Hyaluronic acid derivative and neural stem cells for SCI and PNT regeneration

1. A method of regenerating nervous fibers in spinal cord or in peripheral nerve injury, said method comprising:

a) preparing a three-dimensional parallel non-woven fiber scaffold comprising a biodegradable hyaluronic acid ester, wherein the scaffold fibers are spatially-oriented along the longitudinal axes of regenerating nervous fibers;

b) submerging said scaffold into a coating solution capable of promoting Adult Neuronal Stem Cells adhesion, branching, proliferation and differentiation;

c) contacting said coated scaffold obtained from step b) with Adult Neuronal Stem Cells, wherein Adult Neuronal Stem Cells are adhered onto said coated scaffold; and

d) culturing and expanding adhered cells in the presence of growth or neurotrophic factors selected from bFGF (basic fibroblast growth factor), CNTF (ciliary neurotrophic factor), BDNF (brain derived neurotrophic factor) and GDNF (glial derived neurotrophic factor) or mixtures thereof for 2 days in vitro before the implantation;

e) preparing the implant site;

f) implanting the cell scaffold obtained from steps a-d) at the injury site prepared in step e);

g) covering the implanted cell scaffold with a lamina or a tube to isolate the regeneration site from the surrounding tissues;

h) suturing or sealing the implanted cell scaffold to the host tissue with fibrin glue, wherein said cell scaffold promotes the regeneration of said nervous fibers in spinal cord or in peripheral nerve injury.

2. The method according to claim 1 , wherein said hyaluronic acid ester is a benzyl ester.

3. The method according to claim 2 , wherein the hyaluronic acid ester has an esterification degree ranging between about 75% and 100%.

4. The method according to claim 1 , wherein said coating solution comprises laminin or fibronectin, or laminin and fibronectin.

5. The method according to claim 4 , wherein the laminin concentration is about 25 μg/ml.

6. The method according to claim 4 , wherein the fibronectin concentration is about 50 μg/ml.

7. The method according to claim 1 , wherein said Adult Neuronal Stem Cells are used in a range from about 9500 to 35000 cells/cm 3 .

8. The method according to claim 1 , wherein said growth or neurotrophic factors are used preferably with a concentration of about 20 ng/ml.

9. The method according to claim 1 , wherein said scaffold fibers are in form of non-woven meshes and fibers, and woven tubular structures having porous structures.

10. The method according to claim 9 , wherein said scaffold fibers have a diameter ranging from about 20 to 50 μm.

11. The method according to claim 1 , wherein said lamina is square.

12. The method according to claim 1 , wherein said cell scaffold is inserted into tubes made of esterified hyaluronic acid.

13. The method according to claim 12 , wherein said tube is sutured with the device to both nerve stubs of the host tissue.

14. A method of manufacturing a cell scaffold comprising:

a) preparing a three-dimensional parallel non-woven fiber scaffold comprising a biodegradable hyaluronic acid ester, wherein the scaffold fibers are spatially-oriented along the longitudinal axes of regenerating nervous fibers;

b) submerging said scaffold into a coating solution capable of promoting Adult Neuronal Stem Cells adhesion, branching, proliferation and differentiation;

c) contacting said coated scaffold obtained from step b) with Adult Neuronal Stem Cells, wherein Adult Neuronal Stem Cells are adhered onto said coated scaffold; and

d) culturing and expanding adhered cells in the presence of growth or neurotrophic factors selected from bFGF (basic fibroblast growth factor), CNTF (ciliary neurotrophic factor), BDNF (brain derived neurotrophic factor) and GDNF (glial derived neurotrophic factor) or mixtures thereof; and

e) obtaining said cell scaffold.