressed as mean values with 0073 FIG. 2. Type III collagen fibrillogenesis after neu SD. tralisation in the presence of transglutaminases. Collagen (3 0078 FIG.7. Collagen (A) and gelatin (B) zymography of mg/ml) was neutralised as in the methods and was treated HFDF cell culture supernatants after 24h growth on different with 0, 50 or 250 g/ml of mTG (A) or tTG (B). 500 uM media. Lane 1: molecular weight markers (BioRad 161 inhibitor R281 was used to confirm that the effects were due 0317); lane 2: Supernatant after growth on GPL tTG treated to transglutaminase activity. The absorbance at 325 nm was collagen; lane 3: Supernatant after growth on mTO treated measured using a PYE Unicam SP1800 UV spectrophotom collagen; lane 4: Supernatant after growth on untreated col eter. The temperature was controlled at 25°C. using a Techne lagen; lane 5: Supernatant after growth in the absence of C-85A circulator. Results are from the average of 3 indepen collagen. dent experiments. (0079 FIG. 8. Residual protein concentration (after 72 0074 FIG. 3. HOB cell mediated collagen degradation hours) of cross-linked and fibronectin-incorporated collagen monitored using Coomassie blue staining. Collagen (6 gels following culture of HOB and HFDF cells. Collagen (6 mg/ml) was pre-treated with either 50 ug/ml tTG or mTO mg/ml), incorporated with 5ug/ml or 50 ug/ml offibronectin, (activities: tTG: 11500 Units/mg. mTO: 16000 Units/mg) was pre-treated with either 100 g/ml tTG or mTO (activities: US 2008/03055.17 A1 Dec. 11, 2008
tTG: 11500 Units/mg. mTO: 16000 Units/mg). HOB (FIGS. and then viewed under a light microscope. Pictures were then 5A and 5B) and HFDF (FIGS. 5C and 5D) cells were then taken and spread cells analysed using ScionImageTM Soft seeded at 2000 cells/well onto the different substrates, using ware. Where by attached and spread cells were distinguished complete media, in a humidified-atmosphere incubator at 37° and characterised based upon the deviations of their cyto C. with 5% CO. After 72 hours, the cells were removed and plasm—as previously described by Jones et al., (1997). the residual collagen-substrates, if any, were washed twice Results are from four independent experiments and represent with PBS and distilled water. Further treatment with micro the 1-hour and 6-hour time points respectively. Each experi bial collagenase and trypsin for 24 hours was performed as ment is with triplicate samples, and are expressed as mean described in the Materials and Methods section. The protein values with SD. Spreading characteristics are represented concentrations of these were determined by the Lowry assay. by the (% average spread cells per field) derived from the Results are from two independent experiments, each with average spread cells divided by total cells in the field of triplicate samples, and are expressed as mean values with vision. The field of vision corresponds to the visible area SD. observed at an x400 magnification with cell numbers ranging from 50-100 cells per field. 0080 FIG.9. Proliferation of HOB and HFDF cells when I0083 FIG. 12. Attachment and spreading characteristics cultured on native and TG-treated collagen substrates (6A of HOB cells on tTG-treated collagen. Collagen (6 mg/ml) and 6B correspond to 50 lug/ml of TG; 6C to 6F corresponds was pre-treated with either tTG or mTO at 50-100 ug/ml to 100 ug/ml of TG). Collagen (6 mg/ml) was pre-treated with (activities: tTG: 11500 Units/mg. mTO: 16000 Units/mg). either a combination of 50 or 100 ug/ml tTG, 50 or 100 ug/ml HOB and HFDF cells were then initially seeded at 2000 of mTO, or 5 or 50 lug/ml of fibronectin (activities: tTG: cells/well of a 96 well plate and cultured on the different 11500 Units/mg. mTO: 16000 Units/mg). HOB (FIGS. 9A, Substrates, using complete media, in a humidified-atmo 9C and 9E) and HFDF (FIGS. 9B, 9D and 9F) cells were sphere incubator at 37°C. with 5% CO, for the relevant time initially seeded at 2000 cells/well of a 96 well plate and points. Cells were fixed using 3.7% (w/v) paraformaldehyde cultured on the different Substrates, using complete media, in before being stained with May-Grunwald and Giemsa stains a humidified-atmosphere incubator at 37°C. with 5% CO, and then viewed with an Olympus C2 microscope before for the relevant time points. Proliferation rates were deter pictures were taken with an Olympus DP10 digital camera. mined by treatment of the samples with CelTiter AQ solution Figures are from a field of vision, under x400 magnification, as described in the Materials and Methods section. Results and indicate the 6 hour time point with cell numbers ranging represent the mean value and SD from four independent from 50-100 cells per field. experiments, each having triplicate samples. I0084 FIG. 13. Alkaline phosphatase activity of HOB cells 0081 FIG. 10. Attachment characteristics of HOB and cultured on TG-treated collagen substrates. Collagen (6 HFDF cells on native, TG-treated and TG-FN incorporated mg/ml) was pre-treated with either 50-250 ug/ml tTG or mTO collagen substrates (10A and 10B correspond to 50 lug/ml of (activities: tTG: 11500 Units/mg. mTO: 16000 Units/mg). TG; 10C to 10F corresponds to 100 ug/ml of TG). Collagen (6 Cells were initially seeded at 2000 cells/wellofa 96 well plate mg/ml) was pre-treated with eithera combination of 50 or 100 and cultured on the different Substrates, using complete ug/ml tTG, 50 or 100 ug/ml of mTO, or 5 or 50 g/ml of media, in a humidified-atmosphere incubator at 37°C. with fibronectin (activities: tTG: 11500 Units/mg. mTO: 16000 5% CO., for the relevant time points. 50 ul of combined Units/mg). HOB (FIGS. 10A, 10C and 10E) and HFDF triplicate supernatant samples were taken and the ALP levels (FIGS. 10B, 10D and 10F) cells were then initially seeded at determined using the ALP Optimized Alkaline Phosphatase 2000 cells/well of a 96 well plate and cultured on the different EC3131 Colorimetric Lit (Sigma) as described in the Mate Substrates, using complete media, in a humidified-atmo rials and Methods section. Results represent the mean value sphere incubator at 37°C. with 5% CO, for the relevant time and SD from three independent experiments. points. Cells were fixed using 3.7% (w/v) paraformaldehyde before being stained with May-Grunwald and Giemsa stains EXAMPLES and then viewed under a light microscope. Pictures were then taken and attached cells analysed using ScionImageTM Soft Methods and Materials ware. Results are from four independent experiments, each I0085 All water used was de-ionised using an Elgastat with triplicate samples, and are expressed as mean values System 2 water purifier (ELGALtd. UK) and a Milli-Q water with +SD. Attachment characteristics are represented by the purifier (Millipore Waters, UK). All chemicals were pur (% average attached cells perfield) derived from the attached chased from Sigma-Aldrich, Poole, UK, unless otherwise average cells divided by total attached cells at 6 hours. The stated. Sterile preparation of stock solutions and chemicals field of vision corresponds to the visible area observed at an were performed either by filtration through a 0.22 Lum What x400 magnification with cell numbers ranging from 50-100 mann sterile filter and/or autoclaving at 121° C. at 15 psi for cells per field. 1 h. Centrifuges and other handling equipment were cleaned I0082 FIG. 11. Spreading characteristics of HOB and EFDF cells on native, TG-treated and TG-FN incorporated with 70% ethanol prior to use. collagen substrates (11A and 11B correspond to 50 lug/ml of TG; 8C to 11F corresponds to 100 ug/ml of TG). Collagen (6 Cell Culture mg/ml) was pre-treated with eithera combination of 50 or 100 I0086 Human osteoblast (HOB) cells, isolated from ug/ml tTG, 50 or 100 ug/ml of mTO, or 5 or 50 g/ml of explants of trabecular bone dissected from femoral heads fibronectin (activities: tTG: 11500 Units/mg. mTO: 16000 following orthopaedic surgery, as described by DiSilvio Units/mg). HOB (FIGS. 11A, 11C and 11E) and HFDF (1995) were kindly supplied by Professor S. Downes and Dr. (FIGS. 11B, 11D and 11F) cells were then initially seeded at S. Anderson (School of Biomedical Sciences, University of 2000 cells/well of a 96 well plate and cultured on the different Nottingham) and used during this investigation. Human fore Substrates, using complete media, in a humidified-atmo skin dermal fibroblast (HFDF) cells isolated from human sphere incubator at 37°C. with 5% CO, for the relevant time neonatal foreskin (Mr. P. Kotsakis, School of Science, Not points. Cells were fixed using 3.7% (w/v) paraformaldehyde tingham Trent University) were also used. Both cell lines before being stained with May-Grunwald and Giemsa stains were used during their low-passage number, ranging from US 2008/03055.17 A1 Dec. 11, 2008
between 11 to 25 passages. Cell lines were cultured and facturers instructions and analysed using a Beckmann maintained, in vitro, as monolayers in T-flasks using DMEM, DU530 UV/Vis Spectrophtometer. supplemented with 10% heat-inactivated (56° C. for 1 h) FCS, 1% non-essential amino acids and 2 mM L-glutamine. Transglutaminase Periodic additions of 1% penicillin-streptomycin were used 0092 Tissue transglutaminase (tTG) was isolated and to avoid bacterial contamination. Flasks were kept in a purified from guinea pig livers following a modification of the humidified-atmosphere incubator at 37°C. and with 5% CO. Leblanc et al. (1999) involving both anion exchange, gel Cells were routinely passaged and allowed to reach greater filtration and affinity chromatography. Commercial samples than 90% confluency at any one time. For detachment, stan of TG were also used during this investigation: tTG from dard trypsinisation was performed using 0.25% (w/v) guinea pig liver (Sigma-Aldrich, Poole, UK. Cat no. T5398) trypsin/2 mM EDTA solution in PBS solution. and microbial transglutaminase, mTO, (Ajinomoto Corpora tion Inc. Japan), isolated from Streptoverticillium mobarae Cell Viability and Proliferation nase, as the commercially available product, ActivaTMWM. This required further purification steps to remove the incor 0087 Cell counts and viability estimations were per porated maltodextrin: briefly, the ActivaTM WM was dis formed using the standard trypan blue exclusion technique by solved in ice-cold 20 mM phosphate buffer, 2 mM EDTA pH means of a 0.22 um sterile filtered 0.5% (w/v) trypan blue 6.0 and filtered, before being loaded onto a 100 ml solution and a haemocytometer. Non-viable cells stained blue SP-Sepharose FF column overnight at a flow rate of 5 ml/min due to the loss of their membrane integrity and, hence, by recycling. The column was then washed and proteins allowed the passage of dye into the cell. Viable cells remained eluted with a 0-1000 mM gradient of NaCl in 20 mM phos colourless. phate buffer, 2 mM EDTA pH 6.0 over 80 min, collecting 5 ml fractions. Fractions were assayed for protein using the Bio 0088 Cell proliferation and viability were also measured Rad DC protein assay (Bio-Rad Laboratories, Hertfordshire, using the CelTiter AQ One Solution Cell ProliferationTM UK. Cat no. 500-0120)—a modification of the Lowry method assay kit (Promega, Southampton, UK. Cat no. G3580). This (Lowry et al., 1951). Fractions containing mTO were pooled, reagent contains a novel tetrazolium compound (MTS) and an aliquoted, freeze dried and stored at -70° C. Before immedi electron coupling reagent (PES). The MTS tetrazolium com ate use, tTG was pre-treated in 2 mM DTT in 50 mM Tris pound is bioreduced by cells into a coloured formazan prod buffer (pH 7.4) for 10 minutes at room temperature, before uct that is soluble in tissue culture medium. This conversion is addition to a final buffered solution containing 5 mM CaCl2 accomplished by NADPH or NADH produced by dehydro and, a minimum of 1 mM DTT in Tris buffer. Typical activi genase enzymes in metabolically active cells. Assays were ties for the transglutaminases used in this investigation were performed, in the dark, simply by the addition of 20 ul of as follows: tTG: 11500-13000 Units/mg and mTO: 16000 CellTiter AQ reagent into the relevant samples in 100 ul of 25000 Units/mg. culture medium. These samples were then incubated in a humidified-atmosphere incubator at 37° C. and with 5% CO2 Transglutaminase Activity for 90 minutes before the absorbance was read at 490 nm. using a SpectraFluor R. plate reader. 0093. The incorporation of 14-C-putrescine into N,N'- dimethylcasein, as described earlier by Lorand et al. (1972) was used to assay for TG activity and monitor the effects of Attachment and Spreading the inhibitors. Unit of transglutaminase activity is 1 nmol of 0089 Cells were seeded on the relevant substrate at a putrescine incorporated per hour. density of 625 cells/mm. After allowing cells to proliferate, they were fixed in 3.7% (w/v) paraformaldehyde, permeabi Collagen lised by the addition of 0.1% (v/v) Triton X-100 in PBS, 0094 Commercial calf skin collagen type I (Sigma-Ald before staining with May-Grunwald (0.25% (w/v) in metha rich, Poole, UK. Cat no. C9791) was used during this inves nol) and Giemsa stains (0.4% (w/7) in methanol, diluted 1:50 tigation. Native collagen samples were solubilised in 0.2M with water). Cells were then viewed under a x400 magnifi acetic acid (Fisher Scientific, Loughborough, UK. Cat no. cation using an Olympus CK2 microscope. Three separate A/0400/PB17) at 4° C. with constant stirring for 24 hours fixed-size random fields per sample were photographed with before use. Neutralisation of the collagen mixture was per an Olympus DP10 digital camera. formed using a 8:1:1 ratio of collagen: 10xDMEM: 0.2M 0090 Pictures were analysed using Scion ImageTM soft HEPES buffer respectively to a final of pH 7.2. Tissue culture ware (Scion Corporation, Maryland, USA) whereby attached plastic was then covered using this collagen mix (recom and spread cells were distinguished and characterised based mended at 6-10 ug/cm) before being placed into a humidi upon the deviations of their cytoplasm—as previously fied-atmosphere incubator for 12 hours to allow gelation to described by Jones et al., (1997). occur. In general, 50ll of the collagen mix was added to each well of a 96 well plate. Plates were used within 48 hours of the Alkaline Phosphatase Activity collagen matrix formation. 0091. The ALP Optimized Alkaline Phosphatase EC 3.1. Modified Collagen by Transglutaminase 3.1 Colorimetric Test(R) kit (obtained from Sigma-Aldrich, Poole, UK. Cat no. DG 1245-K) was used to quantify the ALP 0.095 Neutralised collagen mixture was subjected to treat activity. Serum ALP hydrolyses p-nitrophenyl phosphate to ment by both tissue and microbial TG. Samples of the neu p-nitrophenol and inorganic phosphate. The hydrolysis tralised collagen were treated with 50-1000 ug/ml of tTG, in occurs at alkaline pH and the p-nitrophenol formed shows an a reaction mix consisting of2 mMDTT and 5 mM CaCl in 10 absorbance maximum at 405 nm. The rate of increase in mM Tris buffer (pH 7.4). The reaction mixture for the micro absorbance at 405 nm is directly proportional to ALPactivity bial enzyme simply consisted of 10 mM Tris buffer (pH 7.4). in the sample. Samples were treated according to the manu Incorporated fibronectin (Sigma-Aldrich, Poole, UK. Cat no. US 2008/03055.17 A1 Dec. 11, 2008
F0895) was used at concentrations of 5ug/ml and 50 g/ml. rate fixed-size random fields per triplicate samples were pho Transglutaminase was always added last to the collagen tographed using an Olympus microscope and digital camera. reaction mix to minimise any self-imposed cross-linking. Protein Concentration Controls using 10 mM EDTA (to block tTG activity) and an 0098. The total protein contents of the collagen samples active site-directed inhibitor 1,3-dimethyl-2-(2-oxopropyl were determined by the Lowry method (Lowry et al., 1951) sulfanyl)-3H-1,3-diazol-1-ium-chloride (R283, Notting using the Bio-Rad DC protein assay kit (Bio-Rad Laborato ham Trent University, UK) were also included in each assay. ries, Hertfordshire, UK. Cat no. 500-0120). When using buff For 96 well plates, 50 ul of the pre-treated collagen mixture ers containing a high percentage of SDS or other detergents, was added to each well before being placed into a humidified the Bicinchoninic acid assay kit (Sigma-Aldrich, Poole, UK. atmosphere incubator, at 37° C. and with 5% CO2, for 8 Cat no. BCA-1) was used (Brown et al., 1989). hours. On removal, wells were washed twice with sterile distilled water and used immediately. Collagenase Degradation of Matrices 0099 Collagen substrates were subjected to digestive Determination of e-(Y-glutamyl)lysine Cross-Link treatment with both a 100 ul of a 1 mg/ml microbial collage 0096 Cross-linked and native samples of collagen were nase solution (Clostridium histolyticum, Sigma-Aldrich, proteolytically digested by a modification of the methods of Poole, UK. Cat no. C9891) followed by 100 ul 0.25% (w/v) Griffin and Wilson (1984), which included an initial digestion trypsin/2 mM EDTA solution in PBS solution for 24 hours at with microbial collagenase (Clostridium histolyticum, 1 37° C. Samples were washed twice with PBS followed by a mg/ml, Sigma-Aldrich, Poole, UK. Cat no. C9891), prior to wash with distilled water before the enzymatic digestion the addition of further proteases. After digestion, Samples treatment. were freeze-dried and then resuspended in 0.1M HCl and Zymography sonicated for 2 minto aid dispersion. An aliquot (10-90 ml) 0100 Gelatin and collagen zymography were carried out was mixed with loading buffer (0.2M lithium citrate, 0.1% according to the following method, adapted from Herronetal, phenol pH 2.2) and loaded onto a Dionex DC-4A resin col 1986. Resolving gels were mixed with the following compo umn 0.5 cmx20 cm using a Pharmacia Alpha Plus amino acid nents, in order: 1 ml of 5 mgml-1 Type I collagen Solution analyser. The buffer elution profile was as shown in the table (Sigma C9791) in 20 mMacetic acid (for collagen zymogra below. Derivatisation was performed post column using phy)/1 ml of 5 mgml-1 porcine gelatin (Sigma G2625) in HO o-pthaldialdehyde (0.8M boric acid, 0.78M potassium (for gelatin zymography), 3.1 ml HO, 2.5 ml of 1.5M Tris hydroxide, 600 mg/ml o-phthaldialdehyde, 0.5% (v/v) HCl pH 8.8, 3.33 ml of 29% acrylamide/1% N,N'-methylene methanol, 0.75% (v/v) 2-mercaptoethanol, 0.35% (v/v) Brij bisacrylamide, 50 ul of 10% ammonium persulphate, 10ul of 30) and the absorbance was measured at 450 nm. Dipeptide N.N.N',N'-tetramethylethylenediamine (TEMED). SDS was was determined by addition of known amounts of e(Y- found to cause precipitation of the collagen and so was not glutamyl)lysine to the sample and comparing peak areas. added to the resolving gel. Stacking gels were poured in the usual way ie. 0.65 ml of 29% acrylamide/1% N,N'-methylene bisacrylamide, 3 ml HO, 1.25 ml 0.5M Tris HCl pH 6.8, 50 ul of 10% SDS, 25ul of 10% ammonium persulphate, 5ul of Time (min) Buffer Column temperature TEMED. O-9 1 25o C. 0101 Samples containing MMPs were diluted 1:1 with 9-32 2 25o C. loading buffer (1M Tris HCl pH 6.8, 50% glycerol, 0.4% 32-67 3 25o C. bromophenol blue) and electrophoresed at 100 V in standard 67-107 3 25o C. 107-123 6 75o C. Laemmli running buffer (24 mM Tris HCl, 192 mM glycine, 123-13S 1 75o C. 3.47 mMSDS, pH 8.3), avoiding overheating (approx 4-5 h). 135-147 1 650 C. After electrophoresis, gels were washed twice, with shaking, 147-159 1 35o C. for 30 min each in 200 ml of 2.5% Triton X-100, to remove 159-171 1 25o C. SDS and recover MMP activity. The gels were then placed in Buffer 1: 0.2M lithium citrate, 0.1% phenol, 1.5% (v/v) propan-2-ol pH 2.8. digestion buffer (100 mM Tris HCl, 5 mM CaCl, 0.005% Buffer 2: 0.3M lithium citrate, 0.1% phenol, 1.5% (v/v) propan-2-ol pH 3.0. Brij-35, 1 uM ZnCl, 0.001% NaN, pH 8) for 16-48 h at 37° Buffer 3: 0.6M lithium citrate, 0.1% phenol pH 3.0. C. Gels were stained with 0.2% Coomassie brilliant blue Buffer 6: 0.3M lithium hydroxide. R-250 in 50% ethanol, 10% acetic acid for 2 hand destained by microwaving for 15 min (full power 850W) in 3 changes of deionised H2O. Coomassie Blue Staining Assay for Cell Culture Determination of Collagen Fibril Formation Rate 0097 Native and pre-treated collagen samples gels were 0102 Collagen fibrillogenesis was monitored by measur plated out at 50 pulper wellofa 96-well plate. 100 ulofa 2x10' ing the absorbance (turbidity) at 325 nm using a PYE Unicam cells/ml cell homogenate, cultured in complete media, were SP1800 UV spectrophotometer. added to the wells in triplicates. Plates were then kept in a humidified-atmosphere incubator for the relevant time point Statistical Analysis of Data (s). After incubation, cells were removed from the collagen 0103 Differences between datasets (shown as mean+S. matrix by addition of 0.5% (w/v) Na-deoxycholate in 10 mM D.) were determined by the Student's t-test at a significance Tris-HC1. A rinse with distilled water was performed before level of p-0.05. the collagen samples were stained with a 0.1% (w/v) Coo Results massie Brilliant blue stain solution (50% (v/v) methanol: 10% (v/v) acetic acid; 40% (v/v) dHO). Samples were Cross-linking of Collagen by Microbial and Tissue Trans allowed to stain for 5 minutes before a further rinse with glutaminases distilled water. Unstained areas, which appeared lighter blue, 0104 Native collagen (type I) was treated with tTG and give an indication of collagen degradation by cells. Two sepa mTG to catalyse the formation of e-(Y-glutamyl)lysine cross US 2008/03055.17 A1 Dec. 11, 2008
linking. Table 1 documents the results from the ion exchange Resistance of Native and Cross-Linked Collagento HOB Cell analyses of the native and TG-treated collagen, giving the Mediated Degradation extent of cross-linking for each of the TG treatments. Treat 0106 The capacity of HOB cells to degrade collagen, via ment of collagen with increasing concentrations of TG leads endogenous proteases was assessed. FIG. 3 presents a selec to a corresponding increase of the amount of e-(Y-glutamyl) tion of digital photographs of the native and TG-treated col lysine bonds present—with up to 1 mol of cross-link per mol lagen gels, when cultured with HOB cells for up to a 72-hour of collagen monomer. Treatment with mTG, gave a much period, and the collagen then stained with Coomassie blue greater increase (almost two-fold) of the amount of isopep after removal of cells. Degradation of collagen occurs just 24 tide formed for the equivalent (ug of protein) TG concentra hours after the HOB cells were seeded onto the collagen. In tion used. It can also be seen that on incorporating fibronectin contrast, with both the tTG and mTO-pre-treated collagen into the collagen via TG, an increase in isopeptide bonds samples, degradation is at a much slower rate, with a higher occurs with the corresponding increase offibronectin concen amount of residual collagen remaining as judged by the tration. However, interestingly, there appears to be a decrease amount of Coomassie blue staining. Hence, collagen treated in the total amount of isopeptide formed for the fibronectin with 50 ug/ml TG (activities: tTG: 11500 Units/mg. mTO: variants as compared to the equivalent collagen-TG only 16000 Units/mg) showed a greater resistance to cell mediated samples. degradation as compared to the native collagen. Comparison of the residual blue staining Suggests that the mTO treated TABLE 1. collagen shows slightly more resilience to HOB-cell degra dation than the tTG-treated variant. When residual protein Transglutaminase mediated cross-linking of collagen type I concentration remaining was assessed following proteolytic and incorporation of fibronectin. Collagen samples were initially digestion, this confirmed the significant increased resistance prepared at 6 mg/ml. Both tTG and mTO were used at concentrations of 50-1000 g/ml (activities: tTG: 11500-13000 Units/mg: of the TG cross-linked collagen to cell mediated degradation mTO: 16000-25000 Units/mg). Fibronectin was incorporated at (p<0.05). However, very little differences can be seen 5 g/ml and 50 g/ml. The cross-linking reaction was between the collagen cross-linked by the different trans allowed to proceed in a humidified-atmosphere incubator overnight glutaminases (FIG. 4). at 37° C. and with 5% CO2.
nmol of cross Resistance of Native and Cross-Linked Collagen to HFDF link-relative Cell Mediated Degradation change mg protein mol cross TG conc. Sample to native link/mol 0107 The capacity of HFDF cells to degrade collagen, via Sample (g/ml) collagen of collagen endogenous proteases was also assessed. FIG. 5 presents a Collagen O16 O.O2 selection of digital photographs of the native and TG-treated Coll-tTG 50 1.09 6.81 O.13 collagen gels, when cultured with HFDF cells for up to a Coll-tTG 100 240 1S.OO O.29 72-hour period. The collagen was then stained with Coo Coll-tTG 2OO 4.6O 28.75 0.55 massie blue after removal of the cells. Degradation of the Coll-tTG 500 S.40 33.75 O.65 collagen occurs just 24 hours after the HFDF cells were Coll-tTG 1OOO 8.90 55.63 1.07 seeded onto the collagen with almost negligible residual gel Coll-mTO 10 O.90 S.63 O.11 Coll-mTO 50 2.00 12.5 O.24 remaining after 72 hours—much greater activity than the Coll-mTO 2OO 4.90 30.63 O.S9 HOB cells. In contrast, with both the tTG and mTO-pre Coll-mTO 500 840 52.50 1.OO treated collagen samples, degradation is a much slower rate Coll-tTG-Fn (5 g/ml) 100 O49 3.06 O.O6 and with a much higher amount of residual collagen remain Coll-tTG-Fn (50 g/ml) 100 1.02 6.38 O.12 ing as judged by the amount of Coomassie blue staining. Coll-mTG-Fn (5 g/ml) 100 O.74 4.63 O.09 Hence, collagen treated with 50 g/ml TG (activities: tTG: Coll-mTG-Fn (50 g/ml) 100 O.78 4.88 O.09 11500 Units/mg; mTG: 16000 Units/mg) showed a much 'Mw collagen: 120 kD greater resistance to HFDF cell mediated degradation as com *native collagen = 0.16 nmol crosslink pared to the native collagen. As found with HOB cells, when TG activity: tTG = 11500-13000 Units/mg. mTO = 16000-25000 Units/mg residual protein concentration remaining was assessed fol lowing proteolytic digestion, this confirmed the increased resistance of cross-linked collagen to cell mediated degrada tion. However, in this case, a significant difference p-0.05) Effect of Transglutaminases on Collagen Fibril Formation can be seen between the collagen cross-linked by the different 0105 To determine the effect of transglutaminase on col TGs (FIG. 6); it appears that mTO treated collagen shows a lagen fibrillogenesis, fibril formation after neutralisation was slightly more resilience to cell mediated degradation than the monitored by measuring absorbance at 325 nm, as a measure tTG-treated variant. of turbidity. In the case of collagen types I and III (see FIGS. (0.108 Matrix Metalloproteinases Secreted by HFDF Cells 1 and 2, respectively), addition of either 50 lug or 250 ug of Grown on Transglutaminase Collagen Matrices mTG or tTG resulted in a significant reduction in the time 0109 Following growth on type I collagen, fibroblasts taken to achieve gel formation. However, type I collagen gels showed an induction of a wide array of collagenases and reached a lower final level of turbidity after treatment with gelatinases when compared with growth on tissue culture transglutaminases compared to untreated gels, whereas type plasticware alone (FIG. 7). After growth on transglutaminase III collagen gels reached a higher final level of turbidity after crosslinked type I collagen, the induction of active MMP1 (45 treatment with transglutaminases compared to untreated gels. kDa) is much less pronounced compared to growth on native Inhibition of transglutaminase activity with the active site collagen, whereas the induction of active MMP2 (66 kDa) directed inhibitor N-Benzyloxycarbonyl-L-phenylalanyl-6- and MMP9 (86 kDa) was increased. Transglutaminase dimethylsulfonium-5-oxo-L-nor-leucine bromide salt crosslinking appeared to alter the MMP expression profile in (R281, a synthetic CBZ-glutaminylglycine analogue) abol a manner consistent with an increase in gelatin character. It is ished these effects. probable that transglutaminase crosslinked collagen matrix is US 2008/03055.17 A1 Dec. 11, 2008
interpreted in a different manner by the fibroblasts and leads make no significant difference (p<0.05) to the cell viability of to an alternative cellular response, probably explaining its both HOB and HFDF cells throughout culture. resistance to cellular degradation. Attachment Characteristics of HOB and HFDF Cells on Resistance of Cross-Linked and Fibronectin-Incorporated Native, TG-Treated and TG-FN Incorporated Collagen Sub Collagen to HOB and HFDF Cell Mediated Degradation Strates 0110. The capacity of HOB and HFDF cells to degrade the 0114 FIGS. 10A to 10F show the short-term cell-attach TG-treated and fibronectin incorporated collagen, via endog ment characteristics of HOB and HFDF cells when cultured enous proteases was also assessed. On removal of the cells, on native, TG-treated and TG-FN incorporated collagen sub after a 72-hour culture period, and staining with Coomassie strates (10A and 10B correspond to 50 g/ml of TG; 10C to blue, it can be seen that differences exist on comparing the 10F corresponds to 100 g/ml of TG; activities: tTG: 11500 TG-cross-linked collagen and the fibronectin-TG-incorpo Units/mg. mTO: 16000 Units/mg) as monitored using light rated collagen (100 ug/ml of TG at activities of activities: microscopy followed by May-Grunwald/Giemsa staining. tTG: 11500 Units/mg. mTO: 16000 Units/mg). FIG. 8 pre Increased numbers of cells can be seen to be attached when sents the residual protein concentration of the samples fol cultured on transglutaminase cross-linked collagen. For the lowing the cell mediated proteolytic digestion. It can be seen HOB cells, comparable cell attachment characteristics can be that significant differences exist for both tTG-Fn and mTO seen on both 50 and 100 g/ml TG-treated collagens (FIGS. Fn treated matrices compared to the normal TG-treated col 10A and 10C) giving a significant increase of about ~20% in lagen (p<0.05) after 72 hours of culture. Interestingly, how attached cells for the corresponding time points over the ever, there appears to be no considerable difference in the non-crosslinked collagen (p<0.05). Comparable enhance resistance of the substrate when fibronectin concentration is ment in cell attachment on the cross-linked collagens were increased. also observed for the HFDF cells (p<0.05) (FIGS. 10B and 10D). In general, matrices incorporated with fibronectin Proliferation Rates of HOB and HFDF Cells on Native, TG show a slight enhancement in the attachment characteristics Treated and TG-EN Incorporated Collagen Substrates for both HOB and HFDF cells (p<0.05) during short-term culture with the exception of matrices treated with 50 ug/ml 0111. The number of viable HOB and HFDF cells on FN; these showing no significant changes (p<0.05) (FIGS. native, TG-treated and TG-EN incorporated collagen matri 10E and 10F). ces (50-100 g/ml of TG; activities: tTG: 11500 Units/mg, mTG: 16000 Units/mg) were monitored using the CellTiter Spreading Characteristics of HOB and HFDF Cells on reagent assay kit according to the manufacturer's instruc Native, TG-Treated and TG-FN Incorporated Collagen Sub tions. It can be seen from FIG. 9A that proliferation for the HOB cells is enhanced on the 50 g/ml TG-treated collagen Strates Substrates—both variants showing a higher cell density over 0115 FIGS. 11A to 11F show the short-term cell-spread 72 hours than that found with native collagen. For long term ing characteristics of HOB and HFDF cells when cultured on survival, the HOB cells also remained more viable after 168 when cultured on native, TG-treated and TG-FN incorporated hours of culture on the TG-treated collagens. In comparison, collagen substrates (11A and 11B correspond to 50 lug/ml of the HFDF cells (FIG. 9B) demonstrated a significantly TG; 11C to 11F corresponds to 100 g/ml of TG; activities: greater increase (p<0.05) in cell number on the TG-treated tTG: 11500 Units/mg; mTG: 16000 Units/mg) as monitored collagens, especially during the initial 72 hours of culture. using light microscopy followed by May-Grunwald/Giemsa However, for long term survival there is very little difference staining (FIG. 12). Increased numbers of cells can be seen to between the different collagens as the cells reach confluency be spread when cultured on 50 ug/ml transglutaminase cross with greater loss of cell viability in the tTG cross-linked linked collagen. In the case of the HOB cells, a comparable collagen. increase of ~5% in the spreading of the HOB cells, at each 0112. It can be seen from FIG.9C that proliferation for the time point, can be seen on both of the TG-treated collagens HOB cells is also enhanced on the 100 g/ml TG-treated (FIG. 11A). In contrast, the HFDF cells show much more collagen Substrates—both variants showing a higher cellden distinct and significant spreading characteristics on the 50 sity than that of native collagen with the tissue trans ug/ml TG-treated collagen increases of at least 10% can be glutaminase variant providing the optimum after 60 hours. In noted for both of the TG-treated variants (FIG.11B)(p<0.05). both cases, enhancement of the long term culture viability is 0116. A further increase in the number of spread cells can experienced with the TG-treated collagens. In comparison, be identified on 100 g/ml transglutaminase cross-linked col the HFDF cells (FIG.9D) demonstrated a considerable dif lagen. In the case of HOB cells, a comparable difference of ference during the initial 48 hours of culture (p<0.05). The ~5% increase ion spread cells can be noted (FIG. 11C)—this TG-treated collagen substrates allow a greater rate of cell behaviour increases with time for extended culture. In con viability to be achieved throughout the 196-hour culture trast for the HFDF cells, although there is still an increase in period; increasing cell density rates by 15%. Microbial the spreading characteristics on the TG-treated collagen, a treated collagen shows a slight advantage compared to the much more distinct and significant behaviour can be identi tissue-TG treated collagen. In general, significant improve fied on the tissue enzyme treated collagen; spreading charac ments are observed when the transglutaminase concentration teristics increase by 15% for many of the time points. Con is increased. trastingly, the microbial-treated collagen shows only a slight 0113. The number of viable HOB and HFDF cells on improvement in the spreading characteristics (FIG. 11D) cross-linked collagen Substrates incorporated with fibronec (p<0.05). tin (5 g/ml and 50 ug/ml) can be seen from FIGS. 9E and 9F 0117. In the case of TG-FN incorporated matrices, it can respectively. In both cases, FN-incorporated matrices show a be seen that a significant enhancement of the spreading char significant improvement (p<0.05) in the cell density during acteristic is noted on 5 ug/ml FN substrates for HOB and the early hours of culture (24 hours). However, interestingly, HFDF cells (p<0.05) (FIGS. 11E and 11F respectively). collagen substrates treated with 50 lug/ml of FN appears to However, for both cases of TG-FN (50 ug/ml), a decrease in US 2008/03055.17 A1 Dec. 11, 2008 the spreading characteristics is noted when compared to the transglutaminases are able to crosslink native collagen type I normal TG-cross-linked substrate. by catalysing the formation ofisopeptide bonds. 0.126 Here, it is demonstrated that TG-modified collagen Alkaline Phosphatase Activity of HOB Cells Cultured on demonstrates greater resistance to cell secreted proteases and, Native and TG-Treated Collagen as a consequence, improved resistance to cell mediated deg radation from cultured HOB and HFDF cells. Crosslinking of 0118 FIG. 13 shows ALP activity of HOB cells cultured the collagen alters the MMP expression profile of HFDF cells on native and TG-treated collagen (50-250 ug/ml of tTG and grown on these modified Substrates, with a reduction of active mTG; activities: tTG: 11500 Units/mg. mTO: 16000 Units/ MMP1 and a corresponding increase in active MMP2 when mg). Increases in ALP activity were observed in all the TG compared to growth on unmodified collagen. This is probably crosslinked collagens—the greatest increase seen with the due to altered signalling of the nature of the extracellular collagen-tTG substrate followed by the collagen-mTG. Typi matrix caused by transglutaminase modification, with cells cally, an increase in the concentration of TG improved the recognising it less as fibrillar collagen. Indeed, transglutami ALP activity of the HOB cells (p<0.05). Interestingly how nase treatment of type I collagen results in a gel that does not ever, the collagen treated with the higher concentration of achieve as high a turbidity as untreated collagen, possibly mTG (250 lug/ml) appears to reduce the corresponding indicating a reduction in fibrillar form. In contrast, type III amount of ALP activity when compared to tTG. collagen shows an increased turbidity with transglutaminase treatment. SUMMARY & CONCLUSIONS 0127. It has also been demonstrated that the modified col 0119 The above results demonstrate the following: lagen is more biocompatible to a wide variety of cells, as I0120 Both microbial and tissue transglutaminases are shown using HOB and HFDF cells. Not only does it enhance able to crosslink type I collagen. the proliferation rates of the cells, but cell attachment and cell 0121 Crosslinking of collagen results in an improve spreading of these cells is also increased when compared to ment in the resistance to degradation by different cell native collagen gels. Additionally, long-term growth and Sur types. vival are maintained with respect to applications in bone repair. Importantly, HOB cells are able to differentiate more I0122) Cells show improved attachment, spreading and quickly on TG-modified collagens as demonstrated by the proliferation when cultured on collagen treated with corresponding increases in ALP activities. Furthermore, on either microbial or tissue transglutaminases; this effect incorporating fibronectin into the collagen Substrates, further is enhanced when fibronectin is also crosslinked to the enhancement of cell properties of proliferation, spreading collagen. and attachment are experienced. I0123 Treatment of type I and type III collagens with 0128. In conclusion, transglutaminase-mediated cross either microbial or tissue transglutaminases immedi linking of collagen has the potential to improve the physical ately after neutralisation from acidic solution, causes an and mechanical properties of native collagen by the formation increase in gelation/fibrillogenesis rate. of stabilising cross-links. Importantly, however, TG increases 0124. These data, taken together, show that transglutami the resistance of the collagen to cell degradation and, in nase treated collagen or collagen/fibronectin matrices offer a addition, enhances the biocompatibility of the substrate by significant advantage over Standard collagen as biomaterials facilitating increased cell enhancing proliferation and also for in vivo use with regard to both biological and physical allowing greater attachment and spreading of cells. stability, and biocompatibility. 0.125 Collagen, with its superior biocompatibility com REFERENCES pared to other natural polymers, and its excellent safety due to its biological characteristics, such as biodegradability and I0129 Aeschlimann, D. and Paulsson, M. (1994): Trans weak antigenicity, has made collagen the primary resource in glutaminases: Protein Cross-Linking Enzymes in Tissues medical applications (Lee et al., 2001). Collagen isolated and Body Fluids. Thrombosis and Haemostasis, 71 (4), from rat tail tendon or foetal calf skin has frequently been 402-425. used successfully as a Support and adhesion Substance in 0.130 Barbani N, Giusti P. Lazzeri L, Polacco G & Pizzi many tissue culture systems for many types of cell lines rani G (1995). Bioartificial materials based on collagen: 1. including osteoblasts (Schuman et al., 1994; Lynch et al., Collagen cross-linking with gaseous glutaraldehyde. Jour 1995) and fibroblasts (Ivarsson et al., 1998). Additionally, nal of Biomaterials Science-Polymer Edition 7 (6): 461 Mizuno et al. (1997) have also reported that type I collagen 469 matrices offer a favourable environment for the induction of I0131 Bell E. Ehrlich P. Buttle DJ & Nakatsuji T (1981). osteoblastic differentiation invitro. However, the use of natu Living tissue formed in vitro and accepted as skin-equiva ral polymers as potential biomaterials, matrices or scaffolds lent of full thickness. Science 221: 1052 for cell based applications in tissue engineering is often (0132 Ben-Slimane S, Guidoin R, Marceau. D. Merhi Y. restricted by its poor mechanical characteristics and loss of King M. W. Sigot-Luizard M. F. 1988. J Eur. Surj. Res. biological properties during formulation (Hubbell, 1995). The major deciding factor, and primary disadvantage, of 20:18-28 many biocomposites concerns the requirement for chemical (0.133 Bradley W G & Wilkes G L (1977). Some mechani cross-linking of the constituent monomers to increase stabil cal property considerations of reconstituted collagen for ity and physical performance during manufacture, thus rais drug release supports. Biomaterials Medical Devices and ing concerns about the issues of toxicity due to residual cata Artificial Organs 5: 159-175 lysts, initiators and unreacted or partially reacted cross I0134 Burke J. F. Yannas IV, Quimby W C, Bondoc CC & linking agents in the final polymer (Coombes et al., 2001). Jung W K (1981). Successful use of a physiologically Collagen, like many natural polymers once extracted from its acceptable artificial skin in the treatment of extensive burn original source and then reprocessed, Suffers from weak injury. Annals of Surgery 194: 413-448 mechanical properties, thermal instability and ease of pro I0135 Chvapil M, Speer D P. Holubec H, Chvapil T A, teolytic breakdown. However, it has been demonstrated that King D H (1993). Collagen-fibers as a temporary scaffold US 2008/03055.17 A1 Dec. 11, 2008
for replacement of ACL in goats. Journal of Biomedical 0152 Lee C H, Singla A & Lee Y (2001). Biomedical Materials Research 27 (3): 313-325 applications of collagen. International Journal of Pharma 0.136 Collighan R, Cortez J & Griffin M (2002). The bio ceutics 221 (1-2): 1-22 technological applications of transglutaminases. Minerva 0153. Lorand et al. (1972) A filter paper assay for transa Biotecnologica 14 (2): 143-148 midating enzymes using radioactive amine Substrates. 0.137 Coombes AGA, Verderio E. Shaw B, Li X, Griffin Alnalytical Biochemistry 50, 623 M. Downes S. (2001). Biocomposites of non-crosslinked 0154) Lowry O H. Rosebrough NJ, Farr A L, Randall RJ natural and synthetic polymers. Biomaterials 23: 21 13 (1951). Journal of Biological Chemistry 193: 265-275 2118 (O155 Lynch MP Stein J. L. Stein G. S. Lian J. B. (1995). 0.138. Dunn M. W. Stenzel K H, Rubin A L., & Miyata T The influence of type I collagen on the development and (1969). Collagen implants in the vitreous. Archives of maintenance of the osteoblast phenotype in primary and Ophthamology 82: 840-844 passaged rat calvarial osteoblasts: modification of expres 0139 Einerson NJ, Stevens K R & Kao WY J (2003). sion of genes Supporting cell growth, adhesion and extra Synthesis and physicochemical analysis of gelatin-based cellular matrix mineralization. Experimental Cell hydrogels for drug carrier matrices. Biomaterials 24(3): Research 216: 35-45 509-523 0156 Matsuda S, Iwata H, Se N & Ikada Y (1999). Bio 0140 Gentile et al., (1991) Isolation and characterization adhesion of gelatin films crosslinked with glutaraldehyde. of cDNA clones to mouse macrophage and human endot Journal of Biomedical Materials Research 45 (1): 20-27 helial cell tissue transglutaminases. J. Biol. Chem. 266(1) (O157 Miyata T, Sohde T. Ruben AL & Stenzel I H (1971). 478-483 Effects of ultraviolet radiation on native and telopeptide 0141 Goo HC, HwangYS, Choi YR, Cho HN & Suh H poor collagen.biochimica et biophysica acta 229: 672-280 (2003). Development of collagenase-resistant collagen 0158 Mizuno M, Shindo M. Kobayashi D, Tsuruga E, and its interaction with adult human dermal fibroblasts. Amemiya A, KubokiY (1997). Osteogenesis by bone mar Biomaterials 24 (28): 5099-5113 row stromal cells maintained on type I collagen matrix gels 0142 Gorham SD, Light N D, Diamond AM, Willins M in vivo. Bone 20(2): 101-107 J. Bailey A. J. Wess T J & Leslie N J (1992). Effect of 0159. Nimni M. E. Cheung D, Strates B. Kodama M & chemical modifications on the Susceptibility of collagen to Sheild K (1998). Bioprosthesis derived from crosslinked proteolysis: 2. Dehydrothermal cross-linking. Interna and chemically modified collagenous tissue. In: Nimni, M tional Journal of Biological Macromolecules 14(3): 129 E (Ed.), Collagen Biotechnology, vol.III. CRC Press, Boca 138 Raton, Fla., p. 1-38 0143 Greenberg, C. S., Birckbichler, P.J. and Rice, R. H. (0160 Petite H, Rault I, Huc A. Menasche P& Herbage D (1991): Transaglutaminase: Multifunctional Crosslinking (1990). Use of the acyl azide method for cross-linking Enzymes that Stabilise Tissues. FASEB, 5,3071-3077 collagen-rich tissues such as pericardium. Journal of Bio 0144. Griffin M, Wilson J (1984). Detection of e-(y- medical Materials Research 24 (2): 179-187 glutamyl)lysine. Molecular and Cellular Biochemistry 58: (0161 Rhee S H, Hwang M H, Si HJ & Choi JY (2003). 37-49 Biological activities of osteoblasts on poly(methyl meth (0145 Grundmann et al. (1986) Characterization of cDNA acrylate)/silica hybrid containing calcium salt. Biomateri coding for human factor XIIa. Proc. Natl. Acad. Sci. USA als 24 (6): 901-906 83(21), 8024-8028 (0162 Richardson et al., 1995, Methods in Molecular Biol 0146 Harkness RD (1966). Collagen. Scientific Progress ogy Vol 39, J Walker ed., Humana Press, Totowa, N.J. (Oxford) 54; 257-274 (0163 Ruderinan R. J. Wade C W. R. Shepard W D & 0147 Hubbel J A (1995). Biomaterials in tissue engineer Leonard F (1973). Prolonged resorption of collagen ing. Biotechnology 13: 565-576 sponges: vapor-phase treatment with formaldehyde. Jour 0148 Ito A, Mase A, Takizawa Y. Shinkai M., Honda H, nal of Biomedical Materials Research 7: 263-265 Hata K., Ueda M & Kobayashi T (2003). Transglutaminase (0164 Sachlos E. Reis N, Ainsley C, Derby B & Czer mediated gelatin matrices incorporating cell adhesion fac nuszka JT (2003). Novel collagen scaffolds with pre tors as a biomaterial for tissue engineering. Journal of defined internal morphology made by solid freeform fab Bioscience and Bioengineering 95 (2): 196-199 rication. Biomaterials 24 (8): 1487-1497 0149 Ivarsson M, McWhirter A, Borg T K, Rubin K. 0.165 Saiki etal (1988) Primer-directed enzymatic ampli (1998). Type I collagen synthesis in cultured human fibro fication of DNA with a thermostable DNA polymerase. blasts: regulation by cell spreading, platelet-derived Science 239, 487-491 growth factor and interactions with collagen fibers. Matrix 0166 Sambrook & Russell, 2001, Molecular Cloning, A Biology 16: 409-425 Laboratory Manual. Third Edition, Cold Spring Harbor, 0150. Johnson TS, Skill NJ, El Nahas AM, Oldroyd SD, N.Y. Thomas GL, Douthwaite JA, Haylor JL, Griffin M (1999). (0167 Schmedlen KH, Masters K S & West SL (2002). Transglutaminase transcription and antigen translocation Photocrosslinkable polyvinyl alcohol hydrogels that can in experimental renal scarring. J. Am. Soc. Nephrol., 10: be modified with cell adhesion peptides for use in tissue 2146-2157 engineering. Biomaterials 23 (22): 4325-4332 0151. Jones RA, Nicholas B, Mian S, Davies PJA, Griffin (0168 Stenzel KH, Dunn M. W. Ruben A L & Miyata T M (1997). Reduced expression of tissue transglutaminase (1969). Collagen gels: design for vitreous replacement. in a human endothelial cell line leads to changes in cell Science 164: 1282-1283 spreading, cell adhesion and reduced polymerisation of 0169 Schuman L, Buma P. Verseyen D. deMan B. van der fibronectin. J. Cell Sci 110: 2461-2472 Kraan PM, van der Berg W B, Homminga GN (1995). US 2008/03055.17 A1 Dec. 11, 2008 13
Chondrocyte behaviour within different types of collagen 20. A method according to claim 1 wherein the trans gel in vitro. Biomaterials 16(10): 809–814 glutaminase is a microbial transglutaminase. 0170 Tu R. Lu C L Thyagarajan K. Wang E. Nguyen H, 21. A method according to claim 20 wherein the trans Shen S. Hata C & Quijano R C (1993). Kinetic-study of glutaminase is derived or prepared from the group consisting collagen fixation with polyepoxy fixatives. Journal of Bio of Streptoverticillium mobaraenase, Streptoverticillium medical Materials Research 27(1): 3-9 ladakanum, StreptoverticilHum cinnamoneum, Bacillus sub tilis and Phytophthora cactorum. 1. A method for producing a biocompatible biomaterial 22. A method according to claim 1 wherein the trans comprising crosslinking collagen using a transglutaminase. glutaminase is a recombinant transglutaminase. 2. A method according to claim 1 wherein the biocompat 23. A method according to claim 1 wherein the trans ible biomaterial exhibits an enhanced ability to support cell glutaminase is a variant transglutaminase. attachment, cell spreading, cell proliferation and/or differen 24. A method according to claim 1 wherein the collagen is tiation compared to non-crosslinked collagen. neutralised prior to treatment with the transglutaminase. 3. A method according to claim 1 wherein the biomaterial 25. A method according to claim 1 wherein the trans exhibits an enhanced ability to Support attachment, spread glutaminase is provided at a concentration of between 50 and ing, proliferation and/or differentiation of osteoblasts com 1000 g per ml of reaction mixture. pared to non-crosslinked collagen. 26. A method according to claim 1 wherein the collagen is 4. A method according to claim 1 wherein the biocompat provided at a concentration of 3 to 6 mg/ml of reaction mix ible biomaterial exhibits enhanced resistance to cell-medi ture. ated degradation compared to non-crosslinked collagen. 27. A method according to claim 1 wherein the treatment of 5. A method according to claim 4 wherein the biocompat collagen with the transglutaminase is performed in the pres ible biomaterial exhibits enhanced resistance to one or more ence of a reducing agent. protease enzymes produced by mammalian cells. 28. A method according to claim 1 wherein the treatment of 6. A method according to claim 1 wherein the biocompat collagen with the transglutaminase is performed in the pres ible biomaterial consists of Substantially pure collagen. ence of calcium ions. 7. A method according to claim 1 wherein the biocompat 29. A method according to claim 1 wherein the treatment of ible biomaterial comprises a cell adhesion factor. collagen with the transglutaminase is performed in the pres 8. A method according to claim 7 wherein the cell adhesion ence of buffering agent which buffers the reaction mixture at factor is selected from the group consisting a fibronectin, pH 7.4. fibrin, fibrillin, glycoSoaminoglycans, hyaluronic acid lami 30. A method according to claim 1 wherein treatment with nin, vitronectin and elastin. the transglutaminase is performed at 37-0>C. 9. A method according to claim 7 wherein the cell adhesion 31. A biomaterial comprising crosslinked collagen factor is fibronectin. obtained or obtainable by a method according to claim 1. 10. A method according to claim 1 wherein the biocompat 32. A biomaterial according to claim 31 which is substan ible biomaterial comprises one or more additives. tially free of catalysts, initiators and/or unreacted or partially 11. A method according to claim 10 wherein the additive is reacted crosslinking agents, wherein the unreacted or par selected from the group consisting of polylactic acid, poly tially reacted crosslinking agent is not a transglutaminase. hydroxybutyrate, poly(epsilon-caprolactone), polygflycolic 33. Use of a biomaterial according to claim 31 in the acid, polysaccharides, chitosans and silicates. manufacture of a medical implant or wound dressing. 12. A method according to claim 10 wherein the additive is 34. A medical implant comprising a biomaterial according selected from the group consisting of metals, bioceramics, to claim 31. glass, silk and biostable polymers. 35. A medical implant according to claim 34 wherein the 13. A method according to claim 12 wherein the biostable medical implant is artificial bone. polymer is selected from the group consisting of polypropy 36. A medical implant according to claim 34 comprising a lene, polyurethane, polytetrafluoroethylene, poly(vinyl chlo bio material according to claim 31 or 32 which is coated, ride), polyamides, poly(methylmethacrylate), polyacetal, impregnated, covalently linked or otherwise mixed with one polycarbonate, poly(-ethylene terphthalate), polyetherether or more additional biomaterials. ketone, and polysulfone. 37. A medical implant according to claim 36 wherein the 14. A method according to claim 1 wherein the trans additional biomaterial is selected from the group consisting glutaminase is a tissue transglutaminase. of material, bioceramics, glass or biostable polymers. 15. A method according to claim 1 claims wherein the 38. A medical implant according to claim 37 wherein the transglutaminase is a plasma transglutaminase. biostable polymer is selected from the group consisting of 16. A method according to claim 1 wherein the trans polyethylene, polypropylene, polyurethane, polytetrafhioro glutaminase is prepared from mammalian tissue or cells. ethylene, poly(vinyl chloride), polyamides, polymethyl 17. A method according to claim 16 wherein the trans methacrylate), polyacetal, polycarbonate, poly(-ethylene ter glutaminase is guinea pig liver tissue transglutaminase. phthalate), polyetheretherketone, and polysulfone. 18. A method according to claim 16 wherein the trans 39. A wound dressing comprising a biomaterial according glutaminase is prepared from human tissue or cells. to claim 31. 19. A method according to claim 18 wherein the human 40. A medical implant according to claim 34 or a wound tissue or cells are selected from the group consisting of lung, dressing according to claim 39 wherein the medical implant liver, spleen, kidney, heart muscle, skeletal muscle, eye lens, or wound dressing is provided in a sealed package. endothelial cells, erythrocytes, Smooth muscle cells, bone and 41. A medical implant or wound dressing according to macrophages. claim 40 wherein the package is sterile. US 2008/03055.17 A1 Dec. 11, 2008
42. A kit for producing a biomaterial according to claim 31 47. A medical implant or wound dressing according to comprising collagen and a transglutaminase. claim 46 wherein the package is sterile. 43. A kit according to claim 42 further comprising a cell 48-50. (canceled) adhesion factor. 51. A wound dressing substantially as hereinbefore 44. A kit according to claim 43 wherein the cell adhesion described with reference to the description. factor is fibronectin. 52. A kit for producing a biomaterial substantially as here 45. (canceled) inbefore described with reference to the description. 46. A kit according to claim 42 wherein the kit is provided in a sealed package. c c c c c