Anterior cruciate ligament xenografts

Abstract

The invention provides an article of manufacture comprising a substantially non-immunogenic ligament or tendon xenograft for implantation into humans. The invention further provides a method for preparing a ligament xenograft by removing at least a portion of a ligament from a non-human animal to provide a xenograft; washing the xenograft in saline and alcohol; subjecting the xenograft to at least one treatment selected from the group consisting of exposure to ultraviolet radiation, immersion in alcohol, ozonation, freeze/thaw cycling, and optionally chemical crosslinking. In addition to or in lieu of the above treatments, the methods include a cellular disruption treatment and either digestion of the carbohydrate moieties of the xenograft with a glycosidase in a range of about 1 mU/ml to about 1000 U/ml or glycosidase digestion followed by treatment for sialylation. The invention also provides articles of manufacture produced by one or more of the above-identified methods of the invention. The invention further provides a ligament xenograft for implantation into a human including a portion of a ligament from a non-human animal, wherein the portion includes extracellular components and substantially only dead cells having substantially no surface α-galactosyl moieties and having sialic acid linked to at least a portion of surface carbohydrate moieties. Each of the xenografts of the invention is substantially non-immunogenic and has substantially the same mechanical properties as the respective native ligament.

What is claimed is:  
     
         1 . A method of preparing a ligament xenograft for implantation into a human, which comprises: 
 (a) removing at least a portion of a ligament from a non-human animal to provide a xenograft;    (b) washing the xenograft in water and alcohol;    (c) subjecting the xenograft to a cellular disruption treatment; and    (d) digesting the xenograft with a glycosidase to remove substantially a plurality of first surface carbohydrate moieties from the xenograft,    wherein the glycosidase has a concentration in a range of about 1 mU/ml to about 1000 U/ml, and    whereby the xenograft has substantially the same mechanical properties as a corresponding portion of a native ligament.    
     
     
         2 . The method of    claim 1   , further comprising the step of: 
 subsequent to the glycosidase digesting step, treating a plurality of second surface carbohydrate moieties on the xenograft with a plurality of capping molecules to cap at least a portion of the second surface carbohydrate moieties,    whereby the xenograft is substantially non-immunogenic.    
     
     
         3 . The method of    claim 2   , wherein the capping step comprises: 
 treating the second surface carbohydrate moieties on the xenograft with the capping molecules having a concentration in a range of about 0.01 mM to about 100 mM.    
     
     
         4 . The method of    claim 2   , wherein at least a portion of the capping molecules are sialic acid molecules.  
     
     
         5 . The method of    claim 1   , wherein the glycosidase is a galactosidase.  
     
     
         6 . The method of    claim 5   , wherein the galactosidase is an α-galactosidase.  
     
     
         7 . The method of    claim 1   , wherein the cellular disruption treatment comprises freeze/thaw cycling.  
     
     
         8 . The method of    claim 1   , wherein the cellular disruption treatment comprises exposure to gamma radiation.  
     
     
         9 . The method of    claim 1   , wherein the removing step comprises removing with the portion a first block of bone attached to a first end of the portion.  
     
     
         10 . The method of    claim 9   , wherein the removing step comprises removing with the portion a second block of bone affixed to a second end of the portion opposite the first end.  
     
     
         11 . The method of    claim 1    further comprising the step of following step (c), exposing the xenograft to a crosslinking agent in a vapor form.  
     
     
         12 . A method of preparing a meniscal xenograft for implantation into a human, which comprises: 
 (a) removing at least a portion of a ligament from a non-human animal to provide a xenograft;    (b) washing the xenograft in water and alcohol;    (c) subjecting the xenograft to a cellular disruption treatment;    (d) digesting the xenograft with a glycosidase to remove substantially a plurality of first surface carbohydrate moieties from the xenograft; and    (e) treating a plurality of second surface carbohydrate moieties on the xenograft with a plurality of sialic acid molecules to cap at least a portion of the second surface carbohydrate moieties,    whereby the xenograft is substantially non-immunogenic and has substantially the same mechanical properties as a corresponding portion of a native ligament.    
     
     
         13 . The method of    claim 12   , wherein the capping step comprises: 
 treating the second surface carbohydrate moieties on the xenograft with the sialic acid molecules having a concentration in a range of about 0.01 mM to about 100 mM.    
     
     
         14 . The method of    claim 12   , wherein at least the glycosidase is a galactosidase.  
     
     
         15 . The method of    claim 14   , wherein at least the galactosidase is an α-galactosidase.  
     
     
         16 . The method of    claim 12   , wherein the cellular disruption treatment comprises freeze/thaw cycling.  
     
     
         17 . The method of    claim 12   , wherein the cellular disruption treatment comprises exposure to gamma radiation.  
     
     
         18 . The method of    claim 12   , wherein the removing step comprises removing with the portion a first block of bone attached to a first end of the portion.  
     
     
         19 . The method of    claim 18   , wherein the removing step comprises removing with the portion a second block of bone affixed to a second end of the portion opposite the first end.  
     
     
         20 . The method of    claim 12    further comprising the step of: 
 following step (c), exposing the xenograft to a crosslinking agent in a vapor form.  
 
     
     
         21 . An article of manufacture comprising a substantially non-immunogenic ligament xenograft for implantation in to a human, produced by 
 (a) removing at least a portion of a ligament from a non-human animal to provide a xenograft;    (b) washing the xenograft in water and alcohol;    (c) subjecting the xenograft to a cellular disruption treatment; and    (d) digesting the xenograft with a glycosidase to remove substantially a plurality of first surface carbohydrate moieties from the xenograft,    wherein the glycosidase has a concentration in a range of about 1 mU/ml to about 1000 U/ml, and    whereby the xenograft has substantially the same mechanical properties as a corresponding portion of a native ligament.    
     
     
         22 . The article of manufacture of    claim 21   , further produced by: 
 subsequent to the glycosidase digesting step, treating a plurality of second surface carbohydrate moieties on the xenograft with a plurality of capping molecules to cap at least a portion of the second surface carbohydrate moieties on the xenograft,    whereby the xenograft is substantially non-immunogenic.    
     
     
         23 . The article of manufacture of    claim 22   , wherein the capping molecules have a concentration in a range of about 0.01 mM to about 100 mM.  
     
     
         24 . The article of manufacture of    claim 22   , wherein at least a portion of the capping molecules are sialic acid molecules.  
     
     
         25 . The article of manufacture of    claim 21   , wherein the glycosidase is a galactosidase.  
     
     
         26 . The article of manufacture of    claim 25   , wherein the galactosidase is an α-galactosidase.  
     
     
         27 . The article of manufacture of    claim 21   , wherein the cellular disruption treatment comprises freeze/thaw cycling.  
     
     
         28 . The article of manufacture of    claim 21   , wherein the cellular disruption treatment comprises exposure to gamma radiation.  
     
     
         29 . The article of manufacture of    claim 21   , wherein the removing step comprises removing with the portion a first block of bone attached to a first end of the portion.  
     
     
         30 . The article of manufacture of    claim 29   , wherein the removing step comprises removing with the portion a second block of bone affixed to a second end of the portion opposite the first end.  
     
     
         31 . The article of manufacture of    claim 21    further comprising the step of following step (c), exposing the xenograft to a crosslinking agent in a vapor form.  
     
     
         32 . An article of manufacture comprising a substantially non-immunogenic ligament xenograft for implantation in to a human, produced by: 
 (a) removing at least a portion of a ligament from a non-human animal to provide a xenograft;    (b) washing the xenograft in water and alcohol;    (c) subjecting the xenograft to a cellular disruption treatment;    (d) digesting the xenograft with a glycosidase to remove substantially a plurality of first surface carbohydrate moieties from the xenograft; and    (e) treating a plurality of second surface carbohydrate moieties on the xenograft with a plurality of sialic acid molecules to cap at least a portion of the second surface carbohydrate moieties,    whereby the xenograft is substantially non-immunogenic and has substantially the same mechanical properties as a corresponding portion of a native meniscus.    
     
     
         33 . The article of manufacture of    claim 32   , wherein the sialic acid molecules have a concentration in a range of about 0.01 mM to about 100 mM.  
     
     
         34 . The article of manufacture of    claim 32   , wherein the glycosidase is a galactosidase.  
     
     
         35 . The article of manufacture of    claim 34   , wherein the galactosidase is an α-galactosidase.  
     
     
         36 . The article of manufacture of    claim 32   , wherein the cellular disruption treatment comprises freeze/thaw cycling.  
     
     
         37 . The article of manufacture of    claim 32   , wherein the cellular disruption treatment comprises exposure to gamma radiation.  
     
     
         38 . The article of manufacture of    claim 32   , wherein the removing step comprises removing with the portion a first block of bone attached to a first end of the portion.  
     
     
         39 . The article of manufacture of    claim 38   , wherein the removing step comprises removing with the portion a second block of bone affixed to a second end of the portion opposite the first end.  
     
     
         40 . The article of manufacture of    claim 32    further comprising the step of: 
 following step (c), exposing the xenograft to a crosslinking agent in a vapor form.  
 
     
     
         41 . A ligament xenograft for implantation into a human comprising: 
 a portion of a ligament from a non-human animal, wherein the portion includes a plurality of extracellular components and a plurality of substantially only dead cells, the extracellular components and the dead cells having substantially no surface α-galactosyl moieties and having a plurality of sialic acid molecules linked to at least a portion of a plurality of surface carbohydrate moieties on the xenograft,    whereby the portion of the ligament is substantially non-immunogenic and has substantially the same mechanical properties as a corresponding portion of a native ligament.    
     
     
         42 . The ligament xenograft of    claim 41   , wherein the portion of the ligament has a first block of bone attached to a first end thereof.  
     
     
         43 . The ligament xenograft of    claim 42   , wherein the portion of the ligament has a second block of bone affixed to a second end thereof opposite the first end.