Intervertebral disc implant
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.
1. 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;
further comprising prior to (a), forming a 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 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.
2. The method of claim 1 , 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.
3. 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,
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; and
iii. 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.
4. The method of claim 3 , 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.
5. The method of claim 1 , wherein the substrate is bone or an engineered biomaterial.
6. The method of claim 5 , wherein the engineered biomaterial is a bone substitute.
7. The method of claim 6 , wherein the bone substitute is CPP.
8. The method of claim 1 , wherein the scaffold is a polymeric scaffold or a synthetic polymeric scaffold.
9. The method of claim 1 , wherein the scaffold comprises one or more of:
(i) polyurethane fibres;
(ii) polycarbonate urethane (PU) fibres; and
(iii) low molecular weight polymer additives containing hydroxyl/carboxylic acid groups that enhance cell and/or protein interactions with the scaffold.
10. The method of claim 3 , wherein the substrate is bone or an engineered biomaterial.
11. The method of claim 10 , wherein the engineered biomaterial is a bone substitute.
12. The method of claim 11 , wherein the bone substitute is CPP.
13. The method of claim 3 , wherein the scaffold is a polymeric scaffold or a synthetic polymeric scaffold.
14. The method of claim 3 , wherein the scaffold comprises one or more of:
(i) polyurethane fibres;
(ii) polycarbonate urethane (PU) fibres; and
(iii) low molecular weight polymer additives containing hydroxyl/carboxylic acid groups that enhance cell and/or protein interactions with the scaffold.