Three-dimensional bioprinting of biosynthetic cellulose (BC) implants and scaffolds for tissue engineering

• US 8,691,974 B2
• Filed: 09/28/2010
• Issued: 04/08/2014
• Est. Priority Date: 09/28/2009
• Status: Active Grant

• Current Assignee: Virginia Tech Intellectual Properties, Inc.
• Original Assignee: Virginia Tech Intellectual Properties, Inc.
• Inventors: Gatenholm, Paul, Backdahl, Henrik, Tzavaras, Theodore Jon, Davalos, Rafael V., Sano, Michael B.
• Primary Examiner(s): Olson, Eric S
• Assistant Examiner(s): Henry, Michael C
• Application Number:
  US13/498,657
• Publication Number:
  US 20120190078A1
• Time in Patent Office: 1,288 Days
• Field of Search: 536/56, 536/124, 514/57, 435/170, 435/179
• US Class Current: 536/56
• CPC Class Codes: A61L 2400/12 Nanosized materials, e.g. nanofibres, nanoparticles, nanowires, nanotubes; Nanostructured surfaces
• CPC Class Codes: A61L 27/20 Polysaccharides
• CPC Class Codes: A61L 27/38 containing added animal cells organs or tissue containing native cells A61L27/36
• CPC Class Codes: A61L 27/56 Porous materials, e.g. foams or sponges
• CPC Class Codes: C08B 1/00 Preparatory treatment of cellulose for making derivatives thereof , e.g. pre-treatment, pre-soaking, activation
• CPC Class Codes: C08L 1/02 Cellulose; Modified cellulose
• CPC Class Codes: C12N 11/12 Cellulose or derivatives thereof
• CPC Class Codes: C12N 2533/78 Cellulose
• CPC Class Codes: C12N 5/0062 General methods for three-dimensional culture
• CPC Class Codes: C12P 19/04 Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds

Abstract

A novel BC fermentation technique for controlling 3D shape, thickness and architecture of the entangled cellulose nano-fibril network is presented. The resultant nano-cellulose based structures are useful as biomedical implants and devices, are useful for tissue engineering and regenerative medicine, and for health care products. More particularly, embodiments of the present invention relate to systems and methods for the production and control of 3-D architecture and morphology of nano-cellulose biomaterials produced by bacteria using any biofabrication process, including the novel 3-D Bioprinting processes disclosed. Representative processes according to the invention involve control of the rate of production of biomaterial by bacteria achieved by meticulous control of the addition of fermentation media using a microfluidic system. In exemplary embodiments, the bacteria gradually grew up along the printed alginate structure that had been placed into the culture, incorporating it. After culture, the printed alginate structure was successfully removed revealing porosity where the alginate had been placed. Porosity and interconnectivity of pores in the resultant 3-D architecture can be achieved by porogen introduction using, e.g., ink-jet printer technology.

Claims

1. A method of producing 3-D nano-cellulose based structures comprising:

providing bacteria capable of producing nano-cellulose;

providing media capable of sustaining the bacteria for the production of nano-cellulose;

controlling microbial production rate by administering media with a microfluidic device, for a sufficient amount of time, and under conditions sufficient for the bacteria to produce nano-cellulose at a desired rate;

continuing the administering of the media until a target three-dimensional structure with a target thickness and target strength is formed which has a morphology defined by a network of multiple layers of interconnected biosynthetic cellulose.

2. The method of claim 1, wherein the administering of the media involves controlling flow rate and volume of the media using a microfluidic device.

3. The method of claim 1, further comprising providing a microporous mold as a template for forming the target 3-D nano-cellulose based structure.

4. The method of claim 1 comprising introducing porosity to the target 3-D structure with porogens added in a computer-controlled manner.

5. The method of claim 3, wherein providing the porous mold comprises submerging the mold in a bioreactor for growth of the target 3-D structure.

6. The method of claim 4, wherein the porogens are alginate or wax particles capable of being removed following biofabrication of the 3-D structure.

7. The method of claim 1, further comprising seeding the three-dimensional nano-cellulose based structure with cells.

8. The method of claim 1, wherein the resultant 3-D nano-cellulose-based structure of claim has a tensile strength of about 1 MPa or higher or a modulus of about 2.5 MPa or higher.