Tissue-engineered intervertebral disc with living cells

Abstract

The present invention relates to a tissue-engineered intervertebral disc (IVD) suitable for total disc replacement in a mammal and methods of fabrication. The IVD comprises a nucleus pulposus structure comprising a first population of living cells that secrete a hydrophilic protein and an annulus fibrosis structure surrounding and in contact with the nucleus pulposus structure, the annulus fibrosis structure comprising a second population of living cells and type I collagen. The collagen fibrils in the annulus fibrosis structure are circumferentially aligned around the nucleus pulposus region due to cell-mediated contraction in the annulus fibrosis structure. Also disclosed are methods of fabricating tissue-engineered intervertebral discs.

1 . (canceled) 
     
     
         2 . A method of fabricating a tissue-engineered intervertebral disc (IVD), said method comprising:
 providing a first gel comprising a first population of living cells;   forming the first gel into a predetermined shape and size;   providing a second different gel comprising a second different population of living cells and collagen;   contacting the formed first gel with the second gel at a region that extends circumferentially around the first gel; and   storing the first and second gels under conditions effective for the collagen in the second gel to align circumferentially around the first gel by self-assembly of collagen due to cell-mediated gel contraction in the second gel, wherein the first gel forms a nucleus pulposus structure and the second gel forms an annulus fibrosus structure surrounding and in contact with the nucleus pulposus structure, thereby fabricating a tissue-engineered IVD suitable for total disc replacement in a mammal.   
     
     
         3 . The method according to  claim 2 , wherein the first gel is an alginate gel comprising about 0.5% to about 10% (w/v) alginate. 
     
     
         4 . The method according to  claim 2 , wherein the first population of cells is present in the first gel at a concentration of about 1×10 6  to about 50×10 6  cells/ml. 
     
     
         5 . The method according to  claim 2 , wherein the first population of cells secrete a proteoglycan. 
     
     
         6 . The method according to  claim 2 , wherein the first population of cells comprises nucleus pulposus cells. 
     
     
         7 . The method according to  claim 2 , wherein the first gel further comprises type II collagen. 
     
     
         8 . The method according to  claim 2 , wherein the nucleus pulposus has an isotropic structure. 
     
     
         9 . The method according to  claim 2 , wherein the second population of cells comprises annulus fibrosus cells. 
     
     
         10 . The method according to  claim 2 , wherein the second population of cells are present in the second gel at a concentration of about 0.1-5.0×10 7  cells/ml. 
     
     
         11 . The method according to  claim 2 , wherein the second gel comprises collagen at a concentration of about 1 mg/ml to about 30 mg/ml. 
     
     
         12 . The method according to  claim 2 , wherein the annulus fibrosus has an anisotropic structure. 
     
     
         13 . The method according to  claim 2 , wherein the IVD is permeable to allow nutrient transport to developing tissue. 
     
     
         14 . The method according to  claim 2  further comprising:
 cross-linking the first gel. 
 
     
     
         15 . The method according to  claim 2 , wherein the second gel is formed at a temperature of about 25° C. to about 37° C. 
     
     
         16 . The method according to  claim 2 , wherein said storing is carried out for about 3 to about 28 days. 
     
     
         17 . The method according to  claim 2 , wherein said forming is carried out by injection molding or by cutting samples from a sheet. 
     
     
         18 . A method of fabricating a tissue-engineered intervertebral disc (IVD), said method comprising:
 providing a first gel comprising a first population of living cells;   providing a second different gel comprising a second different population of living cells and collagen;   forming the second gel around a central mandrel structure;   storing the second gel under conditions effective for the collagen in the second gel to align circumferentially around the central mandrel structure by self-assembly of collagen due to cell-mediated gel contraction in the second gel;   replacing the central mandrel structure with the first gel, wherein the first gel forms a nucleus pulposus structure and the second gel forms an annulus fibrosus structure surrounding and in contact with the nucleus pulposus structure, thereby fabricating a tissue-engineered IVD suitable for total disc replacement in a mammal.   
     
     
         19 . The method according to  claim 18 , further comprising:
 forming the first gel into a predetermined shape and size.   
     
     
         20 . The method according to  claim 18 , wherein the central mandrel structure comprises plastic or metal. 
     
     
         21 . The method according to  claim 18 , wherein said forming is carried out by injection molding or by cutting samples from a sheet.