Multiscale Porosity in Compressible Cryogenically 3D Printed Gels for Bone Tissue Engineering

Abstract

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.

Microchannelled chitosan sponge grafting with hydrophobic alkyl chain for enhanced noncompressible hemorrhage and tissue regeneration

“…Interconnected pores of the hemostatic sponge could endow itself with the ability to concentrate blood clotting factors and rapidly recover initial shape 5,10,29 . Moreover, they were able to provide a comfortable niche to support host cell in ltration, vascularization, and tissue ingrowth 30 . Micro-CT images showed that the alkylated CS sponges with different porosity (MACS-1/2/3) fabricated by a combination of the template leaching method and freeze-drying possessed a uniform microchannel structure with an increased microchannel density (Fig.…”

Recent progress in extrusion 3D bioprinting of hydrogel biomaterials for tissue regeneration: a comprehensive review with focus on advanced fabrication techniques

“…The challenge of using hydrogels for the fabrication of the musculoskeletal system via 3D bioprinting should be seriously considered since a stiff and coherent hydrogel-based construct would be required for implantation in the human body [133]. Accordingly, different strategies have been developed to enhance the strength of hydrogel-based bioprinted constructs, including utilizing toughened hydrogels and reinforcement of printed hydrogels with thermoplastic polymers [134][135][136][137][138][139][140][141] or bioceramics [142][143][144][145], nanofibers, nanoparticle [146][147][148][149], microparticles, and microcarriers [150,151]. Moreover, the crosslinking of bioprinted constructs by UV-rays and chemical agents does not only improve their mechanical properties, but it could also increase the stiffness, longevity, and thermal stability of 3D printed constructs [128,152,153].…”

“…While the outer shape and macroporosity were controlled by the 3D printer, desirable rough surface morphology and the microporous structure were obtained through lyophilization. Their result showed that the incorporation of bulk and surface porosity could be lead to increasing the water uptake ratio, cell retention capability, cell infiltration, attachment, proliferation, (Alkaline phosphatase (ALP) level, and mineralization [147]. However, microvasculature as a major challenge in engineering large bone graft substitutes [154] is receiving considerable attention regarding that bone comprises of an extensive vascular system in the medullary cavity that infiltrates into the bone containing osteocytes within 100 μm distance.…”

Combined Porogen Leaching and Emulsion Templating to produce Bone Tissue Engineering Scaffolds

“…Inclusion of this multiscale porosity when producing bone tissue engineering scaffolds is often overlooked with pore sizes typically confined to a single order of magnitude. However, it has been demonstrated that a multiscale porosity enhances in vitro and in vivo performance of scaffolds[ 2 - 4 ]. The reason for this is that different size pores promote different functions.…”