One‐Step Bioprinting of Multi‐Channel Hydrogel Filaments Using Chaotic Advection: Fabrication of Pre‐Vascularized Muscle‐Like Tissues

Abstract

The biofabrication of living constructs containing hollow channels is critical for manufacturing thick tissues. However, current technologies are limited in their effectiveness in the fabrication of channels with diameters smaller than hundreds of micrometers. It is demonstrated that the co-extrusion of cell-laden hydrogels and sacrificial materials through printheads containing Kenics static mixing elements enables the continuous and one-step fabrication of thin hydrogel filaments (1 mm in diameter) containing dozens of hollow microchannels with widths as small as a single cell. Pre-vascularized skeletal muscle-like filaments are bioprinted by loading murine myoblasts (C2C12 cells) in gelatin methacryloyl -alginate hydrogels and using hydroxyethyl cellulose as a sacrificial material. Higher viability and metabolic activity are observed in filaments with hollow multi-channels than in solid constructs. The presence of hollow channels promotes the expression of Ki67 (a proliferation biomarker), mitigates the expression of hypoxia-inducible factor 1-alpha , and markedly enhances cell alignment (i.e., 82% of muscle myofibrils aligned (in ±10°) to the main direction of the microchannels after seven days of culture). The emergence of sarcomeric 𝜶-actin is verified through immunofluorescence and gene expression. Overall, this work presents an effective and practical tool for the fabrication of pre-vascularized engineered tissues.

Structural and biological engineering of 3D hydrogels for wound healing

“…However, more investigations should be conducted to increase the currently limited number of polymers used for in situ fabrication strategies and to make these strategies more holistic, cost-effective, robust, and versatile. Structuring the hydrogels by creating hollow channels [ 237 ] or cavities [ 238 ], for example, could also significantly facilitate the wound vascularization process. Vascularization remains an enormous challenge yet to be overcome in tissue engineering (in general) and wound healing (in particular).…”

Supplementation of GelMA with Minimally Processed Tissue Promotes the Formation of Densely Packed Skeletal-Muscle-Like Tissues

“…71,72 Myoblast alignment has previously been achieved using chemical 73 (e.g., growth factors) or physical cues, 74 or both. 62,75,76 A number of strategies, including shear-induced alignment using flows 16 or extrusion bioprinting, 77 topographical cues provided by patterned surfaces, 78 or controlled cell responses provided by fabricating compartments within a 3D construct, 11,12 have been proposed for the biofabrication of aligned engineered tissues. However, oriented domains spontaneously emerge when a high cell density is reached, causing cell confinement and the formation of oriented domains.…”

“…5,8 Significant advances have been reported for repairing or fabricating functional muscle tissues in vitro using several biofabrication technologies, such as three-dimensional (3D) bioprinting, 9−13 4D bioprinting, 14 and in situ bioprinting. 15 A wide spectrum of "smart" materials have been used in these applications, including anisotropic materials, 12,16,17 conductive biomaterials; 18−20 biomaterials that significantly enhance musculoskeletal-muscle injury repair, 21 materials that release cells into injured muscle, 19,20 and materials that promote the integration of the engineered tissue into the natural tissue. 22,23 One approach that has often been successful is the direct use of the extracellular matrix (ECM) derived from skeletal muscle as scaffolding material or as a hydrogel supplement to promote myoblast proliferation and maturation or to repair muscle injuries in vivo.…”

“…Significant advances have been reported for repairing or fabricating functional muscle tissues in vitro using several biofabrication technologies, such as three-dimensional (3D) bioprinting, − 4D bioprinting, and in situ bioprinting . A wide spectrum of “smart” materials have been used in these applications, including anisotropic materials, ,, conductive biomaterials; − biomaterials that significantly enhance musculoskeletal-muscle injury repair, materials that release cells into injured muscle, , and materials that promote the integration of the engineered tissue into the natural tissue. , …”

Hybrid biofabrication of neurosecretory structures as a model for neurosecretion

“…Irrespective of the type of bioprinting technology, the purpose is to deposit cells, biomaterials, and cytokines with high precision and to accurately control the spatial geometry, density, and controllable distribution of loaded cells to realize 3D construction of a 3D-bioprinted structure[ 12 , 13 ]. 3D bioprinting tissues has been reported, including 3D-bioprinted liver tissue[ 14 - 16 ], glomerulus[ 17 ], myocardial[ 18 ], brain[ 19 ], and skeletal muscle[ 20 ] and has been shown to maintain cell bioactivity, tissue morphological structure, and organoid function. The tissue-like structures produced by the organisms also showed varying degrees of biological functions.…”