Methods and compositions to facilitate repair of avascular tissue

Abstract

Compositions and methods are provided for repairing damaged avascular zones, including intervertebral disc, in a patient in need thereof.

What is claimed is: 
     
         1 . A method for treating an avascular zone in a patient in need thereof, the method comprising:
 an isolation and expansion process consisting of:
 obtaining whole marrow from the patient in need thereof; 
 centrifuging the whole marrow to separate nucleated cells from red blood cells; 
 removing the nucleated cells from the centrifuged whole marrow; 
 washing and counting the nucleated cells; 
 culturing the nucleated cells in a culture medium under a selective pressure of about 1% to about 10% oxygen for 1-28 days; 
 selecting viable mesenchymal stem cells capable of growth in the culture medium under the selective pressure of about 1% to about 10% oxygen; 
   an implantation process comprising:
 providing the selected, viable, mesenchymal stem cells for implantation in the avascular zone; 
 administering a compound in the avascular zone, wherein the compound is selected from the group consisting of VEGF-A, PIGF, VEGF-B, VEGF-C, VEGF-D, TGF-β, Ang-1, Ang-2, IGF, HGF, FGF, Tie2, PDGF, CCL2, Alpha-V Beta-5, Alpha-5 Beta-1, VE-cadherin, PECAM-1, plasminogen activator, nitrogen oxide synthase, and combinations thereof; 
 wherein the administering the compound in the avascular zone is prior to, during, or after implanting the selected, viable, mesenchymal stem cells. 
   
     
     
         2 . The method of  claim 1  wherein the selective pressure includes culturing the nucleated cells in from about 3 to about 7% oxygen. 
     
     
         3 . The method of  claim 1  wherein the selective pressure further includes culturing the nucleated cells in from about 2 to about 10% carbon dioxide. 
     
     
         4 . The method of  claim 2  wherein the selective pressure further includes culturing the nucleated cells in from about 2 to about 10% carbon dioxide. 
     
     
         5 . The method of  claim 1 , wherein the culture medium comprises 10% to 20% human platelet lysate. 
     
     
         6 . The method of  claim 1 , further comprising maintaining the pH of the culture medium at about 6.6 to about 7. 
     
     
         7 . The method of  claim 1 , further comprising maintaining the osmolarity of the culture medium at about 350 mOsm to about 600 mOsm. 
     
     
         8 . The method of  claim 1 , wherein the selected, viable mesenchymal stem cells are implanted into a posterior disc annulus of a degenerative intervertebral disc. 
     
     
         9 . The method of  claim 1  wherein the compound is selected from the group consisting of VEGF-B, TGF-β, FGF, PDGF, HGF, IGF, and combinations thereof. 
     
     
         10 . The method of  claim 1  wherein the compound is PDGF. 
     
     
         11 . The method of  claim 1  wherein the compound is FGF. 
     
     
         12 . The method of  claim 1  wherein the compound is VEGF-B. 
     
     
         13 . The method of  claim 1  wherein the compound is TGF-β. 
     
     
         14 . A method for treating an avascular zone in a patient in need thereof, the method comprising:
 an isolation and expansion process consisting of:
 obtaining whole marrow from the patient in need thereof; 
 centrifuging the whole marrow to separate nucleated cells from red blood cells; 
 removing the nucleated cells from the centrifuged whole marrow; 
 washing and counting the nucleated cells; 
 culturing the nucleated cells in a culture medium under a selective pressure of about 10% oxygen and then culturing the nucleated cells under decreasing levels of oxygen until about 1% oxygen, wherein the culturing from about 10% oxygen to about 1% oxygen is for 1-28 days; 
 selecting viable mesenchymal stem cells capable of growth in the culture medium under the selective pressure of about 10% to about 1% oxygen; 
 an implantation process comprising: 
 providing the selected, viable mesenchymal stem cells for implantation in the avascular zone; 
 administering a compound in the avascular zone, wherein the compound is selected from the group consisting of VEGF-A, PIGF, VEGF-B, VEGF-C, VEGF-D, TGF-β, Ang-1, Ang-2, IGF, HGF, FGF, Tie2, PDGF, CCL2, Alpha-V Beta-5, Alpha-5 Beta-1, VE-cadherin, PECAM-1, plasminogen activator, nitrogen oxide synthase, and combinations thereof; 
 wherein the administering the compound in the avascular zone is prior to, during, or after implanting the selected, viable, mesenchymal stem cells. 
   
     
     
         15 . The method of  claim 14 , wherein the nucleated cells are cultured in a culture medium under a selective pressure of about 10% oxygen and then cultured under decreasing levels of oxygen until about 5% oxygen, wherein the culturing from about 10% oxygen to about 5% oxygen is for 1-28 days. 
     
     
         16 . The method of  claim 14  wherein the compound is selected from the group consisting of VEGF-B, TGF-β, FGF, PDGF, HGF, IGF, and combinations thereof. 
     
     
         17 . The method of  claim 14  wherein the compound is PDGF. 
     
     
         18 . The method of  claim 14  wherein the compound is FGF. 
     
     
         19 . The method of  claim 14  wherein the compound is VEGF-B. 
     
     
         20 . The method of  claim 14  wherein the compound is TGF-β.