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Three-dimensional (3D) printing technology has seen several refinements when introduced in the field of medical devices and regenerative medicines. However, it is still a challenge to 3D print gels for building complex constructs as per the desired shape and size. Here, we present a novel method to 3D print gelatin/carboxymethylchitin/hydroxyapatite composite gel constructs of a complex shape. The objective of this study is to fabricate a bioactive gel scaffold with a controlled hierarchical structure. The hierarchy ranges from 3D outer shape to macroporosity to microporosity and rough surface. The fabrication process developed here uses 3D printing in a local cryogenic atmosphere, followed by lyophilization and crosslinking. The gel instantly freezes after extrusion on the cold plate. The cooling action is not limited to the build plate, but the entire gel scaffold is cooled during the 3D printing process. This enables the construction of a stable self-sustaining large-sized 3D complex geometry. Further, lyophilization introduces bulk microporosity into the scaffolds. The outer shape and macroporosity were controlled with the 3D printer, whereas the microporous structure and desirable rough surface morphology were obtained through lyophilization. With cryogenic 3D printing, up to 90% microporosity could be incorporated into the scaffolds. The microporosity and pore size distribution were controlled by changing the cross-linker and total polymer concentration, which resulted in six times increase in surface open pores of size <20 μm on increasing the cross-linker concentration from 25 to 100 mg/mL. The introduction of bulk microporosity was shown to increase swelling by 1.8 times along with a significant increase in human umbilical cord mesenchymal stem cells and Saos-2 cell attachment (2×), proliferation (2.4×), Saos-2 cell alkaline phosphatase level (2×), and mineralization (3×). The scaffolds are spongy in nature in a wet state, thus making them potential implants for bone cavities with a small opening. The application of these cryogenically 3D printed compressible gel scaffolds with multiscale porosity extends to a small-as well as a large-sized open/partially open patient-specific bone defect.

Regeneration of injured neuronal areas is a big challenge owing to the complex structure and function of the nervous system along with the limited regeneration capacity of neural cells. Recent reports show that patterned and functionalized scaffolds could control neural cell directional growth. In this study, aligned nanofibers (ANFs) were fabricated using a versatile and cost-effective approach, electrospinning, and further processed to make a patterned hybrid scaffold (HANF). The patterned scaffold had circular rings of ANFs reinforced in a biocompatible gellan–gelatin hydrogel matrix to provide adequate mechanical strength and contact guidance for adhesion and growth of neural cells in vitro . Quercetin was loaded into the nanofibrous scaffold to provide a functional agent that supported regeneration of neural cells. The reinforced ANFs enhanced the mechanical strength of the scaffold and provided a cylindrical nerve conduit structure to support neuronal cell growth. The influence of scaffold topology on cell behavior was assessed in in vitro cell culture conditions that revealed that the functionalized patterned scaffolds favored directed neurite cell growth/extension with favored cell culture morphology and showed no cytotoxicity toward neural cells. The results ultimately indicated that the fabricated scaffold has potential for guiding nerve tissue growth and can be used as nerve regeneration scaffolds.

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