IL154208A - Plasma protein matrices and methods for preparation thereof - Google Patents

Description

limn? ΠΙΟ'ΒΠ πατ7ΰ 'τα7Π 7 ninoa Plasma Protein Matrices and Methods for Preparation Thereof PRO/025/IL ABSTRACT A porous homogeneous freeze-dried fibrin matrix, useful as an implant for tissue engineering as well as in biotechnology, and methods of producing said matrix are provided. The incorporation of glycosaminoglycans and bioactive agents during the formation of the matrix results in a sponge having advantageous biological, mechanical and physical properties. The matrices according to the present invention may be used clinically, per se or as a cell-bearing implant.

PLASMA PROTEIN MATRICES AND METHODS FOR PREPARATION THEREOF FIELD OF THE INVENTION The present invention concerns glycosaminoglycan-enriched biomatrices comprising fibrin and at least one bioactive agent useful for clinical applications including as implants for tissue engineering. The matrices according to the present invention may be used clinically, per se or as cell-bearing implants.

BACKGROUND OF THE INVENTION Tissue engineering may be defined as the art of reconstructing mammalian tissues, both structurally and functionally (Hunziker, 2002). Tissue engineering generally includes the delivery of a polymeric scaffold that serves as an architectural support onto which cells may attach, proliferate, and synthesize new tissue to repair a wound or defect.

An example of a tissue that is prone to damage by disease and trauma is the articular cartilage, one of several types of cartilage in the body, found at the articular surfaces of bones. Damage to cartilage may result from an inflammatory disease such as rheumatoid arthritis, from a degenerative process such as osteoarthritis or from trauma such as intraarticular fracture or following ligament injuries. Cartilage lesions are often associated with pain and reduced function and generally do not heal without medical intervention.

Current therapeutic strategies for repairing damaged cartilage encompass procedures that induce spontaneous repair response and those which reconstruct the tissue in a structural and functional manner. The former, including surgical techniques such as abrasion artheroplasty, microfracture or subchondral micro-drilling, expose the subchondral region of bone thereby allowing the formation of a blood clot and infiltration of plunpotent stem cells to initiate the healing response. Often the induced tissue is not durable and the clinical improvements are short lived.

Transplantation of chondral or osteochondral tissue from either autologous or allogeneic sources relies on the rationale that the proliferative and tissue-differentiation activities of these cells result in the formation of neocartilage. In fact, this technique shows high variability and limited clinical success. 1 Matrices useful for tissue regeneration and/or as biocompatible surfaces useful for tissue culture are well known in the art. These matrices may therefore be considered as substrates for cell growth either in vitro or in vivo. Suitable matrices for tissue growth and/or regeneration include both biodegradable and biostable entities. Among the many candidates that may serve as useful matrices claimed to support tissue growth or regeneration, are gels, foams, sheets, and numerous porous particulate structures of different forms and shapes.

Porous materials formed from synthetic and/or naturally occurring biodegradable materials have been used in the past as wound dressings or implants. A porous material provides structural support and a framework for cellular in-growth and tissue regeneration. Preferably, the porous material is gradually absorbed as the tissue regenerates. Typical bioabsorbable materials for use in the fabrication of porous wound dressings or implants include both synthetic polymers and biopolymers such as structural proteins and polysaccharides. The biopolymers may be either selected or manipulated in ways that affect their physico-chemical properties. For example biopolymers may be cross-linked either enzymatically, chemically or by other means, thereby providing greater or lesser degrees of flexibility or susceptibility to degradation.

Many natural polymers have been disclosed to be useful for tissue engineering or culture, including various constituents of the extracellular matrix including fibronectin, various types of collagen, and laminin, as well as keratin, fibrin and fibrinogen, hyaluronic acid, heparan sulfate, chondroitin sulfate and others.

Fibrin is a major plasma protein which participates in the blood coagulation process. The coagulation of blood is a complex process comprising the sequential interaction of a number of plasma proteins, in particular of fibrinogen (factor I), factor II, factor V, factor VII, factor VIII, factor IX, factor X, factor XI, factor XII and factor XIII. Other plasma proteins such as Von Willebrand factor (vWF), albumin, immunoglobulins, coagulation factors, and complement components may also play a part in the formation of protein networks or blood clots.

Fibrin is known in the art as a tissue adhesive medical device and can be used in wound healing and tissue repair. Lyophilized plasma-derived protein concentrate (containing Factor XIII, fibronectin, and fibrinogen), in the presence of thrombin and 2 calcium ions forms an injectable biological sealant (fibrin glue). US 5,411,885 discloses a method of embedding and culturing tissue employing fibrin glue.

US 4,642,120 discloses the use of fibrinogen-containing glue in combination with autologous mesenchymal or chondrocytic cells to promote repair of cartilage and bone defects. US 5,260,420 discloses a method for preparation and use of biological glue comprising plasma proteins for therapeutic use. US 6,440,427 provides an adhesive composition consisting essentially of fibrin forming components and a viscosity enhancing polysaccharide such as hyaluronic acid.

US 6,074,663 discloses a cross-linked fibrin sheet-like material for the prevention of adhesion formation. PCT application WO 00/51538 discloses a bioadhesive, porous PEG-crosslinked albumin and fibrin scaffold, useful for wound healing. A freeze-dried fibrin-antibiotic clot for the slow release of an antibiotic is described by Itokazu (Itokazu et al., Infection 25:359-63, 1997).

Protein-polysaccharide matrices have been described, as well. US 5,972,385 discloses a lyophilized cross-linked collagen-polysaccharide matrix that is administered alone or in combination with other therapeutics, such as growth factors, for tissue repair. The invention also discloses the cross-linked collagen-polysaccharide matrix in combination with fibrin. US 5,206,023 and 5,368,858 disclose a method and composition for inducing cartilage repair comprising dressing the site with a biodegradable matrix formed by mixing matrix forming material with a proliferative agent and a transforming factor.

A freeze-dried fibrin web for wound healing has been disclosed in US 6,310,267 and 6,486,377. The preparation of said web necessitates a single- or multistage dialysis of the fibrinogen solution. According to that disclosure, the single-stage or multistage dialysis of the fibrinogen solution changes crucially its composition by reducing the concentration of salts and amino acids. The dialysis is carried out in an aqueous solution of a physiologically compatible inorganic salt and an organic complexing agent.

A fibrin sponge containing a blood clotting activator for hemostasis, tissue adhesion, wound healing and cell culture support is disclosed in WO 99/15209.

According to that disclosure, the restoration of moisture or water content following lyophilization is crucial for obtaining a soft, adaptable, absorbent sponge. The sponge 3 may be impregnated with additives such as a blood clotting activator, stabilizers, preservatives and other agents.

US 5,736,372 discloses a biodegradable synthetic polymeric fibrous matrix containing chondrocytes for in vivo production of a functional cartilaginous structure to be used in joint lining. US 5,948,429 discloses a method of preparing a biopolymer foam comprising forming a biopolymer solution, cross-linking said solution with ultraviolet radiation and subsequently freeze-drying to form a lattice.

US 5,466,462 and 5,700,476 disclose heteromorphic sponges comprising a biopolymer matrix structure, at least one substructure and at least one pharmacologically active agent. The substructures allow the incorporation of one or more active agents into the final product for phasic release resulting in a sponge that is structurally inhomogeneous.

US 5,443,950 relates to a method for implantation of a variety of cell types growing on a three dimensional cell matrix which has been inoculated with stromal cells to form a three dimensional stromal matrix. US 5,842,477 discloses a method of in vivo cartilage repair by implanting a biocompatible three-dimensional scaffold in combination with periosteal/perichondrial tissue and stromal cells, with or without bioactive agents, for the production of new cartilage at the site of implantation. The scaffold is selected from a group consisting of biodegradable or non-biodegradable materials.

There remains a need for a material having superior biological and physical properties for treatment of tissue defects including but not limited to those found in diseased or injured cartilage and bone. In particular, there remains a need for a biocompatible material having enhanced biological properties and added therapeutic agents in addition to exhibiting superior physical and mechanical properties. Existing freeze-dried implants for tissue engineering purposes are prepared by admixing or by impregnating various preformed biopolymers with bioactive agents. This presents additional awkward steps for the medical practitioner. The art has not heretofore provided a stable freeze-dried fibrin sponge capable of delivering a bioactive agent contained therein ab initio. 4 SUMMARY OF THE INVENTION It is an object of the present invention to provide a porous, homogeneous fibrin matrix comprising at least one glycosaminoglycan and at least one bioactive agent having superior biological, physical and mechanical properties that is useful as a support for growth of cells, both in vitro and in vivo. It is another object of the present invention to provide such a porous fibrin matrix having sustained release of bioactive agents. It is yet another object of the present invention to provide such a porous fibrin matrix that is useful as an implant per se, or as a cell-bearing implant for repairing tissue damaged by disease or trauma. It is yet another object of the present invention to provide such a porous fibrin matrix that requires minimal preparation for use by the medical practitioner. It is a further object of the present invention to provide a method of promoting growth and repair of cartilage and bone in vivo. It is yet a further object of the present invention to provide methods for preparing said matrix.

These and other objects are met by the present invention.

Though numerous biomatrices comprising various plasma or tissue proteins are known in the art to which the present invention pertains, none has proven entirely satisfactory in meeting the criteria required for successful tissue engineering. The present invention discloses a porous homogeneous fibrin matrix, also referred to as a sponge, wherein glycosaminoglycans such as heparin and hyaluronic acid and bioactive agents such as therapeutic proteins or platelets, are incorporated into the matrix forming materials prior to initiation of the enzymatic reaction that induces cross-linking of the matrix structure. The resulting modified fibrin sponge has attributes that make it particularly advantageous for supporting and promoting cell growth both in vivo and in vitro.

Among the advantageous properties of the matrices of the invention: The matrices can be formed successfully with partially purified proteins, such as crude fractions of plasma proteins.

The plasma proteins can be retrieved from autologous material thereby obviating the need for pooled blood sources with the attendant health risks.

The matrices have superior biological properties, controlled by varying the glycosaminoglycans and bioactive agents used in the composition. Desirable properties include biodegradability, controlled release of bioactive agents, lack of immunogenicity, and the capacity to maintain and promote cell growth, proliferation, differentiation and migration.

The matrices have superior physical properties, which may be controlled by the glycosaminoglycans used in the composition. The desirable properties include texture, pore size and interconnecting channels, hydrophilicity, hydrophilicity, adhesion, wettability, adherence and texture.

The matrices have superior mechanical properties, controlled by glycosaminoglycans used in the composition. Desirable properties include tensile strength, elasticity, compressibility, resistance to shear, and moldability.

The matrices of the invention provide all components fundamental for tissue repair, thus facilitating the medical practitioner's task.

It is now disclosed for the first time that the aforementioned desirable properties of the matrices can be controlled by the incorporation of glycosaminoglycans and bioactive agents during the formation of the matrix. Biologically active agents that may be included in the formation of the matrix include growth factors, cytokines, blood platelets, platelet supernatants or extracts and platelet derived proteins, hormones, analgesics, anti-inflammatory agents, anti-microbials or enzymes.

The essential constituents of the matrices of the invention are fibrin, at least one glycosaminoglycan and at least one bioactive agent. Fibrin is obtained by the interaction of the plasma proteins fibrinogen and thrombin in the presence of calcium ions (Ca+2) and Factor XIII or another fibrin stabilizing factor, to form a fibrin clot. It is now disclosed that the incorporation of at least one glycosaminoglycan and at least one bioactive agent prior to the formation of the clot greatly enhances the physical, mechanical and biological attributes of the resulting sponge.

The plasma proteins utilized in the present invention may be purified from a plasma source or may be used from a commercially available source, including native or recombinant proteins. The preferred source of the plasma proteins is allogeneic blood, more preferably autologous blood. The addition of an exogenous anti-fibrinolytic agent is not necessary in the preparation of an allogeneic or autologous plasma protein sponge made from cryoprecipitated or concentrated plasma proteins, due to the presence of endogenous anti-fibrinolytic activity. 6 The present invention encompasses the addition of at least one glycosaminoglycan to the matrix forming materials, which results in a sponge having certain advantageous properties including physical, mechanical and/or biological properties. The incorporation of at least one glycosaminoglycan is shown to impart superior characteristics including elasticity and regular pore size to the sponge. The sponges formed are substantially homogeneous having no particles or interrupting substructures other than the pores and interconnecting channels.

Preferably the matrix is prepared using a glycosaminoglycan selected from the group consisting of crosslinked and uncrosslinked hyaluronic acid, heparin and heparin derivatives and mimetics, chondroitin sulfate, dextran sulfate, dermatan sulfate, heparan sulfate and keratan sulfate. Preferably the matrix is prepared with hyaluronic acid. More preferably the matrix is prepared with hyaluronic acid and heparin.

The at least one bioactive agent added during the preparation of the matrix is selected to enhance the healing process of the injured or diseased tissue. A bioactive agent may be a therapeutic protein belonging to the class of growth factors or cytokines that may encourage more rapid proliferation, chemotaxis, adhesion or differentiation of cells within the implant and the surrounding region or a more rapid vascularization of the implant. Bioactive agents including platelets and platelet supernatant or extract promote the proliferation and differentiation of chondrocytes, osteoblasts and other cell types. Bioactive agents belonging to the class of antimicrobial or anti-inflammatory agents may accelerate the healing process by minimizing infection and inflammation. Enzymes such as chondroitinase or matrix metalloproteinases (MMPs) may be incorporated to aid in the degradation of cartilage, thus stimulating release of cells into the matrix and the surrounding milieu.

According to one embodiment of the present invention the at least one bioactive agent is a therapeutic protein selected from the group consisting of growth factors and their variants. Preferably the growth factor is a fibroblast growth factor (FGF) or bone morphogenetic protein (BMP) or variant thereof. More preferably the FGF is an FGF having the capacity to induce cartilage and bone repair and regeneration and or angiogenesis. The growth factors may be incorporated during the formation of the clot at a wide range of concentrations, depending on the application. Sustained release of the growth factor may depend on several factors including growth factor concentration, type of glycosaminoglycan incorporated and thrombin concentration. 7 One preferred embodiment in accordance with the present invention is a porous homogeneous fibrin matrix comprising crosslinked hyaluronic acid and an FGF.

Another preferred embodiment in accordance with the present invention is a porous homogeneous fibrin matrix comprising crosslinked hyaluronic acid, heparin and an FGF.

In another embodiment of the present invention the bioactive agent is selected from platelets and/or platelet supernatant, preferably from an autologous source.

Platelet rich plasma (PRP) is plasma enriched with platelets (thrombocytes) or platelet supernatant. PRP may be produced from allogeneic or autologous blood using methods known in the art. According to one preferred embodiment, platelet rich plasma provides the source of both the plasma proteins and the bioactive agent. According to another preferred embodiment, the platelet rich plasma derives from an autologous source. The addition of an anti-fibrinolytic agent to the plasma proteins is optional.

The platelets may be present in the plasma protein concentrate or may be added exogenously. An exogenous source of platelets is added during the clot forming process to a final concentration of 0.1% to 30% of final sponge volume, more preferably 5% to 25% of final sponge volume. An exogenous source of platelet supernatant is added during the clot forming process to a final concentration of 0.1% to 30% of final sponge volume, more preferably 1% to 15% of final sponge volume. According to another embodiment the present invention may further include the incorporation of an additional synthetic or natural polymer prior to formation of the clot which may modify certain properties of the sponge including physical, mechanical and/or biological properties. These may impart superior characteristics including elasticity, regular pore size and strength to the sponge. The sponges formed are substantially homogeneous having no interrupting substructures or particles. Non-limiting examples of natural polymers include cellulose, pectin, polyuronic acids, hexuronyl hexosaminoglycan sulfate and inositol hexasulfate.

The synthetic polymers may be non-biodegradable or biodegradable. Examples of non-degradable materials include polytetrafluoroethylene, perfluorinated polymers such as fluorinated ethylene propylene, polypropylene, polyethylene, polyethylene terapthalate, silicone, silicone rubber, polysufone, polyurethane, non-degradable polycarboxylate, non-degradable polycarbonate, non-degradable polyester, polyacrylic, 8 polyhydroxymethacrylate, polymethylmethacrylate, polyamide such as polyesteramide, and copolymers, block copolymers and blends of the above materials.

Examples of degradable materials include hydrolyzable polyesters such as polylactic acid and polyglycolic acid, polyorthoesters, degradable polycarboxylates, degradable polycarbonates, degradable polycaprolactones, polyanhydride, and copolymers, block copolymers and blends of the above materials. Other components include surfactants including lecithin.

The fibrin sponge of the invention may have irregular pores or substantially regular pores. As used herein the term "substantially regular pores" means that the majority of the pores or more preferably substantially all the pores are in the same size range. More preferred matrices according to the invention have pores of a diameter in the range of 50-500 microns. Currently most preferred embodiments according to the present invention are plasma protein sponges with pore sizes in the range of 100-400 microns having interconnecting channels allowing infiltration and migration of the cells into and within the matrix.

The present invention provides a method for preparing a porous homogeneous fibrin matrix. The matrix forming solutions include a thrombin solution and a plasma protein solution. As used herein the thrombin solution comprises a source ofCa+2 ions and thrombin in an amount sufficient to cleave fibrinogen and yield a fibrin clot. The plasma protein solution comprises fibrinogen and factor XIII and may derive from a commercial, allogeneic or autologous source, in the substantial absence of organic chelating agents. The at least one bioactive agent and glycosaminoglycan are added independently to either of the matrix forming solutions, i.e. the plasma protein solution or to the thrombin solution, prior to the formation of the clot or are placed into the mold prior to, concurrently with or following addition of the thrombin solution.

In one currently preferred embodiment of the invention the porous homogeneous fibrin sponge is prepared by transferring the thrombin solution into a mold, adding the plasma protein solution; freezing the clotted mixture and lyophilizing. Alternatively, the plasma proteins are mixed with thrombin in the presence of Ca ions under conditions suitable for achieving clotting; the mixture is cast or mold in a solid support prior to achieving clotting; the clotted mixture is frozen and lyophilized. It is to be understood that the bioactive agent and glycosaminoglycan are added independently to 9 either of the matrix forming solutions, i.e. the plasma proteins or to the thrombin solution, prior to the formation of the clot or are placed into the mold prior to, concurrently with or following addition of the thrombin.

The final concentration of thrombin may be varied in order to produce sponges with distinct biological, physical and mechanical features useful for different applications. Use of a high concentration of thrombin, i.e. about 30 IU/ml (about 1.5 IU/mg total protein), yields a sponge with smaller pores and thicker fibers than use of thrombin at a low concentration, i.e. about 3 IU/ml (0.15 IU/mg total protein).

The addition of an anti-fibrinolytic agent to the plasma proteins is optional.

A method for preparing a porous homogeneous fibrin matrix useful as a scaffold for growing cells, and as a scaffold for implantation in vivo or in situ comprises the following steps: providing a thrombin solution and a plasma protein solution wherein at least one of the plasma protein solution or the thrombin solution contains at least one glycosaminoglycan and at least one of the plasma protein solution or the thrombin solution contains at least one bioactive agent; introducing the thrombin solution and the plasma protein solution into a solid receptacle or mold; incubating under conditions appropriate to achieve clotting; freezing the clotted mixture; and lyophilizing the clotted mixture, to obtain a sponge.

According to another embodiment the matrix of the invention may be prepared by the above method further comprising the steps of: pre-mixing the plasma protein solution with the thrombin solution in the presence of calcium ions, at least glycosaminoglycan and at least one bioactive agent, under conditions suitable for achieving clotting; casting the mixture of plasma proteins and thrombin in a solid receptacle or mold prior to achieving clotting.

A method for preparing a porous homogeneous fibrin matrix useful for support of cell growth after implantation in situ, is provided comprising the steps of: providing a thrombin solution and a plasma protein solution wherein at least one of the plasma protein solution or the thrombin solution contains at least one glycosaminoglycan and at least one of the plasma protein solution or the thrombin solution contains at least one bioactive agent; introducing the thrombin solution and the plasma protein solution into a solid receptacle or mold; incubating under conditions appropriate to achieve clotting; freezing the clotted mixture; lyophilizing the clotted mixture, to obtain a sponge; cutting the sponge into sections of desired shape; and implanting the sections in situ.

The sponge may further comprise cells including stem cells, progenitor cells or other cell types including chondrocytes, osteoblasts, hepatocytes, mesenchymal, epithelial, urothelial, neuronal, pancreatic, renal or ocular cell types.

According to one currently preferred embodiment at least 0.15 international units of thrombin per mg total plasma protein comprising at least one μg FGF is introduced into a solid support. Plasma proteins at a concentration of about 20-40 mg/ml are mixed with hyaluronic acid and or heparin and the mixture is added to the thrombin in the solid support to achieve formation of a clot. The clot is frozen at -70°C for approximately 16 hours and lyophilized for at least 16 hours, preferably 24 hours.

According to another currently preferred embodiment at least 0.15 to 15 units of thrombin per mg total plasma protein is introduced to a receptacle or solid support. Plasma proteins at a concentration of about 20-40 mg/ml comprising a hyaluronic acid and platelets or platelet supernatant are mixed and the mixture added to the thrombin in the solid support to achieve formation of a clot. The clot is frozen at -70°C for approximately 16 hours and lyophilized for at least 16 hours, preferably 24 hours.

In its final form prior to use with cells the sponge is substantially dry and contains less than 15% residual moisture, more preferably less than 10% residual moisture and most preferably less than 5% residual moisture. 1 1 The in vivo uses of the porous homogeneous fibrin matrix are manifold. The porous homogeneous fibrin matrix may function as a scaffold and may be used as an implant per se, for providing mechanical support to a defective or injured site in situ and/or for providing a matrix within which cells from the defective or injured site proliferate and differentiate. For example, for cartilage repair the porous homogeneous fibrin matrix may be used in conjunction with other therapeutic procedures including chondral shaving, laser or abrasion chondroplasty, and drilling or microfracture techniques.

The porous homogeneous fibrin matrix of the invention, being an effective scaffold supporting cell growth, may further be utilized in vivo in reconstructive surgery, for example as a matrix for regenerating cells and tissue including neuronal cells, cardiovascular tissue, urothelial cells and breast tissue. Some typical orthopedic applications include joint resurfacing, meniscus repair, non-union fracture repair, craniofacial reconstruction or repair of an invertebral disc. Furthermore, the porous homogeneous fibrin matrix may be used as a coating on synthetic or other implants such as pins and plates, for example, in hip replacement procedures. Thus, the present invention further provides implants or medical devices coated with the comprising the porous homogeneous fibrin matrix of the invention.

Furthermore, the sponge of the present invention may be used as a component of a two-phase or multi-phase material for tissue repair such as seen in osteochondral defects. In a non-limiting example, one layer may comprise a calcium phosphate material whilst an additional layer may comprise the sponge of the invention.

The porous homogeneous fibrin matrix of the invention is useful, inter alia, as an unexpectedly advantageous support for cellular growth. The matrix of the present invention is a biocompatible surface useful for tissue culture, such as for growing mesenchymal cells, chondrocytes, osteoblasts, epithelial cells, neuronal cells, hepatic, renal, pancreatic and any other cell types which it is desired to culture within a three dimensional support. Preferably the matrix of the invention is prepared from autologous plasma protein components, more preferably from autologous platelet rich plasma.

Further provided by the present invention is a cell bearing implant for transplanting cells in vivo. According to one currently preferred embodiment of the 12 present invention the matrix is a sponge comprising plasma proteins able to support the proliferation of a variety of cell types. Preferably, the sponge is inoculated with cells and the cells allowed to proliferate in vitro prior to in vivo implantation. Alternatively, the sponge is allowed to absorb cells that have been cultured or harvested and the sponge comprising the cells is implanted in vivo.

The implant consists of a porous homogeneous fibrin matrix bearing cells at a density that is at least 104 (ten thousand) cells per cm3, preferably 105 cells per cm3, more preferably 106 cells per cm3. Preferably, a porous homogeneous fibrin matrix for transplanting chondrocytes comprising autologous plasma proteins and autologous chondrocytes is used as an implant for transplantation. According to one currently preferred embodiment two sponges are implanted at the site of injury or disease. In a non-limiting example a dry sponge is placed at the site of injury or disease, autologous chondrocytes are seeded on the implant and another sponge is placed on top, in a sandwich arrangement. Alternatively, a first sponge in which cells were seeded several days prior to implantation is placed on top of the second dry sponge, in sandwich arrangement. The matrix of the invention may be cut into sections of desired size and shape to fit the affected area prior to seeding with cells or prior to implantation.

BRIEF DESCRIPTION OF THE FIGURES The present invention will be understood and appreciated more fully from the following detailed description taken in conjunction with the figures in which: Figures 1 A and IB shows a bar graph of FGF release from plasma protein matrices having FGF and heparin incorporated, ab initio; Figures 2A and 2B represent FGF release from plasma protein matrices having FGF and heparin adsorbed following sponge preparation; Figure 3 represents the FGF release from commercial and allogeneic plasma protein sponges comprising 0.024% hyaluronic acid and FGF, ab initio; Figure 4 shows histochemical sections of a fibrin sponge of the invention prepared from commercial fibrinogen, seeded with porcine chondrocytes.

Figure 5 A-5D show histochemical sections of a fibrin sponge of the invention prepared from human plasma proteins, seeded with porcine chondrocytes. 13 DETAILED DESCRIPTION OF THE INVENTION The present invention provides a porous homogeneous fibrin matrix comprising plasma-derived proteins, at least one glycosaminoglycan and at least one bioactive agent. The porous homogeneous fibrin matrix, also referred to as a sponge, is disclosed wherein at least one glycosaminoglycan and at least one bioactive agent such as a therapeutic protein or platelets, are incorporated into the matrix forming materials prior to initiation of the enzymatic reaction that induces cross-linking of the matrix structure. The resulting modified fibrin sponge has attributes that make it particularly advantageous for supporting and promoting cell growth both in vivo and in vitro.

The matrix according to an embodiment of the invention is useful in methods for regenerating and/or repairing various tissues in vivo, for example in tissue engineering methods, and for growing cells in vitro.

As disclosed in copending international patent publication WO 03/07873 assigned to the assignees of the present invention the addition of selected auxiliary agents may be added in the preparation of the sponge to improve certain physical, mechanical and biological properties of the matrix. It is now revealed that the incorporation of glycosaminoglycans and of bioactive agents during the preparation of the matrices imparts superior biological properties, including cell proliferation, cell differentiation, and sustained release of the bioactive agents.

Definitions For convenience and clarity certain terms employed in the specification, examples and claims are described herein.

"Plasma" as used herein refers to the fluid, non-cellular portion of the blood of humans or animals as found prior to coagulation.

"Plasma protein" as used herein refers to the soluble proteins found in the plasma of normal humans or animals. These include but are not limited to coagulation proteins, albumin, lipoproteins and complement proteins.

"Platelet rich plasma" or "PRP" as used herein refers to a plasma containing platelets. A platelet sample or platelet-derived extract or supernatant may be added 14 exogenously. Alternatively, platelet rich plasma may be prepared by methods known in the art, including those disclosed in US 6,475,175 and 6,398,972.

A "matrix" as used herein, refers to a porous structure, solid or semi-solid biodegradable substance having pores and interconnecting channels sufficiently large to allow cells to populate, or invade the matrix. The matrix-forming materials require addition of a polymerizing agent to form the matrix, such as addition of thrombin in the presence of bivalent calcium ions to a solution containing fibrinogen to form a fibrin clot. The clot is subsequently freeze-dried yielding a porous, homogeneous fibrin matrix. The fibrin matrix of the present invention may be denoted herein as a scaffold or as a sponge, for use as an implant per se, for the culturing of cells or as a cell-bearing tissue replacement implant.

The term "biocompatible" as used herein refers to materials which have low toxicity, acceptable foreign body reactions in the living body, and affinity with living tissues.

The terms "lyophilize" or "freeze drying" refer to the preparation of a composition in dry form by rapid freezing and dehydration in the frozen state (sometimes referred to as sublimation). This process may take place under vacuum at reduced air pressure resulting in drying at a lower temperature than required at full pressure.

The term "residual moisture" as used herein refers to the amount of water remaining in the dried sample. It is referred to as a percent of the weight of the sample. The plasma protein matrices of the invention preferably having less than 15% residual moisture, more preferably having less that 10% residual moisture, most preferably having less than 5% residual moisture.

The term "cell-bearing" as used herein refers to the capacity of the matrix to allow cells to be maintained within the structure being referred to. Preferably, the cells are able to proliferate and invade the pores and channels of the matrix. ¾ '* ' \ ' - ·«' The term "implantation" refers to the insertion of a sponge of the invention into a patient, whereby the implant serves to replace, fully or partially, tissue that has been damaged or removed.

The "biologically active" or "bioactive agents" incorporated into the sponge, for example, growth factors, platelet and platelet extracts, angiogenic factors, and the like, are advantageous to encourage a more rapid growth of the cells within the implant, or a more rapid vascularization of the implant. Such factors have now been shown to be effectively retained within the sponge and form a source or depot of bioactive agent, for sustained release.

The "pore size" of a pore within a plasma protein sponge is determined by using the equation: P=(L x H)1/2 wherein, L and H are the average length and height of the pores, respectively, as determined by microscopic analysis of the various sponges.

The "pore wall thickness" is a parameter that characterizes the distance between the pores within a sponge and is indicative of the microstructure of the sponges. It is determined by measurement at the microscopic level.

The term "substantially homogeneous " refers to a sponge of the invention having no particles or substructures other than pores and interconnecting channels interrupting the matrix.

"Surfactant" refers to a substance that alters energy relationship at interfaces, such as, for example, synthetic organic compounds displaying surface activity, including, inter alia, wetting agents, detergents, penetrants, spreaders, dispersing agents, and foaming agents. Preferable examples of surfactants useful in the present invention are hydrophobic compounds, and include phospholipids, oils, and fluorosurfactants .

An "anionic polysaccharide" as used herein, is a polysaccharide, including non-modified as well as chemical derivatives thereof, that contains one negatively charged group (e.g., carboxyl groups at pH values above about 4.0) and includes salts thereof, such as sodium or potassium salts, alkaline earth metal salts such as calcium or magnesium salts. Examples of anionic polysaccharides include pectin, alginate, galactans, galactomannans, glucomannans and polyuronic acids.

A "glycosaminoglycan" or "GAG" as used herein refers to a long unbranched polysaccharide molecules found on the cell surface or extracellular matrix. Examples of glycosaminoglycan include heparin, chondroitin sulfate, dextran sulfate, dermatan sulfate, heparan sulfate, keratan sulfate, crosslinked or uncrosslinked hyaluronic acid, hexuronyl hexosaminoglycan sulfate, and inositol hexasulfate. Derivatives, salts and mimetics of the above are intended to be included in the invention. 16 The term "cartilage" as used herein, refers to a specialized type of connective tissue that contains chondrocytes embedded in an extracellular matrix. The biochemical composition of cartilage differs according to type but in general comprises collagen, predominantly type II collagen along with other minor types, e.g., types IX and XI, proteoglycans, other proteins, and water. Several types of cartilage are recognized in the art, including, for example, hyaline cartilage, articular cartilage, costal cartilage, fibrous cartilage, meniscal cartilage, elastic cartilage, auricular cartilage, and yellow cartilage. The production of any type of cartilage is intended to fall within the scope of the invention.

The term "chondrocytes" as used herein, refers to cells which are capable of producing components of cartilage tissue.

A "substantial absence of organic chelating agents" refers to a concentration less than 1 mM, for example less than ImM EDTA.

In one currently preferred embodiment of the present invention a porous homogeneous fibrin matrix is provided. The matrix of this type may be produced according to the invention by exposing a plasma protein solution to a thrombin solution, in the presence of at least one glycosaminoglycan and at least one bioactive agent, subjecting said mixture to freezing and lyophilization to produce a sponge-like matrix.

US 6,310,267 discloses a specific process for preparing a biodegradable flexible fibrin web for wound healing. The process necessitates dialyzing a fibrinogen solution with a solution containing chelators and forming the flexible web by the addition of a thrombin solution, freeze drying and lyophilizing the web.

US 5,466,462 discloses a bioabsorbable heteromorphic sponge having a homogeneous structure sufficiently interrupted by macroscopic substructures to facilitate cellular movement and to provide for phasic release of active agents..

In contrast to fibrin webs and heteromorphic sponge of the art the present inventors have discovered that a biocompatible, homogeneous three-dimensional (3D) sponge-like matrix may be prepared from a crude plasma protein solution comprising at least one bioactive agent and at least one glycosaminoglycan, wherein the bioactive agent and glycosaminoglycan are incorporated into the matrix forming materials prior to initiation of the enzymatic reaction that induces cross-linking of the matrix structure.

The resulting porous homogeneous fibrin sponge has attributes that make it particularly advantageous for supporting and promoting cell growth both in vivo and in vitro. A porous homogeneous fibrin matrix, scaffold or sponge with advantageous properties including adherence to tissue, pore size and biocompatibility is obtained without the dialysis or addition of substructures in the form of milled sponges, powders, aggregates or the like.

The plasma protein solution may be from a commercial source, natural or recombinant proteins, or may be prepared from plasma. According to one currently preferred embodiment of the present invention the plasma protein solution derives from allogeneic plasma. According to one currently more preferred embodiment of the present invention, at least one of the components, preferably the plasma proteins, used for preparing the matrix derives from autologous plasma. According to another embodiment of the present invention, all of the plasma components used in preparing the matrix are autologous. The plasma proteins may be isolated by a variety of methods, as known in the art and exemplified herein below, resulting in a plasma protein matrix having substantially similar properties, as measured by pore size, elasticity, compression and cell bearing capabilities. A stable thrombin component may be isolated from autologous plasma, according to methods known in the art for example those disclosed in US Patent No. 6,274,090 and Haisch et al (Med Biol Eng Comput 38:686-9, 2000).

Additionally, the thrombin concentration in the matrix may be varied in order to produce sponges with distinct biological, physical and mechanical features useful for different applications. Use of a high concentration of thrombin, i.e. at least 30 IU/ml (1.5 IU/mg total protein), yields a sponge with smaller pores and thicker fibers than use of thrombin at a low concentration, i.e. about 3 IU/ml (0.15 IU/mg total protein).

The resulting plasma protein matrix exhibits advantageous properties including biocompatibility, pore size compatible with cell invasion and proliferation and ability to be molded or cast into definite shapes.

In a currently preferred embodiment of the present invention, blood is drawn from a patient in need of tissue repair or regeneration, plasma proteins, are isolated from the autologous plasma and a matrix prepared thereof. The platelets are optionally 18 isolated and returned to the plasma. The matrix of the present invention may serve as a support or scaffold per se or as a cell-bearing scaffold for in vivo implantation.

According to one currently preferred embodiment of the present invention a porous homogeneous fibrin sponge produced from a fibrinogen solution, wherein the fibrinogen solution is subjected to dialysis with a solution not requiring a complexing agent, serves as a scaffold for the growth of cells in vitro and in vivo. In another aspect the fibrin sponge is formed by the action of a thrombin solution on the dialyzed fibrinogen solution and subsequently subjected to freeze-drying.

While not wishing to be bound by any particular theory the substantial absence of organic complexing agents may provide the matrix of the present invention with properties beneficial to the proliferation and metabolism of certain cell types. As shown in the examples herein, the matrix of the present invention supports the proliferation of cartilage cells in both in vivo and in vitro systems.

The presence of certain organic complexing agents in a range of 1 to 20 mM, necessary for the production of a flexible fibrin web disclosed in US 6,310,267 for wound healing, may in itself have a detrimental effect on the proliferation of certain cell types. The use of a fibrin web for cell growth and proliferation, in vivo or in vitro, has not been disclosed. Nevertheless, it may be possible to culture certain types of cell types using the webs of the aforementioned patent.

Glycosaminoglycans The porous homogeneous fibrin matrix of the invention comprises components which modulate the mechanical, physical and biological properties including elasticity, pore size, surface adhesion and ability to sustain cell growth and proliferation. These include materials belonging to the family of glycosaminoglycans, including hyaluronic acid, heparin, chondroitin sulfate, dextran sulfate, dermatan sulfate, heparan sulfate and keratan sulfate. It is to be understood that salts, derivatives and mimetics of the above are included in the invention, including high and low molecular weight heparins.

Preferably the sponge is prepared with crosslinked or uncrosslinked hyaluronic acid, more preferably with crosslinked hyaluronic acid and heparin or derivatives thereof.

According to one currently preferred embodiment of the present invention heparin is incorporated into the matrix to a final concentration of 0.01% to 10%, more preferably 0.5% to 5%. 19 According to another currently preferred embodiment of the present invention hyaluronic acid is incorporated into the matrix to a final concentration of 0.001% to .5%, more preferably 0.01% to 0.1%.

According to yet another currently preferred embodiment of the present invention both heparin and hyaluronic acid are incorporated into the matrix at respective concentration ranges.

Surprisingly, in view of the known function of heparin as an anti-coagulant, it is now disclosed that the incorporation of heparin into the matrix does not interfere with either the formation of the matrix or the therapeutic benefits of the matrix. Without wishing to be bound by theory, heparin serves primarily to bind FGF or other therapeutic proteins and creates a depot for sustained release of said proteins. In addition, low molecular weight fragments of heparin released from the matrix may function as anti-inflammatory agents and assist in the healing process of diseased or traumatized tissue (US 5,474,987; 5,686,431; 5,908,837).

Optionally, anti-fibrinolytic agents including tranexamic acid may be included in the matrix of the invention. These compounds prevent fibrinolysis and thus can be used for controlling the rate of degradation of fibrin in vivo. An anti-fibrinolytic agent is preferably added to a matrix forming composition when the plasma protein source is other than a crude plasma protein source. Tranexamic acid may be added to a final concentration ranging between 0.1% to 10%, preferably 1% to 5%. Additional anti-fibrinolytic agents, well known in the art, may be used alone or in combination, including aprotinin, α-2-macroglobulin, α-2-plasmin inhibitor, plasminogen activator inhibitor and other natural or synthetic agents.

Bioactive Agents Bioactive agents, such as cytokines, growth factors and their activators, platelets, bioactive peptides etc., are included in the matrix of the invention. Optionally, they may also be added to the cells to be seeded on the matrix for example, to enhance a therapeutic effect. Incorporation of such agents into the sponge of the present invention provides a slow-release or sustained-release mechanism. For example, growth factors, structural proteins or cytokines which enhance the temporal sequence of wound repair, enhance angiogenesis, alter the rate of proliferation or increase the metabolic synthesis of extracellular matrix proteins are useful additives to the matrix of the present invention. Representative proteins include bone growth factors (BMPs, IGF) and fibroblast growth factors, including FGF2, FGF4, FGF9 and FGF18 for bone and cartilage healing, cartilage growth factor genes (CGF, TGF-β) for cartilage healing, nerve growth factor genes (NGF) and certain FGFs for nerve healing, and general growth factors such as platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF-1), keratinocyte growth factor (KGF), endothelial derived growth supplement (EDGF), epidermal growth factor (EGF) and other proteins which may enhance the action of the growth factors including heparin sulfate proteoglycans (HSPGs) their mimetics such as dextran sulfate, sucrose octa sulfate or heparin, and fragments thereof. Other factors shown to act on cells forming bone, cartilage or other connective tissue include retinoids, growth hormone (GH), and transferrin. Proteins specific for cartilage repair include cartilage growth factor (CGF), FGFs and TGF-β.

The bioactive proteins of the invention are polypeptides or derivatives or variants thereof, obtained from natural, synthetic or recombinant sources, which exhibit the ability to stimulate DNA synthesis and cell division in vitro of a variety of cells, including primary fibroblasts, chondrocytes, vascular and corneal endothelial cells, osteoblasts, myoblasts, smooth muscle and neuronal cells.

According to one currently preferred embodiment of the present invention platelets are incorporated into the matrix. The platelets may be present in the plasma protein concentrate or may be added exogenously. An exogenous source of platelets or platelet supernatant is added to the matrix to a final concentration of 0.1% to 30% final volume, more preferably 1% to 10% final volume.

Additionally, cells genetically engineered to express the aforementioned proteins are including in the present invention. Preferred examples for cartilage repair uses periosteal cells, mesenchymal stem cells or chondrocytes per se or transfected with cartilage growth factor genes selected from a group including transforming growth factor-β (TGF-β), certain FGFs or CGF; bone repair uses periosteal or other mesenchymal stem cells or osteoblasts per se or transfected with bone growth factor genes selected from a group including bone morphogenetic protein (BMP) family genes or fibroblast growth factor family genes; for nerve repair uses neural cells and neural support cells per se or transfected with genes selected from a group including nerve growth factor (NGF) gene or specific FGFs. 21 Furthermore, specific enzymes maybe admixed with the sponge of the invention in order to promote degradation of the proteoglycans and /or proteins present in the cartilage. Without wishing to be bound by theory, chondrocytes of the cartilage are embedded in the thick extracellular matrix (ECM) of the joint. Enzymes known in the art including collagenase, trypsin, chymotrypsin, chondroitinase of the various types, degrade the ECM of the surface of the joint, thereby releasing chondrocytes that are able to invade the sponge of the invention to promote cartilage regeneration.

The matrix of the invention, in certain embodiments may further include one or more antiseptics, such as methylene blue, and/or one or more drugs including antimicrobials such as antibiotics and antiviral agents; chemotherapeutic agents; anti-rejection agents; analgesics and analgesic combinations; anti-inflammatory agents; adhesion protein such as fibronectin or fragments thereof and hormones such as steroids.

Applications The porous homogeneous fibrin matrix of the invention is useful as scaffold for tissue engineering applications. The presence of the bioactive agents and the glycosaminoglycan together provides as an unexpectedly advantageous support for cellular growth in vitro and in vivo.

The in vivo uses of the enriched plasma matrix are manifold. The plasma protein scaffold may be used as an implant per se, for providing mechanical support to a defective or injured site in situ and/or for providing a matrix within which cells from the defective or injured site proliferate and differentiate.

The homogeneous porous fibrin matrix of the present invention can be utilized in reconstructive surgery methods for regenerating and/or repairing tissue that have been damaged for example by trauma, surgical procedures or disease. The present invention provides a matrix for use as an implantable scaffold per se for tissue regeneration. According to a currently preferred embodiment of the invention, the matrix serves as both a physical support and an adhesive substrate for in vivo cell growth. As the cell populations grow and the cells function normally, they begin to secrete their own extracellular matrix (ECM) support. The scaffold polymer is selected to degrade as the need for an artificial support diminishes. 22 Scaffold applications include the regeneration of tissues such as neuronal, musculoskeletal, cartilaginous, tendonous, hepatic, pancreatic, renal, ocular, arteriovenous, urinary or any other tissue forming solid or hollow organs. Some typical orthopedic applications include joint resurfacing, meniscus repair, non-union fracture repair, craniofacial reconstruction or repair of an invertebral disc.

In a certain embodiment of the present invention cells may be cultured on the matrix for subsequent implantation. Stem cells derived from any tissue or induced to differentiate into a specific tissue type may be utilized. Preferably the cells are derived from autologous tissue. For example, for culturing cartilage, chondrocytes or mesenchymal stem cells may be seeded on the matrix. In specific embodiments of the invention, chondrocytes or chondrocyte progenitor cells can be seeded on the matrix prior to implantation or at the site of implantation in vivo. Additionally, the cell of interest may be engineered to express a gene product which would exert a therapeutic effect, for example anti-inflammatory peptides or proteins, growth factors having angiogenic, chemotactic, osteogenic or proliferative effects. A non-limitative example of genetically engineering cells to enhance healing is disclosed in US 6,398,816.

According to certain embodiments of the invention, the plasma protein matrix is used as a support for chondrocyte growth and as a scaffold for neo cartilage formation. However, the plasma matrix of the invention may be used as a surface useful for tissue culture for any suitable cells, such as mesenchymal cells or other tissue forming cells at different levels of potency. For example, cells typically referred to as "stem cells" or "mesenchymal stem cells", are pluripotent, or lineage-uncommitted cells, which are potentially capable of an unlimited number of mitotic divisions to either renew a line or to produce progeny cells with the capacity to differentiate into any cell type can be grown on the matrix of the invention. In addition, lineage-committed "progenitor cells" can be grown on the matrix of the invention. A lineage-committed progenitor cell is generally considered to be incapable of an unlimited number of mitotic divisions and will eventually differentiate only into a specific cell type In yet further embodiments of the invention, the porous homogeneous fibrin matrix can be utilized as a coating of synthetic or other implants or medical devices. The matrix of the invention may be applied to prostheses such as pins or plates by coating or adhering methods known to persons skilled in the art. The matrix coating, 23 which is capable of supporting and facilitating cellular growth, can thus be useful in providing a favorable environment for the implant or prosthesis.

The glycosaminoglycan enriched homogeneous fibrin matrix of the invention is demonstrated as a support per se or as a scaffold for growing various cell types for implantation at a site of diseased or traumatized tissue. A person skilled in the art can adjust the procedures exemplified below in accordance with specific tissue requirements. In one non-limiting example, for cartilage repair the glycosaminoglycan enriched homogeneous fibrin matrix may be used in conjunction with other therapeutic procedures including chondral shaving, laser or abrasion chondroplasty, and drilling or microfracture techniques.

Preferably, the fibrin sponge is implanted as such, and serves as a scaffold for cellular growth in situ. Alternatively, the matrix is seeded with desired cells, the cells allowed to proliferate and the sponge comprising the cells implanted at a site in need of tissue repair or regeneration. The glycosaminoglycan enriched homogeneous fibrin matrix, in its dry form, adheres exceptionally well to tissue surfaces. According to one embodiment of the present invention a dry sponge of the invention, or another type of bioabsorbable matrix, is placed on the area where tissue regeneration is desired. A second sponge, onto which particular cells were cultured, is placed on top of the dry sponge. The wetted sponge of the invention adheres well to the dry sponge of the invention or another matrix. During the healing process, the cells from the sponge onto which the cells were originally seeded migrate into the matrix adhering directly to the area of tissue regeneration. This system obviates the need for biological glue in instances where a wetted sponge does not adhere well.

In the reconstruction of structural tissues like cartilage and bone, tissue shape is integral to function, requiring the molding of the matrix into three dimensional configuration articles of varying thickness and shape. Accordingly, the matrix of the invention may be formed to assume a specific shape including a sphere, cube, rod, tube or a sheet. The shape is determined by the shape of a mold, receptacle or support which may be made of any inert material and may be in contact with the matrix on all sides, as for a sphere or cube, or on a limited number of sides as for a sheet. The matrix may be shaped in the form of body organs or parts and constitute prostheses. Removing portions of the matrix with scissors, a scalpel, a laser beam or any other cutting instrument can create any refinements required in the three-dimensional structure.

The glycosaminoglycan enriched homogeneous matrix must be configured to provide both adequate sites for attachment and adequate diffusion of nutrients from the cell culture to maintain cell viability and growth until the matrix is implanted and vascularization has occurred. Cellular invasion is required by cells which can lay down the tissue to replace the implant and thus repair any defect which the implant is intended to repair.

The matrix according to further embodiments of the invention can be used as a matrix for growing cells or tissue culture in vitro. The matrices of the invention provide a relatively large surface area for cells to grow on and a mechanically improved scaffold for implantation.

The methods for seeding cells on the matrix are manifold. In a non-limiting example, the cells are adsorbed by placing the cells on the surface of the matrix or absorbed into the matrix by placing the sponge in a solution containing cells.

Preferably the matrix is seeded with the desired cells by surface seeding, at a density of 10 cells per cm , more preferably 10 cells per cm .

It will be appreciated that the matrix of the invention can support the growth and/or implantation of any type of cartilage or other suitable tissue. Furthermore, although the invention is directed predominantly to methods for growth and/or implantation of tissue in humans, the invention may also include methods for growth and/or implantation of tissues in any mammal.

Furthermore, the sponge of the present invention may be used as a component of a two-phase or multi-phase material for tissue repair such as seen in osteochondral defects. In a non-limiting example, one layer may comprise a calcium phosphate material whilst an additional layer may comprise the sponge of the invention. Gao et al. (Tissue Engin 8:827-837, 2002) describes a method for the repair of osteochondral defects in rabbit knees using a composite material comprising an injectable calcium phosphate and a hyaluronic acid sponge.

Method of Matrix Preparation The present invention provides a method for preparing a porous homogeneous fibrin matrix. The matrix forming solutions include a thrombin solution and a plasma protein solution. As used herein the thrombin solution comprises thrombin in an amount sufficient to cleave fibrinogen and yield a fibrin matrix in the presence of Ca+2 ions. The plasma proteins may derive from a commercial, allogeneic or autologous source and comprise fibrinogen and factor XIII, in the substantial absence of organic chelating agents. The at least one bioactive agent and glycosaminoglycan are added independently to either of the matrix forming solutions, i.e. the plasma proteins or to the thrombin solution, prior to the formation of the clot or are placed into the mold prior to, concurrently with or following addition of the thrombin.

In one currently preferred embodiment of the invention the porous homogeneous fibrin sponge is prepared by transferring the thrombin solution into a mold, adding the plasma protein solution; freezing the clotted mixture and lyophilizing. Alternatively, the plasma proteins are mixed with thrombin in the presence of Ca+2 ions under conditions suitable for achieving clotting; the mixture is cast or mold in a solid support prior to achieving clotting; the clotted mixture is frozen and lyophilized. It is to be understood that the bioactive agent and glycosaminoglycan are added independently to either of the matrix forming solutions, i.e. the plasma proteins or to the thrombin solution, prior to the formation of the clot or are placed into the mold prior to, concurrently with or following addition of the thrombin.

The final concentration of thrombin may be varied in order to produce sponges with distinct biological, physical and mechanical features useful for different applications. Use of a high concentration of thrombin, i.e. about 30 IU/ml (about 1.5 IU/mg total protein), yields a sponge with smaller pores and thicker fibers than use of thrombin at a low concentration, i.e. about 3 IU/ml (0.15 IU/mg total protein).

The addition of an anti-fibrinolytic agent to the plasma proteins is optional.

A method for preparing a porous homogeneous fibrin matrix useful as a scaffold for growing cells, and as a scaffold for implantation in vivo or in situ comprises the following steps: providing a thrombin solution and a plasma protein solution wherein at least one of the plasma protein solution or the thrombin solution contains at least one glycosaminoglycan and at least one of the plasma protein solution or the thrombin solution contains at least one bioactive agent; introducing the thrombin solution and the plasma protein solution to a solid receptacle or mold; incubating under conditions appropriate to achieve clotting; 26 freezing the clotted mixture; and lyophilizing the clotted mixture, to obtain a sponge.

According to another embodiment the matrix of the invention may be prepared by the above method further comprising the steps of: pre-mixing the plasma protein solution with the thrombin solution in the presence of calcium ions, at least glycosaminoglycan and at least one bioactive agent, under conditions suitable for achieving clotting; casting the mixture of plasma proteins and thrombin in a solid receptacle or mold prior to achieving clotting.

A method for preparing a porous homogeneous fibrin matrix useful for support of cell growth after implantation in situ, is provided comprising the steps of: providing a thrombin solution and a plasma protein solution wherein at least one of the plasma protein solution or the thrombin solution contains at least one glycosaminoglycan and at least one of the plasma protein solution or the thrombin solution contains at least one bioactive agent; introducing the thrombin solution and the plasma protein solution to a solid receptacle or mold; incubating under conditions appropriate to achieve clotting; freezing the clotted mixture; lyophilizing the clotted mixture, to obtain a sponge; cutting the sponge into sections of desired shape; and implanting the sections in situ.

According to one currently preferred embodiment at least 0.15 units of thrombin per mg total plasma protein comprising at least one ug (microgram) FGF is introduced to a solid support. Plasma proteins at a concentration of about 20-30 mg/ml are mixed with hyaluronic acid and the mixture added to the thrombin in the solid support to achieve formation of a clot. The clot is incubated at room temperature for 10 min to about 2 hours and is frozen at -70°C for approximately 16 hours and lyophilized for at least 16 hours, preferably 24 hours. 27 According to another currently preferred embodiment at least 0.15 to 15 units of thrombin per mg total plasma protein is introduced to a solid support. Plasma proteins at a concentration of about 20-30 mg/ml comprising a hyaluronic acid and platelets or platelet supernatant are mixed and the mixture added to the thrombin in the solid support to achieve formation of a clot. The clot is frozen at -70°C for approximately 16 hours and lyophilized for at least 16 hours, preferably 24 hours.

In its final form prior to use with cells the sponge is substantially dry and contains less than 15% residual moisture, more preferably less than 10% residual moisture and most preferably less than 5% residual moisture.

The following examples are intended to be merely illustrative in nature and to be construed in a non-limitative fashion.

EXAMPLES Example 1 : Isolation of Plasma Proteins from Whole Plasma Fresh frozen plasma was received from the blood bank (Tel-Hashomer, Israel). The plasma (220 ml) was thawed in a 4°C incubator over night, followed by centrifugation at 4°C at approximately 1900g for 30 min. The pellet was resuspended in 2.5ml PBS with gentle rolling until a homogenized solution was seen. An anti-fibrinolytic; and arginine (final 2%) are optionally added to the plasma protein fraction.

The total protein concentration was approximately 42-50 mg/ml as estimated by Bradford assay and SDS-PAGE (comparing to a standard).

Although detailed methods are given for the preparation of the plasma protein, it is to be understood that other methods of preparing plasma proteins are known in the art and are useful in the preparation of the matrix of the present invention. A non-limiting example of a protocol for the preparation of a fibrinogen-enriched solution is given by Sims, et al. (Plastic & Recon. Surg. 101 : 1580-85, 1998).

Example 2: Extraction of Plasma Protein Fractions from Allogeneic or Autologous Blood Materials: 1) Sodium citrate, 3.8 % or any other pharmaceutically acceptable anti-coagulant 2) Ammonium sulfate (NH4)2S04, saturated (500g/l) 3) Ammonium sulfate (NH4)2S04, 25% 28 4) Phosphate-EDTA buffer: 50 mM phosphate, 10 mM EDTA, pH 6.6 ) Tris-NaCl buffer: 50 mM Tris, 150 mM NaCl, pH 7.4 6) Ethanol, absolute 4°C 7) Whole blood (Israel Blood Bank, Tel Hashomer Hospital or from patient) Methods: One bag of blood from the blood bank contained 450 ml and contained sodium citrate. To the 450 ml autologous blood, 50 ml of a 3.8% sodium citrate solution was added and the solution was mixed gently.

The blood-sodium citrate was distributed to 50 ml tubes (40 ml/tube) and centrifuged at 2,100g for 20 min. The supernatant plasma was collected into 50 ml tubes and re-centrifuged at 5000g for 15 min. at 4°C. The supernatant plasma was collected into a flask, put on ice, and saturated ammonium sulfate solution was added at a ratio of one volume ammonium sulfate to 3 volumes of supernatant (1:3 volume ratio). A typical amount was 75 ml ammonium sulfate to 225 ml plasma. The solution was kept at 4°C for 1.5 hrs with occasional mild shaking (magnetic stirring is not allowed).

The supernatant plasma was divided into 50 ml tubes (40 ml/tube) and centrifuged at 5000g for 15 min at 4°C. The supernatant was discarded and each pellet washed with 10 ml 25% ammonium sulfate solution (pellet not dissolved).

Each pellet was dissolved in 6-7 ml of the phosphate-EDTA buffer. A sample, typically 100 μΐ of the solution, was kept for SDS-PAGE and clotting analyses. The dissolved pellets were pooled and the ammonium sulfate precipitation was repeated by adding saturated ammonium sulfate to the plasma sample to achieve a 1 :3 volume ratio (Typically, 25 ml ammonium sulfate to 75 ml plasma). The solution was kept at 4°C for 1.5 hrs with occasional mild shaking, divided into 50 ml tubes and centrifuged at 5000g for 15 min.

The supernatant was discarded and the pellets were dissolved in a volume of Tris-NaCl buffer that was equal to or less than the volume of phosphate-EDTA buffer used above. A typical total amount was about 45 ml.

The sample was dialyzed (SnakeSkin™ dialysis tubes, 3.5 kD cutoff, Pierce) for 3-4 hours or overnight at 4°C in 1.5 liters of Tris-NaCl buffer. The dialyzed sample 29 was centrifuged in high-speed resistant tubes at 21,000g for 15 min at 4°C to remove any insoluble material. The supernatant was collected and kept on ice.

The supernatant plasma was divided into 50 ml tubes. Chilled (EtOH) was added to a final concentration of 7%. (for example: 3.7 ml EtOH to 49 ml supernatant) and kept on ice for 30 min. It is essential that the solutions be chilled for the precipitate to form.

The solution was centrifuged at 5000g for 15 min, the supernatant discarded and the pellet dissolved in the same volume (typically amount 45 ml) Tris-NaCl buffer. The solution was dialyzed overnight at 4°C in 1.5 liter of Tris-NaCl Buffer. The dialyzed solution was centrifuged at 21,000, at 4°C for 15 min, to eliminate any non-dissolved material.

Protein concentrations were determined using the Bradford method (Bradford (1976) Anal. Biochem. 72:248-254). The protein yields ranged from 0.2 to 0.6 mg per ml of full blood, with typical results of 0.4 to 0.5 mg/ml.

Clot formation ability was determined by adding 30 μΐ thrombin (100 IU/ml; Omrix) to 70 μΐ plasma product (10 mg/ml), clotting should occur within 30 sec. Protein purity was determined by electrophoretic analysis of 50 μg of the sample on a 5% SDS-polyacrylamide gel and staining using Coommassie blue. The remainder of the supernatant was collected, frozen and lyophilized until dry, 48 hours.

A non-limiting method for the isolation of a platelet-enriched plasma is disclosed in US 6,475,175.

Example 3 : Matrix Preparation Method Materials and Methods: Source of plasma proteins e.g. as prepared in Example 2 or commercial Calcium Chloride 5 mM Thrombin (1000 International Units/ml, Omrix) Hyaluronic acid (Genzyme, Hylan, MW 6 x 106) FGF (^g/sponge) The concentration of thrombin determines the reaction time for the polymerization of the fibrin monomers and contributes to the pore size and fiber thickness of the final sponge. A concentration of 0.15 IU thrombin/mg plasma proteins yielded a sponge with good physical and biological properties. The concentration of 15 IU thrombin/mg plasma proteins and 1.5 IU thrombin/mg plasma proteins was chosen because it gave a fast reaction but allowed the two solutions enough time to mix thoroughly before the reaction completes, but other concentrations are acceptable for obtaining a matrix with substantially similar properties. For convenience, as used herein 1.5 IU thrombin/mg total protein is the equivalent of 30 IU thrombin/ml.

Sponges were made by mixing a plasma protein solution with a thrombin solution, casting, freezing and lyophilizing. Human plasma proteins, from different sources: allogeneic or autologous, with various levels of plasma proteins, were used having a protein concentration between 10-50 mg/ml. Commercial fibrinogen (Omrix) was tested, as well, at a concentration of 10-20mg/ml.

Thrombin ( 1000 IU/ml) was diluted 1 : 10, 1 : 100 or 1 : 1000 in a 5mM calcium chloride solution. The final sponge formulation for plasma proteins from a commercial source included tranexamic acid at a concentration of 5% or 10%. The above two solutions (plasma protein and thrombin) were mixed together in a ratio of 21 :9 respectively (for example 210 μΐ plasma protein and 90 μΐ thrombin solution), in the following order: A 48 well ELISA plate was coated with 90 μΐ of thrombin solution, and the plasma protein solution was added. The mixture was incubated at room temperature (~25°C) for 10 minutes or until the clot formed, followed by freezing at -70 °C overnight (- 16 h), and lyophilization under sterile conditions, -85 °C until dry for at least 16 hours and up to 24h. Note that the final concentration of thrombin was between 0.15 to 15 IU/mg plasma proteins.

Sponges having substantially similar biomechanical features and biocompatibility were obtained from plasma protein solutions isolated from the different sources, allogeneic or autologous blood, whole plasma or commercial fibrinogen. These features include pore size, surface adherence, ability to maintain cell growth and proliferation and biocompatibility. This results shows that for the preparation of a plasma protein sponge of the present invention, a range of methods for preparing the plasma protein and a range of protein concentrations may be utilized. 31 In a preferred embodiment of the present invention the matrix is prepared with certain additives including heparin, glycerol and hyaluronate (hyaluronic acid).

Mechanical and physical parameters were shown to be controlled by incorporating certain additives. All additives were filtered (0.2 μπι) and were added to the plasma protein solution. FGF (1 μg/sponge) was added either to the plasma protein solution or to the thrombin solution. A non-limiting sample list of the additives and concentrations tested are shown in the Table 1 below: Table 1 Experiments are carried out to determine the optimal concentration of the additives in terms of matrix flexibility, elasticity, pore size, sustained release of bioactive agents and cell growth capacity. The additives impart beneficial properties, including surface, mechanical and/or biological properties, to the sponge during its preparation. Optimization is carried out regarding the concentration of the bioactive agents as well. The preferred bioactive agents include growth factors, platelet supernatant, native platelets, platelet membranes and other materials. A currently most preferred embodiment in accordance with the present invention is a sponge comprising heparin and hyaluronic acid with an FGF.

Another currently preferred embodiment of the present invention provides a plasma protein sponge incorporating at least one additive and blood platelets or platelet supernatant. 32 Sponges comprising 0.024% or 0.08% final concentration hyaluronic acid and 1% or 10% final concentration platelet released supernatant or whole platelets were prepared. Platelet supernatant was made by exposing isolated platelets (obtained from the Israel blood bank) to thrombin as described (Gruber et al., Clin Oral Implants Res 13:529-535, 2002), collecting the supernatant and adding it to the plasma protein solution prior to sponge formation. Sponges comprising platelets were made by adding platelets directly to the plasma proteins in the following manner: 73 μΐ platelets and additive (hyaluronic acid to 0.024% or 0.08% final concentration) was added to plasma proteins (30 mg total protein/ml) and the solution brought to 210 μΐ final volume. The sponge was made as described herein.

Example 4: Matrix Morphology and Mechanical Properties In general, matrices for tissue engineering are characterized according to several criteria, including chemical nature, homogeneity, porosity, adhesion, biocompatibility and elasticity, amongst others (Hunziker, Osteoart. Cart., 10:432-465, 2002). Table II of the aforementioned reference lists several of the properties and the biological basis of these properties.

In the lab of the inventors, several of the properties have been measured.

Porosity, important for cell migration and adhesion was investigated by geometrical measurements using the light microscope by sectioning the matrix into thick specimens. Specimens were mounted on slides and were stained by hematoxylin/eosin. An optical micrometer measured the pore size and the distance between neighboring pores.

Scanning Electron Microscope (SEM) Analysis is performed in order to analyze homogeneity and ultrastructure of the matrix.

Moisture and residual moisture are measured using standard tests, known in the art. In its final form prior to use with cells the sponge is substantially dry and contains less than 15% residual moisture, more preferably less than 10% residual moisture and most preferably less than 5% residual moisture.

Mechanical property measurements are performed using a Chatillon TCD200 machine with a digital force gauge DF 12. Each plasma protein sponge is 2.5 cm long, 0.5 cm wide; and is fully lyophilized. 33 Deformation represents the elasticity of the sponge, i.e. the amount of pull as measured in millimeters (mm) that may be exerted until the sponge tears. Force is calculated in kPa and represents the amount of energy required to tear the sponge strips. The thickness is incorporated in to the calculation.

All sponges contain additives and bioactive agents and were prepared from commercially available fibrinogen (approximately 20 fibrinogen mg/ml) or plasma proteins (about 30 mg/ml total protein). Hyaluronic acid (Hylan, Genzyme, MW 6x 106), heparin (Sigma, MW 6,000) or glycerol were added according to Table 1 presented herein below. The sponges prepared from purified fibrinogen and those prepared from crude plasma proteins exhibit substantially identical mechanical properties. The sponges comprising both HA and heparin, and FGF exhibit good FGF release profiles. Each additive appears to impart certain properties to the sponge. Microscopic analysis is performed to determine the pore size and pore uniformity of the sponges comprising the different components. Procedure for preparing sponges with additives is presented herein below.

Example 5: Dissolution rate The rate of sponge dissolution measures the level of crosslinking that the plasma proteins have undergone. Certain storage conditions may enhance crosslinking..

Sponges are stored for one to six months under different conditions: a. Inert atmosphere N2 (g) b. Open air (standard atmosphere). c. Vacuum packaging Following the time allotted, the sponges were tested by dissolving the sponge in 0.75 ml 10M urea or a collagenase solution. A sample is removed every 10 minutes and the amount of protein in the urea or collagenase solution is determined by a standard Bradford assay.

Example 6; Resorbability Assay The capacity of the matrix to be resorbed in the body is attributable to its composition and level of crosslinking. Several assays, both in vivo and in vitro are known in the art to analyze resorbability of implantable composites. In vivo assays include intramuscular, subcutaneous and intraosseous models. 34 Example 7; Release of Bioactive Agents from Matrix One factor which facilitates the regeneration of tissue on the matrices is the delivery of growth factors or other biological agents into the milieu. The incorporation and release of growth factors from these matrices was assessed in vitro and may be assessed in vivo using radiolabeled or tagged growth factors, for example fluorescent-labeled, alkaline phosphatase labeled or horseradish peroxidase-labeled growth factor. The fraction and rate of released agent is measured by following the radioactivity, fluorescence, enzymatic activity or other attributes of the tag. Similarly, release of enzymes from the matrix is determined by analyzing enzymatic activity into the microenvironment in an in vitro or in vivo assay. Specifically, the release of an FGF from the matrix of the invention was performed as described herein: FDCP Assay: The FDCP cell line is a murine immortalized, interleukin 3-dependent cell line of myelocytic bone marrow origin that does not express endogenous FGF Receptors (FGFR). Upon transfection with FGFR cDNA, the FDCP cell line exhibited a dose-dependent proliferative response to FGF that can replace the dependence on IL-3. FGFR transfected FDCP cells can therefore been used to screen for FGFR signaling. FDCP cells response to various ligands is quantitated by a cell proliferation assay with XTT reagent (Cell Proliferation Kit, Biological Industries Co.). The method is based on the capability of mitochondrial enzymes to reduce tetrazolium salts into a colorigenic compound, which can be quantitated and is indicative of cell viability.

Specifically, FDCP cells stably expressing the FGFR1 (FDCP-FGFR1) were grown in "full medium" (Iscove's Medium containing 2ml glutamine, 10% FCS, 100μg/ml penicillin, 100μg/ml streptomycin) supplemented with 5μg/ml heparin.

Cells were split every 3 days and kept in culture no more than one month. One day prior to the experiment the cells were split. Before the experiment the cells were washed 3 times (1000 rpm, 6 min) with full medium. The cells were resuspended and counted with Trypan Blue. Twenty thousand (2 x 104) cells were added to each well of 96-well plate in 50 μΐ full medium with or without heparin. Conditioned medium from the sponges containing FGF or FGF complexed with the various glycosaminoglycans was added in an additional volume of 50 μΐ full medium to bring the final volume to 100 μΐ. The plate was incubated for 48 hours at 37°C. To test cell proliferation, 100 μΐ of PMS reagent was added to 5 ml of XTT reagent and mixed well (according to manufacture protocol). 50 μΐ of the latter solution were aliquoted into each well, and the plates incubated at 37°C for 4 hours and the color developed was read by a spectro-ELISA reader at ^onm- Figures 1 and 2 show the results of this assay for a sponge of the invention made of commercial fibrinogen (Omrix, 20 mg/ml) and either 15 IU or 1.5 IU thrombin/mg fibrinogen. The sponges comprised either 0.5, 1.5 or 2.5 μg/ml heparin and ^g total FGF2 variant (an Nl 11G substitution). Supernatant was tested after 1 and 5 days and results for proliferastion recorded. Figure 1 A shows the release of FGF2 variant from a sponge comprising both heparin and FGF2v. Figure IB shows the percent of total release after 1 and 5 days. Figures 2A and 2B show the release results from a sponge wherein the FGF and heparin were adsorbed, rather than incorporated ab initio. This experiment shows that in the sponges made with 15 IU/mg (left side of charts) show a different release profile than those made with 1.5 IU/mg (right side of charts). In addition, the release profile of FGF is dependent on the concentration of heparin in the sponge. Without wishing to be bound to a particular theory, the heparin may serve to stabilize the released FGF. The sponges which had the heparin and FGF adsorbed (figure 2A and B) show a poor release rate of FGF even after 5 days.

Figure 3 shows the rapid release of FGF from matrices of the invention comprising 0.024% hyaluronic acid and one μg FGF2 variant, incorporated into the matrix ab initio. The left side of the bar graph shows release from a sponge made using a commercial source of fibrinogen (Omrix, 20mg/ml) and the right side of the bar graph shows release from a plasma protein matrix (30 mg/ml total protein).

Example 8: Cell Seeding Different methods of seeding cells onto the sponge may be used. Important to seeding is cell adherence, migratory capacity and proliferation of cells within the matrix. Cells may be suspended in medium, PBS, or any biocompatible buffer alone or in the presence of bioactive agents. Cells may be seeded by placing a drop of liquid containing cells on the sponge and allowing the cells to adsorb into the sponge.

Alternatively, the cells in the liquid may be absorbed into the sponge by placing the sponge in a container holding a suspension of cells. 36 Materials and Methods: Cultured cells are prepared in growth medium (MEM) with or without additional 2 6 growth factors, and placed on top of a sponge at a density of between 10 -10 cells per standard 300μ1 plasma protein sponge (approximately 0.2 cm3) in a microtiter plate having 48 wells. Different volumes of growth media are added and the cells allowed to grow for various time periods. It is to be understood that the sponge of may be of varying sizes, shapes and thickness.

Following a three-day, 6-7 day and three week incubation for the seeded sponges, the sponges are sectioned and the cell invasion and proliferation observed. Cell proliferation is determined as described in Example 10.

Examples of cell growth in the fibrin sponge of the invention are shown in Figures 4 and 5. Figure 4 shows pig chondrocyte cell that have invaded a fibrin sponge made of commercial fibrinogen (Omrix, 20 mg/ml) comprising 0.08% hylan and 1 μg FGF2 variant (FGF2 having an Nl 11G substitution). The left figure ids a sagittal section, the right figures are cross sections. Note the infiltration of the chondrocytes into the sponge following a 6 day incubation.

Figures 5A-5D show pig chondrocytes (0.5 x 106 cells in 30 μΐ) that have been cultured (6 days) on a fibrin sponge made from pooled human plasma (30 mg/ml) comprising 0.024% Hylan and 1 μg FGF variant. Figures 5A and 5B show hematoxylin and eosin (H&E) staining (xlOO magnification). Figure 5C shows a 400x magnification of a sponge section stained with Masson's stain. Note the staining for cells and intracellular matrix surrounding the cells. Figure 5D shows a x200 magnification section of sponge stained with Masson's stain. Note the cells present within many of the pores.

Example 9: Cell Isolation and Culturing Reagents: Collagenase Type 2; Worthington Biochemical Corp. (Cat. #: 4147) Stock solution: 1700 units/ml in medium (in MEM) Minimal Essential Medium (MEM) Gibco BRL (cat: 21090-022) Fetal Bovine Serum (FBS); Gibco BRL (cat: 16000-044) L- Glutamine Solution; Gibco BRL (cat: 25030-024) 37 Complete medium: Minimal Essential Medium (MEM ) supplemented with 10% fetal calf serum (FCS), 2mM L-Glutamine and lOOU/ml penicillin, and 100μ§/πι1 streptomycin Preparation of Implants for Articular Cartilage The sponge of the present invention may be used as a cell bearing scaffold for tissue repair and regeneration. In one aspect, the cells are cultured on the sponge in vitro prior to implantation. In a preferred aspect, the sponge is seeded with cells immediately before implantation and the cells allowed to proliferate in vivo.

Cartilage biopsies from fresh pig cartilage were sectioned into small pieces, approximately of 3-4 mm thick, washed aseptically with PBS and placed in a new tube containing 3 ml MEM medium. The cartilage may be obtained from any vertebrate species, and is preferably allogeneic or autologous.

Collagenase type II was diluted 1 :5 and 1 ml was added to the cartilage pieces and the mixture was shaken gently in a 37 °C incubator over night. When most of the sample was digested, the suspension was poured through sterile gauze to remove matrix debris and undigested material. The filtrate was centrifuged and washed twice to remove residual enzyme.

The number of cells was determined by a hemocytometer and viability was determined by trypan blue exclusion. The cells were plated in 150 cm2 tissue culture flasks in 30 ml of culture medium at a concentration of 5x 106 cells/ml. Flasks were placed in a 37°C incubator at 5% C02 atmosphere and 95% humidity. The culture medium was changed every three to four days. The cells adhere and become confluent following one week incubation.

At confluence, the cell medium was removed and 3ml trypsin-EDTA solution were added. Thirty ml MEM+ FBS was added, the solution was centrifuged at 800g for 10 minutes. The supernatant was removed, the pellet dispersed and the cells were counted. To create a cell-bearing matrix, 102 - 106 cells were seeded on a plasma protein scaffold of 9mm in diameter and a thickness of 2mm (approximately 0.2 cm3). The matrices were placed in a 37 °C incubator for 1 hour and 1ml of fresh medium was added to each. The medium was replaced with fresh medium and every few days the matrices were taken to cell proliferation and differentiation analysis. 38 Furthermore, the cell population grown on the above matrices expresses several of the chondrocyte differentiation markers. One of several phenotypes expressed during chondrocyte differentiation is glycosaminoglycan (GAG) production. The production of GAGs iss identified in histological staining using Acian blue and quatitated using the DMB (3,3'-dimethoxybenzidine dihydrochloride) Dye method.

Example 10: Cell Proliferation Assay Proliferation of the cartilage cells on the matrix of the invention was quantitated by one of two methods, CyQUANT® (Molecular Probes) or XTT reagent (Biological Industries, Co.). The plasma protein matrix was dissolved in collagenase or other enzymes and the cells collected by centrifugation and subjected to analysis according to manufacturer's protocols.

In one experiment, human articular chondrocytes (104-106 cells/30-100 μΐ) are grown in the presence of the matrix of the invention and collagenase in microwell plates. The cells were grown overnight in MEM, 34 U collagenase was added and the cells or cells+sponge incubated for four hours. XTT reagent was added for 3-4 hours and the plates were read in an ELISA reader at A490 mm. Results are shown in Figure 6. As can be seen the proliferation rate of the cells was not impaired by the presence of the sponge nor by the addition of the collagenase.

Example 11: Goat Articular Cartilage Repair Model A comparative study to evaluate the use of implanted sponges in knee injuries in goats is performed.

The goals of the study are to compare the sponges of the invention to commercially available sponges and sponges without additives and or bioactive agents in terms of inflammatory response, ability to adhere to the injured surface, to test the capacity of the sponges to induce the growth of hyaline cartilage.

Materials and Methods: 6 goats are randomly assigned to one of the treatment groups.

In one particular experimental system the different sponges (matrices) tested include: 1 : glycosaminoglycan enriched fibrin sponge prepared with HyA and FGF 39 2: glycosaminoglycan enriched fibrin sponge prepared with HyA and heparin and FGF 3: glycosaminoglycan enriched fibrin sponge, no FGF 4: glycosaminoglycan enriched fibrin sponge prepared with HyA and FGF and a calcium phosphate layer : commercial sponge 6: sham operated Antibiotics: 2ml of amoxycillin is injected EM immediately before the procedure and once a day for 4 days after the procedure.

Anesthesia: Pre-medication: 0.2ml xylazine 2% per 25 kg body weight was administered EM.

Induction: Once the goats are dazed an I.V. venflon is inserted.

Formulation of 1ml valium (lmg) + 2ml Ketamine (lOOmg/ml) 1 ml/25 kg BW bolus was administered through the venflon. An additional 0.5ml can be added as needed. For procedures longer than 30 minutes, intubations and gas (Haloten, or isofloran) anesthesia was administered.

Procedures for performing the wound: Animals are shaved in both knees and washed with disinfectant. Knees are exposed on the lateral side and the patella dislocated laterally and the cartilage exposed.

Four partial thickness defects (6mm) are created in the joint surface of the femoral condyles of the knee using a scalpel without penetrating the sub-chondral bone. The defect area is washed using saline. In another experimental procedure , full thickness osteochondral defects are created.

Procedure for transplanting the sponge: The lyophilized matrices are implanted and adhered to the defect. Optionally a fibrin-based biological glue may be used. The patella is relocated and the sinovia and skin closed using Vicryll sutures. The skin is cleaned with an iodine ointment.

Analgesia: 0.05mg/kg buprenorphine is injected SC (subcutaneous) just before the procedure and at the end of the same day Follow up and histological evaluation: At the end of the follow up period (6 or 10 weeks) goats are sacrificed and tissue taken for histological evaluation: Knee's are 40 evaluated for mobility, and appearance. Histological sections are prepared from the injured area and from 2-3mm margins. The tissue is de-calcified and slides prepared in a standard procedure.

Slides are stained with hematoxylin-eosin and alcian-blue staining.

Immunohistological analyses are performed, including immunohistochemistry using antibodies to cell type markers or in situ RNA hybridization using RNA or DNA probes.

Histological evaluation is performed to measure the following parameters: Characteristics of the neo-formed tissue, regularity of the joint surface of the regenerated tissue, structural integrity and thickness of the regenerated tissue, endochondral ossification and state of the cells in the remaining cartilage.

Example 12: One-Step Procedure for Treating Damaged Cartilage: Suitable For Arthroscopy or Hemi-Arthrotomy Autologous chondrocyte implantation has proven clinically effective in restoring hyaline-like cartilage to isolated chondral defects of the knee. The present therapies include three major steps: 1. Diagnostic Arthroscopy and biopsy of healthy cartilage. 2. Cultivation of cells. 3. Injection of cultured chondrocytes into the lesion under a periosteal flap, which is taken from the tibia and sutured over the lesion.

A variation of this technique provides implant of the matrix of the present invention. A less traumatic method is presented herein, wherein the patient undergoes a single surgical procedure for cartilage repair.

Procedure: A patient with a cartilage defect is called to the physician's office for a consultation several days prior to the arthroscopy or hemi-arthrotomy. Blood (approximately 100-250 ml) is drawn and plasma proteins and platelets are isolated. A plasma protein matrix, or several matrices, is prepared, labeled and stored aseptically until the day of the surgery.

Optionally, on the day of the surgery, cartilage from the patient's joint is removed, cut into small pieces and placed in a test tube containing collagenase, hyaluronidase or other cartilage degrading enzymes, or combinations thereof. 41 In the meantime, the surgeon will treat the defective region of the joint by removing damaged tissue, cleansing and preparing the area for an implant. The prepared matrix is removed from its container and cut to fit the defective domain. Optionally, following approximately 20-30 minutes of enzymatic treatment, the cells and small pieces of cartilage are spun down in a tabletop centrifuge, rinsed in PBS and resuspended in a small amount (50μ1-1000μ1) of PBS. The surgeon seeds the cells onto the sponge, in situ. Alternatively the cells are absorbed into the sponge and the cell-bearing sponge implanted into the defective joint region. Optionally, extracellular matrix degrading enzymes and or other bioactive agents including growth factors and/or anti-inflammatory compounds are added to the sponge. In certain instances the surgeon will place a dry sponge directly onto the injured area, optionally add enzyme solution to said sponge and place a second, cell-bearing sponge on top of the first sponge. The joint is closed and is treated as customary for an arthroscopic or hemi-arthrotomy procedure and the patient is released to recover.

Kit A kit comprising the components useful for practicing the method of the invention, will allow for the convenient practice of the method of the invention in a surgical setting. In a preferred embodiment, a kit of the invention will provide sterile components suitable for easy use in the surgical environment including, sterile solutions (saline, enzymes) a sterile, cell-free matrix material suitable for supporting autologous chondrocytes that are to be implanted into an articular joint surface defect and instructions for use. Although the matrix may be of any material that is biocompatible, non-immunogenic and has the ability to maintain cell growth and proliferation, the matrix is preferably prepared from allogeneic plasma, more preferably from autologous plasma.

Example 13; Bone Fracture Repair Model Suitable animal models are used to create bilateral osteotomies to demonstrate the efficacy of the novel variants of the present invention. In a rabbit model a 4-6 mm osteotomy is created in New Zealand Rabbits in compliance with the Animal Care Committee of the Hebrew University. The ulna was chosen because it is only slightly weight-bearing and allows the creation of a bone defect without requiring a cast or other immobilization treatment. In addition, this gap constitutes a spontaneously 42 healing defect that allows the evaluation of the tested agent. The primary indices of fracture healing are accelerated duration of healing and callus formation. The test compounds consist of a matrices of the invention and control matrices.

Surgery procedure: Animals are anesthetized according to standard protocol. Gap formation is performed in the mid ulna bone. A sponge of the invention is placed into the gap area in each limb and the fracture is closed. Animals are treated with analgesic for 3 days post operation. The duration of the experiment is 6 weeks.

Healing time and quality asessment: X-ray grading provides fracture healing status assessment. Rabbits are X-rayed every other week for 5 weeks after surgery. X-rays are scored by two orthopedic surgeons in a blinded manner according to a standard grading scale protocol.

Quality evaluation: at the end of the experiment rabbits are sacrificed and fracture area is sent for histological and mechanical strength evaluation. Histology is scored by a pathologist for evaluation of histological changes during the healing process using standard staining methods, using hematoxylin and eosin for cytoplasm and nucleus. Indigo-carmin staining is also applied for detection of newly generated callus. Mechanical strength evaluation is performed using the "4 points bending" method.

The treatments groups are: sham osteotomy, osteotomy treated with fibrin sponge alone, osteotomy treated with glycosaminoglycan enriched sponge alone, osteotomy treated with glycosaminoglycan enriched sponge containing FGF-2 or FGF-9 and osteotomy treated with sponge containing FGF-2 or FGF-9 variant.

Another example of an animal model for bone repair is presented in Cook et al., (Am J. Vet Res 64:2-20, 2003).

While the present invention has been particularly described, persons skilled in the art will appreciate that many variations and modifications can be made. Therefore, the invention is not to be construed as restricted to the particularly described embodiments, rather the scope, spirit and concept of the invention will be more readily understood by reference to the claims which follow. 43

Claims (9)

1. A porous homogeneous freeze-dried fibrin matrix comprising plasma proteins, the plasma proteins comprising fibrinogen, thrombin and Factor ΧΓΠ substantially devoid of organic chelating agents, further comprising at least one glycosaminoglycan and at least one bioactive agent and having residual moisture below 5%.

2. The porous homogeneous fibrin matrix according to claim 1 wherein at least one of the plasma proteins is autologous.

3. The porous homogeneous fibrin matrix according to claim 1 wherein all the plasma proteins are autologous.

4. The porous homogeneous fibrin matrix according to claim 1 wherein the at least one glycosaminoglycan is selected from a group consisting of hyaluronic acid, heparin, heparan sulfate, chondroitin sulfate, dextran sulfate, dermatan sulfate, keratan sulfate and derivatives or mimetics thereof.

5. The porous homogeneous fibrin matrix according to claim 4 wherein the glycosaminoglycan is crosslinked or uncrosslinked hyaluronic acid.

6. The porous homogeneous fibrin matrix according to claim 4 wherein the glycosaminoglycan is heparin or a derivative or mimetic thereof.

7. The porous homogeneous fibrin matrix according to claim 1 wherein the at least one bioactive agent is selected from the group consisting of growth factors, cytokines, platelets, platelet supernatant and platelet derived proteins, hormones, analgesics, anti-inflammatory agents, anti-microbials or enzymes.

8. The porous homogeneous fibrin matrix according to claim 7 wherein the at least one bioactive agent is a growth factor.

9. The matrix according to claim 8 wherein the growth factor is a fibroblast growth factor or variant thereof. 44 The porous homogeneous fibrin matrix according to claim 1 wherein the at least one bioactive agent is a fibroblast growth factor or variant thereof and the at least one glycosaminoglycan is crosslinked or uncrosslinked hyaluronic acid. The porous homogeneous fibrin matrix according to claim 1 wherein the at least one bioactive agent is a fibroblast growth factor or variant thereof and the at least one glycosaminoglycan is heparin or a derivative or mimetic thereof. The porous homogeneous fibrin matrix according to claim 7 wherein the at least one bioactive agent is platelets or platelet supernatant. The porous homogeneous fibrin matrix according to claim 12 wherein the platelets or platelet supernatant comprise at least 0.1% of the matrix. The porous homogeneous fibrin matrix according to claim 1 further comprising an exogenous anti-fibrinolytic agent. The porous homogeneous fibrin matrix according to claim 14 wherein the anti-fibrinolytic agent is tranexamic acid. The porous homogeneous fibrin matrix according to claim 15 comprising at least 5% tranexamic acid. The porous homogeneous fibrin matrix according to claim 1 wherein the thrombin is at least 0.5 international units of thrombin per mg protein. The porous homogeneous fibrin matrix according to claim 1 wherein the thrombin is at least 0.05 international units of thrombin per mg protein. The porous homogeneous fibrin matrix according to claim 1 further comprising cells. The porous homogeneous fibrin matrix according to claim 19 wherein the cells are stem cells or progenitor cells. The porous homogeneous fibrin matrix according to claim 20 wherein the cells are selected from chondrocytes, osteoblasts, hepatocytes, mesenchymal, epithelial, urothelial, neuronal, pancreatic, renal or ocular cell types. 45 22. The porous homogeneous fibrin matrix according to claim 1 having pores in the size range of 50-500 microns. 23. The porous homogeneous fibrin matrix according to claim 22 wherein the pores are in the size range of 100-400 microns. 24. A porous homogeneous freeze-dried fibrin matrix comprising plasma proteins, the plasma proteins comprising fibrinogen, thrombin and Factor XIII substantially devoid of organic chelating agents, further comprising at least one glycosaminoglycan and at least one bioactive agent and having residual moisture below 5% useful for implantation. 25. The porous homogeneous fibrin matrix according to claim 24 wherein at least one of the plasma proteins is autologous. 26. The porous homogeneous fibrin matrix according to claim 24 wherein all the plasma proteins are autologous. 27. The porous homogeneous fibrin matrix according to claim 24 wherein the at least one glycosaminoglycan is selected from a group consisting of hyaluronic acid, heparin, heparan sulfate, chondroitin sulfate, dextran sulfate, dermatan sulfate, keratan sulfate and derivatives or mimetics thereof. 28. The porous homogeneous fibrin matrix according to claim 27 wherein the glycosaminoglycan is crosslinked or uncrosslinked hyaluronic acid. 29. The porous homogeneous fibrin matrix according to claim 27 wherein the glycosaminoglycan is heparin or a derivative or mimetic thereof. 30. The porous homogeneous fibrin matrix according to claim 24 wherein the at least one bioactive agent is selected from a group consisting of growth factors, cytokines, platelets, platelet supernatant and platelet derived proteins, hormones, analgesics, anti-inflammatory agents, anti-microbials or enzymes. 31. The porous homogeneous fibrin matrix according to claim 30 wherein the at least one bioactive agent is a growth factor. 46 32. The porous homogeneous fibrin matrix according to claim 31 wherein the growth factor is a fibroblast growth factor or variant thereof. 33. The porous homogeneous fibrin matrix according to claim 24 wherein the at least one bioactive agent is a fibroblast growth factor or variant thereof and the at least one glycosaminoglycan is crosslinked or uncrosslinked hyaluronic acid. 34. The porous homogeneous fibrin matrix according to claim 24 wherein the at least one bioactive agent is a fibroblast growth factor or variant thereof and the at least one glycosaminoglycan is heparin or a derivative or mimetic thereof. 35. The porous homogeneous fibrin matrix according to claim 30 wherein the at least one bioactive agent is platelets or platelet supernatant. 36. The porous homogeneous fibrin matrix according to claim 35 wherein the platelets or platelet supernatant comprise at least 0.1% of the matrix. 37. The porous homogeneous fibrin matrix according to claim 24 further comprising an exogenous anti-fibrinolytic agent. 38. The porous homogeneous fibrin matrix according to claim 37 wherein the anti- fibrinolytic agent is tranexamic acid. 39. The porous homogeneous fibrin matrix according to claim 38 comprising at least 5% tranexamic acid. 40. The porous homogeneous fibrin matrix according to claim 24 wherein the thrombin is at least 0.5 international units of thrombin per mg protein. 41. The porous homogeneous fibrin matrix according to claim 24 wherein the thrombin is at least 0.05 international units of thrombin per mg protein. 42. The porous homogeneous fibrin matrix according to claim 24 further comprising cells. 43. The porous homogeneous fibrin matrix according to claim 42 wherein the cells are stem cells or progenitor cells. 47 44. The porous homogeneous fibrin matrix according to claim 43 wherein the cells are selected from chondrocytes, osteoblasts, hepatocytes, mesenchymal, epithelial, urothelial, neuronal, pancreatic, renal or ocular cell types. 45. The porous homogeneous fibrin matrix according to claim 24 having pores in the size range of 50-500 microns. 46. The porous homogeneous fibrin matrix according to claim 45 wherein the pores are in the size range of 100-400 microns. 47. A method for preparing a porous homogeneous fibrin matrix useful as a scaffold for growing cells the method comprising the steps of: providing a thrombin solution and a plasma protein solution wherein at least one of the plasma protein solution or the thrombin solution contains at least one glycosaminoglycan and at least one of the plasma protein solution or the thrombin solution contains at least one bioactive agent; introducing the thrombin solution and the plasma protein solution into a solid receptacle or mold; incubating under conditions appropriate to achieve clotting; freezing the clotted mixture; and lyophilizing the clotted mixture, to obtain a sponge. 48. The method according to claim 47 wherein at least one of the plasma proteins is autologous. 49. The method according to claim 47 wherein all the plasma proteins are autologous. 50. The method according to claim 47 wherein the at least one glycosaminoglycan is selected from a group consisting of hyaluronic acid, heparin, heparan sulfate, chondroitin sulfate, dextran sulfate, dermatan sulfate, keratan sulfate and derivatives or mimetics thereof. 48 51. The method according to claim 50 wherein the at least one glycosaminoglycan is crosslinked or uncrosslinked hyaluronic acid. 52. The method according to claim 50 wherein the glycosaminoglycan is heparin or a derivative or mimetic thereof. 53. The method according to claim 47 wherein the at least one bioactive agent is selected from the group consisting of growth factors, cytokines, platelets, platelet supernatant and platelet derived proteins, hormones, analgesics, antiinflammatory agents, anti-microbials or enzymes. 54. The method according to claim 53 wherein the at least one bioactive agent is a growth factor. 55. The method according to claim 54 wherein the growth factor is a fibroblast growth factor or variant thereof. 56. The method according to claim 47 wherein the at least one bioactive agent is a fibroblast growth factor or variant thereof and the at least one glycosaminoglycan is crosslinked or uncrosslinked hyaluronic acid. 57. The method according to claim 47 wherein the at least one bioactive agent is a fibroblast growth factor or variant thereof and the at least one glycosaminoglycan is heparin or a derivative or mimetic thereof. 58. The method according to claim 53 wherein the at least one bioactive agent is platelets or platelet supernatant. 59. The method according to claim 58 wherein the platelets or platelet supernatant comprise at least 0.1% of the matrix. 60. The method according to claim 47 further comprising an exogenous anti- fibrinolytic agent. 61. The method according to claim 60 wherein the anti-fibrino lytic agent is tranexamic acid. 62. The method according to claim 61 comprising at least 1% tranexamic acid. 49 63. The method according to claim 47 wherein the thrombin is at least 0.5 international units of thrombin per mg protein. 64. The method according to claim 47 wherein the thrombin is at least 0.05 international units of thrombin per mg protein. 65. The method according to claim 47 further comprising premixing the thrombin solution and the plasma protein solution. 66. A method for preparing a porous scaffold useful for implantation in vivo, comprising the steps of: providing a thrombin solution and a plasma protein solution wherein at least one of the plasma protein solution or the thrombin solution contains at least one glycosaminoglycan and at least one of the plasma protein solution or the thrombin solution contains at least one bioactive agent; introducing the thrombin solution and the plasma protein solution into a solid receptacle or mold; incubating under conditions appropriate to achieve clotting; freezing the clotted mixture; and lyophilizing the clotted mixture, to obtain a sponge. 67. The method according to claim 66 wherein at least one of the plasma proteins is autologous. 68. The method according to claim 66 wherein all the plasma proteins are autologous. 69. The method according to claim 66 wherein the at least one glycosaminoglycan is selected from a group consisting of hyaluronic acid, heparin, heparan sulfate, chondroitin sulfate, dextran sulfate, dermatan sulfate, keratan sulfate and derivatives or mimetics thereof. 70. The method according to claim 69 wherein the at least one glycosaminoglycan is crosslinked or uncrosslinked hyaluronic acid. 50 71. The method according to claim 69 wherein the glycosaminoglycan is heparin or a derivative or mimetic thereof. 72. The method according to claim 66 wherein the at least one bioactive agent is selected from the group consisting of growth factors, cytokines, platelets, platelet supernatant and platelet derived proteins, hormones, analgesics, antiinflammatory agents, anti-microbials or enzymes. 73. The method according to claim 72 wherein the at least one bioactive agent is a growth factor. 74. The method according to claim 73 wherein the growth factor is a fibroblast growth factor or variant thereof. 75. The method according to claim 66 wherein the at least one bioactive agent is a fibroblast growth factor or variant thereof and the at least one glycosaminoglycan is crosslinked or uncrosslinked hyaluronic acid. 76. The method according to claim 66 wherein the at least one bioactive agent is a fibroblast growth factor or variant thereof and the at least one glycosaminoglycan is heparin or a derivative or mimetic thereof. 77. The method according to claim 72 wherein the at least one bioactive agent is platelets or platelet supernatant. 78. The method according to claim 77 wherein the platelets or platelet supernatant comprise at least 0.1% of the matrix. 79. The method according to claim 66 further comprising an exogenous anti- fibrinolytic agent. 80. The method according to claim 79 wherein the anti-fibrinolytic agent is tranexamic acid. 81. The method according to claim 80 comprising at least 1% tranexamic acid. 82. The method according to claim 66 wherein the thrombin is at least 0.5 international units of thrombin per mg protein. 51 83. The method according to claim 66 wherein the thrombin is at least 0.05 international units of thrombin per mg protein. 84. The method according to claim 66 further comprising premixing the thrombin solution and the plasma protein solution. 85. A method for preparing a porous homogeneous fibrin matrix useful for support of cell growth after implantation in situ, comprising the steps of: providing a thrombin solution and a plasma protein solution wherein at least one of the plasma protein solution or the thrombin solution contains at least one glycosaminoglycan and at least one of the plasma protein solution or the thrombin solution contains at least one bioactive agent; introducing the thrombin solution and the plasma protein solution into a solid receptacle or mold; incubating under conditions appropriate to achieve clotting; freezing the clotted mixture; lyophilizing the clotted mixture, to obtain a sponge; cutting the sponge into sections of desired shape. 86. The method according to claim 85 wherein at least one of the plasma proteins is autologous. 87. The method according to claim 85 wherein all the plasma proteins are autologous. 88. The method according to claim 85 wherein the at least one glycosaminoglycan is selected from a group consisting of hyaluronic acid, heparin, heparan sulfate, chondroitin sulfate, dextran sulfate, dermatan sulfate, keratan sulfate and derivatives or mimetics thereof. 52 89. The method according to claim 88 wherein the at least one glycosaminoglycan is crosslinked or uncrosslinked hyaluronic acid. 90. The method according to claim 88 wherein the glycosaminoglycan is heparin or a derivative or mimetic thereof. 91. The method according to claim 85 wherein the at least one bioactive agent is selected from the group consisting of growth factors, cytokines, platelets, platelet supernatant and platelet derived proteins, hormones, analgesics, antiinflammatory agents, anti-microbials or enzymes. 92. The method according to claim 91 wherein the at least one bioactive agent is a growth factor. 93. The method according to claim 92 wherein the growth factor is a fibroblast growth factor or variant thereof. 94. The method according to claim 85 wherein the at least one bioactive agent is a fibroblast growth factor or variant thereof and the at least one glycosaminoglycan is crosslinked or uncrosslinked hyaluronic acid. 95. The method according to claim 85 wherein the at least one bioactive agent is a fibroblast growth factor or variant thereof and the at least one glycosaminoglycan is heparin or a derivative or mimetic thereof. 96. The method according to claim 91 wherein the at least one bioactive agent is platelets or platelet supernatant. 97. The method according to claim 96 wherein the platelets or platelet supernatant comprise at least 0.1% of the matrix. 98. The method according to claim 85 further comprising an exogenous anti- fibrinolytic agent. 99. The method according to claim 98 wherein the anti-fibrinolytic agent is tranexamic acid. 100. The method according to claim 99 comprising at least 1% tranexamic acid. 53 101. The method according to claim 85 wherein the thrombin is at least 0.5 international units of thrombin per mg protein. 102. The method according to claim 85 wherein the thrombin is at least 0.05 international units of thrombin per mg protein. 103. The method according to claim 85 further comprising premixing the thrombin solution and the plasma protein solution. 104. The method according to claim 85 further comprising cells. 105. The method according to claim 104 wherein the cells are stem cells or progenitor cells. 106. The method according to claim 105 wherein the cells are selected from chondrocytes, osteoblasts, hepatocytes, mesenchymal, epithelial, urothelial, neuronal, pancreatic, renal or ocular cell types. 107. An implant prepared according to any one of claims 47, 66, or 85. 108. The implant according to claim 107 for use in treating injured tissue. 109. The implant according to claim 108 wherein the tissue is skeletal tissue. 1 10. The implant according to claim 109 wherein the skeletal tissue is cartilage. 111. A porous coating for an implant comprising the matrix according to any one of claims 47, 66 or 85. 54