Mineralized Three-Dimensional Bone Constructs

Abstract

The present disclosure provides ex-vivo derived mineralized three-dimensional bone constructs. The bone constructs are obtained by culturing osteoblasts and osteclast precursors under randomized gravity vector conditions. Preferably, the randomized gravity vector conditions are obtained using a low shear stress rotating bioreactor, such as a High Aspect Ratio Vessel (HARV) culture system. The bone constructs of the disclosure have utility in physiological studies of bone formation and bone function, in drug discovery, and in orthopedics.

1 . A method for producing mineralized three-dimensional bone constructs, the method comprising: culturing osteoclast precursors and osteoblasts in a matrix-free culture medium using a low shear rotational three-dimensional tissue culture technique under conditions effective to form aggregates comprising said osteoblasts and said osteoclast precursors, and culturing said aggregates using said low shear rotational three-dimensional tissue culture technique in a matrix-free mineralization culture medium that promotes mineralization of said aggregates. 
     
     
         2 . A method for producing mineralized three-dimensional bone constructs, the method comprising:
 (a) introducing osteoclast precursors and osteoblasts into a cylindrical culture vessel that rotates about a central horizontal axis, said cylindrical culture vessel comprising a matrix-free culture medium;   (b) culturing said osteoblasts and said osteoclast precursors in said cylindrical culture vessel during horizontal rotation at a rate effective to create low shear conditions and to promote the formation of aggregates comprising said osteoclast precursors and said osteoblasts; and   (c) introducing a matrix-free mineralization culture medium into said cylindrical culture vessel and culturing said aggregates during horizontal rotation at a rate effective to create low shear conditions, wherein said mineralized three-dimensional bone constructs are formed.   
     
     
         3 . The method of  claim 2 , wherein said cylindrical culture vessel is a High Aspect Ratio Vessel (HARV) having a diameter greater than its height. 
     
     
         4 . The method of  claim 2  wherein the horizontal rotation during step (b) is at a lower average rate than the rotation during step (c). 
     
     
         5 . The method of  claim 2  wherein the ratio of osteoblasts:osteoclast precursors introduced into said cylindrical culture vessel in step (a) is about 2:1. 
     
     
         6 . The method of  claim 2  wherein between about 1 million and about 8 million osteoblasts are introduced into said cylindrical culture vessel in step (a). 
     
     
         7 . The method of  claim 2  which further comprises between (b) and (c): (b)(1) further culturing said aggregates in said cylindrical culture vessel during horizontal rotation at a rate effective to create low shear conditions. 
     
     
         8 . The method of  claim 7  wherein the average rate of horizontal rotation in step (b) is lower than in step (b)(1) and step (c). 
     
     
         9 . The method of  claim 8  wherein step (b) is allowed to proceed for between about 24 hours and about 48 hours. 
     
     
         10 . The method of  claim 8  wherein step (b)(1) is allowed to proceed for between about 5 days and about 7 days. 
     
     
         11 . The method of  claim 8  wherein step (c) is allowed to proceed for between about 14 days and about 21 days. 
     
     
         12 . The method of  claim 2  wherein said cylindrical culture vessel has a diameter of about 10 cm and a height of about 1 cm, and wherein the average rate of horizontal rotation in step (b) is between about 2 revolutions per minute and about 4 revolutions per minute. 
     
     
         13 . The method of  claim 12  wherein the average rate of horizontal rotation in step (b)(i) is between about 9 revolutions per minute and about 16 revolutions per minute and wherein the average rate of horizontal rotation in step (c) is between about 9 revolutions per minute and about 20 revolutions per minute.