A METHOD FOR MAKING A POROUS SCAFFOLD SUITABLE FOR USE IN REPAIR OF OSSEOUS, CHONDRAL, OR OSTEOCHONDRAL DEFECTS IN A MAMMAL

Abstract

A method for making a porous devitalised scaffold suitable for use in repair of osseous, chondral, or osteochondral defects in a mammal comprises the steps of providing micronized extracellular matrix (ECM) tissue, mixing the micronized extracellular matrix with a liquid to provide a slurry, and freeze-drying the slurry to provide the porous scaffold. A porous scaffold suitable for use in repair of osseous, chondral, or osteochondral defects in a mammal and comprising a porous freeze-dried matrix formed from micronised decellularised extracellular matrix tissue is also described.

Claims

1.-65. (canceled)

66. A method for making a porous scaffold suitable for use in repair of osseous, chondral, or osteochondral defects in a mammal, the method comprising the step of: providing micronized extracellular matrix (ECM) tissue; mixing the micronized extracellular matrix with a liquid to provide a slurry; and freeze-drying the slurry to provide the porous scaffold, wherein the extracellular matrix is treated to reduce the GAG content to less than 90% of the GAG content of untreated ECM, and wherein the ECM tissue is cartilage ECM.

67. A method according to claim 66 in which the micronized cartilage extracellular matrix tissue has a mean particle size of 10-200 microns.

68. A method according to claim 66 in which the slurry comprises 100-400 mg/ml micronized cartilage ECM tissue.

69. A method according to claim 66 in which the micronized cartilage extracellular matrix tissue is cryomilled cartilage extracellular matrix tissue.

70. A method according to claim 66 in which the porous scaffold is cross-linked.

71. A method according to claim 66 in which the cartilage extracellular matrix is hyaline cartilage ECM or growth plate ECM.

72. A method according to claim 66 in which the cartilage extracellular matrix is decellularised before or after micronizing.

73. A method according to claim 66 in which the method of the invention includes an additional step of seeding the scaffold with a biological material selected from cells or a biological growth factor.

74. A method according to claim 73 in which: the cells are selected from the group consisting of stem cells, chondrocytes, mesenchymal cells and osteoblasts; and/or in which the biological growth factor is selected from the group consisting of one or more of the TGF-β superfamily or cannabinoids.

75. A method of making a multilayer scaffold comprising the steps of making a first layer comprising a porous scaffold according to a method of claim 66, making a second layer comprising a porous scaffold according to a method of claim 66, wherein the first layer is attached to the second layer.

76. A method according to claim 75 in which the process includes a step of attaching the first layer to the second layer to form the multilayer scaffold, in which the first layer comprises hyaline cartilage ECM and the second layer comprises growth plate ECM.

77. A porous scaffold suitable for use in repair of osseous, chondral, or osteochondral defects in a mammal and comprising a porous freeze-dried matrix formed from micronised decellularised extracellular matrix tissue, wherein the extracellular matrix tissue comprises less than 90% of the GAG content of natural ECM, and wherein the extracellular matrix tissue is cartilage extracellular matrix tissue.

78. A porous scaffold according to claim 77 in which the porous scaffold is cross-linked.

79. A porous scaffold according to claim 77 in which the cartilage extracellular matrix is hyaline cartilage extracellular matrix or growth plate extracellular matrix.

80. A multilayer scaffold suitable for repair of osteochondral defects in a mammal and having a first layer comprising a porous scaffold according to claim 77 in which the cartilage extracellular matrix is hyaline cartilage extracellular matrix and a second layer comprising a porous scaffold according to claim 77 in which the cartilage extracellular matrix is growth plate extracellular matrix, in which the first layer is attached to the second layer.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0063] FIG. 1: Concentration modulates scaffold morphology. Helium ion (HIM) micrographs showed different architecture in scaffolds when cartilage ECM slurry concentration was altered: (A) 250 mg/ml; (B) 500 mg/ml; (C) 1000 mg/ml. Mean pore size decreased with increased concentration of ECM.

[0064] FIG. 2: Distribution of live cells. Confocal microscopy at day 1 and day 28 of human infrapatellar fat pad derived stem cells seeded in ECM derived scaffolds: (A) 250 mg/ml, (B) 500 mg/ml and 1000 mg/ml. Picture represents cross-section of ECM derived scaffolds. Poor cellular penetration at day 1 was observed in the (B) 500 mg/ml and (C) 1000 mg/ml scaffolds, which contrasts with the 250 mg/ml scaffold where a homogeneous cellular infiltration was observed (A). At day 28, scaffold with 1000 mg/ml of ECM continued to show poor stem cells infiltration (F).

[0065] FIG. 3: sGAG deposition for day 0, 7, 14 and 28. Histological images of glycosaminoglycans (GAG) (alcian blue) and cell nuclei (nuclear fast red) staining for ECM derived scaffolds—250, 500 and 1000 mg/ml—at day 0, 7, 14 and 28 of culture (A). In (A) it is possible to observe strong GAG deposition and cell distribution for the 250 mg/ml scaffold. With high magnification it is possible to observe the superior GAG deposition for 250 (B), followed by 500 (C) and 1000 mg/ml scaffold (D).

[0066] FIG. 4: TGF-β3 release profile for constructs with or without EDAC crosslinking. ELISA results for TGF-β3 release into the media from TGF-β3 loaded ECM derived scaffold indicates slower release rate for scaffolds with EDAC crosslinking, with significant difference at day 4 (n=6, *p<0.05).

[0067] FIG. 5: Tailored sGAG scaffold morphology. Helium ion (HIM) micrographs showed altered pore size and architecture in tailoring GAG concentration of scaffolds: (A) 5% GAG; (B) 50% GAG; (C) 100% GAG. Mean pore size increased with decreasing GAG concentration.

[0068] FIG. 6: sGAG Histology. GAG staining on decellularized tailored GAG cartilage explants. micrographs showed in tailoring GAG concentration: (A) 5% GAG; (B) 50% GAG; (C) 100% GAG.

[0069] FIG. 7: TGF-β3 release profile from tailored GAG cartilage ECM scaffolds. ELISA results for TGF-β3 release into the media from TGF-β3 loaded ECM derived scaffold 4 (n=4). By removing sGAGs from the ECM prior to scaffold fabrication, it is possible to slow the release of growth factors from the construct.

[0070] FIG. 8: Biochemical assay for DNA and GAG content of tailored GAG scaffold.

[0071] FIG. 9: Biochemical assays performed on cartilage tissues engineered in vitro using tailored GAG scaffolds seeded with human stem cells

[0072] FIG. 10: Histological staining for sGAG (Alcian blue) and Collagen (Picrosirius red) of cartilage tissues engineered in vitro using ECM derived scaffolds seeded with human stem cells.

[0073] FIG. 11: Gross appearance of Fibrin-ECM particle constructs post-gelation

[0074] FIG. 12: TGF-β3 release profile for Fibrin hydrogel loaded with ECM particles. ELISA results for TGF-β3 release into the media from TGF-β3 loaded ECM derived particles (n=4).

[0075] FIG. 13: GAG content of cartilage tissues engineered in vitro using ECM particle loaded hydrogels. Fibrin hydrogels containing ECM particles loaded with TGF-β3 showed higher GAG accumulation than constructs where TGF-β3 was either added directly to the media or added to control gelatine microparticles (MPs).

[0076] FIG. 14: Histology. GAG and Collagen staining of cartilage tissues engineered in vitro within fibrin hydrogels loaded with ECM derived particles

[0077] FIG. 15: Gross morphology after of tissues generated in vivo using proposed injectable construct.

[0078] FIG. 16: Histology. GAG and Collagen staining for tissues generated in vivo.

[0079] FIG. 17. A representative PDMS mould used to control freeze-drying of growth plate ECM to specific dimensions

[0080] FIG. 18: Schematic outlining the steps involved in endochondral ossification, whereby a cartilage template is converted to a mature bone

[0081] FIG. 19: Histological analysis of constructs at day 0 and following 28 days of culture in either chondrogenic or osteogenic medium, demonstrating the deposition of the main constituents of cartilage, sGAG and collagen.

[0082] FIG. 20: MSC-seeded growth plate ECM constructs with positive collagen type II and collagen type X staining

[0083] FIG. 21: Mineral deposition was observed in MSC-seeded growth plate ECM scaffolds cultured in either chondrogenic or osteogenic medium, in comparison to MSCs seeded on a cartilage ECM construct which only mineralised in osteogenic culture conditions.

[0084] FIG. 22: An image of a bi-layered construct containing cartilage ECM in the top layer and growth plate ECM in the bottom layer and histological analysis of tissue deposited by MSCs seeded onto osteochondral scaffolds after 28 days in culture.

[0085] FIG. 23: Reconstructed images of growth plate scaffold treated and untreated cranial defects at 4 and 8 weeks showing the level of mineralisation achieved

[0086] FIG. 24: Reconstructed images of growth plate scaffold treated and untreated femoral defects at 4 and 8 weeks showing the level of mineralisation achieved

[0087] FIG. 25: Histological analysis of repair tissue formed across the cranial defect after 4 and 8 weeks either (a) untreated or (b) treated with the growth plate scaffold. (c) Higher power images of the repair tissue, demonstrating de novo bone forming both upon and within the original growth plate ECM tissue.

[0088] FIG. 26: Biochemical assays for DNA and GAG content of solubilised ECM scaffold (a). Scaffolds were generated using micronized ECM (High GAG) or solubilised ECM. Macroscopic images of wet and dry solubilised ECM scaffolds (b).

[0089] FIG. 27: Biochemical assays performed on cartilage tissues engineered in vitro using solubilised or micronized ECM scaffolds seeded with human stem cells (a). Scaffolds were generated using micronized ECM (High GAG) or solubilised ECM. Gross morphology of tissues generated using scaffolds (b).

DETAILED DESCRIPTION OF THE INVENTION

Definitions

[0090] In this specification, the term “porous” as applied to a scaffold should be understood to mean having a porosity of at least 90% as determined using the method of Gleeson et al (J. P. Gleeson, N. A. Plunkett, F. J. O'Brien—Addition of hydroxyapatite improves stiffness, interconnectivity and osteogenic potential of a highly porous collagen-based scaffold for bone tissue regeneration—Eur Cell Mater, 20 (2010), pp. 218-223) In one embodiment, the scaffold (or each layer in the scaffold) has a porosity of at least 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98%. Ideally, the scaffold has a porosity of at least 98%, ideally at least 98.5%.

[0091] In this specification, the term “osseous defect” should be understood to mean any defect within bony tissue.

[0092] In this specification, the term “chondral defect” should be understood to mean any defect within the articular surface of a joint that does not penetrate through the subchondral bone.

[0093] In this specification, the term “osteochondral defect” should be understood to mean a defect to the articular surface that affects both the articular cartilage and the underlying bone.

[0094] In this specification, the term “extracellular matrix tissue” or “extracellular matrix” or “ECM” should be understood to mean a collection of extracellular molecules secreted by cells that provides structural and biochemical support to the surrounding cells. The ECM may be obtained from a mammal, for example a human or a non-human mammal, or it may be engineered in-vitro using published techniques, for example Vinardell et al (Vinardell, T., Sheehy, E., Buckley, C. T., Kelly, D. J. A comparison of the functionality and in vivo phenotypic stability of cartilaginous tissues engineered from different stem cells sources. Tissue Engineering Part A, 18(11-12), 1161-1170, 2012) and Buckley et al (Buckley, C. T., Vinardell, T., Kelly, D. J. Oxygen Tension Differentially Regulates the Functional Properties of Cartilaginous Tissues Engineered from Infrapatellar Fat Pad Derived MSCs and Articular Chondrocytes. Osteoarthritis and Cartilage, 18 (10), 1345-1354, 2010). Examples of extracellular matrix for the purpose of the present invention include cartilage ECM (obtained from porcine articular cartilage tissue) and growth plate ECM (typically obtained from the epiphysial plate of porcine tibia or femora).

[0095] In this specification, the term “hyaline cartilage ECM” should be understood to mean ECM obtained from hyaline cartilage which is a tissue found, for example, in the ear and nose and on joint surfaces. It is mostly composed of type II collagen and chondroitin sulphate.

[0096] In this specification, the term “articular cartilage ECM” should be understood to mean ECM obtained from articular cartilage, which is a form of hyaline cartilage found at the articular end of joints.

[0097] In this specification, the term “growth plate ECM” or “growth plate tissue ECM” should be understood to mean ECM obtained from growth plate tissue of developing bones, typically developing long bones. This could include the epiphyseal plate in the metaphysis of a long bone, or articular cartilage from skeletally immature joints as this tissue is also known to act as a surface growth plate during development and skeletal maturation.

[0098] In this specification, the term “micronised” as applied to ECM should be understood to mean provided in a particulate form, in which the particles of ECM have a mean particle size of less than 200 microns as determined using routine light microscopy. Preferably, the micronised ECM has a mean particle size of less than 150 or 100 microns. Ideally, the micronized ECM has a mean particle size between 20 and 200 microns, 20 and 150 microns, 20 and 100 microns, 20 and 70 microns, 30 and 70 microns, 30 and 60 microns, 40 and 60 microns, and ideally about 50 microns. Methods of micronisation include milling, cryomilling,

[0099] In this specification, the term “cryomilled” should be understood to mean a process in which a material is cryogenically frozen and then milled. Examples of cryomilling machines include the RETCH CRYOMILL™.

[0100] In this specification, the term “solubilised” should be understood to mean a process by which ECM tissue is digested, ideally enzymatically digested, to become soluble in an aqueous solvent. Suitably solubilising agents will be known to the person skilled in the art, and include enzymes and denaturing agents such as urea. An example of an enzyme that can be used to digest ECM tissue to become soluble is a protease, for example pepsin, or a collagense. Preferably, the solubilised ECM will be a purified collagen with substantial removal of GAG and xenogeneic DNA. Ideally, the solubilised ECM will have greater than 50%, 60%, 70%, 80% or 90% removal of GAG and DNA when compared to native ECM tissue.

[0101] In this specification, the term “freeze-drying” as applied to a slurry refers to a process in which the slurry is frozen, typically to a final freezing temperature of from −10° C. to −70° C. and then sublimated under pressure. In one embodiment, the desired final freezing temperature is between −10° C. and −70° C. Suitably, the desired final freezing temperature is between −30° C. and −50° C. Typically, the desired final freezing temperature is between −35° C. and −45° C., ideally about −40° C. In one embodiment of the invention, freezing or freeze-drying is carried out at a constant cooling rate. This means that the rate of cooling does not vary by more than +/−10% of the target cooling rate, i.e. if the desired rate of cooling is 1.0° C./min, and the actual rate of cooling varied between 0.9° C./min and 1.1° C./min, this would nonetheless still be considered to be a constant cooling rate. Typically, the constant cooling rate is between 0.1° C./min to 10° C./min. Preferably, freeze-drying is carried out at a constant cooling rate of between 0.5° C./min to 1.5° C./min. More preferably, freezing or freeze-drying is carried out at a constant cooling rate of between 0.8° C./min to 1.1° C./min. Typically, freezing or freeze-drying is carried at a constant cooling rate of about 0.9° C./min. The temperature of the freeze-drying chamber at a start of the freeze-drying process (i.e. when the slurry is placed in the chamber) is usually greater than 0° C., preferably at about ambient temperature. The sublimation step is generally carried out after the final freezing temperature is reached. This step involves heating the freeze-drying chamber to a sublimation temperature (generally about 0° C.), preferably at a constant heating rate. The process typically includes a final sublimation step where an ice phase in the formed scaffold is sublimated under vacuum for a suitable period of time.

[0102] In this specification, the term “slurry” should be understood to mean a suspension of micronized ECM in a solvent, suitably an aqueous solvent, for example water. Typically, the slurry comprises less than 500, 400, 300 mg/ml micronized ECM. Suitably, the slurry comprises 100-500, 100-400, 200-300, 230-270, and ideally about 250 mg/ml micronized ECM.

[0103] In this specification, the term “cross-linked” should be understood to mean treated to introduce cross-links between different polymeric molecules in the ECM. The ECM may be micronised ECM or solubilised ECM. Crosslinking may be performed on the solubilised ECM or on the formed freeze-dried scaffold. Typically, the scaffold is cross-linked by one or more of the means selected from the group comprising: dehydrothermal (DHT) cross-linking; and chemical cross-linking. When crosslinking is be performed on the solubilised ECM, the crosslinking agent is typically a chemically crosslinking agent. Suitable chemical cross-linking agents and methods will be well known to those skilled in the art and include a glyoxal, 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDAC) or Glutaraldehyde. Ideally, the scaffold is cross-linked using DHT and EDAC cross-linking. Cross-linking can be carried out at any stage of the fabrication process. In a preferred embodiment, scaffold pore symmetry can be controlled by varying the degree of cross-linking within each respective layer using cross linking methods familiar to one skilled in the art. Similarly, in another embodiment, scaffold permeability or flow conductivity can be varied by varying the mechanical properties of the scaffold using either cross linking or other stiffness improvement methodologies known to one skilled in the art.

[0104] In this specification, the term “GAG” should be understood to mean glycosaminoglycan, particularly sulphated glycosaminoglycans.

[0105] In this specification, the term “reduced GAG content” as applied to ECM from a given source should be understood to mean a GAG content that is reduced compared to natural ECM from the same source, for example less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or 5% GAG content of natural ECM. Methods of reducing GAG content include the use of buffers, detergents (such as Sodium dodecyl sulfate or Triton-X or Sodium deoxycholate) or other chemicals (e.g. chondroitinase ABC) known to reduce the sGAG content of tissues.

[0106] In this specification, the term “decellularised” or “devitalised” as a applied to a material (for example ECM, a scaffold, or a gel) should be understood to mean that the cellular content of the material is reduced partially or preferably completely. Method of decellularising a material include chemical nucleic acid digestion, possibly following partial or total removal of matrix components from the ECM.

[0107] In this specification, the term “seeding” as applied to a scaffold should be understood to mean incorporating a biological material into a scaffold. Method of seeding a scaffold include soaking the scaffold in a solution containing the biological material for a sufficient time to allow the biological material infiltrate the pores of the scaffold.

[0108] In this specification, the term “cells” should be understood to mean any type of cell, particularly stem cells, chondrocytes, and osteoblasts. Preferably, the cells are mesenchymal stem cells.

[0109] In this specification, the term “biological material” should be understood to mean proteins, peptides, nucleic acid, nucleic acid constructs, nucleic acid vectors, or chemical molecules having biological activity. Preferably, the biological material comprises a biological growth factor, for example one or more of the TGF-β superfamily, (IFG, FGF, BMP, PDGF, EGF) or cannabinoids.

[0110] In this specification, the term “cannabinoids” should be understood to mean a biological compound which can be naturally or synthetically derived and that acts on the cannabinoid receptor types 1 and/or 2 (CB.sub.1 and CB.sub.2), for example Δ9-tetrahydrocannabinol (Δ9-THC).

[0111] In this specification, the term “gel base” should be understood to mean a matrix having both solid and liquid properties. An exemplary gel base is an agarose gel.

[0112] In this specification, the term “injectable” should be understood to mean that the gel is sufficiently deformable to enable it to be injected into a defect in cartilage or bone.

EXPERIMENTAL

[0113] Development of Decellularized ECM Derived Scaffolds with a Uniform Pore Size.

[0114] Cartilage used in the fabrication of ECM derived scaffolds was harvested, in sterile conditions, from the femoral condyles of female pigs (3 months old) shortly after sacrifice. The cartilage was first broken up into small pieces using a scalpel. Cartilage particles were then broken up using a cryogenic mill (6770 Freezer/Mill, SPEX, UK). These small pieces of cartilage where then homogenized in distilled water (dH2O) using a homogenizer (IKAT10, IKA Works Inc, NC, USA) to create a cartilage slurry (250 mg/ml). The slurry was transferred to custom made moulds (containing wells 5 mm in diameter and 3 mm in height) and freeze-dried (FreeZone Triad, Labconco, KC, USA) to produce porous scaffolds. Briefly, the slurry was frozen to −30° C. (1° C./min) and kept at that temperature for one hour. The temperature was then increased to −10° C. (1° C./min), followed by a hold of 24 hours and then finally increased to room temperature (0.5° C./min). Next, two different crosslinking techniques were applied to the scaffolds. The scaffolds underwent DHT and 1-Ethyl-3-3dimethyl aminopropyl carbodiimide (EDAC) crosslinking The DHT process was performed in a vacuum oven (VD23, Binder, Germany), at 115° C., in 2 mbar for 24 hours. The EDAC (Sigma-Aldrich, Germany) crosslinking consisted of chemical exposure for 2 hours at a concentration of 6 mM in the presence of N-Hydroxysuccinimide (NHS) (Sigma-Aldrich, Germany). A molar ratio of 2.5 M EDAC/M N-Hydroxysuccinimide was used. After EDAC crosslinking the scaffolds were washed twice in sterile PBS (Sigma-Aldrich, Germany).

Development of Decellularized ECM Derived Scaffolds with Controlled Pore Size and Tailored Growth Factor Release Rates.

[0115] Articular cartilage was harvested from femoral condyles of female 4 months old pigs under sterile condition shortly after sacrifice. All steps of the decellularization and tailoring GAG protocol were performed in 2 mL working volume at room temperature. This protocol consists of three phases. In Phase I, the 50 and 5% GAG groups were incubated in basic buffer (10 mM Tris-HCl (pH 8.0)) containing 100 mM DTT, 2 mM MgCl2, and 10 mM KCl for 24 hrs; and anatomically adjacent pieces of cartilage subjected to 1 min incubations for 100% GAG group. The 5% GAG groups were additionally subjected to 0.5% SDS treatment with basic buffer containing 100 mM DTT, 2 mM MgCl2, and 10 mM KCl for 24 hrs. Following sGAG removal, nucleic acid digestion (2.5 Kunitz units/mL deoxyribonuclease I, 7.5 Kunitz units/mL ribonuclease A, 0.15 M NaCl, 2 mM MgCl.sub.2 (H2O) in 10 mM Tris-HCl (pH 7.6)) was performed for 24 h and washout (10 mM Tris-buffered saline (pH 7.5)) for 48 h. In phase II, the cartilage tailored GAG-ECM scaffolds were prepared by cryo-milling followed by DHT+EDAC crosslinking as described in section 1 above.

Development of Solubilised ECM Derived Scaffolds

[0116] Cartilage used in the fabrication of ECM derived scaffolds was harvested, in sterile conditions, from the femoral condyles or growth plates of female pigs (3 months old) shortly after sacrifice. The cartilage was first broken up into small pieces using a scalpel. ECM tissue was then transfer to sterile containers. ECM tissue was then pre-treated with 0.2M NaOH for 24 hours at 4° C. After washing and removal of pre-treatment solution, the ECM tissue was then digested with pepsin in 0.5 M Acetic Acid. Pepsin is added at a concentration of ˜1500 units pepsin per 50 mg ECM tissue. The ECM was then incubated in the pepsin solution for 24 hours at <20° C. with rotation at a speed of 4 rpm. Salt precipitation was then performed to extract purified collagen using concentration of NaCl between 0.1M-5M. In order to remove any remaining salt, acid or pepsin, dialysis can be performed on the solubilised collagen. Dialysis was performed against 0.02 M Na.sub.2HPO.sub.4 (pH 9.4) for 24 h at 4° C. The solubilised collagen can then be freeze-dried. To generate scaffolds, the freeze dried collagen was rehydrated in an aqueous solution at a concentration range of 1 mg/ml to 200 mg/ml preferably, 20 mg/ml. Once rehydrated the collagen can then be cross-linked to form a gel with Glyoxal at a concentration between 1 mM and 50 mM preferably, 10 mM. The solution is then incubated for 30 minutes at 37° C. to allow cross-linking to take place. After incubation the gel can then be transferred to moulds and freeze-dried to create scaffolds.

Development of Injectable Decellularized ECM Derived Particles as Growth Factor Delivery Systems.

[0117] Particulated cartilage ECM is fabricated as described in 1 or 2 above. Instead of freeze-drying these particles to produce a porous scaffold, it is also possible to combine these particles with a hydrogel to develop an injectable chondroinductive composite biomaterial that also acts as a growth factor delivery system.

[0118] One manifestation of this invention would be to combine ECM particles with a fibrin hydrogel. The particulated cartilaginous material is incorporated into the hydrogel by mixing directly with the fibrinogen, with the desired ratio. Gelation occurs by adding thrombin to the fibrinogen/ECM-particles slurry. Appropriate mixing ensures a homogeneous distribution of bioactive cartilage ECM-derived micro-particles within the hydrogel.

Development of Decellularized Growth Plate ECM Derived Scaffolds.

[0119] Growth plate used in the fabrication of ECM derived scaffolds was harvested, in sterile conditions, from the femur, fibula and tibia of female pigs (3 months old) shortly after sacrifice. The growth plate was first broken up into small pieces using a scalpel, and then broken up using a cryogenic mill (6770 Freezer/Mill, SPEX, UK). These small pieces of growth plate were then homogenized in distilled water (dH.sub.2O) using a homogenizer (IKAT10, IKA Works Inc, NC, USA) to create a slurry (250 mg/ml). The slurry was transferred to custom made moulds and freeze-dried (FreeZone Triad, Labconco, KC, USA) to produce porous scaffolds. Briefly, the slurry was frozen to −30° C. (1° C./min) and kept at that temperature for one hour. The temperature was then increased to −10° C. (1° C./min), followed by a hold of 24 hours and then finally increased to room temperature (0.5° C./min). Next, two different crosslinking techniques were applied to the scaffolds. The scaffolds underwent DHT and 1-Ethyl-3-3dimethyl aminopropyl carbodiimide (EDAC) crosslinking. The DHT process was performed in a vacuum oven (VD23, Binder, Germany), at 115° C., in 2 mbar for 24 hours. The EDAC (Sigma-Aldrich, Germany) crosslinking consisted of chemical exposure for 2 hours at a concentration of 6 mM in the presence of N-Hydroxysuccinimide (NHS) (Sigma-Aldrich, Germany). A molar ratio of 2.5 M EDAC/M N-Hydroxysuccinimide was used. After EDAC crosslinking the scaffolds were washed twice in sterile PBS (Sigma-Aldrich, Germany).

[0120] Results obtained from both in vitro and in vivo characterisation of the growth plate scaffold will be presented below, and demonstrate its potential for use in bone tissue regeneration. Also, we will display the ability of the growth plate scaffold layer to be combined with a cartilage ECM layer to generate an osteochondral graft which can be potentially applied to repair both bone (osteo) and cartilage (chondral) layers simultaneously.

[0121] The invention is not limited to the embodiments hereinbefore described which may be varied in construction and detail without departing from the spirit of the invention.