One of the most promising techniques for treating severe peripheral artery disease is the use of cellular tissue-engineered vascular grafts (TEVGs). This study proposes an inverse-gravity (IG) extrusion technique for creating long double-layered cellular TEVGs with diameters over 3 mm. A three-layered coaxial laminar hydrogel flow in an 8 mm-diameter pipe was realised simply by changing the extrusion direction of the hydrogel from being aligned with the direction of gravity to against it. This technique produced an extruded mixture of human aortic smooth muscle cells (HASMCs) and type-I collagen as a tubular structure with an inner diameter of 3.5 mm. After a 21 day maturation period, the maximal burst pressure, longitudinal breaking force, and circumferential breaking force of the HASMC TEVG were 416 mmHg, 0.69 N, and 0.89 N, respectively. The HASMC TEVG was endothelialised with human umbilical vein endothelial cells to form a tunica intima that simulated human vessels. Besides subcutaneous implantability on mice, the double-layered blood vessels showed a considerably lower adherence of platelets and red blood cells once exposed to heparinised mouse blood and were considered nonhaemolytic. The proposed IG extrusion technique can be applied in various fields requiring multilayered materials with large diameters.

Background: Erythropoietin (EPO) plays important roles in neuroprotection in central nervous system injury. Due to the limited therapeutic time window and coexistence of hematopoietic/extrahematopoietic receptors displaying heterogenic and phylogenetic differences, fast, targeted delivery agents, such as nanobots, are needed. To confirm the feasibility of EPO-nanobots (ENBs) as therapeutic tools, the authors evaluated controlled EPO release from ENBs and compared the neuroprotective bioequivalence of these substances after preconditioning sonication. Methods: ENBs were manufactured by a nanospray drying technique with preconditioning sonication. SH-SY5Y neuronal cells were cotreated with thapsigargin and either EPO or ENBs before cell viability, EPO receptor activation, and endoplasmic reticulum stress-related pathway deactivation were determined over 24 h. Results: Preconditioning sonication (50–60 kHz) for 1 h increased the cumulative EPO release from the ENBs (84% versus 25% at 24 h). Between EPO and ENBs at 24 h, both neuronal cell viability (both > 65% versus 15% for thapsigargin alone) and the expression of the proapoptotic/apoptotic biomolecular markers JAK2, PDI, PERK, GRP78, ATF6, CHOP, TGF-β, and caspase-3 were nearly the same or similar. Conclusion: ENBs controlled EPO release in vitro after preconditioning sonication, leading to neuroprotection similar to that of EPO at 24 h.

Volumetric skeletal muscle injuries are prevalent, highlighting the imperative need for scaffolds to facilitate the healing process of such wounds. Human skeletal muscle is composed of multiple fascicles, which are parallel bundles of muscle fibres surrounded by a layer of connective tissue that contains blood vessels and nerves. Replicating these structures presents a considerable challenge. Here, we developed a method to fabricate multifascicle human skeletal muscle scaffolds that mimic the natural structure of human skeletal muscle bundles using a seven-barrel nozzle. To form the core material to generate the fascicle structure, human skeletal myoblasts were encapsulated in Matrigel with calcium chloride. Meanwhile, to create the shell that plays a role as the connective tissue structure, human fibroblasts and human umbilical vein endothelial cells within a mixture of porcine muscle decellularized extracellular matrix and sodium alginate at a 95:5 ratio was used. We assessed four types of extruded scaffolds monolithic-monoculture (Mo-M), monolithic-coculture (Mo-C), multifascicle-monoculture (Mu-M), and multifascicle-coculture (Mu-C) to determine the structural effect of muscle mimicking scaffold. The Mu-C scaffold demonstrated cell proliferation, differentiation, vascularization, mechanical properties, and functionality that were superior to those of the other scaffolds. Furthermore, in an in vivo mouse model of volumetric muscle loss, the Mu-C scaffold effectively regenerated the tibialis anterior muscle defect, demonstrating its potential for volumetric muscle transplantation. The multibarrel nozzle device was applied to create functional Mu-C muscle scaffolds that structurally mimicked human skeletal muscle. Our nozzle will be further used to produce other volumetric functional tissues, such as tendons and peripheral nerves.

Chronic liver disease remains a leading cause of global mortality, yet organ shortages and transplant complications limit the efficacy of orthotopic liver transplantation. While extracorporeal support systems serve as temporary bridges, they fail to restore long-term patient autonomy or replicate complex biosynthetic functions. This systematic review, conducted in accordance with PRISMA 2020 guidelines, evaluates recent advancements in implantable artificial livers (IALs) designed for permanent functional integration. We analyzed 71 eligible studies, assessing cellular sources, fabrication strategies, maturation processes, and functional readiness. Our findings indicate significant progress in stem-cell-derived hepatocytes and bioactive scaffolds, such as decellularized extracellular matrix (dECM). However, a critical technological gap remains in scaling current sub-centimeter prototypes toward clinically relevant volumes (~200 mL). Key engineering challenges include integrating hierarchical vascular networks, requiring primary vessels exceeding 2 mm in diameter for surgical anastomosis, and functional biliary systems to prevent cholestatic injury. Furthermore, while micro-vascularization and protein synthesis are well documented, higher-order functions such as spatial zonation and coordinated metabolic stability remain underreported. Future clinical translation necessitates advancements in multi-cellular patterning, microfluidic-driven maturation, and autologous reprogramming. This review provides a comprehensive roadmap for bridging the gap between biofabricated constructs and organ-scale hepatic replacement, emphasizing the need for standardized functional benchmarks to ensure long-term success.

Engineering a transplantable artificial liver requires reconstituting the hepatic lobule, whose densely cellular parenchyma is organized around a hierarchically branched vascular network. Achieving high cellular density and hierarchical vascularization within a single fabrication step remains a major challenge. Here, we present an ultrasonic standing-wave (USW)-based strategy that concurrently organizes vascular and parenchymal compartments during a single extrusion. HepG2–endothelial cell aggregates were fabricated as building blocks and co-extruded with single endothelial cells through a glass capillary under USW actuation in a liver-derived decellularized extracellular matrix–alginate hydrogel, enabling rapid, ultraviolet-free crosslinking. Due to their size difference, the single cells were patterned from one supplying line in the acoustic pressure node to four microvascular lines while the aggregates localized away from the acoustic pressure node, reconstituting the basic microvascular–parenchymal unit of the lobule. Aggregate size increased with seeding density while maintaining uniformity and over 90% viability, and the patterned endothelial cells formed perfusable lumina through which 5 µm microparticles flowed freely. Compared with hepatocyte-only cultures, the vascularized constructs showed a 1.7-fold increase in urea production, and 2.7- and 6.2-fold increases in CYP1A2 and CYP3A4 expression, respectively. Following implantation into the mouse liver, the scaffolds showed favorable biocompatibility, sustained proliferation over 21 days, and progressive host integration, with the smallest blood-containing lumina maturing from 26.1 µm to 7.9 µm in diameter and increasing collagen deposition at the graft–host interface. This approach provides a template-free route to hierarchically vascularized hepatic units and a promising step toward implantable liver tissue.