Treatment of collagen defects using protein solutions

Abstract

Methods of stimulating collagen production, including stimulation of chondrocyte production, at the site of a defect. Methods include administering to the site of a defect at least two proteins from the group IL-1ra, sTNF-RI, sTNF-RII, IGF-I, EGF, HGF, PDGF-AB, PDGF-BB, VEGF, TGF-β1, and sIL-1RII.

What is claimed is: 
     
         1 . A method for stimulating chondrocyte production at a site of a collagen defect in a mammalian subject, comprising administering to the site a composition comprising at least two proteins selected from the group consisting of IL-1ra, sTNF-RI, sTNF-RII, IGF-I, EGF, HGF, PDGF-AB, PDGF-BB, VEGF, TGF-β1, and sIL-1RII, wherein the concentration of each protein in the composition is greater than the concentration of the protein in normal blood. 
     
     
         2 . The method according to  claim 1 , wherein the blood derived composition is autologous to the subject. 
     
     
         3 . The method according to  claim 1 , wherein the blood derived composition comprises
 a) at least about 10,000 pg/ml IL1-ra;   (b) at least about 1,200 pg/ml sTNF-RI; and   (c) a protein selected from the group consisting of sTNF-RII, IGF-I, EGF, HGF, PDGF-AB, PDGF-BB, VEGF, TGF-β1, and sIL-1RII, and mixtures thereof, wherein the protein has a concentration higher than the protein's baseline concentration in normal blood.   
     
     
         4 . A method for stimulating chondrocyte production at a site of a collagen defect in a mammalian subject, comprising administering to the site a blood-derived composition comprising:
 (a) interleukin-1 receptor antagonist (IL-1ra), at a concentration at least 3 times greater than the concentration of IL-1ra in normal blood;   (b) soluble tissue necrosis factor-r1 (sTNF-r1), at a concentration at least 2 times greater than the concentration of IL-1ra in normal blood;   (c) cytokine-producing cells at a concentration at least 2 times greater than the concentration of cytokine-producing cells in normal blood; and   (d) platelets, at a concentration at least 2 times greater than the concentration of platelets in normal blood.   
     
     
         5 . The method according to  claim 4 , wherein the concentration of IL-1ra is from about 10,000 pg/ml to about 50,000 pg/ml. 
     
     
         6 . The method according to  claim 4 , wherein the concentration of TNF-r1 from about 1,200 pg/ml to about 4,000 pg/ml. 
     
     
         7 . The method according to  claim 6 , wherein the concentration of sTNF-r1 about 2000 pg/ml or greater. 
     
     
         8 . The method according to  claim 4 , wherein the composition further comprises a protein selected from the group consisting of sTNF-RII, IGF-I, EGF, HGF, PDGF-AB, PDGF-BB, VEGF, TGF-β1, and sIL-1RII, and mixtures thereof, wherein the concentration of the protein in the composition is greater than the concentration of the protein in normal blood. 
     
     
         9 . The method according to  claim 4 , wherein the composition further comprises concentrated bone marrow aspirate. 
     
     
         10 . A method for stimulating chondrocyte production at a site of a collagen defect in a mammalian subject, comprising:
 (a) obtaining a cytokine cell suspension from the subject;   (b) fractionating the liquid to produce an autologous protein solution comprising interleukin-1 receptor antagonist;   (c) administering the autologous protein solution to the site of the defect in the subject.   
     
     
         11 . The method according to  claim 10 , wherein the cytokine cell suspension comprises whole blood, bone marrow aspirate, adipose tissue, urine, fractions thereof, and mixtures thereof. 
     
     
         12 . The method according to  claim 11 , wherein the liquid is platelet rich plasma. 
     
     
         13 . The method according to  claim 12 , wherein the fractionating comprises placing blood in a container a separator operable to separate the blood into two or more fractions; and centrifuging the separator to create a platelet-rich plasma fraction. 
     
     
         14 . The method according to  claim 13 , wherein the fractionating further comprises contacting the platelet-rich plasma with polyacrylamide beads, and separating the polyacrylamide beads from the platelet-rich plasma to form the autologous protein solution. 
     
     
         15 . The method according to  claim 10 , wherein the fractionating comprises:
 (1) loading tissue comprising whole blood, bone marrow aspirate, or both, and an anticoagulant into a tube comprising a buoy disposed in the tube, wherein the buoy has a density such that the buoy reaches an equilibrium position upon centrifugation of the tissue in the tube, the equilibrium position being between a first fraction and a second fraction comprising cytokine-producing cells, the second fraction having a concentration of cytokine-producing cells greater than the concentration of cytokine-producing cells in the first fraction;   (2) centrifuging the tube so that the buoy defines an interface between the first fraction and the second fraction; and   (3) collecting the second fraction;   (4) loading the second fraction into a concentrator assembly comprising a solid extraction material and incubating the second fraction in contact with the solid extraction material; and   (5) rotating the concentrator assembly at centrifugal speeds to separate from the solid extraction material the solution rich in interleukin-1 receptor antagonist having a concentration of interleukin-1 receptor antagonist greater than that of the whole blood.   
     
     
         16 . The method according to  claim 15 , wherein the solid extraction material is selected from the group consisting of corundum, quartz, titanium, dextran, agarose, polyacrylamide, polystyrene, polyethylene, polyvinyl chloride, polypropylene, and combinations thereof. 
     
     
         17 . The method according to  claim 14 , wherein the solid extraction material comprises polyacrylamide. 
     
     
         18 . The method according to  claim 15 , wherein the solid extraction material comprises a form selected from the group consisting of a bead, fiber, powder, porous material, and combinations thereof. 
     
     
         19 . The method according to  claim 10 , further comprising:
 (d) obtaining bone marrow aspirate from the subject;   (e) concentrating the bone marrow aspirate to produce a concentrated bone marrow aspirate (cBMA);   (f) administering the cBMA to the subject.   
     
     
         20 . The method according to  claim 19 , wherein the administering of the cBMA is performed concurrently with the administering of the autologous protein solution. 
     
     
         21 . The method according to  claim 20 , wherein APS and the cBMA are in an APS:cBMA ratio of from about 10:1 to about 1:10. 
     
     
         22 . The method according to  claim 20 , wherein the bone marrow aspirate is mixed with the blood during or prior to the fractionating of the blood. 
     
     
         23 . The method according to  claim 22 , wherein fractionating comprises placing the blood and the bone marrow aspirate in a container a separator operable to separate the blood and bone marrow aspirate into two or more fractions; and centrifuging the separator to create a platelet-rich plasma fraction comprising cBMA. 
     
     
         24 . The method according to  claim 10 , wherein the mammalian subject is human. 
     
     
         25 . The method according to  claim 10 , wherein the mammalian subject is a companion, working, or sports animal. 
     
     
         26 . The method according to  claim 10 , wherein the cytokine cell suspension is autologous to the mammalian subject. 
     
     
         27 . The method according to  claim 10 , wherein the collagen defect is associated with osteoarthritis. 
     
     
         28 . The method according to  claim 10 , further comprising administering a concomitant therapy comprising topically administering to the site a composition comprising hyaluronic acid, collagen, and combinations thereof.