Enzyme-mediated modification of fivrin for tissue engineering

Details Enzyme-mediated modification of fivrin for tissue engineering Enzyme-mediated modification of fivrin for tissue engineering

2000-10-24

2003-08-19

Lankford, Jr., Leon B. (Department: 1651)

Drug, bio-affecting and body treating compositions

Preparations characterized by special physical form

Implant or insert

C435S193000, C514S002600, C530S300000, C530S350000, C530S402000

Reexamination Certificate

active

06607740

ABSTRACT:

BACKGROUND OF THE RELATED ART
Fibrin is a natural gel with several biomedical applications. Fibrin gel has been used as a sealant because of its ability to bind to many tissues and its natural role in wound healing. Some specific applications include use as a sealant for vascular graft attachment, heart valve attachment, bone positioning in fractures and tendon repair (Sierra, D. H.,
Journal of Biomaterials Applications
, 7:309-352, 1993). Additionally, these gels have been used as drug delivery devices, and for neuronal regeneration (Williams, et al.,
Journal of Comparative Neurobiology
, 264:284-290, 1987).
The process by which fibrinogen is polymerized into fibrin has also been characterized. Initially, a protease cleaves the dimeric fibrinogen molecule at the two symmetric sites. There are several possible proteases that can cleave fibrinogen, including thrombin, reptilase, and protease III, and each one severs the protein at a different site (Francis, et al.,
Blood Cells
, 19:291-307, 1993). Each of these cleavage sites have been located (FIG.
1
). Once the fibrinogen is cleaved, a self-polymerization step occurs in which the fibrinogen monomers come together and form a non-covalently crosslinked polymer gel (Sierra, 1993). A schematic representation of the fibrin polymer is shown in FIG.
2
. This self-assembly happens because binding sites become exposed after protease cleavage occurs. Once they are exposed, these binding sites in the center of the molecule can bind to other sites on the fibrinogen chains, these sites being present at the ends of the peptide chains (Stryer, L.
In Biochemistry
, W. H. Freeman & Company, N.Y., 1975). In this manner, a polymer network is formed. Factor XIIIa, a transglutaminase activated from factor XIII by thrombin proteolysis, may then covalently cross-link the polymer network. Other transglutaminases exist and may also be involved in covalent crosslinking and grafting to the fibrin network.
Once a crosslinked fibrin gel is formed, the subsequent degradation is tightly controlled. One of the key molecules in controlling the degradation of fibrin is &agr;2-plasmin inhibitor (Aoki, N.,
Progress in Cardiovascular Disease
, 21:267-286, 1979). This molecule acts by crosslinking to the &agr; chain of fibrin through the action of factor XIIIa (Sakata, et al.,
Journal of Clinical Investigation
, 65:290-297, 1980). By attaching itself to the gel, a high concentration of inhibitor can be localized to the gel. The inhibitor then acts by preventing the binding of plasminogen to fibrin (Aoki, et al.,
Thrombosis and Haemostasis
, 39:22-31, 1978) and inactivating plasmin (Aoki, 1979). The &agr;-2 plasmin inhibitor contains a glutamine substrate. The exact sequence has been identified as NQEQVSPL (SEQ ID NO: 15), with the first glutamine being the active amino acid for crosslinking.
The components required for making fibrin gels can be obtained in two ways. One method is to cryoprecipitate the fibrinogen from plasma. In this process, factor XIII precipitates with the fibrinogen, so it is already present. The proteases are purified from plasma using similar methods. Another technique is to make recombinant forms of these proteins either in culture or with transgene animals. The advantage of this is that the purity is much higher, and the concentrations of each of these components can be controlled.
Cells interact with their environment through protein-protein, protein-oligosaccharide and protein-polysaccharide interactions at the cell surface. Extracellular matrix proteins provide a host of bioactive signals to the cell. This dense network is required to support the cells, and many proteins in the matrix have been shown to control cell adhesion, spreading, migration and differentiation (Carey,
Annual Review of Physiology
, 53:161-177, 1991). Some of the specific proteins that have shown to be particularly active include laminin, vitronectin, fibronectin, fibrin, fibrinogen and collagen (Lander,
Journal of Trends in Neurological Science
, 12:189-195, 1989). Many studies of laminin have been conducted, and it has been shown that laminin plays a vital role in the development and regeneration of nerves in vivo and nerve cells in vitro (Williams,
Neurochemical Research
, 12:851-869, 1987; Williams, et al., 1993), as well as in angiogenesis.
Some of the specific sequences that directly interact with cellular receptors and cause either adhesion, spreading or signal transduction have been identified. This means that the short active peptide sequences can be used instead of the entire protein for both in vivo and in vitro experiments. Laminin, a large multidomain protein (Martin,
Annual Review of Cellular Biology
, 3:57-85, 1987), has been shown to consist of three chains with several receptor-binding domains. These receptor-binding domains include the YIGSR (SEQ ID NO: 1) sequence of the laminin B1 chain ( Graf, et al.,
Cell
, 48:989-996, 1987; Kleinman, et al.,
Archives of Biochemistry and Biophysics
, 272:39-45, 1989; and Massia, et al.,
J. of Biol. Chem
., 268:8053-8059, 1993), LRGDN (SEQ ID NO: 2) of the laminin A chain (Ignatius, et al.,
J. of Cell Biology
, 111:709-720, 1990) and PDGSR (SEQ ID NO: 3) of the laminin B1 chain (Kleinman, et al., 1989). Several other recognition sequences for neuronal cells have also been identified. These include IKVAV (SEQ ID NO: 4) of the laminin A chain (Tashiro, et al.,
J. of Biol. Chem
., 264:16174-16182, 1989) and the sequence RNIAEIIKDI (SEQ ID NO: 5) of the laminin B2 chain (Liesi, et al.,
FEBS Letters
, 244:141-148, 1989). The receptors that bind to these specific sequences have also often been identified. A subset of cellular receptors that has shown to be responsible for much of the binding is the integrin superfamily (Rouslahti, E.,
J. of Clin. Investigation
, 87:1-5, 1991). Integrins are protein heterodimers that consist of &agr; and &bgr; subunits. Previous work has shown that the tripeptide RGD binds to several &bgr;1 and &bgr;3 integrins (Hynes, R. O.,
Cell
, 69:1-25, 1992; Yamada, K. M.,
J. of Biol. Chem
., 266:12809-12812, 1991), IKVAV(SEQ ID NO: 4) binds to a 110 kDa receptor (Tashiro, et al.,
J. of Biol. Chem
., 264:16174-16182, 1989; Luckenbill-Edds, et al.,
Cell Tissue Research
, 279:371-377, 1995), YIGSR (SEQ ID NO: 1) binds to a 67 kDa receptor (Graf, et al., 1987) and DGEA (SEQ ID NO: 6), a collagen sequence, binds to the &agr;
2
,&bgr;
1
integrin (Zutter & Santaro,
Amer. J. of Pathology
, 137:113-120, 1990). The receptor for the RNIAEIIKDI (SEQ ID NO: 5) sequence has not been reported.
Work has been done in crosslinking bioactive peptides to large carrier molecules and incorporating them within fibrin gels. By attaching the peptides to the large carrier polymers, the rate of diffusion out of the fibrin gel will be slowed down. In one series of experiments, polyacrylic acid was used as the carrier polymer and various sequences from laminin were covalently bound to them to confer neuroactivity (Herbert, C. in Chemical Engineering 146) to the gel. The stability of such a system was poor due to a lack of covalent or high affinity binding between the fibrin and the bioactive molecule.
Very little work has been done in incorporating peptide sequences and other bioactive factors into fibrin gels and even less has been done in covalently binding peptides directly to fibrin. However, a significant amount of energy has been spent on determining which proteins bind to fibrin via enzymatic activity and often determining the exact sequence which binds as well. The sequence for fibrin &ggr;-echain crosslinking has been determined and the exact site has been located as well (Doolittle, et al.,
Biochem
. &
Biophys. Res. Comm
., 44:94-100, 1971). Factor XIIIa has also been shown to crosslink fibronectin to fibronectin (Barry & Mosher,
J. of Biol. Chem
., 264:4179-4185, 1989), as well as fibronectin to fibrin itself (Okada, et al.,
J. of Biol. Chem
., 260:1811-1820, 1985). This enzyme also crosslinks von Willebrand factor (Hada, et al., Blood, 68:95-101, 1986), as well as &agr;-2 plasmin

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