Cellulose nanofibrillar bioink for 3d bioprinting for cell culturing, tissue engineering and regenerative medicine applications

Abstract

The present invention relates to biomaterial in the form of dispersion of cellulose nanofibrils with extraordinary shear thinning properties which can be converted into desire 3D shape using 3D Bioprinting technology. In this invention cellulose nanofibril dispersion, is processed through different mechanical, enzymatic and chemical steps to yield dispersion with desired morphological and rheological properties to be used as bioink in 3D Bioprinter. The processes are followed by purification, adjusting of osmolarity of the material and sterilization to yield biomaterial which has cytocompatibility and can be combined with living cells. Cellulose nanofibrils can be produced by microbial process but can also be isolated from plant secondary or primary cell wall, animals such as tunicates, algae and fungi. The present invention describes applications of this novel cellulose nanofibrillar bioink for 3D Bioprinting of tissue and organs with desired architecture.

1 . A cellulose nanofibril bioink comprising:
 a dispersion of cellulose nanofibrils in a liquid media, wherein the cellulose nanofibrils have a length of about 1-100 microns and a width of about 10 nanometers to 20 microns;   a viscosity of between 1 and 50 Pa*s at 100 s −1  at room temperature; and   a solids content of up to about 5% by weight of the dispersion.   
     
     
         2 . The bioink of  claim 1 , wherein the cellulose nanofibrils have an average length of about 1-20 microns and an average width of about 10-30 nanometers. 
     
     
         3 . The bioink of  claim 1 , further comprising one or more biopolymers chosen from collagen or elastin. 
     
     
         4 . The bioink of  claim 3 , wherein one or more of the biopolymers is collagen. 
     
     
         5 . The bioink of  claim 1 , wherein the solids content is up to about 3% by weight of the dispersion. 
     
     
         6 . The bioink of  claim 5 , wherein the solids content is up to about 1% by weight of the dispersion. 
     
     
         7 . The bioink of  claim 1 , further comprising cells. 
     
     
         8 . The bioink of  claim 7 , wherein the cells are adapted to be human cells. 
     
     
         9 . The bioink of  claim 7 , wherein the cells are chosen from bovine fibroblasts, human chondrocytes, and/or induced pluripotent stem cells. 
     
     
         10 . The bioink of  claim 1 , further comprising a crosslinking agent, alginate, or both. 
     
     
         11 . The bioink of  claim 10 , wherein the ratio of cellulose nanofibrils to alginate is about 80:20. 
     
     
         12 . The bioink of  claim 1 , wherein the viscosity is about 10 Pa·s at 100 s-1. 
     
     
         13 . The bioink of  claim 1 , wherein the cellulose nanofibrils are chosen from animal, algae, plant, tree, fungus, wood, tunicate, and/or bacteria type cellulose nanofibrils. 
     
     
         14 . The bioink of  claim 1 , wherein the cellulose nanofibrils are chosen from one or more of  Acetobacter -,  Acromobacter -,  Agrobacterium -,  Alacaligenes -,  Azotobacter -,  Pseudomonas -,  Rhizobium -,  Sarcina -,  Gluconacetobacter xylinus -,  Acetobacter xylinum -,  Lactobacillus mali -,  Agrobacterium tumefaciens -,  Rhizobium leguminosarum  bv.  Trifolii -,  Sarcina ventriculi -,  enterobacteriaceae Salmonella  spp.-,  Escherichia coli -,  Klebsiella pneumoniae -,  cyanobacteria -,  Tracheophyta -, and/or  Tracheobionta -type cellulose nanofibrils. 
     
     
         15 . A method comprising:
 providing a cellulose nanofibril bioink comprising:
 a dispersion of cellulose nanofibrils in a liquid media, wherein the cellulose nanofibrils have a length of about 1-100 microns and a width of about 10 nanometers to 20 microns; 
 a viscosity of between 1 and 50 Pa*s at 100 s −1  at room temperature; and 
 a solids content of up to about 5% by weight of the dispersion; and 
   bioprinting a 3D construct using the cellulose nanofibril bioink.   
     
     
         16 . The method of  claim 15 , wherein the solids content of the cellulose nanofibril bioink is up to about 3% by weight of the dispersion. 
     
     
         17 . The method of  claim 15 , wherein the solids content of the cellulose nanofibril bioink is up to about 1% by weight of the dispersion. 
     
     
         18 . The method of  claim 15 , further comprising 3D bioprinting with the cellulose nanofibril bioink in a manner to prepare one or more of scaffolds, tissues, and/or organs. 
     
     
         19 . The method of  claim 18 , further comprising implanting one or more of the scaffolds, tissues, and/or organs into a human or animal. 
     
     
         20 . The method of  claim 15 , wherein the cellulose nanofibrils are chosen from one or more of  Acetobacter -,  Acromobacter -,  Agrobacterium -,  Alacaligenes -,  Azotobacter -,  Pseudomonas -,  Rhizobium -,  Sarcina -,  Gluconacetobacter xylinus -,  Acetobacter xylinum -,  Lactobacillus mali -,  Agrobacterium tumefaciens -,  Rhizobium leguminosarum  bv.  Trifolii -,  Sarcina ventriculi -,  enterobacteriaceae Salmonella  spp.-,  Escherichia coli -,  Klebsiella pneumoniae -,  cyanobacteria -,  Tracheophyta -, and/or  Tracheobionta -type cellulose nanofibrils. 
     
     
         21 . The bioink of  claim 7 , wherein the cells are adapted to be living cells. 
     
     
         22 . The bioink of  claim 1 , wherein the cellulose nanofibrils have an aspect ratio of 200 or less. 
     
     
         23 . The bioink of  claim 22 , wherein the cellulose nanofibrils have an average length of about 1-10 microns and an average width of about 10-20 nanometers. 
     
     
         24 . The bioink of  claim 23 , wherein the cellulose nanofibrils are wood derived cellulose nanofibrils. 
     
     
         25 . The bioink of  claim 23 , wherein the cellulose nanofibrils are bacterial cellulose nanofibrils. 
     
     
         26 . The bioink of  claim 1 , further comprising hyaluronic acid or hyaluronic acid and alginate. 
     
     
         27 . The method of  claim 15 , wherein the 3D construct is a skin-like construct.