WO2008157608A1

WO2008157608A1 - Composite scaffolds for tissue regeneration

Info

Publication number: WO2008157608A1
Application number: PCT/US2008/067352
Authority: WO
Inventors: Vivek Shenoy; Lu Liu; Sara Fermanian; Blanka Sharma
Original assignee: Cartlix, Inc.
Priority date: 2007-06-18
Filing date: 2008-06-18
Publication date: 2008-12-24

Abstract

The present invention provides a porous multi-layer composite scaffold useful for tissue regeneration and a method of fabricating the same. The porous multi-layer composite scaffold comprises a first layer comprising crosslinked collagen and a polysaccharide; a second layer of crosslinked collagen and calcium based minerals, which is covalently bonded to the first layer; and a third layer of crosslinked collagen and a polysaccharide, which is covalently bonded to the second layer. Preferably, the second layer further comprises a polysaccharide. The ratio of collagen to polysaccharide in each of the three layers is from about 3:1 to about 1:1 by weight. The porous multi-layer composite scaffold may further comprises a biologically active agent.

Description

Attorney Docket No. CART-007/01WO

COMPOSITE SCAFFOLDS FOR TISSUE REGENERATION

CROSS REFERENCE TO RELATED APPLICATION

[001] The present application claims the benefit of U.S. Provisional Application

No. 60/944,609, filed on June 18, 2007 and entitled "Composite Scaffolds For Tissue Regeneration", which is herein incorporated by reference in its entirety for all purposes.

FIELD OF INVENTION

[002] The present invention relates to porous composite scaffolds useful for tissue regeneration and methods of fabricating these porous composite scaffolds.

BACKGROUND

[003] Tissue regeneration refers to the regrowth or repair of cells, tissues, and organs and restoration of their functions via the interplay of living cells, an extra-cellular matrix, and cell communicators. Compared to conventional medical treatments, such as, drugs, hormones, vaccines, prosthetics, surgery, or organ transplantation, tissue regeneration provides an advantageous approach to treat diseases. For example, drugs often have undesirable side effects, prosthetics are not biologically active and do not integrate or remodel into the body, surgery is invasive, and organ transplantation is limited by donor availability and toxic immunosuppressive cocktails. In contrast, tissue regeneration delivers living tissue and stimulates the body's own natural healing process by activating the body's inherent ability to repair and regenerate. Thus, tissue regeneration becomes an increasingly important therapy to heal or reconstruct diseased tissue and support the regeneration of diseased or injured organs. Doctors now use tissue regeneration to speed up healing and to help injuries that will not heal or repair on their own. Tissue regeneration has been used to treat heal broken bones, severe burns, chronic wounds, heart damage, nerve damage, and many other diseases. "Tissue Regeneration", "Tissue Engineering", and "Regenerative Medicine" are related terms and sometimes used interchangeably.

[004] Tissue regeneration therapy is often carried out by using a scaffold simulating the body anatomy. One major challenge facing tissue regeneration is the lack

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of appropriate biomaterials for the scaffold. It is required that the scaffold used for tissue regeneration be biocompatible and bioabsorbable. In addition, the scaffold needs to be conformable/compressible as well as robust/incompressible.

Conformability/compressibility allow the scaffold to conform to the biological structure without inducing detrimental stress, while mechanical strength/incompressibility allows the scaffold to withstand handling during fabrication and implantation without compromising its structural integrity. It is also preferable to use porous scaffolds since they can retain aqueous medium and living cells which are critical for tissue growth. Thus, an ideal scaffold for tissue regeneration has composite properties which are well balanced among flexibility/compressibility, mechanical stiffness/incompressibility, porosity, and other physiochemical properties.

[005] The prior art has extensively utilized collagen-based scaffolds as an analog for the extracellular matrix in tissue engineering. To improve the mechanical properties and degradation rate of collagen scaffolds, cross-linking of collagen itself or crosslinking of collagen with chondroitin sulfate (CS) have been employed. See, for examples, Powell, H.M. et al, Biomaterials, 2006, 27, 5821-5827; Pieper, J.S. et al, Biomaterials, 2000, 21, 581-593; Pieper, J.S. et al, Biomaterials, 1998, 20, 847-858; and U.S. Patent Application Publication No. 2003/0100739 to Tsai et al.; and However, these collagen- CS scaffolds lack the desired mechanical strength, i.e., incompressibility, thereby not able to retain their porosity when pressed within a tissue space. Furthermore, a single layer scaffold does not possess composite properties desirable for biomaterials used in tissue engineering.

[006] Lynn et al. (WO 2006/095154) disclose a process for preparing a composite biomaterial wherein at least one layer comprises a triple co-precipitate of collagen, one or more glycosaminoglycans, and calcium phosphate brushite. Lynn et al. state that the microstructure of co-precipitate is substantially different from a material prepared from mechanical mixing of its components. Additionally, Lynn et al. assert that the material prepared by the mechanical mixing process, although biocompatible, produces limited in-growth of natural bone when in the human or animal body. To obtain a co-precipitate of Lynn et al, it is necessary to maintain an acidic condition for the mixture as well as low ratio of glycosaminoglycan in the mixture. In fact, the ratio of

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collagen to the glycosaminoglycan in the biomaterial of Lynn et al. is from 8: 1 to 30: 1 by weight and the preparation of the biomaterial is carried out at pH between 2.5 to 6.5, i.e., under acidic condition, to enable co-precipitation of the components. As stated by Lynn et al. and as would be inherent in a mixture containing a precipitate where components have undergone co-precipitation, the biomaterial of Lynn et al. is heterogeneous in comparison to a mechanically mixed homogeneous mixture. It is important to note that heterogeneous biomaterials containing precipitated components compromise the physical integrity of the biomaterials.

[007] Tsai et al. (US 2003/0100739) disclose a method for producing biomaterials of crosslinked hyaluronic acid and collagen at a pH value between 3 and 11. However, the biomaterial does not have the necessary incompressibility for tissue regeneration application. Furthermore, the method is carried out in presence of organic solvents. It is well known in the art that organic solvents are detrimental to biological tissues, thus any residue of organic solvents in a composite scaffold can be harmful to the recipient of the scaffold. Moreover, the crosslinking step of Tsai et al. is performed after the lyophilizing step thereby resulting in uneven crosslinking of the collagen and hyaluronic acid.. Therefore, the method of Tsai et al. is not ideal for producing biomaterials used in tissue regeneration.

[008] Boyce (US 5,273,900) discloses a bilayer membrane with a porous layer and a non-porous laminating layer, wherein the porous layer contains co-precipitates of collagen and mucopolysaccharide. Lu et al. (US 2006/0036331) disclose a multi-layer scaffold wherein the first layer is a non-porous hydrogel. Notably, lack of porosity in a biomaterial can diminish its ability to retain aqueous medium and living cells which are critical for tissue growth. Moreover, both Boyce and Lu et al. cast the layers using mixtures in acidic conditions. It is observed that crosslinking reaction of collagen and/or glycosaminoglycan under an acidic condition is much faster than that under a basic condition. For a scaffold having more than one layer, it would be advantageous to have adjoining layers crosslinked with each other since crosslinking between adjoining layers enhances the integrity and monolithic strength of the multi-layer scaffold. However, it is difficult to accomplish crosslinking between adjoining layers by using acidic mixtures because the earlier cast layer likely becomes substantially crosslinked when another layer

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is cast later thereby allowing little further crosslinking between the adjoining layers. Furthermore, acidic mixtures of collagen and glycoaminoglycan result in co-precipitation of the components thus forming heterogeneous microstructures.

[009] Yamamoto et al. (US 6,764,517) disclose a porous single layer scaffold with mineralized collagen and a binder wherein the mineral has diameter size less than 5 microns. Inasmuch as the Yamamoto scaffold is single layered, it does not possess composite properties desirable for biomaterials used in tissue engineering. [0010] Stone et al. (US 5,306,311) disclose a two-component scaffold with a conical rigid base layer consisting of collagen and calcium phosphate granules, and a porous top layer consisting of crosslinked collagen and polysaccharide wherein the ratio of collagen to the polysaccharide is above 3:1 by weight. However, the two layers of Stone et al. are disparate and lack monolithic integrity. The unique shape and specific composition of the Stone scaffold may also limit its other uses in tissue regeneration. [0011] Spiro et al. (US 6,773,723) disclose a bilayer scaffold consisting of collagen and/or polysaccharide. However, mere variation of the concentration of collagen and/or polysaccharide and adjustment of the ratio of collagen to polysaccharide cannot impart sufficiently balanced properties of flexibility/compressibility and mechanical toughness to the resulting scaffold.

[0012] Thus, there is still a strong need for a scaffold having desirable physiochemical properties to be applied in tissue regeneration.

SUMMARY OF THE INVENTION

[0013] Accordingly, the present invention provides a porous multi-layer composite scaffold wherein different layers have different physiochemical properties synergistically combining the conformability, robustness, and other desirable properties. [0014] In one embodiment, the present invention provides a method of fabricating a porous multi-layer composite scaffold. The method comprises: applying, to a substrate, a first mixture comprising collagen, a polysaccharide, and a crosslinker to form a first layer of mixture, wherein the ratio of collagen to polysaccharide is from about 3:1 to about 1 : 1 by weight, and the pH of the first mixture is more than 7; applying a second mixture comprising collagen, calcium based minerals, and a crosslinker over the first

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layer of mixture to form a second layer of mixture before the first layer of mixture is substantially crosslinked, wherein the pH of the second mixture is more than 7; applying a third mixture comprising collagen, a polysaccharide, and a crosslinker over the second layer to form a third layer of mixture before the first and second layers of mixture are substantially crosslinked, wherein the ratio of collagen to polysaccharide is from about 3: 1 to about 1 : 1 by weight, and the pH of the third mixture is more than 7; allowing the first, second, and third layers of mixture to crosslink for about 3 hours or more to form a substantially crosslinked multi-layer composite scaffold; and lyophilizing the substantially crosslinked multi-layer composite scaffold to form a porous multi-layer composite scaffold. Preferably, the second mixture further comprises a polysaccharide, and the ratio of collagen to polysaccharide is from about 3: 1 to about 1 : 1 by weight. [0015] In another embodiment, the present invention provides a porous multilayer composite scaffold, which comprises: a first layer comprising crosslinked collagen and a polysaccharide, wherein the ratio of collagen to polysaccharide is from about 3:1 to about 1 : 1 by weight; a second layer comprising crosslinked collagen and calcium based minerals, wherein the second layer is attached to the first layer through covalent bonds; and a third layer comprising crosslinked collagen and a polysaccharide, wherein the third layer is attached to the second layer through covalent bonds, and the ratio of collagen to polysaccharide is from about 3: 1 to about 1 : 1 by weight. Preferably, the second mixture further comprises a polysaccharide, and the ratio of collagen to polysaccharide is from about 3: 1 to about 1 : 1 by weight.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIG. 1 is a partial sectional view of a tri-layer composite scaffold illustrating the porous multi-layer composite scaffold of the present invention. [0017] FIG. 2 is a partial sectional view of a tri-layer composite scaffold with two thin layers of calcium phosphate granules.

DETAILED DESCRIPTION OF THE INVENTION

[0018] The present invention provides a method of fabricating a porous multilayer composite scaffold that can be used for tissue regeneration.

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[0019] In the inventive method, a first mixture is applied to a substrate by known means, such as spinning, casting, and dipping, to form a first layer on the substrate. The substrate can be a mould; a medical device, such as an implant; or a supporting surface. The term "mould" as used herein denotes any container or object capable of shaping, holding, or supporting the first mixture. The mould may be in any desired shape, and may be fabricated from any suitable material including polymers, metals, ceramics, glass ceramics, and glasses. Preferably, the mould is a tray.

[0020] The first mixture comprises collagen, a polysaccharide, and a crosslinker.

By "collagen", it is meant any of a family of extracellular, closely related proteins occurring as a major component of connective tissue, giving it strength and flexibility. At least 14 types of collagen exist, each composed of tropocollagen (q.v.) units that share a common triple -helical shape but that vary somewhat in composition between types, with the types being localized to different tissues, stages, or functions. In some types, including the most common, Type I, the tropocollagen rods associate to form fibrils or fibers; in other types the rods are not fibrillar but are associated with fibrillar collagens, while in others they form nonfibrillar, nonperiodic but structured networks. Collagen useful for the present invention includes both fibrillar and nonfibrillar types. The term collagen as used herein encompasses recombinant human (rh) collagen. [0021] By "polysaccharide", it is meant a carbohydrate that can be decomposed by hydrolysis into two or more molecules of monosaccharides. Preferably, the polysaccharide of the present invention comprises carboxylic acid groups. More preferably, the polysaccharide of the present invention is a long unbranched polysaccharide comprising carboxylic acid groups. In one embodiment, the polysaccharide is a glycosaminoglycan (GAG), also known as mucopolysaccharide. Glycosaminoglycan is a heteropolysaccharide containing an N-acetylated hexosamine and a hexose or hexuronic acid as a characteristic repeating disaccharide unit, wherein either or both of N-acetylated hexosamine and a hexose or hexuronic acid may be sulfated. The combination of the sulfate group and the carboxylate groups of the uronic acid residues gives them a very high density of negative charge. Examples of the polysaccharide of the present invention include, but are not limited to chondroitin sulfate, hyaluronic acid, dermatan sulfate, keratan sulfate, heparin, heparan sulfate, their

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derivatives, and combinations thereof. In one embodiment of the present invention, the polysaccharide is chondroitin sulfate, its derivatives, or combinations thereof. The composite scaffold of the present invention has a high polysaccharide content. That is, the ratio of collagen to polysaccharide in the mixtures is less than 3: 1. Preferably, the ratio of collagen to polysaccharide in the first mixture is from about 3:1 to about 1: 1 by weight. More preferably, the ratio of collagen to polysaccharide in the first mixture is from about 2: 1 by weight.

[0022] The derivative of chondroitin sulfate denotes a molecule that has a chondroitin sulfate core and one or more additional functional groups that enable the molecule to be crosslinked by polymerization. The polymerization can be initiated by photo, redox, ionic, and other polymerization initiators. Examples of the functional groups include, but are not limited to methacrylate, acrylamide, aldehyde, and combinations thereof. Derivatives of other polysachharides described above can also be used in the present invention.

[0023] By "crosslinker", it is meant a chemical agent that can activate certain functional groups of a polymer, protein, or other complex organic molecule and thereby facilitate the formation of covalent bonds linking the chains of atoms in the polymer, protein, or other complex organic molecule. Crosslinkers suitable for the present invention include, but are not limited to carbodiimide agents, aldehyde based crosslinkers, or combinations thereof. A carbodiimide agent is a chemical agent containing a functional group of the formula N=C=N. Carbodiimide agents are often used to activate carboxylic acids towards amide or ester formation. Examples of the carbodiimide agent include, but are not limited to l-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC), N,N'-dicyclohexylcarbodiimide (DCC), N,N'- diisopropylcarbodiimide (DIC), and combinations thereof. An aldehyde based crosslinker is a compound containing an aldehyde group at the terminal position of the compound structure. Examples of the aldehyde based crosslinker include, but are not limited to formaldehyde, acetaldehyde, glutaraldehyde, and combinations thereof. In one embodiment of the present invention, the crosslinker of the first mixture is EDC. The amount of the crosslinker, such as EDC, used in the first mixture depends on the desired extent of the crosslinking. The term "crosslinking" as used herein denotes formation of

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covalent bonds linking one chain or portion of a polymer, protein, or other complex organic molecule to another. In general, the higher concentration of the crosslinker, the more extensive the mixture of collagen and polysaccharide is crosslinked. It is noted that the crosslinking as referred to in the present invention includes crosslinking among different chains or portions of collagen, crosslinking among different chains or portions of the polysaccharide, and crosslinking among different chains or portions of collagen and the polysaccharide. In one embodiment of the present invention, the concentration of EDC ranges from about 1 to about 200 mM with the range from about 5 to about 100 mM more preferred.

[0024] The first mixture is prepared by mechanically mixing collagen, the polysaccharide, and the crosslinker. In one embodiment of the present invention, collagen, the polysaccharide, and the crosslinker are mechanically mixed in an aqueous media. Preferably, the aqueous media is a water solution, suspension, or slurry. The mechanical mixing is a process applying mechanical means to mix the components, such as blending, stirring, shaking, or other agitating method. The pH of the first mixture is more than 7. Preferably, the pH of the first mixture ranges from about 8 to about 12. The pH of the first mixture can be adjusted by one or more inorganic or organic bases known to those skilled in the art. The base can be hydroxide base, such as NaOH, KOH, Ca(OH)₂, Ba(OH)₂, and the like; or nonhydroxide base, such as NaHCO₃, NaCO₃, KHCO₃, KCO₃, ammonia, and the like. The bases used for adjusting the pH of the mixture are compatible with and not detrimental to each of collagen, the polysaccharide, and the crosslinker.

[0025] The first mixture can be cast to form the first layer in various thickness as desired. Preferably, the layers in the present composite scaffold have a thickness ranging from about 0.5 to about 20 mm with the range from about 1 to about 10 mm more preferred.

[0026] When collagen and a polysaccharide are mixed in a basic condition, i.e., at pH of more than 7, the resulting mixture is more homogenous than that in a acidic or neutral condition. That is, a basic mixture of collagen and the polysaccharide is evenly and stably distributed in the solvent and forms homogenous microstructures. Thus, a mixture of collagen and a polysaccharide in a basic condition do not form a co-

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precipitate. Homogeneity of such a basic mixture allows a high ratio of the polysaccharide therein, such as a ratio of collagen to the polysaccharide being less than 3: 1. The pH of such a mixture is more than 7, preferably ranges from about 8 to about 12. In one embodiment of the present invention, the ratio of collagen to chondroitin sulfate in a mixture is about 2: 1. Higher ratio of polysaccharide in the mixture of collagen and a polysaccharide increases the resulting scaffold's retention of fluid under pressure. Furthermore, a basic condition slows down the crosslinking reaction of collagen and the polysaccharide compare to a neutral or acidic condition, i.e., a pH of 7 or less. The slow crosslinking process of the present invention allows casting additional layers without the earlier cast layers becoming substantially crosslinked. This enables crosslinking of adjoining layers and thereby increases the integrity and mechanical strength of the resulting scaffold.

[0027] A second mixture comprising collagen, calcium based minerals, and a crosslinker is then applied over the first layer of mixture to form a second layer of mixture before the first layer of mixture is substantially crosslinked. By "before substantially crosslinked", it is meant less than 90% of the crosslinkers have been consumed by the crosslinking process. The terms "collagen" and "crosslinker" have the same meaning as described above. By "calcium based minerals", it is meant inorganic salts containing calcium ions. It is preferred that the calcium based minerals are in granular forms. It is also preferred that the calcium based minerals are porous. Examples of the calcium based minerals include, but are not limited to calcium phosphate, calcium sulfate, calcium silicate, silicate substituted calcium phosphate, calcium carbonate, calcium citrate, calcium malate, calcium gluconate, calcium oxide, calcium chloride, calcium hydroxide, and combinations thereof. Preferably, the calcium based minerals are calcium phosphate, calcium sulfate, calcium silicate, silicate substituted calcium phosphate, and combinations thereof. Examples of the calcium phosphate include, but are not limited to β-tri-calcium phosphate, hydroxyapatite, biphasic β-tri-calcium phosphate and hydroxyapatite, and combinations thereof. Examples of the calcium sulfate include, but are not limited to calcium sulfate hemihydrate, calcium sulfate dihydrate, and combinations thereof. The term "silicate" denotes a compound containing an anion in which one or more central silicon atoms are surrounded by electronegative

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ligands. When calcium sulfate hemihydrate is used in the second mixture, the amount of calcium sulfate hemihydrate can be adjusted so that the second layer sets and hardens after it is cast on the first layer. In one preferred embodiment, the second mixture further comprises a polysaccharide. The term "polysaccharide" has the same meaning as described above.

[0028] The second mixture is prepared by mechanically mixing collagen, calcium based minerals, and the crosslinker. When the second mixture comprises a polysaccharide, the second mixture is prepared by mechanically mixing collagen, the polysaccharide, calcium based minerals, and the crosslinker. The ratio of collagen to polysaccharide in the second mixture is from about 3: 1 to about 1 : 1 by weight. More preferably, the ratio of collagen to polysaccharide in the second mixture is from about 2: 1 by weight. The pH of the second mixture is more than 7. Preferably, the pH of the second mixture ranges from about 8 to about 12. The conditions of preparing the second mixture is the same as described above for preparing the first mixture. [0029] Next, applying a third mixture comprising collagen, a polysaccharide, and a crosslinker over the second layer to form a third layer of mixture before the first and second layers of mixture are substantially crosslinked. The ratio of collagen to polysaccharide in the third mixture is from about 3: 1 to about 1 : 1 by weight. More preferably, the ratio of collagen to polysaccharide in the third mixture is from about 2: 1 by weight. The pH of the third mixture is more than 7. Preferably, the pH of the third mixture ranges from about 8 to about 12. The third mixture is prepared by mechanically mixing collagen, a polysaccharide, and a crosslinker. The conditions of preparing the third mixture is the same as described above for preparing the first mixture. The collagen in each of the three mixtures can be the same or different. The polysaccharide in each of the three mixtures can be the same or different. Preferably, the polysaccharide in each of the three mixtures is chondroitin sulfate, its derivatives, or combinations thereof. The crosslinker in each of the three mixtures can be the same or different. Preferably, the crosslinkers in the three mixtures are carbodiimide, such as EDC. To further increase yields and decrease side reactions of the crosslinking process, one or more crosslinking additives may be added to any one of the three mixtures. Examples of the crosslinking

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additive include, but are not limited to N-hydroxysuccinimide (NHS), hydroxybenzotriazole (HOBT), hydroxyazotriazole (HOAT), and combinations thereof. [0030] Different layers of the present composite scaffold have different physiochemical properties, such as conformability/flexibility, mechanical stiffness/incompressibility, and the ability to retain fluid. Particularly, the middle layer has different physiochemical properties from the top and bottom layers. The physiochemical properties of the composite scaffold can be adjusted by changing the concentrations of collagen and/or the polysaccharide, the ratio of collagen to the polysaccharide, and other conditions. The ratio of collagen to polysaccharide in the first and third mixtures may be the same or different ranging about 3: 1 to about 1 : 1 by weight. When the second mixture comprises a polysaccharide, the ratio of collagen to the polysaccharide may be the same as or different from that of the other two mixtures ranging from about 3:1 to about 1:1 by weight. When the ratio of collagen to the polysaccharide is in the range described in the present invention, the resulting composite scaffold has the desirable compressibility as well as ability to retain fluid under mild compressive loads. That is, when implanted into a tissue space, the composite scaffold is sufficiently conformable/flexible to alter its shape in accordance to the structure of the surrounding tissue space without causing stress thereto, and the composite scaffold is also able to retain such a level of fluid content to facilitate the growth of body tissues within the composite scaffold. Preferably, the ratio of collagen to the polysaccharide in the present invention is about 2:1 by weight. In the present invention, the concentration of collagen typically ranges from about 5 to about 80 mg/ml with the range from about 10 to about 60 mg/ml more preferred, while the concentration of the polysaccharide typically ranges from about 2 to about 80 mg/ml with the range from about 5 to about 60 mg/ml more preferred.

[0031 ] The calcium based minerals in the second mixture imparts desirable firmness and incompressibility to the second layer. Depending on the type of calcium based minerals, their concentrations in the mixture, and their granular sizes, the firmness and incompressibility of the second layer changes accordingly. Typically, for example, the granules of the calcium based minerals are porous and have diameter sizes ranging from about 50 microns to about 10 millimeters with the range from about 75 microns to

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about 7.5 millimeters more preferred and the range from about 100 microns to about 5 millimeters most preferred. Typically, for example, the concentration of the calcium based minerals ranges from about 5 to about 1500 mg/ml with the range from about 10 to about 1000 mg/ml more preferred. In one embodiment, additional layers can be formed by the steps described above.

[0032] Next, the first, second, and third layers of mixture are allowed to crosslink for about 3 hours or more to form a substantially crosslinked multi-layer composite scaffold. By "substantially crosslinked", it is meant 90% or more of the crosslinkers have been consumed by the crosslinking process. Preferably, the first, second, and third layers of mixture are allowed to crosslink for about 4 to about 24 hours to form a substantially crosslinked multi-layer composite scaffold. The crosslinking kinetics are dependent on the concentration of the components, the temperature as well as the pH of the mixture. In general, higher concentration of the components, particularly the crosslinkers, and higher temperature speed up the crosslinking process, while higher pH slows down the crosslinking process. The crosslinking of the first, second, and third layers of mixture yields a substantially crosslinked monolithic composite scaffold. In other words, the step of substantial crosslinking not only allows further crosslinking within each layers, but also causes crosslinking between the layers which are in direct contact. Thus, the substantial crosslinking step allows formation of covalent bonds between the first and second layers as well as between the second and third layers, and thereby enhances the integrity and mechanical strength of the multi-layer composite scaffold. In one embodiment of the present invention, the first, second, and third layers of mixture are allowed to stand still at the room temperature, i.e., from about 20° to about 25°C, for about 4 to about 24 hours to form a substantially crosslinked multi-layer composite scaffold. If the substantial crosslinking process is desired to be faster, the first, second, and third layers of mixture can be placed at a temperature higher than the room temperature, but preferably lower than about 45°C.

[0033] The substantially crosslinked multi-layer composite scaffold is then lyophilized to form a porous multi-layer composite scaffold. By "porous", it is meant full of pores, vessels, or holes and able to absorb fluid. The pores, vessels, or holes may be of various sizes, shapes, and aspect ratios. Lyophilization, also known as freeze-drying, is a

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dehydration process that involves freezing the material and then reducing the surrounding pressure with or without additional heat to allow the frozen water in the material to sublime directly from the solid phase to gas. Typically, high vacuum is applied in the lyophilization process. In the present invention, the substantially crosslinked multi-layer composite scaffold may be lyophilized by any methods known to those skilled in the art. In one embodiment of the present invention, the sublimation step comprises reducing the pressure in the environment around the mould and frozen the three layers to below the triple point of the water/ice/water vapor system, followed by elevation of the temperature to greater than the temperature of the solid- vapor transition temperature at the achieved vacuum pressure. The ice in the product is directly converted into vapor via sublimation as long as the ambient partial liquid vapor pressure is lower than the partial pressure of the frozen liquid at its current temperature. The temperature is typically elevated to 0⁰C or above. The freeze-drying parameters may be adjusted to control pore size and aspect ratio as desired. In general, slower cooling rates and higher final freezing temperatures yields large pores with higher aspect ratios, while faster cooling rates and lower final freezing temperatures causes the formation of small equiaxed pores. It is noted that each and every layer of the present multi-layer composite scaffold is porous. [0034] Thus, the present method affords a porous multi-layer composite scaffold that can be used in the field of tissue engineering. For example, the inventive porous multi-layer composite scaffold can enhance tissue regeneration in musculoskeletal tissues as well as osteochondral tissues. Particularly, different regions/layers of the porous multi-layer composite scaffold have different physiochemical properties in such a way that the conformability/flexibility, mechanical strength/incompressibility, and ability to retain fluid under mild compressive loads are synergistically balanced. For example, the compressibility of the upper and lower regions and incompressibility of the middle region allow the composite scaffold to effectively fill voids and mold to surfaces with irregular contours while providing a scaffold with mechanical rigidity thereby retaining its porosity.

[0035] The present composite scaffolds of varying compressibility can overcome the limitations of prior art scaffolds that are either compressible or non-compressible. The compressible scaffolds have limited applicability since, when subjected to

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mechanical stresses, the compressible graft is structural altered thereby changing its porosity. The performance of incompressible scaffolds is hindered by its inability to be in close apposition to the surrounding tissue when it is used to fill voids and placed adjacent to surfaces with irregular contours. When using the present composite scaffolds, the compressibility allows the scaffolds to be in close apposition to surrounding tissue which can enhance the integration of the regenerative tissue, while the incompressible region can help withstand moderate mechanical stresses without compromising the structural integrity of the scaffold. The presence of calcium based minerals in the collagen/CS scaffold can aid in regeneration of bony tissue while the combination of collagen and a polysaccharide like chondroitin can aid in the regeneration of chondral tissue. Furthermore, a crosslinked scaffold of a fibrous collagen with hydrophilic chondroitin sulfate provides a unique combination of fluid retention properties which results in mechanical properties similar to chondral tissue.

[0036] In one embodiment of the present invention, the porous multi-layer composite scaffold is rinsed with water after the lyophilizing step, and then re-lyophilized as described above. The rinsing step can remove the unreacted crosslinkers, if any, and/or the side products of the crosslinkers. For example, the carbodiimide agents become urea compounds after the crosslinking process. If desired, the rinsing and re- lyophilizing steps may be repeated.

[0037] Depending on the intended use, the porous multi-layer composite scaffold can be in various shape or size. In one embodiment of the present invention, the first mixture is cast in such a way to form a spherical core, then the second mixture is applied over the spherical core to form the second layer. Next, the third mixture is applied over the second layer to form a third layer. The resulting spherical shaped multi-layer composite scaffold can be substantially crosslinked and lyophilized as the steps described above.

[0038] In another aspect, the present invention provides a porous multi-layer composite scaffold, which comprises: a first layer comprising crosslinked collagen and a polysaccharide; a second layer comprising crosslinked collagen and calcium based minerals; and a third layer comprising crosslinked collagen and a polysaccharide. The second layer is attached to the first layer through covalent bonds, and the third layer is

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attached to the second layer through covalent bonds. The ratio of collagen to the polysaccharide in the first and third layers can be the same or different ranging from about 3: 1 to about 1 : 1 by weight. In one embodiment of the present invention, the second layer further comprises a polysaccharide and the ratio of collagen to the polysaccharide may be the same as or different from that of other two layers ranging from about 3:1 to about 1 : 1 by weight. The terms "collagen", "polysaccharide", and "calcium based minerals" have the same meaning as described above. In the present porous multi-layer composite scaffold, collagen and polysaccharide are crosslinked both intra-molecularly and inter-molecularly.

[0039] In one embodiment of the present invention, the porous multi-layer composite scaffold is a porous tri-layer composite scaffold. As described herein, the first, second, and third layers are also referred to as the upper, middle, and bottom layers/regions, respectively. In some instances, the first and third layers are referred to as the outer layers/regions.

[0040] The porous multi-layer composite scaffold of the present invention may comprise additional layers. The additional layers may be fabricated by the inventive method described above or by other methods known to those skilled in the art. For example, the crosslinking of the additional layers may be carried out either before or after lyophilization. In one embodiment, a first, second, third, and fourth mixtures, each of which comprises one or more crosslinkers, are cast in sequence to form four layers. Then the four layers are allowed to substantially crosslink to form a crosslinked tetra-layer composite scaffold, which is subsequently lyophilized. In another embodiment, a fourth mixture that does not comprise any crosslinker is cast over a tri-layer composite scaffold prepared as described above, and then lyophilized to form a porous fourth layer, and then the lyophilized fourth layer is soaked in a solution containing one or more crosslinkers thereby accomplishing the crosslinking process.

[0041] The porous multi-layer composite scaffold of the present invention can be used to deliver one or more biologically active agents. The porous multi-layer composite scaffold of the present invention may further comprise one or more biologically active agents. Examples of the biologically active agent include, but are not limited to nutrient; pharmaceutical agents; growth factors; cells, such as blood cells, chondrocytes, and stem

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cells; peptides; osteoinductive agents; chondroinductive agents; saccharide molecules; polynucleotides, such as DNA; and combinations thereof. Additional cells that can be used for the present invention include, but are not limited to chondrocytes, osteoblasts, other cells that form bone, muscle cells, fibroblasts, hepatocytes, islet cells, cells of intestinal origin, cells of kidney origin, stem cells, and other cells acting primarily to synthesize and secrete, or to metabolize materials as described in U. S. Patent 6,224, 893 Bl to Langer et al. , the entire disclosure of which is incorporated herein by reference. Preferably, the biological agent is a bone marrow aspirate, growth factors, chondrocytes, osteoblasts, fibroblasts, hepatocytes, mesenchymal stem cells, and any combination thereof. The biologically active agents may be present in any of the layers of the present composite scaffold, and may be attached to the porous multi-layer composite scaffold covalently or non-covalently. In one embodiment, a biologically active agent is crosslinked to the porous multi-layer composite scaffold via amide formation with the carboxylic acid groups of collagen and/or the polysaccharide. In another embodiment, a biologically active agent is covalently attached to the porous multi-layer composite scaffold via crosslinking with the functional group of a polysaccharide derivative. In yet another embodiment, the porous multi-layer composite scaffold is immersed in a solution of the biologically active agent thereby soaked with the biologically active agent. [0042] The porous multi-layer composite scaffold of the present invention may be implanted into a tissue space with or without a biologically active agent. Before being implanted, the fabricated composite scaffold may be sterilized by the methods known to those skilled in the art, such as gamma irradiation, ebeam irradiation, ethylene oxide, and other methods. The sterilization can be performed before or after the attachment of one or more biologically active agents.

[0043] An illustrative example of the fabrication process is described herein.

Collagen, preferably fibrous collagen, is mechanically mixed with chondroitin sulfate (CS) and l-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) to form a first mixture. The concentration of collagen can range from 10 - 60 mg/ml, the concentration of CS can range from 5 - 60 mg/ml, and the concentration of EDC can range from 5 - 100 mM. The pH of the mixture is adjusted to above 7. N-

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hydroxysulfosuccinimide (NHS) may also be added to the mixture to increase the crosslinking efficiency of the EDC. The first mixture is then cast into a tray. [0044] Collagen, preferably fibrous collagen, is mixed with tri-calcium phosphate granules (TCP) and l-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) to form a second mixture. The concentration of collagen can range from 10 - 60 mg/ml, the concentration of TCP can range from 10 - 1000 mg/ml, and the concentration of EDC can range from 5 - 100 mM. The pH of the mixture is adjusted to above 7. N- hydroxysulfosuccinimide (NHS) may also be added to the mixture to increase the crosslinking efficiency of the EDC. The TCP granules are preferably porous and the particle size preferably ranges from 100 microns up to 5 millimeters. The addition of granules increases the compressive strength of the lyophilized scaffold. The second mixture may also contain CS in the range of 5 - 60 mg/ml. The second mixture is immediately cast on top of the first mixture.

[0045] A third mixture similar to the first mixture is immediately cast on top of the second mixture. The mixtures are allowed to crosslink for about 4 to 24 hours at about 20⁰C to form a crosslinked composite scaffold . The crosslinked composite scaffold is then lyophilized to obtain a porous composite scaffold. As illustrated in FIG. 1, regions 1 and 3 are the layers of crosslinked collagen and CS having flexibility and compressibility, while region 2 is the layer of collagen, CS, and TCP granules having mechanical strength and incompressibility. Regions 1, 2, and 3 are all porous. [0046] Furthermore, during the fabrication of the composite scaffold as described above, a thin layer of calcium phosphate granules, preferably ranging from 50 to 250 microns can be layered on the first mixture before casting the second mixture containing collagen, CS, and TCP. Similarly, another thin layer of TCP granules, can be layered on the second mixture before casting the third mixture containing collagen and CS. The addition of the thin layers of TCP provides increased mechanical rigidity between the different regions of the composite scaffold as well as a transition between the region containing larger granules of TCP and regions without TCP. FIG. 2 illustrates a tri-layer composite scaffold wherein there is a thin layer 5 of TCP between the layer 4 of collagen and CS and the layer 6 of collagen, CS, and TCP granules, and a thin layer 7 of TCP

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between the layer 8 of collagen and CS and the layer 6 of collagen, CS, and TCP granules.

[0047] In addition, a thin layer of crosslinking solution may also be applied on top of the first layer before casting the second layer and on top of the second layer before casting the third layer. The lyophilized scaffold may be sterilized using commons methods like ebeam irradiation, gamma irradiation, ethylene oxide etc. To minimize any detrimental effect of the sterilization process, the scaffold could be sterilized in the absence of oxygen or under a blanket of inert gas, and/or under controlled humidity and temperature.

[0048] Other polysaccharides which have a carboxylic acid group, such as, for example, hyaluronic acid, may be used as the sole polysaccharide in the mixture or as one of the polysaccharides in combination with chondroitin sulfate. Granules of hydroxyapatite (HA), biphasic HA/TCP, or other calcium based minerals may be added to the mixture instead of TCP. The composite scaffolds may be fabricated with each layer having different compositions, for example; the upper, middle, and lower region may have different concentrations of collagen or different ratios of collagen and polysaccharide.

[0049] The composite scaffolds described in the present invention can be used as a bone graft in orthopedic applications. For example, the composite scaffolds can be soaked in bone marrow aspirate and used to fill bony voids. The compressible outer regions of the scaffold will enable the graft to effectively fill irregular bone voids or areas within spinal interbody cages in spinal fusion applications. The compressibility of the upper and lower regions and incompressibility of the middle region allow the scaffold to effectively fill voids and mold to surfaces with irregular contours while providing a scaffold with mechanical rigidity thereby retaining its porosity.

[0050] For posterior spinal fusion applications, the scaffold fabricated in the form of a strip can be used across the spinal processes, with the compressible outer regions enabling the graft to adapt to the contours of the bone surface while the incompressible middle region provides mechanical rigidity to the scaffold.

[0051] The composite scaffolds can also be used in interface applications, such as, for example, osteochondral defects. The graft made from the composite scaffold

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could be retained in the defect by a mechanical fit or an adhesive that can attach the composite scaffold to the defect surface. The adhesive can be a polymer having reactive groups that react with both the collagen on the defect surface and the collagen or polysaccharide in the composite scaffold. An example of an adhesive would be an oxidized polysaccharide like chondroitin sulfate or hyaluronic acid which reacts with the lysine groups on the collagen in the defect and the lysine groups on the collagen in the composite scaffold. Other reactive groups which react with collagen or the polysaccharide may also be used.

[0052] The polysaccharide could be added to the formulation as crosslinked particles. The cross-linked particles of polysaccharide may be derived from photo and/or redox-initiated polymerization of derivatized polysaccharides. The polysaccharide can be derivatized by reacting it with glycidyl methacrylate. The composite scaffolds containing the polysaccharide particles are fabricated as described above. The pore structure of the scaffold could be modulated by the addition of cross-linked polysaccharide particles. The presence of the polysaccharide particles in the composite scaffold could also enhance its cellular interactions.

[0053] Derivatized polysaccharides can be added to the formulation solely or in combination with polysaccharides. The polysaccharide may be derivatized by reacting it with glycidyl methacrylate. Biomolecules, such as, for example, growth factors, peptides, and etc., can be covalently cross-linked to the scaffold using the methacrylate chemistry and a photo and/or redox initiator. Oxidized polysaccharide may also be used.

[0054] The following examples are provided to illustrate the processes for fabricating the multi-layer composite scaffolds.

Example 1

[0055] Fibrous bovine collagen (Devro , Australia) was mixed with bovine chondroitin sulfate (Kraeber™ GMBH, Germany) in water. The pH of the collagen and chondroitin sulfate mixture was adjusted to pH 9.4. Then a solution of l-Ethyl-3-[3- dimethylaminopropyl] carbodiimide hydrochloride (EDC) (Sigma Aldrich) in water was added to the collagen and chondroitin sulfate mixture to obtain a final composition of 40

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mg/g collagen, 20 mg/g chondroitin sulfate, and 50 mg/g EDC. This process afforded

Mixture A.

[0056] Tri-calcium phosphate granules (TCP) (Berkeley Advanced Biomaterials) was mixed with collagen, chondroitin sulfate, and EDC to obtain a composition of 27 mg/g collagen, 13 mg/g chondroitin sulfate, 6.69 mg/g EDC, and 330 mg/g TCP to obtain

Mixture B.

[0057] Next, Mixture A was cast in a tray as a 4 mm thick bottom layer. Mixture

B was then cast as a 6 mm thick middle layer on top of the bottom layer. Finally, a 4 mm thick top layer was cast on top of the middle layer using mixture A.

[0058] The tri-layer composite scaffold was allowed to crosslink at 20⁰C for about 12 hours. The crosslinked composite scaffold was then lyophilized over 72 hours to obtain a porous matrix. The lyophilized composite scaffold was cut into different shapes (squares, rectangles, and cylinders) of varying sizes.

Example 2

[0059] In a separate experiment, the porous matrix as prepared in Example 1 was rinsed with water to remove any reactants or byproducts. The rinsed scaffold was lyophilized again. The re-lyophilized composite scaffold was cut into different shapes (squares, rectangles, and cylinders) of varying sizes.

[0060] All publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. [0061 ] While the present invention has been particularly shown and described with respect to preferred embodiments thereof, it will be understood by those skilled in the art that the foregoing and other changes in forms and details may be made without departing from the spirit and scope of the invention. It is therefore intended that the present invention not be limited to the exact forms and details described and illustrated but fall within the scope of the appended claims.

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Claims

Attorney Docket No. CART-007/01WOWe claim:

1. A method of fabricating a porous multi-layer composite scaffold comprising: applying, to a substrate, a first mixture comprising collagen, a polysaccharide, and a crosslinker to form a first layer of mixture, wherein the ratio of collagen to polysaccharide is from about 3: 1 to about 1 : 1 by weight, and the pH of the first mixture is more than 7; applying a second mixture comprising collagen, calcium based minerals, and a crosslinker over the first layer of mixture to form a second layer of mixture before the first layer of mixture is substantially crosslinked, wherein the pH of the second mixture is more than 7; applying a third mixture comprising collagen, a polysaccharide, and a crosslinker over the second layer to form a third layer of mixture before the first and second layers of mixture are substantially crosslinked, wherein the ratio of collagen to polysaccharide is from about 3: 1 to about 1 : 1 by weight, and the pH of the third mixture is more than 7; allowing the first, second, and third layers of mixture to crosslink for about 3 hours or more to form a substantially crosslinked multi-layer composite scaffold; and lyophilizing the substantially crosslinked multi-layer composite scaffold to form a porous multi-layer composite scaffold.

2. The method of claim 1 further comprising rinsing the porous multi-layer composite scaffold after the lyophilizing step; and relyophilizing the rinsed porous multilayer composite scaffold.

3. The method of claim 1 , wherein the first mixture is prepared by mechanically mixing collagen, a polysaccharide, and a crosslinker; the second mixture is prepared by mechanically mixing collagen, calcium based minerals, and a crosslinker; and the third mixture is prepared by mechanically mixing collagen, a polysaccharide, and a crosslinker.

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4. The method of claim 1, wherein the second mixture further comprises a polysaccharide, and the ratio of collagen to polysaccharide is from about 3: 1 to about 1 : 1 by weight.

5. The method of claim 4, wherein the second mixture is prepared by mechanically mixing collagen, a polysaccharide, calcium based minerals, and a crosslinker

6. The method of claim 1 or 4, wherein the pH of each of the three mixtures is from about 8 to about 12.

7. The method of claim 1 or 4, wherein the first, second, and third layers of mixture are allowed to crosslink for about 4 to about 24 hours to form a substantially crosslinked multi-layer composite scaffold.

8. The method of claim 1 or 4, wherein the polysaccharide comprises carboxylic acid groups.

9. The method of claim 8, wherein the polysaccharide is selected from the group consisting of chondroitin sulfate, hyaluronic acid, dermatan sulfate, keratan sulfate, heparin, heparan sulfate, and a combination thereof.

10. The method of claim 1, wherein the crosslinkers of the first, second, and third mixtures is a carbodiimide agent, an aldehyde based crosslinker, or a combination thereof.

11. The method of claim 1 , wherein the crosslinkers of the first and third mixtures are carbodiimide agents, and the crosslinker of the second mixture is an aldehyde based crosslinker.

12. The method of claim 1, wherein the crosslinkers of the first, second, and third mixtures are carbodiimide agents.

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13. The method of claim 10, 11, or 12, wherein the carbodiimide agent is selected from a group consisting of l-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC), N^'-dicyclohexylcarbodiimide (DCC), N,N'- diisopropylcarbodiimide (DIC), and a combination thereof.

14. The method of claim 10 or 11, wherein the aldehyde based crosslinker is selected from a group consisting of formaldehyde, acetaldehyde, glutaraldehyde, and a combination thereof.

15. The method of claim 1, wherein the calcium based minerals are granular.

16. The method of claim 1 , wherein the granular calcium based minerals are porous.

17. The method of claim 1 , wherein the calcium based minerals are selected from the group consisting of calcium phosphate, calcium sulfate, calcium silicate, silicate substituted calcium phosphate, calcium carbonate, calcium citrate, calcium malate, calcium gluconate, calcium oxide, calcium chloride, calcium hydroxide, and combinations thereof.

18. The method of claim 17, wherein the calcium phosphate is selected from the group consisting of β-tri-calcium phosphate, hydroxyapatite, biphasic β-tri-calcium phosphate and hydroxyapatite, and a combination thereof.

19. The method of claim 17, wherein the calcium sulfate is selected from the group consisting of calcium sulfate hemihydrate, calcium sulfate dihydrate, and a combination thereof.

20. The method of claim 1, wherein at least one of the three mixtures further comprises a crosslinking additive selected from the group consisting of N-

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hydroxysuccinimide (NHS), hydroxybenzotriazole (HOBT), hydroxyazotriazole (HOAT), and a combination thereof.

21. The method of claim 1 , wherein the ratio of collagen to polysaccharide in each of the three mixtures is about 2:1 by weight.

22. The method of claim 1 , wherein the substrate is a mould.

23. A porous multi-layer composite scaffold, comprising: a first layer comprising crosslinked collagen and a polysaccharide, wherein the ratio of collagen to polysaccharide is from about 3: 1 to about 1 : 1 by weight; a second layer comprising crosslinked collagen and calcium based minerals, wherein the second layer is attached to the first layer through covalent bonds; and a third layer comprising crosslinked collagen and a polysaccharide, wherein the third layer is attached to the second layer through covalent bonds, and the ratio of collagen to polysaccharide is from about 3: 1 to about 1 : 1 by weight.

24. The porous multi-layer composite scaffold of claim 23, wherein the second layer further comprises a polysaccharide.

25. The porous multi-layer composite scaffold of claim 23 or 24, wherein the polysaccharide comprises carboxylic acid groups.

26. The porous multi-layer composite scaffold of claim 23 or 24, wherein the polysaccharide is selected from the group consisting of chondroitin sulfate, hyaluronic acid, dermatan sulfate, keratan sulfate, heparin, heparan sulfate, and a combination thereof.

27. The porous multi-layer composite scaffold of claim 23, wherein the calcium based minerals are granular.

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28. The porous multi-layer composite scaffold of claim 23, wherein the granular calcium based minerals are porous.

29. The porous multi-layer composite scaffold of claim 23, wherein the calcium based minerals are selected from the group consisting calcium phosphate, calcium sulfate, calcium silicate, silicate substituted calcium phosphate, calcium carbonate, calcium citrate, calcium malate, calcium gluconate, calcium oxide, calcium chloride, calcium hydroxide, and combinations thereof.

30. The method of claim 29, wherein the calcium based minerals are calcium phosphate.

31. The porous multi-layer composite scaffold of claim 30, wherein the calcium phosphate is selected from the group consisting of β-tri-calcium phosphate, hydroxy apatite, biphasic β-tri-calcium phosphate and hydroxyapatite, and a combination thereof.

32. The method of claim 29, wherein the calcium based minerals are calcium sulfate.

33. The method of claim 32, wherein the calcium sulfate is selected from the group consisting of calcium sulfate hemihydrate, calcium sulfate dihydrate, and a combination thereof.

34. The porous multi-layer composite scaffold of claim 23, wherein the ratio of collagen and polysaccharide in each of the three layers is the same or different.

35. The porous multi-layer composite scaffold of claim 23, wherein the ratio of collagen and polysaccharide in each of the three layers is about 2: 1 by weight.

36. The porous multi-layer composite scaffold of claim 23, wherein the polysaccharide in each of the three layers is the same or different.

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37. The porous multi-layer composite scaffold of claim 23 further comprises a biologically active agent.

38. The porous multi-layer composite scaffold of claim 37, wherein the biologically active agent is selected from the group consisting of bone marrow aspirate, growth factors, chondrocytes, osteoblasts, fibroblasts, hepatocytes, mesenchymal stem cells, and a combination thereof.

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