Bioprinting of Cartilaginous Auricular Constructs Utilizing an Enzymatically Crosslinkable Bioink

Abstract

Bioprinting of functional tissues could overcome tissue shortages and allow a more rapid response for treatments. However, despite recent progress in bioprinting, and its outstanding ability to position cells and biomaterials in a precise 3D manner, its success has been limited, due to insufficient maturation of constructs into functional tissue. Here, a novel calcium‐triggered enzymatic crosslinking (CTEC) mechanism for bioinks based on the activation cascade of Factor XIII is presented and utilized for the biofabrication of cartilaginous constructs. Hyaluronan transglutaminase (HA‐TG), an enzymatically crosslinkable material, has shown excellent characteristics for chondrogenesis and builds the basis of the CTEC bioink. The bioink supports tissue maturation with neocartilage formation and stiffening of constructs up to 400 kPa. Bioprinted constructs remain stable in vivo for 24 weeks and bioprinted auricular constructs transform into cartilaginous grafts. A major limitation of the current study is the deposition of collagen I, indicating the maturation toward fibrocartilage rather than elastic cartilage. Shifting the maturation process toward elastic cartilage will therefore be essential in order for the developed bioinks to offer a novel tissue engineered treatment for microtia patients. CTEC bioprinting furthermore opens up use of enzymatically crosslinkable biopolymers and their modularity to support a multitude of tissues.

3D‐Printed Reinforcement Scaffolds with Targeted Biodegradation Properties for the Tissue Engineering of Articular Cartilage

“…Previous studies, [27][28][29][30][31] reported a significant maturation of the HA-TG hydrogels in the first three weeks of culture due to the deposition of ECM from the cells leading to an increase in overall stiffness of the samples. Nevertheless, the very low stiffness of the HA-TG hydrogel reported at day one implies that implantation at this early stage could be at risk of failure.…”

“…Semiquantitative Evaluation: Semiquantitative evaluation of the histological and immunohistological staining was performed as previously described. [27] Briefly, images were decomposed in channels in ImageJ (National Institutes of Health, USA, v1.52n) using color deconvolution. The channels represented the hematoxylin and DAB stain for Collagen I and Collagen II, while the hematoxylin and stain of glycosaminoglycans for the SafraninO staining.…”

“…[52,54] For this reason, a viability test was performed to evaluate the viability of cells in the presence of the reinforcement material once embedded in a HA-TG hydrogel. Since previous studies have shown that HA-TG is an excellent hydrogel for cartilage regeneration, [27][28][29][30][31] any reduction in viability would be linked to the presence of the reinforcement material alone. A viability analysis was performed to evaluate the biocompatibility of the reinforcement structure when combined with the HA-TG hydrogel over 21 d (Figure S4B,C, Supporting Information).…”

“…In particular, hyaluronan and hyaluronan based hydrogels have shown to be optimal for tissue engineering since hyaluronan is one of the main components of the extracellular matrix, [25] in particular cartilage, where together with proteoglycans it forms aggregates, responsible for the extraordinary mechanical properties of cartilage and has shown to be a crucial component for cell proliferation and migration. [26] Recent works [27][28][29][30][31] have shown the potential of enzymatically crosslinking HA for tissue engineering, where excellent biocompatibility, cell proliferation and tissue maturation can be observed. Furthermore, the enzymatic crosslinking process involves FXIII, a multifunctional protein involved in a variety of mechanisms for regulation and repair processes, such ECM stabilization and wound healing.…”

Computer vision-aided bioprinting for bone research

“…The restricted application of bioprinting for bone implantation is related to its low resolution in the spatial material structure. An accurate scaffold width is momentous for in vivo investigations, such as in enzymatically cross-linkable materials, 54 neuron regeneration and connection, 55 and cell delivery. 56 This example suggests why precise control of the bone scaffold width plays a vital role in macroscopic organ function as well as in microscopic cell differentiation and growth.…”