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

Abstract

Immobilizing microorganisms inside 3D printed semi-permeable substrates can be desirable for biotechnological processes since it simplifies product separation and purification, reducing costs, and processing time. To this end, we developed a strategy for synthesizing a feedstock suitable for 3D bioprinting of mechanically rigid and insoluble materials with embedded living bacteria. The processing route is based on a highly particle-filled alumina/chitosan nanocomposite gel which is reinforced by (a) electrostatic interactions with alginate and (b) covalent binding between the chitosan molecules with the mild gelation agent genipin. To analyze network formation and material properties, we characterized the rheological properties and printability of the feedstock gel. Stability measurements showed that the genipin-crosslinked chitosan/alginate/alumina gels did not dissolve in PBS, NaOH, or HCl after 60 days of incubation. Alginate-containing gels also showed less swelling in water than gels without alginate. Furthermore, E. coli bacteria were embedded in the nanocomposites and we analyzed the influence of the individual bioink components as well as of the printing process on bacterial viability. Here, the addition of alginate was necessary to maintain the effective viability of the embedded bacteria, while samples without alginate showed no bacterial viability. The experimental results demonstrate the potential of this approach for producing macroscopic bioactive materials with complex 3D geometries as a platform for novel applications in bioprocessing.

Biomimetic Nanomaterials: Diversity, Technology, and Biomedical Applications

“…Finally, by analyzing the most recent literary sources, it was possible to list the main challenges that stand in the way of the development of BNM technology: (i) there remains much room for the improvement of BNMs in terms of their stability and biocompatibility since BNMs have been used only in animal studies, but they have not been widely used in clinical practice [ 22 ]; (ii) understanding the energetic contributions that rule interactions at the nano–bio interface (i.e., where NPs meet biological barriers, specifically cell membranes) is very complex due to the high compositional heterogeneity of biomembranes and the intrinsic variability of the biological environment [ 34 ]; (iii) the clinical application of BNMs encounters complex fabrication processes, unsuitable large-scale production, low yields, and difficult preservation [ 24 ]; (iv) the utilization of biosynthesis (e.g., engineered bacteria) requires scaled-up manufacturing, dose determination, and potential biosafety studies [ 23 ]; (v) the processing of biomaterials (e.g., bioprinting) that incorporate living cells is still very challenging, especially considering that the production of mechanically rigid and insoluble substrates usually requires nonbiocompatible processes, such as chemical cross-linking or sintering [ 65 ].…”

“… Classification of biomimetic nanomaterials (BNMs) based on the literature data, including information on biomimetic structure [ 16 , 34 , 35 , 36 , 37 , 38 ], biomimetic synthesis [ 32 , 39 , 40 , 41 , 42 , 43 ], biogenic components [ 31 , 33 , 44 , 45 , 46 , 47 ], magnetic BNMs [ 48 , 49 , 50 , 51 , 52 , 53 , 54 ], metal and metal oxide BNMs [ 55 , 56 , 57 , 58 , 59 , 60 , 61 ], organic, ceramic and hybrid BNMs [ 62 , 63 , 64 , 65 , 66 , 67 ]. …”

“…Due to the wide variety of proposed structures and compositions of BNMs, the use of magnetic, metal, and metal oxide materials, as well as organic, ceramic, and hybrid (multicomponent) structural elements, are considered separately. The proposed classification with some examples from the literature, including information on biomimetic structure [ 16 , 34 , 35 , 36 , 37 , 38 ], biomimetic synthesis [ 32 , 39 , 40 , 41 , 42 , 43 ], biogenic components [ 31 , 33 , 44 , 45 , 46 , 47 ], magnetic BNMs [ 48 , 49 , 50 , 51 , 52 , 53 , 54 ], metal and metal oxide BNMs [ 55 , 56 , 57 , 58 , 59 , 60 , 61 ], organic, ceramic and hybrid BNMs [ 62 , 63 , 64 , 65 , 66 , 67 ], is shown in Figure 2 .…”

Graphene Oxide–Carbamoylated Chitosan Hydrogels with Tunable Mechanical Properties for Biological Applications

“…7 Mainardi et al synthesized gels using CHalginate polyelectrolyte complex with alumina nanoparticles, followed by covalent cross-linking using genipin. 8 Marapureddy et al have reported the preparation of CH-based hydrogels by carbamoylation using potassium cyanate (KCNO), which replaces the free amine in CH with a carbamyl group and induces hydrogen bonding to form a 3D network. 9 While CH has proved to be an excellent candidate in biological applications, pure CH-based materials are limited to being used as standalone scaffolds because of their low mechanical strength and stability.…”

“…CH can be functionalized to synthesize cross-linked networks using quarterization, N-acylation, oxidation, or cross-linking reactions . Mainardi et al synthesized gels using CH-alginate polyelectrolyte complex with alumina nanoparticles, followed by covalent cross-linking using genipin . Marapureddy et al have reported the preparation of CH-based hydrogels by carbamoylation using potassium cyanate (KCNO), which replaces the free amine in CH with a carbamyl group and induces hydrogen bonding to form a 3D network …”