Isolation and Identification of Glycosaminoglycans

Abstract

The isolation and identification of glycosaminoglycans capable of binding to proteins having a heparin-binding domain is disclosed, as well as the use of the glycosaminoglycans isolated in the growth and/or development of tissue.

1 . A method of isolating glycosaminoglycans capable of binding to a protein having a heparin-binding domain, the method comprising:
 (i) providing a solid support having polypeptide molecules adhered to the support, wherein the polypeptide comprises a heparin-binding domain;   (ii) contacting the polypeptide molecules with a mixture comprising glycosaminoglycans such that polypeptide-glycosaminoglycan complexes are allowed to form;   (iii) partitioning polypeptide-glycosaminoglycan complexes from the remainder of the mixture;   (iv) dissociating glycosaminoglycans from the polypeptide-glycosaminoglycan complexes;   (v) collecting the dissociated glycosaminoglycans.   
     
     
         2 . A method of identifying glycosaminoglycans capable of stimulating or inhibiting the growth and/or differentiation of cells and/or tissues, the method comprising:
 (i) providing a solid support having polypeptide molecules adhered to the support, wherein the polypeptide comprises a heparin-binding domain;   (ii) contacting the polypeptide molecules with a mixture comprising glycosaminoglycans such that polypeptide-glycosaminoglycan complexes are allowed to form;   (iii) partitioning polypeptide-glycosaminoglycan complexes from the remainder of the mixture;   (iv) dissociating glycosaminoglycans from the polypeptide-glycosaminoglycan complexes;   (v) collecting the dissociated glycosaminoglycans;   (vi) adding the collected glycosaminoglycans to cells or tissues in which a protein containing the amino acid sequence of the heparin-binding domain is present;   (vii) measuring one or more of: proliferation of the cells, differentiation of the cells, expression of one or more protein markers.   
     
     
         3 . The method of  claim 1  or  2  wherein the mixture comprising glycosaminoglycans contains extracellular matrix material. 
     
     
         4 . The method of  claim 3  wherein the extracellular matrix material is derived from connective tissue or connective tissue cells. 
     
     
         5 . The method of any one of  claims 1  to  4  wherein the mixture comprising glycosaminoglycans contains one or more of a dextran sulphate, a chondroitin sulphate, a heparan sulphate. 
     
     
         6 . The method of any one of  claims 1  to  5  wherein the mixture comprising glycosaminoglycans has been enriched for one of dextran sulphate, chondroitin sulphate, heparan sulphate. 
     
     
         7 . The method of any one of  claims 1  to  6  wherein the method further comprises subjecting the collected glycosaminoglycans to further analysis in order to determine structural characteristics of the GAG. 
     
     
         8 . The method of any one of  claims 1  to  7  wherein the glycosaminoglycan-polypeptide complexes are contacted with a lyase. 
     
     
         9 . The method of any one of  claims 1  to  8  wherein the polypeptide is, or comprises, one of SEQ ID NO.s: 1-13 or 17. 
     
     
         10 . Heparan sulphate HS/BMP2. 
     
     
         11 . Culture media comprising HS/BMP2. 
     
     
         12 . A pharmaceutical composition or medicament comprising HS/BMP2. 
     
     
         13 . The pharmaceutical composition or medicament of  claim 12  further comprising a pharmaceutically acceptable carrier, adjuvant or diluent. 
     
     
         14 . The pharmaceutical composition or medicament of  claim 12  or  13  further comprising BMP2 protein. 
     
     
         15 . A pharmaceutical compositions or medicament according to any one of  claims 12  to  14  for use in the prevention or treatment of injury or disease. 
     
     
         16 . Use of HS/BMP2 in the manufacture of a medicament for the prevention or treatment of injury or disease. 
     
     
         17 . The pharmaceutical composition or use of  claim 15  or  16  wherein the prevention or treatment is chosen from: repair, regeneration or replacement of connective tissue and wound healing. 
     
     
         18 . A method of preventing or treating injury or disease in a patient in need of treatment thereof, the method comprising administering an effective amount of HS/BMP2 to the patient. 
     
     
         19 . The method of  claim 18  wherein the administered HS/BMP2 is formulated as a pharmaceutical composition or medicament. 
     
     
         20 . The method of  claim 19  wherein the pharmaceutical composition or medicament further comprises BMP2 protein. 
     
     
         21 . A method of promoting or inhibiting osteogenesis comprising administering HS/BMP2 to bone precursor cells or bone stem cells. 
     
     
         22 . The method of  claim 21  wherein the bone precursor cells or bone stem cells are contacted with HS/BMP2 in vitro. 
     
     
         23 . The method of  claim 21  wherein the bone precursor cells or bone stem cells are contacted with HS/BMP2 in vivo. 
     
     
         24 . The method of any one of  claims 21  to  23  wherein the bone precursor cells or bone stem cells are contacted with BMP2 simultaneously with HS/BMP2. 
     
     
         25 . A method of promoting or inhibiting the formation of cartilage tissue comprising administering HS/BMP2 to cartilage precursor cells or cartilage stem cells. 
     
     
         26 . The method of  claim 25  wherein the cartilage precursor cells or cartilage stem cells are contacted with HS/BMP2 in vitro. 
     
     
         27 . The method of  claim 25  wherein the cartilage precursor cells or cartilage stem cells are contacted with HS/BMP2 in vivo. 
     
     
         28 . The method of any one of  claims 25  to  27  wherein the cartilage precursor cells or cartilage stem cells are contacted with BMP2 simultaneously with HS/BMP2. 
     
     
         29 . A method for the repair, replacement or regeneration of connective tissue in a human or animal patient in need of such treatment, the method comprising:
 (i) culturing mesenchymal stem cells in vitro in contact with HS/BMP2 for a period of time sufficient for said cells to form connective tissue;   (ii) collecting said connective tissue;   (iii) implanting said connective tissue into the body of the patient at a site of injury or disease to repair, replace or regenerate connective tissue in the patient.   
     
     
         30 . The method of  claim 29  further comprising contacting the mesenchymal cells in culture with exogenous BMP2. 
     
     
         31 . Connective tissue obtained by in vitro culture of mesenchymal stem cells in the presence of HS/BMP2. 
     
     
         32 . Connective tissue as claimed in  claim 31 , wherein the mesenchymal cells are cultured in the presence of exogenous BMP2, and optionally in the presence of BMP2. 
     
     
         33 . A pharmaceutical composition comprising stem cells and HS/BMP2. 
     
     
         34 . The pharmaceutical composition of  claim 32  wherein the stem cells are mesenchymal stem cells. 
     
     
         35 . The pharmaceutical composition of  claim 33  or  34  wherein the composition further comprises BMP2. 
     
     
         36 . A pharmaceutical composition according to any of  claims 33  to  35  for use in the treatment of injury or disease. 
     
     
         37 . A method for the treatment of injury or disease in a patient in need of treatment thereof comprising administering to the patient a pharmaceutical composition comprising stem cells and HS/BMP2. 
     
     
         38 . The method of  claim 37  wherein the stem cells are mesenchymal stem cells. 
     
     
         39 . The method of  claim 37  or  38  wherein the method further comprises administering BMP2 to the patient. 
     
     
         40 . Use of HS/BMP2 in the growth of connective tissue in vitro. 
     
     
         41 . A method for growing connective tissue in vitro comprising culturing mesenchymal stem cells in contact with exogenously added HS/BMP2. 
     
     
         42 . A biological implant comprising a solid or semi-solid matrix material impregnated with HS/BMP2. 
     
     
         43 . The biological implant of  claim 42  further impregnated with BMP2. 
     
     
         44 . The biological implant of  claim 42  or  43  further impregnated with mesenchymal stem cells. 
     
     
         45 . A kit comprising a predetermined amount of a glycosaminoglycan having high affinity for a protein having a heparin-binding domain and a predetermined amount of said protein. 
     
     
         46 . The kit of  claim 45  wherein the glycosaminoglycan is HS/BMP2 and the protein is BMP2.