The present invention relates to porous freeze-dried fibrin matrices substantially devoid of external anti-fibrinolytic agents, and methods of producing such matrices. Resilient matrices, also known as sponges, that are particularly beneficial for supporting three dimensional cell growth are obtained from plasma proteins substantially devoid of plasminogen or from partially purified plasma proteins, thus obviating the need for exogenous anti-fibrinolytic agents. Furthermore, incorporation of glycosaminoglycans and bioactive agents during the formation of the matrix results in a sponge having advantageous biological, mechanical and physical properties. The compositions of the present invention are useful clinically, without cells or as a cell bearing implants.

Description of the Invention

SUMMARY OF THE INVENTION

The present invention relates to biomatrices substantially devoid of external anti-fibrinolytic agents, which have been shown to be deleterious to cells and tissue and which may induce adverse reactions in patients. It is now disclosed for the first time that resilient, non-brittle, matrices, also known as sponges, that are particularly beneficial for supporting three dimensional cell growth may be obtained from plasma proteins substantially devoid of plasminogen, thus obviating the need for external anti-fibrinolytic agents. It is further disclosed that unexpectedly biomatrices obtained from partially purified plasma proteins also obviates the need for exogenous anti-fibrinolytic agents. The compositions and methods of the present invention are effective for in vivo and in vitro applications including as biocompatible implants for tissue engineering as well as in biotechnology for the in vitro culturing and differentiation of cells. The matrices according to the present invention are three-dimensional (3D) and may be used clinically, per se or as cell-bearing implants. The present invention provides all components fundamental for tissue repair, thus facilitating the medical practitioner's task and providing a superior alternative for tissue reparation and regeneration in a patient.

The present invention provides a porous, freeze-dried fibrin matrix formed from plasma proteins comprising fibrinogen, thrombin and Factor XIII, the matrix having less than 10% residual moisture and being substantially devoid of exogenous anti-fibrinolytic agents and of organic chelating agents, which exhibits superior characteristics. The present invention is based in part on the unexpected finding that a matrix comprising plasma proteins substantially devoid of exogenous anti-fibrinolytic agents and plasminogen imparts superior stability and significantly improves cell seeding and cell dispersion while retaining other positive attributes of these matrices. The plasminogen-free matrices in particular exhibit reduced resorbability compared to fibrin matrices-known in the art, providing a long lasting implant with enhanced stability and endurance for successful tissue growth, repair and regeneration.

In one aspect, the present invention relates to a porous freeze-dried fibrin matrix formed from plasma proteins substantially devoid of exogenous anti-fibrinolytic agents and of organic chelating agents. According to one embodiment the plasma proteins are purified from a plasma source or may be used from a commercially available source, including native or recombinant proteins, in the substantial absence of exogenous anti-fibrinolytic agents and of organic chelating agents. According to another embodiment the plasma protein source is selected from total blood, blood fractions, blood derivative, cryoprecipitate, recombinant proteins, plasma and plasma fractions. The plasma proteins may be selected from xenogeneic, allogeneic and autologous plasma sources. According to one embodiment the plasma source is autologous.

In another aspect, the present invention provides a porous freeze-dried fibrin matrix formed from plasma proteins comprising fibrinogen, thrombin and Factor XIII, the matrix having less than 10% residual moisture and being substantially devoid of exogenous anti-fibrinolytic agents, plasminogen and of organic chelating agents. In one embodiment substantially devoid of plasminogen refers to the plasma protein solution comprising less than about 20% of plasminogen normally present in blood plasma, preferably less than about 10% of the plasminogen normally present in plasma and more preferably less than about 5% of the plasminogen normally present in plasma. The inventors have discovered that a porous freeze-dried fibrin matrix comprising plasma proteins substantially devoid of plasminogen provides a superior matrix for clinical and biotechnological applications. In addition to eliminating the need for exogenous anti-fibrinolytic agents and their concomitant detrimental effects, the inventors now show that the fibrin matrix of the present invention is superior as a scaffold for cell seeding, growth and differentiation and for use in tissue repair and regeneration.

The fibrin matrix of the invention may be used per se, comprising plasma proteins substantially devoid of exogenous anti-fibrinolytic agents and of organic chelating agents, for clinical and biotechnological applications. It may however, further comprise additives that impart additional advantageous biological, physical and mechanical characteristics to the matrix. The present invention encompasses the incorporation into the matrix of at least one additive to provide a matrix having improved biological, mechanical and/or physical properties.

Copending international patent application WO 03/007873 by some of the applicants of the present invention discloses a fibrin matrix comprising plasma proteins and at least one anti-fibrinolytic agent, optionally further comprising agents such as polysaccharides, anionic polysaccharides, glycosaminoglycans, or synthetic polymers added in the preparation to improve certain physical, mechanical and biological properties of the matrix. The requirement for an anti-fibrinolytic agent has now been removed or overcome by the substantial absence of plasminogen in the matrix.

In one embodiment, the present invention is related to a porous fibrin matrix substantially devoid of exogenous anti-fibrinolytic agents, plasminogen and of organic chelating agents further comprising at least one additive selected from the group consisting of polysaccharides, glycosaminoglycans (GAGs) and synthetic polymers that is useful as a support for growth and differentiation of cells, both in vitro and in vivo.

According to one embodiment the additive may be added ab initio, i.e., during formation of the clot. According to alternative embodiments the additive is introduced to the matrix any time following formation of the matrix. According to various embodiments of the present invention, the matrix is prepared using at least one glycosaminoglycan selected from the group consisting of crosslinked hyaluronic acid, non-crosslinked hyaluronic acid, heparin and heparin derivatives and heparin mimetics, chondroitin sulfate, dextran sulfate, dermatan sulfate, heparan sulfate and keratan sulfate.

The glycosaminoglycan is added to the matrix at a final concentration that imparts suppleness and elasticity to the matrix and precludes the need for adjusting the moisture content of the final composition. According to one embodiment of the present invention the glycosaminoglycan is selected from crosslinked and non-crosslinked hyaluronic acid. In one embodiment the concentration of non-crosslinked hyaluronic acid is about 0.005% to about 0.5% final (V/V) more preferably about 0.05% to about 0.1%. In another embodiment the concentration of crosslinked hyaluronic acid is about 0.001% to about 0.1% and more preferably about 0.05% to about 0.09% final (V/V). According to another embodiment the glycosaminoglycan is selected from heparin and heparin derivatives.

The present invention further encompasses a fibrin matrix comprising at least one bioactive agent selected from the group consisting of therapeutic proteins, platelets and platelet supernatant, analgesics, anti-microbial or anti-inflammatory agents and enzymes.

According to one embodiment the present invention provides a freeze-dried porous matrix comprising plasma proteins substantially devoid of exogenous anti-fibrinolytic agents, plasminogen and of organic chelating agents, further comprising at least one glycosaminoglycan and at least one bioactive agent.

According to another 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. In one aspect, the growth factor is selected from a fibroblast growth factor (FGF) and variants thereof. In another aspect, the FGF is an FGF having the capacity to induce or enhance cartilage and bone repair and regeneration and or angiogenesis. The growth factors may be incorporated at a wide range of concentrations, depending on the potency of the factor and the intended application.

For certain applications, sustained or phasic release of a bioactive agent may be preferred. In one embodiment, the at least one growth factor is incorporated in to the fibrin matrix directly, ab initio. In another embodiment, the at least one growth factor is bound to a carrier molecule such as heparin and is incorporated into the matrix ab initio. Sustained release of a bioactive agent depends on several factors including growth factor concentration, type of glycosaminoglycan incorporated and fibrin and thrombin concentration.

In contrast to the bioabsorbable heteromorphic sponge of the art, the present inventors now disclose a freeze-dried fibrin sponge compromising plasma proteins substantially devoid of exogenous anti-fibrinolytic agents, plasminogen and of organic chelating agents optionally comprising at least one additive selected from the group consisting of polysaccharides, glycosaminoglycans and synthetic polymers and optionally further comprising at least one bioactive agent.

According to one embodiment, the present invention provides a porous freeze-dried fibrin matrix comprising plasma proteins substantially devoid of exogenous anti-fibrinolytic agents, plasminogen and of organic chelating agents, further comprising at least one glycosaminoglycan and at least one bioactive agent. According to another embodiment of the invention the at least one glycosaminoglycan is selected from heparin and derivatives thereof, the at least one bioactive agent is a therapeutic protein selected from the FGF family of growth factors and variants thereof. This sponge provides phasic release of the FGF from the matrix and may be beneficial in certain therapeutic applications.

According to another embodiment the present invention provides a porous freeze-dried fibrin matrix comprising plasma proteins substantially devoid of exogenous anti-fibrinolytic agents, plasminogen and of organic chelating agents further comprising hyaluronic acid, heparin and at least one bioactive agent. The hyaluronic acid is selected from crosslinked and non-crosslinked hyaluronic acid. Preferably, the hyaluronic acid and the heparin or heparin derivative are incorporated into the sponge ab initio. The bioactive agent such as a growth factor may be incorporated into the sponge per se or heparin bound. Preferably the growth factor is selected from the family of FGF therapeutic molecules.

Another aspect of the invention provides a method of preparing the porous fibrin matrix. A method for preparing a porous freeze-dried fibrin matrix having less than 10% residual moisture and being substantially devoid of exogenous anti-fibrinolytic agents and of organic chelating agents comprises the following steps: providing a thrombin solution and a plasma protein solution wherein the plasma protein solution is substantially devoid of exogenous anti-fibrinolytic agents and of organic chelating agents; introducing the thrombin solution and the plasma protein solution to a solid receptacle or mold in the presence of calcium ions; incubating under conditions appropriate to achieve clotting; freezing the clotted mixture; and lyophilizing the clotted mixture, to obtain a sponge, and optionally seeding the sponge with cells prior to implantation.

According to one embodiment of the present invention the plasma proteins are partially purified plasma proteins. According to another embodiment of the present invention the plasma proteins are devoid of plasminogen. According to yet another embodiment the plasma protein solution comprising less than about 20% of plasminogen normally present in blood plasma, preferably less than about 10% of the plasminogen normally present in plasma and more preferably less than about 5% of the plasminogen normally present in plasma.

According to one embodiment of the invention the porous fibrin sponge is prepared by transferring the thrombin solution into a mold or solid receptacle, adding the plasma protein solution to achieve clot formation; freezing the clotted mixture and lyophilizing. Alternatively, the plasma proteins are mixed with thrombin in the presence of calcium ions under conditions suitable for achieving clotting; the mixture is cast in a solid support prior to achieving clotting; the clotted mixture is frozen and lyophilized. It is to be understood that when incorporated, additives and bioactive agents are added independently of each other to either of the matrix forming solutions, i.e. the plasma proteins or the thrombin solution, prior to the formation of the clot or are placed into the mold or solid receptacle prior to, concurrently with or following addition of the thrombin.

In one embodiment the invention provides a heterogeneous porous fibrin matrix wherein particulate matter is incorporated into the sponge ab initio. Particulate matter may include materials such as calcium phosphate particles, bone chips or glass fibers that are able to impart certain advantageous properties to the matrix including strength, additional porosity or phasic release.

According to various embodiments of the present invention plasma proteins at a concentration of about 10 mg/ml to about 50 mg/ml, substantially devoid of anti-fibrinolytic agents, plasminogen and of organic chelating agents, are mixed with at least one glycosaminoglycan such as hyaluronic acid and/or heparin, the mixture is incubated and added to the thrombin solution in the solid support to achieve formation of a clot. The clot is subsequently frozen and lyophilized.

In one embodiment, prior to implantation or use with cells, the sponge is substantially dry and contains less than 15% residual moisture, preferably less than 10% residual moisture. Surprisingly, this property of the sponge has been shown to be particularly advantageous for cell seeding and attachment.

Another aspect of the present invention provides a method of treatment and use of the freeze-dried fibrin matrix substantially devoid of exogenous anti-fibrinolytic agents and of organic chelating agents for tissue regeneration and repair of injured, diseased or traumatized tissue, including cartilage and bone defects and other tissue types including but not limited to liver, pancreas, and cardiac tissue. The method of treatment described herein is advantageous in that it requires minimal preparation for use by the medical practitioner. Other advantageous properties derive from the absence of exogenous anti-fibrinolytic agent such as tranexamic acid and aprotinin, which may be detrimental to the patient and the tissue surrounding the implant. Additionally, the absence of an exogenous anti-fibrinolytic agent renders the sponge a superior scaffold for in vivo or in vitro cellular attachment, growth, proliferation, infiltration and differentiation.

According to one embodiment the sponge is implanted per se. In another embodiment the sponge is cut into at least one section of desired shape.

In one embodiment the sponge further comprises cells. According to another embodiment the cells are selected from stem cells or progenitor cells. According to yet another embodiment the cells are selected from chondrocytes, osteoblasts, hepatocytes, or mesenchymal, endothelial, epithelial, urothelial, endocrine, neuronal, pancreatic, renal and ocular cell types.

The in vivo uses of the porous fibrin matrix are manifold. The porous fibrin matrix may function as a scaffold for in vitro culturing of cells or 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 invade, proliferate and/or differentiate. The matrix is useful in treating articular cartilage defects of any type, including chondral and subchondral defects, arising from trauma such as an accident or sports injury or disease such as osteoarthritis. The porous fibrin matrix may be used per se or in combination with other therapies. For example, for cartilage repair the porous fibrin matrix is useful in conjunction with other therapeutic procedures including chondral shaving, laser or abrasion chondroplasty, and drilling or microfracture techniques.

Other typical orthopedic applications include joint resurfacing, meniscus repair, non-union fracture repair, craniofacial reconstruction or repair of an invertebral disc. Furthermore, the porous fibrin matrix is useful 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 a finish comprising the porous fibrin matrix of the invention.

The porous fibrin matrix of the invention is useful, inter alia, as an unexpectedly advantageous support for cellular growth. The absence of exogenous anti-fibrinolytic agents results in a fibrin matrix that is fully compatible with in vitro and in vivo cell growth, proliferation and differentiation. An additional advantage of the fibrin matrix of the invention is its improved ability to absorb cells and maintain their viability. The need to hydrate or rinse the sponge of the invention prior to cell seeding is precluded by the absence of exogenous anti-fibrinolytic agents, thus rendering a sponge with superior cell incorporation capacity.

The porous fibrin matrix of the invention, being an effective scaffold supporting cell growth, may be utilized in vivo in reconstructive surgery, for example as a matrix for regenerating tissue comprising neuronal cells, hepatic cells, urothelial cells, osteoblasts, cardiovascular tissue and mammary tissue or any other cell types which it is desired to culture within a three dimensional support. Thus, the matrix of this invention may be used to construct living tissue equivalents, including but not limited to liver, pancreas, nerve, glands, tendons, skin, blood vessels, bone, tendon, ligaments, and other organ equivalents, among many others.

According to one embodiment of the present invention the matrix is a sponge or scaffold able to support the proliferation of a variety of cell types. In one aspect, the sponge is inoculated with cells and the cells are allowed to proliferate in vitro prior to in vivo implantation. Alternatively, the sponge is seeded with cells that have been cultured or harvested and the sponge comprising the cells is implanted in situ. In one embodiment the porous fibrin matrix useful as an implant for transplantation comprises autologous plasma proteins and autologous chondrocytes.

According to one embodiment the present invention provides a method of treating or repairing injured, diseased or traumatized tissue, the method comprising the step of implanting a porous freeze-dried fibrin matrix formed from plasma proteins comprising fibrinogen, thrombin and Factor XIII, the matrix having less than 10% residual moisture and being substantially devoid of exogenous anti-fibrinolytic agents and of organic chelating agents to the site of injury, disease or trauma. The tissue is selected from cartilage, bone, liver, mesenchymal, endothelial, epithelial, urothelial, endocrine, neuronal, pancreatic, renal and ocular tissue types. According to another embodiment the porous freeze-dried fibrin matrix formed from plasma proteins comprising fibrinogen, thrombin and Factor XIII, the matrix having less than 10% residual moisture and being substantially devoid of exogenous anti-fibrinolytic agents, plasminogen and of organic chelating agents is implanted into the site of injury disease or trauma.

Further provided is the use of an implant of the present invention for the treatment or repair of injured, diseased or traumatized tissue, the use comprising the step of implanting a matrix of the present invention to the site of injury, disease or trauma. The tissue is selected from cartilage, bone, liver, mesenchymal, endothelial, epithelial, urothelial, endocrine, neuronal, pancreatic, renal and ocular tissue types.

DETAILED DESCRIPTION OF THE INVENTION

Though numerous biomatrices comprising 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 and tissue reparation. The present invention discloses a porous fibrin matrix, also referred to as a sponge, comprising plasma proteins substantially devoid of plasminogen and of organic chelating agents. The absence of plasminogen in the matrix obviates the need for external anti-fibrinolytic agents. It is further disclosed that unexpectedly biomatrices comprising partially purified plasma proteins also obviate the need for the addition of exogenous anti-fibrinolytic agents. The compositions and methods of the present invention are effective for in vitro and in vivo applications including as cell-bearing implants for tissue engineering and reparation.

The resulting fibrin, or plasma protein, sponge has attributes that make it particularly advantageous for supporting and promoting cell growth both in vivo and in vitro. Plasminogen is a plasma protein which is enzymatically converted to an active serine protease, plasmin, having fibrinolytic activity. This activity results in the rapid degradation of fibrin in fibrin glue and matrices. Anti-fibrinolytic agents such as tranexamic acid and aprotinin are typically incorporated into fibrin glue, sponges and matrices in order to maintain the integrity of the substrate. The sponges of the present invention are stable and exhibit reduced bioresorbability and overcome the need to add exogenous anti-fibrinolytic agents.

Among the advantageous properties of the matrices of the invention:

The fibrin matrices exhibit superior biological properties including reduced biodegradability, an absence of immunogenicity or other adverse reactions, the capacity to maintain and promote high levels of cell growth, proliferation, differentiation and migration and controlled release of bioactive agents.

The matrices have superior mechanical properties, controlled by varying the additives used in the composition. Desirable properties include suppleness, elasticity and durability.

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

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

The matrices of the invention provide all components fundamental for tissue repair, thus facilitating the medical practitioner's task. In addition, the composition of the sponge renders it suitable for minimally invasive surgery of articular cartilage. The sponge may be implanted in a mini-arthrotomy or arthroscopy procedure, thus avoiding the multiple site surgeries and a full arthrotomy used for ACT.

Embodiments of the Invention

The present invention relates to porous, freeze-dried fibrin matrices comprised of plasma proteins substantially devoid of exogenous anti-fibrinolytic agents useful for supporting cell growth. The present invention relates to the unexpected finding that a porous, freeze-dried fibrin matrix comprised of plasma proteins substantially devoid of plasminogen exhibits superior biological characteristics, in particular cell viability and cell proliferation. Plasminogen is a plasma protein which is enzymatically converted to an active serine protease, plasmin, having fibrinolytic activity. This activity results in the rapid degradation of fibrin in fibrin clots and matrices. Anti-fibrinolytic agents are typically incorporated into fibrin clots and matrices in order to maintain the integrity of the substrate. The matrices of the present invention lack plasminogen thus obviating the need for exogenous anti-fibrinolytic agents, which have been shown to be deleterious to cells and tissue and which may induce adverse reactions in patients. It is now further disclosed that matrices comprising partially purified plasma proteins also obviate the need for exogenous anti-fibrinolytic agents. The compositions and methods of the present invention are effective in in vivo and in vitro applications including as fully biocompatible implants for tissue engineering as well as in biotechnology. The matrices according to the present invention may be used clinically, per se or as cell-bearing implants. They are true three-dimensional structures capable of providing support and of maintaining cell growth and differentiation.

In one aspect, the present invention relates to a freeze-dried fibrin matrix comprising plasma proteins substantially devoid of exogenous anti-fibrinolytic agents, plasminogen and of organic chelating agents. Substantially devoid of plasminogen refers to plasma proteins comprising less than about 20% plasmin or plasminogen normally present in plasma, preferably less than about 10% of plasminogen normally present in plasma, more preferably less than about 5% of plasminogen normally present in plasma.

The inventors have discovered that a porous freeze-dried fibrin matrix comprising plasma proteins substantially devoid of exogenous anti-fibrinolytic agents, plasminogen and of organic chelating agents provides a superior matrix for clinical and biotechnological applications. In addition to eliminating the need for exogenous anti-fibrinolytic agents and their concomitant detrimental effects, the inventors now show that the fibrin matrix of the present invention is superior as a scaffold for cell seeding, growth and differentiation and tissue repair and regeneration.

According to one embodiment of the present invention, the fibrin matrix comprises plasma proteins, the major protein being fibrin. Fibrin is obtained by the interaction of the plasma proteins fibrinogen (Factor I) and thrombin in the presence of calcium ions (Ca.sup.+2) and Factor XIII or another fibrin stabilizing factor, to form a fibrin clot. 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, in the substantial absence of organic chelating agents. Total blood, blood fractions, blood derivative, cryoprecipitate, recombinant proteins, plasma or plasma fractions may serve as a plasma protein source for the fibrin sponge of the present invention. The plasma source may be allogeneic or autologous. Another source of the plasma proteins, specifically of fibrinogen, includes fibrinogen variants, including the high molecular weight (HMW), the low molecular weight (LMW) and the LMW derivative (LMW') variants, for example as disclosed in PCT patent application WO 03/087160.

The plasma proteins are substantially devoid of plasminogen. Plasminogen may be removed from the plasma by methods known in the art. In one non-limiting example, the plasminogen is removed from plasma by affinity purification. Epsilon amino carboxylic acid (EACA) ligands as well as lysine resin have been used to purify plasminogen from whole plasma. PCT patent application WO 02/095019 discloses a method for specifically removing plasminogen and plasmin in the presence of fibrinogen from a mixture such as blood or cryoprecipitate. The method requires contacting the mixture comprising plasminogen with a rigid amino acid, such as tranexamic acid, wherein the amino group and carboxylic group are about 7 angstroms apart and the rigid amino acid is covalently bound to the support via the amino group. PCT patent application WO 95/25748 discloses a topical fibrinogen complex essentially free of plasminogen whereby the plasminogen was removed using a Sepharose.RTM.-lysine column. Alternatively, some or all of the plasma proteins may be recombinant and consequentially devoid of plasminogen, for example as disclosed in PCT publication WO 99/56797.

The plasma proteins are further substantially devoid of exogenous anti-fibrinolytic agents, which have been shown to be detrimental to cell growth and may induce adverse reactions in patients. Surprisingly, a matrix comprising partially purified plasma proteins also obviates the need for exogenous anti-fibrinolytic agents.

The fibrin matrix of the invention may be used per se, comprising plasma proteins substantially devoid of exogenous anti-fibrinolytic agents, plasminogen and of organic chelating agents, for clinical and biotechnological applications. It may however, further comprise additives that impart other advantageous biological, physical and mechanical characteristics to the matrix. Copending international patent application WO 03/007873 of some of the inventors of the present invention discloses a fibrin matrix comprising plasma proteins and at least one anti-fibrinolytic agent, optionally further comprising agents such as polysaccharides, anionic polysaccharides, glycosaminoglycans, or synthetic polymers added in the preparation to improve certain physical, mechanical and biological properties of the matrix. The incorporation of at least one such agent was shown to impart superior characteristics including elasticity and regular pore size to the sponge.

In one embodiment, the present invention is related to a porous fibrin matrix substantially devoid of exogenous anti-fibrinolytic agents, plasminogen and of organic chelating agents further comprising at least one additive selected from the group consisting of polysaccharides, glycosaminoglycans and synthetic polymers that is useful as a support for culturing or growth of cells, both in vitro and in vivo. The incorporation of at least one additive to the matrix forming materials, 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 to the sponge. The sponges formed are substantially homogeneous having no particles or interrupting substructures other than the pores and interconnecting channels.

In one embodiment the additive may be added ab initio, during formation of the clot. In another embodiment the additive may be introduced to the matrix anytime following formation of the sponge. According to various embodiments of the present invention, the matrix is prepared using at least one glycosaminoglycan selected from the group consisting of crosslinked hyaluronic acid, non-crosslinked hyaluronic acid, heparin and heparin derivatives and mimetics, chondroitin sulfate, dextran sulfate, dermatan sulfate, heparan sulfate and keratan sulfate. In one aspect the glycosaminoglycan is incorporated into the matrix during initial formation of the clot. In one embodiment the glycosaminoglycan is hyaluronic acid. The glycosaminoglycan is added to a final concentration that imparts suppleness and elasticity to the sponge and precludes the need for adjusting the moisture content of the final composition. Hyaluronic acid may be crosslinked or non-crosslinked, having a variety of different molecular weights and may originate from an animal source or a recombinant source. According to one embodiment the concentration of non-crosslinked hyaluronic acid is about 0.005% to about 0.5% final (v/v) more preferably about 0.05% to about 0.1%. In another embodiment the concentration of crosslinked hyaluronic acid is about 0.001% to about 0.1% and more preferably around 0.05% to about 0.09% final concentration. According to one embodiment the glycosaminoglycan is selected from heparin and a derivative thereof.

According to yet 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. Non-limiting examples of natural polymers include cellulose, pectin, polyuronic acids, hexuronyl hexosaminoglycan sulfate and inositol hexasulfate.

The synthetic polymers useful for the present invention 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, 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.

In one embodiment, the invention provides a heterogeneous sponge comprising particulate matter such as calcium phosphate crystals or other particles. The particulate matter may be incorporated ab initio in order to provide a matrix having physical or biological characteristics advantageous for certain applications.

Bioactive Agents

In one embodiment the matrix of the invention further comprises at least one bioactive agent, such as a cytokine, a growth factor and their activators, platelets, a bioactive peptide etc. Without wishing to be bound by theory, incorporation of such agents into the sponge of the present invention provides a slow-release or sustained-release mechanism. As the matrix degrades in vivo, the bioactive agents are released into the surrounding milieu. 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. 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 or differentiation of a variety of cells, including primary fibroblasts, embryonal stem cells (ESC), adult stem cells, chondrocytes, vascular and corneal endothelial cells, osteoblasts, myoblasts, smooth muscle and neuronal cells. Representative proteins include bone growth factors (BMPs, IGF) and fibroblast growth factors and their variants, including FGF2, FGF4, FGF9 and FGF18 for bone and cartilage healing, cartilage growth factor genes (CGF, TGF-.beta.) 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-.beta..

Other biologically active agents that may be included into the matrix include blood platelets, platelet supernatants or extracts and platelet derived proteins, hormones, analgesics, anti-inflammatory agents, anti-microbials or enzymes. Bioactive agents including platelets and platelet supernatant or extract promote the proliferation and differentiation of skeletal cells including chondrocytes and osteoblasts and of other cell types including but not limited to hepatocytes and endothelial cells. Bioactive agents belonging to the class of anti-microbial 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. In one non-limiting example, the at least one bioactive agent, added ab initio or at any stage following preparation, may be selected to enhance the healing process of the injured or diseased tissue.

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. In one embodiment, the growth factor is a fibroblast growth factor (FGF) or bone morphogenetic protein (BMP) or variant thereof. In another embodiment, the FGF is an FGF or FGF variant having the capacity to induce cartilage and bone repair and regeneration and or angiogenesis. The growth factors may be incorporated at a wide range of concentrations, depending on the application. For certain applications sustained release of a bioactive agent is preferred. Sustained release of a bioactive agent may depend on several factors including growth factor concentration, type of glycosaminoglycan incorporated and thrombin concentration.

In contrast to the bioabsorbable heteromorphic sponge of the art, the present inventors now disclose a freeze-dried homogenous fibrin sponge compromising plasma proteins substantially devoid of plasminogen and of organic chelating agents further comprising at least one additive selected from the group consisting of polysaccharides, glycosaminoglycans and synthetic polymers and at least one bioactive agent providing phasic release of said bioactive agent.

According to various specific embodiments of the present invention the porous fibrin matrix comprising plasma proteins substantially devoid of antifibrinolytic agents, plasminogen further comprises at least one glycosaminoglycan and at least one bioactive agent, wherein the bioactive agent is a therapeutic protein belonging to the FGF family of growth factors. In one embodiment a porous fibrin matrix comprising plasma proteins substantially devoid of plasminogen and of organic chelating agents further comprises hyaluronic acid, heparin and an FGF. In one aspect the hyaluronic acid and the heparin or heparin mimetic are incorporated into the sponge ab initio.

According to one non-limiting example the present invention provides a porous homogenous freeze-dried fibrin matrix comprising plasma proteins substantially devoid of plasminogen, substantially devoid of organic chelating agents, further comprising at least one glycosaminoglycan and at least one bioactive agent, wherein the at least one glycosaminoglycan is heparin and the at least one bioactive agent is a therapeutic protein belonging to the FGF family of growth factors or a variant thereof. This sponge provides phasic release of the FGF from the matrix and may be beneficial in certain therapeutic applications. Optionally, at least one bioactive agent may be added to the cell, either in culture or during seeding, for example, to enhance a therapeutic effect.

Additionally, cells genetically engineered to express the aforementioned proteins are including in the present invention. According to one aspect, periosteal cells, mesenchymal stem cells or chondrocytes are used per se or are transfected with cartilage growth factor genes selected from a group including transforming growth factor-.beta. (TGF-.beta.), certain FGFs or CGF for cartilage repair and regeneration; for bone repair periosteal or other mesenchymal stem cells or osteoblasts are used per se or are 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 neural cells and neural support cells are used per se or are transfected with genes selected from a group including nerve growth factor (NGF) gene or specific FGFs.

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. Chondrocytes of the cartilage are embedded in the thick extracellular matrix (ECM) of the joint. Without wishing to be bound by theory enzymes known in the art including collagenase, trypsin, chymotrypsin, chondroitinase of the various types, are able to 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.

According to one embodiment the at least one bioactive agent is platelets or platelet supernatant. 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.

Applications

The porous homogeneous fibrin matrix of the invention is useful as scaffold for tissue engineering applications. The absence of plasminogen obviates the need for external anti-fibrinolytic agents and thus results in a sponge that is fully biocompatible. The optional 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 plasma matrix are manifold. The fibrin 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 cells may be stem cells or progenitor cells or may be specialized cells such as chondrocytes, osteoblasts, hepatocytes, or mesenchymal, endothelial, epithelial, urothelial, endocrine, neuronal, pancreatic, renal or ocular cell types.

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 one aspect 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.

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.

The porous fibrin matrix of the invention is useful, inter alia, as an unexpectedly advantageous support for cellular growth. The absence of exogenous anti-fibrinolytic agents results in a fibrin matrix that is fully compatible with in vitro and in vivo cell growth, proliferation and differentiation. An additional advantage of the fibrin matrix of the invention is its improved ability to absorb cells and retain them. The need to wet or wash the sponge of the invention prior to cell seeding is precluded by the absence of exogenous anti-fibrinolytic agents. In one embodiment the matrix of the invention serves as a scaffold for the growth, proliferation and/or differentiation of cells including stem cells, progenitor cells or other cell types including chondrocytes, osteoblasts, hepatocytes, mesenchymal, epithelial, urothelial, neuronal, pancreatic, renal or any other cell types which it is desired to culture within a three dimensional support.

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. The sponge is useful for the delivery of cells in situ to a specific site in the body, such as dopamine expressing cells to Parkinson's patients.

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 U.S. Pat. No. 6,398,816.

According to certain embodiments of the invention, the fibrin 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. Cell types include chondrocytes, osteoblasts, hepatocytes, or mesenchymal, endothelial, epithelial, urothelial, endocrine, neuronal, pancreatic, renal or ocular cell types.

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, which is capable of supporting and facilitating cellular growth, can thus be useful in providing a favorable environment for the implant or prosthesis.

A person skilled in the art can adjust the procedures exemplified below in accordance with specific tissue requirements. For example, for cartilage repair the porous, homogeneous freeze-dried fibrin matrix of the invention 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 per se, 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.

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 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. The matrix may be seeded with the desired cells by surface seeding, at a density of about 10.sup.4 cells per cm.sup.3, more preferably about 10.sup.5 cells per cm.sup.3.

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 calcium ions (Ca.sup.+2) ions. The plasma proteins may derive from a commercial, xenogeneic, allogeneic or autologous source and comprise fibrinogen and factor XIII, substantially devoid of plasminogen and in the substantial absence of organic chelating agents. The plasma protein solution may comprise fibrinogen variants such as the high molecular weight or low molecular weight variants.

According to one embodiment of the present 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 calcium 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 when incorporated, additives and bioactive agents 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.

A method for preparing a porous freeze-dried fibrin matrix formed from plasma proteins having less than 10% residual moisture and being substantially devoid of exogenous anti-fibrinolytic agents and of organic chelating agent comprises the following steps: providing a thrombin solution and a plasma protein solution wherein the plasma protein solution is substantially devoid of exogenous anti-fibrinolytic agents and of organic chelating agents; introducing the thrombin solution and the plasma protein solution to a solid receptacle or mold in the presence of calcium ions; incubating under conditions appropriate to achieve clotting; freezing the clotted mixture; and lyophilizing the clotted mixture, to obtain a sponge.

According to one embodiment of the present invention the plasma proteins are partially purified plasma proteins. According to another embodiment of the present invention the plasma proteins are devoid of plasminogen. According to yet another embodiment the plasma protein solution comprising less than about 20% of plasminogen normally present in blood plasma, preferably less than about 10% of the plasminogen normally present in plasma and more preferably less than about 5% of the plasminogen normally present in plasma.

According to one embodiment the matrix of the invention may be prepared by sequential introduction of the thrombin solution and plasma protein solution into the mold or solid receptacle. Either solution may be introduced first. According to another embodiment of the present invention the thrombin solution and the plasma protein solution are mixed together and subsequently introduced into a mold. The resulting sponges are different in their porosity and cell dispersion.

A method for preparing a porous freeze-dried fibrin matrix having less than 10% residual moisture and being substantially devoid of exogenous anti-fibrinolytic agents, and of organic chelating agents further comprising at least one additive selected from the group consisting of polysaccharides, glycosaminoglycans and synthetic polymers comprises the following steps: providing a plasma protein solution substantially devoid of exogenous anti-fibrinolytic agents and of organic chelating agents and a thrombin solution and wherein at least one of the plasma protein solution or the thrombin solution contains at least one additive selected from the group consisting of polysaccharides, glycosaminoglycans and synthetic polymers; 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;

The sponge may further comprise at least one bioactive agent, added ab initio to either the thrombin solution or the plasma protein solution.

According to one embodiment of the present invention the plasma proteins are partially purified plasma proteins. According to another embodiment of the present invention the plasma proteins are devoid of plasminogen. According to yet another embodiment the plasma protein solution comprising less than about 20% of plasminogen normally present in blood plasma, preferably less than about 10% of the plasminogen normally present in plasma and more preferably less than about 5% of the plasminogen normally present in plasma.

According to various embodiments of the present invention plasma proteins at a concentration of about 20 mg/ml to about 50 mg/ml, substantially devoid of exogenous anti-fibrinolytic agents, plasminogen and of organic chelating agents are mixed with hyaluronic acid and/or heparin and the mixture is added to the thrombin solution in the solid support to achieve formation of a clot. The clot is frozen and lyophilized.

According to another embodiment a plasma protein solution comprising plasma proteins at a concentration of about 20 to about 50 mg/ml, substantially devoid of antifibrinolytic agents and substantially in the absence of organic chelating agents, comprising hyaluronic acid and heparin bound to FGF are mixed and the mixture added to the thrombin solution in the solid support to achieve formation of a clot. The clot is frozen and lyophilized.

The final concentration of thrombin may be varied in order to produce sponges with distinct biological, physical and mechanical features useful for different applications. Thrombin concentrations of about 0.5 IU/ml to about 2 IU/ml provide sponges with similar properties in terms of cell viability and growth. Other concentrations, as low as 0.15 IU/ml may be useful as well, depending on the application.

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.

Yet another aspect of the present invention provides methods of treatment and use of the fibrin matrix of the invention for treating injured or traumatized tissue, including cartilage and bone defects. The method of treatment described herein is advantageous in that it requires minimal preparation for use by the medical practitioner. The in vivo uses of the porous fibrin matrix are manifold. The porous 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 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 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, osteochondral defect repair or repair of an invertebral disc. The fibrin matrix of the invention may serve to treat defects resulting from disease such as osteoarthritis. The components of the matrix may be cast into a mold specifically designed for a distinct lesion or defect. In a non-limiting example, the mold may be prepared by computer aided design. In other instances the medical practioner may have to cut or slice the sponge to fit a particular lesion or defect. The matrix of the invention is particularly beneficial for minimally invasive surgical techniques such as a mini-arthrotomy or arthroscopy and overcomes the need for fully open joint surgery.

In one embodiment, the porous 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 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 plasma protein solution may be from a commercial source, natural or recombinant proteins, or may be prepared from plasma. According to one embodiment of the present invention the plasma protein solution derives from allogeneic plasma. According to another embodiment of the present invention, at least one of the components, preferably the plasma proteins, used for preparing the matrix derives from autologous plasma or recombinant proteins. 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 fibrin 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 U.S. Pat. No. 6,274,090 and Haisch et al (Med Biol Eng Comput 38:686-9, 2000).

The resulting fibrin 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 one aspect, 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 isolated and returned to the plasma. The matrix of the present invention may serve as an implant for use as a scaffold per se or as a cell-bearing scaffold for in vivo implantation.

According to one embodiment of the present invention a porous 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. According to another embodiment 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 U.S. Pat. No. 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.

According to one embodiment of the present invention heparin is incorporated into the matrix to a final concentration of about 0.1 ug/ml to about 1 mg/ml. In another embodiment the concentration of heparin is about 1 ug/ml to about 50 ug/ml. As used herein ug/ml refers to a microgram per milliliter.

According to another embodiment of the present invention crosslinked hyaluronic acid is incorporated into the matrix to a final concentration of about 0.001% to about 0.1%, more preferably about 0.05% to about 0.09%.

According to another embodiment of the present invention non-crosslinked hyaluronic acid is incorporated into the matrix to a final concentration of about 0.005% to about 0.5%, more preferably about 0.05% to about 0.1%.

According to yet another 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 (U.S. Pat. Nos. 5,474,987; 5,686,431; 5,908,837).
 

Claim 1 of 16 Claims

1. A porous freeze-dried fibrin matrix formed from plasma proteins comprising fibrinogen cleaved by the action of thrombin at varying concentrations sufficient to cleave said fibrinogen and Factor XIII, the matrix having less than 10% residual moisture and being devoid of exogenous anti-fibrinolytic agents and of organic chelating agents, wherein the plasma proteins comprise partially purified plasma proteins that are devoid of plasminogen.

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