Method for producing tissue scaffold having aligned fibrils

• US 7,727,441 B2
• Filed: 02/25/2008
• Issued: 06/01/2010
• Est. Priority Date: 06/04/2003
• Status: Active Grant

• Current Assignee: University of South Carolina
• Original Assignee: University of South Carolina
• Inventors: Goldsmith, Edie C., Gore, C. Michael, Goodwin, Richard L., Yost, Michael J., Terracio, Louis
• Primary Examiner(s): Huson; Monica A
• Application Number:
  US12/072,167
• Publication Number:
  US 20080217820A1
• Time in Patent Office: 827 Days
• Field of Search: None
• US Class Current: 264/209.1
• CPC Class Codes: B29C 48/09 Articles with cross-sections having partially or fully enclosed cavities, e.g. pipes or channels
• CPC Class Codes: B29C 48/33 with parts rotatable relative to each other
• CPC Class Codes: C12M 21/08 for producing artificial tissue or for ex-vivo cultivation of tissue prostheses A61F2/00, grafts A61L27/00
• CPC Class Codes: C12M 25/14 Scaffolds; Matrices in general C12N5/0068

Abstract

A tubular tissue scaffold is described which comprises a tube having a wall, wherein the wall includes biopolymer fibrils that are aligned in a helical pattern around the longitudinal axis of the tube where the pitch of the helical pattern changes with the radial position in the tube wall. The scaffold is capable of directing the morphological pattern of attached and growing cells to form a helical pattern around the tube walls. Additionally, an apparatus for producing such a tubular tissue scaffold is disclosed, the apparatus comprising a biopolymer gel dispersion feed pump that is operably connected to a tube-forming device having an exit port, where the tube-forming device is capable of producing a tube from the gel dispersion while providing an angular shear force across the wall of the tube, and a liquid bath located to receive the tubular tissue scaffold from the tube-forming device. A method for producing the tubular tissue scaffolds is also disclosed. Also, artificial tissue comprising living cells attached to a tubular tissue scaffold as described herein is disclosed. Methods for using the artificial tissue are also disclosed.

Claims

1. A method of producing a tubular tissue scaffold, the method comprising:

feeding a gel dispersion comprising a biopolymer into an extruder, wherein the extruder has counter-rotating members configured to produce a tube from the gel dispersion while providing a radial shear force across the wall of the tube, and wherein the extruder has a gas channel connecting a gas source with the luminal space of the tubular tissue scaffold as it exits the tube-forming device;

forming the gel dispersion into a tube that is substantially free of distinct layers; and

causing the gel dispersion to solidify, thereby forming a tubular tissue scaffold comprising a tube wall having biopolymer fibrils that are aligned in a helical pattern around the longitudinal axis of the tube and where the pitch of the helical pattern changes with the radial position in the tube wall.

2. The method according to claim 1, wherein the method comprises:

feeding the gel dispersion into a counter-rotating cone extruder having an annular-shaped exit port and having a gas channel connecting a gas source with the center of the annular-shaped exit port and opening into the luminal space of the tubular tissue scaffold as it exits the extruder;

extruding the gel dispersion to form a tube that is substantially free of distinct layers; and

causing the gel dispersion to solidify, thereby forming a tubular tissue scaffold comprising a biopolymer having fibrils in the tube wall that are aligned in a helical pattern around the longitudinal axis of the tube and where the pitch of the helical pattern on the luminal surface of the tube is different from the pitch of the helical pattern on the outside surface of the tube.

3. The method according to claim 2, wherein the biopolymer comprises collagen, fibronectin, laminin, elastin, fibrin, proteoglycans, hyaluronan, or combinations thereof.

4. The method according to claim 3, wherein the biopolymer comprises collagen.

5. The method according to claim 4, wherein the biopolymer comprises type 1 collagen.

6. The method according to claim 5 further comprising preparing the gel dispersion of collagen according to a method comprising:

removing the hair from the hide of a bovine;

washing the hide sequentially in water, water containing NaHCO₃ and surfactant, and water;

contacting the hide with an aqueous solution containing NaHCO₃, Ca(OH)₂, and NaHS;

washing the hide in water;

treating the hide with an aqueous solution of Ca(OH)₂;

rinsing the hide with water and trimming the hide of any remaining skin tissue and fat;

placing the hide in an aqueous NaCl solution and adding hydrochloric acid solution until the pH is stable between about 6.0 and 8.0;

washing the hide in water;

placing the hide in an aqueous solution of acetic acid with or without pepsin;

mixing and allowing the hide to swell;

placing the swollen hide in a mill and processing into a gel dispersion;

filtering the gel dispersion to remove undissolved particles;

centrifuging the gel dispersion to remove small undissolved particles;

adding NaCl to the gel dispersion in an amount sufficient to precipitate collagen from the gel dispersion;

filtering the collagen precipitate and resuspending it in deionized water;

adding NaOH to bring the pH of the collagen dispersion to a pH between about 6 and about 8;

dialyzing the collagen dispersion against phosphate buffered saline solution;

resuspending the collagen in deionized water;

centrifuging the collagen dispersion to concentrate solid collagen gel as a pellet; and

resuspending the pellet in aqueous HCl.

7. The method according to claim 4 further comprising preparing the gel dispersion of collagen according to a method comprising:

removing the hair from the hide of a freshly killed bovine;

washing the hide sequentially in water, water containing 0.2% NaHCO₃ and 0.02% surfactant, percent by weight, and water;

contacting the hide with an aqueous solution containing 0.6% NaHCO₃, 2% Ca(OH)₂ and 4.3% NaHS, percent by weight, for a period of from about 0.1 hrs to about 10 hrs;

washing the hide in three changes of deionized water;

treating the hide with an aqueous solution of 2% Ca(OH)₂, percent by weight, for a period of from about 1 to about 24 hours;

rinsing the hide with water and trimming the hide of any remaining skin tissue and fat;

placing the hide in an aqueous 2 M NaCl solution and adding hydrochloric acid solution until the pH is stable between about 6.8 and 7.0;

washing the hide in three changes of deionized water;

placing the hide in an aqueous solution of 0.5 N acetic acid with or without pepsin added to give a 1;

100 weight ration based on wet hide weight;

mixing for a period of from about 1 to about 24 hours and allowing the hide to swell;

placing the swollen hide in a high-shear mill with ice and processing into a gel dispersion;

filtering the gel dispersion to remove undissolved particles;

centrifuging the gel dispersion to remove small undissolved particles;

adding NaCl to the gel dispersion in an amount sufficient to give a 2 M concentration, which will cause the precipitation of collagen from the gel dispersion;

filtering the collagen precipitate and resuspending it in deionized water;

adding NaOH to bring the pH of the collagen dispersion to pH 7.2;

dialyzing the collagen dispersion against phosphate buffered saline solution for a period of from about 1 to about 24 hours;

resuspending the collagen in three changes of deionized water over a period of about 24 hours;

centrifuging the collagen dispersion to concentrate solid collagen gel as a pellet; and

resuspending the pellet in 0.012 N HCl to a final collagen concentration of about 20 mg/ml.

8. The method according to claim 2, further comprising feeding a gas from the gas source to the luminal space of the tubular tissue scaffold as it exits the extruder.

9. The method according to claim 8, wherein the gas comprises a mixture of air and ammonia gas.

10. The method according to claim 9, wherein the mixture of air and ammonia is about a 50:

50 mixture by volume.

11. The method according to claim 8, wherein the outside surface of the tubular tissue scaffold is contacted with a mixture of air and ammonia gas as it exits the extruder.

12. The method according to claim 11, wherein the tubular tissue scaffold exiting the extruder falls into a liquid bath located to receive the tubular tissue scaffold from the extruder.

13. The method according to claim 12, wherein the liquid bath is located a defined distance beneath the exit port of the extruder.

14. The method according to claim 13, wherein the liquid bath is a aqueous ammonia bath at a pH of about 10.

15. The method according to claim 13, wherein the biopolymer gel dispersion is fed to the extruder at a defined feed rate, and the defined feed rate and the defined distance are selected so that the gel dispersion solidifies to form a tubular tissue scaffold comprising a biopolymer having fibrils in the tube wall that are aligned in a helical pattern around the longitudinal axis of the tube and where the pitch of the helical pattern on the luminal surface of the tube is different from the pitch of the helical pattern on the outside surface of the tube.

16. The method according to claim 15, wherein the outside diameter of the annular-shaped exit port is about 5 mm and the width of the annular space is about 0.5 mm, and wherein the defined feed rate is sufficient to provide a tube extrusion rate of about 150 cm/mim. and the defined distance is between about 10 cm and about 20 cm.

17. The method according to claim 13, where the tubular tissue scaffold remains in the bath for about 15 min.

18. The method according to claim 3, wherein the step of causing the gel to solidify includes immersing the tube in an aqueous solution containing 0.3 % sodium bicarbonate, percent by weight.

19. The method according to claim 3, further comprising sterilizing the tubular tissue scaffold with gamma or UV radiation.

20. The method according to claim 3, wherein the counter-rotating cone extruder comprises an external rotating member having a cone-shaped cavity therethrough and an internal rotating cone which fits within the cone-shaped cavity of the external rotating member, and wherein the external rotating member is driven to rotate in one direction at a speed of from about 1 to about 1800 rpm and the internal rotating cone is driven to rotate in the opposite direction at a speed of from about 1 to about 1800 rpm.

21. The method according to claim 20, wherein the external rotating member is driven to rotate in one direction at a speed of from about 150 to about 900 rpm and the internal rotating cone is driven to rotate in the opposite direction at a speed of from about 150 to about 900 rpm.