AU2004321311B8

AU2004321311B8 - Neo-cartilage constructs and methods of preparation thereof

Info

Publication number: AU2004321311B8
Application number: AU2004321311A
Authority: AU
Inventors: Akihiko Kusanagi; Shuichi Mizuno; Robert Lane Smith; Laurance J. B. Tarrant; Toshimasa Tokuno
Original assignee: Histogenics Corp
Current assignee: Ocugen Inc
Priority date: 2004-07-30
Filing date: 2004-07-30
Publication date: 2009-11-19
Anticipated expiration: 2024-07-30
Also published as: CA2541827A1; EP1796592A1; AU2004321311A8; WO2006022671A1; JP2008507971A; AU2004321311A1; AU2004321311B2; CA2541827C; EP1796592A4

Description

WO 2006/022671 PCT/US2004/024902 METHOD FOR IN SITU REPAIR OF INJURED, DAMAGED, DISEASED OR AGED ARTICULAR CARTILAGE USING NEO-CARTILAGE CONSTRUCTS AND A METHOD FOR PREPARATION THEREOF 5 BACKGROUND OF THE INVENTION Field of Invention The current invention concerns a method for treatment of injured, damaged, diseased or aged articular cartilage using neo-cartilage constructs implanted into 10 a joint cartilage lesion in situ. The method is particularly useful for repair and restoration of function of the injured, traumatized, aged or diseased cartilage. In particular, the invention concerns a method where the implantation of the construct of the 15 invention initiates and achieves incorporation of neo cartilage into a native surrounding cartilage including a formation of a new superficial cartilage layer overgrowing and eventually completely sealing the lesion in the joint cartilage. The neo-cartilage construct of 20 the invention comprises at least chondrocytes incorporated into a support matrix processed according to the algorithm of the invention. The construct is implanted into the joint cartilage lesion typically below one layer or between two layers of biologically 25 acceptable sealants. The method for the treatment of articular cartilage comprises preparation of -the neo cartilage construct from the autologous or heterologous chondrocytes ex vivo, preparing the lesion for implantation of said construct including an optional step 30 of depositing a first sealant compound at the bottom of the lesion for sealing the joint cartilage lesion and protecting the construct from effects of blood-borne agents, implanting the construct of the invention on the top of the first sealant and depositing a second sealant 35 compound over the construct. The invention further concerns a method for repair and restoration of the injured, damaged, diseased or aged cartilage into its full functionality and for treatment 2 of injured or diseased cartilage by implanting the neo-cartilage construct between two layers of biologically acceptable sealants. Additionally, the invention concerns a method for generation of the neo-cartilage and the neo-cartilage construct of the invention. 5 BACKGROUND AND RELATED DISCLOSURES Damage to the articular cartilage which occurs in active individuals and older generation adults as a result of either acute or repetitive traumatic injury or aging is quite common. Such damaged cartilage leads to pain, affects mobility and results in debilitating disability. 10 Typical treatment choices, depending on lesion and symptom severity, are rest and other conservative treatments, minor arthroscopic surgery to clean up and smooth the surface of the damaged cartilage area, and other surgical procedures such as microfracture, drilling, and abrasion. All of these may provide symptomatic relief, but the benefit is usually only temporary, especially if the person's pre-injury activity level is is maintained. For example, severe and chronic forms of knee joint cartilage damage can lead to greater deterioration of the joint cartilage and may eventually lead to a total knee joint replacement. Approximately 200,000 total knee replacement operations are performed annually. The artificial joint generally lasts only 10 to 15 years and the operation is, therefore, typically not recommended for people under the age of fifty. 20 It would, therefore, be extremely advantageous to have available a method for in situ treatment of these injuries which would effectively restore the cartilage to its pre injury state. Attempts to provide means and methods for repair of articular cartilage are disclosed, for example, in U.S. patents 5,723,331; 5,786,217; 6,150,163; 6,294,202; 6,322,563 and in the U.S. patent application Ser. No. 09/896,912 (Publication No. 2002 25 0082220 Al), filed on June 29, 2001 3 U.S. patent 5,723,331 describes methods and compositions for preparation of synthetic cartilage for the repair of articular cartilage using ex vivo proliferated denuded chondrogenic cells seeded ex vivo, in the wells containing adhesive surface. These cells redifferentiate and begin to secrete cartilage-specific extracellular matrix thereby 5 providing an unlimited amount of synthetic cartilage for surgical delivery to a site of the articular defect. U.S. patent 5,786,217 describes methods for preparing a multi-cell layered synthetic cartilage patch prepared essentially by the same method as described in '331 patent except that the denuded cells are non-differentiated, and culturing these cells for a 1o time necessary for these cells to differentiate and form a multi cell-layered synthetic cartilage. U.S. application Ser. No. 09/896,912 (Publication No. 2002-0082220 Al), filed on June 29, 2001 concerns a method for repairing cartilage, meniscus, ligament, tendon, bone, skin, cornea, periodontal tissues, abscesses, resected tumors and ulcers by is introducing into tissue a temperature dependent polymer gel in conjunction with at least one blood component which adheres to the tissue and promotes support for cell proliferation for repairing the tissue. None of the above cited references results in repair and regeneration of cartilage in situ including de novo formation of the superficial cartilage layer sealing a joint 20 cartilage lesion in situ. It is thus a primary objective of this invention to provide a method and means for regeneration of injured or traumatized cartilage by forming, in the injured lesion of the cartilage, a cavity, by administering at least one but typically two separate layers of a biologically acceptable glue sealant and implanting a neo-cartilage containing construct 25 under the one layer or into said cavity. The method according to the invention results in the growth of the superficial cartilage layer over the lesion and sealing the lesion.

4 All patents, patent applications and publications cited herein are hereby incorporated by reference. SUMMARY One aspect of the current invention is a method for repair and restoration of 5 damaged, injured, diseased or aged cartilage to a functional cartilage, said method comprising steps: a) preparing a neo-cartilage construct comprising autologous or heterologous chondrocytes incorporated into a sponge, porous scaffold or thermo reversible gelation hydrogel (TRGH) matrix support and subjected to the algorithm of the io invention; b) optionally introducing a first layer of a first biologically acceptable sealant into a cartilage lesion; c) implanting said construct into said lesion or into said cavity over the first layer of said first sealant; 15 d) introducing a second layer of a second biologically acceptable sealant over said construct wherein said second sealant may or may not be the same as the first sealant and wherein a combination of said construct and said second sealant results in formation and growth of a superficial cartilage layer sealing the cartilage lesion in situ. In another aspect, the present invention provides a method for treatment of 20 cartilage lesions and defects comprising steps: a) isolating autologous or heterologous chondrocytes or providing stem cells that can be differentiated into chondrocytes; b) expanding said isolated chondrocytes or stem cells in a growth medium; c) suspending said expanded chondrocytes or stem cells in a collagen 25 containing solution; d) providing biodegradable support matrix containing plurality of pores; e) incorporating the suspension obtained in step c) into said support matrix thereby producing a seeded support matrix; f) preparing an implantable construct for implantation into said cartilage 30 lesion by activating said chondrocytes or stem cells by subjecting said seeded support matrix to conditions promoting activation and propagation of said chondrocytes or stem cells within said support matrix, wherein said conditions for activation and propagation of chondrocytes comprise perfusing said seeded support matrix with a culture medium at a flow rate from 35 about 1 pl/min to about 500 gl/min and 4a during said perfusing, applying to said seeded support matrix a cyclic hydrostatic pressure from about 0.01 MPa to about 10 MPa above atmospheric pressure at about 0.01 to about 1 Hz for from about 1 to about 8 hours followed by applying a static atmospheric pressure for from 16 to about 23 hours; 5 g) implanting said construct into said cartilage lesion; and h) depositing a biodegradable polyethylene glycol cross-linked with methylated collagen over said construct, wherein said implanting of said construct into said cartilage lesion and said deposition of said polyethylene glycol cross-linked with methylated collagen over said 1o implanted construct results, within three months, in a growth and maturing of said chondrocytes or stem cells and in integration of said construct seeded with said chondrocytes or stem cells into a native cartilage of the lesion and in formation of a superficial cartilage layer that overgrows said construct implanted within said lesion, wherein said superficial cartilage layer is an outermost layer of cartilage that is forms the layer of squamous-like flattened superficial zone chondrocytes covering a layer of said polyethylene glycol cross-linked with methylated collagen and overgrowing the lesion, and wherein said biodegradable support matrix and said polyethylene glycol cross-linked with methylated collagen are both degraded within said three months. 20 Another aspect of the current invention is a method for repair and restoration of damaged, injured, diseased or aged cartilage to a functional cartilage, said method comprising steps: a) obtaining autologous or heterologous chondrocytes; b) culturing said chondrocytes ex vivo into a neo-cartilage, said neo 25 cartilage comprising autologous or heterologous chondrocytes incorporated into a sponge or TRGH matrix support subjected to the algorithm of the invention; c) optionally introducing a first layer of a first biologically acceptable sealant into a cartilage lesion; d) depositing a space-holding thermo-reversible WO 2006/022671 PCT/US2004/024902 5 gel (SHTG) or TRGH into a lesion or into a cavity formed above the first sealant layer thereby permitting sufficient time for growth and differentiation of ex vivo cultured neo-cartilage, said space holding thermo 5 reversible gel (SHTG) deposited into said cavity as a sol at temperatures between about 5 to about 25*C, wherein within said cavity and at the body temperature said SHTG converts from the fluidic sol into a solid gel and in this form SHTG holds the space for subsequent 10 introduction of the neo-cartilage cultured ex vivo, and provides protection against cell and blood-borne agents migration into the cavity from the subchondral space and from the synovial capsule and wherein its presence further provides a substrate for and promotes in situ 15 formation of a de novo superficial cartilage layer covering the cartilage lesion; e) depositing a second layer of a second biologically acceptable sealant over the cartilage lesion; 20 f) removing said SHTG by cooling said lesion to change SHTG into sol; f) depositing said neo-cartilage cultured ex vivo into the cavity formed between two layers of sealants and under the de novo formed superficial cartilage layer; 25 g) removing said SHTG or TRGH from the cavity after the neo-cartilage integration into a native cartilage under the formed superficial cartilage layer by cooling said lesion to from about 5 to about 15 0 C to convert the solid gel into fluidic sol and removing said sol or, in 30 alternative, leaving said SHTG or TRGH to disintegrate and be removed naturally. Another aspect of the current invention is a method for repair and restoration of damaged, injured, diseased or aged cartilage to a functional cartilage, said method 35 comprising steps: a) preparing an intact and discreet piece of neo cartilage by culturing autologous or heterologous chondrocytes ex vivo, suspending said cultured WO 2006/022671 PCT/US2004/024902 6 chondrocytes in a thermo-reversible gelation hydrogel (TRGH) and warming said suspension of chondrocytes to temperature above 30*C in order to convert TRGH into a solid gel and subjecting the solid gel to the algorithm 5 of the invention; b) introducing a first and a second layer of a first and a second biologically acceptable sealant into a cartilage lesion; c) cooling said TRGH/neo-cartilage to 5-15*C to 10 sol state; d) depositing said neo-cartilage suspended in the TRGH into a cavity formed between two layers of sealants as a sol at temperatures between about 5 to about 25*C wherein, within said cavity and at the body temperature, 15 said TRGH converts from the sol state into the solid gel and in this state provides protection for and enables integration of deposited neo-cartilage into a native surrounding cartilage and wherein the presence of TRGH further provides a substrate and promotes in situ 20 formation of de novo superficial cartilage layer covering the cartilage lesion; e) leaving said TRGH in the lesion until its disintegration or, in alternative, removing said TRGH from the cavity as a sol by cooling said lesion to 25 temperature between 5 and 15 0 C after the neo-cartilage integration and formation of superficial cartilage layer; and d) evaluating incorporation of said neo-cartilage into a surrounding native cartilage. 30 Still another aspect of the current invention is a method for repair and restoration of damaged, injured, diseased or aged cartilage to a functional cartilage, said method comprising steps: a) preparing neo-cartilage or a neo-cartilage 35 containing construct comprising autologous cultured chondrocytes incorporated into a gel or thermo-reversible gel matrix support ex vivo and subjected to the algorithm of the invention; WO 2006/022671 PCT/US2004/024902 7 b) introducing a first layer of a first biologically acceptable sealant into a cartilage lesion; c) depositing said construct or said neo-cartilage over the first layer of the first sealant; and 5 d) depositing a layer of a second biologically acceptable sealant either over the neo-cartilage construct or the neo-cartilage deposited into a cartilage lesion and covering the lesion with said second sealant, wherein in time said neo-cartilage is integrated into 10 the native cartilage and wherein the presence of the neo cartilage construct and the second sealant promotes in situ formation and growth of de novo superficial cartilage layer covering the cartilage lesion. Another aspect of the current invention is a neo 15 cartilage construct suitable for implantation into a cartilage lesion in situ. Yet another aspect of the current invention is a neo-cartilage construct implanted under one or between two layers of biologically acceptable sealants within a 20 cartilage lesion. Still another aspect of the current invention is a neo-cartilage construct implanted in situ into a cartilage lesion between two layers of sealants wherein a first sealant is deposited at the bottom of a cartilage 25 lesion and the second sealant is deposited over the implanted construct on the top of the cartilage lesion and wherein the second sealant leads to formation and growth of superficial cartilage layer which seals said cartilage lesion. 30 Another aspect of the current invention is a method for fabrication of a three-dimensional neo-cartilage construct of the invention comprising steps of: a) preparing a support matrix structure; b) harvesting a piece of cartilage from a donor for 35 isolation of chondrocytes; c) culturing and expanding the chondrocytes; d) suspending the expanded chondrocytes in a suspension fluid; WO 2006/022671 PCT/US2004/024902 8 e) incorporating said suspended chondrocytes into said matrix; and f) propagating said chondrocytes into two or three dimensional neo-cartilage construct using the algorithm 5 of the invention. Still another aspect of the current invention is a method for generation of an autologous type of neo cartilage construct by generating a carrier support for autologous chondrocytes cultured into neo-cartilage 10 wherein said support is a biologically acceptable cell carrier thermo-reversible polymer gel or a thermo reversible gelation hydrogel (CCTG, TRGH or VITROGEN*), wherein said neo-cartilage is suspended within the CCTG and wherein a resulting CCTG/neo-cartilage or TRGH/neo 15 cartilage or a suspension thereof is injected into the cartilage lesion. Still another aspect of the current invention is a method for generation and maintaining integrity of the lesion cavity for the introduction of neo-cartilage, a 20 neo-cartilage gel, a neo-cartilage suspension or neo cartilage construct from a synovial capsule and for blocking the migration of subchondral and synovial cells and cell and blood products into said cavity and for providing a substrate for a formation of superficial 25 cartilage layer overgrowing the lesion by introducing a biologically acceptable space-holding thermo-reversible gel (SHTG) into a cleaned lesion for a duration of culturing autologous chondrocytes into neo-cartilage before introducing said neo-cartilage or neo-cartilage 30 construct or suspension into the lesion. Still another aspect of the current invention is a method for treatment of damaged, injured, diseased or aged cartilage by utilizing any of the methods listed above to implant the neo-cartilage construct into the 35 lesion. BRIEF DESCRIPTION OF DRAWINGS Figure 1 shows a construct comprising neo-cartilage. Figure 1A is a schematic drawing of the sponge made of WO 2006/022671 PCT/US2004/024902 9 sol/gel showing the distribution of chondrocytes within the collagen sponge. Figure 1B is a micrograph of the actual neo-cartilage construct held in the forceps having 4 mm in diameter and thickness of 1.5 mm. Seeding density 5 of the construct is 300,000 chondrocytes per 25 pl of collagen solution (12,000,000 cells/ml). Figure 2A shows a diagram of hydrostatic pressure culture system. Figure 2B shows a TESS culture processor unit. 10 Figure 3A is a graph representing S-GAG accumulation in cell constructs subjected to static atmospheric (control) or cyclic hydrostatic pressure (test). Figure 3B is a photomicrograph of Safranin-O'staining for S-GAG on paraffin sections in 18 days subjected to static 15 pressure. Figure 3C is a photomicrograph of Safranin-O staining for S-GAG on paraffin sections in cell constructs subjected to cyclic hydrostatic pressure for 6 days followed by 12 days of static pressure. Figure 4 illustrates effect of cyclic and constant 20 hydrostatic pressure on production of S-GAG (Figure 4A) and DNA (Figure 4B). Figure 5A shows S-GAG accumulation in cell constructs under continued culture conditions of static culture (control), medium perfusion (COMPa), cyclic 25 hydrostatic pressure (Cy-HP) combined with medium perfusion (control) and constant hydrostatic pressure combined with medium perfusion (constant-HP) . Figure 5B illustrates DNA content at day 6 and day 18 in cells constructs submitted to static conditions (control), 30 medium perfusion only (COMPa), cyclic hydrostatic pressure (Cy-HP) and constant hydrostatic pressure (constant-HP). Figure 6A is a photomicrograph of Safranin-O staining for S-GAG on paraffin sections in 18 days cell 35 constructs subjected to static atmospheric pressure. Figure 6B is a photomicrograph of Safranin-O staining for S-GAG on paraffin sections in cell constructs subjected to cyclic hydrostatic pressure for 6 days followed by 12 WO 2006/022671 PCT/US2004/024902 10 days of static pressure. Figure 6C is a photomicrograph of type II collagen immunohistochemistry on paraffin sections in 6 days cell constructs subjected to static atmospheric pressure. Figure 6D is a photomicrograph of 5 type II collagen immunohistochemistry on paraffin sections in cell constructs subjected to cyclic hydrostatic pressure for 6 days. Figure 7A is a graph illustrating effect of the medium perfusion flow rate on cell proliferation (DNA 10 content) by cell constructs subjected to a medium flow rate of either 0.005 or 0.05 ml/min. Figure 7B illustrates effect of flow rate on production of S-GAG. Figure 8 shows accumulation detected histologically by toluidine S-GAG blue staining after 15 days culture 15 submitted to perfusion (Figure 8A), cyclic hydrostatic pressure (Figure 8B) and constant hydrostatic pressure (Figure 8C). Figure 9 illustrates effect of low oxygen tension on S-GAG production (Figure 9A) and cell proliferation 20 (Figure 9B). Figure 10A shows an arthroscopic observation of the control empty defect site 2 weeks after creating empty defect. Figure 10B shows an arthroscopic observation of the porcine neo-culture (Porcine-NeoCart") implant site 25 2 weeks after the implantation. Figure 11 shows the control lesion without treatment with porcine neo-cartilage where the proliferation of fibrocartilage within the defect site is clearly visible after 4 months. Figure 11A shows a defect site vis-a-vis 30 subchondral bone with a site of formation of fibro cartilage. Figure 11B shows a defect site synovium and synovial migration. Figure 11C shows the defect site and formation of fibrocartilage. Figure 12A and 12B shows integration of porcine neo 35 cartilage into the lesion within the host's cartilage after 3 months. Figure 12C shows the regenerated hyaline-like cartilage in the porcine neo-cartilage implanted site. Figure 12D shows the integration between WO 2006/022671 PCT/US2004/024902 11 the porcine neo-cartilage and the host cartilage laterally and at the subchondral bone. Figure 13A shows S-GAG production in cell constructs subjected to cyclic hydrostatic pressure and to 5 atomospheric pressure (control) with medium perfusion. Figure 13B shows DNA content in cell constructs subjected to cyclic and constant hydrostatic pressure with medium perfusion. Figure 14 shows histological evaluation of cell 10 constructs by Safranin-O. Figure 14A shows S-GAG accumulation at day 0 (initial) . Figure 14B shows accumulation of S-GAG on day 21 in cell constructs subjected to atmospheric pressure (control). Figure 14C shows accumulation of S-GAG on day 21 in cell constructs 15 subjected to 7 days of cyclic hydrostatic pressure (Cy HP#1) followed by 14 days of to atmospheric pressure. Figure 14D shows accumulation of S-GAG on day 21 in cell constructs subjected to 14 days of cyclic hydrostatic pressure (Cy-HP#2) followed by 7 days of to atmospheric 20 pressure. Figure 14E shows accumulation of S-GAG on day 21 in cell constructs subjected to 7 days of constant hydrostatic pressure (Constant-HP) followed by 14 days of atmospheric pressure. DEFINITIONS 25 As used herein: "Chondrocyte" means a nondividing cartilage cell which occupies a lacuna within the cartilage matrix. "Isogenous chondrocytes" means clones of cartilage cell derived from one cell of division. Isogenous 30 chondrocytes occur in clusters called isogenous nests. "Autologous chondrocytes" means chondrocytes isolated from a donor's own healthy articular cartilage. "Heterologous chondrocytes" means chondrocytes derived from a donor of a different species or from a 35 donor of the same species but not the recipient individual or a donor tissue that is derived from the recipient individual but is non-articular cartilage isolated from a cartilage of the different species.

WO 2006/022671 PCT/US2004/024902 12 "Support matrix" means biologically acceptable sol gel or sponge scaffold suitable for seeding expanded chondrocytes that provides a structural support for growth and three-dimensional propagation of chondrocytes. 5 The support matrix is prepared from such materials as Type I collagen, Type II collagen, Type IV collagen, gelatin, agarose, cell-contracted collagen containing proteoglycans, glycosaminoglycans or glycoproteins, fibronectin, laminin, bioactive peptide growth factors, 10 cytokines, elastin, fibrin, synthetic polymeric fibers made of poly-acids such as polylactic, polyglycolic or polyamino acids, polycaprolactones, polyamino acids, polypeptide gel, copolymers thereof and combinations thereof. The gel solution matrix may be a polymeric 15 thermo-reversible gelling hydrogel. The support matrix is preferably biocompatible, biodegradable, hydrophilic, non-reactive, has a neutral charge and be able to have or has a defined structure. "Neo-cartilage" means an immature hyaline cartilage 20 wherein the ratio of extracellular matrix to chondrocytes is lower than in mature hyaline cartilage. "Mature hyaline cartilage" means cartilage consisting of groups of isogenous chondrocytes located within lacunae cavities which are scattered throughout an 25 extracellular collagen matrix. "Autologous Cultured Neo-Cartilage" means a hyaline neo-cartilage tissue grown ex vivo from chondrocytes isolated from a donor's own healthy articular cartilage. "Neo-cartilage construct", "NEOCART"" or "'NeoCart"" 30 means a 3-dimensional structural composition comprising chondrocytes incorporated into a matrix support treated by or subjected to the algorithm of the invention. Neo cartilage construct thus means a discrete piece of hyaline neo-cartilage formed from cultured chondrocytes 35 for implantation into lesion of a damaged, aged or diseased cartilage wherein, after implantation, the neo cartilage is integrated into a native cartilage within the lesion. NeoCart* cartilage is manufactured by and is 13 proprietary of Histogenics Corporation, Easthampton, MA. "TESSTM"I means Tissue Engineering Support System which is available as TESS culture processor unit for culturing of chondrocytes prepared from arthroscopic biopsy samples. The unit permits changes in hydrostatic pressure, including cyclic 5 hydrostatic pressure changes and controls other physical parameters such as temperature, gas concentration, medium perfusion rate and such other parameters as may be needed. Relevant detailed information is found in US patents 6,432,713 B2, patent application Serial No.: 09/895,162 (Patent No. 6,607,917), Serial No.: 09/895,161 (Patent No. 6,599,734), PCT JPOI/01516 Japanese patent applications 2001-126543 and 2001 10 261556, incorporated herein by reference. "Sealant" means a biologically acceptable typically rapid-gelling formulation having a specified range of adhesive and cohesive properties. Sealant is thus a biologically acceptable rapidly gelling synthetic compound having adhesive and/or gluing properties, and is typically a hydrogel, such as derivatized is polyethylene glycol (PEG) which is preferably cross-linked with a collagen compound, typically alkylated collagen. Examples of suitable sealants are tetra-hydrosuccinimidyl or tetra-thiol derivatized PEG, or a combination thereof, commercially available from Cohesion Technologies, Palo Alto, CA under the trade name CoSealT , described in J. Biomed. Mater. Res Appl. Biomater, 58: 545-555 (2001) or two-part polymer 20 compositions that rapidly form a matrix where at least one of the compounds is polymeric, such as, polyamino acid, polysaccharide, polyalkylene oxide or polyethylene glycol and two parts are linked through a covalent bond, as described in US patent 6,312,725B1, herein incorporated by reference, and cross-linked PEG with methyl collagen, such as a cross-linked polyethylene glycol hydrogel with methyl-collagen. The 25 sealant of the invention typically gels and/or bonds rapidly upon contact with tissue, particularly with tissue containing WO 2006/022671 PCT/US2004/024902 14 collagen. "First sealant" means a biologically acceptable tissue sealant which is deposited at the bottom of the lesion. 5 "Second sealant" means a biologically acceptable sealant which is deposited above and over the neo cartilage construct implanted into a lesion. The second sealant may or may not be the same as the first sealant and is preferably a cross-linked polyethylene glycol 10 hydrogel with methyl-collagen. "Hydrostatic pressure" means pressure measured above the atmospheric pressure. "Cyclic hydrostatic pressure" or "Cy-HP" means the application of repeated, two or multiplicity periods of 15 applied hydrostatic pressure within a defined loading interval which creates a sine wave form of measured pressure. "Constant hydrostatic pressure", "constant-HP" or "CHP" means the application of a non-fluctuating or non 20 cyclic pressure load over a period of time. "Loading" or "loading interval" means a period of applied cyclic hydrostatic pressure load followed by a return to atmospheric pressure where no external pressure is applied. 25 "Resting phase" means a variable length of time wherein cells are maintained in culture at atmospheric pressure after exposure to or culturing under cyclic hydrostatic pressure. "De novo" or "de novo formation" means the new 30 production of cells, such as chondrocytes, fibroblasts, fibrochondrocytes, tenocytes, osteoblasts and stem cells capable of differentiation, or tissues such as cartilage connective tissue, fibrocartilage, tendon, and bone within a support structure, such as multi-layered system, 35 scaffold or collagen matrix or formation of superficial cartilage layer. "Superficial cartilage layer" means an outermost layer of cartilage that forms the layer of squamous-like WO 2006/022671 PCT/US2004/024902 15 flattened superficial zone chondrocytes covering the layer of the second sealant and overgrowing the lesion. "Thermo-reversible" means a compound or composition changing its physical properties such as viscosity and 5 consistency, from sol to gel, depending on the temperature. The thermo-reversible composition is typically completely in a sol (liquid) state at between about 5 and 15 0 C and in a gel (solid) state at about 30 0 C and above. The gel/sol state in between shows a lesser 10 or higher degree of viscosity and depends on the temperature. When the temperature is higher than 150C, the sol begins to change into gel and with the temperature closer to 30-370 the sol becomes more and more solidified as gel. At lower temperatures, typically 15 lower than 15 0 C, the sol has more liquid consistency. "TRGH" means thermo-reversible gelation hydrogel material in which the sol-gel transition occurs on the opposite temperature cycle of agar and gelatin gels. Consequently, the viscous fluidic phase is in a sol stage 20 and the solid phase is in a gel stage. TRGH has very quick sol-gel transformation which requires no cure time and occurs simply as a function of temperature without hysteresis. The sol-gel transition temperature can be set at any temperature in the range from 5 0 C to 70 0 C by 25 molecular design of thermo-reversible gelation polymer (TGP), a high molecular weight polymer of which less than 5 wt% is enough for hydrogel formation. "SHTG" means space holding thermo-reversible gel. "Sol-gel solution" means a colloidal suspension 30 which, under certain conditions, transitions from a liquid (sol) to a solid material (gel). The "sol" is a suspension of aqueous collagen that is transitioned, by heat treatment, into a gel. " G A G " m e a n s glycosaminoglycan. 35 "S-GAG" means sulfated glycosaminoglycan. "MMP" means matrix metalloproteinase, an enzyme associated with cartilage degeneration in an injured or diseased joint.

WO 2006/022671 PCT/US2004/024902 16 "DMB" means dimethylene blue used for staining of chondrocytes. "MPa" means MegaPascal. One MPa is equal to 145 psi. 5 "Superficial zone cartilage" means the flattened outermost layer. of chondrocytes covering the extracellular matrix intermediate zone and deeper zone of mature articular cartilage in which non-dividing cells are dispersed. 10 "Connective tissue" means tissue that protect and support the body organs, and also tissues that hold organs together. Examples of such tissues include mesenchyme, mucous, connective, reticular, elastic, collagenous, bone, blood, or cartilage tissue such as 15 hyaline cartilage, fibrocartilage, and elastic cartilage. "The algorithm" means variable defined conditions, such as variable pressure or non-pressure conditions, variable perfusion rate, different medium, different cell density, different temperature, variable time, different 20 oxygen and carbon dioxide conditions, etc., to which a cellular construct of neo-cartilage is subjected in order to convert it to a mature neo-cartilage construct. "Adhesive strength" means a peel bond strength measurement, which can be accomplished by bonding two 25 plastic tabs with an adhesive formulation. The tabs can be formed by cutting 1 x 5 cm strips from polystyrene weighing boats. To the surface of the boat are bonded (using commercial cyanoacrylate Superglue), sheets of sausage casing (collagen sheeting, available from butcher 30 supply houses). The sausage casing is hydrated in water or physiological saline for 20 min to one hour and the adhesive is applied to a 1 x 1 cm area at one end of the tab; the adhesive is cured. Then, the free ends of the tab are each bent and attached to the upper and lower 35 grips, respectively, of a tensile testing apparatus and pulled at 10 mm/min strain rate, recording the force in Newtons to peel. A constant force trace allows estimation of N/m, or force per width of the strip. A minimum force WO 2006/022671 PCT/US2004/024902 17 per width of 10 N/m is desired; 10ON/m or higher is more desirable. Alternatively, the same tab can be bonded (a single tab) over a 1 x 1 cm area to tissue, either dissected or 5 exposed tissue in a living animal, during surgery. The free end of the tab is then gripped or attached through a perforation to a hook affixed to a hand-held tensile test device (Omega DFG51-2 digital force gauge; Omega Engineering, Stamford, CT) and pulled upward at 10 approximately 1 cm/sec. The maximum force required to detach the tab from the tissue is recorded. The minimum force desired in such measurements would be 0.1 N to detach the tab. Forces or 0.2 to 1 N are more desirable. "Cohesive strength" means the force required to 15 achieve tensile failure and is (pulling in extension); measured using a tensile test apparatus. The glue or adhesive can be cured in a "dog-bone"-shaped mold. The wide ends of the formed solid adhesive can then be affixed, using cyanoacrylate (Superglue) to plastic tabs, 20 and gripped in the test apparatus. Force at extensional failure should be at least 0.2 MPa (2 N/cm2) but preferably 0.8 to 1 MPa or higher. "Lap shear measurements" means a test of bonding strength, in which the sealant formulation is applied to 25 overlapping tabs of tissue, cured, and then the force to pull the tabs apart is measured. The test reflects adhesive and cohesive bonding; strong adhesives will exhibit values of 0.5 up to 4-6 N/cm 2 of overlap area. DETAILED DESCRIPTION OF THE INVENTION 30 This invention is based on finding that when neo cartilage or a neo-cartilage construct is deposited into a lesion of injured, traumatized, aged or diseased cartilage or under the top sealant or between layers of a first (bottom) and a second (top) sealant, within time 35 the neo-cartilage is incorporated into the surrounding native cartilage and that under these circumstances, the second top sealant promotes in situ formation of de novo superficial cartilage layer over the cartilage lesion WO 2006/022671 PCT/US2004/024902 18 wherein the neo-cartilage is implanted. The invention thus, in its broadest scope, concerns a method for repair and restoration of damaged, injured, traumatized or aged cartilage to its full functionality, 5 and a method for treatment of injuries or diseases caused by damaged cartilage due to the trauma, injury, disease or age by implanting a neo-cartilage construct and further includes a method for preparation of neo cartilage from chondrocytes harvested from a donor's 10 tissue, a method for formation of a support matrix, a method for fabrication of a neo-cartilage construct and additionally a method for de novo formation of a superficial cartilage layer in situ. Briefly, the invention comprises preparation of neo 15 cartilage from harvested autologous or heterologous chondrocytes, culturing and expansion of chondrocytes, seeding the chondrocytes within a collagenous or thermo reversible gel support matrix and propagating said chondrocytes in two or three-dimensions. To achieve the 20 chondrocyte propagation, the seeded support matrix is subjected to the algorithm of variable conditions, such as static atmospheric pressure, constant or cyclic hydrostatic pressure, temperature changes, oxygen and/or carbon dioxide level changes and changes in perfusion 25 flow rate of the culture medium in the presence of various supplements, such as, growth factors, donor's serum, ascorbic acid, ITS, etc. The chondrocyte-seeded support matrix treated as above becomes a neo-cartilage construct (neo-cartilage) suitable for implanting into a 30 joint cartilage lesion. The neo-cartilage construct is implanted into the lesion under a top sealant, or into a cavity formed by two layers of adhesive sealants. The first layer of the sealant is deposited at and covers the bottom of the 35 lesion and its function is to protect the integrity of said lesion from cell migration and from effects of various blood and tissue metabolites and also to form a bottom of the cavity into which the neo-cartilage WO 2006/022671 PCT/US2004/024902 19 construct is deposited. In one embodiment, after the neo-cartilage construct is emplaced into the lesion cavity, the second adhesive layer is deposited on the top of the neo-cartilage 5 construct and within several months results in formation of the superficial cartilage layer completely sealing the lesion. In the alternative embodiment, two adhesive layers may be deposited concurrently with or before the 10 construct is implanted into the cavity between them. In such an instance, in the interim, said cavity may be filed with a space holding thermo-reversible gel (SHTG). Both sealant layers and the construct or space holding gel are left within the lesion cavity for a certain 15 predetermined period of time, typically from one week to several months, or in case of the space holding gel, until the neo-cartilage construct is prepared ex vivo and ready to be implanted. The second layer deposited on the top and over the lesion promotes formation of a 20 superficial cartilage layer which covers the lesion on the outside and eventually overgrows the lesion completely thereby resulting in complete or almost complete sealing of the lesion and of the neo-cartilage construct deposited within said lesion leading to 25 incorporation of neo-cartilage into a native cartilage and resulting in healing of the injured or damaged cartilage. In alternative, the thermo-reversible gel may serve as an initiator for promotion of formation of the superficial cartilage layer. 30 Both the support matrix of the neo-cartilage construct or the space holding thermo-reversible gel deposited into the lesion are materials which are biodegradable and permit and promote formation of the superficial cartilage layer and integration of the 35 chondrocytes from the neo-cartilage construct into the native cartilage within the lesion cavity. Such integration begins within several weeks or months following the implanting and may continue for several WO 2006/022671 PCT/US2004/024902 20 months and involves a growth and maturing of neo cartilage into normal cartilage integrated into the healthy cartilage. The top sealant layer promotes an overgrowth of the lesion with the superficial cartilage 5 layer typically in about two-three months when the sealant is itself degraded. In the alternative embodiment, the lesion cavity is filled. with a space-holding gel until the outer superficial cartilage layer is formed at which time the 10 neo-cartilage construct comprising ex vivo propagated chondrocytes suspended in a thermo-reversible sol is introduced at a temperature between 50 and 150 C. After it is introduced into the lesion as a liquid sol, the introduced thermo-reversible sol-gel is converted into a 15 solid gel at body temperatures of 37 0 C or at the same or similar temperature as the temperature of the synovial cavity. The neo-cartilage construct introduced into the lesion is integrated into the native cartilage surrounding the cavity and is completely covered with the 20 superficial cartilage layer. In the alternative, the neo-cartilage construct is deposited into a lesion of injured, traumatized, aged or diseased cartilage over the first (bottom) sealant layer and the thermo-reversible gel of the neo-cartilage 25 construct promotes in situ formation of the superficial membrane without a need to add the second sealant. The method for treatment of injured, traumatized, diseased or aged cartilage comprises treating the injured, traumatized, diseased or aged cartilage with an 30 implanted neo-cartilage construct prepared by methods described above and/or by any combination of steps or components as described. I. Preparation of Neo-Cartilage Constructs Preparation of neo-cartilage constructs for 35 implanting into the cartilage lesion involves harvesting and culturing chondrocytes, seeding them in the support matrix and preparation thereof, and propagating the chondrocytes either ex vivo, in vitro, or in vivo.

WO 2006/022671 PCT/US2004/024902 21 A. Cartilage and Neo-Cartilage Cartilage is a connective tissue covering joints and bones. Neo-cartilage is immature cartilage which eventually, upon deposition into the lesion according to 5 this invention, is integrated into and acquires properties of mature cartilage. Differences between the two types of cartilage is in their maturity. Cartilage is a mature tissue comprising metabolically active but non-dividing chondrocytes; neo-cartilage is an immature 10 cartilage comprising metabolically and genetically activated chondrocytes which are able to divide and multiply. This invention utilizes properties of neo cartilage in achieving repair and restoration of damaged cartilage into the full functionality of the healthy 15 cartilage by enabling the neo-cartilage to be integrated into the mature cartilage surrounding the lesion and in this way repair the defect. a) Cartilage Cartilage is a connective tissue characterized by 20 its poor vascularity and a firm consistency. Cartilage consist of mature non-dividing chondrocytes (cells), collagen (interstitial matrix of fibers) and a ground proteoglycan substance (glycoaminoglycans or mucopolysaccharides). Later two are cumulatively known 25 as extracellular matrix. There are three kinds of cartilage, namely hyaline cartilage, elastic cartilage and fibrocartilage. Hyaline cartilage found primarily in joints has a frosted glass appearance with interstitial substance containing fine 30 type II collagen fibers obscured by proteoglycan. Elastic cartilage is a cartilage in which, in addition to the collagen fibers and proteoglycan, the cells are surrounded by a capsular matrix surrounded by an interstitial matrix containing elastic fiber network. 35 The elastic cartilage is found, for example, in the central portion of the epiglottis. Fibrocartilage contains Type I collagen fibers and is typically found in transitional tissues between tendons, ligaments or bones.

WO 2006/022671 PCT/US2004/024902 22 The articular cartilage of the joints, such as the knee cartilage, is the hyaline cartilage which consists of approximately 5% of chondrocytes (total volume) embedded in approximately 95% extracellular matrix (total 5 volume). The extracellular matrix contains a variety of macromolecules, including collagen and proteoglycan. The structure of the hyaline cartilage matrix allows it to reasonably well absorb shock and withstand shearing and compression forces. Normal hyaline cartilage has also an 10 extremely low coefficient of friction at the articular surface. Healthy hyaline cartilage has a contiguous consistency without any lesions, tears, cracks, ruptures, holes or shredded surface. Due to trauma, injury, 15 disease such as osteoarthritis, or aging, however, the contiguous surface of the cartilage is disturbed and the cartilage surface shows cracks, tears, ruptures, holes or shredded surface resulting in cartilage lesions. Partly because hyaline cartilage is avascular, the spontaneous 20 healing of large defects is not believed to occur in humans and other mammals and the articular cartilage has thus only a limited, if any, capacity for repair. A variety of surgical procedures have been developed and used in attempts to repair damaged cartilage. These 25 procedures are performed with the intent of allowing bone marrow cells to infiltrate the defect and promote its healing. Generally, these procedures are only partly successful. More often than not, these procedures result in formation of a fibrous cartilage tissue 30 (fibrocartilage) which does fill and repair the cartilage lesion but, because it is qualitatively different being made of Type I collagen fibers, it is less durable and less resilient than the normal articular (hyaline) cartilage and thus has only a limited ability to 35 withstand shock and shearing forces than does healthy hyaline cartilage. Since all diarthroid joints, particularly knees joints, are constantly subjected to relatively large loads and shearing forces, replacement WO 2006/022671 PCT/US2004/024902 23 of the healthy hyaline cartilage with fibrocartilage does not result in complete tissue repair and functional recovery. b) Neo-Cartilage 5 Neo-cartilage is an immature hyaline cartilage where the ratio of extracellular matrix to chondrocytes is lower than in mature hyaline cartilage. Mature hyaline cartilage has the ratio of the extracelluar matrix to chondrocytes approximately 95:5. The neo-cartilage has 10 a lower ratio of the extracelluar matrix to chondrocytes than mature cartilage and thus comprises more than 5% of chondrocytes. In the process of development of this invention, it was discovered that under the conditions described below, 15 the older inactive chondrocytes could be activated from static non-dividing stage to an active stage where they divide, multiply, promote growth of the extracellular matrix and develop into new cartilage (neo-cartilage). The neo-cartilage thus contains chondrocytes which were 20 rejuvenated and are surrounded by a newly synthesized extracellular-matrix macromolecules. A process for activation was found to require certain period of time, typically from about 1 week to about 3 months and it is thus preferred that the neo-cartilage be prepared ex vivo 25 where nutrients needs and mechanical loading are well defined. B. Preparation of Neo-Cartilage Neo-cartilage prepared according to the current invention is grown ex vivo from chondrocytes isolated 30 from the mammalian donor's source. In the alternative, neo-cartilage may also be grown in situ or in vivo under conditions described below. Typical donor sources of mammalian chondrocytes are swine or humans. Neo-cartilage of the invention for human 35 use is preferably grown from autologous chondrocytes obtained from the patient during arthroscopy. While it is preferred that for human use chondrocytes are autologous, it is to be understood that chondrocytes WO 2006/022671 PCT/US2004/024902 24 obtained from other mammalian sources are equally suitable for preparation of neo-cartilage for treatment of damaged, diseased or aged cartilage. The use of both autologous and heterologous chondrocytes is intended to 5 be within the scope of the invention. a) Isolation of Chondrocytes Specific procedures used for isolation of mammalian chondrocytes generally using swine cartilage as an -example are described in Example 1. The isolation of 10 human chondrocytes and preparation of autologous human neo-cartilage is according to procedures described in Example 2. Briefly, the donor cartilage is obtained either by arthroscopic biopsy from the human donor or from a joint 15 or bone, such as, for example, the femur of the slaughtered animal and processed according to Example 1 or 2. The cartilage is preferably digested by collagenase, a strong protease, most preferably Type I collagenase, in a solution containing preferably about 20 0.15% of collagenase. The digestion is run for several hours to several days, preferably for about 18 hours. In alternative, the extracellular matter can be digested with proteases or sugar lyases including but not limited to heparitinase, heparinase, chondroitinase ABC, 25 chondroitinase B and chondroitinase AC. The lyases are added in admixture with collagenase or in a sequential enzyme digestion steps. These lyases promote further isolation of the chondrocytes from the extracellular matrix ECM including disruption the glycosaminoglycans of 30 the pericellular environment such that the chondrocytes do not receive inhibitory signals that prevent them from dividing or producing healthy new extracellular matrix. This finding is especially important for osteoarthritic chondrocytes which have very slow division rates and 35 reduced ability to produce extracellular matrix. This is especially important for osteoarthritic chondrocytes which have very slow division rates and reduced ability to produce ECM. US patent 5,916,557 WO 2006/022671 PCT/US2004/024902 25 shows that application of chondroitinase ABC to chondrocytes in vitro resulted couterintuitively in the promotion of new cartilage production. The ability to free the chondrocytes from all extra 5 and pericellular inhibitory material and thereby to promote cell expansion and differentiation is especially important in autologous osteoarthritic tissue where the growth is otherwise slow because these chondrocytes have reduced ability to produce ECM where neo-cartilage 10 formation in the TESS processor under pressure is greatly improved by this early step of the process. Furthermore, this method of stimulating chondrocyte growth and differentiation is relatively benign compared to the application of growth factors or other chemical stimuli 15 at a later stage of the formation of neo-cartilage, since the cells are washed free of the enzymes before culturing. b) Expansion of Chondrocytes The isolated chondrocytes are then expanded by any 20 method suitable for such purposes such as, for example, by incubation in a suitable growth medium, for a period of several days, typically from about 3 to about 30 days, preferably for 14 days, at about 37 0 C. Any kind of culture or incubation apparatus or chamber may be used 25 for expanding chondrocytes. The expansion of the cells is preferably associated with the removal of dead chondrocytes, residual native extracellular matrix and other cellular debris before the chondrocytes are selected for culturing and multiplying. Selected 30 chondrocytes are collected and isolated using trypsinization process or any other suitable method. Expanded chondrocytes are then suspended in a suitable solution and seeded into a support matrix to form a seeded matrix. The seeded matrix is typically 35 processed in a tissue processor. c) Suspension and Seeding of Chondrocytes in the Support Matrix Following the expansion, chondrocytes are suspended WO 2006/022671 PCT/US2004/024902 26 in any suitable solution, preferably collagen containing solution. For the purposes of this invention such solution is typically a gel, preferably sol-gel transitional solution which changes the state of the 5 solution from liquid sol to solid gel above room temperature. The most preferred such solution is the thermo-reversible gelation hydrogel or a thermo reversible polymer gel. The thermo-reversible property is important both for immobilization of the chondrocytes 10 within the support matrix and for implanting of the neo cartilage construct within the cartilage lesion. One characteristic of the sol-gel is its ability to be cured or transitioned from a liquid into a solid form. This property may be advantageously used for solidifying 15 the suspension of chondrocytes withing the support matrix for delivery, storing or preservation purposes. Additionally, these properties of sol-gel also permit its use as a support matrix by changing its sol-gel transition by increasing or decreasing temperature, as 20 described in greater detail below for thermo-reversible gelation hydrogel, or exposing the sol-gel to various chemical or physical conditions or ultraviolet radiation. In one embodiment the expanded chondrocytes are suspended in a collagenous sol-gel solution before 25 incorporation (seeding) into the support matrix. The sol-gel viscosity permits easy mixing of chondrocytes avoiding need to use shear forces. One example of the suitable sol-gel solution is the solution substantially composed of Type I collagen, commercially available under 30 trade name VITROGEN* from Cohesion Corporation, Palo Alto, CA. VITROGEN is a purified pepsin-solubilized bovine collagen dissolved in 0.012N HCl. Sterile collagen for tissue culture may be additionally obtained from other sources, such as, for example, Collaborative 35 Biomedical, Bedford, MA, and Gattefosse, SA, St Priest, France. When using a VITROGEN solution, the cell density is approximately 5-10 x 106 cells/ml. However, both the WO 2006/022671 PCT/US2004/024902 27 density of the cells, the volume for their seeding and strength of the solution are variables within the algorithm , and the higher or lower number of chondrocytes may be suspended in a larger or lower volume 5 of the suspension solution, depending on the size of the support matrix and the size of the cartilage lesion. Seeding of the suspended chondrocytes into the support matrix is by any means which permit even distribution of the chondrocytes within said support 10 matrix. Seeding may be achieved by bringing the suspension and the support matrix into close contact and seeding the cells by wicking or suction of the suspension into the matrix by capillary action, by inserting the support matrix into the suspension, by using suction, 15 positive or negative pressure, injection or any other means which will result in even distribution of the chondrocytes within said support matrix. In alternative embodiment, the chondrocytes are suspended in the thermo-reversible gelation hydrogel or 20 gel polymer at temperature between 5 and 150C. At that temperature, the hydrogel is at a liquid sol stage and easily permits the chondrocytes to be suspended in the sol. Once the chondrocytes are evenly distributed within the sol, the sol is subjected to higher temperature of 25 about 30-370C at which temperature, the liquid sol solidifies into solid gel having evenly distributed chondrocytes within. The gelling time is from about several minutes to several hours, typically about 1 hour. In such an instance, the solidified gel may itself become 30 and be used as a support matrix or the suspension in sol state may be loaded into a separate support matrix, such as a sponge or honeycomb support matrix. Other means of generating suspending gels, not necessarily thermo-reversible, are also available and 35 suitable for use. Polyethylene glycol (PEG) derivatives, in which one PEG chain contains vinyl sulfone or acrylate end groups, and the other PEG chain contains free thiol groups will covalently bond to form thio-ether linkages.

WO 2006/022671 PCT/US2004/024902 28 If one or both partner PEG molecules are branched (three or four-armed), the coupling results in a network, or gel. If the molecular weight of the PEG chains is several thousand Daltons (500 to 10,000 Daltons along any linear 5 chain segment), the network will be open, swellable by water, and compatible with living cells. The coupling reaction can be accomplished by preparing 5 to 20% (w/v) solutions of each PEG separately in aqueous buffers or cell culture media. Chondrocytes can be added to the 10 thiol-PEG solution. Just prior to incorporation into the support matrix, the cells plus thiol PEG and the acrylate or vinyl sulfone PEG are mixed and infused into the matrix. Gelation will begin spontaneously in 1 to 5 minutes; the rate of gelation can be modulated somewhat 15 by the concentration of PEG reagent and by pH. The rate of coupling is faster at pH 7.8 than at pH 6.9. Such gels are not degradable unless additional ester or labile linkages are incorporated into the chain. Such PEG reagents may be purchased from Shearwater Polymers, 20 Huntsville, AL, USA; or from SunBio, Korea. In a second alternative, alginate solutions can be gelled in the presence of calcium ions. This reaction has been employed for many years to suspend cells in gels or micro-capsules. Cells can be mixed with a 1-2% (w/v) 25 solution of alginate in culture media devoid of calcium or other divalent ions, and infused into the support matrix. The matrix can then be immersed in a solution containing calcium chloride, which will diffuse into the matrix and gel the alginate, trapping and supporting the 30 cells. Analogous reactions can be accomplished with other polymers which bear negatively charged carboxyl groups, such as hyaluronic acid. Viscous solutions of hyaluronic acid can be used to suspend cells and gelled by diffusion of ferric ions. 35 Suspension loaded into the support matrix or gelled into the solid support is processed using the algorithm of the invention. Such processing is performed in a processing apparatus, such as a TESS processor.

WO 2006/022671 PCT/US2004/024902 29 C. Preparation of Support Matrix The support matrix for seeding expanded chondrocytes provides a structural support for growth and three dimensional propagation of chondrocytes. Generally, the 5 support matrix is biologically biocompatible, hydrophilic and has preferably a neutral charge. Typically, the support matrix is a two or three dimensional structural composition, or a composition able to be converted into such structure, containing a 10 plurality of pores dividing the space into a fluidically connected interstitial network. In some embodiments the support matrix is a sponge-like structure or honeycomb like lattice. In general, any polymeric material can serve as the 15 support matrix, provided it is biocompatible with tissue and possesses the required geometry. Polymers, natural or synthetic, which can be induced to undergo formation of fibers or coacervates, can then be freeze-dried as aqueous dispersions to form sponges. Typically, such 20 sponges must be stabilized by crosslinking, such as, for example, ionizing radiation. Practical exmaple includes preparation of freeze-dried sponges of poly-hydroxyethyl methacrylate (pHEMA), optionally having additional molecules, such as gelatin, entrapped within 25 advantageously. Such types of sponges can advantageously function as support matrices for the present invention. Incorporation of agarose, hyaluronic acid, or other bio active polymers can be used to modulate cellular responses. A wide range of polymers may be suitable for 30 the fabrication of support matrix sponges, including agarose, hyaluronic acid, alginic acid, dextrans, polyHEMA, and poly-vinyl alcohol above or in combination. Typically, the support matrix is prepared from a collagenous gel or gel solution containing Type I 35 collagen, Type II collagen, Type IV collagen, gelatin, agarose, cell-contracted collagens containing proteoglycans, glycosaminoglycans or glycoproteins, fibronectin, laminin, bioactive peptide growth factors, WO 2006/022671 PCT/US2004/024902 30 cytokines, elastin, fibrin, synthetic polymeric fibers made of poly-acids such as polylactic, polyglycotic or polyamino acids, polycaprolactones, polyamino acids, polypeptide gel, copolymers thereof and combinations 5 thereof. Preferably, the support matrix is a gel solution, most preferably containing aqueous Type I collagen or a polymeric, preferably thermo-reversible, gel matrix. The gel or gel solution used for preparation of the 10 support matrix is typically washed with water and subsequently freeze-dried or lyophilized to yield a sponge like matrix able to incorporate or wick the chondrocytes suspension withing the matrix. The cellular support matrix of the current invention acts like a 15 sponge when infiltrated with the chondrocyte suspension wherein the cells are evenly distributed. One important aspect of the support matrix is the pore size of the support matrix. Support matrices having different pore sizes permit faster or slower infiltration 20 of the chondrocytes into said matrix, faster or slower growth and propagation of the cells and, ultimately, the higher or lower density of the cells in the neo-cartilage construct. Such pore size may be adjusted by varying the pH of the gel solution, collagen concentration, 25 lyophilization conditions, etc. Typically, the pore size of the support matrix is from about 50 to about 500 p1, preferably the pore size is between 100 and 300 i and most preferably about 200 p1. The support matrix may be prepared according to 30 procedures described in Example 3, or by any other procedure, such as, for example, procedures described in the U.S. Patent 6,022,744; 5,206,028; 5,656,492; 4,522,753 and 6,080,194 herein.incorporated by reference. One preferred type of support matrix is Type-I 35 collagen support matrix fabricated into a sponge, commercially available from Koken Company, Ltd., Tokyo, Japan, under the trade name Honeycomb Sponge. An exemplary neo-cartilage support matrix made of WO 2006/022671 PCT/US2004/024902 31 collagen and seeded with chondrocytes is seen in Figure 1, wherein Figure 1A is a schematic drawing of the sponge made of sol/gel showing the distribution of chondrocytes within the collagen sponge. Figure 1B shows a 5 microphotograph of the actual neo-cartilage construct (Neo-Cart") having 4 mm in diameter and thickness of 1.5 mm. The seeding density of this construct is 300,000 375,000 chondrocytes per 25 pl of collagen solution corresponding to about 12,000,000 - 15,000,000 cells/ml. 10 a) Honeycomb Cellular Support Matrix In one embodiment of the invention, the support matrix is a honeycomb-like lattice matrix providing a cellular support for activated chondrocytes, herein described as neo-cartilage. The honeycomb-like matrix 15 supports a growth platform for the neo-cartilage and permits three-dimensional propagation of the neo cartilage. The honeycomb-like matrix is fabricated from a polymerous compound, such as collagen, gelatin, Type I 20 collagen, Type II collagen or any other polymer having a desirable properties. In the preferred embodiment, the honeycomb-like matrix is prepared from a solution comprising Type I collagen. The pores of the honeycomb-like matrix are evenly 25 distributed within said matrix to form a sponge-like structure able to taking in and evenly distributing the neo-cartilage suspended in a viscous solution. b) Sol-Gel Cellular Support Matrix In another embodiment, the support matrix is 30 fabricated from sol-gel materials wherein said sol-gel materials can be converted from sol to gel and vice versa by changing temperature. For these materials the sol-gel transition occurs on the opposite temperature cycle of agar and gelatin gels. Thus, in these materials the sol 35 is converted to a solid gel at a higher temperature. Sol gel material is a material which is a viscous sol at temperatures of below 150 and a solid gel at temperatures around and above 370*. Typically, these materials change WO 2006/022671 PCT/US2004/024902 32 their form from sol to gel by transition at temperatures between about 150 and 370 and are in transitional state at temperatures between 15 0 C and 370*. The most preferred materials are Type I collagen containing gels and a 5 thermo-reversible gelation hydrogel (TRGH) which has a rapid gelation point. In one embodiment, the sol-gel material is substantially composed of Type I collagen and, in the form of 99.9% pure pepsin-solubilized bovine dermal 10 collagen dissolved in 0.012N HCl, is commercially available under the tradename VITROGEN@ from Cohesion Corporation, Palo Alto, CA. One important characteristic of this sol-gel is its ability to be cured by transition into a solid gel form wherein said gel cannot be mixed or 15 poured or otherwise disturbed thereby forming a solid structure containing immobilized chondrocytes. Type I collagen sol-gel is generally suitable for suspending the chondrocytes and for seeding them into a separately prepared support matrix in the sol form and 20 gel the sol into the solid gel by heating the support matrix to a proper temperature, usually around 30-37* and, in this form, processing the seeded support matrix. This type of sol-gel can also be used as a support matrix for purposes of processing the gel containing 25 chondrocytes in the processor of the invention into a neo-cartilage construct. In another embodiment, the sol-gel is thermo reversible gelation hydrogel (TRGH). Sol-gel thermo reversible material for preparation of sol-gel support 30 matrix is a material which is a viscous sol at temperatures of below 150 and solid gel at temperatures above 370*. The primary characteristic of the thermo reversible gelation hydrogel (TRGH) is that it gels at body temperature and sols at lower than 15-25 0 C 35 temperature that upon its degradation within the body it does not leave biologically deleterious material and that it does not absorb water at gel temperatures.. TRGH has a very quick sol-gel transformation which requires no WO 2006/022671 PCT/US2004/024902 33 cure time and occurs simply as a function of temperature without hysteresis. The sol-gel transition temperature can be set at any temperature in the range from 5 0 C to 70 0 C by the molecular design of the thermo-reversible 5 gelation polymer (TGP), a high molecular weight polymer of which less than 5 wt% is enough for hydrogel formation. The typical TRGH is generally made of blocks of high molecular weight polymer comprising numerous hydrophobic 10 domains cross-linked with hydrophilic polymer blocks. TRGH has low osmotic pressure and is very stable as it is not dissolved in water when the temperature is maintained above the sol-gel transition temperature. Hydrophilic polymer blocks in the hydrogel prevent macroscopic phase 15 separation and separation of water from hydrogel during gelation. These properties make it especially suitable for safe storing and extended shelf-life. The thermo-reversible gel, particularly a space holding thermo-reversible gel (SHTG), should be a 20 compressively strong and stable at 37 0 C and below till about 320C, that is to about temperature of the synovial capsule of the joint which is typically below 370C, but should easily solubilize below 30-310C to be able to be conveniently removed from the cavity as sol. The 25 compressive strength of the SHTG or TRGH must be able to resist compression by the normal activity of the joint. In this regard, the thermo-reversible hydrogel is an aqueous solution of thermo-reversible gelation polymer 30 (TGP) which turns into hydrogel upon heating and liquefies upon cooling. TGP is a block copolymer composed of temperature responsive polymer (TRP) block, such as poly(N-isopropylacrylamide) or polypropylene oxide and of hydrophilic polymer blocks such as 35 polyethylene oxide. Thermally reversible hydrogels consisting of co polymers of polyethylene oxide and polypropylene oxide are available from BASF Wyandotte Chemical Corporation WO 2006/022671 PCT/US2004/024902 34 under the trade name of Pluronics. In general, thermo-reversibility is due to the presence of hydrophobic and hydrophilic groups on the same polymer chain, such as in the case of collagen and 5 for copolymers of polyethylene oxide and polypropylene oxide. When the polymer solution is warmed, hydrophobic interactions cause chain association and gelation; when the polymer solution is cooled, the hydrophobic interaction disappears and the polymer chains are dis 10 associated, leading to dissolution of the gel. Any suitably biocompatible polymer, natural or synthetic, with such characteristics will exhibit the same reversible gelling behavior. This type of thermo-reversible gelation hydrogel is 15 particularly preferred for preparation of neo-cartilage constructs for implantation of the construct into the lesion. In such an instance, the harvested chondrocytes are suspended in the TRGH sol, then warmed to about 37 0 C into the solid gel which thus itself becomes a seeded 20 support matrix, then submitting said seeded matrix to the processing in the tissue processor using the algorithm of the invention, including resting period as described below, thereby resulting in a formation of neo-cartilage construct, then submitting said construct to cooling to 25 change its form into a sol and in this form injecting the neo-cartilage into the lesion wherein upon warming to body temperature the sol is immediately converted into the gel containing neo-cartilage. In time, the delivered neo-cartilage is integrated into the existing cartilage 30 and the TRGH is subsequently degraded leaving no undesirable debris behind. D. Processing Neo-Cartilage and Tissue Processors In order to promote three-dimensional growth and propagation of chondrocytes and/or neo-cartilage, it is 35 advantageous and/or necessary in certain instances to facilitates such growth and propagation by changing conditions of their growth. Such facilitation may be initiated either ex vivo, in vitro or in vivo.

35 This process is, in the current invention, achieved by subjecting either the suspended expanded chondrocytes or the support matrix incorporated with suspended chondrocytes to certain protocol (the algorithm) of conditions which were found to promote such propagation. Such conditions are, for example, application of constant or 5 cyclic hydrostatic pressure, resting periods, recirculation and changing flow rate of media, regulation of oxygen or carbon dioxide concentrations, cell density, control pH, availability of nutrients and co-factors, etc. Typically, this process is performed in the apparatus, preferably in the TESSTM tissue processor, permitting changing of the conditions, as stated above. 10 a) Neo-Cartilage Tissue Processor The general design of the tissue processor is the apparatus for culturing chondrocytes comprising a culture unit having a culture chamber containing culture medium and a supply unit for the continuous and intermittent delivery of the culture medium, a pressure generator for applying atmospheric or constant or cyclic hydrostatic 15 pressure above the atmospheric pressure to chondrocytes in the tissue chamber, said generator having means for changing the pressure, timing, or applying the atmospheric, constant or cyclic hydrostatic pressure at predetermined periods and, optionally, a means capable of delivering and/or absorbing gases such as nitrogen, carbon dioxide and oxygen. Additionally, the processor typically comprises a hermetically sealed space 20 including a heating, cooling and humidifying means. An exemplary scheme of the tissue processor suitable for applying hydrostatic pressure, changing flow rate of the medium and regulating gas concentration delivered to the seeded support system suitable for purposes of this invention is seen in Figure 2A. The tissue processor, seen in Figure 2B, known as Tissue Engineering Support System 25 (TESS) is described in the U.S. patent 6,432,713 issued on August 13, 2002, and also in the U.S. application 09/895,162 (Patent No. 6,607,917), both hereby incorporated by WO 2006/022671 PCT/US2004/024902 36 reference. b) Biochemical and Histological Testing of Neo-Cartilage Constructs The neo-cartilage constructs are tested for their 5 metabolic activity, genetic activation and histological appearance. Typically, the constructs are harvested at days 6 and 18. For histological evaluation of the immature and mature cartilage matrix, 4% paraformaldehyde-fixed 10 paraffin sections are stained with Safranin-O and Type II collagen antibody. For biochemical analysis, neo cartilage constructs are digested in papain at 60 0 C for 18 hours and DNA is measured using, for example, Hoechst 33258 dye method as described in Anal. Biochem., 174:168 15 176 (1988). The production of glycoaminoglycan (GAG) or sulfated-glycosaminoglycan (S-GAG) indicating a metabolic activity of the chondrocyte culture is tested using, for example, modified dimethylene blue (DMB) microassay according to Connective Tissue Research, 9:247-248 20 (1982). c) Conditions for Propagation of Chondrocytes, Preparation of Neo-Cartilage and Neo-Cartilage Constructs Neo-cartilage construct, as used herein, means a 25 matrix seeded with chondrocytes and processed according to the invention. Neo-cartilage constructs may be produced as 3 dimensional patches comprising neo-cartilage having an approximate size of the lesion into which they are 30 deposited or they may be produced as 3-dimensional sheet for use in repairs of extensive cartilage injuries. Their size and shape is determined by the shape and size of the support matrix. Their functionality is determined by the conditions (the algorithm) under which they were 35 processed. Conditions for three-dimensional propagation of chondrocytes in the support matrix into neo-cartilage construct are variable and are adjusted according to the WO 2006/022671 PCT/US2004/024902 37 intended use and/or function of the neo-cartilage and depend on the type of used thermo-reversible hydrogel and on the density of the seeded cells. Thus for production of small neo-cartilage constructs, the conditions will be 5 different from those needed for production of large constructs or for production of extensive neo-cartilage sheets for partial or total replacement of extensively damaged or diseased, for example osteoarthritic, cartilage. 10 i) Processing Neo-Cartilage under Variable Flow One aspect of this invention is the discovery that if the support matrix seeded with chondrocytes is perfused under varying medium flow rates, the cell proliferation, measured by increased accumulation of the 15 extracellular matrix, can be advantageously increased or decreased. Generally, the lower medium flow rate results in the higher extracellular matrix accumulation. Perfusion is an important variable condition for culturing chondrocytes incorporated into support 20 matrices. Using a faster perfusion flow rate may slow down extracellular matrix accumulation affecting growth and propagation of chondrocytes, as measured by production of sulfated glycosaminoglycan (S-GAG). A slower perfusion rate, on the other hand, results in 25 higher production of S-GAG. These results are important for controlling the neo-cartilage growth and for, for example, storage, preservation, transport and shelf-life of neo-cartilage constructs. The perfusion flow rate suitable for purposes of 30 this invention is from about 0.001 to about 0.5 ml/min, preferably from about of 0.005 to about 0.05 ml/min. At the medium perfusion rate 0.005 ml/min the accumulation of extracellular matrix is significantly (p-<0.05) increased compared to accumulation of extracellular 35 matrix observed following perfusion at rate 0.05 ml/min. The optimum flow rate depends upon the total number of cells in the culture chamber.

WO 2006/022671 PCT/US2004/024902 38 ii) Processing Neo-Cartilage Under Different Types of Pressure Subjecting the seeded support matrix to hydrostatic pressure, in conjunction with a decreased perfusion flow, 5 is an integral part of the culture processing system according to this invention. Different types of hydrostatic pressure have a significant effect on glycosaminoglycan production and thus on extracellular matrix accumulation compared to atmospheric pressure 10 alone. The hydrostatic pressure, particularly cyclic hydrostatic pressure applied according to this invention has been found to stimulate chondrocyte proliferation and metabolism which contributes to extracellular matrix accumulation. 15 Hydrostatic pressure suitable for processing chondrocytes seeded within the support matrix is either a constant or cyclic hydrostatic pressure, such pressure being the pressure above the atmospheric pressure. The cyclic hydrostatic pressure suitable for use in 20 processing of the seeded support matrix is from about 0.01 to about 10.0 MPa, preferably from about 0.5 to about 5.0 MPa and most preferably at about 3.0 MPa at 0.01 Hz to about 2.0 Hz, preferably at about 0.5 Hz, applied for about 1 hour to about 30 days, preferably 25 about 7 days, with or without resting period. Typically, the period of hydrostatic pressure is followed by the resting period, typically from about 1 day to about 30 days, preferably about 16-18 days. Studies performed in support of this invention 30 indicate that cell viability is not affected by the hydrostatic pressure and is maintained with chondrocytes distributed uniformly within the support matrix. Following the treatment with hydrostatic pressure, accumulations of both DNA and S-GAG are significantly 35 increased compared to cultures not experiencing applied load, indicating that chondrocyte activation and metabolic and genetic activity can be controlled by the culture environment.

WO 2006/022671 PCT/US2004/024902 39 iii) Processing Neo-Cartilage Under Reduced Oxygen Concentration Another variable in the processing of seeded support matrices is the concentration of oxygen, carbon dioxide 5 and nitrogen. The chondrocytes-seeded support matrix described above may be further cultured under reduced 02 concentration (i.e. less than 20% saturation) during formation of neo-cartilage in the TESS processor. The 10 reduced oxygen concentration of cartilage has been observed in vivo, and such reduction may be due to its normal lack of vascularization which produces a lower oxygen partial pressure, as compared to the adjacent tissues. In this set of studies, chondrocytes seeded in 15 support matrix or neo-cartilage were cultured under oxygen concentration between about 0% and about 20% saturation or under dioxide concentration about 5%. E) Determination of Conditions for Optimization of The Algorithm 20 The ultimate aim of this invention was to find and confirm conditions (the algorithm) for preparation of neo-cartilage constructs for implantation into cartilage lesions, which in conjunction with deposition of one or two sealant layers, would lead to healing of the damaged, 25 injured, diseased or aged cartilage by a) growth of superficial cartilage layer completely overgrowing and covering the lesion and protecting implanted neo cartilage construct; b) integration of neo-cartilage implanted into the lesion as the neo-cartilage construct; 30 and c) subsequent degradation of the construct and sealant materials. The underlying studies, described below, show that a properly designed and optimized culture conditions utilizing hydrostatic pressure with medium perfusion 35 followed by constant culture result in fabrication of neo-cartilage constructs which are integrated into the native cartilage when implanted under the one layer or in between two layers of sealants according to the WO 2006/022671 PCT/US2004/024902 40 invention. General design for a method for preparation of neo cartilage constructs comprises steps: a) isolation of chondrocytes from a donor tissue; 5 b) expanding the chondrocytes for about 3-28 days; c) seeding chondrocytes in a thermo-reversible or collagen gel or collagen sponge support matrix; d) subjecting the seeded gel or sponge to constant or cyclic hydrostatic pressure above 10 atmospheric pressure (about 0.5-3.0 MPa at 0.5 Hz) with medium perfusion rate of 5pl/min for several (5-10) days; and e) subjecting the seeded gel or sponge to resting period for ten to fourteen days at constant 15 (atmospheric) pressure. Neo-cartilage constructs obtained by the above outlined conditions and method show that the combined algorithm of hydrostatic pressure and constant pressure has advantage over conventional culture methods by 20 resulting in higher cell proliferation and extracellular matrix accumulation. Use of thermo-reversible or collagen gel or collagen sponge support matrix maintains uniform cell distribution within the support matrix and also provides support for newly synthesized extracellular 25 matrix. Obtained 3-dimensional neo-cartilage construct is easy to handle and manipulate and can be easily and safely implanted in a surgical setting. Combination of a period of cyclic hydrostatic pressure under low medium perfusion rate followed up with 30 a period of constant culture (resting period) results in increased cell proliferation, increased production of Type II collagen, increased DNA content and increased S GAG accumulation. Increased cell proliferation shows that the 35 harvested inactive non-dividing chondrocytes have been activated into neo-cartilage containing active, dividing and multiplying chondrocytes. Increased level of DNA shows genetic activation of inactive chondrocytes.

WO 2006/022671 PCT/US2004/024902 41 Increased production of Type II collagen and S-GAG shows that production of the extracellular matrix has been activated using the algorithm described above. Although the optimized algorithm described above is 5 preferred, it is to be understood that this algorithm may be advantageously changed using variations of ranges of cyclic hydrostatic pressure, flow rate, duration of the pressure and resting period as disclosed above in detail description of each condition. All variations of all 10 conditions and combinations thereof are intended to be within the scope of this invention. F. Supporting Experimental Studies In order to test effects of different conditions on the propagation of chondrocytes within the support matrix 15 for fabrication of the neo-cartilage construct, studies combining conditions described above for process optimization were performed during development of this invention. Results are shown in Figures 3-9 and in Tables 1-3. 20 For all following studies, the experimental design was as follows with changes in studies conditions. Cartilage was harvested under sterile conditions from the trachea of the swine hind limbs, minced and digested, as described in Example 7. Chondrocytes were 25 expanded for 5 days at 37 0 C and suspended in VITROGEN* (300,000/30 pl). The suspension was absorbed into a support matrix, usually a collagen sponge (4 mm in diameter and 2 mm in thickness) as seen in Figure 1, commercially available from Koken Co., Tokyo, Japan. The 30 sponges seeded with chondrocytes were pre-incubated for 1 hour at 37 0 C to gel the collagen, followed by incubation in culture medium at 37 0 C, 5% CO 2 and cultured in the Tissue Engineering Support System (TESS' m ) processor seen in Fig. 2. 35 a) Evaluation of Effect of Hydrostatic Pressure To evaluate the effect of the pressure and/or medium perfusion rate, the cell seeded sponges were subjected to medium perfusion at 5 pl/min (0.005 ml/min) or 50 pl/min WO 2006/022671 PCT/US2004/024902 42 (0.05 ml/min) under the cyclic (Cy-HP) or constant hydrostatic pressure (constant-HP) of 0.5 MPa at 0.5 Hz for 6 days in the TESS processor. Resting period under atmospheric pressure followed for 12 days. Some seeded 5 sponges served as controls. These were incubated under atmospheric pressure and without perfusion at 37 0 C for a total of 18 days in culture. Sponges harvested 24 hours after seeding with cells (day 0) served as an initial control. More detailed conditions are to be found in 10 Examples and in the following text. At the end of culture period, the support matrices were harvested for biochemical and histological analysis. Sulfated glycosaminoglycan production was measured using a modified dimethylmethylene blue microassay. 15 Histological analysis utilized Safranin-O staining. More detailed conditions are to be found in Examples. The first study was directed to determination of effect of constant (atmospheric), cyclic or constant hydrostatic pressure on production of S-GAG. 20 At the end of the culture period, both control and test matrices were harvested for biochemical and histological analysis. For biochemical analysis, production of sulfated glycosaminoglycan (S-GAG pg/cell construct) was measured using a modified 25 dimethylmethylene blue (DMB) and DNA microassays described in Example 7. Results are seen in Tables 1 and 2 and Figures 3-6. Results of some studies are seen in Tables 1 and 2 showing a numerical representation of observed increase 30 in S-GAG production in matrices treated with the algorithm of the invention. Table 1 Pressure Conditions WO 2006/022671 PCT/US2004/024902 43 Group In TESS In Incubator Total S-GAG Production (n=6) (3 MPa Cyclic (Atmospheric days in (pg/cell construct) Pressure,@0.5 Hz) Pressure) Culture (Mean±SD) Initial - 0 day 0 12.56 ± 0.99 Control - 18 days 18 57.73 6.43 5 Test 6 days 12 days 18 *76.32 + 4.12 (*: p<0.05, compared to Control) Table 1 summarizes results obtained from seeded matrices (n=6) subjected either to atmospheric pressure in an incubator for 18 days (control) or to processing in 10 TESS processor under 3 MPa cyclic hydrostatic pressure at 0.5 Hz for 6 days, followed by 12 days in incubator at atmospheric pressure (test). As seen in Table 1, S-GAG production (pg/cell construct) per seeded matrix was significantly increased 15 to 132% for test compared to 100% control (Figure 3A). Histological results seen in Figure 3B and 3C (Safranin-O staining for S-GAG) were consistent with the results obtained biochemically. Figure 3B is a photomicrograph of Safranin-O staining for S-GAG on paraffin sections in 20 18 days subjected to static pressure. Figure 3C is a photomicrograph of Safranin-O staining for S-GAG on paraffin sections in cell constructs subjected to cyclic hydrostatic pressure for 6 days followed by 12 days of static culture. 25 As seen in Figure 3B, when the cell constructs are subjected to static atmospheric pressure (Figure 3B), there is much lower S-GAG accumulation in the construct than when it is subjected to a cyclic hydrostatic pressure for 6 days, followed by 12 days of static 30 atmospheric pressure (Figure 3C). To determine the effect of the hydrostatic pressure on chondrocyte proliferation stimulation and WO 2006/022671 PCT/US2004/024902 44 matrix accumulation, cartilage was harvested under sterile conditions as described above. Chondrocytes were expanded for 5 days at 37*C and suspended in VITROGEN* (300,000/30 pl) . The suspension was absorbed into a 5 honeycomb support matrix or collagen sponge as seen in Figure 1. The cell constructs were incubated in culture medium at 37 0 C, 5% CO 2 and 20% 02, at 0.5 MPa cyclic hydrostatic pressure or 0.5 MPa constant hydrostatic pressure for 6 days followed by incubation for 12 days at 10 atmospheric pressure in the Tissue Engineering Support System (TESS'M) processor seen in Figure 2. The remaining cell matrices comprising the control group were incubated at atmospheric pressure for 18 days at 37 0 C, 5% CO 2 ans 20%02 15 At the end of the culture period, the matrices were harvested for biochemical analysis. Results are seen in Table 2. Glycosaminoglycan production was measures using a modified dimethylmethylene blue (DMB) microassay. Cell proliferation was measured using a 20 modified Hoechst Dye DNA assay. Formation of neo-tissue was evaluated by Safranin-O staining. Results are seen in Figures, 4A, 4B, 5A and 5B and in Table 2. Table 2 25 Group Pressure Conditions S-GAG DNA (n=7) In TESS Days in Total GAG Production Time Incubator days (pg/cell Type of Days (Atmospheric In construct) DNA Index Pressure Pressure) culture (Mean ± SD) (Control=1) 30 Control ~ 18 18 59.85 ± 7.69 i Cycli 0.5 MPa d 12 18 *91.05± 10.68 1.49 Cyclic days Const-HP 0.5 MPa 6 12 18 *9785 5.53 1.74 Constant 35 (*: p<0.05, compared to Control) All cultures were incubated at 37 0 C, 5% CO 2 and 20% WO 2006/022671 PCT/US2004/024902 45 02. In TESS culture, the medium flow rate was 0.05 ml/min. Two cell matrices from each group were harvested for histological analysis. 5 As seen in Table 2, the matrices subjected to conditions listed in groups control, cyclic hydrostatic pressure (Cy-HP) and constant hydrostatic pressure (const HP) resulted in production of 59.85, 91.05 and 97.85 pg/cell construct of S-GAG and 1, 1.49 and 1.74 (control=1) 10 of DNA Index, respectively. These results clearly show that neo-cartilage cultured under hydrostatic pressure, whether cyclic or constant, followed by static culture is more genetically and metabolically active than one treated under static atomospheric conditions (controls) . These 15 results are graphically illustrated in Figure 4 which shows effect of hydrostatic pressure on production of sulfated glycosaminoglycan (Figure 4A) and DNA content (Figure 4B). Figure 4A is a graphical representation of results enumerated in Table 2 and shows the sulfated 20 glycosaminoglycan production in pg/cell construct wherein control represents seeded matrices subjected to atmospheric pressure, Cy-HP represents seeded matrices subjected to cyclic hydrostatic pressure (0.5 MPa) and constant-HP represent matrices subjected to constant hydrostatic 25 pressure (0.5 MPa). Results seen in Table 2 are illustrated graphically in Figure 4A, under the conditions described above. There was significant increase in S-GAG production for both the cyclic (Cy-HP) and constant hydrostatic pressure (constant 30 HP) groups compared to atmospheric pressure (control) group. Specifically, the production of S-GAG in group Control was 59.85 pg/cell construct. In group Cy-HP the production was 91.05 pg/cell construct. In group constant HP cell construct production was 97.85 pg/cell construct 35 resulting in increase of S-GAG production to 152% for group Cy-HP and by 162% for group constant-HP compared to group WO 2006/022671 PCT/US2004/024902 46 control. Figure 4B shows DNA production with corresponding results presented in Table 2 for DNA, likewise showing increased production of DNA in constructs processed under 5 cyclic or constant hydrostatic pressure. Figure 5A is a graph comparing effect of constant atmospheric pressure (Control) and zero MPa hydrostatic pressure (OMPa) serving as pressure controls, 0.5 MPa cyclic hydrostatic pressure (Cy-HP) and 0.5 MPa constant 10 hydrostatic pressure (constant-HP) at day 6 and 18 on support matrices subjected to processing in the TESS processor. All matrices were incubated at 37 0 C for 18 days. The Cy-HP and constant-HP were applied for the first 6 days followed by 12 days of incubation at atmospheric pressure. 15 Results seen in Figure 5A show that combination of Cy-HP or constant-HP with resting period of atmospheric pressure incubation resulted in significant (p<0.05) increase of S-GAG production in the processed matrices compared to S-GAG production observed in matrices processed 20 at atmospheric pressure with perfusion only. Figure 5B shows the Index of DNA content (Initial=1) in matrices subjected to static (Control) , zero hydrostatic (OMPa), cyclic (Cy-HP) or constant (Constant-HP) hydrostatic pressure for 6 day and 12 days of atmospheric 25 pressure culture. Increase in DNA content in matrices subjected to the algorithm conditions is clearly shown in both cyclic and constant hydrostatic pressure groups. Comparison to initial and control DNA level to constant DNA levels in all three groups subjected to hydrostatic 30 pressure reveals that the DNA level in constructs subjected to the cyclic hydrostatic pressure at day 6 then at day 18 and the DNA level constructs subjected to constant hydrostatic pressure is lower at day 6 than at day 18. High levels of DNA is observed in matrices submitted to 35 constant hydrostatic pressure at day 18. Figures 6A and 6B show histological evaluation of WO 2006/022671 PCT/US2004/024902 47 matrices by Safranin-O. Figure 6A shows accumulation of S GAG on day 18 in matrices subjected to atmospheric pressure. Figure 6B shows accumulation of S-GAG in matrices subjected to 6 days of cyclic hydrostatic pressure 5 (Cy-HP), followed by 12 days of atmospheric pressure. The greater S-GAG accumulation in Cy-HP culture matrices is evident from the increased density of the photomicrograph clearly visible in the construct. Figure 3C shows accumulation of Type II collagen in matrices subjected to 10 the atmospheric pressure or to the cyclic hydrostatic pressure (Figure 6D) . Larger accumulation of Type II collagen in Figure D is clearly seen. These results demonstrate that chondrocytes may be placed in culture to coalesce into a neo-cartilage 15 construct with accumulated extracellular matrix macro molecules, such as sulfated glycosaminoglycan (S-GAG). b) Evaluation of Effect of Perfusion Flow The second type of study was performed in order to determine the effect of perfusion flow rate on chondrocyte 20 proliferation (DNA content) and production of extracellular matrix (S-GAG accumulation). Results are seen in Table 3 and Figures 7A and 7B. Figure 7 describes determination of effect of the perfusion flow rate on cell proliferation measured by level 25 of DNA (Figure 7A), S-GAG accumulation (Figure 7B) at day 0, 6 and 18. Figure 7A shows that the lower perfusion rate (5 pl/min) results in higher DNA content index used as a measure for determination of cell proliferation. 30 Specifically, the DNA content index compared to the initial DNA content equal to 1 increased by about 50% to about 1.5 when the culture perfusion rate was 5 pl/min. The higher perfusion rate (50 pl/min) resulted in much smaller increase in DNA content index to about 1.2. 35 Table 3 shows the effect of perfusion flow rate on the S-GAG production in matrices treated as outlined above WO 2006/022671 PCT/US2004/024902 48 where the flow rate was either 0.05 ml/min (50 pl/min) or 0.005 ml/min (5 pl/min). Table 3 Culture duration Medium In TESS Total GAG Production In Incubator 5 Group Perfusion (0.5 MPa , days (pg/cell (n=7) Flow Rate Cyclic (Atmospheric in construct) Pressure) (ml/min) Pressure culture (Mean + SD) A 0.05 ml/min 6 days 12 days 18 days 78.75 + 6.84 B 0.005 ml/min 6 days 12 days 18 days 107.33 + 8.53 All cultures were incubated at 37 0 C, 5% CO 2 and 20% 10 02. In the culture, 0.5 MPa cyclic pressure at 0.5 Hz was applied to the cell matrices. Two matrices from each group were harvested for histological analysis. As seen in Table 3, the lower perfusion rate (5 pl/min) resulted in approximately 1.5 higher production of 15 S-GAG than the higher perfusion rate (50 pl/min). These results are seen in graphical form in Figure 7B. Figure 7B is graph showing differences between S-GAG production by seeded support matrices subjected to a medium perfusion flow rate of 0.005 ml/min compared to matrices 20 subjected to a medium perfusion flow rate of 0.05 ml/min at days 6 and 18. As seen in Figure 7B, increase in S-GAG production up to 136% (p<0.05) in matrices subjected to a slower rate of 0.005 ml/min. The results summarized in Figures 7A and 7B clearly 25 show a significant increase in both the DNA index and S-GAG production in the cell construct at a flow rate of 0.005 ml/min compared to the flow rate 0.05 ml/ml. There is no significant difference in the amount of S-GAG released into the medium between the two flow rates. It is therefore 30 possible to use lower flow rate and avoid shear.

WO 2006/022671 PCT/US2004/024902 49 Determination whether the combination of the perfusion flow rate with cyclic or constant hydrostatic pressure leads to increased formation of extracelluar matter was also studied. Results are seen in Figure 8. 5 Figure 8 illustrates formation of extracellular matrix after 15 days culture determined in matrices treated with perfusion (0.005 ml/min) only (Figure 8A), cyclic hydrostatic pressure 2.8 MPa at 0.015 Hz (Figure 8B) and constant hydrostatic pressure 2.8 MPa at 0.015 Hz (Figure 10 8C) as determined by toluidine blue staining. This figure clearly shows that hydrostatic pressure and medium perfusion enhances production of extracellular matrix. c. Evaluation of Effect of Low Oxygen Tension The third type of study was performed in order to 15 determine the effect of low oxygen tension on chondrocyte proliferation (DNA content) and production of extracellular matrix (S-GAG accumulation). Results are seen in Table 4 and Figure 9A and 9B. Table 4 Total UAG Production 20 Group Oxygen days (pg/cell (n=8) concentration in construct) M Culture duration culture (Mean ± SD) In TESS (0.5 MPa In Incubator Cyclic (Atmospheric Pressure) Pressure) 21 A 20% 7 days 14 days days 60.89 + 6.02 21 B 2% 7 days 14 days days *105.59 ± 10.95 25 (*: p<0.05, compared to group A) All cultures were incubated at 37 0 C, at 5% CO 2 . In TESS culture, the medium flow rate was 0.005 ml/min. Two cell matrices from each group were harvested for histological analysis. 30 As seen in Table 4, the lower oxygen tension (2% 02 WO 2006/022671 PCT/US2004/024902 50 concentration) resulted in approximately 1.7 higher production of S-GAG than higher oxygen concentration (20%) corresponding to atmospheric 02 concentration. These results are seen in graphical form in Figure 9A. 5 Figure 9A is a graph showing differences between S GAG production by cell constructs subjected to 2% oxygen concentration (Cy-HP) and to cyclic hydrostatic pressure followed by static culture compared to cell constructs subjected to 20% oxygen concentration and Cy-HP followed by 10 static culture. As already seen in Table 4, at 2% oxygen concentration compared to 20% concentration, the production of S-GAG rose by approximately 70%. Figure 9B shows the DNA content index (initial=1) in cell constructs subjected to 2% or 20% oxygen concentration 15 with Cy-HP culture followed by static culture. There are no significant differences in the DNA content index between 2% oxygen concentration and 20% oxygen concentration. These results indicate that the lower oxygen tension stimulates S-GAG production in cell constructs when 20 combined with the cyclic hydrostatic culture followed by static culture. However, the cell proliferation, expressed as DNA content index, is not affected by changes in oxygen tension. The algorithm of the invention thus comprises 25 combination of the low perfusion flow rate and low oxygen tension with a certain predetermined period of hydrostatic pressure followed by the period of a resting culture period in the absence of externally applied load. d) General Applicability of Algorithm to Various 30 Cell Types The algorithm described above for chondrocytes is similarly applicable to other types of cell and tissue, such as fibroblasts, fibrochondrocytes, tenocytes, osteoblasts and stem cells capable of differentiation, or 35 tissues such as cartilage connective tissue, fibrocartilage, tendon and bone. The algorithm conditions WO 2006/022671 PCT/US2004/024902 51 may be the same or different but would be generally within the above described ranges. II. Neo-Cartilage Composition Construct The neo-cartilage composition construct is a 5 multilayered three-dimensional structure comprised at least of living chondrocytes incorporated into a cellular support matrix. The support matrix is seeded with living chondrocytes. The construct is fabricated in vitro and ex vivo 10 prior to implanting into the cartilage lesion. The construct is fabricated using the method and conditions, cumulatively called the algorithm, described above, with all conditions being variable within the given ranges and depending on the intended use or on the method of delivery. 15 In one embodiment, the autologous or heterologous chondrocytes are cultured as described, seeded into the support matrix and processed into the neo-cartilage construct using predetermined medium perfusion flow rate, cyclic or constant hydrostatic pressure and reduced or 20 increased concentration of oxygen and/or carbon dioxide. The neo-cartilage construct is delivered into the cartilage lesion cavity and deposited between two layers of sealant and left in situ to be integrated into the native cartilage. 25 I. Method for Formation of Superficial Cartilage Laver The primary aspect of this invention is a finding that when the neo-cartilage, neo-cartilage construct or seeded support matrix produced according to procedures and 30 conditions described above is implanted into a cartilage lesion cavity and covered with a biocompatible adhesive sealant, the resulting combination leads to a formation of a superficial cartilage layer completely overgrowing said lesion. 35 The method is based on producing a neo-cartilage and neo-cartilage construct comprising support matrix seeded WO 2006/022671 PCT/US2004/024902 52 with expanded chondrocytes processed according to the algorithm of the invention. Chondrocytes are typically suspended in a collagen sol which is thermo-reversible and easily changes from sol to gel at the body temperature 5 thereby permitting external preparation of and delivery of the neo-cartilage construct into the lesion in form of the sol which changes its state into gel upon delivery to the lesion and warming to the body temperature. The neo-cartilage construct is implanted into the 10 lesion and covered by a layer of a biologically acceptable adhesive sealant. Optionally, the first layer of the sealant is introduced into the lesion and deposited at the bottom of the lesion. This first sealant's function is to prevent entry and to block the migration of subchondral and 15 synovial cells of the extraneous components, such as blood borne agents, cell and cell debris, etc., into the cavity and their interference with the integration of the neo cartilage therein. The second sealant layer is placed over the surface of the construct. The presence of both these 20 sealants in combination with the neo-cartilage construct results in successful integration of the neo-cartilage into the joint cartilage. The method may be practiced in several modes and each mode involves generic steps outlined below in variable 25 combinations. General way to practice the method for repair and restoration of damaged, injured, diseased or aged cartilage to a functional cartilage is to follow steps: a) Preparing Neo-Cartilage, Neo-Cartilage Construct 30 or Chondrocyte Support Matrix This step involves preparation of neo-cartilage, neo cartilage comprising constructs and support matrix comprising autologous or heterologous chondrocytes incorporated therein. Preparation of any of the three 35 entities named above is described in greater detail above in sections I.B-D.

WO 2006/022671 PCT/US2004/024902 53 b) Depositing the First and Second Sealant into the Lesion This step involves introducing a first and a second layer of a first and a second biologically acceptable 5 sealant into a cartilage lesion. The first and second sealants may be the same or different. It is to be understood that the utilization of the first bottom layer is optional and that the method for a formation of the superficial cartilage layer is enabled without the first 10 layer. Specifically, this step involves deposition of the first sealant at the bottom of the lesion and of the second sealant over the lesion. The first and the second sealants can be the same or different, however, both the first and 15 the second sealants must have certain definite properties to fulfill their functions. The first sealant, deposited into the cavity before the neo-cartilage is deposited, acts as a protector of the lesion cavity integrity, that is, it protects the lesion 20 cavity not only from extraneous substances but it also protect this cavity from formation of the fibrocartilage in the interim when the cavity is filled with a space-holding gel in expectation of implantation of the neo-cartilage after processing. The second sealant acts as a protector 25 of the lesion cavity on the outside as well as a protector of the neo-cartilage construct deposited within a cavity formed between the two sealants and as well as an initiator of the formation of the superficial cartilage layer. 1. First Sealant 30 The optionally deposited first sealant forms an interface between the introduced neo-cartilage construct and the native cartilage. The first sealant, deposited at the bottom of the lesion, must be able to protect the construct from and prevent chondrocyte migration into the 35 sub-chondral space. Additionally, the first sealant prevents the infiltration of blood vessels and undesirable WO 2006/022671 PCT/US2004/024902 54 cells and cell debris into the neo-cartilage construct and it also prevents formation of the fibrocartilage. 2. Second Sealant The second sealant acts as a protector of the neo 5 cartilage construct or the lesion cavity on the outside and is typically deposited over the lesion either before or after the neo-cartilage is deposited therein and in this way protects the integrity of the lesion cavity from any undesirable effects of the outside environment, such as 10 invading cells or degradative agents and seals the space holding gel in place before the neo-cartilage is deposited therein. The second sealant also acts as a protector of the neo-cartilage construct implanted within a cavity formed between the two sealants. In this way, the second 15 sealant may be deposited after the neo-cartilage is deposited over the first sealant and seal the neo-cartilage within the cavity or it may be deposited over the space holding gel. The third function of the second sealant is as an initiator or substrate for the formation of a 20 superficial cartilage layer. Studies performed during the development of this invention discovered that when the second sealant was deposited over the cartilage lesion, a growth of the superficial cartilage layer occurred as an extension of the native superficial cartilage layer. This 25 superficial cartilage layer is particularly well-developed when the lesion cavity is filled with the space-holding or thermo-reversible gel thereby leading to the conclusion that such a gel might provide a substrate for the formation of such superficial cartilage layer. 30 3. First and Second Sealant Properties The first or second sealant of the invention must possess the following characteristics: Sealant must be biologically acceptable, easy to use and possess required adhesive and cohesive properties. 35 The sealant is biologically compatible with tissue, be non-toxic, not swell excessively, not be extremely rigid WO 2006/022671 PCT/US2004/024902 55 or hard, as this could cause abrasion of or extrusion of the sealant from the tissue site, must not interfere with the formation of new cartilage, or promote the formation of other interfering or undesired tissue, such as bone or 5 blood vessels and must resorb and degrade by an acceptable pathway or be incorporated into the tissue. The sealant must rapidly gel from a flowable liquid or paste to a load-bearing gel within 3 to 15 minutes, preferably within 3-5 min. Longer gelation times are not 10 compatible with surgical time constraints. Additionally, the overall mode of use should be relatively simple because complex procedures will not be accepted by surgeons. Adhesive bonding is required to attach the sealant formulation to tissue and to seal and support such tissue. 15 Minimal possessing peel strengths of the sealant should be at least 3N/m and preferably 10 to 30 N/m. Additionally, the sealant must itself be sufficiently strong so that it does not break or tear internally, i.e., it must possess sufficient cohesive strength, measured as tensile strength 20 in the range of 0.2 MPa, but preferably 0.8 to 1.0 MPa. Alternatively, a lap shear measurement may be given to define the bond strength of the formulation should have values of at least 0.5 N/cm 2 and preferably 1 to 6 N/cm 2 . Sealants possessing the required characteristics are 25 typically polymeric. In the un-cured, or liquid state, such sealant materials consist of freely flowable polymer chains which are not cross-linked together, but are neat liquids or are dissolved in physiologically compatible aqueous buffers. The polymeric chains also possess side chains or 30 available groups which can, upon the appropriate triggering step, react with each other to couple, or cross-link the polymer chains together. If the polymer chains are branched, i.e., comprising three or more arms on at least one partner, the coupling reaction leads to the formation 35 of a network which is infinite in molecular weight, i.e., a gel.

WO 2006/022671 PCT/US2004/024902 56 The formed gel has cohesive strength dependent on the number of inter-chain linkages, the length (molecular weight) of the chains between links, the degree of inclusion of solvent in the gel, the presence of 5 reinforcing agents, and other factors. Typically, networks in which the molecular weight of chain segments between junction points (cross-link bonds) is 100-500 Daltons are tough, strong, and do not swell appreciably. Networks in which the chain segments are 500-2500 Daltons swell 10 dramatically in aqueous solvents and become mechanically weak. In some cases the latter gels can be strengthened by specific reinforcer molecules; for example, the methylated collagen reinforces the gels formed from 4-armed PEGs of 10,000 Daltons (2500 Daltons per chain segment). 15 The gel's adhesive strength permits bonding to adjacent biological tissue by one or more mechanisms, including electrostatic, hydrophobic, or covalent bonding. Adhesion can also occur through mechanical inter-lock, in which the uncured liquid flows into tissue irregularities 20 and fissures, then, upon solidification, the gel is mechanically attached to the tissue surface. At the time of use, some type of triggering action is required. For example, it can be the mixing of two reactive partners, it can be the addition of a reagent to 25 raise the pH, or it can be the application of heat or light energy. Once the sealant is in place, it must be non-toxic to adjacent tissue, and it must be incorporated into the tissue and retained permanently, or removed, usually by 30 hydrolytic or enzymatic degradation. Degradation can occur internally in the polymer chains, or by degradation of chain linkages, followed by diffusion and removal of polymer fragments dissolved in physiological fluids. Another characteristic of the sealant is the degree 35 of swelling it undergoes in the tissue environment. Excessive swelling is undesirable, both because it creates WO 2006/022671 PCT/US2004/024902 57 pressure and stress locally, and because a swollen sealant gel loses tensile strength, due to the plasticizing effect of the imbibed solvent (in this case, the solvent is physiological fluid) . Gel swelling is modulated by the 5 hydrophobicity of the polymer chains. In some cases it may be desirable to derivatize the base polymer of the sealant so that it is less hydrophilic. For example, one function of methylated collagen containing sealant is presumably to control swelling of the gel. In another example, the 10 sealant made from penta-erythritol tetra-thiol and polyethylene glycol diacrylate can be modified to include polypropylene glycol diacrylate, which is less hydrophilic than polyethylene glycol. In a third example, sealants containing gelatin and starch can also be methylated both 15 on the gelatin and on the starch, again to decrease hydrophilicity. 4. Suitable and Non-suitable Sealants Sealants suitable for purposes of this invention include the sealants prepared from gelatin and di-aldehyde 20 starch triggered by mixing aqueous solutions of gelatin and dialdehyde starch which spontaneously react and gel. The gel bonds to tissue through a reaction of aldehyde groups on starch molecules and amino groups on proteins of tissue, with an adhesive bond strength to up to 100 N/m and 25 an elastic modulus of 8 x 106 Pa, which is a characteristic of a relatively tough, strong material. After swelling in physiological fluids this cohesive strength declines. The gelled sealant is degraded by enzymes that cleave the peptide bonds of gelatin and the glycosidic bonds of 30 starch. Another acceptable sealant is made from a copolymer of polyethylene glycol and poly-lactide or -glycolide, further containing acrylate side chains and gelled by light, in the presence of some activating molecules. The 35 linkage is formed by free-radical chemistry. The gel bonds to tissue by mechanical interlock, having flowed into WO 2006/022671 PCT/US2004/024902 58 tissue surface irregularities prior to curing. The sealant degrades from the tissue by hydrolytic cleavage of the linkage between polyethylene glycol chains, which then dissolve in physiological fluids and are excreted. 5 The acceptable sealant made from periodate-oxidized gelatin remains liquid at acid pH, because free aldehyde and amino groups on the gelatin cannot react. To trigger gelation, the oxidized gelatin is mixed with a buffer that raises the pH, and the solution gels. Bonding to tissue is 10 through aldehyde groups on the gelatin reacting with amino groups on tissue. After gelation, the sealant can be degraded enzymatically, due to cleavage of peptide bonds in gelatin. Still another sealant made from a 4-armed 15 pentaerythritol thiol and a polyethylene glycol diacrylate is formed when these two neat liquids (not dissolved in aqueous buffers) are mixed. The rate of gelation is controlled by the amount of a catalyst, which can be a quaternary amino compound, such as tri-ethanolamine. A 20 covalent linkage is formed between the thiol and acrylate, to form a thio-ether bond. The final gel is firm and swells very little. The tensile strength of this gel is high, about 2 MPa, which is comparable to that of cyanoacrylate acceptable Superglue. Degradation of such gels in vivo is 25 slow. Therefore, the gel may be encapsulated or incorporated into tissue. Another example is the composition, preferred for use in this invention, that contains 4-armed tetra-succinimidyl ester or tetra-thiol derivatized PEG, plus methylated 30 collagen. The reactive PEG reagents in powder form are mixed with the viscous, fluid methylated collagen (previously dissolved in water); this viscous solution is then mixed with a high pH buffer to trigger gelation. The tensile strength of this cured gel is about 0.3 MPa. 35 Degradation presumably occurs through hydrolytic cleavage of ester bonds present in the succinimidyl ester PEG, WO 2006/022671 PCT/US2004/024902 59 releasing the soluble PEG chains which are excreted. In general, a sealant useful for the purposes of this application has adhesive, or peel strengths at least 1ON/m and preferably 100 N/cm; it needs to have tensile strength 5 in the range of 0.2 MPa to 3 MPa, but preferably 0.8 to 1.0 MPa. In so-called "lap shear" bonding tests, values of 0.5 up to 4-6 N/cm 2 are characteristic of strong biological adhesives. Such properties can be achieved by a variety of 10 materials, both natural and synthetic. Examples include: 1) gelatin and di-aldehyde starch (International Patent Publication Number WO 97/29715; 21 Aug. 1997;); 2) 4-armed penta-erythritol tetra-thiol and polyethylene glycol diacrylate (International Patent Application Number WO 15 00/44808; 3 Aug 2000; example 14) ; 3) photo-polymerizable polyethylene glycol-co-poly(a-hydroxy acid) diacrylate macromers (US Pat No. 5,410,016; Apr. 25, 1995); 4) periodate-oxidized gelatin (US Pat No. 5,618,551, Apr. 8, 1997); 5) serum albumin and di-functional polyethylene 20 glycol derivatized with maleimidyl, succinimidyl, phthalimidyl and related active groups (International Patent Publication Number WO 96/03159, Feb 8, 1996) and 6) 4-armed polyethylene glycols derivatized with succinimidyl ester and thiol, plus methylated collagen, referred to as 25 "CT3" (US Pat No 6,312, 725B1, Nov. 6, 2001). Various other sealant formulations are available commercially or are described in the literature. However, the majority of these are not suitable for practicing this invention for a variety of reasons. 30 For example, fibrin sealant is unsuitable because it interferes with the formation of cartilage. Cyanoacrylate, or Superglue, is extremely strong but it might exhibit toxic reactions in tissue. Un-reinforced hydrogels of various types typically 35 exhibit tensile strengths of lower than 0.02 MPa, which is too weak to support the adhesion required for the purpose WO 2006/022671 PCT/US2004/024902 60 of this application because such gels will swell too much, tear too easily, and break down too rapidly. It is worth noting that it is not the presence or absence of particular protein or polymer chains, such as 5 gelatin or polyethylene glycol, which necessarily govern the mechanical strength and degradation pattern of the sealant. The mechanical strength and degradation pattern are controlled by the cross-link density of the final cured gel, by the types of degradable linkages which are 10 present, and by the types of modifications and the presence of reinforcing molecules, which may affect swelling or internal gel bonding. 5. Preferred Sealants The first or second sealant of the invention must be 15 a biologically acceptable, typically rapidly gelling synthetic compound having adhesive, bonding and/or gluing properties, and is typically a hydrogel, such as derivatized polyethylene glycol (PEG) which is preferably cross-linked with a collagen compound, typically alkylated 20 collagen. Sealant should have a tensile strength of at least 0.3 MPa. Examples of suitable sealants are tetra hydrosuccinimidyl or tetra-thiol derivatized PEG, or a combination thereof, commercially available from Cohesion Technologies, Palo Alto, CA under the trade name CoSeal' m , 25 described in J. Biomed. Mater. Res (Appl. Biomater.), 58:545-555 (2001) . Other compounds suitable to be used are the rapid gelling biocompatible polymer compositions described in the U.S. patent 6,312,725 B1, herein incorporated by reference. Additionally, the sealant may 30 be two or more-part polymers compositions that rapidly form a matrix where at least one of the compounds is polymer, such as, polyamino acid, polysaccharide, polyalkylene oxide or polyethylene glycol and two parts are linked through a covalent bond and cross-linked PEG with methyl collagen, 35 commercially available. The sealant of the invention typically gels rapidly WO 2006/022671 PCT/US2004/024902 61 upon contact with tissue, particularly with tissue containing collagen. The second sealant may or may not be the same as the first sealant. Both the first and the second is preferably a cross-linked polyethylene glycol 5 hydrogel with methyl-collagen, which has adhesive properties. c) Implanting the Neo-Cartilage Construct Next step in the method of the invention comprises implanting said neo-cartilage into a lesion cavity formed 10 under the second sealant or between two layers of sealants, said cavity either filled with neo-cartilage construct deposited therein or, optionally, with a space holding thermo-reversible gel (SHTG) deposited into said cavity as a sol at temperatures between about 5 to about 25 0 C 15 wherein, within said cavity and at the body temperature, said SHTG converts the sol into gel and in this form the SHTG holds the space for introduction of the neo-cartilage construct and provides protection for the neo-cartilage and wherein its presence further promotes in situ formation of 20 de novo superficial cartilage layer covering the cartilage lesion. The above step is versatile in that the neo-cartilage may be deposited into said lesion cavity after the first sealant is deposited but before the second sealant is 25 deposited over it or the first and second sealants may be deposited first and the cavity is filled with the space holding thermo-reversible gel for the interim period when the neo-cartilage is cultured and processed or it may be deposited into the lesion cavity without the first sealant 30 and covered with the second sealant. The neo-cartilage is either autologous or heterologous and is prepared as described above in sections I.B. a-c. a) Removing the Space-Holding or Thermo-Reversible 35 Gel from the Lesion Cavity The neo-cartilage is deposited into the cavity either WO 2006/022671 PCT/US2004/024902 62 before or after the formation of the superficial cartilage layer. In all cases, when the first sealant is used, the first sealant is deposited first. In one embodiment, the neo-cartilage construct containing, typically, the 5 heterologous neo-cartilage might be deposited on the top of the first sealant layer and immediately covered by the second sealant layer. In such an instance, the neo cartilage is left in the cavity until the superficial cartilage layer is formed and the neo-cartilage is 10 integrated into the surrounding cartilage. Then, depending on the material used for neo-cartilage construct, the sponge gel or thermo-reversible gelling hydrogel are left in the cavity to disintegrate. In the instance when the two sealants are deposited 15 first, the space within the lesion cavity is optionally filled with a polymer gel, such as the space-holding thermo-reversible gel. Such gel is left in the cavity until the neo-cartilage construct is cultured, processed and ready to be implanted. Since such thermo-reversible gel 20 might or might not be completely or partially degraded during this time, it may be removed from the cavity by cooling the lesion to about 5 0 C at which temperature the gel becomes a sol, and by removing said sol from the cavity, for example, by injection. Using the same process, 25 that is by cooling the solid gel of the neo-cartilage, the process may be reversed for introduction of the neo cartilage construct into said lesion cavity wherein, after the sol is warmed into the body temperature, the sol is converted into a solid gel. 30 Thus, the primary premise of this process is that the removal and/or introduction of the space holding gel or introduction of neo-cartilage construct proceeds at the cold temperature where the composition is in the sol state and converts into solid gel at warmer temperatures. In 35 this way the gel may be removed from the cavity as the sol after the neo-cartilage integration and formation of WO 2006/022671 PCT/US2004/024902 63 superficial cartilage layer. e. Generation of the Superficial Cartilage Layer A combination of the neo-cartilage construct comprising the neo-cartilage suspended in the thermo 5 reversible gel or support matrix seeded with chondrocytes with the adhesive polymeric second sealant leads to overgrowth and complete or almost complete sealing of the lesion cavity. In alternative, depending on the surface chemistry of 10 the thermo-reversible gel, the superficial layer could grow directly over the neo-cartilage construct if such surface chemistry is propitious to such growth. Typically, a biologically acceptable second sealant, preferably a cross-linked PEG hydrogel with methyl collagen 15 sealant is deposited either over the neo-cartilage construct implanted into the lesion cavity or is deposited over the lesion before the neo-cartilage construct is deposited therein. The second sealant acts as an initiator for formation of the superficial cartilage layer which in 20 time completely overgrows the lesion. The superficial cartilage layer in several weeks or months completely covers the lesion and permits integration of the neo cartilage of the neo-cartilage construct or chondrocytes seeded within the support matrix into the native 25 surrounding cartilage substantially without formation of fibrocartilage. Formation of the superficial cartilage layer is a very important aspect of the healing of the cartilage and its repair and regeneration. 30 I. In vivo Studies in Swine of Weight-Bearing Region of the Knee The method according to the invention was tested and confirmed in in vivo studies wherein the generation of the superficial cartilage layer has been confirmed in a three 35 month study performed in a swine model in order to evaluate porcine neo-cartilage construct integration into the WO 2006/022671 PCT/US2004/024902 64 surrounding cartilage. The neo-cartilage construct prepared according to the method of the invention was implanted into an artificially generated lesion in a pig's knee. Detailed conditions of 5 the study are described in Example 8. Results of this study are illustrated in Figures 10, 11 and 12 depicting histological evaluation using Safranin-O staining method of artificially created cartilage lesions. Briefly, the study comprised of an open arthrotomy of 10 the right knee joint performed on all animals. A biopsy of the cartilage was obtained. Chondrocytes were isolated from the cartilage biopsy and cultured within a collagen matrix in a Tissue Engineering Support System (TESS m ) as described in detail above to produce porcine neo-cartilage 15 construct for subsequent implantation. A defect was created in the medial femoral condyle of the right knee. This defect, which served as a control, was not implanted with the neo-cartilage construct. The empty defect is seen in Figure 10A. Following surgery, the 20 joint was immobilized with an external fixation device for a period of about two weeks. Three weeks after the arthrotomy on the right knee was performed, an open arthrotomy was performed on the left knee and the same defects were created in this medial femoral condyle. The 25 porcine neo-cartilage was implanted within defects in this knee which was similarly immobilized. The porcine implant site is seen in Figure 10B which also show initiation of formation of a superficial cartilage layer two weeks after implantation. 30 The operated sites were periodically viewed via arthroscopy at monthly intervals. Subsequently, approximately 3 months after porcine neo-cartilage implantation, animals were euthanized and joints harvested and prepared for histological examination. The implanted 35 sites were prepared and examined histologically. Comparison of Figure 11 (control at four months after arthrotomy) and WO 2006/022671 PCT/US2004/024902 65 Figure 12 shows test knee three month following arthrotomy and neo-cartilage implantation according to the invention. This figure shows that in the control knee there is a visible formation of fibrocartilage, in the test group 5 (Figures 12A-D), the implanted porcine neo-cartilage construct resulted in production of dense regenerating hyaline cartilage and in the same test group, there was clearly visible cell integration (Figures 12C and 12D) and formation of the superficial cartilage layer (Figures 12A 10 and 12B). Figures 11A-llC thus shows the control lesion at 4 months following the surgery without a treatment with the neo-cartilage construct. Noticeable in Figure 11A is the proliferation of fibrocartilage within the defect site. 15 Figure 11 shows the control without porcine-NeoCart m at 4 months. The proliferation of fibrocartilage in the defect site is clearly noticeable. Also seen is synovial tissue that has infiltrated into the subchondral space. Figures 12A-12D, on the other hand, show that after 20 3 months post implantation in a weight bearing region of the knee, the porcine neo-cartilage has produced dense hyaline-like cartilage and has integrated with the host cartilage laterally and at the interface of the subchondral bone. 25 Figure 12 shows formation of regenerated hyaline-like cartilage in the implant site. Figure 12B illustrates the beginning of integration between the porcine neo-cartilage and the native cartilage laterally and at the subchondral bone. 30 The porcine neo-cartilage was delivered to the defect by implantation of neo-cartilage construct between two layers of sealant. The newly formed superficial cartilage layer formed over the defect at three months following the implantation is clearly visible. 35 Figure 12 shows and confirms that 3 months after implantation in a weight-bearing region of the knee, the WO 2006/022671 PCT/US2004/024902 66 porcine-NeoCart" has produced dense hyaline-like cartilage and has integrated with the host cartilage laterally and at the interface of the subchondral bone. These results confirm that the damaged, injured, 5 diseased or aged cartilage may be repaired by using neo cartilage implants preparing according to the algorithm of the invention. V. Human Osteoarthritic Cartilage Articular cartilage is a unique tissue with no 10 vascular, nerve, or lymphatic supply. The lack of vascular and lymphatic circulation may be one of the reasons why articular cartilage has such a poor intrinsic capacity to heal, except for formation of fibrous or fibrocartilaginous tissue. Unique mechanical functions of articular cartilage 15 are never reestablished spontaneously after a significant injury or disease, such as osteoarthritis (OA). In osteoarthritis, disruption of the structural integrity of the matrix by the degeneration of individual matrix proteins leads to reduced mechanical properties and 20 impaired function. Currently, the only available treatment of severe osteoarthritis of the knee is a total knee replacement in elderly patients. In young and middle aged patients, however, there is no optimal treatment. 25 In order to evaluate suitability of the current invention for treatment of osteoarthritis, studies using algorithm of the invention including a TESS culture system using neo-cartilage scaffold construct and algorithm of the invention (hydrostatic pressure and medium perfusion) on 30 human OA chondrocytes, cell proliferation and extracellular matrix accumulation in OA chondrocytes was investigated. Results are seen in Table 5 and in Figures 13-15.

WO 2006/022671 PCT/US2004/024902 67 Table 5 Group Total S-GAG Production DNA content (n=7) days (pg/cell construct) (pg/cell Pressure Conditions In culture (Mean ± SD) construct) (Mean ± SD) 51 1 In TESS In Incubator Iype ot Pressure Time Initial ~0 day 0 day 19.23 ±0.87 1.88 ±0.40 Control ~ ~ 21 days 21 days 23.81 ± 2.61 2.34 ± 0,32 0.5 MPa Cy-HP#1 . 7 days 14 days 21 days *29.53 ± 1/60 2.33 ± 0.12 Cyclic -. 3 MPa 10 Cy-HP#2 14 days 7 days 21 days *34.39 ±0.99 2.35 ±0.09 Cyclic 0.5 MPa Const-HP Consta 7 days 14 days 21 days 26.94 ±5.14 **2.65± 0.28 Constant I (*: p<0.05, compared to Control in S-GAG production) (**: p<0.05, compared to Initial in DNA content) In the TESS processor, the medium flow rate was 0.005 15 ml/min and the hydrostatic pressure as indicated. Two cell matrices from each group were harvested for histological analysis. As seen in Table 5 and Figure 13A, S-GAG production in cell constructs subjected to cyclic hydrostatic pressure 20 with medium perfusion was significantly greater than those subjected to atmospheric pressure (control). Especially, S-GAG production (ig/cell construct) was significantly increased (144%) for Cy-HP#2 where the cyclic hydrostatic pressure was used for 14 days followed by 7 days of static 25 atmospheric pressure compared to control. Figure 13B shows DNA content index with corresponding results presented in Table 5 for DNA, likewise showing increased production of DNA. Increase in DNA content index in cell constructs using the neo-cartilage construct 30 subjected to constant hydrostatic pressure was clearly shown in a comparison to initial level. DNA level in cell constructs subjected to constant hydrostatic pressure with WO 2006/022671 PCT/US2004/024902 68 medium perfusion was significantly increased to 142% compared to initial DNA level index. , Figured 14A-14E show histological evaluation of cell constructs by Safranin-O. Figure 14A shows S-GAG 5 accumulation at day 0 (initial). Figure 14B shows accumulation of S-GAG on day 21 in cell constructs subjected to atmospheric pressure (control). Figure 14C shows accumulation of S-GAG on day 21 in cell constructs subjected to 7 days of cyclic hydrostatic pressure (Cy 10 HP#1) followed by 14 days of to atmospheric pressure. Figure 14D shows accumulation of S-GAG on day 21 in cell constructs subjected to 14 days of cyclic hydrostatic pressure (Cy-HP#2) followed by 7 days of to atmospheric pressure. Figure 14E shows accumulation of S-GAG on day 21 15 in cell constructs subjected to 7 days of constant hydrostatic pressure (Constant-HP) followed by 14 days of atmospheric pressure. The greater S-GAG accumulation in both cell constructs subjected to cyclic hydrostatic pressure (7 and 14 days) is evident from the increased 20 density of the photomicrograph clearly visible in the Figure 14C and 14D. These results demonstrate that hydrostatic pressure with medium perfusion promotes both cell proliferation and neo-cartilage phenotypic activity, that is, cartilage 25 extracellular matrix production, in the scaffold neo cartilage constructs seeded with human OA chondrocytes. This evidence indicates that the algorithm of invention using TESS culture system and hydrostatic pressure combined with medium perfusion regenerates human OA chondrocytes and 30 transforms the OA cartilage into the healthy hyaline cartilage. VI. Method for Treatment of Cartilage Lesions The method for treatment of damaged, injured, diseased or aged cartilage according to the invention is 35 suitable for healing of small lesion due to acute injury as well as healing of the large lesions caused by WO 2006/022671 PCT/US2004/024902 69 osteoarthritis or other joint degenerative diseases and/or transforming the diseased OA cartilage into the healthy hyaline cartilage. The method generally encompasses five novel features, 5 namely, employing a biologically acceptable thermo reversible polymer gel as a carrier support matrix for neo cartilage generated from autologous chondrocytes, producing the autologous neo-cartilage by a process of the invention, employing a biologically acceptable thermo-reversible gel 10 as a space-holding means for the interim period when the autologous neo-cartilage is produced, depositing one or two adhesive sealants to the lesion and, following depositing the sealants and implantation of the neo-cartilage within a cavity generated thereby, a formation of the superficial 15 cartilage layer covering the lesion and protecting the integrity of the neo-cartilage deposited therein. The method generally comprises steps: a) debriding an articular cartilage lesion and during the debriding harvesting a small quantity (50-4000 mg) of 20 non-osteoarthritic hyaline cartilage; b) fabrication and processing of the neo-cartilage construct according to the above described procedures; c) preparing the lesion for implantation of the neo cartilage construct by depositing the one or two sealant 25 layers, the first (optional) at the bottom of the lesion and the second one over and on the top of the lesion, and, using all variation already described above, depositing either the neo-cartilage construct within the cavity formed below the top seal and/or between the two sealant layers or 30 depositing the space holding thermo-reversible polymer gel into the cavity between the two layers to uphold the integrity of the cavity in the interim when the neo cartilage construct is being prepared; d) implanting the neo-cartilage construct into said 35 cavity formed between the two sealant layers to allow for integration of the neo-cartilage into the surrounding WO 2006/022671 PCT/US2004/024902 70 native intact cartilage and formation of the superficial cartilage layer; and e) optionally removing the space holding polymer gel from the cavity before the neo-cartilage implantation. 5 In the alternative method for treatment, expanded and differentiated chondrocytes may be deposited directly into a joint lesion in a suitable typically thermo-reversible gelation hydrogel solution. There are several advantages of the current method. 10 First, the method is very versatile and any of the variations may be advantageously utilized for treatment of a specific injury, damage, aging or disease. The method permits generation of autologous neo cartilage by providing alternative means for maintaining a 15 space between two sealant layers until the autologous neo cartilage is prepared. The method permits generation of more dense neo-cartilage and three-dimensional expansion of chondrocytes and extracellular matrix. The deposition of the second top sealant layer 20 resulting in formation of superficial cartilage layer constitutes a substitute for synovial membrane and provides the outer surface of healthy articular cartilage overgrowing, protecting, containing and providing critical metabolic factors aiding in growth and incorporation of 25 autologous neo-cartilage in the lesion. Deposition of the first bottom sealant layer protects the integrity of the lesion after cleaning during surgery and prevents migration of subchondral and synovial cells and cell products thereby creating milieu for formation of 30 healthy hyaline cartilage from the neo-cartilage and also preventing formation of the fibrocartilage. The method further permits deposition of the space holding gel or thermo-reversible polymer gel to be deposited whether alone or with suspended processed neo 35 cartilage into the lesion at temperature between 5 and 250C as a sol. Selection of thermo-reversible gel may be WO 2006/022671 PCT/US2004/024902 71 crucial as certain TRGH may function as a promoter for growth of the superficial. cartilage layer without a need to apply the second sealant. The method further permits said thermo-reversible 5 hydrogel be enhanced with hyaluronic acid, typically added in about 5 to about 50%, preferably about 20% (v/v), wherein such hyaluronic acid acts as an enhancer of the matrix-forming characteristics of the gel and to act as a hydration factor in the synovial space in general and 10 within the lesion cavity in particular. Additionally, the gel acts as a slow-release unit for hyaluronic acid, greatly increasing a period of hydration within the cavity and also as a substrate for formation of the superficial cartilage layer and it can 15 also be conveniently removed, if needs be, by cooling the lesion so that the solid gel formed at 37 0 C is converted to sol and can be removed by injection or otherwise. For treatment of the cartilage, a subject is treated, according to this invention, with a prepared autologous or 20 heterologous neo-cartilage or neo-cartilage construct implanted into the lesion, the neo-cartilage or the construct is left in the lesion for two-three months and typically, it does not need any further intervention as during these three months, the neo-cartilage is fully 25 integrated into the native cartilage and becomes a fully functional cartilage covered with a superficial cartilage layer which eventually grows into or provides the same type of surface as a synovial membrane of the intact joint. Finally, the diseased, osteoarthritic cartilage may 30 be fully replaced by the regenerated hyaline-like cartilage when processed according to the algorithm of this invention. The algorithm and/or implantation protocol may assume any variation described above or possible within the realm 35 of this invention. It is thus intended that every and all variations in the treatment protocol (algorithm of the WO 2006/022671 PCT/US2004/024902 72 cartilage) are within the scope of the current invention. EXAMPLE 1 Isolation of Chondrocytes from Source Tissue This example describes the procedure used for 5 isolation of chondrocytes from swine cartilage. Chondrocytes were enzymatically isolated from cartilage harvested under sterile conditions from the hind limbs of 6-month old swine. The femur was detached from the tibia and the trachea head exposed. Strips of 10 cartilage were removed from the trachea using a surgical blade. The cartilage was minced, digested in a 0.15% collagenase type I solution in DMEM/Nutrient Mixture F-12 (DMEM/F-12) 1:1 mixture with 1% penicillin-streptomycin 15 (P/S) and gently rotated for 18 hours at 37 0 C. Chondrocytes were collected and rinsed twice by centrifugation at 1500 rpm for 5 min. Chondrocytes were re-suspended in DMEM/F-12 containing 1% penicillin streptomysin and 10% FBS. 20 Chondrocytes were expanded for about 5 days at 37 0 C. EXAMPLE 2 The Production of Human Neo-Cartilage Construct This example describes conditions for production of neo-cartilage for human use. 25 The patient undergoes arthroscopic biopsy of a small (200-500 mg) piece of healthy cartilage from the ipsilateral knee. The biopsy is taken from the non-weight bearing portion of the femoral condyle or from the femoral notch as deemed most appropriate for the patient. The 30 biopsy sample is placed into a sterile, non-cytotoxic, non pyrogenic specimen container which is packaged and shipped to the laboratory. At the laboratory the biopsy sample is examined against acceptance criteria and then transferred to the 35 chondrocyte isolation and expansion area. Samples from the biopsy specimen transport buffer are tested for sterility WO 2006/022671 PCT/US2004/024902 73 and for mycoplasma. The expanded chondrocytes are suspended in VITROGEN@ gellable collagen solution, commercially available from Cohesion Corp., Palo Alto, CA. A pre-formed collagen sponge (22x22 mm square and 2-4 mm in 5 thickness, wherein the thickness depends on the thickness of patient's cartilage), commercially available from Koken Co., Japan or honeycomb matrix produced according to this invention is placed into the resulting chondrocyte suspension which absorbs the chondrocyte/collagen 10 suspension into this matrix. The resulting chondrocyte-loaded matrix is warmed to 37 0 C to gel the VITROGEN in order to spatially secure the chondrocytes within the support matrix. The loaded support matrix is then placed into Tissue Engineering Support 15 System (TESS'

M

) culture unit. Typical time for cell expansion from removal of a biopsy sample to placement of the chondrocyte loaded culture matrix in the TESS m culture unit is 10-40 days. Within the TESS m culture unit, cyclic or constant hydrostatic pressure is used to induce the 20 chondrocytes to begin growing and expressing their cartilage generating program for about 1 hour to about 30 days. The still developing new cartilage is transferred to a constant, resting culture phase. The neo-cartilage 25 production process requires a minimum time of 10 days in resting culture. After this minimum 10-day period the neo cartilage, hereinafter called neo-cartilage construct, undergoes final inspections and is packaged for return to the clinic to be implanted. At the time of release, tests 30 for sterility, endotoxin, and mycoplasma contamination must be negative for microbial and mycoplasma contamination and must show <0.5 EU/ml of endotoxin. EXAMPLE 3 Preparation of Support Matrices 35 This example illustrates preparation of the cellular support matrix, also called the TESS matrix.

WO 2006/022671 PCT/US2004/024902 74 300 grams of a 1% aqueous atelocollagen solution (VITROGEN*), maintained at pH 3.0, is poured into a 10 x 20 cm tray. This tray is then placed in a 5 liter container. A 50 ml open container containing 30 ml of a 3% aqueous 5 ammonia solution is then placed next to the tray, in the 5 liter chamber, containing 300 grams of said 1% aqueous solution of atelocollagen. The 5 liter container containing the open trays of atelocollagen and ammonia is then sealed and left to stand at room temperature for 12 10 hours. During this period the ammonia gas, released from the open container of aqueous ammonia and confined within the sealed 5 liter container, is reacted with the aqueous atelocollagen resulting in gelling said aqueous solution of atelocollagen. 15 The collagenous gel is then washed with water overnight and, subsequently, freeze-dried to yield a sponge like matrix. This freeze dried matrix is then cut into squares, sterilized, and stored under a sterile wrap. Alternatively, the support matrix may be prepared as 20 follows. A porous collagen matrix, having a thickness of about 4 mm to 10 mm, is hydrated using a humidity-controlled chamber, with a relative humidity of 80% at 25 0 C, for 60 minutes. The collagen material is compressed between two 25 Teflon sheets to a thickness of less than 0.2 mm. The compressed material is then cross-linked in a solution of 0.5% formaldehyde, 1% sodium bicarbonate at pH 8 for 60 minutes. The cross-linked membrane is then rinsed thoroughly with water, and freeze-dried for about 48 hours. 30 The dense collagen barrier has an inner construction of densely packed fibers that are intertwined into a multi layer structure. In alternative, the integration layer is prepared from collagen-based dispersions or solutions that are air 35 dried into sheet form. Drying is performed at temperatures ranging from approximately 4 to 40 0 C for a period of time WO 2006/022671 PCT/US2004/024902 75 of about 7 to 48 hours. EXAMPLE 4 Seeding Cells in the TESS Matrix This example describes procedures used for seeding 5 cells in the TESS matrix. Isolated chondrocytes were incubated for a period of five days at 37 0 C in a standard incubator. Cells were then collected by trypsinization. A cell suspension of 150,000 cells in 18 Il of 10 VITROGEN solution was seeded per matrix having an approximate volume of 19 pl, with nine matrices per group. The seeded matrix (collagen sponge 4mm in diameter and 1.5 mm in thickness) may be scaled-up to an increased volume, where approximately 1 pl of the above described cell 15 suspension is seeded in 1 pl of matrix. The control group matrices were incubated in a 37 0 C incubator and the test group was incubated in the TESS. In alternative set-up, isolated chondrocytes were incubated for a period of five days at 37 0 C in a standard 20 incubator. Cells were then collected by trypsinization. A cell suspension of 300,000 cells in 18 p of VITROGEN solution was seeded per matrix having an approximate volume of 19 pl with eight matrices per group. EXAMPLE 5 25 Effect of Cyclic Hydrostatic Pressure This example describes procedures used for determination of effect of cyclic hydrostatic pressure in vitro formation of chondrocyte-seeded support matrices. Swine articular chondrocytes (sACs) were 30 enzymatically isolated from cartilage with type I collagenase. The cells were suspended in collagen (VITROGEN) as described above and wicked into the honeycombed sponge element of the cellular support matrix. The cells seeded in the support matrix were incubated at 35 37 0 C, 5% CO 2 and 20% 02. After 24 hours, some of these cells matrices were transferred to the TESSTm processor and WO 2006/022671 PCT/US2004/024902 76 incubated at 0.5 or 3.0 MPa cyclic or constant hydrostatic pressure with medium perfusion (0.05 ml/min) as described above for 6 or 7 days followed by a 12 or 14 day resting phase. The control group comprised of chondrocytes seeded 5 in matrices incubated for 18 or 21 days at atmospheric pressure, at 37 0 C, 5% CO 2 and 20% or 2% 02. At the end of the culture period (18 or 21 days), the matrices were harvested for biochemical and histological analysis. For biochemical analysis, sulfated 10 glycosaminoglycan (S-GAG) production was measured using a modified dimethylmethylene blue (DMB) microassay. Two matrices from each group were harvested for histological analysis. EXAMPLE 6 15 Effect of Medium Flow Rate on Extracellular Matrix Accumulation of Chondrocytes in Collagen Sponges This example described conditions used to determine effect of medium flow on production and accumulation of extracellular matrix by chondrocytes seeded into collagen 20 sponges. Chondrocyte Isolation Swine legs were obtained from a local abattoir. Within 4-6 hours after slaughter, cartilage was harvested under sterile conditions from the trochlea of the hind 25 limbs. The cartilage was minced and digested in 0.15% collagenase type I in DMEM/F-12 containing 1% penicillin streptomycin (P/S) for 18 'hours at 37 0 C. Isolated swine articulate chondrocytes (sACs) were collected, rinsed, and resuspended in DMEM/F-12 supplemented with 10% fetal bovine 30 serum (FBS) and 1% P/S. sACs then were expanded for 5 days at 37 0 C. Cell Seeding in Collagen Sponges sACs were harvested with Trypsin EDTA and cell viability was measured by trypan-blue exclusion. Three 35 hundred thousand sACs were suspended in 30 pul of a neutralized 0.25% collagen solution (VITROGEN*, Cohesion WO 2006/022671 PCT/US2004/024902 77 Corp., Palo Alto, CA) , and the suspension was absorbed into a collagen sponge, 4 mm in diameter and 2 mm in thickness, commercially available from Koken Co., Japan. Seeded sponges were pre-incubated for 1 hour at 37 0 C to gel the 5 collagen, followed by incubation in culture medium at 37 0 C in 5% CO 2 . Tissue Engineering Support System (TESS' m ) Culture Following the incubation, the seeded sponges were transferred to and cultured in the Tissue Engineering 10 Support System (TESS'

M

) processor. To evaluate the effect of medium perfusion rate, sponges were subjected to medium perfusion at 5 pl/min or 50 pl/min. Cyclic hydrostatic pressure (Cy-HP) 0 - 0.5 MPA pressure at 0.5 Hz applied was for 6 days. Some sponges were incubated under constant 15 conditions at atmospheric pressure and no perfusion at 37 0 C for a total of 18 days in culture. Sponges harvested 24 hours after seeding with cells (day 0) served as an initial control. Histological and Biochemical Analysis 20 Cell constructs were harvested after 6 and 18 days of culture. For histological evaluation, 4% paraformaldehyde fixed, paraffin sections were stained with Safranin-O (Saf 0) and Type II collagen antibody. 25 For biochemical analysis, seeded sponges were digested in papain at 60 0 C for 18 hours and DNA content was measured using the Hoechst 33258 dye method. Sulfated glycosaminoglycan (S-GAG) accumulation was measured using a modified dimethylmethylene blue (DMB) microassay. 30 EXAMPLE 7 Biochemical and Histological Assays This example describes assays used for biochemical and histological studies (DMB assay). Biochemical (DMB) Assay 35 At the end of the culture six matrices from each group were used in the biochemistry assay.

WO 2006/022671 PCT/US2004/024902 78 The matrices were transferred to microcentrifuge tubes and digested in 300 pl of papain (125 pg/ml in 0.1 M sodium phosphate, 5 mM disodium EDTA, and 5 mM L-cysteine HCl) for 18 hours at 60 0 C. GAG production in the matrices 5 was measured using a modified dimethylene blue (DMB) microassay with shark chondroitin sulfate as a control Connective Tissue Research, 9: 247-248 (1982). DNA content was determined by Hoechst 33258 dye method according to Anal. Biochem., 174:168-176 (1988). 10 Histological Assay The remaining matrices from each group were fixed in 4% paraformaldehyde. The matrices were processed and embedded in paraffin. 10 pm sections were cut on a microtome and stained with Safranin-O (Saf 0). 15 EXAMPLE 8 Evaluation of Porcine Neo-Cartilage Integration in a Swine Model This example describe the procedure and results of study performed for evaluation of integration of porcine 20 neo-cartilage in a swine model. An open arthrotomy of the right knee joint was performed on all animals, and a biopsy of the cartilage was obtained. Chondrocytes were isolated from the cartilage biopsy 25 and cultured within a collagen matrix in a Tissue Engineering Support System (TESS") to produce porcine Neocart for subsequent implantation. A defect was created in the medial femoral condyle of the pig's right knee. This defect (control) was not 30 implanted with porcine-NeoCart' m . Following surgery, the joint was immobilized with an external fixation construct for a period of about two weeks. Three weeks after the arthrotomy on the right knee was performed, an open arthrotomy was performed on the left knee and defects were 35 created in this medial femoral condyle. The porcine NeoCart m was implanted within the defect(s) in this knee WO 2006/022671 PCT/US2004/024902 79 which was similarly immobilized. The operated sites were subsequently viewed via arthroscopy two weeks after implantation or defect creation and thereafter at monthly intervals. Animals were euthanized and the joints 5 harvested and prepared for histological examination approximately 3 months after porcine-NeoCart m implantation. The implanted sites were prepared and examined histologically. Results are seen in Figures 10-12. Figure 10 shows 10 results of the arthroscopic examination. The empty defect is seen in Figure 10A. The porcine NeoCart T implant site is seen in Figure 10B which also shows still-evident absorbable sutures and the superficial cartilage layer growing over the porcine NeoCart' m . 15 EXAMPLE 9 Protocol for In Vivo, Ex Vivo or In Vitro Growth of Porcine Neo-Cartilage Autologous porcine chondrocytes are seeded into the cellular support matrix and incubated under cyclic 20 hydrostatic pressure at 37 0 C and 5% CO 2 . Cyclic hydrostatic pressure is either 0.5 or 3.0 MPa at 0.5 Hz. The duration of said cyclic pressure is approximately 6 days followed by a resting phase of 12 days in an incubator maintained at 37 0 C at atmospheric pressure. At the end of 25 this resting phase, the matrices were harvested for biochemical and histological analysis. In the alternative protocol, the algorithm for the growth cells of in vivo and in vitro, the application of hydrostatic pressure is used on isolated in situ cartilage, 30 or application of hydrostatic pressure for about 1-8 hours followed by about 16-23 hours of recovery period. EXAMPLE 10 Regeneration of Human Chondrocytes This example describes the procedure used for 35 regeneration of human chondrocytes. Chondrocytes from osteoarthritic (OA) patients (40 WO 2006/022671 PCT/US2004/024902 80 years old) were expanded for 18 days in monolayer culture at 37 0 C and suspended in VITROGEN@ (300,000 cells/30 f~l). The cell suspension was absorbed into a support matrix, usually a collagen honeycomb sponge (4 mm in diameter and 5 2 mm in thickness, Koken Co. , Japan) . The cell constructs were incubated in culture medium supplemented with 10% FBS and 1% ITS (Insulin-transferrin-sodium selenite, Sigma) at 37 0 C, 5% CO 2 and 20% 02, at 0.5 MPa cyclic hydrostatic pressure (Cy-HP) or 0.5 MPa constant hydrostatic pressure 10 (Constant-HP) for 7 or 14 days in the TESS M processor followed by incubation for 7 or 14 days at atmospheric pressure for 7 or 14 days in an CO 2 incubator at 37 0 C. The remaining cell constructs compromising the control group were incubated atmospheric pressure for 21 days at 37 0 C, 5% 15 CO 2 and 20% 02 Before starting the culture, some cell constructs were harvested for biochemical and histological analysis as an initial condition. At the end of the culture period, the cell constructs were harvested for biochemical and 20 histological analysis. Sulfated glycosaminoglycan production was measured using a modified dimethylmethylene blue (DMB) micro assay. Cell proliferation was measured using a modified Hoechst Dye DNA assay. Formation of neo tissue was analyzed by Safranin-O staining. 25

Claims

1. A method for treatment of cartilage lesions and defects comprising steps: a) isolating autologous or heterologous chondrocytes or providing stem s cells that can be differentiated into chondrocytes; b) expanding said isolated chondrocytes or stem cells in a growth medium; c) suspending said expanded chondrocytes or stem cells in a collagen-containing solution; d) providing biodegradable support matrix containing plurality of pores; 10 e) incorporating the suspension obtained in step c) into said support matrix thereby producing a seeded support matrix; f) preparing an implantable construct for implantation into said cartilage lesion by activating said chondrocytes or stem cells by subjecting said seeded support matrix to conditions promoting activation and propagation of said chondrocytes or stem 15 cells within said support matrix, wherein said conditions for activation and propagation of chondrocytes comprise perfusing said seeded support matrix with a culture medium at a flow rate from about I pd/min to about 500 p1/min and during said perfusing, applying to said seeded support matrix a cyclic hydrostatic 20 pressure from about 0.01 MPa to about 10 MPa above atmospheric pressure at about 0.01 to about 1 Hz for from about 1 to about 8 hours followed by applying a static atmospheric pressure for from 16 to about 23 hours; g) implanting said construct into said cartilage lesion; and h) depositing a biodegradable polyethylene glycol cross-linked with 25 methylated collagen over said construct, wherein said implanting of said construct into said cartilage lesion and said deposition of said polyethylene glycol cross-linked with methylated collagen over said implanted construct results, within three months, in a growth and maturing of said chondrocytes or stem cells and in integration of said construct seeded with said 30 chondrocytes or stem cells into a native cartilage of the lesion and in formation of a superficial cartilage layer that overgrows said construct implanted within said lesion, wherein said superficial cartilage layer is an outermost layer of cartilage that forms the layer of squamous-like flattened superficial zone chondrocytes covering a layer 82 of said polyethylene glycol cross-linked with methylated collagen and overgrowing the lesion, and wherein said biodegradable support matrix and said polyethylene glycol cross-linked with methylated collagen are both degraded within said three months. s

2. The method according to claim I further comprising step i) wherein a tissue sealant is deposited into said cartilage lesion before said construct is implanted therein, wherein said sealant is selected from the group consisting of gelatine, a copolymer of polyethylene glycol and poly-lactide or poly-glycolide, periodate-oxidized to gelatine, 4-armed pentaerythritol thiol and a polyethylene glycol diacrylate, 4-armed tetra-succinimidyl ester or tetra-thiol derivatized PEG, photo-polymerizable polyethylene glycol-co-poly (a-hydroxy acid) diacrylate macromer, 4-armed polyethylene glycol derivatized with succinimidyl ester and thiol further cross-linked with methylated collagen, derivatized polyethylene glycol, polyethylene glycol (PEG) cross-linked with 15 alkylated collagen, tetra-hydrosuccinimidyl or tetra-thiol derivatized PEG, PEG cross-linked with methylated collagen, and a combination thereof.

3. The method according to claim I or 2 wherein said support matrix is in the form of a sponge, honeycomb or lattice prepared from a compound selected from the group consisting of a Type I collagen, Type II collagen, Type IV collagen, gelatine, 20 agarose, collagen containing proteoglycan, collagen containing glycosaminoglycan and collagen containing glycoprotein.

4. The method according to any one of claims 1 to 3 wherein said support matrix is prepared from Type I collagen.

5. The method according to any one of claims I to 4 wherein said 25 hydrostatic cyclic pressure is applied from about 0.05 MPa to about 3 MPa at 0.1 to about 0.5 Hz.

6. The method according to any one of claims I to 5 wherein said perfusion flow rate is from about 5pL/min to about 50ptUmin.

7. The method according to claim 6 wherein said perfusion flow rate is 30 about 5ptUmin. 83

8. The method according to any one of claims 1 to 7 wherein cell activation is performed under oxygen concentration from about I % to about 20%.

9. The method according to claim 8 wherein cell activation is performed under oxygen concentration from about 2% to about 5%. 5

10. The method according to any one of claims 1 to 9 wherein said formation of superficial cartilage layer constitutes a substitute synovial membrane.

11. A method for treatment of cartilage lesions and defects substantially as hereinbefore described with reference to any one of the examples. Dated 3 September, 2008 10 Histogenics Corporation Patent Attorneys for the Applicant/Nominated Person SPRUSON & FERGUSON