Growth factor modified protein matrices for tissue engineering

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DESCRIPTION

Growth factor modified protein matrices 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 to the use of fusion proteins or peptides in the repair and regeneration of tissues, and in the controlled release of the PTH.

Background of the invention

Parathyroid hormone (PTH) is an 84 amino acid peptide that is made and secreted by the parathyroid gland. This hormone plays a primary role in controlling serum calcium levels through its action on various tissues, including bones. Studies in humans with various forms of PTH have shown an anabolic effect on bones, which makes them interesting for the treatment of osteoporosis and related bone disorders (US 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. This receptor is known to be found on osteoblasts, the cells that are responsible for the formation of new bones.

The N-terminal 34 amino acid domain of human hormone has been reported to be biologically equivalent to full-length hormone. PTH 1-34 and its mode of action was first reported in US Patent No. 4,086,196. Because research has been conducted on PTH 1-34 and other truncated versions of the natural human form of PTH, such as PTH1-25, PTH1-31, and PTH1-38 (see, for example, Rixon RH, et al., JBone Miner. Res., 9 (8): 1179-89 (August 1994).

The mechanism by which PTH influences. in 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, it will decrease bone density. In contrast, 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 mature bone, the PTH receptor has only been shown to be present on the surface of cells of the osteoblast lineage, but not on osteoclasts. The role of PTH in bone remodeling is directed through the osteoblasts as opposed to the osteoclasts. However, cells at different stages of the osteoblast lineage respond differently when they bind to PTH. Therefore, the dramatic differences that are observed when PTH is administered using different methods can be taken into account for understanding the different effects that the same molecule has on different cells within the osteoblast lineage.

When PTH binds to a mesenchymal hemocytoblast, the cell is induced to differentiate into a proteoblast. Thus, by adding PTH to the system, there is an increase in the proteoblast population. However, these proteoblast cells also have the PTH receptor, and subsequent binding of PTH to the receptor on these cells leads to a different response. When PTH binds to the proteoblast, this results in two separate consequences that lead to bone resorption. First, it inhibits the further differentiation of proteoblasts into osteoblasts. Second, it increases the secretion of Interleukin 6 (IL-6) from proteoblasts. IL-6 both inhibits proteoblast differentiation, as well as increases proteoclast differentiation within osteoclasts. This dual response of cells within the osteoblast lineage is that it provides the complex reaction between bone remodeling and PTH exposure. If PTH is dosed periodically for short periods of time, then mesenchymal blood cells are induced to differentiate into osteoblasts. Short dosing periods then prevent newly formed proteoblasts from producing IL-6, preventing osteoclast activation. Therefore, during dosing intervals, these newly formed preosteoblasts can further differentiate into osteoblasts, resulting in bone formation. However, if a constant dose of PTH is applied, then the preosteoblasts will have the opportunity to initiate IL-6 production, thus activating and inhibiting osteoclasts, leading to the opposite effect: bone resorption.

For tissue repair or regeneration, cells must migrate into a wounded mass, proliferate, express the matrix components or form the extracellular matrix, and constitute a final tissue conformation. Multiple cell populations must often participate in this morphogenetic response, frequently including vascular and nerve cells. For this to happen, matrices have been shown to improve quite a bit and have been found to be essential in some cases. Procedures have been developed to develop matrices from natural or synthetic origins or a mixture of both. The growing matrices of natural cells undergo remodeling by cellular influences, all based on proteolysis, for example, by plasmin (degrading fibrin) and matrix metalloproteinases (degrading collagen, elastin, etc.). This degradation is quite localized and occurs only at the moment of direct contact with the migrating cell. Furthermore, the delivery of specific cell signaling proteins, such as, for example, growth factors, is tightly controlled. In the natural model, macroporous cell growth matrices are not used, instead of microporous matrices the cells can be degraded, locally and on demand, as the cells

ES 2 301 697 T3 cells migrate into the matrix. Due to issues relating to immunogenicity, expensive production, limited availability, variability, and batch purification, matrices have been developed based on synthetic precursor molecules, such as, for example, modified polyethylene glycol in and / or on the body.

While much work has been done studying the systemic effects of PTH, research has not explored local or topical administration of PTH. As PTH has a direct anabolic effect on the osteoblast cell lineage, it must have significant potential to heal bone defects in addition to influencing bone density if adequately presented within a defective site. Once the defect has been filled with preosteoblasts, if the PTH signal is abrogated, the newly formed preosteoblasts can then differentiate into osteoblasts and initiate conversion of the injured mass, first into injured bone tissue and then into a mature bone structure.

Therefore, an objective of the present invention is to provide a PTH in a form that can be bound to a matrix for tissue repair, regeneration and remodeling.

A further objective is to present a PTH in a form suitable for topical or local administration to a patient to cure bone defects.

Summary of the invention

Set out herein, 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 matrices, and kits containing these fusion proteins or peptides . Fusion proteins are covalently linked to natural or synthetic materials to form matrices, they can be used to heal bone defects. Optionally, all components for the formation of the matrix are applied to a bone defect and the matrix is ​​formed at the site of application. 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 bond between the first and second domains containing hydrolytic or enzymatic cleavage sites. In particular, the fusion peptide contains PTH in one domain, a substrate domain capable of being covalently cross-linked to a matrix in a second domain, and a degradation site between the first and second domains. Preferably, the PTH is human PTH, although PTH from other sources, such as, for example, bovine PTH, may be suitable. 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, i.e., bone formation , similar to the previous one. The site of degradation allows the rate of delivery to vary to different locations within the matrix depending on 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 release that allows a greater percentage of the drug to be released at the time of greatest cellular activity.

Brief description of the drawings or figures

Figure 1 is a chromatogram for fluorescence detection of fibrin gels containing plasmin degraded peptide and free peptide. Size exclusion chromatography of a fibrin gel degraded with α-peptide is shown.₂PIi-₇-ATIIIi₂i_i₃₄ incorporated (-), with the same free peptide added to the degraded fibrin gel that contains the incorporated peptide (· · ·), and the free peptide alone (-). The N-terminal leucine residue was dansylated (abbreviated dL). The free peptide eluted at longer times, which corresponds to a lower molecular weight, than did the peptide incorporated into the fibrin gel during coagulation, demonstrating covalent binding to fibrin degrades and thus covalent incorporation via the action of Factor XIIIa activity.

Figure 2 is a graph of the incorporation of dLNQEQVSPLRGD (SEQ ID NO: 1) in fibrin gels with added exogenous Factor XIII. When 1 U / mL was added, the level of incorporation increased such that more than 25 moles of peptide / moles of fibrinogen could be reached.

Figure 3 is a graph of the incorporation of the bidomain peptide dLNQEQVSPLRGD (SEQ ID NO: 1), in undiluted fibrin gum. Three separate sets of reagents 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 mM, possibly due to diffusion limitations within the rather dense fibrin matrix that is created. The level of incorporation was very consistent, with each kit of reagents providing a similar incorporation profile.

Detailed description of the invention

Products and methods for hard tissue repair, regeneration or remodeling, in particular for bone growth, using natural and synthetic matrices having the distributable PTH incorporated herein are described herein. Natural matrices are biocompatible and biodegradable and can be formed in vitro

ES 2 301 697 T3 or in vivo, at the time of implantation. PTH can be incorporated into matrices and maintain its full bioactivity. PTH can be incorporated in a distributive way, using techniques that provide control over the shape and timing and to what degree the 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", as used herein, refers to a material intended to interface with biological systems to evaluate, treat, augment, or replace any tissue, organ, or function of the body depending on the material, whether it be permanently or temporarily. The terms "biomaterial" and "matrix" are used interchangeably herein and mean a degraded polymeric network which, depending on the nature of the matrix, can increase with water but not dissolve in water, that is, form a hydrogel that it remains in the body for a certain period of time fulfilling certain support functions for damaged or traumatized hard tissue.

"PTH fusion peptide" as generally used herein refers to a peptide containing 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. An enzymatic or hydrolytic degradation site can also be present between the first and second domains.

"Strong nucleophile", as generally used herein, refers to a molecule that is capable of donating an electron pair to an electrophile in a polar bond forming reaction. Preferably, the strong nucleophile is more nucleophilic than water at physiological pH. Examples of strong nucleophiles are thiols and amines.

"Conjugated unsaturated bond", as generally used herein, refers to the alternation of multiple carbon-carbon, carbon-heteroatom, or heteroatom-heteroatom bonds with individual bonds, or the attachment of a functional group to a macromolecule, such as, for example, a synthetic polymer or a protein. These bonds can undergo addition reactions.

"Conjugated unsaturated group", as generally used herein, refers to a molecule or region of a molecule, containing an alternation of carbon-carbon, carbon-heteroatom, or heteroatom-heteroatom bonds, with single bonds, which have a multiple bond that can undergo addition reactions. Examples of conjugated unsaturated groups include but are not limited to: vinyl sulfones, acrylates, acrylamides, quinones, and vinylpyridiniums, eg, 2- or 4-vinylpyridinium and itaconates.

"Synthetic precursor molecules", as generally used herein, refers to molecules that do not exist in nature.

"Nature-occurring precursor components or polymers", as generally used herein, refers to molecules that could be found in nature.

"Functionalize", as generally used herein, refers to modifying a molecule in a way that results in the binding of a functional group or entity. For example, a molecule can have a functional group by introducing a molecule that makes the molecule a strong nucleophile by 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 efficiently integrated into functional groups by partially or completely reducing disulfide bonds to create free thiols.

"Functionality" as generally used herein refers to the number of reactive sites on a molecule.

"Branch point functionality", as generally used herein, refers to the number of branches that extend from a point in the molecule.

"Adhesion site or cell binding site", as generally used herein, refers to a peptide sequence to which a molecule, for example, an adhesion-stimulating receptor, binds on the surface of a cell. Examples of adhesion sites include but are not limited to: the RGD sequence from fibronectin, and the YIGSR sequence (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" as generally used herein refers to functional events elicited by a protein of interest. In some embodiments, this includes the events analyzed by measuring interactions of one 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 dephosphorylation, transcription or translation.

ES 2 301 697 T3 "Sensitive biological molecule" as 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 adversely affecting sensitive biological materials.

"Regenerate", as it is generally used herein, means to regrow a portion or all of something, such as, for example, hard tissue, particularly bone.

"Multifunctional", as generally used herein, refers to more than one electrophilic and / or nucleophilic functional group per molecule (ie, monomer, oligo, and polymer).

"Self-selective reaction" as it is generally used herein, 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 at reaction site. As used herein, the nucleophile preferably binds to an electrophile and an electrophile preferably binds to a strong nucleophile, rather than other biological compounds.

"Degradation", as generally used herein, means the formation of covalent bonds. However, it can also refer to bonding. non-covalent, such as, for example, ionic bonds, or combinations of covalent and non-covalent bonds.

"Polymeric network", as it is generally used herein, means the product of a process in which virtually all monomers, oligo or polymers are joined by intermolecular covalent bonds through their available functional groups to produce a large molecule.

"Physiological", as generally used herein, means the conditions as they can be found in living vertebrates. In particular, physiological conditions, refers to the conditions in the human body such as, for example, pH temperature, etc. Physiological temperatures means in particular a temperature variation between 35 ° C to 42 ° C, preferably about 37 ° C.

"Density of degradation" as generally used herein refers to the average molecular weight between two degradations (Me) of the respective molecules.

"Equivalent weight" as generally used herein refers to mmol of the functional group / g of the substance.

"Bulking" as generally used herein refers to the increase in volume and mass through the proportion of water by the biomaterial. The terms "water absorption" and "bulking" are used interchangeably throughout this application.

"Steady state", as generally used herein, means the state in which a hydrogel does not experience an increase or decrease in mass when stored under constant conditions in water.

I. Matrices and PTH

A. Materials for the matrix

The matrix is ​​formed by ionically, covalently, or by combinations thereof, the precursor molecules for a polymeric network or by bulking up one or more polymeric materials, i.e., the matrices, to form a polymeric network that has sufficient separation. inter-polymeric 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, into which the matrix will be implanted. A particularly preferred matrix protein is fibrin, although matrices made from other proteins can also be used, such as, for example, collagen or gelatin. 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 binding.

Fibrin matrices

Fibrin is a natural material that has been reported for various biomedical applications. Fibrin has been described as a material for cell ingrowth matrices in US Patent No. 6,331,422 to Hubbell et al. Fibrin gels have been used as sealants due to their ability to bind to many tissues and their natural function is wound healing. Some specific applications include use as a sealant for vascular graft junction, heart valve junction, bone placement in fractures, and tendon repair (Sierra, DH, Journal of Biomaterials Applications, 7: 309-352 (1993) ). Additionally, these gels have been used as devices for drug delivery, and for neuronal regeneration (Williams, et al.,

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Journal of Comparative Neurobiology, 264: 284-290 (1987)). Although fibrin provides a solid support for tissue regeneration and cell ingrowth, there are few active sequences in the monomer that directly enhance 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, reptylase, 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 coalesce and form a non-covalently degraded polymer gel (Sierra, 1993). This self-assembly occurs because the binding sites are exposed after cleavage of the protease occurs. Once exposed, these binding sites in the center of the molecule can bind to other sites on fibrinogen chains, which are present at the ends of peptide chains (Stryer, L. In Biochemistry, WH Freeman & Company , NY, 1975). In this way, a polymeric network is formed. Factor XIIIa, a transglutaminase activated from Factor XIII by thrombin proteolysis, can then covalently degrade the polymer network. Other transglutaminases exist and may also be involved in covalent degradation and graft formation with the fibrin network.

Once a degraded fibrin gel is formed, subsequent degradation is rigorously controlled. One of the key molecules to control fibrin degradation is the inhibitor a2-plasmin (Aoki, N., Progress in Cardiovascular Disease, 21: 267-286,1979). This molecule works by degrading the fibrin α chain through the action of Factor XIIIa (Sakata, et al., Journal of Clinical Investigation, 65: 290-297, 1980). By binding itself to the gel, a high concentration of the inhibitor can be located with the gel. The inhibitor then acts by preventing the binding of plasminogen to 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.

Bidominium peptides, which contain a Factor XIIIa substrate sequence and a bioactive peptide sequence, have been shown to be degradable in fibrin gels and that this bioactive peptide maintains its cellular activity in vitro (Schense, JC, et al. (1999) Bioconj. Chem. 10: 75-81).

Synthetic matrices

Degradation reactions for the formation of synthetic matrices for the application which include (i) free radical polymerization between two or more precursors containing unsaturated double bonds, as described in Hern et al., J Biomed Mater . Beef. 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 set forth in US Patent No. 5,874,500 to Rhee et al., (Iii) the condensation and addition reactions and (iv) the Michael-type addition reaction between a strong nucleophile and a conjugated or bonding unsaturated group (such as a strong electrophile). In particular, the reaction between a precursor molecule having a thiol or amine group as the nucleophilic group and precursor molecules including acrylate or vinyl sulfone groups as the electrophilic groups is preferred. The thiol group is most preferred as the nucleophilic group. Michael-type addition reactions are described in Hubbell et al. WO 00/44808, the content thereof is incorporated herein by reference. Michael-type addition reactions allow 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 self-selectively react to form a degraded three-dimensional biomaterial.

Preferably, the conjugated unsaturated groups or conjugated unsaturated bonds are acrylates, vinylsulfones, methacrylates, acrylamides, methacrylamides, acrylonitriles, vinyl sulfones, 2- or 4-vinylpyridinium, maleimides, or quinones.

Preferred nucleophilic groups are thiol groups, amino groups, or hydroxyl groups. Thiol groups are practically more reactive than unprotonated amine groups. As stated above, pH is important in this consideration: deprotonated thiol is practically more reactive than protonated thiol. Therefore, addition reactions that include 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 better carried out more rapidly 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 using a thiol as the first precursor molecule, a conjugate structure that is selective in its reactivity for the thiol relative to amines is quite desirable.

Suitable first and second precursor molecules include proteins, peptides, polyoxyalkylenes, poly (vinyl alcohol), poly (ethylene-co-vinyl alcohol), 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 poly (ethylene oxide) -co- block copolymers poly (propylene oxide). A particularly preferred precursor molecule is polyethylene glycol.

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Polyethylene glycol (PEG) provides a convenient building block. Linear (meaning double-ended) or branched (meaning more than two-ended) PEGs can be easily acquired or synthesized and then functional groups added to the PEG end groups, to introduce any strong nucleophiles, 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 matrix formation, especially matrices that are desired to degrade peptides in vivo, they provide a very convenient building block. It is easy 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 tri-vinyl sulfone PEG (a PEG that has three branches with vinyl sulfones on each of its branches) at or slightly higher physiological pH (e.g. , 8 to 9). Gelling, too, can proceed well at even higher pH, albeit 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 are carried by the nodes of the network, with the peptides as connecting links. . Peptides can be selected as protease substrates, in order to make the network capable of being infiltrated and degraded by cells, as is done in a protein-based network, such as for example, in a fibrin matrix. . 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 available. limited to this sequence. A particularly useful sequence is a substrate for 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 which is degradable by collagenase, but not by plasmin, or by plasmin but not by collagenase. Furthermore, 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 K_m okay_cat, or both, of the enzymatic reaction. In this way, a biomaterial can be elaborated that is biomimetic, and that is capable of being remodeled through the normal remodeling characteristics of cells. For example, this study shows the substrate sites for the important plasmin protease. The gelling 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 into the matrix is ​​important when forming the matrix from PEG vinyl sulfone. Apart from matrices formed from the reaction of PEG acrylates and PEG thiols, 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. Physiological saline can serve as the solvent. Minimal heat is generated by the reaction. Therefore, gelation takes place in vivo or in vitro, in direct contact with the tissue, without detrimental toxicity. In this way, polymers other than PEG can be used, either telehelically modified or modified in their side groups.

For most health indications, the rate of cell ingress or migration of cells in the matrix in combination with an adapted degradation rate of the matrix is ​​decisive for the overall healing response. The potential for hydrolytically non-degradable matrices to be invaded by cells is primarily a function of 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. Healing matrices found in nature, such as fibrin matrices, which are formed in 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 cell adhesion ligands 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 formation of the polymeric network. In order to achieve a sufficiently short gelation time (between 3 to 10 minutes at a pH between 7 and 8 and at a temperature ranging from 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 high enough. Under these conditions, bulking could take place 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. Thus, increasing the volume of the polymeric network is important to lengthen and increase the space between the branch points.

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Regardless of the initial concentration of the precursor molecules, hydrogels produced from the same synthetic precursor molecules, such as, for example, a four-branched vinyl sulfone PEG and a SH-group peptide, will increase in volume at the same water content in the steady state. This means that the higher the initial concentration of the precursor molecules, the greater the final volume of the hydrogel will be when it reaches its equilibrium state. If the available space in the body is too small to allow a sufficient bulking and in particular if the bond formed from the precursor components is not hydrolytically degradable, the cellular infiltration rate and the healing response will decrease. As a consequence, the optimum must be found between the two contradictory requirements for application to 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 substantially similar branches in molecular weight and a second precursor molecule that is at least one bifunctional molecule. The equivalent weight index of the functional groups of the first and second precursor molecules is between 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 branch points are selected such that the water content of the resulting polymeric network is between% by weight of equilibrium and 92% by weight of the total weight of the polymeric network after completion of the water absorption. Preferably, the water content is between 93 and 95% by weight of the total weight of the polymeric network and the water after completion of 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 for an additional volume increase. Therefore it is preferred that the selection of the initial concentrations of the precursor components is as low as possible. This is true for all bulking matrices although in particular for those matrices that undergo degradation supplied by cells that do not contain hydrolytically degradable bonds in the polymeric network.

The balance between gel time and low starting 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, that is, the functionality of the branch 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 cysteine ​​groups. then the molecular weight of the branches of the first precursor molecule and the molecular weight of the second precursor molecule are preferably selected such that the bonds between the branch points after network formation have a molecular weight in the range between 10 to 13 kD (under the conditions in which the bonds are linear, unbranched), 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 network formation). 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 the 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 linear to a three or four branched precursor component, the molecular weight, that is, 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, that is, each branch having a molecular weight of 5 kD and as the second precursor molecule , a linear bifunctional molecule with a molecular weight in the range of 0.5 to 1.5 kD, even more preferably about 1 kD. Preferably, the first and second precursor components is a polyethylene glycol.

In a preferred embodiment, the first precursor component includes as functional groups the conjugated unsaturated groups or bonds, an acrylate or a vinylsulfone is more preferred and the functional groups of the second precursor molecule include a nucleophilic group, preferably a thiol group. or amino. In another preferred embodiment of the present invention, the first precursor molecule is a 20 kD four-branch polymer (each branch has a molecular weight of 5k Da) that has functional groups at the terminus of each branch and the second precursor molecule is a molecule linear bifunctional with 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 cysteine ​​groups. In both preferred embodiments, the initial concentration of the sum of the first and second precursor molecules ranges from 8 to 11% by weight, preferably between 9 and 10% by weight of the total weight of the first and second precursor molecules and water (previously of the formation of the polymeric network), preferably between 5 and 8% by weight until reaching a gelation time of less than 10 minutes. These compositions have a gel time at pH 8.0 and 37 ° C of approximately 310 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 onset, although in an aqueous environment the bonds will hyrolyze and the network will separate, to allow cell infiltration. With an increase in the degree of total branching of the polymeric network, the molecular weight of the entanglements, that is, the length of the bonds, must increase.

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B. Cell binding sites

Cells interact with their environment through protein-protein interactions. Protein-oligosaccharide and protein-polysaccharide on the cell surface. Extracellular matrix proteins provide a host for bioactive signals to the cell. This dense network is required to support cells, and many proteins in the matrix have been shown to control cell adhesion, dispersion, migration, and 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 studies of laminin 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, scattering or signal transduction have been identified.

Laminin, a large multidomain protein (Martin, Annual Review of Cellular Biology, 3: 57-85, 1987), has been shown to consist of three chains with various receptor-binding domains. These receptor-binding domains include the YIGSR sequence (SEQ ID NO: 2) of the laminin B1 chain (Graf, et al., Cell, 48: 989-99E, 1987; Kleinman, et al., Archives of Biochemistry and Biophysics, 272: 39-45, 1989; and Massia, et al, J: ofBiol. Chem., 268: 8053-8059, 1993), LRGDN (SEQ ID NO: 3) of laminin A chain (Ignatius, et al., J: of Cell Biology, 111: 709-720, 1990) and PDGSR ( SEQ ID NO: 4) of the laminin B1 chain (Kleinman, et al., 1989). Various other recognition sequences have also been identified. These include IKVAV (SEQ ID NO: 5) from the lamin A chain (Tashiro, et al., J; ofBiol. Chem., 264: 16174-16182, 1989) and the rNiAEIIKDI sequence (SEQ ID NO: 6) of laminin B2 chain (Liesi, et al., FEBS Letters, 244: 141-148, 1989). Receptors that bind to these specific sequences have also often been identified. A subset of cellular receptors that has been shown to be responsible for the majority of binding is the integrin superfamily (Rouslahti, E., J. of Clin. Investigation, 87:15, 1991). Integrins are protein heterodimers consisting of α and β subunits. Previous work has shown that the RGD tripeptide binds to various β1 and β3 integrins (Hynes, RO, Cell, 69: 1-25, 1992; Yamada, KM, J; ofBiol. Chem., 266: 12809-12812, 1991 ), IKVAV (SEQ ID NO: 5) binds to a 110 kDa receptor (Tashiro, et al., J ofBiol. 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 integrin α₂, βι (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 for cell adhesion are incorporated into the matrix, namely peptides that bind to receptors to stimulate adhesion on cell surfaces in the biomaterials of the present invention. These adhesion stimulating peptides can be selected from the group as described above. In particular the RGD sequence derived from fibronectin, the YIGSR sequence (SEQ ID NO: 2) derived from laminin are preferred. The incorporation of binding sites is a particularly preferred embodiment with synthetic matrices, although it can also be included with some of the natural matrices. Incorporation can be accomplished, for example, simply by mixing a cysteine-containing cell-binding peptide with the parent molecule that includes the conjugated unsaturated group, such as for example, PEG acrylate, PEG acrylamide, or PEG vinyl sulfone a few minutes before mixing. with the remainder 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 multithiol is added to the system, a degraded network will form. Another important implication of the way networks are prepared here is the efficiency of incorporation of pendant bioactive ligands such as, for example, adhesion signals. This step must 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 a large stoichiometric excess (minimum: 40 times) of multibranched electrophilic precursors on thiols and therefore definitely quantitative. Aside from avoiding unwanted inhibition, this achievement is even more biologically significant: cell behavior is extremely sensitive to small changes in ligand densities, and a precise knowledge of incorporated ligands helps to design and understand cell matrix interactions. In summary, the concentration of the covalently bound adhesion sites on the matrix significantly influences the rate of cellular infiltration. For example, for a given hydrogel, a RGD concentration can be incorporated into the matrix with supports for cell ingrowth, and cell migration in an optimal way. The optimal concentration variation of the RGD-like adnesion sites 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 term of water absorption.

Excellent bone healing results have been achieved by maintaining the rate of cell migration and the rate of degradation of the matrix in the fasted state. Regarding the matrix design (in particular covalently linked PTH 1-34), a four-branched polyethylene glycol with a molecular weight of approximately 20,000 Da degraded with a GCRPQGIWGQDRC protease degradation site (SEQ ID NO: 8) and 0.050 mM GRGDSP (SEQ ID NO: 9) provides particularly good cell ingrowth results and healing of bone defects. The initial concentration of PEG and inner peptide at 10% by weight of the total weight of the molecules and water (before the increase in

ES 2 301 697 T3 volume). The gels have a usable consistency and allow osteoblasts and precursor cell to easily infiltrate the matrix.

The matrix material is preferably biodegradable by naturally occurring enzymes. The rate of degradation can be manipulated by the degree of degradation and the inclusion of protease inhibitors in the matrix.

C. Degradable substrate domains

The PTH fusion peptide can be degraded and covalently attached to matrices through the degradable substrate domain of the PTH fusion peptide. The type of substrate domain depends on the nature of the matrix. Transglutaminase substrate domains are particularly preferred for incorporation into fibrin matrices. The transglutaminase substrate domain may be a Factor XIIIa substrate domain. This Factor XIIIa substrate domain can include GAKDV (SEQ ID NO: 10), KKKK (SEQ ID NO: 11), or NQEQVSPL (SEQ ID NO: 12). Coupling between PTH and the transglutaminase substrate domain can be accomplished by chemical synthesis.

The transglutaminase substrate domain may be a substrate for a transglutaminase other than Factor XIIIa. The most preferred Factor XIIIa substrate domain has an amino acid sequence of NQEQVSPL (SEQ ID NO: 12) (herein referred to as "TG"). Other proteins that recognize transglutaminase, such as, for example, fibronectin, could be coupled to the peptide of the transglutaminase substrate.

TABLE 1

Transglutaminase substrate domains

<td>SEQ ID NO: 13</td><td>'ÍRGVTIGEGQQHHLGG (SEQ ID NO: 13) A peptide with glutamine at the transglutaminase docking site in the fibrinogen chain</td>

<td>SEQ ID NO: 14</td><td>GAKDV (SEQ ID NO: 14) A peptide that mimics the lysine docking site on the fibrinogen chain</td>

<td>SEQ ID NO: 11</td><td>KKKK (SEQ ID NO: 11) A peptide with a polylysine in a random docking site</td>

<td>SEQ ID NO: 12</td><td>NQEQVSPL (SEQ ID NO: 12) A peptide that mimics the site of degradation in an a2 plasmin inhibitor (abbreviated TG)</td>

For the incorporation of PTH into a matrix formed from synthetic precursor components, the fusion peptide is PTH or any other peptide to be incorporated must be synthesized with at least one additional cysteine ​​group (-SH) preferably at the N-terminus. of PTH as the degradable substrate domain. Cysteine ​​can either bind directly to PTH or through a linker sequence. The linking sequence additionally

ES 2 301 697 T3 can include an enzymatically degradable amino acid sequence, such that PTH can be cleaved from the matrix by enzymes practically in the natural way. The free cysteine ​​group reacts with the conjugated unsaturated group of the parent component in a Michael-type addition reaction. In the case of PTH 1-34, binding to a synthetic matrix for PTH 1-34 is made possible by linking 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 plasmin sequence is attached as a linker between the cysteine ​​and the peptide. The GYKNR sequence (SEQ ID NO: 15) between the first domain and the second domain, cysteine, renders the plasmin bond degradable.

In this way, the PTH fusion peptides can be further modified to contain a degradable site between the binding site, i.e. the second domain (i.e. the Factor XIIIa or cysteine ​​substrate domain) and the PTH, that is, the first domain. These sites can be degraded either by non-specific hydrolysis (ie, an ester bond) or they can be substrates for specific enzymatic degradation (either proteolytic or polysaccharide degradation). These degradable sites allow the engineering of the more specific release of PTH from fibrin gel-like matrices. For example, degradation based on enzyme activity allows the release of PTH that will be controlled by a cellular process rather than diffusion of the factor through the gel. The degradable or binding site is cleaved by enzymes released from cells invading the matrix.

The degradation sites allow PTH to be released with little or no modification with the primary peptide sequence, which can result in increased factor activity. In addition, it allows the release of the factor to be controlled by specific cellular processes, such as, for example, localized proteolysis, instead of diffusion from some porous materials. This allows the factors to be released at different rates within the same material depending on the location of the cells within the material. This also reduces the amount of total PTH needed, since its release is controlled by cellular processes. Preservation of PTH and its bioavailability are distinct advantages of exploiting cell-specific proteolytic activity over the use of diffusion controlled release devices. In a possible explanation for the good healing of a bone defect with PTH covalently bound to a matrix, it seems important that PTH is administered locally over a prolonged period of time (i.e., not just a single pulsed dose) but not in a single dose. Continuous form. This is accomplished by slow degradation, through either enzymatic cleavage or hydrolytic cleavage from the matrix. In this way, the molecule is then delivered through a pseudo-pulsed effect that occurs over a sustained period of time. When a parent cell infiltrates the matrix, it will find a PTH molecule and can differentiate into a proteoblast. However, if this particular cell does not continue to release the bound PTH from the matrix, it will effectively turn into an osteoblast and initiate bone matrix production. Lastly, the therapeutic effects of the peptide are localized to the defective region and subsequently enhanced.

The enzymes that could be used for proteolytic degradation are many. Proteolytically degradable sites could include substrates for collagenase, plasmin, elastase, stomelysin, or plasminogen activators. Example substrates are listed below. P1-P5 denotes amino acid positions 1-5 towards the amino terminus of the protein from the site where proteolysis occurs. P1'-P4 'denote amino acid positions 1-4 towards the carboxy terminus of the protein from which proteolysis occurs.

(Table goes to next page)

ES 2 301 697 T3

TABLE 2

Sample substrate sequences for protease

<td>Protease</td><td>P</td><td>Q4</td><td>Q3</td><td>P2</td><td>Pl</td><td>Pl '</td><td>P2 '</td><td>P3 '</td><td>P4 '</td><td>Reference</td>

<td>Plasmin</td><td></td><td></td><td>L</td><td>I</td><td>K</td><td>M</td><td>K</td><td>P</td><td></td><td>Takagi and Doolittle, (1975) Biochem. 14: 5149-5156</td>

<td>Plasntina</td><td></td><td></td><td>N</td><td>F</td><td>K</td><td>S</td><td>Q</td><td>L</td><td></td><td>Takagi and Doolittle, 1975</td>

<td>Stromelysin</td><td>Ac</td><td>G</td><td>P</td><td>L</td><td>TO</td><td>L</td><td>T</td><td>TO</td><td>L</td><td>Smith et al., (1995). J. Bio. Chem. 270: 6440- 6449</td>

<td>Stromelysin</td><td></td><td>Ac</td><td>P</td><td>F</td><td>AND</td><td>L</td><td>R</td><td>TO</td><td>nh₂</td><td>Smith et al., 1995</td>

<td>Elastase</td><td></td><td></td><td>z-</td><td>TO</td><td>TO</td><td>F</td><td>TO</td><td>nh₂</td><td></td><td>Besson et al., (1996) Analytical Biochemistry 237: 216-223</td>

<td>Colagenaza</td><td></td><td>G</td><td>P</td><td>L</td><td>G</td><td>I</td><td>TO</td><td>G</td><td>P</td><td>NetzelArnett et al., (1991) J. Biol. Chem. 266: 67476755</td>

<td>t-PA</td><td>P</td><td>H</td><td>Y</td><td>G</td><td>R</td><td>S</td><td>G</td><td>G</td><td></td><td>Coombs et al. , 1998. J. Biol. Chem. 273: 43234328</td>

<td>u-PA</td><td>P</td><td>G</td><td>s</td><td>G</td><td>R</td><td>S</td><td>TO</td><td>S</td><td>G</td><td>Coombs et al., 1998</td>

In another preferred embodiment, an oligo-ester domain could be inserted between the first and second domains. This could be done using an oligo-ester such as, for example, lactic acid oligomers.

The substrate for non-enzymatic degradation could consist of any bond that undergoes hydrolysis by an acid or base catalyzed mechanism. These substrates can include oligo-esters such as for example,

ES 2 301 697 T3 lactic or glycolic acid oligomers. The rate of degradation of these materials can be controlled through the choice of oligomer.

D. PTH

The term "PTH" as used herein includes the human sequence of PTH 1-84 and all truncated allelic and modified versions of PTH that exhibit bone-building properties when used. covalently bind to biodegradable natural or synthetic 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 the 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 factors

In a preferred embodiment for incorporation of a PTH into the matrix, the matrix includes fibrin which is formed from fibrinogen, a source of calcium and thrombin and the PTH fusion peptide 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, one domain, the second, is a substrate for a degrading enzyme such as for example Factor XIIIa. Factor XIIIa is a transglutaminase that is active during clotting. This enzyme, formed naturally from Factor XIII through thrombin cleavage, functions to link fibrin chains together via amide bonds, formed between glutamine side chains and lysine side chains. The enzyme also functions to bind other peptides to fibrin during clotting, for example the cellular binding sites provided also include a Factor XIIIa. Specifically, the sequence NQEQVSP (SEQ ID NO: 16) has been shown to function as an effective substrate for Factor XIIIa. As described hereinabove, it either binds directly to PTH or can include a degradation site between PTH (first domain) and the sequence NQEQVSP (SEQ ID NO: 16) (second domain). As such, the PTH fusion peptide can be incorporated into fibrin during coagulation via a Factor XIIIa substrate.

Design of fusion proteins for incorporation

The PTH fusion peptide including a first domain including PTH, a second domain including a substrate domain for a degradation enzyme, and optionally a degradation site between the first and second domains can be incorporated into fibrin gels. using several different schemes. Preferably, the second domain includes a transglutaminase substrate domain and even more preferably includes a Factor XIIIa substrate domain. Most preferably the substrate domain of Factor XIIIa includes NQEQVSP (SEQ ID NO: 16). When this PTH-fusion peptide is present during the polymerization of fibrinogen, that is, during the formation of the fibrin matrix, it is incorporated directly into the matrix.

The site of degradation between the first and second domain of the PTH fusion peptide can be an enzymatic degradation site as described above. Preferably, the site of degradation can be cleaved by an enzyme selected from the group consisting of plasmin and matrix metalloproteinase. By careful selection of K_m and K_cat of this site of enzymatic degradation, 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 placement of the site of degradation that is prepared to each type of protein and application. This PTH fusion peptide could be directly degraded in the fibrin matrix as described above. However, the incorporation of an enzymatic degradation site alters the release of PTH during proteolysis. When 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 released PTH, which could now be nearly free of any engineered fusion sequences, as well as any degraded fibrin.

II. Method of use

The matrices can be used for tissue repair, regeneration, or remodeling, and / or the release of PTH, before or at the time of implantation. In some cases it will be desirable to induce degradation at the site of administration to conform the matrix to the tissue at the implantation site. In other cases, it will be desirable to prepare the matrix prior to implantation.

Cells can also be added to the matrix before or at the time of implantation, or even after implantation, either at the time of or after degradation of the polymer to form the matrix. This can be done in addition to or in place of matrix degradation to produce interstitial separation designed to stimulate cell proliferation or ingrowth.

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 adhesion formation after surgery.

ES 2 301 697 T3

III. Application methods

In the preferred embodiment, the material gels in situ within or on the body. In another embodiment, the matrix can be formed outside the body and then applied in the preformed shape. 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 application of the mixture to the body to avoid combining or contacting each other under conditions that allow polymerization or gelation of the components. For contacting prior to administration, a reagent kit can be used to separate the compositions from one another. Upon mixing under 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 instantly after mixing. This rapid gelation makes injection, that is, pressure removal of the gelled material through the injection needle almost impossible.

In one embodiment, the matrix is ​​formed from fibrinogen. Fibrinogen, through a cascade of several reactions, gels to form a matrix, when it comes into contact with thrombin and a source of calcium at the appropriate temperature and pH. The three components, fibrinogen, thrombin, and the calcium source, must be stored separately. However, as long as at least one of the three components is kept separate, the other two components can be combined prior to 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 from a solution of thrombin in a calcium chloride buffer (eg, at a concentration ranging from 40 to 50 mM). The fibrinogen buffer can be a histidine buffer at a preferred concentration of 50mM further including NaCl at a preferred concentration of 150mM or TRIS buffered saline (preferably at a concentration of 33mM).

In a preferred embodiment, a reagent kit is provided, containing a fusion protein, fibrinogen, thrombin, and a source of calcium. Optionally, the reagent kit may contain a degrading enzyme, such as Factor XIIIa for example. 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 can be present in either the fibrinogen or thrombin solution. 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 by pressing the contents of both syringes 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 can contain the fusion protein. Before use, Tris or histidine buffer is added to fibrinogen, the buffer may additionally contain aprotinin. The lyophilized thrombin dissolves in the calcium chloride solution. Subsequently, the fibrinogen and thrombin solutions are placed in separate vial / syringe containers and mixed by a two-way connecting device, such as, for example, a two-way syringe. Optionally, the vial / syringe containers are divided into two parts thus having two chambers separated by an adjustable partition that is perpendicular to the wall of the syringe body. One chamber contains lyophilized fibrinogen or thrombin, while the other chamber contains a suitable buffer. When the plunger is pressed down, the split moves and releases the buffer inside the fibrinogen chamber to dissolve the fibrinogen. Once both fibrinogen and thrombin are dissolved, both two-part syringe barrels are attached to a two-way connecting device and the contents are mixed by pressing them out 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 approximately 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 multithiol) only reacts with the multiaceptor 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. multi-receptor. Both the multiaceptor and multithiol component are stored as a solution in buffers. The two compositions can include the cell binding site and additionally the bioactive molecule. Thus, the first composition of the system for example can include the solutions of the nucleophilic component and the second composition of the system can include the solution of the multi-receptor component. Either or both compositions can include the base. In another embodiment, the multiaceptor and multithiol can be included as a solution in the first composition and the second composition can include the base. The connection and mixing are presented in the same way as described above for fibrinogen. The two-part syringe body is equally suitable for synthetic precursor components. Instead of fibrinogen and thrombin, the multiaceptor

ES 2 301 697 T3 and the multithiol components are stored in powder form in one of the chambers and the other chamber contains the basic buffer.

The following examples are included to demonstrate preferred embodiments of the invention. While the compositions and methods have been described in preferred terms and embodiments, it will be apparent to one of ordinary skill in the art that variations can be applied to the composition, methods and in the steps or sequence of steps of the method described in the present without departing from the concept spirit and scope of the invention.

Example 1

Arrays Containing Covalently Bound TGPTH

Synthesis of TGPTH

PTH 1-34-mer peptide showing similar activity with whole protein, and proteins of this length can be synthesized by standard solid state peptide synthesis methods.

All peptides were synthesized on solid resin using an automated peptide synthesizer using standard 9-fluorenylmethyloxycarbonyl chemistry. The peptides were purified by c18 chromatography and analyzed using reverse phase chromatography via HPLC to determine purity, as well as mass spectroscopy (MALDI) to identify the molecular weight of each product. Using this method, the following peptide, (referred to herein as "TGPTH") was synthesized:

NHj-Asn-Gln-Glu-Gln-Val-Ser-Pro-Leu-Ser-Val-Ser-GluIle-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-SerMet- Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-AspVal-His-Asn-Phe-COOH (SEQ ID NO: 17)

In vivo results

TGPTH activity to enhance bone regeneration was tested on a TISSUCOL matrix^® in a defect by hole drilled in a sheep. Eight mm and 12 mm deep holes were created in the proximal and distal femur and humerus of a sheep. These holes were filled with an in situ polymerizing fibrin gel. Defects were left empty, filled with TISSUCOL® or TGPTH was added to fibrin TISSUCOL® at 400 jUg / mL before polymerization. In each example in which TISSUCOL was used^®, this 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 healing period, the animals were sacrificed, and the bone samples were removed and analyzed by microcomputer topography (μΟΓ). The percentage of defective volume filled with calcified bone tissue was then determined. When the defects were left empty, there was no calcified tissue formation within the fibrin matrix. When only a fibrin gel was added, there was practically no bone healing. However, with the addition of 400 g / mL TGPTH, the level of healing increased dramatically, with the defect filling 35% with calcified bone.

Example 2

Healing response with modified PTH 1-34 attached to a fibrin matrix

Materials

The modified version of PTH_1-34 that can be incorporated into a fibrin matrix has been tested for the healing response in critically sized cranial defect in rats.

A fibrin gel was made from fibrin sealer precursor components from the TISSUCOL® Reagent Kit (Baxter AG, CH-8604 Volketswil / ZH). Fibrinogen was diluted in sterile 0.03M buffered Tris solution (TBS, pH 7.4) to form a solution of approximately 8 mg / mL and thrombin was diluted in CaCl solution.₂ 50 mM sterile to form a 2 U / mL solution. 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-PTH was added_1-34 or TGPTH, ₃₄ to thrombin, and mixed to form a homogeneous concentration.

ES 2 301 697 T3

To form the fibrin gene, the diluted precursors were mixed together by injecting fibrinogen into a tube containing the thrombin. In the case of the perforated defect in a sheep (as will be described later), this mixture was then immediately injected into a perforated effect created in a cancellous bone of the sheep, where a fibrin gel was formed in 1-5 minutes. In the first series of animal experiments, the efficacy of a fusion protein containing PTH was tested_1-34 as the bioactive factor (NQEQVSPLYKNRSVSEIQLMHNLGKHLNS MERVEWLRKKLQDVHNF, SEQ ID NO: 18) in the healing of cortical bones in a small animal model. The sequence YKNR (SEQ ID NO: 19) makes the plasmin bond degradable ("TG-pl-PTH₁_₃₄). TG-pl-PTH_1-34 It is produced by chemical synthesis. Purification was carried out via reverse phase HPLC (Column C18) using a TFA as the counter ion which resulted in a final product which was a TFA salt. The purity of TG-pl-PTH1_34 was determined to be 95%.

The healing response was explored at both short (3 weeks) and long (7 weeks) healing times to determine if an improvement in healing could be observed.

Critically sized cranial defect in rats

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

Results

When TG-pl-PTH1_34 was studied at the time of 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 as other potent morphogens, including rhBMP-2, showed a significant 3-week healing effect. In contrast, the healing effect for TG-pl-PTH_1-34, could not be observed at this early time point. However, when the longest time (7 weeks) was analyzed, a moderate dose-dependent improvement in healing in the critically sized cranial defect in rats was observed with the addition of PTH_1-34 modified 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

Healing response in cranial defect in rats with modified PTH

<td>Sample</td><td>Dose</td><td>Weather</td><td>Healing (% of defect</td>

<td></td><td>(pg / mL)</td><td>(days)</td><td>stuffed with bone)</td>

<td>Fibrin</td><td> 0</td><td> 63</td><td> 38</td>

<td>TG-pl - PTHi-34</td><td> 10</td><td> 63</td><td> 43</td>

<td>TG-pl-PTHi-34</td><td> 200</td><td> 63</td><td> 50</td>

<td>TG-pl-PTHi-34</td><td> 500</td><td> 63</td><td> 62</td>

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 piercing defect in sheep

TG-pl-PTH_1-34 It was also tested in a model with a large bone defect to test the effect of this hormone on bone healing. In the sheep perforation defect model, 8 mm cylindrical perforation defects that were approximately 15 mm deep were placed in both the distal and 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 µl) containing various doses of TG-µl-PTH._1-34, or TGPTH1-34. The animals were allowed to cure for eight weeks and then euthanized. The defect was analyzed with juCT and histology.

ES 2 301 697 T3

For this series of experiments, three types of compositions were tested. First, TG-pl-PTH was tested_1-34over a large variation in concentration. Second, another PTH was used_1-34 modified, the TGPTH_1-34(NQEQVSPLSVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF; SEQ ID NO: 17) which only had a transglutaminase sequence at the amino terminus, and a degradation site. In this way, the TGPTH_1-34 it could only be released by degradation of the fibrin matrix itself. TGPTH_1-34 produced and purified in a manner similar to TG-pl-PTH_1-34. Purity was determined to be 95%. TGPTH_1-34 It was tested at various concentrations that were similar to TG-pl-PTH1-34 concentrations to compare efficacy. Finally, the matrices were produced in the presence of granular material, with either tGpTH_1-34 or TG-pl-PTH_1-34. The granular material was a standard tricalcium phosphate / hydroxyapatite mixture that was incorporated into the matrix during gelling. The effect of adding these granules on the efficacy of PTH was explored_1-34. As a control, unmodified fibrin was tested.

Results

When any of the PTH molecules_1-34 modified was placed in long bone defects, a significant improvement in healing responses was observed over the use of fibrin matrices (control) alone. The use of fibrin alone resulted in poor healing, where only 20% of the original defect was filled with newly formed bone.

TG-pl-PTH was tested_1-34, in a concentration series of 20-1000 pg / ml · .. For each dose tested, a significant increase in the healing response was observed. For example, when 100 pg / mL of TG-pl-PTH was used_1-34, the cure rate was increased by almost 60%.

In a second series of experiments, TGPTH was tested_1-34. The use of TGPTH_1-34 bone healing also increased. 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 sequence of PTH_1-34 Modified resulted in a larger healing response than the control.

Finally, when any PTH molecule_1-34 Modified was bound to the matrix and a mixture of pellets / matrix was used, the efficacy of PTH1-34 was maintained. This was tested for both TG-pl-PTH1-34 (see Table 4) and TGPTH_1-34 (see Table 5).

TABLE 4

Healing of a perforation defect in sheep with TG-pl-PTH, ₃₄ incorporated in a fibrin matrix; 8 week cure time

<td>Sample</td><td>dose (g / mL)</td><td>Healing (% of defect stuffed with bone)</td>

<td>Fibrin (control)</td><td> 0</td><td> 20</td>

<td>TG-pl-PTHx-34</td><td> 50</td><td> 31, 3</td>

<td>TG-pl-PTHi-34</td><td> 100</td><td> 59, 7</td>

<td>TG-pl-PTHi-34</td><td> 400</td><td> 73</td>

<td>TG-pl-PTHi-34</td><td> 1000</td><td> 77</td>

<td>TG-pl-PTHi-34 400TCP</td><td> 400</td><td> 68</td>

ES 2 301 697 T3

TABLE 5

Healing of a perforation defect in sheep with PTH_1-34 attached to a fibrin matrix; 8 week cure time

<td>Sample</td><td>dose (g / mL)</td><td>Healing (% of defect stuffed with bone)</td>

<td>Fibrin</td><td> 0</td><td> 20</td>

<td>TGPTHr-34</td><td> 400</td><td> 40</td>

<td>TGPTHi-34</td><td> 1000</td><td> 65</td>

<td>TGPTH1-34 400TCP</td><td> 400</td><td> 71</td>

Histological evaluation showed high infiltration in the original defect of spindle and osteoblast progenitor cells supported on an extracellular matrix. Active osteoids with large rounded osteoblasts were common, and indochondral dosing signals (chondrocytes) were observed. For eight weeks, osteoclasts and healthy signs of remodeling could be found. Although unlike the results obtained from continuous exposure to systemic PTH, no overt response was observed from osteoclasts and new bone formation was significantly greater than absorption in the defect area and around the bone. herself. 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 precursor components for synthetic matrices

Preparation of PEG-vinylsulfones

Commercially available branched PEGs (4-branch PEG, molecular weight 14,800, 4-branch PEG, molecular weight 10,000, and 8-branch PEG, molecular weight 20,000; Shearwater Polimers, Huntsville, AL, USA) were functional groups in the OH term.

The PEG vinyl sulfones were produced under argon atmosphere by reacting a dichloromethane solution of the precursor polymers (previously dried over molecular sieves) with NaH and then, after evolution with hydrogen, with divinylsulfone (molar ratios: OH 1: NaH 5 : divinylsulfone 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 clear. The derived polymer was isolated by precipitation in ice cold diethyl ether. The product was redissolved in dioloromethane and reprecipitated from diethyl ether (with rigorous washing) twice to remove all excess divinylsulfone. Finally, the product was dried under vacuum. The derivation was confirmed with Ή NMR. The product showed characteristic vinylsulfone peaks at 6.21 ppm (two hydrogen) and 6.97 ppm (one hydrogen). The degree of conversion of the final group was found to be 100%.

Preparation of PEG-acrylates

PEG acrylates were produced under argon atmosphere by reacting an azeotropically seated toluene solution of the precursor polymers with acryloyl chloride, in the presence of triethylamine (molar ratios: 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%; OH to acrylate conversion: 100% (from 'H-NMR analysis)¹H-NMR (CDCl₃): 3.6 (341H (14800 4 branches: 337H theoretical), 230 (10000 4 branches 227H theoretical), or 210H (20,000 8 branches: 227H theoretical), PEG chain protons), 4.3 (t, 2H, -CH₂CH₂-O-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 (v CH), 1724 (v C = O), 1460 (v_s CH2), 1344, 1281, 1242, 1097 (v_ace COC), 952, 842 (v_sCOC) cm^-1.

ES 2 301 697 T3

Peptide synthesis

All peptides were synthesized on solid resin using an automated peptide synthesizer (9050 Pep Plus Synthesizer, Millipore, Framingham, USA) with standard 9-fluorenylmethyloxycarbonyl chemistry. Hydrophobic scavengers and cleaved protecting groups were removed by precipitating the peptide in cold diethyl ether and dissolving 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 size exclusion column with TBS, pH 7.0 as the damper in progress.

Matrix formation by conjugate addition reactions

MMP-sensitive gels were formed by conjugated addition to a peptide-linked nucleophile and a PEG-linked conjugated unsaturation that allows migration of proteolytic cells. The synthesis of the gels is carried out entirely 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 Ac-GCGYGRGDSPG-NH2 (SEQ ID NO: 20) peptide) were pendently attached to a multi-branched PEG-vinylsulfone and then this precursor was degraded with a dithiol-containing peptide (for example, the MMP substrate Ac-GCRDGPQGIAGFDRCG-NH2 (SEQ ID NO: 21)). In a typical gel preparation for three-dimensional in vitro studies, 4-branch PEG-vinyl sulfone (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-GCGYGRGDSPGNH2 (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 degrading peptide Ac-GCRDGPQGIWGQDRCG-NH₂ (SEQ ID NO: 21) was mixed with the previous solution and the gels were synthesized. Gelation occurred within a few minutes, however, the degradation reaction was carried out for one hour at 37 ° C to ensure complete reaction.

Non-MMP sensitive gels were formed by the addition of conjugates with a PEG-linked nucleophile and a PEG-linked conjugated unsaturation that allows non-proteolytic cell migration.

The synthesis of gels was also carried out entirely through the Michael-type addition reaction of thiol-PEG on PEG with vinyl sulfone functional group. In a first step, the adhesion peptides were pendently linked (for example, the Ac-GCGYGRGDSPG-NH2 (SEQ ID NO: 20) peptide) to a multi-branched vinyl sulfone PEG 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-branch vinyl sulfone PEG (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 cell adhesive gels, the dissolved peptide Ac-GCGYGRGDSPG-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 PEG-dithiol precursor was mixed with the above solution and the gels were synthesized. Gelation occurred within a few minutes, however, the degradation reaction was carried out for one hour at 37 ° C to ensure complete reaction.

Matrix formation by condensation reactions

MMP-sensitive gels were formed by condensation reactions with a peptide X-linker containing multiple amines and an electrophilically active PEG that allows 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 difunctional double ester PEG-N-hydroxysuccinimide (Shearwater polymers) (NHS-HBS-CM-PEG-CM-HBA-NHS). In a first step, one of the adhesion peptides (for example, the Ac-GCGYGRGDSPG-NH peptide₂) (SEQ ID NO: 20) was reacted with a small fraction of NHS-HBSCM-PEG-CM-HBA-NHS and then this precursor was degraded to a network when mixing with the peptide Ac-GKGPQGIAGQKG PQGIAGQKG-NH₂ (SEQ ID NO: 22) that carries three ε-amines (and one primary 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% (w / w) solution and the hydrogels were formed in less than one hour.

In contrast to the hydrogels present formed by the Michael-type reaction, the desired self-selectivity was not guaranteed in this procedure, because the amines present in cell-like or tissue-like biological materials would also react with the activated difunctional double esters. This is also true for other PEGs bearing electrophilic functional groups such as, for example, PEG-oxycarbonylimidazole (CDI-PEG), or nitrophenyl PEG carbonate.

MMP-insensitive hydrogels were formed by condensation reactions with a PEGamine degrader and an electrophilically active PEG that allows non-proteolytic cell migration.

Hydrogels were also formed by conducting a condensation reaction between commercially available branched PEG-amines (Jeffamines) and the same difunctional double ester PEG-N-hydroxysuccinimide (NHS-HBSCM-PEG-CM-HBA-NHS). In a first step, the adhesion peptides (for example, the Ac-GCGYGRGDSP G-NH₂) (SEQ ID NO: 20) were reacted with a small fraction of NHS-HBS-CM-pEG-CM-HBA-NHS

ES 2 301 697 T3 and then this precursor was degraded to a network by mixing with the multi-branched 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% (w / w) solution and hydrogels were formed in less than one hour.

Again, in contrast to the hydrogels present formed by the Michael-type reaction, the desired autoselectivity was not guaranteed in this procedure, because the amines present in cell-like or tissue-like biological materials also did not react with the activated difunctional double esters. This is also true for other PEGs bearing electrophilic functional groups such as, for example, PEG-oxycarbonylimidazole (CDI-PEG), or PEG nitrophenyl PEG carbonate.

Example 4

Bone regeneration with synthetic enzymatically degradable matrices

Two different starting concentrations of enzymatic degradable gels were used. In each of these, the concentration of RGD and active factor (CplPTH at 100 jUg / mL) were kept constant. The polymer network was formed from a four-branch functional group PEG with four vinyl sulfone end groups of molecular weight of 20 kD (molecular weight of each branch 5kD) and dithiol peptide of the following sequence 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 the functional group PEG (first precursor molecule) and 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 components + 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 components + triethanolamine solution) which corresponds 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 thiols 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 polymeric network plus water, in this way 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 polymeric network plus water, thus having an aqueous content of 92.6.

In order to explore the effect of this change, these materials were tested on the perforation defect in a sheep. Here, a 750 pL defect was placed in the cancellous bone of the femoral shaft and humerus of the sheep and filled with an in situ gelatinizing enzyme gel. The following amount of calcified tissue was obtained, determined via juCT, with each group at N = 2.

Initial gel concentration

Calcified tissue

12.6%

9.5%

2.7%

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.

So 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 how they linked, via enzymatic degradation sites, the density of the linkers, and various other variables were crucial in enabling a functional healing response. These differences were observed very clearly in the perforation defect model in the sheep.

Example 5

Bone formation with synthetic hydrolytically degradable matrices

A fusion peptide with PTH 1-34 was tested on a synthetic gel as well as the perforation defect model in a sheep exactly as described for the fibrin matrices. A hydrogel network was created by co-mixing acrylated 4-branch polyethylene glycol, MW 15,000 (Peg 4 * 15 Acr) with a linear polyethylene glycol dithiol of MW 3400. Through a Michael-type 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 acrylate to create a covalent bond. By mixing the multifunctional precursors together such that the combined multifunctionality was greater than or equal to five, a hydrogel was formed. Furthermore, 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

ES 2 301 697 T3 binds to the matrix by adding a cysteine, the amino acid-containing thiol, 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 PTH1 34 sequence and both were attached to the matrix via the acrylates in the degrading. Later, 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 perforation defect model in a sheep to test the ability of matrix-bound C-PTH1 34 to enhance bone development. In order to determine the amount of enhancement, 0.100 and 400 jug / mL of the fusion peptide with PTH were added to the matrix.₁ _₃₄. When this was done, an increase in bone formation was observed with the addition of iPTH_₃₄. In each test, the healing response was measured in the same time of eight weeks. This was compared to the defects that were left empty. When the synthetic hydrolytically degradable matrix was used without the PTH fusion peptide, the healing response was measured at approximately 40%. This means that 40% of the original defect volume was filled with newly formed bone tissue. Later, when 400 jug / mL of the fusion peptide were used with PTH ^₃₄ modified, the healing response increased to approximately 60%. In comparison, when the defects were left empty, approximately 10% 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

<td>Treatment</td><td>Healing (% calcified tissue)</td>

<td>Empty</td><td> 10</td>

<td>Hydrolytic gel</td><td> 40</td>

<td>Hydrolytic gel with</td><td> 54</td>

<td>100 pg / mL CPTH</td><td></td>

<td>Hydrolytic gel with</td><td> 60</td>

<td>400 pg / mL CPTH</td><td></td>

Compared to 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 PTH 1-34 bound to this matrix, healing increased even more with the level that was up to 50% higher than when the matrix was used alone and 500% higher than the level of healing obtained when the defect it was left empty.