Decellularized Biomaterial and Method for Formation

Abstract

Methods for developing a decellularized tissue and biomaterials for use as biomimetic grafts or in vitro cellular scaffolds formed with the decellularized tissue are described. The biomaterials are particularly well suited for use as an intervertebral disc graft. The decellularized tissue is formed from an intervertebral disc source tissue and can be substantially decellularized and substantially free of potential immunogenic material (e.g., DNA and RNA), while maintaining ECM materials including both glycosaminoglycan and collagen.

What is claimed is: 
     
         1 . A method for forming a biomaterial, the method comprising:
 contacting a tissue with a decellularization solution, the decellularization solution include a non-ionic surfactant and a protease inhibitor;   subjecting the tissue while in contact with the decellularization solution to ultrasonication;   agitating the tissue while in contact with the decellularization solution; and   contacting the tissue with an enzyme solution.   
     
     
         2 . The method of  claim 1 , wherein the non-ionic surfactant is a polyethylene oxide based surfactant or a polysorbate based surfactant. 
     
     
         3 . The method of  claim 1 , wherein the decellularization solution includes the non-ionic surfactant in an amount of from about 0.5 v/v % to about 2 v/v %. 
     
     
         4 . The method of  claim 1 , wherein the protease inhibitor comprises N-ethylmaleimide, phenylmethylsulfonylfluoride, ethylenediamine tetraacetic acid, ethylene glycol-bis-(2-aminoethyl(ether)NNN′N′-tetraacetic acid, ammonium chloride, elevated pH, aprotinin or leupeptin. 
     
     
         5 . The method of  claim 1 , the decellularization solution further comprising one or more of an ionic surfactant, a salt, a buffer, an antibiotic, or an antimycotic. 
     
     
         6 . The method of  claim 1 , wherein the ultrasonication is carried out for a period of from about 5 minutes to about 60 minutes. 
     
     
         7 . The method of  claim 1 , wherein the ultrasonication is carried out at a power level of about 10 Watts or greater and at a frequency of about 20 kilohertz or greater. 
     
     
         8 . The method of  claim 1 , further comprising repeating the ultrasonication step periodically. 
     
     
         9 . The method of  claim 8 , further comprising replacing the decellularization solution with a fresh decellularization solution periodically in conjunction with each ultrasonication step. 
     
     
         10 . The method of  claim 1 , wherein the enzyme solution comprises one or more nucleases. 
     
     
         11 . The method of  claim 1 , further comprising crosslinking one or more components of the tissue. 
     
     
         12 . The method of  claim 1 , wherein the tissue comprises intervertebral disc tissue. 
     
     
         13 . The method of  claim 12 , wherein the tissue comprises nucleus pulposus tissue. 
     
     
         14 . The method of  claim 12 , wherein the tissue comprises annulus fibrosus tissue. 
     
     
         15 . The method of  claim 12 , wherein the tissue comprises an entire intervertebral disc. 
     
     
         16 . The method of  claim 1 , the tissue comprising glycosaminoglycan and nucleic acid, wherein following them method, the tissue has a glycosaminoglycan content of about 200 micrograms or greater per milligram dry weight of the tissue and has a nucleic acid content of about 50 nanograms or less per milligram dry weight of the tissue. 
     
     
         17 . The method of  claim 1 , further comprising seeding the tissue with extrinsic cells. 
     
     
         18 . The method of  claim 1 , wherein the method forms an implantable graft biomaterial. 
     
     
         19 . The method of  claim 18 , wherein the implantable graft biomaterial is an intervertebral disc graft biomaterial, an articular cartilage graft biomaterial, or an osteochondral graft biomaterial. 
     
     
         20 . The method of  claim 1 , wherein the method forms a scaffolding biomaterial.