Method for producing a collagen membrane and uses thereof
The present invention relates to a method of producing a collagen membrane that has particular mechanical properties. In particular, the present invention relates to a method A of producing a collagen membrane comprising the steps of (i) isolating a collagen-containing tissue and incubating same in an ethanol solution; (ii) incubating the collagen-containing tissue from step (i) in a first solution comprising an inorganic salt and an anionic surfactant in order to denature non-collagenous proteins contained therein; (iii) incubating the collagen-containing tissue produced in step (ii) in a second solution comprising an inorganic acid until the collagen in said material is denatured; and (iv) incubating the collagen-containing tissue produced in step (iii) in a third solution comprising an inorganic acid with simultaneous mechanical stimulation for sufficient time to enable the collagen bundles in said collagen-containing tissue to align; wherein the mechanical stimulation comprises applying tension cyclically to the collagen-containing tissue.
1. A method of producing a collagen-containing membrane, the method comprising the steps of:
(i) incubating an isolated collagen-containing tissue comprising collagen bundles in an ethanol solution;
(ii) incubating the collagen-containing tissue from step (i) in a solution comprising an inorganic salt selected from the group consisting of trimethylammonium chloride, tetramethylammonium chloride, sodium chloride, lithium chloride, perchlorate and trifluoromethanesulfonate and an anionic surfactant selected from the group consisting of alkyl sulfates, alkyl ether sulfates, alkyl sulfonates, and alkyl aryl sulfonates in order to denature non-collagenous proteins contained therein;
(iii) incubating the collagen-containing tissue produced in step (ii) in a solution comprising HCl until the collagen in said tissue is denatured; and
(iv) manipulating the collagen-containing tissue produced in step (iii) using simultaneous mechanical stimulation for sufficient time to enable the collagen bundles in said collagen-containing tissue to align and incubating said tissue in a solution comprising an HCl until the collagen-containing membrane is produced;
wherein the mechanical stimulation comprises applying tension cyclically to the collagen-containing tissue.
2. The method of claim 1 , wherein the ethanol solution used in step (i) comprises greater than 70% ethanol.
3. The method of claim 1 , wherein the inorganic salt used in step (ii) is lithium chloride (LiCl).
4. The method of claim 1 , wherein the anionic surfactant used in step (ii) is an alkyl sulfate, wherein the alkyl sulfate is sodium dodecyl sulphate (SDS).
5. The method of claim 1 , wherein the solution in step (ii) comprises an alkyl sulfate, wherein the alkyl sulfate is SDS and the solution comprises 1% (v/v) SDS and 0.2% (v/v) LiCl.
6. The method of claim 1 , wherein the incubation in step (ii) is undertaken at 4° C.
7. The method of claim 1 , wherein the incubation in step (ii) is undertaken for at least 12 hours.
8. The method of claim 1 , wherein the solution in step (iii) comprises 0.5% (v/v) HCl.
9. The method of claim 1 , wherein the incubation in step (iii) is undertaken for 30 minutes.
10. The method of claim 1 , wherein the incubation in step (iii) is undertaken with shaking.
11. The method of claim 1 , wherein the solution in step (iv) comprises 1% (v/v) HCl solution.
12. The method of claim 1 , wherein the incubation in step (iv) is undertaken for 12 to 36 hours.
13. The method of claim 1 , wherein the incubation in step (iv) is undertaken for 24 hours.
14. The method of claim 1 , wherein the incubation in step (iv) is undertaken with shaking.
15. The method of claim 1 , wherein between steps (ii) and (iii) and/or between steps (iii) and (iv) the collagen-containing tissue is contacted with glycerol in order to visualize and facilitate the removal of fat and/or blood vessels.
16. The method of claim 15 , wherein the glycerol is contacted with the collagen-containing tissue for at least 10 minutes.
17. The method of claim 1 , wherein between steps (ii) and (iii) and/or between steps (iii) and (iv) the collagen-containing tissue is washed.
18. The method of claim 17 , wherein the wash used between steps (ii) and (iii) is carried out using acetone to remove the denatured proteins.
19. The method of claim 18 , wherein the collagen-containing tissue is further washed in a solution comprising NaOH and NaCl during producing said collagen-containing membrane.
20. The method of claim 18 , wherein the collagen-containing tissue is further washed during producing said collagen-containing membrane after the acetone with sterile water.
21. The method of claim 1 , wherein after step (iv) the collagen-containing tissue is washed during producing said collagen-containing membrane with sterile water.
22. The method of claim 1 , wherein:
in step (i) the isolated collagen-containing tissue is incubated in an ethanol solution comprising greater than 70% (v/v) ethanol;
in step (ii) the collagen-containing tissue from step (i) is incubated in a solution comprising 1% (v/v) SDS and 0.2% (v/v) LiCl at a temperature of 4° C. for at least 12 hours in order to denature non-collagenous proteins contained in the collagen-containing tissue;
in step (iii) the collagen-containing tissue produced in step (ii) is incubated in a solution comprising 0.5% (v/v) HCl for 30 minutes until the collagen in said tissue is denatured; and
in step (iv) after the manipulating the collagen-containing tissue produced in step (iii), incubating said collagen-containing tissue in a solution comprising 1% (v/v) HCl solution for 12 to 36 hours until the collagen-containing membrane is produced.