WO2008086147A1

WO2008086147A1 - Compositions and methods for the repair and regeneration of cartilage and/or bone

Info

Publication number: WO2008086147A1
Application number: PCT/US2008/050185
Authority: WO
Inventors: Bohdan Pomahac
Original assignee: The Brigham And Women's Hospital, Inc.
Priority date: 2007-01-05
Filing date: 2008-01-04
Publication date: 2008-07-17

Abstract

The present invention is related to methods and compositions for the repair of bone and cartilage defects. In particular embodiments, the present invention relates to the use of a cell coated substrate comprising cells which develop into cartilage for the repair of defects of cartilage, and the use of a cell coated substrate comprising cells which develop into bone for the repair of bone defects. In some embodiments, the present invention relates to methods and compositions for the repair of large bone and cartilage defects. In particular embodiments, the substrate of the cell coated substrate comprises a copolymer such as, but not limited to, a copolymer of polylatic acid (PLA) and poly glycolic acid (PGA).

Description

COMPOSITIONS AJVD METHODS FOR THE REPAER AND REGENERATION OF

CARTELAGE AND/OR BONE

CROSS REFERENCED APPLICATIONS [1] This application claims benefit under 35 U.S.C. 119(e) of U.S. Provisional Application Serial No: 60/878,971 filed on January 5^th 2007, the contents of which are incorporated herein in their entirety by reference.

FIELD OF THE INVENTION [2] The present invention relates generally to methods and compositions for the repair of cartilage and/or bone defects.

BACKGROUND OF THE INVENTION

[3] Arthritis is a worldwide health problem. In the USA alone, an estimated 43 million people suffer from arthritis. According to American Arthritis Foundation statistics, this condition is currently a number one cause of disability. Recent estimates place the direct medical cost related to arthritis in the USA at 15.2 billion dollars per year. Medical treatment has successfully slowed progression of the disease however, cure is still unavailable. Surgical options are directed towards pain relieve, and preservation of joint mobility. [4] The fundamental problem lies in poor healing of cartilaginous joint defects. Small defects can heal spontaneously, while larger ones fill with fibrocartilagenous tissue of mechanically inferior quality. Ongoing joint degenerative process is accompanied by pain, and usually leads to therapeutic intervention due to impending loss of joint function. [5] Articular cartilage injury and degeneration present medical problems to the general population which are constantly addressed by orthopedic surgeries. Every year in the United

States, over 500,000 arthroplastic or joint repair procedures are performed. These include approximately 125,000 total hip and 150,000 total knee arthroplastic and over 41,000 open arthroscopic procedures to repair cartilaginous defects of the knee. [6] Three types of cartilage are present in mammals and include: hyaline cartilage; fibrocartilage and elastic cartilage. Hyaline cartilage is predominantly found on the articulating surfaces of articulating joints and 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. Fibrocartilage is softer and mechanically inferior to hyaline cartilage. Elastic cartilage also is similar to hyaline cartilage except that it contains fibers of elastin and is present in the pinna of the ears, the epiglottis, and the larynx. [7] The surfaces of articulating bones in mammalian joints are covered with hyaline articular cartilage. The articular cartilage prevents direct contact of the opposing bone surfaces and permits the near frictionless movement of the articulating bones relative to one another. The articular cartilage lesions are classified into two groups according to their severity: partial thickness defect and full thickness defect.

[8] Partial thickness defect is the injury or erosion on the cartilage tissue of articular surface that does not reach the subchondral bone. Partial thickness defect is the injury or erosion on the cartilage tissue of articular surface that does not reach the subchondral bone, they 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 arrangements 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. Partial thickness defect of articular cartilage lesions can be treated by surgery and arthroscopic methods such as abrasion arthroplasty, debridement and lavage, high tibial osteotomy, microfracturing, and drilling.

[9] Full thickness defect is the injury or erosion on the cartilage tissue that penetrates 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. However, general arthroscopy cannot be used to cure full thickness defects where the damaged area is much wider and/or deeper than that of partial thickness defect. As a result, patients are faced with the only choice of undergoing both joint excision and replacement with an artificial joint to relieve pain and regain function of joints. It is estimated that over 150,000 knee replacement operations caused by full thickness defect are performed annually in the U.S., and the number of such operations is rising year after year. Because artificial joints are expensive, medical costs for hospitalization and surgery are high. Furthermore, because artificial joints are made of metals that last only about 10 to 20 years after being transplanted into human bodies, young patients must suffer from the pain of going through another surgical procedure in the future, while older patients, who, more often than not, are unable to go through another surgery, become handicapped and walking impaired, placing heavy burdens on both families and society. Therefore, there is a great need for the development of a technique for treating full- thickness defects. [10] Current clinical treatments for symptomatic full thickness cartilage defects involve techniques aimed at 1) removing surface irregularities by shaving and debridement 2) penetration of subchondral bone by drilling, fracturing or abrasion to augment the natural repair response described above (i.e. the family of bone-marrow stimulation techniques) 3) joint realignment or osteotomy to use remaining cartilage for articulation 4) pharmacological modulation 5) tissue transplantation and 6) cell transplantation (Newman, 1998; Buckwalter and Mankin, 1997). Most of these methods have been shown to have some short term benefit in reducing symptoms (months to a few years), while none have been able to consistently demonstrate successful repair of articular cartilage defects after the first few years.

[11] The cell transplantation approach possesses some potential advantages over other cartilage repair techniques in that they 1) minimize additional cartilage and bone injury, 2) reduce reliance on donors by ex vivo cell production, 3) could mimic natural biological processes of cartilage development, and 4) can provide tailored cell types to execute better repair.

[12] One commercially available cell transplantation method of cartilage repair is the product Carticel®, which is a commercial process to culture a patients own cartilage cells for use in the repair of cartilage defects in femoral condyle marketed by Genzyme Biosurgery. The procedure uses an initial arthroscopy procedure to take a biopsy of the patients own (autologous) chondrocytes from a healthy non-load bearing or less loaded area of articular cartilage.

Enzymatic digestion of the harvested tissue releases the cells that are sent to a laboratory where they are grown for a period ranging from 2-5 weeks. Once cultivated, the cells are injected during a complicated open and extensive knee procedure into areas of defective cartilage where it is hoped that they will facilitate the repair of damaged tissue. An autologous periosteal flap with cambium layer is used to seal the transplanted cells in place and act as a mechanical barrier.

Fibrin glue and sutures are used to seal the edges of the flap. This technique preserves the subchondral bone plate and is described in US 5,786,217, which is incorporated herein in its entirety by reference (Tubo et al 1998), which discloses a method of growing a complete intact piece of cartilage outside the body, using in vitro methods and a pre-shaped growing well, and then surgically implanting the piece of cartilage into the defect, using sutures and/or adhesives to anchor it. US 5,842,477 which is incorporated herein in its entirety by reference (Naughton et al, 1998), discloses the use of certain types of periosteal and/or perichondrial tissue in conjunction with chondrocyte implants, to promote the migration of chondrocyte, progenitor, or stromal cells into the area being repaired. [13] Several major drawbacks are characteristic for Carticel®, include (i) a two stage operation, firstly to harvest of the chondrocytes and their in vitro expansion; and a second complicated surgery for the implantation of expanded chondrocytes, (ii) the need for complete joint exposure (open knee surgery) for chondrocyte implantation that includes significant morbidity for the patient, (iii) the technique limited to knee joint only, and (iv) securing the chondrocytes with a periosteal flap typically with sutures and a fibrin glue. Further, while suturing or tacking the implant can aid retention, sutures are known to further injure the articular surface (Breinan et al., 1997). Biological glues have been attempted with limited success (Kandel et al., 1995; Jurgenson et al., 1997). [14] As with cell sources for cartilage repair, there have been numerous published patents for delivery vehicles in cartilage repair ranging from gel matrices, sutures and fibers, to screw type devices (Schwartz, 1998), and magnetic systems (Halpern, 1997). Most of the patents in this field tend to concur that the best way to promote cartilage regeneration inside a joint involves the use of a "resorbable" matrix. One such example is collagen, where the collagen fibers are slowly and gradually digested which is matched by gradual secretion of new collagen fibers by cells in the tissue, resulting in a process of turnover and replacement that helps keep tissue flexible, healthy, and strong. Examples of resorbable collagen matrix are described in US patents

4,846,835 (Grande 1989), 4,880,429 (Stone 1989), 5,007,934 (Stone et al 1991), 5,306,311 (Stone et al 1994), 5,206,023 (Hunziker 1993), and 5,518,680 (Cima et al 1996), which are incorporated herein in their entirety by reference. [15] Various alternatives to collagen for in biodegradable matrices have been used however a major limitation of their use includes remodeling and loss of biomechanical strength with time.

Further limitations include loss of chondrocyte phenotype and phenotypic alteration of cells (chondrocytes' behavioral changes lead to their conversion into less specialized cells), stress- shielding, hindrance of neotissue organization, and degradation product toxicity. In particular, the chondrocytes fail to retain their structural morphology and the cartilage coated substrate fails to retain their structural shape and undergoes histological ossification (Christophel et al, Arch

Facial Plast Surgery; 2006;8:l 17-22).

[16] In techniques where cell transplantation for assisted cartilage repair where cells are grown ex vivo with or without a substrate matrix, press-fitting can be used by preparing an implant that is slightly larger than the defect and forcing it therein. However, press-fitting necessitates the use of a tissue that is formed ex vivo and thus not optimized for the geometric, physical, and biological factors of the site in which it is implanted.

[17] One of the limitations of current cartilage or chondrocyte transplantation methodologies arises from the fact that under the current state of the art, chondrocyte cell transplants can only be used to repair small cartilage defects and are often only applicable to repair of the knee. While efforts to achieve successful chondrocyte transplantation for larger defect areas have been extensively tried, success rates decrease with increasing size of the cartilage defect, and where the defect size is 1 square centimeter or more, the success rate is extremely low. Accordingly, although chondrocyte transplants are useful for treating many types of sports injuries and other types of mechanical trauma or injury (such as automobile or bicycling accidents, falls, etc.), they are severely limited, and in most cases ineffective, for treating elderly patients, patients suffering from osteoarthritis, and various other types of patients with defects larger than about 1 to about 1.5 square centimeters. Therefore, the development of a technique for treating full-thickness defects that is more versatile to multiple joints (i.e. not limited to the knee joint) and has a less invasive implantation of the chondrocytes, and is anchorage independent would be highly desirable.

SUMMARY OF THE INVENTION [18] The present invention is directed towards the repair of bone and cartilage defects. In particular, the invention relates to the use of cell coated substrate for the repair of defects of bone and cartilage. The cells coating the substrate are selected depending on the defect to be repaired, which will be become apparent in the application, for example, but not limited to the repair of a bone defect or a cartilage defect.

[19] One aspect of the invention, the cell coated substrate is a cartilage coated substrate for the repair of cartilage defects. In such embodiments, the cells coating the substrate are chondrocytes, chondrocyte progenitors, chondrocyte precursors any that develops to become cartilage. One embodiment of the present invention utilizes a cartilage coated substrate that fits to, adheres and fills the defect, permits proper cell differentiation and the production of mechanically functional articular cartilage extracellular matrix. In one embodiment, the invention provides a cartilage repair technique comprising chondrocytes with increased structural integrity for repair of large articular cartilage defects, while maintaining their phenotype. The invention is versatile to multiple joints (i.e not limited to the knee joint) and is less invasive implantation than methods currently used in the art.

[20] In one aspect, the methods relate to the treatment or repair of cartilage defects, in particular large defects of articular cartilage, for example defects at least lcm², or at least 1.5cm², or greater than 1.5cm² or 2.0cm² in diameter or surface area. In the current art, cell transplantation methods for the repair of large defects have major limitations, including loss phenotype of implanted cells and limited applicability to the knee joints and invasive, potentially life threatening surgical procedures. The present inventors have discovered that a substrate coated with cartilage cells (herein referred to as "cartilage coated substrate") can be prepared using a substrate that permits the chondrocytes to generate their own extracellular matrix. The cell coated substrate is further capable of being molded into a configuration to fit into an irregular defect. In one embodiment the molded cell coated substrate is implanted into the cartilage defect and permitted to integrate into the site of the defect.

[21] Accordingly, one aspect of the present invention relates to methods and compositions for the treatment (for example, but not limited to, the repair) or reduction of risk of cartilage defects in a subject, in particular articular cartilage defects. In some embodiments, the cartilage defect is a large defect, for example, a defect of lcm square in diameter or more, for example defects at least lcm , or at least 1.5cm , or greater than 1.5cm or 2.0cm in diameter or surface area. In some embodiments, the subject is a mammal, and in some embodiments the mammal is a human or a non-human animal. In particular, the invention relates to improved methods and compositions for implantation of cell coated substrate with multiple advantages over existing techniques. For example, but not limited to, the cell coated substrate of the invention can be used to repair cartilage defects on multiple different types of joints with minimal invasive surgery procedures and without the need for a periosteal flap, as well as advantages of no loss of structural integrity and no loss of phenotype of the implanted chondrocytes. [22] In another aspect of the present invention relates to the treatment of bone defects. In particular, the methods relate to use of a cell coated substrate used to repair defects in bones, for example broken or fractured bones. In some embodiments, the cell coated substrate comprises cells such as, but are not limited to, osteoblasts, or osteoblast progenitors, osteoblast precursors or stem cells, or a mixture of such cells. In some embodiments, the cell coated substrate as disclosed herein is molded to wrap around the circumference of the bone defect if the defect is to a bone which permits such placement of the cell coated substrate. In alternative embodiments, the cell coated substrate is placed over and/or molded to fit the bone defect.

[23] In some embodiments, the substrate of the cell coated substrate is a copolymer of polylactic acid (PLA) and poly glycolic acid (PGA). In some embodiments, the substrate is poly(lactic-co- glycolic) acid or variants or analogues thereof, and in a specific embodiment, the cell coated substrate is 82% polylactic acid (PLA) and 18% poly glycolic acid (PGA). In some embodiments, the cells are seeded onto the substrate in a substantially smooth configuration, and after sufficient time for the cells to generate extracellular matrix, for example chondrocytes or osteoblasts are cultured for sufficient amount of time for generation of extracellular matrix associated with cartilage and bone respectively. The cell coated substrate is molded for the approximate dimension of the cartilage and/or bone defect. In some embodiments, the cell coated substrate is molded to a configuration of pre-determined size and shape of the cartilage and/or bone defect. In such embodiments, the size and shape of the cartilage and/or bone defect can be determined by methods known by persons skilled in the art, for example but not limited to MRI, x-rays etc. In some embodiments, the molding is done prior to or during the surgery for implantation of the cell coated substrate. In some embodiments, the substrate is molded prior to seeding with cells. In alternative embodiments, the substrate is molded after seeding of cells with cells.

[24] The cell coated substrate can be implanted into the defect using known procedures. In some embodiments the cell coated substrate is trimmed to fit the defect. For example, where the cell coated substrate is a cartilage coated substrate for the repair of cartilage defects, the cartilage coated substrate is trimmed to fit the cartilage defect. In some embodiments, the cell coated substrate is frozen prior to implantation in a subject. In related embodiments, the cell coated substrate is molded before or after it is frozen. [25] In some embodiments, the cells are chondrocytes, and in other embodiments, the cells are chondrocyte progenitors, chondrocyte precursors, for example stem cells. In some embodiments, the chondrocytes are denuded chondrocytes.

[26] In alternative embodiments, the cells are osteblasts. In alternative embodiments, the osteoblasts are osteoblast progenitors, osteoblast precursors, osteoprogenitors, bone marrow cells, mesenchymal cells or stem cells, for example adult stem cells or embryonic stem cells. [27] In some embodiments, the substrate is coated with cell adhesion molecules, for example, but not limited to polylysine prior to being coated with cells, for example chondrocyte cells. In some embodiments, the cells are autologous, and in other embodiments the cells are allogenic. [28] In some embodiments, the cell coated substrate comprises other bioresorbable materials. In some embodiments, the cell coated substrate also comprises chondrocyte inductive factors and/or chondroinducive factors, for example, but not limited to growth factors. In some embodiments, the cell coated substrates also comprises osteoblast inductive factors, for example but not limited to growth factors. In such an embodiments, the chondrocyte inductive factors and/or osteoblast inducive factors are released into the proximity of the attached cells as the cell coated substrate degrades over a certain period of time.

BRIEF DESCRIPTION OF FIGURES

[29] Figure 1 shows an example of cells adhering to the substrate, a cancer cell line expressing LacZ was seeded in a ring and cultured on the surface of the substrate of poly(lactic-co-glycol) acid (shown in figure IA) and stained with beta-galactosidease for LacZ expression (blue staining). Microscopically, the cells attach to the resorbable substrate (figure 1C), and a greater attachment is seen when the resorbable substrate is coated with polylysine for increased cell adhesion (figure IB). Figure ID shows an example of a cartilage coated substrate, wherein 500,000 chondrocytes are seeded no a substrate of 4mm in diameter. In such an example, the chondrocytes appear like a pellet and can be implanted into the cartilage defect to be repaired. In some instances, the chondrocytes of such a cartilage coated substrate can contact the defect with the substrate on the superficial outermost edge. In alternative instances, the cartilage coated implant can be implanted into the defect where the substrate contacts the defect and the cells or cartilage is on the outermost superficial layer of the defect. Figure IE shows a resorbable substrate comprising chondrocytes is molded into the shape of a cone (figure IE). A resorbable substrate seeded with 500,000 chondrocytes prior to insertion into the cartilage defect. In some embodiments the cone can be molded so the cells are on the outside and will contact with the cartilage defect, and in other embodiments, the cone can be molded so the cells are on the inside and the substrate contacts the cartilage defect. Figure IF shows an example where the substrate is molded so that the cells are on the inside of the cone. [30] Figure 2 shows a resorbable substrate seeded with 500,000 chondrocytes and molded into the shape of a cone was inserted into the cartilage defect with the chondrocytes facing the exposed defect and cut flush with the surrounding cartilage. Figure 2A shows the cartilage defect to be repaired, and Fig 2B shows the same defect after implantation of the cartilage coated substrate into the cartilage defect, where the cartilage coated substrate has been molded into the shape of a cone. Once implanted in the desired position at the desired defect, the cartilage coated substrate is cut flush with the surrounding endogenous cartilage (Figure 2C). In an experimental control, the defect is left unfilled as shown in Figure 2A. The cone was filled with chondrocytes in the experimental group (panel 2C), and left empty in control group (panel 2A).

[31] Figure 3 shows the control defect (with no cell coated substrate) filled with fibrinous/blood clot 2 weeks following wounding (Fig 3A), whereas the defect where the cartilage coated substrate which was molded in a cone configuration was inserted shows cartilage regeneration (Figure 3B and 3C) and the presence of white cartilage and gross morphological characteristics of cartilage tissue (Fig 3B and 3C). It should be noted that the cartilage from the implant in panel 3B is whiter than the surrounding normal cartilage, which is due to the fact that the mass of chondrocyte is a lot thicker than regular cartilage. [32] Figure 4 shows the morphological and histological characteristics of cross sections spanning the cartilage defect from the cartilage defect which was implanted with the cartilage coated substrate is shown in panel 4A. In the experiment in panel 4A, the cartilage coated substrate was implanted with chondrocyte cells facing the bone defect, and the substrate on the superficial outer most edge furthers from the surface of the defect. Panel 4B shows a cartilage defect from the control group which was not implanted with the cartilage coated substrate. The cartilage is stained with H&E, and panel 4 A shows chondrocytes in a typical configuration for hyaline cartilage spanning the defect, whereas panel 4B shows fibrous tissue in the area of cartilage defect. In both images, the length of the cartilage defect is the area located between the two arrows shown, with the naive cartilage located on the outsides of the arrows. [33] Figure 5 shows morphology characterization of the new cartilage in the implant. Panel 5 A shows hematoxylin / eosin (H&E) staining of the explanted cartilage from the cartilage defect 2 weeks following implantation with cartilage coated substrate and molded in a cone configuration. The structure of the cartilage closely resembles naive hyaline cartilage of typical conformation which is found in a rib or joint. Panel 5B shows safranin staining (a cartilage- specific stain) of cartilage derived from cartilage coated substrate, where the substrate is substantially smooth surface of the substrate poly(lactic-co-glycolic) acid polymer. The safranin staining resembles closely the hyaline cartilage found in a rib or joint. DETAILED DESCRIPTION

[34] Broadly, in some embodiments the present invention comprises a method for preparing a cell coated substrate for the repair of defects in a subject. In some embodiments, the defect is a cartilage defect, and in alternative embodiments, the defect is a bone defect. In some embodiments, the method comprises: (1) seeding cells on a substantially smooth substrate; and (2) culturing the cells on the substrate for a time sufficient to permit the cells to secrete an extracellular matrix thereby forming a cell coated substrate. In some embodiments, the defect is cartilage defect. In such embodiment, the cells are for example, but not limited to chondrocytes, progenitors, chondrocyte progenitors or stem cells or a mixture thereof. In some embodiments, the defect is a bone defect. In such embodiments, the cells are for example, but not limited to, osteoblasts, bone progenitor cells, osteoprogenitor cells or stem cells or a mixture thereof. In other embodiments the cells are progenitor cells, stem cells, mesenchymal cells, bone marrow cells, or differentiated or genetically modified cells thereof. In some embodiments, the cell coated substrate can be molded to fit the defect to be repaired. Repair of cartilage defects using a cell coated substrate as disclosed herein results in the generation of cartilage comprising chondrocyte cells dispersed within an endogenously produced and secreted extracellular matrix. Such cell coated substrates can be used for the repair of an articular cartilage defect in a subject. In some embodiments, the present invention further includes a composition for the repair of a cartilage defect, in particular an articular cartilage defect.

Definitions

[35] For convenience, certain terms employed in the specification, examples, and appended claims are collected here. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[36] The term "defect" as used herein refers to an imperfection that impairs worth or utility or the absence of something necessary for completeness or perfection; or a deficiency in function. The term defect as used herein is not limited to acquired defects, for example defects from damage, injury or wear, but the term defects also encompasses defects due to non-acquired or existing defects, for example congenial or developmental defects.

[37] The term "chondrocytes" or "chondrocyte cells" or "cartilage cells" are used interchangeably herein refers to cells that are capable of expressing characteristic biochemical markers of chondrocytes, including but not limited to collagen type II, chondroitin sulfate, keratin sulfate and characteristic morphologic markers, limited to the rounded morphology observed in culture, and able to secrete collagen type II, including but not limited to the generation of tissue or matrices with hemodynamic properties of cartilage in vitro. As used herein, the term "chondrocyte cell", is understood to mean any cell which, when exposed to an appropriate stimuli, can 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. [38] As used herein, the term "cartilage" is not limited to a class of cartilage known as "hyaline" or "articulating" cartilage. This is a relatively stiff form of cartilage, which exists in articulating joints, such as knees, hips, shoulders, etc. Other types of cartilage, which exist in parts of the body such as ears and noses, are not relevant herein. [39] The terms "damaged cartilage" and "cartilage defect" are used interchangeably herein, and are used in a broad sense. Either term refers to a segment of cartilage that suffers from any type of damage or defect that appears to be amenable to repair or improvement using transplanted chondrocyte cells or the implantation of the cell coated substrate of the invention, regardless of whether the problem was caused by mechanical trauma, a disease such as arthritis or osteoarthritis, etc. [40] As used herein, the term "cartilage" in the context of 'cartilage coated substrate" is understood to include any cartilage tissue produced in vitro that contains chondrocyte 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. [41] As used herein, the term "articular cartilage", is understood to mean any cartilage tissue, either in vivo or produced in vitro that biochemically and morphologically resembles the cartilage normally found on the articulating surfaces of mammalian joints. [42] Reference herein to "articulating surface" refers to the fact that in a healthy joint, two cartilage-covered surfaces on two different bones will rub, slide, roll, or otherwise move while in contact with each other, as the joint is flexed or extended. This interaction between two surfaces is referred to as articulation, and the two cartilage surfaces that contact and press against each other as a joint is moved are said to "articulate".

[43] 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 can be enzymatically and/or mechanically disaggregated in order to release the denuded cells.

[44] As used herein the terms "chondroinductive agent" or "chondroinductive factor" refers to any natural or synthetic, organic or inorganic chemical or biochemical compound or combination or mixture of compounds, or any mechanical or other physical device, container, influence or force that can be applied to any cells, progenitor cells or stem cells so as to effect their in vitro differentiation, for example into chondrocytes or cartilage or the production of cartilaginous tissue. The chondroinductive agent is preferably selected, individually or in combination, from the group consisting of a glucocorticoid such as dexamethasone; a member of the transforming growth factor-β superfamily such as a bone morphogenic protein (preferably BMP-2 or BMP-4), TGF-βl, TGF-β2, TGF-β3, insulin-like growth factor (IGF), platelet derived growth factor (PDGF), epidermal growth factor (EGF), acidic fibroblast growth factor (aFBF), basic fibroblast growth factor (bFBF), hepatocytic growth factor (HGF), keratocyte growth factor (KGF), osteogenic proteins (OP-I, OP-2, and OP-3), inhibin A or chondrocyte stimulating activity factor (CSA); a component of the collagenous extracellular matrix such as collagen I (particularly in the form of a gel); and a vitamin A analogue such as retinoic acid and; ascorbate or other related vitamin C analogue. [45] The term "bone" as used herein is intended to refer to bone that is cortical, cancellous or cortico-cancellous of autogenous, allogenic, xenogenic or transgenic origin. [46] The term "tissue" as used herein is intended to refer to a collection of any cells, (i.e. more than one cell) and is intended to include intact cells, blood, blood preparations such as plasma and serum, bones, joints, muscles, smooth muscles, and organs, and also includes a collection of heterologous cells as well as any collection of cells that form part of a transplantable or implantable tissue.

[47] The term "transplant" and "implant" are used interchangeably herein, refer to tissue, material or cells exogenous or allogenic) which can be introduced into the body of a subject to replace or supplement the structure or function of the endogenous tissue. [48] The terms "autologous" and "autograft" are used interchangeably herein, refers to tissue or cells which originate with or are derived from the recipient subject, whereas the terms 'allogenic" and "allograft" refer to cells and tissues which originate with or are derived from a donor of the same species as the recipient. The terms "xenogenic" and "xenograft" refer to cells or tissue, which originate with or are derived from a species other than that of the recipient. The term "autologous" as used herein refers to cells removed from a donor and administered to a recipient, wherein the donor and recipient are the same individual.

[49] Terms such as "repair" are also used broadly, and when used in the context of cartilage and/or bone and other tissues is intended to mean without limitation repair, regeneration, reconstitution, reconstruction or bulking of tissues. It is also intended to refer to a surgical or arthroscopic procedure, which improves the condition of a segment of damaged cartilage or bone, even if such improvement does not rise to the level of a total and perfect cure. [50] The term "regeneration" means regrowth of a cell population, organ or tissue after disease or trauma. [51] The term "polymer" in the present application is intended to mean without limitation a polymer solution, polymer suspension, a polymer particulate or powder and a polymer micellar suspension. [52] The term "bioresorbable" refers to the ability of a material to be reabsorbed in vivo. The absorbable polymer material can is selected from the group consisting of polyglycolic acid (PGA), polylactic acid (PLA), poly(lactic-co-glycolic) acid (PLGA), polyanhydride, polycapralactone (PCL), polydioxanone and polyorthoester. The bioabsorbable polymer material also can be composite material that comprises an absorbable polymer material and other materials.

[53] As used herein, the term "glycolide" is understood to include polyglycolic acid. Further, the term "lactide" is understood to include L-lactide, D-lactide, blends thereof, and lactic acid polymers and copolymers. [54] The term "polyglycolic acid", "poly(glycolic) acid and "PGA" are used interchangeably herein, refer to a polymer of glycolic acid. The term "polylactic acid", "poly(lactic) acid" and "PLA" are used interchangeably herein, refer to a member of the polyester family, in particular the poly(α-hydroxyl acid) family, and refers to a polymer of lactic acid molecules. The terms PLA and polylactic acid are intended to encompass all isometric forms of poly(lactic)acid, for example d(-), 1(+) and racimic (d,l) and the polymers are usually abbreviated to indicate the chirality. PoIy(I)LA and poly(d)LA are semi-crystalline solids.

[55] The term "poly(lactic-glycol acid)" and "PLGA" are used interchangeably herein, is intended to refer to all copolymers of PLA and PGA, for example but not limited to (1)LA/GA and (dl)LA/GA and different ratios of PLA:PGA. [56] The term "smooth" as used herein, is used to refer to the surface of the substrate, which is covered with cartilage. The term "substantially smooth" is used to refer to the surface of the substrate whereby the substrate is mostly smooth, but will have minor indentations and convex and/or concave configurations in the surface, but does not substantially distract from the smooth surface. The "substantially smooth" surface has an average macro-roughness not exceeding 3mm. Additionally, the substrate is free of auxiliary matrices. However, auxiliary matrices, separate from the substrate, can be combined with the coated substrate in clinical use. [57] The term "porous" as used herein, refers to small indentations or void spaces on the surface of the substrate in which cells and other materials can adhere to. For the purposes of the present application, the void spaces are on the surface and substantially located near the surface of the substrate.

[58] The term "progenitor cell" as used herein, refers to an undifferentiated cell which is capable of proliferation and giving rise to more progenitor cells having the ability to generate a large number of mother cells that can in turn give rise to differentiated cells, or daughter cells which can undergo subsequent differentiation. Stated another way, the term "progenitor cell" refers to a cell with a cellular phenotype that is more primitive (i.e., is at an earlier step along a developmental pathway or progression than is a fully differentiated cell). Often, progenitor cells also have significant or very high proliferative potential, and can give rise to multiple distinct differentiated cell types or to a single differentiated cell type, depending on the developmental pathway and on the environment in which the cells develop and differentiate. Like stem cells, it is possible that cells that begin as progenitor cells might proceed toward a differentiated phenotype, but then "reverse" and re-express the progenitor cell phenotype, a process known as

"dedifferentiation" or "reprogramming". The progeny of progenitor cells, often referred to as daughter cells, can be induced to proliferate and produce progeny that subsequently differentiate into one or more mature cell types, while also retaining one or more cells with parental developmental potential. An example of a progenitor cell is a stem cell. In one embodiment, the term progenitor cell or stem cell refers to a generalized mother cell whose descendants (progeny) specialize, often in different directions, by differentiation, e.g., by acquiring completely individual characters, as occurs in progressive diversification of embryonic cells and tissues. Cellular differentiation is a complex process typically occurring through many cell divisions. A differentiated cell can derive from a multipotent cell which itself is derived from a multipotent cell, and so on. While each of these multipotent cells can be considered stem cells, the range of cell types each can give rise to can vary considerably. Some differentiated cells also have the capacity to give rise to cells of greater developmental potential. Such capacity can be natural or can be induced artificially upon treatment with various factors, hi many biological instances, progenitor cells are also "multipotent" because they can produce progeny of more than one distinct cell type, but this is not required for "stem-ness." Self-renewal is the other classical part of the stem cell definition, and it is essential as used in this document. In theory, self-renewal can occur by either of two major mechanisms. Progenitor cells can divide asymmetrically, with one daughter retaining the progenitor state and the other daughter expressing some distinct other specific function and phenotype. Alternatively, some of the progenitor cells in a population can divide symmetrically into two stems, thus maintaining some progenitor cells in the population as a whole, while other cells in the population give rise to differentiated progeny only. Formally, it is possible that cells that begin as progenitor cells might proceed toward a differentiated phenotype, but then "reverse" and re-express the progenitor cell phenotype, a term often referred to as "dedifferentiation". [59] The term "cartilage stem cell" refers to a stem cell which is capable of proliferation and giving rise to more progenitor cells having the ability to generate a large number of mother cells that can in turn give rise to differentiated or differentiable daughter cells which can eventually terminally differentiate into cartilage cells and other cells of the articular cartilage. [60] As used herein, the term "bone progenitor cells" refers to cells that have the capacity to ultimately form, or contribute to the formation of, new bone tissue. This includes various cells in different stages of differentiation, such as, for example, stem cells, macrophages, fibroblasts, vascular cells, osteoblasts, chondroblasts, and osteoclasts. Bone progenitor cells also include cells that have been isolated and manipulated in vitro, e.g., subjected to stimulation with agents such as cytokines or growth factors or even genetically engineered cells. The particular type or types of bone progenitor cells that are stimulated are not important, so long as the cells are stimulated in such a way that they are activated and, in the context of in vivo embodiments, ultimately give rise to new bone tissue.

[61] The term "bone progenitor cell" is also used to particularly refer to those cells that are located within, are in contact with, or migrate towards (i. e., "home to"), bone progenitor tissue and which cells directly or indirectly stimulate the formation of mature bone. As such, the progenitor cells can be cells that ultimately differentiate into mature bone cells themselves, i.e., cells that

"directly" form new bone tissue. Cells that, upon stimulation, attract further progenitor cells or promote nearby cells to differentiate into bone- forming cells (e.g., into osteoblasts, osteocytes and/or osteoclasts) are also considered to be progenitor cells as their stimulation "indirectly" leads to bone repair or regeneration. Cells affecting bone formation indirectly can do so by the elaboration of various growth factors or cytokines, or by their physical interaction with other cell types.

[62] In terms of bone progenitor cells, these can also be cells that are attracted or recruited to an area of bone tissue or bone damage. In some embodiments, bone progenitor cells can be cells that are present within an artificially created osteotomy site in an animal model. Bone progenitor cells can also be isolated from animal or human tissues and maintained in an in vitro environment. Suitable areas of the body from which to obtain bone progenitor cells are areas such as the bone tissue and fluid surrounding a fracture or other skeletal defect (whether or not this is an artificially created site), or indeed, from the bone marrow. Isolated cells can be stimulated and then be returned to an appropriate site in an animal where bone repair is to be stimulated. In such cases, the cells can be used as therapeutic agents. Such ex vivo protocols are well known to those of skill in the art

[63] The term "differentiation" in the present context means the process of the formation of cells expressing markers known to be associated with cells that are more specialized and the process of a cell becoming more closely related to a terminally differentiated cell such as differentiated cells incapable of further division or differentiation. The pathway along which cells progress from a less committed cell, to a cell that is increasingly committed to a particular cell type, i.e. the development of a cell from an uncommitted cell (for example, a stem cell), to a cell with an increasing degree of commitment to a particular differentiated cell type, and finally to a terminally differentiated cell is known as "progressive differentiation" or "progressive commitment". Cell which are more specialized (e.g., have begun to progress along a path of progressive differentiation) but not yet terminally differentiated are referred to as partially differentiated. Differentiation is a developmental process whereby cells assume a specialized phenotype, e.g., acquire one or more characteristics or functions distinct from other cell types. In some cases, the differentiated phenotype refers to a cell phenotype that is at the mature endpoint in some developmental pathway (a so called terminally differentiated cell). In many, but not all tissues, the process of differentiation is coupled with exit from the cell cycle. In these cases, the terminally differentiated cells lose or greatly restrict their capacity to proliferate. However, we note that in the context of this application, the term "differentiation" or "differentiated" refers to cells that are more specialized in their fate or function than at a previous point in their development, and includes both cells that are terminally differentiated and cells that, although not terminally differentiated, are more specialized than at a previous point in their development.

[64] In the context of cell ontogeny, the adjective "differentiated", or "differentiating" is a relative term. Accordingly, a "differentiated cell" is a cell that has progressed further down the developmental pathway than the cell it is being compared with. Thus, stem cells can differentiate to lineage-restricted precursor cells (such as mesenchymal cells or progenitors), which in turn can differentiate into other types of precursor cells further down the pathway (such as an osteoprogenitor), and then to an end-stage differentiated cell, which plays a characteristic role in a certain tissue type, and can or can not retain the capacity to proliferate further. [65] The term "marker" as used herein is used to describe the characteristics and/or phenotype of a cell. Markers can be used for selection of cells comprising characteristics of interests. Markers will vary with specific cells. Markers are characteristics, whether morphological or biochemical

(enzymatic), particular to a cell type, or molecules expressed by the cell type. Preferably, such markers are proteins, and more preferably, possess an epitope for antibodies or other binding molecules available in the art. However, a marker may consist of any molecule found in a cell including, but not limited to, proteins (peptides and polypeptides), lipids, polysaccharides, nucleic acids and steroids. Additionally, a marker may comprise a morphological or functional characteristic of a cell. Examples of morphological traits include, but are not limited to, shape, size, and nuclear to cytoplasmic ratio. Examples of functional traits include, but are not limited to, the ability to adhere to particular substrates, ability to incorporate or exclude particular dyes, ability to migrate under particular conditions, and the ability to differentiate along particular lineages. Markers may be detected by any method available to one of skill in the art.

[66] The term "embryonic stem cell" is used herein is used to refer to the pluripotent stem cells of the inner cell mass of the embryonic blastocyst (see U.S. Patent Nos. 5,843,780, 6,200,806, which are incorporated herein in their entirety by reference). Such cells can similarly be obtained from the inner cell mass of blastocysts derived from somatic cell nuclear transfer (see, for example, U.S. Patent Nos. 5,945,577, 5,994,619, 6,235,970, which are incorporated herein in their entirety by reference). The distinguishing characteristics of an embryonic stem cell define an embryonic stem cell phenotype. Accordingly, a cell has the phenotype of an embryonic stem cell if it possesses one or more of the unique characteristics of an embryonic stem cell such that that cell can be distinguished from other cells. Exemplary distinguishing embryonic stem cell characteristics include, without limitation, gene expression profile, proliferative capacity, differentiation capacity, karyotype, responsiveness to particular culture conditions, and the like.

[67] The term "adult stem cell" is used to refer to any multipotent stem cell derived from non- embryonic tissue, including fetal, juvenile, and adult tissue. Stem cells have been isolated from a wide variety of adult tissues including blood, bone marrow, brain, olfactory epithelium, skin, pancreas, skeletal muscle, and cardiac muscle. Each of these stem cells can be characterized based on gene expression, factor responsiveness, cell markers, and morphology in culture.

Exemplary adult stem cells include but are not limited to, neural stem cells, neural crest stem cells, mesenchymal stem cells, hematopoietic stem cells, and pancreatic stem cells. As indicated above, stem cells have been found resident in virtually every tissue. Accordingly, the present invention appreciates that stem cell populations can be isolated from virtually any animal tissue or any animal or organism.

[68] The term "osteoimplant" as used herein refers to any bone-derived implant prepared in accordance with the embodiments of this invention and therefore is intended to include expressions such as bone membrane, bone graft, etc. [69] The term "osteogenic" as applied to the osteoimplant of this invention shall be understood as referring to the ability of the osteoimplant to enhance or accelerate the ingrowth of new bone tissue by one or more mechanisms such as osteoinduction and/or osteoconduction. [70] The term "osteoinductive" as used herein shall be understood to refer to the ability of a substance to recruit and transform cells from the host which have the potential for repairing bone tissue. [71] The term "osteoinductive" as used herein shall be understood to refer to the ability of a substance to provide biologically inert surfaces which are receptive to the growth of new host bone.

[72] The terms "subject" and "individual" are used interchangeably herein, and refer to an animal or mammal, for example a human, to whom treatment, including prophylactic treatment, by implantation with the cell coated substrate of the present invention can be performed. The term

"subject" as used herein refers to human and non-human animals. The term "non-human animals" and "non-human mammals" are used interchangeably herein includes all vertebrates, e.g., mammals, such as non-human primates, (particularly higher primates), horses, sheep, dog, rodent (e.g. mouse or rat), guinea pig, goat, pig, cat, rabbits, cows, and non-mammals such as chickens, amphibians, reptiles etc. In one embodiment, the subject is human. In another embodiment, the subject is an experimental animal or animal substitute as a disease model. In other embodiment, the subject is a horse. [73] The expression "mechanically shaping" or expressions of similar import as used herein shall be understood as referring to the application of external forces, e.g., compressive, lateral, etc., to the cell coated substrate or substrate of the invention through any suitable means, e.g., pressing, rolling, etc. [74] The term "biodegradable" as used herein denotes a composition that is not biologically harmful and can be chemically degraded or decomposed by natural effectors (e.g., weather, soil bacteria, plants, animals). [75] The term "bioresorbable" refers to the ability of a material to be resorbed over time in the body (i.e. in vivo) so that its original presence is no longer detected once it has been resorbed. [76] The term "genetically modified" as used herein refers to a cell or entity, by human manipulation such as chemical, physical, viral or stress-induced or other means that has undergone mutation or selection; or that an exogenous nucleic acid has been introduced to the cell or entity through any standard means, such as transfection; such that the cell or entity gas acquired a new characteristic, phenotype, genotype, and/or gene expression product, including but not limited to a gene marker, a gene product, and/or a mRNA, to endow the original cell or entity, at a genetic level, with a function, characteristic, or genetic element not present in non- genetically modified, non-selected counterpart cells or entities. [77] As used herein, "implanting" is defined as when an artificially fabricated material is surgically implanted in a living body. [78] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "a cell coated substrate" means one cell coated substrate or more than one cell coated substrate. [79] The term "comprising" means "including principally, but not necessary solely". Furthermore, variation of the word "comprising", such as "comprise" and "comprises", have correspondingly varied meanings.

[80] Cells [81] In some embodiments of the invention, the cells that coat the substrate of the present invention are cells that can differentiate or transdifferentiate or dedifferentiate into cartilage cells or cartilage-like cells, for example but not limited to, progenitor cells, stem cells, chondrocytes, mesenchymal cells, embryonic stem cells, cells lines and genetically modified versions thereof etc. [82] hi alternative embodiments of the invention, the cells that coat the substrate of the invention are cells that can differentiate or transdifferentiate or dedifferentiate into a bone cells or bone- like cell, for example but not limited to, progenitor cells, bone progenitor cells, stem cells, osteoblasts, osteoprogenitor cells, bone marrow cells, mesenchymal stem cells, cell lines and genetically modified version thereof etc. In some embodiments, the osteoblasts and osteoporgenitor cells can be differentiated cells obtained by culturing the mesenchymal stem cells derived from a living subject.

[83] In some embodiments where the cells are chondrocyte cells, the chondrocytes useful in the practice of the present invention can be sampled from a variety of sources in a subject, for example a mammal that contain such cells, for example, pre-existing cartilage tissue, perichondrial tissue or bone marrow. In one embodiment, the chondrocytes are from articular cartilage (from either weight bearing (also referred to load-bearing) or non-weight bearing joints). In alternative embodiments, the cartilage is from costal cartilage, nasal cartilage, auricular cartilage, tracheal cartilage, epiglottic cartilage, thyroid cartilage, arytenoid cartilage and cricoid cartilage are useful sources of chondrocyte cells for the present invention. [84] In some embodiments, chondrocyte cells can be isolated from human cartilage tissue, for example but not limited to, 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; from human perichondrial tissue, i.e., perichondrial tissue sampled from the surface of 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; or from human bone marrow. See for example U.S Patent 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 herein in their entirety by reference. [85] Although, the herein disclosed invention has been characterized as using chondrocytes, it can be embodied using any cells that secrete extracellular matrix components suitable for causing the cells to become cartilage or bone or other tissue. Such cells can also include, for example, osteoblasts, myoblasts, fibroblasts such as those harvested from tendon, ligament, skin, meniscus or disk of the temporomandibular joint, or multi-potent stem cells that are capable of differentiating into matrix-producing cells, including but not limited to mesenchymal stem cells, progenitors and/or stem cells from muscle, skin, bone marrow stroma or embryonic stem cells, and fused or hybrid or genetically modified cells thereof. It is to be understood that the current technology can be extended to other organs such as liver, kidney, pancreas, etc. [86] In some embodiments, where the cell coated substrate is useful for the repair of a bone defects, the cells coating the substrate are cells which assist in the formation of bone or bone matrix, either exclusively or with other cells. Examples of such cells include, but are not limited to, at least one kind of cells selected from marrow cells, mesenchymal stem cells, osteoblasts, osteoprogenitor cells, osteoclasts, osteocytes and fused cells or modified variants thereof. [87] In some embodiments the cells are obtained from biopsy, where appropriate, and in other embodiments, the cells are obtained upon autopsy. Biopsy samples of articular cartilage or bone can 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. [88] In some embodiments, the cells are derived from perichondrial tissue, which 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 subjects suffering from osteoporosis provides a source of chondrocyte cells when the normal articular cartilage is diseased or unavailable. Biopsy samples of perichondrial tissue can 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). Bone Constr. Surg. 72-. 1003-1007, the disclosures of which are incorporated by reference herein. [89] It is also contemplated that cells, for example chondrocyte cells or bone progenitor cells and/or mesenchymal cells useful in the practice of the invention can 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. [90] In alternative embodiments, the cells are denuded chondrocyte cells. Protocols for preparing denuded chondrocyte cells from cartilage tissue, perichondrial tissue, and bone marrow are well known in the art, for example, see U.S. patent Applications 5,786,217, 5,842,447 and 5,759,190 etc, which are specifically incorporated herein in their entirety by reference. [91] It is contemplated that growth factors can be added to the cells, for example chondrocyte cells, which can be added prior to, or after, or concurrent with, seeding the cells on the substrate of the present invention to enhance or stimulate the production of articular cartilage specific proteoglycans and/or collagen (Luyten & Reddi (1992) in "Biological Regulation of the Chondrocytes", CRC Press, Boca Raton, Ann Arbor, London, and Tokyo, p.p. 227-236). In some embodiments, the growth factors include, but are not limited to transforming growth factor-A (TGF-A), insulin-like growth factor (IGF), platelet derived growth factor (PDGF), epidermal growth factor (EGF), acidic fibroblast growth factor (aFBF), basic fibroblast growth factor (bFBF), hepatocytic growth factor, (HGF) keratinocyte growth factor.(KGF), the bone morphogenic factors (BMPs) i.e. , BMP-I, BMP-2, BMP-3, BMP-4, BMP-5 and BMP-6 and the osteogenic proteins (OPs), i.e. OP-I, OP -2 and OP-3. In such embodiments, concentrations of TGF-β, TGF-A, IGF, PDGF, EGF, aFBF, bFBF, FGF-2, FGF-5, HGF, and KGF, range from about 1 to 100 ng/ml, and in some instances from about 5 to about 50 ng/ml, and in other instances from about 10 to about 20 ng/ml. In some embodiments, concentrations of the BMP's and range from about 1 to about 500 ng/ml, and sometimes from about 50 to about 300 ng/ml, and in alternative instances from about 100 to about 200 ng/ml. However, these particular growth factors are not limiting. Any polypeptide growth factor capable of stimulating or inducing the production of cartilage specific proteoglycans and collagen can be useful in the practice of the present invention

[92] Another aspect of the present invention provides the steps of in vitro culturing cells to form a cell coated substrate. In some embodiments, the cells, for example chondrocytes or osteoprogenitors or mesenchymal stem cells are obtained from a living subject. The present invention then provides methods for adhering the cells, for example bone progenitor cells, chondrocytes, mesenchymal stem cells etc to the surface of the substrate, and in some embodiments differentiating the adhered cells, for example osteoprogenitors or mesenchymal stem cells to at least one kind of cells selected from chondrocytes, osteoblasts and osteoprogenitor cells. As a result, in some embodiments, the surface of the substrate is coated with cartilage and in alternative embodiments the surface is coated with bone matrix produced in vitro by differentiation of the cells. The osteoblasts and the osteoprogenitor cells can be differentiated cells obtained by culturing, for example, bone progenitors and/or mesenchymal stem cells derived from a living subject. [93] The osteoblasts or the osteoprogenitor cells can be obtained by culturing cells, for example bone progenitors, stem cells and/or mesenchymal stem cells in the presence of differentiating inducing factor (dexamethasone). In some embodiments, the bone progenitor cells and/or mesenchymal stem cells can be proliferated by separating and culturing marrow cells obtained from a living subject, which is scheduled to be implanted with the cell coated substrate, therefore preventing adverse problems such as occurrence of rejection caused by autoimmunity after the implantation.

[94] In some embodiments, the cells, for example cartilage progenitors and/or osteoprogenitors are differentiated into chondrocytes and osteoblasts in advance to seeding on the substrate. In alternative embodiments the differentiation of cells into cartilage or bone is concurrent with seeding or after seeding the cells on the surface of the substrate, and therefore the differentiation of the cells occurs when they are adhered to the substrate. [95] In some embodiments, the cells can be genetically modified to produce gene products beneficial for transplantation, for example anti-inflammatory factors, e.g anti-GM-CSF, anti- TNF, anti-ILl and anti-ILs etc. Alternatively, the chondrocytes can be genetically modified to "knock-out" expression of native gene products that promote inflammation, for example GM- CSF, TNF, EL-I , IL-2 or "knock-out" expression of MHC in order to reduce the risk of rejection.

In addition, the chondrocytes can be genetically modified for used in gene therapy to adjust the level of a gene activity in a patient to assist or improve the results of cartilage transplantation. [96] In some embodiments, the cells are seeded on a substrate of the invention. The cells are cultured on the substrate under conventional culture conditions well known in the art from 1 to 90 days. Ln some embodiments, the cells are cultured from 5 to 90 days and in some embodiments from 10 to 30 days. The duration of the culture can be determined by persons skilled in the art, and is sufficient for enable the production and secretion of extracellular matrix. Ln some embodiments, cells, for example, chondrocyte cells, can be cultured and expanded in vitro prior to seeding on the substrate. Ln alternative embodiments, the cells are seeded directly on the resorbable substrate. Ln some embodiments, the substrate is coated to enhance attachment of cells, such as, for example, chondrocyte cells.

[97] Ln some embodiments, the cell coated substrate can be cryopreserved for subsequent use using techniques well known in the art. See for example, Pollack et al (1975) in "Readings in Mammalian Cell Culture' Cold Spring Harbor laboratory Press, Cold Spring Harbor, the disclosure of which is incorporated herein in its entirety by reference. Any tissue culture technique that is suitable for the propagation of chondrocytes or bone progenitors from biopsy specimens can be used to expand the cells to practice the invention. Techniques well known to those skilled in the art can be found in R. I. Freshney, Ed., ANIMAL CELL CULTURE: A PRACTICAL APPROACH (LRL Press, Oxford, England, 1986) and R. I. Freshney, Ed., CULTURE OF ANIMAL CELLS: A MANUAL OF BASIC TECHNIQUES, Alan R. Liss &

Co., New York, 1987), which are hereby incorporated herein in their entirety by reference. [98] Substrates

[99] The desired substrate useful in the invention is malleable, to enable its molding into desired shape and dimension of the defect to be repaired, for example cartilage defects or bone defects. In some embodiments, the support is biodegradable and/or bioresorbable so that it will be absorbed and/or replaced by the subjects own tissue.

[100] Ln some embodiments, the substrate is bioresorbable and/or biodegradable. Further, in some embodiments the substrate is biocompatible and bioreplacable. As noted above, the substrate has a substantially smooth surface. Ln further embodiments, the substrate is mechanically strong and also malleable. Ln some embodiments, the substrate is malleable under non-physiological conditions, for example but not limited to by temperature above body temperature, and for example by pressures exceeding normal physiological pressures, for example, by mechanical manipulation or mechanical shaping or by an altered surrounding environment, for example excessive heat, pressure or acidic or alkali conditions. In some embodiments, the substrate is malleable under non-physiological conditions, for example, where substrate is heated to be malleable, for example heated to 50-80⁰C, the substrate is molded prior to seeding of the cells.

[101] hi alternative embodiments, the substrate is molded by physiological conditions, for example by body temperature. Such an embodiment is useful for enabling the substrate, or cells coated substrate to be molded into the defect at the time it is implanted. In such an embodiment where the substrate is malleable by physiological conditions, the substrate is seeded with cells prior to, or after being molded.

[102] hi some embodiments, the substrate can be moldable in a putty form that will eventually harden (i.e the substrate is self-setting) and be able to withstand normal physiological stresses. Conventional materials of this type have been characterized as bone cements, although bone cements that are a self-setting putty type and have the required mechanical properties are generally not resorbable and eventually fail after prolonged implantation. By using biodegradable substrate of the present invention, the dynamics of bone resorption and new bone formation following cell coated substrate implantation can be modulated and controlled and, hence, avoid problems associated with their implantation. This step- wise degradation of the substrate enables decreasing reinforcement with concurrent increase in bone repair by the cells coated substrate of the invention, which is important for appropriate growth and formation of bone matrix in newly formed bone, and thus for appropriate repair of the bone defect. Since bone is a dynamic tissue that responds to changes in stress, gradual loading of the regenerating bone stimulates further bone formation without causing stress damage to the implanted cell coated substrate. Thus, the cell coated substrate for bone repair is multifunctional. Furthermore, its overall rate of incorporation into host bone can be more predictable based on the degradation rates of the polymer matrixes used.

[103] In one embodiment, substrate comprises materials with organic and malleable properties and can be used to create a substrate comprising a smooth surface, hi some embodiments the substrate is a naturally occurring or synthetic substrate, or derivative thereof. [104] hi alternative embodiments, the substrate is an aliphatic polyesters or polymers, for example polylactic (PLA or PLLA) and polyglycolic acid (PGA). hi some embodiments, the substrate is poly(glycol-co-lactic) acid (PGLA), a copolymer of PLA and PGA. hi some embodiments, the polylactic acid and polyglycolic acid (PGLA) polymer comprises about 82% m lactide or polylactic acid (PLA) and about 18% m glycolide or polyglycolic acid (PGA), also known as Lactsorb™ (see U.S. Patent Application 5,569,250 and 5,868,746). Lactsorb™ can be prepared as described in U.S. Patent Applications 5,569,250, 4,523,591 and 6,096,885 the entire disclosures are incorporated herein in its entirety by reference. Alternatively, Lactsorb™ is available commercially from Lorenz surgical (Biomet Inc. Jacksonville, FL). Other examples of copolymerization methods for producing poly(D,L-lactide-co-glycolide) and other random copolymer of resorbable material are disclosed in U.S. Patent No. 4,157,437 and the International patent Publication No. WO97/36553, which are disclosed herein in its entirety by reference. Other materials comprising polylactic acid and polyglycolic acid are known to persons skilled in the art and are useful in the present invention, for example polyglycolic acid polylactic acid (PGLA) sutures, Vicryl™ (Ethicon Inc., Sommerville, NJ, U.S.A). In further embodiments, the polymer of poly-lactic and Poly-glycolic acid is polycaprolactone (PCL) (see for example, Gunatillake et al, 2003; Eur Cells & Materials, 5; 1 -16, Griffith et al, 2000, polymeric biomaterials, Acta. Mater, 48;263-277, and Hayashi T et al, prog Polym Sci, 19;663- 702).

[105] The cell coated substrate of the invention can comprise any substrate including bioabsorbable materials such as polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone (PCL), polydioxanone (PDO), trimethylene carbonate (TMC), polyvinyl alcohol (PVA), copolymers or derivatives or blends thereof. In one embodiment, the substrate is a polylactic acid and polyglycolic acid copolymer at a 95:5 mole ratio.

[106] In one embodiment, the substrate is biocompatible, and biodegrades or autocatalytically degrades in vivo into biocompatible byproducts. Not to be bound by theory, but prevailing mechanism for polymer degradation is chemical hydrolysis of the hydrolytically unstable backbone of the PLGA polymers. This occurs in two phases. In the first phase, water penetrates the polymer, preferentially attacking the chemical bonds in the amorphous phase and converting long polymer chains into shorter water- soluble fragments. Because this occurs initially in the amorphous phase, there is a reduction in molecular weight without a loss in physical properties since the polymer matrix is still held together by the crystalline regions. The reduction in molecular weight is soon followed by a reduction in physical properties, as water begins to fragment the material. In the second phase, enzymatic attack and metabolization of the fragments occurs, resulting in a rapid loss of polymer mass. This type of degradation, when the rate at which water penetrates the substrate material exceeds that at which the polymer is converted into water- soluble materials (resulting in erosion throughout the substrate), is termed

"bulk erosion" (Hubbell and Langer, 1995). The rate of degradation of PLGA's can be controlled, in part by the copolymer ratio with higher glycolide or lactide ratios favoring longer degradation times. Polymers of varying copolymer ratios including PLA, PLGA75:25, and PLGA50:50 have different degradation rates, with PLGA50:50 degrading the quickest, followed by PLGA 75:25 then PLA. Therefore, with increasing percentage of PGA and concurrent decrease in percentage of PLA in a co-polymer of PLGA increases the rate of degradation compared to PLA alone, and thus the rate of degradation can be tailored to the desired use. Any ration of PLA:PGA copolymer is encompassed for use in the present invention. [107] In some embodiments, the substrate comprises at least one of polyglycolic acid (PGA), polylactic acid (PLA), poly(lactic-co-glycolic) acid (PLGA), polyanhydride, polycapralactone (PCL), polydioxanone and polyorthoester. One of the most common polymers used as a biomaterial is the polyester copolymer poly(lactic acid-glycolic acid) (PLGA). PLGA is highly biocompatible, degrades into biocompatible monomers and has a wide range of mechanical properties making this copolymer and its homopolymers, PLA and PGA, useful in skeletal repair and regeneration. The substrate can be porous or non-porous comprising these polymers for use in bone repair have been prepared using various techniques.

[108] The substrate of the present invention can also be a material that comprises an absorbable polymer material and other materials. In some embodiments, other materials can be selected to be used as the resorbable material, which can be selected from the group consisting of hydroxyapatite (HAP), tricalcium phosphate (TCP), tetracalcium phosphate (TTCP), dicalcium phosphate anhydrous (DCPA), dicalcium phosphate dihydrate (DCPD), octacalcium phosphate

(OCP), calcium pyrophosphate (CPP), collagen, gelatin, hyaluronic acid, chitin, and poly(ethylene glycol). In alternative embodiments, the substrate can also comprise additional material, for example, but are not limited to calcium alginate, agarose, types I, II, IV or other collagen isoform, fibrin, hyaluronate derivatives or other materials (Perka C. et al. (2000) J. Biomed. Mater. Res. 49:305-311; Sechriest V F. et al. (2000) J. Biomed. Mater. Res. 49:534-

541 ; Chu C R et al. (1995) J. Biomed. Mater. Res. 29:1147-1154; Hendrickson D A et al. (1994) Orthop. Res. 12:485-497).

[109] In some embodiments, the substrate composed of a poly(lactic acid-co-glycolic acid) [PLGA], can be prepared as a composite with other materials. For example, other materials include for example, but not limited to calcium phosphate ceramic, for example as HA, for engineering of surface modifications of cortical bone allografts, and in some embodiments, the PLGA can be prepared in conjunction with an osteoconductive buffering agent such as HA. Such materials can also be used as fillers or bulking agents, or buffering compounds. HA is a buffering compound since it neutralizes acidic breakdown products of biodegradable polymers such as lactic acid and glycolic acid containing polymers, thereby diminishing the likelihood these materials could cause cytotoxicity, separation of the implant and sepsis. [110] In some embodiments, the substrate of the present invention can additionally provide controlled release of bioactive factors to the seeded cells, for example, growth factors and other agents to sustain or control subsequent cell growth and proliferation of the cells coated on the substrate of the present invention. In such a way, the cells are supplied with a constant source of growth factors and other agents for the duration of the lifetime of the cell coated substrate. In some embodiments where the cell coated substrate is used for cartilage repair, the growth factors and other agents are condroinducive agents. Examples of chondroinducive agents include, for example but not limited to, transforming growth factor-A (TGF-A), insulin-like growth factor (IGF), platelet derived growth factor (PDGF), epidermal growth factor (EGF), acidic fibroblast growth factor (aFBF), basic fibroblast growth factor (bFBF), hepatocytic growth factor, (HGF) keratinocyte growth factor.(KGF), the bone morphogenic factors (BMPs) i.e. , BMP-I, BMP-2, BMP-3, BMP-4, BMP-5 and BMP-6 and the osteogenic proteins (OPs), i.e. OP-I, OP-2 and OP- 3, and combinations and variants thereof. [Ill] In alternative embodiments, where the cells coated substrate is used for the repair of bone defects, the growth factor and agents have effects which stimulate bone ingrowth (i.e., osteoconductive agents) and/or bone cell recruitment (i.e.. osteoinductive agents), or as a scaffolding for bone progenitor cells that are seeded onto or into the matrix, prior to or at the time of implantation, which can themselves produce osteoconductive and/or osteoinductive agents. [112] Examples of stimulatory molecules include for example, but not limited to, regulatory factors involved in bone repair, such as hormones, cytokines, growth factors, and other molecules that regulate growth and differentiation; and osteoinductive agents such as bone morphogenetic or morphogenic proteins (BMPs). The latter are also referred to as osteogenic bone inductive proteins or osteogenic proteins (OPs). Several BMP (or OP) genes have now been cloned, and the common designations are BMP-I through BMP-8. Although the BMP terminology is widely used, it can prove to be the case that there is an OP counterpart term for every individual BMP (Alper. 1994). BMPs 2-8 are generally thought to be osteogenic, although BMP-I is a more generalized morphogen (Shimell et al., 1991). BMP-3 is also called osteogenin (Luyten et al., 1989) and BMP-7 is also called OP-I (Ozkaynak et al., 1990). BMPs are related to, or part of, the transforming growth factor-beta (TGF-beta) superfamily, and both TGF beta 1 and TGF beta

2 also regulates osteoblast function (Seitz et al., 1992). Several BMP (or OP) nucleotide sequences and polypeptides have been described in U.S. patents, e.g.. U.S. Patent Nos. 4,795,804, 4,877,864, 4,968,590, and 5,108,753; including, specifically, BMP-I disclosed in U.S. Patent No. 5,108,922; BMP-2A in U.S. Patent Nos. 5,166,058 and 5,013,649; BMP-2B disclosed in U.S. Patent No. 5,013,649; BMP-3 in U.S. Patent No. 5,116,738; BMP-5 in U.S.

Patent No.. 5,106,748; BMP-6 in U.S. Patent No. 5,187,076; BMP-7 in U.S. Patent Nos. 5,108,753 and 5,141,905; and OP-I, COP-5 and COP-7 in U.S. Patent No. 5,011,691. [113] Other growth factors or hormones that have been reported to have the capacity to stimulate new bone formation include acidic fibroblast growth factor (Jingushi et al., 1990); estrogen (Boden et al., 1989); macrophage colony stimulating factor (Horowitz et al., 1989); and calcium regulatory agents such as parathyroid hormone (PTH) (Raisz & Kream, 1983). Several groups have investigated the possibility of using bone stimulating proteins and polypeptides, particularly recombinant BMPs, to influence bone repair in vivo. For example, recombinant BMP-2 has been employed to repair surgically created defects in the mandible of adult dogs (Toriumi et al., 1991), and high doses of this molecule have been shown to functionally repair segmental defects in rat femurs (Yasko et al., 1992). Chen and colleagues showed that a single application of 25-100 ng of recombinant TGF-beta 1 adjacent to cartilage induced endochondral bone formation in the rabbit ear full thickness skin wounds (Chen et al., 1991). It has also been reported that an application of TGF-beta 1 in a 3% methylcellulose gel was able to repair surgically induced large skull defects that otherwise heal by fibrous connective tissue and never form bone (Beck et al., 1991).

[114] In some embodiments, osteotropic proteins also include, for example, transforming growth factor, fibroblast growth factor, granulocyte/macrophage colony stimulating factor, epidermal growth factor, platelet derived growth factor, insulin-like growth factor, and leukemia inhibitory factor. [115] In a further embodiment, instead of a protein growth factor or agent released by the substrate on degradation, a gene or other nucleotide molecule encoding the stimulatory factor can be released. For example but not limited to, the nucleotide molecule can be DNA (double or single- stranded) or RNA (e.g.. mRNA, tRNA, rRNA), or it can be an antisense nucleic acid molecule, such as antisense RNA that can function to disrupt gene expression or growth factors themselves including TGF-beta 1 and 2, and IGF-I . The nucleic acid segments can be genomic sequences, including exons or introns alone or exons and introns, or coding cDNA regions, or any nucleic acid construct, for example genes or gene fragments that one desires to transfer to a bone progenitor cells or cells coating the substrate, for example chondrocytes. Suitable nucleic acid segments can also be in virtually any form, such as naked DNA or RNA, including linear nucleic acid molecules and plasmids, or nucleic acid analogues, such as peptide nucleic acid (PNA), pseudo-complementary nucleic acid (pc-PNA), locked nucleic acid (LNA) and other agents, such as peptides, aptamers, RNAi etc, or as a functional insert within the genomes of various recombinant viruses, including viruses with DNA genomes and retroviruses. [116] As noted above, stimulatory factors, including chondroinductive agents, osteoinductive and osteoinductive agents, can be absorbed into, onto or coupled to the substrate of the present invention, with the cells also seeded onto the substrate. Thus, in some embodiments, growth factors and agents, for example chondroinductive agents, osteoinducive agents and osteoconducive agents and/or solids can be added to the substrate of the present invention. In some embodiments, the added agents and factors can not distribute equally throughout the substrate, and therefore regions of the substrate will be created that will have a different composition of the added agents and/or solids. Alternatively, the agents and/or solids can be more concentrated in desired regions (i.e., near the top, sides, or bottom) of the substrate, for example, the agents and/or solids are concentrated in selected locations.

[117] In some embodiments, the solids are of a type that will not react with the substrate. Generally, the added solids have an average diameter of less than about 1.0 mm and preferably will have an average diameter of about 50 to about 500 microns. Preferably, the solids are present in an amount such that they will constitute from about 1 to about 50 volume percent of the total volume of the particle and polymer-solvent mixture (wherein the total volume percent equals 100 volume percent). Exemplary solids include, but are not limited to, particles of demineralized bone, calcium phosphate particles, Bioglass particles, calcium sulfate, or calcium carbonate particles for bone repair, leachable solids for pore creation and particles of bioabsorbable polymers that are effective as reinforcing materials or to create pores as they are absorbed, and non-bioabsorbable materials. Suitable leachable solids include nontoxic leachable materials such as salts (e.g., sodium chloride, potassium chloride, calcium chloride, sodium tartrate, sodium citrate, and the like), biocompatible mono and disaccharides (e.g., glucose, fructose, dextrose, maltose, lactose and sucrose), polysaccharides (e.g., starch, alginate, chitosan), water soluble proteins (e.g., gelatin and agarose). The leachable materials can be removed by immersing the substrate with the leachable material in a solvent in which the particle is soluble for a sufficient amount of time to allow leaching of substantially all of the particles, but which does not detrimentally alter the substrate. In one embodiment, the solvent is water, for example distilled- deionized water. Such a process is described in U.S. Pat. No. 5,514,378, which is incorporated herein in its entirety by reference.

[118] In some embodiments, the substrate of the present invention can be a smooth surface which also has pores on the surface, allowing for the easy adherence and stable fixation of cells, for example chondrocytes, mesenchymal stem cells, osteoblasts, and osteoprogenitor cells in pores of the surface. Importantly, in the methods of the invention provide a substrate with pores on the surface but not interdispersed throughout the entire substrate. In addition, at least part of the substrate can be calcified. Pores on the surface of the substrate can be created by methods commonly known by persons skilled in the art. Representative methods include, for example, solvent evaporation, where the substrate or polymer is dissolved in a solvent. Examples of organic solvents which can be used to dissolve the substrate are well known in the art and include for example, glacial acetic acid, methylene chloride, chloroform, tetrahydrofuran, and acetone. Accurate control over pore size in the substrate is desired in order to have adherence of the cells on the surface of the substrate without their penetration into the substrate itself. In some embodiments, the desired pore size of pores on the surface of the substrate is about 150-250 μm (Hulbert et al., J. Biomed. Mat. Res. 1970 4:443). [119] In another embodiment of this invention, the pores on the surface of the substrate are filled with calcium phosphate, or a calcium phosphate based material. In another embodiment, the pores on the surface of the substrate are filled an osteoinductive material with or without a buffering or osteoconductive filler. In some embodiments, the substrate is mixed with the osteoinductive agent with or without the filler such as the calcium phosphate based material.

[120] In some embodiments, suitable fillers include graphite or pyrolytic carbon; bioceramics; bone powder; fully mineralized and partially or fully demineralized cortical and cancellous bone in any form, including particles such as demineralized bone powder (or "demineralized bone solid" as it can also be known); sheets and shaped bone pieces; bioglass or other bioceramic or natural or synthetic polymers, e.g., bioabsorbable polymers such as polyglycolide, polylactide, glycolide-lactide copolymer, and the like; nonbioabsorbable materials such as starches, polymethyl methacrylate, polytetrafiuoroethylene, polyurethane, polyethylene and nylon; anorganic bone (i.e., bone mineral only, with the organic constituents removed), dentin tooth enamel, aragonite, calcite, nacre, amorphous Calcium phosphate, hydroxyapatite, Tricalcium phosphate and other Calcium phosphate materials; Calcium salts; etc. and mixtures of any of the foregoing. When employed, filler will typically represent from about 1 to about 50 weight percent of the bone particle containing composition, calculated prior to forming the shaped material. In some embodiments, the fillers are ceramics, particularly hydroxyapatite and mineralized cortical bone powder. [121] In some embodiments, the substrate is also coated with, or combined with biostatic or biocidal agents. Suitable biostatic/biocidal agents include for example, but not limited to antibiotics, povidone, sugars, mucopolysaccharides, chlorobutanol, quarternary ammonium compounds such as benzalkonium chloride, organic mercurials, parahydroxy benzoates, aromatic alcohols, halogenated phenols, sorbic acid, benzoic acid, dioxin, EDTA, BHT, BHA, TBHQ, gallate esters, NDGA, tocopherols, gum guaiac, lecithin, boric acid, citric acid, p-

Hydroxy benzoic acid esters, propionates, Sulfur dioxide and sulfites, nitrates and nitrites of Potassium and Sodium, diethyl pyrocarbonate, Sodium diacetate, diphenyl, hexamethylene tetramine o-phenyl phenol, and Sodium o-phenylphenoxide, etc. When employed, biostatic/biocidal agent will typically represent from about 1 to about 25 weight percent of the substrate, calculated prior to forming the shaped material. In some embodiments, the biostatic/biocidal agents are antibiotic drugs. [122] In some embodiments, the substrate is pretreated prior to seeding with cells and/or chondrocyte cells or osteoprogenitor in order to enhance the attachment of cells to the substrate. For example, prior to seeding with cells, the substrate can be treated with, for example, but not limited to, 0.1M acetic acid and incubated in polylysine, polylysine, PBS, collagen, poly-laminin and other cell adhesive substances known to persons skilled in the art. [123] Suitable surface active agents include the biocompatible nonionic, cationic, anionic and amphoteric surfactants and mixtures thereof. When employed, surface active agent will typically represent from about 1 to about 20 weight percent of the substrate, calculated prior to forming the shaped material. It will be understood by those skilled in the art that the foregoing list of optional substances is not intended to be exhaustive and that other materials can be admixed with substrate within the practice of the present invention.

[124] Any of a variety of medically and/or surgically useful optional substances can be incorporated in, or associated with, the substrate either before, during, or after preparation of the cell coated substrate. Thus for example, one or more of such substances can be introduced into the substrate, e.g., by soaking or immersing the substrate in a solution or dispersion of the desired substance(s), by adding the substance(s) to the carrier component of the cell coated substrate or by adding the substance(s) directly to cell coated substrate. Medically/surgically useful substances include physiologically or pharmacologically active substances that act locally or systemically in the host subject. [125] The medically/surgically useful substances are, for example but not limited to bioactive substances which can be readily combined with the cell coated substrate of this invention and include, e.g., demineralized bone powder as described in U.S. Pat. No. 5,073,373 the contents of which are incorporated herein by reference; collagen, insoluble collagen derivatives, etc., and soluble solids and/or liquids dissolved therein; antiviricides, particularly those effective against HIV and hepatitis; antimicrobials and/or antibiotics such as erythromycin, bacitracin, neomycin, penicillin, polymycin B, tetracyclines, biomycin, Chloromycetin, and streptomycins, cefazolin, ampicillin, azactam, tobramycin, clindamycin and gentamycin, etc.; biocidal/biostatic sugars such as dextran, glucose, etc.; amino acids; peptides; vitamins; inorganic elements; co-factors for protein synthesis; hormones; endocrine tissue or tissue fragments; synthesizers; enzymes such as alkaline phosphatase, collagenase, peptidases, oxidases, etc.; polymer cell scaffolds with parenchymal cells; angiogenic agents and polymeric carriers containing such agents; collagen lattices; antigenic agents; cytoskeletal agents; cartilage fragments; living cells such as chondrocytes, bone marrow cells, mesenchymal stem cells; natural extracts; genetically engineered living cells or otherwise modified living cells; expanded or cultured cells; DNA delivered by plasmid, viral vectors or other means; tissue transplants; demineralized bone powder; autogenous tissues such as blood, serum, soft tissue, bone marrow, etc.; bioadhesives; bone morphogenic proteins (BMPs); osteoinductive factor (IFO); fibronectin (FN); endothelial cell growth factor (ECGF); vascular endothelial growth factor (VEGF); cementum attachment extracts (CAE); ketanserin; human growth hormone (HGH); animal growth hormones; epidermal growth factor (EGF); interlenkins, e.g., interleukin-1 (IL-I), interleukin-2 (IL-2); human alpha thrombin; transforming growth factor (TGF -beta); insulin-like growth factors (IGF-I, IGF-2); platelet derived growth factors (PDGF); fibroblast growth factors (FGF, BFGF, etc.); periodontal ligament chemotactic factor (PDLGF); enamel matrix proteins; growth and differentiation factors (GDF); hedgehog family of proteins; protein receptor molecules; small peptides derived from growth factors above; bone promoters; cytokines; somatotropin; bone digestors; antitumor agents; cellular attractants and attachment agents; immuno-suppressants; permeation enhancers, e.g., fatty acid esters such as laureate, myristate and stearate monoesters of polyethylene glycol, enamine derivatives, alpha-keto aldehydes, etc.; and nucleic acids. The amounts of such optionally added substances can vary widely with optimum levels being readily determined in a specific case by routine experimentation. [126] It will be understood by those skilled in the art that the foregoing list of medically/surgically useful substances is not intended to be exhaustive and that other useful substances can be admixed with substrate and/or the cell coated substrate within the practice of the present invention. [127] The total amount of such optionally added medically/surgically useful substances will typically range from about 0 to about 95, or about 1 to about 60, or about 1 to about 40 weight percent based on the weight of the entire composition prior to compression of the composition, with optimal levels being readily determined in a specific case by routine experimentation. In some embodiments, a medically/surgically useful substance is bone morphogenic proteins. [128] In some embodiments, the substrate is sterilized prior to or after the seeding chondrocytes. General sterilization methods can be used, for example, but not limited to ethylene oxide or irradating with an electron beam, and in some embodiments, where the effect of the sterilization is toxic to the cells coated on, or to be coated on the substrate, alternative sterilization methods are sought or compensatory methods adopted, for example, additional TGFβ added to the cultured chondrocytes to reduce chondrocytes detaching from the substrate prior to forming extracellular matrix due to the use of irradiation sterilization.

[129] In some embodiments, the surface of the substrate can preferably be thickly coated with a cartilage or bone matrix to improve the biocompatibility after the implantation. In some embodiments, a part of the cell coated substrate can be calcified. In addition, the cell coated substrate comprising a bone matrix can preferably contain a growth factor such as bone morphogenetic protein, which is secreted by at least one of cells selected from marrow cells, mesenchymal stem cells, osteoblasts, and osteoprogenitor cells. Since the growth factor promotes physiological adhesion, proliferation, and differentiation of cells into their differentiated cell type, the addition of growth factors to the cell coated substrate can improve the speed of cartilage and/or bone repair and biocompatibility of the implanted cell coated substrate. [130] Molding of cell coated substrate

[131] In one embodiment, the cell coated substrate comprises a pliable or moldable support that permits the attachment of cells, for example see Figure IE. It is appreciated that any pliable, malleable or moldable bioresorbable support that allows the attachment of cells, for example chondrocyte cells or osteoblasts or osteoprogenitors can be useful in the practice of the present invention. In some embodiments, the size and shape and dimension of the bioresorbable material is molded, for example, but not limited to, the size and shape and dimension of the defect to be repaired, for example cartilage or bone defect to be repaired. [132] In some embodiments, the substrate of the present invention can be molded or mechanically shaped by any known procedures in the art. As discussed above, the molding methods depend on the material of the substrate, for example malleable under non-physiological or physiological conditions as discussed in the section entitled 'substrate" above. In some embodiments, such shaping or molding can be accomplished through the application of compressive and, optionally, simultaneous lateral force(s). Because the substrate can display viscoelastic properties, the force required to deform the mass is sensitive to the rate of application and is readily determined by routine experimentation. The application of this force(s) can be accomplished by a variety of methods, e.g., pressing, extruding, rolling, etc. When necessary to prevent the substrate from sticking to the work surface and/or rollers), the substrate can be placed between two flexible stick-resistant surfaces, e.g., Tyvek™ sheets, or a Teflon™ coated surface [133] In some embodiments, the substrate can be molded to assume a determined or regular form or configuration, for example, but not limited to a sheet, plate, disk, tunnel, cone, tube, to name but a few. Prefabricated geometry would include, but not be limited to, a crescent apron, I-shape, a rectangluar bib, neutralization plates, reconstructive plates, buttress plates, T-buttress plates, spoon plates, clover leaf plates, condylar plates, compression plates, bridge plates, wave plates, etc. Partial tubular as well as flat plates can also be fabricated from the substrate of this invention. Such plates can include for example such conformations as, e.g., concave contoured, bowl shaped, defect shaped, etc. Importantly, the substrate can be molded or shaped by any suitable mechanical shaping means, and in some embodiments computerized modeling can, for example, be employed to provide for the intricately-shaped architecture of the smooth substrate which is custom-fitted to the cartilage and/or bone repair site with great precision.

[134] Optionally, the substrate can be formed into a laminate. Advantages of a laminate of the substrate include: i) allowing the laminate to be shaped in three dimensions, as in the introduction of a concave surface shape, etc.; ii) each layer of the laminate would be continuous, without requiring binding of the joints between the pieces. A laminate prepared according to the invention herein would provide a more uniform and stronger laminate implant for the repair of bone defects (ie osteoimplants) or for the repair of cartilage defects than those that are available utilizing prior art methods.

[135] In some embodiments, the substrate is in a smooth configuration. In some embodiments, the substrate is in a substantially smooth configuration. In some embodiments, the thickness of the substrate ranges from 0.1mm to 5mm. In some embodiments the thickness ranges from approximately 0.5-2.0mm, which is sufficiently thin to be moldable, but sufficiently thick for structural integrity. In some embodiments, the substrate is 0.5mm, 0.8mm or 1.2mm in thickness. In some embodiments, the cell coated substrate of this invention for the used in bone repair is much thinner than prior art implants for bone repair or other osteoimplants. For example, the substrate of the present invention can have a thickness for example ranging from about 50 microns to about 2000 microns. In one embodiment, the substrate can be contoured to a specific three-dimensional architecture that is retained after implantation. This form holding embodiment of the invention is able to be incorporated as a graft into the cartilage or bone defect and retains its architecture even during implantation. [136] In each of the different embodiments of the invention, the cell coated substrate prepared according to the method of this invention is not limited as to its final size by the anatomic constraints of the cartilage or bone defect to be repaired, but rather, it is capable of being made to any size so long as an appropriate amount of starting material is available. The term "flexible" as utilized herein refers to the ability of the substrate to be deformed by the application of a force or combination of forces, e.g., compressive, flexural, etc.

[137] The area of the substrate varies depending on the size of the defect. In some embodiments, the area of the substrate ranges from 2.0mm²- 10.0cm². In some embodiments, the dimensions of the substrate to repair a small cartilage defect are in the range, for example but not limited to, from 2mm²-lcm². In alternative embodiments, the dimensions of the substrate to repair a large cartilage defect are in the range of, for example but not limited to, lcm²-5.0cm². In some embodiments, the substrates can be greater than 5.0cm² in dimensions. The dimensions and the thickness of the substrate can be made in various dimensions depending on the size of the defect being treated. [138] In some embodiments, the size of the defect to be repaired is determined, for example, by MRI and a three dimensional map of the cartilage and cartilage defect can be determined, for example, see U.S. Patent Application 2002/0087274 and U.S. Patent Application 2002/0157676, the disclosures of which are incorporated herein in their entirety by reference. In some embodiment of the invention, the tissue is created by a technique known in the art that builds a complex 3-dimensional (3D) object from 3D structures in a flat or different configuration. In some embodiments, the size of the defect can be determined by alternative methods, for example, but not limited to inferentially by computer aided tomography (CAT scanning), X-ray examination, magnetic resonance imaging (MRI), analysis of synovial fluid or serum markers or by any other procedures known in the art. [139] In some embodiments, the cell coated substrate prepared according to methods of the present invention can be "trimmed" to a pre-selected size and shape and dimensions by the surgeon performing the surgical repair of the cartilage defect or bone defect, m an alternative embodiment, the cell coated substrate can be trimmed at the time of implantation (for example see Figure 6b as an exemplary example). Trimming can be performed with the use of a sharp cutting implement, for example a scalpel, a pair of scissors, or an arthroscopic device fitted with a cutting edge, using procedures well known in the art. [140] The composition, thickness, and porosity of the resorbable substrate can be controlled to provide the desired mechanical and biological characteristics. For example, the bioabsorption rate of the cell coated substrate can be selected to provide a longer or shorter bioabsorption profile depending on the composition of the material of the substrate, the structural integrity required to sustain repeated mechanical forces that can be applied to the cell coated substrate after implantation. Generally, the cell coated substrate has the thickness in the range of about

0.1mm to 5mm. In some embodiments, the thickness is from 0.5mm to 2.0mm. In some embodiments, the thickness of the substrate is 0.5mm, 0.8mm or 1.2mm in thickness. Alternatively, in some embodiments, for example for some applications such as for the repair of large cartilage defects or large bone defects, the cell coated substrate has a thickness greater than about 5 mm.

[141] Use of cell coated substrate for cartilage defect repair.

[142] In some embodiments, the cell coated substrate as disclosed herein can be used to replace or augment existing cartilage and/or bone tissue, to introduce new or altered tissue, to modify artificial prosthesis, or to add to biological tissues or structures, hi some embodiments, the cell coated substrate useful for the repair of cartilage defects is a cartilage coated substrate. For example, but not limited to, particular embodiments of the invention include the used of cell coated substrates, for example cartilage coated substrates for i) hip prosthesis ii) knee reconstruction and iii) prosthesis of other joints requiring reconstruction and/or replacement of articular cartilage. In some embodiments, the joints in need of repair are for example but not limited to, knee, hip, elbow, ankle, glenohumeral joint. The cartilage coated substrate can also be employed in minor and major reconstructive surgery for different types of joints. Detailed procedures have been described in Resnick, D. and Niwayama G eds., 1988, Diagnosis of Bone and Joint Disorders, 2d ed., W.B. Sanders Co. [143] The cell coated substrates, for example a cartilage coated substrate as disclosed herein are especially useful for, but are not limited to, the repair of large defects in cartilage. As used herein, a "large" defect generally refers to a cartilage defect that covers or adversely affects a surface area larger than about 1 square centimeter (cm), for example, at least lcm², or at least 1.5cm² or at least 2cm² or greater than 2cm² or any surface area size or diameter in between lcm² and 2cm². As noted above, success rates using transplanted chondrocyte cells to repair cartilage defects drop off sharply when the defect is larger than about 1 square cm. The present invention however, is not limited to treating defects larger than 1 square centimeter. In some embodiments, the compositions as disclosed herein can also be used for treating smaller cartilage defects, such as defects in the size range of about 0.5 to about 1 square cm, or less than 0.5cm². In some embodiments the cell coated substrate, for example the cartilage coated substrate can be useful for the repair of articular cartilage defects. In some embodiments the defects are human articular cartilage defects.

[144] In some embodiments, cartilage defects in mammals are readily identifiable visually during arthroscopic examination or during open surgery of the joint. In alternative embodiments, cartilage defects can also be identified inferentially, for example, by using computer aided tomography (CAT scanning), X-ray examination, magnetic resonance imaging (MRI), analysis of synovial fluid or serum markers or by any other procedures known in the art. In some embodiments, treatment of the defects can be effected during an arthroscopic or open surgical procedure using the methods and compositions disclosed herein. [145] Accordingly, once the defect has been identified, the defect can be treated by the following steps of (1) surgically implanting at the pre- determined site, a piece of cell coated substrate prepared by the methodologies described herein, and (2) permitting the cartilage or the cell coated substrate to integrate into predetermined site. The cell coated substrate optimally has a size and shape such that when the cell coated substrate is implanted into the defect, the edges of the implanted tissue contact directly the edges of the defect. In addition, the cell coated substrate can be molded to fit the defect prior to or during the implantation procedure.

[146] Surgical procedures for effecting the repair of articular cartilage defects are well known in the art. See for example: Luyten & Reddi (1992) in "Biological Regulation of the Chondrocytes", CRC Press, Boca Raton, Ann Arbor, London, & Tokyo, p.p. 227-236, the disclosure of which is incorporated by reference herein. [147] Use of cell coated substrate for Bone Defect Repair

[148] The cell coated substrate of the present invention can be used to replace or repair damaged bone tissue, to introduce new tissue, to modify artificial prosthesis, or to repair bone defects or add to biological tissues or structures. In some embodiments, the cell coated substrate of the present invention is useful for the repair of a large number of essentially intact, but defected bone structures, including for example but not limited to femur, femur head, distal end of femur, proximal end of femur, fibula, tibia, ilia, mandibular, humerus, radius, ulna, vertebrae, ribs, scapula, foot bones and hand bones, prior to subsequent processing into small specific cut-bone grafts and of being usable on small cut-bone grafts, including iliac crest wedges, Cloward dowels, ribs, cancellous cubes, fibular struts. The process involves repairing the bone defect with a cell coated substrate comprising cells which have become or are capable of becoming or differentiating into bone tissue and bone matrix and/or bone.

[149] The bone to be repaired can be cortical, cancellous, or cortico-cancellous of autogenous, allogenic, xenogenic or transgenic origin. In some embodiments, the bone defect to be repaired is brought about during the course of surgery, infection, malignancy or developmental malformation. The cell coated substrate of the present invention can be utilized in a wide variety of orthopedic, periodontal, neurosurgical, oral and maxillofacial surgical procedures such as for example but not limited to, the repair of simple and compound fractures and non-unions; external and internal fixations; joint reconstructions such as arthrodesis; general arthroplasty; cup arthroplasty of the hip; femoral and humeral head replacement; femoral head surface replacement and total joint replacement; repairs of the vertebral column including spinal fusion and internal fixation; tumor surgery, e.g., deficit filling; discectomy; laminectomy; excision of spinal cord tumors; anterior cervical and thoracic operations; repairs of spinal injuries; scoliosis, lordosis and kyphosis treatments; intermaxillary fixation of fractures; mentoplasty; temporomandibular joint replacement; alveolar ridge augmentation and reconstruction; inlay osteoimplants; implant placement and revision; sinus lifts; cosmetic procedures; etc. [150] In some embodiments, the specific bones which can be repaired or replaced with the cell coated substrate of the invention, are for example but not limited to, the ethmoid, frontal, nasal, occipital, parietal, temporal, mandible, maxilla, zygomatic, cervical vertebra, thoracic vertebra, lumbar vertebra, sacrum, rib, sternum, clavicle, scapula, humerus, radius, ulna, carpal bones, metacarpal bones, phalanges, ilium, ischium, pubis, femur, tibia, fibula, patella, calcaneus, tarsal and metatarsal bones.

[151] In some embodiments, the cell coated substrate of the present invention is useful for clinical applications, such as for example, the treatment of traumatic fractures, pathologic fractures, stress fractures, congential defects or fractures, or operative defects in any bone of the body that would be treated with plate fixation. Fracture categories treated with the cell coated substrate can include but not be limited to intraarticular or periarticular fractures; metaphyseal fractures; transverse, oblique, comminuted, and fragmented fractures; repair to non-fractured sites; defects due to periodontal disease or surgery; and other bone defects. The cell coated substrate can also be used to repair bone defects currently treated with cancellous bone grafting, for example but not limited to segmental bone loss. [152] In some embodiments, the cell coated substrate can be used in various shapes as a replacement of the bone tissue. For example, the artificial material can include an artificial joint such as a hip joint, a knee joint, a finger joint, a shoulder joint, an elbow joint, and an ankle joint; a metallic artificial bone; an artificial bone made of synthetic resin; an artificial bone made of ceramics; volts (screw) for coupling bone tissues; prosthesis materials; dental implant materials; and bone connecting structures. [153] In some embodiments, at the time just prior to when the cell coated substrate as disclosed herein is to be placed in a bone defect site, optional materials, e.g., autograft bone marrow aspirate, autograft bone, preparations of selected autograft cells, autograft cells containing genes encoding bone promoting action, etc., can be combined with the cell coated substrate of this invention. [154] In some embodiments, the cell coated substrate can be implanted at the bone repair site, if desired, using any suitable affixation means, e.g., sutures, staples, bioadhesives, screws, pins, rivets, other fasteners and the like or it can be retained in place by the closing of the soft tissues around it. [155] Cell coated substrate implantation. [156] In some embodiments the cell coated substrate, for example a cartilage coated substrate can be molded into the appropriate shape to fit and/or conform to the size and shape of the defect to be repaired. In alternative embodiments, the cell coated substrate, for example cartilage coated substrate is flat, non-molded configuration. In further embodiments, the cell coated substrate is molded into a cone configuration or any other configuration that is appropriately shaped for repair of the defect to be repaired. In some embodiments, the cell coated substrate is implanted into the subject so that the cells coating the contact the defect. In other words, going from the superficial outermost edge of the cell coated substrate towards the defect, the substrate is on the outer most-edge, followed by the cells, for example chondrocyte cells which are in contact with the defect. In alternative embodiments, the cell coated substrate, for example cartilage coated substrate is implanted into the subject so that the substrate is in contact with the defect. In other words, going from the superficial outermost edge of the cell coated implant towards the defect, the cells, for example chondrocyte cells are on the outer most-edge, followed by the substrate which is in contact with the defect. [157] In an alternative embodiment, two or more cell coated substrates can be implanted into a subject to repair a defect. In some embodiments, two cell coated substrates are joined together.

In some embodiments, cell coated substrates can be joined together with the cell layers on the outer most edges, and the substrate layers on the inside, and thus when implanted to repair the defect, the cells are in contact with the defect and also on the superficial outermost layer, with the joined substrate layers on the inside. In other words, going from the superficial outermost edge of the joined cell coated implant towards the defect, the cells from one cell coated substrate are on the superficial outermost edge, followed by the substrate of the same cell coated substrate, then the substrate of another cell coated substrate, the cells of the same cell coated substrate which are in contact with the defect.

[158] In alternative embodiments, cell coated substrates can be joined together with the cell layers on the inside, and the substrates on the outer most edges, and thus when implanted to repair the defect, the substrate is in contact with the defect and is also on the superficial outermost layer, with the cells sandwiched between the substrates on the inside. In other words, going from the superficial outermost edge of the joined cell coated implants towards the defect, the substrate from one cell coated substrate is on the superficial outermost edge, followed by the cells of the same cell coated substrate, then the cells of another cell coated substrate, followed by the substrate of the same cell coated substrate which is in contact with the defect. In some embodiments, multiple cell coated substrates can be joined together and in any joining combination, for example joining by cell-to-cell or substrate-to-substrate of the cell coated substrates as discussed above. [159] In some embodiments, the joined cell coated substrates can comprise cells of different origins, for example one cell coated substrate can be coated with cells for cartilage repair, for example chondrocytes, and the other cell coated substrate can be coated with cells for bone repair, for example bone progenitors or osteoblasts or osteoprogenitors etc.

[160] In one embodiment, the substrate of the present invention can be molded to the size of the defect to be repaired prior to seeding with cells, for example chondrocytes or osteoblasts or osteoprogenitors etc. Alternatively, the cell coated substrate can be molded after seeding with cells, for example chondrocytes. In an alternative embodiment, once access is made into the affected anatomical site (whether by minimally invasive or open surgical technique), the cell coated substrate can be molded once placed in the desired position of the defect to be repaired, for example cartilage defect or bone defect. The time of molding of the substrate, for example pre- or post-seeding with cells, is determined by the material of the substrate, for example if it is malleable under physiological or non-physiological conditions, as discussed above, and also determined by the defect to be corrected and also on the decision by the physician performing the implantation of the cell coated substrate. [161] In some embodiments, the cell coated substrate, for example cartilage coated substrate need not use any chemical and/or mechanical fasteners for attachment. In such embodiments, the placement of the cell coated substrate can be accomplished through an interference fit of the cell coated substrate with an appropriate site in the tissue to be treated.

[162] In an alternative embodiment, once the cell coated substrate, for example cartilage coated substrate is molded into the desired configuration and is located in the desired position, it can be optionally affixed by using a suitable technique commonly known to persons skilled in the art.

In one aspect, the implanted cell coated substrate can be affixed by a chemical and/or mechanical fastening technique. In alternative embodiments, where the defect is a large defect, for example lcm² or greater, the cell coated substrate can be secured in place by any means known to persons skilled in the art. For example, such methods involve use of staples, screws and sutures etc, as well as suitable chemical fasteners include glues and/or adhesive such as fibrin glue, fibrin clot, cyanoacrylate, and other known biologically compatible adhesives.

Examples of suitable binders and biological adhesives include, but are not limited to, fibrin glue, fibrinogen, thrombin, mussel adhesive protein, silk, elastin, collagen, casein, gelatin, albumin, keratin, chitin or chitosan; cyanoacrylates; epoxy-based compounds; dental resin sealants; bioactive glass ceramics (such as apatite- wollastonite); dental resin cements; glass ionomer cements; nonbioabsorbable polymer such as polyacrylate, polymethyl methacrylate, polytetrafluroethylene, polyurethane, polyamide; gelatin-resorcinol-formaldehyde glues; collagen-based glues; acrylic resins; bioabsorbable polymers such as starches, polyglycolide, polylactide, glycolide-lactide copolymers, polycaprolactone, polycarboxylic acids and their copolymers, polycarbonates, polyorthoesters, polyamino acids, polycyanoacrylates, polyhydroxybutyrate, polyhydroxyvalyrate, polyphosphazenes, polyvinylpyrrolidone, polypropylene fumarate, poly-propylene fumarate-diacrylate, poly (propylene glycol-co-fumaric acid), tyrosine-based polycarbonates, pharmaceutical tablet binders (such as Eudragit® binders available from Hulls America, Inc.), polyvinylpyrrolidone, cellulose, ethyl cellulose, micro- crystalline cellulose and blends thereof; nonbioabsorbable polymers such as polyacrylate, polymethylmethacrylate, polytetrafluroethylene, polyurethane and polyamide; etc., derivatives and blends of the foregoing are also suitable, as well as glycolide-lactide copolymer In some embodiments, the cell coated substrate can be secured in position using suitable mechanical fasteners, including for example but not limited to sutures, staples, tissue tacks, suture anchors, darts, screws, and arrows. It is understood that combinations of one or more chemical and/or mechanical fasteners can be used. For example but not limited to, the cell coated substrate can be surgically fixed into the defect with biodegradable sutures, i.e., (Ethicon, Johnson & Johnson) and/or by applying a bioadhesive to the region interfacing the patch and the defect. In some embodiments, the bioadhesives include, but are not limited to, fibrinthrombin glues similar to those disclosed in France Patent No. 2 448 900; France Patent No. 2 448 901 and European Patent No: 88401961.3 and synthetic bioadhesives similar to those disclosed in

U.S. Pat. No. 5,197,973, the contents of which are incorporated herein in their entirety by reference. In alternative embodiments, the cell coated substrate can be secured in place by use of screws comprising the same bioresorbable material as the substrate, for example but not limited, to screws comprising poly-lactic acid and poly-glycolic acid, or poly(lactic-co-glycolic) acid or co-polymers and variants or derivatives thereof. It is contemplated, however, that alternative types of sutures and biocompatible glues can be useful in the practice of the invention. In some instances, damaged cartilage and/or damaged bone maybe surgically excised prior to implantation of the cell coated substrate. Additionally, the adhesion of the cell coated substrate to the articular cartilage defect can be enhanced by treating the defect with transglutaminase (Ichinose et al. (1990) J. Biol. Chem. 265(3):13411- 13414; Najjar et al. (1984). [164] One of ordinary skill in the art will appreciate that the requirement to secure the cell coated substrate can be determined by a surgeon, based on principles of medical science and the applicable treatment objectives. [165] In some embodiments, the cell coated substrate can be molded into a variety of configurations. For example but not limited to, the cell coated substrate, for example cartilage coated substrate can be folded or stacked in multiple laminates or it can be rolled into the shape or a tube-like structure.

[166] In some embodiments, the cell coated substrate is allogeneic. In some embodiments, the cell coated substrate is also autogenic nature. Accordingly, allogeneic cell coated substrate can be prepared from biopsy tissue isolated from a mammal belonging to the same species as the intended recipient. Autogenic cell coated substrate can be prepared from biopsy tissue derived from the intended recipient. In additional embodiments, the cell coated substrate as disclosed herein can be useful in the repair of human articular cartilage defects, or in the repair of human bone defects. [167] In further embodiments, the cell coated substrate comprising the chondrocytes can be prepared in advance to the surgical procedure. In some embodiments, the cell coated substrate is frozen and cryoprotected for use in a surgical procedure at a later date is also within the scope of the invention.

[168] In some embodiments, the cell coated substrate as disclosed herein can be molded before, during or after implantation depending material of the substrate. Following molding, one embodiment of the cell coated substrate is that it is flexible yet retains its shape and much of its tensile strength. The cell coated substrate can be packaged in either the molded state or non molded state, and also pre- or post-seeding with cells, and cryoprotected and stored for subsequent application, hi some embodiments, the cell coated substrate with cells attached, as disclose herein can be packaged in an appropriate packaging so that it is ready for immediate use at the surgical site. Optional materials can also be added prior to packaging. In some embodiments, the cell coated substrate as disclosed herein is frozen prior to storage. [169] The practice of the present invention employs, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Molecular Cloning A Laboratory Manual,

2nd Ed., ed. By Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D.N. Glover ed., 1985); Oligonucleotide Synthesis (MJ. Gait ed., 1984); Mullis er al, U.S. Patent No: 4,683,195; Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds. 1984); Transcription and Translation (B.D. Hames & SJ. Higgins eds. 1984); Culture of Animal Cells (R.I. Freshley, Alan R. Liss, Inc., 1987); Immobilized cells and Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide to Molecular Cloning (1984); the treatise, Methods in Enzymology (Academic Press, Inc., N. Y.); Gene Transfer vectors for Mammalian Cells (J.H. Miller and M.P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods in Enzymology, VoIs. 154 and 155 (Wu et al, eds.), Immunochemical methods In Cell and Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook of Experimental Immunology, Volumes I-IV (D.M Weir and CC. Blackwell eds., 1986);

Manipulating the Mouse Embryo (Cold Spring Harbor Press: Cold Spring Harbor, N. Y., 1986). [170] The present invention has been described in terms of particular embodiments found or proposed by the present inventor to comprise preferred modes for the practice of the invention. It will be appreciated by those of skill in the art that, in light of the present disclosure, numerous modifications and changes can be made in the particular embodiments exemplified without departing from the intended scope of the invention. For example, due to codon redundancy, changes can be made in the underlying DNA sequence without affecting the protein sequence. Moreover, due to biological functional equivalency considerations, changes can be made in protein structure without affecting the biological action in kind or amount. All such modifications are intended to be included within the scope of the appended claims.

[171]Throughout this application, various publications are referenced. The disclosures of all of the publications and those references cited within those publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains. The following non-limiting examples are illustrative of the principles and practice of this invention. Numerous additional embodiments within the scope and spirit of the invention will become apparent to those skilled in the art, and any variations in the exemplified methods which occur to the skilled artisan are intended to fall within the scope of the present invention.

EXAMPLES

[172]The examples presented herein relate to cell coated substrates for the repair of bone and cartilage defects. In the examples, cartilage coated substrate is used as exemplary cell coated substrate. Further, chondrocytes are used as an exemplary cell, although the methods of the invention are applicable to any cell. Furthermore, the cartilage coated substrate used for the repair of a cartilage defect is used as an exemplary repair of a defect, although the methods of the invention are also applicable to cell coated substrates for the repair of bone defects. Furthermore, the copolymers of poly(lactic) acid and poly(glycolic) acid, in particular poly(lactic-co-glycolic) acid (PLGA) for is used as an exemplary substrate in the invention, although the method of the invention are applicable to the use of other resorbable substrates. Throughout this application, various publications are referenced. The disclosures of all of the publications and those references cited within those publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains. The following examples are not intended to limit the scope of the claims to the invention, but are rather intended to be exemplary of certain embodiments. Any variations in the exemplified methods which occur to the skilled artisan are intended to fall within the scope of the present invention. [173] METHODS

[174] Chrondrocytes and expansion: Chondrocytes were isolated from 2-3 months old New Zealand white rabbits by procedures described previously Lee et al, 1996, J cell Biochem, 60;508-520; Lee et al, 2003; Tissue Engineering, 9;703-711; Ballock et al, Dev Biol, 1994;

158;414-429, Kato et al, Proc Natl Acad Sci, USA, 1998;85;9552-9556. Briefly, Three to four week old New Zealand rabbits were anesthetized using intramuscular injection of Ketamine 45 mg/kg and xylazine 5 mg/kg and under sterile conditions the right 5^th - 8^th rib were exposed. Parts of cartilage away from the osteochondral junction of all the ribs were removed and immediately transferred to Dulbecco's Modified Eagles medium (DMEM) with 10% penicillin streptomycin neomycin (PSN) at 37⁰C. The tissues was then cut into lmm cube pieces and subjected to digestion with 0.2% collagenase IA overnight. Chondrocytes were then isolated for culture and seeded at 1x10E6 per 100mm dish. Alternatively, both Auricular cartilages were harvested, and their chondrocytes isolated according to the above protocol. Chondrocytes are passed once or several times once reaching confluence and seeded onto PGA/PLLA template plate (Lactsorb®) in concentration0.5xl0⁶ml per 12 square millimeters. The chondrocytes were allowed to affix to the resorbable template in vitro for a sufficient amount of time to enable them to generate extracellular matrix. This time ranged from overnight incubation to 8 weeks of incubation. 5 days was typically sufficient for the production of extracellular matrix formation. [175] Chondrocyte culture conditions. The chondrocytes are grown in HyQ Ham's F12 medium containing the following additives: ITS, FBS, Penicillin/Streptomycin, Gentamycin, Amphotericin, L-Ascorbic acid-2 -Phosphate, TGF-betal, Non-essential amino acids, and L- glutamine. [176] Animal surgery. 2-3 months old New Zealand White rabbits were used, and all animal procedures and handling approved by Harvard Standing Committee on Animals. Briefly, medial parapatellar incision was used for access of the knee joint, and articular defect created using a slow motion drill bit of a size 5mm. The resorbable substrate comprising chondrocytes was inserted into the defect in the knee, (see Leung et al, J Clinical Biomech, 2002;17;594-602 and Leung et al, J Clini Exp Rheumatol, 1999; 17;579-600). Identical lesions were created in control rabbits with insertion of substrate without cells or left empty. All operations were performed under intramuscular general anesthesia using combination of Ketamine 45 mg/kg and xylazine 5 mg/kg. Postoperatively free cage activities were allowed. The animals were sacrificed after 2, 4, 6, and 8 weeks. Tissue samples for histological examination were removed. The specimens were immersed in mixture of formic acid and sodium formate (6.8g/ml) for 48 hours. This way demineralized specimen was cut in half through the defect, paraffin embedded and the cellular morphology and tissue phenotype assessed following 4 micrometer cuts by staining with hematoxylin/eosin. The presence of sulfated proteoglycans in the extracellular matrix was assayed by staining the particles with 1% alcain blue in hydrochloric acid. The cell coated substrate interaction with the tissue was evaluated by optical microscopy.

EXAMPLE l

[177] Transplantation of cartilage coated substrate to a cartilage defect of critical size.

[178] In the following examples, the inventors demonstrate the use of cartilage coated substrates for the repair of articular cartilage defects, in particular large articular cartilage defects. For the production of a cartilage coated substrate, chondrocyte cells were seeded on the surface of a smooth substrate and allowed to create their own extracellular matrix. This is the major and fundamental difference compared with the use of matrices and/or scaffolds that have been used in the past. [179] In the following examples, the smooth support used comprised of a co-polymer of poly(lactic) acid and poly(glycolic) acid, onto which the cells attached, propagated and organized to form their own extracellular matrix.. The use of a smooth substrate enables the generated cartilage to withstand greater mechanical stress than in the absence of a substrate, and therefore protecting the chondrocytes from mechanical trauma or shear during implantation, as well as creating a cartilage implant that easily withstands shear forces of weight bearing post-implantation. The substrate also allows transmission of pressure that is important for chondrocyte growth and proper differentiation (Elder et al., Chondrocyte differentiation is modulated by frequency and duration of cyclic compressive loading. Annals of Biomedical Engineering 2001; 29(6):476-82).

Furthermore, the use of a substrate also enables the implantation of the cartilage coated substrate without the need to secure or anchor the implant in place.

[180] In the examples, the cartilage coated substrate is implanted into the cartilage defect site arthroscopically, another key advantage to the present invention over existing methods of cartilage transplantation methods. [181] Also demonstrated in the examples, the cartilage coated substrate can be shaped and molded into any configuration, for example but not limited to contour to the dimensions and shape of the cartilage defect. Methods to determine the dimensions and shape of the defect are well known in the art, for example one such example is using the patient's MRI, CT scan or imprint of the defect obtained during arthroscopy as a guide. In this way, the cartilage coated substrate can be configured to contour any defect of virtually any joint, and therefore can be used for the repair of cartilage defects in such joints.

[182] Chondrocytes can be isolated from its native cartilage, and grown in vitro following well established protocols. The inventors show chondrocyte cells attach and grow on the surface of the resorbable plate, shown by beta-galactosidase staining for LacZ expression (fig 2a and 2c).

The strength of attachment is further improved by coating the plate with polylysine (figure 2b).

[183] One of the major limitations of transplantation of chondrocyte on a matrix or scaffold is the occurrence of loss of chondrocyte phenotype, loss of structural integrity and ossification of the chondrocyte implant post implantation (as discussed in Chrisophel et al, Arch Facial Plast Surg, 2006, 8; 117-122). Chrisophel et al. demonstrates the typical loss of structural integrity of chondrocyte implants that occurs 8 weeks following transplantation of synthetic cartilage. In these experiments, chondrocytes were seeded into the matrix and cultured in vitro. Following several weeks they were implanted subcutaneously into a rabbit. Eight weeks following implantation there is gross and complete loss of morphology, decrease in mass, as well as signs of chondrocyte de-differentiation, foreign body reaction and ossification.

[184] To overcome these issues, the inventors have discovered a method for chondrocyte implant using a smooth substrate, wherein the chondrocytes are cultured on the smooth substrate to enable them to produce their own extracellular matrix, at which point the substrate is molded to conform to the dimensions of the cartilage defect and implanted into the defect. In the examples, the smooth substrate is a resorbable substrate. The smooth substrate can be molded to conform with the size and shape of the cartilage defect after the culturing on the surface the chondrocytes, or alternatively, the molding can be performed during chondrocyte culturing or before the seeding of the chondrocyte cells. [185] The inventors demonstrate the use of cartilage coated substrate for the delivery of chondrocyte cells to a critical size joint defect (spontaneously non-healing defect) and maintain the chondrocyte survival. In this example, the chondrocytes were harvested from the cartilaginous part of ribs, released from its substance using collagenase, and seeded on a standard 100mm plate. Ham's F12 medium with standard additives was used for culture. Once the cells reached confluence, the chondrocytes were released with trypsin, and centrifuged to a pellet using a Becket centrifuge at 1200 rpm for 2 minutes. The cells were then grown in a pellet culture for 6 weeks on the substrate. The cartilage coated substrate has a structural integrity such that it can be handled with forceps (as shown in figure ID), and resembles both grossly (Figure ID and 3C) and microscopically, cartilaginous tissue (Figures 4A, 4B, 5 A and 5B). The cartilage coated substrate was molded into a cone shape (Figure IE) for implantation into the critical size joint defect (figure 2B and data not shown). The inventors generated a rabbit knee cartilaginous defect by anaesthetizing a rabbit using standard procedures and a parapatellar incision was made on the medial side of the knee joint. The critical defect size (5mm) was made using a drill at controlled slow speed (data not shown).. The rabbit knee model was used for implantation of the chondrocytes, due to less interfering factors as opposed to subcutaneous space. The size of the knee defect is so called "critical size defect" that has been extensively shown in the literature as not spontaneously able to heal (data not shown). The implant was inserted into the critical size defect (Fig 2B) and cut flush with the surrounding cartilage (Fig 2C). The rabbit was sacrificed 2 weeks following the implantation. Gross examination revealed cartilage regeneration in the defect (Figure 3B and 3C) compared to the control (Figure 3A), where no cell or cartilage coated substrate was implanted. The control defect also demonstrated a fibrinous blood clot at the site of the critical cartilage defect (Fig 3A), whereas the critical cartilage defect is completely repaired after two weeks in the experimental group and exhibits gross cartilage tissue (Figure 3B). This appears whiter than surrounding cartilage due to it is thicker than naive surrounding cartilage. Histological analysis by hematoxylin/eosin (H&E) staining indicates the implanted chondrocytes have typical configuration of hyaline cartilage spanning the defect, and form a continuous layer spanning the defect (Figure 4A), compared with the absence of chondrocytes spanning the defect in the control group (Figure 4B). Further, the chondrocytes implanted on the cartilage coated substrate are partially integrated with the naϊve cartilage at the transition zone in the experimental group (data not shown), as determined by analysis of a cross section spanning the cartilage defect in which a cartilage coated substrate had been implanted, with the transition zone between native cartilage and transplanted chondrocytes on the resorbable plate completely integrated (data not shown). Further integration is likely to occur as analysis was only done at the two week post- implantation time and is likely to be an insufficient time for complete integration to occur. Importantly, Figure 5 A and Figure 5B show the morphological phenotype of the implanted chondrocytes demonstrating that the chondrocytes have a structure which resembles naϊve hyaline cartilage that is typically found in a rib or joint (Figure 5A). Figure 5B is the cartilage coated substrate cultured in vitro stained with Safranin (a cartilage-specific stain) also showing the chondrocytes closely resembles hyaline cartilage found in a joint or rib.

REFERENCES The references cited herein and throughout the application are incorporated herein by reference.

Claims

We claim:

1. A method for preparing a cell coated substrate, the method comprising the steps of:

(a) seeding cells on a substantially smooth substrate which allows for attachment of the cells,

(b) culturing the cells for a time sufficient to allow the cells to differentiate.

2. The method of claim 1, further comprising culturing the cells for a time sufficient to enable them to endogenously produce an extracellular matrix.

3. The method of claims 1 or 2, wherein the cell coated substrate is cartilage coated substrate.

4. The method of claims 1, 2 or 3, wherein the cell coated substrate is bone or bone progenitor or osteoblast or osteoprogenitor coated substrate.

5. The method of claims 1, 2 3, or 4, wherein the cells are chondrocyte cells.

6. The method of claims 1 , 2, 3, 4 or 5, wherein the cells are cells involved in bone repair and/or bone growth.

7. The method of claims 1 , 2, 3, 4, 5 or 6, wherein the cells are progenitor cells, pluripotent cells, multipotent progenitor cells, stem cells, embryonic stem (ES) cells, adult stem cells, postnatal stem cells, fetal stem cells, embryoid bodies or precursors or differentiated cells, or a mixture thereof.

8. The method of claim 1 , 2, 3, 4, 5, 6 or 7, wherein the progenitors are chondrocyte progenitors or bone progenitor or oestoprogenitors, mesenchymal cells, bone marrow cells or differentiated cells or a mixture thereof.

9. The method of claim 1, further comprising molding the substrate.

10. The method of claims 1 or 9, wherein the molding is performed prior to, or after seeding with cells.

11. The method of claims 1, 2, 3 or 4, wherein the substrate is a biodegradable.

12. The method of claims 1 or 11, wherein the biodegradable substrate is a polymer.

13. The method of claims 1, 11 or 12, wherein the polymer comprises of poly(lactic-co-glycolic) acid, and/or polylactic acid and/or poly-glycolic acid and/or poly-lactic acid, and/or suture- like material.

14. The method of claims 1, 11, 12 or 13, wherein the biodegradable material comprises polylactic acid and poly-glycolic acid.

15. The method of claims 1, 11, 12, 13 or 14, wherein the polyglycolic acid is poly L-glycolic acid.

16. The method of claims 1, 11, 12, 13, 14 or 15, wherein the biodegradable material comprises 82% poly-lactic acid and 18% polyglycolic acid.

17. The method of any of the above claims, wherein the substrate comprises adhesion molecules.

18. The method of claim 17, wherein the adhesion molecules are polylysine, polylaminin, gelatin, collagen type I, III; hyaluronic acid and its derivatives, dextran or polymers and derivatives and modified versions thereof.

19. The method of any of the above claims, wherein the substrate further comprises one or more of the group consisting of sodium hyalurinate, hyaluronic acid and its derivatives, gelating, collagen, chitosan, alginate, buffered PBS, Dextran or polymers.

20. The method of any of the above claims, wherein the substrate comprises pores.

21. The method of any of the above claims, wherein the chondrocytes cells are derived from cartilage.

22. The method of claim 21, wherein the cartilage is articular cartilage.

23. The method of claim 22, wherein the cartilage is selected from a group consisting of costal cartilage, nasal cartilage, auricular cartilage and cricoid cartilage.

24. The method of claim 5, wherein the chondrocytes cells are denuded chondrocytes.

25. The method of any of the above claims, wherein the cells are derived from human tissue.

26. The method of any of the above claims, wherein the cells are genetically modified cells.

27. The method of any of the above claims, wherein the cell coated substrate is autogenic or allogenic.

28. The method of any of the above claims, wherein the extracellular matrix comprises at least one of type II collagen, proteoglycan, chrondroitin-6-sulfate or keratan sulfate.

29. The method of any of the above claims, wherein the cartilage coated substrate comprises articular cartilage.

30. The method of any of the above claims, wherein the culturing further comprises culturing the cells in the presence of a growth factor, hormones, cytokines, bone morphogenic proteins or other agents and molecules that regulate growth, differentiation and proliferation of the cells.

31. The method of claim 30, wherein the growth factor is a chondrocyte stimulating factor.

32. The method of claims 30 or 31, wherein growth factor is one or more of the group consisting of growth factors, fibroblast growth factors (FGF-2, FGF-5), insulin like growth factor- 1 (IGFl), transforming growth factor beta (TGF-β), bone morphogenic proteins (BMPl -8) platelet derived growth factor (PDGF), vascular endothelial growth factor (VEGF), endothelial growth factor (EGF), hepatocyte growth factor (HGF), karatinocyte growth factor, oestogenic protein- 1 (OP-I or BMP-7),and fibroblast growth factor (FGF), acidic fibroblast growth factor, estrogen, macrophage colony stimulating factor, calcium regulatory agents, parathyroid horomone (PTH), TGF-alpha, TGF-beta and mixtures, homologues or variants thereof.

33. The method of claims 30, 31 or 32, wherein the growth factor or agent is a peptide, protein, nucleic acid, aptamer, small molecule or analogues and fragments thereof.

34. The method of claim 33, wherein the nucleic acid is DNA or RNA or derivatives or analogues thereof.

35. The method of claims 33 or 34, wherein the nucleic acid is DNA, DNA analogues, peptide nucleic acid (PNA), pseudo-complementary PNA (pcPNA), locked nucleic acid (LNA), RNA, RNAi or derivatives analogues thereof.

36. The method of any of the above claims, wherein the cell further comprises human allogenic or autologous chondrocytes, human allogenic or autologous bone marrow cells or stem cells.

37. The method of any of the above claims, further comprising freezing the cartilage coated substrate.

38. The method of claim 37, wherein the cartilage coated substrate is molded prior to being frozen.

39. The method of claim 37, wherein the cartilage coated substrate is molded after to being frozen.

40. A composition of comprising substantially smooth substrate coated with cells.

41. The composition of claim 40, wherein the substrate is coated with cartilage.

42. The composition of claims 40 or 41, wherein the substrate is coated with cell is involved in bone repair and/or bone growth.

43. The composition of claims 40, 41 or 42, wherein the substrate comprises poly-lactic acid and poly-glycolic acid or analogues or polymers or copolymers thereof.

44. The composition of claim 40, 41 , 42 or 43, wherein the cartilage is articular cartilage.

45. A method for treating an articular cartilage defect in a subject, the method comprising implanting at the site of the defect a cartilage coated substrate produced by the method of any of the above claims, having an approximate dimension of the cartilage defect.

46. A method for treating an articular cartilage defect in a subject, the method comprising implanting at the site of the defect a composition of claim 40.

47. A method for treating a bone defect in a subject, the method comprising implanting at the site of the defect a cell coated substrate produced by the method of any of the above claims, having an appropriate dimension for the repair of the bone defect.

48. A method for treating a bone defect in a subject, the method comprising implanting at the site of the defect a composition of claim 40.

49. The method of any of the claims 45-48, wherein the site is a predetermined site.

50. The method of any of the above claims, wherein the cartilage coated substrate is molded into a configuration that interfϊts with the cartilage defect.

51. The method of any of the above claims, wherein the treating is repairing the articular cartilage defect.

52. The method of any of the above claims, wherein the defect is a partial thickness or full- thickness defect.

53. The method of any of the above claims, wherein the method comprises an additional step of excising defective articular cartilage from the subject prior to implanting the synthetic cartilage.

54. The method of any of the above claims, wherein the subject is mammalian.

55. The method of any of the above claims, wherein the mammal is human.