ES2423904T3 - Lyophilized fibrin matrices and methods of preparation thereof - Google Patents

Abstract

A lyophilized porous fibrin matrix is formed from plasma proteins composed of defibrinogen, thrombin and factor XIII, the matrix contains less than 10% residual moisture, without added exogenous antifibrinolytic agents and comprises less than 1 mM of organic chelating agents, where plasma proteins they comprise less than 20% plasminogen normally present in plasma.

Description

Lyophilized fibrin matrices and methods of preparation thereof

TECHNICAL FIELD

[0001] The present invention describes lyophilized biomatrices composed of plasminogen-free plasma protein for clinical applications such as tissue engineering implants. In addition, lyophilized plasma protein biomatrices are substantially free of exogenous antifibrinolytic agents as well as organic chelating agents. The matrices, according to the present invention, have clinical utility, per se or as cell-carrying implants.

PREVIOUS DESIGN

Tissue Engineering

[0002] Tissue engineering can be defined as the technique of tissue reconstruction or regeneration in mammals, both structurally and functionally (Hunziker, Osteoarth. Cart. 10: 432-63,2002). Tissue engineering generally includes the release of a natural or synthetic support that serves as an architectural support on which cells can be fixed, proliferate and synthesize new tissues for the reconstruction of a wound or defect.

[0003] An example of tissue prone to damage due to ailments and trauma is articular cartilage, one of the many types of cartilage in the body, which can be found on the surface of the bones. Damage to the cartilage can be the result of an inflammatory disease such as rheumatoid arthritis, a degenerative process such as osteoarthritis or trauma such as an intra-articular fracture or the consecutive ligament injury. Cartilage lesions are often accompanied by pain and reduced mobility, usually do not heal and without medical intervention may require total osteoarticular replacement.

[0004] Current therapeutic strategies to repair damaged cartilage include procedures that induce a spontaneous repair response and those that reconstruct the tissue in a structural and functional manner. These strategies include surgical techniques that expose the subchondral bone, thus allowing infiltration of bone marrow progenitor cells to initiate the reconstruction response. Frequently, induced tissue is a type of combined fibrocartilage without durability and whose clinical improvements are very limited. The last strategy includes the transplantation of chondral or osteochondral cells or tissues, whether of autologous or allogeneic origin. Autologous Chondrocyte Transplantation (CAT) refers to autologous chondrocyte transplantation in a cartilage lesion that has been isolated from a biopsy of the patient's cartilage and expanded in vitro. In fact, this technique requires a complicated procedure that involves two surgical works and shows enormous variability and limited clinical success.

[0005] In this discipline, it is known that matrices are practical for tissue regeneration as well as to act as biocompatible surfaces for them. These matrices can also be considered as substrates for cell growth, either in vitro or in vivo. Matrices for tissue growth and / or regeneration include both biodegradable and biostable entities. Among the many possibilities that can be used as support matrices for tissue growth or regeneration are gels, foams, plates and certain porous structures of different shapes and sizes.

[0006] In the past, porous materials formed from synthetic materials and / or biodegradable materials of natural origin have been used as dressings or implants. A porous material provides structural support and support for intracellular growth and tissue regeneration. Preferably, the porous material degrades gradually, being absorbed as regenerated tissue. Bioreabsorbable materials that are often used in the manufacture of porous dressings or implants include polymers and synthetic biopolymers such as structural proteins and polysaccharides. The biopolymers can be selected or modified so that their physicochemical properties are modified to provide greater flexibility or less vulnerability to degradation.

[0007] It has been found that numerous natural polymers are useful in tissue or crop engineering, including various constituents of the extracellular matrix such as fibronectin, some types of collagen and laminin, such as keratin, fibrin and fibrinogen, acid hyaluronic acid, heparan sulfate and chondroitin sulfate among others. US 6,425,918 and 6,334,968 describe a bioreabsorbable lyophilized polysaccharide sponge and its use as a matrix or support for implantation in a patient.

Fibrin

[0008] Fibrinogen is an important plasma protein that participates in the blood coagulation process. Once the blood vessel is damaged, the fibrinogen becomes insoluble fibrin that serves as a structure for the clot. Blood coagulation is a complex process that involves the sequential interaction of a number of plasma proteins, particularly fibrinogen (factor I), prothrombin (factor II), factor V and factors VII-

XIII Other plasma proteins such as von Willebrand factor, immunoglobulins, coagulation factors and complement components are also part of the blood clot formation.

[0009] In this discipline, fibrin is known as a tissue adhesive for effective medical use for wound reconstruction and tissue repair. The lyophilized protein concentrate derived from plasma (fibrinogen compound, factor XIII and fibronectin), in the presence of thrombin and calcium ions gives rise to an injectable biological sealant (fibrin gum). US Patent 5,411,885 describes a method of embedding and culturing tissues with the use of fibrin gum.

[0010] US Patent 4,642,120 explains the use of fibrinogen-containing gums in combination with autologous or chondrocytic mesenchymal cells as a method to favor the repair of defects in cartilage and bone. US 5,260,420 describes a method for the preparation and use of biological glues composed of therapeutic plasma proteins. US Patent 6,440,427 describes an adhesive compound formed mostly of fibrin precursor components and a polysaccharide such as hyaluronic acid that improves viscosity. Itokazu (Itokazu et al., Infection 25: 359-63, 1997) described a lyophilized fibrin clot for prolonged release of antibiotics.

[0011] US Patent 5,972,385 discloses a lyophilized matrix of crosslinked collagen polysaccharide that is administered alone or in combination with other tissue repair therapeutics. US Patents 5,206,023 and US 5,368,858 describe a cartilage repair composition consisting of a biodegradable matrix, a proliferation or and a transformation factor.

[0012] In US 4,442,655 and EP 0068149 a lyophilized fibrinogen structure with mesh structure is described which can be used for the treatment of wounds, as a filling material for bone cavities

or as a support for active substances for controlled release. The structure is prepared by the mixture of fibrinogen and thrombin solution, then see it in a mold, freeze and lyophilize.

[0013] In US 6,310,267 and 6,486,377, a lyophilized fibrin membrane for wound healing is described. The preparation of this membrane requires a dialysis of one or several stages of the fibrinogen solution. According to this disclosure, the dialysis of one or several stages of the fibrinogen solution radically changes its composition by reducing the concentration of salts and amino acids. This dialysis is carried out in an aqueous solution of a physiologically compatible inorganic salt and an organic complexing agent.

[0014] In WO 99/15209 a fibrin sponge is described which contains a blood coagulation activator for hemostasis, tissue adhesion, wound healing and cell culture. According to this publication, the restoration of moisture or water content after lyophilization is crucial to obtain a soft, adaptable and absorbent sponge. The sponge may be impregnated with additives such as blood coagulation activators, stabilizers, preservatives and other agents.

[0015] US Patents 5,466,462 and 5,700,476 describe a bioreabsorbable heteromophic sponge composed of a biopolymeric matrix, 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 physical release. US Patent 5,443,950 is related to the growth of cells derived from a desired tissue in a previously established stromal matrix. In addition, US 5,842,477 describes a method of reconstructing cartilage in vivo by implanting a three-dimensional biocompatible framework in combination with periosteal / perichondral tissue and stromal cells, with or without bioactive agents.

Fibrinolysis

[0016] Current lyophilized fibrin implants intended for tissue engineering are prepared using fibrinogen or plasma protein solutions, resulting in proteases that can compromise the stability of certain plasma proteins and lead to matrix degradation. Plasminogen is an important plasma protein that binds to fibrin during clot formation. Within the clot or matrix, the plasminogen is enzymatically converted to plasmin, the result of degradation of the clot or matrix, which acts as a fibrinolytic agent. Normally, this process is delayed by adding antifibrinolytic agents to the composition, including, among others, aprotinin, £ -aminocaproic acid or tranexamic acid. These agents can cause harmful effects on growth, proliferation and / or cell differentiation, or they can cause adverse reactions in patients. This discipline has so far not given a lyophilized stable fibrin matrix substantially free of exogenous antifibrinolytic agents.

[0017] The international patent application in process WO 03/007873 made by some of the inventors of the present invention, reveals a lyophilized plasma protein matrix composed of plasma proteins and, at least one antifibrinolytic agent, may also be composed of other excipients to improve certain physical, mechanical and biological properties of the matrix.

SUMMARY OF THE INVENTION

[0019] The present invention describes biomatrices substantially free of external antifibrinolytic agents, as it has been shown that they are harmful to cells and tissues and can cause adverse reactions in patients. It is now when it is first disclosed that flexible and resistant matrices, also known as sponges, which are particularly beneficial as support for three-dimensional cell growth, can be obtained from plasminogen-free plasma proteins, thereby avoiding the need for external antifibrinolytic agents. It is also described below that biomatrices, which were obtained unexpectedly from partially purified plasma proteins, also avoid the need for exogenous antifibrinolytic agents. The compositions and methods of the present invention are effective in both in vivo and in vitro applications, including biocompatible implants in tissue engineering and biotechnology for in vitro culture and cell differentiation. The matrices, according to the present invention, are three-dimensional (3D) and can be used clinically per se or as cell-bearing implants. The present invention explains all the fundamental components for tissue reconstruction, thus facilitating the task of the medical professional and providing a better alternative for the reconstruction and regeneration of patient tissues.

[0020] The present invention discloses a lyophilized porous fibrin matrix formed from plasma proteins that include fibrinogen, thrombin and Factor III. The matrix has less than 10% residual moisture and is free of exogenous antifibrinolytic agents and organic chelating agents, thus showing better characteristics. The present invention is based partially on the unexpected finding that a matrix composed of plasma proteins free of exogenous and plasminogen antifibrinolytic agents transmits more stability and significantly improves the inoculation and dispersion of cells, but maintains other positive properties of these matrices. The matrices, in particular those that are free of plasminogen, show reduced resorbability if compared with the fibrin matrices known so far, resulting in a long-term implant with improved stability and resistance for the correct development, reconstruction and regeneration of tissues.

[0021] The present invention discloses a lyophilized porous fibrin matrix formed from plasma proteins that include fibrinogen, thrombin and Factor III. The matrix has less than 10% residual moisture and is free of exogenous antifibrinolytic agents, plasminogen and organic chelating agents. In an embodiment substantially free of plasminogen it refers to the plasma protein solution containing less than 20% of the plasminogen normally present in the blood plasma, less than 10% of the plasminogen normally present in the plasma and more preferably less than 5% of the plasminogen normally present in plasma. The inventors have discovered that a lyophilized porous fibrin matrix composed of plasminogen-free plasma proteins has an improved matrix for clinical and biotechnological applications. In addition to eliminating the need for exogenous antifibrinolytic agents and their consequent adverse effects, the inventors now show that the fibrin matrix of the present invention is better as a framework for inoculation, growth and cell differentiation, as well as for use in tissue regeneration and reconstruction.

[0022] The fibrin matrix of the invention can be used per se, including plasma proteins substantially free of exogenous antifibrinolytic agents and chelating agents, for clinical and biotechnological applications. It is worth mentioning, however, additives that improve the biological, physical and mechanical characteristics of the matrix. The present invention includes incorporation into the matrix of at least one additive to produce a matrix with improved biological, mechanical and / or physical properties.

[0023] The international patent application in process WO 03/007873 made by some applicants of the present invention discloses a fibrin matrix composed of plasma proteins and at least one antifibrinolytic agent, optionally also composed of agents such as polysaccharides , anionic polysaccharides, glycosaminoglycans or synthetic polymers added to the preparation to improve certain physical, mechanical and biological properties of the matrix. In this way, the need for an antifibrinolytic agent is eliminated or overcome by the substantial absence of plasminogen in the matrix.

[0024] In one embodiment, the present invention is related to a porous fibrin matrix substantially free of exogenous antifibrinolytic agents, plasminogen and organic chelating agents comprising at least one additive selected from a group consisting of polysaccharides, glycosaminoglycans (GAGs) and Synthetic polymers that serve as support for cell growth and differentiation both in vitro and in vivo.

[0025] According to one embodiment, the additive can be added ab initio, for example, during clot formation. According to other alternative embodiments, the additive is introduced into the matrix at any time after its formation. According to various embodiments carried out during the present invention, the matrix is prepared using at least one glycosaminoglycan selected from the group consisting of crosslinked hyaluronic acid, uncrosslinked hyaluronic acid, heparin, heparin derivatives and heparin mimetics, chondroitin sulfate, dextran sulfate, dermatan sulfate, heparan sulfate and keratan sulfate.

[0026] Glycosaminoglycan is added to the matrix as the final concentration that provides flexibility and elasticity to the matrix and discards the need to adjust the moisture content of the final composition. According to an embodiment carried out during the present invention, the glycosaminoglycan is selected from the crosslinked and uncrosslinked hyaluronic acid. In one embodiment the concentration of uncrosslinked hyaluronic acid is 0.005% to a final 0.5% (V / V) but it is preferable to be 0.05% to 0.1%. In another embodiment the concentration of cross-linked hyaluronic acid is 0.001% to 0.1% and it is preferable that it be 0.05% to a final 0.09% (V / V). According to another embodiment, the glycosaminoglycan is selected from heparin and its derivatives.

[0027] The present invention further includes a fibrin matrix composed of at least one bioactive agent selected from the group consisting of therapeutic proteins, platelets and platelet supernatant, analgesics, antimicrobial or anti-inflammatory agents and enzymes.

[0028] According to one embodiment, the present invention provides a lyophilized porous matrix composed of plasma proteins substantially free of exogenous antifibrinolytic agents, plasminogen and organic chelating agents, in addition to being composed of at least one glycosaminoglycan and at least one bioactive agent.

[0029] According to another embodiment carried out in the present invention, at least one bioactive agent is a therapeutic protein selected from the group consisting of growth factors and their derivatives. On the one hand, the growth factor is selected from a fibroblast growth factor (FGF) and its variants. On the other hand, FGF is an FGF capable of inducing or improving the reconstruction and regeneration of cartilage and bone and / or angiogenesis. Growth factors can be incorporated in a wide range of concentrations, depending on the power of the factor and the desired application.

[0030] For certain applications, prolonged or phased release of a bioactive agent is preferred. In one embodiment, the at least one growth factor is incorporated directly into the fibrin matrix, ab initio. In another embodiment, the at least one growth factor binds to a transport molecule such as heparin and is incorporated into the ab initio matrix. The prolonged release of a bioactive agent depends on various factors, such as the concentration of the growth factor, the type of glycosaminoglycan that is incorporated, and the concentration of fibrin and thrombin.

[0031] Compared to current bioreabsorbable heteromorphic sponges, the present invention discovers a lyophilized fibrin sponge composed of plasma proteins substantially free of exogenous antifibrinolytic agents, plasminogen and organic chelating agents, which may be composed in addition to at least one selected additive from the group consisting of polysaccharides, glycosaminoglycans and synthetic polymers, in addition to at least one bioactive agent.

[0032] According to the embodiment, the present invention provides a lyophilized porous fibrin matrix composed of plasma proteins substantially free of exogenous antifibrinolytic agents, plasminogen and organic chelating agents, in addition to at least one glycosaminoglycan and a bioactive agent. According to another embodiment carried out during the present invention, the at least one glycosaminoglycan is selected from heparin and its derivatives, the bioactive agent is a therapeutic protein selected from the family of growth factors FGF and its derivatives. This sponge provides a gradual release of matrix FGF and may be beneficial in certain therapeutic applications.

[0033] According to another embodiment, the present invention discloses a lyophilized porous fibrin matrix composed of plasma proteins substantially free of exogenous antifibrinolytic agents, plasminogen and organic chelating agents, in addition to hyaluronic acid, heparin and at least one bioactive agent. Hyaluronic acid is selected from crosslinked and uncrosslinked hyaluronic acid. Preferably, hyaluronic acid and heparin or heparin derivatives are incorporated into the ab initio sponge. The bioactive agent, as a growth factor, can be incorporated into the sponge per se or together with heparin. Preferably, the growth factor is selected from the family of therapeutic FGF molecules.

[0034] Another aspect of the invention provides a method of preparing the porous fibrin matrix. The method of preparing a lyophilized porous fibrin matrix with less than 10% residual moisture and substantially free of exogenous antifibrinolytic agents, plasminogen and organic chelating agents comprises the following steps:

providing a thrombin solution and a plasma protein solution in which it is substantially free of exogenous antifibrinolytic agents, plasminogen and organic chelating agents;

Introduce the thrombin solution and the plasma protein solution into a solid support or template in the presence of calcium ions;

Incubate under appropriate conditions to achieve coagulation;

Freeze the coagulated mixture; and lyophilize the coagulated mixture, to obtain a sponge, and

optionally, cultivate the sponge with the previous cells for the implant.

[0035] According to an embodiment carried out during the present invention, plasma proteins are partially purified plasma proteins. Plasma proteins are free from plasminogen, that is, the plasma protein solution has less than 20% of the plasminogen normally present in the blood plasma, preferably less than 10% of plasminogen normally present in the plasma and more preferably less than 5% of plasminogen normally present in plasma.

[0036] According to an embodiment carried out during the invention, the porous fibrin sponge is prepared by transferring thrombin solution to a solid mold or support, adding the plasma protein solution to achieve coagulation; freezing the coagulated mixture and lyophilizing it. Alternatively, plasma proteins are mixed with thrombin in the presence of calcium ions under the appropriate conditions to achieve coagulation; the mixture is melted on a solid support before coagulation; The coagulated mixture is frozen and lyophilized. It should be noted that once incorporated, additives and bioactive agents are added independently to each of the fibrin solutions being made, that is, the plasma proteins or thrombin solution, before clot formation, are placed in a solid mold or support before the addition of thrombin, simultaneously or afterwards.

[0037] In one embodiment, the invention provides a heterogeneous porous fibrin matrix in which the particulate material is incorporated into the ab initio sponge. The particulate material may include materials such as calcium phosphate particles, bone chips or glass fibers capable of providing certain improved properties to the matrix, such as strength, supplementary porosity or gradual release.

[0038] According to various embodiments of the present invention, plasma proteins in concentration of 10mg / ml to 50mg / ml, substantially free of antifibrinolytic agents, plasminogen and 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 clot formation. The clot is subsequently frozen and lyophilized.

[0039] In one embodiment, prior to implantation or use with cells, the sponge is considerably dry and contains less than 15% residual moisture, preferably less than 10% residual moisture. Surprisingly, it has been shown that this property of the sponge is particularly beneficial for cell culture and adhesion.

[0040] Another aspect of the present invention describes a method for the treatment and use of the lyophilized fibrin matrix substantially free of exogenous antifibrinolytic agents, plasminogen and organic chelating agents in the regeneration and reconstruction of damaged, diseased or traumatized tissues, including cartilage and bone defects and other types of tissue that include, but are not limited to, the liver, pancreas and heart tissue. The treatment method described here is an advantage since it requires minimal preparation by the medical professional. Other advantageous properties derive from the absence of exogenous antifibrinolytic agents such as tranexamic acid and aprotinin, which can be harmful to the patient and the tissue of the area surrounding the implant. In addition, the absence of an exogenous antifibrinolytic agent infers the sponge with an improved framework for adhesion, growth, proliferation, infiltration and cell differentiation, both in vivo and in vitro.

[0041] According to one embodiment, the sponge is implanted per se. In another embodiment, the sponge is cut into at least one section of the desired shape.

[0042] In one embodiment, the sponge is also composed of cells. According to another embodiment, the cells are selected from stem cells or progenitor cells. According to another embodiment, the cells are selected from chondrocytes, osteoblasts, hepatocytes or mesenchymal, endothelial, epithelial, urothelial, endocrine, neuronal, pancreatic, renal or ocular type cells.

[0043] There are various in vivo uses of the porous fibrin matrix. The porous fibrin matrix can function as a framework for in vivo cell culture or as an implant per se, to provide mechanical support in situ to a defective or damaged area and / or to provide a matrix in which the cells of the damaged area or Defective can develop, proliferate and / or differentiate. The matrix is suitable in the treatment of articular cartilage defects of any type, including chondral and subchondral defects, both those resulting from trauma, such as an accident or sports injuries, to diseases such as osteoarthritis. The porous fibrin matrix can be used per se or combined with other therapies. For example, the porous fibrin matrix can be used for cartilage reconstruction together with other therapeutic procedures such as chondral debridement, laser or abrasion chondroplasty and perforation or microfracture techniques.

[0044] Other typical orthopedic applications include joint arthroplasty, meniscus reconstruction, reconstruction of vicious consolidation in fractures, craniofacial reconstruction or intervertebral disc reconstruction. In addition, the porous fibrin matrix is suitable as a coating of synthetic material or other implants such as nails or plates, for example, in hip arthroplasty. Thus, the present invention further provides implants or medical instruments with a composite finish of the porous fibrin matrix of this invention.

[0045] The porous fibrin matrix of the invention has inter alia utility, as an unexpectedly beneficial support for cell growth. The absence of exogenous antifibrinolytic agents results in a fibrin matrix fully compatible with cell growth, proliferation and differentiation, both in vitro and in vivo. An additional advantage of the fibrin matrix of the invention is the improved ability to absorb cells and maintain their viability. The need to hydrate or rinse the sponge of the invention before cell culture is ruled out by the absence of exogenous antifibrinolytic agents, thus achieving a sponge with a superior cell incorporation capacity.

[0046] The porous fibrin matrix of the invention, being an effective support framework for cell growth, can be used in surgical reconstruction in vivo, for example as a matrix for the regeneration of tissues composed of hepatic, neuronal cells, urothelial, osteoblasts, cardiovascular tissue and breast tissue

or any other class of cells that are desired to grow on a three-dimensional support. Thus, the matrix of this invention can be used to make tissue equivalent to living tissue of, among many others, liver, pancreas, nerves, glands, tendons, skin, blood vessels, bone and ligaments.

[0047] According to an embodiment of the present invention, the matrix is a sponge or framework capable of supporting proliferation in a wide variety of cell types. In fact, the sponge is inoculated with cells and the cells can proliferate in vitro before implantation in vivo. Alternatively, the sponge is cultured with cells that have been cultured or extracted and the sponge, cells included, is implanted in situ. In one embodiment, the use of the porous fibrin matrix as an implant for transplantation is composed of autologous plasma proteins and autologous chondrocytes.

[0048] According to one embodiment, the present invention discloses a method for the treatment or reconstruction of damaged, diseased or traumatized tissue. The method comprises implantation in the area of damaged, diseased tissue.

or traumatized by a lyophilized porous fibrin sponge formed from plasma proteins composed of fibrinogen, thrombin and Factor III. The matrix has less than 10% residual water and is substantially free of exogenous antifibrinolytic agents, plasminogen and organic chelating agents. The tissue is selected from cartilaginous, bone, liver, mesenchymal, endothelial, epithelial, urothelial, endocrine, neuronal, pancreatic, renal and ocular tissue.

[0049] The invention also presents the use of an implant for the treatment or reconstruction of damaged, diseased or traumatized tissue. This use includes the implantation of a matrix of the present invention in the area of damaged, diseased or traumatized tissue. The tissue is selected from cartilaginous, bone, liver, mesenchymal, endothelial, epithelial, urothelial, endocrine, neuronal, pancreatic, renal and ocular tissue.

[0050] This and other embodiments are clarified below by means of figures, detailed descriptions and examples.

BRIEF DESCRIPTION OF THE FIGURES

[0051] The present invention will be better understood and fully appreciated thanks to the following detailed description and the accompanying figures:

Figure 1A shows a graph of the viability of porcine chondrocytes in the matrix substantially free of plasminogen compared to a standard sponge composed of an exogenous antifibrinolytic agent. Figure 1B shows the viability of human chondrocytes in a matrix substantially free of plasminogen compared to a standard sponge composed of exogenous antifibrinolytic agents. Figure 1C shows the viability of human chondrocytes cultured in matrices substantially free of plasminogen, with or without hyaluronic acid. Figures 1D and 1E show photographs of the matrices of the invention, dried and cultured with cells, respectively.

Figures 2A, 2B and 2C show histological sections of the distribution of chondrocytes in fibrin sponges substantially free of plasminogen, in cultures of one week.

Figures 3A and 3B show the degradation rate of the sponge substantially free of plasminogen compared to the sponge composed of tranexamic acid, in urea or collagenase.

Figures 4A and 4B depict the release of FGF from fibrin matrices substantially free of plasminogen composed of 0.08% cross-linked hyaluronic acid and different amounts of heparin prepared in two different ways. Figure 4C shows a bar graph of FGF release of plasma protein matrices composed of tranexamic acid (commercial) and human plasma protein matrix composed of partially purified plasma proteins and 0.024% hyaluronic acid, FGF and heparin, added ab initio. Figure 4D shows the FGF release pattern of plasma protein matrices substantially free of exogenous antifibrinolytic agents, composed of various concentrations of thrombin and heparin. Figure 4E represents the release of FGF from commercial and plasma protein sponges composed of FGF, ab initio.

Figures 5A, 5B, 5C and 5D show histochemical sections of a plasma protein composed of partially purified human plasma proteins cultured with porcine chondrocytes.

Figure 6A shows rat primary hepatocytes incubated for three days in a lyophilized porous plasma protein matrix, substantially free of plasminogen. Figures 6B and 6C show the CHO and L8 cell lines incubated for three days in a lyophilized porous plasma protein matrix substantially free of plasminogen.

Figure 7A shows the insertion of a lyophilized porous plasma protein carrier carrying substantially plasminogen-free cells into the subcutaneous pocket of a naked mouse. Figure 7B shows the neocartilage that developed.

Figures 8A, 8B and 8C show cross histological sections of a neocartilage nodule. Figure 8A shows the cell matrix formed after one week, such as those stained with toluidine blue and fast red. Figures 8B and 8C show histological sections of the H&E stained neocartilage nodule, with magnifications of 200x and 400x respectively.

Figures 9A and 9B show cross histological sections of the H&E stained neocartilage nodule, with increases of 10x and 100x.

Figure 10 depicts cell viability in plasma protein sponges prepared either by mixing the plasma protein and thrombin solutions beforehand or by mixing the solutions during casting. Figures 10B and 10C show cross histological sections of sponges carrying substantially free plasminogen cells prepared by previously mixing the plasma protein and thrombin solutions (10B) or mixing the plasma protein and thrombin solutions during the smelting stage.

DETAILED DESCRIPTION OF THE INVENTION

[0052] Although numerous biomatrices composed of plasma or tissue proteins are already known in the discipline to which the present invention belongs, none has fully and satisfactorily demonstrated compliance with the criteria required for success in tissue engineering and reconstruction. The present invention describes a porous fibrin matrix, also called a sponge, composed of plasma proteins substantially free of plasminogen and organic chelating agents. The absence of plasminogen in the matrix avoids the need for external antifibrinolytic agents. It is further described that, unexpectedly, biomatrices composed of partially purified plasma proteins also avoid the need to add exogenous antifibrinolytic agents. The compositions and methods of the present invention are effective both in vitro and in vivo in applications such as cell-bearing implants in tissue engineering and reconstruction.

[0053] The resulting fibrin, or plasma protein sponge, has properties that make it particularly beneficial for stimulating and facilitating cell growth both in vivo and in vitro. Plasminogen is a plasma protein enzymatically converted into an active serine protease, plasmin, which has fibrinolytic activity. This activity is the result of rapid degradation of fibrin in fibrin gum and matrices. Antifibrinolytic agents such as tranexamic acid and aprotinin are incorporated into fibrin gum, sponges and matrices to maintain the integrity of the substrate. The sponges of the present invention are stable and show reduced bioreabsorbability, avoiding the need to add exogenous antifibrinolytic agents.

[0054] Among the beneficial properties of the matrices of the invention:

Fibrin matrices show better biological properties including reduced biodegrabability, absence of immunogenicity or other adverse reactions, the ability to maintain and promote high rates of cell growth, proliferation, differentiation and migration and controlled release of bioactive agents.

[0055] The matrices have better mechanical properties, controlled by varying the additives used in the composition. Desirable properties include flexibility, elasticity and durability.

[0056] The matrices have better physical properties, controlled by varying the additives used in the composition. Desirable properties include texture, pore size and interconnection channels, hydrophilicity, adhesion, wettability, adhesion and texture.

[0057] Plasma proteins can be recovered from autologous or recombinant material, thus avoiding the need to extract it from the blood concentrate, as it carries health risks.

[0058] The matrices of the invention have all the fundamental components for tissue reconstruction, thus facilitating the work of the medical professional. In addition, the composition of the sponge makes it suitable for minimal invasive surgery of the articular cartilage. The sponge can be implanted during a miniarthrotomy or an arthroscopy, thus avoiding multiple surgery and total arthrotomy used for ACT.

Definitions

[0059] Certain terms, examples and statements used in the specification are described and clarified below.

[0060] The term "plasma" used herein refers to the liquid part and without human or animal blood cells in their state prior to coagulation.

[0061] The term "plasma protein" used herein refers to soluble proteins found in the plasma of a human being or a common animal. These include, but are not limited to, coagulant proteins, albumin, lipoproteins and complement proteins. The most important plasma protein is fibrinogen, which after division by thrombin in the presence of calcium ions and Factor XIII becomes fibrin. A fibrin matrix can be exchanged for a plasma protein matrix.

[0062] The term "plasminogen" used herein refers to plasminogen and plasmin. The terms "substantially free of plasminogen" or "without plasminogen" refer to plasma proteins with less than 20% of the plasminogen normally present in the plasma, preferably less than 10% of the plasminogen normally present in the plasma and preferably less than 5% of the Plasminogen normally present in plasma. Plasma is usually composed of approximately 200mg of plasminogen per liter of fresh plasma (approximately 2 Imoi / iitro). Piasminogen is the precursor of the active plasmin enzyme.

[0063] The term "substantial absence of organic chelating agents" or "substantially free of external antifibrinolytic agents" used herein refers to a concentration of less than 1mm of a Quranic chelating agent such as EDTA or other organic chelating agents known in the discipline. .

[0064] The term "substantially free of exogenous antifibrinolytic agents" or "substantially free of external antifibrinolytic agents" used herein refers to a solution of plasma or fibrinogen proteins to which no antifibrinolytic agent has been added. Many examples of antifibrinolytic agents include tranexamic acid (ASD), aprotinin and -aminocaproic acid (EACA). Fits

It should be noted that the small amount of exogenous antifibrinolytic agents may occur in plasma proteins due to the methods of processing it.

[0065] The terms "platelet rich plasma" or "PRP" used herein refer to platelet-composed plasma. A simple platelet extract, derivative or a platelet supernatant can be added exogenously. Alternatively, platelet-rich plasma can be prepared from methods already known in the discipline, including those presented in US Patents 6,475,175 and US 6,398,972.

[0066] The "matrix" used herein refers to a substance of porous structure, solid or semi-solid and biodegradable with pores and interconnecting channels wide enough to allow cells to colonize or invade the matrix. The fibrin matrix of the invention may have irregular or substantially regular pores. As used herein, the term "substantially regular pores" means that most pores

or more preferably substantially all pores are in the same size range. The materials that form the matrix require the addition of a polymerizing agent to form the matrix, such as the addition of thrombin in the presence of bivalent calcium ions to a solution composed of fibrinogen to form a clot. Next, the clot is lyophilized producing a porous fibrin matrix. The fibrin matrix of the present invention can be referred to herein as a framework, biomatrix or sponge, for use as an implant per se, for cell culture or as an implant of cell bearing tissue. Although the examples presented here refer to the use of the matrix for cartilage reconstruction, it should be understood that the matrix can be used for the reconstruction and regeneration of many other tissues such as bone, breast, epithelial, neuronal, liver and the endothelial.

[0067] The term "stem cells" used herein refers to an undifferentiated cell capable of proliferation. Stem cells are capable of producing both new stem cells and cells called "progenitors" that differentiate to produce specialized cells found in mammalian tissues and organs.

[0068] The term "biocompatible" used herein refers to materials that have low toxicity, clinically acceptable levels of reaction to foreign bodies in the living body and affinity with living tissue. [0069] The term "lyophilized" used herein refers to the preparation of a dry composition formed by rapid freezing and thawing in the frozen state (sometimes referred to as sublimation). This process can be done under vacuum at a reduced air pressure resulting from drying at a temperature lower than that required at maximum pressure.

[0070] The term "residual moisture" used herein refers to the amount of moisture remaining in the dried sample. It is established as a percentage of the sample weight. In fact, the fibrin matrices of the invention have left less than 15% residual moisture, preferably less than 10% residual moisture.

[0071] The term "cell carrier" used herein refers to the ability of the matrix to allow cells to be maintained within their structure. In fact, the cells are capable of invading the pores or channels of the matrix and can undergo proliferation and / or differentiation.

[0072] The term "implantation" used herein refers to the insertion into a patient of a sponge of the invention through which the implant serves to replace, totally or partially, damaged, diseased tissue or tissue that has been removed.

[0073] "Biologically active" or "bioactive agents" incorporated into the sponge, for example, growth factors, platelets and platelet extract and angionic factors among others, stimulate rapid growth or differentiation of cells in the implant, as well as a faster vascularization of it. As shown, the factors are effectively preserved in the sponge and form a source, or reservoir, of bioactive agent for prolonged release. Other bioactive agents include antibiotics, enzymes, additional plasma proteins or combinations thereof.

[0074] The "pore size" of a pore of a plasma protein sponge is determined by the equation: P = (L x H) 1/2 in which L and H are, respectively, the average length and height of the pores, as determined by the microscopic analysis of various sponges.

[0075] The term "polysaccharides" used herein refers to complex carbohydrates made from more than one saccharide. The definition includes anionic polysaccharides, including unmodified ones, such as chemical derivatives thereof, such as potassium or sodium salt, alkaline earth metal salt, such as calcium or magnesium salts. Many examples of anionic polysaccharides include pectin, alginate, galactans, galactomannans, glucomannan and polyuronic acids.

[0076] A "glycosaminoglycan" or "GAG" refers to unbranched long polysaccharides found on the cell surface or in the extracellular matrix. Many examples of glycosaminoglycan include heparin, chondroitin sulfate, dextran sulfate, dermatan sulfate, heparan sulfate, keratan sulfate, crosslinked and uncrosslinked hyaluronic acid, hexuronyl hexosaminoglycan sulfate and inositol hexasulfate. Also included in the invention are derivatives, mimetics and salts of the aforementioned, including low molecular weight heparin. Although it is not desirable to be conditioned by a particular theory, the presence of GAGs, in particular heparin, helps in the immobilization of growth factors, in particular by joining growth factors such as those of the Fibroblast Growth Factor family. (FGF).

[0077] The term "cartilage" used herein refers to a specialized type of connective tissue that contains chondrocytes embedded in an extracellular matrix. The biochemical composition of cartilage is different depending on the type, but in general it is composed of collagen, mainly type II collagen, among others that are in smaller quantities, such as types IX and XI, proteoglycans, other proteins and water. Numerous types of cartilage are known in this discipline, including, for example, hyaline, articular, costal, fibrous (fibrocartilage), meniscal, elastic, atrial and yellow cartilage. The production of any type of cartilage falls within the scope of the invention. The term "chondrocytes" used herein refers to cells capable of producing components of the cartilaginous tissue.

[0078] The term "variant" used herein refers to a polypeptide sequence that has certain modified structural properties of the matrix protein or in the wild. For example, the variant may be truncated either at the N-terminal end or at the C-terminal end or at both terminal ends or they may have deleted, inserted or substituted amino acids. The variant may have similar or altered activity when compared to that of the protein in the wild.

Embodiments of the Invention

[0079] The present invention relates to lyophilized porous fibrin matrices composed of plasma proteins substantially free of exogenous antifibrinolytic agents that function as support for cell growth. The present invention relates to the unexpected discovery that a lyophilized porous fibrin matrix composed of plasminogen-free plasma proteins shows superior biological characteristics, in particular cell viability and proliferation. Plasminogen is a plasma protein that is enzymatically converted into an active serine protease, plasmin, with fibrinolytic activity. This activity is the result of the rapid degradation of fibrin in clots and matrices. Antifibrinolytic agents are usually incorporated into the matrices and fibrin clots to preserve the integrity of the substrate. The matrices of the present invention lack plasminogen, thus obviating the need for exogenous antifibrinolytic agents, which have been found harmful to cells and tissue and which can unravel adverse reactions in patients. It has now been revealed that matrices composed of partially purified plasma proteins also obviate the need for exogenous antifibrinolytic agents. The compositions and methodologies of the following invention are effective in both in vivo and in vitro applications, including biocompatible implants in tissue engineering and biotechnology. The matrices, according to the present invention, can be used clinically, per se or as cell-carrying implants. They are true three-dimensional structures capable of providing support and conservation in cell growth and differentiation.

[0080] In fact, the present invention describes a lyophilized fibrin matrix composed of plasma proteins substantially free of exogenous antifibrinolytic agents, plasminogen and organic chelating agents. Substantially free from plasminogen refers to plasma proteins composed of less than 25% plasmin or plasminogen normally present in the plasma, preferably less than 10% plasminogen normally present in the plasma and more preferably less than 5% plasminogen normally present in the plasma

[0081] The inventors have discovered that a lyophilized fibrin matrix composed of plasma proteins substantially free of exogenous antifibrinolytic agents, plasminogen and organic chelating agents results in an improved matrix for clinical and biotechnological use. In addition to eliminating the need for exogenous antifibrinolytic agents and their consequent adverse effects, the inventors now show that the fibrin matrix of the present invention works best as a framework for cell culture, growth and differentiation, as well as in reconstruction and tissue regeneration

[0082] According to an embodiment of the present invention, the fibrin matrix is composed of plasma proteins, with fibrin being the main protein. Fibrin is obtained by the interaction of fibrinogen (Factor I) and thrombin in the presence of calcium ions (CA + 2) and Factor XIII or another fibrin stabilizing factor, to form a fibrin clot. The plasma protein used in the present invention can be purified if it comes from plasma concentrate or can be used from a commercial concentrate, including native or recombinant proteins, with a substantial absence of organic chelating agents. Whole blood, blood fractions, blood derivatives, cryoprecipitates, recombinant proteins, plasma or plasma fractions can serve as a source of plasma protein for the fibrin sponge of the present invention. The plasma concentrate can be allogeneic or autologous. Another source of plasma protein, specifically fibrinogen, includes fibrinogen variants, such as high molecular weight (HMW), low molecular weight (LMW) and LMW (LMW ') derivatives, as described, by example, in PCT patent application WO 03/087160.

[0083] Plasma proteins are substantially free of plasminogen. Plasminogen can be removed from plasma by methods already known in the discipline. Among the many examples, plasminogen is removed from plasma by affinity purification. Epsilon aminocaproic acid (EACA) ligands, like the lysine resin, are used to purify plasma plasminogen. PCT patent application WO 02/095019 describes a method to specifically eliminate plasminogen and plasmin in the presence of fibrinogen from a mixture such as blood or cryoprecipitate. The method requires contact of the plasminogen compound mixture with a hard amino acid, such as tranexamic acid, in which the amino group and the carboxylic group are approximately 7 angstroms away and the hard amino acid is linked by a covalent bond to the support through the amino group. In PCT patent application WO 95/25748 a topical plasminogen-free fibrinogen complex is described in which the plasminogen was removed using Sepharosa® C-lysine. Alternatively, some or all plasma proteins can be recombinant and plasminogen free, as described, for example, in PCT patent application WO 99/56797.

[0084] Plasma proteins are also free of exogenous antifibrinolytic agents, which have been shown to be detrimental to cell growth and that may lead to adverse reactions in the patient. Surprisingly, a matrix composed of partially purified plasma proteins also avoids the need for exogenous antifibrinolytic agents.

[0085] The fibrin matrix of the invention composed of plasma proteins substantially free of exogenous antifibrinolytic agents, plasminogen and organic chelating agents, can be used per se, for clinical and biotechnological use. It can also be composed of additives that add other biological, physical and mechanical advantages to the matrix. The international patent application in process WO 03/007873 made by some applicants of the present invention reveals a fibrin matrix composed of plasma proteins and at least one antifibrinolytic agent, also, optionally, composed of agents such as polysaccharides, anionic polysaccharides, glycosaminoglycans, or synthetic polymers that are added to the preparation to improve certain physical, mechanical and biological properties of the matrix. The incorporation of at least one of these agents was shown to improve characteristics such as elasticity and regular pore size of the sponge.

[0086] In one embodiment, the present invention is related to a porous fibrin matrix substantially free of exogenous antifibrinolytic agents, plasminogen and organic chelating agents composed, in addition, of at least one supplement selected from the group consisting of polysaccharides, glycosaminoglycans and synthetic polymers It acts as a support for the culture or growth of cells, both in vitro and in vivo. The incorporation of at least one additive to the materials that form the matrix results in a sponge with certain improved physical, mechanical and / or biological properties. The incorporation of at least one glycosaminoglycan has been proven to confer better characteristics, including the elasticity of the sponge. The resulting sponges are substantially homogeneous and have no particles or substructures beyond the pores and interconnecting channels.

[0087] In one embodiment the additive can be added ab initio, during clot formation. In another embodiment, the additive can be introduced into the matrix at any time after the formation of the sponge. According to various embodiments carried out during the present invention, the matrix is prepared using at least one glycosaminoglycan selected from the group consisting of crosslinked and uncrosslinked hyaluronic acid, heparin, derivatives and mimetics of heparin, chondroitin sulfate, dextran sulfate, dermatan sulfate, heparan sulfate and keratan sulfate. In fact, glycosaminoglycan is incorporated into the matrix during the initial formation of the clot. In one embodiment the glycosaminoglycan is hyaluronic acid. Glycosaminoglycan is added to a final concentration that gives flexibility and elasticity to the sponge and discards the need to add moisture to the final composition. Hyaluronic acid can be crosslinked or uncrosslinked and have a variety of different molecular weights and can be of animal origin or from a recombinant source. According to one embodiment, the concentration of uncrosslinked hyaluronic acid is 0.005% to 0.5% final (v / v), more preferably 0.05% to 0.1%. In another embodiment the concentration of crosslinked hyaluronic acid is 0.001% to 0.1% and more preferably 0.05% to 0.09% final concentration. According to one embodiment, the glycosaminoglycan is selected from heparin and its derivatives.

[0088] According to another embodiment, the present invention may also include the incorporation of an additional synthetic or natural polymer prior to clot formation that can modify certain physical, mechanical and / or biological properties of the sponge. These can give the sponge better characteristics including elasticity, regular pore size and strength. Many examples of natural polymers include cellulose, pectin, polyuronic acids, hexuronyl hexosaminoglycan sulfate and inositol hexasulfate.

[0089] The synthetic polymers used in the present invention can be biodegradable or non-biodegradable. Examples of non-biodegradable materials include polytetrafluoroethylene, perfumed polymers such as fluorinated ethylene propylene, polypropylene, polyethylene, polyethylene terephthalate, silicone, silicone rubber, polysulfone, polyurethane, non-degradable polycarboxylate, non-degradable polycarbonate, non-degradable polyester, polyacrylic methacrylate , polymethylmethacrylate, polyamides such as polyesteramide and copolymers, block copolymers and combinations of all these materials.

[0090] Some examples of degradable materials include hydrophilic polyesters such as polylactic acid and polyorthoester acid, degradable polycarboxylates, degradable polycarbonates, degradable polycaprolactones, polyanhydrides and copolymers, block copolymers and combinations of all these materials. Other components include surfactants such as lecithin.

Bioactive Agents

[0092] In one embodiment, the matrix of the invention is further composed of at least one bioactive agent, such as cytokine, a growth factor and its activators, platelets, a bioactive peptide, etc. Although it is not desirable to be conditioned by any particular theory, the incorporation of such agents into the sponge of the present invention provides a slow or controlled release mechanism. When the matrix degrades in vivo, bioactive agents are released in the surrounding environment. For example, growth factors, structural proteins or cytokines are additives used in the matrix of the present invention, improving the temporal sequence of wound healing, angiogenesis, altering the rate of proliferation or increasing the metabolic synthesis of the matrix. of extracellular proteins. The bioactive proteins of the invention are polypeptides or derivatives or variants thereof, of natural, synthetic or recombinant origin, which show the ability to stimulate DNA synthesis and cell division or differentiation of a variety of cells, including primary fibroblasts, embryonic stem cells. (ESC), adult stem cells, chondrocytes, vascular and corneal endothelial cells, osteoblasts, myoblasts, smooth muscles and neuronal cells. Representative proteins include bone growth factors (BMPs, IGF) and cartilage growth factor genes (CGF, TGF-�) for healing, nerve growth factor genes (PDGF), vascular endothelial growth factor ( VEGF), insulin growth factor (IGF-1), keratinocyte growth factor (KGF), supplement for endothelium-derived growth factor (EDGF), epidermal growth factor (EGF) and other proteins that can improve the action of growth factors including heparan sulfate proteoglycans (HSPGs) and their mimetics such as dextran sulfate, sucrose or heparin octasulfate and fragments thereof. It has been proven that other factors that act in cells in bone, cartilaginous or other connective tissue formation include retinoids, growth hormone (GH) and transferrin. Specific proteins for cartilage reconstruction include cartilage growth factor (CGF), FGFs and TGF-�.

[0093] Other biologically active agents that may be included in the matrix include blood platelets, supernatant or platelet extract and proteins derived from platelets, hormones, analgesics, anti-inflammatory agents, antimicrobials or enzymes. Bioactive agents composed of platelets, platelet extract or supernatant promote the proliferation and differentiation of bone cells including chondrocytes and osteoblasts, as well as other cell types including, among others, hepatocytes and endothelial cells. Bioactive agents belonging to the class of antimicrobial or anti-inflammatory agents can accelerate the healing process by minimizing infection and inflammation. Enzymes such as chondroitinase or metalloproteinase matrix (MMPs) can be incorporated to aid cartilage degradation, thus stimulating the release of cells in the matrix and the surrounding area. Among many other examples, the at least one bioactive agent, added ab initio or at any other stage of the preparation, can be selected to improve the healing process of damaged or diseased tissues.

[0094] According to an embodiment carried out during 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 a morphogenetic bone protein (BMP) or a variant thereof. In another embodiment, the FGF is an FGF or a variant that has the capacity to cause reconstruction, regeneration or angiogenesis of bone and cartilage. Growth factors can be added to a wide range of concentrations, depending on their use. For certain uses, prolonged release of the bioactive agent is preferable. The prolonged release of a bioactive agent can depend on various factors such as the concentration of the growth factor, the type of glycosaminoglycan incorporated and the concentration of thrombin.

[0095] In contrast to the heteromorphic bioabsorbable sponge already known in the discipline, the present inventors now reveal a lyophilized homogeneous fibrin sponge composed of plasminogen-free plasma proteins and organic chelating agents, composed in addition to at least one additive selected from the group consisting of polysaccharides, glycosaminoglycans and synthetic polymers and at least one bioactive agent provided that the release of said bioactive agent is gradual.

[0096] According to several specific embodiments of the present invention, the porous fibrin matrix composed of plasma proteins substantially free of antifibrinolytic and plasminogen agents, further comprises at least one glycosaminoglycan and at least one bioactive agent in which the bioactive agent is a protein therapeutic belonging to the family of growth factors FGF. In one embodiment, a porous fibrin matrix composed of plasma proteins substantially free of plasminogen and organic chelating agents, is further composed of hyaluronic acid, heparin and an FGF. Hyaluronic acid and heparin or their mimetics are incorporated into the sponge ab initio.

[0097] According to one of the multiple examples, the invention features a homogeneous lyophilized porous fibrin matrix composed of plasminogen-free plasma proteins and organic chelating agents, it is further composed of at least one glycosaminoglycan and at least one bioactive agent, in the that the at least one glycosaminoglycan is heparin and the at least one bioactive agent is a therapeutic protein belonging to the family of FGF growth factors or one of its variants. This sponge presents a gradual release of matrix FGF and may be beneficial in certain therapeutic applications. Optionally, at least one bioactive agent can be added to the cell during culture to, for example, improve a therapeutic effect.

[0098] In addition, genetically produced cells to extract the aforementioned proteins are included in the present invention. Periosteal cells, mesenchymal stem cells or chondrocytes are used per se or by transfection with cartilage growth factor genes selected from a group consisting of TGF- �� some FGFs or CGF for cartilage reconstruction and regeneration; For periodic bone repair, other mesenchymal stem cells or osteoblasts are used per se or transferred with bone growth factor genes selected from a group consisting of bone morphogenetic protein (BMP), family genes, or family genes of the fibroblast growth factor family. ; for nerve repair, neuronal and neuronal support cells are used per se or transferred with genes selected from a group consisting of the nerve growth factor (NGF) gene or specific FGFs.

[0099] In addition, specific enzymes can be incorporated into the sponge of the invention to improve the degradation of proteoglycans and / or the proteins present in cartilage. The cartilage chondrocytes are embedded in the thick extracellular matrix (ECM) of the joint. Although it is not desirable to be conditioned by any particular theory, enzymes known in the discipline, including collagenase, trypsin, cyotripsin and controlinase of various types, are capable of degrading the ECM from the surface of the joint, thus releasing chondrocytes capable of invading the sponge of the invention to improve cartilage regeneration.

[0100] The matrix of the invention, in certain embodiments, may also include one or more antiseptics, such as methylene blue and / or one or more drugs including antimicrobials such as antibiotic and antiviral agents; chemotherapeutic agents; anti-rejection, analgesic and combination agents thereof; anti-inflammatory agents; Adhesion proteins such as fibronectin or fragments thereof, and hormones such as steroids.

[0101] According to one embodiment the at least one bioactive agent is platelets or platelet supernatant. Platelets may be present in the plasma protein concentrate or may be added exogenously. An exogenous source of platelets is added during the clot formation process at a final concentration of 0.1% to 30% of the final volume of the sponge, more preferably 5% to 25% of the final volume of the sponge. An exogenous source of platelet supernatant is added during the clot formation process at a final concentration of 0.1% to 30% of the final volume of the sponge, more preferably 1% to 15% of the final volume of the sponge.

Applications

[0102] The homogeneous porous fibrin matrix of the invention is useful for both use and framework in tissue engineering. The absence of plasminogen avoids the need for external antifibrinolytic agents, resulting in a fully biocompatible sponge. The optional presence of bioactive agents together with glycosaminoglycan provides an unexpected and advantageous support for cell growth in vitro and in vivo.

[0103] The in vivo uses of the plasma matrix are multiple. The fibrin shell can be used as an implant per se or to provide mechanical support to a defective or damaged area in situ and / or a matrix in which the cells of a defective or damaged area proliferate and differentiate. The cells can be stem cells, progenitor cells or specialized cells such as chondrocytes, osteoblasts, hepatocytes or mesenchymal, endothelial, epithelial, urothelial, endocrine, pancreatic, renal or ocular neuronal cells.

[0104] The homogeneous porous fibrin matrix of the present invention can be used in surgery as a method of reconstruction and / or repair of tissues that have been damaged, for example, in trauma, in a disease or in surgical procedures. The invention has a matrix that can be used as an implantable framework per se or for tissue regeneration. According to one aspect of the invention, the matrix serves both as a physical support and as an adhesive substrate for cell growth in vivo. When the population grows and the cells function normally, they begin to secrete their own extracellular matrix (ECM) support.

[0105] Uses as a framework include the regeneration of tissues such as neuronal, musculoskeletal, cartilaginous, tendon, hepatic, pancreatic, renal, ocular, arteriovenous, urinary or any other type of tissue that forms hard or hollow organs. Some typical orthopedic uses include joint arthroplasty, meniscus reconstruction, reconstruction of vicious consolidation in fractures, craniofacial reconstruction or intervertebral disc reconstruction.

[0106] The porous fibrin matrix of the invention is useful, inter alia, as an unexpected advantageous support for cell growth. The absence of exogenous antifibrinolytic agents results in a fibrin matrix fully compatible with cell growth, proliferation and differentiation in vivo and in vitro. An additional advantage of the fibrin matrix of the invention is the demonstrated ability to absorb or retain cells. The need to moisten or wash the sponge of the invention before cell culture is ruled out by the absence of exogenous antifibrinolytic agents. In one embodiment, the matrix of the invention serves as a framework for cell growth, proliferation and / differentiation, including stem cells, progenitor cells or other cell types including chondrocytes, osteoblasts, hepatocytes, mesenchymal, epithelial type cells. , urothelial, neuronal, pancreatic, renal or other types of cells suitable for culture on a three-dimensional support.

[0107] In a certain embodiment of the present invention the cells can be cultured in the matrix for subsequent implantation. Stem cells derived from any tissue or produced to differentiate into a specific type of tissue can also be used. Preferably cells derived from autologous tissue. For example, for cartilage culture, chondrocytes or mesenchymal stem cells can be cultured in the matrix. In specific embodiments of the invention, chondrocytes or chondrocyte progenitor cells can be cultured in the matrix before implantation or in the implantation area in vivo. The sponge is useful for the release of cells in situ in a specific area of the body, such as dopamine-producing cells in Parkinson's patients.

[0108] Additionally, the cell of interest can be produced to express a genetic product that would exert therapeutic effect, for example peptides or aintiinflamarian proteins, growth factors with angiogenic, chemotactic, osteogenic or proliferative effect. A large number of examples of genetically produced cells to improve healing are disclosed in US Patent 3,698,816.

[0109] According to certain embodiments of the invention, the fibrin matrix is used as a support for the growth of chondrocytes and as a framework for the formation of new cartilage. However, the plasma matrix of the invention can be used as a useful surface for tissue culture of any cell, such as mesenchymal or other tissue-forming cells at different power levels. For example, cells typically called "stem cells" or "mesenchymal stem cells", which are pluripotent or independent cells, can be developed in the matrix of the invention, with an unlimited potential capacity to divide mitotically or to renew or produce progenitor cells capable of differentiate in any type of cell. In addition, cells responsible for lineage can develop in the matrix of the invention. A cell responsible for the lineage is normally considered incapable of dividing mitotically in an unlimited way and will differ only to a specific cell type. Such types include chondrocytes, osteoblasts, hepatocytes or mesenchymal, endothelial, epithelial, urothelial, endocrine, neuronal, pancreatic, renal or ocular cells.

[0110] In another embodiment of the invention, the lyophilized homogeneous porous fibrin matrix can be used as a coating of synthetic material, implants or medical material. The matrix of the invention can be applied to prostheses such as nails or plates by coating or adhesion, methods already known to those skilled in the art. The matrix coating, which is capable of supporting and facilitating cell growth, can thus be useful for presenting a favorable environment for the implant or prosthesis.

[0111] A person skilled in the discipline can adapt the procedures exemplified below according to the specific requirements of the fabric. For example, for cartilage reconstruction, the lyophilized homogeneous porous fibrin matrix of the invention can be used in conjunction with other therapeutic procedures such as chondral debridement, laser or abrasion chondroplasty and perforation or microfracture techniques.

[0112] Preferably, the fibrin sponge is implanted per se and serves as a framework for cell growth in situ. Alternatively, the matrix is cultured with the desired cells, which proliferate, and the sponge containing these cells is implanted in the area that needs tissue reconstruction or regeneration. The homogenous enriched fibrin and glycosaminoglycan matrix, in its dry state, adheres exceptionally well to the tissue surface. According to an embodiment of the present invention, a dry sponge of the invention, or another type of bioabsorbable matrix, is located in the area where tissue regeneration is sought. A second sponge, in which particular cells have been cultured, is placed on top of the dry sponge. The wet sponge of the invention adheres well to the dry sponge of the invention or to another matrix. During the healing process, the sponge cells in which the cells were originally grown migrate to the matrix, adhering directly to the tissue regeneration area.

[0113] In the reconstruction of structural tissues such as cartilage and bone, the shape of the tissue is essential in function, requiring a matrix molding in products of three-dimensional configuration of different shape and thickness. Consequently, the shape is determined by the shape of a mold, receptacle or support that can be made of any inert material and can be in contact with the matrix on all sides, be it sphere or cube, with a limited number of faces or as a sheet. The matrix may be in the form of an organ of the body or parts of a prosthesis. By removing portions of the die with scissors, scalpel, laser or other sharp instruments, any detail that requires the three-dimensional structure can be created.

[0114] The matrix, according to various embodiments of the invention, can be used as a matrix for cell growth or for tissue culture in vitro. The matrices of the invention have a relatively wide surface for cell growth and a mechanically improved framework for implantation.

[0115] The methods for culturing cells in the matrix are numerous. Among the many examples, cells are absorbed by placing the cells on a matrix surface or absorbed by the matrix by placing the sponge in a solution composed of cells. The matrix can be cultured with the desired cells by surface culture, at a density of approximately 104 cells per cm3, more preferably about 105 cells per cm3.

[0116] It will be appreciated that the matrix of the invention can support the growth and / or implantation of any type of cartilage or tissue. In addition, although the invention is predominantly directed to methods for the growth and / or implantation of tissues in humans, the invention may also include methods for the growth and / or implantation of tissues in other mammals.

[0117] In addition, the sponge of the present invention can be used as a component of a biphasic or multiphase material for tissue reconstruction as seen in osteochondral defects. Among the many examples, a layer may contain calcium phosphate material although a supplementary layer may contain the sponge of the invention. Gao et al. (Tissue engin. 8: 827-837, 2002) describes a method of reconstruction of osteochondral defects in the knees of a rabbit using a composite material formed by injectable calcium phosphate and a hyaluronic acid sponge.

Matrix Preparation Method

[0118] The present invention presents a method for the preparation of a homogeneous porous fibrin matrix. Matrix formation solutions include a thrombin solution and a plasma protein solution. The thrombin solution used herein is composed of a sufficient amount of thrombin to cleave fibrinogen and produce a fibrin matrix in the presence of calcium ions (Ca + 2). Plasma proteins can be derived from a commercial, xenogenic, allogeneic or autologous source comprising fibrinogen and factor XIII substantially free of plasminogen and in considerable absence of organic chelating agents. The plasma protein solution may contain fibrinogen derivatives such as high molecular weight or low molecular weight variants.

[0119] According to an embodiment carried out during the present invention, the homogeneous porous fibrin sponge is prepared by transferring the thrombin solution to a mold, adding plasma protein solution, freezing and lyophilizing the coagulated mixture. Alternatively, the plasma proteins are mixed with thrombin in the presence of calcium ions under appropriate conditions to achieve coagulation, the mixture is melted in a solid mold or support before coagulation, the coagulated mixture is frozen and lyophilized. It should be taken into account that once the mixture is incorporated, additives and bioactive agents are added independently to each of the matrix formation solutions, that is, the plasma proteins or the thrombin solution, before the formation of the Clot, are placed in a solid mold or support before the addition of thrombin, simultaneously or after.

[0120] A method for the preparation of a lyophilized porous fibrin matrix formed from plasma proteins that have less than 10% residual moisture and that are substantially free of exogenous antifibrinolytic agents, plasminogen and organic chelating agents, comprises the following steps :

provide a thrombin solution and a plasma protein solution where the plasma protein solution is substantially free of exogenous antifibrinolytic agents, plasminogen and organic chelating agents.

Introduce the thrombin solution and the plasma protein solution into a solid support or template in the presence of calcium ions.

Incubate under appropriate conditions to achieve coagulation.

Freeze the coagulated mixture.

Lyophilize the coagulated mixture to obtain a sponge.

[0121] According to an embodiment carried out during the present invention, plasma proteins are partially purified plasma proteins. Plasma proteins are free from plasminogen, that is, the plasma protein solution includes less than 20% plasminogen normally present in the blood plasma, preferably less than 10% plasminogen normally present in the plasma and more preferably less than 5% of Plasminogen normally present in plasma.

[0122] According to an embodiment carried out during the present invention, the matrix can be prepared by sequential introduction of thrombin solution and plasma protein solution into a solid mold or support. Either solution can be poured first. According to other embodiments carried out during 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 terms of porosity and cell dispersion.

[0123] A method for the preparation of a lyophilized porous fibrin matrix with less than 10% residual moisture and substantially free of exogenous antifibrinolytic agents, plasminogen and organic chelating agents and containing at least one additive selected from the group consisting of polysaccharides, glycosaminoglycans and synthetic polymers, comprising the following steps:

Present a plasma solution substantially free of exogenous, plasminogen and antifibrinolytic agents. organic chelating agents, a thrombin solution and where at least the plasma protein solution or the thrombin solution contains at least one additive selected from the group consisting of polysaccharides, glycosaminoglycans and synthetic polymers. Introduce the thrombin solution and the plasma protein solution into a solid support or mold. Incubate under appropriate conditions to achieve coagulation. Freeze the coagulated mixture. Lyophilize the coagulated mixture to obtain a sponge.

[0124] The sponge can be composed of at least one bioactive agent, added ab initio either to the thrombin solution or to the plasma protein solution.

[0125] According to an embodiment carried out during the present invention, plasma proteins are partially purified plasma proteins. Plasma proteins are free from plasminogen, that is, the plasma protein solution contains less than 20% plasminogen normally present in the blood plasma, preferably less than 10% plasminogen normally present in the plasma.

[0126] According to other embodiments carried out during the present invention, plasma proteins substantially free of exogenous antifibrinolytic agents, plasminogen and organic chelating agents, in a concentration of 20 mg / ml to 50 mg / ml, is mixed with hyaluronic acid and / or heparin and the mixture is added to the thrombin solution in the solid support for the formation of a clot. The clot is frozen and lyophilized. [0127] The final concentration of thrombin can be varied in order to produce sponges with different biological, physical and mechanical characteristics useful for different applications. Thrombin concentrations of 0.5 IU / ml at 2 IU / ml produce sponges with similar properties in terms of viability and cell growth. Other concentrations as low as 0.15 IU / ml may also be useful, depending on the application.

[0128] In its final form before use with cells, the sponge is substantially dry and contains less than 15% residual moisture, more preferably less than 10% residual moisture.

[0129] However, another aspect of the present invention presents methods of treatment and use of the fibrin matrix of the invention for the treatment of damaged or traumatized tissue, including cartilage and bone defects. The treatment method described in this document provides an advantage, since it requires minimal preparation by the medical professional for its use. The in vivo uses of the porous fibrin matrix are multiple. The porous fibrin matrix can function as a framework and can be used as an implant per se, providing mechanical support to a defective or damaged area in situ and / or providing a matrix within which cells from the area proliferate and differentiate damaged and defective. For example, the porous fibrin matrix can be used for cartilage reconstruction together with other therapeutic procedures such as chondral debridement, laser or abrasion chondroplasty and perforation or microfracture techniques.

[0130] Being an effective framework that supports cell growth, the porous fibrin matrix of the invention can also be used in in vivo reconstructive surgery, for example, as a matrix for the regeneration of cells and tissues, including neuronal cells, cardiovascular tissue, urothelial cells and breast tissue. Other typical orthopedic applications include joint arthroplasty, meniscus reconstruction, reconstruction of vicious consolidation in fractures, craniofacial reconstruction or intervertebra disc reconstruction. The fibrin matrix of the invention can be used for the treatment of defects resulting from diseases such as osteoarthritis. The matrix components melt into a mold designed specifically for a different lesion or defect. Among the many examples, the mold can be prepared by computer-aided design. In other cases, the doctor will have to cut or make a fine cut on the sponge to reconstruct a particular injury or defect. The matrix of the invention is particularly beneficial in minimally invasive surgery, such as miniarthrotomy or arthroscopy and avoids the need for open joint surgery.

[0131] In addition, the porous fibrin matrix is suitable as a coating of synthetic material or other implants such as nails or plates, for example, in hip arthroplasty. Thus, the present invention further provides implants or medical instruments with a composite finish of the porous fibrin matrix of the invention.

[0132] On the other hand, the sponge of the present invention can be used as a component of a biphasic or multiphase material for tissue reconstruction, as verified in osteochondral defects. Among the many examples, a layer may contain calcium phosphate material although a supplementary layer may contain the sponge of the invention.

[0133] The plasma protein solution may come from a commercial source, from natural or recombinant proteins, or it may be prepared from plasma. According to an embodiment carried out during the present invention, the plasma protein solution is derived from allogeneic plasma. According to another embodiment carried out during the present invention, at least one of the components, preferably plasma proteins, are used for the preparation of the matrix from autologous plasma or recombinant proteins. According to another embodiment carried out during the present invention, all plasma components used in the preparation of the matrix are autologous. Plasma proteins can be isolated following a variety of methods, known in the discipline and exemplified below, resulting in a fibrin matrix with substantially similar properties, measured by pore size capabilities, elasticity, compression and capabilities. of cell carriers. A stable thrombin component can be isolated from autologous plasma, according to methods known in the discipline, for example those described in US Patent No. 6,274,090 and Haisch et al (Med. Biol. Eng. Comput 38: 686- 9.2000).

[0134] The resulting fibrin matrix shows advantageous properties including biocompatibility, pore size compatible with cell invasion and proliferation and the ability to be molded or molten to achieve a certain shape.

[0135] In fact, blood is drawn from a patient according to the need for tissue reconstruction or regeneration, plasma proteins are isolated from the autologous plasma and from a matrix prepared therefrom. Platelets are optionally isolated and returned to plasma. The matrix of the present invention can serve as an implant for use as a framework per se or as a cell-bearing framework for implantation in vivo.

[0136] According to an embodiment carried out during the present invention, a porous fibrin sponge produced from a fibrinogen solution, where the fibrinogen solution is dialyzed with a solution that does not require a complexing agent, serves as a framework for cell growth in vitro and in vivo. According to another embodiment, the fibrin sponge is formed by the action of a thrombin solution in the dialyzed fibrinogen solution and subsequently subjected to lyophilization.

[0137] Although it is not desirable to be conditioned by any particular theory, the substantial absence of organic complexing agents, can provide the matrix of the present invention with beneficial properties for the proliferation and metabolism of various cell types. As shown in the examples of this document, the matrix of the present invention maintains the proliferation of cartilage cellular systems both in vivo and in vitro.

[0138] The presence of certain organic complexing agents in a range of 1 to 20 mM, necessary for the production of a flexible fibrin network, as described in US 6,310,267 for wound healing, may itself have a detrimental effect on the proliferation of certain types of cells. The use of a fibrin network for cell growth and proliferation in vivo or in vitro has not been disclosed, however, it may be possible for certain cultures of different cell types using the networks of the aforementioned patent.

[0139] According to an embodiment carried out during the present invention, heparin is incorporated into the matrix with a final concentration of 0.1 ug / ml to 1 mg / ml. In another embodiment, the heparin concentration is from 1 ug / ml to 50 ug / ml. As indicated herein, ug / ml refers to one microgram per milliliter.

[0140] According to another embodiment carried out during the present invention, the crosslinked hyaluronic acid is incorporated into the matrix with a final concentration of 0.001% to 0.1%, more preferably 0.05% to 0.09%.

[0141] According to another embodiment carried out during the present invention, uncrosslinked hyaluronic acid is incorporated into the matrix with a final concentration of 0.005% to 0.5%, more preferably 0.05% to 0.1%.

[0142] According to another embodiment carried out during the present invention, both heparin and hyaluronic acid are incorporated into the matrix at respective concentration ranges.

[0143] To the surprise, in view of the known function of heparin as an anticoagulant, it has now been revealed that the incorporation of heparin into the matrix does not interfere with either the formation of the matrix or the therapeutic benefits thereof. Without wishing to be conditioned by theory, heparin has as its main function to bind FGF or other therapeutic proteins and create a reservoir for the prolonged release of said proteins. In addition, the low molecular weight fragments of the heparin released from the matrix can function as anti-inflammatory agents and assist in the healing process of diseased or traumatized tissue (US patents 5474987; 5686431; 5908837).

[0144] The following examples are intended to be merely illustrative in nature and should not be construed in a limiting manner.

EXAMPLES

Example 1: Preparation of a fibrin matrix

[0145] Although methods for the preparation of plasma protein are given in detail, it should be understood that other methods of preparation of plasma proteins useful in the preparation of the matrix of the present invention are known in the discipline. One of the many examples of protocol for the preparation of an enriched fibrinogen solution has been described by Sims, et al. (Plastic & Recon. Surg. 101: 1580-85, 1998). Any source of plasma proteins can be used, as long as the plasma proteins are processed to be substantially free of antifibrinolytic agents, plasminogen and organic chelating agents. See examples 2 and 3 of the methods for preparing plasma protein below.

Materials and methods:

[0146] Source of plasma proteins, for example, plasminogen-free fibrinogen (Omrix, IL) approximately 50-65 mg / ml of stock solution.

5 mM calcium chloride

Thrombin (1,000 international units / ml, Omrix, IL.

[0147] Optional: Hyaluronic acid, cross-linked (Hylan (Synvisc), approx. MW 6 x 106, Genzyme, US) or non-crosslinked (approx. MW 8 x 105, MTF, US; approx. MW 3.6 x 106, BTG, IL).

[0148] The thrombin concentration determines the reaction time for the polymerization of the fibrin monomers and contributes to the pore size and thickness of the fibers of the final sponge. A concentration of 0.15 IU at 15 IU of thrombin / mg of plasma proteins produced a sponge with good physical and biological properties. Concentrations of 1 IU at 1.5 IU of thrombin / mg of plasma proteins were chosen as it resulted in a rapid reaction, but allows sufficient time to pour the two solutions (plasma protein and thrombin) before the completion of the reaction. It should be noted that other concentrations are acceptable for obtaining a matrix with substantially similar properties. For simplicity, as shown herein 1.5 IU of thrombin / mg of total protein equals 30 IU of thrombin / ml.

[0149] The plasma protein solution and the thrombin solution are mixed together in a 2: 1 ratio (for example 210 μl of plasma protein and 90 μl of thrombin solution) in the following order: A 48-well plate of ELISA it was coated with 90 μl of thrombin solution, and the plasma protein solution was added. On the other hand, to a 96-well plate of ELISA, about 19.5 ul of thrombin solution was added, adding 45.5 ul of plasma protein, or for a slightly thicker matrix about 24 ul of solution was added of thrombin and 51 ul of plasma proteins. The mixture was incubated at room temperature (~ 25 ° C) for approximately 10 minutes or until clot formation, freezing it with an oscillation between 60 ° C and -90 ° C several days for 30 minutes. The sponges of the 48-well plates were lyophilized for 5 hours while the sponges of the 96-well plates were lyophilized for 4 hours. The 96-well plate produced sponges of about 5 mm in diameter and the 48-well plates produced approximately 10 mm (1 cm) in diameter (about 0.8 cm2).

[0150] A 35 mm diameter sponge was prepared for the reconstruction of larger defects, such as those that may develop in osteoarthritis. A 35 mm (9.5 cm2, or about 2 to about 2.5 cm3) sponge was prepared by mixing 2 ml of plasma protein solution with 1 ml of thrombin, melted in an appropriate mold, such as a petri dish of 35 mm or a 6-well cell culture plate, being frozen and lyophilized at -40 ° C for 12 hours. Fibrin sponges were prepared under aseptic conditions. It should be noted that the solutions can be melted in a mold in the desired way. The resulting sponge was a matrix with mesh structure.

[0151] According to another embodiment carried out during the present invention, the matrix was prepared with certain additives including polysaccharides, glycosaminoglycans and synthetic polymers. It has been shown that biological, mechanical and physical parameters can be controlled by the incorporation of additives. All additives were filtered (0.2 μm) and added to the plasma protein solution. When the hyaluronic acid was incorporated into the matrix, the plasma protein solution and the hyaluronic acid solution, they were incubated together before casting or molding. Below are several examples of additives and concentrations tested in Table 1:

Table 1

Additive

% final concentration

Glycerol

 0.005; 0.01; 0.05; 0.1; 0.5; one

Crosslinked AH (X-linked)

0.0024; 0.012; 0.024; 0.05; 0.10; 0.5

AH Non-X-linked

0.002; 0.02; 0.05; 0.07; 0.08; 0.09; 0.1; 0.11; 0.13

Heparin

0.05; 0.1; 0.5; 1.0; 2.5; 10 ug / ml final

Heparin + Reticulated AH

Combinations above

Heparin + AH Non-X-linked

Combinations above

Glycerol + AH

Combinations above

Note: non-X-linked refers to uncrosslinked hyaluronic acid.

[0152] A therapeutic protein, FGF (sponge 1 to 10 ug / 0.2cm2) was added either to the plasma protein solution or mixed with heparin and then added to the plasma protein or thrombin solutions. Experiments have been carried out to determine the optimum concentration of the additives in terms of matrix flexibility, elasticity, pore size, prolonged release of bioactive agents and cell growth capacity. The additives confer beneficial properties, including surface, mechanical and / or biological properties to the sponge during its preparation. Optimization is also carried out with respect to the concentration of bioactive agents. In one embodiment, the bioactive agents include growth factors, platelet supernatant, native platelets, platelet membranes and other materials. According to an embodiment carried out during the present invention, it has a matrix composed of heparin or a derivative thereof and compound hyaluronic acid, in addition to FGF or FGF variants. Below, the examples are shown.

Example 2: Isolation of partially purified plasma proteins from whole plasma

[0153] Plasma protein can be prepared from different sources, such as fresh plasma, frozen fresh plasma, recombinant and xenogenic proteins and allogeneic or autologous blood. Fresh frozen plasma was received from the blood bank (Tel-Hashomer, Israel). The plasma (220 ml) was thawed in an incubator at 4 ° C overnight, followed by centrifugation at 4 ° C and 1900g for 30 min. The pellet was resuspended in 2.5ml of PBS with a gentle vibratory motion until a homogenized solution was seen. The total protein concentration was approximately 42-50 mg / ml according to the Bradford assay and the SDS-PAGE (compared to a standard). Plasma can also be treated to eliminate plasminogen, using methods known in the discipline. In PCT patents WO 02/095019 and WO 95/25748 numerous examples of useful methods for the removal of plasminogen from blood or derivatives thereof such as plasma or cryoprecipitate are described.

[0154] It should be understood that the source of plasma protein can be xenogeneic, allogeneic or autologous blood. Preferably, the plasma protein source is allogeneic or autologous. In US 6475175 one of the many examples for the isolation of platelet rich plasma is described.

[0155] Another embodiment carried out during the present invention features a plasma protein sponge that incorporates at least one additive and blood platelets or platelet supernatant. The sponges are composed of 0.024% or 0.08% of the final concentration of hyaluronic acid and 1% or 10% is prepared with the final concentration of released platelet supernatant or with the platelet set. The platelet supernatant was made by exposing the isolated platelets (obtained from the blood bank of Israel) to thrombin (Gruber et al., Clin Oral Implants Res. 13: 529-535, 2002), collecting the supernatant and adding it to the plasma protein solution before sponge formation. Sponges containing platelets were prepared by directly adding platelets to plasma proteins as follows: 73 ul platelets and additives (0.024% hyaluronic acid or 0.08% final concentration) were added to the plasma proteins ( 30 mg total protein / ml) and the solution was brought to a final volume of 210 ul. The sponge was made as described above using partially purified plasma proteins.

Example 3: Extraction of allogeneic or autologous plasma protein fractions

Materials:

[0156]

1) 3.8% sodium citrate or any other pharmaceutically acceptable anticoagulant

2) Ammonium sulfate (NH4) 2SO4, saturated (500g / l)

3) 25% Ammonium Sulfate (NH4) 2SO4

4) EDTA-phosphate regulatory solution: 50 mM phosphate, 10 mM EDTA, pH 6.6

5) Tris-NaCl regulatory solution: 50 mM Tris, 150 mM NaCl, pH 7.4

6) Ethanol, absolute 4 ° C

7) Total blood (Blood Bank of Israel, Tel Hashomer Hospital or patient)

Methods:

[0157] This method can be used to produce plasma proteins that can be treated for plasminogen removal by methods known in the discipline, including affinity chromatography. Plasma proteins are isolated according to standard methods. To a 450 ml blood bag from the blood bank containing sodium citrate, 50 ml of a 3.8% sodium citrate solution was added and the solution was mixed gently.

[0158] The blood-sodium citrate was centrifuged at 2100 g for 20 min. The plasma supernatant was collected and centrifuged again at 5000 g for 15 min at 4 ° C. The plasma supernatant was placed on ice, and saturated ammonium sulfate solution was added to a ratio of one volume of ammonium sulfate per 3 volumes of supernatant (volume ratio 1: 3). The solution was stored at 4 ° C for 1.5 hours with occasional moderate oscillation (magnetic stirring is not allowed). The plasma supernatant was centrifuged at 5000 g for 15 min at 4 ° C. The supernatant was discarded and each pellet was washed with 10 ml of 25% ammonium sulfate solution (the pellet does not dissolve). Each pellet was dissolved in 6-7 ml of EDTA-phosphate regulatory solution. As an example, 100 ml of the solution was preserved to make an SDS-PAGE and a coagulation analysis. The dissolved pellets were mixed and the ammonium sulfate precipitate was repeated by adding saturated ammonium sulfate to the plasma sample to achieve a 1: 3 volume ratio (generally, 25 ml of ammonium sulfate with 75 ml of plasma). The solution was stored at 4 ° C for 1.5 hours with occasional moderate oscillation, and centrifuged at 5000g for 15 min. The supernatant was discarded and the pellets were dissolved in a volume of Tris-NaCl regulatory solution, which was equal to or less than the volume of EDTA-phosphate regulatory solution used previously. A typical total amount was approximately 45 ml.

[0159] The sample can be dialyzed (SnakeSkin ™ dialysis tubes, cut-off value 3.5 kD, Pierce) about 3 or 4 hours or overnight at 4 ° C in 1.5 liters of Tris-NaCl buffer solution. The dialyzed sample was centrifuged in high speed resistant tubes at 21,000g for 15 min at 4 ° C, to remove any remaining insoluble material. The supernatant was collected and preserved on ice.

[0160] The supernatant was precipitated with ethanol at a final concentration of 7% and stored on ice for 30 min. The solution was centrifuged at 5000g for 15 min, the supernatant was discarded and the pellet was dissolved in the same volume of Tris-NaCl regulatory solution (generally an amount of 45 ml). The solution was dialyzed overnight at 4 ° C in 1.5 liters of Tris-NaCl regulatory solution. The dialyzed solution was centrifuged at 21,000g at 4 ° C for 15 min to remove any trace of insoluble material.

[0161] Protein concentrations were determined using the standard Bradford method. Protein productions range between 0.2 and 0.6 mg per ml of whole blood, with typical results of 0.4 to 0.5 mg / ml. The clot formation capacity was determined by adding 30 μl of thrombin (100 U / ml; Omrix) to 70 μl of plasma product (10 mg / ml); Coagulation should occur in 30 seconds. Protein purity was determined by electrophoretic analysis of 50 μg of the sample on a 5% SDS-polyacrylamide gel and stained Coommassie blue. The rest of the supernatant was collected, frozen and lyophilized until drying, that is, 48 hours.

Example 4: Presence of plasmin and plasminogen in the plasma protein sample.

[0162] Plasminogen-free plasma proteins are composed of 9 to 10 ug of plasmin and plasminogen per milliliter of total protein, identified by the polyclonal antibody that detects both plasminogen and plasmin. Human plasma generally contains 200 mg of plasminogen per liter or about 200 ug / ml.

[0163] This experiment was designed to determine the concentration of plasmin that can be supported in a plasma protein clot. The same experimental design is used to test plasminogen resistance. Plasminogen is the precursor of active serine protease plasmin, capable of degrading fibrin.

[0164] Two plasmin concentrations (Biomedical ICN, 194.198, 20 mg / ml solution), 0.09 mg / ml, 0.045 mg / ml, were added to plasminogen-free plasma proteins (Omrix) before disposal of the solutions. The plasmin concentration of 0.09 mg / ml represents a concentration of plasmin ten times greater than the concentration of plasmin and total plasminogen present in commercial plasma proteins. The plasmin concentration of 0.045 mg / ml represents a fivefold concentration of plasmin. The plasma protein solution for the sponge composed of 0.09 mg / ml of plasmin was prepared by mixing 281 ul of plasma proteins (64 mg / ml), 67.5 ul of hyaluronic acid, 2.7 ul of plasmin and 251, 3 saline solution. The plasma protein solution for the sponge composed of 0.045 mg / ml of plasmin was prepared by mixing 281 ul plasma proteins, 67.5 ul of hyaluronic acid, 1.35 ul of plasmin and 250 ul of saline solution. A control was prepared without the addition of plasmin. Five sponges were prepared from each solution by adding 43 ul of thrombin to a well (1 IU of thrombin / mg of plasma protein) and 87 ul of plasma protein solution and the mixture was allowed to stand at room temperature.

[0165] None of the mixtures containing plasmin formed a clot, while the control without plasmin formed a clot in just a few minutes and a lyophilized sponge formed after freezing and lyophilization. This indicates that plasma proteins can tolerate less than 45 ug / ml of plasmin or less than 22.5% of plasminogen and plasmin normally present in plasma.

Example 5: Matrix morphology and mechanical properties

[0166] Matrices for tissue engineering, in general, are characterized by several criteria, including chemical nature, porosity, adhesion, biocompatibility and elasticity, among others (Hunziker, Osteoart. Cart., 10: 432-465, 2002). Table II of the reference mentioned above lists several of the properties and biological bases thereof.

[0167] In the inventors' laboratory, several of the properties were determined. Porosity, important for cell migration and adhesion is determined by geometric measurements using the light microscope to section the matrix into thick samples. The samples were mounted on the slides and stained with hematoxylin / eosin. An optical micrometer measured the pore size and the distance between adjacent pores.

[0168] The Scanning Electron Microscope (SEM) is used to analyze the homogeneity and ultrastructure of the matrix. The thickness of the fibrin fibers is also measured in this way.

[0169] In its final form before use with cells, the sponge is substantially dry and contains less than 15% residual moisture, more preferably less than 10% residual moisture and more preferably less than 3% residual moisture. This characteristic was measured by methods already known in the discipline. [0170] Mechanical property measurements were carried out using a Chatillon TCD200 machine with a DF12 digital dynamometer. Each plasma protein sponge was 2.5 cm long, 0.5 cm wide; and lyophilized completely. The deformation represents the elasticity of the sponge, that is, the amount of traction was measured in millimeters (mm) exerted until the sponge tears. The force is calculated in kiloPascal (kPa) and represents the amount of energy needed to tear the sponge strips. The thickness is incorporated into the calculation.

Example 6: Cell culture in the matrix

[0171] Different methods of cell culture can be used in the sponge. For cultivation, adhesion, migration capacity and proliferation of cells within the matrix is important. The cells can be suspended in a medium, in PBS, or in any biocompatible regulatory solution alone or in the presence of bioactive agents. The cells can be cultured by placing a drop of the liquid that contains the cells in the sponge and allowing the cells to be absorbed. On the other hand, the cells of the liquid can be absorbed by the sponge by placing the sponge in a container containing a suspension of cells. Other methods that include aerosol seeding have also proven effective.

[0172] A particular advantage of the present invention is the high level of cell viability and the excellent distribution of cells after cell culture directly on a dry sponge. Often, a matrix composed of an exogenous antifibrinolytic agent such as tranexamic acid exhibits a lower cell viability after culture. The cells appear to recover, but exogenous antifibrinolytic agents can be detrimental to the initial growth of the cell. When said sponge is washed, the tranexamic acid is partially or totally removed, improving cell proliferation. It was also found that many cells settle on the periphery of the matrix using a wet sponge, while there is a better distribution of the cells after cultivation in a dry sponge.

Materials and methods:

[0173] Sponges composed of different concentrations of plasma proteins and thrombin were tested. Sponges comprising 10 mg / ml, 15, 16.5 mg / ml, 18 mg / ml, 20 mg / ml, 22 mg / ml, 25 mg / ml, 30 mg / ml and varying concentrations of hyaluronic acid (from about 0.05% to about 1.1%) and 1, 1.5 or 2 IU of thrombin / mg of protein. A total of about 5x105 to about 5x106 chondrocytes were grown in sponges 1 cm in diameter and incubated for three days. Different volumes of growth medium are added and the matrix to which the cells have been added is incubated. It should be understood that the sponge can have different sizes, shapes and thicknesses.

[0174] After incubation of the cultured sponges for three days, 1 week and three weeks, some of the sponges were degraded to collagenase and the cells were counted after staining with trypan blue. Cell proliferation is determined as described in example 8.

[0175] Sponge samples or cell-bearing matrices were integrated in paraffin and sections were prepared using a microtome. The histological sections were further stained using different biological spots including hematoxylin and eosin (H&E), toluidine blue and rapid red, Masson's trichrome staining and others. All sponges showed a similar cellular distribution, with living cells present in all layers of the sponge.

[0176] Examples of the invention of cell growth in the fibrin sponge are shown in Figures 1A, 1B and 2. Each sponge was cultured with 5x106 porcine or human chondrocytes of 30 microliters in volume, incubated one hour and a fresh medium was added. After three days, the sponges were degraded in collagenase and the number of living cells was counted after staining with trypan blue. Figure 1A shows the increased viability of porcine chondrocytes in matrices with (mottled) and without plasminogen (solid) after a three day culture in the incubator. After three days, more than 50% of the cells remained viable when compared to 20% of the cells grown in the standard matrix prepared from plasma proteins composed of tranexamic acid. Figure 1 B shows the viability of human chondrocytes cultured in matrices with (mottled) and without plasminogen (solid-dry) after a three day incubation. Sponges without plasminogen cultured with human chondrocytes showed superior cell viability when compared with sponges composed of tranexamic acid. Figure 1C shows cell viability after three days in three different sponge compositions. All sponges composed of plasminogen-free plasma proteins, were grown with approximately 4x106 human chondrocytes. The mottled bar represents the viability in a 20 mg plasma protein sponge / ml, with no additives present. The solid black bar represents cell viability in a sponge comprising 20 mg / ml and 0.075% hyaluronic acid.

The square bar represents cell viability in a sponge that ranges between 18 mg / ml and 0.75% hyaluronic acid. It can be seen that the three sponges provide a good structure for cell culture. Figures 1D and 1E show photographs of dry sponges one centimeter (1 cm) in diameter and cell-bearing implants, respectively.

[0177] Figures 2A, 2B and 2C show cross-sectional histological sections through the center of a matrix composed of human chondrocytes after 1 week of incubation. The fibrin sponge was made of commercially available plasminogen-free fibrinogen (Omrix, 20 mg / ml) composed of 0.05% hyaluronic acid and 1x106 human cells. It is necessary to take into account the infiltration of the chondrocytes in the sponge. Figures 2A, 2B and 2C show increases of 40x, 100x and 400x, respectively.

Example 7: In vitro Degradation Assay

[0178] An assay was carried out to determine the degradation rate of the sponge of the invention. The differences in the degradation index can be seen between the sponge of the invention and a standard sponge composed of fibrinogen and an exogenous antifibrinolytic such as tranexamic acid.

[0179] The test was performed as follows: three different types of sponges were prepared, each with the same fibrinogen concentration, the same thrombin concentration (1.5U / mg protein) and hyaluronic acid. Differences were seen in the source of fibrinogen and hyaluronic acid.

[0180] A fibrin sponge composed of 10% tranexamic acid, fibrinogen (Omrix, 27 mg / ml), and cross-linked hyaluronic acid (0.08% Syvisc).

[0181] A fibrin sponge was prepared from fibrinogen substantially free of plasminogen (Omrix 27 mg / ml) and cross-linked hyaluronic acid (Synvisc 0.08%).

[0182] A fibrin sponge was prepared from fibrinogen substantially free of plasminogen (Omrix 27 mg / ml) and uncrosslinked hyaluronic acid (0.08% BTG).

[0183] The experiment was performed as follows: Five sponges prepared in 96-well plates were placed in 48-well plates and 750 ul of 10M urea was added to cover the sponges.

[0184] 20 ul samples were obtained from each well at the following points: 1, 2, 3, 4, 5, 8 minutes, 10 minutes, 30 minutes and 1 hour. The protein in each sample was measured in a standard Bradford assay. The results are presented in Figure 3A.

[0185] The sponge, composed of standard fibrinogen and 10% tranexamic acid, underwent rapid degradation as measured by the protein (mg / ml) detected in the supernatant and could not be seen until after 10 minutes , while the sponge composed of plasminogen-free plasma protein remained stable.

[0186] In a similar experiment, the sponges were degraded in collagenase. The test sponges were incubated in 400 ml of collagenase (1.7 mg / ml) diluted 1:10 in DMEM without FBS at 37 ° C until completely dissolved. Samples were collected at different time points and protein concentration was examined by the Bradford assay. The results are presented in Figure 3B. The sponge comprising 10% tranexamic acid and cross-linked hyaluronic acid degrades much faster than sponges containing fibrinogen substantially free of plasminogen also composed of cross-linked or non-crosslinked hyaluronic acid (wo plm x link, wo plm non x link, respectively).

Example 8: Release of matrix bioactive agents

[0187] For certain applications, prolonged release of a bioactive agent as a growth factor may be desirable. The incorporation and release of matrix growth factors of the invention are evaluated in vitro or in vivo using radiolabeled or labeled growth factors, for example, fluorochrome labeling, with alkaline phosphatase or radish peroxidase labeled growth factor. . The fraction and speed of the released agent were measured following radioactivity, fluorescence, enzymatic activity or other peculiarity of the tag. Similarly, the release of enzymes from the matrix is determined by analyzing the enzymatic activity in the microenvironment in an in vitro or in vivo assay. The release of an FGF from the matrix of the invention was carried out as described herein.

[0188] The growth factor release index was determined from sponges prepared in two alternative methods. In one case, FGF2 was adsorbed to heparin and the combined product was added to the plasma protein solution. In the second example, each component was added separately to the individual solutions: heparin was added to the plasma protein solution, while FGF2 was added to the

thrombin solution The sponges were melted with the two mixtures and the release of FGF2 was determined in a FDCP trial, vide supra.

Materials and methods:

[0189] Plasma protein (approximately 20-65 mg fibrinogen / ml; Omrix, without plasminogen).

[0190] Uncrosslinked hyaluronic acid (MTF or BTG), Heparin (Sigma, MW 6000) A sponge of FGF2 (ProChon) 2.5 ug per 75 ul

[0191] Fibrin-free sponges substantially free of plasminogen and organic chelating agents were prepared using the method described in example 1 with the following modification: the plasma protein solution composed of uncrosslinked hyaluronic acid with a final concentration of 0.08%.

[0192] The first set of sponges was prepared by mixing heparin solutions with FGF2 and adding the Mix to the plasma protein solution.

[0193] The second set of sponges was prepared by adding heparin to the protein solution plasma with a final concentration of 0.1, 0.5 or 2.5 ug / ml. FGF2 was added to the thrombin solution to make the final concentration 2.5 ug / per sponge. The sponges were melted as described previously. The release of FGF2 was determined in an FDCP assay as described below.

[0194] FDCP Assay: The FDCP cell line is an interleukin-3 cell line of a bone marrow of origin immortalized murine that does not express endogenous FGF receptors (FGFR). Regarding FGFR transfection FDCP cell line cDNA exhibited a dose-dependent proliferative response of FGF that may replace the dependence on IL-3. The FGFR transferred to FDCP cells can be used for the detection of FGFR The responses of FDCP cells to several ligands is quantified by a cell proliferation assay with XTT reagent (Cell Proliferation Kit, Biologic Industries Co.). The method is based on the ability of the mitochondrial enzymes to reduce tetrazolium salts in a colorigenic compound that can be quantified and that is an indicative of cell viability.

[0195] Specifically, stable FDCP cells expressing FGFRI (FDCP-FGFRI) were grown in "complete medium" (Iscove medium containing 2 ml glutamine, 10% FCS, 100 ug / ml penicillin, ML / ml of streptomycin) supplemented with 5 ug / ml of heparin. The cells were divided every 3 days and were they kept in cultivation no more than a month. One day before the experiment, the cells divide. Before the Experiment the cells were washed 3 times (1,000 rpm, 6 min) in complete medium. The cells were resuspended and they had trypan blue. Twenty thousand cells (2 x 104) were added to each well of the 96 plate wells in 50 μl in complete medium with or without heparin. The conditioned medium of sponges composed of FGF

or FGF complexed with the various glycosaminoglycans was added to an additional volume of 50 μl in complete medium to have a final volume of 100 μl. The plate was incubated for 48 hours at 37 ° C. To test cell proliferation, 100 μl of PMS reagent was added to 5 ml of XTT reagent and mixed well (according to the manufacturing protocol). 50 μl aliquots of the above solution were taken and the plates were incubated at 37 ° C for 4 hours and the color developed was read by a spectrum-ELISA reader at A490 nm. Figures 4A and 4B show the results of this test of the invention for a sponge made of commercial plasma proteins substantially free of plasminogen (Omrix, final 20 mg / ml) composed of 0.08% non-crosslinked hyaluronic acid, either by the addition of heparin and FGF2 variants that have been previously mixed (mixed) to the plasma protein component of the sponge ab initio or by the addition of heparin to the plasma protein solution and the FGF2 variant to the thrombin solution ( SEP), followed by mixing and melting of the clot. The sponges are composed of 0.5, 1.5 or 2.5 ug / ml of heparin and 1 ug total variant of FGF2. The supernatant was tested after several days and the results for proliferation were recorded. Figure 4A shows the release of the FGF2 variant of a sponge composed of heparin and FGF2v after 1, 3 and 5 days. Figure 4B shows the total release percentage of a period between 5 and 37 days. The release profile of FGF depends on the concentration of heparin in the sponge. Without being linked to a particular theory, heparin can serve as a stabilizer of the released FGF. Figure 4C shows the release profile of more than 5 days of a FGF variant of a sponge composed of heparin, commercial fibrinogen and tranexamic acid or plasma protein substantially free of antifibrinolytic agents. The results show an optimal release profile for both compositions.

Example 9: Chondrocyte Isolation and Culture

Reagents:

[0196] Collagenase Type 2; Worthington Biochemical Corp. (Cat. #: 4147) Stock solution: 1700 units / ml in medium (in MEM) Minimum Essential Medium (MEM) Gibco BRL (cat: 21090-022) 30

Bovine Fetal Serum (FBS); Gibco BRL (cat: 16000-044) L-Glutamine Solution; Gibco BRL (cat: 25030-024) Complete medium: Minimum Essential Medium (MEM) supplemented with 10% fetal bovine serum (FCS), L-Glutamine 2mM and 100U / ml penicillin, and 100μg / ml streptomycin

Implant preparation for articular cartilage

[0197] The sponge of the present invention can be used as a cell-bearing framework, for tissue reconstruction and regeneration. On the one hand, the cells are grown in the sponge in vitro before implantation. On the other hand, the sponge is cultured with cells immediately before implantation and the cells are allowed to grow and proliferate in vivo.

[0198] Cartilage biopsies from fresh pig cartilage were divided into sections of small pieces, approximately 3-4 mm thick, washed aseptically with PBS and placed in a new tube with 3 ml of MEM medium. The cartilage can be obtained from any of the vertebrate species, and is preferably allogeneic or autologous.

[0199] Type II collagenase was diluted to 1: 5 and 1 ml was added to the cartilage pieces and the mixture was gently stirred in an incubator at 37 ° C overnight. Most of the sample was assimilated, the suspension was poured through a sterile gauze to remove matrix residues and non-assimilated material. The filtrate was centrifuged and washed twice to remove residual enzyme.

[0200] The number of cells was determined with a hemocytometer and the viability was determined with the exclusion of trypan blue. The cells were inoculated into 150 cm2 tissue culture flasks in 30 ml of culture medium at a concentration of 5x106 cells / ml. The flasks were placed in an incubator at 37 ° C in an atmosphere at 5% CO2 and 95% humidity. The culture medium was changed every three to four days. The cells adhere and converge after one week of incubation.

[0201] The cell medium was removed and 3ml of trypsin-EDTA solution and thirty ml of MEM + FBS were added, the solution was centrifuged at 800g for 10 minutes. The supernatant was removed, the pellet was dispersed and the cells were counted. To create a cell-bearing matrix, 102-106 cells were cultured in a plasma protein framework 9mm in diameter and 2mm thick (approximately 0.2 cm3). The matrices were placed in an incubator at 37 ° C, for 1 hour and 1ml of fresh medium was added to each. The medium was replaced with fresh medium and matrices were taken every day for the differentiation and cell proliferation analysis.

[0202] On the other hand, the adult cell population of the previous matrices expresses several of the differentiation markers of the chondrocytes. One of several phenotypes expressed during chondrocyte differentiation is the production of glycosaminoglycan (GAG). The production of GAGs was identified in histological staining using alcian blue and was quantified using the DMB staining method (3,3'-dimethoxobenzidine dihydrochloride). The extracellular matrix of cartilage can also be identified by staining toluidine blue and fast red.

Example 10: Cell Proliferation Assay

[0203] The proliferation of cartilage cells in the matrix of the invention was quantified by one of these two methods: Cy-QUANT® (Molecular Probes) or XTT reagent (Biological Industries, Co.). The plasma protein matrix was dissolved in collagenase or other enzymes and the cells were collected by centrifugation and subjected to analysis according to the manufacturer's protocols.

[0204] In one experiment, human articular chondrocytes (104-106 cells / 30-100 ul) were grown in matrices substantially free of exogenous antifibrinolytic agents in micro well plates. The cells were grown overnight in MEM, 34 U of collagenase were added and the cells or sponge cells were incubated for four hours. The XTT reagent was added for 3-4 hours and the plates were read in an ELISA reader at A490 mm. The results show that the cell proliferation rate was not affected either by the presence of the sponge or by the addition of collagenase. Figures 5A, 5B, 5C and 5D show porcine chondrocytes (0.5 x 106 cells in 30 ul) that have been cultured (6 days) in a fibrin sponge from human plasma (30 mg / ml) composed of 0.024 % of Hylan 1 ug derived from FGF. Figures 5A and 5B show a staining of hematoxylin and eosin (H&E) (100x magnification). Figure 5C shows a 400x magnification of a section of sponge stained with Masson stain. Note the staining of the cells and the intracellular matrix that surrounds the cells. Figure 5D shows a 200x magnification of a section of a sponge dyed with Masson stain. The cells present in the large number of pores must be taken into account.

Example 11: Culture, growth of cells and cell lines in the fibrin matrix

[0205] In order to determine the ability of the plasma protein matrix to support cell growth, several different types of cells and cell lines were grown. Specifically, rat liver primary hepatocytes were cultured in the matrix. 1cm diameter sponges composed of plasminogen-free plasma proteins (20 mg protein / ml, Omrix), 0.075% hyaluronic acid and 1 IU thrombin / ml were prepared. Approximately 6.6 x105 primary hepatocytes were cultured in the sponges in HDM (hormonally defined medium) without serum and incubated for three days in which histological samples were made and stained with H&E. Figure 6A shows a representative section of a sponge comprising hepatocytes. The good dispersion of cells through the matrix and the presence of typical cells that maintain their liver characteristics must be taken into account.

[0206] Two cell lines were tested to determine the viability and growth within the sponges, the L8 cell line of the rat skeletal muscle and the Chinese CHO hamster ovary cell line. Two million cells were cultured in each matrix and incubated for three days. The CHO cell line was cultured in the middle of Iscove, the L8 line in DMEM cultures. Histological cuts were made and stained with H&E. Figures 6B and 6C show sponge sections with CHO and L8 cells, respectively. In addition, CHO and L8 cells were removed from several sponges and counted using trypan blue. L8 cells showed 57-67% viability, while CHO cells showed more than 85% viability. Both figures show a good distribution and cell viability. The matrix of the invention provides an improved framework for engineering and tissue regeneration.

Example 12: Formation of ectopic cartilage in nude mice

[0207] The assay was designed to determine the ability of isolated chondrocytes to create neocartilages in an ectopic area and to determine the quality of this cartilage if compared to natural cartilage.

[0208] Human and porcine chondrocytes cultured in matrices of the invention were used to induce ectopic cartilage on the backs of nude mice.

[0209] Treatment sections: Study groups include different amounts of cells cultured in the fibrin matrix substantially free of plasminogen. Either human or porcine chondrocyte 10e5 (10 ^ 5) or 10e6 (10 ^ 6) was cultured in a fibrin sponge from a 96-well plate (~ 65 ul). The control group consisted of implanted matrices without cells.

[0210] Preparation of fibrin matrices: The method for sponge preparation consists of mixing a plasminogen-free fibrinogen solution (Omrix), with a thrombin solution (Omrix) in the presence of uncrosslinked hyaluronic acid (BTG), giving thus leading to a final concentration of 20 mg of fibrinogen / ml, 0.5 IU of thrombin / mg of fibrinogen and 0.08% of hyaluronic acid. The solutions were added to a mold (96-well plate, 65 ml volume), where coagulation took place at room temperature. The clot quickly froze at -70 ° C, followed by lyophilization resulting in an implant (matrix, sponge) with spongy texture.

[0211] Culture: The sponges were cultured with human or porcine chondrocytes (105-106/20 ul culture medium in a 96-well plate and incubated at 37 ° C for 1 hour. The culture medium was added to the well and The sponge was incubated for a period of 24-48 hours.The sponge was placed in the subcutaneous incisions made on the back of the nude mice.

[0212] Implantation procedure: Animals were anesthetized with ketamine-xylazine. The skin on the back was shaved and disinfected with alcohol. Two incisions were made on each side of the back, parallel to the spine. In each incision a subcutaneous pocket or a pocket of the muscular fascia was made by blunt dissection. The sponges were implanted in the pockets according to the treatment section. The skin was closed with simple suture. Each treatment was repeated 5 times and in each mouse 4 sponges were implanted. See the table below.

2.

Table 2: Experimental trial

Mouse Nº

Left proximal Left distal Right proximal Right distal 55 Labeling

one

1x10 ^ 5 Human 1x10 ^ 6 Human 1x10 ^ 5 Pigs 1x10 ^ 6 Pigs No tag

2

1x10 ^ 6 Human 1x10 ^ 5 Human 1x10 ^ 6 Pigs Sponge w / o cells 601 right ear

3

1x10 ^ 6 Pigs 1x10 ^ 5 Pigs 1x10 ^ 5 Human 1x10 ^ 6 Human 1 left ear

Four. Five

1x10 ^ 5 Pigs 1x10 ^ 6 Human Sponge w / o cells 1x10 ^ 6 Pigs 1x10 ^ 6 Human Sponge w / o cells 1x10 ^ 5 Human 1x10 ^ 5 Human 65 2 right ear 2 left ear

6

Sponge w / o cells 2626 Sponge w / o cells 1x10 ^ 6 Pigs 1x10 ^ 5 Pigs RIGHT + LEFT

[0213] Evaluation of induced cartilage formation: One or four weeks after implantation, mice were sacrificed by recovering implants with surrounding tissue and prepared for histology evaluation. The microscopic evaluation consists of a complete morphological description of the implant. Additional analyzes include safranine or H&E staining, alcian blue and type II anti-collagen staining.

[0214] Figure 7A shows the implantation procedure. Figure 7B shows the growth of ectopic cartilage derived from a sponge with incorporated cells (cells 105) on the back of a mouse, Figures 8A, 8B and 8C show a section stained with hematoxylin-eosin of the human chondrocytes of a stopper of neocartilage after one week. Figure 8A shows a histological section with some cells that show a strong staining of the cartilage matrix using toluidine blue and fast red. Figures 8B and 8C show histological sections stained with H&E. The good dispersion of the cells and the presence of the cell matrix surrounding the cells must be taken into account. Figures 9A and 9B show a section stained with hematoxylin-eosin of porcine chondrocytes with increases of 40x and 200x after 4 weeks of growth in situ.

[0215] The results of this experiment confirmed that the matrix composed of plasminogen-free plasma proteins is an effective matrix for cartilage formation. The matrices are neither immunogenic nor toxic, and support the growth and differentiation of chondrocytes.

Example 13: Method for matrix preparation

[0216] Sponges were prepared in two different ways and tested for viability and cell dispersion. Both methods comprise the steps of preparing plasma protein and thrombin solutions. One method is also composed of the sequential dispensing of thrombin and fibrinogen solutions in a mold. The second method, "premix," requires that the two solutions be mixed before being placed in a mold. The resulting sponges are different in terms of porosity and cellular absorption capacity. Figure 10A shows the cell viability of chondrocytes in sponges prepared using the two different methods. Cell viability is similar in both types of sponges. A difference in porosity and cell dispersion can be seen. Figure 10B shows the cells that settle in the upper layers of a sponge prepared by the premix method. Figure 10C shows the distribution of cells throughout the matrix in a sponge prepared according to the method in which the solutions are melted in the mold sequentially. Some applications may benefit one type of sponge over another.

Example 14: A sheep's cartilage repair model

[0217] This study was designed to evaluate the ability of the chondrocytes assigned to the fibrin matrix of the invention for cartilage reconstruction in a large animal model. A total of 20 sheep were chosen each with an approximate weight between 60 and 80 kg. Eight of the animals underwent a chondrocyte extraction procedure before implantation. The extracted chondrocytes were enlarged and classified into recombinant human fibrin matrices.

[0218] The housing conditions of the animals were in accordance with the applicable laws and regulations regarding laboratory animals. The experiments were performed according to the principles of local laws for animal experimentation. The animals were examined if signs of illness or lameness were found. Acceptance in the study depended on whether they were disease free, healthy, and without a history of prior use. Osteoarthritis was excluded by preoperative radiography. The animals were conditioned for an adequate period of time as determined by the institution. Each animal is identified with a tattoo and a unique number tag. The animals were assigned to the treatment groups by random assignment of identification numbers. The study design is shown in Table 3 below:

Table 3

# Sheep

Treatment Matrix type

1A-7A

Untreated Untreated

1B-7B

 Microfactura Microfactura

1C-4C

Matrix only TEA + crosslinked AH

5C-8C

Matrix only Without plasminogen + crosslinked AH

9C-12C

Matrix only Plasminogen + non-crosslinked AH

1D-4D

Matrix + cells Without plasminogen + crosslinked AH

5D-8D

Matrix + cells Without plasminogen + uncrosslinked AH

[0219] Animals were observed daily to check their status in the course of the study. In the unlikely event that an animal is damaged, sick or dying, it will be treated following medical practice

Current veterinary If justified, euthanasia will be carried out according to the guidelines established by the AVMA Panel in Euthanasia (JAVMA, March 2000). The veterinarian in charge will carry out a clinical diagnosis and treatment if the animal shows signs of disease.

[0220] All animals were weighed during the quarantine period, before surgery (day 0) and at the end of the study (day 112). [0221] Group A. Untreated defects: In animals 7b (14 defects) chondral defects were created in the condyle and left untreated.

[0222] Group B. Microfracture: In 7 animals (14 defects), the microfracture was performed without further treatment. Four microfractures with special punches were performed on each defect until dotted bleeding was observed.

[0223] Group C Fibrin matrix only: fibrin matrices composed of TEA and cross-linked hyaluronic acid were implanted in 4 sheep (1C-4C). They were implanted in 4 matrix sheep of fibrin from plasminogen-free fibrinogen, cross-linked hyaluronic acid (5C-8C) or non-cross-linked (9C-12C). The matrices were implanted after the creation of the defects as described below.

[0224] Group D cell-bearing fibrin matrix: implanted in 4 fibrin matrix sheep prepared from fibrinogen plasminogen and any cross-linked hyaluronic acid ID-4D)) or non-cross-linked (5D-8D) cultured with chondrocytes and in the knee defects).

[0225] Operation: The left knee joint was covered with a sterile cloth and opened by an anteromedial approach under general anesthesia. The medial condyle was discovered, and small pieces of cartilage were removed from the low weight surfaces of the trochlea and the intercondylar notch. The cartilage was cut superficially with a scalpel to prevent bleeding. The wound was closed in layers. A plaster immobilization was applied to the knee and ankle joint for five days and activity in the cage was limited to reduce joint load in order to prevent displacement of the kneecap. The tissue sample was chopped and washed under sterile conditions and the cells were isolated by collagenase following a standard assimilation protocol. The cells were grown in 75 ml flasks (Corning) and incubated at 37 ° C. The media change was made every day. After 3 weeks, approximately 200,000 cells (2x105) were cultured in the selected fibrin matrices and cultured for 4 days in 6-well plates. The cell matrices were transferred with a sterile cloth to the operating room. The medial condyle of the sheep's right knee was discovered. Using a 4.5 mm punch (Smith & Nephew), two defects were made, 1 and 2.5 cm in the distal intercondylar notch in the medial condyle of the femur. The defects are described with the dermal punch to the subchondral bone and the cartilage was removed with small curettes. An attempt will be made to eliminate all traces of articular cartilage. To do this, the surface of the calcified cartilage must be gently scraped off. It should be noted that there is no bleeding from the subchondral bone. The fibrin matrices were fixed in place with fibrin gum.

[0226] After the defect treatment, the capsule bleeding points were stopped by cauterization and wound closure was performed in layers. Plaster fixation was applied for another five days and activity in the cage was limited to reduce joint load in order to prevent dislocation of reconstructive graft tissue. After having removed the plaster, the sheep were given total freedom in their activity, and had a balanced nutrition twice a day. Until the second day of the postoperative period, 2gr of cefazolin was administered three times a day.

[0227] All animals of group C and D were sacrificed at 16 weeks post-operatively as described below in Mankin, H (NEJM (1974) 291: 1335-1340).

[0228] Necropsy: Animals were sacrificed 16 weeks after the operation. The animals were weighed before the sacrifice. The bleeding subject was induced deep sedation with a mixture of ketamine-xylazine according to the guidelines established by the AVMA Panel in Euthanasia (JAVMA, March 2000).

[0229] The total evaluation and sampling was carried out as described in Table 4. The articular surfaces opposite to the defective areas were examined for any abnormal joint surface. In addition, macroscopic evaluations of the knee joints were performed to determine cartilage reconstruction based on the previous scoring criteria listed in Table 5. Femurs, kneecaps, synovial membrane, and popliteal lymph nodes were collected. and placed in properly labeled containers. Immediately after tissue extraction, macroscopic morphological examinations of the cartilage surface were performed and photographic records of each sample were made.

Table 4: Macroscopic evaluation and sample collection

[0230] Macroscopic Morphological Observations: after collecting the knee joints, the joints were opened, photographed and the area of surface defect was scored as indicated in the table

5. The synovial membrane was examined to determine the degree of inflammation. Joint fluid was collected and analyzed.

Table 5: Scoring criteria for macroscopic morphological evaluations

features

Classification Punctuation

Integration process (relative to the new tissue of the native cartilage)

Full Partial None 2 1 0

Smoothness of the cartilage surface

Soft Intermediate Rugged 2 1 0

Cartilage surface (degree of filling)

Flush Light depression Depression 2 1 0

Cartilage color, opacity or translucency of the neocartilage

Transparent Translucent Opaque 2 1 0

10 [0231] Histology and histological evaluation: The knees were opened under sterile conditions and a culture sample was obtained. The synovium was documented macroscopically, the defects photographed and the joint examined thoroughly. The distal femur was removed and placed in 10% buffered neutral formalin for 12 hours. The trochlea areas that contain the defects and the extracted areas were analyzed and preserved in 10% formalin for 4 days. The samples were subsequently placed in a descaling solution.

15 (Tritriplex 100g (Epignost, Austria) and 33g of Tris-hydroxymethylene-amnomethane (Merck Eurolab, Belgium) per liter) two to four days at room temperature. The decalcified samples were integrated in paraffin and cut with a microtome into sections 5 μm thick.

[0232] Sections were stained with hematoxylin and eosin (H&E), safranine or / rapid green, alcian blue and

20 azán for the evaluation of the types of tissues. Immunohistochemistry with antibodies to type I and type II collagens is carried out according to a standard ABC protocol using antibodies conjugated with HRP. The cartilage and sheep tendons of a healthy animal are used as controls.

[0233] Optical microscopy is performed on a Vanox Olympus research microscope by putting in

A histomorphometric method is used to determine the percentage of the selected tissue types (analysis). A series of cross-sectional histological sections are evaluated from the middle portion of the defect. The defect filling is determined as a percentage of area of reconstructive tissue in the defect, based on the cross-sectional area in a sagittal plane through the center of the lesion. The area, the filling, the height, the base of the defect, and the type of tissue were evaluated. The types of tissues are characterized as follows: 1. fibrous tissue 2. tissue

30 transition 3. hyaline tissue and 4 articular cartilage. A semiquantitative analysis of the defect and the adjacent tissue is performed according to the adapted standard scores of O'Driscoll, Pineda and Frenkel.

Example 15: Human Clinical Trial

[0234] A feasibility study has been presented and approved (by the Ethics Committee of the University Hospital of Vienna) to evaluate the safety and performance of the fibrin matrices of the invention in the treatment of chronic heart defects. Femoral condyle cartilage.

[0235] A phase I study is carried out in patients, a non-randomized open label uses a matrix of

Fibrin or a fibrin cell carrier matrix prepared with plasminogen-free fibrinogen and autologous chondrocytes. Patients who met the entry criteria will undergo an arthroscopic procedure to confirm the diagnosis and to remove a biopsy for chondrocyte growth for future transplantation. Three to six weeks after the cell harvest, patients will be hospitalized for surgery. After surgery, patients will be monitored for their safety as follows: during 5-7

45 days of hospitalization, after discharge at week 2 and week 6, and evaluation of the procedure at week 12, month 6 and month 12.

[0236] The main purpose is to evaluate the safety of a cell-carrying matrix of the invention, in which the matrix serves as a framework for the cultivation and transplantation of autologous chondrocytes in the treatment of a lesion.

50 chronic condyle cartilage.

[0237] The secondary objective is to evaluate the performance of a cell-carrying matrix in the restorative function, according to the improvement in the results of the RMI, a questionnaire of quality of life and score of the articular function. Safety parameters will include vital signs, serum chemistry, hematology and systemic and local adverse events. All parameters are presented, including the inclusion, exclusion and withdrawal criteria of the patient.

Example 16: One-step procedure for the treatment of damaged cartilage: Suitable for arthroscopy or hemiarthrotomy

[0238] Autologous chondrocyte implants have proven clinically effective in rebuilding hyaline cartilage to isolate chondral knee defects. Current therapies include three main steps:

one.

 Diagnostic arthroscopy and healthy cartilage biopsy.

2.

 The culture of cells.

3.

 The injection of chondrocytes grown in the lesion under a periodic flap, which is taken from the tibia and sutured over the lesion.

[0239] The disadvantages of the technique include the need for two separate surgical procedures, the need for a surgical area to isolate a periodic flap and the tendency for cartilage overgrowth due to the presence of the flap. An improved variant of this technique provides a matrix implant of the present invention. A less traumatic method is presented in this document, where the patient undergoes a unique surgical procedure for cartilage reconstruction.

[0240] Procedure: A patient with a cartilage defect can donate autologous plasma several days before arthroscopy or hemi-arthrotomy. The blood (approximately 100 to 250 ml) is extracted and the plasma proteins are purified, eliminating the plasminogen. A plasma protein matrix, or several matrices, are prepared, labeled and stored aseptically until the day of surgery.

[0241] Optionally, on the day of surgery, the cartilage is removed from the patient's joint, cut into small pieces and placed in a test tube containing collagenase, hyaluronidase or other cartilage degradation enzymes or combinations thereof.

[0242] The surgeon treats the defective region of the joint by removing damaged tissues, cleaning and preparing the area for the implant. The prepared matrix is removed from its container and cut to fit in the defective area. Approximately 20-30 minutes after the enzyme treatment, the cells and small pieces of cartilage are centrifuged in a tabletop centrifuge, washed in PBS and resuspended in a small amount (50 ul-1000 ul) of PBS. The surgeon cultivates the cells in the sponge in situ. Alternatively, the cells are absorbed in the sponge and the cell-carrying sponge is integrated into the region of the defective joint. Optionally, degradation enzymes of the extracellular matrix and / or other bioactive agents that include growth factors and / or anti-inflammatory compounds are added. In certain cases, the surgeon will place a dry sponge directly on the injured area, optionally add enzyme solution to said sponge and place a second cell-carrying sponge on top of the first sponge. The joint is closed, treated as usual by an arthroscopic procedure or hemi-arthrotomy and the patient is allowed to recover.

Kit

[0243] A kit composed of components useful for practicing the method of the invention will allow the convenient practice of the method of the invention in a surgical environment. In one embodiment, a kit of the invention will provide sterile components suitable for easy use in the surgical environment including sterile solutions (saline solution, enzymes) and a free sterile matrix that supports autologous chondrocytes prepared to be integrated into the surface defect of the joint and instructions for use.

Example 17: Repair of a bone model

[0244] The plasma protein matrix of the present invention is useful for the treatment of bone defects including osteotomy, particularly in areas of the skeleton that do not support weight. Suitable models of animals are used to create bilateral osteotomies to demonstrate the efficacy of the present invention. A 4 to 6 mm osteotomy was performed on a rabbit specimen from New Zealand in compliance with the Animal Protection Committee. The ulna is chosen because it is only a light weight support and allows the creation of a bone defect without the need for a mold or other immobilization treatment. In addition, this fissure constitutes a spontaneous cure defect that allows the evaluation of the agent tested. The main indicators of fracture healing are the accelerated duration of healing and callus formation. The test compounds are composed of matrices of the invention and control matrices.

[0245] Surgical procedure: Animals are anesthetized according to the standard protocol. The fissure formation is done in the middle of the ulna. A sponge of the invention is placed in the fissure area of each limb and the fracture is closed. The animals are treated with analgesics for 3 days after the operation. The total duration of the experiment is 6 weeks.

[0246] Cure time and quality assessment: The X-ray examination presents the assessment of the state of fracture healing. X-rays are taken of rabbits every week for 5 weeks after surgery. Radiographs are assessed by two blind orthopedic surgeons according to a standard classification scale protocol.

[0247] Quality assessment: at the end of the experiment, the rabbits were sacrificed and the fracture area was sent for a histological and mechanical strength assessment. The histology was scored by a pathologist for the evaluation of histological changes during the healing process using standard staining methods and using hematoxylin and eosin (H&E) for the cytoplasm and nucleus. Indigo-carmine staining is also applied for the detection of new calluses generated. The evaluation of the mechanical resistance is carried out using the method of the "4 inflection points"

[0248] Treatment groups: simulated osteotomy, osteotomy treated only with a fibrin sponge, osteotomy treated with a fibrin sponge composed of glycosaminoglycan, optional heparin growth factors.

[0249] Another example of an animal model for bone reconstruction is described in Cook et al., (Am. J. Vet Res. 64: 2-20,2003).

Claims (16)

one.

A lyophilized porous fibrin matrix is formed from plasma proteins composed of fibrinogen, thrombin and factor XIII, the matrix contains less than 10% residual moisture, without added exogenous antifibrinolytic agents and comprises less than 1 mM of organic chelating agents, where Plasma proteins comprise less than 20% of plasminogen normally present in plasma.

2.

A method for the preparation of a lyophilized porous fibrin matrix containing less than 10% residual moisture, without added exogenous antifibrinolytic agents, containing less than 1 mM of organic chelating agents, and comprising less than 20% of plasminogen normally present in plasma it comprises the following steps:

Provide a thrombin solution and a plasma protein solution, the protein solution plasma without adding added exogenous antifibrinolytic agents, with less than 1 mM of agents organic chelators and comprising less than 20% plasminogen normally present in the plasma. Introduce the thrombin solution and the plasma protein solution in a solid support or in a mold in presence of calcium ions. Incubate under appropriate conditions to achieve coagulation. Freeze the coagulated mixture. Lyophilize the coagulated mixture to obtain a sponge.

3.

The use of a lyophilized porous fibrin matrix according to claim 1 for the preparation of an implant to be implanted in the area of disease or injury for the treatment of a diseased or injured tissue.

Four.

A lyophilized porous fibrin matrix according to claim 1, the method according to claim 2 or the use according to claim 3, wherein the plasma proteins contain less than 10% of the plasminogen present in normal plasma.

5.

A lyophilized porous fibrin matrix according to claim 1, the method according to claim 2 or the use according to claim 3, wherein the plasma proteins contain less than 5% of the plasminogen normally present in the plasma.

6.

A lyophilized porous fibrin matrix, method or use according to any one of claims 1 to 5, wherein the plasma proteins contain purified proteins from a plasma source.

7.

A lyophilized porous fibrin matrix, method or use according to any one of claims 1 to 6, which also contains at least one additive selected from the group consisting of polysaccharides, glycosaminoglycans and synthetic polymers.

8.

A lyophilized porous fibrin matrix, method or use according to claim 7, wherein said glycosaminoglycan is selected from crosslinked and uncrosslinked hyaluronic acid.

9.

A lyophilized porous fibrin matrix, method or use according to claim 7, wherein said glycosaminoglycan is selected from the group consisting of heparin, low molecular weight heparin, heparan sulfate or a combination thereof.

10.

A lyophilized porous fibrin matrix, method or use according to any one of claims 1 to 9, further containing at least one bioactive agent selected from growth factors, cytokines, platelets, platelet supernatant, platelet derived proteins, hormones, analgesics , anti-inflammatory agents, antimicrobial agents and enzymes.

eleven.

A lyophilized porous fibrin matrix, method or use according to claim 10, wherein said growth factor is a fibroblast growth factor selected from the group FGF-9, FGF-2, FGF-4, FGF-18 or a combination thereof.

12.

A lyophilized porous fibrin matrix, method or use according to any one of claims 1 to 11 further containing cells.

13.

A lyophilized porous fibrin matrix, method or use according to claim 12 wherein said cells are selected from stem cells, progenitor cells, chondrocytes, osteoblasts, hepatocytes or mesenchymal, endothelial, epithelial, urothelial, endocrine, neuronal, pancreatic cell types , renal and ocular.

14.

A lyophilized porous fibrin matrix, method or use according to any one of claims 1 to 13, wherein at least one of the plasma proteins is autologous.

fifteen.

A lyophilized porous fibrin matrix, method or use according to claim 14, wherein all plasma proteins are autologous.

16.

A lyophilized porous fibrin matrix, method or use according to any one of claims 1 to 15, wherein at least one of the plasma proteins is recombinant.