MXPA04006021A - Growth factor modified protein matrices for tissue engineering. - Google Patents

Abstract

Proteins are incorporated into protein or polysaccharide matrices for use in tissue repair, regeneration and/or remodeling and/or drug delivery. The proteins can be incorporated so that they are released by degradation of the matrix, by enzymatic action and/or diffusion. As demonstrated by the examples, one method is to bind heparin to the matrix by either covalent or non-covalent methods, to form a heparin-matrix. The heparin then non-covalently binds heparin-binding growth factors to the protein matrix. Alternatively, a fusion protein can be constructed which contains a crosslinking region such as a factor XIIIa substrate and the native protein sequence. Incorporation of degradable linkages between the matrix and the bioactive factors can be particularly useful when long-term drug delivery is desired, for example in the case of nerve regeneration, where it is desirable to vary the rate of drug release spatially as a function of regeneration, e.g. rapidly near the living tissue interface and more slowly farther into the injury zone. Additional benefits include the lower total drug dose within the delivery system, and spatial regulation of release which permits a greater percentage of the drug to be released at the time of greatest cellular activity.

Description

MATRICES OF MODIFIED PROTEIN WITH GROWTH FACTOR FOR TISSUE ENGINEERING FIELD OF THE INVENTION The invention relates to fusion proteins or peptides containing PTH and an amino acid sequence that allows binding interactions with matrices and with the use of fusion proteins or peptides in the repair and regeneration of tissues, and in the controlled release of PTH.

BACKGROUND OF THE INVENTION The parathyroid hormone (PTH) is a peptide of 84 amino acids that is elaborated and. . secreted by the parathyroid gland. This hormone plays a primary role in the control of serum calcium levels through its action on various tissues, including bone. Studies in humans with various forms of PTH have demonstrated an anabolic effect on bones, which makes them interesting for the treatment of osteoporosis and related bone disorders (U.S. Patent No. 5,747,456 to Chorev, et al., And WO. 00/10596 from Eli Lilly &Co.).

Parathyroid hormone acts on cells by binding to a cell surface receptor. It is known that this receptor is found on osteoblasts, the cells that are responsible for the formation of new bones. It has been reported that the N-terminal 34 amino acid domain of the human hormone is biologically equivalent to the full length hormone. PTH 1-34 and its mode of action was first reported in U.S. Patent No. 4,086,196. Because the research has been conducted on PTH 1-34 and other truncated versions of the natural human PTH form, such as, for example, PTH1-25, PTH1-31 and PTH1-38 (see, for example, Rixon RH, et al., J Bone Miner, Res., 9 (8): 1179-89 (August 1994) The mechanism by which PTH influences bone remodeling is complicated, which has led to conflicting results and subsequently-, To a significant number of studies on the exact mechanism involved, it has been shown that if PTH is administered systemically in a continuous manner, bone density will decrease, on the contrary, it has been reported that if the same molecule is administered in a pulsatile manner, bone density will increase (see, for example, WO 99/31137 by Eli Lilly &Co.) · This apparent contradiction can be explained by the mechanism by which PTH modulates bone remodeling and subsequently the visible parameter of bone density Within the mature bone, the r PTH eceptor has only been shown to be present on the surface of cells of the osteoblast lineage, but not on osteoclasts. The role played by PTH in bone remodeling is directed through osteoblasts as opposed to osteoclasts. However, cells in different stages of the osteoblast lineage respond differently when they bind to PTH. Therefore, the dramatic differences observed when PTH is administered using different methods can be taken into account for the understanding of the different effects that the same molecule has on the different cells within the osteoblast lineage. When PTH binds to a mesenchymal hemocytoblast, the cell is induced to differentiate into a proteoblast. Thus, when PTH is added to the system, there is an increase in the proteoblast population. However, these proteoblast cells also have the PTH receptor, and the subsequent binding of PTH to the receptor on these cells leads to a different response. When PTH binds to proteoblast, this results in two separate consequences that lead to bone resorption. First, it inhibits further differentiation of proteoblast cysts in osteoblasts. Second, it increases the secretion of Interleukin 6 (IL-6) from the proteoblasts. IL-6 both inhibits proteoblast differentiation, as well as increases the differentiation of proteoclasts inside the osteoclasts. This dual response of the cells within the osteoblast lineage is that it provides the complex reaction between bone remodeling and exposure to PTH. If the PTH is dosed periodically for short periods of time, then the mesenchymal stem cells are induced to differentiate into osteoblasts. The short dosing periods then prevent the newly formed proteoblasts from producing IL-6, preventing the activation of the osteoclasts. Therefore, during the dosing intervals, these newly formed preoblasts can be further differentiated into osteoblasts, resulting in bone formation. However, if a constant dose of PTH is applied, then the pre-osteoblasts will have the opportunity to initiate the production of IL-6, thus activating the osteoclasts and inhibiting them, leading to the opposite effect: bone resorption. For repair or regeneration of tissues, the cells must migrate into a wounded mass, proliferate, express the matrix components or form the extracellular matrix, and constitute a final tissue conformation. Frequently, multiple cell populations, often including vascular and nervous cells, must participate in this morphogenetic response. For this to happen it has been shown that the matrices improve a lot and it has been found that in some cases they are essential. Procedures have been developed to develop matrices from natural or synthetic sources or a mixture of both. The growing matrices of natural cells are subjected to remodeling by cellular influence, all based on proteolysis, for example, by plasmin (degrading fibrin) and matrix metalloproteinazas (degrading collagen, elastin, etc.). This degradation is quite localized and occurs only at the moment of direct contact with the migrant cell. In addition, the supply of specific cellular signaling proteins, such as, for example, growth factors, is tightly controlled. In the natural model, growth matrices of macroporous cells are not used, instead of the microporous matrices the cells can be degraded, locally and at the time of demand, as the cells migrate to the interior of the matrix. Due to issues related to immunogenicity, expensive production, limited availability, variability and batch purification, matrices based on synthetic precursor molecules have been developed, such as, for example, modified polyethylene glycol in and / or on the body. While much work has been done in the study of the systemic effects of PTH, the research has not explored the local or topical administration of PTH. As PTH has a direct anabolic effect on the cell line of osteoblasts, it must have an important potential to cure bone defects as well as to influence bone density if it occurs properly within a defective site. Once the defect has been filled with pre-osteoblasts, if the PTH signal is canceled, the newly formed pre-osteoblasts can then differentiate into osteoblasts and initiate the conversion of the injured mass, first into wounded bone tissue and then into a Mature bone structure. Therefore, an object of the present invention is to provide a PTH in a form that can be attached to a matrix for tissue repair, regeneration and remodeling. An additional objective is to present a PTH in a form suitable for topical or local administration to a patient to cure bone defects.

SEMIARY OF THE INVENTION Fusion peptides containing a parathyroid hormone (PTH) in one domain and a substrate domain capable of covalently crosslinking to a matrix in another domain and the matrices, and the kits containing these proteins are disclosed herein. of fusion or peptides. The fusion proteins are covalently bound to materials natural or synthetic to form matrices, can be used to cure bone defects. Optionally, all. the components for the formation of the matrix are applied to a bone defect and the matrix is formed at the application site. The fusion peptide can be incorporated into the matrix in such a way that the fusion peptide as a whole or only the respective PTH sequence of the first domain is released by degradation of the matrix, by enzymatic and / or hydrolytic action.

Also, the fusion peptide may contain a degradable linkage. between the first and second domain containing hydrolytic or enzymatic cleavage sites. In particular, the fusion peptide contains the PTH in a domain, a substrate domain capable of being covalently crosslinked to a matrix in a second domain, and a degradation site between the first and the second domain. Preferably, PTH is human PTH, although PTH from other sources may be suitable, such as, for example, bovine PTH. PTH can be PTH 1-84 (natural), PTH 1-38, PTH 1-34, PTH 1-31, or PTH 1-25, or any modified or allelic versions of PTH that exhibit properties, ie, bone formation , similar to the previous one. The degradation site allows the delivery rate to vary to different locations within the matrix depending on the cellular activity at that location and / or within the matrix. Additional benefits include the lower total dose of drugs within the delivery system, and the spatial regulation of the release that allows a greater percentage of the drug to be released at the time of increased cellular activity.

BRIEF DESCRIPTION OF THE DRAWINGS OR FIGURES Figure 1 is a chromatogram for fluorescence detection of fibrin gels containing peptide degranded with plasmin and free peptide. The chromatography by size exclusion of a fibrin gel degraded with the peptide a2 ??? - 7-? ???? 2? -? 34 incorporated (-), with the same free peptide added to the degraded fibrin gel containing the incorporated peptide (| ""), and the free peptide only (-). The N-terminal leucine residue was digested (abbreviated dL). The free peptide eluted at longer times, corresponding to a lower molecular weight, than did the peptide incorporated in the fibrin gel during coagulation, demonstrating the covalent binding to fibrin degrades and in this way the covalent incorporation via the action of Factor XlIIa activity. Figure 2 is a graph of the incorporation of dLNQEQVSPLRGD (SEQ ID NO: 1) into fibrin gels with the exogenous Factor XIII added. When 1 U / mL was added, the level of incorporation increased so that more than 25 moles of peptide / moles of fibrinogen could be reached. Figure 3 is a graph of the bidominium peptide incorporation dLNQEQVSPLRGD (SEQ ID NO: 1), into undiluted fibrin gum. Three separate reagent kits were tested and in each case a high level of incorporation could be observed, reaching 25 moles of peptide / moles of fibrinogen. The concentration of exogenous peptide required for maximum incorporation was at least 5 iriM, possibly due to diffusion limitations within the fairly dense fibrin matrix that is created. The level of incorporation was very consistent, with each reagent kit providing a similar incorporation profile.

DETAILED DESCRIPTION OF THE INVENTION Products and methods for the repair, regeneration or remodeling of hard tissue, in particular for bone growth, are described herein., using natural and synthetic matrices having the PTH that can be distributed incorporated herein. Natural matrices are biocompatible and biodegradable and can be formed in vitro or in vivo, at the time of implantation. PTH · can be incorporated into the matrices and maintain their total bioactivity. PTH can be incorporated in a way that can be distributed, using techniques that provide control over the shape and timing and to what degree PTH is released, so that the matrix can be used to repair tissues directly or indirectly, using the matrix as a controlled release vehicle.

Definitions "Biomaterial", in the sense in which it is used herein, refers to a material intended to be interconnected with biological systems to evaluate, treat, augment, or replace any tissue, organ or function of the body depending on the material either permanently or temporarily. The terms "biomaterial" and "matrix" are used interchangeably herein and signify a degraded polymer network which, depending on the nature of the matrix, may increase with water although not dissolve in the water, that is, form an idrogel that remains in the body during a period of time. a certain period of time fulfilling certain support functions for traumatized or defective hard tissue. "PTH fusion peptide" in the sense in which it is generally used herein, refers to a peptide that contains at least a first and a second domain. One domain contains a PTH (natural or truncated forms, in particular PTH 1-34), and the other domain contains a substrate domain that will be degraded to a matrix. A site of enzymatic or hydrolytic degradation may also be present between the first and the second domain. "Strong nucleophile," in the sense in which it is generally used in the present, refers to a molecule that is capable of donating a pair of electrons to a. electrophile in a polar bond forming reaction. Preferably, the Strong nucleophile is more nucleophilic than water at a physiological pH. Examples of strong nucleophiles are trolls and amines. "Conjugated unsaturated bond", in the sense in which it is generally used in the present, refers to the alternation of multiple carbon-carbon bonds, carbon-he eroatomo or het eroátomo-heteroatorno with individual bonds, or the union of a group functional to a macromolecule, such as, for example, a synthetic polymer or a protein. These links may experience addition reactions. "Conjugated unsaturated group", in the sense in which it is generally used herein, refers to a molecule or region of a molecule, which contains an alternation of carbon-carbon, carbon-heteroatom or heteroatom-heteroatom bonds, with individual links, which have a multiple bond that can experience addition reactions. Examples of conjugated unsaturated groups include, but are not limited to: vinylsulfones, acrylates, acrylamides, quinones, and vinylpyridiniums, for example, 2- or 4-vinylpyridinium and itaconates. "Synthetic precursor molecules", in the sense in which it is generally used herein, refers to molecules that do not exist in nature. "Precursor components or polymers that occur in nature", in the sense in which it is generally used herein, refers to molecules that could be found in nature. "Functional! Zar", in the sense in which it is generally used herein, refers to the modification of a molecule in a manner that results in the union of a functional group or entity. For example, a molecule can have a functional group by introducing a molecule that converts the molecule into a strong nucleophile a conjugated unsaturation. Preferably, a molecule, for example PEG, is provided with a functional group to be converted to a thiol, amine, acrylate, or quinone. Proteins, in particular, can also be effectively integrated with functional groups by partially or completely reducing disulfide bonds to create free thiols. "Functionality", in the sense in which it is generally used herein refers to the number of reactive sites in a molecule.

"Functionality of branch points", in the sense in which it is generally used herein, refers to the number of branches that extend from a point in the molecule. "Adhesion site or cell binding site", in the sense in which it is used in generally herein, refers to a sequence of peptides to which a molecule is attached, for example, an adhesion-stimulating receptor on the surface of a cell. Examples of adhesion sites include, but are not limited to: the RGD sequence from fibronectin, and the sequence YIGSR (SEQ ID NO: 2) from laminin. Preferably, the adhesion sites are incorporated into the biomaterial by including a substrate domain that can be degraded with a matrix. "Biological activity", in the sense in which it is generally used herein refers to functional events caused by a protein of interest. In some embodiments, this includes the events analyzed by measuring the interactions of a polypeptide with another polypeptide. They also include analyzing the effect that the protein of interest has on growth, differentiation, death, migration, adhesion, cellular interactions with other proteins, enzymatic activity, protein phosphorylation or dephosphonylation, transcription or translation. "Sensitive biological molecule" in the sense in which it is generally used herein, refers to a molecule that is found in a cell, or in a body, or that can be used as a therapeutic agent for a cell or a body , which can react with other molecules in its presence. Examples of sensitive biological molecules include, but are not limited to: peptides, proteins, nucleic acids and drugs. Biomaterials can be produced in the presence of sensitive biological materials, without negatively affecting sensitive biological materials. "Regenerate", in the sense in which it is generally used in the present, means to grow again a portion or the whole of something, such as, for example, hard tissue, in particular bones. "Multifunctional", in the sense in which it is generally used herein, refers to more than one electrophilic and / or nucleophilic functional group per molecule (i.e., monomer, oligo and polymer). "Self-selective reaction" in the sense in which it is generally used in the present, means that the first precursor component of a composition reacts much faster with the second precursor component of the composition and vice versa than with other compounds present in a mixture or in reaction site. In the sense in which it is used herein, the nucleophile is preferably attached to an electrophile and an electrophile preferably binds to a strong nucleophile, in place of other biological compounds. "Degradation", in the sense in which it is generally used in the present, means the formation of covalent bonds. However, it can also refer to the formation of non-covalent bonds, such as, for example, ionic bonds, or combinations of covalent and non-covalent bonds. "Polymeric network", in the sense in which it is generally used in the present, means the product of a process in which virtually all monomers, oligo or polymers are bound by intermolecular covalent bonds through their functional groups available to produce a large molecule. "Physiological", in the sense in which it is generally used in the present, means the conditions as they may be found in living vertebrates. In particular, physiological conditions, refers to conditions in the human body such as, for example, pH temperature, etc. Physiological temperatures mean in particular a temperature variation between 35 ° C to 42 ° C, preferably about 37 ° C. "Degradation density", in the sense in which it is generally used herein refers to the average molecular weight between two degradations (Me) of the respective molecules. "Equivalent weight", in the sense in which it is generally used herein, refers to mmol of the functional group / g of the substance. "Volume increase", in the sense in which it is generally used herein refers to the increase in volume and mass by the proportion of water by the biomaterial. The terms "water absorption" and "volume increase" are used interchangeably throughout this application. "Balance state", in the sense in which it is generally used herein, means the state in which a hydrogel does not experience an increase or decrease in mass when stored under constant water conditions.

I. Matrices and PTH A. Materials for the matrix The matrix is formed by ionically degrading, covalently, or by combinations thereof, the precursor molecules for a polymer network or by increasing one or more polymeric materials, ie, the matrices, to form a polymer network having sufficient inter-polymer separation to allow internal growth or migration in the cell matrix. In one embodiment, the matrix is formed of proteins, preferably proteins naturally present in the patient, within which the matrix will be implanted. A particularly preferred matrix protein is fibrin, although matrices made from other proteins, such as, for example, collagen or gelatin, can also be used. Polysaccharides and glycoproteins can also be used to form the matrix. It is also possible to use synthetic polymers that can be degraded by ionic or covalent agglutination.

Fibrin matrices Fibrin is a natural material that has been reported for various biomedical applications. Fibrin has been described as a material for cellular internal growth matrices in U.S. Patent No. 6,331,422 to Hubbell et al. Fibrin gels have been used as sealants because of their ability to bind to many tissues and their natural function is wound healing. Some specific applications include the use as a sealer for the union of vascular grafts, the union of cardiac valves, the placement of bones in fractures and repair of tendons (Sierra, DH, Journal of Eiomaterials Applications, 7: 309-352 (1993)). ). Additionally, these gels have been used as devices for drug delivery, and for neuronal regeneration (Williams, et al., Journal of Comparative Neurobiology, 264: 284-290 (1987)). Although fibrin provides a solid support for tissue regeneration and cellular internal growth, there are few active sequences in the monomer that directly improve these processes. The process by which fibrinogen polymerizes inside fibrin has also been characterized. Initially, a protease cleaves the dimeric fibrinogen molecule at two symmetric sites. There are several possible proteases that can cleave fibrinogen, including thrombin, reptilase, and protease III, and each serves the protein at a different site (Francis, et al., Blood Cells, 19: 291-307, 1993). Once the fibrinogen is cleaved, a self-polymerization step occurs in which the fibrinogen monomers join and form a non-covalently degraded polymer gel (Sierra, 1993). This self-assembly occurs because the binding sites are exposed after the protease cleavage occurs. Once exposed, these binding sites in the center of the molecule can bind to other sites in the fibrinogen chains, which are present at the ends of the peptide chains (Stryer, L. In Biochemistry, WH Freeman & Company, NY, 1975). In this way, a polymer network is formed. Factor XlIIa, a transglutaminase activated from Factor XIII by thrombin proteolysis, can then covalently degrade the polymer network. There are other transglutaminases and they may also be involved in covalent degradation and graft formation with the fibrin network. Once a degraded fibrin gel is formed, subsequent degradation is strictly controlled. One of the key molecules to control fibrin degradation is the a2-plasmin inhibitor (Aoki, N., Progress in Cardiovascular Disease, 21: 267-286, 1979). This molecule acts by degrading the fibrin α chain through the action of Factor XlIIa (Sakata, et al., Journal of Clinical Investigation, 65: 290-297, 1980). By attaching itself to the gel, a high concentration of the inhibitor can be localized with the gel. The inhibitor then acts by preventing the binding of plasminogen with fibrin (Aoki, et al., - Thrombosis and Haemostasis, 39: 22-31, 1978) and by activating plasmin (Aoki, 1979). The a2-plasmin inhibitor contains a glutamine substrate. The exact sequence has been identified as NQEQVSPL (SEQ ID NO: 12), with the first glutamine being the amino acid for degradation. It has been shown that bi-domain peptides, which contain a substrate sequence of Factor XlIIa and a bioactive peptide sequence, can be degraded in fibrin gels and that this bioactive peptide maintains its cellular activity in vitro (Schense, JC, et al. al. (1999) Bioconj Chem. 10: 75-81).

Synthetic matrices The degradation reactions for the formation of synthetic matrices for the application in which are included (i) the polymerization of free radicals between two or more precursors containing unsaturated double bonds, as described in Hern et al., J Biomed Mater. Res. 39: 266-276 (1998), (ii) the nucleophilic substitution reaction such as, for example, between a precursor that includes an amine group and a precursor that includes a succinimidyl group as disclosed in U.S. Pat. No. 5,874,500 of Rhee et al., (Iii) the condensation and addition reactions and (iv) the Michael-type addition reaction between a strong nucleophile and a conjugated unsaturated group or bond (as a strong electrophile). In particular, the reaction between a precursor molecule having a thiol or amine group such as the nucleophilic group and the precursor molecules including acrylate or vinylsulfone groups as electrophilic groups is preferred. The thiol group is most preferred as the nucleophilic group. Michael-type addition reactions are described in WO 00/44808 to Hubbell et al., The content thereof being incorporated herein by reference. Michael-type addition reactions allow the in situ degradation of at least a first and a second precursor component under physiological conditions in a self-selective manner, even in the presence of sensitive biological materials. When one of the precursor components has a functional group of at least two, and at least one of the other precursor components has a functional group greater than two, the system will react self-selectively to form a degraded three-dimensional biomaterial. Preferably, the conjugated unsaturated groups or conjugated unsaturated bonds are acrylates, vinylsulfones, methacrylates, acrylamides, methacrylamides, acrylonitriles, vinylsulfones, 2- or 4-vinylpyridinium, maleimides, or quinones. The nucleophilic groups are preferably thiol groups, amino groups, or hydroxyl groups. The thiol groups are practically more reactive than the non-protonated amine groups. As stated above, pH is important in this regard: the deprotonated thiol is practically more reactive than the protonated thiol. Therefore, addition reactions that include a conjugated unsaturation, such as, for example, an acrylate or a quinone, with a thiol to convert two precursor components into a matrix will often be carried out better faster and self-selectively at a pH of about 8. At the pH of about 8, most of the thiols of interest are deprotonated (and thus are more reactive) and most of the amines of interest are still protonated (and thus are less reactive). When a thiol is used as the first precursor molecule, a conjugate structure that is selective in its reactivity to the thiol relative to the amines is quite convenient. The first and second suitable precursor molecules include proteins, peptides, polyoxyalkylenes, poly (vinyl alcohol), poly (ethylene alcohol, -co-vinyl), poly (acrylic acid), poly (ethylene-co-acrylic acid), poly (ethyloxazoline) ), poly (vinylpyrrolidone), poly (ethylene-co-vinylpyrrolidone), poly (maleic acid), poly (ethylene-co-maleic acid), poly (acrylamide), and block copolymers of poly (ethylene oxide) -co -poly (propylene oxide). A particularly preferred precursor molecule is polyethylene glycol. Polyethylene glycol (PEG) provides a convenient building block. Linear PEGs (which means two ends) or branched PEGs (meaning more than two ends) can easily be acquired or synthesized and then add functional groups to the PEG end groups, to introduce any strong nucleophile, such as for example , a thiol, or a conjugated structure, such as, for example, an acrylate or a vinylsulfone. When these components are mixed either with each other or with a corresponding component in a slightly basic environment, a matrix will be formed by the reaction between the first and second precursor components. A PEG component can be reacted with a component without PEG, and the molecular weight or hydrophilicity of any component can be controlled to manipulate the mechanical characteristics, permeability, and water content of the resulting biomaterial. These materials are generally useful in medical implants, as will be described in more detail below. In the formation of matrices, especially the matrices that are desired to degrade in vivo, peptides, provide a very convenient constituent block. It is simple to synthesize peptides containing two or more cysteine residues, and this component can then easily serve as the first precursor component with the nucleophilic groups. For example, a peptide with two free cysteine residues will easily form a matrix when mixed with a PEG tri-vinylsulfone (a PEG having three branches with vinylsulfones in each of its branches) at a physiological or slightly higher pH (e.g. , 8 to 9). The gelation can also proceed well at an even higher pH, although with the potential loss of self-selectivity. When the two liquid precursor components are mixed together as they react over a period of a few minutes to form an elastic gel, consisting of a network of PEG chains, which carry the network nodes, with the peptides as connecting bonds .

The peptides can be selected as protease substrates, in order to make the network capable of being infiltrated and degraded by the cells, as is done in a protein-based network, such as, for example, in a matrix of proteins. fibrin. Preferably, the sequences in the domains are substrates for enzymes that are involved in cell migration (eg, as substrates for enzymes such as, for example, collagenase, plasmin, metalloproteinase (MMP) or elastases), although suitable domains will not be limited to this sequence. A particularly useful sequence is a substrate for the enzymatic plasmin (see Examples). The degradation characteristics of the gels can be manipulated by changing them from the peptide that serves as the degrading nodes. A gel can be produced that is degradable by collagenase, but not by plasmin, or by plasmin but not by collagenase. In addition, it is possible to cause the gel to degrade faster or slower in response to this enzyme, simply by changing the amino acid sequence to alter the Km or Kcatr or both, of the enzymatic reaction. In this way, a biomaterial can be made that is biomimetic, and that is capable of being remodeled by the normal remodeling characteristics of the cells. For example, this study shows the substrate sites for the important plasmin protease. The gelation of the PEG with the peptide is self-selective. Optionally, biofunctional agents can be incorporated into the matrix to provide chemical bonding with other species (eg, a tissue surface). Having protease substrates incorporated in the matrix is important when forming the matrix from PEG vinylsulfone. Apart from the matrices formed from the reaction of the PEG acrylates and the PEG thiols, the matrices formed from PEG vinyl sulfones and PEG thiols do not contain hydrolytically degradable bonds. Therefore, the incorporation of protease substrates allows the matrix to degrade in the body. Synthetic matrices are operationally simple to form. Two liquid precursors are mixed, one precursor contains a precursor molecule with nucleophilic groups and the other precursor molecule contains the electrophilic groups. As the solvent can serve physiological saline solution. Minimal heat is generated by the reaction. Therefore, gelation is carried out in vivo or in vitro, in direct contact with the tissue, without harmful toxicity. In this way, polymers other than PEG can be used, either telehelical modified or modified in their side groups. For most health indications, the rate of cell internal growth or migration of the cells in the matrix in combination with a rate of degradation adapted from the matrix is decisive for the total healing response. The potential of the hydrolytically non-degradable matrices that will be invaded by the cells is mainly a function of the lattice density. If the space between the branch points or nodes is too small in relation to the size of the cells or if the rate of degradation of the matrix, which results in the creation of more space within the matrix, is very slow, a very limited healing response will be observed. The healing matrices found in nature, such as fibrin matrices, which are formed as a response to damage in the body, are known to consist of a very separate network that can be very easily invaded by cells. Infiltration is promoted by ligands for cell adhesion that are an integrated part of the fibrin network. Matrices made from synthetic hydrophilic precursor molecules, similar to polyethylene glycol, increase in volume in an aqueous environment after the formation of the polymer network. In order to achieve a sufficiently short gel time (between 3 to 10 minutes at a pH between 7 to 8 and a temperature at a variation of 36 to 38 ° C) and a quantitative reaction during the in situ formation of the matrix in the body, the initial concentration of the precursor molecules must be sufficiently high. Under these conditions, the volume increase could be carried out after network formation, and the necessary initial concentrations could lead to matrices too dense for cellular infiltration, when the matrix is not degradable in an aqueous environment. In this way, the increase in volume of the polymer network is important to lengthen and increase the space between the branch points. Regardless of the initial concentration of the precursor molecules, the hydrogels produced from the same synthetic precursor molecules, such as for example, a PEG vinylsulfone of -four branches and a peptide with SH groups, will increase in volume to the same water content in the state of equilibrium. This means that the higher the initial concentration of the precursor molecules, the greater the final volume of the hydrogel when it reaches its equilibrium state. If the available space in the body is too small to allow sufficient volume increase and in particular if the bond formed from the precursor components is not hydrolytically degradable, the rate of cellular infiltration and the healing response will decrease. As a consequence, the optimum must be found between the two contradictory requirements for application in the body. Good cellular infiltration and subsequent healing responses have been observed with a three-dimensional polymeric network formed from the reaction of a trifunctional branched polymer with at least three branches practically similar in molecular weight and a second precursor molecule which is at least one bifunctional molecule. The equivalent weight index of the functional groups of the! First and second precursor molecules are between i 0.9 and 1.1. The molecular weights of the branches of the first precursor molecule, the molecular weight of the second precursor molecule and the functionality of the branching points are selected in such a way that the water content of the resulting polymer network is between% by weight of equilibrium and 92% by weight of the total weight of the polymer network after finishing the water absorption. Preferably, the water content is between 93 and 95% by weight of the total weight of the polymer network and the water after completing the water absorption. The term of water absorption can be reached either when the equilibrium concentration is reached or when the available space in the biomaterial does not allow an additional volume increase. It is therefore preferred that the selection of the initial concentrations of the precursor components be as low as possible. This is true for all matrices that can increase in volume, but in particular for those matrices that undergo degradation, and are supplied by cells that do not contain hydrolytically degradable bonds in the polymer network. The equilibrium between gel time and low initial start concentration in particular for hydrolytically non-degradable gels should be optimized based on the structure of the precursor molecules. In particular, the molecular weight of the branches of the first precursor molecule, the molecular weight of the second precursor molecule and the degree of branching, ie the functionality of the branching points, must be adjusted accordingly. The actual reaction mechanism has a minor influence on this interaction. This first precursor molecule is a polymer with three or four branches with a functional group at the end of each branch and the second precursor molecule is a linear bifunctional molecule, preferably a peptide containing at least two cistern groups, then the weight The molecular branches of the first precursor molecule and the molecular weight of the second precursor molecule are preferably selected in such a way that the bonds between the branching points after network formation have a molecular weight in the range from 10 to 13 kD (under the conditions in which the links are linear, not branched), preferably between 11 and 12 kD. This allows an initial concentration of the sum of the first and second precursor molecules in the range between 8 to 12% by weight, preferably between 9 and 10% by weight of the total weight of the first and second precursor molecules in solution (before the formation of the network). In case the degree of branching of the first precursor component increases to eight and the second precursor molecule is still a linear bifunctional molecule, the molecular weight of the bonds between branching points preferably increases to a molecular weight between 18 to 24 kDa . In case the degree of branching of the second precursor molecule increases from the linear to a precursor component of three or four branches, the molecular weight, ie, the length of the bonds will accordingly increase. In a preferred embodiment of the present invention, a composition is selected to include as the first precursor molecule a 15 kD polymer, with three trifunctional branches, i.e., each branch having a molecular weight of 5 kD and as the second precursor molecule. , a bifunctional linear molecule of a molecular weight in the range between 0.5 to 1.5 kD, even more preferably approximately 1 kD. Preferably, the first and the second precursor component is a polyethylene glycol. In a preferred embodiment, the first precursor component includes conjugated unsaturated groups or bonds as functional groups, an acrylate or a vinylsulfone is more preferred, and the functional groups of the second precursor molecule include a nucleophilic group, preferably, a thiol or amino group. In another preferred embodiment of the present invention, the first precursor molecule is a 20 kD polymer of four branches' (each branch has a molecular weight of 5kDa) having functional groups at the end of each branch and the second precursor molecule is a bifunctional linear molecule of a molecular weight in the range between 1 to 3 kD, preferably between 1.5 and 2 kD. Preferably, the first precursor molecule is a polyethylene glycol having vinylsulfone groups and the second precursor molecule is a peptide having cistern groups. In both preferred embodiments, the initial concentration of the sum of the first and second precursor molecule ranges from 8 to 11% by weight, preferably between 9 and 10% by weight of the total weight of the first and second precursor molecule and water (before of the formation of the polymer network), preferably between 5 and 8% by weight until a gel time of less than 10 minutes is reached. These compositions have a gel time at pH 8.0 and 37 ° C of about 3-10 minutes after mixing. When the matrix contains hydrolytically degradable bonds, formed, for example, by the preferred reaction between acrylates and thiols, the crosslink density with respect to cellular infiltration is especially important at the start, although in an aqueous environment, the bonds will hydrolyze and the network will separate, to allow cellular infiltration. With an increase in the degree of total branching of the polymer network, the molecular weight of the entanglements, i.e., the length of the links must increase.

B. Cell binding sites Cells interact with their environment through protein-protein interactions. Protein-oligosaccharide and protein-polysaccharide on the cell surface. The extracellular matrix proteins provide a host of bioactive signals to the cell. This dense network is required to support the cells, and it has been shown that many proteins in the matrix control adhesion, dispersion, migration and cell differentiation (Carey, Annual Review of Physiology, 53: 161-177, 1991). Some of the specific proteins that have been shown to be particularly active include laminin, vitronectin, fibronectin, fibrin, fibrinogen, and collagen (Lander, Journal of Trends in Neurological Science, 12: 189-195, 1989). Many laminin studies have been conducted, and laminin has been shown to play a vital role in the development and regeneration of in vivo media and nerve cells in vitro (Williams, Neurochemical Research, 12: 851-869, 1987), as well as well as in angiogenesis. Some of the specific sequences that interact directly with cellular receptors and cause either adhesion, dispersion or signal transduction have been identified. It has been shown that laminin, a large multidomain protein (Martin, Annual Review of Cellular Biology, 3: 57-85, 1987), consists of three chains with various receptor binding domains. These receptor binding domains include the sequence YIGSR (SEQ ID NO: 2) of the laminin chain Bl (Graf, et al., Cell, 48: 989-996, 1987; Kleinman, et al., Archives of Biochemistry and Biophysics, 272: 39-45, 1989; and Massia, et al, J: of. Biol. Chem., 268: 8053-8059, 1993), LRGDN (SEQ ID NO: 3) of the laminin A chain (Ignatius , et al., J: of Cell Biology, 111: 709-720, 1990) and PDGSR (SEQ ID NO: 4) of the laminin chain Bl (Kleinman, et al., 1989). Other diverse recognition sequences have also been identified. These include IKVAV (SEQ ID NO: 5) of the sheet chain A (Tashiro, et al., J; of Biol. Chem., 264: 1617 -16182, 1989) and the sequence RNIAEI I DI (SEQ ID NO: 6) of the laminin B2 chain (Liesi, et al., FEBS Letters, 244: 141-148, 1989). Frequently, the receptors that bind to these specific sequences have also been identified. A subset of cellular receptors that have been shown to be responsible for the majority of the binding is the integrin superfamily (Rouslahti, E., J. of Clin.Investigation, 87: 1-5, 1991). Integrins are protein heterodimers that consist of subunits a and β. Previous work has shown that the tripeptide RGD binds to various β-integrins? and ß3 (Hynes, RO, Cell, 69: 1-25, 1992; Yamada, KM, J; of Biol. Chem., 266: 12809-12812, 1991), IKVAV (SEQ ID NO: 5) is attached to a 110 kDa receptor (Tas iro, et al., J of Biol. Chem., 264: 16174-16182, 1989); Luckenbill-Edds, et al., Cell Tissue Research, 279: 371-377, 1995), YIGSR (SEQ ID NO: 2) binds to a 67 kDa receptor (Graf, et al., 1987) and DGEA (SEQ ID. NO: 7), a collagen sequence, binds to the integrin (* 2, ββ (Zutter &Santaro, Amer. J. of Patholody, 137: 113-120, 1990) .The receptor for the RNIAEIIKDI sequence ( SEQ ID NO: 6) has not been reported.In a further preferred embodiment, peptide sites are incorporated for cell adhesion in the matrix, namely the peptides that bind to the receptors to stimulate adhesion on the surfaces of the cells in the cells. The biomaterials of the present invention These peptides for stimulating adhesion can be selected from the group as described above In particular, the RGD sequence from fibronectin, the sequence YIGSR (SEQ ID NO: 2) from laminin, is preferred. of binding sites is a particularly preferred embodiment with synthetic matrices, although It can be included with some of the natural matrices. The incorporation can be carried out, for example, simply by mixing a cell binding peptide containing cysteine with the precursor molecule including the conjugated unsaturated group, such as, for example, PEG acrylate, PEG acrylamide or PEG vinylsulfone a few minutes before mixing with the rest of the precursor component including the nucleophilic group, such as, for example, the thiol-containing precursor component. If the cell binding site does not include a cysteine, it can be chemically synthesized to include one. During this first step, the peptide to stimulate adhesion will be incorporated at one end of the precursor with multiple functional groups and with a conjugated unsaturation.; When the remaining multitiol is added to the system, a degraded network will be formed. Another important implication of the way in which the networks are prepared here is the efficiency of incorporation of the hanging bioactive ligands such as, for example, adhesion signals. This step should be quantitative because, for example, unbound ligands (eg, adhesion sites) could inhibit the interaction of cells with the matrix. As will be described later, the derivation of the precursor with these pendant oligopeptides is conducted in a first step in large stoichiometric excess (minimum: 40 times) of multiradified electrophilic precursors on thiols and therefore definitely quantitative. Apart from avoiding unwanted inhibition, this achievement is even biologically more significant: cellular behavior is extremely sensitive to small changes in ligand densities and precise knowledge of incorporated ligands helps to design and understand the interactions of the cell matrix. In summary, the concentration of adhesion sites covalently bound in the matrix significantly influences the rate of cellular infiltration. For example, for a given hydrogel, an RGD concentration in the matrix can be incorporated with supports for internal cell growth and cell migration in an optimal way. The optimum concentration variation of the adhesion sites similar to RGD is between 0.04 and 0.05 mM and even more preferably 0.05 mM in particular for a matrix having an aqueous content between the equilibrium concentration and 92% by weight after the end of the absorption of water. Excellent results of bone healing have been achieved by maintaining the cell migration index and the rate of degradation of the fasting matrix. With respect to the design of the matrix (in particular PTH 1-34 'covalently bound), a polyethylene glycol with four branches with a molecular weight of about 20,000 Da degraded with a GCRPQGI protease degradation site GQDRC (SEQ ID NO: 8) and 0.050 mM GRGDSP (SEQ ID NO: 9) provides particularly good internal cellular growth results and healing of bone defects. The initial concentration of PEG and internal peptide at 10% by weight of the total weight of the molecules and water (before the increase in volume). The gels have a usable consistency and allow the osteoblasts and the precursor cell to easily infiltrate the matrix. The matrix material is preferably biodegradable by naturally occurring enzymes. The degradation index can be manipulated by the degree of degradation and the inclusion of protease inhibitors in the matrix.

C. Domains of the degradable substrate The PTH fusion peptide can be degraded and covalently linked to the matrices through the degradable substrate domain of the PTH fusion peptide. The type of substrate domain depends on the nature of the matrix. For incorporation into fibrin matrices, transglutaminase substrate domains are particularly preferred. The substrate domain of transglutaminase can be a substrate domain of Factor XlIIa. This substrate domain of Factor XlIIa may include GAKDV (SEQ ID NO: 10), KKK (SEQ ID NO: 11), or NQEQVSPL (SEQ ID NO: 12). The coupling between PTH and the substrate domain of transglutaminase can be carried out by chemical synthesis. The transglutaminase substrate domain can be a substrate for a transglutaminase other than Factor XlIIa. The most preferred Factor XlIIa substrate domain has an amino acid sequence of NQEQVSPL (SEQ ID NO: 12) (herein referred to as WTG "). Other proteins recognizing transglutaminase, such as, for example, fibronectin, could be coupled to the peptide of the transglutaminase substrate.

Table 1: Domains of the transglutaminase substrate SEQ ID NO: 13 YRGOTIGEGQQHHLGG (SEQ ID NO: 13) A peptide with glutamine at the site of coupling with transglutaminase in the fibrinogen chain SEQ ID NO: 14 GAKDV (SEQ ID NO: 14) A peptide that mimics the coupling site with lysine in the fibrinogen chain SEQ ID NO: 11 KKKK (SEQ ID NO: 11) A peptide with a polylysine at a random coupling site SEQ ID NO: 12 NQEQVSPL (SEQ ID NO: 12) A peptide that mimics the site of degradation in a plasmin a2 inhibitor (abbreviated TG) For incorporation of PTH into a matrix formed from synthetic precursor components, the fusion peptide is PTH or any other peptide that will be incorporated should be synthesized with at least one additional cysteine (-SH) group preferably at the N-terminus of PTH as the domain of the degradable substrate. Cysteine can either be linked directly to PTH or through a binding sequence. The linker sequence may additionally include an enzymatically degradable amino acid sequence, such that PTH can be excised from the matrix by enzymes in virtually the native form. The free cysteine group reacts with the conjugated unsaturated group of the precursor component in a Michael-type addition reaction. In the case of PTH 1-34, the link to a synthetic matrix for PTH 1-34 is made possible by the binding of an additional amino acid sequence to the N-terminus of PTH 1-34 containing at least one cysteine. The thiol group of cysteine can react with a conjugated unsaturated bond on the synthetic polymer to form a covalent bond. The possibility that (a) only one cysteine binds to the peptide, in the possibility that (b) an enzymatically degradable, degradable sequence of plasmin binds as a linker between the cysteine and the peptide. The sequence GYKNR (SEQ ID NO: 15) between the first domain and the second domain, cysteine, makes the plasmin bond degradable. In this way, the PTH fusion peptides can be further modified to contain a degradable site between the binding site, ie, the second domain (i.e. the substrate domain of Factor XlIIa or the cysteine) and the PTH, that is, the first domain. These sites can be degraded either by non-specific hydrolysis (i.e., an ester linkage) or can be substrates for specific enzymatic degradation (either proteolytic or polysaccharide degradation). These degradable sites allow the engineering treatment of the more specific release of PTH from matrices similar to fibrin gels. For example, degradation based on enzymatic activity allows the release of PTH that will be controlled by a cellular process instead of diffusing the factor through the gel. The degradable site or bond is cleaved by the enzymes released from the cells that invade the matrix. The degradation sites allow PTH to be released with little or no modification with the primary peptide sequence, which can result in greater factor activity. In addition, it allows the release of the factor to be controlled by specific cellular processes, such as, for example, localized proteolysis, rather than diffusion from some porous materials. This allows the factors to be released at different speeds within the same material depending on the location of the cells within the material. This also reduces the amount of total PTH needed, because its release is controlled by cellular processes. The preservation of PTH and its bioavailability are advantages other than the exploitation of specific cellular proteolytic activity with respect to the use of devices for controlled release of diffusion. In a possible explanation for the good healing of a bone defect with PTH covalently bound to a matrix, it seems important that PTH be administered. locally 'for a prolonged period of time (ie, not just a single pulse dose) but not in a continuous way. This is accomplished by slow degradation, through either enzymatic cleavage or hydrolytic cleavage of the matrix. In this way, the molecule is then delivered through a pseudo-pulsed effect that occurs for a sustained period of time. When a cell. rogenitora infiltrates the matrix, it will find a molecule of PTH and can be differentiated into a proteoblasto. However, if this particular cell does not continue to release the bound PTH from the matrix, it will effectively become an osteoblast and initiate the production of the bone matrix. Finally, the therapeutic effects of the peptide are located in the defective region and are subsequently increased. The enzymes that could be used for proteolytic degradation are many. The proteolytically degradable sites could include substrates for activators of collagenase, plasmin, elastase, stomalisin, or plasminogen. Next, example substrates are listed. P1-P5 denotes the positions 1-5 of amino acids towards the amino terminus of the protein from the. site where proteolysis occurs. Pl'-P4 'denote the 1-4 amino acid positions towards the carboxy terminus of the protein from where the proteolysis occurs.

Table 2: Sample substrate sequences for protease Protease P P4 P3 P2 Pl Pl 'P2' P3 'P4' Reference Plasmin L I K M K P Takagi and Doolittle, (1975) Biochem. 14: 5149-5156 Plasmin N F K S Q L Takagi and Doolittle, 1975 Stromelysin Ac G P L. A L T A L Smith et al., (1995). J. Bio. Chem. 270: 6440-6449 Stromelysin Ac P F E L R A NH2 Smith et al., 1995 Elastase z- A A F A N¾ Besson et al., (1996) ñnalytical Biochemistry 237: 216-223 Colagenaza G P L G I A G P Netzel-Arnett et al., (1991) J. Biol. Chem. 266: 6747-6755 t-PA P H Y G R S G G Coombs et al. , 1998. J. Biol. Chem. 273: 4323-4328 u-PA P G s G R S A S G Coombs et al., 1998 In another preferred embodiment, an oligo-ester domain could be inserted between the first and the second domain. This could be carried out using an oligo-ester such as, for example, the lactic acid oligomers. The substrate for nonenzymatic degradation could consist of any bond that undergoes hydrolysis by a mechanism catalyzed by acid or base. These substrates may include oligo-esters such as, for example, oligomers of lactic or glycolic acid. The degradation rate of these materials can be controlled through the choice of oligomer.

D. PTH The term "PTH" in the sense in which it is used in the present, includes the human PTH 1-84 sequence and all modified and allelic, truncated versions of PTH that exhibit properties for bone formation when they are covalently bound to natural or synthetic biodegradable matrices. Preferred truncated versions of PTH are PTH 1-38, PTH 1-34, PTH 1-31 or PTH 1-25. The most preferred is PTH 1-34. Preferably, PTH is human PTH, although PTH from other sources, such as, for example, bovine PTH, may be suitable.

Methods for. incorporation and / or release of bioactive agents In a preferred embodiment for the incorporation of a PTH within the matrix, the matrix includes fibrin which is formed from fibrinogen, a source of calcium and thrombin and the fusion peptide with PTH It will be incorporated into the fibrin during coagulation. The PTH fusion peptide is designated as a fusion peptide that includes two domains, a first and a second, a domain, the second is a substrate for a degrading enzyme such as, for example, Factor XlIIa. , Factor XlIIa is a transglutaminase that is active during < coagulation. · This enzyme, formed naturally from Factor XIII by thrombin cleavage, works to bind the fibrin chains together via amide bonds, formed between the glutamine side chains and the lysine side chains. The enzyme also functions to bind other peptides to fibrin during coagulation, for example, the cell binding sites provided also include a Factor XlIIa. Specifically, the sequence NQEQVSP (SEQ ID NO: 16) has been shown to function as an effective substrate for Factor XlIIa. As described hereinabove, this either binds directly to the PTH or may include a degradation site between the PTH (first domain) and the sequence NQEQVSP (SEQ ID NO: 16) (second domain). As such, the fusion peptide with PTH can be incorporated into the fibrin during coagulation via a substrate of Factor XlIIa.

Design of fusion proteins for, incorporation The fusion peptide with PTH including a first domain including PTH, a second domain including a substrate domain for a degradation enzyme and optionally a degradation site. Second domain can be incorporated into fibrin gels using various different schemes. Preferably, the second domain includes a transglutaminase substrate domain and even more preferably includes a substrate domain of Factor XlIIa. Most preferably, the substrate domain of Factor XlIIa includes NQEQVSP (SEQ ID NO: 16). When this fusion peptide with PTH is present during the polymerization of fibrinogen, that is, during the formation of the fibrin matrix, it is incorporated directly into the matrix. The degradation site between the first and the second domain of the fusion peptide with PTH can be an enzymatic degradation site as described above. Preferably, the degradation site can be cleaved by an enzyme that is selected from the group consisting of plasmin and matrix metalloproteinase. By careful selection of Km and cat from this enzymatic degradation site, the degradation could be controlled to occur either before or after the protein matrix and / or by using similar or different enzymes to degrade the matrix, with the degradation site placement that is prepared for each type of protein and application. This fusion peptide with PTH could be degraded directly in the fibrin matrix as described above. However, the incorporation of an enzymatic degradation site alters the release of PTH during proteolysis. When the cell-derived proteases reach the sequestered fusion peptide, they can cleave the engineered protein and the newly formed degradation site. The resulting degradation products could include the released PTH, which could now be almost free of any fusion sequences engineered, as well as any degraded fibrin.

II. Method of use The matrices can be used for the repair, regeneration, or remodeling of tissues, and / or the release of PTH, before or at the time of implantation. In some cases it will be convenient to induce degradation at the administration site to conform the matrix to the tissue at the implantation site. In other cases, it will be convenient to prepare the matrix before implantation. The cells can also be added to the matrix before or at the time of implantation, or even after implantation, either at the time or after the degradation of the polymer to form the matrix. This can be done in addition to or instead of the degradation of the matrix to produce the interstitial separation designed to stimulate cell proliferation or internal growth. Although in most cases it will be convenient to implant the matrix to stimulate cell growth or proliferation, in some cases bioactive factors will be used to inhibit the rate of cell proliferation. One specific application is to inhibit the formation of adhesions after surgery.

III. Application methods In the preferred embodiment, the material gels in situ in the interior or on the body. In another embodiment, the matrix can be formed outside the body and then applied in the preformed conformation. The matrix material can be produced from synthetic or natural precursor components. Regardless of the type of precursor component used, the precursor components must be separated prior to the application of the mixture to the body to avoid commingling or contacting each other under conditions that allow the polymerization or gelation of the components. To make the contact before administration, a reagent kit can be used that separates the compositions from each other. At the time of mixing at conditions that allow polymerization, the compositions form a three-dimensional network supplemented with the bioactive factor. Depending on the precursor components and their concentrations, gelation can occur almost instantaneously after mixing. This rapid gelling makes injection almost impossible, that is, the pressure extraction of the gelled material through the injection needle. In one embodiment, the matrix is formed from fibrinogen. The fibrinogen, through a cascade of several reactions gels to form a matrix, when contacted with thrombin and a source of calcium at the appropriate temperature and pH. The three components, fibrinogen, thrombin and calcium source, should be stored separately. However, as long as at least one of the three components is kept separate, the other two components can be combined before administration. In a first embodiment, fibrinogen (which may additionally contain aprotinin to increase stability) is dissolved in a buffer solution at a physiological pH (in the range of pH 6.5 to 8.0, preferably 7.0 to 7.5) and stored separately. of a thrombin solution in a calcium chloride buffer (for example, at a concentration ranging from 40 to 50 mM). The buffer solution for fibrinogen can be a histidine buffer solution at a preferred concentration of 50 mM including additionally NaCl at a preferred concentration of 150 mM or buffered saline TRIS (preferably at a concentration of 33 mM). In a preferred embodiment, a reagent kit is provided, which contains a fusion protein, fibrinogen, -trombine and a calcium source. Optionally, the reagent kit may contain a degrading enzyme, such as, for example, Factor XlIIa. The fusion protein contains a bioactive factor, a substrate domain for a degrading enzyme and a degradation site between the substrate domain and the bioactive factor. The fusion protein may be present in the solution of either fibrinogen or thrombin. In a preferred embodiment, the fibrinogen solution contains the fusion protein.

The solutions are preferably mixed with a two-way syringe device, in which mixing occurs when the contents of both syringes are extracted by pressure through a mixing chamber and / or needle and / or static mixer. In a preferred embodiment, both fibrinogen and thrombin are stored separately in lyophilized form. Either one may contain the fusion protein. Before being used, Tris or histidine buffer is added to the fibrinogen, the buffer may additionally contain aprotinin. The lyophilized thrombin is dissolved in the calcium chloride solution. Subsequently, the fibrinogen and thrombin solutions are placed in separate vials / containers and mixed by a two-way connecting device, such as, for example, a two-way syringe. Optionally, the vial / j eringa containers are divided into two parts thus having two chambers separated by an adjustable division that is perpendicular to the wall of the syringe body. One of the chambers contains freeze-dried fibrinogen or thrombin, while the other chamber contains a suitable buffer solution. When the plunger is pressed down, the division moves and releases the buffer inside the fibrinogen chamber to dissolve the fibrinogen. Once both fibrinogen and thrombin are dissolved, both bodies of the syringe divided into two parts are joined to a two-way connection device and the contents are mixed when they are removed by pressure through the injection needle attached to the device of connection. Optionally, the connecting device contains a static mixer to improve the mixing of the contents. In a preferred embodiment, the fibrinogen is diluted eight times and the thrombin is diluted 20 times before mixing. This ratio results in a gel time of about one minute. In another preferred embodiment, the matrix is formed from synthetic precursor components capable of undergoing a Michael addition reaction. Because the nucleophilic precursor component (the multitiol) only reacts with the multi-receptor component (the conjugated unsaturated group) at basic pH, the three components that must be stored separately before mixing are: the base, the nucleophilic component and the component multiaceptor. Both the multiaceptor and the multitiol component are stored as a solution in shock absorbers. The two compositions can include the cell binding site and additionally the bioactive molecule. In this way, the first composition of the system for example may include the solutions of the nucleophilic component and the second composition of the system may include the solution of the multi-receptor component. Either or both compositions may include the base. In another embodiment, the multi-receptor and the multitiol may be included as a solution in the first composition and the second composition may include the base. The connection and mixing are presented in the same manner as described above for fibrinogen. The body of the syringe divided into two parts is equally suitable for the synthetic precursor components. Instead of fibrinogen and thrombin, the multi-receptor and multitiol components are stored in a sprayed form in one of the chambers and the other chamber contains the basic buffer. The following examples are included to demonstrate the preferred embodiments of the invention. While the compositions and methods have been described in the preferred terms and modalities, it will be apparent to one skilled in the art that variations to the composition, methods and steps or sequence of steps of the method described in present without departing from the concept spirit and scope of the invention.

EXAMPLE 1: Matrices containing covalently linked TGPTH. Synthesis of TGPTH PTH peptide 1-34-mer showing similar activity with the whole protein, and proteins of this length can be synthesized by synthesis methods for standard solid state peptides. All peptides were synthesized on solid resin using an automatic peptide synthesizer using the standard 9-fluorenylmethyloxycarbonyl chemistry. The peptides were purified by cl8 chromatography and analyzed using reverse phase chromatography via HPLC to determine the purity, as well as mass spectroscopy (MALDI) to identify the molecular weight of each product.

Using this method, the following peptide was synthesized (referred to herein as "TGPTH"): NH3-Asn-Gln-Glu-Gln-Val-Ser-Pro-Leu-Ser-Val-Ser-Glu-Ile-Gln -Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val -His-Asn-Phe-COOH (SEQ ID NO: 17) In vivo results The activity of TGPTH to improve bone regeneration was tested in a TISSUCOL® matrix in a hole defect drilled in a sheep. Holes eight mm and 12 mm deep were created in the femur and proximal and distal humerus of a sheep. These orifices were filled with a polymerizing fibrin gel in situ. The defects were left empty, filled with TISSUCOL® or TGPTH was added to the TISSUCOL® fibrin at 400 g / mL prior to polymerization. In each example in which TISSUCOL® was used, it was diluted four times from the available standard concentration, leading to a fibrinogen concentration of 12.5 mg / mL. The defects were allowed to heal for eight weeks. After this curing period, the animals were sacrificed, and the bone samples were removed and analyzed by microcomputerized topography (pCT). Then the percentage of defective volume filled with calcified bone tissue was determined. When the defects were left empty, there was no formation of calcified tissue inside the fibrin matrix. When only one fibrin gel was added, there was practically no bone healing either. However, with the addition of 400 g / mL of TGPTH, the level of healing increased dramatically, with the defect filling to 35% with calcified bone.

EXAMPLE 2: Answer. healing with modified PTH 1-34 bound to a fibrin matrix. Materials The modified version of PTH1-34 that can be incorporated into a fibrin matrix has been tested for the healing response in the critically sized skull defect in rats. A fibrin gel was made from fibrin sealant precursor components of the TISSÜCOL® reagent kit (Baxter AG, CH-8604 Volketswil / ZH). Fibrinogen was diluted in sterile 0.03M Tris buffered (TBS, pH 7.4) to form a solution of about 8 mg / mL and thrombin was diluted in sterile CaCl2 solution 50 mM to form a solution of 2 U / mL. The final fibrinogen concentration was the original TISSUCOL® formulation 1: 8 (approximately 100 mg / mL) and the original TISSUCOL® thrombin concentration 1: 160 (approximately 500 IE / mL). Then a predetermined amount of TG-pl-PTHi-34 or TGPTH1-.34 was added to the thrombin, and mixed to form a homogeneous concentration. To form the fibrin gene, the diluted precursors were mixed together by injecting fibrinogen into a tube containing thrombin. In the case of the perforated defect in a sheep (as will be described later), this mixture was then injected immediately into a perforated effect created on a cancellous bone of the sheep, where a fibrin gel formed in 1-5 minutes. In the first series of animal experiments the efficacy of a fusion protein was tested which contained PTHi_34 as the bioactive facto (NQEQVSPLYKNRSVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF, SEQ ID NO: 18) in healing cortical bones in a small animal model. The sequence YKNR (SEQ ID NO: 19), makes the link degradable plasmin ("TG-pl - ???? - 34) | TG-pl-PTHi-34 is produced by chemical synthesis.The purification was carried out via reverse phase HPLC (C18 column) by using a TFA as the counter ion which resulted in a final product that was a TFA salt. the purity of the TG-pl-PTHi_34 was determined to be 95%. the response of healing was explored in healing times both short (3 weeks) and long (7 weeks) to determine if an improvement in healing could be observed.

Cranial defect of critical size in rats Rats were anesthetized and the cranial spindle exposed. The periosteum on the external surface of the skull retracted in such a way that it could not play a role in the healing process, and an 8 mm round individual defect was created. This defect size was selected since it had previously been determined that defects of 8 mm or greater do not heal spontaneously on their own, and are critical size defects. The defect was then fitted with a preformed fibrin matrix and the animal allowed to cure for 3 and 7 weeks. The defective region was then explanted and analyzed by radiology as well as histology.

.Results When TG-pl-PTHi_34 was studied in 3 weeks, the level of healing was very similar to that observed with a fibrin matrix alone. The 3-week time was selected as an early time according to other potent morphogens, including rhBMP-2, they showed a significant healing effect in 3 weeks. On the contrary, the healing effect for TG-pl-PTHi_34 could not be observed at this early time point. However, when the longer time (7 weeks) was analyzed, an improvement was observed that depends on a moderate dose in the treatment of the critically sized cranial defect in rats with the addition of modified PTH1-.34 to fibrin matrices. . The results are shown in Table 3. When the high dose of modified PTH1-34 was used, the response to healing increased by 65%.

Table 3: Cranial defect healing response in rats with modified PTH These results demonstrate that when PTH1-34 was incorporated into a fibrin matrix, it maintained some activity as evidenced by the 'modest' increase in bone formation.

Bone perforation defect in sheep TG-pl-PTHi-34 was also tested in a model with large bone defect to test the effect of this hormone on bone healing. In the model with a perforation defect in sheep, 8 mm cylindrical perforation defects were placed, which were approximately 15 mm deep in both the distal and the proximal regions of the femur and humerus bones. Because the defect was placed in the epiphysis of long bones, the defect was surrounded by trabecular bone with a thin layer of cortical bone at the edge of the defect. The defects were then filled with an in situ polymerizing fibrin (approximately 750 L) which contained various doses of TG-pl-PTH! -34, or TGPTHi_34. The animals were allowed to cure for eight weeks and then sacrificed. The defect was analyzed with pCT and histology. For this series of experiments, three types of compositions were tested. First, TG-pl-PTHx- ^ was tested on a large concentration variation. Secondly, another modified PTRX-3i, the TGPTHi_34 (NQEQVSPLSVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF; SEQ ID NO: 17) which had only one transglutaminase sequence at the amino terminus, and a degradation site was used. In this way, TGPTH1-34 could only be released by degradation of the fibrin matrix itself. TGPTHi_34 was produced and purified in a manner similar to G-pl-P H! -34. The purity was determined to be 95%. GP H! -34 was tested at various concentrations that were similar to TG-pl-iPTH-34 concentrations to compare efficacy. Finally, the matrices were produced in the presence of granular material, with either TGPTHi-34 or TG-pl-PTHi_34. The granular material was a mixture of standard tricalcium phosphate / hydroxyapatite that was incorporated into the matrix during gelation. The effect of adding these granules on the efficacy of PTH1-34 was explored. As a control, unmodified fibrin was tested.

Results When any of the molecules of Modified PTHi_34 was placed in long bone defects, a significant improvement in healing responses was observed with respect to the use of fibrin (control) matrices alone. The use of fibrin alone resulted in little cure, where only 20% of the original defect was filled with newly formed bone. TG-pl-PTHi-34 was tested in a concentration series of 20-1000 μg / mL. For each dose tested, a significant increase in the healing response was observed. For example, when 100 pg / mL of TG-pl-PTHi_34 was used, the cure rate was increased by almost 60%. In a second series of experiments, TGPTH! -34 was tested. The use of TGPTHi-34 also increased bone healing. For example, the use of 400 pg / mL improved the healing response to 40%, and 1000 pg / mL increased bone healing to 65%. In this way, the addition of any modified PTHi_34 sequence resulted in a more important healing response than the control. Finally, when any modified PTHi-3 molecule was bound to the matrix and a mixture of beads / matrix was used, the efficacy of P Hi_34 was maintained. This was tested for both TG-pl -PTH1-34 (see Table 4) and TGPTHi_3 (see Table 5).

Table 4: Healing of a perforation defect in sheep with TG-pl-PTHi-34 incorporated in a fibrin matrix; 8 weeks healing time Table 5: Cure of a perforation defect in sheep with PTH1-34 bound to a fibrin matrix; -8 weeks healing time The histological evaluation showed high infiltration in the original defect of the fusiform progenitor cells and of osteoblasts supported on an extracellular matrix. Active osteoids with large rounded osteoblasts were common, and indochondral dosing signals (chondrocytes) were observed. Osteoclasts and healthy signs of remodeling may be found for eight weeks. Although unlike the results obtained from the continuous exposure to systemic PTH, no manifest response was observed from osteoclasts and the formation of new bones was significantly greater than that of the absorption in the defect area and around the same No foreign body inflammatory response was detected (ie, there were no giant cells and only a slight presence of monocytes). Granules were still present in the samples with added mineral particles.

Example 3: Preparation of the precursor components for synthetic matrices. Preparation of PEG-vinylsulfones Commercially available branched PEGs (4-branched PEG, molecular weight 14, 800, 4-branched PEG, molecular weight 10,000 and PEG 8 branches, molecular weight 20,000, Shearwater Polimers, Huntsville, AL, USA) were functional groups in the term OH. The PEG vinylsulfones were produced under argon atmosphere by reacting a dichloromethane solution of the precursor polymers (pre-dried on molecular sieves) with NaH and then, after evolution with hydrogen, with divinyl sulfone (molar proportions: OH 1: NaH 5 : divinyl sulfone 50). The reaction was carried out at room temperature for 3 days under argon with constant stirring. After neutralization of the reaction solution with concentrated acetic acid, the solution was filtered through paper until it was clear. The derivatized polymer was isolated by precipitation in ice cold diethyl ether. The product was redissolved in dichloromethane and re-precipitated in diethyl ether (with stringent washing) twice to remove all excess divinyl sulfone. Finally, the product was dried under vacuum. The derivation was confirmed with 1H NMR. The product showed characteristic vinylsulfone peaks at 6.21 ppm (two hydrogen) and 6.97 ppm (one hydrogen). It was found that the degree of conversion of the final group was 100%. Preparation of PEG-acrylates PEG acrylates were produced under argon atmosphere by reacting a toluene solution azeotropically selected from the precursor polymers with acryloyl chloride, in the presence of triethylamine (molar proportions: OH 1: acryloyl chloride 2: triethylamine 2.2) . The reaction proceeded with stirring overnight in the dark at room temperature. The resulting pale yellow solution was filtered through a bed of neutral alumina; after evaporation of the solvent, the reaction product was dissolved in dichloromethane, washed with water, dried over sodium sulfate and precipitated in cold diethyl ether. Yield: 88%; conversion of OH to acrylate: 100% (from the 1 H-NMR analysis) 1 H-NMR (CDC 13): 3.6 (341H (14800 4 branches: 337 H theoretical), 230 (10000 4 branches 227H theoretical), or 210H (20000 8 branches: theoretical 227H), PEG chain protons), 4.3 (t, 2H, -CH2-C¾-0-CO-CH = CH2), 5.8 (dd, 1H, CH2 = CH-COO-), 6.1 and 6.4 (dd, 1H, CH2 = CH-COO-) ppm.

FT-IR (film on an ATR plate): 2990-2790 (? CH), 1724 (or C = 0), 1460 (us CH2), 1344, 1281, 1242, 1097 (uas COC), 952, 842 (? 3 COC) crrT1.

Synthesis of peptides All peptides were synthesized on solid resin using an automatic peptide synthesizer (9050 Pep Plus Synthesizer, Millipore, Framingham, USA) with standard 9-fluorenylmethyloxycarbonyl chemistry. The hydrophobic scavengers and cleaved protecting groups were removed by precipitation of the peptide in cold diethyl ether and dissolution in deionized water. After lyophilization, the peptides were redissolved in 0.03 M Tris-buffered saline (TBS, pH 7.0) and purified using HPLC (Waters).; Milford, USA) on a column of enclusion by size with TBS, pH 7.0 as the buffer in progress.

Formation of the matrix by conjugate addition reactions Mels sensitive to MMP were formed by the conjugate addition with a nucleophile linked by peptides and a conjugated unsaturation linked by PEG allowing the migration of proteolytic cells. The synthesis of the gels is carried out completely through a Michael-type addition reaction of thiol-PEG on PEG with vinyl sulfone functional group. In a first step, adhesion peptides (for example, the peptide Ac-GCGYGi¾GDSPG-NH2 (SEQ ID NO: 20)) were attached in a hanging manner to a multiradified PEG-vinylsulfone and then this precursor was degraded with a peptide containing dithiol. (e.g., the MMP substrate Ac-GCRDGPQG2AG £ TJRCG-NH2 (SEQ ID NO: 21)). In a typical gel preparation for three-dimensional in vitro studies, PEG-vinylsulfone with 4 branches (molecular weight 15000) was dissolved in a TEOA buffer (0.3M, pH 8.0) to provide a 10% (w / w) solution. In order to make the gels adhesive with cells, the dissolved peptide Ac-GCGYG-RGDSPG-NH2 (SEQ ID NO: 20) (same buffer) was added to this solution. The adhesion peptide was allowed to react for 30 minutes at 37 ° C. After this, the Ac-GCRDGPQGJiGGDRCG-NH2 degrading peptide (SEQ ID NO: 21) was mixed with the above solution and the gels were synthesized. The gelation occurred in a few minutes, however, the degradation reaction was carried out for one hour at 37 ° C to guarantee the complete reaction. Gels not sensitive to MMP were formed by the addition of conjugates with a nucleophile linked with PEG and a conjugated unsaturation linked to PEG that allows non-proteolytic cell migration. The synthesis of gels was also carried out completely through the Michael-type addition reaction of the thiol-PEG on PEG with vinyl sulfone functional group. In a first step, the adhesion peptides were attached in a pendant manner (for example, the peptide Ac-GCGyGi? GÜSPG-NH2 (SEQ ID NO: 20)) to a multiradified PEG vinyl sulfone and then this precursor was degraded with a PEG- dithiol (pm 3.4 kD). In a typical gel preparation for three-dimensional in vitro studies, the 4-branched PEG vinyl sulfone (15,000 molecular weight) was dissolved in a. TEOA buffer (0. 3M, pH 8.0) to provide a 10% solution (w / w). In order to make the cell adhesive gels, the dissolved peptide Ac-GCGYGi¾GD5PG-NH2 (SEQ ID NO: 20) (in the same buffer) was added to this solution. The adhesion peptide was allowed to react for 30 minutes at 37 ° C. After this, the precursor PEG-dithiol was mixed with the previous solution and the gels were synthesized. The gelation occurred in a few minutes, however, the degradation reaction was carried out for one hour at 37 ° C to guarantee the complete reaction.

Formation of the matrix by condensation reactions MP-sensitive gels were formed by condensation reactions with a peptide X-linker containing multiple amines and an electrophilically active PEG that allows the proteolytic cell migration. MMP-sensitive hydrogels were also created by conducting a condensation reaction between the MMP-sensitive oligopeptide containing two MMP substrates and three Lys. { Ac-GKGPQGIAGQKGPQGIAGQKG-NH2 (SEQ ID NO: 22) and a commercially available double difunctional ester PEG-N-hydroxysuccinimide (Shearwater polimers) (NHS-HBS-CM-PEG-CM-HBA-NHS). In a first step, one of the adhesion peptides (e.g., the peptide AC-GCGYG.RGDSPG-NH2) (SEQ ID NO: 20) was reacted with a small fraction of NHS-HBS-CM-PEG-CM- HBA-NHS and then this precursor was degraded to a network by mixing with the peptide Ac-GKGPQGIAGQKGPQGIAGQKG-NE2 (SEQ ID NO: 22) which carries three e-amines ((and uria primary amine) .In a typical gel preparation for In three-dimensional in vitro studies, the two components were dissolved in 10 mM PBS at pH 7.4 to provide a 10% solution (w / w) and the hydrogels were formed in less than one hour by contrast - with the present hydrogels formed by the Michael type reaction, the desired self - selectivity in this procedure was not guaranteed, because the amines present in cell - or tissue - like biological materials would also react with the activated difunctional double esters,. This is also true for other PEGs that carry electrophilic functional groups such as, for example, PEG-oxycarbonylimidazole (CDI-PEG), or PEG nitrophenyl carbonate. Hydrogels were formed that were not sensitive to M P by condensation reactions with a PEG-amine degradant and an electrophilically active PEG that allows non-proteolytic cell migration. Hydrogels were also formed by conducting a condensation reaction between the commercially available branched PEG amines (Jeffamines) and the same difunctional double ester PEG-N-hydroxysuccinimide (NHS-HBS-CM-PEG-C -HBA-NHS). In a first step, the adhesion peptides (for example, the peptide Ac-GCGYGi? G £ >; 5PG-NH2) (SEQ ID NO: 20) were reacted with a small fraction of NHS-HBS -CM-PEG-CM-HBA-NHS and then this precursor was degraded to a network by mixing with the multiramide PEG amine. In a typical gel preparation for three-dimensional in vitro studies, the two components were dissolved in 10 mM PBS at pH 7.4 to provide a 10% solution (w / w) hydrogels were formed in less than one hour. Again, in contrast to the present hydrogels formed by the Mic ael reaction, the desired self-selectivity in this procedure was not guaranteed, because the amines present in biological materials similar to cells or tissues would not react with the different activated double esters. This is also true for other PEGs that carry electrophilic functional groups such as, for example, PEG-oxycarbonylimidazole (CDI-PEG), or PEG carbonate nitrophenyl PEG.

Example 4: Bone regeneration with enzymatically degradable synthetic matrices Two different start concentrations of the enzymatically degradable gels were used. In each of these, the concentration of RGD and the active factor (CplPTH at 100 g / mL) remained constant. the polymer network was formed from a PEG with a functional group of four branches with four final vinyl sulfone groups of a molecular weight of 20 kD (molecular weight of each of the 5kD branches) and dithiol peptide of the following Gly- Cys-Arg-Asp- (Gly-Pro-Gln-Gly-Ile-Trp-Gly-Gln) -Asp-Arg-Cys-Gly (SEQ ID NO: 21). The two precursor components were dissolved in 0.3 M triethanolamine. The initial concentration of PEG with functional group (first precursor molecule) and the dithiol peptide (second precursor molecule) were varied. In one case the concentration was 12.6% by weight of the total weight of the composition (first and second precursor component + triethanolamine solution). 12.6% by weight corresponds to a 10% by weight solution when calculated on the basis of only the first precursor component (100 mg / mL of the first precursor molecule). The second starting concentration was 9.5% by weight of the total weight of the composition (first and second precursor component + triethanolamine solution) corresponding to 7.5% by weight based on only the first precursor molecule (75 mg / mL of the first precursor molecule) of the total weight. This had the consequence that the amount of the dithiol peptide was changed in such a way that the molar ratio between the vinylsulfones and the trolls was maintained. The gel that started from an initial concentration of 12.6% by weight increased in volume to a concentration of 8.9% by weight of the total weight of the polymer network plus water, thus the matrix had an aqueous content of 91.1.

The gel that started from an initial concentration of 9.5% by weight increased in volume to a final concentration of 7.4% by weight of the total weight of the polymer network plus water, thus had an aqueous content of 92.6. In order to explore the effect of this change, these materials were tested in the defect of perforation in a sheep. Here, a defect of 750 yL was placed in the cancellous bone of the diaphysis of the femur and humerus of the sheep and was filled with a gelatinising enzymatic gel in situ. The following amount of calcified tissue, determined via μ ??, was obtained with each group at N = 2.

Initial concentration of the gel Calcified tissue 12. 6% 2.7% 9.5% 38.4% By making the gels less dense and easier for cell penetration, the resulting healing response with the addition of an active factor was stronger. The effect of having final solid concentrations less than 8.5% by weight is obvious from these results. Then clearly, the design of the matrix is crucial to allow healing in wound defects. Each of these hydrogels was composed of large polyethylene glycol chains, linked at the end to create a matrix. However, the details of the way they were linked, via enzymatic degradation sites, the density of binders and various other variables were crucial to allow a functional healing response. These differences were observed very clearly in the model of perforation defect in sheep.

Example 5: Bone formation with synthetic hydrolytically degradable matrices A fusion peptide with PTH 1-34 was tested in a synthetic gel as well as in the model of perforation defect in a sheep exactly as described for fibrin matrices. A hydrogel network was created by co-mixing polyethylene glycol with 4 acrylated branches, PM 15,000 (Peg 4 * 15 Acr) with a linear polyethylene glycol of PM 3400. Through an ichael reaction, when the two components were mixed in a 0.3 M triethanolamine buffer at pH 8.0, the resulting thiolates that formed at this pH were then reacted with the conjugated unsaturation of the acrylate to create a covalent bond. By mixing together the multifunctional precursors, in such a way that the combined multifunctionality was greater than or equal to five, a hydrogel was formed. In addition, biactive factors can be added to the matrix through an identical reaction scheme. In this case, bioactive factors, including cell adhesion motifs or morphogenic or mitogenic factors could be bound to the matrix by adding a cysteine, the thiol containing amino acids, to the sequence. Here, a cysteine had to be added to the cell adhesion sequence, RGD, and more specifically, RGDSP (SEQ ID NO: 23), as well as to the PTTI sequence! 34 and both were bound to the matrix via the acrylates. in the degrading. Subsequently, these newly formed hydrogels, then had many esters close to a thiol, which has been shown to be hydrolytically unstable.

This instability allows the gels to slowly degrade and be replaced by newly formed tissue. These particular gels, hydrolytically degradable with RGD and PTH covalently bound to the matrix, were tested in the model of perforation defect in a sheep to test the ability of C-PT H1-.34 attached to the matrix to improve bone development . In order to determine the amount of improvement, 0, 100 and 400 pg / mL of the fusion peptide was added to the matrix with PTH1-34. When this was done, an increase in bone formation was observed with the addition of PTH 1-34. In each test the healing response was measured in the same time of eight weeks. This was compared with the defects that were left empty. When the synthetic hydrolytically degradable matrix was used without the fusion peptide with PTH, the healing response was measured at approximately 40%. This means that 40% of the original defect volume was filled with newly formed bone tissue. Then, when 400 g / mL of the fusion peptide was used with the modified PTH1-34, the healing response increased to approximately 60%. In comparison, when the defects were left empty, approximately 10% was filled with calcified tissue. These data are shown in the following Table 6.

Table 6: Healing response with synthetic matrices with and without modified PTH In comparison with an empty defect, the addition of the hydrolytically degradable peg gel only had a great effect on bone healing, increasing the amount of calcified tissue by 300%. When the 'PTH 1-34 was added to this matrix, the cure increased even more with the level that was up to 50% higher than when the matrix was used alone and at 500% higher than the level of cure obtained when the defect was left empty.

LIST OF SEQUENCES < 110 > Eidgenossiche Te'chnische Hochschule Zurich, University of Zurich, - and Jeffrey A. Hubbell < 120 > Matrices of modified protein with tissue growth factor < 130 > ETH 107 CIP (3) < 150 > 10 / 024,918 < 151 > 2001-12-18 < 150 > PCT / EP02 / 12458 < 151 > 2002-11-07 < 160 > 23 < 170 > Patenln version 3.1 < 210 > 1 < 211 > 12 < 212 > PRT < 213 > Artificial sequence < 220 > < 223 > Bidomin peptide < 220 > < 221 > MOD_RES < 222 > (1) .. (1) < 223 > dansyl leucine < 40Q > 1 . Leu Asn Gln Glu Gln Val Ser Pro Leu Arg Gly Asp 1 5 10 < 210 > 2 < 211 > 5 < 212 > PRT < 213 > Homo sapiens < 400 > 2 Tyr lie Gly Ser Arg 1 5- < 210 > 3 < 211 > 5 < 212 > PRT < 213 > Homo sapiens < 400 > 3 Leu Arg Gly Asp Asn 1 5 < 210 > 4 < 211 > 5 < 212 > PRT < 213 > Homo sapiens < 400 > 4 Pro Asp Gly Ser Arg 1 5 < 210 > 5 < 211 > 5 < 212 > PRT < 213 > Homo sapiens < 400 > 5 lie Lys Val Ala Val 1 5 < 210 > 6 < 211 > 10 < 212 > PRT < 213 > Homo sapiens < 400 > 6 Arg Asn lie Wing Glu lie lie Lys Asp lie 1 5 10 < 210 > 7 < 211 > 4 < 212 > PRT < 213 > Homo sapiens < 400 > 7 Asp Gly Glu Wing 1 < 210 > 8 < 211 > 13 < 212 > PRT < 213 > Homo sapiens < 400 > 8 Gly Cys Arg Pro Gln Gly Lie Trp Gly Gln Asp Arg Cys 1 5 10 < 210 > 9 < 211 > 6 < 212 > PRT < 213 > Homo sapiens < 400 > 9 Gly Arg Gly Asp Ser Pro 1 5 < 210 > 10 < 211 > 5 < 212 > PRT < 213 > Homo sapiens < 400 > 10 Gly Ala Lys Asp Val 1 5 < 210 > 11 < 211 > 4 < 212 > PRT < 213 > Homo sapiens < 400 > 11 Lys Lys Lys Lys 1 < 210 > 12 < 211 > 8 < 212 > PRT < 213 > Homo sapiens < 400 > 12 Asn Gln Glu Gln Val Ser Pro Leu 1 5 < 210 > 13 < 211 > 16 < 212 > PRT < 213 > Homo sapiens < 400 > 13 Tyr Arg Gly Aso Thr lie Gly Glu Gly Gln Gln His His Leu Gly Gly 1 5 10 15 <; 210 > 14 < 211 > 5 < 212 > PRT < 213 > Homo sapiens < 400 > 14 Gly Ala Lys Asp Val 1 5 < 210 > 15 < 211 > 5 < 212 > PRT < 213 > Homo sapiens < 400 > 15 Gly Lys Tyr Asn Arg 1 5 < 210 > 16 < 211 > 7 < 212 > PRT < 213 > Homo sapiens < 400 > 16 Asn Gln Glu Gln Val Ser Pro 1 5 < 210 > 17 < 211 > 42 < 212 > PRT < 213 > Homo sapiens < 400 > 17 Asn Gln Glu Gln Val Ser Pro Leu Ser Val Ser Glu Lie Gln Leu Mefc 1 5 10 15 His Asn Leu Gly Lys. His Leu Asn Ser Met Glu Arg Val Glu Trp Leu 20 25 30 Arg Lys Lys Leu Gln Asp Val His Asn Phe 35 40 < 210 > 18 < 211 > 46 < 212 > PRT < 213 > Homo sapiens < 400 > 18 Asn Gln Glu Gln. Val Ser Pro Leu Tyr Lys Asn Arg Ser Val Ser Glu 1 5 10 15 lie Gln Leu Met His Asn Leu Gly Lys His Leu Asn Ser Met Glu Arg 20 25 30 Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His Asn Phe 35 40 45 < 210 > 19 < 211 > 4 < 212 > PRT < 213 > Homo sapiens < 400 > 19 Tyr Lys Asn Arg 1 < 210 > 20 < 211 > 11 < 212 > PRT < 213 > Homo sapiens < 220 > < 221 > MOD_RES < 222 > (1) .. (1) < 223 > ACETILATION < 220 > < 221 > M0D_RES < 222 > (eleven) - . (11) < 223 > A IDATION < 400 > 20 Gly Cys Gly Tyr Gly Arg Gly Asp Ser Pro Gly 1 5 10 < 210 > 21 < 211 > 16 < 212 > PRT < 213 > Homo sapiens < 220 > < 221 > M0D_RES < 222 > (1) .. (1) < 223 > ACETILATION < 220 > < 221 > MOD_RES < 222 > (16) .. (16) < 223 > AMIDATION < 400 > 21 Gly Cys Arg Asp Gly Pro Gln Gly lie Trp Gly Gln Asp Arg Cys Gly 1 5. 1 ° 15 < 210 > 22 < 211 > 21 < 212 > PRT < 213 > Homo sapiens < 220 > < 221 > OD RES < 222 > (1) · · (i) < 223 > ACETILATION < 220 > < 221 > MOD RES < 222 > (21) .. (21) < 223 > A IDATION < 400 > 22 Gly Lys Gly Pro Gln Gly lie Wing Gly Gln Lys Gly Pro Gln Gly lie 1 5 10 15 Ala Gly Gln Lys Gly 20 < 210 > 23 < 211 > 5 < 212 > PRT < 213 > Homo sapiens < 400 > 23 Arg Gly Asp Ser? Ro 1 5 6

Claims (19)

1. NOVELTY OF THE INVENTION Having described the present invention, it is considered as a novelty and, therefore, the content of the following CLAIMS is claimed as property: 1. A fusion peptide, characterized in that it comprises: a first domain comprising PTH and a second domain comprising a covalently degradable substrate domain. 2. The fusion peptide according to claim 1, further characterized in that it comprises a degradation site between the first and the second domain. 3. The fusion peptide according to claim 1, characterized in that the PTH is selected from the group consisting of PTH 1-84, PTH 1-28 ,. PTH 1-34, - PTH 1-31 and PTH 1-25. 4. The fusion peptide according to claim 3, characterized in that the PTH is PTH 1-34. 5. The fusion peptide according to the rei indication 1, characterized in that the second domain comprises a transglutaminase substrate domain. 6. The fusion peptide according to claim 5, characterized in that the second domain comprises a substrate domain of Factor XlIIa. 7. The fusion peptide according to claim 6, characterized in that the substrate domain of Factor XlIIa comprises SEQ ID NO: 12. 8. The fusion peptide according to claim 1, characterized in that the second domain comprises at least one cysteine. 9. The fusion peptide according to claim 2, characterized in that the degradation site is an enzymatic or hydrolytic degradation site. The fusion peptide according to claim 8, characterized in that the degradation site is an enzymatic degradation site, which is cleaved by an enzyme selected from the group consisting of plasmin and matrix metalloproteinase. 11. A kit of reagents characterized in that it comprises the fusion peptide according to claims 1-10. 12. The reagent kit according to claim 11, further characterized in that it comprises fibrinogen, thrombin and a source of calcium. The reagent kit according to claim 11, characterized in that the reagent kit also comprises a degrading enzyme. 14. A suitable matrix for cell internal growth or growth, comprising the fusion peptide according to claims 1-10, characterized in that the fusion peptide is covalently bound to the matrix. 15. The matrix according to claim 14, characterized in that the matrix is fibrin. The matrix according to claim 14, characterized in that the matrix is formed by a Michael-type addition reaction between a first precursor molecule comprising n nucleophilic groups and a second precursor molecule comprising m electrophilic groups, where n and are at least two and the sum n + m is at least five. 17. The matrix according to claim 16, characterized in that the electrophilic groups are conjugated unsaturated groups and the nucleophilic groups are selected from the group consisting of thiols and amines. 18. The matrix according to claim 14, characterized in that the matrix comprises polyethylene glycol. 19. A method for making a characteristic matrix comprising: providing at least one matrix material capable of forming a degraded matrix, wherein the matrix material is selected from the group consisting of proteins and synthetic materials; adding the fusion peptide according to claims 1-10 to the matrix material, and degrading the matrix material, such that the fusion peptide binds to the matrix through the second domain. SUMMARY OF THE INVENTION Proteins are incorporated into a protein or polysaccharide matrices for use in tissue repair, regeneration and / or remodeling and / or drug delivery. The proteins can be incorporated in such a way that they are released by degradation of the matrix, by action and / or enzymatic diffusion. As demonstrated by the examples, one method is to bind heparin to the matrix by either covalent or non-covalent methods, to form a heparin matrix. Then non-covalent heparin binds to heparin-binding growth factors to the protein matrix. Alternatively, a fusion protein containing a crosslinking region can be constructed such as, for example, a substrate of Factor XlIIa and the natural protein sequence. The incorporation of degradable bonds between the matrix and bioactive factors. it may be particularly useful when a long-term drug supply is desired, for example, in the case of nerve regeneration, where it is desirable to vary the drug release rate spatially as a regeneration function, for example, rapidly close to the interface »of living tissue and more slowly away in the injured area. Additional benefits include the lowest total drug dose within the delivery system, and the spatial regulation of release that allows a greater percentage of the drug to be released at the time of increased cellular activity.