AU2015202319A1 - Method and compositions for repair of cartilage using an in vivo bioreactor. - Google Patents

Abstract

Methods and compositions for the biological repair of cartilage using a hybrid construct combining both an inert structure and living core are described. The inert structure is intended to act not only as a delivery system to feed and grow a living core 5 component, but also as an inducer of cell differentiation. The inert structure comprises concentric internal and external and inflatable/expandable balloon-like bio-polymers. The living core comprises the cell-matrix construct comprised of HDFs, for example, seeded in a scaffold. The method comprises surgically removing a damaged cartilage from a patient and inserting the hybrid construct into the cavity generated after the 10 foregoing surgical intervention. The balloons of the inert structure are successively inflated within the target area, such as a joint, for example. Also disclosed herein are methods for growing and differentiating human fibroblasts into chondrocyte-like cells via mechanical strain. Neural Foramen Spinal Nerve Annulus lntervertebral Disc

Description

I AUSTRALIA Patents Act 1990 (Cth) COMPLETE SPECIFICATION APPLICANT SPINALCYTE, LLC TITLE METHODS AND COMPOSITIONS FOR REPAIR OF CARTILAGE USING AN IN VIVO BIORE ACTOR 2 METHODS AND COMPOSITIONS FOR REPAIR OF CARTILAGE USING AN IN VIVO BIOREACTOR CROSS-REFERENCE TO RELATED APPLICATIONS 5 The present application is a divisional application of Australian Patent Application No. 2013257540 which claims priority to United States Provisional Application Serial No. 60/771,172, entitled Method for Repairing an Intervertebral Disc filed on Febmary 7, 2006 and United States Provisional Application Serial No. 10 60/785,478, entitled MultiLayered Multi-Compartmental Three-Dimensional Po lymberic Scaffold For Intervertebral Disc Repair filed on March 24, 2006, the contents of all of which are incorporated by reference herein in their entirety. FIELD OF THE INVENTION 15 The present invention generally concerns at leas the fields of medicine, surgery, anatomy, biology, cell biology, and/or molecular biology. In particular aspects the pre sent invention concerns the fields of cartilage repair, such as articular cartilage repair. More particularly the field of the invention concerns cell-matrix 20 encapsulation devices for growing, proliferating, and/or differentiating cells into chondrocyte-like cells under mechanical stress. BACKGROUND OF THE INVENTION 25 Typically, articular cartilage is a tissue that is not naturally regenerated once damaged. Recently, efforts have been made to reconstruct damaged biological tissues by regenerating a portion of the damaged tissues in laboratories, This approach, defined as "tissue engineering" has raised tremendous attention. Tissue engineering involves the development of biocompatible materials 30 capable of specifically interacting with biological tissues to produce functional tissue equivalents. Tissue engineering has a basic concept of collecting a desired tissue from a patient, isolating cells from the tissue specimen, proliferating cells, seeding the 3 proliferated cells onto a biodegradable polymeric scaffold, culturing the cells for a predetermined period in vitro, and transplanting hack the cell/polymer construct into the patient After transplantatio the cell in e transplanted scaffold use oxygen and nutrients gained by diffusion of body fluids to proliferate and differentiate to form a ) new tissue, whereas the scaffold has been dis solved, The scaffold used for the regeneration of biological tissue is usually comprised of a material that serves as matrix to allow cells to attach to the surface of the material and form a the dn ensiona tissue. This material should be non-toxic, biocompatible and biodegradable. The most wdely used biodegradable polymers, 10 sati saying the aforementioned physical requirements, include organic polymers such as polyglycolic acid (PGA), polylactiacglycolic cid (PLGA), poly-s-caprolactone (PCL), polyamino acids, polyanhydrides. polyorthoesters; natural hydrogels such as collagen, hyaluronic acid, alginate, agarose, chitosan; synthetic hydrogels such as poly(ethylene oxide) (PE) polyvinyl alcohol) (PVA), poly(acrylic acid) (PAA), 15 poly(propylene fumarate-co-thylene glycol) [P(PF-co-EG) and copolymers thereof. The aforementione polymers have been researched to fabricate porous scaffold. Howev'er conventional fabrication techniques generally result in scaffolds with low porosities tha do not adequately support cell growth. The pores on the surface of the scaffold are often blocked, nutients are not sufficiently supplied to the 20 cells, and cells have difficulties in growing into the scaffold, Recently, the application of micro-fabrication technology in the field of tissue engineering has rendered possible the development of complex seaffol with micron-scale resolution. These scaffolds referred to as "microfluidc scaffolds" present a network of m icro-channels that allow fluid flov within the scaffold This network ofmicro-hannels helps to 25 provide both nutrients and oluble factors to distinct sections of the scaffold. The scaffold can also be encapsulated with a semipermeable membrane. U.S. Patent Public ation No. 2006/0147486 re ates to a porous scaffold enveloped with a semi-penneable membrane. Th is semi-permeable membrane selectively in trod uces nutrients into the scaffold from outside the scaffold as well exereting metabole 30 wastes generated by the tissue cells to the outside of the afford. The publication 4 describes the method to row cells witn this saffbld In vitro fr regenerating a biological tissue. U.S. Pat. No. 6,27422 describes a de ce containing cells in a yarn matrix encapsulated in a semi-perneable membrane. In this case, the seni-permeable 5 membrane al lows implanted cells to receive nutrients but also allows therapeutic molecules produced by the implanted cells to diffuse to host cells. [his device is used for cell therapy: the encapsulated cells secrete endogenous proteins to the host. This device functions as a bioartificial organ (for example, as artificial pancreas by secreting insulin). 10 Despite such progress in the engineering of scaffolds with improved difbsion of nutrients, the scaffold once transplanted to the patient suffers from a limited supply of nutrients, Indeed. in vivo, nutrients and oxygen are delivered to disc cells through blood vessels in the endplates of the vertebrae adjacent to the disc. In degenerative disc disease, the vertebral endplates of vertebrae are not well-functioning P5 and do not allow sufficient diffusion of nutrition to the implanted cell-scaffold, Different cell types can be used to engineer articular cartilage, Primary differentiated cells of articular cartilage (i.e. chondrocytes) from biopsies of existing cartilage can be used. These cells are often procured from an autologous source since the procurement of heterologous cells or cells from cadavers carries the inherent risk 20 of transfer of pathogens. Mesenchymal Stem Cells (MSCs), which are embiyonic-like cells found in bone marrow, are capable of differentiation into different type of mesenchymal tissues and especially cartilaginous tissue; therefore, they are another cell source for cartilage engineering. However, these cell sourcing raise many issues. Chondrocytes from 25 intervertebral disc are difficult to harvest, because the autologous cells are obtained from the patient's disc and therefore it requires an invasive procedure (back surgery) to perform a biopsy. If cells are harvested from a healthy disc it jeopardizes the functioning of the healthy dise. If cells are harvested from a damaged ise during the discectomy, it provides abnormal cells from a degenerated tissue, Moreover, 30 chondrocytes are difficult to expand in culture since they de-differentiate. Regarding chondrocytes from other cartilages, the elastic cartilage front the ear is easy to harvest, 5 but it produces only hyaline cartilage and not fibro-cartilage, as in the disc. MSCs also have some disadvantages, because they require a bone marrow biopsy. While a large quantity of cells is needed for tissue engineering, it is difficult to obtain a large quantity of adult stem cells. 5 Numerous papers have reported the culture conditions that stimulate chondrogenesis of mesenchymal stem cells or de-differentiat chondrocytes, These condition are the fol owing: high density micromass culture; hypoxia; supplementation with growth factors, such a Bone Morhogenetic Proteins (BMP) particularly BMP~24,6, and -7, transforming growth factor beta (TGF-p), and/or 10 insulin growth factor one (JGF4); supplementation with ascorbic acid; onlture on specific matrix such as alginate; culture under mechanical stress such as Intennittent Hydrostatic Pressure (IHP) (Watt, 1988; Dozin et aL, 1992; Sullivan et at, 1994; Denker e at, 1999; Zur Nieden et aL 2005; Zhou et al., 2004; Majumdar et al., 2001; Barry et a,:.2001? Elder et aL, 2005; Mow et at, 1992; Domr et al., 2000). 15 Few studies have reported the conversion of Humnan Dermal Fibroblasts (HDs) into chondroc elike cels. U.S. Patent No. 6, 4& 165 concerns the conversion of HDFs into chondrocyte-like cells under high density micromass culture and hypoxia. French MM et A. (2004) reported the conversion of HDFs into chondrocyte when the cells are grown on the proteoglycan, aggrecan, and 20 supplemented with insulin growth factor one (IGF-). Degenerative Disc Disease Degenerative Disc Disease (DDD) requires 700,000 procedures each year 25 performed by 4,500 spine surgeons, and the majority of disc disorders occur in young patients. Therefore, it is critical to develop effective and saf strategic. to treat this disease. An intervertebral disc (IVD) is a complex structure comprising three distinctive tissues: the annulus, the nucleus, and cartilage endplates. The annulusis a 30 well-organized, mti-layered structure of collagen fibers. The nucleus is comprised mainly of glycosaminoglycan (hydrophilic polymer). The cartilage endplates supply 6 nutrients. The foregoing combination allows the normal disc to perform two conflicting functions: stability and flexibility, The intervertebral disc absorbs shocks, maintains motion, and keeps stability. Similar to other cartilages, the innate repair capacity of the intervertebral 5 disc (which acts as ajoint between two vertebra) is low, because it is vascular and nutritionally supported only by passive diffusion at the endplates. Consequently, once the degenerative process is activated, it is ultimately considered to be an irreversible condition, Once damaged, the degenerated disc may bulge or extrude, and therefore needs to be removed. Currently, the common surgical treatment for patients with chronic low back pain due to degenerative disc disease is either discectomy or spinal fusion Discectomy is an appropriate procedure and is routinely performed to remove the degenerated nucleus through a fenestration within the annulus: it allows removal of both the extruded nucleus (hemniectmy) and the degenerated remaining inter-vertebral 5 nucleus fragments. Although this procedure is ideal for decompressing and relieving the nervous system (root or cauda equine) it is a poor operation for the spine, because it creates a potentially disabling condition that leads to a degenerative cascade that may require an additional invasive surgical procedue, like fusion or arthroplasty, for example. Diseectomy brings a good shor-temn effect in relieving radicular pain, but it 20 causes disc height reduction with neuroforamen stenosis, instability of the treated level, poor result on back pain, and/or complications, such as spinal stenosis or facet pain, for example. Spinal. fusion is the most effective treatment for low back pain, It is a surgical procedure in which an entire disc is removed and the two adjacent vertebrae 25 are united together ("fused") with the interposition of a graft (cages, bone grafts, and/or fixation devices, for example), it is indicated for patients with advanced disc degeneration. Over 200,000 spinal fusions are performed each year in the U.S. alone, but by eliminating the motion, the spinal fusion alters the bionechanical properties of the inter-vertebral disc and increases stress and strain on the discs that are adjacent to 30 the fused disc. In fact, both discectomy and fusion worsen the condition of the affected disc, adjacent discs, and surrounding issues (such as facet ints)leading to further degeneration. The failure of these procedures has led to a search for the development of non-fusion technologies, such as disc or disc nucleus prosthesis, for example. Disc 5 arthroplasty with an artifcial disc is an emerging treatment for patients with disc degeneration. Its advantages are to maintain motion, decrease incidence of adjacent segment degeneration, avoid complications related to fusion, and allow early return to function. Today, two kinds of devices are marketed: the total disc replacement and the nuclear replacement, but both of them have major pitfalls. Total disc replacement is a 10 bulky metallic prosthesis designed to replace the entire disc: annulus, nucleus and endplates. These prostheses use an invasive anterior (trans- or retro-peritoneal) approach that requires the presence of a vascular surgeon. Dislodgements, wear debris, degeneration of adjacent intervertebral discs, facet joint arthrosis, and subsidence of this type of prosthesis have been reported. The artificial nucleus 15 substitute preserves the remaining disc tissues and their functions. Its design allows its implantation through a posterior approach, but the major limitation of such nucleus prosthesis is that it can be used only in patients in whom disc degeneration is at an early or intermediate stage, because it requires the presence of a competent natural annulus. Implant extrusion remains a primary concern. As a hydrogel-based device, it 20 is fragile, and so does not resist the outstanding bio-mechanical constraints of the lumbar spine (shear forces). As inert materials, they may lose their mechanical properties over time, and tears and breakages have been reported. Replacing the nucleus only and leaving in place a damaged annulus generates the conditions for implant extrusion or recidivism of discal herniation, 25 Tissue engineering and regenerative medicine represent a new option for the treatment of DDD. A variety of approaches are used to regenerate tissues These approaches can be categorized into three groups: 1) biomaterials, without additional cells, that are used to send signals to attract cells and promote regeneration; 2) cells alone may be used, to form a tissue; and 3) cells may be used with a biomaterial 30 scaffold that acts as a frame for developing tissues. While Autologous Chondrocyte Transplantation (ACT) has been used for a few years to repair articular cartilage, 8 tissue engineering for isc repair remains in its infancy. Intensive research is currently done, and animal studies have shown the feasibility of tissue-engineered intervertebral disc. More interestingly, recent pilot clinical studies have shown that ACT is an efficient treatment of herniated disc. The main disadvantage of ACT for disc repair is 5 that it requires a disc biopsy. 'Therefore, there is a need for an improved method to restore disc anatomy and improve its functioning, and there thus remains a need for an improved method of cartilage repair. SUMMARY OF THE INWENTION i10 The present invention concerns methods and compositions for biological repair of any kind of cartilage, including intervertebral and joint cartilage, for example. More specially, but not exclusively, the present invention relates to methods and compositions for biological repair of cartlage using an implantable 15 device that is a combination fan inert structure acting as an in vivo bioreactor and a living structure comprised of chondrocytes or &hondrocytciike celIs, for example, such as cells derive ed from the exemplary Human Dennal Fbrobiasts (HD~s), in specific embodiments. More particularly, but not exclusively, the present invention relates to a hybrid construct combining both an iner structure and living core. 'The 20 inert sitrctre acts not only as a delivery system to feed and grow a living core component, but also ats as an inducer of el differentiation, in certain aspects, In embodiments of the invention, this inert structure comprises two expandable balloon like biopolymers, namely, an internal membrane (like a balloon) that is enclosed within an extemal membrane (also like a balloon) Hence, the inert structure 25 comprises two generally concentric inflatable membranes. The two membranes may be further defined as a first enclosed membrane that is structurally within a second enclosed membrane. In specific embodiments, the shapes may be considered to be genera y spherical, generally eliptical, generally rounded, generally orbed, generally discoid, generally spheroidIial, generally globule, balloon-like, and so forth. In 30 additional specific embodiments, the shape is individual-specific and confonns to the 9 shape and size of the remaining cavity in the joint or intervertebral disc region oft te individual, In certain aspects, the invention generates natural tissue in vitro, such as from stem cells, chondrocytes, and so forth. More particularly, but not exclusively, 5 the present invention relates to a method for growing and differentiating Human Fibroblasts into chondrocyte-like cells, for example The cells, which are autologous in certain embodiments, are put into a scaffold matrix made of one or more biopolymers, such as to mimic a natural matrix, The scaffold may be seeded in vitro, and in certain aspects growth factors are provided o the cells, the matrix, or both. The 10 Lscaffold is put into a bioreactor, vhich is a system for perfusion of medium and allows application of mechanical fore t the seaffold. Following delivery of the force, cells are assisted in differentiation, especially for generation of cartilage. In specific embodiment', the invention employs differentiaton of certain cells into chondrocyte-like ells. In specific embodiments, HDFs, fo example, are 15 differentiated into chondrocyte-like cells under particular culture conditionssuch as hypoxia (Nicoll et at, 2001), high density microass culture, and culture on specific matrix, such as aggrecan (French et at, 2004). In specific embodiments factors that mimic the in vivo environment of intervertebral chondrocytes are potent stimulii for chondrogenic differentiation of HDFs, for example; such factors include the 20 following: 1) three dimensionality; 2) low oxygen tension (<5%); and 3) mechanical stress; and 4) intermittent hydrostatic pressure. in specific embodiments, el viability and chondrogenic differentiation of HDFs seeded in threedimens'iona aginate bead cultures are detennined. In another embodiment, the effects of oxygen tension on the differentiation of HUE c'utred in alginate beads are characterized. In an additional 25 specific embodiment, the effects of hydrostatic compression on the differentiation of IFDFs cultured in alginate beads are characterized. Differentiation of cells into chondrocytes or chondrocyte-like cells may occur in any suitable manner, including differentiation in vitro prior to implantation of the device into an individual or differentiation in vitro prior to implantation of the 30 device into an individual and also in vivo following implantation.

10 In specific embodiments the device of the invention prov 'ides a method for in vivo regeneration of a joint, such as an intervertebral disc, e bow, knee, shoulder, hip, ternporo-mandibular joint, and so farth, In certain aspects of the invention a living compartment comprises the cell-matrix construct of chondrocytelke cells, such 5 as are derived fromI HDFs, seeded in a biomaterial. The culture and differentation of the living compartment may be initiated in vitro, in certain embodiments. The living core is seeded in the inert biomaterial and implanted, and the cells continue to proliferate and differentiate in vivo. In certain embodiments, the cartilage that is the focus of application of the 10 invention is intervertebral disc cartilage. In particular aspects of the invention, cells utilized in the invention are subjected to mechanical strain for chondrogenic differentiation. Thus, embodiments of the invention provide an inter-vertebral inert structure acting as an in vivo bioreactor for inducing growth and dufferentiation ofa living core. In further embodiments, the invention provides a hybrid construct 15 combining both an inert structure and living core for implantation into the inter somatic space using a minimaly invasive surgery. It is an advantage hat in one or more embodiments of the present invention there is provided a method intended to repair a degenerated intervertebral disc, e g. restore intervertebr disc anatomy and improve its functioning. In particular 20 aspects of the invention, there is provided a method to repair damaged disc using a hybrid structure made of an inert containing device intended to feed and differentiate an inner Iiving core. Therefore. the inert strcture acts as a delivery system of nutrients and growth factors and as a bioreactor able to differentiate autologous dennal fbroblasts injo chondrcyte-like ells. Under mechanical stress (such as 25 intermittent hydrostatic pressure, and/or fluid shear stress), the cells vll acquire the characteristics of nucleus cells in the central part and ainulus cells in the periphery. Ixemplary tbroblast-derived chondrocytedlike cells may be harvested from skin, such as by a biopsy, and then seeded onto three-dimensional polymer scaffold for use of the repair of the disc. This ould obviate the need fo invasive technique to harvest 30 autologous chondrocytes, in particular aspects An advantage of certain aspects of the inventive hybrid construct that combines both an inert biomaterial acting as a nutrientdelivery system and. living cells easily harvested from skin, for example, is tha it is capable of self-maintenance or remodeling and may restore the disc function using a minimally invasive posterior surgical approach, for example. In certain aspects of the invention, the damaged cartilage from the joint or 5 intervertebral space is removed and the hybrid stucture is installed within the space provided by the foregoing removal In some embodiments of the invention, the device is implanted using a minimally invasive surgical procedure. In specific embodiments, an exemplary surgical technique is employed. In general embodiments for intervertebral discs, when an intervertebral disc must be removed from between two 10 adjacent vertebrae, e.g. in the lumbar pine, it is les invasive to surgically proceed posteriorly from the back of the patient. This minimally invasive procedure allows proceeding with the curettage of the inter-somatic space through a small aperture within the annulus (annulotomy) for removing the degenerated fragments of the disc's nucleus. Using this small annulus opening, the present invention employs a novel 15 intervertebral repairing pack that can be slid through he aforementioned incision and then expanded into the area generated by the nucleus removal within the inter-somatic space, for example, In specific embodiments, the removal of the damaged disc and the installation of the tissue-engneered construct are done in the same posterior operation, thereby minimizing risks, chances of surgical complications and re-interventionsas 20 well as surgery time. In one embodiment of the invention, there is an implantable device comprising a cells/scaffold composition and an encapsulating device, wherein the encapsulating device comprises a first generally concentric membrane: a second generally concentric membrane that is concentrically external to the first generally 25 concentric membrane; a first volume within the first generally concentric membrane; a second volume that is external to the first generally concentric membrane and that is intemal to the second generally concentric membrane; and a strcture for extracting material from the second volume, wherein the first generally concentric membrane is semi-permeable and houses the cell/scaffold composition. A membrane may be 30 considered generally concentric compared to another if the centers of each of the membrane are substanially nearby, 12 In certain aspects of the invention, an 'diidul is provided another therapy in addition to the implantable device of the mention. For exam ple, before, during, and/or after implantation of the device, the individual may receive one or more antibiotics, Exemplary post-operative therapies includes Non Steroida Anti 5 Inflamniatory Drugs (NSAIDs), simple pain killers (analgesics) and/or myo-relaxing medication as needed, and it may be followed by a functional rehabilitation post operatively, such as after the first, second, third or more post-operative week, for example. In some embodiments, there is a hybrid structure for cartilage repair 10 comprising an encapsudating device comprised of inert material and a living core comprised of Thondrocyte-like ce '. This encapsulating device act s an i vivo bioreactor for cartilage engineering, It allows in vio growth and differentiation of cartilage cells by providing growth factors and nutrients and transmitting a physiologic loading regimen, 15 In one embodiment of the invention, there is an implantable device, comprising a cells/scaffold composition; and an encapsulating device that comprises: a first membrane having an inside and an outside; a second membrane having an inside and an outside, wherein the first membrane is encapsudated inlse the second membrane; a first volume disposed inside the first membrane; a second volume that is 20 disposed outside the first membrane and that is disposed inside the second membrane; and a structure for adding fluid to the second volume, removing fluid from the second volume, or both, wherein the cells/scaffold composition is disposed inside the first membrane and the first membrane has one or more of the following characteristics: seni-ermeable; biocompatible; biodegradable; and resorbable, wherein the second 25 membrane has one or more f the following characteristics: biocompatible; hermetic to fluid; permeable to oxygen; resorbable; biodegradable; and expandable. I a specific embodiment, the scaffold is comprised of a synthetic polymer, a natural hydrogen, or a synthetic hydrogel. In an additional specific embodiment, the a;ynthetic poymer is polyglycolic acid, polylactic acid, polylactic-co 30 glycolic acid, poly - caproyactone, or poly(glycerol-Sebacate) (PGS). in another specific embodiment, the synthetic polymer is a polyphosphazene, a polyanhydride, or 13 a poly(orthoester) In paticula embodiments, the natural hydrogel comprises collagen, byaluronic acid, lginate, agarose, chitosan, fibrin, gelatin, or a copolymer thereof. In a further embodiment, the synthetic hydrogel comprises polyethylene oxide), polyvinyl alcohol), poly(acrylic acid), poly(propylene fumarate-co-ethylene glycol). or a copolymer thereof In certain a pects of the invention, the cells in the device are chondrocyte cells or chondrocyte-like cells, such as wherein the chondrocyte cells or chondrocyte like cells secrete a moleeue selected from tie group consisting of aggrecan pe II collagen, Sox-9 protein, carilage link protein, and perlecan. In particular cases, the 10 cells were differentiated from fibroblast c els and/or stem cells. Exemplary fibroblast cells are dermal fibroblasts, tendon fibroblasts, ligament fibroblasts, synovial fibroblasts, foreskin fibroblasts, or a mixture thereof In particular aspects, the first membrane is comprised of a biodegradable, biocompatible, and res orbable polymer. In further aspects, the first rnembrane is 15 comprised of a polyacrylate, a polyvinylidene, a polyvinyl chloride copolymer, a polyurethane, a polystyrene, a polyamnide, a cellulose acetate, a cellulose nitrate, a polysulfone, a polyphosphazene, a polyacrylonitrile, a poly(acrylonitrile/covinyl chloride) or a derivative, copolynmer or mixture thereof In specific aspects' the first membrane is generated by polyelectrolyte complexation. in specific aspects, the 20 second membrane is co mprised of polyglycolic acid (PGA), polylactic acid (PLA), polylactic-co-gly'colic acid (PLGA), poly'.-caprolactone (PCL), polyurethane (PU), polydioxanone (PDO). a polyethylene, poly (glycerol sebacate) (PGS), or a derivaive, copolymer, or mixture thereof In additional embodirments, the rate of resorhability of the seco nd mem brane is slower than the rate of resorbability of the first membrane. 25 In particular embodiments, lie implantable device comprises one or more nutrients, growth factors, and/or medicaments. In some cases, the implantable device may be further defined as comprising a basal cell culture medium comprising te one or more nu trients, growth factors, and/or medicaments. In specific e mbodiments, the medium is suepplmened wth Feta Bovine Serum (FBS), ascorbie acid, and/r 30 dexamethas'one. The- nutrients, growth factors, and/or medicaments may be present in the scaffold, the first volume, the second v'olu mes or a combination thereof in certain 14 cases. The growth factor is selected from the group consisting of bone morphogenetic protein 2(MP2), BMP-4, BMP-6, BM1-7, cartilage-deried morphogentic protein (CDMP) transfonning growth factor beta (T -s), insulin growth factr one (IGF-), fibroblast growth factor (FGFs), basic fibroblast growth factor (bFGF) FGF-2, 5 plateleerived growth factor (PDGF), and a mixture thereof, in specific embodiments. and the medicament may be further defined as e onor more of an antibiotic, antifungal agent, or antiviral agent. In certain aspects of the invention, the structure comprises one or more tubes and/or comprises one or more catheters and/or one o more reservoirs. In 10 particular cases, the structure is further defined as comprising one or more of a first tube; a second tube; optinlly, a first reservoir; and optionally a second reservoir. In a specific embodiment, the first and second tubes respectively comprise firs ends positioned within the second volume, wherein the first and second tubes respectively conmprse second end connected to first and second reservoirs, or both The first 15 and/or second tubes are comprised of the same material as the second membrane, in one exemplary case, and the fir t and/or second tubes are comprised of silicone rubber, in one exemplary ca In one embodiment of t:e invention, there is a method of repairing damaged cartilage in a joint (such as an intervertebral dise) of an individual, 20 comprising delivering a device in accordance with the in vention to the respective joint (such as intervertebral disc) of the individual In a specific aspect, the method further comprises preparing the cells/scaffold composition under table e vivo conditions. In another specific embodiment, preparing the cells/seaffold omposiion is defined as subjecting one or more cells to a scaffold under suitable conditions, Preparing of the 25 cells/scaffold composition may occur for no less than about two to three days, in certain aspects of the invention. In a specific embodiment the suitable conditions allow proliferation of the cells, such as, for example, Howing the stimulation of chondrogenic differentiation. Suitable conditions may be further defined as being under high density micromass culture, being ider low oxygen tension (between 30 about 10%-7.5%), being under mechanical stress, and/or being fed by a medium 15 supplemented with growth factors, ascorbic acid, and/or dexamethasone, in exemplary embodiments. In particular embodiments, the cells/scaffold composition is subjected to mechanical sress, which may be hydrostatic pressure, fluid shear stress, or a 5 combination thereof, for example. In a specific embodiment, the mechanical stress is intermittent, In particular cases, the mechanical stress is fluid shear stress and the scaffold is microfluidic scaffold. In other particular embodiments, the delivering step is defined as implanting the device using minimally invasive surgery. In one exemplary case, 10 following implantation of the device into the individual, the second membrane is inflated to fill a void in the joint, such as an intervertebral disc. In another exemplary case, prior to delivery of the device to an intervertebral disc of the individual, at least part of an endogenous intervertebral disc was removed from the individual. The joint of concern with the invention may be an intervertebral disc, a knee, a shoulder, an 15 elbow, a hip, or a temporo-mandibular joit, in specific embodiments. In certain aspects of the invention he s. structure of the device comprises: a first tube having first and second ends, said first end of the first tube disposed within the second volume; a second tube having first and second ends, said first end of the second tube disposed within the second volume; a first reservoir; and a second 20 reservoir, wherei following delivery of the device to an intervene disc in the individual and following inflation of the second membrane, the secon ends of the first and second tubes are respectively connected to the first and second reservoirs. In a specific embodiment, the first and second reservoirs are subcutaneously positioned in the individual Method of the invention may further comprise sealing the first 25 membrane, sealing the second membrane, or both. In a specific aspect, at least part of the second volume is exchanged. In one exemplary embodiment, the method of the invention further comprises removing at least part of the second volume through the first reservoir. In another specific aspect, the method comprising removing fluid from the first or second reservoir, delivering a fluid to the respective second or first 30 reservoir, or concomitantly removing fluid from the first or second reservoir and delivering a fluid to the respective second or first reservoir, 16 In certain cases, the cella!eaffold composition is inserted into the first membrane prior to delivery of he device into the idividual or wherein the cclIs/scaffold composition is inserted into the first membrane subsequent to delivery of the devieinto the individual In a specific embodiment, the first membrane is 5 inserted into the second membrane prior to delivery of the device into the individual or wherein the first membrane is inserted into the second membrane subsequent to delivery of the device into the individual. In one embodiment of the invention, there is a method of preparing a cells/scaffold composition, wherein the cells are chondrocytes or chondrocyte-like 10 cells, comprising: subjecting cells capable of differentiating into a chondrocytedike cell to the scaffold; subjecting the cells to mechanical stress; and optionally subjecting the cells to one or more growth factors suitable for differentiation to a chondrocyte or chondrocyte-like cell. In a specific embodiment, the mechanical stress is intermittent. In a further embodiment, there is a kit comprising the device of the 15 invention, wherein the device is housed in one or more suitable containers. In specific embodiments, the kit further comprises cells that are chondrocyte cells, chondrocyte like cells, or cells that are capable of differentiating to chondrocyte cells or chondrocyte-like cells. In an additional embodiment, there is an implantable device, comprising: a 20 cells/scaffold composition encapsulated inside a membrane, said membrane having an inside and an outside; and a structure for exchanging at least part of fluid that is inside the membrane, wherein the membrane has one or more of the fbllowi ng characteristics: semi-permeable; biocompatible; biodegradable; and resorbable. In another embodiment, there is a hybrid structure for cartilage repair, 25 comprising: an encapsulating device comprising inert material; and a living core comprising chondroytedike cells, wherein said encapsulating device encapsulates the living core, In an additional embodiment, there is an in vivo bioreactor for cartilage engineering, comprising a device that encapsulates cells, wherein said cells are 30 capable of differentiating to chondrocytes or chondrocyte-like cells, wherein the encapsulation of said cells provides suitable conditions for in vivo growth and 17 differentiation of said cells, wherein said conditions comprise providing a physiologic loading regimen on said cells. In a specific embodiment, the physiologic loading regimen comprises force from a spine of an individual. The foregoing has outlined rather broadly the features and technical 5 advantages of the present invention in order that the detailed description of the invention that follows may be better understood. Additional features and advantages of the invention will be described hereinafter which form the subject of the claims of the invention. It should be appreciated by those skilled in the art that the conception and specific embodiment disclosed may be readily utilized as a basis for modifying or 10 designing other structures for carrying out the same purposes of the present invention. It should also be realized by those skilled in the art that such equivalent constructions do not depart from the spirit and scope of the invention as set forth in the appended claims. The novel features which are believed to be characteristic of the invention, both as to its organization and method of operation, together with further features and 15 advantages will be better understood from the following description when considered in connection with the accompanying figures, It is to be expressly understood, however, that each of the figures is provided for the purpose of illustration and description only and is not intended as a definition of the limits of the present invention. The p resent application refers to a number of references and documents all 20 of which are incorporated herein in their entirety. Throughout this specification, the word "comprise", or variations thereof such as "comprises" r "comprising", will be understood to imply the inclusion of a stated element integer or step, or group of elements integers or steps, but not the exclusion of any other element, integer or step, or group of element, integers or steps. 25 A.ny dicussion of documents, acts, materials, devices articles or the like which has been included in this specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia or elsewhere before the 30 priority date of this application.

is BRIEF DESCRIPTION OF THE DRAWINGS For a more complete understanding of the present invention, reference is now made to the following descriptions taken in conjunction with the accompanying drawings. 5 FIG. I illustrates a cross-section of the exemplary L4-L5 intervertebral space. FIG, 2 shows a posterior approach to an intervertebral space, including exemplary herniated tissue and degenerated discal tissue. FIG. 3 illustrates an exemplary embodiment of the annulus defect location 10 for disc removal. FIG. 4 shows abdominal cross-section and draining system in embodiments of the invention, including an exemplary medline incision and exemplary Iolter-Rickham reservoirs (Codman & Shurtleff, Inc.; Raynham, MA) 15 DETAILED DESCRIPTION OF THE INVENTION As used herein the specification, "a" or "an" may mean one or more. As used herein in the claim(s) when used in conjunction with e word "comprising", the words "a" or "an" may mean one or more than one. As used herein "another" may 20 mean at least a second or more. In specific embodiments, aspects of the invention may "consist essentially of or "consist of' one or more sequences of the invention, for example. Some embodiments of the invention may consist of or consist essentially of one or more elements, method steps, and/or methods o the invention, It is contemplated that any method or composition described herein can be implemented 25 with respect to any other method or composition described herein. . Definitions The term bioreactorr" as used herein refers to a system i wich a 30 biological conversion is effected. Cells are cultivated in a controlled manner and are converted via specific reactions, in specific embodiments. In some aspects of the 19 invention, a bioreactor is capable of regulating one or more of the following parameters: temperature, medium pH, exchanges of gases, mechanical stimuli, pO,, PCO> and humidity. A perfusion system is present in the bioreactor (perfusion bioreactort in specific embodiments, to provide constant supply of nutrients and to 5 remove efficiently the waste products. Mechanical stresses are an important factor of chondrocyte nation, Combinations of mechanical stresses are simultaneously developed during join motion on an intermittent basis that includes cell and tissue deformation, compressive and shear forces, fluid flow, and changes in hydrostatic pressure, for example. These conditions are reproduced with the bioreactor, in certain 10 aspects. The term "catheter" as used herein refers to a hollow t:e, which may be flexible or rigid, that is employed to drain fluid from an area in the body. The term "chondrocyte-like cells" as used herein ref rs to cells that are not primary chondrocytes but are derived from stem cells (such as mesenchymal stein 15 cells) or cells from other lineages (such as fibroblasts). These chondrocyte-like cells have a phenotype of chondrocytes (cells of cartilage). This means th not only do they have a shape of chondrocytes (polygonal and/or rhomboidal cells for example), but also they are able to aggregate and produce cartilage matrix components, such as slfated proteoglycan and type H collagen, for example. Thus exemplary markers of 20 chondrocyte-like cells include one or more of aggrecan, which i a chondroitin sulfate and keratan sulfate proteoglycan, type II collagen, Sox-9 protein, cartilage link protein, and perlecan, which is a heparan sulfate proteoglycan, for example. The term "copolymer" as used herein refers to a polymer comprising two or more different monomers (polymer: a naturally occurring or synthetic compound 25 comprising large molecules made up of a linked series of repeated simple mononers). The term "de-differentiation" as used herein refers to regression of a specialized cell or tissue to a simpler, more embryonic, unspecialized form. When chondrocytes are grown ex vNvo in monolayers, they lack their in vivo environment (and especially the three dimensionality and mechanical stress) and undergo 30 morphological and molecular changes called de-differentiation, This process involves 211 a change in morphology and a change from expression of chondrocyte-specific genes to that of genes that are normally expressed in fibroblasts. The tenn "discectomy" a-' used herein refers to a procedure to remove part or all of a degenerated nucleus through a fenestration within the annulus. It is performed through a minimall invasive approach using an operative microscope. The procedure frees the roots by removing the compressive herniated (extruded) nucleus. It allows removing the degenerated remaining nucleus through a tenotomy (aperture) within the annulus, In picular, a diseetomy is actually a herniectomy with the removal of the degenerated nucleus fagments. 10 The term "encapsulate" or "eneapsulatig" as used herein refers to enclosing within a boundary, such as in a membranous sac. The term "fluid shear stress" refers to the motion of fluids upon a surface, which results in the generation of shear stress. Shear stress a stress state where the stress is parallel to a surface. Microfluidi he 'affold allows fluid flow in the 15 microchannels. This fluid flow induces fluid shear stress on the cells seeding in the scaffold. The term "hermetic" as used herein refers to being made liquid-tight, such as by fusion or sealing, for example. In particular, a hermetic membrane does not allow liquid inside it to exit the membrane, although it allows oxygen and carbon 20 dioxide to cross the membrane (such as oxygen to enter the membrane and carbon dioxide *o leave the membrane). Then term "hydrostatic pressure" refers to the pressure exerted or transmitted by liquid (for example, water) at rest. The intervertebral disc is exposed to wide ranges of intradiscal hydrostatic pressure during different loading exercises and 25 are at their minimum (about 0.25MPa) during dying or elaxed sitting and at maximum (about 2.5 to 5 Ma) during lifting weights with a round back. These different loading magnitudes influence the intervertebral disc b alteration of disc matrix turnover depending on their magnitoes. Numero studies have been done to determine the best regimen tor intenittent hydrostatic pressure (1IP) to be applied in vitro to the 30 cells to induce chondrogenic differentiation of cells in vitro Different regimens have been tested. In these studies, MHP applied is within the amplitude ranges from 0.5MPa 21 to about 5MPa and a tequency range from 0.01 Hz to 1 Hz. The encapsulating device is designed to transmit in vivo hydrostatic pressure to the cellmatrix construct, in specific embodiment'i The ext enal envelop filled with liquid (medium) is compressed during different loading exercises; under this compression some liquid medium 5 difUses through the sem-permeable internal membrane, which allows perfusion of the cek matrix construt and generates hydrostatic pressure within the cell-matrix construct. In this system, the appropriate physiologic hydrostatic pressure is applied to the cell-matrix construct, which is useful for chondrogenic differentiation of the cells. 10 The term "hypoxia" as used herein refers to a deficiency in oxygen. n specific aspects, it refers to oxygen tension that is less than about 20%. The term "joint" as used herein refers to a region in the body wherein two bones of a skeleton join. The term "membrane" as used herein refers to a pliable layer of material 15 that separates different types and/or areas of biological material. It may be comprised of natural and/or synthetic material, and it may be permeable to substances in solution, for example. The term "nicrofluidic scaffold" as used herein refers to a material that comprises a system of microchannels. 20 The tem "minimally invasive surgery" as used herein refr to procedures performed through one or more small incisions in an indi vidual. For example, in certain aspects minimally invasive surgery uses specialized techniques, miniature cameras with microscopes, tiny fiber-optic flashlights and/or high definition monitors. For the individual, minimally invasive surgery means less trauma to the body', less 25 blood loss, smaller surgical scar(s) and less need for pain medication,. when compared to conventional open surgery. Individuals are suited to leave a medical facility sooner after minimally invasive surgery and return to normal activities sooner than with conventional open surgery. The term "reservoir" as used herein refers to a device that acts as an 30 injection chamber. In specific embodiments, the type of reservoir may be one that is routinely used in the art to deliver drugs (antibiotics for example, in case of meningitis 22 or veatriculitis) into the cerebral ventricular system (hence the term of "entriculostony"), into a vein (chemotherapy for oncologic purpose), or into the subarachnoid spinal space (morphine for pain relief), for example. h may be considered to be a kind of drug delivery system, in particular aspects. It is comprised 5 of several pars: I) a silicone-based (called "silastic") material top that allows repeated punctures without losing its waterproof characteristics; 2) a stainless-steel base that avoids the needle to injure the underlying tissues; and 3) a silastic end that connects to a catheter. The catheter may also be made of silastic. Its distal end can be brought to the site and cut at the right size, while its proximal end is connected to the 10 reservoir's end. The exemplary system defines a I to 2 em 3 chamber, hence its name of "reservoir" (tank). The term "scaffold" as used herein refers to a porous biodegradable polymer construct that supports cell growth and/or migration, for example, The tern "seeding" as used herein refers to implanting cells in a scaffold, 15 The cells will attach to the scaffold and then grow and differentiate in the scaffold, IL General Embodiments of the Invention In general embodiments of the invention, there is provided a device and 20 methods of its se, wherein the device comprises cel-ratrix construct of chondrocyte-like cells encapsulated in a mutilayered membrane. Although any tissues may be repaired at least in part by methods f the invention, including any cartilage tissues, ina particular exemplary embodiment, interverebral disc cartilage or joint cartilage is repaired, Exemplary methods of the invention tilize c mbination of a living core and inert core 25 or struure, thereby providing a hybrid structure, in particular aspects of the invention, the living core comprises a cel-matrix construct of ch ondrocytelike cells, such as are derived from HDFs, and the iner structure comprises the living core ad is implanted into a patient using a minimally invasive surgical procedure, for example. The present invention provides a method for biological repair of cartilage 30 using autologous Human Deal Fibroblast (HDFs) as cell sourcing. The present invention also provides device comprising a ceibmatrix constut of cells, such as chondrocyte-like cells, that are encapsulated in a multilayered membrane. In a particular 23 enmbodment, the invention concerns growth and differentiation of cellsin viv using a special device. The chondrogenic differentiation is induced by mechanical stress, and in particular aspects, Intermittent Hydrostatic Pressure (IHP) and/or fluid shear stress, for example. 5 A general embodiment of the invention is to use HDFs as cell sourcing for engineering new cartilage for the intervertebral disc, because these cells are easy to harvest and to grow. The idea is to induce differentiation of these cells into chondrocyte like cells. There is already some evidence for chondrogenic differentiation of HDFs into chondrocyte-like cells. However, these studies are only in vitro and the technique to 10 differentiate the cells is based on the use of specific growth factors, hypoxia, or specific matrix such a' aggrecan. Because of its design, this device allows one or both of the f allowing, for example: 1) iffuio of nutrients and oxygen to the living cells; and/r 2) transfer of he load onto the cells. This mechanical force and especially the IHP is critical for I5 chondrogenic differentiation of fbroblasts, It is known that IHP is the most potent stimulus for induction and maintenance of the chondrocyte phenotype. When chondrocytes are harvested from cartilage to be used to engineer in vitro new cartilage, these cells need to be expanded hut this causes the chondrocytes to dedifferentiate, It has been shown that IHP can redifferentiate the cells nto chondrocytes. People who are using 20 chondrocytes to engineer cartilage in viro ofen use mechanical strains and especially IHP as induce of differentiation However, there is nothing in the literature on the effects of IHP on chondrogenic differentiation of HiDs In embodiments of the invention, there are at least two components to the device: 1) cellqnatrix construct, wherein the cells (HDFs, for example) are seeded into a 25 scaffold (an cells tha do not attach to the scaffold may be washed away); 2) encapsulating device. In specific embodiments, the in vivo encapsulating device is comprised of to concentric membranes, in specific embodiment: 1) an internal membrane is a semipermeable membrane that wraps the cell-scaffold construct (this sernipermeabe membrane is permeable to small molecules and so allows diffusion of 30 nutie ts and oxygen an elininatin of was:e, but it is inipermeable to macromolecules such as collagen and glycosamin glycan-, for example; these macromolecules that form the natural extracellular matrix arc then retained within the scaffold); and 2) an external 24 membrane is hermetic to fuid but permeable to oxygen, and is expandable and inflatable in order to be implanted through a minimally invasive posterior surgical procedure (in specific embodiments, when expanded it will fit the cavity of discectomy, for example exactly fit the cavity). The external membrane is filled with medium which 5 nourish the cells. The fluid enclosed within the nvelop forms a fluid environment that transfers I-IP to the living cells. About the day after surgery, when the individual can stand up and begin to walk again, he applies some load on the spine and especially on the instrumented level. Therefore, he living core receives the right cyclic hydrostatic pressure regimen under physiological load through the envelope that is filled with medium, which 10 is useful fo HDFs growth and conversion. Thus, in certain aspects the individual walks within about one day of implantation of the device, about two days, about three days, about four days, or about five or mare days following implantation of the device. In specific enbodiment, the extema membrane filled with medium is connected to a raining system to regularly chraige d he medium. The chondrogenic 15 differentiation of HIDFs is induced by mechanical stress and especially Intermittent Hydrostatic Pressure -H-P) and/or fluid shear stress in vitro and then in vivo. Exemplary co-culture conditions are as follows: high density micromass culture, supplemental tion with BMPQ a scorbic acid, and hypoxia, for example At least some embodiments described herein may ameliorate problems in the 20 field. The nutrients and growth factors are provided to the celbmatrix construct by the in situ medium. It avoids the problem of diffusion of nutrients from the surrounding natural tissue (endplates) w huh is usually deficient due to deg neration of these structures. Growths factors important for chondrogenic differentiation of HD-s are added to the medium, In specific aspects, HD~s are deployed, which void the use of invasive 25 te h unique to harvest chondrocytes, IDs, r any other cells, are pre-dif ferentiated in v itro for a short period of time and continue to grow and differentiate in vivo. The encapsulating device with its extemal envelop filled with fluid may provide the physiologic loading and compressive forces ideal for chondrogenic differentiation of HDFs.

25 IML The Hybrid Construct Embodiments of the invention can employ a hybrid construct for repair of cartilage in a joint, such as an intervertebral disc. Exemplary embodiments of the hybrid construct are described herein, and in cerai aspects the hybrid construct is an 5 implantable device, for implntation into a mammal, such as a human, dog, cat, horse, pig, sheep, goat, and so forth. In particular aspects a hybrid construct is comprised of at least a living core, comprising cell and a scaffold, and an inert structure. A. Cells/Scaffold Composition The living core, whichi may be referred to as the cells/scaffold composition is 10 aclmatrix construct and compries cells ede in a scaffol (which may be referred to as a matrix). In a specific embodiment, the seaffold comprises alginate beads; a microfluidic scaffold (the Microfluidic scaffold could be made of any bodegradable biopolymer [organic biodegradable polymers: poly(L-lacti acid) (PLA), poly(glycolic acid) (POA), poly-actic-co-glycolie aci (PiLGA) natural hydrogels collagen , HA, 15 alginate, agarose, chitosan, combination collagen/HA, ciiosan/CAG, ollagenGAG); and/or synthetic hydrogels (Poly(ethylenc oxide), (PEO), polyvinyl alcohol) (PVA), poly(acrylic acid) (PAA), polypropylene fumarateneo-ethylene glycol) (P(PFco-EG))], for example. In specific embodiments, cell adhesion ligands such as peptides or polysaccharides are employed, The peptide sequences may be capable of binding to 20 cellular receptors. These peptides could comprise the exemplary amino acid sequences arginine-glycine-aspartic acid (ROD) argininine-glutami acidasparti acid-valine (REDV). tyrosineisoleucine-glycineserinearginine (YISR), or isoleuclysnic valine-alanine-valine (IKVAV) and may be attached to the scaffold, wherein the ligands and/or growth factors may be incorporated to regulate cell fate, In fact, the growth factors 25 can be incorporated in the scaffold or included in the medium in the extemal membrane, for example. The scaftfld materials may be biodegradable, and the rate of biodegradation can be manipulated. In accordance with the invention and as explained above, HDFs are differentiated into chondrocyte-like cells under mechanical stress either i vinro or in vvo 30 or both in vitro followed by in vivo, for example. As explained above, important co culture conditions include high cell density culture; growth factors (BMP-2); and/or 26 ascorbic acid, for example. HDFs can also be differentiated into chondrocyte-like cells under low oxygen tension and culture on aggregan with insulin growth factor one (IGF-). As aforementioned, bioreactors are used to induce in vitro proliferation and differentiation of HDFs. The inert structure of the present invention is used to induce in vivo 5 differentiation, in particular aspects of the invention. HDFs in alginate beads or HDs seeded in a microfluidic scaffold or HDFs seeded in any other polymeric scaffold are encapsulated in a semi-permeable membrane that is part of an inert structure, in specific embodiments of the invention. A function of the semi-permeable membrane is to encapsulate the chondrocyte-matrix construct to concentrate the production of ECM 10 proteins. This membrane allows the passage of 02, nutrients/ waste, and CO 2 , for example. In specific embodiments, scaffold refers to a porous biodegradable polymer construct to support cell growth and/or migration This material is non-toxic, bicompatible and biodegradable, in specific embodiments, 15 In exemplary embodhnents, alginate is employed for the scaffold. Alginate is a natural polysaccharide isolated front seaweed. t is a polysaccharide composed D mannuronate and Lguluronate monomers. When crosslinked with calcium ions, it fan a gel that is bioompatible, biodegradable. Alginate is well established as matrix material for tissue within regenerative medicine It has been used more widely than other hydrogels 20 to assess 'ii potential of hyrogel scaffod for cartilage engineering, Mac. beads of alginate (-3mm in size) or microbeads of alginate (250-500pm) can be used in this invention. Microbeads of alginate are preferred, These smaller beads have the advantage of a higher surface to volume ratio allowing good anport of essential nutrients, they are also less fragile. Algiate is iocompatibe and approved by the U.S. Food and Drug 25 Adninistration for human use. IHD~s may be seeded in alginate macrobeads (as described below) or preferential 'in aginate microbeads. There are different techniques known in the art to generate alginate microbeads. There are usually produced by electrostatic drplet generation. For example, HIDs an be seeded in alginate mierbeads as follows. Alginate 30 powder (Sigma, St Louis, MO) is dissolved in WFI water at a concentration of 2.2% w/w and then mix with a suspension of 1D1s in culture medium to obtain final concentrations of 1.5% Wiw alginate and 10' cell /nl, Alginate microbeads are then produced by 27 electrostatic droplet generation, In brief, cell/alginate suspension is extruded through a positively charged blunt stainless steel needle at a constant flow rate of 14,0 mlh by a syringe pump and resulting droplets are collected in a gelling bath (1.5 w/v CaCl 2 ). As Na* ions are exchanged with Ca-" ions, alginate droplets harden and form insoluble 5 microbeads with entrapped cells. The microbeads are left for 30 min in the gelling bath in order to complete gellation. Microfluidic scaffolds may also be employed, in particular embodiments. They are complex scaffolds with micron-scale resolution. These scaffolds present a network of micro-channels that allow fluid flow within the scaffold. This network of 0 micro-channels helps to provide both nutrients and soluble factors to distt sections of the scaffold. These scaffolds can be made of different biopolymes They can be made of synthetic polymers such as polyglycolic acid (PGA), polylacti acid LA) polylactic-co glycolic acid (PLGA); synthetic hydrogels such as poly(ethyleneoxide) (PEO) poly(vinyl alcohol) (PVA), poly(acrylic acid) (PAA), poly(propylene furmarate-co-ethylene glycol 5 (P(PF-co-EG), or of Poly(glyceorok-Sebacate) (PGS) that is a biodegradable elastomer. In the invention, this microfluidic scaffold is encapsulated with a semipermeable membrane. This semi-permeable membrane allows the perfusion of medium containing nutrients' growth factors within the scaffold. By circulating within the network of micro-channels, the medium ill apply fluid shear tress on the cells seeded in this scaffold. This 20 mechanical ibrce is critical for chondrogenic differentiation of HDFs. B. The Inert Structure In the invention, the hybrid construct employs an inert structure as part of its 25 composition. Functions of the inert structure may be biological (delivery of nutrients and/or growth factors) and/or mechanical (to transfer mechanical forces, such as onto the cells/scaffold composition; such forces may include IHP and/or Fluid shear stress), for example. The inert structure may function as an "in vivo bioreactor" by transferring mechanical strain and by providing medium (by perfusion through the semi-permeable 30 membrane) to the cells/scaffold composition. In certain embodiments, functions of the inert structure include one or more of the following: 1) to hermetically encase the living core; 2) to act as a semi-permeable 28 membrane by allowing certain molecules (for xapie nutrients, growth factor, eca.) to pass through it by diffusion (and occasionally specialized "facilitated diffusion") under certain physico-chemiai conditions (for example, hydrostatic pressure, osmotic concentration, temperature, etc.); 3) to transfer the load and to share the dynamic 5 mechanical stress (hydrostatic pressure) to the living compartment acting as an inducer of cell differentiation; and/or 4) to act as an in vivo bioreactor, The inert structure may be considered an encapsulating device, in specific emubodiments For certain embodiments, it is designed to apply mechanical stres on the cells seeded in the three-imensional scaffold composition n. The external 10 membrane of the encapsulating device is filled with fluid (medium). The fluid enclosed wthin the envelop forms a fluid environment that transfers the cyclic hydrostatic pressure to the living cells. When the patient stands up, for example, he applies ome loa o his spine that is transferred to the Iving cells through the external envelop that is filled with fluid. This membrane provides the physiologic 15 loading and compressive forces suitable for chondrogenic differentiation of cells such as HDLFs. In the case of cells embedded in a microfluidic seaf old, the medium circulating within the micro-channels also apply fluid shear stress on the ells. This fluid shear stress is another force that induces chondrogenic differentiation of cells. In specific embodiments, the membrane is generally alloonshaped, and in 20 additional embodiments the membranes are generally concentric ith one another. In further specific embodiments, the iner structure comprises to expandable balkonlike bio-polymers, namely, internal balloon "-M" which is enclosed within an external balloon "E". Hence, the inert structure comprises two concentric envelopes capable of being successively inflated and having inflation activity. In certain aspects, a number X of 25 membranes may be utilized in the device, where'n X is any whole integer greater than one. That is, X balloons may be instaled concentrically as with layrs in an onion, each of them defining a space with a specific function (for example, for waste, media, oxygen, and/or for connecting the graft to natural tissue). In one embodiment, the external balloon, layer or envelope "E" comprises a 30 biocompatible resilient, inflatable, hermetic, expandable, and/or resorbable (time TI, wherein T is the time for complete resorption of "E") material that is able to be sealed once installed in the cavity. In specific embodiments, external balloon "E" is capable of 29 having or has one or more of the following activities: 1) to receive a second internal balloon or layer or envelope "i" enclosing the cells (in the form of cell-matrix construct, or cell solution or graft); 2) to be inflated wih a media (for example, liquid) or to expand its wall (for example, via swelling) in order to fill the cavity resulting of the discectomy; 5 and 3) to close the annlus defect to prv'ent it from "herniating" or coming out from the inter-somatic space through the tenotomy incision once the construct is under load, In one embodiment, the intemal bon or layer "I" comprises a bio ompatibe, resilient, inflatabe, semi-permeable, and/or resorbable (time T 2 <Ti, wherein T is the time for complete resorption of "1") material that is able to seal the 10 living core once installed into the extemal layer, The internal balloon (envelop, membrane, or layer) "I" is capable of having or has the following activities: l)to hermeticall; wrap up the living 'ore; 2) to act as a semi-permeable membrane by allowing certain molecules (for example, nutrients, growth factors, etc.) to pass through it by diffusion (and occasionally specialized "facilitated dif'fusion") under certain physico 15 chemical conditions (for example hydrostatic pressure, osmotic concentration, temperature, etc.); and 3) to transfer the load to the living core so as to share the dynamic mechanical srain therewith, thereby acting as an induce of cell differentiation. in accordance with an aspect of the invention, the combination of an external compartment media (such as a liquid, for example) or swelled w-all (such s hydrated 20 hydrogel, for instance) "E" and e interal semi-permeable envelope provide a delivery system of nutrients and growth factors capable of feeding an inner living core. These envelopes also transfer the mechanical forces, including hydrostatic pressure to the living core. The inert structure is an encapsulating device intended to wrap up, feed and 25 differentiate a living core made of cells, such as HDFs. In a preferred embodiment, the inert structure comprises two expandable balloon-like bio-polymeric membranes, namely, internal membrane "" which enclosed within an external membrane "E", Hence, the incrt structure comprises two concentric envelops intended to be successively inflated. At the rest position, the two envelopes "" 30 and "E" are flat, deformable, shaped and fit one another. Both can be sealed once implanted. The inert structure composition may be determined by the choice of tissue engineering system, in specific embodiments.

30 The external envelope "E" comprises a material that is inflate (in order to be implanted flat through a minimally invasive posterior approach, then loaded with the 1iving core, and then infated with the media solution); resilient (to transfer load sharing onto the living core); expandable (to allow its expansion and fill the cavity 5 resulting of the disc tomy); permeable to Q, but hernetic to fluids: relative hypoxia is a useful parameter of HIDFs conversion, but 02 tension within the natural disc is appropriately low; biodegradable (to allow the graft to reconnect with the natural remaining disc)' biocompatible (to minimize inflammatory reaction); resorbable (time T );or combination tereot 10 "E may be positioned in a joint, for instance, into the cavity resulting of the curettage of an inter-somatic space between a pair of adjacent vertebme and within the remaining discal tissue, in specific embodiments. It also m'ay be mechanically able to maintain the disc height under loading. In additional embodiments, "E" receives a second internal ballon "I" enclosing the living core. "E" may be inflated with a fluidic solution 15 (for instance the media) to extend in the chamber (cavity resulting of the discectomy) peripherally up to the remaining discal tissue and fill the cavity. "E" is configured such that it allows changing of the media (removal of metabolic wastes and/or replenishment of nutrients and/or growth factors, for example), such as under an isobaric regimen, for example. "E" acts as an in vv bioreactor by transferring the load sharing onto the living 20 core, for example with cyclic hydrostatic pressure (which is useful for differentiating cells into chondrocyte-ike cells, in certain aspects. In particular embodiments, the con figuration of"E" produces relative hypoxia due to its characteristics (Hypoxia or hypoxia-mimicking agent, as lactate, induces HDFs conversion in chondrocytes-like cells). "E"may also close the annulus defect (tenotomy opening) to prevent it from 25 "herniating" or coning out from the inter-somatic space through the tenotomy incision once the onstruct is under load. In certain aspects of the invention, the internal membrane "I" comprises a membrane that is biocompatible; resilient; inflatable (while the media is consumed, the living core grows and expands to the inner wall of the external membrane); semi 30 permeable (controlled release system for nutrients, growth Eactors, etc.); biodegradable (so as not to interfere with the long-term properties of the repaired tissue); and resorbable (time T2 < T), E" must resorb after "I" not only to avoid the leakage and the lost of the 31 media while the living core v not mature yet, but also to keep the fragile T away from any direct mechanical strain), In other certain aspects, "I" hermetically wraps up the living core; acts as a delivery system of nutrients an growth factors capable of feeding an inner living core through a semi-permeable membrane by allowing certain molecules (for 5 example mutrents, growth factor c.) to pass through it by diffusion (and occasionally specialized "facilitated diffusion") ude certain physc-chemical conditions (for example hydrostatic pressure, osmotic concentration, temperature, etc.) These two membranes ("E" and ") define 2 volumes VE and VI. These two distinct volumes may have diterent shapes (spherical, cylindrical, conical, etc.) 10 depending of the contour of the inter-vertebral cavity and the load sharing. In specific embodiments, the device conforms to the shape of the cavity, Volume VE is defied as the space that separates membrane "E" from Membrane "I", It comprises nutrients and growth factors (media) to be delivered to the cells, in specific embodiments, such as through the sei-permeable membrane "I". It also 15 acts as a load-bearing structure capable of transferring mechanical strain, fr example the cyclic hydrostatic pressure regimen or the high fluid shear stress (due to its high content water) to the living core (which induces chondrogenic differentiation of cells, such as HDFs). Volume V 1 is defined as the space that is outwardly limited by the internal 20 semi-permeable membrane "I" and comprises the living core made of chondrocte-like cells, such as cells derived from HDFs. Until the living core has become viable (e.g, capable of self maintenance) the media enclosed in VE may be regularly changed in order to remove any toxic wastes accumulated due to the metabolism (free radicals and/or lactic acid, fbr example), as well 25 as any other cellular scraps or debris' as a esult of the cellular growth. Such a change allows replenishnent of its contents with nutrients and/or growth factors, In specific embodiments, this procedure is performed periodically, such as one or more times per week or month, for example at least once a week, such as twice a week. In additional specific embodiments, it requires to equip "E" with an additional feature for draining VE 30 This draining system may be made of one or more tubes and one or more reservoirs, in certain aspects, and in particular embodiments it comprises two tubes and two reservoirs. The first tube may be employed to remove the used media, and the second tube may be 32 employed to inject the new media. Each of these tubes (or catheters) comprises a proximal end that hermetically connects to V; and a distal end that connects to a reservoir. These catheters could be made either of the same material as "E" or of' sicone rubber, for example. Their length may be of any suitable length so long a they can span from the 5 reservoir to the device. They may be comprised between about 10 and 15 centimeters, and is pre-operatively set up by cutting their distal end at the appropriate length according to the depth of the operative site and the anatomical dat (patients morphology). Their outer diameter may be of any suitable length, but in specific embodihents they are about 2.5 millimeters in length, such as in order to be small enough to exit from the tenotomy 10 aperture, not to compress or injure the adjacent root, and allow a 1,2 millimeter inner diameter. The tubes may be implanted at the end of a discectomy procedure, after implantation, inflation and sealing of the bio-reactor, and before skin closure, They may be connected to the distal end of each tube (catheter). Then, each reservoir may be 15 subcutaneously positioned so that it can be reachable by a needle front the skin (percutaneous puncture). In one embodiment, the engineered living core is pre-encapsulated with "I and then slid into "E", While the media is consumed, the living coe expands to the inner wall of the envelope "E'. The envelop "E" resorbs and the graftbreconnecs with the 20 natural remaining disc. 1. The eternal semi-permeable membrane The internal envelop comprises the living core, includes a controlled release 25 system (in order to allow the feeding of the living core with the media through its semi permeability characteristics), is expandable (while the media is consumed, the living core expands to the inner wall of the external membrane), and/or is biodegradable (so as not to interfere with the long-term properties of the repaired tissue) for example. In specific embodiments, the internal membrane is'a semi-permeable 30 membrane that wraps the cell-scaffold composition. This semi-perneable membrane is permeable to small molecules and so allows diffusion of nutrients and oxygen and elimination of wastes; but this membrane is impermeable to macromolecules such as collagen and glycosaminoglycans. These macromolecules that fom the natural extracellular matrix are then retained within the scaffold, This membrane also isolates the cell-matrix construct from the host environment and protects from inflammatory and immunological response of the host against the biopolymeric scaffold. 5 Various polymers and polymer blends can be used to manufacture this membrane, including but not limited to, polyac rlate (including acrylic copolymers), polyvinylidenes, polyvinyl chloride copolymers. polyurethanes, polystyrenes, polyamides. cellulose acetates, cellulose nitrates, polysulfones (cluding polyether sulfones), polyphosphazenes, polyacrylonitrles, poly(aerylonitrile/covinyl chloride), PTFE, as well 10 as derivatives, copolymers and mixtures of the foregoing, In one embodiment, the semi-ermeable membrane is generated y polyelectrolyte complexation: polyanion (PA) an polycation (PC) Va interations between oppositely charged polymers form polyelectrolyte complex (PEC)k The anionic component may be a biocompatible polymer, such as, but not limited , sodium alginate., 15 cellulose sulfate, carboxymethyl cellulose, or hyaluronic acid, and the Cationic component may be made of a polymer, such as, but not limited to, chitosan poly(Lysine, poly(L ornithine),Poly(methylen-co-guanidie) poly(vinylamine), poly(ethvlenimine), poly(DADMAC), or poly(N-vinylpyrrolidone), for example. To carry out the encapsulation of the cellmatix constroot with a semih 20 permeable PEC membrane, cell matrix-construct is first immersed in the anionic solution and then in the cationic solution. After a reaction time that varies depending on the nature of the anionic and cationic components, a mechanically stable semi-permeable membrane is formed. Depending on the reaction conditions (polymer concentration, reaction time), the scaffold is either tightly wrapped within the membrane or separated from it by a gap. 25 Volume V 1 . is defined as the space that is outwardly limited by the internal semipermeable membrane "" and comprises the living core made of chondrocyte-like cells derived from HDFs, for example. 2. The external membrane 30 The external membrane may be expandable, elastic and/or inflatable in order to be imp anted through a minimally invasive posterior surgical procedure and when 34 expanded to fit exctly the ca'ity of discectomy, Tis membrane is hermec to fluid but penneabe to oxygen and is filled with medium thnt provides nutrients and growth factors to the cells, The fluid enclosed wthin the envelop forms a fluid environment that transfers IHP to the living cells. When the patient stands up, he applies sone load on his spine that 5 is transferred to the lving ells through the membrane that is filled with fluid. This membane is mechanicaly 'resistant to support the load. The external membrane may be made of a biocompatible, biodegradable pdymer. Various polymers can be used o manufacture this membrane including, but not limited to, polyglycoli acid (PGA), polylactic a (PLA), polylactic-co-glvcolic acid 10 (PLGA), po"ye-eaprolactone (PCL), polurethane (PU), polydioxanone (PDO), polyethylenes, poly (gycerol sebacate) (POS) as well as derivatives, copolyners and mixtures of the foregoing, in one embodiment the membrane is comprised of an expandable, biocomnpatible, biodegradable polyurethane. This membrane is in direct contact with the host-surrounding tissue and is 1S biocompatible to avoid inflammatory reaction of the host. Different techniques can be used to improve the membrane biocompatibili such as, but not limited to, coating the membrane with hyaluronic acid, W. Cells Utilized in the Invention 20 In certain embodiments of the invention,' y cell may be employed so long as the cell is capable of differentiating into a chondrocyte or chondrocyte-like cell, i specific embodiments, the cell is in fact a chondrocyte, although it may be derived from a sten cell (for example, mesenchymal stem cell), or a fibroblast cell, 25 such as a dermnal brobast, tendon fibroblast, ligament fibroblast, or synovial fibroblast. Autologous cells may be utilized, although in alternative embodiments allogeneic lIs are employed; in specific embodiments, te allogeneic cells have been assayed for disenae ani are considered suitable for human transmission. In certain aspects of the invention the cell or cells are autologous, although in alternative 30 embodiments the cells are allogeneic. In cases wherein the cells are not autologous, prior to use in the invention the cells may be processed by standard means in the art to remove potentially hazardous materials, pathogens, etc. In particular aspects, the cells 35 ma be transfected with one or more nucleic acids, such as transfected with a growth factor, including BMP-2 A -4 6, and/or fl, for example. In particular aspects, chondrocyte-like differentiation of human dennal fibroblasts may be facilitated by employing one r more f the following: seeding cells in alginate; seeding ces in extracellular matrix proteins such as aggrecan or perlecan, hypoxic conditions (such as hypoxia or one or more hypoxia-minicking agents, for ex ample lactate, Desferrioxamiine mesylate (DFX)t cobalt chloride (Co "l), or nickel, for example); high density micromass culture; presence f one r more growth factors (including, for example, bone morphogenetic proteins (DMPs), 10 including at least BMP-2; transforming growth factor beta (TGF-4); insulin growth factor ne (IGFI); and hroblast growth factors (FGFs) and particularly basic fibroblast gnwth factor (bFGF) and FGF-2, platelet-derived growth facor (PDGF), cailage-derived morphogenetic protein (CDMP)]; presence of ascrbic acid, dexamethasone, parathyroid hormone-related protein (PTHrP), hedgehog proteins: ? sonic hedgehog (SHH) and Indian hedgehog (IHH)). Culture under mechanial stress may be employed. High density micromass culture is a culture technique that mimics the celh ar condensation stage that occurs during the onset of cartilage formation in the developing limb. In particular aspects of the invention, human dermal fibroblasts are 20 employed, at least because they can be non-invasively harvested, such as from a punch biopsy as little as about 3mm in diameter (in specific embodiments) from skin, for example a circular biopsy skin specimen. Also, human denial fibroblasts can expand easily in culture and can differentiate into chondrocyte-like cells under particular culture conditions. 25 In accordance with the invention, autologous HDFs are harvested from punch biopsy of skin tissue (6 mm) from the patient. In the laboratory, subcutaneous fat and deep dermis are dissected away with scissors. The remaining Jissue is minced and incubated overnight in 0,25% trypsin at 4*C, Then, dernal and epidermal frag cents are mechanically separated. The dermal fragments of the biopsy are minced 30 and the pieces are used to initiate explant cultures. Fibroblast harvested frm he explants are grown in Dulbecco's MEM (DMEM) with 10% calf serum at 37*C in 83% 36 CO. These cells are expanded before being differentiated into chondrocytes, in particular aspects. Some aspects may employ HDFs purchased commercially, such as from laboratories (such as Cascade Biologics). The cells can be adult HDFs or neonatal 5 HDFs. Neonatal foreskin fibroblasts are a very convenient source of cells,. for example. These cells are used commercially and are readily available and easy to grow. V. Growing and Differentiating Cells into Chondrocytes or Chondrocyte 10 like Cells Mechanical stress /strain are important factors for ehondrogenesis. The present method uses one or more mechanical strains and, in particular embodiments, uses intemttent hydrostatic pressure (1IHP) as inducer of chondrogenic differentiation of 15 HIDFs. IHP is known as a potent stimulus for induction and maintenance of the chondrocyte phenotype Recent studies have demonstrated that IHP stimulates chondrainduction of urine embryonic fibroblasts cultured with BMP-2, IHP can also induce chondrogenic difetrentiation of HIDFs. It is known that HDFs can differentiate into chondrocyte-ike cells under low oxygen tension. Therefore, in accordance with an 20 embodiment of th present invention, mechanical stress, especially IHP and shear fluid stress, induce chondrogenic differentiation of fibroblasts cultured in a three dimensional matrix and low oxygen tension, for example. Mechanical stress can be performed in vitro, in vivo, ex vivo, in vitro followed by in viva, or a combination thereof. In an embodiment, the differentiation will be 25 initiated n vitro, and the chndrocyte-like cells seeded in the matrix will be then implanted viv and continue to grow and differentiate. The inert structure is intended to provide a physiologic fading regimen to induce in vivo differentiation of HDFs, in specific aspects of the invention. in specific aspects of the invention, cells are induced to undergo 30 differentiation into chronrocytes or chondrocyte-like cells, Such differentiation may occur prior to in va delivery, such as on a scaffold, or subsequent to delivery in vivo. In specific embodiments, the cell is subjected to conditions to facilitate differentiation 37 into chondroc s . In a further specific embodiment, a condition comprises mechanical stress. Regulation of genes by mechanical forces has been studied extensively for vascular endothelial celIs and chondrocytes that are obviously subjected to high fluid shear or pressure load. .n specific embodiments of the 5 invention, mechanical stress stimulates chondogenic differentiation of HDFs. Such mechanical stress may be of any kind, although in specific embodiments it comprises hydrostatic pressure and/or fluid shear stress. In additional specific embodiments, the stress is constant or intermittent In the present invention, mechanical stress, especially cyclic hydrostatic 10 pressure and shear fluid stress induce chondrogenic differentiation of fibroblasts seeded in a three dimensional matrix. The choice of the co-culture conditions to stimulate the chondrogenic differentiation of HDFs is based on data known in the art. Different exemplary factors such as high cell density culture, culture with BMP2 and ascorbic acid, culture in low oxygen tension are known to stimulate chondrogenesis 15 and are used solely as examples in the invention as co-factors in addition to the mechanical stress, Chondrocytes from intervertebral discs are difficult to harvest. The autologous cells are obtained from the patient's disc and so requires an invasive procedure (back surgery) to perform a biopsy. If cells are harvested from a healthy disc, it 20 jeopardizes the functioning of a normal disc, If cells are harvested from a damaged disc during the discectomy, it pro ides abnormal cells from a degenerated tissue. Moreover, chondrocytes are difficult to expand in culture because they de-differentiate. Chondrocytes from other cartilages such as the elastic cartilage from the ear is easy to harvest but produces only hyaline cartilage and not fibro-cartilage as in the disc. Stem 25 cells that are usually used for tissue engine ring also have some disadvantages, because they require a bone marrow biopsy. A large quantity of cells is needed for tissue engineering, and it is difficult to obtai efficient quantity of adult stem cells. The rationale for using autologous HD s as a means of cell sourcing follows from the following: 1) HDFs can be noninvasively harvested front a punch biopsy as 30 little as a 3.0mm diameter circular skin specimen, for example; 2) the risk of contamination from another donor (such as Hepatits B Virus, Human Immunodeficiency Virus, Creutzfeldt-Jakob disease, etc) does not exist.; and 3) HDFs can expand easily in 38 culture and differentiate into chondrocyteiike cells under particular culture conditions. Other fibroblast populations could be used, such as tendon or ligament, for example. In an embodiment, autologous fibroblasts are preferred The choice of the culture conditions to stimulate the hondrogen 5 differentiation of HDFs is based on data known in the art. Different factors support chondrogenesis, such as, for example high cell density culture, culture with BMP-2 and ascorbic acid, and seeding cells in alginate matrix, In viro growth and/or differentiation of the cells in the cells/scaffold composition may comprise at least two or more days prior to use in vivo. In certain cases, the cells may be checked or monitored to ensure that at 10 least some of the cells are dividing, Cells that are not dividing and/or that are not affixed directly or indirectly to the scaffold may be removed. In other embodiments, HDEs are embedded in hydrogel that in specific embodiments is a natural hydrogel such as collagen, hyaluronic acid (HA), a combination of collagen/HA, alginate chitosan; a synthetic hydrogel such as 15 poly(ethyleneoxide) (PEO), poly(vnyl alcohol) (PVA). poly(acrylic acid) (PAA), poly(propylene frmarate-co-ethylene glycol (P(PF-co-EC), arid poly'peptides, or other biodegradable polymers such as poly(Llactic acid) (PLA), poly(glycolic acid) (PGA), poly-lactic-co-glycol acid (PLGA); or a combination of any of these above mentioned polymers. A cycli hydrostatic compression is then applied using any 20 suitable in vitro bioreactor in the art. VL Methods of Repairing Damaged Cartilage In certain embodiments, the invention includes methods of repairing any 25 darnaged cartilage, although in particular aspects the cartilage is in an intervertebral disc or any joint. Generally, for disc embodiments when an intervertebral disc must be removed from between two adjacent vertebrae, e.g. in the lumbar spine, it is less invasive to surgically proceed posteriorly from the back of the patient. This minimally invasive procedure allows one to proceed with the curettage of the inter-sonatic space through a 30 small aperture within the annulus (tenotomy) for removing the degenerated fragm-ent of the disc's nucleus. As the annuls fenestration is smal the present invention provides an intervertebral construct that is slid through the aforementioned incision and then expanded 39 into the room generated by the nucleus removal within the inter-somatic space. The removal of the damaged disc ani the installation of the construct are performed in the same posterior approach. As mentioned above, the inert structure is made of two expandable balloons 5 "1" and "E". At the rest position, the two balloons "1" and "E" are fiat, deformable, shaped and fit one another. Once the balloon "I" is inside the balloon "E" they are both installed into the intervertebral space through the annulus aperture, and then successively inflated so as to define two distinct volumes (V: > V 1 ) of shapes (spherical, cylindrical, conical, etc,) depending on the contour of the inter-vertebral cavity and the load sharing, for 10 example. In particular aspects, the first balloon to be filled is the internal balloon "I" regardless of the volume of the remaining cavity, Volume V 1 represents the core of tle construct that receives and houses the living core. Once filed with the living core, the balloon "I" is hermetically sealed, That is, once the envelope "I" is placed. into the 15 envelope "E", both are positioned into the intervertebral space through the annulIs aperture, and then successively inflated. "I" is the first to be instrumented with the implantation of the living core, and then sealed. Then, the external balloon "E" is inflated with the media solution until its volume VE gets a contour that mates with, or follows, the inner surface of the remaining part of the natural disc after the curettage thereof This 20 inner surface of the remaining disc can be either the remainder of the nucleus tissue or the inner wall of the natural annulus, depending how extensive the curettage has been performed. In a second embodiment to install the intervertebral construct, "E" is positioned into the intervertebral space, then "I" is placed into "E", and they are both 25 filled successively as aforementioned. In a third embodiment, "E" is positioned into the discectomy cavity, then the pre-encapsulated living core is placed into "E", and then "E" is filled with the media. The volume of the cavity resulting from the discectomy may be evaluated prior to the installation of the external balloon "E" such that proper fluid volume can be 30 selected and injected. The cavity volume could be, for instance, measured by introducing a fluid (e.g., water) therein, until the cavity is filled therewith, and by then withdrawing 40 the fluid from the cavity by way of a syringe, thereby substantially exactly measuring the cavity's volume, In certain aspects, the inert structure composition depends on the choice of tissue engineering system that relies on material of fabrication, pare characteristics, 5 absorbability and mechanical properties, for example, such as non-degradabl polymers, degradable polymers or naturally derived hydrogels (for example collagen, fibrin, agarose, alginate, etc.), The living core or compartment (V 1 ) is made of chondrocyte-like cells derived from autologous Human Dermal Fbroblasts (HDFs), for example, such as those 10 harvested from skin of the patient and seeded in a scaffold (such as alginate beads, or micofluidic scaffold, or any other polymric caffold) and fed from the supportive compartment (VE). The advantage of this hybrid construct combining both an inert biomaterial acting as a nutrients delivery system and living cells easily harvested from skin is that it is capable of selfmaintenance or remodeling and may restore the disc 15 function using a minimally invasive posterior surgical approach. Volume VE, is defined as the space that separates layer "E" from layer "I" that comprises nutrients and growth factors (media) to be delivered to the cells (delivery system) This volume can be the result either of its filling by the liquid media, or its swelling from its wall (expandable h ydrophilic biomaterial as hy rogel, for instance) after having been hydrated (the media is 20 made of a high ratio of water). Growth factors may be delivered through the semi-permeable internal membrane "I". Example of growth factors include, for example, cartilage-derived morphogenetic protein (CDMP), bone morphogenetic proteins (BMPs), transforming growth factor beta (TGF-P), and insulin growth factor one (IGF-1), fibroblast growth 25 factors (FFs), platelet-derived growth factor (PDGF), for example. Mechanical strain, such as high fluid shear and/or pressure load, is transferred on internal layer "I", and therefore on V 1 through the external layer "E" and the external area V. This mechanical strain induces chondrogenic differentiation of the cells inside the internal layer, 30 The draining system is then installed, wherein each catheter exiting from the intervertebral space through the tenotomy aperture is carefully kept away from the adjacent root, or at least positioned along the root without any harmful conflict.

41 A trans-muscular path is performed using an introducer from the operative site to a subcutaneous location distant from the skin aperture. Each tube is "tunnelized" into the aforementioned muscular path then connected to the corresponding reservoir. The two reservoirs are distant from the medial skin incision, positioned at 2 or 3 centinmeters from the medial line, sub utaneously placed so that they are easily palpable and identifiable Each skin incision is closed. As it is usual to proceed after such minimally invasive posterior approach, the patient is asked to stand up as early as the day after the surgery, and can begin to walk again. Therefore, the implant receives the right cyclic hydrostatic pressure regimen un der 10 physiologial load, which is critical for HDFs growth and conversion, Periodically, such as one or more fimes per week or month, the media may be changed The drinking system allows one to provide the volume VE with the appropriate amount of new media in order to continue to supply the living core, but also to maintain the adequate olune and therefore the right press sure regimen. The individual lies face 15 down Each reservoir is simultaneously pun cured with a needle. A syringe is plugged in on each of these need es and the new media is slowly injected by pushing down the piston while the same mount of fluid is simultaneously removed from the other syringe by pulling up, so that the internal pressure remains almost the same, and avoids the volume VE to collapse, or, at the contrary to deliver too high of a pressure to the volume V. 20 which could cause irreversible damage to the living core. The procedure is stopped when the color and the aspect of the exiting fluid has becorne identical to the entering fluid, for example. Samples from the removed used media may be taken for bacteriological, pathological and chemical purposes. When the living core is capable of self maintenance and has filled the room of 25 the discectomy, both tubes and reservoirs can be removed. Alternatively, only one or both reservoirs can be removed under local anesthesia while the tubes are tied at their distal end. In another alternative embodiment, both can be let in place, In specific embodiments, a follow'up MRI is performed, such as within weeks or months of the surgery (for example, about (weeks after surgery) to assess the 30 graft growth and to document the disc healing. In another embodiment, the engineered living core is pre-encapsulated and released as aforementioned.

42 These foregoing functions are provided by the inert structure of the invention that relies on two concentric membranes with two different skills, The external cnveop is mechanically able to maintain disc height under loading; is inflatable (in order to be implanted hrough a minimal invasive posterior approach and receive the media 5 solution); is resilient (to transfer load sharing onto the graft); is expandable (to alow its sweling and fill the cavity resulting of the discectomy); is hernetic (to avoid any leak of the he spinal canal, or recidivism of herniation through the annulus defect -tenotomy-); is biodegradable (the envelop resorbs to allow the graft to reconnect with the natural remaining disc); and is biocompatible (to minimize 10 inflammatory reaction). It may be drained with one or several catheters) connected to one or several Rickham reservoir(s) subcutaneously inserted at the end of he surgical procedure, for example. These reservoirs are intended to remove any toxic wastes accumulated with the metabolism (free radicals or lactic acid, for example), as well as any other cellular 15 scraps consecutive to the growth. They also allow providing the volume VE with the appropriate amount of new media in order to continue to supply the living core, but also to maintain the adequate volume and therefore the right pressure regimen. They are removed when the living core is capable of self maintenance and has filed the room of the discectomy. 20 It should be noted that the various components and features of the hybrid structure, as well as the method of repairing damaged cartilage and method for growing HDFs into chondrocyte-like cells described above, can be combined in a variety of ways so as to provide other embodiments within the scope of the invention, 25 VIL Alternative Embodiment of the Invention In another embodiment, instead of having two generally spherically (for example) concentric envelopes, the device could be made of a unique external envelope "E" with the same aforementioned characteristics (especially expandability and/or 30 inflatable properties), and receives an unwrapped living core (non-encapsulated nor wrapped uip with a membrane). Actually, in this embodiment, this living core is a cell 43 matrix cnstrct and is directly positioned into "E". Then, the volume V is expanded with the media lquid solution til mating the cavity. Thus , in another embodiment, instead of having two "concentric" balloons, the device is comprised of a unique external batloon "E" with the same aforementioned 5 characteristics (especially expandability and/or inflatable properties) for housing the engineered living core. Once the lving core is released within the membrane "E", the volume V is expanded with the nedia lquid sohuion until the membrane "E" reaches the boundaries of the cavity. Neither barrier nor membrane wrap up the graft anymore. While the media is consumed, the living core expands to the inner wall of the 10 balloon/Iayer/membrane "E". The envelop resorbs and the graft reconnects with the natural remaining disc. EXAMPLES 15 The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those f skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice, However, those of 20 skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. EXAMPLE 1 25 EXEMILARY MATERIALS AND METHODS Exemplary embodiments of materials and methods for use in the invention are described in this Example. 30 44 Cell Culture Wile autologouis HFIUs harvested from the patient are us,.ed to construct the implant, preliminary studies are performed using neonatL foreskin fibroblasts, as there are convenient sources of c-l for experimental purpose, fr example. Further studies using autologous cells harvested from the patient are performed to demonstrate that the procedure works with these cells. Neonata foresk-ii HDFs are obtained from Cascade Biologics (Porland, Oregon) and expanded in vitro with DMEM (Invitrogen, Carlsbad, CA, USA), 10 conning 10% FBS (Invitrogen) and antibiotics. Suspensions of HDFs are seeded in alginate or in monolayer culture as described below, To generate alginate gel cultures, cells are suspended at high density (107 cells/m) in 2% wt/voI medium viscosity alginate (Signia-Aldrich, St. Louis, MO), and 25mL droplets are cros'slinked in 100 n I CaCl 2 , 0.9% NaCl solution. The resulting 15 alginate beads arc then washed extensively in DMEM containing 10% FBS and antibiotics. Alginate bcads are immersed in DMEM containing 10% FBS and antibiotic supplemented with 100 ng/mI recombinant human Bone Moirphogenic Protein2 (' MP-2) and 50 mg ascorbic acid, Such conditions: high cell density and culture with BMP-2 and ascorbic acid have been chosen because they are known to 20 stimulate chondroinduction (Watt, 1988; Dozin et al, 1992; Sullivan et aL, 1994; Denker et al, 1999; ZurNieden et al., 2005; Zhou et a., 2004). To generate monolayer cultures, HDFs are seeded in plastic flasks with an oxygen level of 20% in DMEM containing 10% FBS without BMP-2 and without ascorbic acid. These cells serve as control 25 Culture under 5% and 20% 02 Cell-embedded a5ginate beads are kept under an atmosphere of 5%02, 5% C0 2 , and 90% N 2 in an Or/C2rregulated incubator (lox oxygen tension or under 30 20%02, 5% CO 2 , and 75% N 2 in an C0 2 -regulated incubate (atmospheric oxygen 45 tension) and cultured for 3 weeks, Then, assessment f chondrogenic differentiation is performed (see Example 3, for example). Hydrostatic compression 5 Cell-embedded alginate beads are divided into pressurized and control groups, and those from each group placed in separate flexible polyethylene/nylon bags permeable to oxygen and carbon dioxide. The bags are filled with 15 ml of medium and are heat-sealed to excide all air. 10 The bags in the pressurized group are placed within a newly developed device designed for the application of cyclic hydrostatic compression (Elder et al., 2005). This device allows the comparison of loading regimes in a wide physiologic range under an equal three-dimensiona culture conditions. It consists of a large cylindrical stainless steel base connected to a lid by bolts that compress an intervening 15 o-ring. A hydraulic cylinder is welded to the lid so that its interior is continuous with that of the chamber. The cylinder and chamber are completely filled with water, so that rapid hydrostatic compression is achieved by a force (generated by a MTS servohydraulic testing machine) applied to the cylinder's piston. A stable 37"C is maintained by immersing the chamber in a temperature-regulated circulating water 20 bath. Bags in the control group a p ilaed in a separate, water-filled stainless steel chamber in the same water bath, The magnitude and frequency of the applied press-re are chosen to be within the physiological ranges (Mow et al., 1992) that have previously been demonstrated to stimulate chondrogenic differentiation of multipotential mesenchyma 25 cells (Eider el at, 2005) and redifferentiation of dedifferentiated chondrocytes (Donm et al, 2000). Short and long duration pressurization are tested and a suecessf chondroinductive hydrostatic pressurization model is determined between the short and long duration hydrostatic pressurization by quantitative and qualitative assessment of chondrogenesis. 30 46 An exemplary regimen comprises the following: 1.0 Hz sinusoidal hydrostatic compression waveform with a minimum applied pressure of 0.3 MPa and a maximum 5.0 MPa. For short duration pressurization, the cells are pressurized 1h/day for 7 days. For long duration 5 pressurizationi, the cells are pressurized 4h/day for 7 days. Each day, immediately upon completion of loading, cultures are removed from the pressure vessel and returned to a water bath inside the tissue culture incubator. Cell viability and chondrogenic differentiation of HDFs under hydrostatic cyclic compression and cultured under specific conditions (such as hypoxia, chondrogenic 10 medium, high cell density) arc assessed using techniques standard in the art In another embodiment, the conversion of fibroblast cells into chondrocytes is induced by hydrostatic cyclic pressure and shear stress, In this case, the cells are seeded into a microfluidic scaffold. Assessment of chondrogenic differ-etiation is performed (see Example 3, 15 for example). EXAMPLE 2 ASSESSMENT OF CELL VIABILITY OF HDFS IN ALGINATE BEADS 20 The viability of HDFs in alginate beads cultured in chondrogenic medium is tested by light microscopy and/or viability test, in specific aspects of the invention. Light microscopy is employed to study morphology and proliferation of 1Ds, In an exemplary viability test, alginate beads are dissolved in dissolving-buffer (0.55 M Na 25 Citrate., 1.5 M NaCl, and 0,5 M EDTA), cells are centrifuged, and the pellet is treated with collagenase for 1 h, Cells are resuspended in DMEM, and viability is determined using a Neubauer chamber and the trypan blue exclusion method, for example. 30 47 EXAMPLE 3 ASSESSMENT OF CHONDROGENIC DIFFERENTIATION 5 In specific embodinents, HDFs are characterized by the production of collagen of type 1, III and V, while chondrocytes are characterized by the production of collagen of type II, IX, XI and he production of sulfated proteoglycaus. Chondrogenic differentiaton is assessed by measuring sulfated glycosaminoglycan (sUAG ) content and collagen I et II production by western 10 blotting. The rate of collagen synthesis is measured by [ 3 H-proline incorporation. Total DNA and sGAG content Cells in alginate beads are recovered from the alginate using 55 mM sodium citrate, 0.9% NaCl solution. Then the cells are py in 300 p of 0.5% v/v 15 Nonidet P-40 buffer (50mM Tris-Cl, 100 mM NaCl, 5 mM MgCl 2 ), The lysate is transferred to microcentrifuge tubes, spun, and DNA is measured n a 100 p supernatant aliquot using the Hoescht dye method (DNA Quantification Kit, Sigma, St. Louis, MO) with calf thymus DNA as standard. The remaining lysis buffer is removed and sGAG digested in 100 pl of 2% v/v papain, 20 mM sodim acetate (pH 20 6) overnight at 60*C. Total sGAG content is then measured by the dimethy methylene blue precipitation method (Blyscan Glycoamninoglycan Assay, Biocolor, Ltd.) using chondroitin 4-sulfate purified from bovine trachea as standard. For each sample, the sGAG content is normalized to the DNA content. 25 Western Blot for collagen type I and type II Five beads of each sample are dissolved in 400 ml buffer (55mIM sodium citrate, 150 mM NaCl). For collagen solubilization, 100 p1 of 0,25 M acetic acid and 100 1 pepsin solution ( ngml 50 mM acetic acid: P-6887, Sigma) are added and the 30 mixture kept at 4* C for 24h. Then, 100 pl of a 10x stock solution TBS (I M Tris, 2M 48 NaCl and 50 mM CaCI 2 , pH 8) and 100 ml pancreatic elastase (1mg/mi TBS; Sigma E-6883) are added and samples incubated for 30 mi at 37"C. The samples are centrifuged for 10 min at 9000xg. The supernatant is collected, 25 ld of bovine collagen type I, bovine collagen type I (Sigma) or sample (each containing 5 mg 5 total protein; quantification with Bio-Rad protein assay) are nixed with 6 d sample buffer, denatured for 5 min at 95*C, and loaded on a 7% acrylamide gel. Electrophoresis is performed. Gel is transferred onto the blotting membrane. The membrane is blocked overnight in blocking-buffer (10% milk powder in TBST buffer) and then incubated with a mouse monoclonal antibody anti-collagen type I antibody 10 (COL-1, ab 6308, Abcam Inc) or a mouse monoclonal antibody anti-collagen type Il (5B2.5, ab3092, Abcam nc) overnight at 4T The membrane is washed with TBST buffer. The goat antimouse biotin conjugated secondary antibody is added (1:500) for 1 h fol lowed by streptavi din-HRP at 1:1000 dilu ion for I hI B ot is developed using ECL from Amersham. Measure of [RJ-proane incorporation In alginate beads in which the rate of collagen production is determined, the medium is removed and replaced with DMEM supplemented with 10% FBS, 20 antibiotics, 25 mg ascorbic acid, [H] proline at 10 pCi/ml, and 100 pg/ml p-amino propionitrile (p-APN) to inhibit collagen cross-link fonation. After a 24 h incubation period, the incorporation of [ 3 H] proline into collagen is measured. Beads are digested at 65"C overnight in 1 ml papain solution [0.125 mg/nl (2,125 units/ml, Sigma), 0.1 M Na 2

HPO

4 , 0.01 M EDTA, pH 6,5]. 200 ml pf each sample are added to 2 ml of 25 scintillation fluid and measured using a scintillation counter. 500 ptl of each sample is mixed with 500 pl PBS and used to detennine the DNA content. Samples and blanks (containing 1 ml PBS) are treated with an ultrasonic beam for 15s. 0.5 ml of RNAse and 0.5 ml pronase are added and incubated at 37"C for 30 min. Then, 0.5 ml of ethidium bromide is added, samples are incubated 30 for 30 min, and they are measured with a flu. orometer. [tHj-proline incorporation is normalized to total DNA content.

49 EXAMPLE 4 EXEMPLARY DESIGN OF STUDIES 5 In specific aspects of the invention, cell viability and chondrogenic differentiation of HDIs seeded are determined in three-dimensional alginate bead cultures. In particular, cell viabili and bondrogenic differentiation of HDFs seeded in alginate beads and cultured in a dhondrogenic medium (medium supplemented with 10 BMP-2 and ascorbic acid) under 20% 02 are compared to HDFs in monolayer cultures in DMEM with 10% FBS under 20% 2 using the exemplary method described above. In another aspect of the invention, the effects of oxygen tension on the differentiation of HDFs cultured in alginate beads is determined. HDs seeded in 15 alginate beads in the chondrogenic medium are cultured for 3 weeks n 2 different oxygen tension: 1) low oxygen tension of 5% 02, 5% CO 2 , and 90% N 2 in an O /COrreg lated incubator; and 2) atmospheric oxygen tension of 20% 02 5% CO2, and 75% N 2 in an C0rregulated incubator. Chiondrogenic differentiation s compared using the exemplary method 20 described above, In an additional embodiment of the invention, the effects of hydrostatic compression on the differentiation of HDFs cultured in alginate beads are detennined, HDFs seeded in alginate beads in the chondrogenic medium are subjected to different stimuli: 1) 1h/day for 7 days hydrostatic pressure (1,0 Hz sinusoidal hydrostatic 25 compression min 0.3 MPa max 5.0 Mpa) and 20% O20; 2) 4h/day for 7 days hydrostatic pressure (1.0 Hz sinusoidal hydrostatic compression min 0.3 MPa max 5.0 Mpa) and 20% 02; 3) 1 h/day for 7 days hydrostatic pressure (1.0 Hz sinusoidal hydrostatic compression min 0.3 MPa max 5,0 Mpa) and 5% 02; 4h/day for 7 days hydrostatic pressure (1.0 Hz sinusoidal hydrostatic compression min 0.3 MPa max 5.0 Mpa) and 30 5% 02.

50 Chondrogenic differentiation may be assessed using the exemplary method described above. REFERENCES 5 All patents and publications mentioned in the specifeaton are indicative of the level of those skilled in the art to which the invention pertains. All patents and publications are herein incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be 10 incorporated by reference. PATENTS US, Patent No. 6,489,165 15 U.S. Patent No. 6,627,422 PUBLICATIONS Barry F, Boyton RE, Li u B et al, Chondrogenic differentiation of 20 mesenchymal stem cells from bone marrow: differentiation-dependent gene expression of matrix components. Exp Cell Res 2001 (268):189-200. Carter DR, Orr TE, Fyhrie DP, Schurman D, Influences of mechanical stress on prenatal and postnatal skeletal development. Clin Orthop 1987(219):237 250, 25 Cui, L., Yin, S., Deng, CL., Yang, GH., Chen, FG., Liu, W., Liu, DL, Cao, YL. Cartilage-derived morphogenetic protein I initiates chondrogenic differentiation of human dermal fibroblasts in vitro. Zhonghua Yi Xue Za Zhi. 2004 Aug 2;84(15):1304-1309, Denker AE, Haas AR, Nicoll SB, Tuan RS. Chondrogenic differentiation 30 of urine C3H10T1/2 multipotential mesenchymal cells: . Stimulation by bone 51 morphogenic protein-2 in high-density micromass cultures Differentiation 1999;64:67-76 Domm C, Fay J, Schunke M, Kurz B. Redifferentiation of dedifferentiated joint cartilage cells in alginate culture. Effect of intermittent hydrostatic pressure and 5 low oxygen partial pressure Orthopade 2000 Feb; 29(2):91-99 Dozin, R. Quarto, G. Campanile & R. Cancedda: In vitro differentiation of mouse embryo chondrocytes: requirement for ascorbic acid. Eur J Cell Biol 1992;58, 390-4 Elder SH, Fulzele KS, McCulley WR. Cyclic hydrostatic compression 0 timulates chondroinduction of C311/10Tl/2 cells. Biomechan Model Mechanobiol 2005(3): 141-146 French MM, Rose S, Canseco J and Athanasiou KA. Chondrogenic differentiation of adult dermal fibroblasts. Annals of Biomedical Engineering 2004; 32 (1):50-56, 15 Hauselmann HJ, Aydelotte MB, Schumacher BL, Kuettner KE, Gitelis S.H,Thonar EJ. Synthesis and tumover of proteoglycans by human and bovine adult articular chondrocytes cultured in alginate beads. Matrix 1992(12): 116-129. Kessler, D., Dethlefsen, S., Haase, L, Ploni"3n, M, Hirche, F., Krieg, T, Eckes, B. Fibroblasts in mechanically stressed collagen lattices assume a "synthetic" 20 phenotype. J. Biol. Chem. 2001 Sept; 276(39)36575-36585. Majumdar MK, Wang E and Morris EA. BMP-2 and BMP-9 promotes chondrogenic differentiation of human multipotential mesenchymal cells and overcomes the inhibitory effect of IL-l, J. Cell. Physiol. 2001(189):275-284. Meisel, II, Alasevic, 0., Hutton, W., Ganey, T. Disc repair with 25 autologous chondrocytes: A pilot clinical study. European Cells and Materials. Vol, 10 Suppl. 3, 2005 (page 39) Watt FM. Effect of seeding density on stability of the dedifferentiated phenotype of pig articular chondrocytes in culture, J Cell Sci 1988; 89:373-378, Mizuno H, Roy AK, Vacanti CA, Kojima K, Ueda M, and Bonassar LJ. 30 Tissue-engineered composites of annulus fibrosus and nucleus pulposus for intervertebral disc replacement. Spine 2004 (29):1290-1298.

52 Mow VC, Ratcliffe A, Poole ARt Cartilage and diarthrodial joints as paradigns for hierarchical materials and structures. Biomaterials 1992; 13(2):67-97. Nicoll SB., Wediychowska, A,, Smith, NR., Bhatnagar, RS. Modulation of proteoglycan and collagen profiles in human dermal fibroblasts by high density 5 micromass culture and treatment with lactic acid suggests change to a chondrogenic phenotype, Connect. Tissue Res. 2001;42(l):59-69. T.A. Sullivan, B. Uschmann, R. Hough & P.S. Leboy: Ascorbate modulation of chondracte gene expression is independent of its role in collagen secretion. J Biol Chem 1994; 36, 22500-6 10 Watt FM4. Effect of seeding density on stability of the dedifferentiated phenotype of pig articular chondrocytes in culture. J Cell Sci 1988; 89:373-378 Zhou S. Glowacki J, and Yates KE. Comparison of TGF-b/BMP pathways signaled by demineralized bone powder and BMP-2 in hunan dermal fibroblasts. J Bone Miner Res 2004; 19:1732-1741, 15 Zur Nieden NI, Kempka G, Rancourt DE, Akhr HJ, Induction of chondro-, osteo- and adipogenesis in embryonic stem cells by bone morphogenic protein-2: effect of cofactors on differentiating lineages. BMC Dev Biol, 2005;Jan 26 (5):1-15 Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can 20 be made herein, without departing from the spirit and scope of the invention as defined by the appended claims. Moreove the scope of the present application is not intended to be limited to the particulJar embodiments of the process, machine, manufacture, composition of matter, means, methods and steps described in the specification. As one of ordinary sll in the art will readily appreciate from the 25 disclosure of the present invention processes machines, manufacture, compositions of matter. means, methods, or steps, presently existing or later to be developed that perform substantially the same fuction or achieve substantially the same result as the corresponding embodiments described herein may be utilized according to the present invention. Accordingly, the appende claims are intended to include within their 30 scope such processes, machinesnanufacture compositions of matter, means, methods, or steps.

Claims (61)

1. An implantable device, comprising: a cells/scaffold composition; and an encapsulating device that comprises: 5 a first membrane having an inside and an outside; a second membrane having an inside and an outside, wherein the first membrane is encapsulated inside the second membrane; 10 a first volume disposed inside the first membrane; a second volume that is disposed outside the first membrane and that is disposed inside the second membrane; and 15 a structure for adding fluid to the second volume, removing fluid from the second volume, or both, wherein the cells/scaffold composition is disposed inside the first membrane and the first 20 membrane has one or more of the following characteristics: semi-permeable; biocompatible; biodegradable; and 25 resorbable, wherein the second membrane has one or more of the following characteristics: 54 biocompatible; hermetic to fluid; permeable to oxygen; resorbable; 5 biodegradable; and expandable.

2. The implantable device of claim 1, wherein the scaffold is comprised of a synthetic polymer, a natural hydrogel, or a synthetic hydrogel.

3. The implantable device of claim 2, wherein the synthetic polymer is 10 polyglycolic acid, polylactic acid, polylactic-co-glycolic acid, poly-s caprolactone, or poly(glycerol-Sebacate) (PGS).

4. The implantable device of claim 2, wherein the synthetic polymer is a polyphosphazene, a polyanhydride, or a poly(orthoester).

5. The implantable device of claim 2, wherein the natural hydrogel comprises 15 collagen, hyaluronic acid, alginate, agarose, chitosan, fibrin, gelatin, or a copolymer thereof

6. The implantable device of claim 2, wherein the synthetic hydrogel comprises poly(ethylene oxide), poly(vinyl alcohol), poly(acrylic acid), poly(propylene fumarate-co-ethylene glycol), or a copolymer thereof 20

7. The implantable device of claim 1, wherein the cells are chondrocyte cells or chondrocyte-like cells.

8. The implantable device of claim 1, wherein the chondrocyte cells or chondrocyte-like cells secrete a molecule selected from the group consisting of aggrecan, type II collagen, Sox-9 protein, cartilage link protein, and 25 perlecan. 55

9. The implantable device of claim 1, wherein the cells were differentiated from fibroblast cells and/or stem cells.

10. The implantable device of claim 9, wherein the fibroblast cells are dermal fibroblasts, tendon fibroblasts, ligament fibroblasts, synovial fibroblasts, 5 foreskin fibroblasts, or a mixture thereof.

11. The implantable device of claim 1, wherein the first membrane is comprised of a biodegradable, biocompatible, and resorbable polymer.

12. The implantable device of claim 1, wherein the first membrane is comprised of a polyacrylate, a polyvinylidene, a polyvinyl chloride copolymer, a 10 polyurethane, a polystyrene, a polyamide, a cellulose acetate, a cellulose nitrate, a polysulfone, a polyphosphazene, a polyacrylonitrile, a poly(acrylonitrile/covinyl chloride) or a derivative, copolymer or mixture thereof.

13. The implantable device of claim 1, wherein the first membrane is generated 15 by polyelectrolyte complexation.

14. The implantable device of claim 1, wherein the second membrane is comprised of polyglycolic acid (PGA), polylactic acid (PLA), polylactic-co glycolic acid (PLGA), poly-s-caprolactone (PCL), polyurethane (PU), polydioxanone (PDO), a polyethylene, poly (glycerol sebacate) (PGS), or a 20 derivative, copolymer, or mixture thereof.

15. The implantable device of claim 1, wherein the rate of resorbability of the second membrane is slower than the rate of resorbability of the first membrane.

16. The implantable device of claim 1, wherein the implantable device 25 comprises one or more nutrients, growth factors, and/or medicaments. 56

17. The implantable device of claim 16, further defined as comprising a basal cell culture medium comprising the one or more nutrients, growth factors, and/or medicaments.

18. The implantable device of claim 17, wherein the medium is supplemented 5 with Fetal Bovine Serum (FBS), ascorbic acid, and/or dexamethasone.

19. The implantable device of claim 16, wherein the nutrients, growth factors, and/or medicaments are present in the scaffold, the first volume, the second volume, or a combination thereof

20. The implantable device of claim 16, wherein the growth factor is selected 10 from the group consisting of bone morphogenetic protein 2 (BMP-2), BMP 4, BMP-6, BMP-7, cartilage-derived morphogenetic protein (CDMP), transforming growth factor beta (TGF-3), insulin growth factor one (IGF-I), fibroblast growth factors (FGFs), basic fibroblast growth factor (bFGF), FGF-2, platelet-derived growth factor (PDGF), and a mixture thereof 15

21. The implantable device of claim 16, wherein the medicament is further defined as one or more of an antibiotic, antifungal agent, or antiviral agent.

22. The implantable device of claim 1, wherein the structure comprises one or more tubes.

23. The implantable device of claim 1, wherein the structure comprises one or 20 more catheters and/or one or more reservoirs.

24. The implantable device of claim 1, wherein the structure is further defined as comprising one or more of: a first tube; a second tube; 25 optionally, a first reservoir; and optionally, a second reservoir. 57

25. The implantable device of claim 24, wherein the first and second tubes respectively comprise first ends positioned within the second volume, wherein the first and second tubes respectively comprise second ends connected to first and second reservoirs, or both. 5

26. The implantable device of claim 24, wherein the first and/or second tubes are comprised of the same material as the second membrane.

27. The implantable device of claim 24, wherein the first and/or second tubes are comprised of silicone rubber.

28. A method of repairing damaged cartilage in a joint of an individual, 10 comprising delivering a device in accordance with claim 1 to the respective joint of the individual.

29. The method of claim 28, wherein the method further comprises preparing the cells/scaffold composition under suitable ex vivo conditions.

30. The method of claim 29, wherein preparing the cells/scaffold composition is 15 defined as subjecting one or more cells to a scaffold under suitable conditions.

31. The method of claim 29, wherein the method further comprises preparing the cells/scaffold composition for no less than about two to three days.

32. The method of claim 29, wherein the suitable conditions allow proliferation 20 of the cells.

33. The method of claim 29, wherein the suitable conditions allow the stimulation of chondrogenic differentiation.

34. The method of claim 28, wherein the cells are chondrocytes or chondrocyte like cells derived from fibroblasts or stem cells. 58

35. The method of claim 34, wherein the fibroblasts cells are dermal fibroblasts, tendon fibroblasts, ligament fibroblasts, synovial fibroblasts, or foreskin fibroblasts.

36. The method of claim 34, wherein the fibroblasts are dermal fibroblasts. 5

37. The method of claim 29, wherein the suitable conditions are further defined as being under high density micromass culture, being under low oxygen tension (between about 1.0%-7.5%), being under mechanical stress, and/or being fed by a medium supplemented with growth factors, ascorbic acid, and/or dexamethasone. 10

38. The method of claim 28, wherein the cells/scaffold composition is subjected to mechanical stress.

39. The method of claim 38, wherein the mechanical stress is defined as hydrostatic pressure, fluid shear stress, or a combination thereof.

40. The method of claim 38, wherein the mechanical stress is intermittent. 15

41. The method of claim 38, wherein the mechanical stress is fluid shear stress and the scaffold is microfluidic scaffold.

42. The method of claim 28, wherein the delivering step is defined as implanting the device using minimally invasive surgery.

43. The method of claim 28, wherein following implantation of the device into 20 the individual, the second membrane is inflated to fill a void in the joint.

44. The method of claim 28, wherein the joint is an intervertebral disc and wherein prior to delivery of the device to the intervertebral disc of the individual, at least part of an endogenous intervertebral disc was removed from the individual. 25 59

45. The method of claim 28, wherein the joint is an intervertebral disc, a knee, a shoulder, an elbow, a hip, or a temporo-mandibular joint.

46. The method of claim 28, wherein at least part of the second volume is exchanged. 5

47. The method of claim 28, wherein the structure of the device comprises: a first tube having first and second ends, said first end of the first tube disposed within the second volume; a second tube having first and second ends, said first end of the second tube disposed within the second volume; 10 a first reservoir; and a second reservoir, wherein following delivery of the device to an intervertebral disc in the individual and following inflation of the second membrane, the second ends of the first and second tubes are respectively connected to the first and 15 second reservoirs.

48. The method of claim 47, wherein the first and second reservoirs are subcutaneously positioned in the individual.

49. The method of claim 28, further comprising sealing the first membrane, sealing the second membrane, or both. 20

50. The method of claim 47, further comprising removing at least part of the second volume through the first reservoir.

51. The method of claim 47, further comprising removing fluid from the first or second reservoir, delivering a fluid to the respective second or first reservoir, or concomitantly removing fluid from the first or second reservoir 25 and delivering a fluid to the respective second or first reservoir. 60

52. The method of claim 47, wherein the cells/scaffold composition is inserted into the first membrane prior to delivery of the device into the individual or wherein the cells/scaffold composition is inserted into the first membrane subsequent to delivery of the device into the individual. 5

53. The method of claim 47, wherein the first membrane is inserted into the second membrane prior to delivery of the device into the individual or wherein the first membrane is inserted into the second membrane subsequent to delivery of the device into the individual.

54. A method of preparing a cells/scaffold composition, wherein the cells are 10 chondrocytes or chondrocyte-like cells, comprising: subjecting cells capable of differentiating into a chondrocyte-like cell to the scaffold; subjecting the cells to mechanical stress; and optionally subjecting the cells to one or more growth factors 15 suitable for differentiation to a chondrocyte or chondrocyte-like cell.

55. The method of claim 54, wherein the mechanical stress is intermittent.

56. A kit comprising the device of claim 1, wherein the device is housed in one or more suitable containers.

57. The kit of claim 56, wherein the kit further comprises cells that are 20 chondrocyte cells, chondrocyte-like cells, or cells that are capable of differentiating to chondrocyte cells or chondrocyte-like cells.

58. An implantable device, comprising: a cells/scaffold composition encapsulated inside a membrane, said membrane having an inside and an outside; and 25 a structure for exchanging at least part of fluid that is inside the membrane, 61 wherein the membrane has one or more of the following characteristics: semi-permeable; biocompatible; 5 biodegradable; and resorbable.

59. A hybrid structure for cartilage repair, comprising: an encapsulating device comprising inert material; and a living core comprising chondrocyte-like cells, 10 wherein said encapsulating device encapsulates the living core.

60. An in vivo bioreactor for cartilage engineering, comprising a device that encapsulates cells, wherein said cells are capable of differentiating to chondrocytes or chondrocyte-like cells, wherein the encapsulation of said cells provides suitable conditions for in vivo growth and differentiation of 15 said cells, wherein said conditions comprise providing a physiologic loading regimen on said cells.

61. The bioreactor of claim 60, wherein the physiologic loading regimen comprises force from a spine of an individual.