JP2009102383A - Growth factor-modified protein matrix for tissue engineering - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To incorporate a protein into a protein or polysaccharide matrix for use in tissue repair, regeneration and/or remodelling and/or drug delivery. <P>SOLUTION: The protein can be incorporated so that it is released by degradation of the matrix, by enzymatic activation and/or diffusion. As demonstrated by the examples, one method involves binding of heparin to the matrix by either a covalent or non-covalent method to form a heparin-matrix. The heparin then non-covalently binds a heparin-binding growth factor to the protein matrix. Alternatively, a fusion protein which contains a crosslinking region such as a factor XIIIa substrate and a native protein sequence can be constructed. Incorporation of a degradable linkage between the matrix and a bioactive factor can be particularly useful when long-term drug delivery is desired (for example in the case of nerve regeneration). <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

(Field of Invention)
The present invention relates to a fusion protein or peptide comprising an amino acid sequence that binds and interacts with PTH and a matrix, and to the use of the fusion protein or peptide in tissue repair and regeneration and controlled release of PTH.

(Background of the Invention)
Parathyroid hormone (PTH) is an 84 amino acid peptide that is made and secreted by the parathyroid body. This hormone plays a major role in regulating serum calcium levels through its action on various tissues (including bone). Studies with different forms of PTH in humans have shown an anabolic effect on bone, which makes these PTH interesting for the treatment of osteoporosis and related bone disorders (US Pat. No. 5, to Chorev et al. , 747, 456 and WO 00 to Eli Lilly & Co.
/ 10596). Parathyroid hormone acts on cells by binding to cell surface receptors. This receptor is known to be found in osteoblasts (cells responsible for forming new bone).

The N-terminal 34 amino acid domain of human hormone has been reported to be biologically equivalent to the full-length hormone. PTH1-34 and its mode of action are described in US Pat. No. 4,086,
It was first reported in 196. Since then, studies have been performed on PTH1-34 and other truncated versions of the native human PTH form (eg, PTH1-25, PTH1-31 and PTH1-38) (eg, Rixon RH et al., J Bone
Miner. Res. 9 (8): 1179-89 (Aug. 1994))
.

The mechanism by which PTH affects bone remodeling is complex, leading to conflicting results, followed by a significant number of studies on the exact mechanism. It has been shown that bone density decreases when PTH is administered systemically in a continuous manner. In contrast, it has been reported that bone density is increased when the same molecule is administered in a pulsatile manner (eg, Eli Li
lly & Co. See WO99 / 31137). This apparent contradiction can be explained by the mechanism by which PTH regulates bone remodeling and subsequently observable parameters of bone density. Within mature bone, the PTH receptor was shown to be present only on the surface of cells of the osteoblast lineage and not on osteoclasts. The role that PTH plays in bone remodeling is directed by osteoblasts, as opposed to osteoclasts. However, cells at various stages of the osteoblast lineage respond differently when they bind to PTH. Thus, the dramatic changes observed when PTH is administered using different methods are explained by understanding the different effects that the same molecule has on different cells within the osteoblast lineage. Can be done.

When PTH binds to mesenchymal stem cells, the cells are induced to differentiate into preosteoblasts. Therefore, adding PTH to the system increases the proportion of preosteoblasts. However, these preosteoblasts have PTH receptors as well, and subsequent binding of PTH to receptors on these cells leads to different responses. When PTH binds preosteoblasts,
It produces two separate results that lead to bone resorption. First, it inhibits further differentiation of preosteoblasts into osteoblasts. Second, it increases the secretion of interleukin 6 (IL-6) from preosteoblasts. IL-6 both inhibits the differentiation of preosteoblasts and increases the differentiation of preosteoblasts into osteoblasts. This dual response from cells within the osteoblast lineage provides a complex response between bone remodeling and PTH exposure. If PTH is dosed periodically over a short period of time,
Mesenchymal stem cells are induced to differentiate into osteoblasts. The short dosing period then inhibits newly formed proosteoblasts from producing IL-6 and inhibits osteoclast activation. Thus, during dose intervals, these newly formed proosteoblasts can further differentiate into osteoblasts, resulting in bone formation. When a fixed volume of PTH is applied, the preosteoblasts become IL-
Has the opportunity to start production of 6, thereby activating osteoclasts, inhibiting themselves and leading to the opposite effect (bone resorption).

For tissue repair or regeneration, cells must move to the wound bed, proliferate, express matrix components, or form an extracellular matrix, and form a final tissue shape. Diverse cell populations (often including vascular and neural cells) often have to participate in this morphological response. The matrix has been shown to be greatly enhanced,
In some cases, it has been found that this response is essential for it to occur. The approach is made in a developing matrix from natural or synthetic origin or a mixture of both. The matrix in which natural cells proliferate is subjected to remodeling by cell sensitization, all based on proteolysis by, for example, plasmin (degrading fibrin) and matrix metalloproteinases (degrading collagen, elastin, etc.). Such degradation occurs only upon direct contact with highly localized and migrating cells. Furthermore, the delivery of certain cell signaling proteins (eg, growth factors) is tightly regulated. In a natural model, a macroporous cellular ingrowth matrix is not used, but rather a microporous matrix from which cells can be degraded locally and optionally when cells migrate into the matrix. used. Due to immunogenicity, costly production, limited availability, batch variability and batch purification concerns, matrices based on synthetic precursor molecules (eg, modified polyethylene glycols) are found in the human body and / or Or developed for tissue regeneration in the human body.

A lot of work has studied the systemic effects of PTH, while studies have not investigated the local or topical administration of PTH. Since PTH has a direct anabolic effect on the osteoblast lineage, it should have a strong potential to heal bone defects in addition to its effect on bone density when properly shown within the defect site. Once the defect is filled with preosteoblasts, if the PTH signal stops, the newly formed preosteoblasts
They differentiate into osteoblasts and begin to convert to a wound bed, first convert to reticular bone tissue and then convert to mature bone structure.

Accordingly, it is an object of the present invention to provide a form of PTH that can be coupled to a matrix for tissue repair, tissue regeneration, and remodeling.

It is a further challenge to give PTH in a form suitable for topical or local administration to a patient to heal a bone defect.

(Summary of the Invention)
A fusion peptide comprising a substrate domain that can be covalently cross-linked to a parathyroid hormone (PTH) of one domain and a matrix of another domain, as well as a matrix, and a kit comprising such a fusion protein or peptide are described herein. Disclosed in the document. The fusion protein is covalently bound to a natural or synthetic material to form a matrix, which can be used to heal bone defects. If necessary, all components to form the matrix are applied to the bone defect and the matrix is formed at the application site. The fusion peptide can be incorporated into the matrix so that it is released either by the entire fusion peptide or only the respective PTH sequence of the first domain, by degradation of the matrix, by enzymatic action and / or by hydrolysis action. Is done. The fusion peptide can also include a degradable bond between the first domain and the second domain, the bond comprising a hydrolytic cleavage site or an enzymatic cleavage site. In particular, the fusion peptide includes a PTH in one domain, a substrate domain that can be covalently crosslinked to a matrix in a second domain, a degradation site between the first and second domains. Preferably, the PTH is human PTH, although PTH from other sources (eg, bovine PTH) may be appropriate. PTH is PTH1-84 (native), PTH1-38, PTH1-34, PTH1-31, or PTH1-2.
Or any modified or allelic version of PTH that exhibits features similar to those described above (ie, osteogenesis). The degradation site allows for a delivery rate that varies at various locations within the matrix, which depends on cellular activity at that location and / or within the matrix. Further advantages include lower total drug dose within the delivery system, spatial control of release allowing a greater percentage of drug to be released at the point of maximum cellular activity. Therefore, the present invention also provides the following.
(1) a fusion peptide, the fusion peptide comprising:
A first domain comprising PTH and
A second domain comprising a covalently crosslinkable substrate domain
including,
Fusion peptide.
(2) The fusion peptide according to item 1, further comprising a degradation site between the first domain and the second domain.
(3) The fusion peptide according to item 1, wherein 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 item 3, wherein the PTH is PTH 1-34.
(5) The fusion peptide according to item 1, wherein the second domain includes a transglutaminase substrate domain.
(6) The fusion peptide according to item 5, wherein the second domain comprises a factor XIIIa substrate domain.
(7) The fusion peptide according to item 6, wherein the factor XIIIa substrate domain comprises SEQ ID NO: 12.
(8) The fusion peptide according to item 1, wherein the second domain contains at least one cysteine.
(9) The fusion peptide according to item 2, wherein the degradation site is an enzymatic degradation site or a hydrolyzable degradation site.
(10) The peptide according to claim 8, wherein the degradation site is an enzymatic degradation site, and the enzymatic degradation site is cleaved by an enzyme selected from the group consisting of plasmin and matrix metalloproteinase.
(11) A kit comprising the fusion peptide according to items 1-10.
(12) The kit according to item 11, further comprising fibrinogen, thrombin and a calcium source.
(13) The kit according to item 11, wherein the kit further comprises a crosslinking enzyme.
(14) A matrix suitable for cell growth or ingrowth, comprising the fusion peptide according to items 1 to 10, wherein the fusion peptide is covalently bound to the matrix.
(15) The matrix according to item 14, wherein the matrix is fibrin.
(16) The matrix according to item 14, wherein the matrix is between a first precursor molecule containing n nucleophilic groups and a second precursor molecule containing m electrophilic groups. A matrix formed by a Michael-type addition reaction, wherein n and m are at least 2 and the sum of n + m is at least 5.
(17) A matrix according to item 16, wherein the electrophilic group is conjugated to an unsaturated group, and the nucleophilic group is selected from the group consisting of thiol and amine.
(18) A matrix according to item 14, wherein the matrix contains polyethylene glycol.
(19) A method for producing a matrix, the method comprising:
Providing at least one matrix material capable of forming a cross-linked matrix, wherein the matrix material is selected from the group consisting of proteins and synthetic materials;
Adding the fusion peptide of items 1-10 to the matrix material; and
Cross-linking the matrix material so that the fusion peptide passes through a second domain
Including a step coupled to
Method.

FIG. 1 is a fluorescence detection chromatogram of plasmin-degrading peptide-containing fibrin gel and free peptide. The incorporated α ₂ PI _1-7 -ATIII _121-134 peptide (−), the same free peptide added to the degraded fibrin gel containing the incorporated peptide (...), and the free peptide alone (- ) Shows size exclusion chromatography of the degraded fibrin gel. The N-terminal leucine residue was dansylated (abbreviated dL). The free peptide elutes in a longer time than the peptide incorporated into the fibrin gel during clotting (which corresponds to a lower molecular weight), which means that it is covalently bound to the degraded fibrin and hence factor XIIIa Covalent incorporation by the action of activity is shown. FIG. 2 is a graph of the incorporation of dLNQEQVSPLRGD (SEQ ID NO: 1) into fibrin gels with added exogenous factor XIII. When 1 U / mL was added, the level of incorporation was increased so that above 25 mol peptide / mol fibrinogen could be achieved. FIG. 3 is a graph of the incorporation of the bidomain peptide dLNQEQVSPLRGD (SEQ ID NO: 1) into undiluted fibrin clew. Three separate kits were tested and in each case a high level of incorporation could be observed, reaching 25 mol peptide / mol fibrinogen. The concentration of exogenous peptide required for maximal incorporation was at least 5 mM, probably due to diffusion limitations within the dense fibrin matrix produced. This level of integration was very consistent and each kit provided a similar integration profile.

(Detailed description of the invention)
Products and methods using natural and synthetic matrices with releasably incorporated PTH therein for hard tissue repair, regeneration or reconstruction, particularly for bone growth, are disclosed herein. It is described in. 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 this matrix and retains its full bioactivity. PTH can be releasably incorporated using technology that provides control over how PTH is released, when PTH is released, and how much PTH is released. As a result, the matrix can be used directly or indirectly for tissue repair using the matrix as a controlled release vehicle.

(Definition)
“Biological material”, as generally used herein, evaluates, treats, enhances or replaces any tissue, organ or function of the body, depending on the material either permanently or temporarily. A material intended to interface with a biological system. The terms “biomaterial” and “
“Matrix” is used interchangeably herein and, depending on the nature of the matrix, can be swollen with water but does not dissolve in water (ie, specific support for traumatic or defective hard tissue) It means a crosslinked polymer network that forms a hydrogel that stays in the body for a specific period of time to function.

A “PTH fusion peptide” as generally used herein is at least a first
And a peptide comprising a second domain. One domain contains PTH (native form or truncated form, in particular PTH 1-34), and the other domain contains a substrate domain to be cross-linked to the 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 can donate an electron pair to a nucleophile in a polar bond formation reaction. Preferably, this 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 a carbon-carbon multiple bond, a carbon-heteroatom multiple bond or a heteroatom-heteroatom bond, or a functional group alternating with a single bond. To a macromolecule (eg, a synthetic polymer or protein). Such a bond can undergo an addition reaction.

“Conjugated unsaturated group”, as generally used herein, refers to a single bond and alternating carbon—
A molecule or region of a molecule or molecule having a carbon multiple bond, carbon-heteroatom multiple bond, or heteroatom-heteroatom bond, and having a multiple bond that can undergo an addition reaction. Examples of conjugated unsaturated groups include vinyl sulfone, acrylate, acrylamide, quinone and vinyl pyridinium (eg, 2- or 4-vinyl pyridinium),
As well as, but not limited to, itaconate.

“Synthetic precursor molecule” as used herein generally refers to a non-naturally occurring molecule.

“Naturally occurring precursor component or polymer” as generally used herein refers to a molecule that can be found in nature.

“Functionalizing” as commonly used herein refers to modifying a molecule in a manner that results in the attachment of a functional group or functional moiety. For example, a molecule can be functionalized by introduction of a molecule that renders the molecule a strong nucleophile or conjugated unsaturation. Preferably, the molecule (eg, PEG) is functionalized to be a thiol, amine, acrylate, or quinone. Proteins can also be efficiently functionalized, in particular by partial or complete reduction of disulfide bonds to generate free thiols.

“Functionality” as commonly used herein refers to the number of reactive sites on a molecule.

As used herein, “branch position functionality” refers to the number of arms extending from one position in a molecule.

An “adhesion site or cell binding site” as generally used herein refers to a molecule (
For example, it refers to a peptide sequence to which an adhesion promoting receptor on the surface of a cell binds. Examples of adhesion sites include fibronectin-derived RGD sequences and laminin-derived YIGSR (
SEQ ID NO: 2) include, but are not limited to, sequences. Preferably, the adhesion site is incorporated into the biomaterial by including a cross-linkable substrate domain in the matrix.

“Biological activity” as commonly used herein refers to a sensory event mediated by a protein of interest. In some embodiments, this includes facts that are assayed by measuring the interaction of one polypeptide with another polypeptide. It also protects the protein of interest from cell proliferation, cell differentiation, cell death, cell migration, cell adhesion, cell interaction with other proteins, enzyme activity, protein phosphorylation or protein dephosphorylation, transcription or translation. It includes assaying the effect of having.

A “susceptible biological molecule” as commonly used herein refers to a molecule that is found in or in the cell or can be used as a therapeutic agent for the cell or body, The therapeutic agent 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 material without adversely affecting the sensitive biological material.

“Regeneration”, as generally used herein, means that some or all of something like hard tissue (especially bone) grows back and comes back to “multifunctional” As generally used herein is one electrophilic and / or nucleophilic functional group per molecule (ie, monomer, oligomer and polymer).
More than one.

“Self-selective reaction”, as generally used herein, is a composition whose first precursor component is more than the other compound present in the mixture or in its reaction site. The second of things
It reacts faster with the precursor components of (and vice versa). As used herein, nucleophilic groups bind preferentially to electrophiles, and electrophiles bind preferentially to strong nucleophilic groups over other biological compounds. To do.

“Crosslinking” as used herein generally means the formation of a covalent bond. However, mention may also be made of the formation of non-covalent bonds (eg ionic bonds) or a combination of covalent and non-covalent bonds.

“Polymer network”, as generally used herein, refers to the fact that substantially all of the monomers, oligomers or polymers are linked by intermolecular covalent bonds through their available functional groups. Means the product of a process that yields two large molecules.

“Physiological”, as used herein, means conditions that can be found in living vertebrates. In particular, physiological conditions refer to conditions in the human body such as temperature, pH and the like. The physiological temperature is in the temperature range between 35 ° C. and 42 ° C., preferably 37 ° C.
Means around.

“Crosslink density”, as generally used herein, refers to the average molecular weight (M _c ) between two crosslinks of each molecule.

“Equivalent”, as generally used herein, is the mm of functional groups per gram of material.
ol.

“Swelling” as used herein refers to an increase in volume and mass due to water uptake by a biomaterial. The terms “water uptake” and “swell” are used synonymously throughout the application.

“Equilibrium state”, as generally used herein, is a state in which a hydrogel does not cause an increase in mass or loss when stored under steady state in water.

(I. Matrix and PTH)
(A. Matrix material)
The matrix may be ionically cross-linked to the polymer network, covalently cross-linked, or a combination thereof, or one or more polymeric materials (
That is, it is formed by swelling the matrix) to form a polymer network with sufficient interpolymer spacing to allow cell ingrowth or migration into the matrix.

In one embodiment, the matrix is formed from a protein, preferably a protein that is naturally present in the patient into which the matrix is implanted. A particularly preferred matrix protein is fibrin, although matrices made from other proteins such as collagen and gelatin can also be used. Polysaccharides and glycoproteins can also be used to form the matrix. It is also possible to use synthetic polymers which can be crosslinked by ionic or covalent bonds.

(Fibrin matrix)
Fibrin is a natural material that has been reported for several biological applications. Fibrin is described as a material for a cell ingrowth matrix in US Pat. No. 6,331,422 to Hubbell et al. Fibrin gels are used as sealants due to their ability to bind to many tissues and their natural role in wound healing. Some specific applications include vascular graft attachment, heart valve attachment, bone placement in fractures, and use as a sealant for tendon repair (Sierrr).
a, D. H. , Journal of Biomaterials Applicati
ons, 7: 309-352 (1993)). In addition, these gels have been used as drug delivery devices and for nerve regeneration (Williams et al., Journal).
al of Comparative Neurobiology, 264: 284-2.
90 (1987)). Fibrin provides a solid support for tissue regeneration and cell ingrowth, but there are few active sequences in monomers that directly enhance these processes.

The process by which fibrinogen polymerizes into fibrin has also been characterized. First, a protease cleaves the dimeric fibrinogen molecule into two symmetrical sites. There are several possible proteases that can cleave fibrinogen (including thrombin, reptilase, and protease III), and each cleaves this protein at a different site (Francis et al., Blood Cells, 19: 291-). 307,
1993). Once fibrinogen is cleaved, a self-polymerization step occurs, in which the fibrinogen monomers come together and form a non-covalently crosslinked polymer gel (Sierra, 1993). This self-assembly occurs because the binding site is exposed after protease cleavage occurs. Once these binding sites are exposed, these binding sites in the center of the molecule can bind to other sites on the fibrinogen chain, which are at the end of the peptide chain (Stryer, L In Biochemistry
y, W. H. Freeman & Company, NY, 1975). In this manner, a polymer network is formed. Factor XIIIa (transglutaminase activated from factor XIII by thrombin proteolysis) can then be covalently crosslinked to this polymer network. Other transglutaminases exist and may be involved in covalent cross-linking and grafting to the fibrin network as well.

Once the crosslinked fibrin gel is formed, subsequent degradation is tightly controlled. One of the major molecules in controlling fibrin degradation is an α2-plasmin inhibitor (Aoki, N., Progress in Cardiovascular Dis
ease, 21: 267-286, 1979). This molecule acts by cross-linking to the α chain of fibrin via the action of Factor XIIIa (Sakata et al., Jour.
nal of Clinical Investigation, 65: 290-297.
, 1980). By attaching itself to the gel, a high concentration of inhibitor can be localized to the gel. This inhibitor then prevents binding of plasminogen to fibrin (Aoki et al., Thrombosis and Haemostasis,
39: 22-31, 1978), and inactivating plasmin (Aoki, 19
79). The α2-plasmin inhibitor contains a glutamine substrate. The exact sequence has been identified as NQEQVSPL (SEQ ID NO: 12), with the first glutamine being the active amino acid for crosslinking.

It has been demonstrated that a two-domain peptide, which includes a factor XIIIa substrate sequence and a bioactive peptide sequence, can crosslink to a fibrin gel and that the bioactive peptide retains its cellular activity in vitro. (Schense, JC et al. (19
99) Bioconj. Chem. 10: 75-81).

(Synthetic matrix)
Crosslinking reactions to form a synthetic matrix for body application include the following: (i) free radical polymerization (eg, Hern) between two or more precursors containing unsaturated double bonds Et al., J. Biomed. Mater. Res. 39: 266-276 (19
98)), (ii) nucleophilic substitution reactions (US Pat. No. 5,874 to Rhee et al., For example, between a precursor containing an amine group and a precursor containing a succinimidyl group).
(Iii) Condensation and addition reactions, and (iv) Michael type addition reactions between strong nucleophiles and conjugated unsaturated groups or bonds (as strong electrophiles). Particularly preferred is a reaction between a precursor molecule having a thiol group or an amine group as a nucleophilic group and a precursor molecule containing an acrylate group or a vinyl sulfone group as an electrophilic group. Most preferred as the nucleophilic group is a thiol group. Mich
The ael type addition reaction is described in WO 00/44808 to Hubbell et al., the contents of which are incorporated herein by reference. Michael-type addition reactions allow in situ crosslinking of at least first and second precursor components in a self-selective manner under physiological conditions, even in the presence of sensitive biological materials To. 1 of the precursor components
When one has at least two functional groups and at least one of the other precursors has more than two functional groups, the system reacts self-selectively to produce a crosslinked three-dimensional biological material. Form.

Preferably, the conjugated unsaturated group or conjugated unsaturated bond is an acrylate, vinyl sulfone,
Methacrylate, acrylamide, methacrylamide, acrylonitrile, vinyl sulfone, 2-vinylpyridinium or 4-vinylpyridinium, maleimide, or quinone.

The nucleophilic group is preferably a thiol group, an amino group, or a hydroxyl group. Thiol groups are substantially more reactive than unprotonated amine groups. As mentioned earlier, pH is important in this regard: Deprotonated thiols are substantially more reactive than protonated thiols. Thus, addition reactions with thiols, including conjugated unsaturation (eg acrylates or quinones), to convert the two precursor components into a matrix are often the most rapid and self-selective at a pH of about 8. In practice. At a pH of about 8, the majority of the desired thiol is deprotonated (thus more reactive) and most of the desired amine remains protonated (and therefore less reactive). is there. When thiols are used as the first precursor molecule, conjugated structures that are selective for the reactivity of the thiol towards the amine are highly 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- Vinylpyrrolidone), poly (maleic acid), poly (ethylene-co-acrylic acid), poly (ethyloxazoline), poly (vinylpyrrolidone), poly (ethylene-co-maleic acid), poly (acrylamide), and poly (acrylamide)
Ethylene oxide) -co-poly (propylene oxide) block copolymers. A particularly preferred precursor molecule is polyethylene glycol.

Polyethylene glycol (PEG) provides a convenient building block. Linear (ie, having two ends) or branched (ie, having more than two ends) PE
G can be easily purchased or synthesized, then the end groups of the PEG are functionalized to introduce either strong nucleophiles (eg thiols) or conjugated structures (eg acrylates or vinyl sulfones). obtain. When these components are mixed in a slightly basic environment, either with each other or with the corresponding components, the matrix is between the first precursor component and the second precursor component. It is formed by the reaction of The PEG component can react with the non-PEG component, and the molecular weight or hydrophilicity of either component depends on the mechanical properties of the resulting biological material,
It can be controlled to manipulate the permeability and water content.

These materials are generally useful in medical implants, as described in further detail below. In forming a matrix, particularly a matrix that is desired to degrade in vivo, the peptides provide a very convenient building block. It is simple to synthesize a peptide containing two or more cysteine residues, and this component can then easily serve as the first precursor component with a nucleophilic group. For example, a peptide having two free cysteine residues can be PEG trivinylsulfone (PEG with three arms each having a vinylsulfone on its arm) and a physiological pH or slightly higher pH (eg, 8 When mixed in ~ 9), a matrix is easily formed. Gelation can also proceed well at higher pH, but potentially at the expense of self-selectivity. When the two liquid precursor components are mixed together, they react in a few minutes to form an elastic gel, which contains a network node with peptides as connecting links, P
It consists of a network of EG chains. The peptide can be selected as a protease substrate to allow the network to be infiltrated or degraded by the cell (as is done in protein-based networks (eg, fibrin matrix)).
Preferably, the sequence in the domain is a substrate for an enzyme involved in cell migration (eg, a substrate for an enzyme such as collagenase, plasmin, metalloproteinase (MMP) or elastase), but suitable domains are those It is not limited to the arrangement of One particularly useful sequence is a substrate for the enzyme plasmin (see examples). The degradability of the gel can be manipulated by changing the details of the peptide that acts as a cross-linking node. Gels can be made that are degradable by collagenase but not degradable by plasmin, or gels degradable by plasmin but not degradable by collagenase. Furthermore, gel degradation can be made faster or slower in response to such enzymes by simply changing the amino acid sequence so as to change the K _m or k _cat or both of the enzymatic reaction. Is possible. Thus, biological material can be made that mimics the organism in that it can be remodeled by the normal remodeling characteristics of the cells. For example,
Such studies indicate a substrate site for the important protease plasmin. Gelation of PEG with peptides is self-selective.

If desired, biofunctional agents can be incorporated into the matrix to provide chemical bonds to other species (eg, tissue surfaces). Incorporating a protease substrate into the matrix
This is important when the matrix is formed from PEG vinyl sulfone. Other than the matrix formed from the reaction of PEG acrylate and PEG thiol, the matrix formed from PEG vinyl sulfone and PEG thiol does not contain hydrolytically degradable linkages. Thus, incorporation of a protease substrate allows this matrix to be degraded in the body.

The composite matrix is simple to form. Two liquid precursors are mixed; one precursor contains a precursor molecule with a nucleophilic group and the other precursor molecule contains an electrophilic group. Physiological saline can serve as a solvent. Minimal heat is generated by the reaction. Thus, gelation can be performed in vivo or in vitro, with direct contact with tissue and without inconvenient toxicity. Therefore, telechelic (telechelic
all) PE, either modified or modified at these side groups
Polymers other than G can be used.

For most healing indications, the rate of cell ingrowth or migration of cells into the matrix is critical to the overall healing response, combined with the adapted degradation rate of the matrix. The possibility that a matrix that is not hydrolytically degradable is invaded by cells is mainly a function of the network density. The space that exists between branch points or nodes is too small for the cell size, or the decomposition of the matrix (which results in the creation of more space within the matrix) is too slow In some cases, a very limited healing response is observed. Healing matrix found in nature (eg fibrin matrix,
It is known that (formed in response to damage in the body) consists of a very loose network that can be very easily invaded by cells. Invasion is facilitated by ligands for cell adhesion, which are integrated as part of the fibrin network.

A matrix made from synthetic hydrophilic precursor molecules swells in an aqueous environment after formation of a polymer network, such as polyethylene glycol. A sufficiently short gel time (for between 3 and 10 minutes at a pH between 7 and 8 and a temperature in the range of 36 to 38 ° C.) and a quantitative reaction is achieved during in situ formation of the matrix in the body. In order to do this, the starting concentration of precursor molecules must be sufficiently high. Under such conditions, swelling after network formation does not occur and the required starting concentration results in a matrix that is too dense for cell infiltration if the matrix is not degradable in an aqueous environment. Therefore, it is important that the swelling of the polymer network expands and widens the space between branch points.

Regardless of the starting concentration of the precursor molecule, hydrogels made from the same synthetic precursor molecule (eg, a peptide having 4-arm PEG vinyl sulfone and SH groups) swell to the same water content at equilibrium. This means that the higher the starting concentration of precursor molecules,
It means that the terminal volume of the hydrogel becomes higher when reaching the equilibrium state. Rate of cell invasion and healing if the space available in the body is too small to allow sufficient swelling, especially when the bonds formed from the precursor components are not hydrolytically degradable Response is slow. As a result, an optimal condition between two contradictory requirements for application in the body must be found. Good cell invasion and subsequent healing response can be achieved by using a trifunctional branched polymer and a second precursor molecule of at least 3 arms of substantially similar molecular weight (
This was observed using a three-dimensional polymer network formed from the reaction with (which is at least a bifunctional molecule). The functional group equivalent ratio of the first precursor molecule to the second precursor molecule is between 0.9 and 1.1. The molecular weight of the arm of the first precursor molecule, the molecular weight of the second precursor molecule, and the functional group at the branch point are such that the water content of the resulting polymer network is complete at the equilibrium weight percent and water uptake. It is chosen to be between 92% by weight of the total weight of the subsequent polymer network. Preferably, the water content is between 93% and 95% by weight of the total weight of the polymer network and water after water uptake. Completion of water uptake
It can be achieved either when an equilibrium concentration is reached or when the space available in the biological material does not allow further volume increase. Therefore, it is preferable to select the starting concentration of the precursor component as low as possible. This is true for all swellable matrices, especially for matrices that are subject to cell-mediated degradation and do not contain hydrolytically degradable bonds in the polymer network.

In particular, for gels that are not hydrolytically degradable, the balance between gelation time and low starting concentration should be optimized based on the structure of the precursor molecules. In particular, the molecular weight of the arm of the first precursor molecule, the molecular weight of the second precursor molecule, and the degree of branching (ie, branch point functionality) must be adjusted accordingly. The actual reaction mechanism has little effect on this interaction.

The first precursor molecule is a three-arm or four-arm polymer having a functional group at the end of each arm, and the second precursor molecule is a linear bifunctional molecule, preferably at least 2 In the case of a peptide containing two cysteine groups, the molecular weight of the arm of the first precursor molecule and the molecular weight of the second precursor molecule are preferably such that after the formation of the network, the bond between the branch points is It is selected to have a molecular weight in the range of 10-13 kD (preferably under linear and unbranched bonds), preferably between 11 kD and 12 kD. This means that the total starting concentration of the first precursor molecule and the second precursor molecule is between 8 and 8 of the total weight of the first and second precursor molecules in the solution (prior to network formation). 12% by weight, preferably 9
It is possible to be in the range of 10% by weight and 10% by weight. When the degree of branching of the first precursor component is increased to 8 and the second precursor molecule is still a linear bifunctional molecule, the molecular weight of the bond between the branch points is preferably Increased to a molecular weight between 18-24 kDa. If the degree of branching of the second precursor molecule is increased from linear to a three-arm or four-arm precursor component, the molecular weight (ie, the length of the bond) increases accordingly. In a preferred embodiment of the present invention, a trifunctional three-arm 15 kD polymer (
Each arm has a molecular weight of 5 kD), and 0.5 as the second precursor molecule
Including bifunctional linear molecules with a molecular weight in the range of ~ 1.5 kD, even more preferably 1 kD,
A composition is selected. Preferably, the first and second precursor components are polyethylene glycol.

In preferred embodiments, the first precursor component comprises as functional group a conjugated unsaturated group or bond, most preferably acrylate or vinyl sulfone, and the functional group of the second precursor molecule is a nucleophilic group. (Preferably, a thiol group or an amino group). In another preferred embodiment of the invention, the first precursor molecule has a functional group at the end of each arm,
A 4-arm 20 kD polymer (each arm has a molecular weight of 5 kDa) and the second precursor molecule is a bifunctional, in the range of 1-3 kD, preferably between 1.5 kD and 2 kD It is a linear molecule. Preferably, the first precursor molecule is a polyethylene glycol having a vinyl sulfone group, and the second precursor molecule is a peptide having a cysteine group. In both preferred embodiments, the total starting concentration of the first precursor molecule and the second precursor molecule is the first and second precursor molecules (before polymer network formation) and water. In order to achieve a gel time of less than 10 minutes, preferably in the range of 8-11% by weight, preferably between 9% and 10% by weight
And 8% by weight. These compositions are at pH 8.0 and 37 ° C., after mixing, about 3 to
Has a gel time of 10 minutes.

If the matrix contains hydrolytically degradable bonds, the density of the network formed (eg due to a favorable reaction between acrylate and thiol) is particularly important early on for cell infiltration, In an aqueous environment, bonds are hydrolyzed and the network loosens, allowing cell infiltration. As the degree of overall branching of the polymer network increases, the molecular weight of the interconnect (ie, the length of the bond) must increase.

(B. Cell attachment site)
Cells interact with their environment at the cell surface via protein-protein interactions, protein-oligosaccharide interactions, and protein-polysaccharide interactions. Extracellular matrix proteins provide a host bioactivity signal to the cell. This dense network is required to support cells, and many proteins in the matrix have been shown to control cell adhesion, diffusion, migration and differentiation (Carey,
Annual Review of Physiology, 53: 161-177,1
991). 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 Neuro
logic Science, 12: 189-195, 1989). Numerous studies of laminin have been carried out, and laminin has been shown to be in vivo in neuronal and in vitro neuronal development and regeneration (Williams, Neurochemical Res.
arch, 12: 851-869, 1987), as well as angiogenesis has been shown to play an important role.

Several specific sequences have been identified that interact directly with cellular receptors and cause either adhesion, diffusion or signaling.

Laminin (large multidomain protein) (Martin, Annual Review)
w of Cellular Biology, 3: 57-85, 1987) has been shown to consist of three chains with several receptor binding domains. These receptor binding domains include the YIGSR (SEQ ID NO: 2) sequence of the laminin B1 chain (Graf
Et al., Cell, 48: 989-996, 1987; Kleinman et al., Archive.
s of Biochemistry and Biophysics, 272: 39-
45, 1989; and Massia et al., J. MoI. of Biol. Chem. 268: 8
053-8059, 1993), LRGDN of laminin A chain (SEQ ID NO: 3) (Ignat
ius et al. of Cell Biology, 111: 709-720, 1990)
As well as the PDGSR of laminin B1 chain (SEQ ID NO: 4) (Kleinman et al., 1989).
Is mentioned. Several other recognition sequences for cells have also been identified. These include laminin A chain IKVAV (SEQ ID NO: 5) (Tashiro et al., J. of B
iol. Chem. , 264: 16174-16182, 1989) and laminin B2.
Chain sequence RNIAEIIKDI (SEQ ID NO: 6) (Liesi et al., FEBS Lette
rs, 244: 141-148, 1989). Receptors that bind to these specific sequences have also often been identified. A subset of cellular receptors that have been shown to be responsible for the majority of this binding is the integrin superfamily (Rou).
slahti, E .; , J .; of Clin. Investigation, 87: 1-5
1991). Integrins are protein heterodimers composed of an α subunit and a β subunit. Previous studies have shown that the tripeptide RGD binds to several β1 and β3 integrins (Hynes, RO, Cell, 6
9: 1-25, 1992; Yamada, K .; M.M. , J .; of Biol. Chem. ,
266: 12809-12812, 1991), IKVAV (SEQ ID NO: 5) is 110 kD
a binding to the receptor (Tashiro et al., J. of Biol. Chem., 2
64: 16174-16182, 1989); Luckenbill-Edds et al., Ce
ll Tissue Research, 279: 371-377, 1995), YIG
SR (SEQ ID NO: 2) binds to the 67 kDa receptor (Graf et al., 1987) and DGEA (SEQ ID NO: 7) (collagen sequence) binds to α ₂ , β ₁ integrin (Zutter and Santaro, Amer. of Pathology, 1
37: 113-120, 1990). The receptor for the RNIAEIIKDI (SEQ ID NO: 6) sequence has not been reported.

In a further preferred embodiment, peptide sites for cell adhesion are incorporated into the matrix. That is, a peptide that binds to an adhesion promoting receptor on the cell surface is incorporated into the biological material of the present invention. Such adhesion promoting peptides can be selected from the group as described above. Particularly preferred are fibronectin-derived RGD sequences, laminin-derived Y
IGSR (SEQ ID NO: 2) sequence. Incorporation of cell attachment sites is a particularly preferred embodiment using a synthetic matrix, but can also be included in some of the natural matrices.
Incorporation, for example, simply mixes a cysteine-containing cell attachment peptide with a precursor molecule containing a conjugated unsaturated group (eg, PEG acrylate, PEG acrylamide, or PEG vinyl sulfone), and after a few minutes, a nucleophilic group ( For example, it can be done by mixing with the remainder of the precursor component comprising a thiol-containing precursor component). If the cell attachment site does not contain cysteine, this site can be chemically synthesized to contain cysteine. During this first step, the adhesion-promoting peptide is incorporated at one end of the conjugated unsaturated, multi-functionalized precursor; a cross-linked network when the remaining multiple thiols are added to the system Is formed. Another important implication of the manner in which the network is prepared here is the efficiency of incorporation of pendant bioactive ligands (eg, adhesion signals). This process is quantitative. For example, unbound ligands (eg, adhesion sites) can inhibit cellular interactions with the matrix. As described below, derivatization of precursors with such pendant oligopeptides is a first step with a large stoichiometric excess (minimum 40 times) of multi-arm electrophiles relative to thiols. It is carried out with the precursor and is therefore quite quantitative.

Aside from preventing unwanted inhibition, this achievement is even more important biologically: cell behavior is extremely sensitive to small changes in ligand density, and the preciseness of the incorporated ligand Knowledge helps to design and understand cell-matrix interactions. In summary, the concentration of adhesion sites covalently bound within the matrix significantly affects the rate of cell infiltration. For example, for a given hydrogel, the RGD concentration range can be incorporated into a matrix while supporting cell ingrowth and cell migration in an optimal manner. RGD
The optimal concentration range for adhesion sites such as is between 0.04 mM and 0.05 mM, and even more preferably water between the equilibrium concentration and 92% by weight, especially after the end of water uptake. For a matrix with content, it is 0.05 mM.

Excellent bone healing results can be achieved by maintaining high cell migration rates and matrix degradation rates. For the matrix design (especially covalently bound PTH1-34), it was cross-linked with the protease degradation site GCRPQGIWGQDRC (SEQ ID NO: 8),
4-arm polyethylene glycol having a molecular weight of about 20,000 Da, and 0.05
0 mM GRGDSP (SEQ ID NO: 9) gives particularly good cell ingrowth results and healing of bone defects. The starting concentration of PEG and peptide is 1% of the total weight of the molecule (before swelling) and water.
Less than 0% by weight. This gel has consistency available and allows osteoblasts and progenitor cells to easily infiltrate the matrix.

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

(C. Crosslinkable substrate domain)
The PTH fusion peptide can be cross-linked and covalently bound to the matrix through the cross-linkable substrate domain of the PTH fusion peptide. The type of substrate domain depends on the nature of the matrix. For incorporation into the fibrin matrix, the transglutaminase substrate domain is particularly preferred. The transglutaminase substrate domain can be a factor XIIIa substrate domain. This factor XIIIa substrate domain is GKDV (SEQ ID NO: 10),
It may include KKKK (SEQ ID NO: 11), or NQEQVSPL (SEQ ID NO: 12). PT
Coupling between H and the transglutaminase substrate domain can be performed by chemical synthesis.

The transglutaminase substrate domain can be a substrate for transglutaminase other than Factor XIIIa. The most preferred Factor XIIIa substrate domain is the amino acid sequence N
QEQVSPL (SEQ ID NO: 12) (referred to herein as “TG”). Other proteins that recognize transglutaminase (eg, fibronectin) can be coupled to a transglutaminase substrate peptide.

  (Table 1: Trusglutaminase substrate domain)

For incorporation of PTH into a matrix formed from a synthetic precursor component, the PTH fusion peptide or any other peptide to be incorporated, together with at least one additional cysteine group (-SH) as a crosslinkable substrate domain (preferably PTH Must be synthesized at the N-terminus. Cysteine can be directly attached to PTH or through a linker sequence. The linker sequence may further comprise an enzymatically degradable amino acid sequence so that the PTH can be cleaved from the matrix by the enzyme in substantially 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 , binding to the synthesis matrix for PTH _1-34 is made possible by attaching 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 in the synthetic polymer to form a covalent bond. Probably (
a) Only cysteine is attached to the peptide and (b) an enzymatically degradable, in particular plasmin degradable sequence, is bound as a linker between the cysteine and the peptide. The sequence GYKNR (SEQ ID NO: 15) between the first and second domains, cysteine makes the linkage plasmin degradable.

Thus, the PTH fusion peptide further includes a degradable site between the adhesion site, ie, the second domain (ie, factor Xllla substrate domain or cysteine) and PTH, ie, the first domain. Can be modified. These sites can be degradable by non-specific hydrolysis (ie, ester linkages) or they can become substrates for specific enzymatic (proteolytic or polysaccharide degradation) degradation. Can be. These degradable sites allow for the manipulation of more specific release of PTH from a matrix such as fibrin gel. For example, degradation based on enzyme activity results in release of PTH,
Allows to be controlled by cellular processes, not by diffusion of factors through the gel. Degradable or binding sites are cleaved by enzymes released from cells that enter the matrix.

Depending on the degradable site, PTH can be released with little or no modification to the first peptide sequence, resulting in higher activity of the factor. Furthermore, the degradation site is
Factor release makes it possible to be controlled by cell-specific processes such as localized proteolysis rather than diffusion from some porous materials. This causes 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 required since its release is controlled by the cellular process. P
Preservation of TH and its bioavailability is achieved in the use of diffusion controlled release devices.
Another advantage for taking advantage of cell-specific proteolytic activity. In one possible explanation for powerful healing of bone defects using PTH covalently bound to the matrix, PTH is localized over a long period of time (ie not a single pulse dose), but in a sustained manner. Is considered important. This is accomplished by gradual degradation by enzymatic or hydrolytic cleavage of the matrix. In this way, the molecule is then delivered through a spurious pulse effect that occurs over the duration. When ancestral cells infiltrate the matrix, they can encounter PTH molecules and differentiate into preosteoblasts. However,
If the particular cell does not continue to release bound PTH from the matrix, the cell is effectively converted to osteoblasts and begins to produce a bone matrix. Eventually, the therapeutic effect of the peptide is localized in the defect region and is consequently magnified.

There are a large number of enzymes that can be used for proteolytic degradation. The proteolytic site can include a substrate for collagenase, plasmin, elastase, stromelysin, or plasminogen activator. Exemplary substrates are shown in the table below. P1 to P5 represent the 1st to 5th amino acids from the site where proteolysis occurs toward the amino terminus of the protein. P1 ′ to P4 ′ are
From amino acid position 1 toward the carboxy terminus of the protein from the site where proteolysis occurs
Represents 4th place.

  (Table 2: Sample substrate sequence for proteases)

In another preferred embodiment, the oligo-ester domain can be inserted between the first domain and the second domain. This is accomplished using an oligo-ester such as an oligomer of lactic acid.

A non-enzymatic degradation substrate can consist of any linkage that undergoes hydrolysis by an acid or base catalytic mechanism. These substrates may include oligo-esters such as oligomers of lactic acid or glycolic acid. The degradation rate of these materials can be controlled by the choice of oligomer.

(D.PTH)
As described herein, the term “PTH” refers to PTH 1-84 as well as all the shortenings of PTH, indicating the characteristics of bone formation when covalently bound to a biologically degradable natural or synthetic matrix. Modified, modified and allelic versions of human sequences. Preferred shortened versions of PTH are PTH1-38, PT
H1 to 34, PTH1 to 31 or PTH1 to 25. Most preferably, PHH1-
34. Preferably, PTH from other sources, such as bovine PTH, where PTH is human PTH may be suitable.

(Methods for uptake and / or release of bioactive factors)
In one preferred embodiment relating to incorporation of PTH into the matrix, the matrix comprises fibrin formed from fibrinogen, a calcium source and thrombin, and the PTH fusion peptide is incorporated into fibrin during clotting. A PTH fusion peptide is designed as a fusion protein comprising two domains, a first domain and a second domain. One domain, the second domain, is a substrate for an enzyme such as Factor XIIIa. Factor XIIIa is a transglutaminase that is activated during clotting. This enzyme is naturally formed from factor XLLLa by cleavage with thrombin and has a function of binding to each other through a fibrin chain through an amide bond formed between a glutamine side chain and a lysine side chain. The enzyme also functions to bind other peptides to fibrin (eg, a cell binding site) during clotting, provided that this cell binding site also includes Factor XIIIa.

In particular, the sequence NQEQVSP (SEQ ID NO: 16) has been shown to function as an effective substrate for Factor VIIIa. As described herein, it includes a degradation site before the sequence directly binds to PTH or between the PTH (first domain) and the NQEQVSP (SEQ ID NO: 16) sequence (second domain). obtain. Thus, the PTH fusion peptide has the number XI.
It can be incorporated into fibrin during clotting through factor IIa substrate.

(Design of fusion protein for uptake)
A PTH fusion peptide comprising a first domain comprising PTH, a second domain comprising a substrate domain for a bridging enzyme, and optionally a degradation site between the first and second domains uses several different schemes. Can be incorporated into the fibrin gel. Preferably, the second domain comprises a transglutaminase substrate domain, and even more preferably, the second domain comprises a factor XIIIa substrate domain. Most preferably, XIIIa
The factor substrate domain includes NQEQVSP (SEQ ID NO: 16). If the PTH fusion peptide is present during fibrinogen polymerization (ie, during the formation of a fibrin matrix), the fusion peptide is incorporated directly into the matrix.

The degradation site between the first and second domains of the PTH fusion peptide can be an enzymatic degradation site as described above. Preferably, the degradation site is cleavable by an enzyme selected from the group consisting of plasmin and matrix melaroproteinase.
By careful selection of K _m and K _cat for the enzymatic degradation site, either before or after the protein matrix and by utilizing the analogous enzymes or different enzyme for degrading / or matrix degradation Can be controlled to occur, and the arrangement of degradation sites is altered for each type of protein and application. This PTH fusion peptide can be cross-linked directly into the fibrin matrix described above. However, incorporating an enzymatic degradation site alters the release of PTH during proteolysis. When cell-derived proteins reach sequestered fusion proteins, they can cleave the engineered protein at the newly formed degradation site. The resulting degradation product contains released PTH, which is now free of any engineered fusion sequence and any degraded fibrin.

(II. How to use)
The matrix may be used prior to or at the time of transplantation for tissue repair, regeneration, or reconstruction and / or for release of PTH. In some cases it is desirable to induce cross-linking at the site of administration in order to adapt the matrix to the tissue at the site of implantation. In other cases, it is convenient to prepare the matrix prior to implantation.

The cells can also be added to the matrix either before or at the time of implantation, or even after implantation, either at the time of crosslinking of the polymer to form the matrix or after crosslinking. This can be in addition to or in place of cross-linking the matrix to create a void space designed to promote cell growth or in-growth.

In most cases, cell growth or proliferation (prolifera)
Although it is desirable to implant a matrix to promote the (ion), in some cases bioactive factors are used to suppress the rate of cell growth. A particular application is to suppress the formation of adhesions after surgery.

(III. Method of application)
In preferred embodiments, the material is gelled in situ or in or on the body. In another embodiment, the matrix can be formed outside the body,
It is then applied in a pre-formed shape. The matrix material can be made from a synthetic or natural precursor composition. Regardless of the type of precursor composition used, the precursor component is:
They should be separated prior to application of the mixture to the body and prevented from mixing or contacting each other under conditions that allow the components to polymerize or gel. To avoid contact prior to administration, kits that separate the compositions from each other can be used. Upon mixing under conditions that allow polymerization, the composition forms a three-dimensional network supplemented with bioactive factors. Depending on the precursor compositions and their concentrations, gelation can occur semi-instantaneously after mixing. Such rapid gelation makes injecting (ie pushing the gelled material through the needle) almost impossible.

In one embodiment, the matrix is formed from fibrinogen. Fibrinogen reacts with the gel through various cascades to form a matrix when contacted with thrombin and calcium sources 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 separated, the other two components can be mixed prior to administration.

In a first embodiment, fibrinogen is dissolved in a buffered solution at physiological pH (pH 6.5-8.0, preferably in the range of pH 7.0-7.5), which increases stability. May further comprise aprotinin, and calcium chloride buffer (eg,
Stored separately from the thrombin solution in the 40-50 mM concentration range). The buffer solution for fibrinogen can be a histidine buffer solution, with a preferred concentration of 50 mM, further comprising NaCl (at a preferred concentration of 150 mM) or TRIS buffered saline (preferably at a concentration of 33 mM). .

In a preferred embodiment, a kit comprising a fusion protein, fibrinogen, thrombin and a calcium source is provided. If necessary, this kit can contain cross-linking enzymes (
For example, Factor XIIIa) may be provided. The fusion protein includes a bioactive factor, a substrate domain for a cross-linking enzyme, and a degradation site between the substrate domain and the bioactive factor. The fusion protein can be present in either fibrinogen solution or thrombin solution. In a preferred embodiment, the fibrinogen solution contains this fusion protein.

These solutions are preferably mixed by a two-way syringe device, where mixing is performed by mixing the contents of both syringes via a mixing chamber and / or a needle and / or a static mixer. It is caused by pushing.

In a preferred embodiment, both fibrinogen and thrombin are stored separately in lyophilized form. Either of these two can include a fusion protein. Prior to use, Tris buffer or histidine buffer is added to fibrinogen,
Aprotinin may further be included. Lyophilized thrombin can be dissolved in a calcium chloride solution. Subsequently, the fibrinogen solution and the thrombin solution are placed in separate containers / vials / syringe bodies and mixed by a two-way connection device (eg, a two-way syringe). Optionally, the container / vial / syringe body has two chambers that are separated by two and are therefore separated by an adjustable compartment perpendicular to the syringe body wall.
One of these chambers contains lyophilized fibrinogen or thrombin, while the other chamber contains a suitable buffer solution. When the plunger is depressed, this compartment moves and releases the buffer into the fibrinogen chamber to dissolve the fibrinogen. Once both fibrinogen and thrombin are dissolved, both 2-minute syringe bodies are attached to the two-way connection device and the contents are mixed by pushing the contents through the injection needle attached to the connection device Is done. If necessary, the connecting device comprises a static mixer to improve the mixing of the contents.

In a preferred embodiment, prior to mixing, fibrinogen is diluted 8 times and thrombin is diluted 20 times. This ratio results in a gel time of about 1 minute.

In another preferred embodiment, the matrix is formed from synthetic precursor components that can undergo a Michael addition reaction. Nucleophilic precursor component (multithiol (multithio
l)) only at a basic pH with a multiacceptor component (
The three components that should be stored separately prior to mixing are the following: base, nucleophilic component, and multi-acceptor component. Both the multi-acceptor component and the multi-thiol component are stored as a solution in buffer. Both ingredients are
It may contain cell adhesion sites and further bioactive molecules. Thus, the first composition of this system can include, for example, a solution of a nucleophilic component, and the second composition of this system can include a solution of a multi-acceptor component. Either or both of these two components can include a base. In another embodiment, the multi-acceptor and multi-thiol can be included as a solution in the first composition, or the second composition can include a base. Connection and mixing occur in the same manner as previously described for fibrinogen. A bipartite syringe body is equally suitable for a synthetic precursor component. Instead of fibrinogen and thrombin, the multi-acceptor component and the multi-thiol component are stored in ground form in one of the chambers, and the other chamber contains a basic buffer.

The following examples are included to demonstrate preferred embodiments of the invention. Although the compositions and methods have been described with reference to preferred embodiments, the compositions and methods described herein, and described herein, without departing from the concept, spirit, and scope of the present invention. It will be apparent to those skilled in the art that variations can be applied to a method step or a series of steps.

(Example 1: Matrix containing covalently bound TGPTH)
(Synthesis of TGPTH)
PTH1-34 mer peptides that exhibit similar activity to the complete protein, and proteins of this length, can be synthesized by standard solid phase peptide synthesis methods.

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

(In vivo results)
The activity of TGPTH to enhance bone regeneration was tested in a sheep drill hole defect in a TISSUCOL® matrix. 12 mm deep 8 mm holes were made in the proximal and distal thighs and upper arms of the sheep. These holes were filled with in situ polymerized fibrin gel. Defects were left empty and filled with TISSUCOL®, or TGPTH was added to TISSUCOL® fibrin at 400 μg / mL prior to polymerization. TISS
In each instance where UCOL® was used, it was diluted 4-fold from the standard available concentration to a fibrinogen concentration of 12.5 mg / mL.

This defect could heal in 8 weeks. After this healing period, the animals were sacrificed and bone samples were removed and analyzed by micro computer topography (μCT). The percentage of defect volume filled with calcified bone tissue was then determined. When the defect remained empty, no calcified tissue formed within the fibrin matrix. Similarly, when only fibrin gel was added, bone was not substantially healed. However, 400 μg / mL TGP
When TH was added, 35% of the defect was filled with calcified bone and the healing level increased dramatically.

Example 2: Healing response with modified PTH 1-34 bound to fibrin matrix
(material)
A modified version of PTH _1-34 that could be incorporated into the fibrin matrix was tested for a healing response in a lethal size rat cranial defect.

Fibrin gel was prepared using a TISSUCOL® kit (Baxter AG, CH-
8604 Volketswil / ZH) fibrin encapsulant precursor component.
Fibrinogen is diluted in 0.03 M sterile Tris buffered solution (TBS, pH 7.4) to produce an approximately 8 mg / mL solution, and thrombin is diluted in 50 mM sterile CaCl ₂ solution to provide a 2 U / mL solution. Was generated. The final concentration of fibrinogen is a 1: 8 original TISSUCOL® formulation (approximately 100 mg / mL) and 1: 160
The original TISSUCOL® thrombin concentration (approximately 500 IE / mL). A pre-determined amount of TG-pl-PTH _1-34 or TGPTH _1-
34 was added to thrombin and mixed to produce a homogeneous concentration.

To produce a fibrin gel, the diluted precursors were mixed together by injecting fibrinogen into a tube containing thrombin. In the case of an ovine drill defect (described below), this mixture was then immediately injected into the drill defect formed in the ovine cancellous bone, where it formed a fibrin gel within 1-5 minutes. In the first series of animal experiments, a fusion protein (NQEQVSPLY containing PTH _1-34 in cortical bone healing)
The efficacy of KRNSVSEIQLMMHNLGGHLNSMERVEWLRKKLQDVHNF, SEQ ID NO: 18) as a bioactive factor was tested in a small animal model. Array YKN
R (SEQ ID NO: 19) yields a degradable linked plasmin (“TG-pl-PTH _1-34 ). TG-pl-PTH _1-34 was made by chemical synthesis. TFA is used as a counter ion. Purification was completed through reverse phase HPLC (C18 column) to give the final product, which is a TFA salt, and the purity of TG-pl-PTH _1-34 was determined to be 95%.

The healing response was investigated with both a single healing time (3 weeks) and a long healing time (7 weeks) to determine if enhanced healing could be observed.

(Cat defect of fatal size in rats)
Rats were anesthetized and the skull was exposed. The periosteum on the external surface of the skull was withdrawn so that the periosteum did not play a role in the healing process and resulted in a single 8 mm circular defect. 8m
Since the defect larger than m was not spontaneously healed by itself and was previously determined to be a fatal size defect, the size of this defect was chosen. The defect was then filled with a pre-formed fibrin matrix and the animals were allowed to heal for 3 and 7 weeks.
The defect area was then explanted and analyzed radiologically and histologically.

(result)
When TG-pl-PTH _1-34 was studied at 3 weeks, the level of healing was very similar to that observed with the fibrin matrix alone. Other potential morphogens (rh
As early as possible when 3) (including BMP-2) showed a strong healing effect by 3 weeks
The time point of the week was selected. In contrast, no curative effect following TG-pl-PTH _1-34 could be observed at this early time point. However, if analyzed at a longer time point (7 weeks),
A moderate dose-dependent improvement in the healing of fatal-sized rat cranial defects with the addition of modified PTH _1-34 to the fibrin matrix was observed. The results are shown in Table 3. When high doses of modified PTH _1-34 were used, the healing response increased to 65%.
(Table 3: Healing response with modified PTH in rat cranial defects)

These results demonstrate that when PTH _1-34 is incorporated into the fibrin matrix, it retains some activity as evidenced by a modest increase in bone formation.

(Sheep bone drill defect)
To investigate TG-pl-PTH _1-34 , the effect of this hormone on bone healing,
A similar test was performed in a long bone defect model. In the sheep drill deficiency model, about 15
An 8 mm cylindrical drill defect with a depth of mm was placed in both the proximal and distal regions of the femur and humerus. Since the defect was placed at the epiphysis of the long bone, the defect was surrounded by trabecular bone with a thin layer of cortical bone at the periphery of the defect. These deficiencies can then be resolved at various doses of TG-p.
Filled with in situ polymerized fibrin (approximately 750 μL) containing l-PTH _1-34 or TGPTH _1-34 . The animals were cured for 8 weeks and then sacrificed. Defects were analyzed by μCT and histology.

For this series of experiments, three types of compositions were tested. First, TG-pl-
PTH _1-34 was tested over a large concentration range. Second, another modified PTH _1-34 TGPT which has no degradation site and has only a transglutaminase sequence at the amino terminus
H _1-34 (NQEQVSPLSVSEIQLMMHNLGGHLN SMERVEWLR
KKLQDVHNF; SEQ ID NO: 17) was used. Therefore, TGPTH _1-34 is
It can be released only by degradation of the fibrin matrix itself. TGPTH _1-34 was prepared and purified in the same manner as TG-pl-PTH _1-34 . The purity was determined to be 95%. To compare efficacy, TGPTH _1-34 was tested at several concentrations that were similar to TG-pl-PTH _1-34 . Finally, TGPTH _1-34 or TG-pl-PT
The matrix was made in the presence of granular material with any of H _1-34 . This granular material was a standard tricalcium phosphate / hydroxyapatite mixture embedded in a matrix during gelation. The effect of the addition of these granules on the efficacy of PTH _1-34 was investigated. As a control, unmodified fibrin was tested.

(result)
When any of the modified PTH _1-34 molecules were placed within a long bone defect, a significant improvement in healing response was observed over the use of fibrin matrix (control) alone.
The use of fibrin alone did not heal and only 20% of the initial defect was filled with newly formed bone.

TG-pl-PTH _1-34 was tested in a concentration series from 20 to 1000 μg / mL. For each dose tested, a significant increase in the healing response was observed. For example, 100 μg
When / mL TG-pl-PTH _1-34 was used, the cure rate increased to almost 60%.

In a second series of experiments, TGPTH _1-34 was tested. The use of TGPTH _1-34 also increased bone healing. For example, the use of 400 μg / mL results in a healing response of 40
% And the use of 100 μg / mL increased bone healing to 65%. Thus, the addition of any of the PTH _1-34 modified sequences resulted in a stronger healing response than the control.

Finally, the potency of PTH _1-34 was maintained when any of the modified PTH _1-34 molecules were bound to the matrix and a granule / matrix mixture was used. This is TG-pl-PT
Both H _1-34 (see Table 4) and TGPTH _1-34 (see Table 5) were tested.

(Table 4: Healing drill defect healing with TG-pl-PTH _1-34 incorporated into fibrin matrix; healing time 8 weeks)

(Table 5: Healedrill deficiency healed by PTH _1-34 bound in fibrin matrix; healing time 8 weeks)

Histological evaluation showed high infiltration in the initial defect of spindle and osteoblast precursor cells supported by the extracellular matrix. Active osteoids with large round osteoblasts were common and signs of ossified tissue (chondrocytes) within the cartilage were observed. By 8 weeks normal signs of osteoclasts and remodeling could be found. However, unlike the results obtained from continuous exposure to synthetic PTH, no overt response from osteoclasts is observed, and new bone formation is significantly greater than resorption within or around the defect area. There were many. No foreign body inflammatory response was observed (ie, there were no giant cells and few hypoallergenic monocytes). Granules are mineral particles (m
The addition of internal particles was still present in the sample.

Example 3: Preparation of precursor components for a synthetic matrix
(Preparation of PEG-vinylsulfone)
Commercially available branch (4 arm PEG, molecular weight 14,800, 4 arm PEG, molecular weight 10,0
00 and 8 arm PEG, molecular weight 20,000; Shearwater Polymer
rs, Huntsville, AL, USA) was functionalized at the OH terminus. Under an argon atmosphere, a dichloromethane solution of the precursor polymer (previously dried through molecular sieve) was reacted with NaH, and then after release of hydrogen, divinyl sulfone (molar ratio: OH 1: N
PEG vinyl sulfone was produced by reacting with aH 5: divinyl sulfone 50). The reaction was carried out at room temperature for 3 days under argon with constant stirring. After neutralizing the reaction solution with concentrated acetic acid, the solution was filtered through paper until clear. The derivatized polymer was isolated by precipitation in ice cold diethyl ether.
The product was redissolved in dichloromethane and reprecipitated twice in diethyl ether (washed well) to remove any excess divinylsulfone. Finally, this product is
Dry under vacuum. This derivatization was confirmed by ¹ H NMR. This product is 6.2
Characteristic vinyl sulfone peaks were exhibited at 1 ppm (2 hydrogens) and 6.97 ppm (1 hydrogen). The degree of end group conversion was measured as 100%.

(Preparation of PEG acrylate)
A toluene solution of the precursor polymer, azeotropically dried under an argon atmosphere, is reacted with acryloyl chloride in the presence of triethylamine (molar ratio: OH 1: acryloyl chloride 2: triethylamine 2.2). ,
PEG acrylate was produced. The reaction was allowed to proceed with stirring overnight in the dark at room temperature. The resulting pale yellow solution is filtered through a neutral alumina bed; after evaporation of the solvent, the reaction product is dissolved in dichloromethane, washed with water, dried over sodium sulfate and in cold diethyl ether. Precipitated.

(Peptide synthesis)
All peptides were converted to an automated peptide synthesizer (9050 Pep Plus S
Synthesized on solid resin using standard 9-fluorenylmethyloxycarbonyl chemistry using ynthesizer, Millipore, Framingham, USA). Hydrophobic scavengers and resolved protecting groups were removed by precipitation of the peptides in cold diethyl ether and dissolution in deionized water. After lyophilization, these peptides were redissolved in 0.03 M Tris-buffered saline (TBS, pH 7.0) and HPLC (Wate) on a size exclusion column using TBS, pH 7.0 as the transfer buffer.
rs; Milford, USA).

(Matrix formation by conjugate addition reaction)
MMP sensitive gels were formed by conjugate addition with peptide chain nucleophilic and PEG chain conjugate unsaturation allowing proteolytic cell migration. Gel synthesis was achieved via a Michael-type addition reaction of thiol PEG over vinylsulfone functionalized PEG. In the first step, the adhesive peptide is attached to the multi-arm PEG vinyl sulfone in a pendant manner (eg, peptide

(SEQ ID NO: 20)), and then the precursor is converted to a dithiol-containing peptide (eg,
Substrate

(SEQ ID NO: 21)). In a representative gel preparation for three-dimensional in vitro studies, 4-arm PEG vinyl sulfone (molecular weight 15000) was added to TEOA buffer (0.3M
, PH 8.0) to give a 10% (w / w) solution. Dissolved peptide to give gel cell adhesion

(SEQ ID NO: 20)) (same buffer) was added to this solution. The adhesion peptide was reacted at 37 ° C. for 30 minutes. Then cross-linked peptide

(SEQ ID NO: 21) was mixed with the above solution to synthesize a gel. This gelation occurred within a few minutes, but the cross-linking reaction was carried out at 37 ° C. for 1 hour to ensure complete reaction.

MMP-insensitive gels were formed by conjugate addition with PEG-linked nucleophiles and PEG-linked conjugate unsaturation allowing non-proteolytic cell migration.

The synthesis of this gel was also achieved via a Michael-type addition reaction of thiol PEG over vinylsulfone functionalized PEG. In the first step, the adhesive peptide is attached to the multi-arm PEG vinyl sulfone in a pendant manner (eg, peptide

(SEQ ID NO: 20)), then this precursor was converted to PEG dithiol (mw 3.4 kD)
And crosslinked. In a typical gel preparation for 3D in vitro studies, 4-arm PE
G vinyl sulfone (molecular weight 15000) was dissolved in TEOA buffer (0.3M, pH 8.0) to obtain a 10% (w / w) solution. Dissolved peptide to give gel cell adhesion

(SEQ ID NO: 20)) (same buffer) was added to this solution. This adhesive peptide was
The reaction was carried out at 30 ° C. for 30 minutes. Then, this PEG dithiol precursor is mixed with the above solution,
A gel was synthesized. This gelation occurred within a few minutes, but the cross-linking reaction was carried out at 37 ° C. for 1 hour to ensure complete reaction.

(Matrix formation by condensation reaction)
An MMP sensitive gel was formed by a condensation reaction of a peptide X linker-containing complex amine and electrophilic active PEG allowing proteolytic cell migration.

The MMP sensitive hydrogel also has an MMP sensitive oligopeptide containing two MMP substrates and three Lys (

(SEQ ID NO: 22)) as well as commercially available (divisional double-ester) PEG-N
-Hydroxysuccinimide (NHS-HBS-CM-PEG-CM-HBA-NHS)
By inducing a condensation reaction between In the first step, an adhesion peptide (eg, peptide

) (SEQ ID NO: 20) is reacted with a small fraction of NHS-HBS-CM-PEG-CM-HBA-NHS, and the precursor is then reacted with three epsilon amines (and one primary amine). Peptides

By mixing with (SEQ ID NO: 22), it was crosslinked to a network structure. In a typical gel preparation for 3D in vitro studies, both components were dissolved in 10 mM PBS pH 7.4 to form a 10% (w / w) solution and formed a hydrogel within at least 1 hour.

In contrast to current hydrogels formed by Michael-type reactions, this approach is because amines present in biological materials such as cells or tissues also react with bifunctional activated double esters. The desired self-selectivity in is not guaranteed. This also means that other PEGs with electrophilic functionality (eg PEG-oxycarbonylimidazole (CD
The same applies to I-PEG) or PEG nitrophenyl carbonate).

An MMP-insensitive hydrogel was formed by a PEG-amine bridge and condensation reaction with electrophilically active PEG, allowing non-proteolytic cell migration. The hydrogel also has a bifunctional double ester PEG-N-hydroxysuccinimide (NHS-HBS-CM-PEG-CM-) similar to commercially available branched PEG-amines (Jeffamines).
Formed by performing a condensation reaction with (HBA-NHS). In the first stage,
Adhesive peptides (eg peptides

) (SEQ ID NO: 20) is reacted with a small fraction of NHS-HBS-CM-PEG-CM-HBA-NHS and then this precursor is mixed with multi-armed PEG-amine to form a network structure. And crosslinked. In a typical gel preparation for three-dimensional in vitro studies, both components were dissolved in 10 mM PBS (pH 7.4) and 10% (w / w)
A solution was obtained and a hydrogel formed within one hour. Again, amines present in biological materials such as cells or tissues also react with bifunctional activated double esters, so
In contrast to current hydrogels formed by Michael-type reactions, the desired self-selectivity in this approach is not guaranteed. This also means that other PEs with electrophilic functionality
The same applies to G (eg PEG-oxycarbonylimidazole (CDI-PEG) or PEG nitrophenyl carbonate).

(Example 4: Bone regeneration using a matrix capable of degrading synthetic enzyme)
Two different starting concentrations of enzymatically degradable gel were used. In each of these, RG
The concentration of D and the active factor (CplPTH at 100 μg / mL) were kept constant. P functionalized with four vinylsulfone end groups of molecular weight 20 kD (molecular weight of each branch is 5 kD)
Four branches branched from the EG and the following sequence Gly-Cys-Arg-Asp- (Gl
y-Pro-Gln-Gly-Ile-Trp-Gly-Gln) -Asp-Arg-C
A polymer network was formed from the dithiol peptide of ys-Gly (SEQ ID NO: 21). Both precursor components were dissolved in 0.3M triethanolamine. The starting concentrations of functionalized PEG (first precursor molecule) and dithiol peptide (second precursor molecule) were varied. In one case, this concentration was 12.6% by weight of the total weight of the composition (first and second precursor components + triethanolamine solution). 12.6 wt% corresponds to a 10 wt% solution (100 mg / mL first precursor molecule) when calculated based on the first precursor component alone. Second
The starting concentration of is 9.5% by weight of the total weight of the composition (first and second precursor components + triethanolamine solution), which is calculated based only on the first precursor component of the total weight Corresponds to 7.5% by weight (75 mg / mL first precursor molecule). This has the result that the amount of dithiol peptide has changed so that the molar ratio between vinyl sulfone and thiol is maintained.

The gel starting from an initial concentration of 12.6% by weight swelled to a concentration of 8.9% by weight of the total weight of the polymer network plus water, so the matrix had a water content of 91.1. The gel started from a starting concentration of 9.5 wt% swelled to a final concentration of 7.4 wt% of the total weight of the polymer network plus water, and thus the matrix had a water content of 92.6.

To examine the effect of this change, these materials were tested in sheep drill defects. Here, 750 μL of the defect was placed in the cancellous bone of the femur and humerus diaphysis and filled with in situ gelling enzyme gel. The following amounts of calcified tissue were obtained (determined by μCT, N = 2 for each group):
Gel starting concentration Calcified tissue 12.6% 2.7%
9.5% 38.4%
By making the gel less dense and making cell penetration easier, the healing response produced by the addition of the active agent was stronger. The effect of having a final solids concentration of less than 8.5% by weight is evident from these results.

Then, clearly, the matrix design is important to allow healing in wound defects. Each of these hydrogels consisted of a large chain of polyethylene glycol and were end-linked to form a matrix. However, details of how they were linked through the density of enzymatic degradation sites, linkers and several other variables were important to enable a functional healing response. These differences were fairly clearly observed in the sheep drilling defect model.

(Example 5. Bone formation with synthetic hydrolyzable matrix)
The PTH1-34 fusion peptide was tested in a synthetic gel and an ovine perforation defect model exactly described for the fibrin matrix. The hydrogel network was acrylated 4-arm polyethylene glycol (MW 15,000 (Peg4 * 15Acr)).
) And linear polyethylene glycol diol of MW 3400. When the two components are mixed in a 0.3 M triethanolamine buffer (pH 8.0) via a Michael-type reaction, the resulting thiolate formed at this pH is then conjugated unbound. It could react with saturated acrylates to form covalent bonds. Multifunctional precursors are mixed together (eg, combined multifunctionality is 5 or more)
As a result, a hydrogel is formed. In addition, bioactive factors can be added to the matrix via the same reaction scheme. In this case, bioactive factors (including cell adhesion motives, or morphology or mitogenic factors) can be bound to the matrix by adding cysteine, thiol-containing amino acids to the sequence. Here, the inventors have identified RGD as a cell adhesion sequence, more specifically, RGDSP (SEQ ID NO: 23) and PTH.
Add a cysteine to the _1-34 sequence, and both via an acrylate in the crosslinker
Combined with the matrix. Subsequently, these newly formed hydrogels then had multiple esters near the thiol, which were shown to be hydrolytically unstable. This instability allows the gel to degrade slowly and be replaced by newly formed tissue.

These particulate gels are hydrolysable by RGD and PTH covalently bound to the matrix, and they are tested in a sheep perforation defect model and the matrix bound C-PT
The ability of H _1-34 to enhance bone growth was tested. 0 μg / m to determine the amount of enhancement
L, 100 μg / mL and 400 μg / mL PTH _1-34 fusion peptide were added to the matrix. After doing this, an increase in bone formation was observed with the addition of PTH _1-34 . In each study, the healing response was measured at the same time point for 8 weeks. This was compared to the deficit remaining empty. When a synthetic hydrolyzable matrix without PTH fusion protein was used, the healing response was measured to be about 40%. This means that 40% of the original defect volume has been filled with newly formed tissue. Then 400 μg /
When mL of modified PTH _1-34 fusion peptide was used, the healing response increased to about 60%. In comparison, if this defect remained empty, about 10% was filled with calcified tissue. This data is shown in Table 6 below.

  Table 6. Healing response by synthetic matrix with or without modified PTH.

Compared to an empty defect, the addition of hydrolyzable peg gel alone had a great effect on bone healing and increased the amount of calcified tissue by 300%. When PTH _1-34 is linked to this matrix, healing is further increased to a level that is 50% higher than when the matrix is used alone, 500% higher than the healing level obtained when the defect remains empty. it was high.

Claims (1)

Invention described in the specification.