Hedgehog signaling promotes the formation of three dimensional cartilage matrices

Abstract

The present invention provides methods and compositions for promoting the formation of three dimensional cartilage matrices. The present invention further provides methods of treating diseases and injuries involving cartilage and bone using the three dimensional cartilage matrices provided herein.

1 . A method for promoting the formation of a three dimensional cartilage matrix from chondrocytes or chondrogenic progenitor cells, wherein the chondrocytes or chondrogenic progenitor cells are contacted with a hedgehog polypeptide, or a functional fragment thereof.  
     
     
         2 . A method for promoting the formation of a three dimensional cartilage matrix from chondrocytes or chondrogenic progenitor cells, wherein the chondrocytes or chondrogenic progenitor cells are contacted with a hedgehog polypeptide and a TGFβ polypeptide, or functional fragments thereof.  
     
     
         3 . A method for promoting the formation of a three dimensional cartilage matrix from chondrocytes or chondrogenic progenitor cells, wherein the chondrocytes or chondrogenic progenitor cells are contacted with at least one hedgehog therapeutic.  
     
     
         4 . A method for promoting cell migration, proliferation, and differentiation of chondrocytes or chondrogenic progenitor cells which results in the formation of a three dimensional cartilage matrix, wherein the chondrocytes or chondrogenic progenitor cells are contacted with a hedgehog polypeptide, or a functional fragment thereof.  
     
     
         5 . A method for promoting cell migration, proliferation, and differentiation of chondrocytes or chondrogenic progenitor cells which results in the formation of a three dimensional cartilage matrix, wherein the chondrocytes or chondrogenic progenitor cells are contacted with a hedgehog polypeptide and a TGFβ polypeptide, or functional fragments thereof.  
     
     
         6 . A method for promoting cell migration, proliferation, and differentiation of chondrocytes or chondrogenic progenitor cells which results in the formation of a three dimensional cartilage matrix, wherein the chondrocytes or chondrogenic progenitor cells are contacted with at least one hedgehog therapeutic.  
     
     
         7 . The method of  claim 6 , wherein the hedgehog therapeutic binds to patched and increases expression of patched and/or gli.  
     
     
         8 . The method of  claim 6 , wherein the hedgehog therapeutic is a small organic molecule which stimulates hedgehog signaling.  
     
     
         9 . The method of  claim 6 , wherein the hedgehog therapeutic alters the localization, protein-protein binding and/or enzymatic activity of an intracellular protein involved in hedgehog signaling.  
     
     
         10 . The method of  claim 6 , wherein the hedgehog therapeutic alters the level of expression of a hedgehog protein, a patched protein or a protein involved in hedgehog signaling.  
     
     
         11 . The method of claims  1 - 6 , wherein the chondrocytes or chondrogenic progenitor cells are cells in culture.  
     
     
         12 . The method of  claim 11 , wherein the cells in culture are selected from embryonic stem cells, adult stem cells, fibroblasts, and primary chondrocytes.  
     
     
         13 . The method of  claim 12 , wherein the adult stem cells are selected from mesenchymal stem cells, neural stem cells, hematopoietic stem cells, and skin-derived progenitor cells.  
     
     
         14 . The method of  claim 11 , wherein the cells in culture are derived from a mammal.  
     
     
         15 . The method of  claim 11 , wherein the cells in culture are derived from a human.  
     
     
         16 . The method of at least one of claims  2  or  5 , wherein the hedgehog polypeptide and the TGFβ polypeptide are co-administered.  
     
     
         17 . The method of at least one of claims  2  or  5 , wherein the hedgehog polypeptide and the TGFβ polypeptide are administered at different times.  
     
     
         18 . The method of at least one of claims  3  or  6 , wherein the cells are treated with more than one hedgehog therapeutic.  
     
     
         19 . The method of  claim 18 , wherein at least one hedgehog therapeutic binds to patched and increases expression of patched and/or gli.  
     
     
         20 . The method of  claim 18 , wherein at least one hedgehog therapeutic is a small organic molecule which stimulates hedgehog signaling.  
     
     
         21 . The method of  claim 18 , wherein at least one hedgehog therapeutic alters the localization, protein-protein binding and/or enzymatic activity of an intracellular protein involved in hedgehog signaling.  
     
     
         22 . The method of  claim 18 , wherein at least one hedgehog therapeutic alters the level of expression of a hedgehog protein, a patched protein or a protein involved in hedgehog signaling.  
     
     
         23 . The method of  claim 18 , wherein the hedgehog therapeutics are co-administered.  
     
     
         24 . The method of  claim 18 , wherein the hedgehog therapeutics are administered at different times.  
     
     
         25 . A three dimensional cartilage matrix produced by contacting chondrocytes or chondrogenic progenitor cells with a hedgehog polypeptide, or a functional fragment thereof.  
     
     
         26 . A three dimensional cartilage matrix produced by contacting chondrocytes or chondrogenic progenitor cells with a hedgehog polypeptide and a TGFβ polypeptide, or functional fragments thereof.  
     
     
         27 . A three dimensional cartilage matrix produced by contacting chondrocytes or chondrogenic progenitor cells with at least one hedgehog therapeutic.  
     
     
         28 . The three dimensional cartilage matrix of  claim 27 , wherein the cells are treated with more than one hedgehog therapeutic.  
     
     
         29 . The three dimensional cartilage matrix of any of claims  25 - 28  combined with a pharmaceutically acceptable excipient.  
     
     
         30 . A method for producing a three dimensional cartilage matrix, comprising 
 (a) establishing a culture of cells with cartilage forming potential; and    (b) contacting the culture of cells with an amount of a hedgehog polypeptide, or functional fragments thereof, sufficient to promote the growth and differentiation of the three dimensional cartilage matrix in the culture of cells.    
     
     
         31 . A method for producing a three dimensional cartilage matrix, comprising 
 (a) establishing a culture of cells with cartilage forming potential; and    (b) contacting the culture of cells with an amount of a hedgehog polypeptide and a TGFβ polypeptide, or a functional fragment thereof, sufficient to promote the growth and differentiation of the three dimensional cartilage matrix in the culture of cells.    
     
     
         32 . A method for producing a three dimensional cartilage matrix, comprising 
 (a) establishing a culture of cells with cartilage forming potential; and    (b) contacting the culture of cells with an amount of at least one hedgehog therapeutic sufficient to promote the growth and differentiation of the three dimensional cartilage matrix in the culture of cells.    
     
     
         33 . A method for producing a three dimensional cartilage matrix, comprising 
 (a) establishing a culture of cells with cartilage forming potential; and    (b) contacting the culture of cells with an amount of more than one hedgehog therapeutic sufficient to promote the growth and differentiation of the three dimensional cartilage matrix in the culture of cells.    
     
     
         34 . The method of any of claims  30 - 33 , further comprising 
 (c) isolating the three dimensional cartilage matrix.    
     
     
         35 . The method of any of claims  30 - 33 , further comprising 
 (c) providing a scaffold to influence the shape of the three dimensional cartilage matrix; and    (d) isolating the three dimensional cartilage matrix.    
     
     
         36 . A method for treating a patient with a condition characterized by cartilage loss, damage, or disease, comprising administering to the patient a therapeutically effective amount of the three dimensional cartilage matrix of  claim 29 .  
     
     
         37 . A method for treating a patient with a condition characterized by cartilage loss, damage, or disease; comprising the method of any of claims  34  or  35 , and further comprising administering a therapeutically effective amount of the three dimensional cartilage matrix to the patient.  
     
     
         38 . A method for treating a condition characterized by cartilage loss, damage, or disease; comprising 
 (a) isolating adult stem cells from a patient;    (b) contacting a culture of the adult stem cells with an amount of a hedgehog polypeptide, or functional fragments thereof, sufficient to promote the growth and differentiation of a three dimensional cartilage matrix in the culture of cells;    (c) isolating the three dimensional cartilage matrix; and    (d) transplanting a therapeutically effective amount of the three dimensional cartilage matrix of step (c) to a patient.    
     
     
         39 . A method for treating a condition characterized by cartilage loss, damage, or disease; comprising 
 (a) isolating adult stem cells from a patient;    (b) contacting a culture of the adult stem cells with an amount of a hedgehog polypeptide and a TGFβ polypeptide, or functional fragments thereof, sufficient to promote the growth and differentiation of a three dimensional cartilage matrix in the culture of cells;    (c) isolating the three dimensional cartilage matrix; and    (d) transplanting a therapeutically effective amount of the three dimensional cartilage matrix of step (c) to a patient.    
     
     
         40 . A method for treating a condition characterized by cartilage loss, damage, or disease; comprising 
 (a) isolating adult stem cells from a patient;    (b) contacting a culture of the adult stem cells with an amount of at least one hedgehog therapeutic sufficient to promote the growth and differentiation of a three dimensional cartilage matrix in the culture of cells;    (c) isolating the three dimensional cartilage matrix; and    (d) transplanting a therapeutically effective amount of the three dimensional cartilage matrix of step (c) to a patient.    
     
     
         41 . A method for treating a condition characterized by cartilage loss, damage, or disease; comprising 
 (a) isolating adult stem cells from a patient;    (b) contacting a culture of the adult stem cells with an amount of more than one hedgehog therapeutic sufficient to promote the growth and differentiation of a three dimensional cartilage matrix in the culture of cells;    (c) isolating the three dimensional cartilage matrix; and    (d) transplanting a therapeutically effective amount of the three dimensional cartilage matrix of step (c) to a patient.    
     
     
         42 . The method of any of claims  38 - 41 , wherein the adult stem cells are isolated from and transplanted to the same patient.  
     
     
         43 . The method of any of claims  38 - 41 , wherein the adult stem cells are isolated from and transplanted to a different patient.  
     
     
         44 . The method of any of claims  38 - 41 , wherein the adult stem cells are mesenchymal stem cells.  
     
     
         45 . A kit for the production of a three dimensional cartilage matrix, comprising 
 (a) a hedgehog polypeptide combined with a pharmaceutically acceptable excipient;    (b) instructions for using (a) to promote the formation of a three dimensional cartilage matrix;    (c) a synthetic scaffold, or precursors for generating a biopolymer carrier or scaffold, to influence the shape of the three dimensional cartilage matrix produced by the instructions of (b).    
     
     
         46 . A kit for the production of a three-dimensional cartilage matrix, comprising 
 (a) a hedgehog polypeptide and a TGFβ polypeptide combined with a pharmaceutically acceptable excipient;    (b) instructions for using (a) to promote the formation of a three dimensional cartilage matrix;    (c) a synthetic scaffold, or precursors for generating a biopolymer carrier or scaffold, to influence the shape of the three dimensional cartilage matrix produced by the instructions of (b).    
     
     
         47 . A kit for the production of a three dimensional cartilage matrix, comprising 
 (a) at least one hedgehog therapeutic combined with a pharmaceutically acceptable excipient;    (b) instructions for using (a) to promote the formation of a three dimensional cartilage matrix;    (c) a synthetic scaffold, or precursors for generating a biopolymer carrier or scaffold, to influence the shape of the three dimensional cartilage matrix produced by the instructions of (b).    
     
     
         48 . A method for conducting a mesenchymal stem cell banking business, comprising 
 (a) harvesting tissue from tissue typed donors;    (b) establishing tissue typed mesenchymal stem cell cultures from the donor tissue;    (c) preserving the tissue typed mesenchymal stem cells for later use.    
     
     
         49 . The method of  claim 48 , wherein the tissue is selected from bone marrow, skin, and skeletal muscle.  
     
     
         50 . The method of  claim 49 , further comprising 
 (d) retrieving the preserved tissue typed mesenchymal stem cells;    (e) providing them to a patient in need thereof.    
     
     
         51 . The method of  claim 50 , further comprising billing the patient or the patient's insurance provider.  
     
     
         52 . A method for conducting a regenerative cartilage business, comprising 
 (a) harvesting tissue from tissue types donors;    (b) establishing mesenchymal stem cells cultures from the harvested tissue;    (c) contacting the mesenchymal stem cell culture with an amount of an agent sufficient to promote the formation of three dimensional cartilage matrices from the mesenchymal stem cells;    (d) providing the three dimensional cartilage matrices to a physician or health care provider for transplantation to a patient in need thereof.    
     
     
         53 . The method of  claim 52 , wherein the agent sufficient to promote three dimensional cartilage formation is selected from at least one of a hedgehog polypeptide, a hedgehog therapeutic, or a hedgehog polypeptide and a TGFβ polypeptide.  
     
     
         54 . The method of  claim 52 , wherein the harvested tissue is selected from bone marrow, skin, and skeletal muscle.  
     
     
         55 . The method of  claim 52 , further comprising 
 (e) banking a portion of the mesenchymal stem cells for later retrieval and use.    
     
     
         56 . The method of  claim 52 , further comprising billing the patient or the patient's insurance provider.