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Abstract
Abstract Herein, we report the first study to create a three-dimensional (3D) bioprinted artificial larynx for whole-laryngeal replacement. Our 3D bio-printed larynx was generated using extrusion-based 3D bioprinter with rabbit's chondrocyte-laden gelatin methacryloyl (GelMA)/glycidyl-methacrylated hyaluronic acid (GMHA) hybrid bioink. We used a polycaprolactone (PCL) outer framework incorporated with pores to achieve the structural strength of printed constructs, as well as to provide a suitable microenvironment to support printed cells. Notably, we established a novel fluidics supply (FS) system that simultaneously supplies basal medium together with a 3D bioprinting process, thereby improving cell survival during the printing process. Our results showed that the FS system enhanced post-printing cell viability, which enabled the generation of a large-scale cell-laden artificial laryngeal framework. Additionally, the incorporation of the PCL outer framework with pores and inner hydrogel provides structural stability and sufficient nutrient/oxygen transport. An animal study confirmed that the transplanted 3D bio-larynx successfully maintained the airway. With further development, our new strategy holds great potential for fabricating human-scale larynxes with in vivo-like biological functions for laryngectomy patients.

Abstract
Cartilage regeneration is challenging because of the poor intrinsic self-repair capacity of avascular tissue. Three-dimensional (3D) bioprinting has gained significant attention in the field of tissue engineering and is a promising technology to overcome current difficulties in cartilage regeneration. Although bioink is an essential component of bioprinting technology, several challenges remain in satisfying different requirements for ideal bioink, including biocompatibility and printability based on specific biological requirements. Gelatin and hyaluronic acid (HA) have been shown to be ideal biomimetic hydrogel sources for cartilage regeneration. However, controlling their structure, mechanical properties, biocompatibility, and degradation rate for cartilage repair remains a challenge. Here, we show a photocurable bioink created by hybridization of gelatin methacryloyl (GelMA) and glycidyl-methacrylated HA (GMHA) for material extrusion 3D bioprinting in cartilage regeneration. GelMA and GMHA were mixed in various ratios, and the mixture of 7% GelMA and 5% GMHA bioink (G7H5) demonstrated the most reliable mechanical properties, rheological properties, and printability. This G7H5 bioink allowed us to build a highly complex larynx structure, including the hyoid bone, thyroid cartilage, cricoid cartilage, arytenoid cartilage, and cervical trachea. This bioink also provided an excellent microenvironment for chondrogenesis of tonsil-derived mesenchymal stem cells (TMSCs) in vitro and in vivo. In summary, this study presents the ideal formulation of GelMA/GMHA hybrid bioink to generate a well-suited photocurable bioink for cartilage regeneration of TMSCs using a material extrusion bioprinter, and could be applied to cartilage tissue engineering.

Abstract
In this present study, we introduce an innovative hybrid 3D bioprinting methodology that integrates fused deposition modeling (FDM) with top–down digital light processing (DLP) for the fabrication of an artificial trachea. Initially, polycaprolactone (PCL) was incorporated using an FDM 3D printer to provide essential mechanical support, replicating the structure of tracheal cartilage. Subsequently, a chondrocyte-laden glycidyl methacrylated silk fibroin hydrogel was introduced via top–down DLP into the PCL scaffold (PCL-Sil scaffold). The mechanical evaluation of PCL-Sil scaffolds showed that they have greater flexibility than PCL scaffolds, with a higher deformation rate (PCL-Sil scaffolds: 140.9% ± 5.37% vs. PCL scaffolds: 124.3% ± 6.25%) and ability to withstand more force before fracturing (3.860 ± 0.140 N for PCL-Sil scaffolds vs. 2.502 ± 0.126 N for PCL scaffolds, ***P < 0.001). Both types of scaffolds showed similar axial compressive strengths (PCL-Sil scaffolds: 4.276 ± 0.127 MPa vs. PCL scaffolds: 4.291 ± 0.135 MPa). Additionally, PCL-Sil scaffolds supported fibroblast proliferation, indicating good biocompatibility. In vivo testing of PCL-Sil scaffolds in a partial tracheal defect rabbit model demonstrated effective tissue regeneration. The scaffolds were pre-cultured in the omentum for two weeks to promote vascularization before transplantation. Eight weeks after transplantation into the animal, bronchoscopy and histological analysis confirmed that the omentum-cultured PCL-Sil scaffolds facilitated rapid tissue regeneration and maintained the luminal diameter at the anastomosis site without signs of stenosis or inflammation. Validation study to assess the feasibility of our hybrid 3D bioprinting technique showed that structures, not only the trachea but also the vertebral bone-disc and trachea-lung complex, were successfully printed.

Abstract
Abstract 3D-printed artificial skeletal muscle, which mimics the structural and functional characteristics of native skeletal muscle, is a promising treatment method for muscle reconstruction. Although various fabrication techniques for skeletal muscle using 3D bio-printers are studied, it is still challenging to build a functional muscle structure. A strategy using microvalve-assisted coaxial 3D bioprinting in consideration of functional skeletal muscle fabrication is reported. The unit (artificial muscle fascicle: AMF) of muscle mimetic tissue is composed of a core filled with medium-based C2C12 myoblast aggregates as a role of muscle fibers and a photo cross-linkable hydrogel-based shell as a role of connective tissue in muscles that enhances printability and cell adhesion and proliferation. Especially, a microvalve system is applied for the core part with even cell distribution and strong cell–cell interaction. This system enhances myotube formation and consequently shows spontaneous contraction. A multi-printed AMF (artificial muscle tissue: AMT) as a piece of muscle is implanted into the anterior tibia (TA) muscle defect site of immunocompromised rats. As a result, the TA-implanted AMT responds to electrical stimulation and represents histologically regenerated muscle tissue. This microvalve-assisted coaxial 3D bioprinting shows a significant step forward to mimicking native skeletal muscle tissue.

Abstract
AbstractThe purpose of this study was to evaluate whether the prior implantation of a 3D-printed polycaprolactone (PCL) artificial trachea in the omentum is beneficial for revascularization of the scaffold and reduces associated complications in the reconstruction of a circumferential tracheal defect. Ten New Zealand rabbits were divided into 2 groups: (1) PCL-OC group (PCL scaffold cultured in omentum for 2 weeks before transplantation) and (2) PCL group. In the PCL-OC group, newly formed connective tissue completely covered the luminal surface of the scaffold with mild inflammation at 2 weeks postoperatively; a minor degree of stenosis was noted at 8 weeks postoperatively. The PCL group showed scaffold exposure without any tissue regeneration at 2 weeks postoperatively, and a moderate degree of luminal stenosis 6 weeks after implantation. Histology revealed highly organized regenerated tissue composed of ciliated respiratory epithelium, and submucosal layer in the PCL-OC group. Neo-cartilage regeneration was noted in part of the regenerated tissue. The PCL group demonstrated severe inflammation and an unorganized structure compared to that of the PCL-OC group. In vivo omentum culture of the tracheal scaffold before transplantation is beneficial for rapid re-epithelialization and revascularization of the scaffold. It also prevents postoperative luminal stenosis.