Methods and compositions for the repair of articular cartilage defects in mammals

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FIELD OF THE INVENTION

This invention relates to methods and compositions for the repair of articular cartilage defects in a mammal. The methods and synthetic cartilage compositions of the invention are particularly useful in treatment of partial-thickness and full-thickness articular cartilage defects.

BACKGROUND OF THE INVENTION

Cartilage is a hyperhydrated structure with water comprising 70% to 80% of its weight. The remaining 20% to 30% comprises type II collagen and proteoglycan. The collagen usually accounts for 70% of the dry weight of cartilage (in "Pathology" (1988) Eds. Rubin & Farber, J. B. Lippincott Company, PA. pp. 1369-1371). Proteoglycans are composed of a central protein core from which long chains of polysaccharides extend. These polysaccharides, called glycosaminoglycans, include: chondroitin-4-sulfate; chondroitin-6-sulfate; and keratan sulfate. Cartilage has a characteristic structural organization consisting of chondrogenic cells dispersed within an endogenously produced and secreted extracellular matrix. The cavities in the matrix which contain the chondrocytes are called cartilage lacunae. Unlike bone, cartilage is neither innervated nor penetrated by either the vascular or lymphatic systems (Clemente (1984) in "Gray's Anatomy, 30.sup.th Edit," Lea & Febiger).

Three types of cartilage are present in a mammal and include: hyaline cartilage; fibrocartilage and elastic cartilage (Rubin and Farber, supra). Hyaline cartilage consists of a gristly mass having a firm, elastic consistency, is translucent and is pearly blue in color. Hyaline cartilage is predominantly found on the articulating surfaces of articulating joints. It is found also in epiphyseal plates, costal cartilage, tracheal cartilage, bronchial cartilage and nasal cartilage. Fibrocartilage is essentially the same as hyaline cartilage except that it contains fibrils of type I collagen that add tensile strength to the cartilage. The collagenous fibers are arranged in bundles, with the cartilage cells located between the bundles. Fibrocartilage is found commonly in the anulus fibrosus of the invertebral disc, tendinous and ligamentous insertions, menisci, the symphysis pubis, and insertions of joint capsules. Elastic cartilage also is similar to hyaline cartilage except that it contains fibers of elastin. It is more opaque than hyaline cartilage and is more flexible and pliant. These characteristics are defined in part by the elastic fibers embedded in the cartilage matrix. Typically, elastic cartilage is present in the pinna of the ears, the epiglottis, and the larynx.

The surfaces of articulating bones in mammalian joints are covered with articular cartilage. The articular cartilage direct contact of the opposing bone surfaces and the near frictionless movement of the articulating bones relative to one another (Clemente, supra).

Two types of articular cartilage defects are commonly observed in mammals and include full-thickness and partial-thickness defects. The two-types of defects differ not only in the extent of physical damage but also in the nature of repair response each type of lesion elicits.

Full-thickness articular cartilage defects include damage to the articular cartilage, the underlying subchondral bone tissue, and the calcified layer of cartilage located between the articular cartilage and the subchondral bone. Full-thickness defects typically arise during severe trauma of the joint or during the late stages of degenerative joint diseases, for example, during osteoarthritis. Since the subchondral bone tissue is both innervated and vascularized, damage to this tissue is often painful. The repair reaction induced by damage to the subchondral bone usually results in the formation of fibrocartilage at the site of the full-thickness defect. Fibrocartilage, however, lacks the biomechanical properties of articular cartilage and fails to persist in the joint on a long term basis.

Partial-thickness articular cartilage defects are restricted to the cartilage tissue itself. These defects usually include fissures or clefts in the articulating surface of the cartilage. Partial-thickness defects are caused by mechanical derangements of the joint which in turn induce wearing of the cartilage tissue within the joint. In the absence of innervation and vasculature, partial-thickness defects do not elicit repair responses and therefore tend not to heal. Although painless, partial-thickness defects often degenerate into full-thickness defects.

Repair of articular cartilage defects with suspensions of isolated chondrocytes has been attempted in a variety of animal models. See for example: Bentley, et al. (1971) Nature 230:385-388; Langer et al. (1974) J. Bone Joint Surg. 56-A:297-304; Green (1977) Clin. Orthop. 124:237-250; and Aston et al. (1986) J. Bone Joint Surg. 68-B:29-35). During transplantation, the cell suspensions may be retained in the defect behind a piece of periosteal tissue that has been previously attached to the surface of the normal cartilage tissue. The rate of successful implantation using cell suspensions was found to be about 40%. It is believed that chondrocytes transplanted in this manner lose their viability during transplantation and that the procedure may result in the formation of fibrocartilage or islands of cartilage embedded in fibrous tissue at the site of the defect.

Three alternative approaches have been developed in an attempt to improve the success rate in treating mammalian articular cartilage defects. In the first approach, synthetic carrier matrices containing dispersed allogeneic chondrocytes may be implanted into the cartilage defect. The implanted chondrocytes hopefully produce and secrete components of the extracellular matrix thereby to form articular cartilage at the site of the defect in situ. In the second approach, synthetic carrier matrices containing chemotactic and mitogenic growth factors may be implanted into the cartilage defect. The growth factors hopefully induce the influx into, and the proliferation of chondrocyte progenitor cells within the matrix. The chondrocyte progenitor cells differentiate subsequently into chondrocytes that in turn secrete components of the extracellular matrix thereby to form articular cartilage at the site of the defect in situ. In the third approach, synthetic cartilage tissue may be grown in vitro and implanted subsequently into the cartilage defect.

In the first approach, the synthetic matrices or biological resorbable immobilization vehicles may be impregnated with allogeneic chondrocytes. A variety of synthetic carrier matrices have been used to date and include: three-dimensional collagen gels (U.S. Pat. No. 4,846,835; Nishimoto (1990) Med. J. Kinki University 15; 75-86; Nixon et al. (1993) Am. J. Vet. Res. 54:349-356; Wakitani et al. (1989) J. Bone Joint Surg. 71B:74-80; Yasui (1989) J. Jpn. Ortho. Assoc. 63:529-538); reconstituted fibrin-thrombin gels (U.S. Pat. Nos. 4,642,120; 5,053,050 and 4,904,259); synthetic polymer matrices containing polyanhydride, polyorthoester, polyglycolic acid and copolymers thereof (U.S. Pat. No. 5,041,138); and hyaluronic acid-based polymers (Robinson et al. (1990) Calcif. Tissue Int. 46:246-253).

The introduction of non-autologous materials into a patient, however, may stimulate an undesirable immune response directed against the implanted material. Such an immune response has been observed in rabbit models (Yoshinao (1990) J. Jpn. Orth. Assoc. 64:835-846. In addition, there is evidence to suggest that neo-cartilage may be formed around the periphery of the implant thereby preventing integration of the implant into the cartilage defect. See for example, Messner (1994) 40.sup.th Annual Meeting Orth. Res. Soc., New Orleans p. 239; and Nixon et al. (1994) 40.sup.th Annual Meeting Orth. Res. Soc., New Orleans p. 241. Monitoring the formation and development of the resulting synthetic cartilage in situ can be difficult to perform and usually involves an arthroscopic or open joint examination. Furthermore, implants containing synthetic polymer components may be unsuitable for repairing large cartilage defects since polymer hydrolysis in situ may inhibit the formation of cartilage and/or its integration into the defect.

In the second approach, the defect may be filled with a biocompatible, biodegradable matrix containing growth factors to stimulate the influx of chondrocyte progenitor cells into the matrix in situ. The matrices optimally contain pores of sufficient dimensions to permit the influx into, and proliferation of the chondrocyte progenitor within the matrix. The matrix also may contain differentiating growth factors to stimulate the differentiation of chondrocyte progenitor cells into chondrocytes. The resulting chondrocytes hopefully secrete extracellular matrix components thereby to form cartilage at the site of the defect in situ. See for example, U.S. Pat. Nos. 5,206,023; 5,270,300; and EP 05 30 804 A1. This approach, however, may have problems similar to those associated with the first approach, hereinabove.

In the third approach, chondrocytes may be cultured in vitro thereby to form synthetic cartilage-like material. The resulting cartilage may be implanted subsequently into the cartilage defect. This type of approach has the advantage over the previous methods in that the development of the synthetic cartilage material may be monitored prior to implantation. In addition, the resulting cartilage may be characterized biochemically and morphologically prior to implantation. Two general procedures have been developed for growing synthetic cartilage in vitro. These include growing chondrogenic cells in either an anchorage-dependent or an anchorage-independent manner.

In the anchorage-independent manner, the chondrogenic cells may be cultured as colonies within an agarose gel. See for example: Benya et al. (1982) Cell 30:215-224; Aydlotte et al. (1990) in Methods and Cartilage Research Chapter 23:pp. 90-92; Aulthouse et al. (1989) In Vitro Cellular and Developmental Biology 25:659-668; Delbruck et al. (1986) Connective Tissue Res. 15:1550-172; and Bohme et al. (1992) J. Cell Biol. 116:1035-1042. Heretofore, only small pieces of cartilage tissue of undefined shape have been prepared using this approach. Furthermore, the resulting cartilage remains embedded within a gel matrix making it unsuitable for implantation into mammals. Alternatively, in another anchorage-independent method, chondrocytes may be cultured as colonies in suspension culture. See for example, Franchimont et al. (1989) J. Rheumatol. 16:5-9; and Bassleer et al. (1990) in "Methods and Cartilage Research", Academic Press Ltd., Chapter 24. As with the gel approach, the resulting particles containing synthethic cartilage-like material may be small and of undefined shape thus making the particles unsuitable for implantation and repair of a predetermined articular cartilage defect.

In the anchorage-dependent method, primary cultures of chondrogenic cells isolated from primary tissue may be grown as monolayers attached to the surface of a cell culture flask. See for example: Yoshihashi (1983) J. Jpn. Ortho. Assoc. 58:629-641; and U.S. Pat. No. 4,356,261. The primary cells derived directly from explant tissue remain capable of producing and secreting extracellular components characteristic of natural cartilage, specifically, type II collagen and sulfated proteoglycans. However, it was observed that after passaging and proliferating the cells as monolayers, by serially passaging the cells, the cells dedifferentiate and lose their ability to secrete type II collagen and sulfated proteoglycans (Schlitz et al., (1973) Cell Differentiation 1:97-108; Mayne et al. (1975) Proc. Natl. Acad. Sci. USA 72:4511-4515; Mayne et al. (1976) Proc. Natl. Acad. Sci. USA 73:1674-1678; Okayama et al. (1976) Proc. Natl. Acad. Sci. USA 73:3224-3228; Pacifici & Holtzer (1977) Am. J. Anat. 150:207-212; Pacifici et al. (1977) Cell 11:891-899; West et al. (1979) Cell 17:491-501; von der Mark (1980) Curr. Top. Dev. Biol. 14:199-225; Oegama and Thompson (1981) J. Biol. Chem. 256:1015-1022; Benya & Schaffer, supra). Consequently, until now it has not been possible to prepare large patches of articular cartilage from small pieces of biopsy tissue using the anchorage-dependent procedures disclosed in U.S. Pat. No. 4,356,261 and Yoshihashi (supra) since the chondrocytes, following the proliferation as monolayers, dedifferentiate and stop secreting a cartilage-specific extracellular matrix.

It is an object of the invention to provide a variety of methods and compositions for the repair of articular cartilage defects in a mammal. Specifically, it is an object of the invention to provide a method for preparing in vitro large quantities of synthetic cartilage from small samples of biopsy tissue for the repair of articular cartilage defects in a mammal. The proliferated but undifferentiated chondrogenic cells may be cultured under conditions that stimulate the secretion of extracellular components characteristic of cartilage. Another object is to provide a method for producing a patch of synthetic cartilage of predetermined volume in vitro. Yet another object is to provide methodologies for preparing synthetic cartilage from chondrocytes isolated from a variety of tissues including pre-existing cartilage tissue and perichondrial tissue. Still another object is to provide methodologies for the repair of articular cartilage defects in a mammal using the compositions described herein.

These and other objects and features of the invention will be apparent from the description, drawings, and claims which follow.

SUMMARY OF THE INVENTION

It has been discovered that large quantities of three-dimensional, multi cell-layered synthetic cartilage may be prepared in vitro from small biopsy samples by the practice of the invention described herein. Also, it has been discovered that synthetic cartilage patches of pre-determined volume may be prepared in vitro by culturing chondrogenic cells in an anchorage-independent manner in a pre-shaped well. Furthermore, it has been discovered that chondrogenic cells useful in the practice of the instant invention may be isolated from a variety of tissues, for example: pre-existing cartilage; perichondrial tissue; or bone marrow, and expanded in vitro prior to cartilage formation. These discoveries enable the preparation of patches of synthetic cartilage for the repair of articular cartilage defects in a mammalian joint.

Broadly, the invention comprises a method for preparing in vitro a synthetic cartilage patch for the repair of a cartilage defect in a mammal. The method includes: (1) seeding denuded chondrogenic cells, proliferated ex vivo, into a pre-shaped well having a cell contacting, cell abhesive surface; and (2) culturing the proliferated chondrogenic cells in the well for a time sufficient to permit the cells to secrete an extracellular matrix thereby to form a three-dimensional, multi cell-layered patch of synthetic cartilage. The resulting synthetic cartilage, preferably synthetic articular cartilage, contains chondrogenic cells dispersed within an endogenously produced and secreted extracellular matrix. The resulting synthetic cartilage patch may be used subsequently for the repair of an articular cartilage defect in a mammal.

As used herein, the term "synthetic cartilage", is understood to mean any cartilage tissue produced in vitro that contains chondrogenic cells dispersed within an endogenously produced and secreted extracellular matrix. The extracellular matrix is composed of collagen fibrils (predominantly fibrils of type II collagen), sulfated proteoglycans, for example, chondroitin-6-sulfate and keratan sulfate, and water.

As used herein, the term "synthetic articular cartilage", is understood to mean any cartilage tissue produced in vitro that biochemically and morphologically resembles the cartilage normally found on the articulating surfaces of mammalian joints.

As used herein, the term "chondrogenic cell", is understood to mean any cell which, when exposed to an appropriate stimuli, may differentiate into a cell capable of producing and secreting components characteristic of cartilage tissue, for example, fibrils of type II collagen, and the sulfated proteoglycans, chondroitin-6-sulfate and keratan sulfate.

As used herein, the term "denuded cell" is understood to mean any cell that has been isolated from a disaggregated tissue containing such a cell. The tissue of interest may be enzymatically and/or mechanically disaggregated in order to release the denuded cells.

As mentioned hereinabove, the cells are cultured in a pre-shaped well having a cell contacting, cell abhesive surface. The cell abhesive surface discourages the chondrogenic cells from attaching to the cell contacting surface of the well. The use of such a well having a cell contacting, cell abhesive surface is a critical aspect of the instant invention. Heretofore, it has been observed that chondrogenic cells expanded by serially passaging the cells as monolayers usually lose their ability to secrete type II collagen and sulfated proteoglycans. It now has been discovered that the undifferentiated, proliferated chondrogenic cells when cultured in such a well redifferentiate and once again start to secrete cartilage specific type II collagen and sulfated proteoglycans.

It is contemplated that the actual dimensions of the well may be pre-determined when the actual size and shape of the cartilage defect to be repaired is known. For example, the well may be dimensioned such that the resulting cartilage may interfit directly into the cartilage defect. Alternatively, the synthetic cartilage patch may be "trimmed" mechanically to the appropriate size and shape by the surgeon prior to insertion into the defect during a surgical procedure. It is appreciated that synthetic cartilage patches prepared in such a manner have the additional advantage over patches prepared as anchorage-dependent primary explant cultures in that the patches may be easily removed from the well obviating the use of enzymatic or other mechanical procedures. Such procedures may affect deleteriously the biochemical and/or biomechanical properties of the resulting cartilage patch.

Cell abhesive surfaces may be prepared by coating the cell contacting surface of a well with a reagent that discourages cell attachment. Preferred reagents include, but are not limited to, silicon based reagents, for example, dichlorodimethylsilane or polytetrafluoroethylene based reagents, for example, Teflon.RTM.. Alternatively, the well may be cast in a material that naturally discourages the attachment of chondrogenic cells. Preferred materials include, but are not limited to, agarose, glass, untreated cell culture plastic and polytetrafluoroethylene, for example, Teflon.RTM.. It is contemplated that any biocompatible material or coating capable of discouraging the attachment of chondrogenic cells may be useful in the practice of the instant invention.

Chondrogenic cells useful in the practice of the invention may be isolated from essentially any tissue containing chondrogenic cells. For example, the chondrogenic cells may be isolated directly from pre-existing cartilage tissue, for example, hyaline cartilage, elastic cartilage, or fibrocartilage. Specifically, chondrogenic cells may be isolated from articular cartilage (from either weight bearing or non-weight bearing joints), costal cartilage, nasal cartilage, auricular cartilage, tracheal cartilage, epiglottic cartilage, thyroid cartilage, arytenoid cartilage and cricoid cartilage. Methods for isolating chondrogenic cells from such tissues are set forth hereinbelow. Alternatively, chondrogenic cells may be isolated from bone marrow. See for example, U.S. Pat. Nos. 5,197,985 and 4,642,120, and Wakitani et al. (1994) J. Bone Joint Surg. 76:579-591, the disclosures of which are incorporated by reference herein.

Once chondrogenic cells have been isolated from the pre-existing tissue they are proliferated ex vivo in monolayer culture using conventional techniques well known in the art. See for example, Pollack (1975) in "Readings in Mammalian Cell Culture", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, the disclosure of which is incorporated by reference herein. Briefly, the population of chondrogenic cells is expanded by culturing the cells as monolayers and by serially passaging the cells. The chondrogenic cells are passaged after the cells have proliferated to such a density that they contact one another on the surface of the cell culture plate. During the passaging step, the cells are released from the substratum. This is performed routinely by pouring a solution containing a proteolytic enzyme, i.e, trypsin, onto the monolayer. The proteolytic enzyme hydrolyzes proteins which anchor the cells on the substratum. As a result, the cells are released from the surface of the substratum. The resulting cells, now in suspension, are diluted with culture medium and replated into a new tissue culture dish at a cell density such that the cells do not contact one another. The cells subsequently reattach onto the surface of the tissue culture and start to proliferate once again. Alternatively, the cells in suspension may be cryopreserved for subsequent use using techniques well known in the art. See for example, Pollack (supra).

The cells are repeatedly passaged until enough cells have been propagated to prepare a piece of synthetic cartilage of pre-determined size. As a result, a population containing a small number of chondrogenic cells originally isolated from a biopsy sample may be expanded in vitro thereby to generate a large number of chondrogenic cells for subsequent use in the practice of the invention.

Following proliferative expansion, the chondrogenic cells are enzymatically released from the substratum to provide a suspension of cells. The cells in suspension then are diluted by the addition of cell culture medium to give a cell density of about 1.times.10.sup.5 -1.times..sup.9 proliferated chondrogenic cells per ml, or more preferably about 1.times.10.sup.6 -5.times.10.sup.8 cells per ml, and most preferably about 3.times.10.sup.6 -2.times.10.sup.8 cells per ml. The cells then are seeded into the pre-shaped well having a cell contacting, cell abhesive surface. About, 1.times.10.sup.3 -1.times.10.sup.7 cells, preferably 1.times.10.sup.4 -1.times.10.sup.6 cells, and most preferably about 5.times.10.sup.4 -5.times.10.sup.5 cells produce a piece of synthetic cartilage 1 mm.sup.3 in volume. Accordingly, the artisan may produce a patch of synthetic cartilage of pre-determined size by seeding an appropriate number of chondrogenic cells into a pre-shaped well. The cells subsequently are cultured in the well under conventional cell culture conditions well known in the art from 1 to 90 days, preferably 5 to 60 days, and most preferably 10 to 30 days thereby to induce the production and secretion of extracellular matrix. The present invention therefore enables the production of large quantities of synthetic cartilage patches from small pieces of biopsied tissue.

In a preferred embodiment, the chondrogenic cells, once proliferated ex vivo, may be seeded into a preshaped well dimensioned to determine the volume of the resulting cartilage tissue. Therefore, using the methodologies described herein, one skilled in the art may prepare synthetic cartilage of pre-determined volume for the repair of articular cartilage defects of pre-determined volume.

In another preferred embodiment, polypeptide growth factors may be added to the chondrogenic cells in the pre-shaped well to enhance or stimulate the production of cartilage specific proteoglycans and/or collagen. Preferred growth factors include, but are not limited to, transforming growth factor-.beta. (TGF-.beta.), insulin-like growth factor (IGF), platelet derived growth factor (PDGF), epidermal growth factor (EGF), acidic or basic fibroblast growth factor (aFBF or bFBF), hepatocytic growth factor (HGF), keratinocyte growth factor (KGF) the bone morphogenic factors (BMPs) including: BMP-1; BMP-2; BMP-3; BMP-4; BMP-5; and BMP-6 and the osteogenic proteins (OPs) including: OP-1; OP-2; and OP-3. In addition, it is contemplated that ascorbate may be added to the chondrogenic cells in the pre-shaped well to enhance or stimulate the production of cartilage specific proteoglycans and collagen. However, these particular compounds are not limiting. Any compound or composition capable of stimulating or inducing the production of cartilage specific proteoglycans and collagen may be useful in the practice of the instant invention.

The invention also provides methodologies for effecting the re of an articular cartilage at a pre-determined site in a mammal. The method comprises the steps of: (1) surgically implanting at the pre-determined site a piece of synthetic cartilage prepared by the methodologies described herein; and (2) permitting the synthetic cartilage to integrate into the pre-determined site. The synthetic cartilage patch may be fixed in place during the surgical procedure. This may be effected by surgically fixing the patch with sutures and/or by applying a biocompatible, bioadhesive to the surface interfacing the cartilage patch and the defect. In some instances, defective cartilage tissue may be removed prior to implantation. Although the shape of the synthetic cartilage may be dimensioned to interfit with the cartilage defect, in specific instances, for example, when the defect is large, it is contemplated that a plurality of synthetic cartilage patches may be surgically implanted into the defect.

In another preferred embodiment, the resulting synthetic cartilage patch is preferably allogenic, and more preferably autogenic in nature. Accordingly, synthetic allogenic cartilage may be prepared from biopsy tissue isolated from a mammal belonging to the same species as the recipient. Synthetic autogenic cartilage may be prepared from biopsy tissue derived from the intended recipient. In another preferred embodiment, the invention provides synthetic articular cartilage for the repair articular cartilage defects in humans. Accordingly, chondrogenic cells may be isolated from human cartilage tissue,i.e., human articular cartilage (from weight bearing and non-weight bearing joints), human costal cartilage, human nasal cartilage, human auricular cartilage, human tracheal cartilage, human epiglottic cartilage, human thyroid cartilage, human arytenoid-cartilage and human cricoid cartilage. Alternatively, the chondrogenic cells useful in the practice of the invention may be derived from human bone marrow.

The methodologies described herein are useful in the treatment of both partial-thickness and full-thickness defects of articular cartilage.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects and features of the invention, as well as the invention itself, may be more fully understood from the following description, when read together with the accompanying drawings, in which:

FIG. 1 shows a flow chart summarizing the steps in the preparation of large amounts of synthetic cartilage from small samples of biopsy tissue for the repair of cartilage defects in a mammal. Initially, tissue containing chondrogenic cells is disaggregated to release denuded chondrogenic cells. The isolated, cells then are proliferated by serially culturing and passaging the cells in monolayer culture. During monolayer culture the chondrogenic cells dedifferentiate and lose their ability to secrete cartilage specific extracellular matrix. Once the appropriate number of cells have been obtained, the proliferated cells are seeded into a pre-shaped well having a cell contacting, cell abhesive surface. The chondrogenic cells are cultured in the well for a time sufficient to allow them to redifferentiate and secrete a cartilage specific extracellular matrix thereby to form synthetic cartilage in vitro.

FIGS. 2a and 2b provide a schematic plan view and a cross-sectional illustration, respectively, of a patch of synthetic cartilage prepared in a pre-shaped well in accordance with the invention.

DETAIL DESCRIPTION OF THE INVENTION

It has been discovered that chondrogenic cells sampled from a mammal and proliferated in monolayer culture ex vivo may be cultured further in a pre-shaped well having a cell contacting, cell abhesive surface thereby to generate a three-dimensional, multi cell-layered patch of synthetic cartilage. In addition, it has been discovered that synthetic cartilage patches of pre-determined volume for use in the surgical replacement of damaged articular cartilage and subsequent integration into the joint of the recipient may be prepared in accordance with the invention. Also, it has been discovered that chondrogenic cells useful in the practice of the instant invention may be isolated from a variety of pre-existing tissues, for example, cartilage tissue and perichondrial tissue or alternatively from bone marrow. These discoveries enable preparation of potentially unlimited quantities of synthetic cartilage patches of pre-determined thickness or volume and thus provides a significant advance in the repair of articular cartilage defects in a mammal.

A flow chart summarizing the steps associated with the preparation of three-dimensional, multi cell-layered patches of synthetic cartilage is shown in FIG. 1. All of the steps described hereinbelow are preferably performed under aseptic conditions.

Briefly, tissue (10) containing chondrogenic cells (12) is disaggregated to release denuded chondrogenic cells (16) from their extracellular matrix (14). The denuded cells then are isolated and proliferated as monolayers in an undifferentiated state ex vivo (18). The passaging procedure may be repeated multiple times (n), for example up to about 7 to 10 passages until enough cells have been propagated to prepare a piece of cartilage of pre-determined size. These steps expand the number of chondrogenic cells in a population that can be used subsequently to form the three-dimensional, multi cell-layered patch of synthetic cartilage.

The proliferated but undifferentiated chondrogenic cells (20) then are seeded into a pre-shaped well (24) having a cell contacting, cell abhesive surface (22). The cell abhesive surface prevents chondrogenic cells cultured in the well from attaching to the surface of the well. The cells, deprived of anchorage, interact with one another and coalesce within hours to generate a cohesive plug of cells. The chondrogenic cells then begin to differentiate, as characterized by the production and secretion of cartilage-specific markers, i.e., type II collagen and sulfated proteoglycans. Type II collagen is found specifically in cartilage. The chondrogenic cells then are cultured in the well for time sufficient to permit the formation of a three-dimensional, multi cell-layered patch of synthetic cartilage (26). The resulting synthetic cartilage patch comprises chondrogenic cells (20) dispersed with a new, endogenously produced and secreted extracellular matrix (28). The extracellular matrix deposited during this procedure is biochemically and morphologically similar to the extracellular matrix found in natural cartilage. Specifically, the synthetic matrix comprises fibers of type II collagen, and sulfated proteoglycans such as chondroitin sulfate and keratan sulfate.

FIG. 2a is a schematic top plan view of a patch of synthetic cartilage (26) prepared in a pre-shaped well (24) in accordance with the invention. FIG. 2b is a schematic cross-sectional view of the patch of cartilage in the well of FIG. 1 taken at lines 2-2. Particulars of methods for making and using the synthetic cartilage are set forth in detail below.

I. Isolation of Tissue Containing Chondrogenic Cells

Chondrogenic cells useful in the practice of the instant invention may be sampled from a variety of sources in a mammal that contain such cells, for example, pre-existing cartilage tissue, perichondrial tissue or bone marrow.

Although costal cartilage, nasal cartilage, auricular cartilage, tracheal cartilage, epiglottic cartilage, thyroid cartilage, arytenoid cartilage and cricoid cartilage are useful sources of chondrogenic cells, articular cartilage (from either weight bearing or non-weight bearing joints) is the preferred source. Biopsy samples of articular cartilage may be readily isolated by a surgeon performing arthroscopic or open joint surgery. Procedures for isolating biopsy tissues are well known in the art and so are not described in detailed herein. See for example, "Operative Arthroscopy" (1991) by McGinty et al.,; Raven Press, New York, the disclosure of which is incorporated by reference herein.

Perichondrial tissue is the membranous tissue that coats the surface of all types of cartilage, except for articular cartilage. Perichondrial tissue provides nutrients to the chondrocytes located in the underlying unvascularized cartilage tissue. Perichondrial tissue sampled from costal (rib) cartilage of patients suffering from osteoporosis provides a source of chondrogenic cells when the normal articular cartilage is diseased or unavailable. Biopsy samples of perichondrial tissue may be isolated from the surface of costal cartilage or alternatively from the surface of auricular cartilage, nasal cartilage and cricoid cartilage using simple surgical procedures well known in the art. See for example: Skoog et al. (1990) Scan. J. Plast. Reconstr. Hand Surg. 24:89-93; Bulstra et al. (1990) J. Orthro. Res. 8:328-335; and Homminga et al. (1990) J. Bone Constr. Surg. 72:1003-1007, the disclosures of which are incorporated by reference herein.

It is contemplated also that chondrogenic cells, specifically mesenchymal cells, useful in the practice of the invention may be isolated from bone marrow. Surgical procedures useful in the isolation of bone marrow are well known in the art and so are not described in detailed herein. See for example, Wakitani et al. (1994) J. Bone Joint Surg. 76: 579-591, the disclosure of which is incorporated by reference herein.

II. Preparation of Denuded Chondrogenic Cells

Protocols for preparing denuded chondrogenic cells from cartilage tissue, perichondrial tissue, and bone marrow are set forth below.

A. From Articular Cartilage

Articular cartilage, both loaded (weight bearing) and unloaded (non-weight bearing), maybe be subjected to enzymatic treatment in order to disaggregate the tissue and release denuded chondrogenic cells from the extracellular matrix. Solutions containing proteolytic enzymes, for example, chondroitinase ABC, hyaluronidase, pronase, collagense, or trypsin may be added to articular cartilage tissue in order to digest the extracellular matrix. See for example, Watt & Dudhia (1988) Differentiation 38:140-147, the disclosure of which is incorporated herein by reference.

In a preferred procedure, articular cartilage is initially cut into pieces of about 1 mm in diameter, or less. This is routinely performed using a sterile scalpel. The minced tissue then is disaggregated enzymatically, for example, by the addition of a solution containing 0.1% collagenase (Boehringer Mannheim GmbH, Germany). Approximately 1 ml of collagenase is added per 0.25 ml equivalents of minced tissue. The sample is then mixed and incubated overnight (up to 16 hours) at 37.degree. C., with agitation. Following the overnight digestion, the residual pieces of tissue are harvested by centrifugation, the supernatant removed, and the remaining cartilage pieces redigested by the addition of a solution containing, for example, 0.25% collagenase and 0.05% trypsin (Sigma Chemical Co., St. Louis). Approximately 1 ml of 0.25% collagenase, 0.05% trypsin is added per 0.25 ml equivalents of residual tissue. The sample then is mixed and incubated for 2-4 hours at 37.degree. C., with agitation. Any remaining tissue is pelleted by centrifugation and the cell suspension harvested. The collagenase, trypsin step is repeated 2-4 times or until the tissue is completely disaggregated.

The enzymatic reaction is terminated by the addition of tissue culture medium supplemented with approximately 10% fetal bovine serum (FBS) (Hyclone, Logan, Utah). A preferred cell culture medium includes, for example, Dulbecco's minimal essential medium (DMEM) (Sigma Chemical Co., St. Louis) supplemented with 10% FBS. An alternative cell culture medium includes a 1:1 (vol/vol) mixture of Medium 199 (Sigma Chemical Co., St. Louis) and Molecular Cell Developmental Biology Medium 202 (MCDB 202) (Sigma Chemical Co., St. Louis), respectively, supplemented with 10% FBS. Alternatively, another cell culture medium useful in the practice of the invention includes a 3:1 (vol/vol) mixture of DMEM and Ham's F-12 (F12) (Sigma Chemical Co., St. Louis), respectively, supplemented with 10% FBS. Fractions containing denuded chondrogenic cells are combined, and the cells inoculated into a cell culture dish at a plating density of about 1.times.10.sup.2 -5.times.10.sup.5 cells/cm.sup.2, preferably about 5.times.10.sup.2 -1.times.10.sup.5 cells/cm.sup.2, and most preferably about 1.times.10.sup.3 -1.times.10.sup.4 cells/cm.sup.2, for cell expansion and testing.

Chondrocytes may be isolated from costal cartilage, nasal cartilage, auricular cartilage, tracheal cartilage, epiglottic cartilage, thyroid cartilage, arytenoid cartilage and cricoid cartilage using the aforementioned procedure.

B. From Perichondrial Tissue

Denuded chondrogenic cells preferably are isolated from perichondrial tissue using the same procedure as described in section II A, hereinabove.

Briefly, the tissue is minced into pieces of about 1 mm in diameter, or less. The minced tissue is repeatedly digested with proteolytic enzymes, for example, trypsin and collagenase. The resulting denuded cells are inoculated into a cell culture dish at a plating density of about 1.times.10.sup.2 -5.times.10.sup.5 cells/cm.sup.2, preferably about 5.times.10.sup.2 to 1.times.10.sup.5 cells/cm.sup.2, and most preferably about 1.times.10.sup.3 -1.times.10.sup.4 cells/cm.sup.2 for cell expansion and testing.

Alternatively, a non-destructive procedure may be used to isolate chondrogenic cells from perichondrial tissue. In this procedure, intact explant tissue is placed in a cell culture dish and incubated in growth medium. The chondrogenic cells located within the tissue migrate out of the tissue and onto the surface of the tissue plate where they begin to proliferate. See for example, Bulstra et al. (1990) J. Orthop. Res. 8:328-335, the disclosure of which is incorporated by reference herein. Briefly, pieces of the minced explant tissue are placed into a tissue culture plate containing tissue culture medium. A preferred cell culture medium comprises DMEM supplemented with 10% FBS. The explant tissues are incubated at 37.degree. C., 5% CO.sub.2 for 3-7 days. During this time the chondrogenic cells migrate out of the explant tissue and onto the surface of the tissue culture dish. After reattaching to the surface of the plate, the cells start to proliferate again.

C. From Bone Marrow

Chondrogenic cells, specifically mesenchymal cells, may be isolated from samples of bone marrow. Procedures useful for the isolation of mesenchymal cells from bone marrow are well known in the art, see for example: U.S. Pat. Nos. 5,197,985; 4,642,120; and Wakitani et al. (1994, supra).

For example, in a preferred method, a plug of bone marrow may be removed surgically from the mammal of interest and added to cell culture medium. Preferred complete growth media are disc