Cell-Laden Nanocellulose/Chitosan-Based Bioinks for 3D Bioprinting and Enhanced Osteogenic Cell Differentiation

Abstract

3D bioprinting has recently emerged as a very useful tool in tissue engineering and regenerative medicine. However, developing suitable bioinks to fabricate specific tissue constructs remains a challenging task. Herein, we report on a nanocellulose/chitosan-based bioink, which is compatible with a 3D extrusion-based bioprinting technology, to design and engineer constructs for bone tissue engineering and regeneration applications. Bioinks were prepared using thermogelling chitosan, glycerophosphate, hydroxyethyl cellulose, and cellulose nanocrystals (CNCs). Formulations were optimized by varying the concentrations of glycerophosphate (80−300 mM), hydroxyethyl cellulose (0−0.5 mg/mL), and CNCs (0−2% w/v) to promote fast gelation kinetics (<7 s) at 37 °C and retain the shape integrity of constructs post 3D bioprinting. We investigated the effect of CNCs and pre-osteoblast cells (MC3T3-E1) on the rheological properties of bioinks, bioink printability, and mechanical properties of bioprinted scaffolds. We demonstrate that the addition of CNCs and cells (5 million cells/mL) significantly improved the viscosity of bioinks and the mechanical properties of chitosan scaffolds post-fabrication. The bioinks were biocompatible and printable at an optimized range of printing pressures (12−20 kPa) that did not compromise cell viability. The presence of CNCs promoted greater osteogenesis of MC3T3-E1 cells in chitosan scaffolds as shown by the upregulation of alkaline phosphatase activity, higher calcium mineralization, and extracellular matrix formation. The versatility of this CNCs-incorporated chitosan hydrogel makes it attractive as a bioink for 3D bioprinting to engineer scaffolds for bone tissue engineering and other therapeutic applications.

Genipin-crosslinked chitosan/alginate/alumina nanocomposite gels for 3D bioprinting

“…For the gel-cast samples, the bioink was cast into small petri dishes (⌀ 35 mm) at room temperature, which were partially covered with Parafilm to avoid significant drying. 3D printed samples were printed into lattice cubes (2 × 2 × 1 cm) using the printer Inkredible (Cellink, Gothenburg, Sweden) in a 6-well plate with a conical precision tip nozzle (⌀ 940 μm) with an extrusion air pressure of 20 ± 5 kPa, a printing speed of 10 mm/s, and a printing temperature of 30 °C—similar as other publications using chitosan-based bioink [ 40 , 50 ]. The numerical controlled programming language G-code with the printing commands was generated using the Cellink HeartWare 2.4.1 software, from Cellink, with a 67% infill density and 0.85 mm layer high.…”

“…Integrating nanoparticles into the chitosan gel to form a composite can significantly enhance mechanical resistance [ 36 ] and printability [ 37 ] and also add extra functionality to the gel, such as conductivity [ 37 ], fluorescence [ 38 ], or antibacterial properties [ 39 ]. For example, Maturavongsadit et al [ 40 ] developed a bioink based on a thermogelling chitosan, glycerophosphate, hydroxyethyl cellulose, and cellulose nanocrystals containing pre-osteoblast cells (MC3T3-E1) for bone tissue engineering. The addition of cellulose nanocrystals into the bioink resulted in a 20% increment on both viscosity and yield stress, as well as nanocrystals promoted a greater osteogenesis of the cells in chitosan scaffolds by higher calcium mineralization and extracellular matrix formation.…”

Chitosan as an Underrated Polymer in Modern Tissue Engineering

“…Chitosan bio-ink has been applied in bioprinting of artificial organs and structures in the human body such as cartilage tissue [277], bone tissue [278], neural connections [279], liver or heart valves [280,281], which shows the versatile use of the material in the bioprinting process [Figure 5]. Table 2 presents the current trends for chitosan application as a bioink composition.…”

Biofabrication Strategies for Musculoskeletal Disorders: Evolution towards Clinical Applications

“…However, natural hydrogels that can be fabricated on a kg-scale at a low price, narrows down the library of possible candidates. Commonly used candidates are thus centred on alginate, chitosan, hyaluronic acid (HA), fibrin, and collagen/gelatin [ 39 , 42 , 49 , 90 , 91 , 92 , 93 , 94 , 95 , 96 , 97 , 98 , 99 , 100 , 101 , 102 , 103 , 104 , 105 , 106 , 107 , 108 , 109 ]. Additional work has been performed with decellularised materials as it offers additional biological complexity and stimulation.…”

“…However, natural hydrogels that can be fabricated on a kg-scale at a low price, narrows down the library of possible candidates. Commonly used candidates are thus centred on alginate, chitosan, hyaluronic acid (HA), fibrin, and collagen/gelatin [39,42,49,[90][91][92][93][94][95][96][97][98][99][100][101][102][103][104][105][106][107][108][109].…”