Scaffolds for soft tissue and uses thereof
The present invention provides tissue scaffolds, methods of generating such scaffolds, and methods of use of such scaffolds to generate aligned and functional tissues for use in methods including regenerative medicine, wound repair and transplantation.
1. A tissue scaffold comprising a polymeric base layer and a patterned metal oxide layer deposited thereon, wherein the metal oxide layer pattern comprises at least two continuous parallel oxide layer stripes deposited on said base layer,
wherein said metal oxide layer stripes are deposited across the polymeric base layer surface, with the continuous length of each stripe layer relative to the width being sufficient to direct the outgrowth of cells aligned parallel to the lengthwise-direction of said metal oxide stripe layer, and with the underlying base layer surface exposed therebetween in continuous parallel stripes to provide an alternating pattern of metal oxide and base layer stripes,
wherein said metal oxide layer stripes are about 10 μm to about 100 μm wide and said base layer stripes are about 10 μm to about 100 μm wide,
wherein the metal oxide stripes further comprise a non-biologic cell adhesive layer disposed on the stripes, and
wherein said scaffold further comprises an extracellular matrix (ECM) layer assembled by a confluent cell layer, where the ECM is oriented across the patterned surface so that ECM fibrils are aligned parallel to the lengthwise-direction of the underlying metal oxide stripes and exposed base layer stripes therebetween.
2. The tissue scaffold of claim 1 wherein the base layer is selected from the group consisting of a hard polymeric surface, a semi-rigid polymeric surface, and a soft polymeric surface.
3. The tissue scaffold of claim 1 wherein the base layer is selected from the group consisting of nylon, polyester, polyamide, polyacrylamide, polyetheretherketone and silicone.
4. The tissue scaffold of claim 1 wherein the base layer is selected from the group consisting of silk and collagen.
5. The tissue scaffold of claim 1 wherein the oxide layer stripes are about 10 μm wide and the base layer stripes are about 10 μm wide, or the oxide layer stripes are about 20 μm wide and the base layer stripes are about 20 μm wide, or the oxide layer stripes are about 30 μm wide and the base layer stripes are about 30 μm wide, or the oxide layer stripes are about 20 μm wide and the base layer stripes are about 10 μm wide.
6. The tissue scaffold of claim 1 wherein the metal oxide is zirconium oxide.
7. The tissue scaffold of claim 1 wherein the non-biologic cell adhesive layer comprises a phosphonate.
8. The tissue scaffold of claim 1 , wherein the cells used for directing outgrowth in the lengthwise-direction of said metal oxide stripe layer are selected from the group consisting of fibroblasts, Schwann cells, stem cells and combinations thereof.
9. The tissue scaffold of claim 1 , wherein the ECM is decellularized.
10. The tissue scaffold of claim 8 , wherein the cells used for directing outgrowth in the lengthwise-direction of said metal oxide stripe layer include human mesenchymal stem cells (hMSCs).
11. The tissue scaffold of claim 8 , wherein the cells used for directing outgrowth in the lengthwise-direction of said metal oxide stripe layer include a combination of Schwann cells and hMSCs.
12. The tissue scaffold of claim 9 , further comprising a plurality of neural cells selected from the group consisting of stem cells, oligopotent cells, differentiated neurons, differentiated glial cells and neural crest cells;
wherein said neural cells form a confluent monolayer across the ECM monolayer while maintaining alignment parallel to said pattern of alternating oxide and base layer stripes and ECM.
13. The tissue scaffold of claim 12 , wherein said neural cells comprise glial support cells.
14. The tissue scaffold of claim 12 , wherein said neural cells comprise human neural cells.
15. The tissue scaffold of claim 12 , wherein said neural cells comprise both human neural cells and glial support cells.
16. A method for making a tissue scaffold comprising:
generating a pattern on a polymeric base layer using photolithography, said pattern comprising at least two continuous parallel protective photoresist compound stripes separated by continuous parallel unprotected base layer stripes to form a pattern of continuous alternating parallel photoresist and base layer stripes;
depositing a metal oxide layer onto the unprotected base layer stripes to form a patterned metal oxide layer deposited across the polymeric base layer surface,
removing the photoresist to expose the underlying base layer surface to provide an alternating pattern of metal oxide and base layer stripes;
wherein the continuous length of each metal oxide stripe layer relative to the width is sufficient to direct the outgrowth of cells aligned parallel to the lengthwise-direction of said metal oxide stripe layer, wherein said metal oxide layer stripes are about 10 μm to about 100 μm wide and said base layer stripes are about 10 μm to about 100 μm wide;
contacting the patterned metal oxide layer with a non-biologic cell adhesive compound to generate a patterned cell adhesive layer disposed on the stripes;
contacting said patterned cell adhesive layer with cells;
culturing said cells under conditions suitable to forming a confluent cell layer and assembling an extracellular matrix (ECM) layer; and
removing said cells to provide a tissue scaffold comprising an ECM layer aligned on the stripes, wherein the ECM is oriented across the patterned surface so that ECM fibrils are aligned parallel to the lengthwise-direction of the underlying metal oxide stripes and exposed base layer stripes therebetween.
17. The method of claim 16 wherein the metal oxide is zirconium oxide.
18. The method of claim 16 wherein the metal oxide layer is deposited onto the patterned base layer by vapor phase deposition of a metal alkoxide.
19. The method of claim 18 wherein the metal alkoxide is zirconium tetra(tert-butoxide).
20. The method of claim 16 wherein said non-biologic cell adhesive compound comprises a phosphonic acid.
21. The method of claim 20 wherein the phosphonic acid is 1,4-butanedi-phosphonic acid.
22. A method for making an artificial tissue comprising living cells attached to a tissue scaffold comprising contacting a tissue scaffold of claim 1 with cells and culturing said cells under conditions suitable for cell growth and/or differentiation.
23. A method of tissue repair or regeneration comprising implanting the tissue scaffold of claim 1 in neural tissue in need of repair or regeneration in a subject in need thereof.
24. A method for identifying a compound that modulates a tissue function comprising contacting the tissue structure of claim 14 with a test compound, and measuring the effect of the test compound on a tissue function in the presence and absence of the test compound, wherein a modulation of the tissue function in the presence of the test compound as compared to the tissue function in the absence of the test compound indicates that the test compound modulates a tissue function, thereby identifying a compound that modulates a tissue function.
25. A method for identifying a compound useful for treating or preventing a tissue disease comprising contacting the tissue structure of claim 14 with a test compound, and measuring the effect of the test compound on a tissue function in the presence and absence of the test compound, wherein a modulation of the tissue function in the presence of the test compound as compared to the tissue function in the absence of the test compound indicates that the test compound modulates a tissue function, thereby identifying a compound useful for treating or preventing a tissue disease.