Device and method for regeneration and repair of cartilage lesions

[0002] This application is a continuation-in-part under 35 U.S.C. § 120 of PCT Application No. PCT/EP 98/05100, entitled “COMPOSITION AND DEVICE FOR IN VIVO CARTILAGE REPAIR COMPRISING NANOCAPSULES WITH OSTEOINDUCTIVE AND/OR CHONDROINDUCTIVE FACTORS”, filed Aug. 12, 1998, which designates the United States and which claims priority from European Application No. EP 97810567.4, entitled “COMPOSITION AND DEVICE FOR IN VIVO CARTILAGE REPAIR”, filed Aug. 14, 1997. The entire disclosures of Application Nos. PCT/EP 98/05100 and EP 97810567.4 are incorporated herein by reference.

[0003] The present invention relates to a cartilage regeneration and repair product that induces cell ingrowth into a bioresorbable material and cell differentiation into cartilage tissue, and to methods of using such a product to repair cartilage lesions.

[0004] Articular cartilage, an avascular tissue found at the ends of articulating bones, has limited natural capacity to heal. During normal cartilage ontogeny, mesenchymal stem cells condense to form areas of high density and proceed through a series of developmental stages that ends in the mature chondrocyte. The final hyaline cartilage tissue contains only chondrocytes that are surrounded by a matrix composed of type II collagen, sulfated proteoglycans, and additional proteins. The matrix is heterogenous in structure and consists of three morphologically distinct zones: superficial, intermediate, and deep. Zones differ among collagen and proteoglycan distribution, calcification, orientation of collagen fibrils, and the positioning and alignment of chondrocytes (Archer et al., 1996, J. Anat.189(1):23-35; Morrison et al., 1996, J. Anat.189(1): 9-22; and Mow et al.,1992, Biomaterials13(2): 67-97). These properties provide the unique mechanical and physical parameters to hyaline cartilage tissue.

[0005] The meniscus, a C-shaped cartilaginous tissue, performs several functions in the knee including load transmission from the femur to the tibia, stabilization in the anterior-posterior position during flexion, and joint lubrication. Damage to the meniscus results in reduced knee stability and knee locking. Over 20 years ago, meniscectomies were performed which permitted immediate pain relief, but were subsequently found to induce the early onset of osteoarthritis (Fairbank, J. Bone Joint Surg.30B: 664-670; Allen et al., 1984, J. Bone Joint Surg.66B:666-671; and Roos et al., 1998, Arth. Rheum.41:687-693). More recently, partial meniscectomies and repair of meniscal tears have been performed (FIGS. 9A-D; Jackson, D., ed., 1995, Reconstructive Knee Surgery Master Techniques in Orthopedic Surgery, ed. R. Thompson, Raven Press: New York). However, partial resection results in the loss of functional meniscus tissue and the early onset of osteoarthritis (Lynch et al., 1983, Clin. Orthop.172:148-153; Cox et al., 1975, Clin. Orthop.109:178-183; King, 1995, J. Bone Joint Surg.77B:836-837). Additionally, repair of meniscal tears is limited to tears in the vascular ⅓ of the meniscus; tears in the semivascular to avascular ⅔ are not repairable (FIGS. 9A-D; Jackson, ibid.). Of the approximately, 560,000 meniscal injuries that occur annually in the United States, an estimated 80% of tears are located in the avascular, irreparable zone. Clearly, a method that both repairs “non-repairable” tears or that can induce regeneration of resected menisci would be valuable for painless musculoskeletal movement and prevention of the early onset of osteoarthritis in a large segment of the population.

[0006] The proximal, concave surface of the meniscus contacts the femoral condyle and the distal, flat surface contacts the tibial plateaus. The outer one-third of the meniscus is highly vascularized and contains dense, enervated, connective tissue. In contrast, the remaining meniscus is semivascular or avascular, aneural tissue consisting of fibrochondrocytes surrounded by abundant extracellular matrix (McDevitt et al., Clin. Orthop. Rel. Res.252:8-17). Fibrochondrocytes are distinctive in both appearance and function compared to undifferentiated fibroblasts. Fibroblasts are elongated cells containing many cellular processes and produce predominantly type I collagen. The matrix produced by fibroblasts does not produce a sufficient mechanical load. In contrast, fibrochondrocytes are round, and are encompassed by lacunae that consists of type I and type II collagen and proteoglycans. These matrix components support compressive forces that are commonly exerted on the meniscus during musculoskeletal movement.

[0007] In the 1960's, demineralized bone matrix was observed to induce the formation of new cartilage and bone when implanted in ectopic sites (Urist, 1965, Science150:893-899). The components responsible for the osteoinductive activities were termed Bone Morphogenetic Proteins (BMP). At least seven individual BMP proteins were subsequently identified from bone (BMP 1-7) and amino acid analysis revealed that six of the seven BMPs were related to each other and to other members of the TGF-β superfamily. During endochondral bone formation, TGF-β family members direct a cascade of events that includes chemotaxis, differentiation of pluripotential cells to the cartilage lineage, maturation of chondrocytes to the hypertrophic stage, mineralization of cartilage, replacement of cartilage with bone cells, and the formation of a calcified matrix (Reddi, 1997, Cytokine& Growth Factor Reviews8:11-20). Although individual, recombinant BMPs can induce these events, the prevalence of multiple TGF-β family members in bone tissue underlies the complexity involved in natural osteogenesis.

[0008] Bone Protein (Sulzer Orthopedics Biologics, Denver, Colo.), also referred to herein as BP, is a naturally derived mixture of proteins isolated from demineralized bovine bones that has osteogenic activity in vitro and in vivo. In the rodent ectopic model, BP induces endochondral bone formation or bone formation through a cartilage intermediate (Damien, C. et al., 1990, J. Biomed. Mater. Res.24:639-654). BP in combination with calcium carbonate promotes bone formation in the body (Poser and Benedict, PCT Publication No. WO95/13767). In vitro, BP has been shown to promote differentiation to cartilage of murine embryonic mesenchymal stem cells (Atkinson et al., 1996, In “Molecular and Developmental Biology of Cartilage”, Bethesda, Md., Annals New York Acad. Sci.785:206-208; Atkinson et al., 1997, J. Cell. Biochem.65:325-339) and of adult myoblast and dermal cells (Atkinson et al., 1998, 44th Annual Meeting, Orthopaedic Research Society, abstract). To ensure chondrogenesis in these in vitro systems, however, culture conditions must be tightly controlled throughout the culture period, including by controlling cellular organization within the culture, optimizing media formulations, and adding exogenous factors that must be carefully established to maximize chondrogenesis over mitogenesis. Such optimization of conditions makes the application of the disclosed in vitro methods to an in vivo system unrealistic and unpredictable. In addition, although in vitro cultures of adult myoblast and dermal cells initially resulted in chondrogenesis, the effect was only transient and over time, the cultures reverted to their original phenotype. Although certain embryonic and precursor cell types showed prolonged chrondrogenesis in vitro in these studies, it would be unpredictable or even impossible in the case of embryonic cells that these specific cell types could be recruited to a site in vivo in an adult patient.

[0009] Atkinson et al., in PCT Application No. PCT/EP/05100, incorporated herein by reference in its entirety, describe a delivery system for osteoinductive and/or chondroinductive mixture of naturally derived factors for the induction of cartilage repair.

[0010] Hunziker (U.S. Pat. Nos. 5,368,858 and 5,206,023) describes a cartilage repair composition consisting of a biodegradable matrix, a proliferation and/or chemotactic agent, and a transforming factor. A two-stage approach is used where each component has a specific function over time. First, a specific concentration of proliferation/chemotactic agent fills the defect with repair cells. Second, a larger transforming factor concentration, preferably provided in conjunction with a delivery system, transforms repair cells to chondrocytes. The second stage delivery of a high concentration of transforming factor in a delivery system (i.e., liposomes) was required to obtain formation of hyaline cartilage tissue at the treatment site.

[0011] Chen and Jeffries (U.S. Pat. No. 5,707,962) describe osteogenic compositions consisting of collagen and sorbed factors to enhance osteogenesis.

[0012] Valee and King (U.S. Pat. No. 4,952,404) describe healing of injured, avascular meniscus tissue by release of the angiogenic factor, angiogenin, over at least 3 weeks.

[0013] Previously, Amoczky et al. described a method using an autogenous fibrin clot to repair an avascular, circular lesion in canine menisci (Amoczky et al., 1988, J. Bone Joint Surg.70A:1209-1217). This approach enhanced repair of meniscal tissue compared to controls lacking the fibrin clot. However, the repair tissue was not meniscus-like tissue, but rather connective scar tissue.

[0014] Hashimoto et al. described a method using fibrin sealant with or without endothelial cell growth factor in avascular, circular meniscal defects in the canine model (Hashimoto et al., 1992, Am. J. Sports Med.20:537-541). The growth factor added a modest benefit compared to healing with fibrin sealant alone and this additional effect was not observed until three months after treatment, indicating an indirect contribution of the growth factor. In addition, the defect was filled with hyaline cartilage-like cells, which are not typically present in normal meniscus tissue.

[0015] Shirakura, et al. describe the use of an autogenous synovium graft sutured into meniscal tears. While the synovium did enhance healing in ⅓ of the animals, the grafts healed with fibrous tissue, not fibrocartilaginous tissue normally observed in meniscus tissue (Shirakura, 1997, Acta. Orthop. Scand.68:51-54). Furthermore, ⅔ of the grafts did not heal.

[0016] The molecular mechanism for cartilage and bone formation has been partially elucidated. Both bone morphogenetic proteins (BMP) and transforming growth factor β (TGFβ) molecules bind to cell surface receptors (i.e., TGFβ/BMP receptors) to initiate a cascade of signals to the nucleus that promotes proliferation, differentiation to cartilage, and/or differentiation to bone (Massague, 1996, Cell85:947-950). In 1984, Urist described a substantially pure, but not recombinant, BMP combined with a biodegradable poly(lactic acid)polymer delivery system for bone repair (U.S. Pat. No. 4,563,489). This system blends together equal quantities of BMP and poly(lactic acid) (PLA) powder (100 μg of each) and decreases the amount of BMP required to promote bone repair.

[0017] Hattersley et al. (WO 96/39170) disclose a two factor composition for inducing cartilaginous tissue formation using a cartilage formation-inducing protein and a cartilage maintenance-inducing protein. Specific recombinant cartilage inducing proteins are specified as BMP-13, MP-52 and BMP-12, and specific cartilage maintenance-inducing proteins are specified as BMP-9. In one embodiment, BMP-9 is encapsulated in a resorbable polymer system and delivered to coincide with the presence of cartilage formation inducing protein(s).

[0018] Laurencin et al. (U.S. Pat. No. 5,629,009) disclose a chondrogenesis-inducing device, consisting of a polyanhydride and polyorthoester, that delivers water soluble proteins derived from demineralized bone matrix, TGFβ, epidermal growth factor (EGF), fibroblast growth factor (FGF) or platelet-derived growth factor (PDGF).

[0019] Previously, Li and Stone (U.S. Pat. No. 5,681,353) have described a Meniscal Augmentation Device that consists of biocompatible and bioresorbable fibers that acts as a scaffold for the ingrowth of meniscal fibrochondrocytes, supports normal meniscal loads, and has an outer surface that approximates the natural meniscus contour. After partial resection of the meniscus to the vascular zone, this device is implanted into the resulting segmental defect. The results have been described in both canines and humans (Stone et al., 1992, Am. J. Sports Med.20:104-111; and Stone et al., 1997, J. Bone Joint Surg.79:17701777).

[0020] The Meniscus Augmentation Device, the research reports described above, and current repair surgeries provide encouraging results in the area of cartilage repair, but are not satisfactory to induce repair of “non-repairable” avascular tears in which the repair tissue is meniscus tissue, and are not satisfactory to produce short patient rehabilitation times and regenerated meniscus tissue in the vascular zone. Furthermore, no reports have been described in which demonstrate enhanced healing rates of “repairable” meniscal tears in vivo.

[0021] The present invention relates to a product and method for repairing and/or regenerating cartilage lesions. The product and method of the present invention are useful for repairing a variety of cartilage lesions, including articular and mensical lesions, including vascular, semivascular and avascular lesions. Moreover, the product and method of the present invention can be used to repair different sizes and shapes of cartilage lesions, including radial tears, bucket handle tears, and segmental defects.

[0022] One embodiment of the present invention is directed to a product for repair of cartilage lesions. The product includes: (a) a cartilage repair matrix; and (b) a cartilage-inducing composition associated with the matrix for provision of chondrogenesis-enhancing proteins. Each of the chondrogenesis-enhancing proteins can be provided as a protein and/or by a recombinant nucleic acid molecule encoding the protein, such recombinant nucleic acid molecule being operatively linked to a transcription control sequence, and mixtures of proteins and recombinant nucleic acid molecules can be provided. The mixture of chondrogenesis-enhancing proteins include at least a first and second transforming growth factor β (TGFβ) superfamily protein, wherein the first and second TGFβ superfamily protein are different, and at least one protein from a family of proteins selected from growth factor proteins and bone matrix proteins. The chondrogenesis-enhancing proteins are further characterized in that, when cultured together with ATDC5 cells for seven days at a concentration of about 100 ng/ml or less, induce a statistically significant increase in A₅₉₅in an Alcian Blue assay performed with said cells.

[0023] The product of the present invention can also be formulated to include: (a) a cartilage repair matrix; and (b) a cartilage-inducing composition associated with the matrix, which includes cells that have been cultured with the above-described mixture of chondrogenesis-enhancing proteins.

[0024] The cartilage repair matrix of a shape and size that conforms to the cartilage defect such that the defect is repaired. As such, the matrix can be configured as a sheet, which is most suitable for repairing cartilage tears, or the matrix can be configured to repair a segmental defect, which can include a tapered shape. The cartilage repair matrix can be formed of any suitable material, including synthetic polymeric material and ground substances. In one embodiment, the matrix is bioresorbable. In another embodiment, the matrix is porous. When the matrix is configured as a sheet, the matrix is preferably not cross-linked, and when the matrix is configured to repair a segmental defect, the matrix is preferably cross-linked.

[0025] The cartilage-inducing composition can be associated with the matrix by any suitable method, including, but not limited to freeze-drying the composition onto a surface of said matrix and suspension within said cartilage repair matrix of a delivery formulation containing said composition. Additionally, the composition can be associated with the matrix ex vivo or in vivo. In one embodiment, a suitable delivery formulation includes nanospheres, wherein the nanospheres are polymer particles having a size of less than 1000 nm and being loaded with between 0.001% and 17% by weight of the cartilage-inducing composition. The nanospheres have an in vitro analytically determined release rate profile with an initial burst of about 10% to about 20% of the total amount of the composition over a first 24 hour period and a long time release rate of a least 0.1% per day during at least seven following days. Yet another suitable delivery vehicle includes a liposome. When the chondrogenesis-enhancing proteins are provided as recombinant nucleic acid molecule, the recombinant nucleic acid molecule can be provided in any suitable form for delivery, including as naked DNA, transformed into a recombinant cell or provided in the form of a recombinant virus.

[0026] In preferred embodiments, the TGFβ superfamily proteins included in the chondrogenesis-enhancing protein mixture include, but are not limited to, TGFβ1, TGFβ2, TGFβ3, bone morphogenetic protein (BMP)-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, cartilage-derived morphogenetic protein (CDMP)-1, CDMP-2, and/or CDMP-3; and more preferably include TGFβ1, TGFβ2, TGFβ3, bone morphogenetic protein (BMP)-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, and/or a cartilage-derived morphogenetic protein (CDMP). The bone matrix proteins included in the chondrogenesis-enhancing protein mixture include, but are not limited to, osteocalcin, osteonectin, bone sialoprotein (BSP), lysyloxidase, cathepsin L pre, osteopontin, matrix GLA protein (MGP), biglycan, decorin, proteoglycan chondroitin sulfate-III (PG-CS III), bone acidic glycoprotein-75 (BAG-75), thrombospondin (TSP) and fibronectin; and more preferably include osteocalcin, osteonectin, MGP, BSP, lysyloxidase and cathepsin L pre. The growth factor proteins included in the chondrogenesis-enhancing protein mixture include, but are not limited to, fibroblast growth factor I (FGF-I), FGF-II, FGF-9, leukocyte inhibitory factor (LIF), insulin, insulin growth factor I (IGF-I), IGF-II, platelet-derived growth factor AA (PDGF-AA), PDGF-BB, PDGF-AB, stromal derived factor-2 (SDF-2), pituitary thyroid hormone (PTH), growth hormone, hepatocyte growth factor (HGF), epithelial growth factor (EGF), transforming growth factor-α (TGFα) and hedgehog proteins; and more preferably include at least fibroblast growth factor I (FGF-I). The chondrogenesis-enhancing proteins can also include one or more serum proteins, including, but not limited to albumin, transferrin, α2-Hs GlycoP, IgG, α1-antitrypsin, β2-microglobulin, Apo A1 lipoprotein (LP) and Factor XIIIb; and more preferably include albumin, transferrin, Apo A1 LP and Factor XIIIb. Particularly preferred mixtures of chondrogenesis-enhancing proteins include TGFβ1, TGFβ2, TGFβ3, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, CDMP, FGF-I, osteocalcin, osteonectin, MGP, BSP, lysyloxidase, and cathepsin L pre, and in another embodiment, TGFβ1, TGFβ2, TGFβ3, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, CDMP, FGF-I, osteocalcin, osteonectin, MGP, BSP, lysyloxidase, cathepsin L pre, albumin, transferrin, Apo A1 LP and Factor XIIIb. Yet another preferred mixture of chondrogenesis-enhancing proteins are bone protein (BP).

[0027] Another embodiment of the present invention relates to a method for repair of cartilage lesions, which includes the steps of implanting and fixing into a cartilage lesion a cartilage repair product of the present invention, as described above. The method of the present invention can be used to enhance the rate and/or quality of repair of vascular cartilage tears and segmental defects, and can provide the ability to repair semivascular and avascular tears and segmental defects that, prior to the present invention, were typically considered to be irreparable. When the lesion is in semivascular or avascular cartilage, the product can additionally include a time controlled delivery formulation.

[0028] In one aspect, the method of the present invention includes the use of two cartilage repair products to repair a segmental defect. The first product includes a cartilage repair matrix, which is configured as a sheet, is associated with the chondrogenesis-enhancing proteins as described above. The second product includes a cartilage repair matrix configured to replace cartilage removed from the segmental defect, which may or may not be associated with the chondrogenesis-enhancing proteins of the present invention.

[0029] FIG. 1A shows a meniscal radial tear.

[0030] FIG. 1B shows a conventional suture repair and resection of the meniscal radial tear illustrated in FIG. 1A.

[0031] FIG. 1C shows a meniscal triple bucket handle tear.

[0032] FIG. 1D shows a conventional suture repair and resection of the meniscal triple bucket handle tear illustrated in FIG. 1C.

[0033] FIG. 2A illustrates implantation of a cartilage repair product of the present invention to a meniscal segmental lesion.

[0034] FIG. 2B illustrates fixation of a cartilage repair product of the present invention to a meniscal segmental lesion.

[0035] FIG. 3A is a diagram illustrating a meniscus cross section having vascular, semi-vascular and avascular zones.

[0036] FIG. 3B illustrates one approximate shape of a cartilage repair product of the present invention.

[0037] FIG. 4A is an illustration of a meniscus having a longitudinal tear in the avascular region as viewed from the femur towards the tibia.

[0038] FIG. 4B is a diagram illustrating a cross section of the meniscus depicted in FIG. 4A containing a cartilage repair product of the present invention.

[0039] FIG. 5 is a line graph showing quantitation of Alcian Blue staining of ATDC5 micromass cultures.

[0040] FIG. 6 is a bar graph showing quantitation of Alcian Blue staining of ATDC5 micromass cultures in Nutridoma-containing media at 7 and 14 days.

[0041] FIG. 7 is a bar graph showing quantitation of Alcian Blue staining of ATDC5 micromass cultures containing HPLC fractions of proteins isolated from demineralized bovine bones.

[0042] FIG. 8 is a diagram illustrating a cross-section view of a combination collagen meniscus implant (CMI) and sheet cartilage repair product of the present invention used to repair a meniscal defect.

[0043] The present application generally relates to a product for repairing and/or regenerating cartilage lesions, and methods of repairing or regenerating cartilage lesions using such a product. The product and methods of the present invention are particularly useful for repairing defects (i.e., lesions) in articular cartilage (e.g., hyaline cartilage) and meniscal cartilage (e.g., fibrocartilage). When used to repair meniscal cartilage, the product and method of the present invention are effective for repairing both vascular and avascular meniscal cartilage lesions. In particular, the product and method of the present invention increase the rate of meniscus repair and induce more normal (i.e., endogenous-type) meniscal tissue than is commonly observed during the conventional repair practiced currently. The cartilage repair product of the present invention can also induce meniscus repair of avascular, “irreparable” tears and, furthermore, fill the defect with meniscus-like tissue. Moreover, the product and method are useful for repairing and regenerating meniscal tissue which has been removed by partial or complete meniscectomy. The product and method of the present invention can enhance blood vessel formation, produce fibrochondrocytes, induce cellular infiltration into the product, induce cellular proliferation, and produce cellular and spatial organization to form a three-dimensional meniscus tissue.

[0044] The ability of the product of the present invention to repair and/or regenerate both vascular and avascular cartilage in vivo has not been achieved by any of the presently known cartilage repair devices/compositions or methods. Moreover, previous devices and methods have been primarily directed to the repair of very small defects and have not been successful in solving problems associated with repair of large, clinically relevant defects. Without being bound by theory, the present inventors believe that the reason that these previous approaches failed to adequately repair cartilage is that they were not able to recapitulate natural cartilage ontogeny faithfully enough, this natural ontogeny being based on a very complicated system of different factors, factor combinations and factor concentrations with temporal and local gradients. A single recombinant factor or two recombinant factors may lack the inductive complexity to mimic cartilage development to a sufficient degree. Similarly, the system used to provide one or two recombinant factors may not have been able to mimic the gradient complexity of the natural system to a satisfactory degree or to maintain a factor concentration for a time that is sufficient to allow a full and permanent differentiation of precursor cells to chondrocytes. Without being bound by theory, the present inventors believe that the repair of certain defects, particularly large defects, requires the maintenance of a sufficient concentration of a particular complex mixture of repair factors at the site for a time sufficient to induce the proper formation of cartilage.

[0045] One aspect of the present invention is directed to a product for repair of cartilage lesions. In one embodiment, such a product includes: (a) a cartilage repair matrix; and, (b) a cartilage-inducing composition associated with the matrix for provision of chondrogenesis-enhancing proteins (described in detail below), whereby each of the chondrogenesis-enhancing proteins is provided as a protein and/or by a recombinant nucleic acid molecule encoding the protein operatively linked to a transcription control sequence.

[0046] According to the present invention, the phrase “cartilage-inducing composition” refers to a formulation which contains a mixture of different chondrogenesis-enhancing proteins which includes: (1) at least a first and second transforming growth factor β (TGFβ) superfamily protein, wherein the first TGFβ superfamily protein is different from the second TGFβ superfamily protein; and, (2) at least one protein from a family of proteins selected from the group of: growth factor proteins and bone matrix proteins. In a preferred embodiment, the cartilage-inducing composition useful in the product of the present invention has an identifying characteristic which includes: an ability to induce cellular infiltration, an ability to induce cellular proliferation, an ability to induce angiogenesis, and/or an ability to induce cellular differentiation to type II collagen-producing chondrocytes, in vivo or under appropriate in vitro conditions.

[0047] According to the present invention, a mixture of chondrogenesis-enhancing proteins having the above-defined minimal protein components is characterized as being capable, when cultured together with ATDC5 cells for seven days at a concentration of about 100 ng/ml or less, of inducing a statistically significant increase in A₅₉₅in an Alcian Blue assay performed with the ATDC5 cells. The specific conditions associated with such an ATDC5/Alcian Blue assay are described in detail below. It is noted that although the mixture of chondrogenesis-enhancing proteins has the above-described characteristic, an individual chondrogenesis-enhancing protein, when isolated from the other proteins in the mixture, is not necessarily chondrogenic. For example, as described below, a bone matrix protein such as osteocalcin is a chondrogenesis-enhancing protein according to the present invention, because when such protein is combined with at least two TGFβ superfamily proteins, the mixture of proteins is capable of inducing a significant increase in A₅₉₅in an ATDC5 Alcian Blue assay. Osteocalcin is not, however, chondrogenic in the absence of the two TGFβ superfamily proteins.

[0048] According to the present invention, the chondrogenesis-enhancing proteins in the cartilage-inducing composition of the present invention include at least two different members of the TGFβ superfamily proteins. In a preferred embodiment, the chondrogenesis-enhancing proteins include at least three different members of the TGFβ superfamily proteins, and in increasing preference, at least four, five, six, seven, eight, nine, and most preferably ten different members of the TGFβ superfamily proteins. As used herein, a “TGFβ superfamily protein” can be any protein of the art-recognized superfamily of extracellular signal transduction proteins that are structurally related to TGFβ1-5. Preferably, a TGFβ superfamily protein suitable for use in the present invention is selected from the following proteins: TGFβ1, TGFβ2, TGFβ3, bone morphogenetic protein (BMP)-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, cartilage-derived morphogenetic protein (CDMP)-1, CDMP-2, and/or CDMP-3. More preferably, the chondrogenesis-enhancing proteins of the present invention include at least two of: TGFβ1, TGFβ2, TGFβ3, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, CDMP-1, CDMP-2, and/or CDMP-3. In one embodiment, the at least first and second TGFβ superfamily proteins in a cartilage-inducing composition of the present invention are selected from at least one of BMP2-8, and more preferably, at least one of TGFβ1-5, and even more preferably, at least one of CDMP1-3.

[0049] According to the present invention, the chondrogenesis-enhancing proteins in the cartilage-inducing composition of the present invention include, in addition to at least two different TGFβ superfamily proteins, at least one bone matrix protein or at least one growth factor protein. In a preferred embodiment, the chondrogenesis-enhancing proteins include at least one bone matrix protein and at least one growth factor protein. In a more preferred embodiment, the chondrogenesis-enhancing proteins include, in increasing preference, at least two, three, four, and most preferably five different bone matrix proteins, and/or at least two growth factor proteins.

[0050] As used herein, “bone matrix proteins” are any of a group of proteins known in the art to be a component of or associated with the minute collagenous fibers and ground substances which form bone matrix. As used herein, a bone matrix protein is not a member of the TGFβ superfamily as described herein, nor a growth factor protein as described herein. Bone matrix proteins can include, but are not limited to, osteocalcin, osteonectin, bone sialoprotein (BSP), lysyloxidase, cathepsin L pre, osteopontin, matrix GLA protein (MGP), biglycan, decorin, proteoglycan-chondroitin sulfate III (PG-CS III), bone acidic glycoprotein (BAG-75), thrombospondin (TSP) and/or fibronectin. Preferably, bone matrix proteins suitable for use with the product of the present invention include one or more of: osteocalcin, osteonectin, MGP, TSP, BSP, lysyloxidase and cathepsin L pre. In one embodiment, the at least one bone matrix protein includes osteocalcin, osteonectin, BSP, lysyloxidase and cathepsin L pre. A particularly preferred bone matrix protein is MGP, and more preferred is osteonectin, and most preferred is TSP.

[0051] As used herein, “growth factor proteins” are any of a group of proteins characterized as an extracellular polypeptide signaling molecule that stimulates a cell to grow or proliferate. Such growth factors may also have other actions besides the induction of cell growth or proliferation. As used herein, a growth factor is not a member of the TGFβ superfamily as defined herein nor is it a bone matrix protein as defined herein. Preferably, growth factor proteins suitable for use with the product of the present invention include one or more of: fibroblast growth factor I (FGF-I), FGF-II, FGF-9, leukocyte inhibitory factor (LIF), insulin, insulin growth factor I (IGF-I), IGF-II, platelet-derived growth factor AA (PDGF-AA), PDGF-BB, PDGF-AB, stromal derived factor-2 (SDF-2), pituitary thyroid hormone (PTH), growth hormone, hepatocyte growth factor (HGF), epithelial growth factor (EGF), transforming growth factor-α (TGFα) and hedgehog proteins. A most preferred growth factor protein for use with the product of the present invention is FGF-I.

[0052] In one embodiment of the present invention, the mixture of chondrogenesis-enhancing proteins can also include one or more serum proteins. As used herein, serum proteins are any of a group of proteins that is known to be a component of serum. A serum protein is not a member of the TGFβ superfamily, a bone matrix protein or a growth factor, as described herein. Preferably, the chondrogenesis-enhancing proteins include, in increasing preference, at least one, two, three, and most preferably four different serum proteins. Serum proteins suitable for use with the product of the present invention include one or more of albumin, transferrin, α2-Hs GlycoP, IgG, α1-antitrypsin, β2-microglobulin, Apo A1 lipoprotein (LP) and Factor XIIIb. Preferably, serum proteins suitable for use with the product of the present invention include one or more of albumin, transferrin, Apo A1 LP and Factor XIIIb.

[0053] In one embodiment of the present invention, a mixture of chondrogenesis-enchancing proteins suitable for use in a cartilage repair product of the present invention includes the following proteins: TGFβ1, TGFβ2, TGFβ3, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, CDMP, FGF-I, osteocalcin, osteonectin, MGP, BSP, lysyloxidase, and cathepsin L pre. In another embodiment, a suitable mixture of chondrogenesis-enchancing proteins includes the following proteins: TGFβ1, TGFβ2, TGFβ3, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, CDMP, FGF-I, osteocalcin, osteonectin, MGP, BSP, lysyloxidase, cathespin L pre, albumin, transferrin, Apo A1 LP and Factor XIIIb. In yet another embodiment, a suitable mixture of chondrogenesis-enchancing proteins includes the mixture of proteins referred to herein as bone protein (BP), which is defined herein as partially-purified protein mixture from bovine long bones as described in Poser and Benedict, WO 95/13767, incorporated herein by reference in its entirety. As described in Poser and Benedict, WO 95/13767: “Bone growth factor was isolated rom the cortical diaphyses of bovine long bones. The marrow and soft tissue was cleaned from the long bones, and the bones were pulverized and demineralized in 1.0 normal (N) hydrochloric acid at a 1:13 weight to volume ratio for 16 hours at 25° C. The bone particles were washed in distilled water and then extracted in a buffered solution comprising of 4 N guanidine hydrochloride buffered with 0.1 N Tris, pH 7.6 at a concentration of 3 milliliters of buffered solution per gram of original powdered bone. The bone was extracted for 48 h at 15° C. The extracted bone particles were then passed through a series of chromatographic purification steps as described in U.S. application Ser. No. 07/689,459 to extract bone growth factor having bone inductive effect at doses less than 35 microgram (μg).”

[0054] According to the present invention, the relative proportions of the proteins in the mixture of chondrogenesis-inducing proteins are any proportions which are sufficient for the mixture, at a concentration of 100 ng/ml or less, to induce a statistically significant increase in A₅₉₅in an Alcian Blue assay performed with ATDC5 cells as described below. In one embodiment, the percentage of TGFβ superfamily members within the mixture ranges between about 0.1% to about 50% of the total mixture, and more preferably, between about 0.5% and about 25%, and even more preferably, between about 1% and about 10% of the total mixture. The percentage of growth factors within the mixture ranges between about 0.01% to about 50% of the total mixture, and preferably, between about 0.05% and about 25%, and even more preferably, between about 0.1% and about 10% of the total mixture. The percentage of serum and bone matrix protein components, either separately or combined, ranges between about 20% to about 98%, and preferably between about 40% to about 98%, and even more preferably between about 80% to about 98%. In another embodiment of the invention, the mixture of chondrogenesis-inducing proteins contains at least BMP-3, BMP-2 and TGFβ1, wherein the quantity of BMP-3 in the mixture is about 2-6 fold greater than the quantity of BMP-2 and about 10-30 fold greater than the quantity of TGFβ1 in the mixture.

[0055] In one embodiment of the cartilage repair product of the present invention, each of the chondrogenesis-enhancing proteins in the cartilage-inducing composition is provided by the composition either: (1) directly as a protein that is associated with the matrix, or (2) as a recombinant nucleic acid molecule associated with the matrix, such recombinant nucleic acid molecule encoding the protein and being operatively linked to a transcription control sequence such that the protein can be expressed under suitable conditions. Therefore, a cartilage-inducing composition of the present invention can include proteins, recombinant nucleic acid molecules, or a combination of proteins and recombinant nucleic acid molecules, such composition providing the chondrogenesis-enhancing proteins described above.

[0056] According to the present invention, a chondrogenesis-enhancing protein can be obtained from its natural source, produced using recombinant DNA technology, or synthesized chemically. As used herein, a chondrogenesis-enhancing protein can be a full-length protein (i.e., in its full-length, naturally occurring form), any homologue of such a protein, any fusion protein containing such a protein, or any mimetope of such a protein. The amino acid sequences for chondrogenesis-enhancing proteins disclosed herein, as well as nucleic acid sequences encoding the same are known in the art and are publicly available, for example, from sequence databases such as GenBank. Such sequences can therefore be obtained and used to produce proteins and recombinant nucleic acid molecules of the present invention.

[0057] A homologue is defined as a protein in which amino acids have been deleted (e.g., a truncated version of the protein, such as a peptide or fragment), inserted, inverted, substituted and/or derivatized (e.g., by glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitation, amidation and/or addition of glycosylphosphatidyl inositol). A homologue of a chondrogenesis-enhancing protein is a protein having an amino acid sequence that is sufficiently similar to a naturally occurring chondrogenesis-enhancing protein amino acid sequence that the homologue has substantially the same or enhanced biological activity compared to the corresponding naturally occurring protein.

[0058] As used herein, a mimetope (also referred to as a synthetic mimic) of a chondrogenesis-enhancing protein according to the present invention refers to any compound that is able to mimic the activity of such a chondrogenesis-enhancing protein, often because the mimetope has a structure that mimics the chondrogenesis-enhancing protein. Mimetopes can be, but are not limited to: peptides that have been modified to decrease their susceptibility to degradation; anti-idiotypic and/or catalytic antibodies, or fragments thereof; non-proteinaceous immunogenic portions of an isolated protein (e.g., carbohydrate structures); and synthetic or natural organic molecules, including nucleic acids. Such mimetopes can be designed using computer-generated structures of naturally occurring chondrogenesis-enhancing protein. Mimetopes can also be obtained by generating random samples of molecules, such as oligonucleotides, peptides or other organic or inorganic molecules, and screening such samples by affinity chromatography techniques using the corresponding binding partner.

[0059] According to the present invention, a fusion protein is a protein that includes a chondrogenesis-enhancing protein-containing domain attached to one or more fusion segments. Suitable fusion segments for use with the present invention include, but are not limited to, segments that can: enhance a protein's stability; enhance the biological activity of a chondrogenesis-enhancing protein; and/or assist purification of a chondrogenesis-enhancing protein (e.g., by affinity chromatography). A suitable fusion segment can be a domain of any size that has the desired function (e.g., imparts increased stability, imparts enhanced biological activity to a protein, and/or simplifies purification of a protein). Fusion segments can be joined to amino and/or carboxyl termini of the chondrogenesis-enhancing protein-containing domain of the protein and can be susceptible to cleavage in order to enable straight-forward recovery of a chondrogenesis-enhancing protein. Fusion proteins are preferably produced by culturing a recombinant cell transformed with a fusion nucleic acid molecule that encodes a protein including the fusion segment attached to either the carboxyl and/or amino terminal end of a chondrogenesis-enhancing protein-containing domain. Preferred fusion segments include a metal binding domain (e.g., a poly-histidine segment); an immunoglobulin binding domain (e.g., Protein a; Protein G; T cell; B cell; Fc receptor or complement protein antibody-binding domains); a sugar binding domain (e.g., a maltose binding domain); and/or a “tag” domain (e.g., at least a portion of β-galactosidase, a strep tag peptide, other domains that can be purified using compounds that bind to the domain, such as monoclonal antibodies).

[0060] Preferably, a mixture of chondrogenesis-enhancing proteins according to the present invention is capable, when cultured together with ATDC5 cells for seven days at a concentration of about 100 ng/ml or less, of inducing a statistically significant increase in A₅₉₅in an Alcian Blue assay performed with the ATDC5 cells. In a preferred embodiment, a mixture of chondrogenesis-enhancing proteins is capable of inducing a significant increase in A₅₉₅in an Alcian Blue assay performed with the ATDC5 cells when cultured under the above conditions at a concentration of about 50 ng/ml or less, and more preferably, about 25 ng/ml or less, and even more preferably, about 10 ng/ml or less. As used herein, a statistically significant increase is defined as an increase in A₅₉₅as compared to a control, in which the probability of such an increase being due to chance is p<0.05, and more preferably, p<0.001, and even more preferably, p<0.005.

[0061] According to the present invention, an ATDC5 Alcian Blue assay is known in the art and is described, for example, in von Schroeder et al., 1994, Teratology50:54-62. For the purposes of determining whether a mixture of chondrogenesis-enhancing proteins meets the requirements of being capable of inducing a significant increase in A₅₉₅in an Alcian Blue assay performed with the ATDC5 cells when cultured with such cells at a given concentration (e.g., 100 ng/ml), the following protocol can be used.

[0062] Murine ATDC5 cells were deposited by T. Atsumi (Deposit No. RCB0565) and are publicly available from the Riken Cell Bank, 3-1-1 Koyadai, Tsukuba Science City, 305 Japan. The ATDCS cells are maintained in 100×20 mm standard tissue culture plates in Dulbecco's modified Eagle's medium (DMEM):Ham's F-12 (1:1) media that contains 5% fetal bovine serum, penicillin (50 U/ml), and streptomycin (50 mg/ml). Cultures are incubated in a humidified incubator at 37° C. and 5% CO₂. Passages 3-8 can be used to assay the activity of the mixture of proteins to be evaluated. To perform the Alcian Blue assay, first, the micromass culture technique is performed as described in Atkinson et al., 1997, ibid., with minor alterations. Briefly, trypsinized cells are resuspended in the ATDC5 culture medium described above at a concentration of about 100,000 cells/25 μl. The 25 μl spot of cells is placed in the center of a 24 well polystyrene microtiter tissue culture dish. After 1.5 hours, 1 ml of the culture media described above is added to the dish. After overnight incubation at 37° C. and 5% CO², media containing various concentrations of the mixture of chondrogenesis-inducing proteins (e.g., 100 ng/ml, 50 ng/ml, 25 ng/ml, 10 ng/ml), 5% FBS, 50 μg/ml ascorbic acid, and 10 mM β-glycerophosphate are added (Day 0), and the incubation is continued. This latter media is then replaced every 3-4 days (for a total of 2 more additions of BP). After incubation with the mixture of proteins to be tested, on Day 7, the culture media is removed and the cultures are washed three times with 1 ml of PBS. The cultures are then fixed with 10% neutral buffered formalin for 15 hours and washed twice with 0.5 N HCl. Cultures are stained for one hour at room temperature with a 0.5% Alcian Blue solution (pH 1.4). The stain is then removed and the cultures are washed with PBS to remove unbound stain. The blue stain is then extracted with guanidium HCl (4M, pH 1.7) at 70° C. for 18 hours, followed by measurement of absorption at 595 nm.

[0063] It is noted that those of skill in the art will be able to make minor modifications to the above protocol and obtain a similar outcome. Such modifications include seeding the bottom of a well with ATDC5 cells (i.e, about 25,000-50,000, not in micromass culture), using a serum substitute in the media, altering the concentration of serum, and/or omitting ascorbic acid and/or β-glycerophosphate from the media. Minor modifications to the Alcian Blue assay itself can include altering the pH of the Alcian Blue solution within the range from about pH 1 to about pH 1.4 and altering the concentration of the Alcian Blue solution within the range from about 0.05% to about 0.5%.

[0064] As discussed above, one or more of the chondrogenesis-enhancing proteins in the cartilage-inducing composition can be provided by the composition as a recombinant nucleic acid molecule associated with the cartilage repair matrix, such recombinant nucleic acid molecule encoding a chondrogenesis-enhancing protein and being operatively linked to a transcription control sequence such that the protein can be expressed under suitable conditions. A recombinant nucleic acid molecule useful in the present invention can include an isolated natural gene encoding a chondrogenesis-enhancing protein or a homologue of such a gene, the latter of which is described in more detail below. A nucleic acid molecule useful in the present invention can include one or more regulatory regions, full-length or partial coding regions, or combinations thereof.

[0065] In accordance with the present invention, an isolated nucleic acid molecule is a nucleic acid molecule that has been removed from its natural milieu (i.e., that has been subject to human manipulation) and can include DNA, RNA, or derivatives of either DNA or RNA. As such, “isolated” does not reflect the extent to which the nucleic acid molecule has been purified. An isolated nucleic acid molecule encoding a chondrogenesis-enhancing protein can be isolated from its natural source or produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification, cloning) or chemical synthesis. Isolated nucleic acid molecules can include, for example, natural allelic variants and nucleic acid molecule homologues modified by nucleotide insertions, deletions, substitutions, and/or inversions in a manner such that the modifications do not substantially interfere with the nucleic acid molecule's ability to encode a chondrogenesis-enhancing protein of the present invention or to form stable hybrids under stringent conditions with natural gene isolates. An isolated nucleic acid molecule can include degeneracies. As used herein, nucleotide degeneracies refers to the phenomenon that one amino acid can be encoded by different nucleotide codons. Thus, the nucleic acid sequence of a nucleic acid molecule that encodes a chondrogenesis-enhancing protein of the present invention can vary due to degeneracies.

[0066] A nucleic acid molecule homologue can be produced using a number of methods known to those skilled in the art (see, for example, Sambrook et al., ibid.). For example, nucleic acid molecules can be modified using a variety of techniques including, but not limited to, by classic mutagenesis and recombinant DNA techniques (e.g., site-directed mutagenesis, chemical treatment, restriction enzyme cleavage, ligation of nucleic acid fragments and/or PCR amplification), or synthesis of oligonucleotide mixtures and ligation of mixture groups to “build” a mixture of nucleic acid molecules and combinations thereof. Nucleic acid molecule homologues can be selected by hybridization with a naturally occurring gene or by screening for the function of a protein encoded by the naturally occurring nucleic acid molecule. Although the phrase “nucleic acid molecule” primarily refers to the physical nucleic acid molecule and the phrase “nucleic acid sequence” primarily refers to the sequence of nucleotides on the nucleic acid molecule, the two phrases can be used interchangeably, especially with respect to a nucleic acid molecule, or a nucleic acid sequence, being capable of encoding a chondrogenesis-enhancing protein.

[0067] Knowing the nucleic acid sequence encoding a naturally occurring chondrogenesis-enhancing protein according to the present invention allows one skilled in the art to, for example, (a) make copies of those nucleic acid molecules, and (b) obtain nucleic acid molecules including at least a portion of such nucleic acid molecules (e.g., nucleic acid molecules including full-length genes, full-length coding regions, regulatory control sequences, truncated coding regions). Such nucleic acid molecules can be obtained in a variety of ways including screening appropriate expression libraries with antibodies; traditional cloning techniques using oligonucleotide probes to screen appropriate libraries or DNA; and PCR amplification of appropriate libraries or DNA using oligonucleotide primers. Techniques to clone and amplify genes are disclosed, for example, in Sambrook et al., ibid.

[0068] According to the present invention, a nucleic acid molecule encoding a chondrogenesis-enhancing protein is operatively linked to one or more transcription control sequences to form a recombinant molecule. The phrase “operatively linked” refers to linking a nucleic acid molecule to a transcription control sequence in a manner such that the molecule is able to be expressed when transfected (i.e., transformed, transduced or transfected) into a host cell.

[0069] Transcription control sequences are sequences which control the initiation, elongation, and termination of transcription. Particularly important transcription control sequences are those which control transcription initiation, such as promoter, enhancer, operator and repressor sequences. Suitable transcription control sequences include any transcription control sequence that can function in at least one of the recombinant cells useful in the product and method of the present invention. A variety of such transcription control sequences are known to those skilled in the art. Preferred transcription control sequences include those which function in mammalian, bacterial, insect cells, and preferably in mammalian cells.

[0070] One or more recombinant nucleic acid molecules encoding a chondrogenesis-enhancing protein can be used to produce the protein. In one embodiment, the protein is produced by expressing a recombinant nucleic acid molecule under conditions effective to produce the protein. A preferred method to produce an encoded protein is by transforming a host cell with one or more recombinant molecules to form a recombinant cell. Suitable host cells to transform include any mammalian cell that can be transformed. Host cells can be either untransfected cells or cells that are already transformed with at least one nucleic acid molecule. Host cells useful in the present invention can be any cell capable of producing a chondrogenesis-enhancing protein. In a preferred embodiment, the host cell itself is useful in enhancing chondrogenesis. A particularly preferred host cell includes a fibrochondrocyte, a chondrocyte, and a mesenchymal precursor cell.

[0071] According to the method of the present invention, a host cell can be transformed with a recombinant nucleic acid molecule encoding a chondrogenesis-enhancing protein in vitro or in vivo. Transformation of a recombinant nucleic acid molecule into a cell in vitro can be accomplished by any method by which a nucleic acid molecule can be inserted into the cell. Transformation techniques include, but are not limited to, transfection, electroporation, microinjection, lipofection, adsorption, and protoplast fusion. The resulting recombinant cell can then be associated with the cartilage repair matrix of the present invention by any suitable method to provide the chondrogenesis-enhancing proteins.

[0072] Recombinant nucleic acid molecules can be delivered in vivo and associated with the cartilage repair matrix in a variety of methods including, but not limited to, (a) administering a naked (i.e., not packaged in a viral coat or cellular membrane) nucleic acid molecule (e.g., as naked DNA or RNA molecules, such as is taught, for example in Wolff et al., 1990, Science 247, 1465-1468); (b) administering a nucleic acid molecule packaged as a recombinant virus or a recombinant cell (i.e., the nucleic acid molecule is delivered by a viral or cellular vehicle), whereby the virus or cell is associated with the cartilage repair matrix; or (c) administering a recombinant nucleic acid molecule associated with the cartilage repair matrix via a delivery vehicle such as a liposome or nanosphere delivery system described herein.

[0073] As discussed above, a recombinant nucleic acid molecule encoding a chondrogenesis-enhancing protein can be associated with the cartilage repair matrix as a recombinant virus particle. A recombinant virus includes a recombinant molecule that is packaged in a viral coat and that can be expressed in an animal after administration. Preferably, the recombinant molecule is packaging-deficient. A number of recombinant virus particles can be used, including, but not limited to, those based on alphaviruses, poxviruses, adenoviruses, herpesviruses, and retroviruses. When administered to an animal, a recombinant virus infects cells at the site of administration of the cartilage repair product and directs the production of a chondrogenesis-enhancing protein.

[0074] Suitable liposomes for use as a delivery vehicle for a recombinant nucleic acid in vivo include any liposome. Preferred liposomes of the present invention include those liposomes commonly used in, for example, gene delivery methods known to those of skill in the art. Methods for preparation of liposomes and complexing nucleic acids with liposomes are well known in the art.

[0075] As described in detail below, a cartilage-inducing composition is associated with a cartilage repair matrix, such that the cartilage repair matrix serves, in one capacity, as a delivery vehicle for the composition to be delivered to the site of a cartilage lesion. Suitable methods for associating a cartilage-inducing composition containing chondrogenesis-enhancing proteins and/or recombinant nucleic acid molecules encoding such proteins with a cartilage repair matrix include any method which allows the proteins and/or recombinant nucleic acid molecules to be delivered to a site of cartilage repair together with a cartilage repair matrix such that the cartilage repair product is effective to repair and/or regenerate cartilage at the site. Such methods of association include, but are not limited to, suspension of the composition within the cartilage repair matrix, freeze-drying of the composition onto a surface of the matrix and suspension within the matrix of a carrier/delivery formulation containing the composition. Additionally, the cartilage-inducing composition can be associated with the matrix prior to placement of the product into a cartilage lesion (i.e., the association of the composition with matrix occurs ex vivo) or alternatively, a cartilage repair matrix can first be implanted into a lesion, followed by association of the cartilage-inducing composition with the matrix, such as by injection into or on top of the matrix (i.e., the association of the composition with matrix occurs in vivo). A cartilage-inducing composition can contain additional delivery formulations or carriers which enhance the association of the composition with the matrix, which enhance the delivery of the composition to the appropriate cells and tissue at the site of the lesion, and which assist in controlling the release of the factors in the composition at the site of the lesion. Suitable delivery formulations include carriers, which, as used herein, include compounds that increase the half-life of a cartilage-inducing composition in the treated animal. Suitable carriers include, but are not limited to, polymeric controlled release vehicles, biodegradable implants, liposomes, bacteria, viruses, oils, cells, esters, and glycols.

[0076] One embodiment of the present invention is a controlled release formulation that is capable of slowly releasing a composition of the present invention into an animal. As used herein, a controlled release formulation comprises a cartilage-inducing composition of the present invention in a controlled release vehicle. Suitable controlled release vehicles include, but are not limited to, biocompatible polymers, other polymeric matrices, capsules, microcapsules, microparticles, bolus preparations, osmotic pumps, diffusion devices, liposomes, lipospheres, and transdermal delivery systems. Other controlled release formulations of the present invention include liquids that, upon association with the matrix or upon administration to an animal, form a solid or a gel in situ. Such controlled release vehicles are preferably associated with the cartilage repair matrix by one of the above-described methods. Preferred controlled release formulations are biodegradable (i.e., bioerodible).

[0077] A preferred controlled release formulation of the present invention is capable of releasing a composition of the present invention at the site of a cartilage lesion of a treated animal at a constant rate sufficient to attain therapeutic dose levels of the chondrogenesis-enhancing proteins provided by the composition to result in enhancement of chondrogenesis at the lesion. A particularly preferred controlled release vehicle according to the present invention is a nanosphere delivery vehicle.

[0078] A nanosphere delivery vehicle according to the present invention includes the nanosphere delivery vehicle described in copending PCT Application No. PCT/EP 98/05100, which is incorporated herein by reference in its entirety. In a preferred embodiment, such a delivery vehicle includes polymer particles having a size of less than 1000 nm and being loaded with between 0.001% and 17% by weight of the cartilage-inducing composition. The nanospheres have an in vitro analytically determined release rate profile with an initial burst of about 10% to about 20% of the total amount of the composition over a first 24 hour period, and a long time release rate of a least 0.1% per day during at least seven following days.

[0079] A cartilage-inducing composition useful in the cartilage repair product of the present invention can also include one or more pharmaceutically acceptable excipients. As used herein, a pharmaceutically acceptable excipient refers to any substance suitable for associating a cartilage-inducing composition with a cartilage repair matrix and maintaining and delivering the components of the composition (e.g., proteins and/or recombinant nucleic acid molecules) to the appropriate cells at a suitable in vivo site (i.e., a cartilage lesion). Preferred pharmaceutically acceptable excipients are capable of maintaining a nucleic acid molecule in a form that, upon arrival of the nucleic acid molecule at the delivery site, the nucleic acid molecule is capable of expressing a chondrogenesis-enhancing protein either by being expressed by a recombinant cell or by entering a host cell at the site of the lesion and being expressed by the cell. A suitable pharmaceutically acceptable excipient is capable of maintaining a protein in a form that, upon arrival of the protein at the delivery site, the protein is biologically active such that chondrogenesis at the site is enhanced. Examples of pharmaceutically acceptable excipients include, but are not limited to water, phosphate buffered saline, Ringer's solution, dextrose solution, serum-containing solutions, Hank's solution, other aqueous physiologically balanced solutions, oils, esters and glycols. Aqueous carriers can contain suitable auxiliary substances required to approximate the physiological conditions of the recipient, for example, by enhancing chemical stability and isotonicity. Particularly preferred excipients include non-ionic diluents, with a preferred non-ionic buffer being 5% dextrose in water (DW5). Suitable auxiliary substances include, for example, sodium acetate, sodium chloride, sodium lactate, potassium chloride, calcium chloride, and other substances used to produce phosphate buffer, Tris buffer, and bicarbonate buffer. Auxiliary substances can also include preservatives, such as thimerosal, m- or o-cresol, formalin and benzol alcohol. Cartilage-inducing compositions of the present invention can be sterilized by conventional methods and/or lyophilized.

[0080] A cartilage-inducing composition is present in the cartilage repair product of the present invention at a concentration that is effective to induce, at the site of a cartilage lesion, one or more of: cellular infiltration, cellular proliferation, angiogenesis, and cellular differentiation to type II collagen-producing chondrocytes. Preferably, a cartilage-inducing composition is present in the cartilage repair product of the present invention at a concentration that is effective to induce cartilage repair and/or regeneration at the site of a cartilage lesion. When the chondrogenesis-enhancing proteins are provided by the cartilage-inducing protein directly as a protein, the cartilage-inducing composition is typically provided at a concentration of from about 0.5% to about 33% by weight of the cartilage repair product. More preferably, the cartilage-inducing composition is provided at a concentration of from about 1% to about 20% by weight of the cartilage repair product. When one or more of the chondrogenesis-enhancing proteins are provided by the composition as a recombinant nucleic acid molecule, an appropriate concentration of a nucleic acid molecule expressing one chondrogenesis-enhancing protein is an amount which results in at least about 1 pg of protein expressed per mg of total tissue protein at the site of delivery per μg of nucleic acid delivered, and more preferably, an amount which results in at least about 10 pg of protein expressed per mg of total tissue protein per μg of nucleic acid delivered; and even more preferably, at least about 50 pg of protein expressed per mg of total tissue protein per μg of nucleic acid delivered; and most preferably, at least about 100 pg of protein expressed per mg of total tissue protein per μg of nucleic acid delivered. One of skill in the art will be able to adjust the concentration of proteins and/or nucleic acid molecules in the composition depending on the types and number of proteins to be provided by the composition, and the delivery vehicle used.

[0081] In another embodiment of the cartilage repair product of the present invention, a cartilage-inducing composition can also contain a factor that non-covalently attaches to one or more of any of the chondrogenesis-enhancing proteins or recombinant nucleic acid molecules in the composition and thus, modify the release rate of the factor. Such factors include, but are not limited to, any ground substance or other polymeric substance. As used herein, a ground substance is defined as the non-living matrix of connective tissue, which includes natural polymers and proteoglycans. Natural polymers include, but are not limited to collagen, elastin, reticulin and analogs thereof. Proteoglycans include, but are not limited to any glycosaminoglycan-containing molecules, and include chondroitin sulfate, dermatan sulphate, heparan sulphate, keratan sulphate and hyaluronan. Preferred ground substances include, but are not limited to, type I collagen, type II collagen, type III collagen, type IV collagen and hyaluronic acid. Preferred other polymeric substances include, but are not limited to, poly(lactic acid) and poly(glycolic acid).

[0082] In a further embodiment, the cartilage-inducing composition can include one or more types of cells which are provided to further enhance chondrogenesis at the site of the cartilage lesion. Such cells include, but are not limited to, fibrochondrocytes, chondrocytes, mesenchymal precursors, and any other cell that can serve as a chondrocyte precursor. Such cells can be associated with the composition and the matrix by any of the methods described above. In one embodiment, at least some of the cells are transformed with a recombinant nucleic acid molecule encoding a chondrogenesis-enhancing protein to form a recombinant cell.

[0083] The cartilage repair product of the present invention also includes a cartilage repair matrix. The cartilage repair matrix is the component of the cartilage repair device which provides a vehicle for delivery of the cartilage-inducing composition to the site of a cartilage lesion and a suitable scaffold upon which cartilage repair and regeneration can occur. In a preferred embodiment, the cartilage repair matrix is bioresorbable.

[0084] According to the present invention, a cartilage repair matrix can be formed of any material that is suitable for in vivo use, and which provides the above-described characteristics of a cartilage repair matrix for use with a cartilage-inducing composition of the present invention. The matrix can be formed of materials which include, but are not limited to, synthetic polymers and/or a ground substance. Preferred ground substances include natural polymers and proteoglycans. Natural polymers include, but are not limited to collagen, elastin, reticulin and analogs thereof. Proteoglycans include, but are not limited to, any glycosaminoglycan-containing molecules. Particularly preferred glycosaminoglycans include chondroitin sulfate, dermatan sulphate, heparan sulphate, keratan sulphate and hyaluronan. Other preferred ground substances include, but are not limited to, type I collagen, type II collagen, type III collagen, type IV collagen and hyaluronic acid. Preferred synthetic polymers include poly(lactic acid) and poly(glycolic acid).

[0085] In one embodiment of the present invention, the cartilage repair matrix includes collagen. Preferably, the matrix contains from about 20% to about 100% collagen by dry weight of the matrix, and more preferably, from about 50% to about 100% collagen by dry weight of the matrix, and even more preferably, from about 75% to about 100% collagen by dry weight of the matrix. In one embodiment, a suitable cartilage repair matrix includes collagen from bovine tendon.

[0086] A cartilage repair matrix suitable for use in the present invention can include a material as described above which is in any suitable form for use in repairing a cartilage lesion, including a sponge, a membrane, a film or a gel. In one embodiment, a suitable cartilage repair matrix includes autograft tissue, allograft tissue and/or xenograft tissue.

[0087] A cartilage repair product of the present invention is useful for repairing a variety of defects in cartilage, including both tears and segmental defects in both vascular and avascular cartilage tissue. The product is particularly useful for repairing defects in hyaline (e.g., articular) and/or fibrocartilage (e.g., meniscal). Examples of various types of cartilage tears and segmental defects for which the cartilage repair product of the present invention can be used are illustrated in FIGS. 1-4. Briefly, FIG. 1 shows a meniscal radial tear (FIG. 1A); a meniscal triple bucket handle tear (FIG. 1C); and a longitudinal tear in the avascular area of a meniscus (FIG. 4A). A meniscal segmental lesion is illustrated in FIGS. 2A and 2B. FIG. 3A additionally schematically illustrates a cross section of the meniscus, which includes vascular, semi-vascular and avascular regions for which, prior to the present invention, only tears in the vascular region were repairable.

[0088] Therefore, since cartilage defects (i.e., lesions) can occur in a variety of shapes, sizes, and locations, a cartilage repair matrix suitable for use in a cartilage repair product of the present invention is of a shape and size sufficient to conform to a specific defect in the cartilage of the patient to be treated. Preferably, the cartilage repair matrix, when used in the repair of a cartilage defect, achieves a geometry at the defect site that is suitable to provide a therapeutic benefit to the patient. Such a therapeutic benefit can be any improvement in a patient's health and well being that is related to a correction of the cartilage defect, and preferably, the therapeutic benefit includes the repair of the defect such that the natural configuration of the cartilage is at least partially restored.

[0089] In the case of a cartilage tear, the cartilage repair matrix is typically configured as a sheet. The sheet is preferably of a shape and size suitable for insertion into the tear and to cover the entire tear surface. One embodiment of a sheet type matrix is schematically illustrated in FIG. 3B. The use of such a matrix to repair an avascular longitudinal tear is schematically illustrated in FIG. 4B. Preferably, the matrix provides an immediate mechanical repair of the tear, a surface for interacting the cartilage-inducing composition with the natural cartilage tissue, and a scaffold upon which chondrogenesis can occur. Additionally, the matrix preferably imparts upon the product a mechanical stability sufficient to allow the product to be anchored into the lesion. Prior to the present invention, a tear in meniscal vascular cartilage as shown in FIGS. 1A and 1C was repaired by suture repair and resection as illustrated in FIGS. 1B and 1D, respectively. Additionally, prior to the present invention, if the tear occurred in avascular cartilage tissue (or in semi-vascular tissue as shown in FIG. 3A), the tear would have been considered “irreparable”. The cartilage repair product of the present invention advantageously allows for the repair of tears in both avascular and vascular cartilage and when used in vascular cartilage, the product enhances the rate and quality of the repair as compared to previously used products and methods, such as shown in FIGS. 1B and 1D.

[0090] In the embodiment in which the cartilage repair matrix is configured as a sheet, the matrix preferably has a thickness of from about 0.1 mm to about 3 mm, and more preferably, from about 0.5 mm to about 2 mm. The thickness can, of course, be varied depending on the configuration of the tear which is to be repaired. In this embodiment, the matrix can be prepared by applying an aqueous dispersion of matrix material into a mold, for example, wherein the mold sets the appropriate thickness for the sheet. Such a method is described in Example 4. In a preferred embodiment, the matrix is prepared from an aqueous dispersion of from about 0.2% to about 4% collagen by weight, and more preferably, the matrix is prepared from an aqueous dispersion of from about 0.5% to about 3% collagen by weight.

[0091] In the case of a segmental defect in cartilage (i.e., any defect that is larger and of a different shape than a tear), the cartilage repair matrix is typically configured to achieve a suitable geometry that repairs the defect, and includes matrices which are configured to replace damaged cartilage which has been removed. Prior to the present invention, repair of a segmental defect or indeed, any defect which occurred in the avascular region of meniscal tissue, typically involved the removal of the damaged tissue, such as by partial or complete excision of the meniscus (i.e., a meniscectomy). Excision was then sometimes followed by a replacement prosthetic meniscus, but until the present invention, such methods were unable to regenerate endogenous-type cartilage in the avascular region of the meniscus. The cartilage repair matrix useful for segmental cartilage defects (i.e., “non-tear” defects) is preferably of a shape and size suitable for providing an immediate mechanical repair of the defect, a surface for interacting the cartilage-inducing composition with the natural cartilage tissue, and a scaffold upon which chondrogenesis can occur. Additionally, the matrix preferably imparts upon the product a mechanical stability sufficient to allow the product to be anchored into the lesion. A cartilage repair matrix suitable for use for repairing segmental defects in the cartilage repair product of the present invention is described in detail in U.S. Pat. No. 5,681,353 to Li et al. which is incorporated herein by reference in its entirety. Other preferred cartilage repair matrices are described in the Examples section. In one embodiment, a cartilage repair matrix used to repair a segmental defect in meniscal cartilage contains a porous ground substance composite which includes collagen and has the shape and mechanical characteristics suitable to repair a meniscus lesion.

[0092] In one embodiment, a cartilage repair matrix suitable for use in the repair of segmental defects has a tapered shape. Such a matrix typically varies in thickness from about 0.5 mm to about 3 mm at its thinnest region to from about 4 mm to about 10 mm at its thickest region. Such a matrix typically has a density of from about 0.07 to about 0.5 grams matrix per cm³, and more preferably, from about 0.1 to about 0.25 grams matrix per cm³, wherein g/cm³represents the number of grams in a cubic centimeter of matrix. FIGS. 2A and 2B illustrate a cartilage repair matrix configured to repair a segmental defect in a meniscus.

[0093] In a preferred embodiment, the cartilage repair matrix of the present invention is porous, which enhances the ability of the matrix to serve as a delivery vehicle for the cartilage-inducing composition and particularly, as a scaffold for chondrogenesis, such as by allowing for the ingrowth of cells into the matrix. Preferably, the pore size is sufficient to maintain the desired mechanical strength of the matrix, while allowing sufficient ingrowth of cells for regeneration of cartilage at the lesion. The porosity of the matrix can vary depending on the configuration of the matrix, but typically, the matrix has a pore size of from about 10 μm to about 500 μm. When the matrix is configured to repair a tear defect, the pore size is typically from about 10 μm to about 100 μm. When the matrix is configured to repair a segmental defect, the pore size is typically from about 50 μm to about 500 μm.

[0094] When the cartilage repair matrix is configured as a sheet, it is preferably not cross-linked. When the cartilage repair matrix is configured to repair a segmental defect, however, the matrix can be cross-linked, such as by artificial cross-linking methods, although such cross-linking is not required. A cartilage repair matrix can be cross-linked by any suitable agent which includes, but is not limited to, formaldehyde, glutaraldehyde, dimethyl suberimidate, carbodiimides, multi-functional epoxides, succinimidyls, Genipin, poly(glycidyl ether), diisocyanates, acyl azide, ultraviolet irradiation, dehydrothermal treatment, tris(hydroxymethyl)phosphine, ascorbate-copper, glucose-lysine and photo-oxidizers. In one embodiment, a cartilage repair matrix is cross-linked with an aldehyde.

[0095] Another embodiment of the present invention relates to a product for the repair of cartilage lesions which includes: (a) a cartilage repair matrix as described in detail above; and, (b) a cartilage inducing composition associated with the matrix which includes cells that have been cultured with a mixture of the chondrogenesis-enhancing proteins as previously described herein. Specifically, the mixture of chondrogenesis-enhancing proteins include: (1) at least a first and second transforming growth factor β (TGFβ) superfamily protein, wherein the first TGFβ superfamily protein is different from the second TGFβ superfamily protein; and, (2) at least one protein from a family of proteins selected from the group of: growth factor proteins and bone matrix proteins. The chondrogenesis-enhancing proteins have been described in detail above. Preferably, the cells to be cultured with the mixture of proteins are cells which are involved in chondrogenesis, and include, but are not limited to, fibrochondrocytes, chondrocytes, mesenchymal precursors, and any other cell that can serve as a chondrocyte precursor. Such cells are preferably cultured in vitro prior to their association with a cartilage repair matrix, under conditions effective to allow the cells to interact with the proteins and initiate chondrogenesis by the cells. Effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH and oxygen conditions that permit interaction of the proteins and cells and initiation of chondrogenesis processes by the cells. An effective, medium refers to any medium in which a cell is cultured provide such a result. Such medium typically comprises an aqueous medium having assimilable carbon, nitrogen and phosphate sources, and appropriate salts, minerals, metals and other nutrients, such as vitamins. Cells can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes, and petri plates. Culturing can be carried out at a temperature, pH and oxygen content appropriate for a the cell. Such culturing conditions are within the expertise of one of ordinary skill in the art. In another aspect of this embodiment of the present invention, a cartilage repair matrix is cultured in vitro together with the cells and mixture of proteins prior to implantation into a cartilage lesion in vivo. In a further embodiment, the cells that have been cultured with the mixture of proteins can be associated with the cartilage repair matrix in conjunction with additional chondrogenesis-enhancing proteins and/or recombinant nucleic acid molecules encoding such proteins as described above.

[0096] Another embodiment of the present invention relates to a product for the repair of vascular and avascular meniscus tears. Such a product includes: (a) a cartilage repair matrix comprising collagen and configured as a sheet; and, (b) a cartilage-inducing composition associated with the matrix for provision of chondrogenesis-enhancing proteins. The chondrogenesis-enhancing proteins include: (i) at least a first and second transforming growth factor β (TGFβ) superfamily protein, wherein the first TGFβ superfamily protein is different from the second TGFβ superfamily protein; and, (ii) at least one protein from a family of proteins selected from the group of growth factor proteins and bone matrix proteins. The chondrogenesis-enhancing proteins, are characterized in that, when cultured together with ATDC5 cells for seven days at a concentration of about 100 ng/ml or less, they induce a statistically significant increase in A₅₉₅in an Alcian Blue assay performed with the cells. Each of the chondrogenesis-enhancing proteins is provided as a protein or by a recombinant nucleic acid molecule encoding the protein operatively linked to a transcription control sequence. In one embodiment, the cartilage repair matrix is formed of a collagen sponge. When the lesion to be repaired is in the avascular region, the product can additionally include a time controlled delivery formulation as described in detail above.

[0097] Yet another embodiment of the present invention relates to a method for repair of cartilage lesions. The method includes the steps of implanting and fixing into a cartilage lesion a product which includes: (a) a cartilage repair matrix; and (b) a cartilage-inducing composition associated with the matrix for provision of chondrogenesis-enhancing proteins. As described in detail above, the chondrogenesis-enhancing proteins include: (i) at least a first and second transforming growth factor β (TGFβ) superfamily protein, wherein the first TGFβ superfamily protein is different from the second TGFβ superfamily protein; and, (ii) at least one protein from a family of proteins selected from: growth factor proteins and/or bone matrix proteins. Also as described above, the chondrogenesis-enhancing proteins, when cultured together with ATDC5 cells for seven days at a concentration of about 100 ng/ml or less, induce a statistically significant increase in A₅₉₅in an Alcian Blue assay performed with the cells. Additionally, each of the chondrogenesis-enhancing proteins is provided as a protein or by a recombinant nucleic acid molecule encoding the protein operatively linked to a transcription control sequence.

[0098] The method of the present invention is useful for repairing any of the cartilage lesions described above, including both articular and meniscal cartilage lesions, and both avascular and vascular defects. The step of implanting is performed using surgical techniques known in the art, and typically involves inserting the repair product directly into the tear when the matrix is configured as a sheet, and involves a more complex process of removing damaged tissue and implanting the repair product when the matrix is configured to repair a segmental defect. The step of fixing can include attaching the product to the cartilage at the site of the lesion by any means suitable for attaching a matrix as described herein to cartilage or tissue surrounding the cartilage in vivo. Such a means for attaching can include, but is not limited to application of bioresorbable sutures, application of non-resorbable sutures, press-fitting, application of arrows, application of nails, or application of a T-fix suture anchor device. Examples 5 and 7-9 describe the method of the present invention when the cartilage repair matrix is configured as a sheet and used to repair meniscal and articular cartilage. Example 6 describes the method of the present invention which is used to repair meniscal and articular cartilage when the repair matrix is configured to repair a segmental defect. FIGS. 2A and 2B illustrate the implantation and fixation of a cartilage repair product into a segmental defect in meniscal cartilage. FIGS. 4A and 4B schematically illustrate the repair of a longitudinal tear in avascular meniscal tissue using a cartilage repair product configured as a sheet.

[0099] In one embodiment of the method of the present invention, a cartilage lesion which is a segmental defect is repaired by using two cartilage repair products of the present invention. In this embodiment, a segmental defect, and preferably a meniscal segmental defect, is repaired by trimming damaged cartilage tissue away to form a suitable interface for implantation of the repair devices. A first cartilage repair product having a matrix configured as a sheet is implanted and fixed along the defect (e.g., along the meniscal rim when the defect is in vascular cartilage and this cartilage has been removed). A second cartilage repair matrix configured to replace the segmental defect, or a cartilage repair product having a matrix configured to replace the segmental defect, is implanted and fixed to the first product configured as a sheet. The sheet provides an interface in which cells can quickly infiltrate and react with the cartilage repair composition. In this embodiment, the cartilage repair product configured as a sheet contains the cartilage repair composition as described herein, and the cartilage repair product configured to repair the segmental defect may or may not be associated with the cartilage repair composition, as deemed necessary by the surgeon. FIG. 8 schematically illustrates the concurrent use of both a cartilage repair product configured as a sheet and a cartilage repair product configured to repair a segmental defect.

[0100] Another embodiment of the method of the present invention relates to a method for repair of avascular meniscus lesions. The method includes the steps of implanting and fixing into a cartilage lesion of the avascular region of a meniscus a cartilage repair product as described herein, wherein the cartilage repair matrix is configured as a sheet, and wherein the cartilage repair product further includes a time controlled delivery formulation which is associated with the matrix in conjunction with the cartilage-inducing composition.

[0101] Another embodiment of the method of the present invention is a method for enhanced repair of vascular meniscus lesions. The method includes the steps of implanting and fixing into a cartilage lesion in the vascular region of a meniscus a cartilage repair product as described herein, wherein the cartilage repair matrix includes collagen and is configured as a sheet. The present inventors have discovered that the use of the cartilage repair product of the present invention to repair vascular lesions measurably enhances the rate of repair of the vascular lesion as compared to the rate of repair of a meniscus lesion repaired in the absence of the product. According to the present invention, a measurable enhancement of the rate of repair is any measurable improvement in the time between Day 0 of the repair (i.e., the day the product is implanted into the patient) and the day on which it is determined that suitable cartilage tissue growth has occurred at the lesion as compared to a vascular lesion that is repaired in the absence of the product of the present invention. Suitable cartilage tissue growth is defined as an initial indication of enhanced blood vessel formation, production of fibrochondrocytes, induction of cellular infiltration into the product, induction of cellular proliferation, and production of cellular and spatial organization to form a three-dimensional tissue that more nearly represents endogenous cartilage tissue from the site of the lesion. In a preferred embodiment, the product of the present invention measurably enhances the rate of repair of a vascular cartilage lesion, as compared to a vascular lesion that is repaired in the absence of the product of the present invention, by at least 25%, and more preferably, by at least about 50%, and more preferably by at least about 100%.

[0102] In addition, the use of the cartilage repair product of the present invention to repair vascular cartilage lesions results in a measurable enhancement of the quality of repair of the vascular lesion as compared to the quality of repair of a lesion repaired in the absence of the product. A measurable enhancement in the quality of repair of the vascular lesion is defined as any measurable improvement in quality of cartilage formation at the site of the lesion, with an improvement being defined as development of a more normal cartilage tissue, which can be indicated by enhanced blood vessel formation, production of fibrochondrocytes, induction of cellular infiltration into the product, induction of cellular proliferation, and production of cellular and spatial organization to form a three-dimensional tissue that more nearly represents endogenous cartilage tissue from the site of the lesion.

[0103] More particularly, the quality of repair of cartilage tissue according to the present invention can be evaluated as follows.

[0104] The quality of meniscal cartilage repair (i.e., fibrocartilage cartilage) can be evaluated by histological analysis of the tissue at the repair site on a scale of 0 to 4 based on the following parameters: I. Histological cartilage staining in the defect, II. Cellular infiltration into the collagen implant, and III. Integration into the endogenous meniscus. These scales according to the present invention are defined as follows:

[0105] Preferably, a measurable enhancement in the quality of repair of a meniscal lesion is defined as a higher score in at least one of the parameters defined above as I, II and III, with a “4” the highest score in each parameter, than a lesion repaired in the absence of the product of the present invention. More preferably, a measurable enhancement in the quality of repair of a meniscal lesion is defined as a score of at least 1, and more preferably at least 2 and more of at least 3, and most preferably at least 4 in at least one of the parameters defined above as I, II and/or III, as compared to a lesion repaired in the absence of the product of the present invention.

[0106] The quality of osteochondral repair (articular cartilage defects) can be evaluated by histological analysis of the tissue at the repair site on a scale of 0 to 4 based on the following parameters: I. defect filling with bone, II. thickness of the repaired cartilage, and III. repaired cartilage integration with the endogenous cartilage. These scales according to the present invention are defined as follows:

[0107] Preferably, a measurable enhancement in the quality of repair of an osteochondral lesion is defined as a higher score in at least one of the parameters defined above as I, II and III, with “4” being the highest score, than a lesion repaired in the absence of the product of the present invention. More preferably, a measurable enhancement in the quality of repair of an osteochondral lesion is defined as a score of at least 1, and more preferably at least 2 and more preferably at least 3, and most preferably at least 4 in at least one of the parameters defined above as I, II or III, as compared to a lesion repaired in the absence of the product of the present invention.

[0108] It is to be noted that the term “a” or “an” entity refers to one or more of that entity; for example, a protein refers to one or more proteins, or to at least one protein. As such, the terms “a” (or “an”), “one or more” and “at least one” can be used interchangeably herein. It is also to be noted that the terms “comprising”, “including”, and “having” can be used interchangeably.

[0109] The following examples are provided for the purposes of illustration and are not intended to limit the scope of the present invention.

[0110] The following example demonstrates that a naturally derived mixture of proteins isolated from demineralized bovine bones (BP) induces spheroid formation and chondrogenesis in vitro in the mesenchymal precursor cell lines, 10T1/2 and C₂C₁₂.

[0111] Murine C3H/10T1/2 (ATCC No. CCL-226) embryonic mesenchymal stem cells and C₂C₁₂adult myoblast cells (ATCC No. CRL-1772; derived from leg muscle) were obtained from the American Type Tissue Collection. 10T1/2 and C₂C₁₂cells were proliferated in the presence of 10% and 15% FBS, respectively. Micromass cultures were performed as follows. Briefly, trypsinized cells were resuspended in media containing FBS at a concentration of 10⁷cells/ml, and 10 μl of cells were placed in the center of a 24 well microtiter tissue culture dish. After 2-3 hours at 37° C., 1 ml of DMEM (for 10T1/2) or 1:1 DMEM:F-12 (for C₂C₁₂) media containing 1% Nutridoma and various concentrations of BP (prepared as described in Poser and Benedict, WO 95/13767, ibid.) were added. BP was present for the initial 48 hours and was not subsequently added.

[0112] The results presented in Table 1 show that BP concentrations greater than or equal to 20 ng/ml induced spheroid formation in >90% of 10T1/2 micromass cultures. In >90% of C₂C₁₂micromass cultures, BP concentrations greater than or equal to 100 ng/ml induce spheroid formation.

[0113] For C₂C₁₂cells, the resulting spheroids were placed in Bouin's fixative for 24 hours and histology and immunocytochemistry was performed. The myosin F1.652 antibody was purchased from the Iowa Hybridoma Bank.

[0114] More specifically, for histology, spheroids were dehydrated and fixed for 20 minutes in absolute methanol at 4° C. Fixed sections were infiltrated and polymerized using the glycol methacrylate embedding technique. The polymerized plugs were then sectioned at 5 mm thickness using a JB-4 Sorvall microtome. Sections were mounted on silate-coated slides and stained with 0.2% Azure II at pH 1.

[0115] For immunocytochemistry, spheroids were snap-frozen in a 100% isopentane/dry ice solution, sectioned at 5 μm thickness using a Reichert-Jung cryostat, and mounted on silane-coated slides. Frozen sections were then fixed in 1% paraformaldehyde for 20 min. rinsed in 0.05 M Tris-Cl (pH 7.4), blocked with 1% BSA for 20 min. at room temperature, and incubated with either goat or mouse primary antibody for 1 hour at room temperature. After rinsing, the sections were blocked with 10% normal rabbit serum for 20 min. at room temperature. The sections were then treated with 1:2,000 biotinylated, rabbit antigoat IgG followed by incubation with a 1:100 streptavidin-conjugated alkaline phosphatase. Each incubation was for 30 min. at room temperature. The mouse-antibody treated slides were incubated with an unlabeled rabbit antimouse (rat absorbed) antibody and then incubated with an alkaline phosphatase antialkaline phosphatase antibody. Each incubation was for 30 minutes at room temperature. The reaction was visualized with an alkaline phosphatase substrate, New Fuchsin. The slides were counterstained with Gill's #1 hematoxylin for 10 sec., dehydrated through graded alcohols, and cleared in Americlear xylene substitute. Polyclonal goat primary antibodies for type II collagen were diluted 1:200 in 0.5 M Tris-HCl (pH 7.4), 1% BSA, and 1% sodium azide prior to use.

[0116] Chondrogenesis is indicated by positive staining of sulfated proteoglycans with Azure at pH 1, morphology of a rounded cell type encompassed by a territorial matrix, and the presence of type II collagen. The collagen quantity was subjectively graded in duplicate samples with a ‘−’ representing no stain detected and increasing ‘+’s reflecting increasing amounts and intensity of stain. Samples lacking the primary antibody received a ‘−’ score.

[0117] Table 2 shows that BP (500 ng/ml) induces chondrogenic markers in 10T1/2 cells over a period of 28 days.

[0118] Table 3 shows that in C₂C₁₂cells, BP (1000 ng/ml) inhibits myosin production (an indicator of muscle) and induces type II collagen production (an indicator of cartilage) after three days in culture.

[0119] The following example demonstrates that a naturally derived mixture of proteins isolated from demineralized bovine bones (BP) inhibits myogenesis in a dose dependent manner in C₂C₁₂cells.

[0120] For myogenesis inhibition experiments, 25,000 C₂C₁₂cells were seeded in triplicate to a 24 well plate in DMEM media that contained 15% FBS. The next day (day 0), this media was replaced with media containing 1% Nutridoma +/− various concentrations of BP. Media was replaced every 2-3 days.

[0121] BP concentrations (0, 10, 20, 60, 100, 400, 1000, or 3000 ng/ml) were tested for the effect on C₂C₁₂myotube formation. As shown in Table 4, a BP concentration of 10 ng/ml produced no morphological differences when compared to cultures lacking BP. However, at 20 and 60 ng/ml, BP substantially decreased the number of myotubes. Complete myotube inhibition was observed at BP concentrations above or equal to 100 ng/ml.

[0122] The following example demonstrates that a naturally derived mixture of proteins isolated from demineralized bovine bones (BP) quantitatively induces chondrogenesis of ATDC5 cells in a dose dependent manner.

[0123] In this experiment, 100,000 ATDC5 cells/25 μl media (DMEM:Ham's F-12 (1:1); 5% FBS; 50 U/ml penicillin; 50 mg/ml streptomycin) were seeded in triplicate to a 24 well plate (micromass culture). ATDC5 cells were deposited by T. Atusmi and are publicly available as Deposit No. RCB0565 from the Riken Cell Bank, 3-1-1 Koyadai, Tsukuba Science City, 305 Japan. After 1½ hour, 1 ml of the media was added. The next day, the same media containing various concentrations of BP, 50 μg/ml ascorbic acid, 5% FBS, and 10 mM β-glycerophosphate were added (day 0). This latter media was replaced every 3-4 days. To those skilled in the art, slight modifications can be made to this protocol to obtain a similar result. For example, fewer cells (e.g., 25,000-50,000) could be seeded to the bottom of the well (e.g., not in micromass culture). Media containing a serum substitute, such as 1% Nutridoma (Boehringer Mannheim) could be used in place of serum. Also, the cultures may contain different concentrations of serum or may contain or lack the ascorbic acid and/or the β-glycerophosphate.

[0124] To measure cartilage matrix production, an Alcian Blue staining method was used as previously described (von Schroeder et al, 1993, ibid.) with minor modifications. Briefly, after incubation with BP as described above, the culture media was removed and the cultures were washed three times with 1 ml of PBS. The cultures were then fixed with 10% neutral buffered formalin for 15 hours and washed twice with 0.5 N HCl. Cultures were stained for 1 hour at room temperature with a 0.5% Alcian Blue solution (pH 1.4). The stain was removed and the cultures were washed with PBS to remove the unbound stain. The blue stain was then extracted with guanidium HCl (4M, pH 1.7) at 70° C. for 18 hours, followed by measurement of absorption at 595 nm. The cultures were performed in triplicate. Using this method, glycosaminoglycan quantification was demonstrated to be proportional to ³⁵SO₄incorporation (Lau, et al., 1993, Teratology47:555-563).

[0125] To determine the effect of BP on chondrogenesis, 0, 10, 20, 60, 100, 400, and 1000 ng/ml BP were added to chondrogenic ATDC5 micromass cultures and, after 7 days, the cultures were stained with Alcian Blue. Qualitative, microscopic evaluation showed that cultures lacking BP contained no positive staining and cultures containing low BP concentrations (10, 20, and 60 ng/ml) showed diffuse staining. With higher doses of BP (100, 400 and 1000 ng/ml BP), both staining intensity and the number of positively stained focal cell areas increased in a dose dependent manner. Cultures treated with 400 and 1000 ng/ml BP contained positively stained focal clusters consisting of rounded cells encompassed by a territorial matrix (data not shown). Negatively stained areas were also present. Quantitation of Alcian Blue staining (FIG. 5) revealed that BP concentrations as low as 10 ng/ml significantly increased Alcian Blue content compared to control cultures lacking BP (p<0.0005). Maximal Alcian Blue staining was observed at 400 ng/ml BP.

[0126] BP also stimulates chondrogenesis in cultures containing a serum substitute, Nutridoma, and this stimulation occurs over 14 days. In the absence of BP, very little Alcian Blue staining was observed at days 7 and 14 (FIG. 6). However, cultures containing 1000 ng/ml BP significantly (p<0.001) stimulated chondrogenesis 8.5 and 11.2 fold at days 7 and 14, respectively, compared to cultures lacking BP (FIG. 6).

[0127] To determine whether other fractions isolated from bone have a similar activity as BP, the following experiment was performed. BP was purified as previously described (Poser and Benedict, PCT Publication No. WO95/13767). However, after the BP containing fractions were collected from the HPLC, two fractions, IBP and PIBP, that were more hydrophobic than BP, were also collected. The chondrogenic activity of BP, IBP and PIBP was tested on ATCD5 cells. FIG. 7 shows that both IBP and PIBP induce significantly less (p<0.005) chondrogenesis than BP.

[0128] To determine whether different lots of BP differed significantly for chondrogenesis, two lots were tested at two concentrations. Table 5 shows that the chondrogenic activity is not significantly different between two different BP lots.

[0129] The following example describes a procedure for preparation of a cartilage repair product of the present invention, configured as a sheet.

[0130] Bovine tendon type I collagen, obtained from ReGen Biologics, Inc., was placed in one syringe. The appropriate volume of 10 mM acetic acid was placed in another syringe. For sponges that contain BP, the appropriate dose of BP was placed in the syringe containing 10 mM acetic acid. The syringes were coupled, and the contents of each syringe were mixed to produce a 2% collagen (w/w) slurry. After an overnight incubation, the preparation was placed in molds of appropriate thickness, frozen at −20° C. for more than 4 hours, and lyophilized until dry. Approximate implant dimensions for the sheet are length: 15.5 mm; width: 4.8 mm; and thickness: 1.2 mm.

[0131] The following example describes the procedure for surgical implantation of a cartilage repair product of the present invention which is configured as a sheet.

[0132] Adult castrated mail Capra hircus goats weighing 50-70 pounds were used in this study. After pre-anesthesia administration, the goats were anesthetized with an inhalation anesthetic (IsoFlurane). The hind limbs of each goat were clipped, scrubbed, and draped in preparation for the surgical procedure. An incision was made over the medial aspect of the knee (stifle) joint. The sartorial fascia was dissected to expose the medial collateral ligament (MCL). A wedge shaped femoral bone block centered on the MCL was created with an oscillating saw and osteotome. The block was then drilled and tapped for later reattachment using a 3.5 mm bicortical screw. The bone block was then elevated to expose the surface of the medial meniscus leaving the coronary ligaments intact. A longitudinal tear was placed in the central, avascular portion of the meniscus using a scalpel. Four treatments were tested in the defect as follows. Group I: nothing was placed in the defect, but the defect was repaired using 1-0 non-absorbable sutures and a horizontal mattress technique (i.e., a conventional repair method); Group II: a collagen sheet was sutured into the defect using the method of Group I; and, Group III: a collagen sheet containing 35 μg BP was sutured into the defect using the method of Group I.

[0133] After reduction and fixation of the bone block with a screw and washer, the sutures were tied outside the capsulate. The subcutaneous tissues were then closed with absorbable suture and subcuticular 3-0 prolene skin closure. After recovery from anesthesia, the goats were placed in holding pens and then placed in an outdoor facility with unrestricted activity.

[0134] After 8 weeks, the animals were euthanized with a pentobarbitol sodium solution (100 mg/kg IV) solution. The menisci were fixed in a 10% neutral buffered formalin solution. Tissue sections for the menisci were then stained with H+E.

[0135] Group III defects that contained 35 μg BP were filled with tissue from the superior to inferior sides of the meniscus and the reparative tissue was integrated into the endogenous meniscus tissue. In addition, the endogenous meniscus tissue immediately adjacent to the defect was more cellular than normal endogenous meniscus, indicating a reparative response. In contrast, Group I and II defects remained empty; no evidence of healing was observed.

[0136] The following example describes the procedure for surgical implantation of a cartilage repair product of the present invention which is configured to replace a segmental meniscal cartilage defect.

[0137] If a meniscal repair can not be accomplished (i.e., such as by using a product of the present invention configured as a sheet) due to the severity of the tear or poor quality of the tissue, then preparation of the meniscal rim is undertaken by removing the torn portions of the cartilaginous tissue. A cartilage repair product of the present invention can be configured to replace the segmental meniscal cartilage defect, thereby serving to both regenerate and repair meniscal cartilage. The surgical procedure, in the absence of the cartilage repair product of the present invention, has been previously described by Stone and Rosenberg (cites). The following procedure describes a modification of the procedure of Stone and Rosenberg, incorporating the use of a cartilage repair product of the present invention.

[0138] Briefly, the torn, fragmented pieces of native meniscal cartilage are removed, and the attachment sites for meniscal horns are anatomically placed or the natural peripheral rim and horns are preserved. Using the surgical techniques described by Stone and Rosenberg, a cartilage repair matrix configured as a collagen meniscus implant (CMI; having the shape and mechanical characteristics of the meniscus), which is associated with a cartilage repair composition according to the present invention, is implanted and fixed to the meniscal rim. During the surgery, the periphery of the meniscal implant must be attached securely enough to permit axial and rotational loads, and the surrounding capsule and ligaments of the knee joint must be neither excessively violated nor constrained by the fixation technique.

[0139] In some cases, a cartilage repair product of the present invention which is configured as a sheet can be fixed to the meniscus rim using the same surgical procedures as described for the CMI product above. This combination use of the cartilage repair product of the present invention is illustrated schematically in FIG. 8. Use of such a sheet optimizes the integration between the meniscal rim and the CMI by providing a thin, porous collagen containing the cartilage repair composition. The sheet provides an interface in which cells can quickly infiltrate and react with the cartilage repair composition. In this embodiment, the cartilage repair product configured as a sheet contains the cartilage repair composition of the present invention, and the cartilage repair product configured as a CMI may or may not be associated with the cartilage repair composition.

[0140] The following experiment demonstrates that a cartilage repair product of the present invention enhances and induces meniscus regeneration in both vascular and avascular meniscal tissue in vivo.

[0141] One hind leg was operated on in 1-2 year old female sheep. Briefly, the surgical approach was to make an incision in the skin and subcutaneous tissues from the distal fourth of the femur distally to the proximal fourth of the tibia. A bone block that contained the origin of the medial collateral ligament was then removed from the femoral condyle. The tibia was abducted and externally rotated. Using a 3 mm dermal biopsy punch (Miltex), two defects were created in the vascular/avascular zone of the medial meniscus. The Collagen Meniscus Implant (CMI) was obtained from ReGen Biologics. Using a 4 mm dermal biopsy punch (Miltex), CMI sponges were cut to size. The 4 mm implants were press-fit into the meniscal defects. CMI either lacked or contained BP (69 μg/mg collagen). To produce CMI containing BP, the following protocol was followed. A saturating volume of 10 mM acetic acid solution that contained BP was added to the 4 mm CMI sponges. The sponges were placed in a humidified environment at room temperature for 30 minutes, frozen at −20° C. for more than 4 hours, and then lyophilized until dry.

[0142] The animals were sacrificed after four weeks and the menisci were fixed in a 10% neutral buffered formalin solution. Tissue sections were stained with Azure B at pH 1 and 4.5. Cells infiltrated the CMI matrix both in the presence or absence of BP, however, histology results demonstrated that only undifferentiated fibroblast cells were present in the implant that lacked BP (data not shown). In contrast, the BP impregnated implant contained differentiated fibrochondrocytes (data not shown). Fibrochondrocytes were identified by rounded cell shape and cells surrounded by positively staining lacunae. In addition, CMI sponges that lacked BP contained cells that did not stain with antibodies against type II collagen. In contrast, CMI sponges that contained BP contained cells that stained positively with antibodies against type II collagen.

[0143] The following example demonstrates that a cartilage repair product of the present invention enhances and induces articular cartilage repair in vivo.

[0144] Five 8 month old (skeletally mature) New Zealand White Rabbits were used in this experiment. Bilateral defects, 8 mm long, 3 mm wide, and 3 mm deep, were produced in the trochlear groove of each knee. For each rabbit, a 2% collagen sponge 8 mm long, 3 mm wide and 3 mm deep, was placed in each knee defect, with one knee receiving the sponge in the absence of BP, and one knee receiving the sponge containing 40-45 μg of BP. Autologous fibrin was prepared from 50 ml of rabbit blood using the alcohol precipitation method according to Kjaergard et al., 1992, Surg. Gynecol. Obst.175(1): 72-73. Approximately 20 μl of fibrinogen was placed on top of the collagen and was polymerized with 10 μl bovine thrombin (Gentrak). After 2 months, the animals were sacrificed.

[0145] A 500 μm slab of the defect area was embedded in glycol methacrylate after fixation in cold methanol and stained with azure B at pH 1 and 4.5. An adjacent 500 μm slab was fixed and decalcified with EDTA in 4% paraformaldehyde and embedded in paraffin. Serial sections were digested with chondroitinase ABC and stained with hemotoxilin and eosin.

[0146] Histologically, defects that contained the collagen sponge only produced more immature tissue than defects that contained collagen+BP. Without added BP, the bone region was filled with fibrocartilage and fibroblastic tissue. In contrast, defects containing BP were filled with osteoblasts and contained a bone morphology. The cartilage area, in the absence of BP, was often 3 mm dep, rather than the normal 0.5-0.7 mm deep. With BP added, the cartilage surface thickness was nearly identical to the endogenous cartilage thickness. In addition, polarized light microscopy revealed that the BP treated defects contained a more mature collagen fiber architecture than defects that did not contain BP.

[0147] While various embodiments of the present invention have been described in detail, it is apparent that modifications and adaptations of those embodiments will occur to those skilled in the art.