Laminous vascular constructs combining cell sheet engineering and electrospinning technologies

• US 10,092,679 B2
• Filed: 10/20/2014
• Issued: 10/09/2018
• Est. Priority Date: 10/18/2013
• Status: Active Grant

• Current Assignee: Wake Forest University Health Sciences
• Original Assignee: Wake Forest University Health Sciences
• Inventors: Ahn, Hyunhee, Ju, Young Min, Atala, Anthony, Lee, Sang Jin
• Primary Examiner(s): Lankford, Jr., Leon B
• Application Number:
  US14/518,352
• Publication Number:
  US 20150112419A1
• Time in Patent Office: 1,450 Days
• Field of Search: None
• CPC Class Codes: A61F 2/06 Blood vessels
• CPC Class Codes: A61F 2/062 Apparatus for the production of blood vessels made from natural tissue or with layers of living cells prostheses made from natural tissue or living cells A61L27/507
• CPC Class Codes: A61F 2/07 Stent-grafts
• CPC Class Codes: A61F 2210/0076 multilayered, e.g. laminated structures
• CPC Class Codes: A61F 2240/001 Designing or manufacturing processes
• CPC Class Codes: A61L 27/26 Mixtures of macromolecular compounds
• CPC Class Codes: A61L 27/3808 Endothelial cells
• CPC Class Codes: A61L 27/3826 Muscle cells, e.g. smooth muscle cells
• CPC Class Codes: A61L 27/507 for artificial blood vessels apparatus for applying cells on a blood vessel prosthesis A61F2/062
• CPC Class Codes: C08L 67/04 Polyesters derived from hydroxycarboxylic acids, e.g. lactones C08L67/06 takes precedence
• CPC Class Codes: C08L 89/06 derived from leather or skin , e.g. gelatin
• CPC Class Codes: C12N 2533/40 Polyhydroxyacids, e.g. polymers of glycolic or lactic acid (PGA, PLA, PLGA); Bioresorbable polymers
• CPC Class Codes: C12N 2533/54 Collagen; Gelatin
• CPC Class Codes: C12N 5/0691 Vascular smooth muscle cells; 3D culture thereof, e.g. models of blood vessels
• CPC Class Codes: D01D 5/0007 Electro-spinning non-woven fabrics produced by electro-spinning D04H1/728

Abstract

Vascular scaffolds and methods of fabricating the same are disclosed for tissue engineering of vascular constructs. By combining electrospun matrices with cell sheet technologies, vascular constructs with more mature cell layers can be obtained for reconstruction of blood vessels, heart valves and the like. A engineered smooth muscle cell sheet, wrapped around an electrospun vascular scaffold, is able to provide a mature SMC layer that expresses strong cell-to-cell junction markers and contractile proteins. In addition, preconditioning of the cell sheet covered vascular scaffold maintained cell viability and infiltration into the scaffold.

Claims

1. A tissue engineered vascular construct, comprising:

an electrospun biocompatible matrix in a tubular configuration; and

a population of smooth muscle cells pre-fabricated as at least one smooth muscle cell sheet, the sheet comprised of smooth muscle cells grown to confluence on a substrate and then detached from said substrate as an intact sheet,

wherein the intact sheet is wrapped around the outside of the matrix to form a vascular construct.

2. The vascular construct of claim 1, wherein at least 2 cell sheets are applied to the outside of the matrix.

3. The vascular construct of claim 1, further comprising a population of endothelial cells seeding onto the inside the tubular matrix.

4. The vascular construct of claim 1, wherein the tubular matrix comprises at least one natural component from about 25 percent to about 75 percent by weight.

5. The vascular construct of claim 1, wherein the tubular matrix comprises an electrospun matrix comprising at least one natural component and at least one synthetic polymer component.

6. The vascular construct of claim 5, wherein the natural component comprises collagen.

7. The vascular construct of claim 5, wherein the synthetic polymer component comprises poly(ε

-caprolactone) (PCL).

8. The vascular construct of claim 1, wherein the smooth muscle cells are derived from progenitor cells isolated from peripheral blood or bone marrow.

9. The vascular construct of claim 1, wherein the population of smooth muscle cells are cultured into one or more patterns.

10. The vascular construct of claim 1, further comprising a population of endothelial cells, applied to the inside of the matrix.

13. A method of producing a tissue engineered vascular construct as recited in claim 1, comprising:

forming an electrospun biocompatible matrix in a tubular configuration;

culturing a cell population comprising smooth muscle cells to form a cell sheet and applying the cell sheet to the outside of the matrix to form a vascular construct.

14. The method of claim 13, wherein at least two cell sheets are applied to the outside of the matrix to form the vascular construct.

15. The method of claim 13, further comprising seeding the inside of the matrix with a population of endothelial cells.

16. The method of claim 13, wherein the tubular matrix comprises at least one natural component from about 25 percent to about 75 percent by weight.

17. The method of claim 13, wherein the tubular matrix comprises an electrospun matrix comprising at least one natural component and at least one synthetic polymer component.

18. The method of claim 17, wherein the natural component comprises collagen.

19. The method of claim 17, wherein the synthetic polymer component comprises poly(ε

-caprolactone) (PCL).

20. The method of claim 13, further comprising:

attaching a first end and a second end of the vascular construct to a first attachment and a second attachment element in a preconditioning chamber, wherein the first attachment and the second attachment elements each have a channel that is fluidly coupled to a fluid flow system; and

preconditioning the construct with the flow system by moving a biological fluid through the construct, wherein a flow-rate and a pulse-rate of the biological fluid is controlled such that a preconditioned vascular construct is produced.

21. The method of claim 20, wherein the step of preconditioning the construct comprises moving the biological fluid through an inside surface of the seeded tubular matrix in a closed fluid flow system.

22. The method of claim 20, wherein the step of preconditioning the seeded tubular matrix further comprises moving a biological fluid having a composition and viscosity that mimics blood through the inside surface of the attached matrix as a pulsed flow to induce a wall shear stress of at least 10 dynes/cm² so that the vascular construct is exposed to fluid flow conditions that mimic flow of blood through a native blood vessel.

23. The method of claim 22, further comprising continuing exposure of the cells on the inside surface of the matrix to the pulsed fluid flow to allow the seeded cells to develop under fluid flow conditions until the matrix can withstand a wall pressure distribution of at least 60 mmHg.

24. The method of claim 20, wherein the pulsed flow has a pulse-rate that is varied over time to induce a wall shear stress in the range of about 10 dynes/cm² to about 45 dynes² .

25. The method of claim 20, wherein the pulsed flow has a pulse-rate that is varied over time to induce a wall pressure distribution in the range of about 60 mmHg to about 200 mmHg.

26. The method of claim 20, wherein the biological fluid is moved through the seeded tubular matrix by a pump.

27. The method of claim 13, wherein the smooth muscle cells are derived from progenitor cells isolated from peripheral blood or bone marrow.

11. A tissue engineered vascular construct, comprising:

an electrospun biocompatible matrix in a tubular configuration comprising at least one natural component and at least one synthetic polymer component, wherein the natural component comprises collagen and wherein the synthetic polymer component comprises poly(ε

-caprolactone) (PCL); and

a population of smooth muscle cells pre-fabricated as at least one smooth muscle cell sheet, the sheet comprised of smooth muscle cells grown to confluence on a substrate and then detached from said substrate as an intact sheet,

wherein the intact sheet is wrapped around the outside of the matrix to form a vascular construct.

12. The vascular construct of claim 11 wherein at least an outer portion of the tubular matrix has fibers that are oriented in a circumferential manner to mimic the orientation of SMCs in native vessels.