The disclosure relates to an intervertebral disc implant comprising a first construct comprising a continuous layer of nucleus pulposus (NP) tissue directly or indirectly on and integrated with a substrate, and a second construct surrounding the first construct comprising one or more continuous laye
The disclosure relates to an intervertebral disc implant comprising a first construct comprising a continuous layer of nucleus pulposus (NP) tissue directly or indirectly on and integrated with a substrate, and a second construct surrounding the first construct comprising one or more continuous layers of annulus fibrosus (AF) tissue on and adherent to a scaffold, wherein the AF tissue, which is composed of single or multiple adherent layers, is integrated with the NP tissue. The disclosure also relates to methods of preparing the multi-tissue intervertebral disc and using the intervertebral disc as an implant.
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1. An intervertebral disc implant comprising (a) a first construct comprising a continuous layer of nucleus pulposus (NP) tissue directly or indirectly on and integrated with a substrate, and(b) a second construct surrounding the first construct comprising one or more continuous layers of annulus fi
1. An intervertebral disc implant comprising (a) a first construct comprising a continuous layer of nucleus pulposus (NP) tissue directly or indirectly on and integrated with a substrate, and(b) a second construct surrounding the first construct comprising one or more continuous layers of annulus fibrosus (AF) tissue on and adherent to a scaffold,wherein the AF tissue is integrated with the NP tissue to provide an intervertebral disc implant that mimics a native intervertebral disc. 2. The intervertebral disc implant of claim 1, wherein the AF tissue is characterized by one or more of the following: i) actin cytoskeleton oriented parallel to the long axis of the cell mimicking the actin organization in AF cells during tissue development and in fully formed tissue;ii) Type I collagen, the major matrix molecule of AF tissue, is present throughout tissue;iii) Type I collagen initially oriented parallel to the cell and fibrillar fibronectin and present in the developing;iv) collagen fibres extending out of the cell in a polarized manner similar to human AF cells in vivo; andv) more than one layer of AF/scaffold is present, layers of AF tissue/scaffold are adherent to each other. 3. The intervertebral disc implant of claim 1, wherein the continuous layer of nucleus pulposus (NP) tissue is directly or indirectly on and integrated with the subsurface of the substrate and the substrate protrudes from the NP and AF tissue. 4. An intervertebral disc implant comprising (a) a first construct comprising a substrate comprising a continuous layer of nucleus pulposus (NP) tissue on and integrated with the subsurface of the substrate, and(b) a second construct surrounding the NP tissue of the first construct and comprising one or more continuous layers of annulus fibrosus (AF) tissue on and adherent to a scaffold,wherein the AF tissue is integrated with the NP tissue. 5. The intervertebral disc implant of claim 1, wherein the substrate is bone or an engineered biomaterial. 6. The intervertebral disc implant of claim 5, wherein the engineered biomaterial is a bone substitute. 7. The intervertebral disc implant of claim 6, wherein the bone substitute is CPP. 8. The intervertebral disc implant of claim 1, wherein the scaffold is a polymeric scaffold or a synthetic polymeric scaffold. 9. (canceled) 10. The intervertebral disc implant of claim 8 wherein the scaffold comprises one or more of: (i) polyurethane fibres;(ii) polycarbonate urethane (PU) fibres;(iii) low molecular weight polymer additives containing hydroxyl/carboxylic acid groups that enhance cell and/or protein interactions with the scaffold. 11-12. (canceled) 13. The intervertebral disc implant of claim 1, further comprising one or more of (i) layers of the AF-scaffold having fibres that are angled obliquely to each other;(ii) the first construct additionally comprising cartilaginous tissue between the NP and the substrate, and wherein the NP tissue is on and integrated with the cartilaginous tissue;(iii) the cartilaginous tissue of (ii) comprising a calcified cartilage tissue or a bizonal cartilage tissue; and(iv) the second construct comprising two or more layers of the AF tissue/scaffold, wherein the layers are adherent to each other and are at oblique angles to each other. 14-16. (canceled) 17. A method for preparing an integrated intervertebral disc implant comprising (a) preparing a first construct by culturing nucleus pulposus (NP) cells or precursors on a substrate to form a continuous layer of NP tissue directly or indirectly on and integrated with the substrate;(b) preparing a second construct by culturing annulus fibrosus (AF) cells or precursors thereof on a scaffold to form two or more continuous layers of AF tissue-scaffold, wherein the layers are adherent to each other to form a single tissue;(c) combining the first construct and second construct so that the second construct surrounds the first construct and the NP tissue and AF tissue are adjacent; and(d) culturing the combined first construct and second construct to prepare the integrated intervertebral disc implant. 18. The method of claim 17, further comprising one or more of: (i) the substrate comprising the continuous layer of nucleus pulposus (NP) tissue fabricated on and integrated with the subsurface of the substrate and the first and second constructs combined such that the second construct surrounds the first construct so that the continuous layers of annulus fibrosus (AF) tissues are adjacent to and integrated with the NP tissue;(ii) forming the scaffold or second construct into a shape configured so that in step (c) the second construct can surround the first construct and the NP tissue and AF tissue are adjacent; wherein the NP tissue and AF tissue are integrated by culturing under suitable conditions in (d);(iii) forming the scaffold or second construct into a donut-shape with a cylindrical hole extending through the center, with the first construct inserted in the cylindrical hole; and(iv) prior to (a), forming a layer of cartilaginous tissue on the substrate. 19-21. (canceled) 22. The method of claim 18((iv), further comprising forming the layer of cartilaginous tissue on the substrate by seeding isolated chondrocytes or chondrocytes obtained from precursor cells grown under chondro-inducing conditions on the substrate; and b culturing the chondrocytes in the presence of a mineralizing agent under suitable conditions to generate a calcified cartilage tissue on the substrate characterized by accumulation of collagen type X, mineral, collagen type II and proteoglycans. 23. The method of claim 22, further comprising culturing additional isolated chondrocytes or stem cell precursors or precursor derived chondrocytes on the calcified cartilage tissue to generate a bizonal cartilage tissue comprising a continuous layer of cartilage tissue engineered on the substrate interfaced with a zone of calcified cartilage tissue. 24. The method of claim 17, wherein (a) further comprises preparing a first construct by: i. seeding isolated chondrocytes or chondrocytes obtained from precursor cells grown under chondro-inducing conditions on a substrate;ii. culturing the chondrocytes in the presence of a mineralizing agent under suitable conditions to generate a calcified cartilage tissue on the substrate characterized by accumulation of collagen type X, mineral, collagen type II and proteoglycans; andiii. culturing NP cells or precursors on the cartilage/substrate produced in ii. to form a continuous layer of NP tissue on and adherent to the cartilaginous layer which is on and integrated with the substrate. 25. The method of claim 24, further comprising in (a) culturing isolated chondrocytes on the engineered calcified cartilage tissue to generate a bizonal cartilage tissue comprising a continuous layer of cartilage tissue engineered on the substrate interfaced with a zone of calcified cartilage tissue after (a)ii. and before (a)iii. 26. A method for producing an engineered calcified cartilage tissue comprising: (a) seeding isolated or differentiated chondrocytes or chondrocytes obtained from precursor cells grown under chondro-inducing conditions on the substrate on a bone or engineered biomaterial substrate; and(b) culturing the chondrocytes in the presence of a mineralizing agent under suitable conditions to generate a calcified cartilage tissue on the substrate characterized by accumulation of collagen type X, mineral, collagen type II and proteoglycans. 27. The method of claim 26 further comprising culturing isolated chondrocytes on the engineered calcified cartilage tissue to generate a bizonal cartilage tissue comprising a continuous layer of cartilage tissue engineered on the substrate interfaced with a zone of calcified cartilage tissue. 28-29. (canceled) 30. A method of treating a damaged or traumatized intervertebral disc in a subject in need thereof comprising replacing part or all of the damaged or traumatized intervertebral disc or adjacent bone of the subject with an intervertebral disc implant of claim 1. 31. The method of claim 30, wherein the subject is a human.
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