3D bioprinting of a hyaluronan bioink through enzymatic-and visible light-crosslinking

Abstract

Extrusion-based three-dimensional bioprinting relies on bioinks engineered to combine viscoelastic properties for extrusion and shape retention, and biological properties for cytocompatibility and tissue regeneration. To satisfy these conflicting requirements, bioinks often utilize either complex mixtures or complex modifications of biopolymers. In this paper we introduce and characterize a bioink exploiting a dual crosslinking mechanism, where an enzymatic reaction forms a soft gel suitable for cell encapsulation and extrusion, while a visible light photo-crosslinking allows shape retention of the printed construct. The influence of cell density and cell type on the rheological and printability properties was assessed correlating the printing outcomes with the damping factor, a rheological characteristic independent of the printing system. Stem cells, chondrocytes and fibroblasts were encapsulated, and their viability was assessed up to 14 days with live/dead, alamar blue and trypan blue assays. Additionally, the impact of the printing parameters on cell viability was investigated. Owing to its straightforward preparation, low modification, presence of two independent crosslinking mechanisms for tuning shear-thinning independently of the final shape fixation, the use of visible green instead of UV light, the possibility of encapsulating and sustaining the viability of different cell types, the hyaluronan bioink here presented is a valid biofabrication tool for producing 3D printed tissue-engineered constructs.

Extrusion 3D Printing of Polymeric Materials with Advanced Properties

“…Other types of hydrogels formed through noncovalent crosslinking, such as by ionic coordination, [ 89 ] electrostatic interactions, [ 90–93 ] host–guest interactions, [ 86,94–99 ] enzymatic crosslinking, [ 95,100 ] self‐assembly, [ 101 ] and hydrogen bonding [ 102 ] also exhibit similar shear‐thinning behavior, allowing easy extrusion of the ink from the nozzle. However, due to the highly dynamic nature of supramolecular interactions, the 3D printed objects may lack structural integrity and can fuse together or disperse in aqueous conditions, compromising the resolution and 3D printability.…”

“…The use of dynamic bonds which undergo reversible breaking, exchange and reformation could be utilized to modify the printability of polymers as well as imparting various advanced functions. [ 41–58,62–67,59,68,60,69,70,72,61,73,71,82,86–88,74–81,83–85,89–133,244,258 , …”

Tissue mimetic hyaluronan bioink containing collagen fibers with controlled orientation modulating cell morphology and alignment

“…In the present work, we propose a technique to introduce anisotropic properties into a THA-Col composite biomaterial. Having two independent cross linking mechanisms, we overcame the difficulty in mixing Col fibrils into a viscoelastic HA-based matrix by combining a previously developed bioink based on the tyramine derivative of HA [29] with acidicsolubilized Col, which was buffered upon mixing, thereby forming fibrils. Since both precursors THA bioink and Col are in liquid form, mixing to homogeneity was easily achieved, as visualized with microscopic techniques.…”

Progressive 3D Printing Technology and Its Application in Medical Materials

“…Multiple bioprinting applications in vascular models, soft-tissue models, and bone models manufactured with extrusion-based printing technology have been well-developed in recent years (Ahlfeld et al, 2017;Paxton et al, 2017;Ahlfeld et al, 2018). One major advantage of its bioprinting application is that the hydrogels of extrusion-based printing is capable to fabricate products with high cell density (> 1 × 10 6 cells ml −1 ) (Petta et al, 2018;Taylor et al, 2018;Chen et al, 2019).…”