New uses of proteoglycans to bind and present growth factors, methods of accelerating wound, tissue or bone repair using such proteoglycans, pharmaceutical compositions of such proteoglycans, and scaffolds coated with such proteoglycans are disclosed. The proteoglycan of the invention is derived from domain I or perlecan.

Description of the Invention

BACKGROUND OF THE INVENTION

The invention provides new uses and compositions of proteoglycans. The proteoglycans are derived from perlecan, an extracellular matrix protein. The inventive proteoglycans retain certain desirable activities of the full-length perlecan molecule, such as the ability to bind growth factors, yet they have a size that allows for effective preparation and application as is not the case for perlecan. Furthermore, large amounts of the inventive proteoglycan can be produced in mammalian cell lines. The inventive proteoglycans can be used as adhesive coatings on scaffolds used for bone and tissue repair to attract and retain growth factors to the repair site. The inventive proteoglycans can also be used to induce differentiation to or maintenance of chondrocyte phenotype.

Chondrogenesis occurs as a multi-step process that is initiated by condensation of mesenchymal stem cells that subsequently undergo a specific program of differentiation. Studies from several laboratories clearly have demonstrated a role for specific soluble signals in this differentiation program that include bone morphogenetic proteins (1), parathyroid hormone related protein (PTIrP)(2), Indian hedgehog (Ihh) (3), and transforming and fibroblast growth factors (4,5). Of interest, several of these are known to interact with heparan sulfate proteoglycans (HSPG), a factor implicated in modulating their bioavailability (6). It has been demonstrated that a large HSPG found in the extracellular matrix (ECM) of developing cartilage, perlecan (Pln, HSPG2), stimulated cells of a murine fibroblast line, C3H10T1/2, to form aggregates in vitro similar to those found in condensing mesenchyme in vivo (7). In addition, Pln maintained the chondrogenic phenotype of adult chonodrocytes in vitro (7).

Consistent with a fundamental role for Pln in endochondral bone formation, targeted disruption of the Pln gene in mice results in severe disorganization of the columnar structure of chondrocytes and defective endochondral ossification (9). Interestingly, the phenotype of the Pln null mice is similar to that caused by activating mutations of fibroblast growth factor receptor 3 (FGFR3), which has been interpreted to mean that these molecules modulate similar signaling pathways in developing cartilage (9).

Pln is a multi-domain protein consisting of five distinct regions, four of which display sequence similarity to other protein families (10). The proteoglycan and its core protein are disclosed in Costell et al. (16). All the perlecan domains are disclosed in Noonan DM et al. (10).

The N-terminal domain I is unique to Pln. Within domain I are three glycosaminoglycan ("GAG") attachment sites, defined by the consensus amino acid triplet Serine- Glycine-Aspartic Acid ("SGD"). While other potential sites for glycosylation exist in the protein core, the N-terminal sites are considered the major site for GAG attachment (11). Domain II contains repeat sequences highly similar to domain IV of the laminin A chain. In mice, domain III contains an Arginine-Glycine-Aspartic acid ("RGD") sequence but in human Pln this sequence is missing (12). Domain IV contains repeats similar to those found in the IgG superfamily member, neural cell adhesion molecule (N-CAM). The C-terminal of domain V shows sequence similarity to G region of the laminin A. chain. There are also epidermal growth factor (EGF)-like sequences spaced between the domain G-like repeats in Pln domain V.

Each domain of Pln previously has been produced as a recombinant protein, and several of these also have been produced in various forms (13-17).

Perlecan has been associated with growth factors. Mongiat et al. reported that perlecan acts as a ligand reservoir for various growth factors, stabilizing them against misfolding and proteolysis (20). Costell et al. reported that perlecan binds and delivers growth factors in two ways (8). Costell et al. reported that perlecan's heparin sulfate and chondroitin sulfate side chains bind growth factors as well as its protein core.

Several groups have studied the interaction between the glycosaminoglycan molecules and the fibroblast growth factor family of heparin-binding growth factors. For example, Walz et al. have found that the biological activities of fibroblast growth factor-1 and fibroblast growth factor-2 depend on their ability to bind cell surfaces and extracellular matrix heparin sulfate side chains, like those found attached to perlecan (27).

Growth factors have been used as coatings for scaffolds implanted to treat numerous skeletal and connective-tissue related disorders. It is of great interest to attract and retain growth factors to the site of bone or tissue repair and thereby accelerate healing.

Although it is known that perlecan is involved in growth factor retention, the intact molecule is too large to exploit commercially as a growth factor adhesive. Perlecan is one of the most complex gene products because of its enormous dimensions and number of posttranslational modifications. Its size does not allow for efficient and cost effective commercial production. The present invention avoids this problem and meets the needs of the art by providing a molecule that can be produced in large amounts in mammalian cell lines and is at least as active as the intact perlecan molecule in binding and presenting heparin-binding growth factors and inducing differentiation to or maintenance of chondrocyte phenotype.

SUMMARY OF INVENTION

The invention is directed to new uses of proteoglycans to bind and present growth factors, pharmaceutical compositions of such proteoglycans, and medical devices coated with such proteoglycans. The proteoglycan of the invention is derived from domain I of perlecan.

DETAILED DESCRIPTION

Proteoglycans Useful in the Invention

Preferred embodiments of the invention involve a proteoglycan of less than 450 kDa, more preferably of less than 100 kDa, still more preferably of less than 2 kDa, still more preferably of about 8 to 10 kDa in molecular weight, comprising the core protein of domain I of a mammalian perlecan to which at least one glycosaminoglycan chain is attached. More preferably, the proteoglycan is substituted with two or three glycosaminoglycan chains. The proteoglycans identified as Pln IA and PlnIB, as defined in the attached Example 1, may be used. The proteoglycan should have at least one and can have more glycosaminoglycan chains, varying in length or composition. The invention also includes uses and compositions of proteoglycans in which the core protein comprises an amino acid sequence having at least about 70-75%, still more preferably at least about 80-85%, and most preferably at least about 90% or more homology to the amino acid sequence of domain I of a mammalian perlecan, preferably murine or human perlecan.

Other preferred embodiments of the invention involve a proteoglycan in which the core protein comprises an amino acid sequence of -- see Original Patent.

Further preferred embodiments involve a proteoglycan comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:2 and is less than 250 amino acids in length and preferably of less than 200 amino acids in length. The invention also includes uses and compositions of proteoglycans in which the core protein comprises an amino acid sequence having at least about 70-75%, still more preferably at least about 80-85%, and most preferably at least about 90% or more homology to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2. Again, as stated before, the proteoglycan should have at least one and can have more glycosaminoglycan chains, varying in length or composition.

To determine the percent homology of two amino acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of one polypeptide for optimal alignment with the other polypeptide). The amino acid residues at corresponding amino acid positions are then compared. When a position in one sequence is occupied by the same amino acid residue as the corresponding position in the other sequence, then the molecules are homologous at that position (i.e., as used herein amino acid "homology" is equivalent to amino acid "identity"). The percent homology between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology=# of identical positions/total # of positions times. 100).

The proteoglycans used in the invention include those molecules having conservative amino acid substitutions at one or more predicted non-essential amino acid residues when compared to a wild-type mammalian perlecan domain I. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in the proteoglycan is preferably replaced with another amino acid residue from the same side chain family such that the proteoglycan retains the ability to bind growth factors. More preferably, the proteoglycans retain the ability to facilitate the formation of aggregates of C3H10T1/2 cells.

In other preferred embodiments, the proteoglycan is a biologically active portion of the perlecan domain I that includes a domain or motif that has growth factor binding ability and/or the ability to support C3H10T1/2 cell agglomeration. Such domains or motifs include the domains associated with glycosaminoglycan attachment to the core polypeptide.

The proteoglycans of the invention may be synthesized in various ways, such as by chemical synthesis, isolation from perlecan, or recombinant production. Preferred is recombinant production. Examples of such production are found in Costell et al. (16). Costell et al. teaches preparation of perlecan domain I from mammalian cell clones on a preparative scale using the pRc/CMV expression vector sold by Invitrogen. The expression vector was cotransfected together with plasmid pSV.sub.pac into human embryonic kidney 293 cells and stable transfectants were selected with puromycin.

The proteoglycans used in the invention may be obtained by derivation from perlecan from any mammalian species, most preferably mouse, rat, or human.

The proteoglycans of the invention may be used to induce differentiation to or maintenance of connective-tissue cells, particularly chondrocytes. More particularly, chondrocyte phenotype can be maintained in cultures using the inventive proteoglycans. The proteoglycans may also be used to bind and present heparin-binding growth factors. The proteoglycans may be used in soluble or insoluble form. They also may be used as a coating for surfaces, particularly surfaces used in tissue engineering or prosthetic devices. For example, scaffolds and medical devices may be coated with the inventive proteoglycans and implanted in a mammalian body. The coating binds growth factors to the surface, which encourages rapid and complete tissue growth at the injured site. Also, the surfaces may be coated with the inventive proteoglycans to which growth factors are attached and then implanted into a mammalian body. These coated scaffolds and devices are expected to provide enhanced recovery for patients suffering from connective tissue disorders, such as bone fractures, cartilage tearing, etc. The inventive proteoglycans increase the adhesion of desirable biological materials such as growth factors to the repair site.

Scaffolds

Devices coated with the proteoglycans of the invention such as implants and scaffolds are also provided. Also provided are implants and scaffolds coated with the proteoglycans of the invention to which growth factors have been attached, either covalently or non-covalently.

Preferably, the scaffold is made of a polymer, a biologically derived material, ceramic, metal, or combinations thereof, that is biologically inert and physiologically compatible with mammalian tissues. The scaffold material preferably does not induce an inflammatory response. The scaffold also preferably is capable of associating with the proteoglycan at sufficient levels to satisfy the intended objective, e.g., increased growth factor adhesion or attraction to the scaffold. The scaffold can bind the proteoglycan either covalently or non-covalently, such as by electrostatic charge or hydrophobic or hydrophilic interactions.

Preferred polymers are polyamides, polypeptides, polyesters, polycarbonates, polyurethanes, polyacetals, polysaccharides, and polyolefins. Specific examples of such polymers include silicone rubber, polyurethane rubber, polyethylene, polyvinyl chloride, poly (hydroxyethyl methacrylate), poly (methyl methacrylate), poly (ethyleneterephthalate), polypropylene, polystyrene, poly (tetrafluoroethylene), polyglycolic acid, cellulose, ethylcellulose, methycellulose, dextran, carboxymethylcellulose, hyaluronic acid, hydroxypropylmethylcellulose, nylon, collagen, and collagen-GAG. Preferred polymers include expanded polytetrafluoroethylene composed of two polymers and having nine billion pores per square inch. Additionally, the scaffold can be a copolymer, composite or blend of the above polymers.

The polymer may have other materials embedded in it, such as carbon fibers embedded in a polyurethane-poly(L-lactide matrix). Additional scaffold materials are disclosed in Sweigart, M. A. (28). Additional scaffold materials are known to those skilled in the art.

Preferred biologically derived materials are matrices comprised of collagen sponge, cortical bone chips, cancellous bone chips, cortico-cancellose bone chips, hydroxyapatite or like ceramics, bioactive glass, growth factors and demineralized bone, which are imbedded or suspended in a carrier material. The carrier material is preferably a fibrin-containing composition that coagulates, collagen formulations, hydroxylapatite, pleuronic polymers, synthetic or natural polymers, carboxymethylcellulose, gelatin, or combinations thereof. More preferably, the carrier is gelatin derived from human or animal tissue. Other preferred biologically derived materials are mammalian tissues, such as perichondral tissue and periosteal tissue.

Methods of Treatment

The proteoglycans of the invention can also be administered directly to injured connective tissue, where growth factors will be attached in vivo and thereby tissue recovery will be enhanced. Growth factors can also be attached to the proteoglycans ex vivo and then the proteoglycan/growth factor product can be administered to a damaged tissue, such as a bone fracture or cartilage tear. The proteoglycan may be in soluble or insoluble form.

The invention provides a method for accelerating wound, tissue, or bone healing in a mammalian subject, e.g., human, by applying to the injured area a therapeutically effective amount of a composition which contains one or more of the proteoglycans of the invention and optionally a heparin-binding growth factor. In addition, the invention provides for pharmaceutical composition comprising one or more of the proteoglycans of the invention or a biologically active portion thereof, a pharmaceutically acceptable carrier, and optionally a heparin-binding growth factor.

The proteoglycans can be used to administer or attract growth factors as treatment for a variety of medical conditions. One important example is in the repair of bone, cartilage, or other connective tissue (such as tendon and ligament). Repair may be needed because of trauma, bone tumor resection, or in the case of joint fusion and spinal fusion for non-healing fractures and osteoporotic lesions. A proteoglycan-coated scaffold also may be used in treating tooth and jaw defects in cases of trauma, bone loss, tooth loss, and gum disease. The scaffolds also are useful in treating cartilage defects such as those which result from rheumatoid arthritis, osteoarthritis and trauma. The scaffolds also may be used to repair defects and damage in skin, muscle and other soft tissues such as results from trauma, burns, ulcers (diabetic ulcers, pressure sores, venus, stasis ulcers, etc.). Likewise, damage to visceral organs including liver damage, heart attack damage, and damage resulting from intestinal cancer or intestinal ulcer may be treated with the scaffolds of the invention.

The proteoglycans of the invention may be administered with growth factors attached or without growth factors attached, such that growth factors already present in the mammalian body can be attracted to and bind to the proteoglycan.

Various heparin-binding growth factors are known in the art or are readily identifiable and can be used in the invention. For example, U.S. Pat. No. 5,876,730 to Brigstock et al. issued Mar. 2, 1999 entitled "Heparin-binding growth factor (HBGF) polypeptides" discloses a group of heparin-binding growth factors isolated from uterine secretory fluids. Another example is transforming growth factor-.beta. Many members of the fibroblast growth factor family ("FGF family") also bind tightly to heparin.

The invention also pertains to in vitro culture of cells with the purpose of creating tissue constructs for repairing tissues and organs in vivo. The scaffolds may be used to promote tissue culture of committed cells and/or differentiation of precursor cells. Thus, the scaffolds of the invention can be used in virtually all instances when it is desirable to provide a substrate for the growth of cells onto or into a tissue replaceable matrix. Scaffolds can also be used with autografts, allografts, and xenografts associated with bone grafts, cartilage grafts, and joint resurfacing implants.
 

Claim 1 of 10 Claims

1. A method for accelerating cartilage repair at a repair site in a mammal comprising applying at the repair site a therapeutically effective amount of a proteoglycan that comprises the amino acid sequence of the core protein of domain I of a mammalian perlecan to which at least one heparan sulfate glycosaminoglycan is attached, wherein the proteoglycan consists of 200 or fewer amino acids and stimulates mesenchymal stem cell differentiation to cartilage.

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