Bioengineered anterior cruciate ligament

Abstract

The present invention provides a method for producing an anterior cruciate ligament ex vivo. The method comprises seeding pluripotent stem cells in a three dimensional matrix, anchoring the seeded matrix by attachment to two anchors, and culturing the cells within the matrix under conditions appropriate for cell growth and regeneration, while subjecting the matrix to one or more mechanical forces via movement of one or both of the attached anchors. Bone marrow stromal cells are preferably used as the pluripotent cells in the method. Suitable matrix materials are materials to which cells can adhere, such as a gel made from collagen type I. Suitable anchor materials are materials to which the matrix can attach, such as Goinopra coral and also demineralized bone. Some examples of tissue which can be produced include other ligaments in the body (hand, wrist, elbow, knee), tendon, cartilage, bone, muscle, and blood vessels.

1 . A method for producing an anterior cruciate ligament ex vivo, comprising the steps: 
 a) providing pluripotent cells, a 3-dimensional matrix of cylindrical form comprised of collagen, and two cylindrically shaped anchors suitable for attachment to the matrix;    b) seeding the cells in the matrix, either pre- or post-matrix formation, by means to uniformly immobilize the cells within the matrix;    c) attaching a face of each respective anchor to either end of the seeded matrix so that the entire surface of each face of the seeded matrix of step b) contacts the face of the respective anchors; and    d) culturing the cells in the anchored matrix of step c) under conditions appropriate for cell growth and regeneration, while subjecting the matrix to one or more mechanical forces via movement of one or both of the attached anchors.    
     
     
         2 . The method of  claim 1  wherein the pluripotent cells are bone marrow stromal cells.  
     
     
         3 . The method of  claim 1  wherein the seeded matrix has a concentration of collagen type I ranging from 2 mg/ml to 6 mg/ml.  
     
     
         4 . The method of  claim 3  wherein the seeded matrix has a final concentration of collagen type I of 2 mg/ml.  
     
     
         5 . The method of  claim 3  wherein the collagen is not cross linked.  
     
     
         6 . The method of  claim 3  wherein the collagen is cross-linked .  
     
     
         7 . The method of  claim 1  wherein the anchors are comprised of Goinopra coral with pore size 500 μm, the coral having been treated by means to convert the calcium carbonate to calcium phosphate.  
     
     
         8 . The method of  claim 7  wherein the Goinopra coral is further infused with fibronectin.  
     
     
         9 . The method of  claim 1  wherein the anchors are comprised of demineralized bone.  
     
     
         10 . The method of  claim 9  wherein the bone is further infused with fibronectin.  
     
     
         11 . The method of  claim 1  wherein the magnitude, duration and combination of mechanical forces are changed over the period of culture to approach that which is experienced by a native ACL in vivo.  
     
     
         12 . The method of  claim 1  wherein the mechanical forces mimic mechanical stimuli experienced by an anterior cruciate ligament in vivo.  
     
     
         13 . The method of  claim 13  wherein the anchored matrix is further cultured under conditions which mimic the chemical stimuli experienced by an anterior cruciate ligament in vivo.  
     
     
         14 . The method of  claim 12  wherein the mechanical force is tension and compression.  
     
     
         15 . The method of  claim 12  wherein the mechanical force is torsion.  
     
     
         16 . The method of  claim 12  wherein the mechanical force is shear.  
     
     
         17 . The method of  claim 12  wherein a combination of mechanical forces are applied to simulate knee joint extension.  
     
     
         18 . The method of  claim 17  wherein the motion of knee joint extension is in the coronal plane.  
     
     
         19 . The method of  claim 17  wherein the motion of knee joint extension is in the sagittal plane.  
     
     
         20 . The method of  claim 12  wherein a combination of mechanical forces are applied to simulate knee joint flexion.  
     
     
         21 . The methods of  claim 12  wherein a combination of mechanical forces are applied which simulate a combination of flexion and extension, the combination of mechanical forces being applied over time to produce an anterior cruciate ligament which has helically organized fibers.  
     
     
         22 . A bioengineered anterior cruciate ligament produced by the method comprising the steps: 
 a) providing pluripotent cells, a 3-dimensional matrix of cylindrical form comprised of collagen, and two cylindrically shaped anchors suitable for attachment to the matrix;    b) seeding the cells in the matrix, either pre- or post-matrix formation, by means to uniformly immobilize the cells within the matrix;    c) attaching a face of each respective anchor to either end of the seeded matrix so that the entire surface of each face of the seeded matrix of step b) contacts the face of the respective anchors; and    d) culturing the cells in the anchored matrix of step c) under conditions appropriate for cell growth and regeneration, while subjecting the matrix to one or more mechanical forces via movement of one or both of the attached anchors.    
     
     
         23 . The bioengineered ligament of  claim 22  wherein the pluripotent cells are bone marrow stromal cells.  
     
     
         24 . The bioengineered ligament of  claim 23  which is characterized by cellular orientation and/or matrix crimp pattern in the direction of the applied mechanical forces of step d).  
     
     
         25 . The bioengineered ligament of  claim 24  which is further characterized by the production of collagen type I, collagen type III, and fibronectin proteins along the axis of mechanical load produced by the mechanical forces of step d).  
     
     
         26 . The bioengineered ligament of  claim 23  wherein the mechanical forces of step d) mimic mechanical stimuli experienced by an anterior cruciate ligament in vivo.  
     
     
         27 . The bioengineered ligament of  claim 26  wherein the ligament fiber bundles are arranged into a helical organization.  
     
     
         28 . A method for producing a predetermined type of ligament ex vivo, comprising the steps: 
 a) providing pluripotent cells, a 3-dimensional matrix to which cells are able to adhere, and two anchors each having a face which is suitable for attachment to the matrix;    b) seeding the cells in the matrix, either pre- or post-matrix formation, by means to uniformly immobilize the cells within the matrix;    c) attaching the face of each respective anchor to opposite ends of the seeded matrix; and    d) culturing the cells in the anchored matrix of step c) under conditions appropriate for cell growth and regeneration, while subjecting the matrix to one or more mechanical forces via movement of one or both of the attached anchors, wherein the mechanical forces mimic one or more mechanical forces experienced by the ligament in vivo.    
     
     
         29 . The method of  claim 28  wherein the pluripotent cells are bone marrow stromal cells.  
     
     
         30 . The method of  claim 28  wherein the matrix has a cylindrical form and is attached to the respective anchor faces at each face of the cylinder.  
     
     
         31 . The method of  claim 28  wherein the matrix is comprised of collagen.  
     
     
         32 . The method of  claim 31  wherein the matrix has a concentration of collagen type I ranging from 2 mg/ml to 6 mg/ml.  
     
     
         33 . The method of  claim 32  wherein the collagen is not cross-linked.  
     
     
         34 . The method of  claim 32  wherein the collagen is cross-linked.  
     
     
         35 . The method of  claim 28  wherein the anchors are further infused with a factor which promotes matrix adhesion to the anchor.  
     
     
         36 . The method of  claim 28  wherein the anchors are comprised of Goinopra coral with pore size 500 μm, wherein the coral has been treated by means to convert the calcium carbonate to calcium phosphate.  
     
     
         37 . The method of  claim 28  wherein the anchors are comprised of demineralized bone.  
     
     
         38 . The method of  claim 28  wherein the ligament produced is an anterior cruciate ligament.  
     
     
         39 . The method of  claim 28  wherein the magnitude, duration and combination of mechanical forces are changed over the period of culture to approach that which is experienced by a native ligament in vivo.  
     
     
         40 . The method of  claim 28  wherein the anchored matrix is further cultured under conditions which mimic the chemical stimuli experienced by a native ligament in vivo.  
     
     
         41 . The method of  claim 28  wherein the mechanical force is tension-compression.  
     
     
         42 . The method of  claim 28  wherein the mechanical force is torsion.  
     
     
         43 . The method of  claim 28  wherein the mechanical force is shear.  
     
     
         44 . The method of  claim 28  wherein a combination of mechanical forces are applied to simulate extension of the joint in which the ligament is located in vivo.  
     
     
         45 . The method of  claim 28  wherein a combination of mechanical forces are applied to simulate flexion of the joint in which the ligament is located in vivo.  
     
     
         46 . The methods of  claim 28  wherein a combination of mechanical forces are applied which simulate a combination of flexion and extension, the combination of forces being applied over time to produce a ligament which has helically organized fibers.  
     
     
         47 . A bioengineered ligament produced by the method comprising the steps: 
 a) providing pluripotent cells, a 3-dimensional matrix to which cells are able to adhere, and two anchors each having a face which is suitable for attachment to the matrix;    b) seeding the cells in the matrix, either pre- or post-matrix formation, by means to uniformly immobilize the cells within the matrix;    c) attaching the face of the respective anchors to opposite ends of the seeded matrix; and    d) culturing the cells in the anchored matrix of step c) under conditions appropriate for cell growth and regeneration, while subjecting the matrix to one or more mechanical forces via movement of one or both of the attached anchors, wherein the mechanical forces mimic forces experienced by the ligament in vivo.    
     
     
         48 . The bioengineered ligament of  claim 47  wherein the pluripotent cells are bone marrow stromal cells.  
     
     
         49 . The bioengineered ligament of  claim 48  which is characterized by cellular orientation in the direction of the applied mechanical forces of step d).  
     
     
         50 . The bioengineered ligament of  claim 49  which is further characterized by collagen III fiber production and fibronectin fiber production along the axis of mechanical load produced by the mechanical forces of step d).  
     
     
         51 . The bioengineered ligament of  claim 47  which is an anterior cruciate ligament, wherein the mechanical forces of step d) mimic mechanical stimuli experienced by an anterior cruciate ligament in vivo.  
     
     
         52 . The bioengineered ligament of  claim 51  wherein the ligament fiber bundles are arranged into an helical organization.  
     
     
         53 . A method for producing a specific tissue type ex vivo, comprising the steps: 
 a) providing pluripotent cells, a 3-dimensional matrix to which cells are able to adhere, and a plurality of anchors each having a face which is suitable for attachment to the matrix;    b) seeding the cells in the matrix, either pre- or post-matrix formation, by means to uniformly immobilize the cells within the matrix;    c) attaching the faces of the respective anchors to the seeded matrix at the appropriate positions; and    d) culturing the cells in the anchored matrix of step c) under conditions appropriate for cell growth and regeneration, while subjecting the matrix to one or more mechanical forces via movement of one or more of the attached anchors, wherein the mechanical forces mimic stresses experienced by the specific tissue type in vivo.    
     
     
         54 . The method of  claim 53  wherein the cells in the anchored matrix are further cultured under conditions which mimic chemical stimuli experienced by the tissue in vivo.  
     
     
         55 . The method of  claim 53  wherein the cells in the anchored matrix are further cultured under conditions which mimic the electromagnetic stimuli experienced by the tissue in vivo.  
     
     
         56 . The method of  claim 53  wherein the tissue type is cartilage.  
     
     
         57 . The method of  claim 53  wherein the tissue type is bone.  
     
     
         58 . The method of  claim 53  wherein the tissue type is blood vessel.  
     
     
         59 . The method of  claim 53  wherein the tissue type is tendon.  
     
     
         60 . The method of  claim 53  wherein the tissue type is muscle.