WO2011115420A2

WO2011115420A2 - Adhesive extracellular matrix mimetic

Info

Publication number: WO2011115420A2
Application number: PCT/KR2011/001831
Authority: WO
Inventors: Jaeyoon Kim; Taehoon G. Lee; Kilwon Cho; Hosun Lim; Seung Goo Lee
Original assignee: Kollodis Biosciences, Inc.; Kollodis Korea, Co., Ltd.
Priority date: 2010-03-16
Filing date: 2011-03-16
Publication date: 2011-09-22
Also published as: WO2011115420A3

Abstract

An extracellular matrix mimetic comprising a hydrogel which is formed by crosslinking a recombinant mussel adhesive protein and a bio-polymer is provided. The three dimensional hydrogel matrix provides biochemical and biophysical microenvironments similar to native extracellular matrix and thus is capable of supporting cell in-growth in vivo or in vitro. The matrix can be tailored to further comprise one or more bioactive peptides. The matrix may also comprise cells encapsulated and dispersed therein, which are capable of proliferating or differentiating upon deposition of the matrix in vivo or in vitro. Methods of preparing the adhesive extracellular matrix and the use of the matrix in vivo or in vitro for tissue engineering or drug delivery applications are also provided.

Description

ADHESIVE EXTRACELLULAR MATRIX MIMETIC

This invention relates to compositions and methods for an adhesive hydrogel to create extracellular matrix mimetic.

A naturally occurring extracellular matrix (ECM) is a three-dimensional molecular complex that varies in composition, and includes bioactive components such as laminin, fibronectin, various collagens and other glycoproteins, hyaluronic acid, proteoglycans, and elastins.

Structurally, ECMs serve as scaffolds for cells, as well as networks of various adhesion ligands and growth factors, which promote cell signaling. For this reason, the ECM has been widely used in cell culture to enhance cellular adhesion. Cellular adhesion to ECM proteins is a fundamental feature of cell development, maintenance of tissue organization, and many pathological conditions. It is, therefore, crucial to understand the adhesion process in order to study cellular functions or tissue engineering applications.

Not only can ECM components influence the cellular behavior, the extracellular microenvironment such as three-dimensionality can contribute to cellular behavior or responses. Over the past few decades, tissue engineers and cell biologists have begun to develop material systems to culture mammalian cells within 3D ECM mimics to circumvent the limitations posed by traditional 2D cell culture.

The realization of the importance of cell-ECM interaction in biomedical applications such as medical device or tissue engineering scaffold has led to a renewed interest in developing ECM constituents and/or an artificial ECM to mimic its function in cell culturing and medical applications.

Reconstituted natural extraceullular matrices such as Matrigel™ from BD Biosciences and GelTrex™ from Invitrogen are extracted from basilar membranes from mouse tumor cells, which are still used by many researchers today. However, their application has been limited due to lack of defined characteristics.

To overcome this defect, many attempts have been made to utilize natural biomaterials or synthetic polymeric materials in order to mimic extracellular matrix with defined physicochemical properties for in vivo and in vitro applications.

Some researchers have described the use of peptides with alternately charged, hydrophobic and hydrophilic amino acids to culture nerve cells, endothelial cells and chondrocytes (See U.S. Patent No. 7,449,180, Shuguang Zhang et al., BioMEMS and Biomedical Nanotechnology, 2007 p39-54, Xiaojun Zhao, et al., Designer Self-Assembling Peptide Materials Macromolecular Bioscience, 2007, p.13-22). Other researchers have demonstrated the use of synthetic amphiphile peptide-containing molecules that can self-assemble into fibrous scaffolds modified with ECM peptide such as RGD that support cell growth and stem cell differentiation (Hossein Hosseinkhania, et al., Biomaterials, 2006, p.4079-4086). These successes illustrate that man-made hydrogels could be useful for forming scaffold materials for 3D cell culture and tissue engineering applications.

However, lack of bioactivity such as poor cell attachment observed in these synthetic extracellular matrices limited their use. To improve the cell attachment on synthetic extracellular matrix, a method of modification of synthetic extracellular matrix with ECM-derived peptide or addition of natural ECM proteins, such as fibronectin or collagen type I, has been developed (See, Byung-Soo Kim, et al., Development of biocompatible synthetic extracellular matrices for tissue engineering, Trends in Biotechnology, December 1998, Pages 224-230). A synthetic ECM was envisaged in which a chemically modified form of collagen was blended with the HA component to create a covalently crosslinked blend of an ECM protein and a GAG component.

Many attempts have been made to create a three-dimensional hydrogel environment incorporating cell adhesion ligands for cell attachment and survival. One example is a crosslinkable hyaluronic acid, alginate or polyethylene glycol based hydrogel with an RGD peptide grafted onto the polymer backbone. This polymer undergoes gelation in the presence of crosslinking agent or UV light. Another is a polyacrylamide system, again with the RGD ligand covalently attached, which is catalytically polymerized prior to any biological interactions (Woerly et al. 1995: Intracerebral implantation of hydrogel-coupled adhesion peptides: tissue reaction. J. Neural Transplant. Plasticity, 5:245-255.; Imen et al., Biomaterials, 27, p3451-3458, 2006; U.S. Pat. Publication No. 20060134050).

All existing technologies require bioactive components such as collagen or fibronectin, which were incorporated physically or chemically into synthetic extracellular matrix, to mimic the physical or chemical attributes of native ECM.

A disadvantage of existing systems is that conversion of the polymers from a liquid to a three dimensional, gel or highly viscous system requires conditions which are detrimental to cell viability, e.g., use of organic solvents and/or elevated temperatures.

The present inventors have developed three dimensional adhesive hydrogel that offers flexibility and customizability to tailor microenvironments for cells that mimic natural in vivo conditions, allow to optimize cell attachment, spreading, proliferation or differentiation.

In light of the aforementioned need in the prior art, the present invention is directed to a hydrogel with customizable ECM matrix in terms of biochemical composition and physical aspects to mimic natural ECM.

One aspect of the present invention is to provide a composition to form ECM mimetic in situ without further requirements to offer a customizable biochemical or biophysical microenvironment. The composition is basically composed of two components; recombinant mussel adhesive protein recombinantly functionalized with extracellular matrix-derived peptide such as RGD and a crosslinker.

In accordance with another aspect of the invention, there are provided an efficient and simple process that allows for customizable hydrogel that is formed in situ as an extracellular matrix mimetic.

Another aspect of the present invention is to provide a three dimensional synthetic extracellular matrix and uses thereof. In accordance with an aspect of the present invention, there is provided a recombinant mussel adhesive protein suitable for the preparation of a synthetic extracellular matrix, comprising a mussel adhesive protein containing N-terminal or C-terminal reactive moiety capable of crosslinking, said recombinant mussel adhesive protein containing a number of bioactive peptide.

In accordance with another aspect of the invention, there is provided a synthetic extracellular matrix comprising a recombinant mussel adhesive protein, a bio-polymer and an aqueous solvent, wherein the mussel adhesive protein and bio-polymer are crosslinked to form a three dimensional matrix such as hydrogel.

In accordance with another aspect of the invention, there are provided uses of the synthetic extracellular matrix as a surface for cell culture, as a scaffold for tissue regeneration, for replacement of damaged or removed tissue in an animal, or for coating surgical implants.

In accordance with another aspect of the invention, there are provided customizable compositions comprising: one or more bioactive peptides or a plurality of cells; a mussel adhesive protein of the invention; a bio-polymer; and an aqueous solvent.

In accordance with another aspect of the invention, there is provided an implant for use in tissue engineering comprising a pre-formed extracellular matrix composition, said matrix comprising an aqueous solvent and a bio-polymer crosslinked with a synthetic co-polymer of the invention.

The present invention provides a three dimensional extracellular matrix mimetic hydrogel (hereinafter can be referred to MAPTrix HyGel™) for life science and medical applications comprising a recombinant mussel adhesive protein, wherein the recombinant mussel protein is genetically engineered with numerous bioactive polypeptides, and a crosslinking agent.

Abbreviations and Definitions

As used herein, the term "bioactive peptide" refers to a biologically active polypeptide or oligopeptide that play a biological role by acting at specific receptor and/or binding sites at different locations in the cells, tissues, or organism.

The term "biological role" refers to the control of biological responses of a cell adhered thereto and/or of a cell in the vicinity of cells adhered thereto. More particularly, the biological response of a cell (adhered to the biofunctional peptide or in the vicinity of the biofunctional peptide) relates to its ability to adhere to a specific substrate, to migrate on this specific substrate, to grow and divide, to grow into a differentiated cell, to express differentiation markers, to form differentiated structures, to respond to a biological stimulus, to communicate with neighboring cells, and/or to organize its cytoskeleton with respect to other cells or with respect to one of the axis of the biofunctional peptide, to express different sets of genes, to express different proteins, to bear different lipids or carbohydrate structure, to adopt different phenotypes, etc.

The term "peptide" includes all moieties containing one or more amino acids linked by a peptide bond. In addition, this term includes within its ambit polymers of modified amino acids, including amino acids which have been post-translationally modified, for example by chemical modification including but not restricted to glycosylation, phosphorylation, acetylation and/or sulphation reactions that effectively alter the basic peptide backbone. Accordingly a peptide may be derived from a naturally-occurring protein, and in particular may be derived from a full-length protein by chemical or enzymatic cleavage, using reagents such as CNBr, or proteases such as trypsin or chymotrypsin, amongst others. Alternatively, such peptides may be derived by chemical synthesis using well known peptide synthetic methods. Included in the scope of the definition of the term "peptide" is a peptide whose biological activity is predictable as a result of its amino acid sequence corresponding to a functional domain. Also encompassed by the term "peptide" is a peptide whose biological activity could have been predicted by the analysis of its amino acid sequence.

The present invention is not limited by the source of the peptide, and clearly extends to peptides and peptide mimetic which are derived from a natural occurring or a non-natural source.

The term "derived from" shall be taken to indicate that a particular peptide or mixture of peptides which has been obtained from a particular protein, protein mixture or protein-containing biological extract, either directly (for example, by proteolytic, chemical or physical digestion of the protein(s) or extract) or indirectly, for example, by chemical synthesis of peptides having amino acid sequences corresponding to naturally-occurring sequences, or peptide variants thereof.

A peptide "derived from" a polypeptide having a particular amino acid sequence is any molecular entity which is identical, substantially homologous, or otherwise functionally or structurally equivalent to that polypeptide. Thus, a molecule derived from a particular polypeptide may encompass the amino acid sequence of the polypeptide, any portion of that polypeptide, or other molecular entity that functions to regulate cell-ECM communications. A molecule derived from such a binding domain will mimic the polypeptide from which it is derived. Such molecular entities may include peptide mimetics and the like.

The term "polypeptide" refers to a polymer of amino acids and does not limit the size to a specific length of the product. However, as used herein, a polypeptide is generally longer than a peptide and may include one or more copies of a peptide of interest. This term also optionally includes post expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like. Included within the definition are, for example, polypeptides containing one or more analogues of an amino acid or labeled amino acids.

The term "extracellular matrix (ECM)" refers to a substrate and/or scaffold in the cell's external environment with which the cells can interact via specific cell surface receptors or binding sites.

The term "ECM proteins" refers to a fibrous protein including fibronectin, laminin, vitronectin, and collagens, whether naturally occurring or synthetic analogs, as long as it is biologically active.

The term "ECM protein segment" refers to any active analogs, fragments or derivatives of ECM proteins.

The term "genetically engineered" or "recombinantly functionalized" refers to the direct manipulation of an organism's gene via genetic introduction and/or manipulation of DNA in the form of a gene which in turn finds expression to produce favorable and/or desirable physical or biofunctional characteristics of a peptide.

The term "mussel adhesive protein" refers to a recombinant mussel adhesive protein selected from FP-1, FP-2, FP-3, FP-4, FP-5, FP-6 and its fragment or fusion of each mussel adhesive protein. The FP-1 comprises an amino acid sequence of SEQ ID NO: 1 tandemly repeated 1 to 80 times. The FP-2 comprises SEQ ID NO: 2, the FP-3 comprises SEQ ID NO: 3, the FP-4 comprises SEQ ID NO: 4, the FP-5 comprises SEQ ID NO: 5, and the FP-6 comprises SEQ ID NO: 6.

The term "functional mussel adhesive protein" refers to a recombinant mussel adhesive protein genetically functionalized with bioactive peptides.

The term "progenitor cell" refers to a stem cell with more specialization and less differentiation potential than a totipotent stem cell. For example, progenitor cells include unipotential cells such as fibroblast or osteoblast.

The term "growth factor mimetic" includes any active analogs, fragments or derivatives of natural growth factors such as NGF, FGF, PDGF, IGF, BDGF, and substance P.

The present invention provides a composition for an extracellular matrix mimetic comprising a mussel adhesive protein and a crosslinking agent. In accordance with one aspect of the invention, there is provided a composition for an extracellular matrix mimetic created in situ, comprising a mussel adhesive protein and a crosslinking agent, wherein said crosslinking agent mediates its crosslinking function chemically via covalent, ionic, hydrogen-bonded, and Van der Waals interactions or physically via molecular entanglement and intertwining or both chemically and physically crosslink under a wide range of pH conditions.

The composition for an extracellular matrix mimetic of the present invention can comprise optionally a biocompatible polymer of natural or synthetic origin. In accordance with one aspect of the present invention, there is provided a composition for an extracellular matrix mimetic created in situ, comprising a mussel adhesive protein, a biocompatible polymer of natural or synthetic origin, and a crosslinking agent, wherein said crosslinking agent mediates its crosslinking function chemically via covalent, ionic, hydrogen-bonded, and Van der Waals interactions or physically via molecular entanglement and intertwining or both chemically and physically crosslink under a wide range of pH conditions.

In addition, the present invention provides a three dimensional extracellular matrix mimetic comprising a mussel adhesive protein and a crosslinking agent. In accordance with onother aspect of the present invention, there is provided a three dimensional extracellular matrix mimetic created in situ, comprising a mussel adhesive protein and a crosslinking agent, wherein said crosslinking agent mediates its crosslinking function chemically via covalent, ionic, hydrogen-bonded, and Van der Waals interactions or physically via molecular entanglement and intertwining or both chemically and physically crosslink under a wide range of pH conditions.

The three dimensional extracellular matrix mimetic of the present invention can comprise optionally a biocompatible polymer of natural or synthetic origin. In accordance with another aspect of the present invention, there is provided a three dimensional extracellular matrix mimetic created in situ, comprising a mussel adhesive protein, a biocompatible polymer of natural or synthetic origin, and a crosslinking agent, wherein said crosslinking agent mediates its crosslinking function chemically via covalent, ionic, hydrogen-bonded, and Van der Waals interactions or physically via molecular entanglement and intertwining or both chemically and physically crosslink under a wide range of pH conditions.

Any suitable mussel adhesive protein based hydrogel may be used as the bioartificial extracellular matrices of this invention. Compositions that form hydrogels are basically composed of two components. The first component is a mussel adhesive protein functionalized with bioactive peptides. The second component is a crosslinkable agent. Examples of commercially available such protein include MAPTrix™ ECM marketed by Kollodis BioSciences, Inc. (Malden, MA). An optional third component is a biocompatible polymer (e.g., polyethylene glycol or polyvinylalcohol), and may be added to the compositions to enhance physicomechanical properties such as adhesive strength of a customizable extracellular matrix mimetic hydrogel.

The MAPTrix™ ECMs, developed by Kollodis BioSciences Inc., are predesigned mussel adhesive proteins based ECM mimetic. The mussel adhesive proteins were recombinantly fuctionalized with a variety of ECM-derived peptides to mimic the bioactivity of naturally occurring ECM, which were demonstrated to have similar bioactivity to natural ECM in primary cell culture as compared to natural or recombinant ECM proteins. The pre-designed ECM mimetics are highly advantageous to create three dimensional ECM microenvironments. For example, it allows to design cell-specific or user-defined regulation of the three dimensional matrix to emulate the native microenvironment in terms of biochemical composition.

A mussel adhesive protein suitable for use in this invention can be prepared from recombinantly or naturally occurring. Most preferred protein that permits presentation of only the desired ECM adhesion molecule or adhesive peptide fragment in 3-D, substantially free of undesired adhesion motifs is a mussel adhesive protein recombinantly functionalized with bioactive peptide.

The mussel adhesive protein can be selected from foot protein FP-1 (SEQ ID NO: 1), FP-2 (SEQ ID NO: 2), FP-3 (SEQ ID NO: 3), FP-4, FP-5 (SEQ ID NO: 4), or FP-6 (SEQ ID NO: 5), or a recombinantly fused mussel adhesive protein with another foot protein, for example, FP-5 fused with FP-1. Preferably a mussel adhesive protein is a fused mussel adhesive protein. The preferred mussel adhesive protein is a fusion protein comprising a first peptide of mussel foot protein selected from mussel FP-2, mussel FP-3 or mussel FP-5 and a second peptide of at least one selected from the group consisting of mussel FP-1, mussel FP-2, mussel FP-3, mussel FP-4, mussel FP-6 and fragment thereof. The most preferred fusion proteins are FP-151 (SEQ ID NO: 6), FP-5 fused with FP-1 and FP-13151 (SEQ ID NO: 7), FP-5 and FP-3 fused with FP-1.

E. coli based protein expression system was commercialized recently to produce a variety of mussel adhesive proteins including FP-151 in an efficient way (see United States Patent No. 7,622,550), and the mussel adhesive proteins are commercially available under Trademarks MAPTrix™ marketed by Kollodis BioSciences, Inc. The method for preparation of mussel adhesive proteins are fully described in the United States Patent No. 7,622,550 which is hereby incorporated by reference for all purposes as if fully set forth herein.

A bioactive peptide can be recombinantly incorporated into C-terminus, N-terminus or C- and N-terminus of a mussel adhesive protein.

Bioactive peptides are necessary for the present invention to mimic the microenvironments of a natural extracellular matrix. Additional components such as growth factors, for example, nerve growth factor or substance P, may also be included to further enhance the beneficial effect of the ECM mimic on cell and tissue culture, medical device and treatment, or any other related application.

Bioactive peptides are natural or synthetic peptides derived from ECM proteins to emulate biochemical or biophysical cues of a natural ECM. The ECM proteins can be fibrous proteins such as collagens, fibronectin, laminin, vitronectin, and the like. ECM proteins can influence integrin activity, and in turn, integrins may activate signaling pathways by coclustering with kinases and adaptor proteins in focal adhesion complexes after their association with polyvalent extracellular matrix (ECM) proteins. For example, a RGD containing peptide segment from fibronectin, laminin or vitronectin to integrins may regulate to its integrin activity.

A suitable peptide fragment of ECM proteins that together forms the ECM mimic are selected from collagen, fibronectin, laminin, vitronectin, bone sialoprotein, entactin, or fibrinogen.

Preferably, a bioactive peptide fragment derived from collagen type I is a GLPGER (SEQ ID NO: 8), KGHRGF (SEQ ID NO: 9), GFPGER (SEQ ID NO: 10), DGEA (SEQ ID NO: 11), GPAGKDGEAGAQG (SEQ ID NO: 12) or GTPGPQGIAGQRDVV (SEQ ID NO: 13) containing peptide.

Preferably, a bioactive peptide fragment derived from collagen type II is a EKGPD (SEQ ID NO: 14), EKGPDP (SEQ ID NO: 15) or EKGPDPL (SEQ ID NO: 16) containing peptide.

Preferably, a bioactive peptide fragment derived from collagen type IV is a TAGSCLRKFSTM (SEQ ID NO: 17), GEFYFDLRLKGDK (SEQ ID NO: 18), TAIPSCPEGTVPLYS (SEQ ID NO: 19), TDIPPCPHGWISLWK (SEQ ID NO: 20) or LAGSCLARFSTM (SEQ ID NO: 21) containing peptide.

Preferably, a bioactive peptide fragment derived from fibronectin is a PHSRN (SEQ ID NO: 22), RGD (SEQ ID NO: 23), GRGDSP (SEQ ID NO: 24), YRVRVTPKEKTGPMKE (SEQ ID NO: 25), SPPRRARVT (SEQ ID NO: 26), WQPPRARI (SEQ ID NO: 27), KNNQKSEPLIGRKKT (SEQ ID NO: 28), EILDVPST (SEQ ID NO: 29) or REDV (SEQ ID NO: 30) containing peptide.

Preferably, a bioactive peptide fragment derived from laminin is a RQVFQVAYIIIKA (SEQ ID NO: 31), SINNTAVMQRLT (SEQ ID NO: 32), IKVAV (SEQ ID NO: 33), NRWHSIYITRFG (SEQ ID NO: 34), TWYKIAFQRNRK (SEQ ID NO: 35) or RKRLQVQLSIRT (SEQ ID NO: 36) containing peptide derived from laminin alpha-1 chain, KNRLTIELEVRT (SEQ ID NO: 37), SYWYRIEASRTG (SEQ ID NO: 38) or DFGTVQLRNGFPFFSYDLG (SEQ ID NO: 39) containing peptide derived from laminin alpha-2 chain, GQLFHVAYILIKF (SEQ ID NO: 40) or KNSFMALYLSKG (SEQ ID NO: 41) containing peptide derived from laminin alpha-3 chain, TLFLAHGRLVFM (SEQ ID NO: 42) containing peptide derived from laminin alpha-4 chain, or GQVFHVAYVLIKF (SEQ ID NO: 43), GIIFFL (SEQ ID NO: 44) or LALFLSNGHFVA (SEQ ID NO: 45) containing peptide derived from laminin alpha-5 chain, RYVVLPR (SEQ ID NO: 46), PDSGR (SEQ ID NO: 47) or YIGSR (SEQ ID NO: 48) containing peptide derived from laminin beta-1 chain, or KAFDITYVRLKF (SEQ ID NO: 49) or RNIAEIIKDI (SEQ ID NO: 50) containing peptide derived from laminin gamma-1 chain.

Preferably, a bioactive peptide fragment derived from vitronectin is a FRHRNRKGY (SEQ ID NO: 51) or KKQRFRHRNRKGYRSQ (SEQ ID NO: 52) containing peptide.

Preferably, a bioactive peptide fragment derived from bone sialoprotein is a FHRRIKA (SEQ ID NO: 53) or KRSR (SEQ ID NO: 54) containing peptide.

In one embodiment of the invention, the mussel adhesive protein is a fusion protein of FP-5 with FP-1 which was recombinantly functionalized with collagen type I-derived peptide GFPGER (SEQ ID NO: 10) to form collagen type I rich extracellular matrix mimetic hydrogel.

In one embodiment of the invention, the mussel adhesive protein is a fusion protein of FP-151 which was recombinantly functionalized with fibronectin-derived peptide GRGDSP (SEQ ID NO: 24) to form fibronectin rich extracellular matrix mimetic hydrogel.

In one embodiment of the invention, the mussel adhesive protein is combination of functional mussel adhesive proteins, mainly composed of FP-151 functionalized with collagen type I-derived peptide IKVAV (SEQ ID NO: 33) which accounts for over 80% of the total ECM-derived peptides, to mimic the biochemical composition of a natural bone ECM.

In one embodiment of the invention, the mussel adhesive protein is combination of functional mussel adhesive proteins with hyaluronic acid, mainly composed of FP-151 functionalized with collagen type I-derived peptide DGEA (SEQ ID NO: 11) which accounts for over 80% of the total ECM-derived peptides, to mimic the biochemical composition of a natural bone ECM.

In another embodiment of the invention, the mussel adhesive protein is combination of functional mussel adhesive proteins, mainly composed of FP-151 functionalized with fibronectin-derived peptide RGD (SEQ ID NO: 23) and FP-151 functionalized with collagen type I-derived peptide DGEA (SEQ ID NO: 11), of two which account for over 60% of the total ECM-derived peptides, to mimic the biochemical composition of a natural basilar lamina.

Hydrogels may be formed either through covalent, ionic or physical bonds introduced through, e.g., chemical crosslinking agents or electromagnetic radiation, such as ultraviolet light, of both natural and synthetic hydrophilic polymers, including homo and co-polymers. Physical (non-covalent) crosslinks may result from, e.g., complexation, hydrogen bonding, desolvation, Van der Waals interactions, or ionic bonding, and may be initiated by mixing components that are physically separated until combined in situ, or as a consequence of a prevalent condition in the physiological environment, such as temperature, pH, and/or ionic strength. Covalent crosslinking may be accomplished by any of a number of mechanisms, including free radical polymerization, condensation polymerization, anionic or cationic polymerization, step growth polymerization, and electrophile-nucleophile reaction.

A chemically crosslinkable agent suitable for use in this invention can be a biocompatible polymer, naturally or synthetically occurring. Preferably a crosslinkable agent is a synthetic polymer which has the appropriate functional groups such that it can be covalently linked directly or through a linker to a mussel adhesive protein. Any polymer meeting the above requirements is useful herein, and the selection of the specific polymer and acquisitions or preparation of such polymer would be conventionally practiced in the art (See The Biomedical Engineering Handbook, ed. Bronzino, Section 4, ed. Park.). Preferred for such crosslinkable polymers are selected from groups comprising poly(alkylene oxides) particularly poly(ethylene glycols), poly(vinyl alcohols), polypeptides, poly(amino acids), such as poly(lysine), poly(allylamines) (PAM), poly(acrylates), polyesters, polyphosphazenes, pluronic polyols, polyoxamers, poly(uronic acids) and copolymers, including graft polymers thereof.

The polymer may be selected to have a wide range of molecular weights, generally from as low as 1,000 up to million. Preferably, a polymer having a molecular weight of less than about 30,000 to 50,000 or one in which the backbone of polymer itself is degradable. Polymers with a degradable polymeric backbone section include those with a backbone having hydrolyzable groups therein, such as polymers containing ester groups in the backbone, for example, aliphatic polyesters of the poly(a-hydroxy acids) including poly(glycolic acid) and poly(lactic acid). When the backbone is itself degradable, it need not be of low molecular weight to provide such degradability.

Furthermore, the present invention provides an adhesive hydrogel comprising a mussel adhesive protein and a crosslinking agent. In accordance with another aspect of the present invention, there is provided an adhesive hydrogel comprising a mussel adhesive protein and a crosslinking agent, wherein said crosslinking agent mediates its crosslinking function chemically via covalent, ionic, hydrogen-bonded, and Van der Waals interactions or physically via molecular entanglement and intertwining or both chemically and physically crosslink under a wide range of pH conditions.

The adhesive hydrogel of the present invention can comprise optionally a biocompatible polymer of natural or synthetic origin. In accordance with another aspect of the present invention, there is provided an adhesive hydrogel comprising a mussel adhesive protein, a biocompatible polymer of natural or synthetic origin, and a crosslinking agent, wherein said crosslinking agent mediates its crosslinking function chemically via covalent, ionic, hydrogen-bonded, and Van der Waals interactions or physically via molecular entanglement and intertwining or both chemically and physically crosslink under a wide range of pH conditions.

In one embodiment of the invention, an extracellular matrix mimetic hydrogel formed in situ composition is provided. The composition is composed of multiple arm PEG and mussel adhesive proteins to mimic the biochemical microenvironments of native ECM. Multiple arm PEG can be selected from the group consisting of 4 arm, 6 arm, 8 arm or 10 arm PEG. Preferred multiple arm PEG is one selected from the group consisting of 4 to 8 arm. The most preferred multiple arm PEG is 6 and 8 arm PEG.

The extracellular matrix mimetic hydrogel does not require a use of organic solvents or elevated temperatures. The extracellular matrix mimetic hydrogel can be formed under physiological conditions.

In a preferred embodiment, the preferred compound is one selected from the group consisting of; 4 to 8-arm PEG-succinic acid, 4 to 8-arm PEG-glutaric acid, 4 to 8-arm PEG-succimidyl succinate, 4 to 8-arm PEG-succimidyl glutarate, 4 to 8-arm PEG-acrylate, or 4 to 8-arm PEG-propion aldehyde.

An extracellular matrix mimetic hydrogel-forming composition comprising the 6-arm PEG-SS or 6-arm PEG-SG can be formed easily with mussel adhesive proteins or the above hydrogel-forming composition comprising the 6-arm PEG-SG can be formed with mussel adhesive proteins or the mixture of mussel adhesive proteins with 6-arm PEG-amine etc.

Many recent reports suggest that biophysical cues such as substrate topography and compliance play an important role in both normal and pathological behaviors (Discher et al., Tissue cells feel and respond to the stiffness of their substrate, Science 310 (2005), pp. 1139 1143, Flanagan et al., Neurite branching on deformable substrates, Neuroreport 13 (2002), pp. 2411 2415). Stem cell lineage specification or cell proliferation and differentiation have been shown to be affected by substrate compliance (Engler et al., Substrate compliance versus ligand density in cell on gel responses, Biophys. J. 86 (2004), pp. 617 628, McDaniel et al., The stiffness of collagen fibrils influences vascular smooth muscle cell phenotype, Biophys. J. 92 (2007), pp. 1759 1769, Boontheekul et al., Regulating myoblast phenotype through controlled gel stiffness and degradation, Tissue Eng. 13 (2007), pp. 1431 1442, Peyton et al., The use of poly(ethylene glycol) hydrogels to investigate the impact of ECM chemistry and mechanics on smooth muscle cells, Biomaterials 27 (2006), pp. 4881 4893). These studies collectively imply that extracellular matrix (ECM) compliance acts as a homeostatic regulator of normal tissue function.

Therefore, tissue engineering scaffold would benefit from engineering user-defined or customizable biophysical cues such as modulus of extracellular matrix mimetic.

One embodiment of the invention provides an extracellular matrix mimetic hydrogel with adjusted modulus to mimic the biophysical properties of native ECM.

Soft extracellular matrix mimetic can be achieved with low degree of crosslinking reaction between MAPTrix™ ECM and multiple arm PEG derivatives. For this goal, the MAPTrix™ concentration should be reduced. However, the molar ratio of lysine residues in the MAPTrix™ ECM to the functional groups in multiple arm PEG may be greater than 2.0 for the crosslinking. Compositions with less the minimum molar ratio failed to form hydrogel. To circumvent the lowest concentration of MAPTrix™ ECM to from extracellular matrix mimetic hydrogel with low elastic modulus less than 10KPa, PEG-NH₂ derivatives could be used to form crosslinked structure with multiple arm PEG-SG.

Another crosslinkable agent is a negatively charged polymer which has the appropriate functional groups such that it can be ionically linked directly or through a linker to a mussel adhesive protein. Any polymer meeting the above requirements is useful herein, and the selection of the specific polymer and acquisitions or preparation of such polymer would be conventionally practiced in the art. Preferably, a crosslinkable agent is a glycosaminoglycans or xanthan gum. Most preferred crosslinkable agent is a hyaluronic acid.

Another crosslinkable agent is physically crosslinked via physical entanglement or crystallization. A non-crosslinked crystallizable, biocompatible polymer can used to form hydrogel extracellular matrix mimetic because the crystallized structure is stable in the physiological conditions. Polyvinyl alcohol (PVA) is a biodegradable and readily water-soluble. PVA-based hydrogels can be fabricated via crystallization. Polyvinyl alcohol films and gels have been reported as ophthalmic inserts, e.g. in the lower conjunctival sac when imbibed with pharmaceuticals such as tetracycline. Such materials are generally in the form of a crosslinked film or gel. See, for example, U.S. Pat. No. 4,559,186,where hydrogel contact lens materials crosslinked with a borate is described. In U.S. Pat. No. 4,619,793, polyvinyl alcohol is annealed to increase the crystallinity thereof and swollen in a swelling solvent.

Another embodimentof the invention provides a hydrogel extracellular matrix mimetic which is structurally supported by the crystallized polyvinyl alcohol and thus is stable under physiological conditions.

Another embodiment of the invention provides uses of the hydrogel extracellular matrix as a surface for cell culture, as a scaffold for tissue regeneration, for replacement of damaged or removed tissue in an animal, or for coating surgical implants.

These and other features and advantages of the invention are evident from the following embodiments when read in conjunction with the accompanying drawings in which;

FIG. 1 illustrates (A) the photographs of the hydrogel in situ formed via chemically crosslinked MAPTrix™ ECM (2wt% solution in PBS buffer) with 8 ARM-SG-20K (2wt% solution in PBS buffer), and (B) the photographs of the hydrogel scaffold in situ formed via chemicvally crosslinked MAPTrix™ ECM (10wt% solution in PBS buffer) with 8 ARM-SG-20K (10wt% solution in PBS buffer).

FIG. 2 illustrates the SEM morphology of ionically crosslinked hydrogels based on MAPTrix™ ECM with hyaluronic acid.

FIG. 3 illustrates (A) the gelation time of chemically crosslinked MAPTrix™ ECM hydrogel with 6 ARM-SG-10K, and (B) the effect of MAPTrix™ ECM concentration on gelation time.

FIG. 4 illustrates the hydrogel scaffold formed via physical crosslinking when the mixture of MAPTrix™ ECM and PVA with boric acid underwent freeze-thaw cycles (4 times).

FIG. 5 illustrates the adhesive strength of MAPTrix HyGel™ applied to glass slide.

FIGs. 6 and 7 illustrate the adhesive strength of MAPTrix HyGel™ applied to rat skin tissue.

FIG. 8 illustrates the effect of MAPTrix HyGel™ containing different bioactive peptides on cellular behavior of HUVEC cells. (A) laminin peptide, IKVAV, containing, (B) fibronectin peptide, REDV, containing, and (C) collagen peptide, GFPGER, containing hydrogel.

The following examples are provided to demonstrate preferred embodiments of the present invention and the invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purposes of exemplification only. Functionally equivalent products, compositions and methods are clearly within the scope of the invention, as described herein.

EXAMPLE 1. MAPTrix HyGel™ created in situ via chemical crosslinking method

To prepare the hydrogels, lysine side chains was reacted with multiarmed polyethylene glycol (PEG) with succinimidyl ester electrophilic functional groups (specifically, succinimidyl glutarate, SG) on the end of each of four arms (4 ARM) having MW of about 10,000 MW polyethylene glycol (referred to herein as 4 AMR-SG-10K) in different concentration.

Essentially identical hydrogels were then made, with the ratios of the electrophilic-nucleophilic functional groups still being 1:1, except that a 6-armed (6 ARM) or 8-armed (8 ARM) PEG with functional groups on the end of each arm with PEG arms having a total MW of about 10,000 (10 k) or 20,000 (20 k) were used instead of the four-armed PEG.

A detailed procedure for making a hydrogel is as follows, using 6 ARM-SG-10K as an example. MAPTrix™ ECM (Kollodis BioSciences, Inc., Malden, MA)was dissolved in distilled water or phosphate buffer solution at a concentration of 20 ㎎/㎖. 6 ARM-SG-10K dissolved in distilled water at 40 ㎎/㎖ were prepared. The two liquid components were mixed by vortex. After 30 minutes, the hydrogel was formed. After 24 hours, the hydrogel was freeze dried, and weighed and swelling ratio was calculated. Gelation time was measured by measuring the viscosity of the mixed solution. ARES rheometer in the cone-plate geometry was used to measure the viscosity. A stopwatch was started at the time of vortexing and stopped when the viscosity exhibited a significant change in magnitude.

Other formulations were similarly made, with varying concentrations and pH: 4 ARM-SG-10K SG (4 arm PEG, with the arms having a total combined MW of 10,000, terminated with succinimidyl glutrate) with 20 ㎎/㎖ concentration; 6 ARM-SG-10K SG (6 arm PEG, with the arms having a total combined MW of 10,000, terminated with succinimidyl glutrate) with 20 ㎎/㎖ concentration; 8 ARM-SG-20K SG (8 arm PEG, with the arms having a total combined MW of 20,000, terminated with succinimidyl glutrate) with 20 ㎎/㎖ concentration. The pH of buffer solution varied from 5.8 to 8.5.

Table 1 shows the results of gelation time obtained for these MAPTrix HyGel™ formulations, and FIG. 1 shows the photographs of chemically crosslinked MAPTrix™ ECM hydrogel using 8 ARM-SG-20K.

Table 1

MAPTrix™ ECM	Crosslinking agent	pH	Gelation time (min)
0.5 ㎎/㎖	8 ARM-SG-20K, 50 ㎎/㎖	6.5	No gelation
0.5 ㎎/㎖	8 ARM-NH₂-20K, 30 ㎎/㎖ 8 ARM-SG-20K, 50 ㎎/㎖	6.5	191
0.5 ㎎/㎖	8 ARM-NH₂-20K, 70 ㎎/㎖ 8 ARM-SG-20K, 50 ㎎/㎖	6.5	57
10 ㎎/㎖	8 ARM-SG-20K, 50 ㎎/㎖	6.5	No gelation
10 ㎎/㎖	8 ARM-NH₂-20K, 20 ㎎/㎖ 8 ARM-SG-20K, 50 ㎎/㎖	6.5	186
10 ㎎/㎖	8 ARM-NH₂-20K, 50 ㎎/㎖ 8 ARM-SG-20K, 50 ㎎/㎖	6.5	175
20 ㎎/㎖	8 ARM-SG-20K, 20 ㎎/㎖	7.5	43
20 ㎎/㎖	8 ARM-SG-20K, 20 ㎎/㎖	6.5	150
17 ㎎/㎖	8 ARM-SG-20K, 20 ㎎/㎖	7.5	No gelation
20 ㎎/㎖	6 ARM-SG-10K, 20 ㎎/㎖	6.5	140
20 ㎎/㎖	6 ARM-SG-10K, 50 ㎎/㎖	6.5	12
50 ㎎/㎖	6 ARM-SG-10K, 20 ㎎/㎖	6.5	14
50 ㎎/㎖	6 ARM-SG-10K, 50 ㎎/㎖	6.5	4
100 ㎎/㎖	6 ARM-SG-10K, 20 ㎎/㎖	6.5	4
100 ㎎/㎖	6 ARM-SG-10K, 50 ㎎/㎖	6.5	3
20 ㎎/㎖	6 ARM-SG-10K, 30 ㎎/㎖	6.5	No gelation
20 ㎎/㎖	6 ARM-SG-10K, 30 ㎎/㎖	7.5	21.2
20 ㎎/㎖	6 ARM-SG-10K, 30 ㎎/㎖	8.5	No gelation
30 ㎎/㎖	6 ARM-SG-10K, 30 ㎎/㎖	6.5	9.3
30 ㎎/㎖	6 ARM-SG-10K, 30 ㎎/㎖	7.5	5.3
30 ㎎/㎖	6 ARM-SG-10K, 50 ㎎/㎖	8.5	4.3
20 ㎎/㎖	4 ARM-SG-20K, 20 ㎎/㎖	7.5	5
20 ㎎/㎖	4 ARM-SG-20K, 20 ㎎/㎖	6.5	10
17 ㎎/㎖	4 ARM-SG-20K, 20 ㎎/㎖	7.5	60
10 ㎎/㎖	4 ARM-SG-20K, 20 ㎎/㎖	7.5	No gelation

EXAMPLE 2. MAPTrix HyGel™ created in situ via ionic crosslinking method

10 milligrams of sodium hyaluronate (HA) with an average molecular weight of 500,000 dalton is dissolved in 1miligrams of water in a beaker under magnetic stirring. After a homogeneous aqueous solution is obtained, 150 microliters of a 1 N HCl is added with agitation to adjust solution pH. Then, 660 microliters of a 5.0% aluminium chloride solution is added with agitation. Finally, 456 microliters of a 1.7N NH4OH solution is added with agitation until a homogeneous gel is obtained with a pH close to neutral. Other formulations were similarly made, with varying concentrations.

Table 2 shows various MAPTrix HyGel™ formulations prepared via ionic crosslinking method.

Table 2

MAPTrix™ ECM	HA	HCl	AlCl₃	NH₄OH
10 ㎎/㎖	5 ㎎/㎖	150 ㎕	660 ㎕	456 ㎕
30 ㎎/㎖	5 ㎎/㎖	150 ㎕	660 ㎕	456 ㎕
10 ㎎/㎖	10 ㎎/㎖	150 ㎕	660 ㎕	456 ㎕
30 ㎎/㎖	10 ㎎/㎖	150 ㎕	660 ㎕	456 ㎕
10 ㎎/㎖	30 ㎎/㎖	150 ㎕	660 ㎕	456 ㎕
30 ㎎/㎖	30 ㎎/㎖	150 ㎕	660 ㎕	456 ㎕

Morphology

MAPTrix HyGel™'s morphology was evaluated by scanning electron microscopy (SEM, Hitachi). FIG. 2 shows the SEM morphology of ionically crosslinked hydrogels based on MAPTrix™ ECM with hyaluronic acid. The hydrogels have numerous pores when inspected with SEM. The typical size of pores in the hydrogels is larger than 10 ㎛, usually in the range of several tens micrometers, and can be up to the millimeter range. The size and number of the pores can be controlled by adjusting the weight ratio of MAPTrix™ ECM concentration to crosslinking agent.

EXAMPLE 3. MAPTrix HyGel™ created in situ via physical crosslinking method

Aqueous solutions of 10wt% PVA were prepared by dissolving PVA (Sigma Aldrich) in distilled water for 6 hours at 60℃ under magnetic stirring. The PVA molecular weight of 30,000∼50,000 was used which had degrees of 99% hydrolysis and polydispersity index of 2.0. MAPTrix™ ECM was dissolved in distilled water or phosphate buffer solution at a concentration of 20 ㎎/㎖. The prepared two solutions were mixed for 20 minutes with under magnetic stirring. The samples were then exposed to three to four cycles of freezing for 8 hours at -20℃ or -85℃ and thawing for 4 hours at 4℃ to get a hydrogel scaffold (FIG 4).

EXAMPLE 4. FORMULATION OF ADHESIVE HYDROGEL COMPOSITION

Adhesive hydrogel preparation

MAPTrix™ ECM was dissolved in distilled water at a concentration of 450 ㎎/㎖. Crosslinking solution was aqueous 4 ARM-SG-10K (MW: 10KDa, JenKem Technology Inc., Allen, Texas USA) solution containing 10% by weight. Two rectangular blocks of surfaces were sprayed lightly with crosslinking solution on the surfaces to be bonded. The surfaces were then coated with MAPTrix™ ECM to a thickness of about 1 ㎜, and again sprayed with crosslinking solution (200 ㎎/㎖ of 4 ARM-SG-10K in PBS buffer solution, pH 7.4). The ratio of MAPTrix™ ECM and crosslinking solution was 1 to 1 by weight. The surfaces were joined within about 10 seconds of the application of crosslinking solution and held in position until cure was complete, generally 10 minutes, dependent on temperature and on the effectiveness of mixing MAP and crosslinking solution.

Testing Protocol and Efficacy of Adhesion

To determine tear strength the glued rectangular block of surface was attached with clamps to a spring balance at one end and to a variable weight on the other. The weight was increased progressively until the bond or adjoining meat broke. Tear strength was recorded in gram force required to break the bond. Tear strengths were determined one minute after joining the surfaces, unless otherwise noted.

When the sequence of application of MAP solution and crosslinking solution is reversed or when MAP and crosslinking solution are applied simultaneously or when MAP and crosslinking solution are pre-mixed immediately prior to application, essentially the same bond strengths are observed.

Adhesive forces and work were measured using a TA-XT2 Texture Analyzer (Texture Technologies Corporation, 18 Fairview Road, Scarsdale, N.Y. 10583) equipped with a 2 inch diameter (flat) probe. Peak adhesive force (maximum force required to pull away from sample) and adhesive work (total force in kilogram over time to pull completely away from sample) were measured by inserting the probe as a constant three kilogram force before pulling the probe up 20 ㎜ (1 ㎜/sec). Test results are summarized in the Table 3 and FIG. 5.

Table 3

Concentration	Crosslinking agent	Adhesive force (gram force)	Substrate
10%	Glutaraldehyde	2599	Glass surface
10%	Glutaraldehyde	1698	Hydrophobic surface
10%	4 ARM-SG-10K	161	Glass surface
20%	4 ARM-SG-10K	4241	Glass surface
30%	4 ARM-SG-10K	4595	Glass surface

EXAMPLE 5. TISSUE ADHESIVE ASSAY

Rat Skin Lap Shear Tensile Strength Assay

The same compositions in above Example 4 were tested for their ability to bond skin together using an in vitro rat skin lap shear tensile strength assay. Adhesives were applied to the subcutaneous side of a strip of harvested rat skin. A second skin strip was overlapped in order to produce an approximate bonding surface of 2 ㎠. A 8 ㎏ weight was applied to the lap joint and the adhesive was allowed to cure for 10 min at room temperature. Adhesive forces and work were measured using a TA-XT2 Texture Analyzer (Texture Technologies Corporation, 18 Fairview Road, Scarsdale, N.Y. 10583) equipped with a 2 inch diameter (flat) probe (see FIG. 5). Peak adhesive force (maximum force required to pull away from sample) and adhesive work (total force in kilogram over time to pull completely away from sample) were measured by inserting the probe as a constant three kilogram force before pulling the probe up 20 ㎜ (1 ㎜/sec). Test results are summarized in the Table 4 and FIG. 6.

Table 4

Concentration	Crosslinking agent	Adhesive force (g. force)
10%	4 ARM-SG-10K	49
20%	4 ARM-SG-10K	62
30%	4 ARM-SG-10K	45

EXAMPLE 6. TUBE FORMATION ASSAY

Endothelial Tube Formation Assay

Endothelial growth media (M199 media), supplemented with 10% fetal bovine serum (FBS) and endothelial cell growth supplement (ECGS, 30 ㎍/㎖; BD Biosciences), was used to seed HUVEC cells.

MAPTrix HyGel™ gel solution was prepared as follows: MAPTrix™ Laminin mimetic containing IKVAV, MAPTrix™ Fibronectin mimetic containing REDV, and MAPTrix Collagen mimetic containing GFPGER (Kollodis BioSciences) were dissolved to the final concentration of 20 ㎎/㎖, respectively, in PBS buffer solution (pH 7.4) was mixed with 10 ㎎/㎖ 4 ARM and 8 ARM PEG-SG, (1:1 (v/v)) . The prepared MAPTrix HyGel™ solutions were added to a 48-well plate (BD Biosciences) and allowed to gel at 37℃ for 2 h. HUVEC cells were washed in serum-free M199 medium by centrifuging at 400g for 1 min, and the washed HUVECs were resuspended in serum-free M199 medium and seeded onto the gel surfaces at the density of 5×10⁴ cells/well with 100 ng/㎖ VEGF and incubated at 37℃ for 24 hours. The morphology of HUVECs was monitored and photographed with a phase contrast microscope at regular intervals (every 6 hours). The effect of MAPTrix™ ECM peptide type on the morphology of HUVEC cells were quite different as seen in the FIG 8 ((A): IKVAV, (B): REDV, and (C): GFPGER). MAPTrix™ ECM containing IKVAV promoted cell adhesion and migration, and induced endothelial tube formation while other peptides (REDV, GFPGER) containing hydrogel promoted cell adhesion, but not tube formation of endothelial cells.

Claims

A composition for an extracellular matrix mimetic comprising a mussel adhesive protein and a crosslinking agent.
A composition for an extracellular matrix mimetic comprising a mussel adhesive protein, a biocompatible polymer and a crosslinking agent.
The composition for an extracellular matrix mimetic of claim 1 or 2, wherein the mussel adhesive protein is selected from the group consisting of FP-1(SEQ ID NO: 1), FP-2 (SEQ ID NO: 2), FP-3 (SEQ ID NO: 3), FP-4, FP-5 (SEQ ID NO: 4), and FP-6 (SEQ ID NO: 5), a fragment thereof and a fusion protein of the mussel adhesive proteins.
The composition for an extracellular matrix mimetic of claim 3, wherein the fusion protein comprises a first peptide of mussel foot protein selected from the group consisting of FP-2 (SEQ ID NO: 2), FP-3 (SEQ ID NO: 3) and FP-5 (SEQ ID NO: 4) and a second peptide of at least one selected from the group consisting of FP-1 (SEQ ID NO: 1), FP-2 (SEQ ID NO: 2), FP-3 (SEQ ID NO: 3), FP-4, FP-6 (SEQ ID NO: 5) and fragment thereof.
The composition for an extracellular matrix mimetic of claim 4, wherein the fusion protein is FP-151 which is FP-5 fused with FP-1.
The composition for an extracellular matrix mimetic of claim 5, wherein the fusion protein is selected from the group consisting of:

a FP-151 (SEQ ID NO: 6), FP-5 fused with FP-1;

a FP-13151 (SEQ ID NO: 7), FP-5 and FP-3 fused with FP-1;

a FP-151 functionalized with collagen type I-derived peptide GFPGER (SEQ ID NO: 10) to form collagen type I rich extracellular matrix mimetic;

a FP-151 functionalized with fibronectin-derived peptide GRGDSP (SEQ ID NO: 24) to form fibronectin rich extracellular matrix mimetic;a combination of hyaluronic acid with FP-151 functionalized with collagen type I-derived peptide DGEA (SEQ ID NO: 11) to mimic the biochemical composition of a natural bone ECM; and

a combination of a FP-151 functionalized with collagen type I-derived peptide GFPGER (SEQ ID NO: 10) and a FP-151 functionalized with laminin derived peptide IKVAV (SEQ ID NO: 33) to mimic the biochemical composition of a natural basilar lamina.
The composition for an extracellular matrix mimetic of claim 1 or 2, wherein a bioactive peptide is incorporated into C-terminus, N-terminus or C- and N-terminus of the mussel adhesive protein.
The composition for an extracellular matrix mimetic of claim 7, wherein the bioactive peptide is a natural or synthetic peptide derived from a extracellular matrix (ECM) protein to emulate biochemical or biophysical cues of a natural ECM.
The composition for an extracellular matrix mimetic of claim 8, wherein the ECM protein is selected from the group consisting of type I, type II or type IV collagen, fibronectin, laminin, vitronectin, bone sialoprotein, entactin and fibrinogen.
The composition for an extracellular matrix mimetic of claim 8 or 9, wherein the bioactive peptide is selected from the group consisting of:

a collagen type I-derived bioactive peptide selected from the group consisting of GLPGER (SEQ ID NO: 8), KGHRGF (SEQ ID NO: 9), GFPGER (SEQ ID NO: 10), DGEA (SEQ ID NO: 11), GPAGKDGEAGAQG (SEQ ID NO: 12) and GTPGPQGIAGQRDVV (SEQ ID NO: 13) containing peptide;

a collagen type II-derived bioactive peptide selected from the group consisting of EKGPD (SEQ ID NO: 14), EKGPDP (SEQ ID NO: 15) and EKGPDPL (SEQ ID NO: 16) containing peptide;

a collagen type IV-derived bioactive peptide selected from the group consisting of TAGSCLRKFSTM (SEQ ID NO: 17), GEFYFDLRLKGDK (SEQ ID NO: 18), TAIPSCPEGTVPLYS (SEQ ID NO: 19), TDIPPCPHGWISLWK (SEQ ID NO: 20) and LAGSCLARFSTM (SEQ ID NO: 21) containing peptide;

a fibronectin-derived bioactive peptide selected from the group consisting of PHSRN (SEQ ID NO: 22), RGD (SEQ ID NO: 23), GRGDSP (SEQ ID NO: 24), YRVRVTPKEKTGPMKE (SEQ ID NO: 25), SPPRRARVT (SEQ ID NO: 26), WQPPRARI (SEQ ID NO: 27), KNNQKSEPLIGRKKT (SEQ ID NO: 28), EILDVPST (SEQ ID NO: 29) and REDV (SEQ ID NO: 30) containing peptide;

a laminin-derived bioactive peptide selected from the group consisting of RQVFQVAYIIIKA (SEQ ID NO: 31), SINNTAVMQRLT (SEQ ID NO: 32), IKVAV (SEQ ID NO: 33), NRWHSIYITRFG (SEQ ID NO: 34), TWYKIAFQRNRK (SEQ ID NO: 35), RKRLQVQLSIRT (SEQ ID NO: 36), KNRLTIELEVRT (SEQ ID NO: 37), SYWYRIEASRTG (SEQ ID NO: 38), DFGTVQLRNGFPFFSYDLG (SEQ ID NO: 39), GQLFHVAYILIKF (SEQ ID NO: 40), KNSFMALYLSKG (SEQ ID NO: 41), TLFLAHGRLVFM (SEQ ID NO: 42), GQVFHVAYVLIKF (SEQ ID NO: 43), GIIFFL (SEQ ID NO: 44), LALFLSNGHFVA (SEQ ID NO: 45), RYVVLPR (SEQ ID NO: 46), PDSGR (SEQ ID NO: 47), YIGSR (SEQ ID NO: 48), KAFDITYVRLKF (SEQ ID NO: 49) and RNIAEIIKDI (SEQ ID NO: 50) containing peptide;

a vitronectin-derived bioactive peptide of FRHRNRKGY (SEQ ID NO: 51) or KKQRFRHRNRKGYRSQ (SEQ ID NO: 52); and

a bone sialoprotein-derived bioactive peptide of FHRRIKA (SEQ ID NO: 53) or KRSR (SEQ ID NO: 54) containing peptide.
The composition for an extracellular matrix mimetic of claim 1 or 2, wherein the crosslinking agent is selected from the group consisting of poly(alkylene oxides), poly(vinyl alcohols), polypeptides, poly(amino acids), poly(allylamines)(PAM), poly(acrylates), polyesters, polyphosphazenes, pluronic polyols, polyoxamers, poly(uronic acids) and copolymers thereof.
The composition for an extracellular matrix mimetic of claim 1 or 2, wherein the crosslinking agent has a molecular weight ranging from 1,000 to 50,000.
The composition for an extracellular matrix mimetic of claim 1 or 2, wherein the crosslinking agent mediates its crosslinking functions by chemically and/or physically crosslinking the mussel adhesive proteins.
The composition for an extracellular matrix mimetic of claim 13, wherein the chemical crosslinking is selected from the group consisting of covalent, ionic, hydrogen-bonded, and Van der Waals interactions.
The composition for an extracellular matrix mimetic of claim 13, wherein the physical crosslinking is molecular entanglement or intertwining.
The composition for an extracellular matrix mimetic of claim 2, wherein the biocompatible polymer is selected from the group consisting of poly(glycolic acid), poly(lactic acid and a graft polymer of PEG (polyethylene glycol) and acetyl-aspartate.
A three dimensional extracellular matrix mimetic prepared by using the composition for an extracellular matrix mimetic of claim 1 comprising a mussel adhesive protein and a crosslinking agent.
A three dimensional extracellular matrix mimetic prepared by using the composition for an extracellular matrix mimetic of claim 1 comprising a mussel adhesive protein, a biocompatible polymer and a crosslinking agent.
The three dimensional extracellular matrix mimetic of claim 17 or 18, wherein the mussel adhesive protein is selected from the group consisting of FP-1, FP-2, FP-3, FP-4, FP-5, FP-6, a fragment thereof and a fusion protein of the mussel adhesive proteins.
The three dimensional extracellular matrix mimetic of claim 19, wherein the fusion protein comprises a first peptide of mussel foot protein selected from the group consisting of FP-2, FP-3 and FP-5 and a second peptide of at least one selected from the group consisting of FP-1, FP-2, FP-3, FP-4, FP-6 and fragment thereof.
The three dimensional extracellular matrix mimetic of claim 20, wherein the fusion protein is FP-151 which is FP-5 fused with FP-1.
The three dimensional extracellular matrix mimetic of claim 21, wherein the fusion protein is selected from the group consisting of:

a FP-151 (SEQ ID NO: 6), FP-5 fused with FP-1;

a FP-13151 (SEQ ID NO: 7), FP-5 and FP-3 fused with FP-1;

a FP-151 functionalized with collagen type I-derived peptide GFPGER (SEQ ID NO: 10) to form collagen type I rich extracellular matrix mimetic;

a FP-151 functionalized with fibronectin-derived peptide GRGDSP (SEQ ID NO: 24) to form fibronectin rich extracellular matrix mimetic;

a combination of hyaluronic acid with FP-151 functionalized with collagen type I-derived peptide DGEA (SEQ ID NO: 11) to mimic the biochemical composition of a natural bone ECM; and

a combination of a FP-151 functionalized with fibronectin-derived peptide RGD (SEQ ID NO: 23) and a FP-151 functionalized with collagen type I-derived peptide DGEA (SEQ ID NO: 11) to mimic the biochemical composition of a natural basilar lamina.
The three dimensional extracellular matrix mimetic of claim 17 or 18, wherein a bioactive peptide is incorporated into C-terminus, N-terminus or C- and N-terminus of the mussel adhesive protein.
The three dimensional extracellular matrix mimetic of claim 23, wherein the bioactive peptide is a natural or synthetic peptide derived from a extracellular matrix (ECM) protein to emulate biochemical or biophysical cues of a natural ECM.
The three dimensional extracellular matrix mimetic of claim 24, wherein the ECM protein is selected from the group consisting of type I, type II or type IV collagen, fibronectin, laminin, vitronectin, bone sialoprotein, entactin and fibrinogen.
The three dimensional extracellular matrix mimetic of claim 24 or 25, wherein the bioactive peptide is selected from the group consisting of:

a collagen type I-derived bioactive peptide selected from the group consisting of GLPGER (SEQ ID NO: 8), KGHRGF (SEQ ID NO: 9), GFPGER (SEQ ID NO: 10), DGEA (SEQ ID NO: 11), GPAGKDGEAGAQG (SEQ ID NO: 12) and GTPGPQGIAGQRDVV (SEQ ID NO: 13) containing peptide;

a collagen type II-derived bioactive peptide selected from the group consisting of EKGPD (SEQ ID NO: 14), EKGPDP (SEQ ID NO: 15) and EKGPDPL (SEQ ID NO: 16) containing peptide;

a collagen type IV-derived bioactive peptide selected from the group consisting of TAGSCLRKFSTM (SEQ ID NO: 17), GEFYFDLRLKGDK (SEQ ID NO: 18), TAIPSCPEGTVPLYS (SEQ ID NO: 19), TDIPPCPHGWISLWK (SEQ ID NO: 20) and LAGSCLARFSTM (SEQ ID NO: 21) containing peptide;

a fibronectin-derived bioactive peptide selected from the group consisting of PHSRN (SEQ ID NO: 22), RGD (SEQ ID NO: 23), GRGDSP (SEQ ID NO: 24), YRVRVTPKEKTGPMKE (SEQ ID NO: 25), SPPRRARVT (SEQ ID NO: 26), WQPPRARI (SEQ ID NO: 27), KNNQKSEPLIGRKKT (SEQ ID NO: 28), EILDVPST (SEQ ID NO: 29) and REDV (SEQ ID NO: 30) containing peptide;

a laminin-derived bioactive peptide selected from the group consisting of RQVFQVAYIIIKA (SEQ ID NO: 31), SINNTAVMQRLT (SEQ ID NO: 32), IKVAV (SEQ ID NO: 33), NRWHSIYITRFG (SEQ ID NO: 34), TWYKIAFQRNRK (SEQ ID NO: 35), RKRLQVQLSIRT (SEQ ID NO: 36), KNRLTIELEVRT (SEQ ID NO: 37), SYWYRIEASRTG (SEQ ID NO: 38), DFGTVQLRNGFPFFSYDLG (SEQ ID NO: 39), GQLFHVAYILIKF (SEQ ID NO: 40), KNSFMALYLSKG (SEQ ID NO: 41), TLFLAHGRLVFM (SEQ ID NO: 42), GQVFHVAYVLIKF (SEQ ID NO: 43), GIIFFL (SEQ ID NO: 44), LALFLSNGHFVA (SEQ ID NO: 45), RYVVLPR (SEQ ID NO: 46), PDSGR (SEQ ID NO: 47), YIGSR (SEQ ID NO: 48), KAFDITYVRLKF (SEQ ID NO: 49) and RNIAEIIKDI (SEQ ID NO: 50) containing peptide;

a vitronectin-derived bioactive peptide of FRHRNRKGY (SEQ ID NO: 51) or KKQRFRHRNRKGYRSQ (SEQ ID NO: 52); and

a bone sialoprotein-derived bioactive peptide of FHRRIKA (SEQ ID NO: 53) or KRSR (SEQ ID NO: 54) containing peptide.
The three dimensional extracellular matrix mimetic of claim 17 or 18, wherein the crosslinking agent is selected from the group consisting of poly(alkylene oxides), poly(vinyl alcohols), polypeptides, poly(amino acids), poly(allylamines)(PAM), poly(acrylates), polyesters, polyphosphazenes, pluronic polyols, polyoxamers, poly(uronic acids) and copolymers thereof.
The three dimensional extracellular matrix mimetic of claim 17 or 18, wherein the crosslinking agent has a molecular weight ranging from 1,000 to 50,000.
The three dimensional extracellular matrix mimetic of claim 17 or 18, wherein the crosslinking agent mediates its crosslinking functions by chemically and/or physically crosslinking the mussel adhesive proteins.
The three dimensional extracellular matrix mimetic of claim 29, wherein the chemical crosslinking is selected from the group consisting of covalent, ionic, hydrogen-bonded, and Van der Waals interactions.
The three dimensional extracellular matrix mimetic of claim 29, wherein the physical crosslinking is molecular entanglement or intertwining.
The three dimensional extracellular matrix mimetic of claim 18, wherein the biocompatible polymer is selected from the group consisting of poly(glycolic acid), poly(lactic acid and a graft polymer of PEG (polyethylene glycol) and acetyl-aspartate.
An adhesive hydrogel prepared by using the composition for an extracellular matrix mimetic of claim 1 comprising a mussel adhesive protein and a crosslinking agent.
An adhesive hydrogel prepared by using the composition for an extracellular matrix mimetic of claim 1 comprising a mussel adhesive protein, a biocompatible polymer and a crosslinking agent.
The adhesive hydrogel of claim 33 or 34, wherein the mussel adhesive protein is selected from the group consisting of FP-1, FP-2, FP-3, FP-4, FP-5, FP-6, a fragment thereof and a fusion protein of the mussel adhesive proteins.
The adhesive hydrogel of claim 35, wherein the fusion protein comprises a first peptide of mussel foot protein selected from the group consisting of FP-2, FP-3 and FP-5 and a second peptide of at least one selected from the group consisting of FP-1, FP-2, FP-3, FP-4, FP-6 and fragment thereof.
The adhesive hydrogel of claim 36, wherein the fusion protein is FP-151 which is FP-5 fused with FP-1.
The adhesive hydrogel of claim 37, wherein the fusion protein is selected from the group consisting of:

a FP-151 (SEQ ID NO: 6), FP-5 fused with FP-1;

a FP-13151 (SEQ ID NO: 7), FP-5 and FP-3 fused with FP-1;

a FP-151 functionalized with collagen type I-derived peptide GFPGER (SEQ ID NO: 10) to form collagen type I rich extracellular matrix mimetic;

a FP-151 functionalized with fibronectin-derived peptide GRGDSP (SEQ ID NO: 24) to form fibronectin rich extracellular matrix mimetic;

a combination of hyaluronic acid with FP-151 functionalized with collagen type I-derived peptide DGEA (SEQ ID NO: 11) to mimic the biochemical composition of a natural bone ECM; and

a combination of a FP-151 functionalized with laminin-derived peptide IKVAV (SEQ ID NO: 33) and a FP-151 functionalized with collagen type I-derived peptide GFPGER (SEQ ID NO: 10) to mimic the biochemical composition of a natural basilar lamina.
The adhesive hydrogel of claim 33 or 34, wherein a bioactive peptide is incorporated into C-terminus, N-terminus or C- and N-terminus of the mussel adhesive protein.
The adhesive hydrogel of claim 39, wherein the bioactive peptide is a natural or synthetic peptide derived from a extracellular matrix (ECM) protein to emulate biochemical or biophysical cues of a natural ECM.
The adhesive hydrogel of claim 40, wherein the ECM protein is selected from the group consisting of type I, type II or type IV collagen, fibronectin, laminin, vitronectin, bone sialoprotein, entactin and fibrinogen.
The adhesive hydrogel of claim 40 or 41, wherein the bioactive peptide is selected from the group consisting of:

a collagen type I-derived bioactive peptide selected from the group consisting of GLPGER (SEQ ID NO: 8), KGHRGF (SEQ ID NO: 9), GFPGER (SEQ ID NO: 10), DGEA (SEQ ID NO: 11), GPAGKDGEAGAQG (SEQ ID NO: 12) and GTPGPQGIAGQRDVV (SEQ ID NO: 13) containing peptide;

a collagen type II-derived bioactive peptide selected from the group consisting of EKGPD (SEQ ID NO: 14), EKGPDP (SEQ ID NO: 15) and EKGPDPL (SEQ ID NO: 16) containing peptide;

a collagen type IV-derived bioactive peptide selected from the group consisting of TAGSCLRKFSTM (SEQ ID NO: 17), GEFYFDLRLKGDK (SEQ ID NO: 18), TAIPSCPEGTVPLYS (SEQ ID NO: 19), TDIPPCPHGWISLWK (SEQ ID NO: 20) and LAGSCLARFSTM (SEQ ID NO: 21) containing peptide;

a fibronectin-derived bioactive peptide selected from the group consisting of PHSRN (SEQ ID NO: 22), RGD (SEQ ID NO: 23), GRGDSP (SEQ ID NO: 24), YRVRVTPKEKTGPMKE (SEQ ID NO: 25), SPPRRARVT (SEQ ID NO: 26), WQPPRARI (SEQ ID NO: 27), KNNQKSEPLIGRKKT (SEQ ID NO: 28), EILDVPST (SEQ ID NO: 29) and REDV (SEQ ID NO: 30) containing peptide;

a laminin-derived bioactive peptide selected from the group consisting of RQVFQVAYIIIKA (SEQ ID NO: 31), SINNTAVMQRLT (SEQ ID NO: 32), IKVAV (SEQ ID NO: 33), NRWHSIYITRFG (SEQ ID NO: 34), TWYKIAFQRNRK (SEQ ID NO: 35), RKRLQVQLSIRT (SEQ ID NO: 36), KNRLTIELEVRT (SEQ ID NO: 37), SYWYRIEASRTG (SEQ ID NO: 38), DFGTVQLRNGFPFFSYDLG (SEQ ID NO: 39), GQLFHVAYILIKF (SEQ ID NO: 40), KNSFMALYLSKG (SEQ ID NO: 41), TLFLAHGRLVFM (SEQ ID NO: 42), GQVFHVAYVLIKF (SEQ ID NO: 43), GIIFFL (SEQ ID NO: 44), LALFLSNGHFVA (SEQ ID NO: 45), RYVVLPR (SEQ ID NO: 46), PDSGR (SEQ ID NO: 47), YIGSR (SEQ ID NO: 48), KAFDITYVRLKF (SEQ ID NO: 49) and RNIAEIIKDI (SEQ ID NO: 50) containing peptide;

a vitronectin-derived bioactive peptide of FRHRNRKGY (SEQ ID NO: 51) or KKQRFRHRNRKGYRSQ (SEQ ID NO: 52); and

a bone sialoprotein-derived bioactive peptide of FHRRIKA (SEQ ID NO: 53) or KRSR (SEQ ID NO: 54) containing peptide.
The adhesive hydrogel of claim 33 or 34, wherein the crosslinking agent is selected from the group consisting of poly(alkylene oxides), poly(vinyl alcohols), polypeptides, poly(amino acids), poly(allylamines)(PAM), poly(acrylates), polyesters, polyphosphazenes, pluronic polyols, polyoxamers, poly(uronic acids) and copolymers thereof.
The adhesive hydrogel of claim 33 or 34, wherein the crosslinking agent is multiple arm PEG.
The adhesive hydrogel of claim 44, wherein the multiple arm PEG is selected from the group consisting of 4 to 10 arm PEG.
The adhesive hydrogel of claim 45, wherein the multiple arm PEG is selected from the group consisting of; 4 to 8-arm PEG-succinic acid, 4 to 8-arm PEG-glutaric acid, 4 to 8-arm PEG-succimidyl succinate, 4 to 8-arm PEG-succimidyl glutarate, 4 to 8-arm PEG-acrylate and 4 to 8-arm PEG-propion aldehyde.
The adhesive hydrogel of claim 33 or 34, wherein the crosslinking agent has a molecular weight ranging from 1,000 to 50,000.
The adhesive hydrogel of claim 33 or 34, wherein the crosslinking agent mediates its crosslinking functions by chemically and/or physically crosslinking the mussel adhesive proteins.
The adhesive hydrogel of claim 48, wherein the chemical crosslinking is selected from the group consisting of covalent, ionic, hydrogen-bonded, and Van der Waals interactions.
The adhesive hydrogel of claim 48, wherein the physical crosslinking is molecular entanglement or intertwining.
The adhesive hydrogel of claim 34, wherein the biocompatible polymer is selected from the group consisting of poly(glycolic acid), poly(lactic acid and a graft polymer of PEG (polyethylene glycol) and acetyl-aspartate.