Biofabrication最新文献

Biliary obstruction prevents normal flow of bile into the duodenum, resulting in the accumulation of hydrophobic bile acids. This causes oxidative stress and subsequent hepatocellular injury. Although biliary stents are commonly used to restore bile flow, their long-term patency is often limited by the risk of restenosis. Ursodeoxycholic acid (UDCA) is widely used to treat cholelithiasis; however, its incorporation into stent-based delivery systems has not been extensively studied. To address this issue, we designed a UDCA-based multilayer drug-eluting membrane for biliary stents to restore bile flow. It comprises an inner UDCA-loaded polycaprolactone (PCL) layer, a central silicone barrier film, and an outer aspirin-loaded PCL layer. The central silicone film provides structural integrity and enables directional drug release, allowing the inward diffusion of UDCA and the outward release of aspirin. UDCA in the inner layer prevents bio-sludge formation within the bile ducts, whereas aspirin in the outer layer helps reduce local inflammation at the stent insertion site. The physicochemical and mechanical properties of the membranes were characterized, and their biocompatibility was evaluated. Aspirin release kinetics were measured using UV spectrophotometry. UDCA release was indirectly assessed based on its regenerative effects in HepG2 cells under oxidative stress, including NRF2 and HO-1 gene expression analysis, using quantitative RT-PCR. The multilayered drug-eluting membrane demonstrated enhanced mechanical strength and enabled sustained directional drug release with a reduced initial burst. These findings suggest that the multilayered drug-eluting membrane holds potential as a platform for localized and controlled drug delivery while addressing the key mechanical and therapeutic limitations of current stents.

Vascularization remains a major challenge in tissue engineering, restricting both the functional integration of grafts as well as the physiological relevance ofin vitromodels. Inspired by the crucial role of Schwann cells (SCs) in guiding vascular development, we investigated their influence on the vascular network formation of human microvascular endothelial cells (HMVECs). Using melt electrowriting (MEW), we fabricated scaffolds consisting of a single layer of parallel fibers to mimic the oriented axons of the peripheral nerve. A suspended seeding approach was carried out to ensure rat-derived SCs adhered specifically to the fibers, creating parallel arrays with SCs exhibiting strong attachment, viability, and upregulation of myelination-, neurotrophic-, and pro-angiogenic-related genes. A customized system was built to co-culture SCs-laden scaffolds with HMVECs seeded on a hydrogel (2D) andina hydrogel (3D). The parallel fiber distances were varied to assess the spacing of the SC arrays that impacted HMVEC organization. The results revealed that SCs on MEW scaffolds exhibited enhanced expression of key genes compared to a 2D monolayer control. Further, these SC-laden scaffolds significantly enhanced HMVEC network formation in both 2D and 3D environments, with reduced fiber distance showing stronger pro-angiogenic responses. To evaluate species differences, human dental pulp stem cell-derived SCs (HDPSC-SCs) were compared with rat primary SCs. HDPSC-SCs not only showed enhanced expression of pro-angiogenic genes on the scaffold but also promoted superior network formation compared to rat SCs. Collectively, our findings highlight the ability of MEW scaffolds to both pattern SC growth and stimulate a pro-regenerative SC phenotype as a strategy to modulate vascular network formation. This provides a foundation for using the patterning of SCs to drive neurovascular organization forin vitromodels and more broadly as an approach of regenerative medicine.

Despite the rapid pace of biomedical engineering research, translating developed products into clinical practice remains challenging due to regulations, manufacturing, and long-termin vivosafety. The eye offers advantageous features to lower translational hurdles, making it an ideal clinical target and an approachable testbed for biofabricated implants. However, eyes also have anatomical and physiological barriers that hinder conventional ophthalmic delivery routes, leading to poor drug bioavailability. Advances in biofabrication and biomaterials used in ophthalmic therapeutic implants have the potential to address the current challenges. This review will explore biomaterials, biofabrication methods, and possible ocular implantation sites from the perspective of developing effective therapeutic implants. It also examines clinically available products and current clinical trials, along with recent advancements and next-generation technologies in ophthalmic therapeutic delivery implants. This review aims to provide insights that facilitate the translation of emerging ocular therapeutics into clinically available treatments.

With the establishment of key principles governing osteochondral structure, function, and reconstruction, researchers have gained an expanded toolkit for the precisein-vitroreconstruction of osteochondral tissues. As a convergence of tissue engineering and microphysiological modeling, the biomechanical heterogeneity of the osteochondral layers, which is critical to joint function, can be precisely engineered within osteochondral unit-on-a-chip (OC-OoCs), making them ideal tools for studying physiological activities. Specifically speaking, OC-OoCs are regarded as a promising platform for investigating the complex physiology of the osteochondral unit and its pathophysiology in disorders such as osteoarthritis (OA) and osteochondritis dissecans (OCDs). In OA, multiple forms of endochondral ossification, including chondrocalcinosis and osteophyte formation, disrupt the normal tissue relationship of cartilage, subchondral bone plate, and subchondral trabecular bone. Additionally, cellular and molecular communication networks between cartilage and subchondral bone are altered due to increased vascularization, porosity, microcracks, and fissures. Recapitulating these key physiological factors is therefore a critical objective in OC-OoC design. However, incorporation of increasing numbers of physiological parameters inevitably elevates system complexity, posing challenges to chip-to-chip reproducibility and batch-to-batch consistency. Robust quality control (QC) and standardization are thus essential to enhance the reliability and translational value of OC-OoC-derived data. This review summarizes the current advancements in OC-OoCs technology for osteochondral research and, from both diseases oriented as well as translational and clinical perspectives, highlights OC-OoCs' potential to advance our understanding of OA and facilitate the development of novel therapeutic strategies.

To replicate key physiological barriers in vitro, we utilized the CELLBLOKS® modular microphysiological system. Specifically, human cerebral microvascular endothelial hCMEC/D3 cells, human retinal pigment epithelial (HRPE) cells, and rat small intestinal IEC-6 cells were grown in CELLBLOKS® to mimic the blood-brain (BBB), blood-cerebrospinal fluid (BCSFB), and intestinal (IB) barriers, respectively. Eugenol is an essential oil component known to permeate the central nervous system (CNS) in vivo after intravenous and oral administrations; it was therefore used for simulated intravenous and oral administrations into the CELLBLOKS® system, using also celiprolol as negative control compound, since it is known for its poor ability to permeate in the CNS from the bloodstream. In particular, the intravenous administration (systemic) of the compounds was simulated by their direct addition to the bloodstream-like lower channel of CELLBLOKS® (basolateral side of both CSFB and IB; apical side for BBB), whereas their oral administration was simulated by apical addition to IEC-6. Permeation measurements, via HPLC, across physiological barriers cultured in CELLBLOKS® demonstrated that, following both simulated oral and systemic administration, eugenol crosses the mimicked BBB and the BCSFB indiscriminately; conversely, the permeation of celiprolol across these barriers results strongly limited in comparison to eugenol. To assess downstream neuroactivity, dopaminergic neuron-like PC12 cells were cultured on NANOSTACKS™ inserts and incorporated into the BBB and BCSFB blocks. After simulated intravenous and oral administrations, significant eugenol-induced dopamine release by PC12 cells was evidenced both in BBB- and BCSFB-delimited neuronal-like compartments. These results validate the CELLBLOKS® and NANOSTACKS™ platforms as robust tools characterized by low costs, high reproducibility and ease of manipulation for in vitro studies of brain targeting of new drugs. This system requires two weeks culture period to be ready for the simulation in vitro of IB, BBB, BCFSB and neuronal tissues, appearing useful in limiting pre-clinical animal testing.

Cell spheroids have been exploited as fundamental engineering units applied as screening platforms or assembled as building blocks for tissue engineering applications. While spheroid encapsulation into hydrogels creates more reliable 3D models, it also brings several constraints, e.g., hydrogel swelling and dynamicity, shading, limitations on depth-resolution, and cell staining strategies for monitoring long real-time imaging. Hence, the objective of this work was to develop a post-imaging automated pipeline for the accurate tracking and measuring of spheroids encapsulated in 3D hydrogels. Using NIS Elements ARv5.30 (Nikon) software, we created a sequence of functions for enhancing spheroid borders, extending the depth of focus, reducing hydrogel shading, and identifying coordinates in an automated manner for time-lapse microscopy analysis up to 70 h. Additionally, we established a method for identifying and tracking migration trajectories of protruded cell clusters that detached from spheroids into the hydrogel. For a comparative hydrogel analysis, fluorescent beads were encapsulated in the ionically crosslinked xanthan gum-alginate (XG-Alg) and photocrosslinked methacrylate hyaluronic acid (HAMA). For pipeline validation, human mesenchymal stem cell spheroids were encapsulated in XG-Alg hydrogel. By employing our pipeline, a high dynamicity and intense swelling effect were detected within XG-Alg, while HAMA remained stable, without noticeable movements up to 60 h. Accurate imaging and tracking detected several spheroid morphological changes, including reversible spheroid-ellipsoid shapes, axis rotational motion, outermost layer movements, spheroid fusion, and a spheroid migration speed of approximately 1.3µm h^-1. Protruded cell clusters were detected in high numbers (83-173 per spheroid), migrating arbitrarily into the hydrogel (16°-311°), with an average speed of approximately 11.4µm h^-1. Our results indicate that this automated pipeline can facilitate the understanding of several cellular dynamic events with high accuracy and low manual interference, which are essential for scaling up tissue engineering and other advanced applications such as drug screening platforms.

The incidence of Parkinson's disease (PD) has been steadily increasing globally, while traditional two-dimensional cell cultures and animal models face significant challenges in effectively elucidating its complex pathological mechanisms and screening potential drugs. Advancedin vitromodels that incorporate patient-specific characteristics and three-dimensional (3D) microenvironments have emerged as powerful alternatives. This review first outlines current perspectives on PD etiology and pathogenesis, highlighting their implications for 3D modeling systems. A systematic comparison evaluates organoid, microfluidic, and 3D bioprinting platforms by leveraging their recent applications in PD mechanistic studies and therapeutic screening. The utilization of these cutting-edge technologies in PD model development not only deepens mechanistic insights but also streamlines therapeutic innovation, paving the way for effective treatments against this debilitating neurodegenerative disorder.

The regeneration of bone tissue depends on the harmonious interaction between blood vessels and nerve fibers, both essential for various physiological and pathological functions in the skeletal system. The key to mimicking the structure and function of natural bone lies in integrating angiogenesis and neurogenesis processes to prepare vascular-nerve-tissue-engineered bone (TEB). Unlike traditional strategies for constructing vascular nerve TEB (such as adding growth factors or cells to scaffolds or preparing composite scaffolds), this study employs a bottom-up approach, using modular microtissue units to construct novel vascular nerve TEB. Initially, vascular-nerve-bone microtissues composed of bone marrow mesenchymal stem cells, endothelial progenitor cells (EPCs), and Schwann cells (SCs) were generated through three-dimensional coculture in microporous array plates. These vascular-neural-bone microtissues were then encapsulated as modular building blocks within gelatin methacrylate (GelMA) hydrogels to construct large-scale vascular-neural TEB. The microtissue-based vascular-neural-TEB construction protocol demonstrated feasibility at the molecular, cellular, and tissue/organ levels. Research findings indicate that the GelMA/MSC/EPC/SC vascular-neural-TEB possesses concurrent capabilities for angiogenesis, neurogenesis, and osteogenesis during bone repair. These findings provide novel insights for the construction of multifunctional bone grafts and lay the foundation for the clinical treatment of bone defects.

Inflammatory signalling is a major contributor to cardiac dysfunction in diseases such as sepsis, myocarditis, and heart failure, yet existingin vitromodels lack the multicellular complexity and physiological relevance needed to accurately recapitulate human cardiac pathology. In this study, we used human cardiac spheroids, three dimensional microtissues composed of cardiomyocytes, fibroblasts, and endothelial cells to study lipopolysaccharide (LPS) induced inflammation. High resolution confocal microscopy confirmed a radial distribution of cell types within the spheroids. Following 24 h LPS stimulation, the spheroids exhibited no significant increase in lactate dehydrogenase release, indicating preserved membrane integrity and absence of overt cytotoxicity. Nevertheless, a robust inflammatory response was observed at both transcriptional and protein levels, including significant upregulation and secretion of TLR2, IL6, TNF, CXCL8, and CCL2. Mitochondrial stress testing revealed significantly reduced basal respiration, ATP production, and maximal respiratory capacity. Functional analyses showed impaired contractility characterized by reduced beat rate, delayed time to peak contraction, and prolonged relaxation time. Together, these findings demonstrate that human cardiac spheroids mount a physiologically relevant, multicellular inflammatory response that compromises both mitochondrial metabolism and mechanical performance. The model offers a powerful platform for investigating innate immune activation and for screening therapeutic interventions targeting inflammation induced cardiac dysfunction.

A major challenge in bone tissue engineering is the embedding of osteocyte-like cells at high density within a mineralized matrix at the micro-scale and a trabecular-like architecture at the macro-scale. Volumetric bioprinting (VBP) enables rapid creation of complex cell-laden constructs through tomographic light projections. However, integrating both high cell densities and inorganic mineral precursors into VBP processes poses challenges due to light scattering, which can compromise print fidelity. In this study, we aim to combine bioinspired polymer-induced liquid-phase precursor (PILP) mineralization with VBP to fabricate cell-laden gelatin methacryloyl hydrogel constructs with amorphous mineral precursors. By stabilizing amorphous mineral precursors with poly-aspartic acid, light scattering is sufficiently reduced to enable printing. Tuning the refractive index of this mineralizing bioresin allows fast VBP of mineralized bone-like constructs with cell densities of up to 3 million cells/ml. The constructs display high cell viability (>90%) and enhanced mineralization when cultured in osteogenic conditions with βglycerophosphate. Encapsulated human mesenchymal stromal cells exhibit an early osteocytic phenotype after 28 days of differentiation. Collectively, this PILP-assisted VBP platform holds promise for the development of advanced in vitro bone models with more physiologically relevant architecture and cellular composition.