WO2008003320A2

WO2008003320A2 - Three-dimensional cell scaffolds

Info

Publication number: WO2008003320A2
Application number: PCT/DK2007/000340
Authority: WO
Inventors: Michael Ulrich-Vinther; Carsten Stengaard; Kjeld SØBALLE; Brian Elmengaard
Original assignee: Region Midtjylland; Aarhus Universitet
Priority date: 2006-07-05
Filing date: 2007-07-04
Publication date: 2008-01-10
Also published as: WO2008003320A3

Abstract

The present invention relates to three-dimensional cell scaffolds that can be used for repairing or regenerating damaged tissue, for example bone and cartilage. The three-dimensional cell scaffold of the present invention comprises a scaffold material and a coating. The coating material comprises a biological agent that aids in the regeneration of cell growth. The present invention further relates to a method of producing three-dimensional scaffolds. Moreover, methods for producing ready-for use implants and methods for regenerating tissue are also within the scope of this invention.

Description

Three-dimensional cell scaffolds

All patent and non-patent references cited in the application, or in the present application, are also hereby incorporated by reference in their entirety.

Field of invention

The present invention relates to cell scaffolds that can be used to repair or regenerate damaged tissue for example bone, cartilage, tendon, ligament among others. The three-dimensional scaffold comprises a scaffold material and a coating. The coating comprises a biological agent. The present invention further relates to a method of producing such scaffolds. Additionally, the invention pertains to a method for producing a ready-for use implant and a method for regenerating tissue.

Background of invention A number of medical conditions are found which can be healed, improved or corrected using three dimensional cell scaffolds that serves as a support system for cells intended to grow and replace missing and/or damaged tissue. The medical conditions can vary from acute trauma to degenerative disease in which tissue structure and function are compromised or lost.

A three dimensional cell scaffold should possess sufficient mechanical strength to maintain its form and structure in response to the pressure exerted by the surrounding tissue upon implantation in situ and to the strain exerted on the scaffold by cells in the interior of the scaffold. The aim of the scaffold structure and cells in the interior of the scaffold or native cells of the surrounding tissue is to perform the function of the native cells or tissue that they are meant to supplement or replace. Consequently, an environment which allows for growth and differentiation of the cells of the scaffold into tissue should be provided by the three dimensional scaffold.

In some contexts it is desirable that the scaffold gradually dissolves as new cellular growth and tissue are developed for example in relation to cartilage defects. However, in other defects involving for example bone defects the scaffold should persist to allow for mechanical strength.

A number of scaffolds for implantation into a recipient are known from the literature. For example US 20040126405 discloses a scaffold inducing cell growth. The scaffold may be used in connection with defects of cartilage and bone. The scaffold is a three- dimensional structure and may be made from a number of polymers for example poly D, L lactic co-glycolic acid. The scaffold may be coated using for example hyaluronic acid. The purpose of coating is to promote adhesion of the scaffold when implanted. It is furthermore disclosed that the scaffold may be seeded with cells. Biologically active agents such as for example growth factors may be comprised in the scaffold.

US2000078077 discloses an implant comprising a biocompatible scaffold and tissue associated hereto. One example of scaffold material is chitosan. The associated tissue is adhered to the scaffold from where individual cells may colonise the scaffold. A biological effector may such as one or more growth factors may be included in the scaffold.

US20050107868 discloses a scaffold for tissue engineering of for example blood vessel. The scaffold may comprise a thermoplastic resin, wherein growth factors may be incorporated. The scaffold may be covered by yet another layer of material for example chitosan.

US20060039947 discloses an implant comprising a surface, a body and a varnish-like abrasion-resistant coating, wherein the coating is adapted to contact bone when implanted. The coating comprises a biodegradable polymer for example poly D, L- lactid acid and may comprise growth factors such as TGF-beta and IGF-1. The body is described as having a base material not being biodegradable as exemplified by stainless steel or titanium. Cells are not pre-seeded into/on the implant prior to implantation.

Presently a number of tissue scaffolds /cell scaffolds are available, however, a need for a tissue scaffold with optimal performance in satisfactorily replacing or regenerating damaged or lost tissue exists. Such scaffold should offer a biocompatible structure which retains adequate mechanical strength and provides optimal conditions for sustaining growth of cells which are seeded on and into the scaffold, or cells from the surrounding tissue which may infiltrate the scaffold.

Summary of invention The present invention discloses a three dimensional cell scaffold with superior characteristics for growth and differentiation of cells pre-seeded on/into the scaffold or for cells populating the scaffold in situ.

In one aspect the present invention relates to a three dimensional cell scaffold, comprising:

(i) at least one biocompatible scaffold material, (ii) at least one coating wherein said at least one coating comprises at least one polymer and/or at least one biological agent.

The three-dimensional cell scaffold offers superior scaffolds with improved regeneration and repair characteristics to be used for the regeneration, repair and healing of a number of injuries to for example bone, joints, and articular cartilage.

Another aspect of the present invention pertains to a method for producing a three- dimensional cell scaffold, comprising the steps of i) providing a three-dimensional cell scaffold ii) producing a mixture of at least one polymer and at least one solvent to form a volatile fluid iii) immersing the three-dimensional cell scaffold of i) into the mixture of ii) to coat the three-dimensional cell scaffold and iv) evaporating said solvent.

Similarly, a third aspect of the invention covers a three-dimensional cell scaffold formed by the steps as defined for the method of producing the three-dimensional cell scaffold.

The present invention also relates to a method for producing a ready-for-use implant as defined herein.

The three-dimensional cell scaffold of the present invention may be used in the regeneration of tissue that has been damaged and therefore one aspect of the invention relates to a method for regenerating tissue in a mammal in need thereof, comprising implanting the cell scaffold of the present invention into said mammal. In a particular aspect the present invention relates to a three-dimensional cell scaffold comprising i) chitosan sponge, and ii) a poly D,L-polylactic acid coating, wherein said coating comprises at least one growth factor.

Description of Drawings

Figure 1 shows TGFbetal -release kinetics from the PDDLA/TGFD1 -coated scaffolds (N=6 in all groups). The scaffolds were either uncoated (Ong TGFbI ), or coated with 50ng TGFbI or 500ng TGFbI The figure demonstrates TGFbI release from the scaffold during a period of 21 days. The growth factor release describes a biphasic kinetic.

Detailed description of the invention

The present invention relates to a three dimensional cell scaffold that finds use in replacing or regenerating damaged or lost tissue. The scaffold offers a biocompatible structure of sufficient mechanical strength. The scaffold provides conditions for sustaining cell growth of both cells seeded on and into the scaffold but also cells from the surrounding tissue which may infiltrate the scaffold. The conditions for sustaining cell growth are obtained by the inclusion of one or more biological agents, for example growth factors and/or hormones,

Cell scaffold

The three-dimensional cell scaffold may be of any material and/or shape that a) allows cells to attach to it (or can be modified by coating to allow cells to attach to it) and b) allows cells to grow in more than one layer and/or orientation. One aspect of the invention relates to a three dimensional cell scaffold, comprising: i) at least one biocompatible scaffold material, ii) at least one coating, wherein said coating comprises at least one polymer and/or at least one biological agent.

Biocompatible polymer The term 'biocompatible' is used to describe materials that are non-toxic to the host organism. Thus, biocompatible materials do not compromise the function of the host organism. During the initial seeding of cells into/on the scaffold it is important that the materials used for the production of the three dimensional scaffold are biocompatible. But also during the degradation of scaffolds or alternatively during long term in situ placement of the scaffold it is important that toxic degradation products do not occur or if they do occur the release of toxic compounds is sufficiently slow to avoid the building up of toxic compounds.

In the present invention the at least one biocompatible scaffold material is biodegradable, biostable or a combination thereof.

Biodegradable scaffold material

In one embodiment of the invention the at least one biocompatible, biodegradable scaffold material is a natural polymer. The natural polymer may be selected from the group consisting of alginate, cellulose, dextran, glycogen, lignin, gellan, gellan gum, hyaluronic acid, xanthan chitosan, agar, carrageenan and chitosan, chitin, collagen, elastin and silk, and copolymers and blends thereof.

For example the natural polymer may be selected from the group consisting of alginate, collagen, elastin, chitosan and silk. However, the natural polymer may be selected from the group consisting of alginate, chitosan, lignin, dextran, glycogen and chitin. Similarly the natural polymer the may be selected from the group consisting of chitosan, collagen, gellan, gellan gum, agar, xanthan chitosan and silk.

In a preferred embodiment of the present invention the scaffold material is chitosan.

According to the present material the natural polymers serving as scaffold material comprises copolymers and blends of any of the listed natural polymers.

In one embodiment of the present invention the biodegradable natural polymer is any of alginate, such as cellulose, for example dextran, such as glycogen, for example lignin, such as gellan, for example gellan gum, such as hyaluronic acid, for example xanthan chitosan, such as agar, for example carrageenan, such as chitosan, for example chitin, such as collagen, for example elastin, such as silk.

In another embodiment of the present invention the at least one biocompatible, biodegradable scaffold material is a synthetic polymer. The synthetic polymer may be selected from the group consisting of poly(lactic acid) (PLA), poly(L-lactic acid), poly(DL-lactic acid), polycaprolactone, poly(glycolic acid) (PGA), polyanhydride, poly(alkylene succinates), poly(hydroxy butyrate) (PHB), poly(butylene diglycolate), poly(.epsilon.-capro1 actone) and copolymers or blends thereof. In one embodiment of the present invention the synthetic polymer may be selected from the group consisting of poly(lactic acid) (PLA), poly(L-lactic acid), poly(DL-lactic acid), polycaprolactone, poly(glycolic acid) (PGA), polyanhydride, poly(alkylene succinates), poly(hydroxy butyrate) (PHB).

In a second embodiment of the present invention the synthetic polymer may be selected from the group consisting of poly(DL-lactic acid), polycaprolactone, poly(glycolic acid) (PGA), polyanhydride, poly(alkylene succinates), poly(hydroxy butyrate) (PHB).

In another embodiment of the present invention the synthetic polymer may be selected from the group consisting of poly(lactic acid) (PLA), poly(L-lactic acid), poly(DL-lactic acid).

In yet another embodiment of the present invention the synthetic polymer may be selected from the group consisting of polycaprolactone, poly(glycolic acid) (PGA), polyanhydride, poly(alkylene succinates), poly(hydroxy butyrate) (PHB), poly(butylene diglycolate), poly(.epsilon.-caproiactone). However, the synthetic polymer may be selected from the group consisting of polycaprolactone, poly(glycolic acid) (PGA), polyanhydride,

In a preferred embodiment the synthetic polymer is poly(DL-lactic acid).

It is understood that any of the synthetic polymers may be used separately. Thus, the synthetic polymer may for example be poly(lactic acid) (PLA), such as poly(L-lactic acid), for example poly(DL-lactic acid), such as polycaprolactone, for example poly(glycolic acid) (PGA), such as polyanhydride, for example poly(alkylene succinates), such as poly(hydroxy butyrate) (PHB), for example poly(butylene diglycolate), such as poly(.epsilon.-caproiactone) and copolymers or blends thereof.

Biostable scaffold materials

In one embodiment the biostable scaffold material according to the present invention is selected from the group consisting of metal, hydroxyapatite, choral, elastomers, acrylic resins, plastics and fluorocarbon polymers. However, the biostable scaffold may be selected from the group consisting of metals, hydroxyapatite, choral and elastomers. Likewise the biostable scaffold may be selected from the group consisting of metal, elastomers, acrylic resins and plastics. In another embodiment of the present invention the biostable scaffold may be selected from the group consisting of acrylic resins and fluorocarbon polymers.

In the present invention suitable metals are thantalum, and titanium, such as thantalum, for example titanium. Suitable plastics comprise for example nylon and polyethylene, such as nylon, for example polyethylene.

Acrylic resins may be used as scaffold material according to the present invention. Also elastomeres represented by for example silicones are suitable scaffold materials. In yet other embodiments the preferred scaffold material is hydroxyapatite. However, also choral may be a suitable scaffold material.

It is appreciated that at least two biocompatible polymers may be a combination of biodegradable and biostable polymers. A combination of using a biostable polymer and a biodegradable polymer may allow for the degradation of the biodegradable polymer over time and subsequently allow for full integration of cellular material in its place. The biostable polymer may in contrast remain, serving as support to the growing, differentiating or differentiated cells or tissue. The embodiment of a combination of biodegradable and biostable is useful when regenerating or repairing body parts such as for example bone where the mechanical strength of the developed tissue is important for the function of for example the bone.

According to the present invention the biocompatible material used as a three dimensional cell scaffold may be selected from the group consisting of natural polymers, synthetic polymers, stable materials or a combination thereof.

In a preferred embodiment the biocompatible polymer is chitosan.

It is understood that it is within the scope of the present invention to include at least one, such as two, for example three, such as four, for example five, such as six, for example seven, such as eight, for example nine, such as ten, for example eleven, such as twelve, for example thirteen, such as fourteen, for example fifteen different biocompatible scaffold materials in the three dimensional cell scaffold.

In one embodiment of the present invention the at least one biocompatible scaffold material of the three dimensional cell scaffold is different from the at least one polymer of the coating. For example in one embodiment the at least one biocompatible scaffold material is a natural polymer and the at least one polymer of the coating is a synthetic polymer. In a preferred embodiment the biocompatible polymer is chitosan and the polymer of the coating is poly (D, L-lactic acid). Said chitosan is in the form of a sponge and adjusted in size to fit the use.

Scaffold shape and pores

The three-dimensional cell scaffold may have a variety of shapes. The shape of the three-dimensional cell scaffold should be suitable to aid in the repair and/or alleviate damage to the defect in question. The three-dimensional cell scaffold has a shape selected from the group consisting of a sheet, a cylinder, a tube, a sphere, a cube, a rectangle, a sponge and an irregular shape.

An example of a suitable scaffold shape is the shape as a sheet which may be suitable for treatment of large dermal defects, fascia defects or other membranes. For example a cylindrical form may be suitable for the repair of focal injuries in articular cartilage. For repair of bone and joint defects the three-dimensional cell scaffold may be in the shape of a sphere. Similarly, a scaffold shape in the form of a rectangle or a cube as well as a sponge-shaped scaffold may be suitable for bone defects. However, the scaffold may be of any irregular shape suitable for a variety tissue defects. The shape of the three-dimensional cell scaffold is not limited to the examples of suitable applications as given above. According to the present invention the shape of the three-dimensional cell scaffold should be suitable for repairing any damage to the tissue as described elsewhere herein.

Pores

Generally speaking the three-dimensional scaffold according to the present invention should be able to accommodate cells that will aid in the repair of the damaged tissue. In order for cells to be cultured inside and on the scaffold the three-dimensional scaffold should comprise cavities suitable in size in which cells should be able to live and multiply. In the following such cavities will be referred to as pores. The three- dimensional cell scaffold therefore has pores. The presence of pores may also allow for the population of the scaffold by cells originating from surrounding tissues by invasion. Thus, in general the size of the pores will range from about one to ten times the diameter of the cells to be seeded in the scaffold. The size of the pores is thus adapted to the type cell to be accommodated within the three dimensional scaffold considering which type of tissue is to be regenerated or repaired. It is important for the pores to be of a sufficiently large size (sufficient pore volume) so as to allow cells (i.e., living cells) to maintain their shape within the structure. Furthermore, a large pore volume is desirable in order to allow a cell suspension to fully penetrate the structure and thus permit cell seeding and/or cell migration throughout the material. In relation to access to nutrients and efficient removal of waste products following cellular metabolism a sufficient pore volume is needed.

According to the present invention the pores of the three-dimensional cell scaffold have a pore size in the range of from about 0.5 μm to 100 μm, such as 5 μm to 100 μm, for example 10 μm to 90 μm, such as 20 μm to 80 μm, for example 25 μm to 75 μm, such as 30 μm to 60 μm, for example 30 μm to 50 μm, such as 0.5 μm to 10 μm, for example 0.5 μm to 20 μm, such as 0.5 μm to 25, for example 0.5 μm to 30 μm, such as 0.5 μm to 40 μm, for example 0.5 μm to 50 μm, such as 10 μm to 75 μm, for example 10 μm to 70 μm, such as 10 μm to 60 μm, for example 10 μm to 50 μm, such as 0.5 μm to 25 μm, for example 0.5 μm to 30 μm, such as 50 μm to 80 μm, for example 50 μm to 75 μm, such as 60 μm to 75 μm, for example 60 μm to 70 μm. such as 50 μm to 100 μm. In one embodiment the pore size is for example 25 μm, such as 30 μm, for example 40 μm, such as 45 μm, for example 50 μm, such as 55 μm, for example 60 μm, such as 65 μm, example 70 μm, such as 75 μm, for example 80 μm, such as 85 μm, for example 90 μm, such as 95 μm, for example 100 μm.

The pores of the three-dimensional cell scaffold should be relatively uniform in size, which ensures that the pores are large enough to accommodate the living cells in a uniform manner throughout the three-dimensional scaffold. Thus, in one embodiment of the invention the pores of three-dimensional cell scaffold are uniform in size.

Coating

In another embodiment the three dimensional cell scaffold according to the present invention may be coated with a suitable material to promote cell growth of the cells of the scaffold but also cells in the surrounding tissue.

According to the present invention the coating consists of a biodegradable polymer. Due to the physiological conditions in the area or at the site of implantation of the three dimensional cell scaffold the coating will gradually degrade. During the degradation process the biological agent will be released to the cells seeded in the scaffold, the cells that have colonized the scaffold and/or the surrounding environment. The gradual degradation of the coating will occur over a period of weeks, months or years.

A biphasic release pattern in which the biological agent is initially released in high amounts and subsequently followed by a low long-lasting release of the biological agent is desired. The initial release of the biological agent in high amounts are sufficient to provide an initial boost to the cells and tissue accelerating the process of regeneration or healing of damaged tissue. The long-lasting stimulus by the biological agent in the second phase of the release pattern will provide a long lasting effect to the regenerating tissue and cells. The present invention provides three-dimensional scaffolds which may be coated with a number of different coatings as described herein which will result in different lengths of initial boost and subsequent low release of biological agents. Furthermore, the ability to vary the release characteristics of the biological agent may be combined with the use of one or more biological agents as described herein. The ability of combining these parameters according to the present invention provides three-dimensional scaffolds which are specifically suited for a given purpose, for example repairing bone or joint defects, or for example focal injuries in articular cartilage, as described elsewhere herein.

Thickness of coating is in the range of nanometers to micrometers, for example 10 μm to 50 μm, such as 10 μm to 30 μm, for example 10 μm to 20 μm. The thickness of the coating depends on the viscosity of the coat-vehicle and the number of repeated coatings that have been added onto the scaffold

The polymer of the coating according to the present invention may be selected from the group consisting of poly-. alpha. -hydroxy acids, polyglycols, polytyrosine carbonates, starch, gelatins, cellulose as well as blends and interpolymers containing these components. Particularly preferred among the poly-. alpha. -hydroxy acids are the polylactides, polyglycol acids, and their interpolymers. One example of a suitable polylactide is marketed by Boehringer-lngelheim under the trade name R 203 and is a racemic poly-D,L-lactide. This racemic compound forms an amorphous layer on the surface of the implant. In one embodiment of the present invention the at least one polymer of the coating is poly (D,L-lactic acid), or mixtures thereof.The formation of crystalline polymer structures in the coating should preferably be avoided, which is why an enantiomerically pure lactide is preferably avoided. Suitable polytyrosene carbonates include for instance p(DTE-co-5% PEG 1000 carbonates) and p(DTE-co- 26% PEG 20000 carbonates). These are copolymers containing the specified amounts of polyethylene glycols.

Biological agent

It is within the scope of the present invention to include biological agents in the coating that will promote outgrowth of the cells seeded on/in the scaffold, ingrowth of cells from the surrounding tissue, tissue development, and cell differentiation of cells within the scaffold. However, it is also within the scope of the present invention to include as biological agents agents which will reduce the risk of infection to the damaged tissue which is to be regenerated or repaired. Such biological agents are antibiotics. In one embodiment the biological agents may be comprised in bioactive nanoparticles. One example of biological agents according to the present invention is tissue specific extracellular matrix (ECM) proteins. ECM proteins may be represented by macromolecules in particulate form or include extracellular matrix molecules deposited by viable cells.

The term extracellular matrix proteins may be one or more of fibronectin, laminin, vitronectin, tenascin, entactin, thrombospondin, elastin, gelatin, collagen, fibrillin, merosin, anchorin, chondronectin, link protein, bone sialoprotein, osteocalcin, osteopontin, epinectin, hyaluronectin, undulin, epiligrin, and kalinin. Other extracellular matrix molecules are described in Kleinman et al., J. Biometer. Sci. Polymer Edn., 5: 1- 11 , (1993), herein incorporated by reference.

Extracellular matrix molecules are commercially available. For example, extracellular matrix from EHS mouse sarcoma tumor is available.

Other biological agents that are suitable for outgrowth of the cells seeded on/in the scaffold, ingrowth, tissue development, and cell differentiation within the scaffold include growth factors, proteoglycans, glycosaminoglycans, bioactive and polysaccharides. These compounds are believed to contain biological, physiological, and structural information for development or regeneration of tissue structure and function. Thus in one embodiment of the present invention the at least one biological agent may be selected from the group consisting of growth factors, proteoglycans, glycosaminoglycans and polysaccharides. Growth factors

The at least one biological agent which is suitable to be included in the coating may be one or more growth factor(s). A number of growth factors exist that are involved in inducing a variety of cellular responses in connection with a variety of cell functions. Some growth factors are for example believed to be osteoinductive, whereas other growth factors are believed to have inductive effect on articular cartilage.

The growth factor of the coating according to the present invention may be selected from the group consisting of platelet derived growth factor (PDGF) AA, PDGF BB; insulin-like growth factors- 1 (IGF-I), IGF-II, acidic fibroblast growth factor (FGF), , basic FGF, .beta.-endothelial cell growth factor, FGF 4, FGF 5, FGF 6, FGF 7, FGF 8, and FGF 9; transforming growth factor TGF-P1 , TGF .beta.1.2, TGF-.beta.2, TGF-.beta.3, TGF-.beta.5; bone morphogenic protein (BMP) 1 , BMP 2, BMP 3, BMP 4, BMP 7, vascular endothelial growth factor (VEGF), placenta growth factor; epidermal growth factor (EGF), amphiregulin, betacellulin, heparin binding EGF, interleukins (IL) -1 , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 , IL-12, IL-13, IL-14, IL-15-18 ; colony stimulating factor (CSF)-G, CSF-GM, CSF-M, erythropoietin; nerve growth factor (NGF), ciliary neurotropic factor, stem cell factor, hepatocyte growth factor.

In another embodiment said coating of the three-dimensional scaffold may comprise a growth factor selected from the group consisting of PDGF AA, PDGF BB, IGF-I, IGF-II, acidic FGF, basic FGF, TGF. beta.1.2, TGF.beta.1.2, TGF-.beta.2, TGF-.beta.3, TGF- .beta.5, BMP 2, BMP 7, IL -1 , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 , IL- 12, IL-13, IL-14, IL-15-18, NGF, ciliary neurotropic factor.

Alternatively, said coating of the three-dimensional scaffold may comprise a growth factor selected from the group consisting of PDGF AA, PDGF BB, IGF-I, IGF-II, acidic FGF, basic FGF, TGF. Beta.1.2, TGF.beta.1.2, TGF-.beta.2, TGF-.beta.3, TGF-.beta.5, BMP 2, BMP 7, or selected from the group consisting of NGF, ciliary neurotropic factor. The growth factor may also be selected from the group consisting of BMP 2, BMP 7, TGF. Beta.1.2, TGF.beta.1.2, TGF-.beta.2, PDGF AA, PDGF BB, IGF-I, IGF-II.

In yet a further embodiment the growth factor is selected from the group consisting of IGF-I, IGF-II, or selected from the group consisting of PDGF AA, PDGF BB. Other embodiments include the growth factor selected from the group consisting of IL - 1 , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 , IL-12, IL-13, IL-14, IL-15-18, or selected from the group consisting of NGF, ciliary neurotropic factor. The growth factor of the coating according to the present invention may be any of platelet derived growth factor (PDGF) AA, such as PDGF BB, for example insulin-like growth factors-1 (IGF-I), such as IGF-II, for example acidic fibroblast growth factor (FGF), such as basic FGF, for example beta. -endothelial cell growth factor, such as FGF 4, for example FGF 5, such as FGF 6, for example FGF 7, such as FGF 8, for example FGF 9, such as transforming growth factor TGF-P1 , for example TGF

.beta.1.2, such as TGF-.beta.2, for example TGF-. beta.3, such as TGF-.beta.5, for example bone morphogenic protein (BMP) 1 , such as BMP 2, for example BMP 3, such as BMP 4, for example BMP 7, such as vascular endothelial growth factor (VEGF), for example placenta growth factor, such as epidermal growth factor (EGF), for example amphiregulin, such as betacellulin, for example heparin binding EGF, such as interleukins (IL) -1, for example IL-2, such as IL-3, for example IL-4, such as IL-5, for example IL-6, such as IL-7, for example IL-8, such as IL-9, for example IL-10, such as IL-11 , for example IL-12, such as IL-13, for example IL-14, such as IL-15-18, for example colony stimulating factor (CSF)-G, such as CSF-GM, for example CSF-M, such as erythropoietin, for example nerve growth factor (NGF), such as ciliary neurotropic factor, for example stem cell factor, such as hepatocyte growth factor, or any combination of the growth factors described herein.

In one embodiment of the present invention TGFbetal stimulates cellular differentiation in for example in case of focal injuries in articular cartilage, whereas IGF-1 stimulates production of matrix during repair of focal injuries in articular cartilage. Therefore, TGFbetal in and IGF-1 can be used in combination for the repair of focal injuries in articular cartilage according to the present invention.

By the term 'glycosaminoglycan' is anticipated at least one of heparan sulfate, chondroitin sulfate, dermatan sulfate, keratan sulfate, hyaluronic acid may be included in the coating according to the present invention. Similarly, the term 'polysaccharide' encompasses at least one of heparin, dextran sulfate, chitin, alginic acid, pectin, and xylan. By the term 'proteoglycan' is meant that at least one of decorin and dermatan sulfate proteoglycans, keratin or keratan sulfate proteoglycans, aggrecan or chondroitin sulfate proteoglycans, heparan sulfate proteoglycans, biglycan, syndecan, perlecan, or serglycin may be comprised in the coating according to the present invention.

However, in another embodiment of the present invention the at least one biological agent may be selected from nutrients, cytokines, hormones, angiogenic factors, vitamins, immunomodulatory factors, and drugs are also expected to provide an environment in the three dimensional scaffold suitable for growth of the cells.

In one embodiment if the present invention the biological agent is biphosphate. In particular the biphosphate is included in the coating of the three-dimensional scaffold during the healing of when bone defects.

In another embodiment of the present invention the at least one biological agent is a hormone which may be selected from the group consisting of Aldosterone, cortisone, hydrocortisone, dexamethasone, Progesterone, testosterone, androsterone, oestrogens, Glucagon, insuline, somatostatin, growth hormone, TSH, oxytocin, prolactin, Thyroxine, thyronine.

In another embodiment of the present invention the hormone is selected from the group consisting of aldosterone, cortisone, hydrocortisone, dexamethasone, oestrogens, insuline.

In yet another embodiment of the present invention the hormone is selected form the group consisting of cortisone, hydrocortisone, dexamethasone, insulin. Preferred embodiments include dexamethasone, or insulin. However, any of

Aldosterone, cortisone, hydrocortisone, dexamethasone, Progesterone, testosterone, androsterone, oestrogens, Glucagon, insuline, somatostatin, growth hormone, TSH, oxytocin, prolactin, Thyroxine, thyronine may be used separately or in combination.

In another embodiment of the present invention the at least one biological agent is a vitamine. The vitamin may be selected from the group consisting of vitamin D (cholecalciferols), vitamin C (Ascorbic acid). However, any of vitamin D, vitamin C may be used separately. In yet a further embodiment of the present invention the at least one biological agent is a cytokine which may be selected from the group consisting of interferones, Tumornecrosis factor-alpha, chemokines. Any of interferones, Tumomecrosis factor- alpha, chemokines may be used separately or in any combination as said at least one biological agent.

The at least one biological agent, for example, growth factors constitute a proportion of the total weight of the coating for example 0.1% to 20% by weight, such as 0.1% to 10%, for example 5% to 15%, such as 8% to 20%, for example 10% to 20%, such as 2% to 15%, for example 1 % to 10%, such as 8% to 20%, for example 10% to

20%,such as 15% to 20% for example 2% to 5%, such as 5% to 8%, for example 0.5 to 8%, such as 1% to 5% by weight, for example 12% to 18%, such as 13% to 20%, for example 17% to 20%, such as 17% to 20% for example 15% to 18%, such as 8% to 15%, by weight of the total weight of the coating.This weight percentage relates to the net amount of the active agent, without counting any pharmaceutical carrier substances. In one embodiment the biological agent is TGFbetal constituting the proportion as mentioned herein. Similarly, in another embodiment the biological agent is IGF-1 constituting the proportion as mentioned herein.

It is anticipated that it is within the scope of the present invention to include at least one, such as two, for example three, such as four, for example five, such as six, for example seven, such as eight, for example nine, such as ten, for example eleven, such as twelve, for example thirteen, such as fourteen, for example fifteen different biological agents in the coating in any combination that will promote outgrowth of the cells seeded on/in the scaffold, in-growth of cells from the surrounding tissue, tissue development, and cell differentiation of cells within the scaffold. Thus, the at least one growth factor may be combined with one or more growth factors. Similarly, the at least one biological agent may be from separate groups (ie. growth factor, nutrients, cytokines, hormones, angiogenic factors, immunomodulatory factors, drugs, ECM, proteoglycans, polysaccharides, glycosaminoglycan) or from separate groups.

Cells

The three-dimensional scaffold may further comprise living cells. Once the cells are inoculated on and/or inside the scaffold the cells will proliferate on and/or inside the scaffold. Thus, the three-dimensional comprising cells can be used in vivo. The three- dimensional cell scaffold according to the present invention will sustain active proliferation of the culture for long periods of time or the desired period of time.

The cells employed in the present invention may be characterized by different donor - recipient relationship. The term 'xenogeneic' means cells or tissue from donor individuals belonging to a different species than the recipient individual. One example is a graft of cells or tissues from a pig which is transplanted or transferred according to the present invention to a human. The term 'allogeneic' refers to cells or tissue from individuals belonging to the same species but from genetically different individuals. The term 'syngeneic' refers to cells or tissue from a donor individual who is genetically identical to the recipient individual, for example where donor and recipient are identical twins belonging to different species. The term 'autologous' refers to cells or tissue from the same individual, for example where cells are from the patient himself. Thus, according to the present invention the three-dimensional scaffold may further comprise cells selected from the group consisting of autologous, xenogeneic, allogeneic and syngeneic cells. In one embodiment the cells may be syngeneic cells in order to avoid rejection of cells due to incompatibility, immunological rejection of the transplant and/or graft versus host disease is likely. Another embodiment is the use of xenogeneic cells, or for example allogeneic cells. In a preferred embodiment the cells are autologous whereby rejection of cells due to incompatibility is avoided.

According to the present invention also genetically modified cells may be used, which have been created to be particularly useful for the regeneration of tissue, such as bone, tendon, ligament and/or cartilage. The cells may be genetically engineered to produce gene products beneficial to transplantation, e.g. anti-inflammatory factors, e.g., anti- GM-CSF, anti-TNF, anti-lL-1 , anti-IL-2, etc. Alternatively, the cells may be genetically engineered to "knock out" expression of native gene products that promote inflammation, e.g., GM-CSF, TNF, IL-1 , IL-2, or "knock out" expression of MHC in order to lower the risk of rejection. In addition, the cells may be genetically engineered for use in gene therapy to adjust the level of gene activity in a patient to assist or improve the results of the cartilage transplantation by use of the three-dimensional cell scaffold according to the present invention. When cells are obtained from a donor or a patient, the cells can be obtained by small biopsies for example 5 mg to 1 ,000 mg of tissue from a patient and/or a donor, such as 50 mg to 1000 mg, for example 100 mg to 1000 mg, such as 200 mg to 1000 mg, for example 300 to 1000 mg, such as 400 mg to 1000 mg, for example 500 mg to 1000 mg, such as 600 mg to 1000 mg, for example 700 to 1000 mg, such as 800 mg to 1000 mg, for example 900 to 1000 mg, such as 5 mg to 100 mg, for example 5 mg to 200 mg, such as 5 mg to 300 mg, for example 5 mg to 400 mg, such as 5 mg to 500 mg, for example 200 mg to 800 mg, such as 200 mg to 700 mg, for example 200 mg to 600 mg, such as 200 mg to 500 mg, for example 200 to 400 mg, such as 400 mg to 1000 mg, for example 400 mg to 900 mg, such as 400 mg to 800 mg, for example 400 to 700 mg, such as 400 mg to 600 mg, for example 50 to 750 mg, such as from 100 to 500 mg, for example from 200 mg to 300 mg. The quantity required for treatment of a tissue defect depends on the volume of the tissue defect, the cellular density in the recipient tissue, and the proliferal potential of the donor cells.

Cartilage cells

In the case of cartilage cells, these may be obtained from any cartilage source in the body, including any articular surface e.g. knee joint, ankle joint, shoulder joint etc.; or from the costal cartilage, rib cartilage, nose cartridge, ear cartridge, symphysis, intervertebral discs, meniscus, soft palate, laryngeal cartilage or tracheal cartilage.

When removing cartilage, bone or muscle cells from the patient or donor, this is advantageously done using an air-tight operation method avoiding donor cell exposure to atmospheric air. By preventing exposure to atmospheric air the cells will produce implants of higher quality (e.g. cartilage implants with a higher content of type Il collagen) than if the cells are exposed to ambient oxygen.

In accordance with the invention, cells are inoculated onto and/or inside the three- dimensional cell scaffold, and grown in culture to form a living tissue material. The cells may be fetal or adult in origin, and may be derived from convenient sources such as cartilage, skin or bone as described in more detail elsewhere herein. Such tissues and/or organs can be obtained by appropriate biopsy or upon autopsy; cadaver organs may be used to provide a generous supply of stromal cells and elements. Alternatively, umbilical cord and placenta tissue or umbilical cord blood may serve as an advantageous source of fetal-type cells, e.g., osteoblast-progenitors, chondrocyte- progenitors and/or fibroblast-like cells for use in the three-dimensional cell scaffold of the invention.

Proliferation of cells The proliferation step, which may precede the inoculation of the three-dimensional cell scaffold may serve two purposes. First and foremost, the cells are proliferated to simply increase their number. In addition to this, cells may also - at least partly - de- differentiate during the proliferation step. In the case of stem cells, differentiation into muscle, bone and/or chondrocyte cells may take place during the proliferation step.

It is convenient that the proliferation is performed in flasks, such as culture flasks, or in Petri discs, or for example rolling flasks, or three-dimensional culture systems. MSC are for example proliferated in monolayer culture. Chondrocytes may be proliferated initially in monolayer for one to two cell divisions and after that transferred and proliferated under growth conditions allowing three-dimensional growth.

The concentration of cells seeded for multiplication preferably is from 5,000 to 10,000 cells per cm². Inoculation may be performed at a temperature from 33 to 37°C, preferably from 36 to 37⁰C.

A suitable medium for proliferation comprises nutrients, buffer and amino-acids. The medium is preferably supplemented with the patient's own serum or serum from another person such as a donor. The medium may contain at least 2 % (v/v) of serum, such as with 3 %, for example at least 4%, such as at least 5 %, for example at least 6 %, such as at least 7 %, for example at least 8 %, such as at least 9 %, for example at least 10 %, such as at least 11 %, for example at least 12 %, such as at least 13 %, for example at least 14 %, such as at least 15 %, for example at least 16 %, such as at least 17 %, for example at least 20 %, such as at least 25 %.

Cells, such as for example chondrocytes or MSCs, are suitably seeded in the scaffold in concentrations of 500,000 to 2,000,000 cells per cm². However, cells may also be suitably seeded in the scaffold in concentrations of 50,000 to 100,000 cells per cm³.

The culture medium used for culturing the cells in the scaffold may be the same as for the proliferation step. A suitable medium may thus comprise nutrients, buffer and amino acids. Preferably, the medium is supplemented with the patient's own serum or serum from another person, such as a donor. The amount of serum in the medium may be at least 2 % (v/v), such as with 3 %, for example at least 4%, such as at least 5 %, for example at least 6 %, such as at least 7 %, for example at least 8 %, such as at least 9 %, for example at least 10 %, such as at least 11 %, for example at least 12 %, such as at least 13 %, for example at least 14 %, such as at least 15 %, for example at least 16 %, such as at least 17 %, for example at least 20 %, such as at least 25 %.

An example of a suitable growth medium is DMEM with 10% Serum.

Many commercially available media, such as DMEM, RPMI 1640, Fisher's Iscove's,

McCoy's, and the like may be suitable for use. It is important that the three-dimensional scaffold be suspended or floated in the medium during the incubation period in order to maximise proliferative activity. In addition, the culture should be "fed" periodically to remove the spent media, depopulate released cells, and add fresh media. The concentration of TGF-β may be adjusted during these steps.

The cells in the scaffold may be caused to divide in the scaffold until cell-cell contact is obtained. Expressed in another way, the cells may be cultured in the scaffold for 1- 60 days, such as 5-50 days, for example 10-40 days, such as 10 to 30 days, for example 10-20 days, such as 20 -60 days, for example 20-50 days, such as 30-50 days, for example 30-60 days, such as 40-60 days, for example 1-20 days, such as 1 -10 days, for example 1-10 days, such as 1-20 days, for example 1-30 days, such as 5-10 days, , or more days if it is required. Cells according to the present invention may be cultured in the scaffold for for example 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 ,12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 100 days, or more days if it is required.

Thus, in one preferred embodiment of the invention the three-dimensional cell scaffold is preseeded with cells before transferring the scaffold to the recipient. However, in another preferred embodiment the three-dimensional cell scaffold is preseeded with cells before transferring the scaffold to the recipient, where the scaffold may subsequently be seeded with cells again.

Sources and isolation of cells Chondrocytes may be derived from articular cartilage, costal cartilage, etc. which can be obtained by biopsy (where appropriate) or upon autopsy. Fibroblasts can be obtained in quantity rather conveniently from foreskin or, alternatively, any appropriate cadaver organ. Fetal cells, including fibroblast-like cells, chondrocyte-progenitors, may be obtained from umbilical cord or placenta tissue or umbilical cord blood. Such fetal cells can be used to prepare a "generic" tissue or cartilaginous tissue. However, a "specific" tissue may be prepared by inoculating the three-dimensional matrix with fibroblasts derived from a particular individual who is later to receive the cells and/or tissues grown in culture in accordance with the three-dimensional system of the invention.

Fibroblasts may be readily isolated by disaggregating an appropriate organ or tissue which is to serve as the source of the fibroblasts. This may be readily accomplished using techniques known to those skilled in the art. For example, the tissue or organ can be disaggregated mechanically and/or treated with digestive enzymes and/or chelating agents that weaken the connections between neighboring cells making it possible to disperse the tissue into a suspension of individual cells without appreciable cell breakage. Enzymatic dissociation can be accomplished by mincing the tissue and treating the minced tissue with any of a number of digestive enzymes either alone or in combination. These include but are not limited to trypsin, chymotrypsin, collagenase, elastase, and/or hyaluronidase, Dnase, pronase, etc. Mechanical disruption can also be accomplished by a number of methods including, but not limited to the use of grinders, blenders, sieves, homogenisers, pressure cells, or sonicators to name but a few. For a review of tissue disaggregation techniques, see Freshney, Culture of Animal Cells. A Manual of Basic Technique, 2d Ed., A. R. Liss, Inc., New York, 1987, Ch. 9, pp. 107-126.

Isolation of substantially single cells may be obtained using mechanical and/or enzymatic separation of cells as described above.

Mechanical separation comprises grinding, blending, sieving, homogenising, exposing to pressure, and/or sonication.

Enzymatic separation of cartilage cells may comprise exposing cells to clostridial collagenase and deoxyribonuclease 1.

Fibroblast-like cells may also be isolated from human umbilical cords (33-44 weeks). Fresh tissues may be minced into pieces and washed with medium or snap-frozen in liquid nitrogen until further use. The umbilical tissues may be disaggregated as described above.

Once the tissue has been reduced to a suspension of individual cells, the suspension can be fractionated into subpopulations from which the cells can be obtained. This also may be accomplished using standard techniques for cell separation including but not limited to cloning and selection of specific cell types, selective destruction of unwanted cells (negative selection), separation based upon differential cell agglutinability in the mixed population, freeze-thaw procedures, differential adherence properties of the cells in the mixed population, filtration, conventional and zonal centrifugation, centrifugal elutriation (counter-streaming centrifugation), unit gravity separation, counter current distribution, electrophoresis and fluorescence-activated cell sorting. For a review of clonal selection and cell separation techniques, see Freshney, Culture of Animal Cells. A Manual of Basic Techniques, 2d Ed., A. R. Liss, Inc., New York, 1987, Ch. 11 and 12, pp. 137-168.

The isolation of chondrocytes, chondrocyte-progenitors, osteocytes, osteoblasts, osteoblast-progenitors, fibroblasts or fibroblast-like cells may, for example, be carried out as follows: Fresh specimens of articular cartilage are carefully dissected from the subchondral bone and finely chopped, washed and digested for example in a two-step procedure using testicular hyaloronidase followed by collagenase. Filtered isolated chondrocytes are then seeded and grown according to standard procedures.

In addition to chondrocytes, chondrocyte-progenitors, osteocytes, osteoblasts, osteoblast-progenitors, fibroblasts or fibroblast-like cells, other cells may be added to form the three-dimensional tissue required to support long term growth in culture. For example, other cells found in loose connective tissue may be inoculated onto the three- dimensional support along with chondrocytes or fibroblasts. Such cells include but are not limited to endothelial cells, pericytes, macrophages, monocytes, plasma cells, mast cells, adipocytes, etc. These stromal cells may readily be derived from appropriate organs including umbilical cord or placenta or umbilical cord blood using methods known in the art such as those discussed above.

The cells which are suitable for inoculating on/into the scaffold according to the present invention are epithelial cells, keratinocytes, adipocytes, hepatocytes, neurons, glial cells, astrocytes, podocytes, Schwann cells, mammary epithelial cells, islet cells, endothelial cells, corneal cells, enterocytes, mesenchymal cells, dermal fibroblasts, mesothelial cells, stem cells, osteoblasts, smooth muscle cells, striated muscle cells, heart muscle cells, cardiac myoblasts, skeletal myoblasts, smooth muscle myoblasts, ligament fibroblasts, tendon fibroblasts, meniscal chondrocytes, articular chondrocytes, discus intervertebrals chondrocytes, odontoblasts, or ameloblasts.

In one embodiment of the invention the three-dimensional cell scaffold comprises cells selected from the group consisting of keratinocytes, adipocytes, hepatocytes, neurons, glial cells, astrocytes, podocytes, Schwann cells, mammary epithelial cells, islet cells, endothelial cells, corneal cells, enterocytes, mesenchymal cells, dermal fibroblasts, mesothelial cells, stem cells, osteoblasts, smooth muscle cells, striated muscle cells, heart muscle cells, cardiac myoblasts, skeletal myoblasts, smooth muscle myoblasts, ligament fibroblasts, tendon fibroblasts, meniscal chondrocytes, articular chondrocytes, discus intervertebralios chondrocytes, odontoblasts, and ameloblasts.

In another embodiment of the invention the three-dimensional cell scaffold comprises cells selected from the group consisting of stem cells, osteoblasts, skeletal myoblasts, ligament fibroblasts, tendon fibroblasts, meniscal chondrocytes, articular chondrocytes, discus invertebralios chondrocytes, neurons, glial cells, astrocytes, Schwann cells.

In a further embodiment said cell scaffold comprises cells selected from the group consisting of stem cells, osteoblasts, skeletal myoblasts, ligament fibroblasts, tendon fibroblasts, meniscal chondrocytes, articular chondrocytes, discus invertebralios chondrocytes.

However, said scaffold comprises cells selected from the group consisting of meniscal chondrocytes, articular chondrocytes, discus invetebralios chondrocytes, or cells selected from the group consisting of ligament fibroblasts, tendon fibroblasts.

Preferred embodiments include a three-dimensional cell scaffold comprising stem cells, osteoblasts, ligament fibroblasts, tendon fibroblasts, meniscal chondrocytes, articular chondrocytes, discus invertebralios chondrocytes.

However, for regenerating tissue connected to glial cells and nerve cells the preferred cells of the scaffold may be selected from the group consisting of neurons, glial cells, astrocytes, Schwann cells. It is within the scope of the present invention to include precursor cells of the cell types mentioned herein. Furthermore, a number of different cell types may be combined, for example two cell types, such as three cell types, for example four cell types, such as five cell types. The different cell types may be co-cultures for example MSC and undifferentiated cells may be co-cultured.

In a preferred embodiment of the present invention the three-dimensional cell scaffold comprises a chitosan scaffold which is coated with PDLLA comprising as biological agent TGFbeta 1 and in which the three-dimensional cell scaffold is seeded with chondrocytes. However, another preferred embodiment is a three-dimensional cell scaffold comprising a chitosan scaffold which is coated with PDLLA comprising IGF-1 and in which the three-dimensional cell scaffold is seeded with chondrocytes. Yet another embodiment of the present invention relates to a three-dimensional cell scaffold comprising a chitosan scaffold which is coated with PDLLA comprising TGFbeta land IGF-1 and in which the three-dimensional cell scaffold is seeded with chondrocytes. Yet a further embodiment of the present invention relates to a three- dimensional cell scaffold comprising a chitosan scaffold which is coated with PDLLA comprising TGFbeta 1 and/or IGF-1 and further an antibiotic and in which the three- dimensional cell scaffold is seeded with chondrocytes.

Method for producing the implant

The present invention also relates to a method for producing a three-dimensional cell scaffold, comprising the steps of i) providing a three-dimensional cell scaffold, ii) producing a mixture of at least one polymer and at least one solvent to form a volatile fluid, iii) immersing the three-dimensional cell scaffold of i) into the mixture of ii) to coat the three-dimensional cell scaffold and iv) evaporating the solvent.

In one embodiment of the invention, the three-dimensional scaffold is a chitosan scaffold. The evaporation step may be performed in a dessicator system at a pressure of -980 mbar. However, the present invention is not limited to the specific system but covers any system that can evaporate the volatile fluid to produce an implant according to the present invention.

In another embodiment of the present invention the at least one solvent is an organic solvent, such as ethyl acetate or for example chloroform. The at least one solvent may a mixture of any solvents compatible with the three-dimensional cell scaffold of the present invention. In yet another embodiment of the invention the mixture comprises 20 mg to 300 mg of a biodegradable polymer per ml of organic solvent. For example the mixture comprises 3-24% PDDLA of the organic solvent volume.

In a further embodiment the mixture further comprises a biological agent selected from any of the biological agents as described elsewhere herein, for example TGF betai , such as IGF-1.

In addition, the method for producing a three-dimensional cell scaffold may further comprise the steps of i) providing a sample of living tissue and/or living cells, ii) processing the sample under sterile conditions, iii) seeding the cells to said scaffold.

A three-dimensional cell scaffold formed by the disclosed steps is also within the scope of the present invention.

Similarly, a method for producing a ready-for use implant as describe herein is also within the scope of the present invention.

Embodiments for use

One aspect of the present invention relates to a method for regenerating tissue in a mammal in need thereof, comprising implanting the three-dimensional cell scaffold of the present invention.

The mammal is for example a goat, mouse, pig, dog, horse, cat, cow or a human. In a preferred embodiment the mammal is a human.

The tissue according to the present invention may be bone, cartilage, tendon, ligament, nerve, skin, vascular, cardiac, pericardial, muscle, ocular, periodontal, breast, pancreatic, esophageal, stomach, kidney, hepatic, mammary, adrenal, urological, and intestinal tissue. Thus, in one embodiment of the invention the tissue that is to be regenerated may be selected form the group consisting of bone, cartilage, tendon, ligament, nerve, skin, vascular, cardiac, pericardial, muscle, ocular, periodontal, breast, pancreatic, esophageal, stomach, kidney, hepatic, mammary, adrenal, urological, and intestinal tissue. In another embodiment the tissue may be selected form the group consisting of bone, cartilage, tendon and ligament. In a particular embodiment the tissue is cartilage. Alternatively, the tissue is bone. The relevance of regenerating the particular tissue types reflects the types of injuries a mammal is in need of treatment for, see elsewhere herein. In one particular embodiment the mammal is a human.

Mammals in need of regenerating tissue comprise mammals wherein injury to tissue has occurred. Surgical intervention is often required to repair the damage. Such surgical repairs can include suturing or otherwise repairing the damaged tissue with known medical devices, augmenting the damaged tissue with other tissue, using an implant, a graft or any combination of these techniques. According to the present invention tissue may be regenerated in a mammal by implanting the three-dimensional scaffold of the present invention.

In one embodiment of the invention the mammal may be suffering from a defect to tissue selected from the group consisting of articular cartilage defects, meniscal defects, discus intervertebralis defects, bone defects, vertebral body fractures, skin wounds, fascial defects, tendon ruptures, ligament ruptures, nerve injuries, spinal cord injuries, blood vessel defects, ear substitution, nasal cartilage defects, muscle defects, heart muscle defects, muscle degeneration, adipose defects, tooth injuries, bladder wall defects, gastric wall defects, intestinal wall defects, pancreatic island transplantation, and eye injuries.

In another embodiment of the present invention the defect to the tissue may be selected from the group consisting of muscle defects, heart muscle defects, muscle degeneration.

In yet another embodiment of the present invention the defect to the tissue may be selected from the group consisting of bladder wall defects, gastric wall defects, intestinal wall defects.

The defect to the tissue may be selected from the group consisting of ear substitution, nasal cartilage defects, or selected from the group consisting of ear substitution, nasal cartilage defects.

In a further embodiment of the present invention the defect to the tissue may be selected from the group consisting of nerve injuries, spinal cord injuries, articular cartilage defects, meniscal defects, discus intervertebralis defects, fascial defects, bone defects, vertebral body fractures, tendon ruptures, ligament ruptures. In a particular embodiment of the present invention the defect to the tissue may be selected from the group consisting of articular cartilage defects, meniscal defects, discus intervertebralis defects, fascial defects, bone defects, vertebral body fractures, tendon ruptures, ligament ruptures, or selected from the group consisting of articular cartilage defects, meniscal defects, discus intervertebralis defects, fascial defects.

Another embodiment of the present invention relates to the defect to the tissue selected from the group consisting of bone defects, vertebral body fractures.

In yet another embodiment of the present invention the defect to the tissue may be selected from the group consisting of tendon ruptures, ligament ruptures.

It is appreciated that any of the separate defects to the tissue such as articular cartilage defects, meniscal defects, discus intervertebralis defects, bone defects, vertebral body fractures, skin wounds, fascial defects, tendon ruptures, ligament ruptures, nerve injuries, spinal cord injuries, blood vessel defects, ear substitution, nasal cartilage defects, muscle defects, heart muscle defects, muscle degeneration, adipose defects, tooth injuries, bladder wall defects, gastric wall defects, intestinal wall defects, pancreatic island transplantation, or eye injuries are within the scope of the present invention.

In the following examples of common injuries are described. However, the present invention is not limited to the described examples.

A common type of tissue injury involves damage to cartilage, which is a non-vascular, resilient, flexible connective tissue. Cartilage typically acts as a "shock-absorber" at articulating joints, but some types of cartilage provide support to tubular structures, such as for example, the larynx, air passages, and the ears. In general, cartilage tissue is comprised of cartilage cells, chondrocytes. Several types of cartilage can be found in the body, including hyaline cartilage, fibrocartilage and elastic cartilage. Hyaline cartilage can appear in the body as distinct pieces, or alternatively, this type of cartilage can be found fused to the articular ends of bones. Hyaline cartilage is generally found in the body as¹ articular cartilage, costal cartilage, and temporary cartilage (i.e., cartilage that is ultimately converted to bone through the process of ossification). Fibrocartilage is a transitional tissue that is typically located between tendon and bone, bone and bone, and/or hyaline cartilage and hyaline cartilage. Elastic cartilage, which contains elastic fibers distributed throughout the extracellular matrix, is typically found in the epliglottis, the ears and the nose. Thus, the present invention relates to mammals suffering from defects to cartilage tissues.

In particular, the present invention relates to a method of regenerating tissue in a mammal suffering from articular cartilage injury comprising implanting the cell scaffold of the present into said mammal. One example of an articular cartilage injury is a traumatic focal articular cartilage defect to the knee. Damaged articular cartilage can severely restrict joint function, can cause debilitating pain and may result in long term chronic diseases such as osteoarthritis, which gradually destroys the cartilage and underlying bone of the joint. Injuries to the articular cartilage tissue often require surgical intervention.

However, another particular embodiment of the invention is the regeneration of cartilage of a type known as fibrocartilage. A common example of cartilage injury is damage to the menisci of a knee joint. There are two menisci of the knee joint, a medial and a lateral meniscus. Each meniscus is a biconcave, fibrocartilage tissue that is interposed between the femur and tibia of the leg. In addition to the menisci of the knee joint, meniscal cartilage can also be found in the acromioclavicular joint and, the joint between the clavicle and the sternum, and in the temporomandibularjoint. The primary functions of meniscal cartilage are to bear loads, to absorb shock and to stabilize a joint. Current conventional treatment modalities for damaged meniscal cartilage include the removal and/or surgical repair of the damaged cartilage.

One other example of damage to fibrocartilage which may be alleviated by the present invention is defects to discus intervertebralis. Thus, another embodiment relates to a method for regeneration in a mammal suffering from damaged discus intervertebralis, comprising implanting the cell scaffold of the present invention.

Yet another embodiment of the present invention relates to a method of regenerating tissue in a mammal suffering from damage to the ligaments and/or tendons comprising implanting the cell scaffold of the present into said mammal. Ligaments and tendons are cords or bands of fibrous tissue that contains soft collagenous tissue. Ligaments connect bone to bone, while tendons connect muscle to bone. Tendons are fibrous cords or bands of variable length that have considerable strength but are virtually devoid of elasticity. Ligaments, in contrast, are generally flexible, to allow the ligament tissue to have freedom of movement. Ligaments and tendons are comprised of fascicles, which contain the basic fibril of the ligament or tendon, as well as the cells that produce the ligament or tendon, known as fibroblasts. The fascicles of the tendon are generally comprised of very densely arranged collagenous fibers, parallel rows of elongated fibroblasts, and a proteoglycan matrix. The fascicles of ligaments also contain a proteoglycan matrix, fibroblasts and collagen fibrils, but the fibrils found in ligament tissue are generally less dense and less structured than the fibrils found in tendon tissue. Thus, the present invention relates to injuries to the ligament, or for example a tendon.

A person skilled in the art will appreciate which cells of the present invention described elsewhere herein may be included in the three-dimensional cell scaffold to regenerate the tissue as described herein.

One example of a ligament injury is a torn anterior cruciate ligament (ACL), which is one of four major ligaments of the knee. The lack of an ACL causes instability of the knee joint and leads to degenerative changes in the knee such as osteoarthritis. The most common repair technique is to remove and discard the ruptured ACL and reconstruct a new ACL using autologous bone-patellar, tendon-bone or hamstring tendons. The present invention offers a solution to the regeneration of ligament reconstruction using the implant of the present invention.

Without limiting the tendon injuries to any particular tendon injury, one example of a tendon injury is a damaged or torn rotator cuff. A rotator cuff is the portion of the shoulder joint that facilitates circular motion of the humerus bone relative to the scapula. The most common injury associated with the rotator cuff is a strain or tear to the supraspinatus tendon. Depending upon the severity of the injury, a torn tendon may require surgical intervention.

Examples

Example 1

Neogenesis of Hyaline Cartilage by Stimulation of Mesenchymal Stem Cells in TG Fbetal -Coated 3-D Scaffolds 3-D scaffolds coated with TGF beta 1 were produced and the release characteristics of TGF betai from PDLLA/TGF betai coated scaffolds were determined in vitro. Also the bioactivity of release of TGFbI was monitored by comparing the proliferation- rate of human adipose-derived MSCs in PDDLA/TGF betai -coated MSC-seeded scaffolds to PDDLA (without TGFbeta1)-coated MSC-seeded scaffolds. Furthermore, the distribution of MSCs in scaffolds was evaluated.

Cultures of Mesenchymal Stem Cells (MSC)

Adipose tissue will be retrieved from lipo-suctions of a healthy human donor after informed consent and ethical approval from Danish Ethical Board. Age, sex and medication will be noted. The cellular component is separated by centrifugation and seeded on T-175 flasks in basic media for culture at standard conditions. After 5 days, non-adherent MSCs are harvested and re-seeded at 1x10e6 cells/T-175 flask. These subcultured putative MSCs are grown in MSC medium until confluence, at which point they are harvested and cryopreserved for later use. Cellular morphology is observed microscopically throughout the culture period to ensure a homogeneous population of large, spindle-shaped cells (typical MSC morphology). For characterization of MSC phenotype relevant CD-expression will be analysed.

Scaffold material fabrication

Chitosan scaffolds are produced according to standard techniques ¹⁰. In brief, high molecular weight chitosan is dissolved in acetic acid and cast in 10ml flat bottom tubes. The tubes are frozen in Styrofoam isolation in -20⁰C overnight and -80°C for three days and finally freeze dried overnight. The scaffold cylinders are sliced in a custom made tool in discs of 2mm or 3mm thickness and cut into 6mm diameter discs using a dermal punch. To remove the acetic acid the scaffolds are rehydrated by incubation for 60 min in each 99%, 96%, 80% and 70% alcohol and sterile PBS overnight. In order to dry the scaffolds, the water is removed by reversing the ethanol series and freeze-drying overnight.

Poly-(d.l-Lactide) (PDLLA)-TGF-betai coating of chitosan scaffolds A 6% PDLLA solution is made in ethyl acetate according to previous description ^u. If growth factors such as TGF- beta 1 are to be incorporated, PDLLA solution is added to freeze dried carrier free human recombinant TGF- beta 1 (R&D systems). Following the PDLLA-growth factor solution is coated onto the chitosan scaffold using a custom- designed vacuum-technique. The coating solution is applied to each scaffold (one to four scaffolds can be coated at a time). The scaffolds evaporated with the PDLLA- growth factor solution are placed in a custom designed dessicator system, in which a vacuum pump is preserving a pressure of -980 mbar. The pressure is continuous monitored on a pressure gauge. The solvent, ethylacetate, evaporates from the scaffolds within seconds after application of vaccum. This technique ensures equal distribution of the growth factor coat throughout the scaffold. After 10 minutes in vacuum, the solvent is completely evaporated, and the scaffold can be stored in a dessicator for weeks before use.

Cell seeding

The growth-factor coated scaffolds are saturated in growth medium. A pellet of high- concentration cell suspension (10⁶ cells per mm scaffold thickness) is added on the top of the scaffold and dragged into the scaffold by hydrodynamic force. This ensures equal distribution of the cell population throughout the scaffold. For culturing, growth medium is changed every two days.

Growth factor release

Release kinetics of TGF-beta1 from TGF- beta 1 -coated scaffolds will be investigated. TGF-beta 1-coated scaffolds with different PDLLA solution (6%, 12% and 24%) in combination with high and low TGF- beta 1 will be covered with PBS in 24-well plates. Multiple samples will be evaluated within a period of 4 weeks.

In vitro chondrogenesis TGF- beta 1-coated scaffolds seeded with human adipose-derived MSCs are cultured in standard medium under standard conditions for up to 4 weeks to evaluated the chondrogenic potential of the construct.

In vivo chondrogenesis The chondrogenic potential of the constructs will be further evaluated in vivo. TGF- beta 1-coated scaffolds seeded with human adipose-derived MSCs will be implanted subcutaneously in SCID mice under general anaestesia. SCID-mice are being used in order to avoid immunogenic response against the xeno-transplanted cells. Each mouse will receive up to 4 implants. MSC differentiation into chondrocyte and synthesis of hyaline cartilage will be monitored after 2, 4 and 8 weeks. Multivalent extraction procedure

The scaffolds are harvested and weighed and immediately flash frozen in liquid nitrogen. Using a Mixermill (Retch) the samples are cryo-homogenized and extraction buffer is added immediately after. The following extraction is performed as described by Hoemann et al ¹². In brief three extractions are made as described herein: Guanidin- HCI extraction: GAG and DNA, Guanidin-Thiocyanate: RNA, Papain-digest: Collagen

DNA measurement: DNA is measured in the supernatant using SYBR-green (Molecular probes) according to manufacture guidelines. A conversion factor of 6.6pg pr cell is used for conversion to number of cells.

GAG measurement: GAG's are measured using Dimethyl-Methylenblue according to Hoemann et al ¹².

Quantitative real time PCR:

Total RNA is measured using Quant-IT Kit (molecular probes). cDNA synthesis is performed on 2μg of total RNA using High Capacity cDNA synthesis (applied biosystems) following the manufactures guidelines. QRT-PCR is performed on Applied

Biosystems 7500 PCR machine. Primers and Probes are designed and produced according to the guidelines of Applied Biosystems. GAPDH and β-Actin will serve as housekeeping genes for normalization. Relative gene expression is performed according to Pffafl ¹³.

Collagen measurement:

Collagen is measured after papain digestion using a commercial kit, Sircol (Biocolor), utilizing the measurement of hydroxyl prolin content.

Histology

Scaffolds for histology are fixed in formalin and embedded in Technovit acryl. Horizontal and vertical sections are performed using stereological sampling of sections ¹¹. Sections are dyed using safranin-0 for detection of GAG's and immunohistochemical stain is used to detect collagen. For general morphological histology, sections are dyed using HE. Furthermore, scaffolds will be evaluated by confocal microscopy after Hoechst staining.

ELISA

The release of TGF-beta 1 is repeatedly measured on cell culture medium during in vivo culturing using ELISA in accordance to manufactures manual (R&D Systems).

Design and statistics

Samples are made in three replicates. The evaluation of the in vitro cultured scaffolds takes place at day 0, 3, 7,14, 21 and 28, whereas scaffolds cultured in vivo will be performed 14 and 28 days after implantation. Samples for histological evaluation are evaluated after 14 days of culture. MSCs from four individuals are used and pooled to overcome inter-individual variation.

Means and standard deviation of three replicates are calculated for relevant data. A normal distribution is expected to describe data distribution. One way ANOVA is used to evaluate whether data are from the same normal distribution. If relevant non-paired two sample T-tests are made with correction for mass significance.

Release characteristics

The release characteristics of TGFbetal from PDLLA/TGFbetai coated scaffolds in vitro

The elution of the coating with incorporated growth factor (TGFbetal) indicated a biphasic kinetic profile. An early release of a large quantity of the incorporated growth factor is observed within the first 12 hours. Following this growth factor boost, the further release occurred at a constant slow rate. After 21 days, approximately 25% of the incorporated TGFbetal was released to the elution fluid (Figure 1).

Bioactivity of release TGFbI

The proliferation-rate of human adipose-derived MSCs were compared in PDDLA/TGFbeta1 -coated MSC-seeded scaffolds and PDDLA(without TGF betai)- coated MSC-seeded scaffolds. The number of cells was determined by microscopy using random cell-counting per field of view. After 14 days, an increase of 20% in the number of cells was observed (p<0.05, N=6).

Distribution of MSCs in scaffolds Distribution of human adipose-derived MSCs were evaluated by microscopy in scaffold-discs with a diameter of 6mm and heights of 1 mm, 2mm, and 3mm. Using the hydrodynamic force method, cells could be evenly distributed on and in the scaffolds.

Example 2

Isolation and proliferation of chondrocytes

The specimens of articular cartilage are brought to the research laboratory from the operating room immediately after harvesting and kept under humid and sterile conditions. The articular cartilage is then carefully dissected from the subchondral bone. Finely chopped fragments of articular cartilage were subsequently washed twice with sterile PBS (pH=7.4). A two step enzymatic digestion is then performed with 1 % testicular hyaluronidase (Sigma, MO) in DMEM/Ham F-12 for 1 hour, and a prolonged collagenase digestion with 1% clostridial collagenase A (Sigma, MO) in DMEM/Ham F- 12 (1 :1 ), 10% serum and 1 % Penicillin / Streptomycin (all Gibco-BRL, MD) for 24 hours in order to dissolve the extra-cellular matrix. These digestions were performed under vigorous shaking at 37⁰C.

The isolated chondrocytes were filtered (Swinnex Filter (20μm pores), Millipore Inc, USA), and resuspended in slightly alkaline DMEM/Ham's F12 media (1 :1) with 10% serum and 1% Penicillin / Streptomycin (all Gibco-BRL, MD). The chondrocytes were seeded onto 24 well plastic plates (Corning Inc., NY) at a density of 10⁵ cells per well and cultured under standard conditions at 37⁰C in a humidified sterile atmosphere of 95% air and 5% CO₂.

References

[l] Ulrich-Vinther M, Maloney MD, O'Keefe RJ, Schwarz EM, 2003:, Articular Cartilage

Biology: J.Am.Acad.Orthop.Surg; v. 11 , p. 421-430.

[2] Brittberg M, Lindahl A, Nilsson A, Ohlsson C, IssaKson O, Petterson L: Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation.

N Engl J Med. 1994 Oct 6;331(14):889-95.

[3] Derrett S, Stokes EA, James M, Bartlett W, Bentley G: Cost and health status analysis after autologous chondrocyte implantation and mosaicplasty: a retrospective comparison, lnt J Technol Assess Health Care. 2005 Summer;21(3):359-67

[4] Halleux C, Sottile V, Gasser JA, Seuwen K, Multi-lineage potential of human mesenchymal stem cells following clonal expansion. J Musculoskelet Neuronal Interact. 2001 Sep;2(1 ):71-6.

[5] Randolph MA, Anseth K, Yaremchuk MJ: Tissue engineering of cartilage. Clin Plast Surg. 2003 Oct;30(4):519-37.

[6] Ushida T, Furukawa K, Toita K, Tateishi T, Three-dimensional seeding of chondrocytes encapsulated in collagen gel into PLLA scaffolds. Cell Transplant. 2002, Cell Transplant; 11(5):489-94.

[7] LeBaron RG, Athanasiou KA. Ex vivo synthesis of articular cartilage. Biomaterials. 2000 Dec. 21(24):2575-87.

[8] Spagnoli A, Longobardi L, O'Rear L: Cartilage disorders: potential therapeutic use of mesenchymal stem cells. Endocr Dev. 2005;9: 17-30.

[9] Stengaard C, Elmengaard B, Soballe K, Ulrich-Vinther M: IGF-coating of polymer stimulates chondrocyte synthesis of collagen and aggrecan. Data in preparation. [10] Elder, S. H., Nettles, D. L., Bumgardner, J. D. "Synthesis and characterization of chitosan scaffolds for cartilage-tissue engineering." Methods MoI. Biol., 2004, 238 41-48.

[H] Gundersen, H. J., Bendtsen, T. F., Korbo, L, Marcussen, N., Moller, A., Nielsen, K., Nyengaard, J. R., Pakkenberg, B., Sorensen, F. B., Vesterby, A. et al. "Some new, simple and efficient stereological methods and their use in pathological research and diagnosis." APMIS, May 1988, 96 (5), 379-394.

[12] Hoemann, C. D., Sun, J., Chrzanowski, V., Buschmann, M. D. "A multivalent assay to detect glycosaminoglycan, protein, collagen, RNA, and DNA content in milligram samples of cartilage or hydrogel-based repair cartilage." Anal.Biochem., January 2002, 300 (1 ), 1-10. [13] Pfaffl, M. W. "A new mathematical model for relative quantification in real-time RT- PCR." Nucleic Acids Res., May 2001 , 29 (9), e45.

[14] Schmidmaier, G., Wildemann, B., Stemberger, A., Haas, N. P., Raschke, M. "Biodegradable poly(D,L-lactide) coating of implants for continuous release of growth factors." J.Biomed.Mater.Res., 2001 , 58 (4), 449-455.

Claims

1. A three dimensional cell scaffold, comprising: i) at least one biocompatible scaffold material, ii) at least one coating wherein said at least one coating comprises at least one polymer and/or at least one biological agent.

2. The three-dimensional cell scaffold according to claim 1 , wherein said scaffold material is biodegradable, biostable, or a combination thereof.

3. The three-dimensional cell scaffold according to claim 1, wherein said scaffold material is biostable.

4. The three-dimensional cell scaffold according to claim 3, wherein said biostable scaffold material is selected from the group consisting of metal, hydroxyapatite, choral, elastomers, acrylic resins, plastics and fluorocarbon polymers.

5. The three-dimensional cell scaffold according to claim 1 , wherein said biodegradable scaffold material is a polymer.

6. The three-dimensional cell scaffold according to claim 5, wherein said scaffold material is a synthetic polymer, a natural polymer, or a combination thereof.

7. The three-dimensional cell scaffold according to claim 5, wherein said scaffold material is a natural polymer selected from the group consisting of alginate, cellulose, dextran, glycogen, lignin, gellan, gellan gum, hyaluronic acid, xanthan chitosan, agar, carrageenan and chitosan, chitin, collagen, elastin and silk, and copolymers and blends thereof.

8. The three-dimensional cell scaffold according to claim 5, wherein said scaffold material is a synthetic polymer selected from the group consisting of poly(lactic acid) (PLA), poly(L-lactic acid), poly(DL-lactic acid), polycaprolactone, poly(glycolic acid) (PGA)₁ polyanhydride, poly(alkylene succinates), poly(hydroxy butyrate) (PHB), poly(butylene diglycolate), poly(.epsilon.- caproiactone) and copolymers or blends thereof.

9. The three-dimensional cell scaffold according to claim 2, wherein said scaffold material is chitosan.

10. The three-dimensional cell scaffold according to claim 2, wherein said scaffold material is in the form of a sponge.

11. The three-dimensional cell scaffold according to claim 2, wherein said scaffold material is a chitosan sponge.

12. The three-dimensional cell scaffold according to claim 1 , wherein said three- dimensional cell scaffold has a shape selected from the group consisting of a sheet, a cylinder, a tube, a sphere, a cube, a rectangle, a sponge and an irregular shape.

13. The three-dimensional cell scaffold according to claim 1 , wherein said scaffold has pores.

14. The three-dimensional cell scaffold according to claim 13, wherein said pores have a pore size in the range of from about 0.5 microns to 100 microns.

15. The three-dimensional cell scaffold according to claim 13, wherein said pores have a pore size in the range of from about 25 μm to 75 μm.

16. The three-dimensional cell scaffold according to claim 13, wherein said pores have a pore size in the range of from about 50 μm to 100 μm.

17. The three-dimensional cell scaffold according to claim 1 , further comprising cells selected from the group consisting of autologous, xenogeneic, allogeneic and syngeneic cells.

18. The three-dimensional cell scaffold according to claim 17, wherein said cells are autologous.

19. The three-dimensional cell scaffold according to any of the preceding claims, wherein said scaffold is pre-seeded with cells.

20. The three-dimensional cell scaffold according to claim 17, wherein said cells are selected from the group consisting of epithelial cells, keratinocytes, adipocytes, hepatocytes, neurons, glial cells, astrocytes, podocytes, Schwann cells, mammary epithelial cells, islet cells, endothelial cells, corneal cells, enterocytes, mesenchymal cells, dermal fibroblasts, mesothelial cells, stem cells, osteoblasts, smooth muscle cells, striated muscle cells, heart muscle cells, cardiac myoblasts, skeletal myoblasts, smooth muscle myoblasts, ligament fibroblasts, tendon fibroblasts, meniscal chondrocytes, articular chondrocytes, discus intervertebralios chondrocytes, odontoblasts and ameloblasts.

21. The three-dimensional cell scaffold according to claim 17, wherein said cells are selected from the group consisting of stem cells, osteoblasts, skeletal myoblasts, ligament fibroblasts, tendon fibroblasts, meniscal chondrocytes, articular chondrocytes, discus invertebralios chondrocytes.

22. The three-dimensional cell scaffold according to claim 17, wherein said cells are selected from the group consisting of meniscal chondrocytes, articular chondrocytes, discus invertebralios chondrocytes.

23. The three-dimensional cell scaffold according to claim 1 , wherein said at least one polymer of said coating comprises poly (D,L-lactic acid), or mixtures thereof.

24. The three-dimensional cell scaffold according to claim 1 , wherein said at least one biological agent of said coating comprises growth factors, proteoglycans, polysaccharides, glucosaminoglycans, ECM, nutrients, cytokines, hormones, drugs.

25. The three-dimensional cell scaffold according to claim 24 , wherein said growth factor is selected from the group consisting of PDGF AA, PDGF BB, IGF-I, IGF- II, acidic FGF, basic FGF, .beta.-endothelial cell growth factor, FGF 4, FGF 5, FGF 6, FGF 7, FGF 8, and FGF 9, TGF-P1 , TGF .beta.1.2, TGF-.beta.2, TGF- .beta.3, TGF-.beta.5; BMP 1 , BMP 2, BMP 3, BMP 4, BMP 7, VEGF, placenta growth factor; EGF, amphiregulin, betacellulin, heparin binding EGF, IL -1 , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 , IL-12, IL-13, IL-14, IL-15-18, CSF-G, CSF-GM, CSF-M, erythropoietin; NGF, ciliary neurotropic factor, stem cell factor, hepatocyte growth factor.

26. The three-dimensional cell scaffold according to claim 24, wherein said growth factor is selected from the group consisting of PDGF AA, PDGF BB, IGF-I, IGF- II, acidic FGF, basic FGF, TGF. Beta.1.2, TGF.beta.1.2, TGF-.beta.2, TGF-

.beta.3, TGF-.beta.5, BMP 2, BMP 7.

27. A method for producing a three-dimensional cell scaffold, comprising the steps of i) providing a three-dimensional cell scaffold ii) producing a mixture of at least one polymer and at least one solvent to form a volatile fluid iii) immersing the three-dimensional cell scaffold of i) into the mixture of ii) to coat the three-dimensional cell scaffold and iv) evaporating said solvent.

28. The method of claim 27, wherein the evaporation step occurs in a dessicator system at a pressure of -980 mbar.

29. The method of claim 27, wherein the solvent is organic.

30. The method of claim 29, wherein the organic solvent is ethyl acetate or chloroform.

31. The method of claim 27, wherein the mixture comprises 20 to 300 mg of biodegradable polymer per ml of organic solvent.

32. The method of claim 31 , wherein the mixture comprises, 3-24% PDDLA of the organic solvent volume.

33. The method of claim 27, wherein said mixture further comprises a biological agent, wherein said biological agent is defined in claim 24.

34. A three-dimensional cell scaffold formed by the steps as defined in claim 27.

35. The method of claim 27 further comprising the steps of i) providing a sample of living tissue and/or living cells, ii) processing said sample under sterile conditions, iii) Seeding said cells to said scaffold

36. A method for producing a ready-for use implant as defined in any of claims 1- 26.

37. A method for regenerating tissue in a mammal in need thereof, comprising implanting the cell scaffold of any of claims 1-26 into said mammal.

38. The method of claim 37, wherein said mammal is a human.

39. The method of claim 37, wherein said tissue is selected from the group consisting of bone, cartilage, tendon, ligament, nerve, skin, vascular, cardiac, pericardial, muscle, ocular, periodontal, breast, pancreatic, esophageal, stomach, kidney, hepatic, mammary, adrenal, urological, and intestinal tissue.

40. The method of claim 37, wherein said tissue is selected from the group consisting of bone, cartilage, tendon, ligament.

41. The method of claim 37, wherein said tissue is cartilage.

42. The method of claim 37, wherein said tissue is bone.

43. A three-dimensional cell scaffold comprising i) chitosan sponge, and ii) a poly D,L-polylactic acid coating, wherein said coating comprises at least one growth factor.

44. The three-dimensional cell scaffold of claim 43, wherein said coating further comprises an antibiotic.

45. The three-dimensional cell scaffold according to claim 43, wherein said coating comprises TGFbetal and/or 1GF-1 , and an antibiotic.