Porous polysaccharide scaffold comprising nano-hydroxyapatite and use for bone formation

Abstract

The present invention relate to three dimensional porous polysaccharide matrices able to induce mineralisation of a tissue in osseous site, as well as in non-osseous site, in the absence of stem cells or growth factors.

1 . A method for preparing a porous polysaccharide scaffold comprising the following steps:
 i) preparing an alkaline aqueous solution comprising at least one polysaccharide, a cross-linking agent and a porogen agent,   ii) transforming the solution into a hydrogel by placing said solution at a temperature from 4° C. to 80° C. for a sufficient time to allow the cross-linking of said at least one polysaccharide,   iii) submerging said hydrogel into a solvent, and   iv) washing the porous polysaccharide scaffold obtained at step iii),   
       wherein the alkaline aqueous solution of step i) further comprises hydroxyapatite. 
     
     
         2 . The method according to  claim 1  wherein the porogen agent is selected from the group consisting of sodium chloride, calcium chloride, ammonium carbonate, ammonium bicarbonate, calcium carbonate, sodium carbonate, sodium bicarbonate and mixtures thereof. 
     
     
         3 . The method according to  claim 1  wherein the weight ratio of the polysaccharide to the porogen agent is in a range from 1:50 to 50:1, preferably from 1:30 to 30:1, preferably from 1:12 to 12:1, preferably 12:14. 
     
     
         4 . A method for preparing a porous polysaccharide scaffold comprising the following steps:
 a) preparing an alkaline aqueous solution comprising at least one polysaccharide, and one cross-linking agent,   b) freezing the aqueous solution of step a), and   c) sublimating the frozen solution of step b),   wherein the alkaline aqueous solution of step a) further comprises hydroxyapatite,   and wherein step b) is performed before cross-linking of the polysaccharide occurs in the solution of step a).   
     
     
         5 . The method of  claim 1 , wherein said polysaccharide is selected from the group consisting of dextran, pullulan, agar, alginic acid, starch, hyaluronic acid, inulin, heparin, fucoidan, chitosan and mixtures thereof. 
     
     
         6 . The method of  claim 1 , wherein said polysaccharide is a mixture of pullulan/dextran in a ratio in a range from 95:5 to 5:95. 
     
     
         7 . The method of  claim 1 , wherein said polysaccharide is a mixture of pullulan/dextran/fucoidan in a ratio in a range from about 70:20:10 to about 50:20:30, preferably from about 70:20:10 to about 50:30:20, and most preferably in a ratio of about 73:22:5 (w/w). 
     
     
         8 . The method according to  claim 1 , wherein said cross-linking agent is selected from the group consisting of trisodium trimetaphosphate (STMP), phosphorus oxychloride (POCl 3 ), epichlorohydrin, formaldehydes, carbodiimides, glutaraldehydes, and mixtures thereof. 
     
     
         9 . The method  claim 17 , wherein said nano-hydroxyapatite is obtained from a solution of phosphoric acid at a concentration between 0.3 to 1M, a solution of calcium hydroxide at a concentration between 0.5 to 1.5M, through chemical precipitation at room temperature. 
     
     
         10 . The method according to  claim 9 , wherein the concentration of nano-hydroxyapatite in the alkaline solution of polysaccharide (w/v) is between 0.01 and 10% (w/v), preferably between 0.1 and 0.5% (w/v), more preferably between 0.1 and 0.3% (w/v). 
     
     
         11 . A porous polysaccharide scaffold obtained by
 i) preparing an alkaline aqueous solution comprising at least one polysaccharide, a cross-linking agent and a porogen agent,   ii) transforming the solution into a hydrogel by placing said solution at a temperature from 4° C. to 80° C. for a sufficient time to allow cross-linking of said at least one polysaccharide,   iii) submerging said hydrogel into a solvent, and   iv) washing the porous polysaccharide scaffold obtained at step iii),   
       wherein the alkaline aqueous solution of step i) further comprises hydroxyapatite. 
     
     
         12 . The porous polysaccharide scaffold of  claim 11 , wherein the size of the pores is from 1 μm to 500 μm and the porosity is from 4% to 75%. 
     
     
         13 - 15 . (canceled) 
     
     
         16 . The method of  claim 1 , wherein said solvent is an aqueous solution. 
     
     
         17 . The method of  claim 1 , wherein said hydroxyapatite is nano-hydroxyapatite. 
     
     
         18 . The method of  claim 3 , wherein said porogen agent is in a range selected from the group consisting of from 1:30 to 30:1, from 1:12 to 12:1, and from 12:14 
     
     
         19 . The method of  claim 4 , wherein said preferably hydroxyapatite is nano-hydroxyapatite. 
     
     
         20 . The method of  claim 6 , wherein said ratio is 75:25 (w/w). 
     
     
         21 . The method of  claim 7 , wherein said ratio is selected from the group consisting of 70:20:10 (w/w), 50:30:20 (w/w), and 73:22:5 (w/w). 
     
     
         22 . The method of  claim 9 , wherein said phosphoric acid concentration is 0.6M and said calcium hydroxide concentration is 1M. 
     
     
         23 . The method of  claim 10 , wherein said nano-hydroxide concentration is between 0.1 and 0.5% (w/v) or between 0.1 and 0.3% (w/v). 
     
     
         24 . The method of  claim 12 , wherein the size of the pores if from 10 to 200 and the porosity is from 4 to 50%. 
     
     
         25 . A method for bone generation comprising the step of administering a porous polysaccharide scaffold obtained by
 i) preparing an alkaline aqueous solution comprising at least one polysaccharide, a cross-linking agent and a porogen agent,   ii) transforming the solution into a hydrogel by placing said solution at a temperature from 4° C. to 80° C. for a sufficient time to allow cross-linking of said at least one polysaccharide,   iii) submerging said hydrogel into a solvent, and   iv) washing the porous polysaccharide scaffold obtained at step iii),   
       wherein the alkaline aqueous solution of step i) further comprises hydroxyapatite. 
     
     
         26 . A method for stimulating ectopic mineralized tissue formation comprising the step of administering a porous polysaccharide scaffold obtained by
 i) preparing an alkaline aqueous solution comprising at least one polysaccharide, a cross-linking agent and a porogen agent,   ii) transforming the solution into a hydrogel by placing said solution at a temperature from 4° C. to 80° C. for a sufficient time to allow cross-linking of said at least one polysaccharide,   iii) submerging said hydrogel into a solvent, and   iv) washing the porous polysaccharide scaffold obtained at step iii),   
       wherein the alkaline aqueous solution of step i) further comprises hydroxyapatite. 
     
     
         27 . A method for treating bone related disorders comprising the step of administering a porous polysaccharide scaffold obtained by
 i) preparing an alkaline aqueous solution comprising at least one polysaccharide, a cross-linking agent and a porogen agent,   ii) transforming the solution into a hydrogel by placing said solution at a temperature from 4° C. to 80° C. for a sufficient time to allow cross-linking of said at least one polysaccharide,   iii) submerging said hydrogel into a solvent, and   iv) washing the porous polysaccharide scaffold obtained at step iii),   
       wherein the alkaline aqueous solution of step i) further comprises hydroxyapatite.